Chemokines bind to sulfatides as revealed by surface plasmon resonance

Chemokines bind to sulfated cell surface glycosaminoglycans and thereby modulate signaling mediated by G-protein-coupled seven-transmembrane domain chemokine receptors. Similar to glycosaminoglycans, sulfated oligosaccharides are also exposed on the cell surface by sulfatides, a class of glycosphingolipids. We have now identified sulfated glycosphingolipids (sulfatides) as novel binding partners for chemokines. Using surface plasmon resonance (SPR), the binding of proinflammatory and homeostatic chemokines to glycosphingolipids, in particular sulfatides, was investigated. Chemokines were immobilized while glycosphingolipids or additional phospholipids incorporated into liposomes were applied as soluble analytes. A specific affinity of the chemokines MCP-1/CCL2, IL-8/CXCL8, SDF-1alpha/CXCL12, MIP-1alpha/CCL3 and MIP-1beta/CCL4 to the sulfatides SM4s, SM3, SM2a and SB2, SB1a was detected. No significant interactions with the chemokines were observed for gangliosides, neutral glycosphingolipids or phospholipids. Chemokine receptors have been associated with the detergent-insoluble fraction supposed to contain 'rafts', i.e., glycosphingolipid enriched microdomains of the cell surface. Accordingly, the data suggest that early chemokine receptor signaling may take place in the vicinity of sulfated glycosphingolipids on the cell surface, whereby these sulfatides could modulate the chemokine receptor-mediated cell activation signal.     

Synthetic chemokines directly labeled with a fluorescent dye as tools for studying chemokine and chemokine receptor interactions

Chemokines constitute a protein family that exhibit a variety of biological activities involved in normal and pathological physiological processes. CCL11 (eotaxin), CCL19 (MIP-3beta), CCL22 (MDC), CXCL11 (I-TAC) and CXCL12 (SDF-1alpha) chemokines, modified with the Alexa Fluor 647 fluorescent dye at specific positions along their sequence, were produced by a chemical route and their biological activities were characterized. In a migration assay, fluorescent chemokines were as biologically active as the unmodified forms. All labeled chemokines specifically stained cell lines transfected with the appropriate human chemokine receptors. The specificity of binding was further established by showing that the unlabeled ligands efficiently competed with the labeled chemokines for binding to their respective receptor. A low molecular weight antagonist of CXCR4 prevented binding of labeled CXCL12 to CXCR4 comparably to a neutralizing anti-CXCR4 antibody. Finally, labeled CCL19 was used for the staining of primary cells, illustrating that this reagent can be used for studying CCR7 expression on different cell types. Together, these results demonstrate that fluorescent synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on intact cells, for applications such as cell-based, high throughput screening, and studies of chemokine receptor expression by primary cells.     

Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL‐6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP‐1 secretion was reduced by CCL15/CCR3. In conclusion, this in‐vitro study identifies chemokine receptor‐ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL‐6 and HGF and reducing the secretion of TIMP‐1.     

Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) presents distinct active clinical forms with different grades of severity, known as localised (LCL), intermediate (ICL) and diffuse (DCL) cutaneous leishmaniasis. LCL and DCL are associated with a polarised T-helper (Th)1 and Th2 immune response, respectively, whereas ICL, or chronic cutaneous leishmaniasis, is associated with an exacerbated immune response and a mixed cytokine expression profile. Chemokines and chemokine receptors are involved in cellular migration and are critical in the inflammatory response. Therefore, we evaluated the expression of the chemokines CXCL10, CCL4, CCL8, CCL11 and CXCL8 and the chemokine receptors CCR3, CXCR3, CCR5 and CCR7 in the lesions of patients with different clinical forms of ACL using immunohistochemistry. LCL patients exhibited a high density of CXCL10+, CCL4+ and CCL8+ cells, indicating an important role for these chemokines in the local Th1 immune response and the migration of CXCR3+ cells. LCL patients showed a higher density of CCR7+ cells than ICL or DCL patients, suggesting major dendritic cell (DC) migration to lymph nodes. Furthermore, DCL was associated with low expression levels of Th1-associated chemokines and CCL11+ epidermal DCs, which contribute to the recruitment of CCR3+ cells. Our findings also suggest an important role for epidermal cells in the induction of skin immune responses through the production of chemokines, such as CXCL10, by keratinocytes.     

C-Terminal Engineering of CXCL12 and CCL5 Chemokines: Functional Characterization by Electrophysiological Recordings

Chemokines are chemotactic cytokines comprised of 70–100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K⁺ channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.     

Profiling heparin-chemokine interactions using synthetic tools.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.     

Interference with Glycosaminoglycan-Chemokine Interactions with a Probe to Alter Leukocyte Recruitment and Inflammation In Vivo

In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo.     

Cutting edge: vasoactive intestinal peptide acts as a potent suppressor of inflammation in vivo by trans-deactivating chemokine receptors

Chemokines mediate trafficking of leukocytes to sites of inflammation and immune responses through activation of G protein-coupled receptors, which thereby provide appealing targets for novel anti-inflammatory agents. Vasoactive intestinal peptide (VIP) is an immunosuppressive neurotransmitter. We show that VIP inhibited the function of chemokine receptors on monocytes and CD4(+) T lymphocytes, with impaired chemotaxis and calcium flux in response to the cognate chemokine ligands CXCL12, CCL3, CCL4, and CCL5. This was mediated by VIP receptor type 1 and was not caused by chemokine receptor internalization. However, VIP caused dose-dependent phosphorylation of the chemokine receptor CCR5. This trans-deactivation process was studied in a murine model of delayed-type hypersensitivity: continuous infusion of VIP resulted in significant abrogation of monocyte and lymphocyte infiltration. Circulating mononuclear cells from VIP-infused mice were unable to respond to chemokines. VIP may provide a novel approach to treatment of inflammatory diseases through inhibition of chemokine-dependent leukocyte recruitment.     

