Role of Chemokines in Endocrine Autoimmune Diseases

Endocr. Rev. 2007 28:492-520 originally published online May 2, 2007; , doi: 10.1210/er.2006-0044

Mario Rotondi, Luca Chiovato, Sergio Romagnani, Mario Serio and Paola Romagnani

Role of Chemokines in Endocrine Autoimmune Diseases

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Role of Chemokines in Endocrine Autoimmune Diseases

Mario Rotondi, Luca Chiovato, Sergio Romagnani, Mario Serio, and Paola Romagnani

Excellence Center for Research, Transfer and High Education De Novo Therapies (M.R., S.R., M.S., P.R.), University ofFlorence, 50121 Florence, Italy; and Unit of Internal Medicine and Endocrinology (M.R., L.C.), Istituto Superiore per laPrevenzione e Sicurezza del Lavoro Laboratory for Endocrine Disruptors, Fondazione Salvatore Maugeri, Istituto diRicovero e Cura a Carattere Scientifico, Chair of Endocrinology, University of Pavia, 27100 Pavia, Italy

Chemokines are a group of peptides of low molecular weightthat induce the chemotaxis of different leukocyte subtypes.The major function of chemokines is the recruitment of leu-kocytes to inflammation sites, but they also play a role intumoral growth, angiogenesis, and organ sclerosis. In the lastfew years, experimental evidence accumulated supportingthe concept that interferon-� (IFN-�) inducible chemokines(CXCL9, CXCL10, and CXCL11) and their receptor, CXCR3,play an important role in the initial stage of autoimmunedisorders involving endocrine glands. The fact that, afterIFN-� stimulation, endocrine epithelial cells secrete CXCL10,which in turn recruits type 1 T helper lymphocytes expressingCXCR3 and secreting IFN-�, thus perpetuating autoimmuneinflammation, strongly supports the concept that chemokinesplay an important role in endocrine autoimmunity. This ar-

ticle reviews the recent literature including basic science,animal models, and clinical studies, regarding the role ofthese chemokines in autoimmune endocrine diseases. The po-tential clinical applications of assaying the serum levels ofCXCL10 and the value of such measurements are reviewed.Clinical studies addressing the issue of a role for serumCXCL10 measurement in Graves’ disease, Graves’ ophthal-mopathy, chronic autoimmune thyroiditis, type 1 diabetesmellitus, and Addison’s disease have been considered. Theprincipal aim was to propose that chemokines, and in partic-ular CXCL10, should no longer be considered as belongingexclusively to basic science, but rather should be used forproviding new insights in the clinical management of patientswith endocrine autoimmune diseases. (Endocrine Reviews 28:492–520, 2007)

I. Introduction

II. The ChemokinesA. Historical notes and nomenclatureB. The CXC chemokine familyC. The IFN-�-inducible CXC chemokines and their re-

ceptor CXCR3III. Main Biological Actions of CXCR3-Binding Chemokines

A. Chemotaxis and regulation of the immune responseB. Angiogenesis

IV. CXCR3-Binding Chemokines in Healthy Subjects and inNonendocrine Immune-Mediated Pathological ConditionsA. CXCR3-binding chemokines in healthy subjects

B. CXCR3-binding chemokines in some immune-medi-ated pathological conditions

V. CXCR3-Binding Chemokines in Endocrine AutoimmuneDiseasesA. Notes on immune effector mechanisms in autoim-

mune diseasesB. Autoimmune thyroid diseasesC. CXCR3-binding chemokines in type 1 diabetes mellitusD. CXCR3-binding chemokines in primary adrenal de-

ficiency (Addison disease)VI. Pharmacological Modulation of Chemokine Secretion and

Biological ActionA. PPAR� agonists in vitro inhibit CXCL10 production

induced by proinflammatory cytokinesB. Corticosteroids in vitro inhibit CXCL10 production

induced by proinflammatory cytokinesVII. Serum Levels of CXCR3-Binding Chemokines: Potential

Applications as Novel Serum Markers in Endocrine Clin-ical Practice

VIII. Future PerspectivesIX. Conclusions

I. Introduction

IN THE LAST FEW YEARS, the role of immune responsesin the pathogenesis of several human diseases has been

demonstrated. Numerous soluble molecules produced by, oractive on, the cells of the immune system were initially iden-tified because of their biological activities and then werecloned. These molecules, which have been named as cyto-kines, act as signaling molecules involved not only in in-flammation, but also in cell differentiation and division, fi-brosis repair, and many other functions. Cytokines differ

First Published Online May 2, 2007Abbreviations: Ab, Antibody or antibodies; ACA, adrenal cortex au-

toantibodies; AD, Addison disease; AITD, autoimmune thyroid disor-ders; APS, autoimmune polyglandular syndrome; BMI, body mass in-dex; 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, lymphocyticchoriomeningitis virus; l-T4, levothyroxine; mAb, monoclonal Ab; Mig,IFN-�-induced monokine; MIP-1�, macrophage inflammatory protein-1�; MMI, methimazole; MS, multiple sclerosis; PBL, peripheral bloodlymphocytes; PPAR�, peroxisomal proliferator-activated receptor-�;RANTES, regulated on activation, normal T cell expressed and secreted;RGZ, rosiglitazone; RIP, rat insulin promotor; SAD, subclinical auto-immune 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.Endocrine Reviews is published by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0163-769X/07/$20.00/0 Endocrine Reviews 28(5):492–520Printed in U.S.A. Copyright © 2007 by The Endocrine Society

doi: 10.1210/er.2006-0044

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from classic hormones because these latter are produced byspecialized cells and released into the bloodstream, thushaving the possibility to act at a distance from their sourcein an “endocrine” fashion. By contrast, cytokines are usuallyproduced by different cell types, and they generally actwithin a short range in a “paracrine” or “autocrine” manner.Because of their many features, cytokines cannot be classifiedin well-defined families. However, a number of them, de-spite their heterogenous functional activity, have beengrouped together under the name of chemokines, whichmeans chemotactic cytokines. A distinctive property of che-mokines is their redundancy, inasmuch as many chemokinesmay have the same receptor, and a single chemokine maybind to different receptors. Exception to this rule is providedby a small group of interferon (IFN)-� inducible chemokines,which interact exclusively with the chemokine receptorCXCR3. All chemokines possess the ability to attract andrecruit distinct types of cells in different organs or tissues. Toexert such a function, many chemokines are released into thebloodstream, where they can be detected and also quanti-tated. Chemokines exhibit their peculiar function of attrac-tion and recruitment of different cell types during physio-logical processes of maturation and trafficking of immunecells throughout different lymphoid organs, but they alsoplay an important role in inducing, maintaining, and am-plifying the inflammatory reactions. Therefore, the ability ofchemokines to attract and recruit different immune cells ininflamed tissues is important for the protection against in-fectious agents. However, chemokines can also have a dan-gerous effect for the body by maintaining and amplifyingchronic inflammatory reactions, when the invading agentcannot be rapidly removed or neutralized, as well as bysustaining chronic immune responses against self-antigenswhich are responsible for autoimmune diseases. For thisreason, the assessment of chemokines in inflamed tissuesmay help with understanding the pathophysiological mech-anisms involved in these disorders. More importantly, che-mokines are produced in the inflamed tissue by both infil-trating and resident cells, with a strict relationship with thephases of inflammation. The enhanced production of che-mokines in the inflamed tissue(s) and the relative blood flowof the inflamed district are both responsible for the increasedconcentrations of the same chemokines in serum and otherbiological fluids. Therefore, it is reasonable to think that atleast in some diseases, the detection and quantitation ofchemokines in biological fluids may provide a useful tool formonitoring the phase and the severity of the disease.

This review will be focused on the possible role of theso-called CXCR3-binding chemokines in autoimmune endo-crine disorders. The main reason for this choice stems fromthe fact that recently CXCR3-binding chemokines were ex-tensively investigated and were found to exhibit strong vari-ations both in inflamed tissues and in the serum during thedifferent phases of autoimmune endocrine diseases. This isprobably 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 inflammatorycells, which express the CXCR3 receptor and are, in turn, ableto produce IFN-�. This sequence of events results in an in-creased production of the same group of chemokines, thus

establishing an important loop for the maintenance and am-plification of inflammatory reactions. Therefore, CXCR3-binding chemokines probably play a pathogenic role in au-toimmune endocrine disorders by influencing thedevelopment and/or by amplifying the inflammatory pro-cess responsible of these diseases. Moreover, due to theirincrease in biological fluids and to the variations of theirlevels according to the different phase of the disease, themeasurement of CXCR3 chemokines in the serum may rep-resent a useful tool for monitoring the activity of the inflam-matory process.

II. The Chemokines

A. Historical notes and nomenclature

The first chemokine was identified in 1977 when Walz etal. (1) sequenced native platelet factor 4, a procoagulant andangiostatic factor stored in platelet �-granules. Subsequently,from 1984 through 1989, cDNAs for structurally related pro-teins, 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 differenti-ation- and activation-associated genes. Thus, the existence ofa gene family was established before identifying their func-tions (9–11). The discovery of the neutrophil-targeted che-mokine IL-8 represented a landmark in immunology becauseit was the first leukocyte subtype-selective chemoattractantto be found (12, 13). The discovery of IL-8 also promoted thesearch for functions of other chemokines on leukocyte che-motaxis as well as the discovery of new family members. Theinterest in the field grew with the subsequent reports ofmacrophage chemotactic protein CCL2, CCL5, and CCL11,the first important chemokines active on monocytes, T cells,and eosinophils, respectively (14–17). As the number of fam-ily members expanded, various short-lived collective termswere used, including “the platelet factor (PF)-4 family” (9),“the small inducible cytokine family” (10), and “the inter-crines” (11). Finally, in 1992 at the Third International Sym-posium on Chemotactic Cytokines in Baden, Germany, theterm “chemokine,” a short neologism for “chemotactic cy-tokines,” was coined and accepted as standard (18). Thenomenclature for chemokines is based on the configurationof a conserved amino-proximal cysteine-containing motif.Based on this system, there are currently four branches of thechemokine family: CXC, CC, CX3C, and C (where X is anyamino acid) (Table 1) (19, 20). The transmission of chemo-kine-encoded messages is mediated by specific cell-surface Gprotein-coupled receptors with seven transmembrane do-mains. At present, the human chemokine receptor systemconsists of 20 different receptors (Table 1). In 2000, a newnomenclature system for chemokines and chemokine recep-tors was approved by the Nomenclature Committee of theInternational Union of Pharmacology (NC-IUPHAR) (Table1) (21).

The main messages of this section are:

Rotondi et al. • Serum Chemokines in Endocrine Autoimmunity Endocrine Reviews, August 2007, 28(5):492–520 493

• Chemokines are a family of small proinflammatory pep-tides with high homology, mediating the recruitment ofdifferent subsets of peripheral blood leukocytes.

• The nomenclature for chemokines is based on the config-uration of a conserved amino-proximal cysteine-contain-ing motif. Based on this system, chemokines are classifiedas CXC, CC, CX3C, and C.

• Chemokines were first named on the basis of their prop-erties or on the cell types from which they were isolated.In 2000, a new nomenclature was introduced.

B. The CXC chemokine family

CXC chemokines have four conserved cysteines and aredistinguished by the presence of one amino acid between the

first and second cysteine. The CXC chemokine subfamilyincludes 14 different members whose encoding genes areclustered on human chromosome 4, with few exceptions (22).Most members of the CXC chemokine family exhibit che-motactic properties toward neutrophils and lymphocytes,and are unique in that they constitute a family showingpositive 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 absenceof the tripeptide motif glutamic acid-leucine-arginine (ELR)N-terminal to the first cysteine residue. Interestingly, asshown by site-directed mutagenesis, the presence or the ab-sence of an ELR motif in the chemokines-amino acid se-quence seems to correlate with their angiogenic or angio-static activity, respectively (24). Thus, ELR� CXCchemokines 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 chemoattrac-tants for lymphocytes. Recently, a novel CXC chemokinereceptor with angiogenic potential was identified and namedas CXCR7 (27).

Another classification scheme was based on the functionof chemokines and their expression pattern. According tothese criteria, two groups of chemokines were identified. Thefirst group includes the so-called inflammatory/induciblechemokines, which are regulated by proinflammatory stim-uli, such as lipopolysaccharide and primary cytokines (i.e.,IL-1 and TNF-�), and orchestrate innate and adaptive im-mune responses. Inflammatory chemokines control the re-cruitment of effector leukocytes in infection and inflamma-tion sites, in tissue injuries, and in tumors.

The second group includes the homeostatic/constitutivechemokines, which are important in lymphocyte and den-dritic cell trafficking, and in immune surveillance. Homeo-static chemokines navigate leukocytes during hematopoiesisin the bone marrow and thymus; during initiation of adap-tive immune responses in the spleen, lymph nodes, andPeyer’s patches; and during immune surveillance in healthyperipheral tissues (19).

The finding that several chemokines cannot be assignedunambiguously to either one of the two functional categoriesled 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 de-fense functions (i.e., are up-regulated under inflammatoryconditions) and also target noneffector leukocytes, includingprecursor and resting mature leukocytes, at sites of leukocytedevelopment and immune surveillance. Many dual-functionchemokines are highly selective for lymphocytes and have arole in T cell development in the thymus, as well as in T cellrecruitment to inflammatory sites.

Genes encoding for inflammatory CXC chemokines aretypically found in a major cluster on human chromosome 4,whereas genes for homeostatic chemokines are located aloneor in small clusters on different chromosomes (23).

Studies on the expression of chemokines in different spe-cies showed that none of the mammalian CXC chemokines,except CXCL12 and CXCL14, possesses orthologs in any

TABLE 1. Family of human chemokines and chemokine receptors

ChemokinesReceptors

New nomenclature Old nomenclature

CXC chemokinesCXCL1 GRO-� CXCR2�CXCR1CXCL2 GRO-� CXCR2CXCL3 GRO-� CXCR2CXCL4 PF4 CXCR3-BCXCL4L1 PF4V1 UnknownCXCL5 ENA-78 CXCR2CXCL6 GCP-2 CXCR1, CXCR2CXCL7 NAP-2 CXCR2CXCL8 IL-8 CXCR1, CXCR2CXCL9 Mig CXCR3-A, CXCR3-BCXCL10 IP-10 CXCR3-A, CXCR3-BCXCL11 I-TAC CXCR3-A, CXCR3-B,

CXCR7CXCL12 SDF-1�/� CXCR4, CXCR7CXCL13 BLC/BCA-1 CXCR5CXCL14 BRAK UnknownCXCL16 CXCR6

C chemokinesXCL1 Lymphotactin-� XCR1XCL2 Lymphotactin-� XCR1

CX3C chemokinesCX3CL1 Fraktalkine CX3CR1

CC chemokinesCCL1 I-309 CCR8CCL2 MCP-1 CCR2CCL3 MIP-1� CCR1, CCR5CCL4 MIP-1� CCR5CCL5 RANTES CCR1, CCR3, CCR5CCL7 MCP-3 CCR1, CCR2, CCR3CCL8 MCP-2 CCR3CCL9/10 MIP-1� UnknownCCL11 Eotaxin CCR3CCL12 MCP-5 CCR2CCL13 MCP-4 CCR2, CCR3CCL14 HCC-1 CCR1CCL15 HCC-2 CCR1, CCR3CCL16 LEC CCR1, CCR8CCL17 TARC CCR4CCL18 PARC UnknownCCL19 ELC CCR7, CCR10CCL20 LARC CCR6CCL21 SLC CCR7, CCR10CCL22 MDC CCR4CCL23 MPIF-1 CCR1CCL24 Eotaxin-2 CCR3CCL25 TECK CCR9CCL26 Eotaxin-3 CCR3CCL27 CTACK CCR10

494 Endocrine Reviews, August 2007, 28(5):492–520 Rotondi et al. • Serum Chemokines in Endocrine Autoimmunity

other vertebrate class, including birds. This finding suggeststhat the fine regulation of inflammatory responses is a recentacquisition in the evolution. Indeed, some orthologs of hu-man CXC chemokines are not represented even in mice (28).

