Selective down-regulation of Th2 cell-mediated airway inflammation in mice by pharmacological intervention of CCR4.

BACKGROUND: The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. OBJECTIVE: The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. METHOD: We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. RESULTS: Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. CONCLUSIONS AND CLINICAL RELEVANCE: There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.

Abundant expression of CXCL9 (MIG) by stromal cells that include dendritic cells and accumulation of CXCR3+ T cells in lymphocyte-rich gastric carcinoma.

The neoplastic environment is generally regarded as an immunosuppressive milieu. However, a group of cancers are characterized by the abundance of tumour-infiltrating lymphocytes (TILs). Here we examined the possible roles of chemokines in the formation of lymphoid stroma in lymphocyte-rich gastric carcinomas (GCs), including EBV(+) cases and conventional GCs. Regardless of EBV positivity, TILs in lymphocyte-rich GCs predominantly expressed CXCR3, while its ligand CXCL9 was abundantly expressed by stromal cells and a portion of cancer cells. CXCL9(+) stromal cells were judged to include dendritic cells, because they partly co-expressed fascin, DC-sign, CD83, DC-lamp or HLA-DR. T cells in close contact with CXCL9(+) cells showed frequent labelling of Ki-67 (approximately 10%), suggesting the immunostimulatory activity of CXCL9(+) stromal cells. The T-cell zone of the regional lymph nodes of lymphocyte-rich GCs also abounded with CXCR3(+) T cells and CXCL9(+) stromal cells. This indicated a close similarity between cancer stroma and regional lymph nodes of lymphocyte-rich GCs. Quantitative RT-PCR also confirmed the strong expression of CXCR3, CXCL9 and IFNgamma in lymphocyte-rich GCs. In contrast, conventional GCs contained less abundant CXCR3(+) T cells and few CXCL9(+) stromal cells. Collectively, the CXCL9-CXCR3 axis plays a pivotal role in the formation of lymphoid stroma in lymphocyte-rich GCs. Given similar findings in the regional lymph nodes, the lymphoid stroma of lymphocyte-rich GCs may represent a tertiary lymphoid tissue with predominantly Th1-shifted immune responses.

Aberrant expression of Fra-2 promotes CCR4 expression and cell proliferation in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Primary ATL cells frequently express CCR4 at high levels. Since HTLV-1 Tax does not induce CCR4 expression, transcription factor(s) constitutively active in ATL may be responsible for its strong expression. We identified an activator protein-1 (AP-1) site in the CCR4 promoter as the major positive regulatory element in ATL cells. Among the AP-1 family members, Fra-2, JunB and JunD are highly expressed in fresh primary ATL cells. Consistently, the Fra-2/JunB and Fra-2/JunD heterodimers strongly activated the CCR4 promoter in Jurkat cells. Furthermore, Fra-2 small interfering RNA (siRNA) or JunD siRNA, but not JunB siRNA, effectively reduced CCR4 expression and cell growth in ATL cells. Conversely, Fra-2 or JunD overexpression promoted cell growth in Jurkat cells. We identified 49 genes, including c-Myb, BCL-6 and MDM2, which were downregulated by Fra-2 siRNA in ATL cells. c-Myb, BCL-6 and MDM2 were also downregulated by JunD siRNA. As Fra-2, these proto-oncogenes were highly expressed in primary ATL cells but not in normal CD4+ T cells. Collectively, aberrantly expressed Fra-2 in association with JunD may play a major role in CCR4 expression and oncogenesis in ATL.

Therapeutic effect of CXCR3-expressing regulatory T cells on liver, lung and intestinal damages in a murine acute GVHD model.

