Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.

Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids

Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180 percent). Removal of the gonads in both males and females (comparison between castrated groups) increased the difference in SOD activity from 83 to 138 percent and reduced the difference in CAT activity from 180 to 86 percent. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48 percent) CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle

Activation of protease activity in rat peritoneal macrophages in protein deficiency: characterization of cathepsin D.

Rat peritoneal macrophages contained high proteolytic activity that was significantly enhanced under the stress induced by protein deficiency. The aspartyl protease cathepsin D which has been known to be the most active protease in endocytic processes was extracted from the macrophages recovered from control (20% protein fed) and protein deficient (4% protein fed) rats and was affinity purified and characterized further. The cathepsin D from the control sample exhibited better recovery, purification and higher specific activity compared to that from the deficient groups. Apparently the pH optima and heat stability of the enzyme from both the groups were similar. The SDS PAGE profile clearly indicated the presence of greater amounts of active forms of cathepsin D in the deficient samples in vivo itself which reflected in a reduction in Km value of the enzyme. Subtle differences observed in the activity of these macrophage proteases in the protein deficient rats may be partly responsible for the enhanced degradation of macrophage membrane proteins reported earlier.

Nature of peritoneal macrophages from DCC immunized mice.

Peritoneal macrophages from mice immunized with the delipidified cell component (DCC) of Mycobacterium leprae showed changes in various parameters such as increased protein synthesis, levels of hydrolytic enzyme and augmented phagocytic ability indicating activation of the cells. Furthermore, the surface structure of the cells were quite different from that of the macrophages of normal mice. These observations indicate that the peritoneal macrophages have been activated to phagocytose and kill M. leprae better in the immunized mice. The ability to kill the pathogen by these cells was reported by us earlier.

Canatoxin induces activation on mice peritoneal macrophages

Canatoxin (CNTX), the toxic protein from Canavalia ensiformis seeds, injected into the peritoneal cavities of mice (10 micrograms/cavity) induced a significant neutrophil migration (10.5 +/- 0.5 x 10(6) cells/cavity) after 4 h. A later migratory effect (48 h) on mononuclear cells, predominantly macrophages, was also observed (controls: 7 +/- 0.9; CNTX: 17 +/- 2.0 x 10(6) cells/cavity). These CNTX-elicited macrophages, when compared to resident cells (R) or cells elicited by thioglycollate (TG), had an increased content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (R: 4.5 +/- 0.5; TG: 7.2 +/- 1.0; CNTX: 20.2 +/- 3.0 mU/10(6) cells) and a greater (> or = 100%) phagocytic activity. The data suggest that CNTX-stimulated macrophages presented some characteristics of activated cells