The role of CXC chemokines in several types of inflam-matory and autoimmune disorders has been largely inves-tigated and was recently reviewed (20). Converging evidencesuggests that a subgroup of CXC chemokines, sharing bind-ing to the same receptor, CXCR3, play a role in the patho-genesis of endocrine autoimmune diseases. Thus, this reviewwill focus mostly on the role of the chemokine receptorCXCR3 and its binding chemokines in endocrine autoim-mune diseases.


• CXC chemokines have four conserved cysteines and aredistinguished by the presence of one amino acid betweenthe first and second cysteine. The CXC chemokine sub-family includes 14 different members.

• Members of the CXC chemokine family exhibit chemo-tactic properties toward neutrophils and lymphocytes andare unique in that they constitute a family exhibiting pos-itive or negative activity on the control of angiogenesis.

• CXC Chemokines have also been classified as “inflamma-tory” or “homeostatic” on the basis of their mainfunctions.

C. The IFN-�-inducible CXC chemokines and theirreceptor CXCR3

Three CXC chemokines were found to share the propertyto be induced by IFN-�. They were initially called “IFN-�-inducible protein 10” (IP-10) (2), Mig (4), and IFN-� inducibleT cell � chemoattractant (I-TAC) (29). In the new nomencla-ture, the three chemokines were named as CXCL10, CXCL9,and CXCL11, respectively (21).

All three chemokines were found to bind a unique receptornamed CXCR3, which was discovered in 1995 on a genomicclone isolated by PCR-based homology hybridization. Thegene was named GPR9, was originally incorrectly mapped tohuman chromosome 8p11.2–12 (30), and was later correctlymapped to chromosome Xq13 (31). The rank order of bindingaffinity is CXCL11 � CXCL10 � CXCL9. Initially, CXCR3was found to be expressed on a subset of circulating T cells,B cells, and natural killer cells, and among T cells, mainly ontype 1 T helper (Th1) cells (32, 33). In subsequent studies, itwas found that CXCR3 was expressed not only by immunecells, but also by resident cells (34) such as human mesangialcells (35), human liver stellate cells, vascular pericytes (36),and human microvascular endothelial cells (37).

More recently, a distinct, receptor, deriving from an al-ternative splicing of the CXCR3 gene was identified andnamed as CXCR3-B (38). CXCR3-B not only binds CXCL10,CXCL9, and CXCL11, but also acts as functional receptor forthe orphan CXC-chemokine CXCL4, which exclusively in-teracts with CXCR3-B. The interaction of chemokines withCXCR3 mediates their chemotactic and immune effects,whereas the binding to the splicing variant CXCR3-B ac-counts for their angiostatic effect (38). To add to the com-plexity of CXCR3 biology, another variant of human CXCR3

has been identified, which is generated by posttranscrip-tional exon skipping. This receptor was named CXCR3-altand binds CXCL11, but its biological role is still unknown(39). The main aim of this review is to discuss the role ofIFN-� inducible chemokines (CXCL9, CXCL10, and CXCL11)and their classic CXCR3 receptor; therefore, the biologicaleffects resulting from the interaction between the alternativevariant of CXCR3 and their ligands will be limited to theirangiostatic effects.


• IP-10, Mig, and I-TAC are CXC chemokines sharing theproperties to be strongly up-regulated by IFN-�.

• IP-10, Mig, and I-TAC have been named as CXCL10,CXCL9, and CXCL11 following the new nomenclature,which will be used throughout this review.

• CXCL9, CXCL10, and CXCL11 share binding to a commonreceptor named CXCR3.

• CXCR3 was first identified on activated T cells, and itsexpression was associated with Th1-mediated immuneresponses.

• CXCR3 is also expressed by cell types different from Tcells, such as endothelial cells, vascular pericytes, andepithelial cells.

• Two splicing variants of the CXCR3 receptor exist, medi-ating different biological functions.

III. Main Biological Actions ofCXCR3-Binding Chemokines

A. Chemotaxis and regulation of the immune response

All three CXCR3-binding chemokines (CXCL9, CXCL10,and CXCL11) have been shown to play a chemotactic role indifferent cells types of the immune system. In particular,activated T cells, B cells, macrophages, and natural killer cellshave been found to express CXCR3 and can be attracted ininflamed tissues by CXCR3-binding chemokines, thus ac-counting for the mononuclear cell infiltrate characteristic ofinflammatory reactions (40). The molecular mechanisms ofthe chemokine-driven cell chemotaxis have been reviewedextensively (19).

T cells were originally divided into two main subsetswhich are named as CD4� T helper (Th) and of CD8�cytotoxic T (Tc) cells. Subsequently, two different types ofCD4� Th cells, known as type 1 Th (Th1) and type 2 Th (Th2),were recognized (Fig. 1). Th1 cells produce cytokines, suchas IL-2, IFN-�, and lymphotoxin-�, which result in the acti-vation 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 aregulatory rather than protective role, inasmuch as cytokinesproduced by these cells (i.e., IL-4 and IL-13) have an inhib-itory effect on the production of Th1 cytokines, as well as onseveral functions of activated macrophages (41). It should benoted, however, that Th1 and Th2 cells do not representclearly distinct lineages of Th cells, as CD4� and CD8� Tcells, but extremely polarized forms of a much more heter-ogenous Th cell response. Moreover, their phenotype of cy-tokine production in humans is not always so clearly polar-ized as in mice.


With regard to the expression of CXCR3, Th1 cells wereinitially described as being specifically equipped with thisreceptor, whereas Th2 cells expressed distinct chemokinereceptors, such as CCR3, CCR4, and CCR8 (32, 33). However,this dichotomy in chemokine receptor expression betweenTh1 and Th2 cells is not so strict as initially thought, thedifference being quantitative rather than qualitative (34).

The fact that Th1 cells produce IFN-�, which induces theproduction by different cell types of CXCL9, CXCL10, andCXCL11, and that these chemokines in turn can attract andrecruit Th1 cells, suggests the existence of a loop betweenIFN-�-producing Th1 cells and resident cells producingCXCR3-binding chemokines (42). Th2 cells express differentchemokine receptors, such as CCR4 and CCR8, thus beingrecruited in target tissues by CCL17, CCL22 (both ligands forCCR4), and CCL1 (ligand of CCR8). Based on these findings,it can be proposed that chemokines interacting with T cellsvia CXCR3 may induce a recruitment of Th1 cells into theinflamed tissues. On the other hand, chemokines interactingwith different chemokine receptors on T cells may recruit Th2cells, which are responsible for allergic inflammation.

Further studies support the concept that the role ofCXCR3-binding chemokines in the regulation of the immuneresponse goes far beyond their powerful chemotactic activityon activated lymphocytes. A large body of experimentalevidence emphasizes the role of CXCL10 in the initiation andamplification of host alloresponses (43). CXCL10-deficientmice have impaired T cell responses, impaired contact hy-persensitivity, and limited inflammatory cell infiltrates. Theyare also unable to control viral infections (44). CXCL10 notonly mediates leukocyte recruitment, but also drives T cellproliferation to allogenic and antigenic stimulation andIFN-� secretion in response to antigenic challenge (42). Ac-cordingly, CXCL10 up-regulates the production of Th1 cy-tokines and down-regulates the production of Th2 cytokines

(45). The final result is a strong up-regulation of inflamma-tory reactions characterized by the production of IFN-�, thusexerting important protective activity against infections sus-tained by intracellular bacteria and some viruses, which isprovided by Th1 cells (Fig. 2). This also results in a down-regulation of allergic inflammation that is provided by Th2responses.


• CXCR3 preferentially mediates chemotaxis of Th1 cells.

FIG. 1. Schematic representation of Th cell differen-tiation and regulation. The production of IL-12 pro-motes the development of Th1 cells producing IFN-�,IL-2, and TNF-�, which activate macrophages and areresponsible for cell-mediated immunity and phago-cyte-dependent protective responses. By contrast, theproduction of IL-4 favors the development of Th2 cellsproducing IL-4, IL-5, and IL-13, which are responsiblefor strong antibody production, eosinophil activation,and inhibition of several macrophage functions, thusproviding phagocyte-independent protective re-sponses. Th1 cells mainly develop after infections byintracellular bacteria and some viruses, whereas Th2cells predominate in response to infestations by gas-trointestinal nematodes. The production of TGF-� andIL-6 promotes the development of Th17 cells, a dis-tinct type of effector T cell that induces tissues dam-age. Once Th17 cells are established, IL-23 also par-ticipates in their maintenance. Treg cells, whichinhibit autoimmunity and protect against tissue in-jury, are induced by TGF-� in the absence of IL-6.Thus, TGF-� functions as a regulator of tissue-dam-aging Th17 cells when collaborating with IL-6 and asan activator of antiinflammatory Treg cells when act-ing without IL-6. Solid lines indicate up-regulation,whereas dotted lines indicate inhibition. [For reviewson the topic, see S. Romagnani: Clin Exp Allergy 36:1357–1366, 2006 (92); and L. Steinman: Nat Med 13:139–145, 2007 (93).]

FIG. 2. Role of CXCL10/CXCR3 interactions in the amplification ofTh1 immune responses. CXCL9, CXCL10, and CXCL11 act as pow-erful chemotactic factors for the recruitment of Th1 cells in inflamedtissues. Furthermore, they act as selective costimulators of IFN-�production by T cells in antigen-dependent responses. BecauseCXCR3 agonists are produced by monocytic, endothelial, and residentepithelial cells in response to IFN-�, this suggests that CXCR3 ligandsand IFN-� production from CD4� T cells have the capacity to form aunique cytokine-/chemokine-positive feedback loop to amplify ongo-ing Th1 immune responses.


• CXCL10 not only mediates leukocyte recruitment, but alsodrives T cell proliferation to allogenic and antigenicstimulation.

• CXCL10 up-regulates the production of Th1 cytokines anddown-regulates the production of Th2 cytokines. The finaleffect is the enhancement of inflammatory reactions char-acterized by the production of IFN-�.

B. Angiogenesis

CXCR3-binding chemokines are powerful inhibitors of an-giogenesis (23). The major receptor mediating the angiostaticeffect of CXC chemokines is CXCR3-B. CXCR3-B is expressedin human microvascular endothelial cells (37, 46) during thelate S-phase of the cell cycle on through mitosis, representingthe first example of a chemokine receptor expression linkedto a particular phase of the cell cycle (37). In vivo, the ex-pression of CXCR3-B in small vessels (37, 47, 48), is higher ininflamed and neoplastic tissues compared with normal tis-sues (37).

Angiostatic CXC chemokines were shown to inhibit an-giogenesis in several experimental models (49–51) and toparticipate in the control of angiogenesis during physiolog-ical repair of tissue injury (52). CXCL9 and CXCL10 arespecifically expressed during the late phase of wound heal-ing repair, to help prevent unlimited vessel growth withoutblocking other repair processes involved in wound healing(23).

CXCR3-binding chemokines are also involved in thepathogenesis of proliferative diabetic retinopathy (53). Thelevels of CXCL10 were found to be significantly higher invitreous samples from patients with inactive, compared withactive, proliferative diabetic retinopathy. This suggests thatdecreased levels of this angiostatic chemokine might favorretinal angiogenesis during diabetic retinopathy (53).

Overall, the angiostatic effect of CXCR3-binding chemo-kines 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 evaluatedin either normal or pathological endocrine glands.


• CXCL10, CXCL9, CXCL11, and CXCL4 are powerful an-giostatic agents.

• The angiostatic effect of CXCL9, CXCL10, CXCL11, andCXCL4 is mediated by their interaction with CXCR3-B.

• CXCR3-B is selectively expressed by endothelial cells onlywhen they are activated and has been observed in endo-thelial cells of inflammatory and neoplastic tissues.

IV. CXCR3-Binding Chemokines in Healthy Subjectsand in Nonendocrine Immune-Mediated

Pathological Conditions

The measurement of the serum levels of CXCL10 is cur-rently performed by commercial solid phase ELISA. Thesekits employ the quantitative sandwich enzyme immunoas-say technique. Early studies measured the serum concentra-tions of CXCL10 by homemade ELISAs. The availability ofthe human recombinant CXCL10 protein and of specific

monoclonal antibodies (mAbs) warrant accurate estimation.The mean minimum detectable dose in human sera is below5.0 pg/ml, whereas the mean coefficients of intra- and in-terassay variations expressed in percentages are below 5.0and 10.0%, respectively. No significant cross-reactivity withother CXCR3-binding chemokines or IFN-� is observed.Commercial ELISA kits for the measurements of CXCL9 andCXCL11 are also available.

A. CXCR3-binding chemokines in healthy subjects

The circulating concentrations of CXCR3-binding chemo-kines in humans have been extensively studied only forCXCL10, both in health and disease, whereas data regardingthe serum levels of the other two chemokines (CXCL9 andCXCL11) are still limited. Studies performed in large seriesof healthy adult subjects found mean serum levels of CXCL10ranging from 70 to 90 pg/ml (54, 55). Variations betweenhealthy subjects may be estimated by sd values of approx-imately 50 pg/ml (54, 55). These figures are comparable tothose reported in the normal subjects used as controls inclinical studies investigating CXCL10 in patients with endo-crine and nonendocrine diseases (which will be quoted whendiscussing the specific disease). Nevertheless, it should benoted that in the latter studies healthy subjects were rarelyscreened for circulating autoantibodies, and therefore it isnot always possible to exclude the presence of subclinicalautoimmune disorders which, at least in some cases, mighthave affected the serum concentrations of CXCL10. It is nowknown that in euthyroid chronic autoimmune thyroiditis(CAT), the most frequent subclinical autoimmune conditionin humans, especially in middle-aged women, the serumlevels of CXCL10 are significantly increased compared withhealthy controls proven to be negative for thyroglobulin (Tg)and thyroid peroxidase (TPO) antibodies (Ab). EuthyroidCAT might represent the most frequently undetected con-dition biasing the results of CXCL10 in apparently healthysubjects. Many other, less easily detectable abnormalities(e.g., other subclinical autoimmune diseases) may lead tosimilar problems. The question arises as to how healthysubjects should be selected when comparing their serumCXCL10 levels to those observed in a specific pathologicalcondition. To complicate the issue further, it should be notedthat there are currently no data regarding fluctuations ofCXCL10 in the serum of individual healthy subjects. As faras our current knowledge permits, we will try to define somephysiological variables, which were found to influence theserum levels of CXCL10.

1. The role of gender. Clinical studies, in which healthy subjectswere screened for excluding subclinical thyroid abnormali-ties by means of thyroid ultrasound (US) and tests for cir-culating Tg Ab and TPO Ab, found no gender-related dif-ferences in circulating concentrations of CXCL10. In theabsence of such a screening for subclinical autoimmune con-ditions, higher serum levels of CXCL10 might be expected infemales, due to the well-known greater prevalence of auto-immunity in women than in men. In clinical studies, thepotential bias resulting from gender-related differences maybe reduced, at least in part, by performing a strict sex-match-ing between the subjects to be evaluated.