Adoptive transfer of CD4+CD25+ regulatory T cells has been shown to have therapeutic effects in experimental graft-vs-host disease (GVHD) models. Chemokines play an important role in the recruitment of alloreactive donor T cells into target organs during GVHD. In this study, we investigated the effectiveness of targeted delivery of CD4+CD25+ regulatory T cells via a transfected chemokine receptor on reduction of organ damage during acute GVHD. High levels of expression of Th1-associated chemokines (CXCL9, CXCL10 and CXCL11) and their receptor CXCR3 were observed in the liver, lung and intestine of GVHD-induced recipient mice. Recipient mice that had undergone transfer of CD4+CD25+Foxp3+ CXCR3-transfected T cells (CXCR3-Treg cells) showed significant amelioration of GVHD changes in the liver, lung and intestine in comparison with recipient mice that had received CD4+CD25+Foxp3+ T cells (Treg cells) or naturally occurring CD4+CD25+ regulatory T cells. This was due to more pronounced migration of CXCR3-Treg cells and their localization for a longer time in Th1-associated chemokine-expressing organs, resulting in stronger suppressive activity. We succeeded in preparing chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression upon accumulation in target organs. This method may provide a new therapeutic approach for organ damage in acute GVHD.

Accumulation of plasma cells expressing CXCR3 in the synovial sublining regions of early rheumatoid arthritis in association with production of Mig/CXCL9 by synovial fibroblasts.

Accumulation of plasma cells in the synovium is one of the diagnostic hallmarks in the histopathological manifestations of rheumatoid arthritis (RA). This seems to be prominent even prior to significant B cell infiltration and/or formation of lymphoid follicles in the synovium. To clarify the mechanism of early plasma cell accumulation, we examined in situ expression of chemokines and their receptors using synovial targeting biopsy specimens, which were obtained under arthroscopy from early RA patients. By immunohistochemical staining, plasma cells were found to express a chemokine receptor CXCR3, while synovial fibroblasts in the synovial sublining regions expressed its ligand, Mig/CXCL9. By reverse transcription-polymerase chain reaction (RT-PCR), using targeted lesions of synovial tissues obtained by laser capture microdissection, expression levels of Mig/CXCL9 in the synovial sublining regions were remarkably high and were likely to be associated with interferon (IFN)-gamma expression. Furthermore, cultured synovial fibroblasts were confirmed to produce Mig/CXCL9 upon stimulation with IFN-gamma. Our results indicate that in the early stage of RA, plasma cells expressing CXCR3 may be recruited directly from the circulation into the synovial sublining regions by its ligand, Mig/CXCL9, produced by synovial fibroblasts.

Augmentation of the migratory ability of DC-based vaccine into regional lymph nodes by efficient CCR7 gene transduction.

Although dendritic cell (DC)-based immunotherapy is considered a promising approach for cancer treatment, a large quantity of DC vaccine is required for effective sensitization/activation of immune cells because of the poor migratory ability of administered DCs into regional lymphoid tissue. In this study, we created a DC vaccine sufficiently transduced with CC chemokine receptor-7 gene (CCR7/DCs) by applying RGD fiber-mutant adenovirus vector (AdRGD), and investigated its immunological characteristics and therapeutic efficacy. CCR7/DCs acquired strong chemotactic activity for CC chemokine ligand-21 (CCL21) and exhibited an immunophenotype similar to mature DCs but not immature DCs with regard to major histocompatibility complex/costimulatory molecule-expression levels and allogenic T cell proliferation-stimulating ability, while maintaining inherent endocytotic activity. Importantly, CCR7/DCs injected intradermally into mice could accumulate in draining lymph nodes about 5.5-fold more efficiently than control AdRGD-applied DCs. Reflecting these properties of CCR7/DCs, DC vaccine genetically engineered to simultaneously express endogenous antigen and CCR7 could elicit more effective antigen-specific immune response in vivo using a lower dosage than DC vaccine transduced with antigen alone. Therefore, the application of CCR7/DCs having positive migratory ability to lymphoid tissues may contribute to reduction of efforts and costs associated with DC vaccine preparation by considerably reducing the DC vaccine dosage needed to achieve effective treatment by DC-based immunotherapy.

CCL22 and CCL17 in rat radiation pneumonitis and in human idiopathic pulmonary fibrosis.

