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

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The rise in cytosolic free Ca2+, shape change, superoxide formation, and granule exocytosis induced in human neutrophils by N-formyl-Met-Leu-Phe (fMLP) and by a newly discovered activating peptide, neutrophil-activating factor, termed NAF, were compared. NAF was effective in the concentration range of 0.1-10 nM and was 10- to 100-fold more potent than fMLP. In qualitative terms, the single responses to either stimulus were remarkably similar: they showed virtually identical onset and initial kinetics, and were all inhibited by pretreatment of the neutrophils with Bordetella pertussis toxin. In addition, the respiratory burst elicited by either stimulus was inhibited by 17-hydroxywortmannin and staurosporine. Two conclusions are drawn from these results: 1) neutrophil activation by NAF (as by fMLP) is dependent on a GTP-binding protein and on protein kinase C; 2) a similar, or even identical, mechanism of signal transduction must be assumed on stimulation of human neutrophils with NAF, fMLP, and other chemotactic agonists. Human monocytes, lymphocytes, and platelets did not show cytosolic free Ca2+ changes when exposed to NAF, which suggests that NAF is selective for the neutrophils.

The physical interaction of particulates with resident mononuclear phagocytes is a consistent feature in certain forms of crystal-induced inflammation. In this study, we observed that monosodium urate crystals stimulated the rapid release of neutrophil chemotactic activity from monocytes, and that this activity steadily increased over 24 hours. Because the release of monocytederived neutrophil chemotactic activity was markedly diminished by pretreatment of the monocytes with cycloheximide, and was completely removed from conditioned media by adsorption to heparin-agarose, we addressed the possibility that monocyte-derived neutrophi1 chemotactic factor/interleukin-8 (IL-8), a heparinbinding neutrophil-activating polypeptide, might modulate these activities. Urate crystal-induced IL-8 secretion from monocytes was verified by radioimmuno- assay. In addition, an IL-%specific antibody markedly inhibited the neutrophil-activating capacity of the conditioned media from monocytes activated by urate crystals, as well as by inflammatory silica crystals. Last, IL-8 was significantly increased in gouty synovial fluids (range 3.0-16.8 ng/ml, mean 8.4 ng/ml, n = 6) relative to osteoarthritic synovial fluids (range 1.1-1.7 nglml, mean 1.5 ng/ml, n = 6) (P = 0.006). We conclude that microcrystal-induced secretion of IL-8 by mononuclear phagocytes may mediate a number of forms of crystalinduced inflammation.