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1.
β-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the β-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG1 to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these β-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the β-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the β-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and β-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the β-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific β-defensin receptor interactions.  相似文献   

2.

Background

The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production.

Methods

A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using in situ hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay.

Results

CCL20 and CCR6 mRNA abundances were increased during active inflammation (CCL20 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; CCR6 1.8 and 2.0, respectively). CCL20 and CCR6 mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and TLR3 silencing reduced CCL20 mRNA and protein levels.

Conclusions

The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.  相似文献   

3.
4.
We previously identified CCL20 as an early chemokine in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis but its functional relevance was unknown. Here we studied the role of CCL20 and its receptor CCR6 in pneumococcal meningitis. In a prospective nationwide study, CCL20 levels were significantly elevated in the CSF of patients with pneumococcal meningitis and correlated with CSF leukocyte counts. CCR6-deficient mice with pneumococcal meningitis and WT mice with pneumococcal meningitis treated with anti-CCL20 antibodies both had reduced CSF white blood cell counts. The reduction in CSF pleocytosis was also accompanied by an increase in brain bacterial titers. Additional in vitro experiments showed direct chemoattractant activity of CCL20 for granulocytes. In summary, our results identify the CCL20-CCR6 axis as an essential component of the innate immune defense against pneumococcal meningitis, controlling granulocyte recruitment.  相似文献   

5.

Objectives

Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.

Methods

A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.

Results

CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.

Conclusion

Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.  相似文献   

6.
CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling.  相似文献   

7.
Chemokines (chemotactic cytokines) drive and direct leukocyte traffic. New evidence suggests that the unusual CCR6/CCL20 chemokine receptor/ligand axis provides key homing signals for recently identified cells of the adaptive immune system, recruiting both pro-inflammatory and suppressive T cell subsets. Thus CCR6 and CCL20 have been recently implicated in various human pathologies, particularly in autoimmune disease. These studies have revealed that targeting CCR6/CCL20 can enhance or inhibit autoimmune disease depending on the cellular basis of pathogenesis and the cell subtype most affected through different CCR6/CCL20 manipulations. Here, we discuss the significance of this chemokine receptor/ligand axis in immune and inflammatory functions, consider the potential for targeting CCR6/CCL20 in human autoimmunity and propose that the shared evolutionary origins of pro-inflammatory and regulatory T cells may contribute to the reason why both immune activation and regulation might be controlled through the same chemokine pathway.  相似文献   

8.
9.
Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APCMIN/+ mice) to generate APCMIN/+ mice with CCR6 knocked out (CCR6KO-APCMIN/+ mice). CCR6KO-APCMIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APCMIN/+ also had normal sized spleens as compared to the enlarged spleens found in APCMIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APCMIN/+ as compared to APCMIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.  相似文献   

10.
11.
We evaluated the role of CCL20 (MIP-3alpha) chemokine in cells directly involved in the remodeling of bone tissue (osteoblasts and osteoclasts) and we confirmed its expression in the subchondral bone tissue of rheumatoid arthritis (RA) patients. The expression of CCL20 and of its receptor CCR6 was evaluated in osteoblasts isolated from bone tissue of post-traumatic (PT) patients. Functional tests were performed to evaluate osteoblast proliferation and matrix protein modulation. Immunohistochemical analysis for CCR6, CCL20, and RANKL was performed on bone samples from RA patients. The role of CCL20 was then analyzed in osteoclast differentiation. We found that in basal conditions CCR6, but not its ligand CCL20, was highly expressed by osteoblasts. Functional analysis on osteoblasts showed that CCL20 significantly increased cellular proliferation but did not affect matrix protein expression. Pro-inflammatory cytokines significantly induced the release of CCL20 and RANKL by human osteoblasts but did not modulate CCR6 expression. Increased expression of CCR6, CCL20, and RANKL was confirmed in RA subchondral bone tissue biopsies. We demonstrated that CCL20 was also an earlier inducer of osteoclast differentiation by increasing the number of pre-osteoclasts, thus favoring cell fusion and MMP-9 release. Our results add new insight to the important role of the CCL20/CCR6, RANKL system in the bone tissue of RA. The contemporary action of CCL20 on osteoblasts and osteoclasts involved in the maintenance of bone tissue homeostasis demonstrates the important role of this compartment in the evolution of RA, by showing a clear uncoupling between new bone formation and bone resorption.  相似文献   

