Requirement of A1-a for bacillus Calmette-Guérin-mediated protection of macrophages against nitric oxide-induced apoptosis.

The role of apoptosis in regulating the course of intracellular microbial infection is not well understood. We studied the relationship between apoptotic regulation and bacillus Calmette-Guérin (BCG) treatment in murine peritoneal exudate macrophages (PEM) and the J774 macrophage cell line. In both PEM and J774 cells, mRNA expression of the anti-apoptotic gene, A1, was selectively induced by BCG treatment as compared with other bcl2 family members (bcl-w, bcl-2, bcl-xl, bcl-xs, bax, bak, bad). In PEM, A1 expression was maximal by 8 h postinfection and was abrogated by the proteasomal inhibitor MG-132. The induction was independent of protein synthesis as well as the p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways and did not require live organism. Three genes encoding closely related isoforms of A1 were all expressed; however, the A1-a isoform displayed the greatest fold induction in PEM. BCG-induced A1 expression was associated with protection of host macrophages from NO-mediated apoptosis in both PEM and J774 cells. BCG-mediated protection was abrogated in PEM derived from A1-a(-/-) mice, indicating a requirement of A1-a for survival of inflammatory macrophages.

Interactive role of nitric oxide and superoxide anion in neutrophil-mediated endothelial cell injury.

This study addresses the interactive role of nitric oxide (NO) and reactive oxygen intermediates (ROI) by direct quantitation of NO and superoxide (O2-) in human neutrophil (PMN)-endothelial cell (EC) co-culture during PMN-mediated EC injury. The results directly demonstrate an inverse correlation between NO and ROI levels in PMN-EC co-culture, which significantly alters the PMN-EC adhesion and PMN-mediated EC killing. N-formylmethionyl-leucyl-phenylalanine (fMLF)-stimulated PMN adhesion to cytokine-treated EC was decreased (> 25%) in the presence of S-nitroso-N-penicillamine, a NO donor. NO also inhibited EC killing by stimulated PMN, suggesting its cytoprotective role. In addition, a significant decrease in NO levels was observed in the PMN-EC co-culture compared with the EC cultured alone (422.45 +/- 35.76 vs. 800.79 +/- 41.69 pmol). The reduced NO levels were restored by the addition of superoxide dismutase, a scavenger of O2-, suggesting that PMN-derived O2- is involved in the neutralization of NO in the co-culture. The results indicate an inverse correlation between NO and O2- in PMN-EC interactions and suggest the need for a critical balance between these two radicals in the regulation of PMN-mediated tissue injury.

AK-5 tumor-induced modulation of host immune function: upregulation of Th-1-type cytokine response mediates early tumor regression.

AK-5, a rat histiocytoma, grows as ascites and undergoes spontaneous regression upon subcutaneous transplantation. Earlier studies from this laboratory have demonstrated that immunogenic rejection of AK-5 tumor is mediated through ADCC involving CD8+ NK cells and anti-AK-5 antibody. Upon subcutaneous transplantation, 55-60% of animals initiated tumor regression between 12-15 days after tumor transplantation (early rejectors), while 40-45% did not evoke regression up to 20-25 days (late rejectors). In order to delineate this differential response among syngeneic animals to the same tumor, we have evaluated the cytokine profiles in circulation of both early and late rejecting animals. Our results show that an increase in IL-2, IFN-gamma, IL-4, IL-12 and TNF-alpha contributed to early regression, suggesting a predominantly Th-1 type of cytokine function being evoked against AK-5 tumor. Hosts with lower circulating levels of these cytokines showed delayed tumor regression. In addition, administration of anti-IL-4/anti-IL-4 + anti-IL-10 lead to a decreased antibody response to AK-5 surface antigens in vivo. Neutralization of IFN-gamma in tumor-bearing animals resulted in inhibition of NK-cell-mediated cytotoxicity against AK-5 cells and delayed the regression process. The present study suggests that early regression of AK-5 tumor depends primarily on the higher levels of circulating Th-1-type cytokines; however, the role of IL-4 and anti-AK-5 antibody in tumor regression cannot be ruled out.

Microbicidal mechanisms of liver macrophages in experimental visceral leishmaniasis.

To study the differential microbicidal potentials of liver macrophages, the oxygen-dependent and oxygen-independent pathways in Kupffer cells and immigrant macrophages of Leishmania donovani-infected BALB/c mice were investigated. Hydrogen peroxide assay was performed using horse radish peroxidase-dependent oxidation of phenol red to quantitate the reactive oxygen species produced. To examine the oxygen-independent pathway, the enzymes N-acetyl-beta-glucosaminidase (NAG) and beta-glucuronidase (beta G) were investigated after exposure of cells to lipopolysaccharide. Hydrogen peroxide release by Kupffer cells was significantly decreased only at 21 days postinfection, whereas hydrogen peroxide release by immigrant macrophages was significantly increased on all postinfection days with a maximum at 21 days postinfection. The pattern of release of NAG and beta G was similar in both cell populations with a peak at 21 days postinfection. The present study therefore suggests that Kupffer cells and immigrant macrophages adopt different pathways to cope with this infection.

