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.

In inflamed tissues, neutrophils are exposed to a variety of stimulatory molecules of different origin that condition their microbicidal and tissue-damaging activities . The best known stimuli are FMLP related to bacterial products (1), C5a formed upon complement activation (2), and two bioactive lipids, plateletactivating factor (PAF)' and leukotriene B4 (LTB4) (3-5). Products like PAF and LTB 4, which are released by stimulated phagocytes and have themselves stimulatory properties, may act as enhancers of antimicrobial defense and inflammation (6, 7). Of particular interest are products of activated mononuclear phagocytes, which are likely to influence neutrophil function at sites of chronic inflammation . We have cultured human blood mononuclear cells in the presence of LPS and different lectins and have tested the conditioned media for the presence of factors acting on the neutrophils . In this paper we describe a neutrophil-activating factor (NAF) produced by stimulated monocytes that induces exocytosis and the respiratory burst. The effects of NAF on human neutrophils are similar to those of FMLP and C5a, but appear mediated by a novel and selective surface rceptor .The results presented here were obtained over the current year using a partially purified preparation of NAF. Only upon completion of the present study did we succeed in purifying NAF to homogeneity . The identification of 32 of 50 presumed residues by amino acid sequencing showed that NAF is a novel polypeptide (8) . Pure NAF has an^-80-fold higher specific activity than the partially purified preparation . Both preparations, however, were qualitatively similar ; they induced exocytosis and a rapid and transient respiratory burst response in human neutrophils, and showed complete cross-desensitization upon repeated application to the cells.Volume

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.

The activities of six synthetic CC chemokines, MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta on human blood monocytes were studied. All CC chemokines elicited a bimodal migration response in vitro. Highest numbers of migrating cells were obtained with the monocyte chemotactic proteins (MCP) and RANTES, somewhat lower numbers with MIP-1 alpha, and only weak migration with MIP-1 beta. The most potent attractants were MCP-1 and MIP-1 alpha which reached maximum efficacy at 0.1 to 1 nM. All CC chemokines also induced the release of N-acetyl-beta-D-glucosaminidase from cytochalasin B-pretreated monocytes. The MCP were most effective (MCP-1 > MCP-3 > MCP-2), RANTES and MIP-1 alpha showed moderate (1/3 of MCP-1 activity), and MIP-1 beta only minimal activity. Cytosolic free Ca2+ changes and exocytosis were used to monitor receptor desensitization. Marked cross-desensitization was observed among MCP-1, MCP-2 and MCP-3 on the one hand, and RANTES, MIP-1 alpha and MIP-1 beta on the other, indicating receptor sharing within these two subgroups of CC chemokines. The responses to RANTES, MIP-1 alpha and MIP-1 beta were also moderately to markedly desensitized by pretreatment with MCP-1, MCP-2 or MCP-3, while the responses to the MCP were virtually unaffected by pretreatment with RANTES, MIP-1 alpha and MIP-1 beta. These results suggest that the MCP also interact with receptors recognized by RANTES, MIP-1 alpha and MIP-1 beta, but not vice versa. Binding studies were performed with radiolabeled MCP-1 or MIP-1 alpha. All MCP competed readily for labeled MCP-1 yielding a concentration-dependent sigmoidal displacement curve. Displacement with RANTES, MIP-1 alpha and MIP-1 beta was observed at higher concentrations, but was not complete. Radiolabeled MIP-1 alpha was displaced efficiently by MIP-1 alpha or MIP-1 beta, but only partially by RANTES. Of the MCP, only MC-3 completely displaced MIP-1 alpha, while only partial displacement was observed with MCP-1 and MCP-2.

