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Excellence Center for Research, Transfer and High Education De Novo Therapies (M.R., S.R., M.S., P.R.), University of Florence, 50121 Florence, Italy; and Unit of Internal Medicine and Endocrinology (M.R., L.C.), Istituto Superiore per la Prevenzione e Sicurezza del Lavoro Laboratory for Endocrine Disruptors, Fondazione Salvatore Maugeri, Istituto di Ricovero e Cura a Carattere Scientifico, Chair of Endocrinology, University of Pavia, 27100 Pavia, Italy
Correspondence: Address all correspondence and requests for reprints to: Mario Rotondi, M.D., Ph.D., Unit of Internal Medicine and Endocrinology, Istituto Superiore per la Prevenzione e Sicurezza del Lavoro Laboratory for Endocrine Disruptors, Fondazione Salvatore Maugeri, Istituto di Ricovero e Cura a Carattere Scientifico, Via S. Maugeri 4, 27100 Pavia, Italy. E-mail: mrotondi{at}fsm.it
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 Top Abstract I. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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(IFN-) inducible chemokines (CXCL9, CXCL10, and CXCL11) and their receptor, CXCR3, play an important role in the initial stage of autoimmune disorders involving endocrine glands. The fact that, after IFN- stimulation, endocrine epithelial cells secrete CXCL10, which in turn recruits type 1 T helper lymphocytes expressing CXCR3 and secreting IFN-, thus perpetuating autoimmune inflammation, strongly supports the concept that chemokines play an important role in endocrine autoimmunity. This article reviews the recent literature including basic science, animal models, and clinical studies, regarding the role of these chemokines in autoimmune endocrine diseases. The potential clinical applications of assaying the serum levels of CXCL10 and the value of such measurements are reviewed. Clinical studies addressing the issue of a role for serum CXCL10 measurement in Graves’ disease, Graves’ ophthalmopathy, chronic autoimmune thyroiditis, type 1 diabetes mellitus, and Addison’s disease have been considered. The principal aim was to propose that chemokines, and in particular CXCL10, should no longer be considered as belonging exclusively to basic science, but rather should be used for providing new insights in the clinical management of patients with endocrine autoimmune diseases.
-inducible CXC chemokines and their receptor CXCR3 agonists in vitro inhibit CXCL10 production induced by proinflammatory cytokines |
I. Introduction |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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inducible chemokines, which interact exclusively with the chemokine receptor CXCR3. All chemokines possess the ability to attract and recruit distinct types of cells in different organs or tissues. To exert such a function, many chemokines are released into the bloodstream, where they can be detected and also quantitated. Chemokines exhibit their peculiar function of attraction and recruitment of different cell types during physiological processes of maturation and trafficking of immune cells throughout different lymphoid organs, but they also play an important role in inducing, maintaining, and amplifying the inflammatory reactions. Therefore, the ability of chemokines to attract and recruit different immune cells in inflamed tissues is important for the protection against infectious agents. However, chemokines can also have a dangerous effect for the body by maintaining and amplifying chronic inflammatory reactions, when the invading agent cannot be rapidly removed or neutralized, as well as by sustaining chronic immune responses against self-antigens which are responsible for autoimmune diseases. For this reason, the assessment of chemokines in inflamed tissues may help with understanding the pathophysiological mechanisms involved in these disorders. More importantly, chemokines are produced in the inflamed tissue by both infiltrating and resident cells, with a strict relationship with the phases of inflammation. The enhanced production of chemokines in the inflamed tissue(s) and the relative blood flow of the inflamed district are both responsible for the increased concentrations of the same chemokines in serum and other biological fluids. Therefore, it is reasonable to think that at least in some diseases, the detection and quantitation of chemokines in biological fluids may provide a useful tool for monitoring the phase and the severity of the disease.
This review will be focused on the possible role of the so-called CXCR3-binding chemokines in autoimmune endocrine disorders. The main reason for this choice stems from the fact that recently CXCR3-binding chemokines were extensively investigated and were found to exhibit strong variations both in inflamed tissues and in the serum during the different phases of autoimmune endocrine diseases. This is probably due to the fact that the production of all CXCR3-binding chemokines by resident cells is stimulated by IFN-. IFN- also induces the local recruitment of inflammatory cells, which express the CXCR3 receptor and are, in turn, able to produce IFN-. This sequence of events results in an increased production of the same group of chemokines, thus establishing an important loop for the maintenance and amplification of inflammatory reactions. Therefore, CXCR3-binding chemokines probably play a pathogenic role in autoimmune endocrine disorders by influencing the development and/or by amplifying the inflammatory process responsible of these diseases. Moreover, due to their increase in biological fluids and to the variations of their levels according to the different phase of the disease, the measurement of CXCR3 chemokines in the serum may represent a useful tool for monitoring the activity of the inflammatory process.
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II. The Chemokines |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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-granules. Subsequently, from 1984 through 1989, cDNAs for structurally related proteins, including IFN--induced protein 10 (IP-10) (2), JE (3), IFN--induced monokine (Mig) (4), regulated on activation, normal T cell expressed and secreted (RANTES) (5), I-309 (6), KC (7), and macrophage inflammatory protein-1 (MIP-1) (8), were cloned by investigators looking for cell differentiation- and activation-associated genes. Thus, the existence of a gene family was established before identifying their functions (9, 10, 11). The discovery of the neutrophil-targeted chemokine IL-8 represented a landmark in immunology because it was the first leukocyte subtype-selective chemoattractant to be found (12, 13). The discovery of IL-8 also promoted the search for functions of other chemokines on leukocyte chemotaxis as well as the discovery of new family members. The interest in the field grew with the subsequent reports of macrophage chemotactic protein CCL2, CCL5, and CCL11, the first important chemokines active on monocytes, T cells, and eosinophils, respectively (14, 15, 16, 17). As the number of family members expanded, various short-lived collective terms were used, including "the platelet factor (PF)-4 family" (9), "the small inducible cytokine family" (10), and "the intercrines" (11). Finally, in 1992 at the Third International Symposium on Chemotactic Cytokines in Baden, Germany, the term "chemokine," a short neologism for "chemotactic cytokines," was coined and accepted as standard (18). The nomenclature for chemokines is based on the configuration of a conserved amino-proximal cysteine-containing motif. Based on this system, there are currently four branches of the chemokine family: CXC, CC, CX3C, and C (where X is any amino acid) (Table 1
) (19, 20). The transmission of chemokine-encoded messages is mediated by specific cell-surface G protein-coupled receptors with seven transmembrane domains. At present, the human chemokine receptor system consists of 20 different receptors (Table 1). In 2000, a new nomenclature system for chemokines and chemokine receptors was approved by the Nomenclature Committee of the International Union of Pharmacology (NC-IUPHAR) (Table 1) (21).
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B. The CXC chemokine family
CXC chemokines have four conserved cysteines and are distinguished by the presence of one amino acid between the first and second cysteine. The CXC chemokine subfamily includes 14 different members whose encoding genes are clustered on human chromosome 4, with few exceptions (22). Most members of the CXC chemokine family exhibit chemotactic properties toward neutrophils and lymphocytes, and are unique in that they constitute a family showing positive or negative activity on the control of angiogenesis (23). CXC chemokines can be further divided into two groups (ELR+ and non-ELR) according to the presence or absence of the tripeptide motif glutamic acid-leucine-arginine (ELR) N-terminal to the first cysteine residue. Interestingly, as shown by site-directed mutagenesis, the presence or the absence of an ELR motif in the chemokines-amino acid sequence seems to correlate with their angiogenic or angiostatic activity, respectively (24). Thus, ELR+ CXC chemokines have been linked to angiogenesis (25, 26), whereas the ELR- CXC chemokines, including CXCL4, CXCL9, CXCL10, and CXCL11, antagonize angiogenesis (23). Furthermore, ELR-CXC chemokines, such as CXCL13, CXCL9, CXCL10, and CXCL11, are powerful chemoattractants for lymphocytes. Recently, a novel CXC chemokine receptor with angiogenic potential was identified and named as CXCR7 (27).
Another classification scheme was based on the function of chemokines and their expression pattern. According to these criteria, two groups of chemokines were identified. The first group includes the so-called inflammatory/inducible chemokines, which are regulated by proinflammatory stimuli, such as lipopolysaccharide and primary cytokines (i.e., IL-1 and TNF-), and orchestrate innate and adaptive immune responses. Inflammatory chemokines control the recruitment of effector leukocytes in infection and inflammation sites, in tissue injuries, and in tumors.
The second group includes the homeostatic/constitutive chemokines, which are important in lymphocyte and dendritic cell trafficking, and in immune surveillance. Homeostatic chemokines navigate leukocytes during hematopoiesis in the bone marrow and thymus; during initiation of adaptive immune responses in the spleen, lymph nodes, and Peyer’s patches; and during immune surveillance in healthy peripheral tissues (19).
The finding that several chemokines cannot be assigned unambiguously to either one of the two functional categories led to the characterization of a third group of chemokines, which were referred to as "dual-function" chemokines (19, 20). Dual-function chemokines participate in immune defense functions (i.e., are up-regulated under inflammatory conditions) and also target noneffector leukocytes, including precursor and resting mature leukocytes, at sites of leukocyte development and immune surveillance. Many dual-function chemokines are highly selective for lymphocytes and have a role in T cell development in the thymus, as well as in T cell recruitment to inflammatory sites.
Genes encoding for inflammatory CXC chemokines are typically found in a major cluster on human chromosome 4, whereas genes for homeostatic chemokines are located alone or in small clusters on different chromosomes (23).
Studies on the expression of chemokines in different species showed that none of the mammalian CXC chemokines, except CXCL12 and CXCL14, possesses orthologs in any other vertebrate class, including birds. This finding suggests that the fine regulation of inflammatory responses is a recent acquisition in the evolution. Indeed, some orthologs of human CXC chemokines are not represented even in mice (28).
The role of CXC chemokines in several types of inflammatory and autoimmune disorders has been largely investigated and was recently reviewed (20). Converging evidence suggests that a subgroup of CXC chemokines, sharing binding to the same receptor, CXCR3, play a role in the pathogenesis of endocrine autoimmune diseases. Thus, this review will focus mostly on the role of the chemokine receptor CXCR3 and its binding chemokines in endocrine autoimmune diseases.
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C. The IFN--inducible CXC chemokines and their receptor CXCR3
Three CXC chemokines were found to share the property to be induced by IFN-. They were initially called "IFN--inducible protein 10" (IP-10) (2), Mig (4), and IFN- inducible T cell chemoattractant (I-TAC) (29). In the new nomenclature, the three chemokines were named as CXCL10, CXCL9, and CXCL11, respectively (21).
All three chemokines were found to bind a unique receptor named CXCR3, which was discovered in 1995 on a genomic clone isolated by PCR-based homology hybridization. The gene was named GPR9, was originally incorrectly mapped to human chromosome 8p11.2–12 (30), and was later correctly mapped to chromosome Xq13 (31). The rank order of binding affinity is CXCL11 > CXCL10 > CXCL9. Initially, CXCR3 was found to be expressed on a subset of circulating T cells, B cells, and natural killer cells, and among T cells, mainly on type 1 T helper (Th1) cells (32, 33). In subsequent studies, it was found that CXCR3 was expressed not only by immune cells, but also by resident cells (34) such as human mesangial cells (35), human liver stellate cells, vascular pericytes (36), and human microvascular endothelial cells (37).
More recently, a distinct, receptor, deriving from an alternative splicing of the CXCR3 gene was identified and named as CXCR3-B (38). CXCR3-B not only binds CXCL10, CXCL9, and CXCL11, but also acts as functional receptor for the orphan CXC-chemokine CXCL4, which exclusively interacts with CXCR3-B. The interaction of chemokines with CXCR3 mediates their chemotactic and immune effects, whereas the binding to the splicing variant CXCR3-B accounts for their angiostatic effect (38). To add to the complexity of CXCR3 biology, another variant of human CXCR3 has been identified, which is generated by posttranscriptional exon skipping. This receptor was named CXCR3-alt and binds CXCL11, but its biological role is still unknown (39). The main aim of this review is to discuss the role of IFN- inducible chemokines (CXCL9, CXCL10, and CXCL11) and their classic CXCR3 receptor; therefore, the biological effects resulting from the interaction between the alternative variant of CXCR3 and their ligands will be limited to their angiostatic effects.