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.     

Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity

Thymic stromal lymphopoietin (TSLP) is said to increase expression of chemokines attracting Th2 T cells. We hypothesized that asthma is characterized by elevated bronchial mucosal expression of TSLP and Th2-attracting, but not Th1-attracting, chemokines as compared with controls, with selective accumulation of cells bearing receptors for these chemokines. We used in situ hybridization and immunohistochemistry to examine the expression and cellular provenance of TSLP, Th2-attracting (thymus and activation-regulated chemokine (TARC)/CCL17, macrophage-derived chemokine (MDC)/CCL22, I-309/CCL1) and Th1-attracting (IFN-gamma-inducible protein 10 (IP-10)/CXCL10, IFN-inducible T cell alpha-chemoattractant (I-TAC)/CXCL11) chemokines and expression of their receptors CCR4, CCR8, and CXCR3 in bronchial biopsies from 20 asthmatics and 15 normal controls. The numbers of cells within the bronchial epithelium and submucosa expressing mRNA for TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in asthmatics as compared with controls (p 相似文献   

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.     

Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.     

Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family

Chemokines coordinate many aspects of leukocyte migration. As chemoattractants they play an important role in the innate and acquired immune response. There is good experimental evidence that N-terminal truncation by secreted or cell surface proteases is a way of modulating chemokine action. The localization of CD26/dipeptidyl peptidase IV on cell surfaces and in biological fluids, its primary specificity, and the type of naturally occurring truncated chemokines are consistent with such a function. We determined the steady-state catalytic parameters for a relevant selection of chemokines (CCL3b, CCL5, CCL11, CCL22, CXCL9, CXCL10, CXCL11, and CXCL12) previously reported to alter their chemotactic behavior due to CD26/dipeptidyl peptidase IV-catalyzed truncation. The results reveal a striking selectivity for stromal cell-derived factor-1alpha (CXCL12) and macrophage-derived chemokine (CCL22). The kinetic parameters support the hypothesis that CD26/dipeptidyl peptidase IV contributes to the degradation of certain chemokines in vivo. The data not only provide insight into the selectivity of the enzyme for specific chemokines, but they also contribute to the general understanding of CD26/dipeptidyl peptidase IV secondary substrate specificity.     

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

Background

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

Methods and Results

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

Conclusions

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.     

CCR9 and CCL25: A review of their roles in tumor promotion

Chemokines constitute a superfamily of small chemotactic cytokines with functions that are based on interactions with their corresponding receptors. It has been found that, among other functions, chemokines regulate the migratory and invasive abilities of cancer cells. Multiple studies have confirmed that chemokine receptor 9 (CCR9) and its exclusive ligand, chemokine 25 (CCL25), are overexpressed in a variety of malignant tumors and are closely associated with tumor proliferation, apoptosis, invasion, migration and drug resistance. This review evaluates recent advances in understanding the role of CCR9/CCL25 in cancer development. First, we outline the general background of chemokines in cancer and the structure and function of CCR9 and CCL25. Next, we describe the basic function of CCR9/CCL25 in the cancer process. Then, we introduce the role of CCR9/CCL25 and related signaling pathways in various cancers. Finally, future research directions are proposed. In general, this paper is intended to serve as a comprehensive repository of information on this topic and is expected to contribute to the design of other research projects and future efforts to develop treatment strategies for ameliorating the effects of CCR9/CCL25 in cancer.     

Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4+ T and CCR7 B and T lymphocytes in thyroid autoimmune diseases

Chemokines and their corresponding receptors are crucial for the recruitment of lymphocytes into the lymphoid organs and for its organization acting in a multistep process. Tissues affected by autoimmune disease often contain ectopic lymphoid follicles which, in the case of autoimmune thyroid disorders, are highly active and specific for thyroid Ags although its pathogenic role remains unclear. To understand the genesis of these lymphoid follicles, the expression of relevant cytokines and chemokines was assessed by real time PCR, immunohistochemistry and by in vitro assays in autoimmune and nonautoimmune thyroid glands. Lymphotoxin alpha, lymphotoxin beta, C-C chemokine ligand (CCL) 21, CXC chemokine ligand (CXCL) 12, CXCL13, and CCL22 were increased in thyroids from autoimmune patients, whereas CXCL12, CXCL13, and CCL22 levels were significantly higher in autoimmune glands with ectopic secondary lymphoid follicles than in those without follicles. Interestingly, thyroid epithelium produced CXCL12 in response to proinflammatory cytokines providing a possible clue for the understanding of how tissue stress may lead to ectopic follicle formation. The finding of a correlation between chemokines and thyroid autoantibodies further suggests that intrathyroidal germinal centers play a significant role in the autoimmune response. Unexpectedly, the percentage of circulating CXCR4(+) T cells and CCR7(+) B and T cells (but not of CXCR5) was significantly reduced in PBMCs of patients with autoimmune thyroid disease when they were compared with their intrathyroidal lymphocytes. This systemic effect of active intrathyroidal lymphoid tissue emerges as a possible new marker of thyroid autoimmune disease activity.