SummaryAfter phagocytosis of yeast opsonized with IgG, neutrophil leukocytes (polymorphonuclear leukocytes [PMN]) expressed high levels of neutrophil-activating peptide 1/interleukin 8 (NAP-1/ID8) mRNA, which peaked after 3-5 h and were still elevated after 18 h. A similar but quantitatively less prominent effect was obtained with lipopolysaccharide (LPS) . After phagocytosis, but not after exposure to LPS, the PMN progressively released considerable amounts of NAP-1/ID8 into the culture medium (18.6-50 ng/ml in 18 h) . The peptide released was biologically active, as indicated by the transient elevation of cytosolic-free calcium in PMN exposed to aliquots of the culture supernatants, and desensitization by prestimulation of the cells with recombinant NAP-1/11A . By producing NAP 1/IIr8 at sites where they phagocytose invading microorganisms, PMN could enhance the recruitment of new defense cells.

Interleukin-8 (IL-8), a pro-inflammatory protein, has been shown by nuclear magnetic resonance (NMR) and x-ray techniques to exist as a homodimer. An IL-8 analog was chemically synthesized, with the amide nitrogen of leucine-25 methylated to selectivity block formation of hydrogen bonds between monomers and thereby prevent dimerization. This analog was shown to be a monomer, as assessed by analytical ultracentrifugation and NMR. Nevertheless, it was equivalent to IL-8 in assays of neutrophil activation, which indicates that the monomer is a functional form of IL-8.

Peptides corresponding to the N-terminal 9 residues of stromal cell-derived factor-1 (SDF-1) have SDF-1 activity. SDF-1, 1-8, 1-9, 1-9 dimer, and 1-17 induced intracellular calcium and chemotaxis in T lymphocytes and CEM cells and bound to CXC chemokine receptor 4 (CXCR4). The peptides had similar activities to SDF-1 but were less potent. Whereas native SDF-1 had halfmaximal chemoattractant activity at 5 nM, the 1-9 dimer required 500 nM and was therefore 100-fold less potent. The 1-17 and a 1-9 monomer analog were 4-and 36-fold, respectively, less potent than the 1-9 dimer. Both the chemotactic and calcium response of the 1-9 dimer was inhibited by an antibody to CXCR4. The basis for the enhanced activity of the dimer form of SDF-1, 1-9 is uncertain, but it could involve an additional fortuitous binding site on the 1-9 peptide in addition to the normal SDF-1, 1-9 site. A 1-9 analog, 1-9[P2G] dimer, was found to be a CXCR4 antagonist. Overall this study shows that the N-terminal peptides are CXCR4 agonists or antagonists, and these could be leads for high affinity ligands.Stromal cell-derived factor-1 (SDF-1) 1 is a member of the chemokine family of structurally related proteins with cell chemoattractant activity (1). Although many chemokines have pro-inflammatory roles, SDF-1 appears to have a fundamental role in the trafficking, export and homing of bone marrow cells (2, 3). It is produced constitutively, and particularly high levels are found in bone marrow stromal cells (4, 5). A basic physiological role is implied by the high level of conservation of the SDF-1 sequence between species (4). In vitro SDF-1 stimulates chemotaxis of a wide range of cells including monocytes and bone marrow-derived progenitor cells (2, 5). Particularly notable is its ability to stimulate a high percentage of resting and activated T lymphocytes (5, 6). It is the only known ligand for CXCR4, a 7-transmembrane receptor that has been variously described as LESTR (7), HUMSTR (8), and fusin (9). CXCR4 is widely expressed on cells of hemopoietic origin and is a major co-receptor for HIV-1 (9). Consistent with this dual role of CXCR4, SDF-1 blocks HIV-1 entry into CD4 ϩ cells (10, 11). The SDF-1 sequence indicates that it belongs to the CXC family of chemokines, but it has only about 22% identity with other chemokines (5). Despite the divergent primary structure, the recently described three-dimensional structure indicates that it has a similar fold to other chemokines (12). Furthermore, structure-activity analysis of SDF-1 (12) indicated the importance of N-terminal residues 1-8 for binding and of residues 1 and 2 for receptor activation. Residues 12-17 located in the loop region also contribute to binding. In the SDF-1 structure, the region N-terminal to the CXC motif is highly disordered, but the loop region immediately following the CXC motif is well defined at least in its backbone atoms. These two regions have been identified as being important in other CC and CXC chemokines (12-15). As with other chemokines, N-terminal modificati...