Krishna Reddy

Morphine promotes apoptosis in Jurkat cells.

Patients with intravenous heroin addiction are prone to recurrent infections and at times these infections are fatal. We evaluated the effect of morphine on the apoptosis of Jurkat cells and freshly isolated human T lymphocytes. Morphine promoted apoptosis of both the Jurkat cells and the freshly isolated T lymphocytes in a dose‐dependent manner. DAGO, a specific p receptor agonist, also promoted Jurkat cell apoptosis. DNA isolated from morphine‐treated Jurkat cells and T lymphocytes also showed integer multiples of 200 base pairs. Superoxide dismutase (SOD) enhanced lymphocyte apoptosis; whereas catalase attenuated the morphine‐induced apoptosis of Jurkat cells as well as of T lymphocytes. Morphine‐treated Jurkat cells also showed a decreased expression of bcl‐2 and an enhanced expression of bax. In addition, morphine‐treated Jurkat cells showed activation of caspase‐3. These results indicate that morphine‐induced T lymphocyte apoptosis may be mediated through the generation of reactive oxygen species. The change in ratio of bax and bcl‐2 seems to tilt the balance toward apoptosis, leading to the activation of caspase‐3. This study provides further support for the hypothesis that morphine may be directly compromising immune function by enhancing apoptosis of T lymphocytes in patients with heroin addiction. J. Leukoc. Biol. 66: 650–658; 1999.

Morphine-induced macrophage apoptosis: the role of transforming growth factor-β

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate‐induced macrophage (Mφ) apoptosis. In the present study, we evaluated the role of transforming growth factor‐β (TGF‐β) in morphine‐induced apoptosis of murine J774 cells and peritoneal Mφ. Mφ harvested from morphine‐treated mice showed greater (P < 0·0001) apoptosis when compared with control Mφ. Morphine also enhanced apoptosis of J774 cells and peritoneal Mφ. Anti‐TGF‐β antibody inhibited (P < 0·001) the morphine‐induced apoptosis in J774 cells (control 0·7 ± 0·4%; 10−6 m morphine 23·5 ± 0·7%; anti‐TGF‐β antibody (Ab) + 10−6 m morphine 8·1 ± 0·7%; apoptotic cells/field) and peritoneal Mφ (control 1·5 ± 0·9%; 10−6 m morphine 29·1 ± 1·4%; 10−6 m morphine + anti‐TGF‐β Ab 19·1 ± 1·8%; apoptotic cells/field). TGF‐β enhanced (P < 0·001) apoptosis of J774 cells and peritoneal Mφ. TGF‐β also promoted Mφ DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mφ cytoplasmic content of TGF‐β. In addition, Western blotting showed increased production of TGF‐β by morphine‐treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine‐induced bax expression was inhibited by anti‐TGF‐β Ab. As both morphine‐induced J774 cell apoptosis and bax expression were inhibited by anti‐TGF‐β Ab, it appears that morphine‐induced J774 cell apoptosis may be mediated through the generation of TGF‐β.

Ethanol-induced neutrophil apoptosis is mediated through nitric oxide.

Clinical reports indicate that acute ethanol intoxication in chronic ethanol abusers is associated with neutropenia. We hypothesize that ethanol accelerates the apoptosis of neutrophils thus decreasing the peripheral blood count of neutrophils. We studied the effect of ethanol on neutrophil apoptosis in vivo as well as in vitro. Human neutrophils harvested from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 0.5 ± 0.25 vs. after, 26.1 ± 2.6% apoptotic neutrophils/field). Peritoneal neutrophils isolated from ethanol‐treated rats also showed increased (P < 0.0001) apoptosis when compared with neutrophils isolated from control rats (control, 0.8 ± 0.2% vs. ethanol, 11.8 ± 0.7% apoptotic neutrophils/field). In in vitro studies, ethanol in concentrations of 50 mM and higher accelerated the apoptosis of human and rat neutrophils. This effect of ethanol on human neutrophils was time dependent. DNA isolated from ethanol‐treated human neutrophils displayed integer multiples of 180 base pairs (ladder pattern), further confirming the effect of ethanol on neutrophil apoptosis. NG ‐monomethyl‐l‐arginine monoac‐etate and NG ‐nitro‐L‐arginine methyl ester, inhibitors of nitric oxide (NO) synthase, attenuated the ethanol‐induced neutrophil apoptosis. Sodium nitroprusside, a NO donor, also promoted neutrophil apoptosis. Moreover, ethanol enhanced neutrophil expression of inducible NO synthase. In addition, ethanol stimulated neutrophil NO generation. These results suggest that ethanol accelerates neutrophil apoptosis. This effect of ethanol on neutrophil apoptosis seems to be mediated through the generation of NO. J. Leukoc. Biol. 66: 930–936; 1999.