12.
CC chemokine ligand (CCL)17 and CCL27 produced by epidermal keratinocytes (KCs) recruit CC chemokine receptor (CCR)4 and CCR10 expressing T cells into the skin, respectively, resulting in enhanced skin inflammation. However, CCR4/CCL17 and CCR10/CCL27 interactions in epidermal KCs have not been investigated. The purpose of this study was to evaluate the role of the CCR4/CCL17 and CCR10/CCL27 loops in cutaneous immune reaction. Normal human KCs (NHKs) and HaCaT KCs expressed both CCR4 and CCR10 at mRNA and protein levels. CCR4 ligand CCL17 but not CCR10 ligand CCL27 induced production of IL-12 p40, granulocyte/monocyte colony-stimulating factor (GM-CSF) and nerve growth factor (NGF) by KCs. Both CCL17 and CCL27 induced migration of KCs in Boyden chamber assay and wound scratch assay. This study revealed that CCR4 and CCR10 are expressed on epidermal KCs and that both are functional in terms of skin cytokine production and/or migration to their ligand CCL17 and CCL27, respectively. Thus this study provided new insight into chemokine/chemokine receptors of KCs.  相似文献   

13.
The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4+CCR4+ lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.  相似文献   

14.
Human chemokine receptor CCR3 (hCCR3) belongs to the G protein-coupled receptors (GPCRs) superfamily of membrane proteins and plays major roles in allergic diseases and angiogenesis. In order to study the structural and functional mechanism of hCCR3, it is essential to produce pure protein with biological functions on a milligram scale. Here we report the expression of hCCR3 gene in a tetracycline-inducible stable mammalian cell line. A cell clone with high hCCR3 expression was selected from 46 stably transfected cell clones and from this cell line pure hCCR3 on a milligram scale was obtained after two-step purification. Circular dichroism spectrum with a characteristic shape and magnitude for α-helix indicated proper folding of hCCR3 after purification. The biological activity of purified hCCR3 was verified by its high binding affinity with its endogenous ligands CCL11 and CCL24, with K D in the range of 10−8 M to 10−6 M.  相似文献   

15.
Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8- /-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.  相似文献   

16.
CCR6 is expressed by memory T cells (mTC) and is a requirement for efficient arrest of a subset of mTC to activated human dermal microvascular endothelial cells (HDMEC) under physiologic shear stress. We now address whether CCR6 alone is sufficient to induce arrest of a model T cell line (Jurkat) that shows low expression of all CCRs tested (CCR1-10). Herein, we transduced Jurkat (JK) T cells expressing fucosyltransferase VII with a chimeric chemokine receptor consisting of CCR6 fused to enhanced green fluorescent protein. In contrast to the starting JK lines, the resulting cell line (JK fucosyltransferase VII-CCR6) migrated 6-fold better to CCL20 in chemotaxis assays, arrested in response to CCL20 that was immobilized to plastic, and demonstrated a 2.5-fold increase in adhesion to activated HDMEC (p = 0.001). Adhesion was blocked by anti-CD18 mAb (p = 0.005) but not by anti-CD49d mAb (p = 0.3). After arrest on recombinant substrates, CCR6 clustered on the surface as detected by real-time observation of enhanced green fluorescent protein fluorescence. Dual-label confocal microscopy revealed that LFA-1 (CD18 and CD11a), but not CXCR4, colocalized with clustered CCR6 in the presence of immobilized CCL20. Thus, the functional expression of CCR6 is sufficient to provide the chemokine signaling necessary to induce arrest of a JK T cell line to activated HDMEC. Clustering of CCR6 and coassociation with critical integrins may serve to strengthen adhesion between T cells and activated endothelial cells.  相似文献   

