Human T cells that are able to produce IL-17 express the chemokine receptor CCR6

Some pathways of T cell differentiation are associated with characteristic patterns of chemokine receptor expression. A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL-17 cytokines and whose differentiation requires the nuclear receptor, RORgammat. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL-17 production for human T cells. Activating cord blood (naive) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9, and CXCR6. Despite these data, we found no strong correlation between the production of IL-17 and expression of CCR9 or CXCR6. By contrast, our analyses revealed that virtually all IL-17-producing CD4+ T cells, either made in our in vitro cultures or found in peripheral blood, expressed CCR6, a receptor found on approximately 50% of CD4+ memory PBL. Compared with CD4+CD45RO+CCR6- cells, CD4+CD45RO+CCR6+ cells contained at least 100-fold more IL-17A mRNA and secreted 100-fold more IL-17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORgammat. CCR6 was likewise expressed on all IL-17-producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL-17-producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL-17-related immune responses and possible targets for preventing inflammatory injury.     

Up-regulation of CCR5 and CCR6 on distinct subpopulations of antigen-activated CD4+ T lymphocytes.

Following infection, naive T cells are activated in the secondary lymphoid tissue, but then need to move to the infected tissue in the periphery to mediate their effector functions. The acquisition of inflammatory chemokine receptors, such as CCR5 and CCR6, may contribute to the efficient relocation of activated T cells to inflamed sites in the periphery. In keeping with this idea, the present study has demonstrated that CCR5 and CCR6 are up-regulated on CD4+ T cells upon activation in the MLR. The observed increase in expression correlated well with the acquisition of an activated/memory phenotype and was largely (CCR5) or completely (CCR6) separated temporally from the initiation of cell division. In contrast, the regulation of two other chemokine receptors, CXCR3 and CXCR4, occurred in close parallel with the cell division process. Increased mRNA levels are likely to contribute to the enhanced surface expression of CCR5 and CCR6, but in the case of CCR6, translocation of intracellular stores of protein to the cell surface may be an additional mechanism of regulation. The up-regulation of CCR5 was more extensive than that of CCR6, as only approximately half the activated CCR5+ T cells coexpressed CCR6. The increased expression of CCR5 resulted in enhanced chemotaxis toward the CCR5 ligand macrophage-inflammatory protein-1beta/CCL4, but up-regulation of CCR6 did not result in altered chemotactic responsiveness to macrophage-inflammatory protein-3alpha/CCL20, suggesting an alternative function for this receptor.     

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.     

The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3

Chemokines and their receptors have been strongly implicated in the inflammatory process. However, their roles in excitotoxic brain injury are largely unknown. In this study we used C-C chemokine receptor 5 (CCR5) knockout (KO) mice to investigate the role of CCR5 in neurodegeneration induced by intranasal administration of the excitotoxin kainic acid (KA). Although KA treatment resulted in an increased CCR5 mRNA level in the hippocampi of wild-type mice, a CCR5 deficiency in KO mice did not affect either the clinical and pathological changes in vivo or the neuronal susceptibilities to KA insult in vitro. KA treatment stimulated mRNA expression of the monocyte chemoattractant protein-2 (MCP-2) in both the wild-type and KO mice. KA treatment did not affect mRNA levels for the macrophage inflammatory protein-1alpha (MIP-1alpha) or the regulated upon activation normal T cells expressed and secreted protein (RANTES) in either wild-type or CCR5 KO mice. CCR2 mRNA expression was undetectable in the hippocampi of wild-type mice regardless of KA treatment. In contrast, CCR5 KO mice showed CCR2 mRNA expression that was remarkably increased after KA treatment. KA treatment did not affect CCR3 mRNA expression in the wild-type mice, whereas KO mice showed both a higher basal level of CCR3 mRNA expression as well as a strong upregulation following KA treatment. These results indicate that CCR5 is not a necessary inflammatory mediator in KA induced neurodegeneration. The roles of CCR5 in excitotoxic injury in CCR5 deficient mice are compensated by increased CCR2 and CCR3 expression, which share the common MCP-2 ligand with CCR5.     

Experimental autoimmune encephalomyelitis on the SJL mouse: effect of gamma delta T cell depletion on chemokine and chemokine receptor expression in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that is a model for multiple sclerosis. Previously, we showed that depletion of gamma delta T cells significantly reduced clinical and pathological signs of disease, which was associated with reduced expression of IL-1 beta, IL-6, TNF-alpha, and lymphotoxin at disease onset and a more persistent reduction in IFN-gamma. In this study, we analyzed the effect of gamma delta T cell depletion on chemokine and chemokine receptor expression. In the CNS of control EAE mice, mRNAs for RANTES, eotaxin, macrophage-inflammatory protein (MIP)-1 alpha, MIP-1 beta, MIP-2, inducible protein-10, and monocyte chemoattractant protein-1 were detected at disease onset, increased as disease progressed, and fell as clinical signs improved. In gamma delta T cell-depleted animals, all of the chemokine mRNAs were reduced at disease onset; but at the height of disease, expression was variable and showed no differences from control animals. mRNA levels then fell in parallel with control EAE mice. ELISA data confirmed reduced expression of MIP-1 alpha and monocyte chemoattractant protein-1 at disease onset in gamma delta T cell-depleted mice, and total T cell numbers were also reduced. In normal CNS mRNAs for CCR1, CCR3, and CCR5 were observed, and these were elevated in EAE animals. mRNAs for CCR2 were also detected in the CNS of affected mice. Depletion of gamma delta T cells reduced expression of CCR1 and CCR5 at disease onset only. We conclude that gamma delta T cells contribute to the development of EAE by promoting an inflammatory environment that serves to accelerate the inflammatory process in the CNS.     

