THE EXPRESSION OF CYTOKINE-INDUCED NEUTROPHIL CHEMOATTRACTANTS (CINC-1 AND CINC-2) IN RAT PERITONEAL-MACROPHAGES IS TRIGGERED BY FC-GAMMA RECEPTOR ACTIVATION - STUDY OF THE SIGNALING MECHANISM

The expression of cytokine-induced neutrophil chemoattractants (CINC-1 and CINC-2) mRNA was studied in rat peritoneal cells stimulated with insoluble IgG/ovalbumin immune complexes. A dose- and time-dependent i nduction was observed in adherent cells, which was more prominent than that induced by the lipid mediator platelet-activating factor (PAF), comparable to that observed in response to 10 mu g endotoxin in the ab sence of lipopolysaccharide (LPS)-binding protein, but lower than that produced by 1 mM dibutyryl cyclic AMP, a compound which stabilizes tr ansiently expressed genes containing AU-rich sequences in the 3' untra nslated region. Analysis of CINC-1 protein by specific enzyme-linked i mmunosorbent assay confirmed the presence of CINC-1 in the supernatant s at concentrations of similar to 4 nM, 4 h after addition of 100 mu g /ml immune complexes. CINC-2 beta protein was detectable at a lower co ncentration (similar to 0.3 nM) under the same conditions. Attempts to relate CINC-1 induction with the pathways for cytoplasmic signaling s howed a dissociation of Ca2+ mobilization and protein kinase C activat ion as judged from the small effect of thapsigargin and the lack of ef fect of phorbol ester. In contrast, these agents produced a marked mob ilization of arachidonate linked to the MAP kinase-dependent activatio n of cytosolic phospholipase A(2). The possible dependence of CINC-1 i nduction on the autocrine generation of lipid mediators was ruled out by a set of experiments including the use of the PAF receptor antagoni st BB823, and the analysis of the effect of free arachidonate and leuk otriene B-4 on CINC-1 induction. Surprisingly, the inhibitor of leukot riene synthesis MK-886 in the range of concentration 1-10 mu M inhibit ed CINC-1 induction by a mechanism that appears to be independent of i ts effect on eicosanoid production. Interestingly, CINC-1 induction ap peared to be related to protein tyrosine phosphorylation reactions on the basis of both the appearance of several tyrosine-phosphorylated pr otein bands in lysates from adherent peritoneal cells treated with imm une complexes and the complete blockade of CINC-1 induction by treatme nt with 1 mu M herbimycin A, an inhibitor of src protein tyrosine kina ses.