Natural killer cell activation-associated induction of target cell DNA fragmentation in a spontaneously regressing rat histiocytoma.

The spontaneous regression of AK-5 histiocytoma is mediated by natural killer (NK) cells through antibody-dependent cell-mediated cellular cytotoxicity (ADCC) and the target cell death involves necrosis and apoptosis. We have studied the NK cell activation and the associated induction of apoptosis in the AK-5 tumour in rats. NK cells from immune animals expressed very low levels of CD16 and CD25 surface receptors, as revealed by Northern hybridization and flow cytometry. Interaction between NK cells and antibody-tagged AK-5 cells triggered the expression of these receptors to a higher level and affected AK-5 killing. Treatment of naive NK cells in vitro with interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-12 also enhanced the expression of these activation markers. Co-culture of NK cells from immune animals with antibody-tagged AK-5 cells induced formation of nuclear bodies in AK-5 and extensive fragmentation of AK-5 cell DNA. NK-mediated apoptosis was inhibited by zinc, actinomycin D and cycloheximide. The in vitro treatment of NK cells with cytokines enhanced their ability to induce apoptosis in AK-5 tumour. These results suggest that the NK cells acquire their ability to induce apoptosis in AK-5 tumour in association with their optimal activation.

Correlation of in vitro and in vivo effects of interferon-gamma with the spontaneous regression of a rat histiocytoma.

Cytokines trigger activation of different lymphocyte populations, resulting in augmentation of humoral and cell-mediated immunity. We have examined the role of IFN-gamma in mediating AK-5 tumor regression. High levels of circulating IFN-gamma and IL-2 marked the process of regression. Moreover, interaction of immune NK cells with antibody-tagged AK-5 resulted in IFN-gamma secretion, providing in vitro evidence for the involvement of this cytokine. IFN-gamma and IL-2 potentiated the cytolytic activity of naive NK cells, suggesting their role in antitumor activity. Furthermore, pretreatment of immune NK cells with protein tyrosine kinase inhibitor downregulated the IFN-gamma release, suggesting that the secretion of IFN-gamma is phosphorylation dependent. Nonimmune cells could be induced to secrete IFN-gamma when exposed to rIL-12, demonstrating IL-12 dependence in inducing IFN-gamma release. In vivo administration of anti-IFN-gamma inhibited the cytotoxic activity and the process of tumor regression, further substantiating the role of IFN-gamma in regulating the rejection of AK-5 tumor. These observations suggest a definitive role for IFN-gamma in AK-5 regression. This cytokine in concert with IL-2 and IL-12 might aid in designing effective anticancer therapy.

AK-5 tumor-induced expression of interleukin-12: role of IL-12 in NK-mediated AK-5 regression.

Spontaneous regression of AK-5, a histiocytic tumor, is mediated by CD3-, CD8+ NK cells through ADCC. The onset of AK-5 regression is associated with the induction of humoral immune response and the augmentation of effector function. The mechanism of tumor cell death involves both necrosis and apoptosis. Interleukin-12, a 75-kDa heterodimeric cytokine, has multiple effects on T and NK cells. We have investigated the role of IL-12 in the NK cell-mediated AK-5 tumor regression process. Subcutaneous transplantation of AK-5 tumor induced the expression of IL-12 (p35 and p40) message by Day 6-8 in the splenocytes of syngenic rats. Similarly, analysis of serum samples from tumor-bearing animals showed the presence of circulating IL-12 around the same time. Interaction of immune cells with antibody-tagged AK-5 cells in vitro also triggered the expression of IL-12 message and protein by 3 hr. The circulating IL-12 in the sera of tumor-rejecting animals, as well as rIL-12, stimulated NK cell proliferation, expression of CD16 and CD25, and the activation of NK cells function. These observations suggest that the ability of the AK-5 tumor to induce the endogenous production of IL-12 may be responsible for keeping the NK cells constantly in an activated state, thus demonstrating an efficient mechanism for the complete regression of the tumor.

Flow cytometric measurement of NK cell immunoconjugates by pulse width processing.