The main messages of this section are:
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III. Main Biological Actions of CXCR3-Binding Chemokines |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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T cells were originally divided into two main subsets which are named as CD4+ T helper (Th) and of CD8+ cytotoxic T (Tc) cells. Subsequently, two different types of CD4+ Th cells, known as type 1 Th (Th1) and type 2 Th (Th2), were recognized (Fig. 1). Th1 cells produce cytokines, such as IL-2, IFN-, and lymphotoxin-, which result in the activation of macrophages, in the production of complement-fixing and -opsonizing antibodies, and also in cytotoxicity (41). By contrast, Th2 cells have been thought to play a regulatory rather than protective role, inasmuch as cytokines produced by these cells (i.e., IL-4 and IL-13) have an inhibitory effect on the production of Th1 cytokines, as well as on several functions of activated macrophages (41). It should be noted, however, that Th1 and Th2 cells do not represent clearly distinct lineages of Th cells, as CD4+ and CD8+ T cells, but extremely polarized forms of a much more heterogenous Th cell response. Moreover, their phenotype of cytokine production in humans is not always so clearly polarized as in mice.
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The fact that Th1 cells produce IFN-, which induces the production by different cell types of CXCL9, CXCL10, and CXCL11, and that these chemokines in turn can attract and recruit Th1 cells, suggests the existence of a loop between IFN--producing Th1 cells and resident cells producing CXCR3-binding chemokines (42). Th2 cells express different chemokine receptors, such as CCR4 and CCR8, thus being recruited in target tissues by CCL17, CCL22 (both ligands for CCR4), and CCL1 (ligand of CCR8). Based on these findings, it can be proposed that chemokines interacting with T cells via CXCR3 may induce a recruitment of Th1 cells into the inflamed tissues. On the other hand, chemokines interacting with different chemokine receptors on T cells may recruit Th2 cells, which are responsible for allergic inflammation.
Further studies support the concept that the role of CXCR3-binding chemokines in the regulation of the immune response goes far beyond their powerful chemotactic activity on activated lymphocytes. A large body of experimental evidence emphasizes the role of CXCL10 in the initiation and amplification of host alloresponses (43). CXCL10-deficient mice have impaired T cell responses, impaired contact hypersensitivity, and limited inflammatory cell infiltrates. They are also unable to control viral infections (44). CXCL10 not only mediates leukocyte recruitment, but also drives T cell proliferation to allogenic and antigenic stimulation and IFN- secretion in response to antigenic challenge (42). Accordingly, CXCL10 up-regulates the production of Th1 cytokines and down-regulates the production of Th2 cytokines (45). The final result is a strong up-regulation of inflammatory reactions characterized by the production of IFN-, thus exerting important protective activity against infections sustained by intracellular bacteria and some viruses, which is provided by Th1 cells (Fig. 2). This also results in a down-regulation of allergic inflammation that is provided by Th2 responses.
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B. Angiogenesis
CXCR3-binding chemokines are powerful inhibitors of angiogenesis (23). The major receptor mediating the angiostatic effect of CXC chemokines is CXCR3-B. CXCR3-B is expressed in human microvascular endothelial cells (37, 46) during the late S-phase of the cell cycle on through mitosis, representing the first example of a chemokine receptor expression linked to a particular phase of the cell cycle (37). In vivo, the expression of CXCR3-B in small vessels (37, 47, 48), is higher in inflamed and neoplastic tissues compared with normal tissues (37).
Angiostatic CXC chemokines were shown to inhibit angiogenesis in several experimental models (49, 50, 51) and to participate in the control of angiogenesis during physiological repair of tissue injury (52). CXCL9 and CXCL10 are specifically expressed during the late phase of wound healing repair, to help prevent unlimited vessel growth without blocking other repair processes involved in wound healing (23).
CXCR3-binding chemokines are also involved in the pathogenesis of proliferative diabetic retinopathy (53). The levels of CXCL10 were found to be significantly higher in vitreous samples from patients with inactive, compared with active, proliferative diabetic retinopathy. This suggests that decreased levels of this angiostatic chemokine might favor retinal angiogenesis during diabetic retinopathy (53).
Overall, the angiostatic effect of CXCR3-binding chemokines is strictly dependent upon the expression of CXCR3-B, the alternatively spliced form of the classic CXCR3 receptor (38). Yet, the expression of CXCR3-B has not been evaluated in either normal or pathological endocrine glands.
The main messages of this section are:
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IV. CXCR3-Binding Chemokines in Healthy Subjects and in Nonendocrine Immune-Mediated Pathological Conditions |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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is observed. Commercial ELISA kits for the measurements of CXCL9 and CXCL11 are also available.
A. CXCR3-binding chemokines in healthy subjects
The circulating concentrations of CXCR3-binding chemokines in humans have been extensively studied only for CXCL10, both in health and disease, whereas data regarding the serum levels of the other two chemokines (CXCL9 and CXCL11) are still limited. Studies performed in large series of healthy adult subjects found mean serum levels of CXCL10 ranging from 70 to 90 pg/ml (54, 55). Variations between healthy subjects may be estimated by SD values of approximately 50 pg/ml (54, 55). These figures are comparable to those reported in the normal subjects used as controls in clinical studies investigating CXCL10 in patients with endocrine and nonendocrine diseases (which will be quoted when discussing the specific disease). Nevertheless, it should be noted that in the latter studies healthy subjects were rarely screened for circulating autoantibodies, and therefore it is not always possible to exclude the presence of subclinical autoimmune disorders which, at least in some cases, might have affected the serum concentrations of CXCL10. It is now known that in euthyroid chronic autoimmune thyroiditis (CAT), the most frequent subclinical autoimmune condition in humans, especially in middle-aged women, the serum levels of CXCL10 are significantly increased compared with healthy controls proven to be negative for thyroglobulin (Tg) and thyroid peroxidase (TPO) antibodies (Ab). Euthyroid CAT might represent the most frequently undetected condition biasing the results of CXCL10 in apparently healthy subjects. Many other, less easily detectable abnormalities (e.g., other subclinical autoimmune diseases) may lead to similar problems. The question arises as to how healthy subjects should be selected when comparing their serum CXCL10 levels to those observed in a specific pathological condition. To complicate the issue further, it should be noted that there are currently no data regarding fluctuations of CXCL10 in the serum of individual healthy subjects. As far as our current knowledge permits, we will try to define some physiological variables, which were found to influence the serum levels of CXCL10.
1. The role of gender.
Clinical studies, in which healthy subjects were screened for excluding subclinical thyroid abnormalities by means of thyroid ultrasound (US) and tests for circulating Tg Ab and TPO Ab, found no gender-related differences in circulating concentrations of CXCL10. In the absence of such a screening for subclinical autoimmune conditions, higher serum levels of CXCL10 might be expected in females, due to the well-known greater prevalence of autoimmunity in women than in men. In clinical studies, the potential bias resulting from gender-related differences may be reduced, at least in part, by performing a strict sex-matching between the subjects to be evaluated.
2. The role of age.
An age-related dysregulation of the immune system has been extensively reported by studies performed in humans and experimental animal models of aging (56, 57, 58, 59). The influence of aging on circulating concentrations of CXCL10 was evaluated in two clinical studies (54, 55). Healthy subjects aged from 10 to 80 yr, proven to be negative for circulating thyroid antibodies and with no evidence of other autoimmune diseases, were studied. In a multiple linear regression model including age, body mass index (BMI), systolic and diastolic blood pressure, glycemia, total high-density and low-density lipoprotein cholesterol, triglycerides, TSH, Tg Ab, and TPO Ab, only age was significantly related to serum levels of CXCL10, a positive correlation being found between the two variables.
3. The role of body weight and BMI.
Although the serum levels of CXCL10 have not been specifically investigated in obesity, studies evaluating healthy subjects reported no change in serum concentrations of CXCL10 in relation to BMI (54). The issue remains open because patients with morbid obesity were not studied.
4. Practical points.
The above-described physiological changes in the serum levels of CXCL10 must be taken into account when this chemokine is measured in different pathological conditions. In diseases such as hepatitis C virus (HCV) hepatitis, primary biliary cirrhosis, or end stage renal diseases, the serum levels of CXCL10 are extremely high; thus, the comparison with healthy subjects might not be biased by gender or age. On the contrary, when studying pathological conditions in which the serum levels of CXCL10 are significantly, but only slightly higher than in healthy controls, such as endocrine autoimmune diseases, the above factors must be considered to reduce potential sources of error. As a consequence, matching patients and controls for gender and age appears critical to avoiding misleading results.
B. CXCR3-binding chemokines in some immune-mediated pathological conditions
In this section, we will briefly review the major findings obtained in nonendocrine diseases, which are either crucial for a better comprehension of the role of chemokines in human pathology or exemplify the applications of their assays in the clinical practice. Consistent with the aim of this review, description of data from basic studies will be limited to essential information, whereas findings obtained in clinical studies will be more extensively described. The latter data support the view that measuring CXCL10 in the serum is useful in several nonendocrine diseases, both autoimmune and nonautoimmune, such as chronic HCV hepatitis. In different clinical settings, the serum levels of CXCL10 proved to be useful as an index predicting the course and severity of the disease, as a marker of its activity, as a predictor of treatment outcome, and as a parameter for choosing the best therapeutic option. The fact that autoimmunity is responsible for most endocrine disease indicates that the results obtained by assaying CXCL10 in autoimmune nonendocrine disorders might be transferred to endocrinopathies. This remains to be done in that, as we will see in the subsequent sections, few clinical studies have been performed so far in endocrine patients.
1. CXCR3-binding chemokines in HCV-induced chronic hepatitis.
The first published clinical study in which the serum levels of CXCL10 were evaluated in human disease is the investigation by Narumi et al. (60), reporting significantly increased circulating concentrations of CXCL10 and CCL2 in patients with HCV compared with healthy subjects. The serum concentrations of both chemokines were found to be significantly higher in patients with chronic active hepatitis C compared with those with chronic persistent hepatitis C. Subsequent studies performed in patients with different types of liver diseases demonstrated that the serum levels of CXCL10 were significantly higher in patients with autoimmune hepatitis, primary biliary cirrhosis, and both hepatitis B virus and HCV chronic hepatitis than in healthy controls (61). Circulating CXCL10 concentrations were found to be significantly correlated with the serum levels of aspartate and alanine aminotransferases. This finding suggested a relationship between the serum levels of CXCL10 and the necroinflammatory activity of hepatitis. Several clinical studies evaluated the changes of serum CXCL10 in patients with HCV hepatitis undergoing IFN- therapy (60, 62). In their first report, Narumi et al. (60) had demonstrated that the serum levels of CXCL10 significantly decreased after IFN- treatment, but only in cured patients, as assessed by the normalization of serum aminotransferases and by the disappearance of HCV from serum for 6–12 months after stopping therapy (60). In nonresponders to IFN-, the basal serum levels of CXCL10 were significantly higher than in responders to therapy and remained high throughout the treatment (60). A subsequent study reported that the serum levels of CXCL10 and CCL4, but not those of CXCL9, decreased significantly in HCV patients showing a virological response to IFN- treatment (63). A recent study simultaneously evaluated the three CXCR3-binding chemokines (CXCL9, CXCL10, and CXCL11) in plasma samples collected at 1 wk before treatment (baseline), 29 d after starting therapy, and 6 months after completion of a course of pegylated IFN, with or without ribavirin (64). The principal interest for this study stems from the fact that it is the only published experience in which the three IFN- inducible chemokines were assessed simultaneously, thus allowing a comparison of the relative importance of CXCL9, CXCL10, and CXCL11. At baseline, the serum concentrations of CXCL9, CXCL10, and CXCL11 were higher in patients with HCV hepatitis compared with healthy controls, the greatest increase being found for CXCL10. After successful antiviral treatment, the serum levels of CXCL10 and CXCL9, but not those of CXCL11, significantly decreased (64). After completion of IFN- treatment, sustained responders had circulating levels of CXCL10 similar to healthy subjects, whereas in nonresponders to therapy, the serum levels of CXCL10 remained elevated. Pretreatment levels of CXCL9 did not differ in responders compared with nonresponders and declined during therapy in both groups. No significant association was found between pretreatment levels of CXCL11 or between its changes in serum and the outcome of treatment.