Pulmonary fibrosis is caused by various known and unknown aetiologies, but the key pathogenic mechanisms are still ill-defined. Chemokines are a large family of chemotactic cytokines that play pivotal roles in various inflammatory diseases. In the present study, the roles of chemokines in a rat model of radiation pneumonitis/ pulmonary fibrosis were examined. Accumulation of inflammatory cells and pneumonitis were observed on day 28, and diffuse alveolar wall thickening with extensive fibrosis was observed on day 56. In addition to the previously reported CCL2 (macrophage chemoattractant protein-1) induction, selective upregulation of CCL22 (macrophage-derived chemokine) and CCL17 (thymus and activation-regulated chemokine) were demonstrated for the first time in the irradiated lung tissues. Immunohistochemically, it was demonstrated that CCL22 and CCL17 were localised primarily to alveolar macrophages, whereas their receptor CC chemokine receptor 4 (CCR4) was detected on alveolar lymphocytes and macrophages. On further analysis of bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis and sarcoidosis, elevated levels of CCL22, but not of CCL17, were observed in the idiopathic pulmonary fibrosis patients. Since these two chemokines play pivotal roles in various type-2 T-helper cell-dominant diseases, it was speculated that CCL22, and probably CCL17, are involved in the pathophysiology of radiation pneumonitis/pulmonary fibrosis and idiopathic pulmonary fibrosis through the recruitment of CC chemokine receptor 4-positive type-2 T-helper cells and alveolar macrophages.

Tumor-suppressive activities by chemokines introduced into OV-HM cells using fiber-mutant adenovirus vectors.

In this study, fiber-mutant adenovirus vectors encoding chemokines, Ad-RGD-mCCL17, Ad-RGD-mCCL21 and Ad-RGD-mCCL22 were constructed. The insertion of integrin-targeting RGD sequence into fiber knob of adenovirus vectors notably enhanced the infection efficiency into tumor cells. Among three chemokine-encoding vectors evaluated, Ad-RGD-mCCL22 showed significant tumor-suppressive activity via transduction into OV-HM cells.

Airway epithelial cells promote transmigration of eosinophils in a new three-dimensional chemotaxis model.

BACKGROUND: Prominent infiltration of eosinophils in airway mucosa is the pathognomonic sign of asthma. The role of airway epithelial cells in eosinophil infiltration, however, has not been fully elucidated. OBJECTIVE: The aim of this study is to develop a new in vitro transmigration system composed of airway epithelial cells and extracellular matrix, and to investigate the role of airway epithelial cells in eosinophil infiltration. METHODS: A layer of type I collagen gel was formed in Netwell, and BEAS-2B bronchial epithelial cells were cultured on the gel. Then the wells covered with epithelial monolayer were filled with medium, inverted, and new upper chambers were constructed on the gel side by applying a ring cap. After further incubation with or without exogenous cytokines for 48 h, eosinophils or neutrophils were loaded in upper chambers (the gel side) and cells transmigrated to lower chambers (the epithelial cell side) were counted. Immunohistochemical analyses were also performed. RESULTS: While a simple collagen gel hardly promoted eosinophil migration even in the presence of eotaxin or RANTES, significant numbers of eosinophils migrated to lower chambers in the presence of the epithelial cells. Replacement of medium in the lower chamber (the epithelial cell side) with fresh medium, addition of exogenous eotaxin or RANTES in the upper chamber (the gel side), or pre-treatment of eosinophils with anti-CCR3 all inhibited transmigration. We found that the epithelial cells produced and deposited extracellular matrix proteins such as type IV collagen onto the type I collagen gel. Separately, we found that type IV collagen itself was capable of enhancing eotaxin-induced eosinophil migration in a standard chemotaxis assay. Neutrophils also efficiently migrated in the present transmigration system. Pre-treatment of epithelial cells with TNF-alpha and IL-4 enhanced eosinophil transmigration, while that of neutrophils was enhanced by TNF-alpha but suppressed by IL-4. CONCLUSION: By utilizing a new in vitro transmigration system mimicking the airway mucosa, we have demonstrated that airway epithelial cells play an essential role in transmigration of eosinophils and that multiple factors such as chemokines, extracellular matrix proteins and exogenous inflammatory cytokines are involved in efficient transmigration.

Increased levels of a TH2-type CC chemokine thymus and activation-regulated chemokine (TARC) in serum and induced sputum of asthmatics.

