T cell chemokine receptor expression in aging

Changes in chemokine receptor expression are important in determining T cell migration and the subsequent immune response. To better understand the contribution of the chemokine system in immune senescence we determined the effect of aging on CD4(+) T cell chemokine receptor function using microarray, RNase protection assays, Western blot, and in vitro chemokine transmigration assays. Freshly isolated CD4(+) cells from aged (20-22 mo) mice were found to express a higher level of CCR1, 2, 4, 5, 6, and 8 and CXCR2-5, and a lower level of CCR7 and 9 than those from young (3-4 mo) animals. Caloric restriction partially or completely restored the aging effects on CCR1, 7, and 8 and CXCR2, 4, and 5. The aging-associated differences in chemokine receptor expression cannot be adequately explained by the age-associated shift in the naive/memory or Th1/Th2 profile. CD4(+) cells from aged animals have increased chemotactic response to stromal cell-derived factor-1 and macrophage-inflammatory protein-1alpha, suggesting that the observed chemokine receptor changes have important functional consequences. We propose that the aging-associated changes in T cell chemokine receptor expression may contribute to the different clinical outcome in T cell chemokine receptor-dependent diseases in the elderly.     

Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK

Thymus-expressed chemokine (TECK) has been reported to chemoattract dendritic cells, thymocytes, and activated macrophages. Here, we show that TECK is a specific agonist for a human orphan receptor called GPR-9-6. We have determined the cDNA sequence of human GPR-9-6 and cloned the corresponding murine cDNA. Human and murine GPR-9-6 expression is very high in the thymus and low in lymph nodes and spleen. RT-PCR analysis of murine GPR-9-6 expression on murine FACS-sorted thymocyte subpopulations showed that this gene is expressed in both immature and mature T cells. Additions of human or murine TECK to HEK 293/human GPR-9-6 and HEK 293/murine GPR-9-6 transfectants provoked intracytoplasmic calcium mobilization. Human TECK also induced the in vitro migration of HEK 293/human GPR-9-6 cells. These results confirm that GPR-9-6 is a specific receptor for TECK. According to the established nomenclature system, we propose to rename GPR-9-6 as CC chemokine receptor 9 (CCR9).     

The chemokine receptor, CCR5

The chemokine receptor, CCR5, is a G protein coupled receptor responsible for some of the effects of the chemokines CCL3, CCL4 and CCL5. It is also one of the co-receptors for the entry of human immunodeficiency virus-1 (HIV-1) into cells. Regulation of CCR5 number on cells is, therefore, important for determining the infection rate by HIV-1.     

Murine CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regulated following pre-TCR signaling

Chemokines are likely to play an important role in regulating the trafficking of developing T cells within the thymus. By using anti-CD3varepsilon treatment of recombinase-activating gene 2 (Rag2-/-) mice to mimic pre-TCR signaling and drive thymocyte development to the double positive stage, we have identified murine GPR-9-6 as a chemokine receptor whose expression is strongly induced following pre-TCR signaling. GPR-9-6 mRNA is present at high levels in the thymus, and by RT-PCR analysis its expression is induced as normal thymocytes undergo the double negative to double positive transition. Furthermore we show that TECK (thymus-expressed chemokine), a chemokine produced by thymic medullary dendritic cells, is a functional ligand for GPR-9-6. TECK specifically induces a calcium flux and chemotaxis of GPR-9-6-transfected cells. In addition, TECK stimulates the migration of normal double positive thymocytes, as well as Rag2-/- thymocytes following anti-CD3varepsilon treatment. Hence, GPR-9-6 has been designated as CC chemokine receptor 9 (CCR9). Our results suggest that TECK delivers signals through CCR9 important for the navigation of developing thymocytes.     

Identification of chemokine receptor CCR4 antagonist

The present study reports the identification and hits to leads optimization of chemokine receptor CCR4 antagonists. Compound 12 is a high affinity, non-cytotoxic antagonist of CCR4 that blocks the functional activity mediated by the receptor.     

