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Laboratory of Experimental Rheumatology and Neuroendocrino-Immunology, Division of Rheumatology, Department of Internal Medicine I, University Hospital, 93042 Regensburg, Germany
Correspondence: Address all correspondence and requests for reprints to: Rainer H. Straub, M.D., Professor of Experimental Medicine, Laboratory of Experimental Rheumatology and Neuroendocrino-Immunology, Department of Internal Medicine I, University Hospital, 93042 Regensburg, Germany. E-mail: rainer.straub{at}klinik.uni-regensburg.de
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Abstract |
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 Top Abstract I. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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This review delineates how the effects of estrogens are dependent on criteria such as: 1) the immune stimulus (foreign antigens or autoantigens) and subsequent antigen-specific immune responses (e.g., T cell inhibited by estrogens vs. activation of B cell); 2) the cell types involved during different phases of the disease; 3) the target organ with its specific microenvironment; 4) timing of 17ß-estradiol administration in relation to the disease course (and the reproductive status of a woman); 5) the concentration of estrogens; 6) the variability in expression of estrogen receptor 
and ß depending on the microenvironment and the cell type; and 7) intracellular metabolism of estrogens leading to important biologically active metabolites with quite different anti- and proinflammatory function. Also mentioned are systemic supersystems such as the hypothalamic-pituitary-adrenal axis, the sensory nervous system, and the sympathetic nervous system and how they are influenced by estrogens.
This review reinforces the concept that estrogens have antiinflammatory but also proinflammatory roles depending on above-mentioned criteria. It also explains that a uniform concept as to the action of estrogens cannot be found for all inflammatory diseases due to the enormous variable responses of immune and repair systems.
B 
—studies in vitro |
I. Introduction |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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In a previous review, the role of the menopause on proinflammatory cytokine activity has been extensively studied (6). This review focused on the increase of proinflammatory cytokines with the menopause (the fall of estrogens and other gonadal steroids), but this review only marginally touched chronic inflammatory diseases. Another review on gonadal steroids and T and B cell immunity was presented 10 yr ago, but since then, a lot of new information has been generated (2). This is particularly true with respect to chronic diseases that formerly have not been allocated to "inflammatory diseases" such as bone resorption. This is important because incidence rates over age for osteoporosis match incidence rates over age for, e.g., rheumatoid arthritis (RA). Furthermore, the progress in animal models of chronic inflammation was immense, and a good portion of this information has been published after 1996.
There is still the unresolved paradox with respect to the immunomodulating role of estrogens. On one side, we recognize inhibition of bone resorption and suppression of inflammation in several animal models of chronic inflammatory diseases. On the other side, we realize the immune supportive role in trauma/sepsis and the proinflammatory effects in some chronic autoimmune diseases in humans (as an initiating or perpetuating factor). This review examines possible causes for this paradox and suggests a solution in the Discussion (Section XV).
Because the author has been confronted by an enormous quantity of literature (approximately 5200 references in the primary retrieval), this review only focuses on estrogens. It is evident that androgens and progesterone are important in inflammation, but presentation of these subjects was not possible due to space constraints. Throughout the text, tables, and figures, the Système International unit for the concentration is given as moles/liter (abbreviated with M).
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II. Estrogen Receptors in Inflammation and Hypoxia |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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and ERß, is of outstanding importance because a preponderance of one ER subtype over the other might change estrogen effects (7, 8). For instance, in synovial tissue of patients with RA, macrophage-like and fibroblast-like synoviocytes were positive for ER (9, 10, 11) and ERß (12). Importantly, one study demonstrated higher density of ERß+ cells than of ER+ cells (13). Others demonstrated a higher density of ERß+ cells in relation to ER+ cells in RA synovial tissue compared with controls (12). ERß preponderance was observed in all three synovial compartments investigated: in the lining cell layer, in fibroblasts, and in inflammatory cells (12). Similarly, the amount of ER was lower in T cells of patients with systemic lupus erythematosus (SLE) than controls, but the quantity of ERß was similar, which indicates a relative increase of ERß in relation to ER in SLE patients (14). In animals subjected to trauma and hemorrhage, a general inflammatory condition, ERß mRNA expression was increased, whereas ER expression was decreased (15). These studies suggest inflammation-dependent up-regulation of ERß relative to ER.
It was further demonstrated that hypoxia, which typically accompanies inflammatory conditions, reduced expression of ER (16, 17), and oxidative stress increased the expression of ERß (18). In endothelial cells (E304), oxidative stress increased ERß relative to ER. In activated macrophages, lipopolysaccharide (LPS) plus interferon (IFN)- in the presence of hypoxia increased expression of ERß but not of ER (18). These studies support a concept of up-regulation of ERß relative to ER under hypoxic conditions, which might lead to a preponderance of signaling through ERß pathways.
The preponderance of ERß relative to ER under inflammatory and hypoxic conditions might influence estrogen effects. One might hypothesize that this depends on the time point of estrogen application in relation to the state of an inflammatory disease. A situation before disease outbreak or at the beginning of a disease with slight tissue inflammation and little hypoxia might be governed by a balance of ER and ERß, whereas in the chronic phase of a disease with much inflammation and a higher degree of hypoxia, ERß is increased relative to ER. In such a situation, ERß-mediated cross-modulation of ER, e.g., demonstrated as ERß-mediated inhibition of ER-stimulated IL-1 secretion (8), might play an important role in the chronic disease course.
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III. Estrogen Modulation of Specific Target Cells |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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A. Estrogens and human peripheral blood mononuclear cells or whole cell cultures
In human peripheral blood mononuclear cells (PBMCs) in the presence of LPS (at 1 ng/ml 
EC50 of LPS), E2 inhibited TNF at concentrations of 10–10 to 10–7 M in male subjects and at 10–8 to 10–7 M in female subjects, but E2 had a stimulating effect in the absence of LPS (20). In whole human blood cultures, E2 at 10–10 to 10–8 M decreased spontaneous secretion of IL-6, TNF, IL-1 receptor antagonist (IL-1ra), IL-1ß, and the ratio of IL-1ß/IL-1ra compared with control, but E2 did not strongly change LPS-stimulated cytokine release (LPS at 500 ng/ml, which is largely higher than the EC50 of LPS) (21). In PBMCs, E2 inhibited TNF release in postmenopausal women with fractures in a dose-dependent manner between 10–12 and 10–6 M but had no consistent effect on PBMCs derived from men or premenopausal women (22).
From these data, it seems that E2 at periovulatory to pregnancy levels inhibited proinflammatory cytokines from PBMCs, which is not unchallenged (23). The contrasting results might be due to divergent effects of E2 on different subtypes of immune cells. Necessarily, it is important to examine the effects of E2 on immune cell subtypes, which is demonstrated in the subsequent sections.
B. Estrogens, the B cell, and antibodies
Mitogen stimulation of T and B lymphocytes resulted in accumulation of IgM-containing and IgM-secreting cells, which was further enhanced by E2 (late follicular to pregnancy levels). This E2 effect was mediated by inhibiting T cell-mediated suppression of B cells (24). A similar phenomenon was observed with human PBMCs stimulated with sheep red blood cells, whereby addition of 10–9 to 10–7 M E2 (maximum at 2 x 10–9 M) augments the antigen-specific immune response by inhibiting CD8+ T cell-mediated suppression of B cells (blocked by tamoxifen, no effect at 5 x 10–10 M) (25). E2 increased antibody-forming cells against sheep erythrocytes (maximum at 1.8 x 10–9 M, no effect at 3.7 x 10–11 M) (26). In PBMCs from normal donors, E2 at pregnancy levels enhanced mitogen-induced generation of antibody-producing cells (27). In human PBMCs, E2 at 10–10 to 10–8 M (IC50, 10–9 M) enhanced IgG and IgM production without altering cell viability and proliferation, and anti-IL-10 antibody partially blocked the E2 effect (28). A similarly enhanced production of anti-double-stranded DNA antibodies was observed with E2 at 10–10 to 10–8 M (IC50, 10–9 M), and E2 increased the B cell-stimulating cytokine IL-10 from monocytes (29). In PBMCs of female rhesus macaques, E2 at 3.7 x 10–9 M increased the frequency of Ig-secreting cells (30).
In women on oral contraceptives (OC), serum levels of IgA and IgG were increased (31). In cycling women, the largest quantities of Ig were detected before ovulation (31). In female rhesus macaques, the frequency of Ig-secreting cells was high in tissues collected from animals in the periovulatory period of the menstrual cycle (30).
In mice, administration of E2 (pregnancy levels) markedly augments the ability of CD5+ B cells to express their autoimmune potential (32). In male MRL lpr/lpr mice (a lupus model), administration of E2 (pregnancy levels) for 31 wk increased the frequency of IgG- and IgM-producing cells (33). In Swiss male mice, administration of E2 (pregnancy levels) for 4 wk increased serum levels of IgG1. In female C3H/N mice, the same treatment increased serum IgG2 levels (33), which was confirmed by others in nonautoimmune C57BL/6J mice with respect to anti-double-stranded DNA antibodies (34). In the collagen-induced arthritis model, E2-treated mice (pregnancy levels) demonstrated an increase in the levels of IgG1 anti-collagen type II antibodies (35).
In Balb/c mice transgenic for the H chain of anti-double-stranded DNA antibodies, E2 (pregnancy levels) rescued naive autoreactive B cells that normally are deleted and caused them to mature to a marginal zone phenotype (36). E2 (at pregnancy levels) further led to the activation of this population, causing an elevation of serum anti-double-stranded DNA antibody titers and renal disease (36). E2 enhanced transitional B cell resistance to apoptosis and expanded the population of marginal zone B cells (36). Estriol (E3) treatment at high pregnancy doses increased IgG1 antibodies in the experimental autoimmune encephalitis (EAE) model in mice most probably by an E3-induced increase of IL-10 production from T cells (37).
From above-mentioned studies, it is clear that E2 can stimulate antibody production by B cells, probably by inhibiting T cell suppression of B cells. In contrast, E2 at high concentrations leads to a suppression of B-lymphocyte lineage precursors (38). In addition, IL-7-responsive B lineage precursors were greatly expanded in genetically hypogonadal female mice. Estrogen replacement in these mice resulted in a dose-dependent reduction in B cell precursors (39). Similarly, pregnancy led to a clear decline in B cell precursors in the bone marrow, which was blocked by ICI 182,780, and an increase was observed in hypogonadal mice (40). E2 at pregnancy levels induced a down-regulation of B lymphopoietic cells in bone marrow of young ovariectomized mice, which is mediated through both ER and ERß (41).
In conclusion, E2 at periovulatory to pregnancy serum levels is able to stimulate antibody secretion under healthy conditions but also in autoimmune diseases, whereas similar serum levels of E2 lead to a suppression of bone marrow B cell lineage precursors. Today we know that autoimmune diseases are not uniform, and sometimes B cells play a major role (as substantiated by the success of anti-CD20 antibody treatment in humans, Rituximab). In chronic inflammatory disorders, where B cells play a decisive role, E2 would promote the disease when autoaggressive B cells are already present, whereas chronically elevated E2 would inhibit initiation of an autoimmune disease when no such B cells are available. This might be a good reason why particularly B cell-dependent diseases such as SLE, mixed connective tissue disease (Sharp syndrome), IgA nephropathy, dermatitis herpetiformis, gluten sensitive enteropathy, myasthenia gravis, and thyroiditis appear in women in the reproductive years, predominantly, in the third or fourth decades of life. It also explains why sometimes these particular diseases are started during or after pregnancy. These ideas are further developed at the end of this review.
C. Estrogens and the T cell
For more than 20 yr, the so-called T helper type 1 (Th1) immune response by CD4-positive T cells was thought to drive cell-mediated immunity leading to tissue damage as in chronic inflammatory autoimmune diseases (42). In contrast, T helper type 2 (Th2) CD4-positive cells drive certain antibody-mediated responses, particularly those that are involved in allergy (42). It is also known that these two pathways reciprocally inhibit each other. Cytokines such as IFN-, IL-12, and TNF were allocated to Th1 reactions, and IL-4, IL-5, and IL-10 to Th2 responses. In recent years, this viewpoint has changed due to the appearance of antiinflammatory T regulatory cells producing TGF-ß and proinflammatory T helper type 17 cells (Th17) producing IL-17 (43). Today, Th17 cells are thought to be the main responsible cells for chronic inflammatory tissue destruction in autoimmune diseases (43). Importantly, this clarifies some unresolved questions such as the ameliorating effects of IFN- in several autoimmune diseases, because IFN- inhibits Th17 cells, which is opposite for TNF that stimulates Th17 cells (Fig. 1
) (43). In addition, IL-4 from Th2-positive cells also inhibits Th17 cells, leading to amelioration of chronic inflammatory autoimmune diseases (Fig. 1) (43). The interested reader is referred to the important recent revision of this subject by L. Steinman (43). With this information in mind, effects of estrogens on T cells need to be revised (Fig. 1). Unfortunately, no direct effects of estrogens on Th17 cells or IL-17 secretion have been described until now. Thus, an influence of estrogens on Th17 cells is deduced from estrogen effects on other T cell cytokines (Fig. 1).
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was also increased at the same dose. In contrast, the effect of E2 on antigen-stimulated secretion of TNF was biphasic, with enhancement occurring at lower doses at 3.6 x 10–9 and 1.8 x 10–8 M, and inhibition present at high concentrations of 3.6 x 10–8 to 3.6 x 10–7 M (44). The same group reported that estrone (E1) and E3 at pregnancy levels enhance secretion of antigen-induced or anti-CD3-stimulated IL-10 and IFN- from CD4+ cells of MS patients, almost identical to that of E2 (45). In addition, E1 and E3, like E2, had a biphasic effect on TNF secretion, with lower concentrations stimulatory and high doses inhibitory (45). In T cells of patients with MS, E3 at pregnancy levels stimulated secretion of IL-10 and inhibited TNF [via inhibition of nuclear factor-B (NF-B)] (46). As compared with the pretreatment situation, relapsing remitting MS patients treated with oral E3 demonstrated a marked decrease in T cell-dependent delayed type hypersensitivity responses to tetanus (47).
These findings were corroborated by studies in mice. In ovariectomized mice, E2 replacement (pregnancy levels) suppressed the T cell-mediated delayed type hypersensitivity reaction (48, 49, 50). Treatment of CD4+ T cells from nonobese diabetic mice with E2 at pregnancy levels increased secretion of IFN- (51). E2 at pregnancy levels increased -galactosylceramide-induced IFN- synthesis by invariant natural killer T cells (52). Ovariectomized C57BL/6 mice were given E2 replacement (pregnancy levels), which increased IFN- mRNA in concanavalin-A-activated enriched splenic T cells but there was no effect on IL-4 mRNA (53). In ovariectomized mice, E2 replacement (proestrus to early pregnancy levels) results in the selective development of IFN--producing cells (via ER) (54). All these experiments clearly demonstrate the positive effect of E2 at pregnancy levels on IFN- production in T cells. Because IFN- has been allocated a Th17-inhibiting role (Fig. 1
), its increase by E2 at pregnancy doses and the E2-mediated inhibition of TNF must be viewed as a favorable effect in chronic inflammation.
Other important indications for the estrogen-meditated inhibition of the proinflammatory TNF came from bone research. Ovariectomy enhanced T cell production of TNF and subsequent bone loss, which was abolished by E2 replacement therapy (proestrus or early pregnancy levels) (55). Ovariectomy-induced bone loss, which was absent in T cell-deficient nude mice, was restored by adoptive transfer of wild-type T cells but not by reconstitution with T cells from TNF-deficient mice (56).
In contrast to the inhibitory effects of E2 on TNF, E2 can increase antiinflammatory T cell responses. In C57BL/6 mice, E2 at pregnancy levels (not at 10–10 or 10–11 M) increased IL-4 secretion and GATA-3 mRNA levels from ER+ CD4+ T cells, whereas this effect was abrogated in ER-deficient T cells (57). Forkhead box P3 (FoxP3) in CD4+CD25+ cells is an important indicator of T regulatory cells (Fig. 1). In C57Bl/6 mice, E2 (pregnancy levels) is capable of augmenting FoxP3 expression in vitro and in vivo and increases CD25+ cell number, which is reduced in ER-deficient animals. Pregnancy stimulated the expression of FoxP3 protein to a similar degree (58), but progesterone might also play a supportive role.
In addition to effects of estrogens on peripheral CD4+ T cells, estrogens have a strong influence on the development and maintenance of thymic function and, thus, on generation of naive CD4+ and CD8+ T cells. ER-deficient mice have smaller thymi than their wild-type littermates; and E2-induced thymus atrophy was reduced in ER-deficient mice (59, 60). In one study, the CD4+/CD8+ phenotype was not changed in ER-deficient mice (59), but a higher frequency of CD4+CD8+ thymocytes was found in another study (60). In ER-intact animals, E2 (pregnancy levels) induced profound thymus involution in normal and adrenalectomized mice (49, 61). However, E2 did not induce lymphocytopenia in the peripheral organs but stimulated extrathymic T cell numbers in the liver and other organs (61). E2 treatment (pregnancy levels) for 4 wk decreased thymus cellularity and the percentage of CD4+ cells in the spleen, which was not observed in double ER-deficient mice (62). Whether this E2-induced thymus involution is positive or negative in chronic inflammation remains to be established because autoaggressive but also autotolerant T cells might be eliminated.
In conclusion, in humans and mice, E2 at periovulatory to pregnancy levels stimulates IL-4, IL-10, and IFN- but inhibits TNF from CD4+ T cells. In humans and mice, E3 and E2, respectively, at pregnancy levels inhibit T cell-dependent delayed type hypersensitivity. In mice, E2 at pregnancy levels increases the Th2-relevant GATA-3 and the T regulatory cell-relevant FoxP3, which might shift the immune response toward tolerance. Because we know that increased IL-4, IL-10, and IFN- in the presence of low TNF support an antiaggressive immune response (Fig. 1), collectively, these data suggest that E2 at periovulatory to pregnancy levels might be a favorable hormone leading to down-regulation of T cell-dependent immunity. The question appears as to what would happen if E2 falls to postmenopausal levels (metestrus) before initiation or during the course of an autoimmune disease. One might assume that the protecting effects of E2 are getting lost under these conditions.
D. Estrogens and the monocyte/macrophage/dendritic cell
In human promonocytic cells (U937), E2 (late pregnancy levels) inhibited LPS-stimulated IL-6 secretion (63). In human monocytic cells (THP-1), the absence of E2 led to an increase of surface CD16 (stimulatory Fc -receptor III), which after cross-linking stimulated secretion of TNF, IL-1ß, and IL-6 (64). This indicates that E2 normally suppresses these cytokines stimulated via cross-linking of CD16.
In mouse splenic macrophages, pretreatment for 16 h with E2 between 3.6 x 10–11 and 1.8 x 10–9 M decreased LPS (1000 ng/ml, EC50 of LPS in mice and rats)-induced TNF production, which was associated with a decreased NF-B-binding activity (65). In murine bone marrow-derived macrophages, preincubation with E2 at pregnancy levels (maximum at 10–8 M) inhibited LPS-induced TNF release (no effect on cells in the absence of LPS) (66). In murine peritoneal macrophages and RAW 264.7 cells, E2 at pregnancy levels inhibited LPS-stimulated nitrite production and TNF secretion (67). In the mouse macrophage cell line RAW 264.7, preincubation with E2 (proestrus levels) and subsequently activated with LPS inhibited nitric oxide (NO) and TNF release (68).
E2 treatment (pregnancy levels) decreased TNF and IL-12 production in mature mouse dendritic cells (69). This study demonstrated an interesting shift toward cytokines such as IL-4 and IL-10. Transfer of E2 (pregnancy levels)-exposed splenic dendritic cells from Lewis rats obtained on d 12 after immunization with myelin basic protein prevented the expansion of CD4+ T cells and increased proportions of regulatory T cells producing IL-10 and CD4+CD28- suppressor T cells, accompanied with increased IL-10 and IFN- (see explanation in Fig. 1), and reduced TNF production (70). This tolerating effect might be mediated by up-regulation of indoleamine 2,3-dioxygenase (70, 71). These inhibitory activities of E2 might be different at lower levels.
Indeed, in human peripheral monocytes, E2 (early follicular to periovulatory levels) resulted in maximal IL-1 stimulation. At higher concentrations of E2 (late pregnancy levels), a significant reduction in IL-1 activity was observed (72). In LPS-stimulated human monocytic cells, E2 (periovulatory levels) increased IL-1ß and IL-1 mRNA expression (73). The mouse macrophage cell line RAW 264.7 was stably transfected with the human ER and an IL-1ß promoter reporter construct. Pretreatment with E2 at 1.8 x 10–8 to 1.8 x 10–6 M (pregnancy levels, no effect at 1.8 x 10–10 M) markedly enhanced LPS-induced IL-1ß promoter-driven activity (blocked by ICI 182,780) (74). In adherent rat peritoneal macrophages, E2 at proestrus levels (little effect at 3.7 x 10–11 M) stimulated secretion of IL-1 with and without LPS, and IL-1 secretion was higher in cells from female compared with male rats (75). These studies demonstrate that lower levels of E2 particularly increase IL-1ß secretion.
