Effects of protein I of Neisseria gonorrhoeae on neutrophil activation: Generation of diacylglycerol from phosphatidylcholine via a specific phospholipase C is associated with exocytosis

Abstract

Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (≤15 s) phase ("triggering") and a slow (≥30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2̇- generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 ± 14% SEM vs. 125 ± 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 ± 10% SEM vs. 107 ± 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2̇- generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP ± protein I (P.I.). Treatment of PMN with FMLP (0.1 μM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 ± 10.2% SEM of resting, 14C-PC = 57 ± 6.4%). Protein I (0.25 μM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 ± 5.6% and [14C]PC = 86 ± 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 ± 19.6 and 88 ± 16.5% vs. 92 ± 9.2 and 91 ± 16% at 60 s). Increases of [3H]- and [14C]phosphatidate (PA) provoked by FMLP were unimpaired in the presence of P.I. (341 ± 99 and 210 ± 73% vs. 358 ± 42 and 187 ± 20%). We conclude that FMLP triggers generation of distinct second messengers for exocytosis and O2̇- generation. Degranulation is tightly correlated with, and may thus require production of the slow DAG from PC by a PC-specific PLC and whereas generation of O2̇- requires phosphatidic acid which can be formed de novo.