Homing and function of human skin gammadelta T cells and NK cells: relevance for tumor surveillance

Normal (noninflamed) human skin contains a network of lymphocytes, but little is known about the homing and function of these cells. The majority of alphabeta T cells in normal skin express CCR8 and produce proinflammatory cytokines. In this study we examined other subsets of cutaneous lymphocytes, focusing on those with potential function in purging healthy tissue of transformed and stressed cells. Human dermal cell suspensions contained significant populations of Vdelta1(+) gammadelta T cells and CD56(+)CD16(-) NK cells, but lacked the subsets of Vdelta2(+) gammadelta T cells and CD56(+)CD16(+) NK cells, which predominate in peripheral blood. The skin-homing receptors CCR8 and CLA were expressed by a large fraction of both cell types, whereas chemokine receptors associated with lymphocyte migration to inflamed skin were absent. Neither cell type expressed CCR7, although gammadelta T cells up-regulated this lymph node-homing receptor upon TCR triggering. Stimulation of cutaneous Vdelta1(+) gammadelta T cell lines induced secretion of large amounts of TNF-alpha, IFN-gamma, and the CCR8 ligand CCL1. In contrast to cutaneous alphabeta T cells, both cell types had the capacity to produce intracellular perforin and displayed strong cytotoxic activity against melanoma cells. We therefore propose that gammadelta T cells and NK cells are regular constituents of normal human skin with potential function in the clearance of tumor and otherwise stressed tissue cells.     

Production of MDC/CCL22 by human intestinal epithelial cells

The intestinal mucosa contains a subset of lymphocytes that produce Th2 cytokines, yet the signals responsible for the recruitment of these cells are poorly understood. Macrophage-derived chemokine (MDC/CCL22) is a recently described CC chemokine known to chemoattract the Th2 cytokine producing cells that express the receptor CCR4. The studies herein demonstrate the constitutive production of MDC/CCL22 in vivo by human colon epithelium and by epithelium of human intestinal xenografts. MDC/CCL22 mRNA expression and protein secretion was upregulated in colon epithelial cell lines in response to proinflammatory cytokines or infection with enteroinvasive bacteria. Inhibition of nuclear factor (NF)-kappaB activation abolished MDC/CCL22 expression in response to proinflammatory stimuli, demonstrating that MDC/CCL22 is a NF-kappaB target gene. In addition, tumor necrosis factor-alpha-induced MDC/CCL22 secretion was differentially modulated by Th1 and Th2 cytokines. Supernatants from the basal, but not apical, side of polarized epithelial cells induced a MDC/CCL22-dependent chemotaxis of CCR4-positive T cells. These studies demonstrate the constitutive and regulated production by intestinal epithelial cells of a chemokine known to function in the trafficking of T cells that produce anti-inflammatory cytokines.     

CCL5-CCR5-mediated apoptosis in T cells: Requirement for glycosaminoglycan binding and CCL5 aggregation

CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.     

Cross-talk between TCR and CCR7 signaling sets a temporal threshold for enhanced T lymphocyte migration

Lymphocyte homing to, and motility within, lymph nodes is regulated by the chemokine receptor CCR7 and its two ligands CCL19 and CCL21. There, lymphocytes are exposed to a number of extracellular stimuli that influence cellular functions and determine the cell fate. In this study, we assessed the effect of TCR engagement on CCR7-mediated cell migration. We found that long-term TCR triggering of freshly isolated human T cells through CD3/CD28 attenuated CCR7-driven chemotaxis, whereas short-term activation significantly enhanced CCR7-mediated, but not CXCR4-mediated, migration efficiency. Short-term activation most prominently enhanced the migratory response of naive T cells of both CD4 and CD8 subsets. We identified distinct roles for Src family kinases in modulating CCR7-mediated T cell migration. We provide evidence that Fyn, together with Ca(2+)-independent protein kinase C isoforms, kept the migratory response of naive T cells toward CCL21 at a low level. In nonactivated T cells, CCR7 triggering induced a Fyn-dependent phosphorylation of the inhibitory Tyr505 of Lck. Inhibiting Fyn in these nonactivated T cells prevented the negative regulation of Lck and facilitated high CCR7-driven T cell chemotaxis. Moreover, we found that the enhanced migration of short-term activated T cells was accompanied by a synergistic, Src-dependent activation of the adaptor molecule linker for activation of T cells. Collectively, we characterize a cross-talk between the TCR and CCR7 and provide mechanistic evidence that the activation status of T cells controls lymphocyte motility and sets a threshold for their migratory response.     

Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.     

Patterns of chemokine receptor expression on peripheral blood gamma delta T lymphocytes: strong expression of CCR5 is a selective feature of V delta 2/V gamma 9 gamma delta T cells

Gammadelta T lymphocytes play an important role in the immune defense against infection, based on the unique reactivity of human Vdelta2Vgamma9 gammadelta T cells toward bacterial phosphoantigens. Chemokines and their corresponding receptors orchestrate numerous cellular reactions, including leukocyte migration, activation, and degranulation. In this study we investigated the expression of various receptors for inflammatory and homeostatic chemokines on peripheral blood gammadelta T cells and compared their expression patterns with those on alphabeta T cells. Although several of the analyzed receptors (including CCR6, CCR7, CXCR4, and CXCR5) were not differentially expressed on gammadelta vs alphabeta T cells, gammadelta T cells expressed strongly increased levels of the RANTES/macrophage inflammatory protein-1alpha/-1beta receptor CCR5 and also enhanced levels of CCR1-3 and CXCR1-3. CCR5 expression was restricted to Vdelta2 gammadelta T cells, while the minor subset of Vdelta1 gammadelta T cells preferentially expressed CXCR1. Stimulation with heat-killed extracts of Mycobacterium tuberculosis down-modulated cell surface expression of CCR5 on gammadelta T cells in a macrophage-dependent manner, while synthetic phosphoantigen isopentenyl pyrophosphate and CCR5 ligands directly triggered CCR5 down-modulation on gammadelta T cells. The functionality of chemokine receptors CCR5 and CXCR3 on gammadelta T cells was demonstrated by Ca(2+) mobilization and chemotactic response to the respective chemokines. Our results identify high level expression of CCR5 as a characteristic and selective feature of circulating Vdelta2 gammadelta T cells, which is in line with their suspected function as Th1 effector T cells.     

CCL21/CCR7 prevents apoptosis via the ERK pathway in human non-small cell lung cancer cells

Previously, we confirmed that C-C chemokine receptor 7 (CCR7) promotes cell proliferation via the extracellular signal-regulated kinase (ERK) pathway, but its role in apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. A549 and H460 cells of NSCLC were used to examine the effect of CCL21/CCR7 on apoptosis using flow cytometry. The results showed that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant decline in the percent of apoptosis. Western blot and real-time PCR assays indicated that activation of CCR7 significantly caused upregulation of anti-apoptotic bcl-2 and downregulation of pro-apoptotic bax and caspase-3, but not p53, at both protein and mRNA levels. CCR7 small interfering RNA significantly attenuated these effects of exogenous CCL21. Besides, PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished these effects of CCL21/CCR7. Coimmunoprecipitation further confirmed that there was an interaction between p-ERK and bcl-2, bax, or caspase-3, particularly in the presence of CCL21. These results strongly suggest that CCL21/CCR7 prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax and caspase-3 potentially via the ERK pathway in A549 and H460 cells of NSCLC.     

Roles of SLC/CCL21 and CCR7 in human kidney for mesangial proliferation, migration, apoptosis, and tissue homeostasis

The release of chemokines by intrinsic renal cells is an important mechanism for the regulation of leukocyte trafficking during renal inflammation. The expression of chemokine receptors by intrinsic renal cells such as mesangial cells (MC) suggests an expanded role for chemokine-chemokine receptor biology in local immunomodulation and potentially glomerular homeostasis. By immunohistochemistry we found the chemokine receptor CCR7 expressed in a mesangial pattern while the CCR7 ligand SLC/CCL21 showed a podocyte-specific expression. CCR7 expression was further characterized by RT-PCR, RNase protection assays, and FACS analysis of cultured human MC, and was found to be constitutively present. Real-time PCR of microdissected glomeruli confirmed the expression of SLC/CCL21. A functional role for CCR7 was demonstrated for human MC migration and proliferation. A protective effect of SLC/CCL21 was shown for MC survival in Fas Ab-induced apoptosis. Finally, "wound healing" was enhanced in the presence of SLC/CCL21 in an in vitro injury model. The constitutive glomerular expression of CCR7 and its ligand SLC/CCL21 in adjacent cell types of the human kidney suggests novel biological functions of this chemokine/chemokine receptor pair and a potential role in processes involved in glomerular homeostasis and regeneration.     