BACKGROUND: Cytokines liberated by TH2 cells play crucial roles in the pathogenesis of bronchial asthma. Recent studies have demonstrated that CC chemokine receptor (CCR)4 is preferentially expressed by TH2 cells. These facts suggest possible involvement of two CCR4-specific ligands i.e., thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), in the pathogenesis of bronchial asthma via recruitment of TH2 cells to inflammatory sites. We investigated the levels of TARC and MDC in the serum and induced sputum of asthmatics. METHODS: The levels of TARC in the serum (46 asthmatics and 26 healthy subjects) and induced sputum (30 asthmatics and 6 healthy subjects) were measured using a highly sensitive ELISA system. The levels of eotaxin and MDC were also measured by ELISA. RESULTS: TARC, but not MDC, was significantly increased in asthmatic sera (P<0.001). Although MDC was undetectable in the sputum of most cases by our assay system, sputum TARC was significantly increased (P=0.027). CONCLUSIONS: The elevated TARC levels in asthmatics might be involved in the pathophysiology of asthma.

Proinflammatory cytokines induce liver and activation-regulated chemokine/macrophage inflammatory protein-3alpha/CCL20 in mucosal epithelial cells through NF-kappaB [correction of NK-kappaB].

Liver and activation-regulated chemokine (LARC)/CCL20 is expressed by surface-lining epithelial and epidermal cells, and is likely to link innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. Here we examined the mechanism of LARC expression in epithelial-type cells. Either IL-1beta or tumor necrosis factor (TNF)-alpha strongly induced LARC mRNA in intestinal cell lines Caco-2 and T84, while both were effective on HEK 293T cells. Induction of LARC was also demonstrated in the intestinal epithelium of BALB/c mice upon treatment with IL-1alpha or TNF-alpha. Transient transfection assays using murine LARC promoter-reporter constructs identified a region essential for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Using site-directed mutagenesis, we demonstrated that an NF-kappaB site located between -96 and -87 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Electrophoretic mobility shift assays demonstrated that p50/p65 heterodimer and p65 homodimer of NF-kappaB bound to this site in 293T cells upon treatment with IL-1beta and TNF-alpha, and p50/p65 heterodimer bound to this site in Caco-2 cells upon treatment with IL-1beta. Co-expression of constitutively active p65 strongly activated the promoter construct carrying the intact NF-kappaB site in 293T and Caco-2 cells. Collectively, LARC expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-alpha primarily through activation of NF-kappaB.

Epigenetic regulation of the KAI1 metastasis suppressor gene in human prostate cancer cell lines.

Expression of the KAI1 gene, a metastasis-suppressor for prostate cancer, is reduced in all foci of prostatic metastasis. The altered regulatory mechanism is not strongly related to mutations or allelic losses of the KAI1 gene in prostate tumors. Since transcriptional silencing of genes has been found to be caused by epigenetic mechanisms, we have investigated the involvement of this epigenetic regulation of KAI1 expression in prostate cancers. The methylation status of the KAI1 promoter region was examined by restriction-enzyme digestion and sequencing, after amplifying a 331-bp fragment in the GC-rich promoter region from 4 human prostate cancer cell lines treated with bisulfite. The same 4 cell lines were also exposed to various concentrations of the demethylating agent, 5-aza-2'-deoxycytidine (5-AzaC) and / or the histone deacetylase inhibitor, trichostatin A (TSA). To clarify the influence of epigenetic modification on reduced KAI1 mRNA expression in the tumor cells, RT-PCR and northern-blot analyses were performed. Bisulfite-sequencing data showed a few methylated CpG islands in the promoter. RT-PCR analysis of 5-AzaC and / or TSA-treated cells indicated reversal of suppression of KAI1 transcription in two cell lines (PC-3 and DU-145), although the expression could not be detected by northern blots. From these results, it is suggested that epigenetic change is not the main mechanism of KAI1 down-regulation, though there remains a possibility that methylation in a more upstream region might be associated with this regulation.

The kinetics of allergen-induced eotaxin level in nasal lavage fluid: its key role in eosinophil recruitment in nasal mucosa.