2. The role of age. An age-related dysregulation of the immunesystem has been extensively reported by studies performedin humans and experimental animal models of aging (56–59).The influence of aging on circulating concentrations ofCXCL10 was evaluated in two clinical studies (54, 55).Healthy subjects aged from 10 to 80 yr, proven to be negativefor circulating thyroid antibodies and with no evidence ofother autoimmune diseases, were studied. In a multiple lin-ear regression model including age, body mass index (BMI),systolic and diastolic blood pressure, glycemia, total high-density and low-density lipoprotein cholesterol, triglycer-ides, TSH, Tg Ab, and TPO Ab, only age was significantlyrelated to serum levels of CXCL10, a positive correlationbeing found between the two variables.

3. The role of body weight and BMI. Although the serum levelsof CXCL10 have not been specifically investigated in obesity,studies evaluating healthy subjects reported no change inserum concentrations of CXCL10 in relation to BMI (54). Theissue remains open because patients with morbid obesitywere not studied.

4. Practical points. The above-described physiological changesin the serum levels of CXCL10 must be taken into accountwhen this chemokine is measured in different pathologicalconditions. In diseases such as hepatitis C virus (HCV) hep-atitis, primary biliary cirrhosis, or end stage renal diseases,the serum levels of CXCL10 are extremely high; thus, thecomparison with healthy subjects might not be biased bygender or age. On the contrary, when studying pathologicalconditions in which the serum levels of CXCL10 are signif-icantly, but only slightly higher than in healthy controls, suchas endocrine autoimmune diseases, the above factors must beconsidered to reduce potential sources of error. As a conse-quence, matching patients and controls for gender and ageappears critical to avoiding misleading results.

B. CXCR3-binding chemokines in some immune-mediatedpathological conditions

In this section, we will briefly review the major findingsobtained in nonendocrine diseases, which are either crucialfor a better comprehension of the role of chemokines inhuman pathology or exemplify the applications of their as-says in the clinical practice. Consistent with the aim of thisreview, description of data from basic studies will be limitedto essential information, whereas findings obtained in clin-ical studies will be more extensively described. The latterdata support the view that measuring CXCL10 in the serumis useful in several nonendocrine diseases, both autoimmuneand nonautoimmune, such as chronic HCV hepatitis. In dif-ferent clinical settings, the serum levels of CXCL10 proved tobe useful as an index predicting the course and severity of thedisease, as a marker of its activity, as a predictor of treatmentoutcome, and as a parameter for choosing the best thera-peutic option. The fact that autoimmunity is responsible formost endocrine disease indicates that the results obtained byassaying CXCL10 in autoimmune nonendocrine disordersmight be transferred to endocrinopathies. This remains to bedone in that, as we will see in the subsequent sections, few

clinical studies have been performed so far in endocrinepatients.

1. CXCR3-binding chemokines in HCV-induced chronic hepatitis.The first published clinical study in which the serum levelsof CXCL10 were evaluated in human disease is the investi-gation by Narumi et al. (60), reporting significantly increasedcirculating concentrations of CXCL10 and CCL2 in patientswith HCV compared with healthy subjects. The serum con-centrations of both chemokines were found to be signifi-cantly higher in patients with chronic active hepatitis C com-pared with those with chronic persistent hepatitis C.Subsequent studies performed in patients with differenttypes of liver diseases demonstrated that the serum levels ofCXCL10 were significantly higher in patients with autoim-mune hepatitis, primary biliary cirrhosis, and both hepatitisB virus and HCV chronic hepatitis than in healthy controls(61). Circulating CXCL10 concentrations were found to besignificantly correlated with the serum levels of aspartateand alanine aminotransferases. This finding suggested a re-lationship between the serum levels of CXCL10 and thenecroinflammatory activity of hepatitis. Several clinical stud-ies evaluated the changes of serum CXCL10 in patients withHCV hepatitis undergoing IFN-� therapy (60, 62). In theirfirst report, Narumi et al. (60) had demonstrated that theserum levels of CXCL10 significantly decreased after IFN-�treatment, but only in cured patients, as assessed by thenormalization of serum aminotransferases and by the dis-appearance of HCV from serum for 6–12 months after stop-ping therapy (60). In nonresponders to IFN-�, the basal se-rum levels of CXCL10 were significantly higher than inresponders to therapy and remained high throughout thetreatment (60). A subsequent study reported that the serumlevels of CXCL10 and CCL4, but not those of CXCL9, de-creased significantly in HCV patients showing a virologicalresponse to IFN-� treatment (63). A recent study simulta-neously evaluated the three CXCR3-binding chemokines(CXCL9, CXCL10, and CXCL11) in plasma samples collectedat 1 wk before treatment (baseline), 29 d after starting ther-apy, and 6 months after completion of a course of pegylatedIFN, with or without ribavirin (64). The principal interest forthis study stems from the fact that it is the only publishedexperience in which the three IFN-� inducible chemokineswere assessed simultaneously, thus allowing a comparisonof the relative importance of CXCL9, CXCL10, and CXCL11.At baseline, the serum concentrations of CXCL9, CXCL10,and CXCL11 were higher in patients with HCV hepatitiscompared with healthy controls, the greatest increase beingfound for CXCL10. After successful antiviral treatment, theserum levels of CXCL10 and CXCL9, but not those ofCXCL11, significantly decreased (64). After completion ofIFN-� treatment, sustained responders had circulating levelsof CXCL10 similar to healthy subjects, whereas in nonre-sponders to therapy, the serum levels of CXCL10 remainedelevated. Pretreatment levels of CXCL9 did not differ inresponders compared with nonresponders and declined dur-ing therapy in both groups. No significant association wasfound between pretreatment levels of CXCL11 or between itschanges in serum and the outcome of treatment.

Taken together, these results demonstrate that the circu-


lating levels of each of the three IFN-�-inducible chemokinesare differently regulated during IFN-� therapy. Of the threechemokines that bind CXCR3, CXCL10 is the one mostclosely associated with the outcome of treatment with IFN-�for HCV-related hepatitis, and its pretreatment levels maypredict the likelihood of a favorable response (64).


• The serum levels of CXCL10 are related to the activity ofHCV hepatitis, showing a significant correlation with theserum concentrations of aminotransferases and with thehistological severity of hepatitis.

• Successful therapy with IFN-� in HCV hepatitis results ina long-lasting normalization of circulating CXCL10, whichis mainly due to a decreased lymphocytic infiltration of theliver.

• Lower pretherapy serum CXCL10 levels can identify thosepatients who will develop a better response to IFN-�treatment.

• Among the CXCR3-binding chemokines (CXCL9,CXCL10, and CXCL11), CXCL10 is the most helpful andreliable serum marker of the therapeutic outcome in HCVpatients.

2. CXCR3-binding chemokines in allograft rejection. Growingevidence suggests that CXCL10 is critical in promoting andamplifying host alloresponses responsible for acute allograftrejection (65–70). In CXCL10- or CXCR3-gene-deficient mice,cardiac transplants are not acutely rejected and undergopermanent engraftment (43, 65). Accordingly, neutralizationof CXCL10 with mAbs prolongs the allograft survival in bothcardiac and small bowel models of allograft rejection (65, 66).Furthermore, the intragraft expression of CXCL10 has beenreported in association with the rejection of renal (67), lung(68), and cardiac (69, 70) allografts. Thus, the importance ofCXCL10–CXCR3 interactions in the pathogenesis of graftfailure appears to be clearly demonstrated in multiorganmodels.

Recent evidence indicates that CXCR3 and CXCL10 arealso highly expressed in conjunction with the developmentof chronic rejection, also named as chronic allograft vascu-lopathy (71). Indeed, in addition to its potent effects on im-mune responses (23, 31, 32, 40, 72–75), CXCL10 also altersvascular endothelial and smooth muscle cell functions (23,35–37, 40, 76, 77), thus promoting the development of chronicallograft nephropathy. Pretransplant serum levels ofCXCL10 were measured in kidney graft recipients to verifyits value in predicting the recipient’s risk for graft rejectionand transplant failure (78, 79). Patients with normally func-tioning grafts showed significantly lower pretransplant se-rum levels of CXCL10 compared with patients who experi-enced graft failure, and lifetime analysis showedsignificantly lower 5-yr survival rates of the grafts with in-creasing pretransplant serum levels of CXCL10. Further-more, frequency of acute rejection episodes in the first monthafter transplant significantly increased in relation to increas-ing pretransplant serum levels of CXCL10. In particular,patients with serum CXCL10 levels greater than 150 pg/mlshowed a nearly 2-fold greater frequency of rejection. Re-jection episodes were not only more frequent, but also more

severe in patients showing high pretransplant serum levelsof CXCL10 (78). Recently, patients developing chronic allo-graft vasculopathy were also shown to have significantlyhigher pretransplant serum concentrations of CXCL10 thanpatients with normally functioning grafts (78, 79). Multivar-iate analyses indicated that high serum levels of CXCL10were a significant risk factor for acute graft rejection and graftfailure (78). Taken together, these results indicate that highpretransplant serum levels of CXCL10 may predict the riskfor the development of acute rejection and chronic allograftvasculopathy. Accordingly, the urinary levels of CXCL9 andCXCL10 are a sensitive and specific predictor for acute re-jection and also mirror the response to antirejection therapy(80, 81). High urinary levels of CXCL10 in the first days aftertransplant also predict acute rejection, as well as short andlong-term graft function (82). Thus, the measurement ofCXCR3-binding chemokines in serum or urine may be usefulto select those patients requiring more aggressive immuno-suppressive regimens.


• High pretransplant serum levels of CXCL10 identify pa-tients with a higher risk for developing acute rejection,chronic allograft vasculopathy, and subsequent graftfailure.

• High pretransplant serum levels of CXCL10 are associatedwith more severe acute rejection, a Th1-mediated reaction.

• Pretransplant levels of serum CXCL10 may be used toidentify patients requiring more aggressive posttransplantimmunosuppression therapy.

3. CXCR3-binding chemokines in multiple sclerosis (MS). Severalchemokine receptors, and among them CXCR3, were shownto be highly expressed in brain samples obtained at autopsyfrom patients with MS (83–85), suggesting that CXCR3 mightbe responsible for the recruitment of autoaggressive T cells.In line with this interpretation, CXCL9 and CXCL10 werefound to be significantly elevated in the cerebrospinal fluid(CSF) of MS patients, being positively correlated with theCSF cell counts. The relevance of elevated levels of CXCL10and CXCL9 in the CSF of MS patients was further supportedby the uniform detection of CXCR3� lymphocytes in theperivascular inflammatory cuffs of brain lesions (83–85). Theaccumulation of these cells was directly related to the de-myelinating process. The demonstration that the chemotacticactivity toward CD4� T cells specific for a myelin basicprotein peptide is mediated by CXCL10 (86) and the notionthat IFN-� is a potent inducer both of CXCL10 and of clinicalrelapses of MS provided evidence for a pathogenetic role ofCXCL10 in this disease.

Th1- and Th2-oriented chemokines were sequentiallymeasured in the serum and the CSF of patients with MS.CXCL10 and CCL2 were chosen as prototype chemokines fora Th-1 and a Th-2 phenotype, respectively. The measurementof CXCL10 and CCL2 in the serum and CSF of MS patientsshowed that these chemokines had a different behavior inrelation to the activity of the disease. CXCL10 was higher inthe serum and the CSF of patients with acute MS and lowerin those with a stable phase of the disease. An oppositepattern characterized the CCL2 secretion profile, with high


levels being found in the serum and the CSF during the activephases of MS and with a decline in the stable phase of thedisease. These findings indicate an involvement of both che-mokines, with reciprocal changes according to the clinicalphase of MS (85, 87, 88). Because CXCL10 is mainly relatedto Th1 responses, the increase of CXCL10 in the serum andthe CSF of patients during the acute phases of MS fits withthe notion that IFN-� mediates the immune changes leadingto an exacerbation of the disease.


• The simultaneous assessment of chemokines associatedwith a Th1 or Th2 immune phenotype may constitute auseful approach in autoimmune diseases with a clinicalcourse characterized by active and stable phases(relapsing/remitting).

• The serum levels of CXCL10 are higher in the active phaseand lower in the stable phase of MS. An opposite behaviorcharacterizes a Th2 chemokine (CCL2).

V. CXCR3-Binding Chemokines in EndocrineAutoimmune Diseases

A. Notes on immune effector mechanisms inautoimmune diseases

Autoimmune diseases are the consequence of an immuneresponse against self-antigens, due to multiple genetic andenvironmental factors that result in a failure of the mecha-nisms devoted to maintaining self-tolerance. The multiplefactors involved in the control of reactivity against self-an-tigens, as well as the mechanisms responsible for their fail-ure, are still partially known and have been widely debatedin recent reviews (89). Failure to maintain self-tolerance re-sults in the activation of both self-reactive T and B cells,which produce chronic inflammatory reactions in target tis-sues. Autoimmune diseases may be organ- or nonorgan-specific. Although the immunopathogenesis of nonorgan-specific autoimmune diseases still remains unclear, theeffector mechanisms involved in organ-specific autoimmu-nity have been mainly related to the activity of CD4� Th andof CD8� Tc cells. In particular, for many years the attentionwas focused on a polarized subset of CD4� T cells, knownas Th1, which are able to produce cytokines, such as IL-2,IFN-�, and lymphotoxin-�, that result in the activation ofmacrophages, production of complement-fixing and -opso-nizing antibodies, and also cytotoxicity. By contrast, anotherpolarized subset of Th cells, known as Th2, has been thoughtto play a protective role, inasmuch as cytokines produced bythese cells (i.e., IL-4 and IL-13), play an inhibitory effect onthe production of Th1 cytokines, as well as on several func-tions of activated macrophages. Th cells able to produce bothTh1 and Th2 cytokines have been named type 0 Th (Th0).

Both Th1 and Th2 cells collaborate with B cells for theproduction of antibodies. However, Th1-induced antibodiesdiffer from those detectable during Th2 responses because ofthe different subclasses. In mice, Th1 lymphocytes induce Bcells to produce mainly IgG2a, whereas Th2 cells induce theproduction of IgG1 and IgE. In humans, the situation is lessclearly dichotomic, but it is known that Th2 responses are

characterized by IgE and IgG4, whereas Th1 responses pro-mote the production of IgG1 and IgG3 subclasses. IgG1,which represent the major subclass of human IgG in theserum, are complement-fixing and -opsonizing antibodies,and therefore they contribute, together with activated mac-rophages, to the phagocyte-dependent protection against in-fectious agents. Usually, IgG1 also represent the major sub-class among autoantibodies, and this is the reason why highlevels of autoantibodies are commonly observed in patientswith diseases characterized by strong Th1 response.

As mentioned in Section III.A, Th1 cells mainly expressCXCR3 as a chemokine receptor and can be recruited intotarget tissues by CXCL9, CXCL10, and CXCL11. Th2 cellsexpress different chemokine receptors, such as CCR4 andCCR8, 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 ofCXCR3-binding chemokines in target tissues of organ-spe-cific autoimmune disorders, including those affecting theendocrine glands, has suggested the existence of an impor-tant pathogenetic loop. The concept is based on the role ofthese chemokines in recruiting Th1 cells and in maintainingand amplifying chemokine production by Th1 cells throughIFN-� production.

It is worth noting that an impressive series of data obtainedboth in experimental animal models and in human diseaseshave shown that when Th1 responses, because of their se-verity and/or chronicity, become dangerous for the body,they can be shifted to a less polarized profile (Th0) or evento responses characterized by the prevalent production ofTh2 cytokines. Likewise, established Th2 responses can beshifted to a less polarized profile or even to a prevalent Th1profile. This phenomenon is known as immune deviation(90).

