No abstract

Interleukin 8, the first chemokine to be characterized, was discovered nearly ten years ago. Today, more than 30 human chemokines are known. They are often upregulated in inflammation and act mainly on leukocytes inducing migration and release responses. The present review deals largely with the new developments of the last three years. Several structural studies have shown that most chemokines form dimers. The dimers, however, dissociate upon dilution, and the monomers constitute the biologically active form. Chemokine activities are mediated by seven-transmembrane-domain, G protein coupled receptors, five of which were discovered in the past three years. The primary receptor-binding domain of all chemokines is near the NH2 terminus, and antagonists can be obtained by truncation or substitutions in this region. Major progress has been made in the understanding of chemokine actions on T lymphocytes that respond to several CC chemokines but also to IP10 and Mig, two CXC chemokines that selectively attract T cells via a novel receptor. Effects of chemokines on angiogenesis and tumor growth have been reported, but the data are still contradictory and the mechanisms unknown. Of considerable interest is the recent discovery that some chemokines function as HIV-suppressive factors by interacting with chemokine receptors which, together with CD4, were recognized as the binding sites for HIV-1.

A PIJfATIVE chemokine receptor that we previously cloned and termed LESTR 1 has recently been shown to function as a coreceptor (termed fusin) for lymphocyte-tropic HIV-1 strains 2 • Cells expressing CD4 became permissive to infection with T -cellline-adapted HIV-1 strains of the syncytium-i.nducing phenotype after transfection with LESTR/fusin complementary DNA. We report here the identification of a human chemokine of the CXC type, stromal cell-derived factor 1 (SDF-1), as the naturaJ ligand for LESTR/fusin, and we propose the term CXCR-4 for this receptor, in keeping with the new cbemokine-receptor nomenclature. SDF-1 activates Chinese hamster ovary (CHO) cells transfected with CXCR-4 eDNA as well as blood leukocytes and lymphocytes. In cell lines expressing CXCR-4 and CD4, and in blood lymphocytes, SDF-1 is a powerful inhibitor of infection by lymphocyte-tropic HIV-1 strains, whereas the CC chemokines RANTES, MIP-1a and MIP-1~, which were shown previously to prevent infection with primary, monocyte-tropic viruses 3 , are inactive. In combination with CC chemokines, which block the infection with monocyte/macrophage-tropic viruses, SDF-1 could help to decrease virus load and prevent the emergence of the syncytium-inducing viruses which are characteristic of the late stages of AIDS 4• LESTR (leukocyte-expressed seven-transmembrane-domain receptor) is an orphan receptor with structural similarity to chemokine receptors. Despite extensive testing of a large number of chemokines, the ligand for LESTR remained elusive 1 • Murine SDF-1 was described as a factor that is produced by bonemarrow stromal cells and shown to induce proliferation of B-cell progenitorsM as well as recruitment of T cells 7 • The human homologue, which was cloned subsequently, is virtually identical to murine SDF-1 (see Methods). SDF-1 is a CXCchemokine with the typical four-cysteine motif and the first two cysteines separated by one amino acid 8 • When human SDF-1 was tested on the CH0-1C2 clone which stably expresses LESTR, a transient rise of cytosolic free Ca 2 + ([Ca 2 +];) was observed (Fig. 1a). This response, which is characteristic of the action of chemokines on blood leukocytes, was not observed with parental CHO cells. Other chemokines, including RANTES (for regulation-upon-activation, normal T expressed and secreted) macrophage inflammatory protein (MIP), MIP-1o: and MIP-1~, were not active. Monocytes, neutrophils and phytohaemagglutinin (PHA)-activated peripheral-blood lymphocytes (PBLs) were also stimulated by SDF-1, as shown by [Ca 2 +]; changes and chemotaxis (Fig. 1b, d). Real-time recordings of Ca 2 + mobilization after sequential stimulation are a reliable way to assess receptor usage by chemokines 8 • Stimulation with a chemokine (at saturating concentrations) causes receptor desensitization, and no response is observed when the cells are restimulated within a short time by a chemokine acting on the same receptor. As shown in Fig. lc, monocytes stimulated with SDF-1 remained fully responsive to subsequent stimulation with ...

