Involvement of protein kinase C and not of NFκB in the modulation of macrophage nitric oxide synthase by tumor-derived phosphatidyl serine

Abstract

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-γ 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-γ resulted in higher levels of NO than those observed with LPS alone, but lower than those of untreated macrophages activated with LPS and IFN-γ. 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 NFκB 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 [Ca 2+ ]i were not observed, while NO was normally produced even under calcium-deprived conditions. In contrast, an effective synergism of IFN-γ with LPS in the production of NO by macrophages required an optimal increase in [Ca 2+ ]i stimulated by IFN-γ. This increment in [Ca 2+ ]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-γ did not alter the latter result, suggesting that IFN-γ 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 NFκB 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 [Ca 2+ ]i promoted by IFN-γ in macrophages, reducing the synergism of this cytokine with LPS and leading to a less effective production of NO.

Blood monocytes from mammary tumor-bearing mice: Early targets of tumor-induced immune suppression?

“…Kinetics of constitutive or induced protein expression may vary over time and initial increased transcriptional expression may ultimately result in protein downregulation due to posttranscriptional or posttranslational mechanisms. Our results in NO downregulation in monocytes from tumor bearing mice match with our previously published results in peritoneal macrophages from tumor bearers, which also show a decrease in NO production upon LPS activation; interestingly monocytes from normal mice constitutively produce NO, in contrast to our results in peritoneal macrophages from normal mice which do not produce measurable amounts of NO unless activated by LPS (13,22).…”

“…Sorted CD115 + monocytes from normal and tumor-bearing mice were plated (3x10 5 cells /96-well flat-bottom) and incubated with and without 10 μg/ml of LPS in complete RPMI for 48 h. Nitrite (NO 2 -) concentration determined in the cell supernatant served as a reflection of NO production and was measured using the colorimetric Griess reaction (21) as previously described (22).…”

Paracrine Interactions between Adipocytes and Tumor Cells Recruit and Modify Macrophages to the Mammary Tumor Microenvironment: The Role of Obesity and Inflammation in Breast Adipose Tissue

“…Nitric oxide was determined in cell supernatants from leptin-pretreated N-PEMs using the Griess colorimetric reaction as previously reported [ 17 , 25 ].…”

Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: Results of a pilot study

“…Phosphatidylserine (PS) has been shown to inhibit the nitric oxide pathway involved in the cytotoxic effect of the macrophages [36]. Importantly, PS also induces anti-inflammatory/immunosuppressive responses in macrophages when expressed by apoptotic cells that need to be silently phagocytosed by macrophages without triggering inflammation [37].…”