Parasite accessory cell interactions in murine leishmaniasis. I. Evasion and stimulus-dependent suppression of the macrophage interleukin 1 response by Leishmania donovani

Interleukin 1 (IL 1) is a principal mediator of the host immune response to microbial challenge. Accessory cells of the monocyte-macrophage series are a major source of this cytokine and are also chronically parasitized by protozoa of the genus Leishmania. This suggests that characterization of the macrophage IL 1 response to Leishmania would increase our understanding of the regulation of host immunity to these organisms. In the present study, the macrophage IL 1 response to Leishmania donovani was examined because infections with this organism have findings consistent with parasite-specific T cell unresponsiveness. Cytokine activity was measured either by direct stimulation or by co-stimulation of thymocytes. Conditioned media from BALB/c resident peritoneal macrophages infected with amastigotes of L. donovani contained no more IL 1 than did supernatant fluids of control cells. In contrast, supernatants from cells stimulated with lipopolysaccharide or heat-killed Listeria monocytogenes had significantly increased cytokine content. Resident cells infected with L. donovani for 4 hr before being stimulated with Listeria demonstrated a suppressed IL 1 response (approximately 40% of Listeria alone) to this secondary particulate stimulus. In contrast, the secondary response of leishmania-preinfected cells to lipopolysaccharide was not affected. To examine whether accessory cell nonresponsiveness to L. donovani (with respect to IL 1) was related to the state of macrophage activation, elicited peritoneal macrophages obtained by injection of proteose peptone were also studied. These cells responded to stimulation with lipopolysaccharide and fixed Staphylococcus aureus with increases in intracellular, membrane, and secreted cytokine activities. In contrast, L. donovani failed to induce any of these activities. This was found to be the case irrespective of whether amastigotes were alive or killed or opsonized with specific antibodies. Elicited cells preinfected with Leishmania responded normally to secondary stimulation with lipopolysaccharide, but not S. aureus (64% of Staphylococcus alone). In addition, attachment and penetration of L. donovani promastigotes and their subsequent conversion to amastigotes within macrophages failed to induce IL 1 synthesis. The findings of this study indicate that L. donovani has the ability to both evade and suppress the macrophage IL 1 response. Because this monokine provides an obligatory signal during macrophage-dependent T cell activation, evasion of signal transduction for IL 1 synthesis may be related to defects in cell-mediated immunity which occur during infections with this organism.     

Arachidonic acid metabolism by murine peritoneal macrophages infected with Leishmania donovani: in vitro evidence for parasite-induced alterations in cyclooxygenase and lipoxygenase pathways

Leishmania donovani is an obligate intracellular protozoan that resides within mononuclear phagocytes of infected mammals. Affected human and rodent hosts commonly show abnormalities of T cell function, which may be related to altered macrophage physiology resulting from intracellular parasitism. To examine this possibility, we studied the metabolism of endogenous arachidonyl-phospholipids and [3H]-arachidonyl-phospholipids by murine peritoneal exudate macrophages infected with amastigotes of L. donovani. Our results indicated that infected cells synthesized increased amounts of both cyclooxygenase and lipoxygenase metabolites of arachidonic acid. Increased synthesis of immunoreactive prostaglandin (PG)E2 was evident as early as 1 to 4 hr after infection, was correlated with the fraction of cells infected, and was inhibited by sodium meclofenamate (0.2 and 20 microM) but not nordihydroguaiaretic acid (3 microM). As determined by thin-layer chromatography, infected cells also produced markedly increased amounts of prostaglandin F2 alpha (also inhibited by sodium meclofenamate) with insignificant increases in thromboxane B2 and the stable metabolite of prostacyclin, 6-oxo-PGF1 alpha. In contrast, stimulation of cells with opsonized zymosan resulted in significantly increased synthesis of all four eicosanoids. L. donovani infection was also found to induce marked increases in synthesis of lipoxygenase metabolites of arachidonic acid by infected cells. This was evidenced by increased amounts of [3H]-labeled material in cell extracts that co-migrated with authentic standards of 5 and 12/15-hydroxy-eicosate-traenoic acids in thin-layer chromatograms. Increased synthesis of these products was largely inhibited by both NDGA (3 microM) and sodium meclofenamate (20 and 0.2 microM). Additional evidence for augmentation of 5-lipoxygenase by Leishmania was provided by the demonstration of increased leukotriene-C4 in conditioned medium from infected cells. These results indicate that macrophages infected with L. donovani produce increased amounts of arachidonic acid metabolites with the potential for influencing cellular immune function and the inflammatory response to infection.     

The kinetics of inhibition of erythrocyte cholinesterase by monomethylcarbamates

1. The kinetics of the interaction of erythrocyte cholinesterase with 1-naphthyl N-methylcarbamate, 2-isopropoxyphenyl N-methylcarbamate and phenyl N-methylcarbamate were studied. Rate constants for inhibition and rate constants for spontaneous reactivation were determined. The calculated rate constants for spontaneous reactivation agreed well with those obtained experimentally. 2. The degree of inhibition obtained after preincubation of enzyme and inhibitor was found to be independent of both the substrate concentration and the dilution of the inhibited enzyme. 3. The reaction between the enzyme and the inhibitor was consistent with carbamates being regarded as poor substrates of cholinesterases. There was no evidence for the formation of a reversible complex between the enzyme and the carbamate.