In the last few years, a novel subset of Th cells has beendiscovered and named Th17 or ThIl-17 (Fig. 1) (91). Thesecells appear to be distinct from Th1 and Th2 cells because ofpeculiar mechanisms of development and possible functions(92, 93). Although Th1 cells mainly develop in response toIL-12 produced by dendritic cells and Th2 cells develop dueto the early presence of IL-4, Th17 cells develop in responseto the production of IL-23, IL-6, and TGF�1 by dendritic cells.Th17 cells have been recently suggested to play a pathogenicrole in autoimmune diseases on the basis of data obtained inanimal models, such as experimental autoimmune enceph-alomyelitis (which is considered as the equivalent of MS) andcollagen-induced arthritis (a model of rheumatoid arthritis).Their role in human endocrine autoimmune diseases re-mains to be established (94). Thus, in our discussion, we willonly take into account the body of experimental evidencesuggesting that in these disorders the effector responses areapparently mediated by Th1 cells.

B. Autoimmune thyroid diseases

1. Background. The thyroid is a major target for autoimmu-nity. Human autoimmune thyroid disorders (AITD) arecharacterized by reactivity to self-thyroid antigens, whichmay be expressed as destructive inflammatory or antirecep-tor autoimmunity (95) and encompass the clinical spectrum


of Graves’ disease and CAT (96–98). Graves’ disease sharesmany immunological features with CAT, both diseases beingcharacterized by lymphocytic infiltration of the gland, whichcan result in tissue destruction (99, 100). One of the his-topathological hallmarks of thyroid glands affected by AITDis leukocytic infiltration, mainly by mononuclear cells, in-cluding T and B lymphocytes and macrophages (95, 101). InAITD, the lymphocytic infiltrate is also an important site ofthyroid autoantibody synthesis (95, 102). Lymphocytes me-diate important inflammatory effects, such as the release ofcytokines (95). The cellular makeup of the infiltrate varieswith the type of AITD, the stage of the disease, and thetherapy used, but it is also patient-dependent. This cellularinfiltrate sometimes organizes itself into germinal centersthat share many of the features of lymph node germinalcenters (101, 103, 104). Intrathyroidal lymphocytes play acentral role in the pathogenesis of AITD, but the mechanismsby which different lymphocytic subsets are recruited andarrested in the thyroid tissue are only partially understood.To the best of our knowledge, the recruitment of lympho-cytes in AITD is a multistep process involving adherence andmigration across the endothelium, trafficking through theinterstitium, and finally moving toward the thyroid follicularcells (105, 106). Leukocyte extravasation involves the com-bined action of adhesion molecules, such as selectins andintegrins, and chemotactic factors, mainly chemokines (107).In AITD, infiltrating lymphocytes and endothelial cells bearan enhanced expression of various adhesion molecules,pointing to lymphocyte function-associated antigen-1/inter-cellular adhesion molecule-1, very late antigen-4/vascularcell adhesion molecule-1, and selectin/selectin ligands ad-hesion pathways as predominant in lymphocyte migrationto the thyroid (108). Studies evaluating cytokines in AITDhave demonstrated the production of IL-1, IL-2, IL-6, IL-10,IFN-�, and TNF-� by infiltrating T cells and macrophages(109–115). However, the specific role of these molecules inthe pathogenesis of AITD is still debated (115). In addition,the thyroid follicular cells themselves produce many cyto-kines (116–120).

2. Chemokines in AITD. In 1992, Weetman et al. (121) firstdescribed the production of chemokines by cultured thyroidfollicular cells. They demonstrated that thyrocytes stimu-lated by IFN-�, TNF-�, or IL-1� produce IL-8, a CXC che-mokine (121). A subsequent study showed that human thy-rocytes in primary culture, upon stimulation with IL-1�,TNF-�, or IFN-�, produce CCL2 (122). Although the highlyorganized lymphomononuclear cell infiltration present inAITD suggested an involvement of chemokines in theirpathogenesis, several years passed before endocrinologistspointed their attention toward these new molecules. It wasnot until 2000 that the expression of chemokines in AITD wasstudied in detail and evidence was provided as to theirpathogenetic role, at least in the initial phases of thesedisorders.

The interest in IFN-� inducible chemokines (CXCL9,CXCL10, and CXCL11), and their receptor (CXCR3), origi-nated from an investigation aimed at evaluating the antian-giogenetic effects of these molecules. To this purpose, theexpression by human endothelial cells of CXCR3 and its

ligands was studied in normal tissues and in specimens fromdiseased organs (37). CXCR3 was detected in a small numberof vascular wall cells from normal tissue specimens includ-ing thymus, liver, kidney, and gut. Thyroid specimens wereobtained from normal tissue and from Graves’ glands. Byboth immunohistochemistry and in situ hybridization, ahigher signal for the protein and the mRNA of CXCR3 wasdetected in endothelial cells from Graves’ glands, but notfrom normal thyroids (37).

In 2001, Garcia-Lopez et al. (123) first demonstrated theproduction of CXCR3-binding chemokines by human thy-rocytes in primary cultures after stimulation with IFN-�. Inthe same culture system, CCL2 and CCL5 were secreted inresponse to TNF-�. In basal conditions, CXCL10 and CXCL9were not detected in the surnatants from thyroid follicularcells, but their secretion was induced by IFN-� and syner-gistically increased by TNF-� addition. As compared withautologous peripheral blood lymphocytes (PBL), intrathy-roidal lymphocytes from AITD patients showed a higherexpression of CXCR3 and of the receptors for CCL5 andCCL2, CCR2, and CCR5, respectively. T lymphoblasts ex-pressing CXCR3 showed an increased migration to super-natants of IFN-� stimulated thyroid follicular cells, whichwas abolished by neutralizing antibodies directed to CXCL9and CXCL10, as well as to their receptor, CXCR3. Takentogether, these data suggested a role for thyroid follicularcells, through the production of CXCL10, CXCL9, and CCL5,in the recruitment of specific subsets of activated lympho-cytes (123).

By using immunohistochemistry, a statistically significantincrease of CXCL10 and CXCL9 was found in thyroid tissuespecimens obtained from Hashimoto’s glands, comparedwith normal thyroid tissue (123). By contrast, in patients withGraves’ disease, the intrathyroidal chemokine expressionpattern was highly variable, with only a few subjects ex-pressing high levels of CXCL10 and CXCL9, as assessed byimmunohistochemistry (123).

A clear-cut demonstration that CXCL9 and CXCL10 werehyperexpressed in Graves’ glands was obtained using com-bined in situ hybridization and immunohistochemistry (124).The expression of the mRNAs for CXCL10 and CXCL9 innormal thyroids, as well as in thyroids from patients withautoimmune and nonautoimmune hyperthyroidism(Graves’ disease and toxic adenoma), was assessed by in situhybridization (Fig. 3). The quantitative evaluation ofCXCL10 and CXCL9 mRNAs, performed by a computerizedvideo image analysis system, provided evidence that theexpression of the mRNAs for the two chemokines was sig-nificantly higher in thyroid glands from Graves’ patientscompared with normal thyroids or toxic adenoma glands.The wide variability in the expression of chemokines re-ported by Garcia-Lopez et al. (123) in Graves’ disease wasconfirmed and was related to the duration of the disease. Astatistically significant increase of CXCL10 expression wasfound 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 thatobserved in normal thyroid specimens (124).

The findings obtained by in situ hybridization were con-firmed by a quantitative RT-PCR analysis of the mRNAs for


IFN-�, CXCL10, and CXCL9 in the same samples. RT-PCRrevealed that the expression of these molecules was highlyheterogeneous, being the mRNA levels for CXCL10 andCXCL9 strictly related to those of the IFN-�. The latter werealso higher in patients with recent-onset (�2 yr) Graves’disease. Multiple double-label immunohistochemistry wasused to identify the cellular source of chemokines andshowed that CXCL10 and CXCL9 were highly expressed byboth thyroid follicular cells and infiltrating mononuclearcells (Fig. 3). The CXCR3 receptor was found only in inflam-matory and endothelial cells (Fig. 4) (124).

By using flow cytometry, it was demonstrated that theexpression of the chemokine receptors on PBL of Graves’patients did not differ from that of normal controls (125). Onthe other hand, there was an enrichment of CXCR3� (thereceptor for CXCL10) and CCR5� (the receptor for CCL2) T

cells among thyroid-derived lymphocytes, compared withPBL. These results were confirmed by using RT-PCR andimmunohistology. The fact that thyroid-derived lympho-cytes showed a different chemokine receptor pattern com-pared with PBL from the same patient suggested a role forCXCR3 and CCR5 in the recruitment of T cells to the thyroidin Graves’ disease (125).

In summary, these early studies demonstrated a role forCXCR3-binding chemokines and their receptor in AITD byevaluating chemokine expression at the mRNA and at theprotein level, both in the thyroid and in primary cultures ofthyrocytes (126, 127). The subsequent steps for defining therole played by chemokines in AITD were provided by clinicalstudies that evaluated the serum levels of CXCL10 in largeseries of patients with Graves’ disease or CAT (124, 128–132).

3. CXCR3-binding chemokines in Graves’ disease. The first ob-servation of increased serum levels of CXCL10 in patientswith Graves’ disease was reported in 2002 (124). Serum sam-ples were collected from 50 unselected Graves’ patients withdifferent duration of their disease, as well as from 25 healthycontrols. All Graves’ patients had been treated with methim-azole (MMI) at variable doses and were euthyroid at the timeof serum analysis. Corticosteroid treatment was an exclusioncriterion. Mean CXCL10 serum levels were significantlyhigher in patients with Graves’ disease compared withhealthy subjects, even if there was a large overlap of CXCL10results between the two groups. The serum concentrations ofCXCL10 were inversely correlated with the duration ofGraves’ disease, the highest levels being found in patientswith recent-onset disease. By contrast, no correlation wasobserved between the serum levels of CXCL10 and otherclinical or biochemical parameters such as sex, age, and titersof circulating Tg Ab or TPO Ab. Interestingly, the reduction

FIG. 4. Expression of the IFN-� inducible chemokine receptor CXCR3in Graves’ disease. Immunohistochemistry reaction was performed on5-�m frozen sections of a thyroid tissue specimen obtained from thesame Graves’ gland shown in Fig. 3. The CXCR3 antibody binding wasrevealed by the avidin-biotin-peroxidase complex system, and theslides were counterstained with Gill’s hematoxylin. The high immu-noreactivity (red) demonstrates an intense protein expression forCXCR3. Original magnification (top panel), �100. High power mag-nification on the same section (bottom panel; original magnification,�400) demonstrates CXCR3 expression (red) by infiltrating inflam-matory cells. [Reprinted from P. Romagnani et al.: Am J Pathol 161:195–206, 2002 (124) with permission from the American Society forInvestigative Pathology.]

FIG. 3. Expression of IFN-� inducible chemokines (CXCL9 andCXCL10) in Graves’ disease. In situ hybridization was performed on10-�m frozen sections from normal and Graves’ thyroid glands hy-bridized with human CXCL10 or CXCL9 antisense mRNA probes (topfour panels). Each probe was hybridized for 16 h, washed, then au-toradiographed and counterstained with hematoxylin-eosin-phloxine(dark-field original magnifications, �100). Normal and Graves’ thy-roid glands showed no signal and high signal, respectively, for theexpression level for mRNAs of CXCL10 and CXCL9. The positivity ofthe signal for the mRNAs encoding both chemokines on thyroid fol-licular cells, at high-power magnification (dark-field original magni-fications, �1000), is shown in the subsequent two panels. The last twopanels show the positivity of the signal for the protein of both che-mokines on thyroid follicular epithelium from the same Graves’ thy-roid. Immunohistochemistry by double-label immunostaining forCXCL10 and CXCL9 (red) and TSH receptor (bluish gray) was per-formed on 5-�m frozen sections, and the corresponding antibodieswere revealed by the avidin-biotin-peroxidase complex system andcounterstained with Gill’s hematoxylin. No counterstain was applied.Original magnification, �400. [Reprinted from P. Romagnani et al.:Am J Pathol 161:195–206, 2002 (124) with permission from the Amer-ican Society for Investigative Pathology.]


of CXCL10 serum levels in long-standing (�2 yr) Graves’disease was associated with a slight increase in the serumconcentrations of CCL22, a chemokine associated with Th2immune responses (133, 134).

The analysis of the CCL22/CXCL10 ratio demonstratedthat a longer duration of Graves’ disease was associated withan increase of the CCL22/CXCL10 ratio in the serum ofGraves’ patients (124). Thus, in the late phase of Graves’disease, an increase in the CCL22/CXCL10 ratio, mainly dueto a CXCL10 decline, is observed both in the thyroid glandand in the serum. This phenomenon parallels the reductionof intrathyroidal IFN-� mRNA expression (124).

a. Changes in serum levels of CXCL10 in relation to thyroidfunction and treatment in Graves’ disease. Following the obser-vation that the serum levels of CXCL10 are increased inGraves’ disease, several clinical trials were designed with theaim of systematically evaluating the serum chemokine statusin Graves’ patients in relation to their thyroid function andtreatment (130–132). The final goal was to relate the findingsof circulating CXCL10 to the clinical phenotype and to eval-uate possible relations between the serum levels of CXCL10and the two major therapeutic strategies used in Graves’disease: medical treatment and thyroid removal. Althoughhigh serum levels of CXCL10 are not a specific feature ofGraves’ disease, having been reported in several endocrineand nonendocrine autoimmune or even nonautoimmune hu-man diseases, the results provided by the following clinicalstudies support the view that measuring CXCL10 serumlevels in Graves’ patients may be useful.

The first study retrospectively evaluated 103 patients withGraves’ disease but with no clinical signs or symptoms ofinflammatory ophthalmopathy (132). Graves’ patients wererecruited irrespective of their thyroid function or drug treat-ment. Thirty of them were hyperthyroid and untreated. Fifty-five patients were on MMI treatment for 1–28 months, andthe remaining patients were euthyroid, being in remissionafter a previous course of MMI. Healthy subjects, patientswith euthyroid CAT, patients with nontoxic nodular goiter,and hyperthyroid patients with toxic nodular goiter servedas controls.

The mean serum levels of CXCL10 were significantlyhigher in Graves’ patients than in healthy subjects or patientswith nontoxic multinodular goiter, but they did not differfrom those found in patients with euthyroid CAT. AmongGraves’ patients, the serum levels of CXCL10 were signifi-cantly higher in those older than 50 yr, in patients with ahypoechoic pattern of the thyroid at US, and in those with anincreased thyroid blood flow. Thyroid volume was unrelatedto circulating CXCL10. No significant correlation was ob-served between the levels of CXCL10 and the titers of Tg Ab,TPO Ab, or TSH-receptor (TR) Ab in serum. However, highserum levels of CXCL10 were mainly observed in Graves’patients who were strongly positive for TR Ab.

Hyperthyroid patients with Graves’ disease had signifi-cantly higher serum CXCL10 levels than those who wereeuthyroid or hypothyroid. Graves’ patients with untreatedhyperthyroidism had significantly higher serum CXCL10levels than those who were hyperthyroid or euthyroid whiletaking MMI (166 � 125, 124 � 41, and 94 � 35 pg/ml,

respectively). The serum levels of CXCL10 did not signifi-cantly differ in hyperthyroid Graves’ patients who were un-treated compared with those who relapsed after a previouscourse of MMI (176 � 125 and 155 � 97 pg/ml, respectively).Euthyroid patients on MMI or in remission after medicaltreatment showed similar serum levels of CXCL10.

This retrospective study confirmed that the serum levelsof CXCL10 are increased in patients with Graves’ disease,being strongly associated with the hyperthyroid phase of thedisease, and do decrease when euthyroidism is restored byMMI treatment (132). In agreement with these findings, highlevels of CXCL10 cosegregated with high TR Ab titers. Fur-thermore, high serum levels of CXCL10 were found to bestrongly associated with a marker of disease activity, such asthe increased thyroid blood flow. In this regard, the questionmight be raised of how the huge blood flow of Graves’ glandswould fit with the high expression of an angiostatic chemo-kine, such as CXCL10. The development of new vessels dur-ing an inflammatory process results from a balance betweenangiogenic and angiostatic factors (23). In Graves’ glands,new vessels develop due to extremely high local concentra-tions of vascular endothelial growth factor produced by thy-roid follicular cells in response to thyroid-stimulating anti-bodies (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 prepon-derant role of vascular endothelial growth factor, an ex-tremely powerful angiogenetic factor (136).

