Lymphocyte-mediated macrophage apoptosis during IL-12 stimulation.

In contrast to the well known immunostimulatory roles of IL-12, little has been known about its immunosuppressive roles. In the present study, IL-12-activated lymphocyte-mediated macrophage apoptosis was investigated by employing murine lymphocyte/macrophage cocultures. IL-12-activated lymphocytes and their culture supernatants induced an inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) synthesis in macrophages. The NO synthesis was markedly inhibited by blocking antibodies to IFN-γ and TNF-α, suggesting the key role of these lymphocyte cytokines in mediating the NO synthesis. The endogenously produced NO inhibited macrophage proliferation, and induced apoptosis in concordance with the accumulation of p53, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and DR5, and the activation of caspase-3, processes that were inhibited by N(G)-monomethyl-l-arginine, aminoguanidine (NO synthase inhibitors) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (an NO scavenger). These results were further supported by the findings obtained from the experiments employing IFN-γ-knockout and iNOS-knockout mice. Our study demonstrated a novel, non-contact-dependent mechanism of macrophage suppression by IL-12-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages due to endogenous NO synthesis induced by cytokines secreted from IL-12-activated lymphocytes.

Glutamine protects mice from acute graft-versus-host disease (aGVHD).

Despite current immunosuppressive therapies, acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT). In the present study, therapeutic effects of intraperitoneal glutamine (Gln) administration (1g/kg/day) in a mouse aGVHD model were evaluated. Gln administration significantly inhibited the GVHD-induced inflammation and tissue injury in the intestine, liver, skin and spleen. Gln therapy improved the score of clinical evidence of aGVHD and prolonged the median survival of aGVHD mice. Gln administration in aGVHD mice increased the fraction of Foxp3+/CD4+/CD25+ cells in the blood measured on day 7, and decreased the serum levels of tumor necrosis factor-α measured on days 7, 14 and 21 after aGVHD induction. These results demonstrated that Gln administration may be useful in protecting the host from aGVHD.

Rosiglitazone prevents graft-versus-host disease (GVHD).

The effect of rosiglitazone, an agonist of peroxisome proliferator-activated receptor-γ (PPARγ), was investigated in a mouse parent-to-F1 GVHD model. Rosiglitazone inhibited mixed lymphocyte reactions, inducing enhanced apoptosis in CD4+, CD8+, and B220+ cells, but not in NK1.1+, Mac-1+, CD4+/CD25+ and CD3+/NK1.1+ cells. Rosiglitazone administration prevented GVHD in the liver, skin, spleen and intestine. Rosiglitazone inhibited GVHD-induced increases in serum levels of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-6, and IL-12, and the GVHD-induced decreases in transforming growth factor-beta and IL-10. Immunophenotyping of splenic leukocytes demonstrated that while rosiglitazone treatment increased the population percentages of both donor and host CD4+/CD25+ and CD3+/NK1.1+ cells, the treatment resulted in lower fractions of both donor and host CD8+ cells. Rosiglitazone inhibited the GVHD-induced decreases in the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the GVHD-induced increase in the splenic p-Akt and nuclear factor-kappa B expression. These results indicate that rosiglitazone and PPARγ activation may be useful in protecting the host from GVHD.

Use of curcumin to decrease nitric oxide production during the induction of antitumor responses by IL-2.

Nitric oxide (NO) synthesis is strongly induced during interleukin (IL)-2 treatment of mice and humans. Although this free radical can act as a cytotoxic effector molecule against cancer cells, immunosuppressive effects have also been suggested. We evaluated the effects of curcumin on IL-2-induced NO synthesis and IL-2-induced antitumor responses in a mouse ascites tumor model. Curcumin inhibited inducible nitric oxide synthase (iNOS) expression and NO production, and thereby enhanced the proliferation and cytotoxic activity of cocultured lymphocytes and macrophages during IL-2 stimulation which we earlier established as an in vitro model of IL-2-induced NO synthesis. Curcumin also decreased apoptosis of cocultured lymphocytes and macrophages during IL-2 stimulation. In contrast, the curcumin-induced changes in proliferation and apoptosis were not observed in cultures of lymphocytes alone, macrophages alone, and cocultured lymphocytes/iNOS-knock out macrophages, all of which produced little nitrite during IL-2 stimulation. In conjunction with IL-2 treatment, oral curcumin administration significantly inhibited IL-2 therapy-induced urinary nitrite/nitrate excretion and iNOS expression of tumor tissues, and further increased the IL-2 therapy-induced prolongation of survival in a murine Meth-A ascites tumor model. Curcumin may be useful as an adjunct to increase the antitumor activity of IL-2 therapy.

Protective effect of Amomi semen extract on alloxan-induced pancreatic beta-cell damage.

The protective effect of Amomi semen extract (ASE) on alloxan-induced pancreatic beta-cell damage was investigated in HIT T-15 cells, a Syrian hamster pancreatic beta-cell line. Alloxan caused pancreatic beta-cell damage through the generation of reactive oxygen species (ROS), the elevation of cytosolic free Ca2+, DNA fragmentation and the decrease of cellular NAD+ and ATP levels. All these effects of alloxan were significantly prevented by pretreatment with a water-soluble extract of Amomi semen. Pretreatment with ASE in pancreatic islets isolated from mice, also significantly abolished the inhibition of glucose-stimulated insulin secretion by alloxan. The results of this study provide evidence that ASE may have a protective activity on alloxan-induced beta-cell damage, and that the protective effect is primarily due to the inhibition of ROS generation by alloxan.

Dimethylsulfoxide induces upregulation of tumor suppressor protein PTEN through nuclear factor-kappaB activation in HL-60 cells.

Dimethylsulfoxide (DMSO) has been known to differentiate HL60 cells into neutrophil like cells. Here, we provide an evidence for the involvement of tumor suppressor PTEN, an antagonist of phosphatidylinositol 3-kinase (PI3K) in the DMSO-induced differentiation of HL60 cells. DMSO upregulated PTEN with unaffecting the expression of PI3K. The upregulation of PTEN by DMSO lead to the decrease of Akt phosphorylation, a downstream of PI3K. The DMSO-induced upregulation of PTEN might be mediated by NF-kappaB activation, which was evidenced by the blockage of DMSO-induced PTEN upregulation with an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC).

A calcium-calmodulin antagonist blocks experimental Vibrio vulnificus cytolysin-induced lethality in an experimental mouse model.

We demonstrated that trifluoperazine, a calcium-calmodulin antagonist, blocked the hyperpermeability induced by Vibrio vulnificus cytolysin in in vitro-modeled endothelium and prevented the deaths of mice. Furthermore, compared to tetracycline alone, tetracycline combined with trifluoperazine enhanced the survival rate of V. vulnificus-infected mice, indicating the role of the cytolysin as an important factor in pathogenesis.

Amomum xanthoides extract prevents cytokine-induced cell death of RINm5F cells through the inhibition of nitric oxide formation.

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.

Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells.

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.

Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin.

The pore-forming cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-gamma. Increased NO production was not affected by polymyxin B, and heat inactivation of cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy-d-[(3)H]glucose. At the higher concentrations of cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFkappaB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.

Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells.

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.

Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes.

The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF-induced LPL suppression is not the result of NO overproduction.