Taken together, these results demonstrate that the circulating levels of each of the three IFN--inducible chemokines are differently regulated during IFN- therapy. Of the three chemokines that bind CXCR3, CXCL10 is the one most closely associated with the outcome of treatment with IFN- for HCV-related hepatitis, and its pretreatment levels may predict the likelihood of a favorable response (64).
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in HCV hepatitis results in a long-lasting normalization of circulating CXCL10, which is mainly due to a decreased lymphocytic infiltration of the liver. treatment.
2. CXCR3-binding chemokines in allograft rejection.
Growing evidence suggests that CXCL10 is critical in promoting and amplifying host alloresponses responsible for acute allograft rejection (65, 66, 67, 68, 69, 70). In CXCL10- or CXCR3-gene-deficient mice, cardiac transplants are not acutely rejected and undergo permanent engraftment (43, 65). Accordingly, neutralization of CXCL10 with mAbs prolongs the allograft survival in both cardiac and small bowel models of allograft rejection (65, 66). Furthermore, the intragraft expression of CXCL10 has been reported in association with the rejection of renal (67), lung (68), and cardiac (69, 70) allografts. Thus, the importance of CXCL10–CXCR3 interactions in the pathogenesis of graft failure appears to be clearly demonstrated in multiorgan models.
Recent evidence indicates that CXCR3 and CXCL10 are also highly expressed in conjunction with the development of chronic rejection, also named as chronic allograft vasculopathy (71). Indeed, in addition to its potent effects on immune responses (23, 31, 32, 40, 72, 73, 74, 75), CXCL10 also alters vascular endothelial and smooth muscle cell functions (23, 35, 36, 37, 40, 76, 77), thus promoting the development of chronic allograft nephropathy. Pretransplant serum levels of CXCL10 were measured in kidney graft recipients to verify its value in predicting the recipient’s risk for graft rejection and transplant failure (78, 79). Patients with normally functioning grafts showed significantly lower pretransplant serum levels of CXCL10 compared with patients who experienced graft failure, and lifetime analysis showed significantly lower 5-yr survival rates of the grafts with increasing pretransplant serum levels of CXCL10. Furthermore, frequency of acute rejection episodes in the first month after transplant significantly increased in relation to increasing pretransplant serum levels of CXCL10. In particular, patients with serum CXCL10 levels greater than 150 pg/ml showed a nearly 2-fold greater frequency of rejection. Rejection episodes were not only more frequent, but also more severe in patients showing high pretransplant serum levels of CXCL10 (78). Recently, patients developing chronic allograft vasculopathy were also shown to have significantly higher pretransplant serum concentrations of CXCL10 than patients with normally functioning grafts (78, 79). Multivariate analyses indicated that high serum levels of CXCL10 were a significant risk factor for acute graft rejection and graft failure (78). Taken together, these results indicate that high pretransplant serum levels of CXCL10 may predict the risk for the development of acute rejection and chronic allograft vasculopathy. Accordingly, the urinary levels of CXCL9 and CXCL10 are a sensitive and specific predictor for acute rejection and also mirror the response to antirejection therapy (80, 81). High urinary levels of CXCL10 in the first days after transplant also predict acute rejection, as well as short and long-term graft function (82). Thus, the measurement of CXCR3-binding chemokines in serum or urine may be useful to select those patients requiring more aggressive immunosuppressive regimens.
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3. CXCR3-binding chemokines in multiple sclerosis (MS).
Several chemokine receptors, and among them CXCR3, were shown to be highly expressed in brain samples obtained at autopsy from patients with MS (83, 84, 85), suggesting that CXCR3 might be responsible for the recruitment of autoaggressive T cells. In line with this interpretation, CXCL9 and CXCL10 were found to be significantly elevated in the cerebrospinal fluid (CSF) of MS patients, being positively correlated with the CSF cell counts. The relevance of elevated levels of CXCL10 and CXCL9 in the CSF of MS patients was further supported by the uniform detection of CXCR3+ lymphocytes in the perivascular inflammatory cuffs of brain lesions (83, 84, 85). The accumulation of these cells was directly related to the demyelinating process. The demonstration that the chemotactic activity toward CD4+ T cells specific for a myelin basic protein peptide is mediated by CXCL10 (86) and the notion that IFN- is a potent inducer both of CXCL10 and of clinical relapses of MS provided evidence for a pathogenetic role of CXCL10 in this disease.
Th1- and Th2-oriented chemokines were sequentially measured in the serum and the CSF of patients with MS. CXCL10 and CCL2 were chosen as prototype chemokines for a Th-1 and a Th-2 phenotype, respectively. The measurement of CXCL10 and CCL2 in the serum and CSF of MS patients showed that these chemokines had a different behavior in relation to the activity of the disease. CXCL10 was higher in the serum and the CSF of patients with acute MS and lower in those with a stable phase of the disease. An opposite pattern characterized the CCL2 secretion profile, with high levels being found in the serum and the CSF during the active phases of MS and with a decline in the stable phase of the disease. These findings indicate an involvement of both chemokines, with reciprocal changes according to the clinical phase of MS (85, 87, 88). Because CXCL10 is mainly related to Th1 responses, the increase of CXCL10 in the serum and the CSF of patients during the acute phases of MS fits with the notion that IFN- mediates the immune changes leading to an exacerbation of the disease.
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V. CXCR3-Binding Chemokines in Endocrine Autoimmune Diseases |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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, and lymphotoxin-, that result in the activation of macrophages, production of complement-fixing and -opsonizing antibodies, and also cytotoxicity. By contrast, another polarized subset of Th cells, known as Th2, has been thought to play a protective role, inasmuch as cytokines produced by these cells (i.e., IL-4 and IL-13), play an inhibitory effect on the production of Th1 cytokines, as well as on several functions of activated macrophages. Th cells able to produce both Th1 and Th2 cytokines have been named type 0 Th (Th0). Both Th1 and Th2 cells collaborate with B cells for the production of antibodies. However, Th1-induced antibodies differ from those detectable during Th2 responses because of the different subclasses. In mice, Th1 lymphocytes induce B cells to produce mainly IgG2a, whereas Th2 cells induce the production of IgG1 and IgE. In humans, the situation is less clearly dichotomic, but it is known that Th2 responses are characterized by IgE and IgG4, whereas Th1 responses promote the production of IgG1 and IgG3 subclasses. IgG1, which represent the major subclass of human IgG in the serum, are complement-fixing and -opsonizing antibodies, and therefore they contribute, together with activated macrophages, to the phagocyte-dependent protection against infectious agents. Usually, IgG1 also represent the major subclass among autoantibodies, and this is the reason why high levels of autoantibodies are commonly observed in patients with diseases characterized by strong Th1 response.
As mentioned in Section III.A, Th1 cells mainly express CXCR3 as a chemokine receptor and can be recruited into target tissues by CXCL9, CXCL10, and CXCL11. Th2 cells express different chemokine receptors, such as CCR4 and CCR8, thus being recruited in target tissues by CCL17, CCL22 (both ligands for CCR4), and CCL1 (ligand of CCR8). The demonstration of IFN--producing T cells and of CXCR3-binding chemokines in target tissues of organ-specific autoimmune disorders, including those affecting the endocrine glands, has suggested the existence of an important pathogenetic loop. The concept is based on the role of these chemokines in recruiting Th1 cells and in maintaining and amplifying chemokine production by Th1 cells through IFN- production.
It is worth noting that an impressive series of data obtained both in experimental animal models and in human diseases have shown that when Th1 responses, because of their severity and/or chronicity, become dangerous for the body, they can be shifted to a less polarized profile (Th0) or even to responses characterized by the prevalent production of Th2 cytokines. Likewise, established Th2 responses can be shifted to a less polarized profile or even to a prevalent Th1 profile. This phenomenon is known as immune deviation (90).
In the last few years, a novel subset of Th cells has been discovered and named Th17 or ThIl-17 (Fig. 1
) (91). These cells appear to be distinct from Th1 and Th2 cells because of peculiar mechanisms of development and possible functions (92, 93). Although Th1 cells mainly develop in response to IL-12 produced by dendritic cells and Th2 cells develop due to the early presence of IL-4, Th17 cells develop in response to the production of IL-23, IL-6, and TGFß1 by dendritic cells. Th17 cells have been recently suggested to play a pathogenic role in autoimmune diseases on the basis of data obtained in animal models, such as experimental autoimmune encephalomyelitis (which is considered as the equivalent of MS) and collagen-induced arthritis (a model of rheumatoid arthritis). Their role in human endocrine autoimmune diseases remains to be established (94). Thus, in our discussion, we will only take into account the body of experimental evidence suggesting that in these disorders the effector responses are apparently mediated by Th1 cells.
B. Autoimmune thyroid diseases
1. Background.
The thyroid is a major target for autoimmunity. Human autoimmune thyroid disorders (AITD) are characterized by reactivity to self-thyroid antigens, which may be expressed as destructive inflammatory or antireceptor autoimmunity (95) and encompass the clinical spectrum of Graves’ disease and CAT (96, 97, 98). Graves’ disease shares many immunological features with CAT, both diseases being characterized by lymphocytic infiltration of the gland, which can result in tissue destruction (99, 100). One of the histopathological hallmarks of thyroid glands affected by AITD is leukocytic infiltration, mainly by mononuclear cells, including T and B lymphocytes and macrophages (95, 101). In AITD, the lymphocytic infiltrate is also an important site of thyroid autoantibody synthesis (95, 102). Lymphocytes mediate important inflammatory effects, such as the release of cytokines (95). The cellular makeup of the infiltrate varies with the type of AITD, the stage of the disease, and the therapy used, but it is also patient-dependent. This cellular infiltrate sometimes organizes itself into germinal centers that share many of the features of lymph node germinal centers (101, 103, 104). Intrathyroidal lymphocytes play a central role in the pathogenesis of AITD, but the mechanisms by which different lymphocytic subsets are recruited and arrested in the thyroid tissue are only partially understood. To the best of our knowledge, the recruitment of lymphocytes in AITD is a multistep process involving adherence and migration across the endothelium, trafficking through the interstitium, and finally moving toward the thyroid follicular cells (105, 106). Leukocyte extravasation involves the combined action of adhesion molecules, such as selectins and integrins, and chemotactic factors, mainly chemokines (107). In AITD, infiltrating lymphocytes and endothelial cells bear an enhanced expression of various adhesion molecules, pointing to lymphocyte function-associated antigen-1/intercellular adhesion molecule-1, very late antigen-4/vascular cell adhesion molecule-1, and selectin/selectin ligands adhesion pathways as predominant in lymphocyte migration to the thyroid (108). Studies evaluating cytokines in AITD have demonstrated the production of IL-1, IL-2, IL-6, IL-10, IFN-, and TNF- by infiltrating T cells and macrophages (109, 110, 111, 112, 113, 114, 115). However, the specific role of these molecules in the pathogenesis of AITD is still debated (115). In addition, the thyroid follicular cells themselves produce many cytokines (116, 117, 118, 119, 120).
2. Chemokines in AITD.
In 1992, Weetman et al. (121) first described the production of chemokines by cultured thyroid follicular cells. They demonstrated that thyrocytes stimulated by IFN-, TNF-, or IL-1 produce IL-8, a CXC chemokine (121). A subsequent study showed that human thyrocytes in primary culture, upon stimulation with IL-1, TNF-, or IFN-, produce CCL2 (122). Although the highly organized lymphomononuclear cell infiltration present in AITD suggested an involvement of chemokines in their pathogenesis, several years passed before endocrinologists pointed their attention toward these new molecules. It was not until 2000 that the expression of chemokines in AITD was studied in detail and evidence was provided as to their pathogenetic role, at least in the initial phases of these disorders.