C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES).

The relationship of expression of the C-C chemokines eotaxin, eotaxin 2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4 to the kinetics of infiltrating eosinophils, basophils, and other inflammatory cells was examined in allergen-induced, late-phase allergic reactions in the skin of human atopic subjects. EG2+ eosinophils peaked at 6 h and correlated significantly with eotaxin mRNA and protein, whereas declining eosinophils at 24 h correlated significantly with eotaxin-2 and MCP-4 mRNA. In contrast, no significant correlations were observed between BB1+ basophil infiltrates, which peaked at 24 h, and expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 or elastase+ neutrophils (6-h peak), CD3+ and CD4+ T cells (24 h), and CD68+ macrophages (72 h). Furthermore, 83% of eosinophils, 40% of basophils, and 1% of CD3+ cells expressed the eotaxin receptor CCR3, while eotaxin protein was expressed by 43% of macrophages, 81% of endothelial cells, and 6% of T cells (6%). These data suggest that 1) eotaxin has a role in the early 6-h recruitment of eosinophils, while eotaxin-2 and MCP-4 appear to be involved in later 24-h infiltration of these CCR3+ cells; 2) different mechanisms may guide the early vs late eosinophilia; and 3) other chemokines and receptors may be involved in basophil accumulation of allergic tissue reactions in human skin.     

Chemokine receptor CCR4 on CD4+ T cells in juvenile rheumatoid arthritis synovial fluid defines a subset of cells with increased IL-4:IFN-gamma mRNA ratios

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-gamma mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4(-) cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-gamma than CD4+/CCR4(-) cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.     

Impact of CCR7 on the gastrointestinal field effect

Standardized intestinal manipulation (IM) leads to local bowel wall inflammation subsequently spreading over the entire gastrointestinal tract. Previously, we demonstrated that this so-called gastrointestinal field effect (FE) is immune mediated. This study aimed to investigate the role of CCR7 in IM-induced FE. Since CCR7 is expressed on activated dendritic cells and T cells and is well known to control their migration, we hypothesized that lack of CCR7 reduces or abolishes FE. Small bowel muscularis and colonic muscularis from CCR7(-/-) and wild-type (WT) mice were obtained after IM of the jejunum or sham operation. FE was analyzed by measuring gastrointestinal transit time of orally given fluorescent dextran (geometric center), colonic transit time, infiltration of MPO-positive cells, and circular smooth muscle contractility. Furthermore, mRNA levels of the inflammatory cytokine IL-6 were determined by RT-PCR. The number of dendritic cells and CD3+CD25+ T cells separately isolated from jejunum and colon was determined in mice after IM and sham operation. There was no significant difference in IL-6 mRNA upregulation in colonic muscularis between sham-operated WT and CCR7(-/-) mice after IM. Contractility of circular muscularis strips of the colon was significantly improved in CCR7(-/-) animals following IM and did not vary significantly from sham-operated animals. Additionally, inflammation of the colon determined by the number of MPO-positive cells and colonic transit time was significantly reduced in CCR7(-/-) mice. In contrast, jejunal contractility and jejunal inflammation of transgenic mice did not differ significantly from WT mice after IM. These data are supported by a significant increase of CD3+CD25+ T cells in the colonic muscularis of WT mice after IM, which could not be observed in CCR7(-/-) mice. These data demonstrate that CCR7 is required for FE and postoperative ileus. CCR7 indirectly affects FE by inhibiting migration of activated dendritic cells and of T cells from the jejunum to the colon. These findings support the critical role of the adaptive immune system in FE.     

Selective diapedesis of Th1 cells induced by endothelial cell RANTES.

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.     

CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-gamma-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8-/- mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.     

Differential secretion of cytokines and adhesion molecules by HUVEC stimulated with low concentrations of bleomycin

Bleomycin (BLM) is known to induce lung inflammation and subsequent fibrosis. Endothelial cells have been reported to play an important role, producing cytokines and adhesion molecules during the inflammatory process in pulmonary fibrosis. To examine the effects of BLM on endothelial cells, we investigated the expression profiles of various cytokines and adhesion molecules produced by endothelial cells stimulated with BLM. Increased expressions of interleukin-8 and monocyte chemoattractant protein-1 measured as protein as well as mRNA by human umbilical vein endothelial cells (HUVECs) were detected after exposure to BLM. Similarly, increased expressions of E-selectin and intercellular adhesion molecule-3 were detected both at the protein and mRNA levels. Under these conditions, a small but significant decrease of [3H]thymidine uptake was detected. These findings indicate that HUVEC were stimulated to secrete cytokines and express adhesion molecules in the presence of low concentrations of BLM which have a mildly inhibitory effect on cellular proliferation.     

