Marcel Loetscher

The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.

T cells infiltrating inflammatory sites are usually of the activated/memory type. The precise mechanism for the positioning of these cells within tissues is unclear. Adhesion molecules certainly play a role; however, the intricate control of cell migration appears to be mediated by numerous chemokines and their receptors. Particularly important chemokines for activated/memory T cells are the CXCR3 ligands IP-10 and Mig and the CCR5 ligands RANTES, macrophage inflammatory protein-1alpha, and macrophage inflammatory protein-1beta. We raised anti-CXCR3 mAbs and were able to detect high levels of CXCR3 expression on activated T cells. Surprisingly, a proportion of circulating blood T cells, B cells, and natural killer cells also expressed CXCR3. CCR5 showed a similar expression pattern as CXCR3, but was expressed on fewer circulating T cells. Blood T cells expressing CXCR3 (and CCR5) were mostly CD45RO+, and generally expressed high levels of beta1 integrins. This phenotype resembled that of T cells infiltrating inflammatory lesions. Immunostaining of T cells in rheumatoid arthritis synovial fluid confirmed that virtually all such T cells expressed CXCR3 and approximately 80% expressed CCR5, representing high enrichment over levels of CXCR3+ and CCR5+ T cells in blood, 35 and 15%, respectively. Analysis by immunohistochemistry of various inflamed tissues gave comparable findings in that virtually all T cells within the lesions expressed CXCR3, particularly in perivascular regions, whereas far fewer T cells within normal lymph nodes expressed CXCR3 or CCR5. These results demonstrate that the chemokine receptor CXCR3 and CCR5 are markers for T cells associated with certain inflammatory reactions, particularly TH-1 type reactions. Moreover, CXCR3 and CCR5 appear to identify subsets of T cells in blood with a predilection for homing to these sites.

Lymphocyte-specific chemokine receptor CXCR3: regulation, chemokine binding and gene localization

Expression of CXCR3, the receptor for the CXC chemokines IFN‐γ‐inducible 10‐kDa protein (IP10) and monokine induced by IFN‐γ (Mig), in human T lymphocytes and their responses to IP10 and Mig were analyzed. About 40 % of resting T lymphocytes (and low numbers of B cells and natural killer cells) stained positive for CXCR3 but these cells did not express CXCR3 transcripts and did not respond to these chemokines. However, treatment with IL‐2 with or without addition of phytohemagglutinin for 10 or more days resulted in cultures of fully responsive, CXCR3‐positive T lymphocytes. Treatment with anti‐CD3 antibodies in the presence or absence of soluble anti‐CD28 antibodies was inhibitory. Addition of chondroitin sulfate C to CXCR3‐expressing murine pre‐B cells allowed the determination of high‐affinity binding for Mig and IP10 with Kd of 0.9 –1.2 nM and 0.2 – 0.3 nM, respectively, and 1.3 × 104 binding sites per cell. The gene for CXCR3 was localized on human chromosome Xq13 which is in clear contrast to all other chemokine receptor genes, suggesting unique function(s) for this receptor and its ligands that may lie beyond their established role in T cell‐dependent immunity.

The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7

Secondary lymphoid‐tissue chemokine, SLC, also known as exodus‐2 and 6Ckine, is a novel CC chemokine with selectivity for T lymphocytes and preferential expression in lymphoid tissues. We have studied its production, receptor usage and biological activities. High levels of SLC mRNA were detected in lymph nodes, the gastrointestinal tract and several gland tissues, but no expression was found by Northern blot analysis in freshly isolated or stimulated blood monocytes and lymphocytes, or neutrophils and eosinophils. In situ hybridization revealed constitutive expression of SLC in the T cell areas and the marginal zone of follicles in lymph nodes and the mucosa‐associated lymphoid tissue, but not in B cell areas or sinuses. Comparison with immunocytochemical staining showed similarity between the in situ expression of SLC and the distribution of interdigitating dendritic cells but not with sinus‐lining dendritic cells, macrophages or T lymphocytes. SLC induced chemotaxis of T lymphocytes and its activity increased considerably when the cells were conditioned with IL‐2 or phytohemagglutinin (PHA). Under optimal conditions SLC had unusually high efficacy and induced the migration of up to 50 % of input T lymphocytes. SLC also induced Ca2+ mobilization in these cells. Similar responses were obtained with EBI1 ligand chemokine (ELC), and sequential stimulation with both chemokines led to cross‐desensitization, suggesting that SLC acts via the ELC receptor, CCR7. This was confirmed using murine pre‐B cells stably transfected with CCR7 which bound SLC with high affinity and showed chemotaxis and Ca2+ mobilization in response to both SLC and ELC. In T lymphocytes PHA and IL‐2, which enhanced chemotactic responsiveness, also markedly enhanced CCR7 expression. In contrast to all known chemokine receptors, up‐regulation of CCR7 by IL‐2 was transient. A maximum was reached in 2 – 3 days and expression returned to initial levels within 8 – 10 days. The present study shows that SLC is constitutively produced within the T cell areas of secondary lymphoid organs and attracts T lymphocytes via CCR7.

Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils.

BACKGROUND The chemokine eotaxin is produced at sites of allergic inflammation, binds selectively to the chemokine receptor CCR3 and attracts eosinophil and basophil leukocytes, which express high numbers of this receptor. Responses of T lymphocytes to eotaxin have not been reported so far. We have investigated the expression of CCR3 in T lymphocytes and analysed the properties and in vivo distribution of T lymphocytes expressing this receptor. RESULTS In search of chemokine receptors with selective expression in T lymphocytes, we have isolated multiple complementary DNAs (cDNAs) encoding CCR3 from a human CD4+ T-cell cDNA library. T-lymphocyte clones with selectivities for protein and non-protein antigens were analysed for expression of CCR3 and production of Th1- and Th2-type cytokines. Of 13 clones with surface CCR3, nine secreted enhanced levels of interleukin-4 and/or interleukin-5, indicating that CCR3 predominates in Th2-type lymphocytes. CCR3+ T lymphocytes readily migrated in response to eotaxin, and showed the characteristic changes in cytosolic free calcium. Immunostaining of contact dermatitis, nasal polyp and ulcerative colitis tissue showed that CCR3+ T lymphocytes are recruited together with eosinophils and, as assessed by flow cytometry, a large proportion of CD3+ cells extracted from the inflamed skin tissue were CCR3+. By contrast, CCR3+ T lymphocytes were absent from tissues that lack eosinophils, as demonstrated for normal skin and rheumatoid arthritis synovium. CONCLUSIONS We show that T lymphocytes co-localizing with eosinophils at sites of allergic inflammation express CCR3, suggesting that eotaxin/CCR3 represents a novel mechanism of T-lymphocyte recruitment. These cells are essential in allergic inflammation, as mice lacking mature T lymphocytes were insensitive to allergen challenge. Surface CCR3 may mark a subset of T lymphocytes that induce eosinophil mobilization and activation through local production of Th2-type cytokines.

Identification of CCR8, the Receptor for the Human CC Chemokine I-309

The nucleotide sequence for a putative chemokine receptor, termed TER1, ChemR1, or CKR-L1, was recently obtained by a polymerase chain reaction-based cloning technique. It encodes a protein of 355 amino acids that shows 32–45% sequence identity with human chemokine receptors. The gene was localized on human chromosome 3p21–24, the site for the genes for the five known CC chemokine receptors, suggesting that the natural ligand may be a CC chemokine. We have stably expressed this receptor in murine pre-B cells 300-19 and have tested their responsiveness to 20 human chemokines and some other potential agonists. The CC chemokine I-309 was the only agonist that selectively induced intracellular Ca2+ mobilization and chemotaxis in receptor-transfected 300-19 cells. Stromal cell-derived factor 1, which binds to murine CXCR4 expressed in parental as well as transfected 300-19 cells, served as positive control in the functional screening. The interaction of I-309 with TER1 was of high affinity as shown by125I-I-309 binding (K d of 1.2 nm) and transient [Ca2+] i changes at subnanomolar concentrations of agonist. Migration responses in receptor-transfected 300-19 cells was typically bimodal with maximal activity at 10 nm of I-309. These data demonstrate that TER1 (ChemR1 or CKR-L1) is the receptor for I-309, and we propose to call this receptor CCR8 in agreement with the current nomenclature for chemokine receptors. The expression of CCR8 in blood leukocytes and lymphocytes was analyzed by Northern blot. No transcripts were found in RNA from freshly isolated blood neutrophils, monocytes, cultured macrophages, and phytohemagglutinin-stimulated T lymphocytes, and a faint hybridization signal corresponding to the RNA species of 4 kb was obtained only with RNA from interleukin-2-treated T lymphocytes. CCR8 is unusual for its selectivity for a single chemokine, previously shown only for CXCR1 and CXCR4, which bind interleukin-8 and stromal cell-derived factor 1, respectively. Identification of the receptor for I-309 represents a significant progress in determining the function of I-309 in inflammation and disease.