Patients with Graves’ disease in remission after a previouscourse of MMI therapy showed serum levels of CXCL10similar to healthy controls or to euthyroid patients withnontoxic multinodular goiter. The reduction of circulatingCXCL10 in patients rendered euthyroid by MMI treatmentcould be ascribed to the well-known immunomodulatoryeffect of antithyroid drugs (137). MMI, besides its ability todecrease thyroid hormone production (138), has been shownto interfere with some immunological abnormalities typicalof Graves’ hyperthyroidism. The immunosuppressive effectof MMI is highlighted by the reduction of circulating thyroidantibodies, which occurs during medical treatment with thisdrug, and by the consistent percentage (nearly 30%) ofGraves’ patients entering prolonged remission after a courseof medical therapy (138, 139). These immunological effects ofMMI might be mediated, at least in part, by an action onchemokine production, resulting in a decreased lymphocyticinfiltration of the gland. Indeed, a milder lymphocytic infil-tration was reported in Graves’ glands after medical treat-ment (140). Patients with newly diagnosed or relapsing hy-perthyroidism had comparable serum concentrations ofCXCL10. The increase in serum concentrations of CXCL10during relapses of hyperthyroidism would be in line with anovel activation of the Th1-mediated immune response andmight be taken as an index of an impending relapse of hy-perthyroidism after MMI treatment. The increase of circu-lating CXCL10 in the active phases of Graves’ disease is inagreement with findings in MS showing that serum levels ofCXCL10 are higher at disease onset and during relapses ofthe neurological disease (85, 88, 141).

The main results of this study can be summarized as in-dicating that CXCL10 is associated with the active phase of


Graves’ disease, in both newly diagnosed and relapsing hy-perthyroid patients, and that the reduction of serum CXCL10levels in Graves’ patients rendered euthyroid by MMI maybe related to an immunomodulatory effect of the drug. Agraphic representation of the mean serum levels of CXCL10at different clinical stages of Graves’ disease (132) is shownin Fig. 5.

The reduction of CXCL10 levels observed in both the thy-roid tissue and serum of Graves’ patients could be in linewith a progressive switch from a Th1 response to a lesspolarized immune response during the course of the disease(142). This shift, which probably reflects a counterregulatorymechanism against inflammation, when the Th1 responsecan become dangerous for the host has been described pre-viously in several experimental animal models of autoim-munity and in chronic inflammatory immune disorders(143–147). Such a shift was also demonstrated to occur inlong-standing Graves’ ophthalmopathy (GO) (142) by study-ing the surface markers of orbital T lymphocytes by flowcytometry. Although no direct proof is available, a generalshift from a Th1 to a Th2 response might occur in long-standing Graves’ disease in an attempt at dampening theinflammatory Th1 response. This concept would be in linewith the observed reduction of intrathyroidal IFN-� and theincreased ratio of CCL22 over CXCL10 in serum. However,given the fact that antithyroid drugs display a well-knownimmunomodulatory effect, the decrease in CXCL10 levelsobserved in long-standing Graves’ disease could also be as-cribed to the use of antithyroid drugs. Indeed, an immunedeviation toward a Th2 phenotype has been observed duringpharmacological treatments in graft vs. host disease (148) andMS (149).

b. Serum levels of CXCL10 and hyperthyroidism. To under-stand the meaning of circulating CXCL10 in Graves’ diseasebetter, a prospective study addressed the question ofwhether hyperthyroidism per se was responsible for the highlevels of serum CXCL10 (130). To this purpose, hyperthyroidpatients with either Graves’ disease or toxic nodular goiterwere enrolled in the study. The serum levels of CXCL10 wereevaluated in hyperthyroid Graves’ patients at the time ofdiagnosis and 3 months after starting medical treatment withMMI. Basal serum levels of CXCL10 were significantly lowerin 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 oc-curred in Graves’ patients after restoration of euthyroidismby MMI. Patients with toxic nodular goiter showed a slight,but not significant, reduction in circulating CXCL10 whenrendered euthyroid by MMI. Thus, the significant decreasein circulating concentrations of CXCL10 in Graves’ patientsafter MMI treatment was interpreted as resulting from animmunomodulatory action of the drug, rather than from therestoration of euthyroidism. Moreover, after accurate sex andage matching, the serum levels of CXCL10 were found to besimilar in healthy subjects, in hyperthyroid patients withtoxic nodular goiter, and in patients receiving levothyroxine(l-T4) at a TSH-suppressive dose for thyroid cancer. A furtherconfirmation of these data is provided by a recent studydemonstrating that Graves’ patients, stratified in relation tocirculating thyroid hormone concentrations, showed similarserum levels of CXCL10 (150). Taken together these findingsindicate that hyperthyroidism per se does not play a role indetermining the increased serum levels of CXCL10 observedin Graves’ disease (130).

c. Site of production of CXCL10 in Graves’ disease. Anotherissue to be elucidated was the site where CXCL10 is pro-duced. Cytokine production in Graves’ disease has beenvariably attributed to thyroid follicular cells (151), to intra-thyroidal lymphocytes (112), or to the activation of humoralimmune reactions in sites other than the thyroid (152, 153).To address this issue, the serum levels of CXCL10 wereevaluated in Graves’ patients submitted to thyroidectomy(131). In a prospective case-control study, 22 Graves’ patientsrendered euthyroid by MMI were submitted to thyroidec-tomy because of a history of relapsing hyperthyroidismand/or for the presence of large goiters. Healthy subjects andpatients with CAT were chosen as controls. A further controlgroup included 20 patients with toxic nodular goiter. Bloodsamples for CXCL10 measurement were collected in hyper-thyroid patients at presentation, when reaching euthyroid-ism on MMI, 3 d after thyroidectomy, and 1 month aftersurgery. Graves’ patients had significantly higher serum lev-els of CXCL10 when hyperthyroid than after reaching eu-thyroidism on MMI treatment. A further and significant re-duction in the serum levels of CXCL10 was observed 3 d afterthyroidectomy. No further significant decrease in the serumlevels of CXCL10 was found at 1 month after thyroidectomy,when the circulating concentrations of CXCL10 were similarto those found in healthy subjects or in patients with toxicnodular goiter, and lower than the concentrations observedin euthyroid patients with CAT (131). Similar findings were

FIG. 5. Schematic representation of the proposed mechanism of lym-phocyte recruitment by CXCR3-binding chemokines in endocrine au-toimmunity. Thyroid follicular cells secrete CXCL9, CXCL10, andCXCL11 upon stimulation with IFN-� and TNF-�. Chemokines, inturn, drive chemotaxis from blood vessels of T cells expressing thechemokine receptor CXCR3. This particular subset of T cells shows aprevalent Th1 immune phenotype and produces IFN-�, thus perpet-uating the inflammatory process. This loop of events supports theactive role played by thyroid follicular cells and in general by cells ofthe glandular epithelium (a similar mechanism has been demon-strated for �-cells and adrenal cells of the zona fasciculata) in deter-mining the specificity of the infiltrating lymphocytes and in main-taining the autoimmune process.


obtained after radioiodine treatment of hyperthyroidism(150). Taken together, the results provided by two studies(131, 150) evaluating the effect of the permanent cure ofhyperthyroidism on circulating CXCL10 concentrations in-dicate that both therapeutic strategies are able to lower serumCXCL10 to the levels observed in healthy subjects. The nor-malization of circulating CXCL10 levels after thyroidectomyor 131I ablation of thyroid tissue can be explained by removalof most intrathyroidal lymphocytes and/or thyrocytes (131,150). These findings support the view that the thyroid is themain source of circulating CXCL10 in patients with Graves’disease and most probably also in patients with CAT (129).


• The serum levels of CXCL10 are increased in patients withGraves’ disease.

• The active phases of Graves’ disease, as assessed by recent-onset thyrotoxicosis or relapsing hyperthyroidism aftermedical treatment, are characterized by the highest cir-culating concentrations of CXCL10.

• There is a significant relationship between the serum lev-els of CXCL10 and US indexes of Graves’ disease activity(hypoechogenicity of the gland at US and increased thy-roid blood flow).

• The serum levels of CXCL10 are not correlated with theserum titers of Tg Ab, TPO Ab, or TR Ab. However,patients with high serum levels of TR Ab are more likelyto show elevated concentrations of CXCL10.

• The increased serum levels of CXCL10 in Graves’ diseaseare not the result of hyperthyroidism per se.

• The significant reduction in circulating levels of CXCL10after restoration of euthyroidism in Graves’ patientstreated with MMI can be ascribed to an immunomodu-latory action of the drug.

• Graves’ patients treated by thyroidectomy or radioiodineablation have serum levels of CXCL10 similar to healthysubjects, supporting the concept that the thyroid is themain source of circulating CXCL10 in Graves’ disease.

4. CXCR3-binding chemokines in GO. There is only one pub-lished study that evaluated the serum concentrations ofCXCL10 in relation to the presence of GO (154). SerumCXCL10 levels were measured in 60 Graves’ patients withoutGO and in 60 age- and sex-matched patients with GO. Thecontrol group included 60 sex- and age-matched healthysubjects. At the time of evaluation, all Graves’ patients withor 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 activityscore (155). A score of 5 out of a maximum of 10, includinga worsening of ophthalmopathy during the previous 2months, indicated active GO. Inactive eye disease was de-fined as no change in eye status over the previous 6 months.A severity eye score was also calculated as the sum of theproducts of each NOSPECS class by its grade (156). Theserum levels of CXCL10 were similar in Graves’ patients withand without ophthalmopathy. Both groups showed signifi-cantly higher circulating CXCL10 levels compared withhealthy controls. Graves’ patients with active ophthalmop-

athy (i.e., higher clinical activity scores) had a more severeeye involvement, a shorter duration of the disease, and sig-nificantly higher mean serum CXCL10 levels than patientswith inactive ophthalmopathy.

The results of this study demonstrated that the circulatinglevels of CXCL10 are elevated in Graves’ patients with oph-thalmopathy when the inflammatory process of the orbit isactive. This finding is in agreement with previous data (112,142, 157–159) showing that in GO the active phase of thedisease is characterized by the presence of proinflammatoryTh1-derived cytokines, whereas other cytokines, and amongthem the Th2-derived ones, do not appear to be associatedwith a specific phase of the eye disease (160). In this scenario,the higher circulating concentrations of CXCL10 observed inGraves’ patients with active, compared with inactive, oph-thalmopathy might be expected.

The above data can be interpreted as indicating thatCXCL10 is transiently involved in the active phase of GO,when the inflammatory process is sustained by a Th1-me-diated immune response, whereas the serum levels of thischemokine decline in long-standing, inactive GO. In agree-ment with this interpretation, data from a previous studydemonstrate that, within 2 yr after the onset of the eye dis-ease, lymphocytes obtained from the orbital tissue of patientswith GO have a prevalent Th1 profile, whereas patients witha disease duration longer than 2 yr show a prevalence of Th2lymphocytes (142). Similar findings have been reported inMS, indicating that high levels of serum CXCL10 are stronglyassociated with the activity of the disease, as assessed byclinical pousses of neurological deterioration (85, 88, 141).

Cytokine production in AITD (161) has been variably in-terpreted as being sustained by thyrocytes (151), intrathy-roidal lymphocytes (112), or the activation of immune reac-tions at sites other than the thyroid (152). Thus, the questionmay be raised of whether the increased serum levels ofCXCL10 in patients with active GO do reflect the immuneprocess involving orbital tissues. In the above-mentionedstudy (154), all patients with GO had a clinical history ofGraves’ hyperthyroidism. However, it is unlikely that theelevation of CXCL10 in their serum resulted from hyperthy-roidism because all patients were euthyroid at the time ofCXCL10 measurements. Furthermore, the increase in serumconcentrations of CXCL10 was more pronounced in GO pa-tients with active inflammatory orbital disease than in thosewith inactive GO. Thus, the presence of orbital inflammationmay be responsible for an increase in circulating concentra-tions of CXCL10.

A major difference between active and inactive GO is thepresence of a lymphocytic infiltrate in orbital tissues (161,162). Thus, the increased production of CXCL10 might besustained by orbital lymphocytes. However, in vitro studiesdemonstrate that in the orbit CXCL10 can also be producedby nonlymphoid cells (154). Both fibroblasts and preadipo-cytes from patients with GO secreted CXCL10 in response toIFN-� stimulation, but not after challenge with TNF-� alone(154). A combination of IFN-� and TNF-� synergisticallyincreased CXCL10 secretion, similar to observations in hu-man thyrocytes (123) and endothelial cells (163). Interest-ingly, the response of orbital fibroblasts and preadipocyteswas similar to that of fibroblasts and preadipocytes of dermal


origin, suggesting that this kind of activation is a generalphenomenon. Taken together, the above-described observa-tions support the view that the production of IFN-� andTNF-� by Th1-activated lymphocytes within the orbit in-duces CXCL10 secretion by orbital fibroblasts and preadi-pocytes. In turn, this chemokine favors the migration of Th1lymphocytes into the orbit, thereby perpetuating the auto-immune cascade.

In conclusion, the serum levels of CXCL10 are increased inpatients with GO, showing a significant association with theactivity of the eye disease. Retrobulbar cells participate in theself-perpetuation of the inflammatory process by releasingchemokines under the influence of proinflammatory cyto-kines. The precise role of the increased or rising CXCL10levels in sera of patients with GO remains to be established.It is worth noting that there are currently no reliable serummarkers of activity in GO; thus, it would be useful confirmingand extending the data on CXCL10 changes in sera of pa-tients with GO.


• The serum levels of CXCL10 are similar in patients withGraves’ disease regardless of the presence ofophthalmopathy.

• Patients with inactive ophthalmopathy show serum levelsof CXCL10 similar to Graves’ patients without ophthal-mopathy, which however are significantly higher thanthose found in healthy controls.

• Graves’ patients with active ophthalmopathy (as assessedby a higher clinical activity score) have significantly higherserum levels of CXCL10 than patients with inactive oph-thalmopathy. The increase in circulating concentrations ofCXCL10 is likely to reflect, at least in part, the presence oforbital inflammation.

• CXCL10 appears to play a role in the initial phase of GO,when the inflammatory process is sustained by a Th1-mediated immune response and then declines in long-standing GO.

• A lymphocytic infiltrate is present in active GO, but not ininactive GO, suggesting that the increased production ofCXCL10 in active GO might be sustained by orbital lym-phocytes. However, in vitro studies demonstrate that in theorbit CXCL10 can be also produced by nonlymphoid cells,such as fibroblasts and preadipocytes.

• The lack of reliable serum parameters reflecting the ac-tivity of GO highlights the importance of further studiesaimed at evaluating whether the measurement of circu-lating CXCL10 might serve this purpose.