In conclusion, in human and mouse/rat monocyte/macrophage-like cells, secretion of IL-1ß is increased at periovulatory/proestrus to early pregnancy levels, whereas IL-1 secretion is inhibited at high pregnancy levels. It is further obvious that proestrus to pregnancy levels of E2 decreased LPS-stimulated TNF secretion in mouse/rat cells. Thus, with respect to TNF, E2 at pregnancy levels exerts similar effects in macrophages compared with T cells, which in both cell types is most probably due to inhibition of NF-B. The dichotomous effect of E2 on IL-1ß and TNF at high and low concentrations is most probably due to inhibition of NF-B at high concentrations (see Section IV.A).
E. Estrogens and macroglial/microglial cells
In primary rat astrocytes, pretreatment with high doses of E2 at 10–6 M reduced the amyloid ß-peptide and LPS-induced activation of NF-B (76). In primary rat astrocytes, E2 at pregnancy levels inhibited the receptor for activated C kinases-1 (RACK-1, the anchoring protein involved in protein kinase C shuttling), protein kinase C activation, LPS-stimulated TNF mRNA, and inducible NO synthase (iNOS) expression (77). In primary rat cortical astrocyte cultures, E2 (proestrus to pregnancy levels) increased neuronal cell survival and both the expression and release of the neuroprotective cytokines TGF-ß1 and TGF-ß2 (blocked by ICI 182,780), which is mediated by a membrane-bound ER (78).
In primary rat microglia and a microglial cell line (N9), pretreatment with E2 at proestrus to pregnancy levels attenuated phorbol-12-myristate-13-acetate-stimulated superoxide release, LPS-stimulated phagocytic activity, and iNOS protein expression, but did not alter NF-B activation (blocked by ICI 182,780). E2 at similar concentrations induced rapid phosphorylation of the p42/p44 MAPK, and the MAPK inhibitor PD 98059 blocked the antiinflammatory effects of E2 (79). In LPS-stimulated microglial cells (N9), E2 at pregnancy levels increased IL-10 while decreasing release of TNF (similar as in T cells). In the same cell type, E2 at pregnancy levels attenuated the LPS-induced increase of membrane major histocompatibility complex class I molecules, CD40, and CD86, and E2 also decreased the percentage of nonstimulated cells positive for major histocompatibility complex class I and II, CD40, and CD152, Fas, and Fas ligand (FasL), whereas CD80 cell surface staining was increased (80).
In primary rat microglia cells, E2 at 10–10 to 10–8 M (proestrus levels, maximum at 10–9 M, minimum at 10–11 M) inhibited iNOS expression and reduced the accumulation of nitrites and nitrates upon various inflammatory stimuli. Under the same conditions, production of prostaglandin E2 and matrix metalloproteinase (MMP)-9 were also inhibited (blocked by ICI 182,780) (81). In murine microglial cells (BV-2), which naturally express only ERß, E2 at 10–13 to 10–9 M (also E3 at 10–10 to 10–8 M) decreased LPS-stimulated NO production and iNOS expression and expression of cyclooxygenase-2 (82).
In conclusion, experiments with mouse and rat macroglial and microglial cells demonstrate that E2 at proestrus to pregnancy levels exerts neuroprotective effects by increasing TGF-ß and by inhibiting iNOS and NO release, and reducing expression of proinflammatory cytokines and prostaglandin E2 production.
F. Estrogens and natural killer cells
An early study in BALB/c mice described that E2 treatment (proestrus to pregnancy levels) led to unresponsiveness of natural killer cells to poly I.C., which was not mediated by changes of IFN (83). Others demonstrated that E2 treatment (pregnancy levels) inhibits IFN--stimulated natural killer cell activity (84). This was corroborated by a study in different mouse strains, which demonstrated that E2 (pregnancy levels) reduced natural killer cell cytotoxicity. A high degree of suppression was observed in C3H/N, DBA/1, and NZB/W mice, whereas other strains displayed a low degree of suppression (C57BL/6 and MRL lpr/lpr mice) (33). In rhesus monkeys, E2 deprivation by ovariectomy (serum levels, 3.7 x 10–11 M, postmenopausal) reduced natural killer cell activity (85). These studies indicate that E2 has bimodal effects inhibiting natural killer cells at pregnancy levels but stimulating these cells at diestrus to proestrus levels.
G. Estrogens and fibroblast
In human uterine fibroblasts, E2 at 10–10, 10–8, and 10–6 M (no effect at 10–12 M) increases the concentration of basic fibroblast growth factor (86). In human cultured fibroblast-like synoviocytes from patients with RA, E2 at pregnancy levels had no effect on constitutive production of IL-6, but E2 increased IL-1ß-induced IL-6 production (87). Because E2 has some stimulatory effects on IL-1ß secretion of monocyte/macrophages at low levels (see Section III.D), the E2-stimulated IL-1ß-induced IL-6 secretion might be a proinflammatory signal.
In primary human adipose fibroblasts, E2 at pregnancy levels increased TNF receptor 1 mRNA and protein levels (no effect at 10–11 to 10–9 M), whereas E2 at the same concentration decreased TNF receptor 2 mRNA (increase at 10–11 to 10–10 M) (88). Assuming that a similar modulation of TNF receptor 1 and TNF receptor 2 also happens in other cell types, and because the TNF receptor 1 is mainly responsible for tissue-specific autoimmunity and the TNF receptor 2 is mainly responsible for proproliferative effects and tissue damage (89), E2 at pregnancy levels might initiate an autoimmune disease, whereas E2 at postmenopausal levels might support tissue damage. This needs to be investigated in B/T cells and macrophages/dendritic cells.
In human endometrial stromal cells (fibroblasts), E2 at pregnancy levels inhibited the secretion of monocyte chemoattractant protein (MCP)-1 with an IC50 of 10–8 M (slight increase at 10–12 M, no inhibition at 10–11 M and 10–10 M, maximum inhibition at 10–6 M) (90). In murine fibroblasts, E2 (maximum at 3.7 x 10–12 M and 9.9 x 10–10 M, no effect in the middle range of 3.7 x 10–11 M) inhibited serum-stimulated MCP-1 (91). In a mouse stromal cell line (ST-2), E2 (pregnancy levels) induced up-regulation of osteoprotegerin, an osteoprotective factor. The effect was dependent on ER, but not of ERß. Moreover, estrogen withdrawal after 5-d pretreatment, mimicking the event occurring in vivo at menopause, dramatically diminished the expression of osteoprotegerin (92). In rabbit uterine cervical fibroblasts, E2 at pregnancy levels (not at 10–10 M) decreased secretion of procollagenase (precursor of MMP-1) and prostromelysin (precursor of MMP-3), whereas the natural antagonists, the tissue inhibitor of MMPs (TIMP), increased in the presence of E2 (93).
In conclusion, in normal human and mouse/rat fibroblasts, E2 exerts tissue-protective effects by up-regulation of basic fibroblast growth factor, osteoprotegerin, and TIMP, and by down-regulation of the proforms of MMP-1 and MMP-3. In contrast, in synovial fibroblast of patients with RA, E2 stimulates the proinflammatory cytokine IL-6 in the presence of IL-1ß, the latter is most probably not derived from fibroblasts. This demonstrates that a change of the environmental context with an increase of IL-1ß from macrophages might render E2 a proinflammatory factor at lower levels.
H. Estrogens and vascular smooth muscle cells
In human vascular smooth muscle cells, E2 at 10–10 to 10–8 M (also E3) inhibited mRNA expression of platelet-derived growth factor-A, IL-1, and IL-6 but not of TGF-ß (94). Additionally, E1 sulfate at 10–10 to 10–8 M inhibited mRNA expression of platelet-derived growth factor-A, IL-1, and IL-6 (94). These antiinflammatory effects of E1 sulfate are to be expected in the presence of steroid sulfatase (leading to estrogen activation). In human vascular smooth muscle cells, steroid sulfatase expression levels were found to be higher in female aortas with mild atherosclerotic changes than in those with severe atherosclerotic changes and in male aortas regardless of atherosclerotic changes (95). In contrast, expression levels of estrogen sulfotransferase (leading to estrogen inactivation) were higher in female aortas with severe atherosclerotic changes and in male aortas than in female aortas with mild atherosclerotic changes (95). Furthermore, IL-1ß markedly inhibited the expression of the sulfatase but stimulated the expression of estrogen sulfotransferase. This was accompanied by IL-1ß-induced inhibition of E2 production from E1 sulfate (95). This confirms a similar effect of the proinflammatory cytokine TNF on the sulfatase in macrophages and fibroblasts (96, 97). By way of inhibiting the sulfatase and stimulating the sulfotransferase, these cytokines add to a proinflammatory milieu.
In human coronary artery smooth muscle cells (express ER and ERß), E2 at 10–9 to 10–7 M (not at 10–11 and 10–10 M) inhibited MCP-1 mRNA expression and protein production (98). In primary aortic smooth cells, E2 at 10–9 M (no other concentration tested) inhibited iNOS expression and reduced the accumulation of nitrites and nitrates consequent to various inflammatory stimuli (99).
In rat vascular smooth muscle cells, E2 did not influence stimulated IL-6 production (100). In rat vascular smooth muscle cells, E2 at pregnancy levels inhibited cell proliferation and both constitutive and IL-1ß-stimulated NF-B activation (101).
In conclusion, E2 at pregnancy levels by way of several pathways contributes to an antiinflammatory milieu in vascular smooth muscle cells. However, presence of a proinflammatory cofactor such as IL-1ß might change availability of E2 by inhibiting E2 allocation and stimulating E2 degradation.
I. Summary
Figure 2 summarizes the most important effects of high and low concentrations of estrogens on different types of cells involved in chronic inflammation. At pregnancy levels, E2 inhibits important proinflammatory pathways such as TNF, IL-1ß, IL-6, MCP-1, iNOS expression, production of MMPs, and activity of natural killer cells, whereas E2 at the same concentration stimulates antiinflammatory pathways such as IL-4, IL-10, TGFß, TIMP, and osteoprotegerin. At lower concentrations, E2 stimulates TNF, IFN-, IL-1ß, and activity of natural killer cells. This dichotomy of E2 at high vs. low concentrations is not observed for B cells, because antibody production is stimulated throughout the concentration range (Fig. 2). Consequences of these E2 effects for chronic inflammation are summarized in the discussion (Section XV).
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IV. Estrogens and Inflammatory Factors |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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BB is an important factor in proinflammatory signaling that can interact with ER pathways (summarized in Ref. 102). Since the overview of McKay and Cidlowski (102), a substantial amount of new data have been generated in cells relevant for inflammation.
In mouse monocytic cells (RAW 264.7), E2 (pregnancy level) blocked LPS-induced DNA binding and transcriptional activity of the p65 subunit of NF-B by preventing its nuclear translocation (103). Similarly, in primary rat astrocytes, pretreatment with high doses of E2 at 10–6 M reduced the LPS-induced activation of NF-B (76). In lymphocytic HeLa cells, E2 (pregnancy levels) almost completely inhibited phorbol-12-myristate-13-acetate-induced NF-B activation and inhibitor of NF-B (IB) protein degradation (104). In rat vascular smooth muscle cells, E2 (pregnancy levels) inhibited both constitutive and IL-1ß-stimulated NF-B activation (101). In human aorta endothelial cells, E2 at 10–10 to 10–7 M (IC50, 10–9 M) inhibited NF-B-mediated luciferase reporter activity and IL-6 secretion (105). In different cell types, it was demonstrated that ER impairs TNF-induction of IL-6 by preventing binding of c-rel and, to a lesser extent, RelA proteins to the NF-B site on the IL-6 promoter (106). This was corroborated by others who demonstrated that E2 inhibited IL-6 production by interfering with the function of NF-B at the IL-6 promoter (107). In macrophages, E2 blocked LPS-induced DNA binding and transcriptional activity of c-rel (103). All these studies clearly show reciprocal inhibition of ER and NF-B pathways in different cell types. In most occasions, periovulatory to pregnancy levels of E2 have been used. However, other important proinflammatory pathways might be linked to ERs as well.
NF-B activity is suppressed in PBMCs from pregnant women compared with age-matched nonpregnant women (108). In a burn injury model in vivo, E2 treatment resulted in a decrease in splenic NF-B activation (109). In rats receiving transient middle cerebral artery occlusion in vivo, substantial apoptosis and inflammatory responses were observed, including IB phosphorylation, NF-B activation, and iNOS overexpression (110). In this model, E2 treatment (short-term pregnancy levels) produced strong protective effects by reducing infarct volume and neuronal apoptosis and by inhibiting NF-B activation and iNOS overexpression (110). In cerebral arteries of ovariectomized rats, E2 treatment at pregnancy levels reduced cerebrovascular NF-B activity (111).
In conclusion, E2 at periovulatory to pregnancy levels inhibits NF-B activation, which must be viewed as an antiinflammatory signal. The necessary E2 levels needed for NF-B inhibition have been analyzed in human cells infected with an adenoviral NF-B luciferase reporter. It was shown that E2 concentrations equal to or above 10–10 M are necessary to inhibit NF-B activation. At 10–11 M or below (postmenopausal level), no effects were observed (112). Necessarily, only at these higher E2 concentrations is the antiinflammatory effect to be expected.
B. Adhesion molecules
Adhesion molecules are important factors during inflammation because they enable inflammatory cells to migrate to inflamed compartments. Many studies with human and mice/rat cells in vitro demonstrated that estrogens inhibited expression of adhesion molecules at pregnancy levels, which seems to be opposite at low estrogen levels.
For instance, cultured human umbilical vein endothelial cells (HUVECs) were propagated in steroid hormone-free medium and were pretreated with E2 (pregnancy levels) for 48 h before IL-1 activation. In these cells, E2 strongly inhibited IL-1-induced expression of membrane E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) (113). In HUVECs, E2 at pregnancy levels decreased the expression of TNF-induced VCAM-1 (114). The E2 metabolite 17-epiestriol, which can be generated in vivo, is much more potent than E2 in inhibiting VCAM-1 expression in these cells (U-shaped response curve, no effect at 10–11 and 10–8 M, maximum at 3 x 10–10 M) (attenuated by ICI 182,780) (114).
In an ER overexpressing endothelial cell line (ECV-304), E2 at pregnancy levels inhibited the effects of TNF on VCAM-1 expression and U937 cell adhesion (115). In normal human female iliac artery endothelial cells, E2 at pregnancy levels decreased IL-1ß-stimulated VCAM-1 and ICAM-1 membrane expression (116). In human umbilical cord endothelial cells (ECV-304), the E-selectin promoter is down-regulated by E2 (no effect at 10–11 M, increasing effect between 10–10 and 10–5 M) (117). In cultured brain endothelial cells, E2 at suprapregnancy levels inhibited the basal and IL-1ß-stimulated expression of ICAM-1 (118).
In a functional assay with human monocytic THP-1 cells, 48-h pretreatment with E2 at suprapregnancy doses inhibited nonstimulated and cytokine-stimulated adhesion to human aortic endothelial cells (119). E2 at pregnancy levels decreased adhesion of U937 promonocytes to TNF-stimulated HUVECs (120). In an in vitro model of the vasculature (parallel plate laminar flow chamber), E2 at pregnancy levels potently inhibited monocyte adhesion, and, in parallel, E2 down-regulated Rac1 GTPase activity in monocytes (121). Transfection of monocytic cells with dominant-negative Rac1 significantly decreases adhesion to human endothelial cells (121).
In a rat myocardial ischemia reperfusion injury model, E2 treatment (proestrus levels) lowered myocardial staining of ICAM-1 (122). In a colitis model in mice, E2 treatment (pregnancy levels) decreased expression of ICAM-1 (123). In a rat model of carrageenan-induced pleurisy, E2 (pregnancy levels) inhibited expression of P-selectin and ICAM-1 (124). These studies clearly demonstrate the inhibitory effect of E2 on expression of various adhesion molecules, which appears at proestrus/ovulatory to pregnancy levels. However, the situation might be different at low concentrations of estrogens.
For instance, E2 (diestrus levels) has been shown to up-regulate L-selectin and 4-integrin dependent adhesion pathways in endothelium and lymphocytes (125). Postmenopausal women with coronary artery disease demonstrated increased serum levels of soluble E-selectin, ICAM-1, and VCAM-1, which is an indication of up-regulated membrane-bound adhesion molecules. An increase of E2 serum levels from approximately 3 x 10–11 to 1.5 x 10–10 M decreased serum levels of these soluble adhesion molecules (126). A similar effect of hormone replacement therapy was corroborated by others (127, 128, 129, 130, 131, 132). In addition, treatment of ovariectomized monkeys with conjugated equine estrogens (CEE) decreased serum levels of soluble VCAM-1 but not of soluble E-selectin (133).
In conclusion, E2 at periovulatory/proestrus to pregnancy levels decreased membrane expression of adhesion molecules and cell adhesion to endothelial cells. Some studies indicate that diestrus to ovariectomy/postmenopausal levels of E2 even increased these adhesion molecules. It is interesting that the small increase of E2 from 3 x 10–11 to 1.5 x 10–10 M under estrogen replacement therapy (ERT) in postmenopausal women already reduced soluble forms of adhesion molecules, which most probably indicates also down-regulation of membrane-bound adhesion molecules. Again, a dichotomy of estrogen effects at low and high levels can be observed.
C. IL-1, IL-2, IL-6, IL-8, IL-12, TNF, IFN-—studies in vitro
E2 effects on cytokine secretion depend on cell types, conditions in the milieu, and estrogen concentrations. Tables 1–4 delineate the effects of estrogens on proinflammatory cytokines (particularly of E2 in vitro). Aspects in humans in vivo are extensively demonstrated in Section XII.
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). Generally, E2 effects are only observed at midfollicular (diestrus) to pregnancy levels. Five studies definitely mention that estrogens at low doses (postmenopausal/metestrus levels) did not lead to marked effects (74, 75, 134) or even exert stimulatory effects on IL-1 (72, 135). Particularly, the latter two studies indicate that E2 at low concentrations might stimulate IL-1ß secretion, whereas it inhibits its secretion at pregnancy levels. This indicates that the well-known dichotomy of estrogen effects is also present here. Only two of 15 studies demonstrate no effects of E2 on IL-1 secretion at all (23, 136). With respect to IL-8, E2 at periovulatory to pregnancy levels inhibited secretion of this chemotactic IL as demonstrated in five studies (Table 1), and no opposite results were reported.
The situation is relatively homogenous with respect to IL-6, where most studies indicate a suppressive effect of E2 at periovulatory to pregnancy levels but little or no effects at early follicular or postmenopausal levels (Table 2). This is substantiated by the fact that in human osteoblastic cells the IL-6 promoter is inhibited by E2 in the absence of a functional ER binding site, which is mediated via down-regulation of NF-B and C/EBP (137). Interestingly, E2 had stimulatory effects on IL-6 secretion in synoviocytes (normal or RA), which happens at pregnancy concentrations (Table 2). Only two of 20 studies demonstrated no effects of E2 on IL-6 secretion at all (23, 136).
Similar to IL-6, most studies demonstrated inhibitory effects of estrogens on TNF secretion at midfollicular to pregnancy levels (Table 3). Of the five studies that demonstrate stimulatory effects of E2 on TNF secretion, two studies delineate that stimulation appears only at lower levels of E2, whereas high pregnancy levels inhibit TNF secretion (44, 45). The latter two studies have often been cited to explain the dual role of estrogens at low vs. high concentrations. Only one of 17 studies demonstrated no effect of E2 at all (23).
E2 effects on IFN- secretion are heterogeneous. A unifying concept seems to exist because E2 stimulates IFN- from T cells but inhibits IFN- from macrophages and dendritic cells (Table 4). This separation might be important because quite different effects of estrogens are to be expected depending on the immune cell involved.
In conclusion, important proinflammatory cytokines are typically inhibited at periovulatory (proestrus) to pregnancy levels of E2, which is evident for IL-6, IL-8, and TNF. Nevertheless, low E2 concentrations were demonstrated to have no or even stimulatory effects. This renders a woman in the postmenopausal phase to a more proinflammatory situation, which might well contribute to the manifestation of chronic inflammatory diseases after the menopause.
D. IL-4, IL-10, TGF-ß—studies in vitro
The interleukins IL-4, IL-10, and TGF-ß are assumed to exert antiinflammatory effects as long as tissue-specific autoimmune diseases are considered. However, these cytokines might have a proinflammatory role in B cell-oriented autoantibody-driven autoimmunity, allergic diseases, or diseases with an overshooting fibrotic repair process (TGF-ß).
It is important to mention that some chronic inflammatory disease can demonstrate different types of immune or inflammatory reactions, whereby T cells, B cells, fibroblasts, or macrophages might play a decisive role depending on the time point of inspection or the subtype of the disease. This has been nicely demonstrated in patients with RA and MS (164, 165, 166, 167). With these prerequisites, we might better understand the role that IL-4, IL-10, and TGF-ß play in chronic inflammatory diseases. The respective immune reaction at a certain time point, at a certain location, and in a given patient defines the set of cytokines used.
With respect to IL-4, all available studies demonstrated that E2 stimulated IL-4 secretion (Table 5). Of the seven studies on IL-10, five demonstrated a stimulating effect of E2 at midfollicular to pregnancy levels. The two studies that report an inhibitory effect on IL-10 (at pregnancy levels) also delineated that E2 at periovulatory doses had no effect (Table 5). All studies devoted to TGF-ß demonstrated that E2 at pregnancy levels stimulated secretion of this cytokine (Table 5).