The interest in IFN- inducible chemokines (CXCL9, CXCL10, and CXCL11), and their receptor (CXCR3), originated from an investigation aimed at evaluating the antiangiogenetic effects of these molecules. To this purpose, the expression by human endothelial cells of CXCR3 and its ligands was studied in normal tissues and in specimens from diseased organs (37). CXCR3 was detected in a small number of vascular wall cells from normal tissue specimens including thymus, liver, kidney, and gut. Thyroid specimens were obtained from normal tissue and from Graves’ glands. By both immunohistochemistry and in situ hybridization, a higher signal for the protein and the mRNA of CXCR3 was detected in endothelial cells from Graves’ glands, but not from normal thyroids (37).
In 2001, Garcià-Lòpez et al. (123) first demonstrated the production of CXCR3-binding chemokines by human thyrocytes in primary cultures after stimulation with IFN-. In the same culture system, CCL2 and CCL5 were secreted in response to TNF-. In basal conditions, CXCL10 and CXCL9 were not detected in the surnatants from thyroid follicular cells, but their secretion was induced by IFN- and synergistically increased by TNF- addition. As compared with autologous peripheral blood lymphocytes (PBL), intrathyroidal lymphocytes from AITD patients showed a higher expression of CXCR3 and of the receptors for CCL5 and CCL2, CCR2, and CCR5, respectively. T lymphoblasts expressing CXCR3 showed an increased migration to supernatants of IFN- stimulated thyroid follicular cells, which was abolished by neutralizing antibodies directed to CXCL9 and CXCL10, as well as to their receptor, CXCR3. Taken together, these data suggested a role for thyroid follicular cells, through the production of CXCL10, CXCL9, and CCL5, in the recruitment of specific subsets of activated lymphocytes (123).
By using immunohistochemistry, a statistically significant increase of CXCL10 and CXCL9 was found in thyroid tissue specimens obtained from Hashimoto’s glands, compared with normal thyroid tissue (123). By contrast, in patients with Graves’ disease, the intrathyroidal chemokine expression pattern was highly variable, with only a few subjects expressing high levels of CXCL10 and CXCL9, as assessed by immunohistochemistry (123).
A clear-cut demonstration that CXCL9 and CXCL10 were hyperexpressed in Graves’ glands was obtained using combined in situ hybridization and immunohistochemistry (124). The expression of the mRNAs for CXCL10 and CXCL9 in normal thyroids, as well as in thyroids from patients with autoimmune and nonautoimmune hyperthyroidism (Graves’ disease and toxic adenoma), was assessed by in situ hybridization (Fig. 3). The quantitative evaluation of CXCL10 and CXCL9 mRNAs, performed by a computerized video image analysis system, provided evidence that the expression of the mRNAs for the two chemokines was significantly higher in thyroid glands from Graves’ patients compared with normal thyroids or toxic adenoma glands. The wide variability in the expression of chemokines reported by Garcià-Lòpez et al. (123) in Graves’ disease was confirmed and was related to the duration of the disease. A statistically significant increase of CXCL10 expression was found in the thyroid of patients with recent onset (<2 yr) compared with patients with long-standing (>2 yr) disease, in whom the expression of CXCL10 did not differ from that observed in normal thyroid specimens (124).
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, CXCL10, and CXCL9 in the same samples. RT-PCR revealed that the expression of these molecules was highly heterogeneous, being the mRNA levels for CXCL10 and CXCL9 strictly related to those of the IFN-. The latter were also higher in patients with recent-onset (<2 yr) Graves’ disease. Multiple double-label immunohistochemistry was used to identify the cellular source of chemokines and showed that CXCL10 and CXCL9 were highly expressed by both thyroid follicular cells and infiltrating mononuclear cells (Fig. 3). The CXCR3 receptor was found only in inflammatory and endothelial cells (Fig. 4) (124).
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In summary, these early studies demonstrated a role for CXCR3-binding chemokines and their receptor in AITD by evaluating chemokine expression at the mRNA and at the protein level, both in the thyroid and in primary cultures of thyrocytes (126, 127). The subsequent steps for defining the role played by chemokines in AITD were provided by clinical studies that evaluated the serum levels of CXCL10 in large series of patients with Graves’ disease or CAT (124, 128, 129, 130, 131, 132).
3. CXCR3-binding chemokines in Graves’ disease.
The first observation of increased serum levels of CXCL10 in patients with Graves’ disease was reported in 2002 (124). Serum samples were collected from 50 unselected Graves’ patients with different duration of their disease, as well as from 25 healthy controls. All Graves’ patients had been treated with methimazole (MMI) at variable doses and were euthyroid at the time of serum analysis. Corticosteroid treatment was an exclusion criterion. Mean CXCL10 serum levels were significantly higher in patients with Graves’ disease compared with healthy subjects, even if there was a large overlap of CXCL10 results between the two groups. The serum concentrations of CXCL10 were inversely correlated with the duration of Graves’ disease, the highest levels being found in patients with recent-onset disease. By contrast, no correlation was observed between the serum levels of CXCL10 and other clinical or biochemical parameters such as sex, age, and titers of circulating Tg Ab or TPO Ab. Interestingly, the reduction of CXCL10 serum levels in long-standing (>2 yr) Graves’ disease was associated with a slight increase in the serum concentrations of CCL22, a chemokine associated with Th2 immune responses (133, 134).
The analysis of the CCL22/CXCL10 ratio demonstrated that a longer duration of Graves’ disease was associated with an increase of the CCL22/CXCL10 ratio in the serum of Graves’ patients (124). Thus, in the late phase of Graves’ disease, an increase in the CCL22/CXCL10 ratio, mainly due to a CXCL10 decline, is observed both in the thyroid gland and in the serum. This phenomenon parallels the reduction of intrathyroidal IFN- mRNA expression (124).
a. Changes in serum levels of CXCL10 in relation to thyroid function and treatment in Graves’ disease.
Following the observation that the serum levels of CXCL10 are increased in Graves’ disease, several clinical trials were designed with the aim of systematically evaluating the serum chemokine status in Graves’ patients in relation to their thyroid function and treatment (130, 131, 132). The final goal was to relate the findings of circulating CXCL10 to the clinical phenotype and to evaluate possible relations between the serum levels of CXCL10 and the two major therapeutic strategies used in Graves’ disease: medical treatment and thyroid removal. Although high serum levels of CXCL10 are not a specific feature of Graves’ disease, having been reported in several endocrine and nonendocrine autoimmune or even nonautoimmune human diseases, the results provided by the following clinical studies support the view that measuring CXCL10 serum levels in Graves’ patients may be useful.
The first study retrospectively evaluated 103 patients with Graves’ disease but with no clinical signs or symptoms of inflammatory ophthalmopathy (132). Graves’ patients were recruited irrespective of their thyroid function or drug treatment. Thirty of them were hyperthyroid and untreated. Fifty-five patients were on MMI treatment for 1–28 months, and the remaining patients were euthyroid, being in remission after a previous course of MMI. Healthy subjects, patients with euthyroid CAT, patients with nontoxic nodular goiter, and hyperthyroid patients with toxic nodular goiter served as controls.
The mean serum levels of CXCL10 were significantly higher in Graves’ patients than in healthy subjects or patients with nontoxic multinodular goiter, but they did not differ from those found in patients with euthyroid CAT. Among Graves’ patients, the serum levels of CXCL10 were significantly higher in those older than 50 yr, in patients with a hypoechoic pattern of the thyroid at US, and in those with an increased thyroid blood flow. Thyroid volume was unrelated to circulating CXCL10. No significant correlation was observed between the levels of CXCL10 and the titers of Tg Ab, TPO Ab, or TSH-receptor (TR) Ab in serum. However, high serum levels of CXCL10 were mainly observed in Graves’ patients who were strongly positive for TR Ab.
Hyperthyroid patients with Graves’ disease had significantly higher serum CXCL10 levels than those who were euthyroid or hypothyroid. Graves’ patients with untreated hyperthyroidism had significantly higher serum CXCL10 levels than those who were hyperthyroid or euthyroid while taking MMI (166 ± 125, 124 ± 41, and 94 ± 35 pg/ml, respectively). The serum levels of CXCL10 did not significantly differ in hyperthyroid Graves’ patients who were untreated compared with those who relapsed after a previous course of MMI (176 ± 125 and 155 ± 97 pg/ml, respectively). Euthyroid patients on MMI or in remission after medical treatment showed similar serum levels of CXCL10.
This retrospective study confirmed that the serum levels of CXCL10 are increased in patients with Graves’ disease, being strongly associated with the hyperthyroid phase of the disease, and do decrease when euthyroidism is restored by MMI treatment (132). In agreement with these findings, high levels of CXCL10 cosegregated with high TR Ab titers. Furthermore, high serum levels of CXCL10 were found to be strongly associated with a marker of disease activity, such as the increased thyroid blood flow. In this regard, the question might be raised of how the huge blood flow of Graves’ glands would fit with the high expression of an angiostatic chemokine, such as CXCL10. The development of new vessels during an inflammatory process results from a balance between angiogenic and angiostatic factors (23). In Graves’ glands, new vessels develop due to extremely high local concentrations of vascular endothelial growth factor produced by thyroid follicular cells in response to thyroid-stimulating antibodies (135). In this setting, the angiostatic effect of CXCL10, which requires binding to the splicing variant B of CXCR3 (see Section III.B), would be easily overcome by the preponderant role of vascular endothelial growth factor, an extremely powerful angiogenetic factor (136).
Patients with Graves’ disease in remission after a previous course of MMI therapy showed serum levels of CXCL10 similar to healthy controls or to euthyroid patients with nontoxic multinodular goiter. The reduction of circulating CXCL10 in patients rendered euthyroid by MMI treatment could be ascribed to the well-known immunomodulatory effect of antithyroid drugs (137). MMI, besides its ability to decrease thyroid hormone production (138), has been shown to interfere with some immunological abnormalities typical of Graves’ hyperthyroidism. The immunosuppressive effect of MMI is highlighted by the reduction of circulating thyroid antibodies, which occurs during medical treatment with this drug, and by the consistent percentage (nearly 30%) of Graves’ patients entering prolonged remission after a course of medical therapy (138, 139). These immunological effects of MMI might be mediated, at least in part, by an action on chemokine production, resulting in a decreased lymphocytic infiltration of the gland. Indeed, a milder lymphocytic infiltration was reported in Graves’ glands after medical treatment (140). Patients with newly diagnosed or relapsing hyperthyroidism had comparable serum concentrations of CXCL10. The increase in serum concentrations of CXCL10 during relapses of hyperthyroidism would be in line with a novel activation of the Th1-mediated immune response and might be taken as an index of an impending relapse of hyperthyroidism after MMI treatment. The increase of circulating CXCL10 in the active phases of Graves’ disease is in agreement with findings in MS showing that serum levels of CXCL10 are higher at disease onset and during relapses of the neurological disease (85, 88, 141).
The main results of this study can be summarized as indicating that CXCL10 is associated with the active phase of Graves’ disease, in both newly diagnosed and relapsing hyperthyroid patients, and that the reduction of serum CXCL10 levels in Graves’ patients rendered euthyroid by MMI may be related to an immunomodulatory effect of the drug. A graphic representation of the mean serum levels of CXCL10 at different clinical stages of Graves’ disease (132) is shown in Fig. 5.
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and the increased ratio of CCL22 over CXCL10 in serum. However, given the fact that antithyroid drugs display a well-known immunomodulatory effect, the decrease in CXCL10 levels observed in long-standing Graves’ disease could also be ascribed to the use of antithyroid drugs. Indeed, an immune deviation toward a Th2 phenotype has been observed during pharmacological treatments in graft vs. host disease (148) and MS (149).
b. Serum levels of CXCL10 and hyperthyroidism.