5. CXCR3-binding chemokines in CAT. Increased circulatingconcentrations of CXCL10 were first reported in patientswith CAT in 2004 (128). The serum levels of CXCL10 wereretrospectively measured in 223 consecutive patients withCAT, 97 euthyroid controls, and 29 patients with nontoxicmultinodular goiter. The three groups were similar for gen-der distribution and age. Twenty-four percent of the CATpatients had subclinical hypothyroidism. The mean serumlevels of CXCL10 were significantly higher in CAT patients(157 � 139 pg/ml) than in controls (79 � 38 pg/ml) or inpatients with multinodular goiter (90 � 32 pg/ml), although

some overlap was evident in the low range of CXCL10 val-ues. In patients with CAT, a hypoechoic pattern of the thy-roid at US, an age older than 50 yr, and especially a conditionof hypothyroidism were all associated with higher serumlevels of CXCL10. The serum levels of this chemokine did notdiffer in relation to the presence of atrophic or goitrousthyroiditis, TPO Ab or Tg Ab positivity, and the presence orabsence of an increased thyroid blood flow. In a multiplelinear regression model including age, thyroid volume, hy-poechogenicity of the gland at US, increased thyroid bloodflow, serum TSH, free T4, and TPO Ab, only age and TSHwere significantly related to the concentrations of CXCL10 inserum.

The association of high levels of circulating CXCL10 witha hypoechoic pattern of the thyroid at US can be explainedby the presence of a marked lymphocytic infiltration, thehistological hallmark of CAT. This interpretation fits withprevious observations in hepatitis showing that CXCL10plays a role in the accumulation of a massive T cell infiltratein the liver (164). Although a hypoechoic pattern of the thy-roid at US is also strongly associated with thyroid dysfunc-tion (165, 166), the serum concentrations of CXCL10 werefound to be higher in hypothyroid than euthyroid patients,independent of gland hypoechogenicity. Thus, it is possiblethat CXCL10 indicates a stronger inflammatory responseresulting in more extensive tissue destruction.

The observation that among CAT patients, those with hy-pothyroidism, compared with the euthyroid ones, showedalmost 2-fold higher serum CXCL10 levels and that the cir-culating concentrations of CXCL10 were significantly corre-lated with those of TSH gives further support to the hypoth-esis that elevated serum levels of CXCL10 are not onlyassociated with the autoimmune process itself, but also maybe a marker of a more aggressive thyroiditis, eventuallyleading to the destruction of thyroid cells with the attendantfunctional impairment.

Recent observations indicate that, in murine models ofexperimental autoimmune thyroiditis, specific combinationsof cytokines convert the inflammatory process from a non-destructive to a destructive one (167, 168). In this regard, ithas been suggested that one of the main differences betweenexperimental autoimmune thyroiditis in mice and CAT inhumans is the fact that human thyroids display a chronicinflammatory environment, mainly enriched in Th1 cyto-kines such as IFN-� and TNF-� (167, 169, 170). This cytokineenvironment results in an enhanced apoptosis of thyroidcells and severe hypothyroidism (167, 170). In the above-described scenario (167, 168), the CXCL10-induced recruit-ment of Th1 lymphocytes, which secrete IFN-� and in turnstimulate chemokine production by follicular cells, would bea critical event for maintaining and expanding the autoim-mune process (126, 171) (Fig. 6). The fact that endocrineepithelial cells interact with the immune system at severallevels and that these interactions contribute to the develop-ment and perpetuation of the autoimmune process consti-tutes a further aspect of the previously described active roleplayed by these cells in autoimmune diseases (172).

a. Serum levels of Th1 and Th2 chemokines in CAT. The issueof understanding the specific role of CXCL10 in CAT was


complicated by the observation that patients with this dis-ease may display increased serum levels of CCL2 (173). CCL2is a prototype CC chemokine, playing an important role ininnate immunity (174–177). CCL2 is also a crucial factor forthe development of adaptive Th2 responses by directing thedifferentiation of Th0 cells to Th2 in vitro (178). The issue wasfurther complicated by studies demonstrating that the ex-pression of CXCL10 and CCL2 in follicular thyroid cells isstimulated by distinct proinflammatory cytokines: IFN-� andTNF-�, respectively (122, 123).

To understand the meaning of the elevated serum con-centrations of CXCL10 and CCL2, these chemokines weresimultaneously measured in 70 consecutive patients withnewly diagnosed CAT (129). The mean serum levels ofCXCL10 were significantly higher in patients with CAT thanin healthy subjects or in patients with nontoxic multinodulargoiter, used as controls. On the contrary, when the wholegroup of CAT patients was considered, the serum concen-trations of CCL2 were found to be similar to those found inboth control groups. Among patients with CAT, the serumconcentrations of CXCL10 and CCL2 were significantlyhigher in hypothyroid subjects than in the euthyroid ones.The serum levels of CXCL10 were significantly increased inhypothyroid patients, irrespective of their age. On the otherhand, the serum levels of CCL2 were significantly increasedin patients older than 50 yr and only to a lesser degree inthose with hypothyroidism (129). No correlation was foundbetween the circulating concentrations of CXCL10 and CCL2.Multivariate analysis showed that the serum levels ofCXCL10 were associated with the severity of hypothyroid-ism, as assessed by the concentrations of TSH in serum,independently from other confounders. The same analysisrevealed that the circulating concentrations of CCL2 weresignificantly associated only with the patients’ age. Thesefindings are in agreement with data on the intrathyroidal

expression of the mRNA for CCL2, indicating that the con-tent of this mRNA is not different in glands with CAT com-pared with multinodular goiters (126, 127). Taken together,these results would not support a pathogenetic role for CCL2in CAT.

On the basis of such results, the study reporting increasedserum levels of CCL2 in patients with CAT compared withhealthy controls or patients with nontoxic nodular goiter(173) should be reinterpreted. The discrepant results ob-tained in the two studies (129, 173) can be explained, at leastin part, by taking into account that in the investigation byKokkotou et al. (173) CAT patients were significantly olderthan controls. The age factor, as a confounding variable,should be always taken into account when considering theserum levels of chemokines. Indeed, several studies indicatethat the serum levels of CCL2 do increase with age, even innormal subjects (59, 179). Moreover, in the study by Antonelliet al. (129) the serum levels of CXCL10 were higher in patientswith hypothyroidism and with a hypoechogenic pattern ofthe thyroid at US, whereas the serum levels of CCL2 were notcorrelated with thyroid gland echogenicity, again supportinga minor role played by CCL2.

The preponderant role of CXCL10 in the pathogenesis ofsevere CAT resulting in hypothyroidism is in line with cur-rent notions of autoimmunity and with experimental data.CXCL10 is a Th1-oriented chemokine, whereas CCL2 is reg-ulated by IL-4, the cardinal Th2 cytokine, and influences Tcells toward a Th2 commitment. In murine models of auto-immune thyroiditis, experiments performed in vitro demon-strated a differential role for CCL5 (a chemokine known tofavor the attraction of Th1 cells) and for CCL2 (preferentiallyactive on Th2 cells) during the onset, the course, and theremission of the disease (180). Briefly, these experimentssuggest that CCL2 attracts specific immune regulatory cellsthat down-regulate the autoimmune reaction, as shown by adecrease in the proliferative response to Tg and by a milderdegree of lymphocytic infiltration in the thyroid (180). Inhuman CAT, an environment strongly enriched in Th1, dueto CXCL10 stimulation associated with an inadequate Th2response, resulting from a poor effect of CCL2, would leadto a severe nonremitting disease producing hypothyroidism.In this regard, data demonstrating an association betweenincreased serum levels of CXCL10 and a more severe courseand aggressiveness of Th1-mediated autoimmune reactions(e.g., acute kidney graft rejections) should be taken into ac-count (78).

b. Serum levels of CXCL10 and hypothyroidism per se. Thedifferential impact of thyroid autoimmunity and hypothy-roidism on circulating concentrations of CXCL10 was inves-tigated by measuring the serum levels of CXCL10 in hypo-thyroid patients of both autoimmune and nonautoimmuneetiology (130). Patients with CAT (50% being euthyroid and50% hypothyroid) underwent a serum CXCL10 assay at en-try. Hypothyroid patients were then given l-T4 substitutiontherapy to normalize their thyroid function. The serum levelsof CXCL10 were reevaluated 3 months later in untreatedeuthyroid subjects with CAT and in originally hypothyroidpatients rendered euthyroid by l-T4. Also included in thestudy were patients with papillary thyroid cancer who had

FIG. 6. Graphic representation of the mean serum levels of CXCL10as measured in patients at different stages of Graves’ disease. Sig-nificant changes in the mean serum levels of CXCL10 are observed inrelation to the clinical phase of Graves’ disease as reported in across-sectional clinical trial (132). Notably, the highest serum levelsof CXCL10 characterize hyperthyroid Graves’ patients at diseaseonset and at relapse of hyperthyroidism after antithyroid drug treat-ment. In general, patients who are in the active phases of the diseaseshow significantly higher serum levels of CXCL10 compared withhealthy subjects. *, Statistical significance with regard to serum lev-els of CXCL10 levels found in healthy subjects (dotted line).


been treated with total thyroidectomy and 131I ablation ofthyroid residues. In 50% of them, the serum levels of CXCL10were evaluated while hypothyroid, after l-T4 withdrawal fora diagnostic whole body scan. In the remaining thyroid can-cer patients, the serum levels of CXCL10 were evaluated onl-T4 therapy after the injection of recombinant human TSHfor diagnostic procedures. The mean levels of circulatingCXCL10 were significantly higher in CAT patients with hy-pothyroidism than in those who were euthyroid. No signif-icant change in the serum concentrations of CXCL10 wasfound in hypothyroid patients rendered euthyroid by a3-month course of l-T4 or in euthyroid patients with CATevaluated after a 3-month follow-up period. In cancer pa-tients on l-T4, the serum levels of CXCL10 were not signif-icantly different from healthy controls. Hypothyroidism re-sulting from l-T4 withdrawal did not induce any significantchange in circulating CXCL10. Also, no significant change inthe serum levels of CXCL10 was found in cancer patientsgiven recombinant human TSH for diagnostic purposes, sug-gesting that the rise of CXCL10 does not result from a TSHstimulation (130).

Taken together, the above data demonstrate that hypo-thyroidism per se does not significantly influence circulatingCXCL10 and suggest that the increased serum levels ofCXCL10 in CAT are related to the autoimmune process itself(130).


• The serum levels of CXCL10 are increased in patients withCAT, compared with healthy subjects or patients withnontoxic multinodular goiter.

• Among patients with CAT, those with impaired thyroidfunction (overt and subclinical hypothyroidism) show sig-nificantly higher serum levels of CXCL10 compared withthe euthyroid ones.

• Multivariate analysis models demonstrated that the serumlevels of CXCL10 in patients with CAT are associated withthe severity of hypothyroidism, as assessed by the con-centrations of TSH in serum, independently from otherconfounders. This finding suggests that the elevated se-rum levels of CXCL10 in CAT may be a marker of a moreaggressive clinical course.

• In CAT, the serum levels of CXCL10 are significantly re-lated to thyroid hypoechogenicity at US, which may betaken as an index of severe lymphocytic infiltration.

• In patients with CAT, the serum levels of CXCL10 are notcorrelated with the titers of Tg Ab or TPO Ab.

• The increased serum levels of CXCL10 in CAT are not theresult of hypothyroidism per se. No significant changes incirculating concentrations of CXCL10 are observed aftercorrection of hypothyroidism with l-T4.

C. CXCR3-binding chemokines in type 1 diabetes mellitus

1. Results of basic studies. Insulin-dependent (type 1) diabetesmellitus (IDDM) is a T cell-driven autoimmune disease ofunknown etiology that results in the destruction of the isletsof Langerhans in genetically predisposed individuals. Themechanisms by which antigen-specific T cells migrate to theislets, a prerequisite for the specific lysis of �-cells, is largely

unknown. Because the etiological agent of IDDM is still un-known, delineating the process of T cell infiltration wouldprovide a basis for understanding the development of thisautoimmune disease. A recent study demonstrated that dur-ing insulitis, the �-cell itself synthesizes and secretes CXCL9and CXCL10, which are the driving force for the recruitmentof CXCR3� cytotoxic T cells (181). In other words, �-cells, thetarget of autoimmunity in IDDM, are largely responsible forattracting islet-specific cytotoxic T cells, thus facilitating theirown demise. A high IFN-� concentration in the islets ofLangerhans correlated both with the expression of CXCL9,CXCL10, and CXCR3 and with a prominent infiltration by Tcells (181). IFN-� was found to be necessary and sufficient tostimulate CXCL9 and CXCL10 expression by �-cells. Takentogether, these data suggest that the process of islet infiltra-tion by lymphocytes reflects an inflammatory loop caused byT cell-derived IFN-�. IFN-� induces the production of CXCL9and CXCL10 by �-cells, leading to enhanced immigration ofadditional IFN-�-secreting T cells. This process is mediatedthrough the chemokine receptor CXCR3 (171, 181).

2. Virus-induced, immune-mediated autoimmune diabetes. Therole of CXCR3 in the pathogenesis of autoimmune diabeteswas demonstrated in a model of virus-induced diabetes byusing a transgenic mice model that expresses the glycopro-tein (GP) of the lymphocytic choriomeningitis virus (LCMV)in the �-cells of the islets of Langerhans [rat insulin promotor(RIP)-GP mice] (182, 183). It is known that RIP-GP mice, afterLCMV infection, rapidly develop diabetes, as assessed by ablood glucose concentration of greater than 300 mg/dl, amassive T cell infiltration of the islets, and the destruction of�-cells, resulting in low to absent pancreatic insulin (183,184). Concurrent with LCMV-mediated immune diabetes isa profound Th1/Tc1-type response characterized by the re-lease of the proinflammatory cytokines IFN-� and TNF-� inthe target organ (185, 186). This event is followed by thedestruction of the �-cells, which is due to CD8� T lympho-cytes specific for LCMV-GP (183, 184).

A recent study demonstrated that LCMV-infected singlytransgenic mice, at d 8 after infection, show severe T cellinfiltration and �-cell destruction of the pancreatic islets. Incontrast, the islets of transgenic CXCR3-deficient mice werenot invaded, although T cells adorned their rim. Monitoringblood glucose levels revealed a significant delay in the oc-currence of diabetes in transgenic mice deficient for CXCR3(181). These observations demonstrated the importance ofCXCR3 in the pathogenesis of IDDM, compared with otherinflammatory chemokine receptors (CCR5 and CCR2) thatwere investigated in the same study (181). Among the threeCXCR3-binding chemokines, the predominant role in deter-mining insulitis was subsequently ascribed to CXCL10 byanother group of investigators (187). Briefly, in RIP-GP mice,the expression of CXCR3-binding chemokines was demon-strated 18–24 h after LCMV injection (187). The analysis ofchemokine expression by RNase protection assay revealedthat the induction was maximal for CXCL10 (�400-fold vs.uninfected mice). The expression of the mRNAs for CXCL9and 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 clar-ifying the pathogenetic role of CXCR3-binding chemokinesin the development of immune IDDM. After the injection ofneutralizing mAbs to either CXCL10 or CXCL9 in RIP-GPmice infected with LCMV, the incidence of IDDM was sig-nificantly reduced to 31% in mice receiving the anti-CXCL10mAb compared with control animals, which were injectedwith a nonspecific isotype-matched hamster mAb (diabetesincidence � 100%) (187). In contrast with CXCL10 neutral-ization, no significant reduction in the incidence of diabetesor in its onset could be detected in animals treated with theanti-CXCL9 mAb, suggesting distinct roles for CXCL10 andCXCL9 in virus-induced immune-mediated diabetes. More-over, when the anti-CXCL10 and the anti-CXCL9 mAbs werecoinjected, the incidence of diabetes was equivalent to thatobserved after administration of the anti-CXCL10 mAb alone(187). The above-described data indicate that CXCL10, andnot CXCL9, is an essential factor in the initiation of theprocess that results in virus-induced immune-mediated di-abetes. Despite the multiplicity of chemokines and cytokinesreleased during inflammation and the likely redundancy ofthe system, these experiments demonstrate a unique andnonredundant role for CXCL10, whose neutralization is ableto prevent the occurrence of diabetes (187, 188).