Lymphocyte Responses to Chemokines

Today, almost three dozen human chemokines have been identified. The main function of these soluble proteins is the recruitment of leukocytes to sites of infection and inflammation. This review emphasizes the new developments in the field of lymphocyte responses to chemokines. Notably, it was shown that lymphocytes require stimulation to become responsive to chemokines, a process that is closely linked to chemokine receptor expression. As an exception, one chemokine, SDF-1, is a highly effective chemoattractant for non-activated T lymphocytes and progenitor B cells. Of particular interest are the chemokines IP10 and Mig which bind to a receptor with selective expression in activated T lymphocytes and, therefore, may be critical mediators of T lymphocyte migration in T cell-dependent immune-responses. All other chemokines with activities in lymphocytes do also induce responses in monocytes and granulocytes. The involvement of chemokine receptors in HIV infection is briefly mentioned, while other interesting areas in chemokine research, such as hematopoiesis and angiogenesis, are not discussed.

TYMSTR, a putative chemokine receptor selectively expressed in activated T cells, exhibits HIV-1 coreceptor function

BACKGROUND Chemokines bind to specific receptors and mediate leukocyte migration to sites of inflammation. Recently, some chemokine receptors, notably CXCR4 and CCR5, have been shown to be essential fusion factors on target cells for infection by human immunodeficiency virus (HIV); the chemokines bound by these receptors have also been shown to act as potent inhibitors of HIV infection. Here, we describe the isolation of a novel, putative chemokine receptor. RESULTS We have isolated the cDNA for a putative human chemokine receptor, which we have termed TYMSTR (T-lymphocyte-expressed seven-transmembrane domain receptor). The TYMSTR gene is localized to human chromosome 3 and encodes a protein that has a high level of identity with chemokine receptors. TYMSTR mRNA was selectively expressed in interleukin-2-stimulated T lymphocytes but not in freshly isolated lymphocytes and leukocytes or related cell lines. The natural ligand for TYMSTR was not identified among 32 human chemokines and other potential ligands. Cells co-expressing TYMSTR and human CD4 fused with cells expressing envelope glycoproteins of macrophage (M)-tropic HIV-1 as well as T-cell line (T)-tropic HIV-1 isolates. Addition of infectious, T-tropic HIV-1 particles to TYMSTR/CD4-expressing cells resulted in viral entry and proviral DNA formation. CONCLUSIONS Our findings demonstrate that TYMSTR, in combination with CD4, mediates HIV-1 fusion and entry. The high-level expression of TYMSTR in CD4(+) T lymphocytes and the selectivity of this receptor for T-tropic and M-tropic HIV-1 strains indicates that TYMSTR might function as HIV coreceptor at both early and late stages of infection.

Soluble IL‐1 receptor type I binds to human dermal fibroblasts and induces calcium flux

Soluble cytokine receptors appear to modify ligand concentrations by stabilizing ligands or by specifically inhibiting interactions of ligands with their membrane‐bound receptors. Here we describe a new function of the soluble interleukin‐1 receptor type I (IL‐1sR I). This receptor induced a transient rise of intracellular free calcium concentration in human dermal fibroblasts in a dose‐dependent fashion. Mobilization of calcium by IL‐1sR I was abolished in the presence of an equimolar concentration of IL‐1 receptor antagonist (IL‐1ra). Neutralizing antibodies against IL‐1β also abolished calcium mobilization stimulated with IL‐1sR I indicating that IL‐1β is involved. IL‐1sR I bound with high affinity (K d 1–2 nM) to the fibroblasts. In addition, IL‐1sR I enhanced expression of IL‐6 and IL‐8 mRNA. The observation that IL‐1sR I can act as a ligand and agonist for membrane IL‐1 extends the concept of the ligand‐receptor functions of both IL‐1 and IL‐1sR I and adds a new dimension to the cytokine network.