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.

Leukocyte traffic through secondary lymphoid tissues is finely tuned by chemokines. We have studied the functional properties of a human T cell subset marked by the expression of CXC chemokine receptor 5 (CXCR5). Memory but not naive T cells from tonsils are CXCR5+ and migrate in response to the B cell–attracting chemokine 1 (BCA-1), which is selectively expressed by reticular cells and blood vessels within B cell follicles. Tonsillar CXCR5+ T cells do not respond to other chemokines present in secondary lymphoid tissues, including secondary lymphoid tissue chemokine (SLC), EBV-induced molecule 1 ligand chemokine (ELC), and stromal cell–derived factor 1 (SDF-1). The involvement of tonsillar CXCR5+ T cells in humoral immune responses is suggested by their localization in the mantle and light zone germinal centers of B cell follicles and by the concomitant expression of activation and costimulatory markers, including CD69, HLA-DR, and inducible costimulator (ICOS). Peripheral blood CXCR5+ T cells also belong to the CD4+ memory T cell subset but, in contrast to tonsillar cells, are in a resting state and migrate weakly to chemokines. CXCR5+ T cells are very inefficient in the production of cytokines but potently induce antibody production during coculture with B cells. These properties portray CXCR5+ T cells as a distinct memory T cell subset with B cell helper function, designated here as follicular B helper T cells (TFH).

SummaryA human receptor that is selective for the CXC chemokines IP10 and Mig was cloned and characterized. The receptor cDNA has an open reading frame of 1104-bp encoding a protein of 368 amino acids with a molecular mass of 40,659 dalton. The sequence includes seven putative transmembrane segments characteristic of G-protein coupled receptors. It shares 40.9 and 40.3% identical amino acids with the two IL-8 receptors, and 34.2-36.9% identity with the five known CC chemokine receptors. The IPl0/Mig receptor is highly expressed in IL-2-activated T lymphocytes, but is not detectable in resting T lymphocytes, B lymphocytes, monocytes and granulocytes. It mediates Ca 2+ mobilization and chernotaxis in response to IP10 and Mig, but does not recognize the CXC-chemokines IL-8, GROom, NAP-2, GCP-2, ENA78, PF4, the CC-chemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-lot, MIP-I[~, RANTES, I309, eotaxin, nor lymphotactin. The exclusive expression in activated T-lymphocytes is of high interest since the receptors for chemokines which have been shown so far to attract lymphocytes, e.g., MCP-1, MCP-2, MCP-3, MIP-lot, MIP-I[3, and R_ANTES, are also found in monocytes and granulocytes. The present observations suggest that the IP10/Mig receptor is involved in the selective recruitment of effector T cells.

In contrast to the remarkable chemokine responses of phagocytes and monocytes that were documented early on, lymphocytes have been considered for a long time to be poor targets for chemokine action. This view has changed dramatically with the discovery that peripheral blood T cells need to be activated before they can migrate in response to inflammatory chemokines. These chemokines do not act on the bulk of resting T cells that are in circulation. The identification of a new group of chemokines that selects resting, as opposed to effector, T and B cells was very exciting. These inflammation-unrelated chemokines affect transendothelial migration and localization of progenitor and mature lymphocytes in lymphoid and nonlymphoid tissues. Here, we summarize the current view of chemokine-mediated lymphocyte traffic and focus on the molecular mechanisms by which T cell responses to chemokines are modulated. Recent developments in this area justify the hypothesis that the distinct migration patterns of lymphocytes throughout their life cycle--that is, during lymphopoiesis, antigen-dependent priming, inflammation and immune surveillance--are finely tuned by changing sets of chemokines that are selective for developmentally regulated chemokine receptors. Thus, the chemokine system assures that cell traffic during inflammatory responses occurs in the proper spatial and temporal fashion and disturbance of this system, therefore, can lead to inflammatory disease.

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2–activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-γ–inducible 10-kD protein (IP-10) and IFN-γ– induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-γ and IL-1 together results in an ∼400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1–stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.