To understand the meaning of circulating CXCL10 in Graves’ disease better, a prospective study addressed the question of whether hyperthyroidism per se was responsible for the high levels of serum CXCL10 (130). To this purpose, hyperthyroid patients with either Graves’ disease or toxic nodular goiter were enrolled in the study. The serum levels of CXCL10 were evaluated in hyperthyroid Graves’ patients at the time of diagnosis and 3 months after starting medical treatment with MMI. Basal serum levels of CXCL10 were significantly lower in patients with toxic nodular goiter than in Graves’ patients, despite an accurate matching for serum free T3 and free T4. A significant reduction in the chemokine serum levels occurred in Graves’ patients after restoration of euthyroidism by MMI. Patients with toxic nodular goiter showed a slight, but not significant, reduction in circulating CXCL10 when rendered euthyroid by MMI. Thus, the significant decrease in circulating concentrations of CXCL10 in Graves’ patients after MMI treatment was interpreted as resulting from an immunomodulatory action of the drug, rather than from the restoration of euthyroidism. Moreover, after accurate sex and age matching, the serum levels of CXCL10 were found to be similar in healthy subjects, in hyperthyroid patients with toxic nodular goiter, and in patients receiving levothyroxine (L-T4) at a TSH-suppressive dose for thyroid cancer. A further confirmation of these data is provided by a recent study demonstrating that Graves’ patients, stratified in relation to circulating thyroid hormone concentrations, showed similar serum levels of CXCL10 (150). Taken together these findings indicate that hyperthyroidism per se does not play a role in determining the increased serum levels of CXCL10 observed in Graves’ disease (130).
c. Site of production of CXCL10 in Graves’ disease.
Another issue to be elucidated was the site where CXCL10 is produced. Cytokine production in Graves’ disease has been variably attributed to thyroid follicular cells (151), to intrathyroidal lymphocytes (112), or to the activation of humoral immune reactions in sites other than the thyroid (152, 153). To address this issue, the serum levels of CXCL10 were evaluated in Graves’ patients submitted to thyroidectomy (131). In a prospective case-control study, 22 Graves’ patients rendered euthyroid by MMI were submitted to thyroidectomy because of a history of relapsing hyperthyroidism and/or for the presence of large goiters. Healthy subjects and patients with CAT were chosen as controls. A further control group included 20 patients with toxic nodular goiter. Blood samples for CXCL10 measurement were collected in hyperthyroid patients at presentation, when reaching euthyroidism on MMI, 3 d after thyroidectomy, and 1 month after surgery. Graves’ patients had significantly higher serum levels of CXCL10 when hyperthyroid than after reaching euthyroidism on MMI treatment. A further and significant reduction in the serum levels of CXCL10 was observed 3 d after thyroidectomy. No further significant decrease in the serum levels of CXCL10 was found at 1 month after thyroidectomy, when the circulating concentrations of CXCL10 were similar to those found in healthy subjects or in patients with toxic nodular goiter, and lower than the concentrations observed in euthyroid patients with CAT (131). Similar findings were obtained after radioiodine treatment of hyperthyroidism (150). Taken together, the results provided by two studies (131, 150) evaluating the effect of the permanent cure of hyperthyroidism on circulating CXCL10 concentrations indicate that both therapeutic strategies are able to lower serum CXCL10 to the levels observed in healthy subjects. The normalization of circulating CXCL10 levels after thyroidectomy or 131I ablation of thyroid tissue can be explained by removal of most intrathyroidal lymphocytes and/or thyrocytes (131, 150). These findings support the view that the thyroid is the main source of circulating CXCL10 in patients with Graves’ disease and most probably also in patients with CAT (129).
The main messages of this section are:
4. CXCR3-binding chemokines in GO.
There is only one published study that evaluated the serum concentrations of CXCL10 in relation to the presence of GO (154). Serum CXCL10 levels were measured in 60 Graves’ patients without GO and in 60 age- and sex-matched patients with GO. The control group included 60 sex- and age-matched healthy subjects. At the time of evaluation, all Graves’ patients with or without GO were clinically and biochemically euthyroid, either on antithyroid drugs or on L-T4 after thyroidectomy, or in remission after medical treatment. In patients with GO, the eye disease activity was assessed by a clinical activity score (155). A score of 5 out of a maximum of 10, including a worsening of ophthalmopathy during the previous 2 months, indicated active GO. Inactive eye disease was defined as no change in eye status over the previous 6 months. A severity eye score was also calculated as the sum of the products of each NOSPECS class by its grade (156). The serum levels of CXCL10 were similar in Graves’ patients with and without ophthalmopathy. Both groups showed significantly higher circulating CXCL10 levels compared with healthy controls. Graves’ patients with active ophthalmopathy (i.e., higher clinical activity scores) had a more severe eye involvement, a shorter duration of the disease, and significantly higher mean serum CXCL10 levels than patients with inactive ophthalmopathy.
The results of this study demonstrated that the circulating levels of CXCL10 are elevated in Graves’ patients with ophthalmopathy when the inflammatory process of the orbit is active. This finding is in agreement with previous data (112, 142, 157, 158, 159) showing that in GO the active phase of the disease is characterized by the presence of proinflammatory Th1-derived cytokines, whereas other cytokines, and among them the Th2-derived ones, do not appear to be associated with a specific phase of the eye disease (160). In this scenario, the higher circulating concentrations of CXCL10 observed in Graves’ patients with active, compared with inactive, ophthalmopathy might be expected.
The above data can be interpreted as indicating that CXCL10 is transiently involved in the active phase of GO, when the inflammatory process is sustained by a Th1-mediated immune response, whereas the serum levels of this chemokine decline in long-standing, inactive GO. In agreement with this interpretation, data from a previous study demonstrate that, within 2 yr after the onset of the eye disease, lymphocytes obtained from the orbital tissue of patients with GO have a prevalent Th1 profile, whereas patients with a disease duration longer than 2 yr show a prevalence of Th2 lymphocytes (142). Similar findings have been reported in MS, indicating that high levels of serum CXCL10 are strongly associated with the activity of the disease, as assessed by clinical pousses of neurological deterioration (85, 88, 141).
Cytokine production in AITD (161) has been variably interpreted as being sustained by thyrocytes (151), intrathyroidal lymphocytes (112), or the activation of immune reactions at sites other than the thyroid (152). Thus, the question may be raised of whether the increased serum levels of CXCL10 in patients with active GO do reflect the immune process involving orbital tissues. In the above-mentioned study (154), all patients with GO had a clinical history of Graves’ hyperthyroidism. However, it is unlikely that the elevation of CXCL10 in their serum resulted from hyperthyroidism because all patients were euthyroid at the time of CXCL10 measurements. Furthermore, the increase in serum concentrations of CXCL10 was more pronounced in GO patients with active inflammatory orbital disease than in those with inactive GO. Thus, the presence of orbital inflammation may be responsible for an increase in circulating concentrations of CXCL10.
A major difference between active and inactive GO is the presence of a lymphocytic infiltrate in orbital tissues (161, 162). Thus, the increased production of CXCL10 might be sustained by orbital lymphocytes. However, in vitro studies demonstrate that in the orbit CXCL10 can also be produced by nonlymphoid cells (154). Both fibroblasts and preadipocytes from patients with GO secreted CXCL10 in response to IFN- stimulation, but not after challenge with TNF- alone (154). A combination of IFN- and TNF- synergistically increased CXCL10 secretion, similar to observations in human thyrocytes (123) and endothelial cells (163). Interestingly, the response of orbital fibroblasts and preadipocytes was similar to that of fibroblasts and preadipocytes of dermal origin, suggesting that this kind of activation is a general phenomenon. Taken together, the above-described observations support the view that the production of IFN- and TNF- by Th1-activated lymphocytes within the orbit induces CXCL10 secretion by orbital fibroblasts and preadipocytes. In turn, this chemokine favors the migration of Th1 lymphocytes into the orbit, thereby perpetuating the autoimmune cascade.
In conclusion, the serum levels of CXCL10 are increased in patients with GO, showing a significant association with the activity of the eye disease. Retrobulbar cells participate in the self-perpetuation of the inflammatory process by releasing chemokines under the influence of proinflammatory cytokines. The precise role of the increased or rising CXCL10 levels in sera of patients with GO remains to be established. It is worth noting that there are currently no reliable serum markers of activity in GO; thus, it would be useful confirming and extending the data on CXCL10 changes in sera of patients with GO.
The main messages of this section are:
5. CXCR3-binding chemokines in CAT.
Increased circulating concentrations of CXCL10 were first reported in patients with CAT in 2004 (128). The serum levels of CXCL10 were retrospectively measured in 223 consecutive patients with CAT, 97 euthyroid controls, and 29 patients with nontoxic multinodular goiter. The three groups were similar for gender distribution and age. Twenty-four percent of the CAT patients had subclinical hypothyroidism. The mean serum levels of CXCL10 were significantly higher in CAT patients (157 ± 139 pg/ml) than in controls (79 ± 38 pg/ml) or in patients with multinodular goiter (90 ± 32 pg/ml), although some overlap was evident in the low range of CXCL10 values. In patients with CAT, a hypoechoic pattern of the thyroid at US, an age older than 50 yr, and especially a condition of hypothyroidism were all associated with higher serum levels of CXCL10. The serum levels of this chemokine did not differ in relation to the presence of atrophic or goitrous thyroiditis, TPO Ab or Tg Ab positivity, and the presence or absence of an increased thyroid blood flow. In a multiple linear regression model including age, thyroid volume, hypoechogenicity of the gland at US, increased thyroid blood flow, serum TSH, free T4, and TPO Ab, only age and TSH were significantly related to the concentrations of CXCL10 in serum.
The association of high levels of circulating CXCL10 with a hypoechoic pattern of the thyroid at US can be explained by the presence of a marked lymphocytic infiltration, the histological hallmark of CAT. This interpretation fits with previous observations in hepatitis showing that CXCL10 plays a role in the accumulation of a massive T cell infiltrate in the liver (164). Although a hypoechoic pattern of the thyroid at US is also strongly associated with thyroid dysfunction (165, 166), the serum concentrations of CXCL10 were found to be higher in hypothyroid than euthyroid patients, independent of gland hypoechogenicity. Thus, it is possible that CXCL10 indicates a stronger inflammatory response resulting in more extensive tissue destruction.
The observation that among CAT patients, those with hypothyroidism, compared with the euthyroid ones, showed almost 2-fold higher serum CXCL10 levels and that the circulating concentrations of CXCL10 were significantly correlated with those of TSH gives further support to the hypothesis that elevated serum levels of CXCL10 are not only associated with the autoimmune process itself, but also may be a marker of a more aggressive thyroiditis, eventually leading to the destruction of thyroid cells with the attendant functional impairment.
Recent observations indicate that, in murine models of experimental autoimmune thyroiditis, specific combinations of cytokines convert the inflammatory process from a nondestructive to a destructive one (167, 168). In this regard, it has been suggested that one of the main differences between experimental autoimmune thyroiditis in mice and CAT in humans is the fact that human thyroids display a chronic inflammatory environment, mainly enriched in Th1 cytokines such as IFN- and TNF- (167, 169, 170). This cytokine environment results in an enhanced apoptosis of thyroid cells and severe hypothyroidism (167, 170). In the above-described scenario (167, 168), the CXCL10-induced recruitment of Th1 lymphocytes, which secrete IFN- and in turn stimulate chemokine production by follicular cells, would be a critical event for maintaining and expanding the autoimmune process (126, 171) (Fig. 6). The fact that endocrine epithelial cells interact with the immune system at several levels and that these interactions contribute to the development and perpetuation of the autoimmune process constitutes a further aspect of the previously described active role played by these cells in autoimmune diseases (172).