Opposite fate of endocytosed CCR7 and its ligands: recycling versus degradation

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play a crucial role for the homing of lymphocytes and dendritic cells to secondary lymphoid tissues. Nevertheless, how CCR7 senses the gradient of chemokines and how migration is terminated are poorly understood. In this study, we demonstrate that CCR7(-GFP) is endocytosed into early endosomes containing transferrin receptor upon CCL19 binding, but less upon CCL21 triggering. Internalization of CCR7 was independent of lipid rafts but relied on dynamin and Eps15 and was inhibited by hypertonic sucrose, suggesting clathrin-dependent endocytosis. After chemokine removal, internalized CCR7 recycled back to the plasma membrane and was able to mediate migration again. In contrast, internalized CCL19 was sorted to lysosomes for degradation, showing opposite fate for endocytosed CCR7 and its ligand.     

A potential immune escape mechanism by melanoma cells through the activation of chemokine-induced T cell death

The immune system attempts to prevent or limit tumor growth, yet efforts to induce responses to tumors yield minimal results, rendering tumors virtually invisible to the immune system [1]. Several mechanisms may account for this subversion, including the triggering of tolerance to tumor antigens [2, 3], TGF-alpha or IL-10 production, downregulation of MHC molecules, or upregulation of FasL expression [4, 5]. Melanoma cells may in some instances use FasL expression to protect themselves against tumor-infiltrating lymphocytes (TIL) [4, 5]. Here, we show another, chemokine-dependent mechanism by which melanoma tumor cells shield themselves from immune reactions. Melanoma-inducible CCL5 (RANTES) production by infiltrating CD8 cells activates an apoptotic pathway in TIL involving cytochrome c release into the cytosol and activation of caspase-9 and -3. This process, triggered by CCL5 binding to CCR5, is not mediated by TNFalpha, Fas, or caspase-8. The effect is not unique to CCL5, as other CCR5 ligands such as CCL3 (MIP-1alpha) and CCL4 (MIP-1beta) also trigger TIL cell death, nor is it limited to melanoma cells, as it also operates in activated primary T lymphocytes. The model assigns a role to the CXC chemokine CXCL12 (SDF-1alpha) in this process, as this melanoma cell-produced chemokine upregulates CCL5 production by TIL, initiating TIL cell death.     

Selective induction of Th2-attracting chemokines CCL17 and CCL22 in human B cells by latent membrane protein 1 of Epstein-Barr virus

Chemokines are likely to play important roles in the pathophysiology of diseases associated with Epstein-Barr virus (EBV). Here, we have analyzed the repertoire of chemokines expressed by EBV-infected B cells. EBV infection of B cells induced expression of TARC/CCL17 and MDC/CCL22, which are known to attract Th2 cells and regulatory T cells via CCR4, and also upregulated constitutive expression of MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, which are known to attract Th1 cells and cytotoxic T cells via CCR5. Accordingly, EBV-immortalized B cells secreted these chemokines, especially CCL3, CCL4, and CCL22, in large quantities. EBV infection or stable expression of LMP1 also induced CCL17 and CCL22 in a B-cell line, BJAB. The inhibitors of the TRAF/NF-kappa B pathway (BAY11-7082) and the p38/ATF2 pathway (SB202190) selectively suppressed the expression of CCL17 and CCL22 in EBV-immortalized B cells and BJAB-LMP1. Consistently, transient-transfection assays using CCL22 promoter-reporter constructs demonstrated that two NF-kappa B sites and a single AP-1 site were involved in the activation of the CCL22 promoter by LMP1. Finally, serum CCL22 levels were significantly elevated in infectious mononucleosis. Collectively, LMP1 induces CCL17 and CCL22 in EBV-infected B cells via activation of NF-kappa B and probably ATF2. Production of CCL17 and CCL22, which attract Th2 and regulatory T cells, may help EBV-infected B cells evade immune surveillance by Th1 cells. However, the concomitant production of CCL3, CCL4, and CCL5 by EBV-infected B cells may eventually attract Th1 cells and cytotoxic T cells, leading to elimination of EBV-infected B cells at latency III and to selection of those with limited expression of latent genes.     

