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 induction 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 micrograms endotoxin in the absence of lipopolysaccharide (LPS)-binding protein, but lower than that produced by 1 mM dibutyryl cyclic AMP, a compound which stabilized transiently expressed genes containing AU-rich sequences in the 3' untranslated region. Analysis of CINC-1 protein by specific enzyme-linked immunosorbent assay confirmed the presence of CINC-1 in the supernatants at concentrations of approximately 4 nM, 4 h after addition of 100 micrograms/ml immune complexes. CINC-2 beta protein was detectable at a lower concentration (approximately 0.3 nM) under the same conditions. Attempts to relate CINC-1 induction with the pathways for cytoplasmic signaling showed a dissociation of Ca2+ mobilization and protein kinase C activation as judged from the small effect of thapsigargin and the lack of effect of phorbol ester. In contrast, these agents produced a marked mobilization of arachidonate linked to the MAP kinase-dependent activation of cytosolic phospholipase A2. The possible dependence of CINC-1 induction on the autocrine generation of lipid mediators was ruled out by a set of experiments including the use of the PAF receptor antagonist BB823, and the analysis of the effect of free arachidonate and leukotriene B4 on CINC-1 induction. Surprisingly, the inhibitor of leukotriene synthesis MK-886 in the range of concentration 1-10 microM inhibited CINC-1 induction by a mechanism that appears to be independent of its effect on eicosanoid production. Interestingly, CINC-1 induction appeared to be related to protein tyrosine phosphorylation reactions on the basis of both the appearance of several tyrosine-phosphorylated protein bands in lysates from adherent peritoneal cells treated with immune complexes and the complete blockade of CINC-1 induction by treatment with 1 microM herbimycin A, an inhibitor of src protein tyrosine kinases.