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E. Estrogens and growth factors
Studies on TGF-ß mentioned in the previous section are not listed here (78, 171, 172, 174). Similar stimulatory effects of E2 at pregnancy levels were demonstrated for TGF-ß3 in osteosarcoma cells (175, 176) and TGF-ß in human kidney carcinoma cells (177). E2 at follicular to pregnancy levels (not at 10–12 M) increased the concentration of basic fibroblast growth factor (86) and keratinocyte growth factor (178). E2 treatment (diestrus to pregnancy levels) caused a dose- and time-dependent increase in keratinocyte growth factor levels in peripubertal (5-wk-old) and mature (11-wk-old) mice (179). In mouse mesenchymal stem cells (C3H10T1/2), E2 at pregnancy levels activated bone morphogenic protein-2 promoter mainly through ER and only little via ERß, which requires a classical ER element site, but not activator protein (AP)-1 or Sp-1 sites (180). In human osteoblast cell lines, E2 at pregnancy concentration increased steady-state levels of bone morphogenic protein-6 (181).
In human macrophages, E2 at follicular to pregnancy levels and membrane-impermeable BSA-conjugated E2 enhanced nerve growth factor secretion and mRNA expression (via AP-1 binding sites on the promoter) (182).
In conclusion, most of these studies indicate that E2 at periovulatory to pregnancy levels increased important growth factors such as TGF-ß, basic fibroblast growth factor, keratinocyte growth factor, and bone morphogenic proteins. This is in agreement with the important growth-supporting role of E2 during pregnancy. However, several studies also demonstrated that E2 at postmenopausal levels had no effect. In inflammatory diseases, in which the overshooting fibrotic repair response plays a decisive pathogenic role, an E2-stimulated increase of these growth factors might be harmful, whereas it can be positive for tissue repair.
F. Estrogens and nitric oxide
These aspects have been extensively covered in previous reviews from 1997 (183) and 2002 (184). In general, it has been identified that estrogens enhance NO production by the endothelial isoform of NO synthase (NOS) due to increases in expression and level of activation (184). These effects are mediated via ERs, but they are independent of ER element action (nongenomic) (184). Furthermore, it has been demonstrated that E2 increases other NOS isoforms (185, 186, 187, 188, 189). This presentation here only examines relevant aspects with respect to inflammatory diseases.
The known stimulatory effect of E2 on NO production at pregnancy levels (little at 10–10 M) was also observed in human granulocytes and monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium (190, 191). In these studies, results were independent of a proinflammatory stimulus. This might be largely different in a situation when a proinflammatory stimulus is added to the system.
For instance, in LPS-stimulated mouse macrophages (RAW 264.7), preincubation with E2 at proestrus to pregnancy levels inhibited NO and TNF release (68). Similarly, E2 at pregnancy levels inhibited IL-1ß-induced NO production in rabbit lacrimal gland acinar cells (192). In primary microglia, aortic smooth cells, and in pleural cells after induction of carrageenan pleurisy, E2 at pregnancy levels inhibited iNOS expression and reduced the accumulation of NO consequent to various inflammatory stimuli (99). E2 and E3 at pregnancy levels inhibit LPS-induced TNF and NO production in primary rat microglia and mouse N9 microglial cells via iNOS inhibition (193). Transient middle cerebral artery occlusion in rats led to a strong inflammatory response with iNOS overexpression, which was strongly inhibited by E2 injection 2 h before occlusion (pregnancy levels) (110). In a pleurisy model, the close interrelation between TNF and IL-1ß and iNOS has been demonstrated, and E2 treatment (pregnancy levels) inhibited production of all three molecules (124). This was also demonstrated in rat primary astrocytes (77).
From this point of view, E2 exerts quite different effects depending on absence or presence of additional inflammatory stimuli such LPS or IL-1ß. In the absence of inflammatory stimuli, E2 increases NO production by stimulating the expression and activity of different isoforms of NOS. However, in the presence of an inflammatory stimulus, E2 at pregnancy levels typically inhibits NO production elicited by cytokine-stimulated iNOS activity. In the latter situation, this might reflect the direct effect of E2 on NF-B activation and proinflammatory cytokine secretion.
G. Estrogens and oxygen radicals
The generation of reactive oxygen species (ROS) is an important proinflammatory factor evolutionarily conserved to overcome infections. However, the generation of ROS is also found in proinflammatory diseases. Thus, it is important to know the effects of E2 on ROS generation.
Several studies using different cell types or species have described that ovariectomy levels of E2 or ovariectomy increased ROS production, whereas treatment with E2 at pregnancy levels decreased ROS production (194, 195, 196, 197, 198, 199). E2 at pregnancy levels protected cardiac cells expressing ERß from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 increased the expression of glutaredoxin as well as -glutamylcysteine synthase, a rate-limiting enzyme for the synthesis of glutathione (blocked by ICI 182,780) (200). In primary rat microglia and a microglial cell line (N9), pretreatment with E2 at proestrus to pregnancy levels attenuated phorbol-12-myristate-13-acetate-stimulated superoxide release (79). A relevant factor for the generation of oxygen radicals is the NADPH oxidase. In different cell lines, E2 at pregnancy levels decreased expression of the NADPH oxidase, which significantly decreased their capacity to generate ROS (201, 202). Another important factor in generating oxygen radicals is the myeloperoxidase. E2 at pregnancy levels blocked stimulated myeloperoxidase expression (203).
In conclusion, the overwhelming majority of studies demonstrate that E2 at proestrus to pregnancy levels inhibits ROS formation. It might well be that this inhibitory activity of E2 might be reverted depending on the cell type and the microenvironmental conditions leading to catechol estrogen formation (204). In addition, ROS formation might well happen below periovulatory/proestrus to pregnancy levels, which lead to a more proinflammatory situation at low concentrations of E2 in the postmenopausal state.
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V. Estrogens and Apoptosis |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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In human peripheral T lymphocytes, E2 at periovulatory to pregnancy levels for 24 h reduced TNF-induced lactate dehydrogenase release and caspase 3/7 activities, which must be viewed as an antiapoptotic effect (205). In human promonocytic cells (U937), E2 at 10–10 to 10–7 M (not at 10–13 to 10–11 M) increased survival and prevented apoptosis induced by TNF in both undifferentiated and macrophage-like phorbol-12-myristate-13-acetate differentiated cells (206). In human monocytic cells (THP-1), E2 at pregnancy levels prevented apoptosis (207). In Balb/c mice transgenic for the H chain of anti-dsDNA antibodies, E2 (at pregnancy levels) rescues naive autoreactive B lymphocytes normally deleted (36). Support of survival of these proinflammatory cell types is most probably an unfavorable signal in autoimmune diseases.
In chronic brain inflammation, the effect of E2 on neuronal or macroglial/microglial cell apoptosis might be important. In rat primary cortical neurons, 24-h pretreatment with E2 and E3 at pregnancy levels reduced N-methyl-D-aspartate-induced toxicity and the N-methyl-D-aspartate-induced apoptotic changes by enhancing Bcl-2 levels (208). In primary cortical neurons, E2 at pregnancy levels reduced the number of apoptotic cells and reduced the number of neurons containing active caspase-3 (blocked by ICI 182,780) (209). In rat primary cortical neurons after glutamate exposure, pretreatment with E2 at pregnancy levels prevented glutamate-induced apoptosis, attenuated calpain up-regulation, shifted the Bax:Bcl-2 ratio toward Bcl-2, and decreased caspase 3 activation (210). In murine microglial cells (N9), E2 at 10–9 M decreased the basal percentage of cells positive for Fas and FasL (80). All these effects in brain cells are of antiapoptotic nature and might protect the tissue from inflammation-induced destruction.
Similar antiapoptotic effects are observed in vivo; e.g., in immature hypophysectomized rats, E2 leading to pregnancy levels prevented apoptosis of granulosa cells, and testosterone had exactly opposite effects (211). In ovariectomized female mice, after myocardial infarction, E2 replacement (pregnancy levels) increased activation of the prosurvival kinase, Akt, and decreased cardiomyocyte apoptosis (212). In cultured neonatal rat cardiomyocytes in vitro, E2 at 10–10 to 10–9 M caused a rapid 2.7-fold increase in Akt phosphorylation and a decrease in apoptosis (reversed by ICI 182,780) (212). Thus, most of these studies demonstrated the antiapoptotic effects of E2.
Nevertheless, some cell types such as osteoclasts behave completely different. In human osteoclasts, E2 at 10–7 M (not at 10–8 M) inhibited IL-1-promoted cell survival, i.e., E2 increased apoptosis (143). In contrast, E2 did not alter IL-1 responsiveness in primary human osteoblasts or bone marrow stromal cells (143). In osteoclast precursors derived from a monoblastic leukemia cell line (FLG29.1), E2 at 10–8 and 10–7 M (not at 10–9 M) induced cell apoptosis (accompanied by caspase 3 activation and DNA fragmentation) (213). This clearly demonstrates that E2 effects must be dependent on additional factors present in different cell types.
An interesting solution for this paradox might be given by a study of Acconcia et al. (214): in different epithelial cell lines, the E2-ER complex rapidly activates multiple signaling pathways (i.e., ERK/MAPK, phosphotidylinositol 3-kinase/Akt) committed to both cell cycle progression and prevention of apoptotic cascades. On the other hand, the E2-ERß complex induced the rapid and persistent phosphorylation of p38/MAPK, which in turn was involved in caspase 3 activation and cleavage of poly(ADP-ribose) polymerase, driving cells into the apoptotic cycle. E2 at 10–8 M promotes cell survival through ER nongenomic signaling and cell death through ERß nongenomic signaling (214). Thus, the influence of E2 would be dependent on the abundance of either ER or ERß (see Section II).
In conclusion, most of these studies demonstrated the antiapoptotic effects of E2. However, E2 at pregnancy levels can also have proapoptotic effects (osteoclasts) and cytopenic effects on hematopoiesis (41, 61, 215, 216, 217, 218, 219, 220). Given the fact that E2 decreases apoptosis of immune cells, this particular aspect of estrogens must be considered a proinflammatory effect.
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VI. Estrogens and Migration of Leukocytes |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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Indeed, in T cells of patients with MS, E3 at pregnancy levels inhibited T cell migration in a transwell model (46). RANTES (regulated on activation normally T cell expressed and secreted) attracts T lymphocytes and macrophages, and its production is enhanced in human keratinocytes. E2 (IC50, 10–9 M) inhibited TNF or IL-1ß-induced RANTES secretion (221). In human keratinocytes, E2 (IC50, 10–9 M) inhibited constitutive and stimulated secretion of MCP-1 and its promoter activity (222). The Sp-1 element and the AP-1 element on the MCP-1 promoter are required for transcription, and they are involved in transrepression by E2. The results further suggest that E2-bound ERß may inhibit MCP-1 gene expression by inhibiting Sp-1 and AP-1 transcriptional activities in keratinocytes (needs the activation function 2 of ERß) (222).
E2 at 10–12 to 10–6 M (maximum at 10–10 M) inhibited the migration of human monocytes exposed to MCP-1 (blocked by tamoxifen and clomiphene) (223). E2 inhibited the secretion of MCP-1 from endometrial stromal cells (fibroblasts) with a IC50 of 10–8 M (slight increase at 10–12 M) (90). In IL-1-stimulated human breast cancer cells (MCF-7), addition of E2 at 10–12 M to 10–9 M (IC50, 5 x 10–11 M) inhibited MCP-1 production in a dose-dependent manner (224). Treatment with E2 at 10–10 and 10–11 M decreased the IL-1-inducible MCP-1 promoter luciferase activity (224). In human coronary artery endothelial cells, E2 at pregnancy levels inhibited MCP-1 (225). In murine macrophages, E2 at 10–10 and 10–9 M (not at 10–11 M) suppressed LPS-stimulated expression of MCP-1 mRNA (226). In murine fibroblasts, E2 inhibited serum-stimulated MCP-1 (bell-shaped curve, maximum at 3.7 x 10–12 and 9.9 x 10–10 M, no effect in the middle range of 3.7 x 10–11 M) (91).
Postmenopausal women were randomized to receive either no hormone replacement therapy (HRT) or 1 mg E2 continuously plus sequential progestin over 1 yr (227). MCP-1 levels in serum gradually decreased after 3, 6, and 12 months with HRT. At 18 months, in women discontinuing HRT, MCP-1 levels rose again, but remained lowered in women continuing HRT (227).
At this point the question appears whether or not a similar inhibitory effect of E2 on migration can be observed in vivo. In ovariectomized rats, after sc implantation of perforated Teflon cylinders in a model of experimental inflammation, E2 pretreatment (proestrus levels) decreased accumulation of exudate, inflammatory cells, and formation of granulation tissue (228). A local skin inflammation was induced by intradermal injection of olive oil and cholera toxin in several strains of castrated mice (induces a fast inflammatory response independent of T and B cells) (229). Treatment with a single injection of E2 (proestrus to pregnancy levels) given up to 4 d before the induction of inflammation suppressed the inflammatory response, as measured by footpad swelling and documented by the infiltration of PBMCs into the inflamed area (229). In a rat model of ischemia and reperfusion injury of the myocardium by occlusion of the left coronary artery, E2 at proestrus to pregnancy levels lowered myocardial necrosis and myeloperoxidase activity, a marker of polymorphonuclear leukocyte accumulation (122). In the model of balloon injury of the right carotid artery in ovariectomized rats, E2 (proestrus to pregnancy levels) reduced granulocyte and monocyte/macrophage migration to the adventitial and periadventitial domains of injured vessels but increased T cells (230). In the same model, E2 (proestrus to pregnancy levels) inhibited expression of mRNA for cytokine-induced neutrophil chemoattractant-2ß and MCP-1 (231).
In carrageenan-induced pleurisy in the ovariectomized rat, prior exposure to E2 leading to proestrus to pregnancy levels induced a decrease in neutrophil migration as shown by cell counting and myeloperoxidase measurement (232). In a model of LPS-induced uveitis in adult male, female, and ovariectomized Lewis rats, cellular infiltration was more marked in male than in female rats, and ovariectomy increased cellular infiltration (233). Treatment with E2 at proestrus to pregnancy levels reduced the cell number in male and ovariectomized female rats, which was accompanied by inhibition of E-selectin in the iris-ciliary body (233).
Apart from effects on MCP-1, these antimigratory effects might depend on changes of chemokines and their receptors. E2 (pregnancy levels) decreased disease-mediating chemokine receptors such as CCR1, CCR2, CXCR2, CCR3, CCR4, CCR5, CCR7, CCR8, and CXCR3 in the central nervous system in a model of EAE (234). E2 treatment of mice for 3 d (pregnancy levels) reduced expression of CCR2 and CXCR3 on murine blood monocytes (235). All above-mentioned studies indicate that E2 at periovulatory/proestrus to pregnancy levels inhibits migration, which was related to a decrease in inflammation.
Interestingly, E2 had completely opposite effects in the uterus, which shows that effects of E2 are specific for the location. Numbers of macrophages increase in the uterus during proestrus in mice, and macrophages migrate into the uterus after implantation (236). Treatment with E2 (pregnancy levels) for 4 d induced a strong recruitment of macrophages and neutrophils into the mouse uterus (not B lymphocytes) (237). This interesting prochemotactic effect in the uterus might depend on E2-induced up-regulation of RANTES, MCP-1, and IL-8 in endometrium cells (238, 239). The total uterine leukocyte population increased significantly when women received oral E2 (periovulatory levels), which was due to a significant increase in both the uterine natural killer cells and the macrophage populations (not T cells) (240).
A possible explanation for increased migration into the uterus might come from E2 regulation of chemokines in uterine cells. In an in vitro organ culture system with human endometrium, E2 at 10–9 M (not at 10–10 or 10–11 M) induces expression of chemokines CXCL10 and/or CXCL11 (blocked by ICI 182,780) (241). In rat uterine epithelial cells, E2 pretreatment at pregnancy levels enhanced stimulated LPS-secretion of CCL20 (blocked by ICI 182,780) but E2 inhibited its constitutive secretion (159). Another explanation might come from the fact that E2 at pregnancy levels increased uterine microvascular endothelial cell monolayer permeability and transiently redistributes interendothelial junction-forming proteins in endothelial cells. Concomitantly, adherens junction proteins are disconnected from the cytoskeleton (blocked by ICI 182,780 and provoked by E2-BSA) (242).
In conclusion, E2 has a strong inhibitory effect on MCP-1 as demonstrated in many nonuterine cell types. In addition, E2 in vivo demonstrated a marked inhibitory effect on migration of leukocytes into inflamed areas, particularly neutrophils and monocytes (innate immune cells). E2-mediated inhibition of leukocyte migration was accompanied by less severe inflammation. Interestingly, E2 had completely opposite effects in the uterus, which might be necessary for normal implantation. The reasons for these site-specific effects are presently not known but might be found in regulation of chemotactic factors/receptors. This section demonstrates that E2 at high levels inhibits migration of innate immune cells to the site of inflammation. This phenomenon probably inhibits the immune/inflammatory response to a tissue autoantigen in women with high levels of estrogens.
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VII. Estrogens and Angiogenesis |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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Whereas it is obvious that E2 at periovulatory/proestrus to pregnancy levels enhances VEGF under normoxia conditions in vitro, it was demonstrated in bovine retinal vascular endothelial cells that E2 under hypoxia conditions inhibited VEGF (246). Others have demonstrated that E2 preincubation at ovariectomy levels (not at pregnancy levels) increased LPS-stimulated VEGF secretion from the mouse macrophage cell line RAW 264.7 (68). These studies demonstrate the dual behavior of E2 in different microenvironments and concentration ranges.
It is important to mention that endogenous downstream conversion products of E2 can have opposite effects on angiogenesis. Indeed, 2-methoxy-E2 is a potent inhibitor of endothelial cell proliferation and migration as well as angiogenesis in vitro (IC50, 1 x 10–8 M; Ref. 247) (248, 249, 250). Moreover, when administered orally in mice, it strongly inhibits the neovascularization of solid tumors and suppresses their growth (247, 248, 249, 250). Thus, the role of E2 is also regulated by possible conversion to downstream metabolites, which might be largely regulated by environmental factors. These concepts are further developed in Section X.
In conclusion, recent studies confirmed the proangiogenic effect of E2, but it was also shown that the role of E2 is determined by environmental factors such as oxygen tension, E2 concentration, and endogenous E2 conversion to metabolites.
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VIII. Estrogens Modulate Inflammatory Processes: Diseases and Models |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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A. Arthritis
In arthritis, effects of estrogens have been extensively studied, and several important reviews have been presented (4, 251, 252, 253, 254, 255). When investigating effects of estrogens on arthritis, three important aspects need to be mentioned primarily.
First, in experimental studies in arthritic animals, most often E2 at proestrus to pregnancy levels has been used. Second, when immune cells of animals or patients were investigated, most often E2 at periovulatory/proestrus to pregnancy levels was used as the sole estrogen (no combinations were applied). Third, and in contrast to items one and two, in observational studies in humans the focus is not solely on E2; e.g., in pregnancy or during the reproductive period, not only E2 increases, and in the postmenopausal phase, not only E2 decreases. Furthermore, in experimental studies in women or men with arthritis, most often mixtures of estrogens have been applied (E2 was only a minor part, and often a gestagen plays an additional role; Ref. 256). Additionally, E2 serum levels were not markedly increased by these therapies. For example, HRT in postmenopausal women increases E2 serum levels from approximately 3 x 10–11 to 1.5 x 10–10 M (early follicular levels) (126), which is not a strong increase in relation to periovulatory and pregnancy levels (1 x 10–9 to 1 x 10–7 M). Thus, the type of estrogen, coadministration of a gestagen, and the concentration of estrogens were largely different in animal experiments compared with studies in humans.
1. Experimental arthritis in animals.
Ovariectomy of female DBA/1 mice increased arthritis induced with type II collagen (257). Ovariectomy-induced loss of endogenous estrogens and inflammation contributed concertedly to osteoporosis in this model (220). In this model, E2 treatment before immunization (proestrus levels) decreased levels of IgG anti-type II collagen antibodies and T cell proliferation, whereas levels of IgM antibodies are increased (257), and it is independent of the thymus (258). This was similar in a rat model of arthritis, which was also independent of the thymus and the CD8+ T cell subpopulation (259, 260). Other groups confirmed the positive effects of E2 at proestrus to pregnancy levels in experimental arthritis (35, 261, 262, 263, 264), and the favorable effect of E2 was also confirmed in male animals with experimental arthritis (265). Collagen type II-induced arthritis can also be elicited by transfer of collagen type II-specific monoclonal antibodies, and E2 treatment (proestrus levels) starting before immunization decreased the severity of arthritis (266). Furthermore, E3 treatment leading to pregnancy levels has an even stronger ameliorating effect on experimental arthritis than E2 (267).
Treatment with the ER/ERß antagonist ICI 182,780 triggered an earlier onset of arthritis during the period when the proestrus cycle was blocked (268). The arthritis-modulating effect of ICI 182,780 was even obtained at doses that were insufficient to block proestrus cycling (268), which indicates that relatively low levels of E2 (diestrus levels) can induce an ameliorating effect. Interestingly, a highly specific agonist of the ERß (called ERB-041) had a strong beneficial effect in the Lewis rat adjuvant-induced arthritis model (269).
In addition, the endogenous metabolite of E2, 2-methoxy-E2, also showed an antiarthritic effect in the collagen type II model in DBA/1 mice but it did not show a feminizing effect on the reproductive tract or inhibition of leukocyte development in hematopoietic or lymphoid organs (270). Neither the T cell-dependent delayed type hypersensitivity nor the B cell-dependent production of anti-type II collagen antibodies was suppressed by 2-methoxy-E2 (270). This indicates that 2-methoxy-E2 exerts its effects directly in inflamed tissue by affecting bystander inflammation.