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and TNF-, respectively (122, 123). To understand the meaning of the elevated serum concentrations of CXCL10 and CCL2, these chemokines were simultaneously measured in 70 consecutive patients with newly diagnosed CAT (129). The mean serum levels of CXCL10 were significantly higher in patients with CAT than in healthy subjects or in patients with nontoxic multinodular goiter, used as controls. On the contrary, when the whole group of CAT patients was considered, the serum concentrations of CCL2 were found to be similar to those found in both control groups. Among patients with CAT, the serum concentrations of CXCL10 and CCL2 were significantly higher in hypothyroid subjects than in the euthyroid ones. The serum levels of CXCL10 were significantly increased in hypothyroid patients, irrespective of their age. On the other hand, the serum levels of CCL2 were significantly increased in patients older than 50 yr and only to a lesser degree in those with hypothyroidism (129). No correlation was found between the circulating concentrations of CXCL10 and CCL2. Multivariate analysis showed that the serum levels of CXCL10 were associated with the severity of hypothyroidism, as assessed by the concentrations of TSH in serum, independently from other confounders. The same analysis revealed that the circulating concentrations of CCL2 were significantly associated only with the patients’ age. These findings are in agreement with data on the intrathyroidal expression of the mRNA for CCL2, indicating that the content of this mRNA is not different in glands with CAT compared with multinodular goiters (126, 127). Taken together, these results would not support a pathogenetic role for CCL2 in CAT.
On the basis of such results, the study reporting increased serum levels of CCL2 in patients with CAT compared with healthy controls or patients with nontoxic nodular goiter (173) should be reinterpreted. The discrepant results obtained in the two studies (129, 173) can be explained, at least in part, by taking into account that in the investigation by Kokkotou et al. (173) CAT patients were significantly older than controls. The age factor, as a confounding variable, should be always taken into account when considering the serum levels of chemokines. Indeed, several studies indicate that the serum levels of CCL2 do increase with age, even in normal subjects (59, 179). Moreover, in the study by Antonelli et al. (129) the serum levels of CXCL10 were higher in patients with hypothyroidism and with a hypoechogenic pattern of the thyroid at US, whereas the serum levels of CCL2 were not correlated with thyroid gland echogenicity, again supporting a minor role played by CCL2.
The preponderant role of CXCL10 in the pathogenesis of severe CAT resulting in hypothyroidism is in line with current notions of autoimmunity and with experimental data. CXCL10 is a Th1-oriented chemokine, whereas CCL2 is regulated by IL-4, the cardinal Th2 cytokine, and influences T cells toward a Th2 commitment. In murine models of autoimmune thyroiditis, experiments performed in vitro demonstrated a differential role for CCL5 (a chemokine known to favor the attraction of Th1 cells) and for CCL2 (preferentially active on Th2 cells) during the onset, the course, and the remission of the disease (180). Briefly, these experiments suggest that CCL2 attracts specific immune regulatory cells that down-regulate the autoimmune reaction, as shown by a decrease in the proliferative response to Tg and by a milder degree of lymphocytic infiltration in the thyroid (180). In human CAT, an environment strongly enriched in Th1, due to CXCL10 stimulation associated with an inadequate Th2 response, resulting from a poor effect of CCL2, would lead to a severe nonremitting disease producing hypothyroidism. In this regard, data demonstrating an association between increased serum levels of CXCL10 and a more severe course and aggressiveness of Th1-mediated autoimmune reactions (e.g., acute kidney graft rejections) should be taken into account (78).
b. Serum levels of CXCL10 and hypothyroidism per se.
The differential impact of thyroid autoimmunity and hypothyroidism on circulating concentrations of CXCL10 was investigated by measuring the serum levels of CXCL10 in hypothyroid patients of both autoimmune and nonautoimmune etiology (130). Patients with CAT (50% being euthyroid and 50% hypothyroid) underwent a serum CXCL10 assay at entry. Hypothyroid patients were then given L-T4 substitution therapy to normalize their thyroid function. The serum levels of CXCL10 were reevaluated 3 months later in untreated euthyroid subjects with CAT and in originally hypothyroid patients rendered euthyroid by L-T4. Also included in the study were patients with papillary thyroid cancer who had been treated with total thyroidectomy and 131I ablation of thyroid residues. In 50% of them, the serum levels of CXCL10 were evaluated while hypothyroid, after L-T4 withdrawal for a diagnostic whole body scan. In the remaining thyroid cancer patients, the serum levels of CXCL10 were evaluated on L-T4 therapy after the injection of recombinant human TSH for diagnostic procedures. The mean levels of circulating CXCL10 were significantly higher in CAT patients with hypothyroidism than in those who were euthyroid. No significant change in the serum concentrations of CXCL10 was found in hypothyroid patients rendered euthyroid by a 3-month course of L-T4 or in euthyroid patients with CAT evaluated after a 3-month follow-up period. In cancer patients on L-T4, the serum levels of CXCL10 were not significantly different from healthy controls. Hypothyroidism resulting from L-T4 withdrawal did not induce any significant change in circulating CXCL10. Also, no significant change in the serum levels of CXCL10 was found in cancer patients given recombinant human TSH for diagnostic purposes, suggesting that the rise of CXCL10 does not result from a TSH stimulation (130).
Taken together, the above data demonstrate that hypothyroidism per se does not significantly influence circulating CXCL10 and suggest that the increased serum levels of CXCL10 in CAT are related to the autoimmune process itself (130).
The main messages of this section are:
C. CXCR3-binding chemokines in type 1 diabetes mellitus
1. Results of basic studies.
Insulin-dependent (type 1) diabetes mellitus (IDDM) is a T cell-driven autoimmune disease of unknown etiology that results in the destruction of the islets of Langerhans in genetically predisposed individuals. The mechanisms by which antigen-specific T cells migrate to the islets, a prerequisite for the specific lysis of ß-cells, is largely unknown. Because the etiological agent of IDDM is still unknown, delineating the process of T cell infiltration would provide a basis for understanding the development of this autoimmune disease. A recent study demonstrated that during insulitis, the ß-cell itself synthesizes and secretes CXCL9 and CXCL10, which are the driving force for the recruitment of CXCR3+ cytotoxic T cells (181). In other words, ß-cells, the target of autoimmunity in IDDM, are largely responsible for attracting islet-specific cytotoxic T cells, thus facilitating their own demise. A high IFN- concentration in the islets of Langerhans correlated both with the expression of CXCL9, CXCL10, and CXCR3 and with a prominent infiltration by T cells (181). IFN- was found to be necessary and sufficient to stimulate CXCL9 and CXCL10 expression by ß-cells. Taken together, these data suggest that the process of islet infiltration by lymphocytes reflects an inflammatory loop caused by T cell-derived IFN-. IFN- induces the production of CXCL9 and CXCL10 by ß-cells, leading to enhanced immigration of additional IFN--secreting T cells. This process is mediated through the chemokine receptor CXCR3 (171, 181).
2. Virus-induced, immune-mediated autoimmune diabetes.
The role of CXCR3 in the pathogenesis of autoimmune diabetes was demonstrated in a model of virus-induced diabetes by using a transgenic mice model that expresses the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) in the ß-cells of the islets of Langerhans [rat insulin promotor (RIP)-GP mice] (182, 183). It is known that RIP-GP mice, after LCMV infection, rapidly develop diabetes, as assessed by a blood glucose concentration of greater than 300 mg/dl, a massive T cell infiltration of the islets, and the destruction of ß-cells, resulting in low to absent pancreatic insulin (183, 184). Concurrent with LCMV-mediated immune diabetes is a profound Th1/Tc1-type response characterized by the release of the proinflammatory cytokines IFN- and TNF- in the target organ (185, 186). This event is followed by the destruction of the ß-cells, which is due to CD8+ T lymphocytes specific for LCMV-GP (183, 184).
A recent study demonstrated that LCMV-infected singly transgenic mice, at d 8 after infection, show severe T cell infiltration and ß-cell destruction of the pancreatic islets. In contrast, the islets of transgenic CXCR3-deficient mice were not invaded, although T cells adorned their rim. Monitoring blood glucose levels revealed a significant delay in the occurrence of diabetes in transgenic mice deficient for CXCR3 (181). These observations demonstrated the importance of CXCR3 in the pathogenesis of IDDM, compared with other inflammatory chemokine receptors (CCR5 and CCR2) that were investigated in the same study (181). Among the three CXCR3-binding chemokines, the predominant role in determining insulitis was subsequently ascribed to CXCL10 by another group of investigators (187). Briefly, in RIP-GP mice, the expression of CXCR3-binding chemokines was demonstrated 18–24 h after LCMV injection (187). The analysis of chemokine expression by RNase protection assay revealed that the induction was maximal for CXCL10 (>400-fold vs. uninfected mice). The expression of the mRNAs for CXCL9 and CXCL11 showed only a 30-fold and 3-fold increase, respectively.
The prevalent role of CXCL10 was not merely a question of quantity, as demonstrated by experiments aimed at clarifying the pathogenetic role of CXCR3-binding chemokines in the development of immune IDDM. After the injection of neutralizing mAbs to either CXCL10 or CXCL9 in RIP-GP mice infected with LCMV, the incidence of IDDM was significantly reduced to 31% in mice receiving the anti-CXCL10 mAb compared with control animals, which were injected with a nonspecific isotype-matched hamster mAb (diabetes incidence = 100%) (187). In contrast with CXCL10 neutralization, no significant reduction in the incidence of diabetes or in its onset could be detected in animals treated with the anti-CXCL9 mAb, suggesting distinct roles for CXCL10 and CXCL9 in virus-induced immune-mediated diabetes. Moreover, when the anti-CXCL10 and the anti-CXCL9 mAbs were coinjected, the incidence of diabetes was equivalent to that observed after administration of the anti-CXCL10 mAb alone (187). The above-described data indicate that CXCL10, and not CXCL9, is an essential factor in the initiation of the process that results in virus-induced immune-mediated diabetes. Despite the multiplicity of chemokines and cytokines released during inflammation and the likely redundancy of the system, these experiments demonstrate a unique and nonredundant role for CXCL10, whose neutralization is able to prevent the occurrence of diabetes (187, 188).
The mechanisms by which CXCL10 neutralization abrogates the development of immune IDDM were identified in a decreased lymphocyte infiltration into the islets of Langerhans with consequent preservation of insulin production. This phenomenon resulted from the inhibition of the clonal expansion of Ag-specific CD8+ T lymphocytes and/or of their migration into the pancreas.
When viruses that are tropic for the pancreas and cause diabetes were compared with viruses that are not tropic for the pancreas and do not cause diabetes, the conclusion was drawn that the increased expression of the CXCL10 mRNA in the islets was not a general phenomenon, occurring after any viral infection (187). Indeed, the infection of mice with viruses that are tropic for the pancreas and are known to cause diabetes, such as Coxsackie virus B4 or the encephalomyocarditis virus variant B, resulted in an increased pancreatic expression of CXCL10. In contrast, the infection with Theiler’s murine encephalomyelitis virus, a virus with no pancreatic tropism and not causing diabetes, did not result in the expression of significant levels of CXCL10 in the pancreas (187).
The above-described findings suggest that a viral tropism for the pancreas and the alteration of the pancreatic milieu by induction of selected chemokines, such as CXCL10, may be the initial step that imprints a pattern for the subsequent development of organ-specific autoimmune diseases caused by viruses. The dominant role played by CXCR3-binding chemokines after a viral infection is not unexpected because CXCR3 is expressed predominantly on activated T cells (31, 189), which function to eliminate virus-infected cells and to control viral infections.
CXCL10 may also have a dual effect in IDDM, being involved both in the initiation and maintenance of the autoimmune process, as well as in the abrogation of autoimmunity. The abrogative rather than enhancing effect of CXCL10 is mainly due to its expression outside the pancreas (190). The administration to LCMV-infected mice of an additional virus infection, at the time when the autodestructive process of the ß-cells was already ongoing, caused the recruitment of T lymphocytes away from the islets (190). Thus, the following scenario for disease abrogation can be postulated: autoaggressive lymphocytes follow the highest bidder, which in this case was the pancreatic-draining lymph node, and leave their original target site. Once arrived at the location with the highest CXCL10 concentrations, the already activated lymphocytes encounter an additional activating inflammatory milieu that pushes them over the edge toward activation-induced cell death. This finding does not hamper, but rather reinforces, the role of CXCL10 in the pathogenesis of IDMM, although it suggests that the CXCL10-mediated effects may be a question of time and location of its expression (190).