The mechanisms by which CXCL10 neutralization abro-gates the development of immune IDDM were identified ina decreased lymphocyte infiltration into the islets of Lang-erhans with consequent preservation of insulin production.This phenomenon resulted from the inhibition of the clonalexpansion of Ag-specific CD8� T lymphocytes and/or oftheir migration into the pancreas.

When viruses that are tropic for the pancreas and causediabetes were compared with viruses that are not tropic forthe pancreas and do not cause diabetes, the conclusion wasdrawn that the increased expression of the CXCL10 mRNAin the islets was not a general phenomenon, occurring afterany viral infection (187). Indeed, the infection of mice withviruses that are tropic for the pancreas and are known tocause diabetes, such as Coxsackie virus B4 or the encepha-lomyocarditis virus variant B, resulted in an increased pan-creatic expression of CXCL10. In contrast, the infection withTheiler’s murine encephalomyelitis virus, a virus with nopancreatic tropism and not causing diabetes, did not resultin the expression of significant levels of CXCL10 in the pan-creas (187).

The above-described findings suggest that a viral tropismfor the pancreas and the alteration of the pancreatic milieuby induction of selected chemokines, such as CXCL10, maybe the initial step that imprints a pattern for the subsequentdevelopment of organ-specific autoimmune diseases causedby viruses. The dominant role played by CXCR3-bindingchemokines after a viral infection is not unexpected becauseCXCR3 is expressed predominantly on activated T cells (31,189), which function to eliminate virus-infected cells and tocontrol viral infections.

CXCL10 may also have a dual effect in IDDM, being in-volved both in the initiation and maintenance of the auto-immune process, as well as in the abrogation of autoimmu-nity. The abrogative rather than enhancing effect of CXCL10is mainly due to its expression outside the pancreas (190). The

administration to LCMV-infected mice of an additional virusinfection, at the time when the autodestructive process of the�-cells was already ongoing, caused the recruitment of Tlymphocytes away from the islets (190). Thus, the followingscenario for disease abrogation can be postulated: autoag-gressive lymphocytes follow the highest bidder, which in thiscase was the pancreatic-draining lymph node, and leave theiroriginal target site. Once arrived at the location with thehighest CXCL10 concentrations, the already activated lym-phocytes encounter an additional activating inflammatorymilieu that pushes them over the edge toward activation-induced cell death. This finding does not hamper, but ratherreinforces, the role of CXCL10 in the pathogenesis of IDMM,although it suggests that the CXCL10-mediated effects maybe a question of time and location of its expression (190).

3. Serum levels of CXCR3-binding chemokines in human type 1diabetes mellitus. Type 1 diabetes mellitus (T1DM) was thefirst endocrine autoimmune condition in which the serumlevels of CXCL10 were investigated (191) and were found tobe increased compared with healthy subjects. These resultswere confirmed by another clinical study (192), but not bytwo subsequent reports (171, 193). Currently, the issue re-mains somehow controversial, as we will see later in thissection.

Shimada et al. (191) measured the serum levels of CXCL10and the titers of anti-glutamic acid decarboxylase (GAD) andIA-2 Ab in 74 patients with T1DM. Patients were divided intwo groups according to negative (Ab� type 1 group) orpositive tests for either or both auto-Ab (Ab� type 1 group).The latter group also included Ab� diabetic patients withresidual �-cell function, the so-called latent autoimmune di-abetes in adults (LADA) (194), or slowly progressive insulin-dependent diabetes (195). Healthy subjects and patients withAb� type 2 diabetes served as controls. The serum levels ofCXCL10 were significantly higher in both Ab� and Ab� type1 groups, compared with the levels found in healthy controls.However, among T1DM patients, only Ab� patients, but notthe Ab� ones, had significantly higher serum levels ofCXCL10 than the type 2 diabetic patients. No significantdifference in CXCL10 levels was observed between the clas-sical variant of T1DM and the LADA groups.

In the same study, a significant positive correlation wasreported between the serum concentrations of CXCL10 andthose of IFN-� (191). A significant positive correlation wasalso found between the serum levels of CXCL10 and thenumber of GAD-reactive IFN-�-producing CD4� cells (191).These findings are in line with the fact that CXCL10 is achemoattractant for Th1 lymphocytes (196) and that IFN-� isa Th1 type cytokine involved in the destruction of pancreatic�-cells in vitro (197). The correlation between serum CXCL10and IFN-� levels was restricted to Ab� type 1 patients andwas still evident when data from GAD Ab-positive and fromIA-2 Ab-positive patients were analyzed separately. No sucha relationship was observed in the Ab� type 1 group, in theAb� type 2 diabetic patients, and in healthy subjects (191).

In the study by Shimada et al. (191), the serum levels ofCXCL10 were significantly higher in recent-onset Ab� type1 patients (disease duration � 3 yr) than in patients withlong-standing disease (disease duration � 3 yr). A significant


negative correlation was found between serum CXCL10 lev-els and disease duration. Because more severe insulitis isgenerally expected in younger subjects, the authors also an-alyzed the relationship between serum levels of CXCL10 andage, demonstrating a significant negative correlation be-tween these two variables. All these relationships were spe-cific for autoimmune diabetes (Ab� type 1).

The issue of serum chemokine status in prediabetic pa-tients was addressed by another clinical study evaluating theserum levels of IFN-� and CXCL10 in patients with eithernewly diagnosed or long-standing T1DM and in theirhealthy first-degree relatives (192). The latter group was di-vided into those at “low” and “high” risk for the develop-ment of diabetes, depending on whether subjects were neg-ative or positive for islet cell and GAD Ab. The serum levelsof CXCL10 were significantly higher in patients with newlydiagnosed IDDM and in subjects with a high risk for devel-oping the disease. In the latter group, the serum concentra-tions of CXCL10 correlated with the levels of IFN-� (191, 192,198). The results of this study demonstrated that the serumlevels of CXCL10 are increased in patients with T1DM, butonly 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 ob-servations (191, 192). Indeed, in two different series of dia-betic patients (171, 193) the serum levels of CXCL10 werefound to be similar in patients with T1DM and in healthycontrols. The few patients showing increased serum levels ofCXCL10 were all positive for circulating islet cell Ab (171).

This observation was further extended by evaluating alarge series of diabetic patients (193) identified by a regionalT1DM registry in central Italy (199). All serum samples werecollected within 6 wk after starting insulin therapy. No sig-nificant difference in the serum levels of CXCL10 was foundbetween T1DM patients at the clinical onset of their diseaseand healthy subjects. The serum concentrations of CXCL10were not related to the patients’ age at clinical diagnosis ofT1DM. However, when patients were subdivided in relationto gender, the serum concentrations of CXCL10 were signif-icantly higher in women with T1DM than in healthy controlwomen or males with T1DM. The latter group of patients hadserum levels of CXCL10 comparable to those of healthy malesubjects (193).

Given the strong evidence provided by basic studies for aphysiopathological role for CXCL10 in the pathogenesis ofT1DM, the observation that healthy subjects and T1DM pa-tients displayed similar levels of serum CXCL10 was unex-pected. To explain this finding, it should be considered thatthe expression of CXCL10 in the islets of Langerhans duringautoimmune diabetes might not be registered when CXCL10is measured in the serum of patients with T1DM. In line withthis hypothesis is the demonstration that in nonobese dia-betic mice, the serum levels of CXCL10 did not correlate withits mRNA expression in the pancreas, being rather associatedwith the expression levels of the mRNAs for CXCL10 andCXCR3 in pancreatic lymph nodes (200). These findingsmight be explained by the minimal blood flow of the isletsof 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 tissueexpression.

The problem remains that in different studies the serumlevels of CXCL10 were found to be either increased ornormal even in patients with recent-onset T1DM (171, 192,193). The reason for these discrepant results is still a matterof debate. Besides differences in the assay methods usedfor CXCL10 measurements, which are unlikely to be thecause of the different results, some considerations may behelpful in identifying possible confounders. The discrep-ancy may result, at least in part, from the different female/male ratio of the patients investigated in the above-de-scribed studies (192, 193). It is well known that the markersof thyroid autoimmunity are 2- to 3-fold more prevalentin women with autoimmune diabetes than in men (201,202), and that the serum concentrations of CXCL10 aresignificantly increased in several autoimmune conditions,irrespective of age and gender (124). Furthermore, at thetime when these studies were performed, the role of agingin determining increased serum levels of CXCL10 had notbeen demonstrated yet (54, 55), and strict age matchingbetween patients and controls may not have been per-formed systematically.

In conclusion, there is currently no definite consensus onthe fact that the serum levels of CXCL10 are increased inpatients with T1DM. Despite straightforward evidence ob-tained in basic studies and experimental animal models ofIDDM, the results provided by clinical studies remain con-troversial and in some cases difficult to interpret. In thisscenario, future clinical studies aimed at clarifying this issue,should enroll diabetic patients proven to be negative forother autoimmune diseases, known to be associated withincreased serum levels of CXCL10. A strict age and sexmatching between patients and controls will also be neces-sary to reach firm conclusions.


• In murine models of insulitis, the �-cell itself synthesizesand secretes CXCL9 and CXCL10, which drive the accu-mulation of CXCR3� cytotoxic T lymphocytes.

• High IFN-� concentrations in the islets of Langerhanscorrelate with the expression of CXCL9, CXCL10, andCXCR3, and a prominent infiltration by T lymphocytes.

• The role of CXCR3 in the pathogenesis of virus-inducedimmune diabetes was clearly demonstrated in transgenicmice.

• Among the three IFN-� inducible chemokines, CXCL10has a predominant role in the development of virus-in-duced immune diabetes in mice.

• CXCL10 neutralization abrogates the development of vi-rus-induced immune diabetes in mice by decreasing thelymphocyte infiltration into the islets of Langerhans, withconsequent preservation of insulin production.

• The measurement of CXCL10 in sera of patients withT1DM provided conflicting results. Some studies reportedelevated circulating concentrations of CXCL10 in thesepatients compared with healthy subjects, whereas otherstudies reported similar serum levels of CXCL10.


D. CXCR3-binding chemokines in primary adrenaldeficiency (Addison disease)

Addison disease (AD) can occur as an isolated conditionor in association with other endocrine and nonendocrineautoimmune diseases, leading to the clinical picture of au-toimmune polyglandular syndrome (APS)-1 and APS-2(203–205). Clinical similarities, common human leukocyteantigen association, and high prevalence of thyroid, gastric,and islet cell Ab suggested that the isolated form of AD maybe a clinical variant of APS-2 (205). Although high rates ofpositivity for adrenal cortex autoantibodies (ACA) and 21-hydroxylase autoantibodies have been reported in patientswith AD, these antibodies probably play a minor role intissue damage and more likely reflect the ongoing autoim-mune response (205–207). Consistent with this view, thehistological picture of an affected gland shows diffuse cor-tical atrophy and lymphocytic infiltration, similar to the his-tological changes commonly observed in other endocrineautoimmune conditions, such as autoimmune thyroiditis(205). Experimental and clinical evidence supports the con-cept that a cell-mediated immune response may representthe pathogenetic mechanism leading to adrenal destruction(208–210). Although the autoimmune etiology currently ac-counts for most of the cases of AD diagnosed in developedcountries (203, 204), there is only one published study eval-uating chemokines in adrenal autoimmunity (211); thus, con-clusions should be drawn with caution.

In the only report concerning the role of serum CXCL10 inAD, 93 patients with overt or subclinical AD were investi-gated (211). Among them, 64 patients had clinically evidentautoimmune primary adrenal insufficiency: 25 with isolatedAD, and 39 with APS. Twenty patients had subclinical au-toimmune AD (SAD), being identified by screening patientswith extraadrenal autoimmune diseases for the presence ofadrenal autoantibodies (212–214). All 20 SAD patients werepositive for both 21-hydroxylase Ab and ACA Ab. Ninepatients with nonautoimmune adrenal failure served as con-trols (215). A further control group included 48 age- andsex-matched healthy subjects, proven to be negative for se-rum thyroid and adrenal Ab.

Compared with healthy subjects and patients with non-autoimmune adrenal failure, the serum levels of CXCL10were significantly increased in both clinically evident andsubclinical AD. No significant difference was found betweenpatients with nonautoimmune AD and controls. The serumlevels of CXCL10 were highly variable among both AD andSAD patients and did not show any significant relationshipwith gender, age, disease duration, or serum titers of 21-hydroxylase Ab and ACA Ab. Patients with isolated AD orAPS showed similar median serum levels of CXCL10. Similarserum levels of CXCL10 were found in SAD patients, irre-spective of normal or impaired cortisol response to ACTH.No gender-dependent difference in the serum levels ofCXCL10 was found in AD. The absence of a gender-relatedeffect in autoimmune AD, either isolated or occurring withinAPS-2, would suggest that autoimmune adrenalitis by itselfis responsible for the high circulating levels of CXCL10 (193).

Contrary to observations in Graves’ disease or type 1 di-abetes (124, 191, 192), the serum levels of CXCL10 were not

found to be correlated with the time elapsed since the di-agnosis of adrenal deficiency. This lack of correlation prob-ably depends upon the peculiar course of AD. Due to thelarge functional reserve of the adrenal glands, clinical symp-toms of adrenal deficiency become manifest only when mas-sive adrenal destruction has occurred (203). It is thereforereasonable to assume that at variance with Graves’ disease,the time since diagnosis does not precisely reflect the dura-tion of the autoimmune process (124, 128, 191).


• The serum levels of CXCL10 are increased in patients withboth overt and SAD, compared with healthy subjects orpatients with nonautoimmune adrenal failure.

• The serum levels of CXCL10 are similar among patientswith autoimmune AD, occurring either isolated or as acomponent of APS.

• The serum levels of CXCL10 are similar in patients withovert or SAD. In the latter patients, similar serum levels ofCXCL10 were found irrespective of normal or impairedcortisol response to ACTH.

VI. Pharmacological Modulation of ChemokineSecretion and Biological Action

Pharmacological strategies aimed at attenuating inflam-mation without inducing generalized immunosuppressionhave focused their attention on chemokines. However, thetask of developing drugs that block chemokine activity washampered by the pleiotropic biological functions displayedby these molecules. For example, CXCL9, CXCL10, andCXCL11 have the ability of recruiting different leukocytesubsets as well as antitumor effects, which are mediated bytheir receptor CXCR3 (see Section III). Because of their pleio-tropic biological effects, these chemokines have been pro-posed as possible therapeutic targets in cancer, in allograftrejection, in glomerulonephritis, in diabetes mellitus, in MS,and in autoimmune disorders of the thyroid.

Since the discovery of the chemokine family, the strategyof selectively blocking the leukocyte recruitment to the siteof inflammation has been validated by several approaches.Despite the redundancy reported by in vitro studies on li-gand-receptor binding and activation, in vivo the system actsthrough a coordinated and perhaps sequential chain ofevents, with temporal and spatial control mechanisms com-ing into play. Thus, interfering with an essential link in thechain, such as CXCR3 in the case of some autoimmune dis-orders, or organ allograft transplant (65, 216), or diabetes(181) might result in a complete inhibition of the inflamma-tory process, as demonstrated in CXCR3�/� models (20). Inthis view, a growing number of antagonists of several che-mokine receptors including CXCR3 are being developed (20).Furthermore, the recent discovery of distinct receptors gen-erated by alternative splicing of the CXCR3 gene wouldallow obtaining selective inhibitors of the two receptors, thuslimiting undesired side effects. Otherwise, a combined acti-vation of the two receptors might be useful in some clinicalconditions, such as cancer, where both enhancement of theimmune response and inhibitory effects on angiogenesis andtumor cell growth are useful. Studies were aimed at evalu-


ating the possibility of mAbs neutralizing chemokines ortargeting their receptor and at investigating the possibility tomodulate the proinflammatory cytokine-induced chemokinesecretion. The first category of studies mainly used animalmodels, either knockout or wild type, whereas the latterexperiences were mainly obtained by using cell cultures inwhich different agents were tested for their capacity to re-duce chemokine secretion. In this review, we will mainlyfocus on in vitro studies performed in endocrine cells.