Female mice similar to women are sheltered from RA flares during pregnancy (see Section XI). Interestingly, an E2 maintenance dose (pregnancy levels) during a short period immediately after parturition protects the mouse from a postpartum flare of the disease, but progesterone and hydrocortisone had no clinical effect (271). A similar postpartum effect of E2 was confirmed by others (272).
In another model of joint disease independent of T cells and B cells and most probably dependent on monocyte/neutrophil invasion, da Silva et al. (273) demonstrated that female mice showed a higher ability than males to degrade cartilage in the cotton-pellet cartilage implant model, which leads to granuloma formation. Gonadectomy resulted in a significant acceleration of cartilage damage in both sexes, which was reverted by E2 replacement (pregnancy levels) (273). This demonstrates a biphasic effect where low to normal levels of E2 exerted an unfavorable effect, whereas pregnancy levels inhibited granuloma formation. It is expected that the granuloma mainly exists of neutrophils and monocytes/macrophages, which are suppressed by E2 at high levels.
Another model makes use of a very fast plasma extravasation response into the synovial cavity within minutes and hours (274). The magnitude of bradykinin-induced synovial plasma extravasation is markedly less in female rats. Ovariectomy of female rats increased bradykinin-induced synovial plasma extravasation, and administration of E2 (proestrus to pregnancy levels) to ovariectomized female rats reconstituted the female phenotype (274).
In conclusion, most studies reported a beneficial effect of E2 at proestrus to pregnancy levels in experimental arthritis.
2. Arthritis in humans.
Female sex has a deteriorating effect on RA disease activity, but the menopausal state with its lower steroid hormone levels is responsible for the major part of the differences in outcome between men and women (275). RA women had a higher disease activity score and health assessment questionnaire scores than RA men (276). RA women below 50 yr of age at study entry had milder disease than older women (close to that of men). At 2-yr follow-up, RA women still had higher disease activity scores and health assessment questionnaire scores compared with men, who had achieved remission in a higher frequency. A radiology damage score showed no sex difference either at study entry or after 2 yr (276).
Not only the biological sex but also the number of gestations had an influence on arthritis. An interesting study was carried out by Jorgensen et al. (277), who demonstrated that having more than three children increased the risk of developing severe disease 4.8-fold when adjusted for age and use of OC. One might speculate that higher fertility, which is most probably also dependent on higher estrogens levels, is linked to a more autoaggressive immune response. However, the increased risk may also be related to the higher probability of immunization with allogenic or semiallogenic material and, thus, repeated triggering of clonal expansion of B cells (278). Nevertheless, these results point to a possible importance of estrogen levels in patients with RA.
The question appears to be whether or not serum or synovial fluid levels of estrogens are different in healthy subjects compared with patients with RA. Most studies indicate that serum levels of E2 and E1 are similar in patients with RA compared with controls (Table 6 and Ref. 279). In addition, 12 wk of anti- TNF antibody treatment did not change serum levels of E2 (280). One study in premenopausal women with RA demonstrated higher E2 serum levels in patients with than without anticardiolipin antibodies (3.6 x 10–10 vs. 2.0 x 10–10 M), but the difference was marginal (281). In male RA patients, E2 levels were higher compared with healthy men, and E2 levels were positively correlated with indices of inflammation (Table 6) (282). Interestingly, this relatively uniform situation is largely different for levels of androgens such as dehydroepiandrosterone sulfate and testosterone, which are markedly lower in inflammatory diseases. This loss of androgens was thought to play an unfavorable role in chronic inflammatory diseases (not subject of this review) (summarized in Ref. 5).
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-hydroxylated estrogens and, in addition, low levels of 2-hydroxy-estrogens (284). The 16-hydroxylated estrogens serve as mitogenic and proliferative estrogens, and 2-hydroxylated forms might play an opposite role due to conversion to O-methylated estrogens (Section X).
Importantly, a similar increase of 4- and 16-hydroxylated forms was detected in the synovial fluid of patients with RA compared with trauma controls (Table 6) (285). This later study also found an increased molar ratio of estrogens to androgens in synovial fluid of patients with RA compared with trauma controls. In conclusion, whereas serum levels of estrogens in patients with RA are similar to levels in healthy controls, local levels of estrogens in inflamed synovial tissue and synovial fluid are markedly higher, particularly in relation to androgens. In addition, conversion of E2 to downstream metabolites provides mitogenic estrogens (see Section X). Both factors must be considered as proinflammatory phenomena in RA.
Another important determining factor of arthritis might be a polymorphism in the ER. For instance, one study demonstrated an influence of an ER polymorphism on the course of RA in Japanese patients: female patients with the ER genotype PPxx (homozygote of Px) tended to have developed RA at a younger age, whereas those with PPXX and ppxx (lack of Px haplotype) developed RA at an older age (no association in men) (293). Others studied ER microsatellites in Japanese patients with RA (294). The ER genotype was classified according to number of dinucleotide (thymine-adenine) repeats between 10 and 27. The frequency of allele 14 (14 repeats) was significantly increased in patients vs. controls but not with disease severity (294). Presently, the functional consequence of these genetic variations is not fully understood. The distribution of ER and ERß in patients with RA was already reported in Section II (an increase of ERß+ cells in the synovial tissue was mentioned).
Effects of OC or HRT on the risk to develop arthritis and on the course of arthritis are extensively demonstrated in Section XII, and the conclusions are shortly summarized here. Almost all studies in women with vs. without OC demonstrated no increased risk to develop RA. In addition, OC use was not associated with changes in the disease course in patients with RA. During OC administration, serum levels of estrogens and progestins only increase to a small extent, and, thus, no change of risk and disease course is to be expected. Prior HRT did not increase the risk to develop RA. HRT or sole ERT markedly improved bone mineral density and reduced bone resorption markers in RA. In addition, HRT had marginal beneficial effects on the articular index, pain score, and morning stiffness, particularly when patients with higher E2 treatment levels were separately analyzed (increase of serum E2 from 5 x 10–11 to 2 x 10–10 M) (295). Because women under HRT do not have serum levels of E2 in the periovulatory or pregnancy range, a strong E2 effect as in animal models is not to be expected. In addition, HRT also includes therapy with gestagens, which might contribute to the observed effects by neutralizing or supporting effects of estrogens.
B. Systemic lupus erythematosus
This subject has been reviewed several times by different groups (296, 297, 298, 299, 300, 301). The general concept is summarized as follows:
The prevalence of SLE is far higher in females than in males, particularly during the reproductive phase. Animal models demonstrated that E2 treatment accelerates the disease process (3, 302, 303). However, it is important to mention that not all lupus pathologies are accelerated in a similar way within the same animal (304). Pregnancy doses of E2 accelerate immune-complex glomerulonephritis but ameliorate focal sialadenitis, renal vasculitis, and periarticular inflammation (304). One can concur with the authors that E2 stimulates antibody production and immune complex phenomena but inhibits T cell-mediated organ pathologies.
In the B cell compartment, E2 is an immune stimulator that affect maturation, selection, and antibody secretion (see also Section III.B) (299, 301). Its impact may be based on the capacity to allow autoreactive B cells to escape the normal mechanisms of tolerance and to accumulate in sufficient numbers to cause clinically apparent disease (36, 299, 301). This outstanding research has demonstrated that E2 leads to the survival and activation of autoreactive B cells eliciting T cell-independent autoreactive marginal zone B cells (36, 299, 301). BALB/c mouse transgenic for the heavy chain of an anti-double-stranded DNA antibody are prone to develop lupus after exposure to exogenous sex hormones (301). These are very good arguments that an E2-enriched environment can stimulate a B cell-dependent autoimmune milieu.
In addition, important studies supported a direct effect of E2 on SLE immune cells, which is most probably unfavorable during the course of the disease. E2 at 10–10 M to 10–8 M (IC50, 10–9 M) enhanced production of IgG anti-double-stranded DNA antibodies as well as total IgG in PBMCs from SLE patients (29). E2 also enhanced total IgG, but not anti-double-stranded DNA, production in the PBMCs of normal donors. E2 increased the B cell-stimulating IL-10 production by monocytes (29). In cultured T cells from female SLE patients, E2 at periovulatory to pregnancy levels increased calcineurin, which was not observed in T cells from control women (305). Calcineurin expression in T cells from patients with vasculitis and RA taking similar medications was unaffected by E2 (305). In peripheral blood T cells from patients with SLE, 2-fluoro-E2 at 10–7 M showed a significant increase in the amount of CD40 ligand (CD40L) on the cell surface (blocked by ICI 182,780), which was not observed on the surface of T cells in normal women (306). These are excellent arguments that E2 not only generates an environment to propagate autoimmune B cells, but also supports several key pathways of already generated autoimmune B cells. It seems that these effects are particularly stimulated at periovulatory to pregnancy levels of E2.
At this point, the question appears to be whether or not estrogen serum or urinary levels of patients with SLE are altered (Table 7). The conclusion is that estrogen serum levels are not different in healthy controls compared with SLE patients, which is different with respect to urinary levels of estrogens. In early studies of the 1980s, increased levels of 16-hydroxylated estrogens were detected in the urine of patients with SLE compared with healthy controls (307, 308). Recently, these studies were confirmed in patients with SLE and, in addition, low levels of 2-hydroxy-estrogens were detected (284). Because 16-hydroxylated estrogens serve as mitogenic and proliferative estrogens and 2-hydroxylated forms might play an opposite role, this altered hormone pattern indicates a proinflammatory milieu.
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C. Brain inflammation
The role of estrogens in chronic inflammatory brain diseases such as EAE and MS was reviewed in 2004 (311). This review emphasized the antiinflammatory effects of E2 and E3. The subsequent text adds new information to this earlier discussion.
As early as 1994, it was reported that ovariectomy led to an earlier EAE onset (267). Long-term treatment with proestrus levels of E2 delayed onset of disease. In contrast, treatment with E3 (achieving proestrus levels) delayed the disease onset for a longer time. Five times higher doses of E2, compared with those seen during pregnancy, were required to obtain similar effects as the low E3 dose (267). Although E3-treated mice had higher levels of serum antibodies of the IgG1 specific for myelin basic protein, E3 reduced the severity of EAE, and antigen-specific T lymphocyte produced higher amounts of IL-10 (37). E2 at pregnancy levels decreased EAE incidence, which was not demonstrated for lower serum levels (<1 x 10–9 M) (312). Higher E2 levels were accompanied by lower ratios of IFN-/IL-10. This was confirmed in several other studies with E2 or E3 at proestrus to pregnancy levels (312, 313, 314, 315). Others demonstrated that E2 suppressed EAE in mice deficient in IL-4, IL-10, and IFN-, and there was a profound decrease in the frequency of TNF-producing cells in the central nervous system and the periphery (316).
Additionally, E2 treatment (pregnancy levels) drastically decreased the recruitment of total inflammatory cells as well as TNF-positive macrophages and T cells into the central nervous system at disease onset (317). E2 treatment (pregnancy levels) decreased frequency of CD11b+/CD11c+ dendritic cells in the brain of EAE mice (69). E2 decreased the frequency of CD11c+/CD8+ dendritic cells in the spleen in mice with EAE, which produced TNF and IFN-. E2 pretreatment (pregnancy levels) was found to suppress the ability of cultured dendritic cells bearing a mature phenotype to present antigen to specific T cells. E2 decreased TNF, IFN-, and IL-12 production in mature dendritic cells (shift toward production of Th2 cytokines IL-4 and IL-10) (69).
Transfer of E2-exposed (3.7 x 10–8 M) splenic dendritic cells from Lewis rats obtained on d 12 after immunization with myelin basic protein prevented the expansion of CD4+ T cells and increased the proportions of regulatory T cells producing IL-10 and CD4+CD28- suppressor T cells, accompanied with increased IL-10 and IFN-, and reduced TNF production (70). E2 up-regulated the expression of indoleamine 2,3-dioxygenase, which promotes tolerogenic properties of dendritic cells (70).
Importantly, the protective effect of E2 in animals with EAE was abrogated in ER-deficient mice but not in ERß-deficient mice (318, 319). This was supported in a brain injury model of stroke (320). Additionally, treatment of mice with EAE, with an ER agonist (10 mg propyl pyrazole triol/kg body weight·d) before the induction of disease resulted in suppression of clinical symptoms of disease, whereas treatment with an ERß-selective agonist (WAY-202041, 10 mg/kg body weight·d) had no effect (321). However, others demonstrated that ER was dispensable for the E2-induced amelioration in an EAE model (322).
In a recent important study in patients with relapsing remitting MS, the favorable effects of E3 (pregnancy levels, 8 mg/d for 6 months) were demonstrated (47). E3 treatment decreased delayed type hypersensitivity responses to tetanus, IFN- levels in PBMCs, and the number and volume of inflammatory lesions on monthly cerebral magnetic resonance images (47). Oral E3 treatment in patients with MS (pregnancy levels) led to increased levels of IL-5 and IL-10 and decreased TNF in stimulated PBMCs (323). These changes in cytokines correlated with reductions of inflammatory lesions on magnetic resonance imaging in relapsing remitting MS. The increase in IL-5 was primarily due to an increase in CD4+ and CD8+ T cells, and the increase in IL-10 was primarily due to an increase in CD64+ monocytes/macrophages, whereas the decrease in TNF was primarily due to a decrease in CD8+ T cells (323). In summary, almost all studies indicate that E2 or E3 at periovulatory/proestrus to pregnancy levels ameliorates EAE or relapsing remitting MS. The question appears to be whether or not this is similar in other inflammatory models of brain inflammation.
In an inflammatory/injury model of the brain, E2 treatment (pregnancy levels) decreased IL-1ß expression in the olfactory bulb in young adult females but paradoxically enhanced its expression in reproductive senescent females (324). In an LPS model, systemic administration of E2 (proestrus levels) 6 h before LPS injection prevents the activation of microglia and the recruitment of peripheral monocytes (325). This effect occurs by limiting the expression of neuroinflammatory mediators, such as the MMP-9, MCP-1, CXCL2, TNF, lysosomal enzymes, and complement C3 receptor (via ER and not via ERß) (325, 326).
Using the APP23 mice, an animal model of Alzheimer’s disease with chronic neuroinflammation, ovariectomy increased microglia activation at ß-amyloid deposits (326). Long-term administration of E2 at proestrus to pregnancy levels reverted the effects of ovariectomy and decreased microglia reactivity (326). Rats receiving transient middle cerebral artery occlusion demonstrated substantial apoptosis and inflammatory responses, including IB phosphorylation, NF-B activation, and iNOS overexpression (110). In ovariectomized rats, E2 treatment leading to proestrus levels (one injection 2 h before occlusion) produced strong protective effects by reducing infarct volume, neuronal apoptosis, and inflammatory responses (110). Thus, also in other models of brain inflammation, E2 at proestrus to pregnancy levels exerts beneficial effects. The question appears to be how this translates into brain cell cultures.
In primary cultures of rat microglia, E2 at proestrus to pregnancy levels (maximum 10–9 M, minimum 10–11 M) inhibited LPS-induced production of iNOS, prostaglandin E2, and MMP-9 (blocked by ICI 182,780) (81). E2 at pregnancy levels inhibited the basal and IL-1ß-mediated expression of ICAM-1 and NF-B activation in cultured brain endothelial cells. Authors concluded that decreased expression of adhesion molecules may account for the capacity of E2 to reduce adhesion of leukocytes in cerebral endothelium in vivo (118). E2 administration at 1 x 10–7 M (only one concentration) to serum-deprived cortical neurons of rats induced neuroprotection (327). In rat primary cortical neurons after glutamate exposure, pretreatment with 10–8 M E2 prevented apoptosis (210). E2 decreased the neurotoxicity of gp120 in mixed neuronal/glial cultures (328).
In conclusion, E2 and E3 at periovulatory/proestrus to pregnancy levels demonstrated many beneficial effects in MS, models of brain inflammation, and cell culture assays. Most of the models are T cell mediated or innate immunity mediated. As such, an important similarity to experimental arthritis exists, where E2 and E3 at higher levels also exerts favorable antiinflammatory effects. The situation might be opposite in brain inflammation dominated by the B cell.
D. Thyroiditis
From the above-mentioned effects of E2 on SLE, it seems obvious that other B cell-dependent diseases are stimulated by E2 in a very similar way. This might also apply for thyroiditis, which appears much more frequent in women in the reproductive years compared with men. However, in thyroiditis, E2 effects have not been studied in great detail.
Interestingly, ERß is a candidate locus in a linkage analysis study in 45 multiplex families with thyroiditis, but the functional significance remains unclear (329). In patients already affected by thyroiditis, estrogen use was associated with a lower rate of hyperthyroidism [relative risk (RR), 0.169; 95% confidence interval (CI), 0.06–0.52], whereas having been pregnant was associated with a higher RR for hyperthyroidism (RR, 6.88; 95% CI, 1.50–30.96) (330). In a fibrotic form of thyroiditis with little inflammation, treatment with tamoxifen was demonstrated to have favorable effects, which might demonstrate the growth-promoting effects of E2 on fibroblasts (331). In experimental autoimmune thyroiditis, administration of E2 at pregnancy levels increased antibody titers in male but not in female mice and in male castrated mice (332). Ovariectomized female animals have a higher incidence of experimental thyroiditis, and testosterone reduces the incidence (333). Female PVG/c rats are more susceptible to the induction of autoimmune thyroiditis initiated by thymectomy and irradiation than similarly treated males (334). Most of these studies demonstrate a supportive effect of E2 on thyroiditis.
Because 2-methoxy-E2 appeared to be an antiinflammatory endogenous metabolite of E2 in other diseases, it might also be an interesting therapeutic principle in thyroiditis. One study has demonstrated that 2-methoxy-E2 decreased the viability of thyroid follicular cells (335). Flow cytometric analysis showed that 2-methoxy-E2 halted cell proliferation by arresting the cells in the G2/M cell-cycle phase, and prolonged exposure to 2-methoxy-E2 led to apoptosis (not blocked by ER antagonist) (335).
E. Intestinal and liver inflammation
1. Intestinal inflammation.
Irritable bowel syndrome, a situation with low-grade intestinal inflammation, is more prevalent in women than in men (336), but we do not know whether E2 directly affects intestinal inflammation in these women. Ovariectomized rats treated with E2 (diestrus levels) exhibited visceral hypersensitivity after partial restraint stress (337). The authors concluded that stress-induced visceral hypersensitivity in female rats is E2-dependent and mediated through neurokinin 1 receptor activation (337). Thus, it seems that pain pathways are up-regulated in women, a subject that is further developed in Section XIII.B. The role of E2 in human inflammatory bowel diseases has not been studied.
In dinitrobenzene sulfonic acid colitis, E2 (pregnancy levels), but not E2 plus tamoxifen or 17-E2, reduced macroscopic and histological scores, myeloperoxidase activity, malondialdehyde levels, and expression of ICAM-1, IFN-, and IL-13 mRNA compared with placebo (123). HLA-B27 transgenic rats with chronic inflammation were treated with ethinyl E2 (proestrus levels) for 5 d (338). E2 treatment improved stool scores, histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon, local myeloperoxidase levels, and mast cell proteases 1, 3, and 4 mRNA (338). In the same model of inflammatory bowel disease, a highly selective ERß agonist (ERB-041) has a good beneficial effect (269), which was confirmed using another ERß agonist (105). In contrast, E2 increased the macroscopic and histological scores compared with placebo in mice with acute dextrane sodium sulfate colitis (123). In this latter model, bacteria influence the disease severity and E2 might up-regulate LPS-induced antiinfectious proinflammatory pathways (see also Section VIII.L).
These results demonstrate antiinflammatory and proinflammatory effects of E2 in three different models of experimental colitis. The reasons for these discrepant results are presently not known but may be related to the different immune cells responsible or the different triggering factors.
2. Liver inflammation.
In a model of hepatic fibrosis with a single dose of dimethylnitrosamine, the fibrotic response of the male liver was stronger than that of the female liver (339). In the male dimethylnitrosamine model, E2 (proestrus or pregnancy levels) reduced hepatic mRNA for type I and III procollagens, TIMP-1, deposition of type I and III collagen protein, total hepatic collagen, and malondialdehyde, a product of lipid peroxidation (339). Ovariectomy in the female dimethylnitrosamine model had a fibrogenic effect. In rat hepatic stellate, E2 at high pregnancy levels reduced cell number, type I collagen production, and -smooth muscle actin expression (339).
In tetrachloric acid-induced liver fibrosis, E2 treatment for 8 wk (proestrus levels) reduced aspartate aminotransferase, alanine aminotransferase, hyaluronic acid, and type IV collagen in sera; suppressed hepatic collagen content; decreased the areas of hepatic stellate cells positive for -smooth muscle actin; and lowered the synthesis of hepatic type I collagen significantly in both sexes (blocked by tamoxifen) (340).
In a liver ischemia/reperfusion injury model, E2 treatment (pregnancy levels) significantly reduced liver necrosis, disintegration of hepatic cords, neutrophil infiltration, and serum TNF (blocked by ICI 182,780) (341). Female C57BL/6 mice are protected to a much greater extent from ischemia/reperfusion injury to the liver than male mice (342). Pretreatment with E2 (pregnancy levels) of C57BL/6 mice subjected to 45 min of warm ischemia decreased liver injury and apoptosis in male mice (partially reversed by ICI 182,780) (342). E2 down-regulated c-Jun N-terminal kinase and p38 MAPK activities, whereas ischemia/reperfusion injury promoted p38ß MAPK and ERK 1/2 activation (342). These results suggest that the cytoprotective effects of E2 are associated with selective modulation of MAPK.