Eotaxin (CCL11) is a potent eosinophil chemoattractant belonging to the C-C chemokine. To evaluate the role of eotaxin in eosinophilic inflammation in nasal mucosa, we investigated the levels of eosinophil chemoattractants in nasal lavage fluids obtained after antigen challenge, compared with eosinophil counts and eosinophil protein X (EPX) levels. In subjects with allergic rhinitis, allergen challenge led to parallel increases in eosinophil counts, levels of EPX, and eotaxin concentrations in nasal lavage fluid. The levels of eotaxin in lavage samples showed strong correlation with lavage levels of eosinophil counts and EPX. Normal subjects had few, if any, eosinophils and EPX as well as the measured parameters in their nasal lavage fluids before and after antigen challenge. In our experiments of eosinophil endothelial transmigration (TEM) assay using the nasal microvascular endothelial cells, eotaxin showed the most potent effect among various eosinophil chemoattractants. In addition, treatment of eosinophils with anti-CCR-3 mAb significantly blocked eosinophil TEM induced by homogenate of nasal mucosa. These results indicate that eotaxin has an important role in eosinophil-dependent inflammation in nasal mucosa and suggest that blocking eotaxin or CCR-3 might be useful for new therapeutic tools of allergic rhinitis.

Chemokine receptors in human basophils: inducible expression of functional CXCR4.

We examined the expression profile of chemokine receptors in human basophils and their regulation by cytokines. Basophils expressed transcripts of CC chemokine receptors (CCR)1, CCR2, CCR3, and CCR5 and CXC chemokine receptors (CXCR)1, CXCR2, and CXCR4. In contrast to the other receptors, surface-CXCR4 expression was not detected in fresh- and whole-blood basophils, but it became apparent gradually during incubation. Among 16 chemokines tested, eotaxin induced the most potent basophil migration. SDF-1 also induced a strong, migratory response comparable with that induced by eotaxin in 24-h, cultured basophils, but it failed to induce degranulation. IL-3 abrogated CXCR4 expression completely, and it only down-regulated CCR2 and CCR3 expression slightly. IL-5, GM-CSF, and IL-4 also down-regulated CXCR4 expression. Thus, expression of CXCR4 was the most strongly affected by cytokines, and this may represent an alternative mechanism for control of cell-specific, biological responses to SDF-1.

Human CC chemokine liver-expressed chemokine/CCL16 is a functional ligand for CCR1, CCR2 and CCR5, and constitutively expressed by hepatocytes.

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEC also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEC was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEC to CCR8 was much less significant. In spite of its binding to CCR5, LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3--4 nM). Taken together, LEC is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses.

Chemokines in immunity.

Chemokines are a superfamily of small, heparin-binding cytokines that induce directed migration of various types of leukocytes through interactions with a group of seven-transmembrane G protein-coupled receptors. At present, over 40 members have been identified in humans. Until a few years ago, chemokines were mainly known as potent attractants for leukocytes such as neutrophils and monocytes, and were thus mostly regarded as the mediators of acute and chronic inflammatory responses. They had highly complex ligand-receptor relationships and their genes were regularly mapped on chromosomes 4 and 17 in humans. Recently, novel chemokines have been identified in rapid succession, mostly through application of bioinformatics on expressed sequence tag databases. A number of surprises have followed the identification of novel chemokines. They are constitutively expressed in lymphoid and other tissues with individually characteristic patterns. Most of them turned out to be highly specific for lymphocytes and dendritic cells. They have much simpler ligand-receptor relationships, and their genes are mapped to chromosomal loci different from the traditional chemokine gene clusters. Thus, the emerging chemokines are functionally and genetically quite different from the classical "inflammatory chemokines" and may be classified as "immune (system) chemokines" because of their profound importance in the genesis, homeostasis and function of the immune system. The emergence of immune chemokines has brought about a great deal of impact on the current immunological research, leading us to a better understanding on the fine traffic regulation of lymphocytes and dendritic cells. The immune chemokines and their receptors are also likely to be important future targets for therapeutic intervention of our immune responses.

A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.

Organization of the chemokine genes in the human and mouse major clusters of CC and CXC chemokines: diversification between the two species.