3. Serum levels of CXCR3-binding chemokines in human type 1 diabetes mellitus.
Type 1 diabetes mellitus (T1DM) was the first endocrine autoimmune condition in which the serum levels of CXCL10 were investigated (191) and were found to be increased compared with healthy subjects. These results were confirmed by another clinical study (192), but not by two subsequent reports (171, 193). Currently, the issue remains somehow controversial, as we will see later in this section.
Shimada et al. (191) measured the serum levels of CXCL10 and the titers of anti-glutamic acid decarboxylase (GAD) and IA-2 Ab in 74 patients with T1DM. Patients were divided in two groups according to negative (Ab– type 1 group) or positive tests for either or both auto-Ab (Ab+ type 1 group). The latter group also included Ab+ diabetic patients with residual ß-cell function, the so-called latent autoimmune diabetes in adults (LADA) (194), or slowly progressive insulin-dependent diabetes (195). Healthy subjects and patients with Ab– type 2 diabetes served as controls. The serum levels of CXCL10 were significantly higher in both Ab+ and Ab– type 1 groups, compared with the levels found in healthy controls. However, among T1DM patients, only Ab+ patients, but not the Ab– ones, had significantly higher serum levels of CXCL10 than the type 2 diabetic patients. No significant difference in CXCL10 levels was observed between the classical variant of T1DM and the LADA groups.
In the same study, a significant positive correlation was reported between the serum concentrations of CXCL10 and those of IFN- (191). A significant positive correlation was also found between the serum levels of CXCL10 and the number of GAD-reactive IFN--producing CD4+ cells (191). These findings are in line with the fact that CXCL10 is a chemoattractant for Th1 lymphocytes (196) and that IFN- is a Th1 type cytokine involved in the destruction of pancreatic ß-cells in vitro (197). The correlation between serum CXCL10 and IFN- levels was restricted to Ab+ type 1 patients and was still evident when data from GAD Ab-positive and from IA-2 Ab-positive patients were analyzed separately. No such a relationship was observed in the Ab– type 1 group, in the Ab– type 2 diabetic patients, and in healthy subjects (191).
In the study by Shimada et al. (191), the serum levels of CXCL10 were significantly higher in recent-onset Ab+ type 1 patients (disease duration < 3 yr) than in patients with long-standing disease (disease duration 
3 yr). A significant negative correlation was found between serum CXCL10 levels and disease duration. Because more severe insulitis is generally expected in younger subjects, the authors also analyzed the relationship between serum levels of CXCL10 and age, demonstrating a significant negative correlation between these two variables. All these relationships were specific for autoimmune diabetes (Ab+ type 1).
The issue of serum chemokine status in prediabetic patients was addressed by another clinical study evaluating the serum levels of IFN- and CXCL10 in patients with either newly diagnosed or long-standing T1DM and in their healthy first-degree relatives (192). The latter group was divided into those at "low" and "high" risk for the development of diabetes, depending on whether subjects were negative or positive for islet cell and GAD Ab. The serum levels of CXCL10 were significantly higher in patients with newly diagnosed IDDM and in subjects with a high risk for developing the disease. In the latter group, the serum concentrations of CXCL10 correlated with the levels of IFN- (191, 192, 198). The results of this study demonstrated that the serum levels of CXCL10 are increased in patients with T1DM, but only during the early and subclinical stages of the disease.
4. Controversies regarding high serum levels of CXCL10 in T1DM.
Subsequent studies did not confirm the above-described observations (191, 192). Indeed, in two different series of diabetic patients (171, 193) the serum levels of CXCL10 were found to be similar in patients with T1DM and in healthy controls. The few patients showing increased serum levels of CXCL10 were all positive for circulating islet cell Ab (171).
This observation was further extended by evaluating a large series of diabetic patients (193) identified by a regional T1DM registry in central Italy (199). All serum samples were collected within 6 wk after starting insulin therapy. No significant difference in the serum levels of CXCL10 was found between T1DM patients at the clinical onset of their disease and healthy subjects. The serum concentrations of CXCL10 were not related to the patients’ age at clinical diagnosis of T1DM. However, when patients were subdivided in relation to gender, the serum concentrations of CXCL10 were significantly higher in women with T1DM than in healthy control women or males with T1DM. The latter group of patients had serum levels of CXCL10 comparable to those of healthy male subjects (193).
Given the strong evidence provided by basic studies for a physiopathological role for CXCL10 in the pathogenesis of T1DM, the observation that healthy subjects and T1DM patients displayed similar levels of serum CXCL10 was unexpected. To explain this finding, it should be considered that the expression of CXCL10 in the islets of Langerhans during autoimmune diabetes might not be registered when CXCL10 is measured in the serum of patients with T1DM. In line with this hypothesis is the demonstration that in nonobese diabetic mice, the serum levels of CXCL10 did not correlate with its mRNA expression in the pancreas, being rather associated with the expression levels of the mRNAs for CXCL10 and CXCR3 in pancreatic lymph nodes (200). These findings might be explained by the minimal blood flow of the islets of Langerhans, compared with the liver and/or the thyroid. In the latter organs, a high blood flow would account for the relationship between serum levels of CXCL10 and its tissue expression.
The problem remains that in different studies the serum levels of CXCL10 were found to be either increased or normal even in patients with recent-onset T1DM (171, 192, 193). The reason for these discrepant results is still a matter of debate. Besides differences in the assay methods used for CXCL10 measurements, which are unlikely to be the cause of the different results, some considerations may be helpful in identifying possible confounders. The discrepancy may result, at least in part, from the different female/male ratio of the patients investigated in the above-described studies (192, 193). It is well known that the markers of thyroid autoimmunity are 2- to 3-fold more prevalent in women with autoimmune diabetes than in men (201, 202), and that the serum concentrations of CXCL10 are significantly increased in several autoimmune conditions, irrespective of age and gender (124). Furthermore, at the time when these studies were performed, the role of aging in determining increased serum levels of CXCL10 had not been demonstrated yet (54, 55), and strict age matching between patients and controls may not have been performed systematically.
In conclusion, there is currently no definite consensus on the fact that the serum levels of CXCL10 are increased in patients with T1DM. Despite straightforward evidence obtained in basic studies and experimental animal models of IDDM, the results provided by clinical studies remain controversial and in some cases difficult to interpret. In this scenario, future clinical studies aimed at clarifying this issue, should enroll diabetic patients proven to be negative for other autoimmune diseases, known to be associated with increased serum levels of CXCL10. A strict age and sex matching between patients and controls will also be necessary to reach firm conclusions.
The main messages of this section are:
concentrations in the islets of Langerhans correlate with the expression of CXCL9, CXCL10, and CXCR3, and a prominent infiltration by T lymphocytes. inducible chemokines, CXCL10 has a predominant role in the development of virus-induced immune diabetes in mice.
D. CXCR3-binding chemokines in primary adrenal deficiency (Addison disease)
Addison disease (AD) can occur as an isolated condition or in association with other endocrine and nonendocrine autoimmune diseases, leading to the clinical picture of autoimmune polyglandular syndrome (APS)-1 and APS-2 (203, 204, 205). Clinical similarities, common human leukocyte antigen association, and high prevalence of thyroid, gastric, and islet cell Ab suggested that the isolated form of AD may be a clinical variant of APS-2 (205). Although high rates of positivity for adrenal cortex autoantibodies (ACA) and 21-hydroxylase autoantibodies have been reported in patients with AD, these antibodies probably play a minor role in tissue damage and more likely reflect the ongoing autoimmune response (205, 206, 207). Consistent with this view, the histological picture of an affected gland shows diffuse cortical atrophy and lymphocytic infiltration, similar to the histological changes commonly observed in other endocrine autoimmune conditions, such as autoimmune thyroiditis (205). Experimental and clinical evidence supports the concept that a cell-mediated immune response may represent the pathogenetic mechanism leading to adrenal destruction (208, 209, 210). Although the autoimmune etiology currently accounts for most of the cases of AD diagnosed in developed countries (203, 204), there is only one published study evaluating chemokines in adrenal autoimmunity (211); thus, conclusions should be drawn with caution.
In the only report concerning the role of serum CXCL10 in AD, 93 patients with overt or subclinical AD were investigated (211). Among them, 64 patients had clinically evident autoimmune primary adrenal insufficiency: 25 with isolated AD, and 39 with APS. Twenty patients had subclinical autoimmune AD (SAD), being identified by screening patients with extraadrenal autoimmune diseases for the presence of adrenal autoantibodies (212, 213, 214). All 20 SAD patients were positive for both 21-hydroxylase Ab and ACA Ab. Nine patients with nonautoimmune adrenal failure served as controls (215). A further control group included 48 age- and sex-matched healthy subjects, proven to be negative for serum thyroid and adrenal Ab.
Compared with healthy subjects and patients with nonautoimmune adrenal failure, the serum levels of CXCL10 were significantly increased in both clinically evident and subclinical AD. No significant difference was found between patients with nonautoimmune AD and controls. The serum levels of CXCL10 were highly variable among both AD and SAD patients and did not show any significant relationship with gender, age, disease duration, or serum titers of 21-hydroxylase Ab and ACA Ab. Patients with isolated AD or APS showed similar median serum levels of CXCL10. Similar serum levels of CXCL10 were found in SAD patients, irrespective of normal or impaired cortisol response to ACTH. No gender-dependent difference in the serum levels of CXCL10 was found in AD. The absence of a gender-related effect in autoimmune AD, either isolated or occurring within APS-2, would suggest that autoimmune adrenalitis by itself is responsible for the high circulating levels of CXCL10 (193).
Contrary to observations in Graves’ disease or type 1 diabetes (124, 191, 192), the serum levels of CXCL10 were not found to be correlated with the time elapsed since the diagnosis of adrenal deficiency. This lack of correlation probably depends upon the peculiar course of AD. Due to the large functional reserve of the adrenal glands, clinical symptoms of adrenal deficiency become manifest only when massive adrenal destruction has occurred (203). It is therefore reasonable to assume that at variance with Graves’ disease, the time since diagnosis does not precisely reflect the duration of the autoimmune process (124, 128, 191).
The main messages of this section are:
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VI. Pharmacological Modulation of Chemokine Secretion and Biological Action |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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Since the discovery of the chemokine family, the strategy of selectively blocking the leukocyte recruitment to the site of inflammation has been validated by several approaches. Despite the redundancy reported by in vitro studies on ligand-receptor binding and activation, in vivo the system acts through a coordinated and perhaps sequential chain of events, with temporal and spatial control mechanisms coming into play. Thus, interfering with an essential link in the chain, such as CXCR3 in the case of some autoimmune disorders, or organ allograft transplant (65, 216), or diabetes (181) might result in a complete inhibition of the inflammatory process, as demonstrated in CXCR3–/– models (20). In this view, a growing number of antagonists of several chemokine receptors including CXCR3 are being developed (20). Furthermore, the recent discovery of distinct receptors generated by alternative splicing of the CXCR3 gene would allow obtaining selective inhibitors of the two receptors, thus limiting undesired side effects. Otherwise, a combined activation of the two receptors might be useful in some clinical conditions, such as cancer, where both enhancement of the immune response and inhibitory effects on angiogenesis and tumor cell growth are useful. Studies were aimed at evaluating the possibility of mAbs neutralizing chemokines or targeting their receptor and at investigating the possibility to modulate the proinflammatory cytokine-induced chemokine secretion. The first category of studies mainly used animal models, either knockout or wild type, whereas the latter experiences were mainly obtained by using cell cultures in which different agents were tested for their capacity to reduce chemokine secretion. In this review, we will mainly focus on in vitro studies performed in endocrine cells.