Ovariectomized mice fed an atherogenic diet had increased hepatic levels of active NF-B and numerous up-regulated inflammatory genes, including VCAM-1, TNF, and RANTES (343). Treatment with ethinyl E2 (proestrus levels) strongly blocked induction of these genes but had no effect on their basal expression levels (blocked by ICI 182,780) (343). In hepatocytes, the oxidative stress-induced activation of NF-B was inhibited by E2 at pregnancy levels through the attenuation of hepatocyte oxidative bursts, which led to decreased extracellular levels of lactate dehydrogenase (344). IL-1ß treatment induced the expression of approximately 75 genes in the liver of ovariectomized mice (345). Ethinyl E2 pretreatment for 5 d (pregnancy levels) reduced the IL-1ß induction (after 1 h) of approximately one third of these genes (proinflammatory in nature). ER was required for this inhibitory activity, because E2 inhibition of IL-1ß-stimulated gene expression occurred in ERß knockout mice but not in ER knockout mice (345).
Above-mentioned studies obviously demonstrate an antiinflammatory role of E2 on inflammatory models of liver disease including fibrosis models. Interestingly, quite opposite effects of E2 were observed in the enteral ethanol feeding model. Ethanol increased serum alanine transaminase levels, liver weight, fat accumulation, and infiltrating leukocytes, which was blocked by ovariectomy and reversed by E2 replacement (proestrus levels) (346). Blood endotoxin and Kupffer cell TNF were increased by ethanol, which was blocked in ovariectomized rats and elevated by estrogen replacement (346). This indicates that E2 at proestrus levels might sensitize Kupffer cells to LPS. Similarly, in rats given E3 (proestrus to pregnancy levels), authors found that E3 treatment in vivo sensitizes Kupffer cells to LPS via mechanisms dependent on increases in CD14 (347). This is most likely due to elevated portal blood LPS caused by increased gut permeability. This largely increased mortality and liver inflammation (347). Toremifene (ER antagonist) treatment did not affect ethanol-increased steatosis, but significantly reduced inflammation and necrosis (348). Ethanol increased the expression of CD14 and TNF mRNA and also the production of TNF by isolated Kupffer cells, but toremifene had no significant counteracting effect. However, toremifene significantly alleviated both ethanol induction of the prooxidant enzyme CYP2E1 and ethanol reduction of the oxidant-protective enzyme selenium-dependent-glutathione peroxidase (348).
In conclusion, these experiments in liver inflammation models demonstrate that E2 has antifibrotic and antiinflammatory effects, which is similar in renal inflammation (see next section). However, it also demonstrates that Kupffer cells in the presence of ethanol are sensitized to LPS, which would be an unfavorable signal in LPS-dependent liver inflammation.
F. Renal inflammation
It is well known that men with chronic renal failure show more rapid decline in renal function with time than do women (349). In salt-sensitive rats (Dahl), ovariectomy increased the severity of glomerulosclerosis and cortical tubulointerstitial fibrosis after 12 months (350), which was attenuated by E2 replacement (proestrus levels). Ovariectomy was also associated with increased deposition and expression of laminin and TGF-ß1 and decreased activity of cortical MMP-9, and E2 replacement (proestrus levels) opposed these effects in the glomerulum (350).
In a model of chronic allograft nephropathy (351), animals were investigated 24 wk after transplantation for histological and immunohistological studies as well as for molecular analysis. E2 treatment (proestrus levels) improved graft function, reduced glomerulosclerosis, and diminished cellular infiltration. Renal allograft damage paralleled intragraft mRNA expression of TGF-ß1, which was reduced in E2-treated animals (351). In mesangial cells, selective ER modulators such as tamoxifen and LY-117018 (an analog of raloxifene) suppressed mesangial type IV and type I collagen protein synthesis (10–7 to 10–10 M, not 10–11 M) with a potency identical to that of E2 (tested at 10–9 M, periovulatory level) (352).
Because TGF-ß1 stimulates extracellular matrix of the mesangium in several renal diseases, the beneficial influence of female gender on the progression of chronic renal diseases may be explained by an inhibitory effect of E2 on secretion of TGF-ß1 and on generation of extracellular matrix (353). In transgenic mice expressing active TGF-ß1 in the glomerulum, E2 treatment (pregnancy levels) attenuated interstitial fibrosis (353), which was confirmed in another model. In transgenic mice, which overexpress TGF-ß1 exclusively in the liver (with high circulating concentrations of TGF-ß1) and develop proteinuria and progressive glomerulosclerosis, E2 at pregnancy levels attenuated mesangial expansion with less glomerular deposition of type I collagen, type IV collagen, and TIMP-2. E2 also prevented proteinuria (349). However, TGF-ß levels were higher in E2-treated mice compared with placebo-treated mice, which underlines that TGF-ß1 is not the primary target of E2 (349). The primary target is most probably a direct influence of E2 on protein kinase CK2 (349). This confirms the stimulatory influence of E2 on TGF-ß, which was also demonstrated in several other cell types (Section IV.D), but this E2-stimulated increase of TGF-ß is not decisive for disease outcome in these glomerulosclerosis models.
In a model of chronic spontaneous inflammation in the MRL lpr/lpr mouse, pregnancy doses of E2 accelerated immune-complex glomerulonephritis but ameliorated renal vasculitis and periarticular inflammation (304). This demonstrates that the same hormone can exert quite different effects depending on the major pathways switched on during chronic inflammation.
In conclusion, male gender is associated with a more rapid progression of chronic renal disease. In various experimental models of renal injury or inflammation, manipulation of the hormonal milieu can replicate the effects of gender on the course of renal disease. Although TGF-ß is an important factor in glomerulosclerosis, the direct stimulating effect of E2 on TGF-ß does not explain the positive effects of E2 in this disease. Positive effects of E2 are mediated by a direct antifibrotic and antiinflammatory activity, which has been described in liver fibrosis in a similar way (Section VIII.E).
G. Inflammation of the lung and heart
Ovariectomy enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration, lung myeloperoxidase activity, lipid peroxidation, and activity/expression of P-selectin, ICAM-1, nitrotyrosine, poly (ADP-ribose) synthase, and iNOS in rats subjected to carrageenan-induced pleurisy (124). The increase of iNOS activity was correlated with a marked enhancement in the production of TNF and IL-1ß. E2 replacement (pregnancy level) 1 h before challenge counteracted lung inflammation (124). This was confirmed by another group in the same model (354): Ovariectomy amplified inflammation, whereas E2 (proestrus levels) attenuated inflammation, tissue damage associated with pleurisy, polymorphonuclear cell infiltration, and cyclooxygenase-2 and iNOS expression in inflamed areas (354).
LPS-induced lung injury was increased in male and ovariectomized mice compared with normal female mice (355). Bronchoalveolar lavage fluids demonstrated enhanced levels of IL-1ß in ovariectomized and male mice. A similar increase for levels of IL-6 and ICAM-1 was observed in lung homogenates from ovariectomized and male mice compared with intact female mice (355). E2 treatment of ovariectomized mice (pregnancy levels, for 6 h) reduced numbers of polymorphonuclear cells, which was accompanied by reduced IL-1ß, IL-6, and ICAM-1 levels (355).
In rats, myocardial ischemia plus reperfusion produced marked myocardial necrosis, increased serum creatinine phosphokinase activity, enhanced cardiac myeloperoxidase activity, increased macrophage and serum TNF, and increased ICAM-1 staining in the myocardium (122). E2 (proestrus to pregnancy levels), injected im 5 min after induction of myocardial ischemia-reperfusion injury, lowered myocardial necrosis and myeloperoxidase activity, reduced serum and macrophage TNF, and decreased serum creatinine phosphokinase activity and staining of ICAM-1 in the injured myocardium (122). E2 added in vitro to peritoneal macrophages collected from untreated rats subjected to myocardial ischemia-reperfusion injury reduced TNF production (122).
In conclusion, in all available inflammatory models of the lung and heart, E2 was demonstrated to have an antiinflammatory effect at proestrus to pregnancy levels.
H. Eye inflammation
Concerning the role of E2 in Sjögren syndrome, this was reviewed in 1997 (356). This review reports about the proinflammatory role of estrogens in Sjögren syndrome, and it also mentioned the antiinflammatory role of androgens.
In a model of endotoxin-induced uveitis in adult male, female, and ovariectomized Lewis rats, cellular infiltration was more marked in male than in female rats, and ovariectomy increased cellular infiltration (233). Treatment with E2 (proestrus to pregnancy levels) reduced the cell number in male and ovariectomized female rats, and E2 inhibited E-selectin and IL-6 expression in the iris-ciliary body (233).
Lewis rats were implanted with E2 pellets (pregnancy levels), and 4 d later were immunized with the retinal antigen interphotoreceptor retinoid binding protein peptide (357). However, E2 therapy in female and male rats did not ameliorate this autoimmune form of uveitis (357). In retinal soluble antigen-induced experimental autoimmune uveoretinitis, incorporated tamoxifen into polyethylene glycol-coated nanoparticles injected 1–2 d before expected onset of the disease resulted in significant inhibition of inflammation, which was reversed by coinjection of E2 (358). Diminished infiltration by major histocompatibility complex class II-positive inflammatory cells and low expression of TNF, IL-1ß, and RANTES mRNA were noted in eyes treated with the tamoxifen nanoparticles (358). Interestingly, autoimmune uveitis was related to the menstrual cycle because disease severity increased in the premenstrual phase (359). In women with uveitis, disease activity decreased after the first pregnancy trimester but flared in the early postpartum period (360).
I. Inflammation in the pancreas
Type 2 diabetes is characterized by loss of ß-cell mass and concomitant deposition of amyloid derived from islet amyloid polypeptide. In human islet amyloid polypeptide transgenic mice, only the males routinely develop diabetes (361). E2 treatment (pregnancy levels) of young prediabetic transgenic mice blocked the progression to hyperglycemia with normal islet morphology with no apparent deposition of islet amyloid. E2 treatment (pregnancy levels) of 1-yr-old diabetic males rapidly reverses obesity and hyperglycemia (361).
Although inflammation in the pancreas has not been studied in humans with type 2 diabetes mellitus on HRT, one study demonstrated that HRT (2 months of 0.625 mg CEE/d, followed by 4 months 0.625 mg CEE/d and medroxyprogesterone 5 mg/d) resulted in improved glycemic control (glycosylated hemoglobin), which was accompanied by reductions in waist-to-hip ratio and central abdominal fat (362). However, this has not been confirmed in other studies (363, 364). Because we do not have pancreas tissue of HRT-treated and -untreated type 1 or type 2 diabetic patients, the animal studies were not confirmed in humans.
In isolated human pancreatic islets stimulated with a cocktail of IFN-, IL-1ß, and TNF, E2 at pregnancy levels decreased cytokine-induced cell death, cell apoptosis, caspase-9 activity, and NF-B activity (365). Euglycemia was achieved in six of 12 animals that received E2-treated islets compared with one of 12 control animals (365). In conclusion, the studies in animals show a beneficial effect of E2 on experimental pancreas inflammation.
J. Prostatitis
In most of the above-mentioned inflammatory diseases, E2 at proestrus to pregnancy levels exerts beneficial effects. Interestingly, this is completely opposite in the prostate. Short-term administration of E2 at pregnancy levels was shown to induce an inflammatory response specific to the lateral prostate of the castrated male rat (366). These changes are histologically similar to a spontaneously arising nonbacterial prostatitis. When the same hormones were administered on a chronic basis, the intensity of inflammation increased with time and was accompanied by a fibromuscular proliferation that consisted of fibroblasts, smooth muscle cells, and collagen (366). Others have demonstrated that this treatment stimulated transcript levels of IL-1ß, IL-6, macrophage inflammatory protein-2, and iNOS in the lateral prostate leading to a proinflammatory reaction (367). Under the same treatment, others confirmed E2-induced lateral prostate inflammation and observed an up-regulation of MMP-2 and MMP-9 and strong leukocyte infiltration (368). These MMP activities were strongly attenuated by cotreatment with testosterone (368).
It can be summarized that the lateral prostate lobe might be a very specific organ in which pregnancy levels of E2 in male castrated rats induce a proinflammatory reaction. This model is used to study prostatitis, and it seems that effects of E2 are highly tissue-specific.
K. Endometriosis
A striking influence of E2 on inflammation can be observed in women with endometriosis (369, 370, 371). In a proportion of women (1–7%), refluxing endometrial cells are not destroyed, although retrograde menstruation is a common phenomenon in menstruating women (>90%). These endometrial cells colonize the peritoneal cavity but also other organs in the abdomen such as the gut, the bladder, and others. These endometrial cells produce abnormal quantities of chemotactic and angiogenic factors, which attract monocytes, macrophages, T cells, B cells, and other immune cells and induce neovascularization (369, 370, 371). In addition, these lesions produce E2, which is locally converted from precusor hormones as demonstrated in Section X.
Once the endometrial cells have taken up residence in ectopic areas, growth factors establish a micromilieu that allows the colonization of these cells in a mesenchymal environment (369, 370, 371). The established dysbalance induces local inflammation and pain. Because inflammation and pain are highly linked to the different phases of the menstrual cycle with a peak during ovulation, the influence of E2 can be directly observed. Necessarily, therapeutic interventions need to suppress production of E2 by the ovary and inhibit E2 synthesis by the lesions. These interventions include androgens, GnRH agonists, progestins, OC, and aromatase inhibitors (372). Endometrial cellular components are responsive to E2 throughout the cycle, and interestingly, there is resistance to progesterone action on these cells in the postovulatory phase (373). Thus E2 has a dual role—as a mitogen for endometrial cells and as a stimulator of proinflammatory cytokines. Hence, therapies are also tailored to the inflammatory component of the disorder and include nonsteroidal antiinflammatory drugs, danazol, and inhibitors of prostaglandin synthesis (372).
Although we have to keep in mind that endometrial cells are particularly responsive to E2 (and this is not the same in other tissues), endometriosis is an example where the proinflammatory role of E2 at follicular to periovulatory levels on immune and other bystander cells is outstandingly visible.
L. Injury: wound repair, trauma, and shock
This subject was reviewed in 2002 (374). These authors concluded that there is a naturally occurring gender difference in immune responses that persists after traumatic injury. Physiological levels of E2 are immunostimulatory, whereas high pregnancy and superphysiological levels are immunosuppressive (374). Evidence suggest that the gender difference in immune responses after injury is mediated in part by alterations in the circulating levels of gonadal steroid hormones through modulation of production of inflammatory and immunoregulatory cytokines (375).
1. Wound repair.
In an excellent review, Kanda and Watanabe (376) demonstrated that estrogens have the potential to accelerate cutaneous wound healing stimulating nerve growth factor and VEGF production in macrophages, granulocyte macrophage colony-stimulating factor in keratinocytes, basic fibroblast growth factor and TGF-ß1 in fibroblasts, which leads to improved wound reinnervation, reepithelialization, and granulation tissue formation.
An age-related decline in wound healing in healthy females was corrected by HRT (377, 378). Topical administration of E2 to the wound in rodents accelerated wound healing by up-regulation of TGF-ß1. Ovariectomized mice have large wound areas, which was not observed in mice deficient for the macrophage migration inhibitory factor (378). LPS-stimulated macrophage migration inhibitory factor from murine peritoneal macrophages was inhibited by E2 at 10–11 to 10–8 M (maximum 10–8 M) (378). These studies support the growth-promoting effects of E2 at proestrus to pregnancy levels, which leads to an acceleration of wound healing (compare also Section IV.E).
2. Trauma and hemorrhage.
One study in humans with severe trauma demonstrated that male patients were at increased risk to develop injury-related sepsis and other sequelae compared with female patients (379). This indicates that similar to sepsis in noninjured patients, men are more prone to sepsis than women. However, no systematic studies that demonstrate the positive effects of E2, ERT, or HRT in these patients have been carried out.
In a review on animal experiments of the year 2002, Chaudry and colleagues (380) demonstrated the beneficial effects of proestrus levels of estrogens on trauma and hemorrhage (380). They noted that several studies have shown marked immunosuppression in males after trauma and hemorrhage, which was not evident in diestrus to proestrus female rats.
Male C3H/HeN mice were castrated and treated with pellets containing either vehicle or E2 (proestrus levels) for 14 d before soft tissue trauma (i.e., laparotomy) and hemorrhagic shock were performed (381). In Kupffer cell and splenic/peritoneal macrophage cultures, E2 treatment reduced IL-10 secretion but did not change IL-6 (381). In ovariectomized mice, trauma plus hemorrhage markedly inhibited splenocyte proliferation, splenocyte release of IFN-, IL-2, and IL-3, peritoneal macrophage IL-1ß and IL-6 release, and splenic macrophage IL-6 and IL-12 release (382). Trauma-hemorrhage-induced suppression of these factors was reverted by a single injection of E2 (proestrus levels) (382). Two hours after trauma plus hemorrhage, splenocyte proliferation, IL-2, IL-3, and IFN- release, and splenic macrophage IL-6 release were maintained in control animals but not in animals receiving an ER antagonist (EM-800) (383). In addition, Kupffer cell TNF release and circulating TNF were increased only in ER antagonist-treated females (383).
Male mice were pretreated with E2 (proestrus levels) or vehicle for 3 d before induction of hypoxemia and again immediately before induction of hypoxia (5% O2) (384). In vehicle-treated mice, splenocyte proliferation, IL-2 and IL-3 production, and splenic macrophage IL-6 and IL-10 production were depressed after hypoxemia. E2-pretreated animals displayed no such depression in splenic cell parameters after hypoxemia (384).
In a model of burn-induced remote organ injury in castrated male rats, E2 treatment (pregnancy levels) inhibited burn-induced elevation in serum TNF levels and the increase of myeloperoxidase activity in liver and lung (385). In male mice after combined ethanol and burn injuries, suppression of cellular immunity was observed (109). E2 (diestrus levels) restored the delayed-type hypersensitivity and splenocyte-proliferative responses, reduced macrophage IL-6, and increased survival after bacterial challenge (109). E2 treatment resulted in a decrease in splenic NF-B activation in injured mice (109). After burn injury, in placebo-treated, aged mice, there was a greater than 75% suppression in the delayed-type hypersensitivity response relative to placebo-treated, sham-injured, aged mice (375). E2 supplementation (diestrus levels) before injury yielded a partial recovery in this response. The increase in circulating levels of IL-6 in burn-injured, aged mice was lowered by nearly 50% in aged, E2-treated mice (and E2 increased survival from 42 to 70% in aged, burn-injured mice) (375). In burn-injured mice, E2 restoration (diestrus levels) is paralleled by a recovery in IFN- production by splenocytes, but not IL-4 production (386).
In conclusion, injury-induced suppression of various immune responses such as delayed-type hypersensitivity, splenocyte proliferation, and cytokine secretion were normalized by E2 treatment at diestrus to proestrus levels. In contrast, pregnancy levels decreased TNF secretion and NF-B activation after injury. These studies demonstrate a bimodal role of E2 because at lower concentrations (diestrus to proestrus levels) E2 stimulates suppressed immune responses, whereas at high levels (pregnancy levels) inhibitory effects were observed. It is interesting that these important studies in animals never triggered HRT or ERT studies in human subjects with trauma and hemorrhage.
3. Endotoxin shock.
Rats given E2 (proestrus levels) ip 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died (387). Peak serum TNF, plasma nitrite, CD14 expression on Kupffer cells, and mRNA for TNF, iNOS, LPS binding protein in the liver were increased after LPS in the estrogen-treated group compared with controls, which was interpreted as Kupffer cell sensitization (347, 387). In mice administered a sublethal dose of LPS, pretreatment with E3 for 7 d (pregnancy levels) increased serum TNF in both control and autoimmune MRL lpr/lpr mice (blocked by tamoxifen) (388). E3-treated mice also exhibited a rapid elevation in serum IL-6 levels after LPS challenge occurring 1 h after LPS, whereas in the placebo group maximal serum IL-6 levels were detected at 3 h postchallenge (blocked by tamoxifen) (388).
In conclusion, these studies demonstrate an endotoxin-sensitizing effect of E2 and E3 at proestrus to pregnancy levels when given shortly before the challenge.
M. Bone resorption
E2 inhibits bone resorption via changes in cytokine and growth factor levels, T cell activation, and oxygen radical production (389, 390, 391, 392, 393, 394), all of which have already been discussed (Sections III.C and IV.C, IV.D, IV.E, and IV.G). These factors play an important role in chronic inflammatory diseases. Thus, it is likely that studies on E2 and bone resorption will unravel important effects of E2 in a concentration range of E2 between postmenopausal and follicular to periovulatory levels. It is accepted that the activation of inflammatory pathways during bone resorption is much smaller than in chronic inflammatory diseases. Nevertheless, studies on bone resorption demonstrate that the fall of estrogen levels from 3 x 10–10 to 3 x 10–11 M is already sufficient to stimulate local inflammation in the bone. Because some chronic inflammatory diseases start in the postmenopausal phase, bone resorption might also be a good model to demonstrate important pathogenetic aspects for chronic inflammatory diseases (e.g., the postmenopausal activation of T cells!).
