Nitric oxide (NO) is a short-lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (M phi) could be induced to synthesize nitrite (NO2-), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like M phi, these clones were found to release high levels of NO2- in response to recombinant interferon-gamma (rIFN-gamma) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor-alpha (rTNF-alpha). As previously demonstrated for M phi, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO2- production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the M phi-like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser 92 and Phe 93, in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.Myristoylated alanine-rich C kinase substrate (MARCKS) 1 and MARCKS-related protein (MRP), also known as Mac-MARCKS, are members of a highly acidic myristoylated family of protein kinase C (PKC) substrates (1, 2). The primary structures of MARCKS and MRP exhibit significant homology, including a highly basic stretch of amino acid residues known as the effector domain (also as the phosphorylation site domain), which contains the serine residues subject to PKC-dependent phosphorylation as well as binding sites for calmodulin and actin. Whereas MARCKS is widely distributed in diverse cell types, MRP is present primarily in brain and reproductive tissue (3, 4) as well as in macrophages, where it was first characterized (5). The biologic functions of MARCKS proteins are unknown. Due to their high effector domain homology, it is also possible that MRP and MARCKS play overlapping roles in some cells. In macrophages, both proteins colocalize in the cytosol in association with components of the actin cytoskeleton (6 -9) and consequently are thought to participate in major cellular responses such as phagocytosis, motility, and membrane trafficking.The expression of MARCKS proteins appears to be highly regulated, and in vitro studies have demonstrated up-or downregulation of MARCKS at both the transcriptional and posttranscriptional levels (5, 10, 11). One mechanism of post-transcriptional regulation involves proteolytic degradation. Spizz and Blackshear (12) recently identified cathepsin B as a cellular MARCKS-cleaving enzyme in fibroblasts. They suggested that cleavage might occur within lysosomes as a result of specific lysosomal targeting sequences identified within the MARCKS primary sequence. At least one c...

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-gamma (IFN-gamma)-stimulated macrophages (M phi) by preventing the secretion of tumor necrosis factor-alpha (TNF-alpha) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-gamma together with exogenous TNF-alpha to induce NO synthesis by bone marrow-derived M phi. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO2-) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO2- production by M phi stimulated in the presence of optimal concentrations of prostaglandin E2, a positive modulator of M phi activation by IFN-gamma/TNF-alpha. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-gamma/TNF-alpha cultures and enhanced NO2(-)-release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on M phi function are more complex than previously recognized.

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