Morphine Induces Splenocyte Apoptosis and Enhanced mRNA Expression of Cathepsin-B

Morphine has been demonstrated to modulate immune function. We studied whether morphine modulates apoptosis of splenocytes. Splenocytes were isolated from control and morphine treated rats. Splenocytes isolated from morphine treated rats showed increased percentage (P < 0.001) of apoptosis when compared to splenocytes isolated from untreated rats (control, 4.7 ± 1.0% apoptotic splenocytes/field vs. morphine, 47.8 ± 3.4% apoptotic splenocytes/field). These results were further confirmed by gel electrophoresis as well as by end-labeling DNA of splenocytes isolated from control and morphine treated rats. Splenocytes from morphine treated rats showed a classical ladder pattern with integer multiples of 180 base pairs. Splenocytes from morphine treated rats also showed increased mRNA expression of cathepsin-B, a gene associated with active cell death. These results suggest that morphine may also be modulating immune function by enhancing apoptosis of splenocytes.

Role of Heme Oxygenase–1 in Morphine-Modulated Apoptosis and Migration of Macrophages

We examined the role of heme oxygenase (HO)-1 in morphine-induced decrease in macrophage migration. Morphine promoted expression of HO-1 in murine macrophages. Morphine-receiving mice (MRCs) showed decreased (P<.001) macrophage migration and increased (P<.001) occurrence of macrophage apoptosis. In in vitro studies, peritoneal macrophages harvested from MRCs also showed decreased (P<.001) migration, compared with those from control mice. Bone marrow cells isolated from MRCs showed not only decreased (P<.001) migration but also increased apoptosis. Pretreatment of MRCs with hemin not only decreased migration of macrophages further but also enhanced the apoptosis of peritoneal macrophages. On the other hand, pretreatment of MRCs with zinc protoporphyrin attenuated the effect of morphine on both macrophage migration and the occurrence of apoptosis. In in vitro studies, pretreatment of macrophages with hemin exacerbated morphine-induced apoptosis, whereas pretreatment with zinc protoporphyrin attenuated morphine-induced macrophage apoptosis.

Ethanol promotes T cell apoptosis through the mitochondrial pathway

Clinical reports suggest that acute ethanol intoxication is often associated with lymphopenia. Previously, ethanol was reported to invoke thymocyte apoptosis. We studied the effect of ethanol on T cell apoptosis. In addition, we evaluated the molecular mechanism of ethanol‐induced T cell apoptosis. Human T cells harvested from healthy subjects after an alcohol drinking binge showed enhanced T cell apoptosis (before, 0·4 ± 0·2% versus after, 19·6 ± 2·5% apoptotic lymphocytes/field; P < 0·001). In in vitro studies, ethanol in a concentration of 50 mm and higher enhanced the apoptosis of Jurkat cells. DNA isolated from ethanol‐treated Jurkat cells displayed integer multiples of 180 base pairs. Ethanol decreased Jurkat cell expression of Bcl‐2, whereas ethanol increased Jurkat cell expression of Bax. Jurkat cells treated with ethanol also showed translocation of cytochrome C into cytosol. Moreover, a caspase‐9 inhibitor partially inhibited ethanol‐induced Jurkat cell apoptosis. In in vivo studies, after binge drinking, T cell expression of Bcl‐2 also decreased. In addition, binge drinking induced the cleavage of caspase‐3, suggesting activation of caspase‐3 in T cells. These results suggest that ethanol promotes T cell apoptosis through the activation of intrinsic or mitochondrial pathway.

HIV-1 gp120 envelope protein modulates proliferation of human glomerular epithelial cells.

Glomerular epithelial cells (GEC) have been demonstrated to undergo morphological alterations in human immunodeficiency virus (HIV)‐associated focal glomerulosclerosis. In the present study, we evaluated the effect of HIV‐1 gp120 envelope protein on the growth of cultured human (H) GEC. gp120 protein enhanced (P < 0.001) the proliferation of HGEC at lower concentrations. The mitogenic effect of gp120 protein on HGEC was further confirmed by enhanced accumulation of proliferating nuclear cell antigen (PCNA) by gp120 protein‐treated cells, as compared with control cells. On the contrary, gp120 protein at higher concentrations suppressed (P < 0.001) the growth of HGEC. To evaluate the mechanism of gp120 protein‐induced HGEC growth suppression, we examined the effect of gp120 protein on HGEC apoptosis. gp120 protein at higher concentrations promoted the apoptosis of HGEC. At higher concentrations, gp120 protein also enhanced DNA fragmentation of HGEC. Anti‐gp120 antibody attenuated the proliferative as well as the apoptotic effects of gp120 protein on HGEC. Because protein kinase C as well as tyrosine kinase inhibitors partially inhibited gp120‐induced proliferation, gp120 appears to be activating both the protein kinase C and tyrosine kinase pathways. In addition, gp120 protein at lower concentrations enhanced mRNA expression of c‐fos and at higher concentrations promoted mRNA expression of c‐jun. We conclude that gp120 has a bimodal effect on proliferation of HGEC. This effect may be mediated through the activation of early growth genes. J. Cell. Biochem. 76:61–70, 1999.