Most of the estrogenic effects of E2 on bone cells are mediated via ER (7, 269), and E2 induces osteoclast apoptosis and inhibits osteoblast apoptosis (see also Section V) (394, 395). E2 deficiency leads to up-regulation of cytokines such as IL-1, IL-7, TNF, IFN-, and IL-6, which contribute to the activation of osteoclasts (394, 396). Particularly, TNF from T cells is an important cofactor in bone resorption because this cytokine supports osteoclast activation mediated by the system of receptor activator of NF-B (RANK)/RANK ligand (RANKL) and c-Fms/macrophage colony stimulating factor (55, 56, 394).
Another important factor in osteoporosis is TGF-ß. Because E2 at 10–12 to 10–8 M stimulates TGF-ß secretion from osteoblasts (see Section IV.D) (171, 397, 398), the inhibitory influence of this cytokine on T cells is lost during menopause or after ovariectomy (394). Ovariectomy up-regulates IFN--induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells (399). The resulting T cell expansion and bone loss are prevented in vivo by both blockade of antigen presenting cell-induced T cell activation and silencing of IFN- receptor signaling (399). A further important factor is IL-7 because this cytokine is important to stimulate T cell pools, which are necessary to elicit bone-resorbing activities (400, 401). Because E2 decreases IL-7-dependent T cell pools in the thymus, increased availability of T cells after ovariectomy can lead to a higher degree of osteoclastogenesis. In addition, IL-7 suppresses bone-forming osteoblasts, while stimulating formation and function of osteoclasts (402).
Additionally, ROS are suppressed by E2 (see Section IV.G), and ROS activate osteoclastogenesis (403, 404, 405). Thus, E2 deficiency necessarily increases ROS-stimulated osteoclastogenesis. An additional factor for postmenopausal bone resorption is the loss of E2-stimulated growth factors such as bone morphogenic proteins and matrix proteins (180, 181, 406).
It is obvious that most of these experiments have been carried out in animals undergoing ovariectomy, but many studies have also been carried out using cells in vitro. E2 inhibits important bone-resorbing factors at periovulatory/proestrus to pregnancy levels, which is true for the inhibiting effect on TNF, IL-1ß, soluble IL-1 receptor type I, and IL-6 (139, 143, 151, 152, 407). Similarly, treatment of postmenopausal or oophorectomized women with HRT resulted in decreased ex vivo secretion of IL-1ß or decreased serum levels of IL-1 and IL-6 (408, 409). These favorable effects of hormone replacement were also observed in patients with chronic inflammatory diseases such as RA (410, 411).
In conclusion, during the last two decades, the study of postmenopausal or ovariectomy-induced E2 deficiency has revealed important effects on bone formation and resorption. Importantly, this involves many aspects of an activated immune system. An activated immune system is the hallmark of chronic inflammatory disease, which is visible in the form of activated immune cells, circulating and locally produced proinflammatory cytokines, and generation of ROS. In comparison to the postmenopausal situation in healthy women, these bone-resorbing factors are increased to a much higher extent in chronic inflammatory diseases. This necessarily leads to accelerated bone resorption independent of glucocorticoid therapy. These studies further demonstrate that E2 at follicular to periovulatory levels is an outstandingly important factor to inhibit a gently activated immune system responsible for an increased postmenopausal bone resorption. However, after initiation of a chronic inflammatory disease, E2 at follicular to periovulatory levels is insufficient to inhibit a strongly activated immune system. Only pregnancy levels of E2/E3 exert strong enough antiinflammatory activities to inhibit activated monocytes, macrophages, dendritic cells, T cells, and neutrophils (47, 323).
N. Summary
Depending on the prevailing factors and dominant cell types involved, different roles of estrogens are to be expected in a given disease. Figure 3 summarizes the role of estrogens in inflammatory disease. If B cells are dominant in an inflammatory disease, E2 at low to high levels stimulates the disease process (Fig. 3). This is opposite in chronic inflammatory disease where monocytes, macrophages, dendritic cells, T cells, fibroblasts, and neutrophils play a dominant role (Fig. 3). This figure also demonstrates the dual role of estrogens depending on the concentration.
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IX. Timing of Estrogen Administration in Relation to the Time Point of an Inflammatory Disease |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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In Alzheimer’s disease, HRT only demonstrates beneficial effects when treatment is started before disease outbreak (414, 415, 416), whereas it had no effects in women already affected by the disease (417). Very similar concepts were developed in atherosclerosis research. Observational studies demonstrate that endogenous E2 in women has beneficial effects on development of atherosclerosis by delaying the disease onset (256). In contrast, HRT or ERT increased the frequency of atherosclerotic events in elderly women (256). In ovariectomized primates, a 70% reduction of atherosclerosis was observed when HRT was initiated simultaneously with an atherosclerotic diet (418), but when HRT was started 2 yr after initiation of an atherosclerotic diet, no protection was observed (418). Similarly, administration of E2 before or during, but not 7 d after, balloon injury inhibited neointima formation in rats (419).
In a model with EAE, treatment of mice with E2 (proestrus to pregnancy levels) at immunization markedly delayed disease onset and reduced disease severity (312). However, when E2 treatment (proestrus to pregnancy levels) was started at disease onset, severity of the established disease was not changed (and E3 treatment had a mild but nonsignificant beneficial effect) (312). Interestingly, ethinyl E2 (pregnancy levels) inhibited disease severity when given before immunization and also at disease onset, but the reasons for these contrasting effects are not known (234). One might speculate that ethinyl E2 other than E2 can be converted to an important favorable metabolite.
In ovariectomized DBA/1 mice with collagen type II arthritis, E2 treatment (proestrus levels) from 4 d before to 8 d after immunization and between d 21 and d 33 (before disease outbreak) delayed arthritis onset, but severity was not markedly changed. When mice were treated beginning at disease onset, severity was markedly reduced (420). This also demonstrates a time-dependent effect of E2 administration.
In the antibody-dependent myasthenia gravis model in mice, E2 treatment for 3 wk before immunization (pregnancy levels) was necessary and sufficient to promote acetylcholine receptor-specific T cell expansion in vivo and to increase disease severity (421). In contrast, when E2 treatment was started at immunization, no increase in antigen-specific T cells was observed (421).
In conclusion, E2 most probably has time-dependent effects on inflammatory diseases. In some diseases, E2 administration delays the onset of the disease but has no significant effect after onset or vice versa. This subject has not been extensively studied, and thus, no firm conclusions can be drawn.
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X. The Role of Estrogen Metabolites in Inflammation |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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In the last decade, the possible role of downstream metabolites of E1 and E2 were shown to have important effects relevant to inflammation. Particularly, the growth-inhibiting effects of 2-hydroxy-E2 and 2-methoxy-E2 came into the focus of recent research. Interestingly, several of these effects are independent of ERs, and relatively high concentrations of hormones between 10–8 and 10–6 M were used (429, 430, 431).
In normal cycling women, plasma concentrations of 2-hydroxy-E1 plus 2-hydroxy-E2 are 8 x 10–10 to 1.5 x 10–9 M, and circulating levels of 2-methoxy-E1 plus 2-methoxy-E2 are 9.4 x 10–10 to 1.2 x 10–9 M (430). In pregnant women, plasma levels of the latter two hormones can increase up to 3.9 x 10–8 M. Binding affinity of 2-methoxy-E1 plus 2-methoxy-E2 to classical ERs is lower compared with E2, but there is a higher binding affinity to SHBG compared with E2 (430). These latter findings suggest that these compounds have a longer half-life than E2. The question that arises is whether or not these metabolites exert pro- or antiinflammatory activities. In this respect, cytostatic or proapoptotic effects might be considered as antiinflammatory.
The mitogenic and growth-arresting effects of 2-methoxy-E2 were demonstrated in maturing oocytes (432). In breast cancer cells (MCF-7), 2-hydroxy-E2 at 1 x 10–8 M enhanced cell growth more than did E2, whereas 2-methoxy-E2 inhibited cell growth (433). 2-Methoxy-E2 inhibited DNA synthesis and mitosis, increased cAMP concentration in early S-phase, and decreased it during mitosis. The results of this study indicate that 2-hydroxy-E2 may be the more potent mitogen, whereas 2-methoxy-E2 acts as a cytostatin (433). In the same breast cancer cells (MCF-7), others demonstrated that 2-methoxy-E2 at 10–8 M inhibited the growth-promoting activities of E2 and 16-hydroxy-E1 at 10–8 M (434). These results further show that 2-methoxy-E2 can down-regulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to the cyclin D1 promoter (434).
In rat aortic smooth muscle cells, endogenous metabolites of E2 differentially inhibit serum-induced DNA synthesis, proliferation, and collagen synthesis in the following order of potency: 2-methoxy-E2 > 2-hydroxy-E2 > E2 
4-methoxy-E2 (430). In contrast, E1, E3, 16-hydroxy-E1, 2-hydoxy-E1, and 4-methoxy-E1 are significantly less potent (only at high concentrations above 10–6 M) (430). These antimitogenic effects of 2-hydroxy-E2 and 2-methoxy-E2 are not blocked by ICI 182,780 (430). In addition, 2-methoxy-E2 inhibited the platelet-derived growth factor-BB-induced MAPK activity in both rat and human aortic smooth muscle cells, and 2-hydroxy-E2 and 2-methoxy-E2 inhibited the mitogenic effects of IGF-I (430). Importantly, catecholamines such as norepinephrine, epinephrine, and isoproterenol at 10–9 to 10–7 M abrogated the inhibitory effects of 2-hydroxy-E2 on cell number, DNA synthesis, collagen synthesis, and cell migration (not blocked by - and ß-adrenergic receptor antagonists!). This is most probably due to competition of catecholamines and 2-hydroxy-E2 for the catechol-O-methyltransferase leading to lower 2-methoxy-E2 levels (435).
In adult dermal fibroblasts, 10–7 M 2-methoxy-E2 bis-sulfamate but not 2-methoxy-E2 caused a reversible morphology change and induced G2/M arrest but no subsequent apoptosis (436). In contrast, treatment of HUVECs did not induce morphology change or G2/M arrest (436). Others demonstrated that 2-methoxy-E2 bis-sulfamate was a potent inhibitor of HUVEC proliferation (IC50, 5 x 10–8 M) (247). Similar results were observed in a coculture system with HUVECs and human dermal fibroblasts leading to an inhibition of tube formation. 2-Methoxy-E2 bis-sulfamate induced Bcl-2 phosphorylation, p53 protein expression, and apoptosis in HUVECs (247).
In three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, bone marrow cells cocultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony-stimulating factor), 2-methoxy-E2 at 2 x 10–6 M reduced osteoclast number by more than 95% and induced apoptosis (437). 2-Hydroxy-E2, the immediate precursor to 2-methoxy-E2, exhibited less cytotoxicity, and 2-methoxy-E1, the E1 analog of 2-methoxy-E2, was not cytotoxic (437). Interestingly, the 2- methoxy-E2-mediated decrease in cell survival was partially inhibited by antilymphotoxin ß-antibodies, suggesting that 2-methoxy-E2-dependent effects involve lymphotoxin ß (437).
In multiple myeloma cells, 2-methoxy-E2 triggered an early transient induction of genes known to stimulate apoptosis and repression of growth/survival-related genes (438). In rat sarcoma cells, 2-methoxy-E2 induced translocation of the proapoptotic protein Bax to mitochondria as an initial apoptotic event that was accompanied by a decrease in mitochondrial transmembrane potential and the formation of ROS followed by mitochondrial release of apoptosis-inducing factor and endonuclease G (439). In addition, 2-methoxy-E2 treatment caused up-regulation of death receptor ligands FasL and TNF and induced caspase 8 activation. A caspase inhibitor did not suppress apoptotic cell death, indicating that the major proapoptotic effect of 2-methoxy-E2 is mediated by a caspase-independent mechanism (439). From these data, it seems that 2-methoxy-E2 has strong proapoptotic and cytostatic activities, but does this also imply that this endogenous E2 metabolite has any effects on relevant pathways in inflammation?
2-Methoxy-E2 inhibited endothelial cell proliferation and migration as well as angiogenesis in vitro (248). Moreover, when administered orally in mice, it strongly inhibits the neovascularization of solid tumors and suppresses their growth (248). Because neoangiogenesis is an important mechanism in inflammation, 2-methoxy-E2 can exert antiinflammatory activities by inhibiting vessel formation. An antiangiogenic effect of 2-methoxy-E2 was confirmed in highly neoangiogenic prolactin-secreting tumors (440).
In the disease model of type II collagen-induced arthritis in ovariectomized DBA/1 mice, 2-methoxy-E2 suppressed development of arthritis (270). In contrast to E2 (proestrus levels), 2-methoxy-E2 exerted neither detectable feminizing effects on the reproductive tract nor inhibition of leukocyte development in hematopoietic organs or lymphoid organs. In contrast, E2 induced a clear decrease of CD4+, CD8+, Ig+ cells in the spleen, and a decrease of CD4+, CD8+, and CD4+CD8+ cells in the thymus. Neither the T cell-dependent delayed type hypersensitivity nor the B cell-dependent production of anti-type II collagen antibodies was suppressed by 2-methoxy-E2 (270). These results are highly interesting because they demonstrate that 2-methoxy-E2 might be a safe antiinflammatory drug without strong immunosuppressive activities.
In chronic puromycin aminonucleoside-induced nephropathy in rats (441), 2-hydroxy-E2 improved glomerular filtration, reduced proteinuria, and attenuated elevated blood pressure. 2-Hydroxy-E2 had no effect on renal tubular cell TGF-ß, but it reduced glomerular proliferating cell nuclear antigen and collagen IV, and glomerular and interstitial macrophage infiltration (not blocked by ICI 182,780) (441). The question arises whether or not proapoptotic or cytostatic effects play a role in human disease.
A case-control analysis using pretreatment urine samples suggested that a lower 2-hydroxy-estrogen/16-hydroxyestrogen ratio was associated with an increased risk of premenopausal and postmenopausal breast cancer diagnosis among Chinese women (442). This might indicate that the cytostatic effect of 2-hydroxy-estrogen-derived 2-methoxy-E2 is missing. Because inflammatory signals are unpleasant in breast cancer development, the loss of 2-methoxy-E2 is thought to play an important unfavorable role (443). This might be similar in inflammatory diseases.
Urinary concentration and total urinary loss of 2-hydroxyestrogens was 10 times higher in healthy subjects compared with patients with either SLE or RA, irrespective of prior prednisolone treatment or sex (284). The urinary concentration and loss of 16-hydroxy-E1 did not differ between healthy subjects and patients with RA/SLE. The ratio of urinary 16-hydroxy-E1/2-hydroxyestrogens was more than 20 times higher in RA and SLE than healthy subjects, irrespective of prior glucocorticoid treatment or sex, which indicates that production of 2-hydroxyestrogens is largely down-regulated in these patients (284). Interestingly, women who developed knee osteoarthritis, which is also an inflammatory disease, were more likely to have a ratio of 16-hydroxy-E1 to 2-hydroxy-E1 in the highest tertile and 2-hydroxy-E1 in the lowest tertile (444). The loss of 2-hydroxyestrogens is obviously a proinflammatory signal, which might be unfavorable in breast cancer development and inflammatory diseases.
An unwanted shift from 2-hydroxyestrogens to 4-hydroxyestrogens might be an additional proinflammatory signal because these endogenous metabolites can be converted to 3,4-quinones, which lead to depurination and mutation in vivo (summarized in Ref. 431). The 3,4-quinones produce ROS, which stimulate inflammation, whereas methylation and sulfation are detoxification pathways, which can inhibit these negative tissue-destroying sequelae (431). Others confirmed that 4-hydroxy-E2 undergoes 2-electron oxidation to quinone via semiquinone, and during this process, ROS can be generated to cause DNA damage and cell death (204). A similar effect of E2 conversion was confirmed in hepatocytes (445). In contrast to the prooxidant activities of 4-hydroxy-E2, 2-hydroxy-E2 is a potent antioxidant (more potent than vitamin E) and thereby protects membrane phospholipids and cells against peroxidation (446). Thus, 4-hydroxy-E2 in contrast to 2-hydroxy-E2 induces carcinogenic and proinflammatory effects (430).
Importantly, cytochrome P4501B1 (CYP1B1) is a dioxin-inducible gene that is active in the formation of 4-hydroxy-E2 (447). In human peripheral blood lymphocytes, absolute CYP1B1 mRNA levels varied more than 30-fold in uncultured mononuclear cells obtained from 10 individuals. In vitro treatment of mitogen-stimulated lymphocytes with dioxin for 1–5 d of culture resulted in a peak induction of CYP1B1 after 3 d. These observations indicate that CYP1B1 exhibits variable constitutive expression and is inducible in vitro by dioxin in human lymphocytes (447). Presently, we do not know whether proinflammatory mediators induce a shift from 2-hydroxyestrogens to 4- and 16-hydroxylated estrogens.
In conclusion, there is clear evidence that 2-methoxy-E2 is a proapoptotic and cytostatic endogenous compound, which can inhibit neoangiogenesis and can attenuate inflammation in animal models. These effects are not mediated via the classical ERs, and they do not lead to feminizing effects. In contrast, 4-hydroxylated estrogens might exert proinflammatory roles by inducing ROS and DNA damage. Thus, stimulation of one or the other pathway is possibly decisive in chronic inflammation.
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XI. Pregnancy and Inflammation |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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. The role of progesterone is particularly mentioned because a separate effect of one single steroid hormone cannot be derived from studies with pregnant women. Thus, the following discussion must take into account these particular circumstances during pregnancy.
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SLE tends to flare during pregnancy, and the puerperium and, not rarely, SLE starts during pregnancy. Most flares are mild, and cutaneous and joint disease are the most common manifestations (455, 456). SLE flares are associated with increased prematurity (457), and active nephritis is an independent factor for fetal mortality (458). SLE disease activity scores varied during pregnancy, being increased in the second trimester and decreased in the third trimester (459). Similarly, pregnancy also affects the disease course in mice. In female MRL lpr/lpr mice, one observes a postpartum exacerbation of their mild spontaneous arthritis (272). Administration of E2 (pregnancy levels) for 2 wk after Freund’s complete adjuvant injection prevented adjuvant-enhanced arthritis, reducing the incidence from 67% to the baseline level of 21% (272). In contrast, the same dose increased proteinuria and mortality rate, which demonstrates completely different effects on joint disease compared with nephritis (272).
Observations in the 19th century (Trousseau 1871, Charcot 1881, Bannatyne 1896) indicated that pregnancy is favorable in RA as summarized in the Nobel Prize Lecture of the rheumatologist Philip S. Hench. He wrote: "... after 1931, records of these cases [of cases with RA] were more carefully made and assembled ... because of my growing belief that this phenomenon of relief [from arthritic disability] was analogous to, if not identical with, that which may occur during jaundice, and that the same agent might be responsible for the relief both during pregnancy and jaundice, although the mechanism ... might be different." (460). A pregnancy-induced remission is experienced by 75% of patients with RA, but a flare-up occurs in more than 60% after delivery (461).
The first indication that T cell pathways are suppressed in pregnant patients with RA was provided by Russell et al. in 1997 (462). LPS-stimulated whole blood cultures were demonstrated to release less IL-2 but more soluble TNF receptor p55 and p75 during pregnancy. In this study, serum TNF and IL-1ß concentrations were unchanged (462). Others demonstrated decreased IFN- and IL-12 production by lymphocytes after phytohemagglutinin stimulation in third-trimester pregnant women with RA compared with healthy pregnant women (463). This study was complemented by data in RA patients (third-trimester pregnancy) that demonstrated that ex vivo monocytic IL-12 production was about 3-fold and TNF production was approximately 40% lower than postpartum values (464). In a prospective longitudinal study, elevated IL-10 levels were found in RA and SLE pregnant women compared with healthy pregnant controls (465), which also fits to the stimulating effects of E2 on this cytokine (Section IV.D). In addition, the concomitantly increasing progesterone has very similar effects (Table 8).
Significantly higher concentrations of soluble TNF receptor 2 and IL-1ra were measured in pregnant compared with nonpregnant women (466). An increase of IL-1ra from the second to the third trimester correlated with improvement of disease activity in both RA and ankylosing spondylitis patients. Compared with nonpregnant patients and other pregnant women, RA patients showed markedly elevated levels of soluble CD30 during pregnancy (466). CD30 (Ki-1) antigen has been considered to be expressed on hematopoietic cells (e.g., Reed-Sternberg cells of Hodgkin’s disease) but also in nonhematopoietic cells such as human decidual cells. It is thought that CD30 is a relatively specific marker for Th2 lymphocytes (449). All these data in pregnant women with inflammatory arthritis demonstrate a Th2 dominance that might be even more pronounced compared with healthy pregnant women. These studies also demonstrate that important antiinflammatory factors such as soluble TNF receptors and IL-1ra are additionally up-regulated (462, 466). Furthermore, these studies indicate the favorable changes during pregnancy to reverse after delivery leading to increased disease activity. These effects are most probably mediated by estrogens and progesterone together.
Others studied 254 women with MS during 269 pregnancies in 12 European countries (467). The women were followed during their pregnancies and for up to 12 months after delivery. In women with MS, the rate of relapses declined during pregnancy, especially in the third trimester, and increased during the first 3 months postpartum before returning to the prepregnancy rate (467).
In conclusion, on the basis of effects of E2 and progesterone on innate and adaptive immune responses (see sections above and Table 8), it is expected that B cell/antibody-driven diseases exacerbate, whereas T cell-driven diseases with cytotoxic and innate immune responses improve. In some diseases, both aspects of immune responses might be present at the same time (272, 304). In such a situation, antibody-dominated pathways are supported, and cytotoxic and innate immunity pathways are inhibited.
