Involvement of protein kinase C and not of NF kappa B in the modulation of macrophage nitric oxide synthase by tumor-derived phosphatidyl serine.
Nitric oxide (NO) is one of the main cytotoxic effector molecules involved in the killing of tumor cells by macrophages. In macrophages, lipopolysaccharide (LPS) alone or in combination with IFN-gamma causes the generation of NO by an inducible form of NO synthase (iNOS). We have previously reported that macrophages from mammary tumor bearers have a downregulation of their NO production leading to a diminished cytotoxic activity. Further studies lead to the isolation and characterization of phosphatidyl serine (PS) as a NO inhibitory factor produced by mammary tumor cells. Pretreatment of macrophages with PS was shown to downregulate their cytotoxic potential and NO production upon stimulation with LPS. Activation of PS-pretreated macrophages with LPS and IFN-gamma resulted in higher levels of NO than those observed with LPS alone, but lower than those of untreated macrophages activated with LPS and IFN-gamma. These results correlated with the levels of iNOS RNA as detected by Northern blot analyses. A study of the expression and binding activity of the transcription factor NF kappa B in macrophages pretreated with PS revealed no differences with untreated macrophages. Investigation of the possible signaling pathways leading to the induction of iNOS revealed that in LPS-stimulated macrophages, increases in internal calcium concentration [Ca2+]i were not observed, while NO was normally produced even under calcium-deprived conditions. In contrast, an effective synergism of IFN-gamma with LPS in the production of NO by macrophages required an optimal increase in [Ca2+]i stimulated by IFN-gamma. This increment in [Ca2+]i was significantly reduced in PS-pretreated macrophages. Further experiments demonstrated that pretreatment of macrophages with PS did not change the normal pattern of tyrosine phosphorylation stimulated by LPS but strikingly inhibited PKC activity. Combinations of LPS and IFN-gamma did not alter the latter result, suggesting that IFN-gamma enhances LPS-induction of iNOS through a pathway other than activation of PKC. Importantly, expression of PKC isozymes in both untreated and PS-pretreated macrophages stimulated with LPS remained constant. Out data suggest that, in tumor bearers, PKC and not NF kappa B is the main target for PS to exert its NO inhibitory action on LPS-activated macrophages. An excess of PS in PS-PKC interaction may be responsible, at least in part, for this type of PKC inhibition. Furthermore, PS also appears to downregulate the rise in [Ca2+]i promoted by IFN-gamma in macrophages, reducing the synergism of this cytokine with LPS and leading to a less effective production of NO.
Calderon
Cesar L
CL
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33236, USA.
Torroella-Kouri
Marta
M
Dinapoli
Michael R
MR
Lopez
Diana M
DM
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007371:Interferon-gamma / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000502:physiology*; D009369:Neoplasms / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism
D007476:Ionophores; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010718:Phosphatidylserines; D012333:RNA, Messenger; D009569:Nitric Oxide; D000001:Calcimycin; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D011493:Protein Kinase C; D002118:Calcium