Granulocyte-macrophage colony stimulating factor up-regulates CCR1 in human neutrophils

Neutrophils (polymorphonuclear leukocytes; PMN) are phagocytic cells instrumental in the clearance of infectious pathogens. Human PMN are commonly thought to respond primarily to chemokines from the CXC family. However, recent findings suggest that under specific cytokine activation conditions, PMN can also respond to some CC chemokines. In this study, the effect of GM-CSF, a well-characterized PMN priming and maturation factor, on CC-chemokine receptor (CCR) expression in PMN was investigated. Constitutive expression of CCR1 and CCR3 mRNA in PMN was detected by ribonuclease protection assay. Following incubation of PMN with GM-CSF (0.01-10 ng/ml; 6 h) CCR1 mRNA expression was rapidly (approximately 1 h) up-regulated. In contrast, no significant induction of CCR2, CCR3, CCR4, or CCR5 mRNA was observed. CCR1 protein was also up-regulated by GM-CSF stimulation. GM-CSF-induced up-regulation of CCR1 showed functional consequences because GM-CSF-treated PMN, but not control cells, responded to the CC chemokines macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-3, and RANTES in assays of chemotactic migration and intracellular calcium mobilization. These results suggest that PMN activated by the proinflammatory cytokine GM-CSF can change their receptor expression pattern and become responsive to CC chemokines.     

Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver

The role played by chemokines in regulating the selective recruitment of lymphocytes to different tissue compartments in disease is poorly characterized. In hepatitis C infection, inflammation confined to portal areas is associated with a less aggressive course, whereas T cell infiltration of the liver parenchyma is associated with progressive liver injury and cirrhosis. We propose a mechanism to explain how lymphocytes are recruited to hepatic lobules during bursts of necroinflammatory activity in chronic hepatitis C infection. We report here that lymphocytes infiltrating hepatitis C-infected liver express high levels of the chemokine receptors CCR5 and CXCR3. However, whereas the CCR5 ligands macrophage inflammatory protein-1alpha and -1beta were largely confined to vessels within portal tracts, the CXCR3 ligands IFN-inducible protein-10 and monokine-induced by IFN-gamma were selectively up-regulated on sinusoidal endothelium. In vitro, human hepatic sinusoidal endothelial cells secreted IFN-inducible protein-10 and monokine-induced by IFN-gamma in response to stimulation with IFN-gamma in combination with either IL-1 or TNF-alpha. This suggests that intrahepatic Th1 cytokines drive the increased expression of IFN-inducible protein-10 and monokine-induced by IFN-gamma and thereby promote the continuing recruitment of CXCR3-expressing T cells into the hepatic lobule in chronic hepatitis C infection.     

Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire

CD56, an adhesion molecule closely related to neural cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLS: Although CD56(+) cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56(+) populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16(+) NK cells from CD16(-) NK cells. CD16(+) NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1beta. CD16(-) NK cells were the only CD56(+) population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.     

Glomerular Endothelial Cell Injury and Damage Precedes That of Podocytes in Adriamycin-Induced Nephropathy

The role of podocytes in the development and progression of glomerular disease has been extensively investigated in the past decade. However, the importance of glomerular endothelial cells in the pathogenesis of proteinuria and glomerulosclerosis has been largely ignored. Recent studies have demonstrated that endothelial nitric oxide synthatase (eNOS) deficiency exacerbates renal injury in anti-GBM and remnant kidney models and accelerates diabetic kidney damage. Increasing evidence also demonstrates the importance of the glomerular endothelium in preventing proteinuria. We hypothesize that endothelial dysfunction can initiate and promote the development and progression of glomerulopathy. Administration of adriamycin (ADR) to C57BL/6 mice, normally an ADR resistant strain, with an eNOS deficiency induced overt proteinuria, severe glomerulosclerosis, interstitial fibrosis and inflammation. We also examined glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice, an ADR susceptible strain, by immunostaining, TUNEL and Western blotting. Interestingly, down-regulation of eNOS and the appearance of apoptotic glomerular endothelial cells occurred as early as 24 hours after ADR injection, whilst synaptopodin, a functional podocyte marker, was reduced 7 days after ADR injection and coincided with a significant increase in the number of apoptotic podocytes. Furthermore, conditioned media from mouse microvascular endothelial cells over-expressing GFP-eNOS protected podocytes from TNF-α-induced loss of synaptopodin. In conclusion, our study demonstrated that endothelial dysfunction and damage precedes podocyte injury in ADR-induced nephropathy. Glomerular endothelial cells may protect podocytes from inflammatory insult. Understanding the role of glomerular endothelial dysfunction in the development of kidney disease will facilitate in the design of novel strategies to treat kidney disease.