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XII. The Contraceptive Pill, Hormone Replacement Therapy, and Inflammation |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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), the discussion below must be viewed with this premise.
A. The contraceptive pill
The influence of OC on RA was studied in population-based samples in which the relative risk to develop RA is investigated in women with vs. without OC treatment before disease outbreak. When compared with women who had never used OC, no increase of the age-adjusted relative risk was found for past users in several independent studies (277, 468, 469, 470, 471, 472, 473, 474, 475, 476). There were only three studies that found a decreased risk for RA in women who had been administered OC before disease outbreak compared with women without OC treatment (477, 478, 479), but the effects were not very marked.
As early as 1972, the influence of OC on women with RA was investigated (480). RA women under OC compared with RA women using other contraceptive methods did not demonstrate changes in levels of antinuclear antibodies, rheumatoid factor, and erythrocyte sedimentation rate as markers of disease activity (480). Only two studies reported improvements by OC treatment with respect to swollen joint count and severity (277, 481), but the long-term effects of OC treatment are not marked (482).
In three case-control studies, OC use before diagnosis was not associated with an increased disease risk in patients with SLE (483, 484, 485). Global disease activity, maximum SLE disease activity index, incidence of flares, time to first flare, and incidence of adverse events were similar among women with SLE independent of OC use (486). In a recent study, it was confirmed that OC did not influence the course of SLE (487).
Similarly, prior use of OC did not change the risk for MS (488, 489, 490)
In conclusion, almost all studies in women with vs. without OC demonstrated no increased risk to develop a chronic inflammatory disease such as RA, SLE, or MS. In addition, OC use was not associated with changes in the disease course in patients with RA and SLE (it is not known for MS). During OC administration, serum levels of estrogens and progestins only increase to a small extent, and, thus, no change of risk is to be expected in a woman with premenopausal estrogen levels.
B. Hormone replacement therapy
HRT is a combination therapy of naturally occurring or synthetic estrogens together with progestins or synthetic progestins. Thus, often a separate analysis of estrogen and progestin effects is impossible to perform, and the effects observed must be assigned to the combination of estrogens and progestins. In addition, HRT makes use of different types of estrogens, and most often CEE or synthetic E2 derivatives were used. CEE are a mixture of many different types of conjugated estrogens, which might have different effects and different pharmacodynamics and pharmacokinetics compared with E2 (256). In contrast, in animal studies most often E2 was used, but rarely a synthetic E2 derivative (ERT) was administered in women as the sole therapy.
Because the sections above mentioned exact E2 concentrations, it is important to point out that serum levels of E2 during hormonal therapy only increase to a relatively small extent; for instance, serum levels of E2 increased from 7 x 10–11 M to 2.5–4.3 x 10–10 M in orally treated women (2 mg/d E2 for 22 d, 1 mg/d E2 for 6 d combined with 1 mg/d norethisterone during the last 10 d of the cycle) and from 7 x 10–11 M to 1.5–1.6 x 10–10 M in transdermally treated women (continuously 50 µg/d E2 combined with 10 mg/d oral medroxyprogesterone during the last 10 d of the cycle) (491). With this information in mind, the following text should be carefully studied.
1. TNF, IL-1ß, IL-6, and C-reactive protein.
In whole blood assays, prior HRT (E2 + progestins) decreased the levels of TNF and thromboxane B2, and of tissue factor from LPS-stimulated monocytes (491). In supernatants of PBMCs, prior HRT (E2 + progestins) induced a decrease in levels of IL-1ß, IL-6, soluble IL-6 receptor, and TNF (23). In LPS-stimulated whole-blood cells, supernatant levels of IL-1ß and TNF were reduced after prior HRT (CEE + progestin) compared with untreated postmenopausal women (492). In a prospective study, HRT decreased serum levels of IL-1ß and TNF (492). In postmenopausal women, treatment with transdermal E2 (50 µg/d) for 12 months decreased spontaneous IL-6 production by PBMCs (147). This inhibiting effect on IL-6 was confirmed by others, but the effect was abrogated by coadministration of medroxyprogesterone (493). In 196 healthy postmenopausal women, 6 months of HRT (oral E2 plus progestin) did not change serum levels of IL-6 and TNF (494). In healthy postmenopausal women, 12 wk of oral E2 treatment did not change serum levels of IL-6 (495). In postmenopausal women, after a low dose of endotoxin, transdermal E2 (0.1 mg, serum levels 3.7 x 10–10 M compared with 2.7 x 10–11 M in controls) attenuated the endotoxin-induced release of TNF, IL-1ra, and IL-6 (496). In postmenopausal women, HRT (no exact type is given) had lower serum IL-6 levels compared with subjects without HRT, which was independent of age, antihypertensive therapy, smoking habits, and blood pressure (497). Thus, most studies with E2 alone or E2 plus progestins showed an inhibiting effect or no effect on proinflammatory cytokines such as TNF, IL-1ß, and IL-6. This situation might be different when CEE with/without progestins are used.
In postmenopausal women, prior HRT (different preparations with CEE and progestins) induced a higher mitogen-induced T cell proliferation, and higher levels of induced TNF from PBMCs (498). CEE plus progestin increased the stimulated secretion of TNF and IL-6 (499). Similarly, in healthy postmenopausal women, HRT (CEE only during 6 wk) resulted in increased levels of IL-6 (130). Others demonstrated that CEE increased TNF secretion from LPS-stimulated PBMCs but did not change IL-6 secretion (499).
In healthy postmenopausal women (with baseline E2 below 9 x 10–11 M, mean = 7.9 x 10–11 M), HRT (CEE only, during 6 wk) resulted in increased levels of C-reactive protein (130). Similarly, CEE increased plasma levels of C-reactive protein (499). In postmenopausal women, HRT (CEE + progestin) increased C-reactive protein levels (131). In healthy postmenopausal women, 6 months of oral HRT (oral E2 plus progestin) increased C-reactive protein serum levels compared with both placebo and transdermal E2 plus progestin (494).
In conclusion, most studies with E2 alone or E2 plus progestins showed an inhibiting effect or no effect on proinflammatory cytokines, but in contrast, studies with CEE with/without progestin most often demonstrated stimulating effects. The different effects of E2 with/without progestins vs. CEE with/without progestins may be due to the mixture of many different types of conjugated estrogens in drugs with CEE, which do not contain the pure E2 (256). As demonstrated above, this mixture of conjugated estrogens might even have unwanted proinflammatory effects. The comparison of E2 with/without progestins vs. CEE with/without progestins also demonstrates that other estrogens than E2 might well have a proinflammatory potential. Furthermore, the dose and route of estrogens might play an additionally important role because only oral but not transdermal therapy leads to an increase of C-reactive protein. This likely demonstrates that hepatic conversion of E2 to downstream estrogens produces proinflammatory metabolites.
2. MCP-1 and adhesion molecules.
In postmenopausal women on 1 mg E2 continuously plus sequential progestin over 1 yr, serum levels of MCP-1, soluble ICAM-1, soluble VCAM-1, and soluble E-selectin gradually decreased after 3, 6, and 12 months (227). HRT (CEE + progestin) for 2 months decreased serum levels of MCP-1, soluble ICAM-1, soluble VCAM-1, soluble E-selectin, tissue factor antigen, and plasminogen activator inhibitor type 1, but increased tissue factor activity (129). In postmenopausal women, HRT (CEE + progestin) decreased soluble ICAM-1, soluble VCAM-1, soluble E-selectin, and soluble thrombomodulin (131). In healthy postmenopausal women, HRT (CEE only during 6 wk) resulted in decreased levels of soluble E-selectin (130). In ovariectomized rhesus macaques, oral CEE decreased serum levels of soluble VCAM-1, MCP-1, and soluble E-selectin (133). However, others demonstrated no effects of oral E2 treatment for 12 wk on serum levels of soluble ICAM-1 (495). In postmenopausal women with coronary artery disease, ERT (CEE only) increased E2 serum levels from approximately 3 x 10–11 M to 1.5 x 10–10 M and decreased serum levels of soluble E-selectin, soluble ICAM-1, and soluble VCAM-1 (126).
In conclusion, most of the studies demonstrated that E2 plus progestins or CEE with/without progestins decrease serum levels of MCP-1 and important soluble adhesion molecules, which is thought to be a favorable effect on atherosclerosis, and probably on other chronic inflammatory diseases. This thinking is based on the idea that soluble adhesion molecules reflect levels on the cell surface, and that lower cell surface levels are related to decreased migration of leukocytes into inflamed tissue (see also Sections IV.B and VI).
3. T and B cells.
HRT in postmenopausal women reduced the number of circulating natural killer cells, CD4+ memory T cells, and CD8+CD11b+ T cells (500). Similarly, circulating numbers of CD4+ T cells and CD4+CD69+ T cells decreased in E2-treated ovariectomized rhesus macaques, but B cells did not change (E2 serum levels 5 x 10–10 M) (501). In healthy women with surgical menopause (myoma operation), transdermal E2 treatment decreased numbers of circulating CD8+ T cells and increased CD19+ B cells (169). Similarly, HRT (different preparations with CEE and progestins) increased the number of circulating CD19+ B cells (498).
These studies demonstrate that HRT particularly decreased T cells numbers, but B cell numbers are unaffected or up-regulated. This confirms the stimulating effects of estrogens on B cells (see also Section III.B).
4. Arthritis.
The influence of HRT on RA initiation was studied in population-based samples. The relative risk to develop RA was investigated in women with vs. without HRT before disease outbreak. An important disadvantage is the fact that almost all observational studies did not mention the type of HRT used. Above-mentioned data demonstrate that the type of HRT, whether CEE or E2 with/without progestins, can be an important decisive factor.
Compared with postmenopausal women who never used HRT, current users had an age-adjusted RR of 1.3 (95% CI, 0.9–2.0), past users had an age-adjusted RR of 0.7 (95% CI, 0.5–1.2), and ever-users had a RR of 1.0 (95% CI, 0.7–1.4) (473). This study nicely summarized the findings of other groups, which confirmed that HRT did not increase the relative risk to develop RA (468, 479, 502, 503, 504, 505). In addition, it might be interesting to study the influence of HRT or ERT on the course of RA because many antiinflammatory effects of E2 have been described (see sections above).
As early as 1964, the influence of HRT on disease course of RA was investigated, but this treatment had little effect (506). Others demonstrated a beneficial effect of the same HRT (0.45 mg/d mestranol, 30 mg/d norethynodrel) in 33 postmenopausal women, with a complete remission in seven patients and an objective improvement in 11 patients (507). In a prospective double-blind crossover study in RA with ethinyl E2 as the sole therapy (12.5 µg/d), small beneficial effects of E2 were observed as a decrease in 30-m walking time and thrombocytosis, and an increase of hemoglobin concentrations (508). No differences between E2 and placebo therapy were observed for erythrocyte sedimentation rate, C-reactive protein, grip strength, number of swollen joints, and joint tenderness score (508).
HRT with 2 mg E2 valerate plus progesterone for 52 wk has been studied in placebo-controlled investigations in patients with RA (509, 510). No significant difference was found in and between both treatment groups with regard to articular indices, pain score by visual analog scale, erythrocyte sedimentation rate, and health questionnaire score on daily activities before, during, and at the end of the study. However, bone mineral density increased in the lumbar vertebral spine and femoral neck in the E2 group compared with the placebo group (509, 510). Similarly, HRT (50 µg E2, twice weekly, plus 1 mg norethisterone for 10 d on a cyclical basis, for 48 wk) increased spine bone mineral density, but bone mineral density at the femoral neck and distal radius did not change (511). In the HRT group, there was improvement in well-being as assessed by the Nottingham Health Care Profile, but pain score, morning stiffness, erythrocyte sedimentation rate, and C-reactive protein did not change (511).
HRT (50 µg/d transdermal E2 plus 1 mg norethisterone/d for 12 d per month) increased serum E2 in only 58.4% of patients with RA (responders, mean E2 levels, 
2 x 10–10 M) but failed to achieve serum E2 levels lower than 10–10 M in 41.6% of patients (nonresponders) (295). Only in the responders, HRT for 6 months decreased the Ritchie articular index, pain score, morning stiffness, and erythrocyte sedimentation rate (295). In the same study, the authors observed an increase in spinal bone mineral density and a stabilization of femoral bone mineral density (295). HRT or ERT was accompanied by a reduction in bone resorption markers in RA irrespective of steroid usage (410).
In postmenopausal women with RA, HRT with E2 (2 mg E2 with/without 2 mg norethisterone) increased E2 serumlevels (from 1.5 x 10–10 M to 6.5 x 10–10 M) and improved bone mineral density in the hip and lumbar spine (accompanied by an increase of IGF-I and osteoprotegerin serum levels), decreased erythrocyte sedimentation rate and disease activity score, and delayed progression of joint destruction among patients with radiological progressive disease (512, 513). Furthermore, this treatment resulted in a decrease of serum levels of type I collagen, C-terminal telopeptide of type I collagen, bone sialoprotein, and C-terminal propeptide of type I procollagen, and in a down-regulation of urinary levels of collagen type II C-telopeptide degradation fragments and cartilage oligomeric matrix protein at 2 yr, and this was associated with improved bone mineral density (411).
In conclusion, prior HRT did not increase the risk to develop RA. HRT or ERT therapy markedly improved bone mineral density and reduced bone resorption markers. In addition, in some studies HRT had small beneficial effects on the articular index, pain score, and morning stiffness when patients with high treatment levels were separately analyzed (295).
5. SLE.
Because estrogens and progesterone can stimulate B cell activities, the importance of HRT on initiation and disease course of SLE was studied. In a prospective cohort study (Nurses’ Health Study) in postmenopausal women, women were classified as never-users or ever-users (current and past) of HRT (514). With never-users of postmenopausal hormones as the reference group, age-adjusted RR for SLE was 2.1 (95% CI, 1.1–4.0) for ever-users, 2.5 (1.2–5.0) for current users, and 1.8 (0.8–4.1) for past users (514). A proportional increase in the risk for SLE was observed related to the duration of HRT (514). In postmenopausal women, short-term estrogen exposure was not associated with increased risk to develop SLE (515). The adjusted odds ratio (OR) comparing longer term estrogen users and nonusers for all cases (SLE and discoid lupus combined) was 2.8 (95% CI, 1.3–5.8) (515). Interestingly, a difference in risk was found between long-term users of estrogens alone (OR 5.3; 1.5–18.6) and those who used estrogens combined with progestins (OR 2.0; 0.8–5.0) compared with nonusers (515). The authors suggest that progestins may reduce the effect of estrogens on SLE and discoid lupus erythematosus.
In postmenopausal women with SLE, the use of HRT (estrogens alone and estrogens plus progestins combined) does not appear to increase the rate of lupus flares over a 1-yr follow-up (516). In a study with 351 postmenopausal patients with SLE, patients were treated for 12 months with HRT (CEE + progestin) or placebo (517). Severe flares were rare in both treatment groups (not different between groups) (517). Mild to moderate flares were significantly increased in the HRT group: 1.14 flares/person-year for HRT, and 0.86 flare/person-year for placebo (RR, 1.34; P = 0.01) (517).
In conclusion, postmenopausal HRT seems to increase the risk of developing SLE or discoid lupus erythematosus. In addition, HRT increases the risk of mild flares in postmenopausal patients already diagnosed with SLE. These data indicate that the stimulating effects of estrogens (and also progestins) on B cells stimulate SLE in postmenopausal women with low levels of estrogens.
6. MS.
Approximately 40% of women with MS reported worsening of symptoms related to the menopause (518). However, studies that systematically analyzed the effect of HRT on risk and course of MS are rare. One study with a small number of MS patients reported beneficial effects in 75% of patients with HRT (type not given) (519). In another larger retrospective study in women with MS, 87% reported no change after administration of HRT, but only 7% described an improvement after commencement of therapy (518). Both studies have no strong impact due to the retrospective design of the study and the low number of patients.
In a recent important study in patients with relapsing remitting MS, the favorable effects of pregnancy doses of E3 were demonstrated (47, 323). Although this investigation was not designed as a HRT study, it nicely demonstrates the positive effects of estrogen treatment.
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XIII. Estrogens and Systemic Response Systems |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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A. The hypothalamic-pituitary-adrenal axis
The HPA axis is an important regulating element during acute and chronic inflammation because it can down-regulate several proinflammatory pathways by releasing glucocorticoids and adrenal androgens (520, 521, 522, 523, 524, 525). In the 1960s, the stimulating influence of estrogens on the HPA axis was described (526). Treatment with OC increased plasma cortisol 2.5-fold (526). In transsexual men, estrogens (ethinyl E2 100 µg/d) plus antiandrogens increased free cortisol levels in 24-h urine samples, which was opposite in transsexual women on androgens (527). In addition, transdermal E2 activated the HPA axis in men during a stress test (528). Because the CRH promoter contains two estrogen-responsive elements, this might constitute a basis of sexual dimorphism in the expression of the CRH gene in the central nervous system (529, 530, 531). This is the situation under normal noninflamed conditions where cytokine levels are generally low. But what happens during chronic inflammation with elevated levels of circulating cytokines?
Indeed, the situation is markedly different when individuals are challenged with proinflammatory cytokines such as IL-1ß or with LPS. In this situation, the inhibitory effect of E2 on proinflammatory cytokines plays a dominant role. In postmenopausal women, a low dose of LPS in the presence of transdermal E2 (0.1 mg, serum levels 3.7 x 10–10 M compared with 2.7 x 10–11 M in controls) attenuated the release of ACTH and cortisol (496). Because E2 also attenuated the endotoxin-induced release of IL-6 and TNF, the authors concluded that E2 inhibited secretion of proinflammatory cytokines and, thus, E2 suppressed cytokine-induced stimulation of the HPA axis (496). Similarly, in ovariectomized rhesus monkeys, E2 (early-midfollicular levels) blunted the cortisol response to intracerebroventricular infusion of IL-1 (532). In ovariectomized rhesus monkeys, 3 wk of E2 replacement therapy decreased IL-1ß-induced secretion of ACTH (533).
In conclusion, E2 stimulates the HPA axis in individuals without a proinflammatory stimulus, but E2 inhibits the HPA axis under proinflammatory stimuli such as IL-1 or LPS (Fig. 4). The latter effect is in part mediated by an inhibitory influence of E2 on secretion of proinflammatory cytokines. One might speculate that in chronic inflammatory diseases the cytokine-induced activation of the HPA axis might be more inhibited in women than men. This is unfavorable for women due to the lower levels of antiinflammatory glucocorticoids. These effects of E2 on the HPA axis might contribute to the sexual dimorphism in chronic inflammatory diseases.
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To change the activity of the sensory nervous system, receptors for estrogens must be present in respective neurons. ER and ERß immunoreactivity was located in sensory neurons of dorsal root ganglia (537). Both receptor subtypes were present in neurons of the dorsal horn (537). On the basis of these findings, one would expect an influence of estrogens on neurogenic inflammatory pathways.
Estrogen treatment of ovariectomized animals stimulated increased expression of the substance P precursor (538, 539). Compared with control rats, mRNA of the substance P receptor (neurokinin 1 receptor) was increased by 2-fold from rats treated with E2 (540). Similarly, E2 at pregnancy levels (not at proestrus levels) led to a 2.5-fold increase in neurokinin 1 receptor mRNA levels (blocked by tamoxifen) (541). Increased neurokinin 1 receptor mRNA levels in response to E2 were related to increased binding of substance P to its receptor (541). Thus, estrogens might increase neurogenic inflammation and sensitization to painful stimuli by up-regulating substance P and its receptor.
In the 1970s, the enlargement of the genital and pudendal receptive field was demonstrated after estrogen treatment (542, 543), which is a sign of increased sensitization of sensory pathways. With respect to the reproductive tract, the stimulating role of estrogens on sensory neurons and substance P production has been summarized (544). In rats, E2 treatment (proestrus to pregnancy levels) increased sensory innervation of mesenteric arterioles after 7 d (545). E2 increased neurite area per sensory dorsal root ganglion neuron, and therefore it was concluded that E2 acts directly on the sensory neuron by increasing the neurite outgrowth in the absence of other exogenous growth factors (546). These findings suggest that E2 increases sensitization of sensory pathways, which possibly leads to an increase of neurogenic inflammation (Fig. 4).
In addition, female rats experience more pain than male rats upon different stimuli (547), which has been demonstrated in humans, too (548). Most patients with painful bladder disorders are women (549). Similarly, irritable bowel syndrome is more prevalent in women than in men (336). Ovariectomized rats treated with E2 (diestrus levels) exhibited visceral hypersensitivity after partial restraint stress (337). The authors concluded that stress-induced visceral hypersensitivity in female rats is E2-dependent and mediated through neurokinin 1 receptor activation (337). In the rat, E2-stimulated hyperalgesia was antagonized by inhibitors of the cyclooxygenase pathway (550). In rats, E2 (proestrus levels) increased the excitability of the temporomandibular joint sensory neurons (551). E2 plus Freund’s adjuvant demonstrated an additive effect on excitability of the temporomandibular joint sensory neurons (551). Intracerebroventricular injection of E2 increased the amount of licking of the formalin-injected painful paw (inhibited by ICI 182,780). The results demonstrate that centrally acting estrogens influence pain processing (552). Numerous studies of sex differences in opioid analgesia in rodents reported that opioids are more potent in males than in females (summarized in Refs. 553 and 554). Aromatase is expressed and is enzymatically active in pain-sensitive neurons in the dorsal horn (555). Treatment with intrathecal aromatase inhibitors increased pain thresholds, which indicates that estrogens sensitized pain perception (555).
