We have identified an indirect pathway by which the neutrophil chemoattractant IL-8 subsequently attracts mononuclear cells. IL-8 degranulates neutrophils resulting in the release of -defensins 1 and 2 (HNP-1 and HNP-2) which are T-cell chemoattractants and a chemoattractant of monocytes, neutrophils, and cathepsin G. The biological relevance of these in vitro results were reinforced by observations that subcutaneous injections of defensins or cathepsin G result in the local accumulation of inflammatory cells. Furthermore, defensins or cathepsin G, when injected into mice together with antigen, act as adjuvants that augment antibody response to the antigens. Thus, proinflammatory chemokines can amplify their effects by releasing a variety of effector molecules that elicit additional inflammatory and immune cells to engage in host defense. Presumably the immunostimulating effect of -defensins is based in part on its T cell chemotactic activity. In view of the adjuvant effect of -defensins we also investigated the effect of and defensins on antigen presenting cells (APC). We observed both to be chemotactic for ?immature? human, but not ?mature?, dendritic cells (DC). Furthermore the -defensins were chemotactic for the subset of CD4+CD45+RA (resting naive) T cells, while -defensins specifically attracted only CD4+CD45+RO (resting memory) T cells. The latter subpopulation is known to express the CCR6 receptor for the chemokine LARC. We established that ?immature? DC derived from human peripheral blood monocytes when cultured with GM-CSF, IL-4 and TGF also express functional CCR6 and are chemoattracted by LARC. We were able to show ?that (1) preincubation of DC with LARC and -defensins resulted in mutual desensitization, that (2) -defensins compete with I125 LARC for binding sites, (3) anti-CCR6 blocks the chemotactic response of DC to -defensins and to LARC and (4) that -defensins are selectively chemotactic for HEK293 cells transfected to overexpress CCR6. This allowed us to conclude that CCR6 is a receptor for chemokines. Thus chemokines in addition to having antimicrobial activities also have chemokine-like activities and provide a signal originating from epithelial cells that helps initiate adaptive immune responses. We are at present investigating the identity of the receptor for -defensins.Chemokines also participate in the regulation of angiogenesis. ELR+ members of the CXC subfamily of chemokines such as IL-8 have been reported to be angiogenic, while ELR-CXC chemokines have been shown to have angiostatic activities. Since angiogenesis is important in tissue remodeling, wound repair and tumorigenesis, we initiated studies of angiogenesis-dependent effect of chemokines on tumor growth. The basis for the angiogenic and angiostatic effects of chemokines are mysterious, since reports concerning expression of appropriate receptors and chemotactic responses by endothelial cells (EC) are contradictory. We used an RNAse protection assay to detect CXC chemokine receptor expression by HUVEC. We confirmed that HUVEC express only very low levels of mRNA for the receptors for ELR+ CXC chemokines (CXCR1 and 2) or for CXCR3, the receptor for ELR- CXC chemokines. However we did detect modest levels of mRNA expression for CXCR4, which was markedly upregulated by incubating HUVEC with the known angiogenic growth factors, VEGF and or bFGF. FACS analysis and ligand binding studies revealed these growth factors also increased cell surface CXCR4 expression, but not the production of chemokines. These receptors were functional and the capacity of SDF-1 and SDF-1 , the ligands for CXCR4, to induce chemotaxis was markedly augmented by bFGF and VEGF. Conversely SDF-1 induced 7 to 13 fold increased production of VEGF by HUVEC. Neutralizing anti-CXCR4 antibodies inhibited the chemotactic response of HUVEC to SDF-1 , but not to VEGF, suggesting that the SDF-1 -CXCR4 interactions occurs down-stream from the VEGF effects. Injection of human SDF-1 subcutaneously into mice induced neovascularization at the injection site. Consequently, even though SDF-1 is an ELR- CXC chemokine, it is angiogenic. Preliminary studies reveal that about 50% of human breast tumors and melanomas produce SDF-1 . We plan to investigate the effect of inhibiting SDF-1 -CXCR4 interactions by themselves and in combination with other antitumor agents on the growth and metastatic spread of human tumors in SCID mice.Following the identification of CCR5 as the coreceptor for invasion of cells by monotropic strains of HIV-1, genetic analysis revealed that a proportion of resistant individuals were homozygous for the CCR5-delta 32 mutation resulting in failure of their cells to express this receptor. Furthermore, HIV-1 individuals heterozygous for CCR5-32 had a reduced incidence of development of AIDS lymphoma. Genetic analysis also has identified a variety of single amino acid substitutions in CCR5, but the frequency of such mutations was too low to permit population analysis. We therefore analyzed the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of mutants: (1) Mutations in the first extracellular N-terminal domain of CCR5 severely reduced specific ligand binding, chemokine induced chemotaxis and blocked HIV-1 entry. (2) One of the extracellular domain variants near the cell membrane, A29S, when co-expressed with CD4, still supported HIV-1 infection but not chemokine responses. (3) The transmembrane region variants of CCR5 support monotrophic HIV-1 infection. Mutations in the first and second transmembrane domains increase RANTES binding affinity, but did not affect MIP1b binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, while mutations in the transmembrane domain affect only the response to chemokine ligands. Studies of these natural receptor variants are therefore providing relevant and unexpected information concerning the structure-function relationships of receptor initiated signal transduction.

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Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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