17.
Dendritic cells (DCs) play essential roles in both innate and adaptive immune responses. In addition, mutual regulation of the nervous system and immune system is well studied. One of neuropeptides, calcitonin gene-related peptide (CGRP), is a potent regulator in immune responses; in particular, it has anti-inflammatory effects in innate immunity. For instance, a deficiency of the CGRP receptor component RAMP 1 (receptor activity-modifying protein 1) results in higher cytokine production in response to LPS (lipopolysaccharide). On the other hand, how CGRP affects DCs in adaptive immunity is largely unknown. In this study, we show that CGRP suppressed Th1 cell differentiation via inhibition of IL-12 production in DCs using an in vitro co-culture system and an in vivo ovalbumin-induced delayed-type hypersensitivity (DTH) model. CGRP also down-regulated the expressions of chemokine receptor CCR2 and its ligands CCL2 and CCL12 in DCs. Intriguingly, the frequency of migrating CCR2+ DCs in draining lymph nodes of RAMP1-deficient mice was higher after DTH immunization. Moreover, these CCR2+ DCs highly expressed IL-12 and CD80, resulting in more effective induction of Th1 differentiation compared with CCR2 DCs. These results indicate that CGRP regulates Th1 type reactions by regulating expression of cytokines, chemokines, and chemokine receptors in DCs.  相似文献   

18.
Chemokines are key mediators of leukocyte recruitment during pathogenic insult and also play a prominent role in homeostasis. While most chemokine receptors bind to multiple chemokines, CCR6 is unique in that this receptor is one of only a few that can bind only a single chemokine ligand, CCL20. CCR6 is an important receptor that is involved in regulating several aspects of mucosal immunity, including the ability to mediate the recruitment of immature dendritic cells (DCs) and mature DCs, and professional antigen presenting cells (APCs) to the sites of epithelial inflammation. Further, CCR6 mediates the homing of both CD4+ T (T-helper; Th) cells and DCs to the gut mucosal lymphoid tissue. DCs, which are known to be essential immune cells in innate immunity and in the initiation of adaptive immunity, play a central role in initiating a primary immune response. Herein, we summarize the role of CCR6 in immune responses at epithelial and mucosal sites in both the lung and gut based on a review of the current literature.  相似文献   

19.
The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX3CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX3CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX3CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx3cr1 gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX3CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.  相似文献   

20.
Subchondral bone remodeling in osteoarthritis (OA) and rheumatoid arthritis (RA) is mainly characterized by the formation of osteophytes/fibrosis and by the presence of infiltrating cells associated to bone resorption. In this study we analyzed CC (cysteine cysteine motif) chemokine ligand (CCL)20 and CC chemokine receptor (CCR)6 function in subchondral bone tissue and osteoblasts isolated from OA and RA patients. CCL20/CCR6 expression was evaluated by immunohistochemical techniques in bone tissue from OA and RA patients. CCL20‐functional tests were performed on osteoblasts isolated from OA and RA patients to evaluate enzymatic response and cell proliferation. Moreover, we assessed Akt phosphorylation as the major signaling pathway for CCL20. In bone tissue biopsies we found that osteoblasts from both OA and RA patients expressed CCR6 while CCL20 was expressed only by RA osteoblasts. Both CCR6 and CCL20 were highly expressed in osteocytes and mononuclear cells from only RA patients. CCL20‐stimulated OA osteoblasts showed a significant increase in β‐N‐acetylhexosaminidase release compared to RA. Conversely, a significant increase in cellular proliferation was found only in CCL20‐stimulated RA osteoblasts associated to Akt phosphorylation. These data were confirmed in bone tissue biopsies. This study demonstrates a different expression of CCL20‐positive osteoblasts in OA versus RA disease that seem to be associated with the presence of infiltrating mononuclear cells. Moreover, CCL20 stimulation resulted in a greater proliferative response in RA osteoblasts compared to OA osteoblasts, mediated by Akt signaling, while OA osteoblasts showed increased enzymatic activity, thus suggesting a differential role of this chemokine in OA and RA. J. Cell. Physiol. 221: 154–160, 2009. © 2009 Wiley‐Liss, Inc  相似文献   

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