Pius Loetscher

RAPID AND COORDINATED SWITCH IN CHEMOKINE RECEPTOR EXPRESSION DURING DENDRITIC CELL MATURATION

Dendritic cells (DC) migrate into inflamed peripheral tissues where they capture antigens and, following maturation, to lymph nodes where they stimulate T cells. To gain insight into this process we compared chemokine receptor expression in immature and mature DC. Immature DC expressed CCR1, CCR2, CCR5 and CXCR1 and responded to their respective ligands, which are chemokines produced at inflammatory sites. Following stimulation with LPS or TNF‐α maturing DC expressed high levels of CCR7 mRNA and acquired responsiveness to the CCR7 ligand EBI1 ligand chemokine (ELC), a chemokine produced in lymphoid organs. Maturation also resulted in up‐regulation of CXCR4 and down‐regulation of CXCR1 mRNA, while CCR1 and CCR5 mRNA were only marginally affected for up to 40 h. However, CCR1 and CCR5 were lost from the cell surface within 3 h, due to receptor down‐regulation mediated by chemokines produced by maturing DC. A complete down‐regulation of CCR1 and CCR5 mRNA was observed only after stimulation with CD40 ligand of DC induced to mature by LPS treatment. These different patterns of chemokine receptors are consistent with “inflammatory” and “primary response” phases of DC function.

Lymphocyte traffic control by chemokines

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 chemo-kines. 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.

CCR5 is characteristic of Th1 lymphocytes

CD4+ lymphocytes can be assigned to two subsets. Th1 lymphocytes secrete interferon gamma (IFNγ) and lymphotoxin, promoting cell-mediated immunity to intracellular pathogens; and Th2 lymphocytes secrete interleukins 4 and 5 (IL-4 and IL-5), which function in allergy and humoral immunity to parasites. Th2 lymphocytes preferentially express the chemokine receptor CCR3 (refs 2 b3). We have studied the occurrence of two additional chemokine receptors, CCR5 and CXCR3, in human, antigen-specific CD4+ Th1 and Th2 cell clones.

Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1

The three‐dimensional structure of stromal cell‐derived factor‐1 (SDF‐1) was determined by NMR spectroscopy. SDF‐1 is a monomer with a disordered N‐terminal region (residues 1–8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution. Results with analogs showed that the N‐terminal eight residues formed an important receptor binding site; however, only Lys‐1 and Pro‐2 were directly involved in receptor activation. Modification to Lys‐1 and/or Pro‐2 resulted in loss of activity, but generated potent SDF‐1 antagonists. Residues 12–17 of the loop region, which we term the RFFESH motif, unlike the N‐terminal region, were well defined in the SDF‐1 structure. The RFFESH formed a receptor binding site, which we propose to be an important initial docking site of SDF‐1 with its receptor. The ability of the SDF‐1 analogs to block HIV‐1 entry via CXCR4, which is a HIV‐1 coreceptor for the virus in addition to being the receptor for SDF‐1, correlated with their affinity for CXCR4. Activation of the receptor is not required for HIV‐1 inhibition.

High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines.

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.

Interleukin-8 and the chemokine family

Two subfamilies of chemokines are distinguished depending on the arrangement of the first two of four conserved cysteines, which are either separated by one amino acid (CXC chemokines) or adjacent (CC chemokines). IL-8 and the other CXC chemokines act preferentially on neutrophils, while the CC chemokines (MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta) act on monocytes, but not neutrophils, and have additional activities toward basophil and eosinophil granulocytes, and T-lymphocytes. Several chemokine receptors have been identified, all of which belong to the seven-transmembrane-domain type and are coupled to G-proteins. The discovery of chemokines has provided the basis for the understanding of leukocyte recruitment and activation in inflammation and other disturbances of tissue homeostasis.

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.

Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes.

The responses of lymphocytes to six CC chemokines–MCP‐1, MCP‐2, MCP‐3, MIP‐1α, MIP‐1β, and RANTES–were studied using cloned human CD4+ and CD8+ T cells. All CC chemokines tested induced migration of both types of lymphocytes, whereas two CXC chemokines used as controls, IL‐8 and IP‐10, were inactive. The monocyte chemotactic proteins (MCP‐1, MCP‐2, and MCP‐3) showed a typically bimodal concentration dependence, and were considerably more effective than MIP‐1α, MIP‐10, or RANTES. All CC chemokines also induced a rapid and transient rise in cytosolic free Ca2+ in either type of T cell. The rise was prevented by Bordetella pertussis toxin treatment, indicating that G‐protein‐coupled receptors are involved in signaling. It was most pronounced with MCP‐1 and MCP‐3, which is in agreement with the efficacy of these chemokines as chemoattractants. The responses to MCP‐2, MIP‐1α, MIP‐1β, and RANTES were weaker, and no changes were obtained on stimulation with IL‐8 or IP‐10. Freshly isolated human blood lymphocytes were also tested, but neither migration nor Ca2+ changes were observed. Low numbers of high‐affinity receptors for MCP‐1 were found on CD4+ and CD8+ cells (<900 per cell, Kd <1 nM), and desensitization experiments showed that MCP‐1, MCP‐2, and MCP‐3 share receptors. Owing to their superior effectiveness on CD4+ and CD8+ T cells, the monocyte chemotactic proteins could play a major role in the recruitment of activated T lymphocytes.—Loetscher, P., Seitz, M., Clark‐Lewis, I., Baggiolini, M., Moser, B. Monocyte chemotactic proteins MCP‐1, MCP‐2, and MCP‐3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. 8: 1055‐1060; 1994.

Chemokines in inflammation and immunity

Since the discovery of interleukin 8 (IL-8), about 50 chemokines have been characterized. Originally, they were considered as inducible mediators of inflammation, but in recent years, several chemokines have been identified that are expressed constitutively and function in the physiological traffic and homing of leukocytes.

Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection

Publisher Summary This chapter reviews two research areas that attract unprecedented attention in a growing number of laboratories the functions of chemokines, their receptors in lymphocytes and lymphoid tissues, and the role of chemokines in HIV infection. Several new chemokines and receptors have been identified and information on their binding selectivity and on the chromosomal location of their genes has been extended in this chapter. Chemokines are not just the mediators of leukocyte recruitment for host defense (for example in inflammation and immune responses against foreign material), as was believed some years ago. Much information is still needed for the full picture, but the recent findings about new chemokines such as BCA-1, SLC, ELC, and their murine homologues that are expressed constitutively in restricted lymphoid tissue areas and attract functionally defined types of lymphocytes bearing the appropriate receptors are already viewed as paradigms for the homeostatic role of these attractants in the immune system. Application of live, but attenuated retrovirus particles that recognize and kill HIV-l-infected target cells is an attractive alternative strategy.