A. PPAR agonists in vitro inhibit CXCL10 production induced by proinflammatory cytokines
The effect of peroxisomal proliferator-activated receptor- (PPAR) agonists on the secretion of CXCL10 induced by proinflammatory cytokines was investigated in primary cultures of human thyroid follicular cells, fibroblasts, and preadipocytes (154). Cells were stimulated with IFN- and TNF- alone or in combination in the absence or presence of increasing concentrations of the PPAR agonist rosiglitazone (RGZ). In all cell cultures, CXCL10 was undetectable basally. IFN- dose-dependently induced CXCL10 release, and the combination of TNF- and IFN- had a significant synergistic effect on CXCL10 secretion. TNF- alone had no effect. Treatment with RGZ, added concomitantly with IFN- and TNF-, dose-dependently inhibited the cytokine-induced secretion of CXCL10. RGZ alone had no effect. Similar results were obtained for different cell cultures. Furthermore, thyrocytes from normal thyroids and from Graves’ glands as well as fibroblasts or preadipocytes, irrespective of their dermal or retrobulbar origin, harbored the same results, suggesting that this kind of activation is a general physiological phenomenon.
PPAR has recently been shown to be involved in the modulation of inflammatory responses. Treatment of endothelial cells with PPAR activators inhibits the IFN--induced expression of CXCL10, CXCL9, and CXCL11 at the mRNA and protein levels (163). The release of chemotactic activity for CXCR3-transfected lymphocytes was also blocked (163). Thus, PPAR activity may be involved in the regulation of IFN--induced chemokine expression in human autoimmunity by attenuating the recruitment of activated T cells at sites of Th1-mediated inflammation (163, 217, 218). In this regard, there is evidence that PPAR receptors are present in the thyroid (219) and in orbital tissues from Graves’ patients (220). These findings suggest that PPAR agonists might play a role in the treatment of endocrine autoimmune diseases. In line with this hypothesis is the demonstration that PPAR agonists (gemfibrozil and fenofibrate) inhibit the clinical signs of experimental autoimmune encephalomyelitis in mice (221). PPAR agonists were also shown to modulate inflammatory responses in endothelial cells (163, 217, 218), by reducing CXCL10 levels in two murine models of colitis (218, 222), and in dendritic cells (217).
At the present state of the art, two mechanisms might be involved in the inhibition of IFN--induced CXCL10 secretion by PPAR activators: 1) a decrease of CXCL10 promoter activity, thus inhibiting the protein binding to the two nuclear factor-
B sites (163); or 2) a reduction of CXCL10 protein levels in a dose-dependent manner up to concentrations that do not affect mRNA levels or nuclear factor-B activation (163).
The fact that treatment of thyroid follicular cells, orbital fibroblasts, and preadipocytes with a pure PPAR activator, such as RGZ, at near-therapeutic doses significantly inhibits IFN--stimulated CXCL10 secretion suggests that PPAR activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation (154).
B. Corticosteroids in vitro inhibit CXCL10 production induced by proinflammatory cytokines
The effect of corticosteroids on cytokine-induced CXCL10 secretions was studied in primary cell cultures of human zona fasciculata cells (hZFC) from normal adrenal glands (211). CXCL10 was undetectable basally, whereas its secretion was significantly induced in hZFC by stimulation with IFN- or IFN- plus TNF- (Fig. 7). Stimulation of hZFC with TNF- alone was not able to induce chemokine secretion. Nevertheless, TNF- had a significant synergic effect with IFN- in determining CXCL10 production. Increasing concentrations of hydrocortisone progressively and significantly inhibited IFN--induced and IFN-- plus TNF--induced CXCL10 secretion.
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Increasing concentrations of hydrocortisone significantly inhibited the secretion of CXCL10 induced by IFN- or IFN- plus TNF- in hZFC. This effect of glucocorticoids on chemokine production is in line with their antiinflammatory and immunosuppressive actions (223). Indeed, glucocorticoids are able to suppress the production of several cytokines and chemokines by inhibiting the nuclear factor-B and by activating protein-1 transcription factor families (224). Glucocorticoids inhibit the IFN--induced expression of major histocompatibility class II molecules and may also block the expression of IFN--inducible genes, indicating that glucocorticoids can suppress IFN- activity (225, 226).
The main messages of this section are:
agonist, dose-dependently inhibits CXCL10 secretion induced by IFN- and TNF- in human primary cultures of thyrocytes, orbital fibroblasts, and preadipocytes. - and TNF--induced CXCL10 secretion in human adrenal cells is significantly inhibited by hydrocortisone. |
VII. Serum Levels of CXCR3-Binding Chemokines: Potential Applications as Novel Serum Markers in Endocrine Clinical Practice |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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Another interesting field of application for CXCL10 measurement could be represented by Graves’ disease. Previously described studies provided evidence supporting the concept that CXCL10 could be an important factor in mediating or in announcing the relapse of hyperthyroidism after antithyroid drugs (132). Moreover, indirect evidence suggests that the measurement of CXCL10 in serum, at the time when hyperthyroidism is diagnosed, may help to identify more severe and aggressive variants of Graves’ disease, thus supporting a definitive treatment of hyperthyroidism with thyroidectomy or radioiodine (131).
The above-mentioned clinical conditions represent a few examples in which currently available data suggest a clinical application of serum CXCL10 measurements in patients with autoimmune endocrine disorders.
Among the three CXCR3-binding chemokines, only CXCL10 has been extensively studied in human endocrine diseases. The few published studies in which CXCL9, CXCL10, and CXCL11 have been evaluated simultaneously (64) indicate that, despite an overall similar behavior, each of the three CXCR3-binding chemokines undergoes specific changes in different clinical settings. Results have been provided by both basic and clinical studies demonstrating that targeting of one or another CXCR3-binding chemokines may produce different biological effects. For this reason, and also in view of the limited information available on CXCL9 and CXCL11 in endocrine diseases, the first step toward a better comprehension of the clinical significance of CXCR3-binding chemokines would be to investigate whether the results obtained by measuring CXCL10 are confirmed, extended, or denied when CXCL9 and CXCL11 are taken into account. The variations in serum chemokine levels between and within healthy subjects are another aspect deserving a more complete elucidation. The currently available data, which suggest a clinical application of measuring serum chemokines are mainly derived from cross-sectional studies. Prospective longitudinal studies are needed to reach firm conclusions as well as to establish the sensitivity and specificity values for serum chemokines measurements.
Other aspects deserve clarification, in particular the problem regarding serum levels in patients with T1DM, which have been reported as either elevated (191, 192) or normal (171, 193) in different studies. The issue must be resolved by studying patients selected after an accurate screening for other autoimmune diseases, which might coexist in T1DM patients.
There are also several unexplored fields in which a potential clinical application for the measurements of serum chemokines may be expected. Given the previously described changes of serum CXCR3-binding chemokines in patients with HCV-related hepatitis undergoing therapy with IFN- (60) and the involvement of CXCL10 in the pathogenesis of CAT (123, 128), it would be interesting to evaluate the role of CXCL10 in driving the appearance of thyroid autoimmune disorders during IFN- treatment (227). Additional studies might address the issue of serum chemokines and thyroid autoimmunity in patients treated with IFN-ß for MS (228). Another clinical condition that might provide a model for a better comprehension of the role of chemokines in endocrine autoimmune diseases is postpartum thyroiditis (229). The evaluation of serum chemokines during IFNs-induced thyroid autoimmunity and in postpartum thyroiditis would represent a unique model for studying the involvement of these molecules in the development of thyroid autoimmunity since its earliest phase, a situation rarely encountered in the clinical practice.
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VIII. Future Perspectives |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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The endocrine epithelial cells may interact with the immune system at several levels in the development and perpetuation of the autoimmune process, and many of these interactions appear to exacerbate the disease progression (231). In this review, particular emphasis has been given to the active role played by endocrine epithelial cells, through production of chemokines induced by IFN- in the recruitment of specific subsets of lymphocytes to the diseased gland. The understanding of these pathogenetic mechanisms of autoimmune endocrine diseases suggests novel approaches to immunotherapy, directed to targeting lymphocyte trafficking and activation and to inducing lymphocyte anergy.
During the past decade, we have witnessed the development of potent agents to treat inflammation and autoimmune diseases. However, attempts directed to induce sustained reversal of disease by broad immune suppression are not likely to be successful or cost effective, mainly in endocrine autoimmune diseases. Thus, interest has grown in finding a way to interrupt the interactions between chemokines and their receptors as a new therapeutic strategy for inflammation. Despite the difficulties encountered in antagonizing chemokine receptors, which are mainly due to the redundancy of the chemokine/chemokine receptors system, some encouraging results have been reached (20). Given the crucial importance of the movement of leukocytes to inflammatory sites, it seems worth continuing research to address the issue of developing specific chemokine-receptor antagonists. These future fields of research, aimed at developing pharmacological agents for human autoimmune diseases, are a little too far from the aim of this review. We would like to conclude by stressing the concept that although there are currently no new drugs interfering with the function of chemokines in autoimmunity, these molecules should be regarded as a novel marker that could be useful not only for researchers but also for clinicians, and whose fields of potential application have not been fully elucidated.
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IX. Conclusions |
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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In the last few years, experimental evidence accumulated supporting the concept that IFN- inducible chemokines (CXCL9, CXCL10, and CXCL11) play an important role in the initial stage of autoimmune disorders involving endocrine glands (193). The fact that, after IFN- stimulation, several endocrine cells secrete CXCL10, which in turn recruits Th1 lymphocytes expressing CXCR3 and secreting IFN-, strongly supports the concept that chemokines may induce and sustain the autoimmune process in different endocrine glands. The availability of reliable, reproducible, sensible, and inexpensive methods for assessing serum CXCL10 provided the opportunity to perform clinical studies in large series of patients affected by endocrine and nonendocrine autoimmune diseases. The circulating concentrations of CXCL10 have been found to be increased in several endocrine autoimmune diseases, including Graves’ disease, CAT, and AD. Other endocrine diseases could share the same findings. In different clinical settings, including endocrine and nonendocrine diseases, the measurement of the serum levels of CXCL10 may be useful to predict the course and the activity of the disease and the therapeutic outcome, to perform the most appropriate therapeutic choice, to assess the favorable response to treatment, as well as to predict relapses. Of course, the efficacy and usefulness of routine serum CXCL10 measurement may differ in such a wide spectrum of pathological conditions. We do not support the idea that CXCL10 may serve in all patients, but we still think that the currently available results support the concept that CXCL10 may be regarded as a novel serum marker in autoimmune endocrine diseases.
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Footnotes |
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The authors have nothing to disclose.
First Published Online May 2, 2007
Abbreviations: Ab, Antibody or antibodies; ACA, adrenal cortex autoantibodies; AD, Addison disease; AITD, autoimmune thyroid disorders; APS, autoimmune polyglandular syndrome; BMI, body mass index; CAT, chronic autoimmune thyroiditis; CSF, cerebrospinal fluid; ELR, glutamic acid-leucine-arginine; GAD, glutamic acid decarboxylase; GO, Graves’ ophthalmopathy; GP, glycoprotein; HCV, hepatitis C virus; hZFC, human zona fasciculata cells; IDDM, insulin-dependent (type 1) diabetes mellitus; IFN, interferon; IP-10, IFN--induced protein 10; I-TAC, IFN--inducible T cell chemoattractant; LCMV, lymphocytic choriomeningitis virus; L-T4, levothyroxine; mAb, monoclonal Ab; Mig, IFN--induced monokine; MIP-1, macrophage inflammatory protein-1; MMI, methimazole; MS, multiple sclerosis; PBL, peripheral blood lymphocytes; PPAR, peroxisomal proliferator-activated receptor-; RANTES, regulated on activation, normal T cell expressed and secreted; RGZ, rosiglitazone; RIP, rat insulin promotor; SAD, subclinical autoimmune AD; Tc cells, cytotoxic T cells; Tg, thyroglobulin; Th, T helper; Th0, type 0 Th; Th1, type 1 Th; Th2, type 2 Th; TPO, thyroid peroxidase; TR, TSH-receptor; US, ultrasound.
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TopAbstractI. IntroductionII. The ChemokinesIII. Main Biological Actions...IV. CXCR3-Binding Chemokines in...V. CXCR3-Binding Chemokines in...VI. Pharmacological Modulation...VII. Serum Levels of...VIII. Future PerspectivesIX. ConclusionsReferences |
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