A. PPAR� agonists in vitro inhibit CXCL10 productioninduced by proinflammatory cytokines

The effect of peroxisomal proliferator-activated receptor-�(PPAR�) agonists on the secretion of CXCL10 induced byproinflammatory cytokines was investigated in primary cul-tures of human thyroid follicular cells, fibroblasts, and prea-dipocytes (154). Cells were stimulated with IFN-� and TNF-�alone or in combination in the absence or presence of in-creasing concentrations of the PPAR� agonist rosiglitazone(RGZ). In all cell cultures, CXCL10 was undetectable basally.IFN-� dose-dependently induced CXCL10 release, and thecombination of TNF-� and IFN-� had a significant syner-gistic effect on CXCL10 secretion. TNF-� alone had no effect.Treatment with RGZ, added concomitantly with IFN-� andTNF-�, dose-dependently inhibited the cytokine-induced se-cretion of CXCL10. RGZ alone had no effect. Similar resultswere obtained for different cell cultures. Furthermore, thy-rocytes from normal thyroids and from Graves’ glands aswell as fibroblasts or preadipocytes, irrespective of their der-mal or retrobulbar origin, harbored the same results, sug-gesting that this kind of activation is a general physiologicalphenomenon.

PPAR� has recently been shown to be involved in themodulation of inflammatory responses. Treatment of endo-thelial cells with PPAR� activators inhibits the IFN-�-in-duced expression of CXCL10, CXCL9, and CXCL11 at themRNA and protein levels (163). The release of chemotacticactivity for CXCR3-transfected lymphocytes was alsoblocked (163). Thus, PPAR� activity may be involved in theregulation of IFN-�-induced chemokine expression in hu-man autoimmunity by attenuating the recruitment of acti-vated T cells at sites of Th1-mediated inflammation (163, 217,218). In this regard, there is evidence that PPAR� receptorsare present in the thyroid (219) and in orbital tissues fromGraves’ patients (220). These findings suggest that PPAR�agonists might play a role in the treatment of endocrineautoimmune diseases. In line with this hypothesis is thedemonstration that PPAR� agonists (gemfibrozil and feno-fibrate) inhibit the clinical signs of experimental autoimmuneencephalomyelitis in mice (221). PPAR� agonists were alsoshown to modulate inflammatory responses in endothelialcells (163, 217, 218), by reducing CXCL10 levels in two mu-rine models of colitis (218, 222), and in dendritic cells (217).

At the present state of the art, two mechanisms might beinvolved in the inhibition of IFN-�-induced CXCL10 secre-tion by PPAR� activators: 1) a decrease of CXCL10 promoteractivity, thus inhibiting the protein binding to the two nu-clear factor-�B sites (163); or 2) a reduction of CXCL10 proteinlevels 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, orbitalfibroblasts, and preadipocytes with a pure PPAR� activator,such as RGZ, at near-therapeutic doses significantly inhibitsIFN-�-stimulated CXCL10 secretion suggests that PPAR�activators might attenuate the recruitment of activated T cellsat sites of Th1-mediated inflammation (154).

B. Corticosteroids in vitro inhibit CXCL10 productioninduced by proinflammatory cytokines

The effect of corticosteroids on cytokine-induced CXCL10secretions was studied in primary cell cultures of humanzona fasciculata cells (hZFC) from normal adrenal glands(211). CXCL10 was undetectable basally, whereas its secre-tion was significantly induced in hZFC by stimulation withIFN-� or IFN-� plus TNF-� (Fig. 7). Stimulation of hZFC withTNF-� alone was not able to induce chemokine secretion.Nevertheless, TNF-� had a significant synergic effect withIFN-� in determining CXCL10 production. Increasing con-centrations of hydrocortisone progressively and significantly

FIG. 7. hZFC in vitro produce CXCL10 after stimulation with IFN-�.Primary cell cultures of hZFC were cultured for 24 h in the presenceof IFN-� in chamber slides, fixed, and incubated with a rabbit anti-human CXCL10 antibody. To assess simultaneously the epithelialorigin of the cells, a double-label immunofluorescence with a mono-clonal anticytokeratin Ab was performed. The slides, after a nuclearcounterstaining with Topro-3 (blue), were examined by conventionalconfocal microscopy and showed high immunoreactivity for bothCXCL10 (green) and cytokeratin (red). Double-label immunofluores-cence analyzed by laser confocal microscopy shows that the signal forthe protein of CXCL10 and cytokeratin colocalize, thus demonstratingsecretion of the chemokine by adrenal cells of zona fasciculata. (Orig-inal magnification, �40). [Reprinted from: M. Rotondi et al.: J ClinEndocrinol Metab 90:2357–2363, 2005 (211), with permission fromThe Endocrine Society, Copyright 2005.]


inhibited IFN-�-induced and IFN-�- plus TNF-�-inducedCXCL10 secretion.

This study provided evidence that hZFC, when stimulatedwith proinflammatory cytokines, are able to produce che-mokines, as assessed by cell supernatant assay and laserconfocal immunofluorescence (211). These results suggest arole for the inflamed glandular epithelium in the recruitmentof specific subsets of infiltrating lymphocytes within theadrenal gland (Fig. 7).

Increasing concentrations of hydrocortisone signifi-cantly inhibited the secretion of CXCL10 induced by IFN-�or IFN-� plus TNF-� in hZFC. This effect of glucocorti-coids on chemokine production is in line with their anti-inflammatory and immunosuppressive actions (223). In-deed, glucocorticoids are able to suppress the productionof several cytokines and chemokines by inhibiting thenuclear factor-�B and by activating protein-1 transcriptionfactor families (224). Glucocorticoids inhibit the IFN-�-induced expression of major histocompatibility class IImolecules and may also block the expression of IFN-�-inducible genes, indicating that glucocorticoids can sup-press IFN-� activity (225, 226).


• RGZ, a PPAR� agonist, dose-dependently inhibitsCXCL10 secretion induced by IFN-� and TNF-� in humanprimary cultures of thyrocytes, orbital fibroblasts, andpreadipocytes.

• IFN-�- and TNF-�-induced CXCL10 secretion in humanadrenal cells is significantly inhibited by hydrocortisone.

VII. Serum Levels of CXCR3-Binding Chemokines:Potential Applications as Novel Serum Markers in

Endocrine Clinical Practice

Throughout previous sections, we reviewed the currentlyavailable data regarding possible clinical applications ofmeasuring CXCR3-binding chemokines in serum (128, 130–132, 154, 171, 191, 192, 211). The potential applications ofserum CXCL10 assay in the clinical practice stem from theobservation that high circulating concentrations of this che-mokine are likely to predict a more rapid and aggressivecourse of the autoimmune inflammatory processes (78).There are currently no reliable parameters to predict theevolution of these endocrine autoimmune disorders, whichmay remain stable for years or progress to overt hormoneinsufficiency. In this view, it is reasonable to believe that,among euthyroid patients with CAT, those displaying highercirculating levels of CXCL10 would be more likely to un-dergo a more rapid functional deterioration and/or a higherrate of progression to hypothyroidism (128). A similar sce-nario could be hypothesized for other endocrine autoim-mune conditions diagnosed at a subclinical stage, such asSAD or prediabetes.

Another interesting field of application for CXCL10 mea-surement could be represented by Graves’ disease. Previ-ously described studies provided evidence supporting theconcept that CXCL10 could be an important factor in medi-ating or in announcing the relapse of hyperthyroidism afterantithyroid drugs (132). Moreover, indirect evidence sug-

gests that the measurement of CXCL10 in serum, at the timewhen hyperthyroidism is diagnosed, may help to identifymore severe and aggressive variants of Graves’ disease, thussupporting a definitive treatment of hyperthyroidism withthyroidectomy or radioiodine (131).

The above-mentioned clinical conditions represent a fewexamples in which currently available data suggest a clinicalapplication of serum CXCL10 measurements in patients withautoimmune endocrine disorders.

Among the three CXCR3-binding chemokines, onlyCXCL10 has been extensively studied in human endocrinediseases. The few published studies in which CXCL9,CXCL10, and CXCL11 have been evaluated simultaneously(64) indicate that, despite an overall similar behavior, each ofthe three CXCR3-binding chemokines undergoes specificchanges in different clinical settings. Results have been pro-vided by both basic and clinical studies demonstrating thattargeting of one or another CXCR3-binding chemokines mayproduce different biological effects. For this reason, and alsoin view of the limited information available on CXCL9 andCXCL11 in endocrine diseases, the first step toward a bettercomprehension of the clinical significance of CXCR3-bindingchemokines would be to investigate whether the results ob-tained by measuring CXCL10 are confirmed, extended, ordenied when CXCL9 and CXCL11 are taken into account.The variations in serum chemokine levels between andwithin healthy subjects are another aspect deserving a morecomplete elucidation. The currently available data, whichsuggest a clinical application of measuring serum chemo-kines are mainly derived from cross-sectional studies. Pro-spective longitudinal studies are needed to reach firm con-clusions as well as to establish the sensitivity and specificityvalues for serum chemokines measurements.

Other aspects deserve clarification, in particular the prob-lem regarding serum levels in patients with T1DM, whichhave been reported as either elevated (191, 192) or normal(171, 193) in different studies. The issue must be resolved bystudying patients selected after an accurate screening forother autoimmune diseases, which might coexist in T1DMpatients.

There are also several unexplored fields in which a po-tential clinical application for the measurements of serumchemokines may be expected. Given the previously de-scribed changes of serum CXCR3-binding chemokines inpatients with HCV-related hepatitis undergoing therapywith IFN-� (60) and the involvement of CXCL10 in the patho-genesis of CAT (123, 128), it would be interesting to evaluatethe role of CXCL10 in driving the appearance of thyroidautoimmune disorders during IFN-� treatment (227). Addi-tional studies might address the issue of serum chemokinesand thyroid autoimmunity in patients treated with IFN-� forMS (228). Another clinical condition that might provide amodel for a better comprehension of the role of chemokinesin endocrine autoimmune diseases is postpartum thyroiditis(229). The evaluation of serum chemokines during IFNs-induced thyroid autoimmunity and in postpartum thyroid-itis would represent a unique model for studying the in-volvement of these molecules in the development of thyroidautoimmunity since its earliest phase, a situation rarely en-countered in the clinical practice.


VIII. Future Perspectives

Endocrine autoimmune diseases are the result of the in-teraction between genetic and environmental factors that arestill incompletely understood. Experimental data, primarilybased on animal models of human diseases, suggest thatautoimmune endocrine diseases result from dysregulatedimmune responses directed against normal constituents ofendocrine glands. The fact that these diseases often follow aprogressive course, resulting in complete cellular destruc-tion, reflects the perpetuation of the response by the contin-ued presence of the antigen, the impact of local inflamma-tion, epitope spreading, and genetically predeterminedsensitivity to the target tissue. The final result is the failureof physiological mechanisms controlling autoreactive T andB cells, which escape tolerance or ignorance (230). Peripheralevents needed for activation of the autoimmune responseinclude costimulatory signals and the action of cytokines andchemokines whose actions in the recruitment, trafficking,and in situ maintenance of specific subsets of activated lym-phocytes constitute crucial steps for the initiation and per-petuation of autoimmune inflammation.

The endocrine epithelial cells may interact with the im-mune system at several levels in the development and per-petuation of the autoimmune process, and many of theseinteractions appear to exacerbate the disease progression(231). In this review, particular emphasis has been given tothe active role played by endocrine epithelial cells, throughproduction of chemokines induced by IFN-� in the recruit-ment of specific subsets of lymphocytes to the diseasedgland. The understanding of these pathogenetic mechanismsof autoimmune endocrine diseases suggests novel ap-proaches to immunotherapy, directed to targeting lympho-cyte trafficking and activation and to inducing lymphocyteanergy.

During the past decade, we have witnessed the develop-ment of potent agents to treat inflammation and autoimmunediseases. However, attempts directed to induce sustainedreversal of disease by broad immune suppression are notlikely to be successful or cost effective, mainly in endocrineautoimmune diseases. Thus, interest has grown in finding away to interrupt the interactions between chemokines andtheir receptors as a new therapeutic strategy for inflamma-tion. Despite the difficulties encountered in antagonizingchemokine receptors, which are mainly due to the redun-dancy of the chemokine/chemokine receptors system, someencouraging results have been reached (20). Given the crucialimportance of the movement of leukocytes to inflammatorysites, it seems worth continuing research to address the issueof developing specific chemokine-receptor antagonists.These future fields of research, aimed at developing phar-macological agents for human autoimmune diseases, are alittle too far from the aim of this review. We would like toconclude by stressing the concept that although there arecurrently no new drugs interfering with the function of che-mokines in autoimmunity, these molecules should be re-garded as a novel marker that could be useful not only forresearchers but also for clinicians, and whose fields of po-tential application have not been fully elucidated.

IX. Conclusions

Chemokines are a family of small, structurally related,molecules that regulate cell trafficking of various subsets ofleukocytes. Other important functions of chemokines havebeen discovered, including the regulation immune re-sponses, wound-healing repair, control of angiogenesis, or-gan sclerosis, and tumor growth and spread. Studies basedon targeting of chemokines and their receptors have shownthat these molecules are important in different pathologicalconditions.

In the last few years, experimental evidence accumulatedsupporting the concept that IFN-� inducible chemokines(CXCL9, CXCL10, and CXCL11) play an important role in theinitial stage of autoimmune disorders involving endocrineglands (193). The fact that, after IFN-� stimulation, severalendocrine cells secrete CXCL10, which in turn recruits Th1lymphocytes expressing CXCR3 and secreting IFN-�,strongly supports the concept that chemokines may induceand sustain the autoimmune process in different endocrineglands. The availability of reliable, reproducible, sensible,and inexpensive methods for assessing serum CXCL10 pro-vided the opportunity to perform clinical studies in largeseries of patients affected by endocrine and nonendocrineautoimmune diseases. The circulating concentrations ofCXCL10 have been found to be increased in several endo-crine autoimmune diseases, including Graves’ disease, CAT,and AD. Other endocrine diseases could share the samefindings. In different clinical settings, including endocrineand nonendocrine diseases, the measurement of the serumlevels of CXCL10 may be useful to predict the course and theactivity of the disease and the therapeutic outcome, to per-form the most appropriate therapeutic choice, to assess thefavorable response to treatment, as well as to predict re-lapses. Of course, the efficacy and usefulness of routine se-rum CXCL10 measurement may differ in such a wide spec-trum of pathological conditions. We do not support the ideathat CXCL10 may serve in all patients, but we still think thatthe currently available results support the concept thatCXCL10 may be regarded as a novel serum marker in au-toimmune endocrine diseases.

Acknowledgments

Address all correspondence and requests for reprints to: Mario Ro-tondi, M.D., Ph.D., Unit of Internal Medicine and Endocrinology, IstitutoSuperiore per la Prevenzione e Sicurezza del Lavoro Laboratory forEndocrine Disruptors, Fondazione Salvatore Maugeri, Istituto di Ricov-ero e Cura a Carattere Scientifico, Via S. Maugeri 4, 27100 Pavia, Italy.E-mail: [email protected]

The experiments reported in this paper were supported in part byfunds from the Tuscany Region Study on Rosiglitazone (TRESOR) Re-search Project.

The authors have nothing to disclose.

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