Chemokines are a family of small cytokines that play essential roles in the directed migration of various types of leukocytes. Based on the arrangement of the conserved cysteine residues, they are classified into two major subfamilies, CXC and CC, and two minor subfamilies, C and CX3C. So far, more than 40 members of this family have been identified in humans. Strikingly, the majority of CXC chemokine genes and that of CC chemokine genes are closely clustered at chromosomes 4q12-21 and 17q11.2, respectively. Similarly, the mouse major CXC and CC chemokine gene clusters are located on chromosomes 5 and 11, respectively. In order to understand the evolutionary processes that generated large numbers of CXC and CC chemokine genes in the respective chromosomal sites, we have constructed BAC and YAC contigs covering the human and mouse major clusters of CXC and CC chemokine genes. The results reveal that the organizations of CXC and CC chemokine genes in the major clusters are quite diverged between the two species most probably due to very recent gene duplications and rearrangements. Our results provide an important insight into the evolutionary processes that generated the major chemokine gene clusters and also valuable information in assigning the orthologues between human and mouse major cluster chemokines.

Chemokines have diverse abilities to form solid phase gradients.

Chemokines play critical roles in leukocyte recruitment into sites of inflammation such as rheumatoid arthritis (RA). While chemokines immobilized on endothelium (solid-phase), but not soluble chemokines, direct rolling leukocytes to firmly adhere to endothelium, soluble and solid-phase chemokine gradients may play important roles in leukocyte extravasation into the tissue. In this study, we have sought to determine (1) if chemokines can be immobilized on structures in the extravascular space, (2) the mechanisms by which chemokines may be immobilized, and (3) if different chemokines have similar potentials to form solid-phase gradients. While secreted alkaline phosphatase (SEAP)-tagged chemokines SLC (CCL21), TARC (CCL17), and RANTES (CCL5) bound to mast cells and the extracellular matrix (ECM) in RA synovium under physiologic salt conditions, MCP1 (CCL2), MIP1 alpha (CCL3), MIP1 beta (CCL4), and fractalkine (FKN, CX3CL1) fusion proteins did not detectably bind. Chemokine binding to ECM and mast cells in situ and to immobilized heparin was inhibited by high salt and glycosaminoglycans (GAGs) heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, but not by dextran or hyaluronan, indicating that the chemokines bind to highly sulfated GAGs. Chemokine binding to synovial structures correlated strongly with avidity of chemokine binding to heparin (SLC > TARC > RANTES > MIP1 beta > MCP1 > MIP1 alpha > FKN). A RANTES mutant with decreased avidity for heparin was not able to bind to ECM or mast cells. Thus, these data indicate that chemokines can bind to ECM and mast cell granule constituents in situ via interactions with GAGs. Further, only a subset of chemokines were able to bind efficiently to structures in the extravascular space, indicating that chemokines may form different types of gradients based on their GAG binding ability and that chemotactic gradients in tissues may be quite complex.

Macrophage-derived chemokine (MDC/CCL22) and CCR4 are involved in the formation of T lymphocyte-dendritic cell clusters in human inflamed skin and secondary lymphoid tissue.

Our previous study demonstrated formation of T cell-dendritic cell (DC) clusters in inflamed dermis of intraorally autotransplanted skin flaps. Such T cell-DC clusters are supposed to be important for close interactions between T cells and DCs including the specific antigen presentation. Here we show the involvement of the macrophage-derived chemokine (MDC/CCL22) and its specific receptor CC chemokine receptor 4 (CCR4) in the formation of T cell-DC clusters. Reverse transcriptase-polymerase chain reaction analysis revealed high levels of mRNA expression for MDC and CCR4 in inflamed skin and neck lymph nodes (LNs), but not in normal skin. Immunohistochemically, MDC(+) cells and CCR4(+) cells were mainly located within the T cell-DC clusters both in the dermis of inflamed skin and the T cell area of LNs. MDC(+) cells were identified to be DCs both in inflamed skin and LNs. The majority of CCR4(+) cells were CD4(+) T cells, accounting for approximately one-third of total CD4(+) T cells in the inflamed skin. Our data suggest that the MDC-CCR4 system plays an important role in the formation of T cell-DC clusters both in inflamed skin and LNs.