TYROSINE KINASE-DEPENDENT AND KINASE-INDEPENDENT EVENTS INDUCED BY INTERLEUKIN-2 STIMULATION - INTERLEUKIN-2-MEDIATED NO PRODUCTION REQUIRED FOR THE INDUCTION OF LYMPHOKINE-ACTIVATED KILLER-CELL ACTIVITY IN RAT SPLENOCYTES IS TYROSINE KINASE INDEPENDENT

Nitric oxide (NO) has recently been shown to be an indispensable co-fa ctor in the generation of lymphokine-activated killer (LAK) cells indu ced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediate d by protein tyrosine kinases (PTK). In this work we utilized a well-c haracterized PTK inhibitor, genistein (GEN), to address the role of PT K on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferat ion, generation of cytotoxic activity and production of NO upon IL-2 s timulation of rat splenocytes. We report here that GEN displays profou nd inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production , measured as NO2-. In contrast, a specific inhibitor of the NO synthe tic pathway (N-G-monomethyl-L-arginine; NMMA) blocked generation of LA K cells and NO production without affecting cell proliferation. If add ed directly to the cytotoxicity tests, GEN exerted minor inhibitory ef fects, not exceeding 25% of control tests, while NMMA was completely i neffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing s ubstance, restored LAK cell generation in cultures performed in the pr esence of NMMA, but not in those performed in the presence of GEN. The se results indicate that IL-2-induced NO production is a PTK-independe nt event. IL-2-stimulated LAK cell generation obligatorily requires th e concurrent activation of PTK dependent and independent signal transd uction pathways.