Bovine gamma delta T cell subsets express distinct patterns of chemokine responsiveness and adhesion molecules: a mechanism for tissue-specific gamma delta T cell subset accumulation

Subsets of gammadelta T cells localize to distinct tissue sites in the absence of exogenous Ag stimulation or development of effector/memory cells. Selective lymphocyte homing from the blood into tissues is controlled by a multistep process involving vascular and lymphocyte adhesion molecules, and G protein-linked chemokine receptors. The role of these mechanisms in the tissue tropism of gammadelta T cells is still poorly understood. In this study, we demonstrate that a subset of gammadelta T cells, most of which express an antigenically distinct TCR and are characterized by coexpression of CD8, selectively accumulated in tissues that expressed high levels of the mucosal vascular addressin, mucosal addressin cell adhesion molecule 1. These cells expressed higher levels of alpha(4)beta(7) integrins than other gammadelta T cell subsets and selectively migrated to the CCR7 ligand secondary lymphoid-tissue chemokine (CCL21). Integrin activation by CCL21 selectively increased CD8(+)gammadelta T cell binding to recombinant mucosal addressin cell adhesion molecule 1. These results suggest that the tropism of circulating CD8(+)gammadelta T cells for mucosal tissues is due, at least in part, to selective developmental expression of adhesion molecules and chemokine receptors.     

TNF family molecule LIGHT regulates chemokine CCL27 expression on mouse embryonic stem cell-derived dendritic cells through NF-kappaB activation

Cytokine LIGHT is a type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. It plays a role in inducing maturation of dendritic cells, such as upregulating CD80, CD86 expression on dendritic cells. However, whether LIGHT induces CC chemokine expression in DC and promotes their migration remains unknown. In this study, we found that esDC express CCR7 and CCR10 (the receptor of CCL27) upon the LIGHT stimulation. LIGHT also upregulates CCL27, but not CCL19 and CCL21 expression in esDC. The esDC migration potential has been increased in LIGHT activated DCs compared with control cells. LIGHT activated DCs autocrine CCL27 which regulate their migration as Blockage of CCL27 on esDC using neutralizing antibody reduces migration potential. In signaling study, we identified that LIGHT activated NF-kappaB in esDC and inhibition of NF-kappaB activation by specific inhibitor can partly attenuate the effect of LIGHT in regulation of CCL27 expression. Moreover, Shp-2 is required in LIGHT activated NF-kappaB because Knockdown of Shp-2 affects the NF-kappaB activation induced by LIGHT and consequently influences LIGHT mediated CCL27 expression. TRAF6 is critical in DC maturation in recent reports; however, knockdown of TRAF6 expression using siRNA did not alter CCL27 expression in LIGHT matured DCs. Our study demonstrates that LIGHT stimulation enhances CCL27 expression through activation of NF-kappaB in DCs.     

Expression and regulation of CCR1 by airway smooth muscle cells in asthma

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-alpha and, to a lesser extent, IFN-gamma resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein (MIP)-1alpha was identified 15 years ago as the first of now four members of the MIP-1 CC chemokine subfamily. These proteins termed CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL9/10 (MIP-1delta), and CCL15 (MIP-1gamma) according to the revised nomenclature for chemokines are produced by many cells, particularly macrophages, dendritic cells, and lymphocytes. MIP-1 proteins, which act via G-protein-coupled cell surface receptors (CCR1, 3, 5), e.g. expressed by lymphocytes and monocytes/macrophages (MPhi), are best known for their chemotactic and proinflammatory effects but can also promote homoeostasis. The encouraging results of preclinical studies in murine models of inflammation, i.e. asthma, arthritis, or multiple sclerosis, have led to the development of potent CCR3 and 5 antagonists, some of which are currently being tested in first clinical trials.     

CD16+ monocyte-derived macrophages activate resting T cells for HIV infection by producing CCR3 and CCR4 ligands

The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.