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.     

Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4(+) T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4(+) T cells into the CNS of MHV-infected mice was investigated. CD4(+) T cells from immunized CCR5(+/+) and CCR5(-/-) mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5(+/+)-derived CD4(+) T cells to MHV-infected RAG1(-/-) mice resulted in CD4(+)-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4(+) T cells from CCR5(-/-) mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1(-/-) recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5(-/-)-derived CD4(+) T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4(+) T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4(+) T cells to the CNS following MHV infection.     

Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.     

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.     

pH-independent endocytic cycling of the chemokine receptor CCR5

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.     

A role for CCR9 in T lymphocyte development and migration

CCR9 mediates chemotaxis in response to CCL25/thymus-expressed chemokine and is selectively expressed on T cells in the thymus and small intestine. To investigate the role of CCR9 in T cell development, the CCR9 gene was disrupted by homologous recombination. B cell development, thymic alphabeta-T cell development, and thymocyte selection appeared unimpaired in adult CCR9-deficient (CCR9(-/-)) mice. However, competitive transplantation experiments revealed that bone marrow from CCR9(-/-) mice was less efficient at repopulating the thymus of lethally irradiated Rag-1(-/-) mice than bone marrow from littermate CCR9(+/+) mice. CCR9(-/-) mice had increased numbers of peripheral gammadelta-T cells but reduced numbers of gammadeltaTCR(+) and CD8alphabeta(+)alphabetaTCR(+) intraepithelial lymphocytes in the small intestine. Thus, CCR9 plays an important, although not indispensable, role in regulating the development and/or migration of both alphabeta(-) and gammadelta(-) T lymphocytes.     

An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.     

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.     

CCR7 expression and memory T cell diversity in humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.     

Core exploration in optimization of chemokine receptor CCR4 antagonists

The design, synthesis, and SAR studies of 'core' variations led to identification of novel, selective, and potent small molecule antagonist (22) of the CC chemokine receptor-4 (CCR4) with improved in vitro activity and liability profile. Compound 22 was efficacious in a murine allergic inflammation model (ED50 approximately 10 mg/kg).     

Evidence for NK cell subsets based on chemokine receptor expression

To help understand the role of chemokines in NK cell trafficking, we determined the chemokine receptor profiles of three different human NK cell lines and freshly isolated primary human NK cells. The cell lines overlapped in their chemokine receptor profiles: CXCR3 and CXCR4 were expressed by all three lines, whereas CCR1, CCR4, CCR6, CCR7, and CX3CR1 were expressed by only one or two of the lines, and no other chemokine receptors were detected. Freshly isolated primary NK cells were found to express CXCR1, CXCR3, and CXCR4, and to contain subsets expressing CCR1, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR5, and CXCR6. With the exception of CCR4, these chemokine receptors were expressed at higher percentages by CD56(bright) NK cells than by CD56(dim) NK cells. In particular, CCR7 was expressed by almost all CD56(bright) NK cells but was not detected on CD56(dim) NK cells. CCR9 and CXCR6 have not been described previously on primary NK cells. These results indicate that within both the CD56(bright) and CD56(dim) NK cell populations, subsets with the capacity for differential trafficking programs exist, which likely influence their functions in innate and adaptive immunity.     

The CC chemokine CCL20 and its receptor CCR6

CCL20, alternatively named liver and activation-regulated chemokine (LARC), macrophage inflammatory protein-3alpha (MIP-3alpha) or Exodus-1, is the only chemokine known to interact with CC chemokine receptor 6 (CCR6), a property shared with the antimicrobial beta-defensins. The ligand-receptor pair CCL20-CCR6 is responsible for the chemoattraction of immature dendritic cells (DC), effector/memory T-cells and B-cells and plays a role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and rheumatoid arthritis. In this review, the discovery, the gene and protein structure, the in vitro biological activities, the cell and inducer specific expression and the tissue distribution of CCL20 and CCR6 are discussed.     

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.