Pulse width analysis, in flow cytometry, has been widely used for optimal cell size resolution in cell kinetics analysis. Pulses, generated by scattered light or fluorescence of cells, are electronically analyzed for their height and width. The information generated from these two properties of the pulses is utilized to distinguish signals from single cells vs. signals from cell clumps or aggregates. Pulse width, unlike pulse height, is more sensitive to differences in cell diameter, and therefore can discriminate very small differences in it, which pulse height cannot. We have exploited this property of pulse widths to measure immunoconjugates between NK cells and their targets. Discrimination of the free target cells from the conjugated ones is possible by the pulse widths of only light scatter signals, both forward and/or orthogonal. This resolution was not obtained if pulse height of the same signals was visualized. Using this resolution it was possible to distinguish single cells from the aggregates between target and effector cells. We propose that this is a better method for distinguishing conjugates than the method in which prior vital staining of cells is used.

Conjugate formation between effector and target as a function of cytotoxicity in antibody-dependent killing of AK-5 tumor.

The present study was carried out to mechanistically view a possible correlation between the process of, conjugate formation and its relation to target cell death. AK-5 killing is mediated by CD8+ natural killer cells through ADCC. Immune effectors on exposure to antibody primed AK-5 tumor cells formed tight conjugates. Ability of various cell types (NK, T, monocytes and macrophages) to form conjugates was evaluated. Marked increase in the number of NK cells binding to the target as compared to the other cell types was observed. Cytotoxicity of free and bound effectors against antibody tagged AK-5 cells demonstrated a reduced cytotoxic ability of the former in contrast to a significantly high lytic potential of the bound effectors. The results highlight the requirement for priming of NK cells which mediate killing of AK-5 tumor and provide additional evidence that formation of stable conjugates acts as the first signal for triggering lymphocyte activation and effective target cell lysis.

Mechanism of antibody-dependent natural killer cell-mediated AK-5 tumor cell death.

The mechanism of antibody-dependent natural killer (NK) cell-mediated killing of a histiocytic tumor, AK-5, was studied. In vitro exposure of immune NK cells with antibody-primed AK-5 cells induced the formation of stable NK-AK-5 conjugates. The frequency of conjugate formation after 1 h coculture of immune NK cells was severalfold higher than that of normal cells. The occurrence of conjugate formation and the disintegration of conjugated targets only in the presence of anti-AK-5 antibody revealed the antibody dependence in recognition, conjugate formation, and triggering of lytic machinery leading to AK-5 cell death. Coculture of immune NK cells with AK-5 cells in the presence of anti-AK-5 antibody induced the expression of perforin mRNA as well as accumulation of protein in the NK cells and extensive fragmentation of AK-5 cell DNA, thereby indicating the involvement of necrosis and apoptosis in the AK-5 cell death. The antibody-dependent expression of perforin as well as DNA fragmentation indicated these events to be consequent to E-T conjugate formation. The observation of target cell DNA fragmentation revealed that the internal target disintegration is the first event which is followed by membrane damage leading to efficient tumor destruction by the host immune system.

Leishmania donovani: in vitro evidence of hepatocyte damage by Kupffer cells and immigrant macrophages in a murine model.

Infection with Leishmania donovani leads to activation of liver macrophages. The role of different macrophage populations of liver in this infection is not clearly defined. Thus, the mechanism involved in hepatocyte damage was studied by coculturing hepatocytes with two populations of liver macrophages, the kupffer cells and immigrant macrophages. The results indicated maximum tissue damage at peak infection in both the macrophage populations cocultured with hepatocytes (P < 0.001). Kupffer cell-hepatocyte coculture treated with scavengers of reactive oxygen intermediates failed to inhibit the hepatocyte damage (P > 0.05). But with heparin and phenylmethylsulfonyl fluoride, a sharp decrease in the damage was noticed (P < 0.001). In contrast, immigrant macrophage-hepatocyte coculture showed a significant reduction in tissue damage when treated with both the scavengers of reactive oxygen intermediates and enzyme inhibitors (P < 0.001). Therefore the murine infection with L. donovani is speculated to involve two distinct subpopulations of liver macrophages with marked differences in morphology and functional capabilities.

Active oxygen species in copper intrauterine device users.

The mechanism of copper intrauterine device (Cu IUD) in limiting intrauterine infections is poorly understood. Copper ions may enhance the release of reactive oxygen species which are deleterious to the microbes. The present study compares the oxidative responses of adherent cell population of uterus prior to Cu-T insertion and at different post-insertion intervals. Increase in reactive oxygen intermediates was evident at 1 week post-insertion. However, the release of active oxygen species decreased thereafter. Further, these responses were only a local phenomenon as the peripheral blood monocytes failed to produce appreciable change following Cu-T insertion. Results suggest the protective role of active oxygen species in Cu IUD users which lasts for a brief period. The withering of respiratory burst activity later on may possibly prevent endometrial damage.