From the above-mentioned studies, it is probable that E2 can enhance pain perception on different levels (in the periphery, in the spinal cord, and in the central nervous system). However, one has to be careful because some studies clearly demonstrated dose-dependent effects of estrogens on pain perception; e.g., in ovariectomized rats, E2 administration (diestrus levels) restored opioid analgesia, but further E2 (proestrus to early pregnancy levels) suppressed opioid analgesia (556). In ovariectomized rats, proestrus levels of E2 increased morphine-induced analgesia, whereas E2 at the pregnancy level had no effect on morphine-induced analgesia (557). Similarly, it is known that pain thresholds increase throughout late pregnancy, which appears to involve spinal opioid receptors (558). This indicates that E2 at high pregnancy levels inhibits sensory neuronal pathways.
In conclusion, estrogens can enhance pain perception and most probably neurogenic inflammation leading to lower pain thresholds in female rats and women. It appears that E2 affects pain perception according to the concentration of this hormone because E2 might stimulate pain sensitization at metestrus to early pregnancy levels, whereas E2 at high pregnancy levels inhibits pain perception.
C. The sympathetic nervous system
The SNS with its most important neurotransmitter norepinephrine elicits proinflammatory effects via 1- and 2-adrenoceptors but has antiinflammatory effects via ß2-adrenoceptors by directly inhibiting TNF secretion and other proinflammatory pathways (559, 560). Thus, a possible influence of estrogens on the SNS might change the influence of the SNS on inflammation.
The question that remains is how the activity of the SNS can be modulated by estrogens. This would imply that ER and/or ERß receptors are present in neurons of the SNS. Indeed, sympathetic neurons express ER and ERß in different anatomical compartments (561). Similarly, one might ask whether estrogens can modulate adrenergic receptors or signaling pathways. It seems as if there are differences with respect to regulation of the 2-adrenoceptor because administration of clonidine, an 2-adrenergic agonist, resulted in a stronger decrease of plasma norepinephrine in women than men (562). This might demonstrate an elevated 2-adrenergic activity in women.
An increase in 2-adrenoceptor activity may be related to a greater number of 2-adrenoceptors in females than in males as found in the urethra (563). Similar findings of an increased 2-adrenoceptor density were reported from platelets of women compared with men (564). With respect to 2-adrenergic stimulation, E2 replacement enhanced vasoconstriction induced by smooth muscle 2-adrenoceptor activation (565). In ewes, E2 treatment increased 2-adrenergic receptor density in myometrial membranes (566). In rabbit uterine smooth muscle, estrogen treatment increased uterine 1- and 2 adrenoceptor density (567). It was reported that E2 pretreatment increases the responsiveness to norepinephrine and clonidine in isolated rat female aorta, and the authors concluded that E2 pretreatment increases the number of 1-adrenoceptors (568). E2 pretreatment of rats (pregnancy levels) increased 1-adrenoceptor numbers in the nucleus tractus solitarius relative to control (569). If such an estrogen-dependent increase of 1- and 2-adrenergic receptor activity would play a role in immune cells, which is not known, it might be a clear proinflammatory signal because these receptors serve many proinflammatory pathways, which is opposite for ß-adrenergic receptors.
Importantly, E2 at pregnancy levels was able to down-regulate ß1-adrenergic receptors on cardiomyocytes (570). In hypothalamic and preoptic brain slices of rats, E2 treatment reduced the ß-adrenergically mediated increase of cAMP (571). Similarly, in rabbit myometrium, E2 inhibited the ß-adrenoceptor pathway (572). However, the endometrium of the ovariectomized rat was devoid of any ß-adrenoceptors, whereas that of the E2-treated rats was increased. After ovariectomy, there was an increase in ß-adrenoceptor density in the myometrium, but this was not observed in the animals given E2 replacement (573). Particularly, these latter results indicate a site-specific modulation of ß-adrenergic receptors by estrogens. As with -adrenoceptors, we do not know whether ß-adrenergic receptors are modulated on immune cells. A potential estrogen-induced decrease in ß-adrenoceptor density on immune cells would be a proinflammatory signal.
Another question remains whether estrogens influence sympathetic innervation, because a low density of sympathetic nerve fibers must be regarded as a proinflammatory signal (559). Since the late 1970s, it has been known that uterine sympathetic nerve fibers disappear during pregnancy (574, 575). The estrogenic status largely affects sympathetic innervation, which has been demonstrated in the ER-deficient mouse; ER-deficient mice demonstrate a marked hyperinnervation of the uterus, which was not demonstrated for sensory or cholinergic nerve fibers (576). The effect of estrogens on sympathetic innervation was also observed in other tissue; ovariectomized rats showed a 59% elevation in vaginal sympathetic nerve fiber density. E2 treatment for 7 d (proestrus level) reduced nerve fiber density to an extent comparable to the situation during estrus (577). However, only urogenital tissues have been investigated, so a general conclusion cannot be drawn. It might be that under the influence of estrogens, similar to the site-specific regulation of ß-adrenoceptors, a location-specific change of tissue innervation appears.
In conclusion, estrogens enhance 1- and 2-adrenergic receptor signaling but can decrease ß-adrenoceptor signaling. Assuming that estrogens would contribute to the loss of sympathetic innervation in inflamed areas similar to the pregnant uterus, this would be an additional proinflammatory signal due to decreased neurotransmitter concentrations (559). Although these concepts are intriguing, it is important that they are verified in inflammatory diseases.
D. Summary
Estrogens influence systemic response systems by reducing the cytokine-stimulated ACTH and cortisol release, by increasing substance P signaling and sensitization to painful stimuli (increase of neurogenic inflammation), and by increasing signaling through proinflammatory -adrenergic pathways (Fig. 4). These estrogen effects can partly explain the sexual dimorphism in chronic inflammatory diseases.
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XIV. The Dual Role of Estrogens and Consequences for Autoimmune Disease |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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summarizes the bimodal effects of E2.
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Other functional antagonisms were demonstrated with respect to TGF-ß (172), VEGF secretion (246), and IL-1RI and IL-1RII (143). Another functional antagonism is dependent on the presence of ER and ERß. In different epithelial cell lines, the E2-ER complex rapidly activates multiple signaling pathways committed to both cell cycle progression and prevention of apoptotic cascades (214). On the other hand, the E2-ERß complex induced the rapid and persistent phosphorylation of p38 MAPK, which in turn was involved in caspase-3 activation and cleavage of poly(ADP-ribose) polymerase, driving cells into the apoptotic cycle. Thus, E2 promotes cell survival through ER nongenomic signaling and cell death through ERß nongenomic signaling (214). It is also a functional antagonism when E2 stimulates many aspects of B cell immunity but inhibits generation of B cell precursors in the bone marrow (Section III.B).
These bimodal effects of E2 need to be taken into account when discussing proinflammatory diseases in pre- and postmenopausal women. These bimodal effects are relevant in many chronic inflammatory diseases as outlined in Fig. 3. Some concepts are developed in the following section.
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XV. Discussion |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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and 2, Section III.I), on various chronic inflammatory diseases (Fig. 3, Section VIII.N), and on systemic response systems (Fig. 4, Section XIII.D). The following part presents afterthoughts of the author, which are synthesizing the information given above. In some parts, the presented ideas have a hypothetical character, which includes the main aspects "estrogen targets," "estrogen concentration," and "estrogen timing." The discussion is given with the particular focus on chronic inflammatory diseases.
A. Autoimmune diseases: the initiation phase
B cells, T cells, and antigen-presenting cells (macrophages/dendritic cells, B cells) are decisive for the initiation of autoimmune diseases. One might separate two clinically important phases of an autoimmune disease: 1) an asymptomatic phase, and 2) a symptomatic phase (after disease outbreak). Disease outbreak means involvement of a target organ/target tissue leading to the classical signs of visible and functionally relevant inflammation.
Recent important work in the field of RA delineated that autoimmune phenomena are already present more than 10 yr before disease outbreak (586, 587). In the asymptomatic phase, T cells, B cells, and antigen-presenting cells play an important hidden role because clonal expansion of autoreactive T cells and B cells happens without overt disease symptoms. Because the influence of estrogens on these secret players is different from other cell types (Figs. 1 and 2), estrogens can have quite opposite roles depending on involved cells.
If B cells play a center role by antigen presentation, autoantibody production, and/or bystander cytokine secretion, E2 will probably speed up the outbreak of the disease in the early reproductive years (Fig. 5, upper part). One might speculate that pregnancy with high levels of E2 and progesterone will prepare the outbreak of the disease because, during this period, autoreactive B cells, which are normally deleted in the bone marrow, are rescued and come to maturity in the marginal zone of the spleen or in other secondary lymphoid organs (36). Because pregnancy with high levels of estrogens but also progesterone has a tremendously inhibitory influence on most inflammatory pathways, women might be sheltered until parturition but might be prone to disease outbreak thereafter in the vulnerable postpartum phase. Because men never experience these high estrogen or progesterone levels, the apparent gender dimorphism of autoimmune disease during the reproductive period of women can be explained.
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, lower part). Nevertheless, clonal expansion of autoreactive B cells might happen during the reproductive period, but the outbreak is delayed. Preceding pregnancies might have a similar preparing role as mentioned above. This might be the reason why autoantibody production is observed more than 10 yr before disease outbreak in the late reproductive years but the disease breaks out in the postmenopausal period (371, 586). Similarly, if tissue-destructive T cells play the most important role, the onset of the disease is expected in the postmenopausal phase when E2 but also progesterone declines (Fig. 5, lower part). Thus, ovariectomy in animal models or the menopause in women is stimulatory for autoimmune arthritis and other autoimmune diseases of late onset. In such a situation, previous pregnancies might even shelter women from the disease. Today, we know that different subtypes of a given autoimmune disease exist. This has been nicely demonstrated in patients with RA and MS (164, 165, 166, 167). One group of patients demonstrates B cell-dependent pathologies, and another group shows mainly tissue-destructive T cell-dependent autoimmunity. Presently, we do not classify these diseases according to the prevailing autoimmune reaction. In addition, we do not classify the diseases according to the success of anti-B cell therapy, but this might be future thinking. It is suggested that such a classification would lead to a better understanding of the role of estrogens in these diseases (251). Classification in this sense might also explain some confusing results obtained in studies in patients with autoimmune diseases.
B. Autoimmune diseases: the symptomatic phase
The location of tissue inflammation in an autoimmune disease is defined by the major autoantigen (or autoantigens). Once a disease has entered the highly inflammatory symptomatic phase, many other cell types get involved depending on the major location of the autoimmune disease (in the brain, in the joint, in the kidney, etc.), e.g., in the joint, macrophages, fibroblasts, osteoclasts, osteoblasts, adipocytes, endothelial cells; and many more get gradually involved in the inflammatory process. This does not similarly happen in the brain in MS or in the thyroid gland in thyroiditis due to another environment. Depending on the predominant cell types involved, E2 (and also progesterone) might have quite different effects on these bystander inflammatory activities of participating cells (Fig. 2, and Section III). Even during the course of an autoimmune disease the different players might get more or less important so that the character of the disease progressively changes. For example, neoangiogenesis plays an important role in early but not in late RA, which necessarily means that E2 influences this factor more in the early than in the late phase of the disease. These changes of the underlying players are probably important for understanding the different roles of administered E2 at different time points as outlined in Section IX.
All players (including B cells, T cells, and antigen-presenting cells) might have very different capacities to take up and to metabolize estrogens. Metabolism of estrogens depends on transport into cells, desulfation of sulfated estrogens, sulfation of nonsulfated estrogens, androgen aromatization, and estrogen conversion to downstream hydroxylated or methylated estrogens, the role of which is slowly being discovered (Section X). In addition, up- or down-regulation of ER and ERß (in cells or on the cell surface), of coactivators, and of corepressors might well depend on involved cells and microenvironmental conditions such as accompanying hypoxia and the surrounding cytokine cocktail (Section II).
In addition, it has been nicely demonstrated in one animal model that E2 accelerates immune-complex glomerulonephritis but ameliorates focal sialadenitis, renal vasculitis, and periarticular inflammation (304). This indicates that quite different pathologies might even be present in the same individual so that estrogens have beneficial effects on one aspect of the disease but a deleterious influence on other mechanisms. This depends most probably on the involved cell types.
From this point of view, one begins to understand that very high E2 levels (and also other steroid hormones such as progesterone) in pregnancy can be beneficial because so many different aspects of the inflammatory process are inhibited. Even patients with SLE profit from the third trimester of pregnancy most probably due to strong immunosuppression during this time (459). Thus, patients with highly proinflammatory autoimmune diseases would only benefit from pregnancy doses of estrogens as recently demonstrated in MS (47). Studies in the last decade also demonstrated that the small change of estrogen or progesterone levels during therapy with OC or HRT does not have the power to increase the risk or to inhibit an ongoing autoimmune disease (no marked effects).
One also understands that loss of estrogens (but also progesterone) in the late reproductive phase and after the menopause can lead to proinflammatory conditions (see bone resorption and activated T cells outlined in Section VIII.M). This is substantiated by the important fact that estrogens can even stimulate several immune mechanisms at postmenopausal levels due to their bimodal role described (Figs. 3 and 5 and Table 9).
C. Inflammatory diseases with involvement of infectious agents
In diseases with infectious agents, particularly cells of the innate immune system (neutrophils, monocytes, macrophages, natural killer cells, mast cells, etc.) and bystander cells of the tissue such as fibroblasts, myofibroblasts, endothelial cells, smooth muscle cells, osteoclasts/osteoblasts, macroglia, microglia, adipocytes, and others play a decisive role in the early phase.
As outlined in Fig. 2, estrogens can stimulate several important pathways of innate immunity at follicular to periovulatory levels such as IL-1ß from monocytes, the respiratory burst and phagocytosis of monocytes, and the activity of natural killer cells. In addition, estrogens inhibit the LPS-stimulated activation of the HPA axis (inhibition of antiinflammatory cortisol) and estrogens stimulate sensory nerve fibers with the proinflammatory substance P. All these mechanisms help to overcome critical periods during bacterial infections after trauma and shock. Finally, E2 sensitizes Kupffer cells to LPS, which stimulates a strong response. This is most likely a reason why women have a better outcome during bacterial infections and why women have a lower risk to develop sepsis compared with men.
The stimulatory role of estrogens and progesterone on B cells must be particularly important because pregnant women do not often develop an infectious disease. It is hypothesized that, in the presence of many immunosuppressive effects of estrogens at pregnancy levels, estrogen-dependent stimulation of B cells is the most important compensatory mechanism to overcome infection. This might also be true during the normal menstrual cycle, when women experience most infections in the late luteal phase, and during menses when E2 and progesterone levels are lowest. Based on these concepts of estrogen-dependent stimulation of innate immunity (at follicular to ovulatory levels), women are better protected from infections than men. This also suggests that infectious stimuli such as LPS from Gram-negative bacteria or peptidoglycan from Gram-positive bacteria (and others) must play a decisive role in how estrogens influence secretion of proinflammatory cytokines. Indeed, it has been demonstrated that E2 at 10–10 to 10–9 M increased IL-1ß secretion from human Streptococci-stimulated or LPS-stimulated monocytes (73, 142). However, no systematic study is available that has demonstrated the effect of E2 on IL-1ß in the absence or presence of LPS or peptidoglycan in the same cell type. It might well be that these important agonists to Toll-like receptors can switch important estrogen signaling pathways or may differentially regulate ERs.
D. Inflammatory diseases with mild inflammation without infectious agents
Usually bone resorption is not accompanied by overt infection or sepsis, neither is it accompanied by a huge inflammation. It is obvious that estrogen levels of healthy women with a regular menstrual cycle protect from bone resorption. The postmenopausal phase with a marked reduction of estrogen (and progesterone) levels constitutes a proinflammatory milieu in the bone.
This long-lasting process with a slow progression and mild local inflammation cannot be compared with autoimmunity and short-lasting trauma/endotoxin shock. In these diseases, the menopause seems to be a transition point, at which mild proinflammatory conditions start to prevail. In this mild inflammation, HRT inducing a little shift of E2 and progesterone concentrations from postmenopausal to follicular levels can prevent inflammation. Without a doubt, HRT is beneficial to hinder bone resorption. Because we know that T cells play an important role in bone resorption, the T cell has attracted recent attention. Similar to above-mentioned autoimmune diseases of late onset, women of reproductive age are protected due to E2-mediated suppression of T cells. With the loss of E2 (but probably also of progesterone), T cells might be important starters of bone resorption. However, after initiation of the disease other cells get involved in the local inflammatory process and the disease course gradually changes (see above). In such a situation, administered E2 can have new roles depending on involved players and metabolism of E2.
E. Inflammatory fibrogenesis in liver and kidney
In these diseases, E2 exerts beneficial effects by decreasing extracellular matrix. Although E2 is a growth-promoting and antiapoptotic hormone, the effects on inflammation-induced fibrogenesis are obviously favorable. By reducing inflammation, E2 is inhibiting other pathways that are important for production of extracellular matrix. Thus, the growth-promoting effect of E2, e.g., on TGF-ß1, can be compensated for by inhibition of inflammation, finally leading to favorable effects of this hormone. Interestingly, these antifibrotic effects of E2 have been described in organs with a high content of epithelial cells (liver and kidney). The question arises whether regulation of E2 metabolism has specific characteristics in epithelial cells.
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XVI. Conclusions |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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and ERß (and their isoforms) depending on the microenvironment and the cell type, and intracellular metabolism of estrogens all play important roles in inflammatory diseases. In addition, systemic supersystems such as the HPA axis, the sensory nervous system, and the SNS can be influenced by estrogens to establish a proinflammatory milieu.
This review reinforces the concept that estrogens have antiinflammatory but also proinflammatory roles depending on above-mentioned influencing factors. This review also explains that a uniform concept for the action of estrogens cannot be found for all known chronic inflammatory diseases (Figs. 3 and 5). Nevertheless, for strictly B cell-dependent diseases such as those shown in Fig. 5, the female to male preponderance can be explained by the propagating effects of estrogens (but possibly also of progesterone). The smaller the influence of B cells and the bigger the weight of T cells and other cells, the less evident is the sex dimorphism in chronic inflammatory diseases (Fig. 5). In addition, because men never experience high estrogen (or progesterone) levels like women during pregnancy, the apparent gender dimorphism of chronic inflammatory diseases during the reproductive period of women can be explained. In addition, and this was not reported here, higher androgen levels in men most often exert inhibitory effects on many immune phenomena, which is an other important argument for why women with low androgen levels are protected from infectious diseases but more prone to B cell-dependent autoimmunity. Finally, we should not forget that sexual dimorphism of diseases may also depend on factors independent of sex hormones (588).
This review stimulated some important questions for future research (see below). These questions reflect the attitude of the author. It is hoped that the reader may derive some important new ideas, which are necessary to unravel further the complexity of estrogen actions in chronic inflammatory diseases.
Questions and future research directions
and ERß and membrane ERs regulated by E2 and in which cells from inflamed tissue does it happen? Do membrane ERs play a role in inflammatory diseases? Do these membrane receptors switch-on different signaling pathways compared with classical ERs? , and what does it mean? Is there cross-regulation of ER and ERß in cells from inflamed tissue? Does up-regulation of ERß inhibit signaling through ER? and ERß change upon Toll-like receptor stimuli?
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Footnotes |
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First Published Online July 19, 2007
Abbreviations: AP-1, Activator protein-1; CEE, conjugated equine estrogens; CI, confidence interval; E1, estrone; E2, 17ß-estradiol; E3, estriol; EAE, experimental autoimmune encephalitis; ER, estrogen receptor; ERT, estrogen replacement therapy; FasL, Fas ligand; FoxP3, forkhead box P3; HPA, hypothalamic-pituitary-adrenal; HRT, hormone replacement therapy; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule-1; IFN, interferon; IB, inhibitor of NF-B; IL-1ra, IL-1 receptor antagonist; iNOS, inducible NOS; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein; MMP, matrix metalloproteinase; MS, multiple sclerosis; NF-B, nuclear factor B; NO, nitric oxide; NOS, NO synthase; OC, oral contraceptives; OR, odds ratio; PBMCs, peripheral blood mononuclear cells; RA, rheumatoid arthritis; RANK, receptor activator of NF-B; RANKL, RANK ligand; RANTES, regulated on activation normally T cell expressed and secreted; ROS, reactive oxygen species; RR, relative risk; SLE, systemic lupus erythematosus; SNS, sympathetic nervous system; Th1, T helper type 1; Th2, T helper type 2; Th17, T helper type 17; TIMP, tissue inhibitor of MMPs; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor.
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References |
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TopAbstractI. IntroductionII. Estrogen Receptors in...III. Estrogen Modulation of...IV. Estrogens and Inflammatory...V. Estrogens and ApoptosisVI. Estrogens and Migration...VII. Estrogens and AngiogenesisVIII. Estrogens Modulate...IX. Timing of Estrogen...X. The Role of...XI. Pregnancy and InflammationXII. The Contraceptive Pill,...XIII. Estrogens and Systemic...XIV. The Dual Role...XV. DiscussionXVI. ConclusionsReferences |
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, monocyte/macrophage; NK cell, natural killer cell; OPG, osteoprotegerin; PDGF, platelet-derived growth factor; VSMC, vascular smooth muscle cell.
