Prince K. Arora

Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death

The role of inflammatory cytokines in the pathogenesis of neurological diseases is not well understood. The neurotoxic effects of cytokines could be mediated by immunostimulation of glial cells to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. Cultured microglia and meningeal fibroblasts, but not Type 1 astrocytes, were induced by lipopolysaccharides and cytokines to synthesize NO and reactive nitrogen oxides from L-arginine. In co-cultures of immunostimulated microglia and cerebellar granule neurons, neurotoxicity was blocked by an inhibitor of NO synthase, NG-nitroarginine, and by oxyhemoglobin, which inactivates NO. Microglial-induced neurotoxicity was also partially attenuated by the N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801 and 2-amino-5-phosphovalerate (APV). Superoxide dismutase, which stabilizes NO through inactivation of superoxide anion, augmented microglial-mediated neurotoxicity either alone or in combination with MK-801 or APV. Hence, immunostimulated microglia mediate neurotoxicity by NO, reactive nitrogen oxides, superoxide anion and NMDA-like substances. These findings suggest a novel role for microglial-produced NO and reactive nitrogen oxides as a neurotoxic agent in neurodegenerative disease states.

Spatial learning impairment in a murine model of AIDS.

Mice infected with an immunosuppressive murine leukemia virus (MuLV) mixture, LP‐BM5, displayed profound and selective deficits in spatial learning in a modified Morris water maze. These deficits appeared before the appearance of gross neurological impairment or histopathological changes in the central nervous system. Thus, LP‐BM5‐infected mice displayed deficits in several aspects of trained performance compared to controls. Furthermore, a failure to exhibit any evidence of task acquisition in this maze was observed almost twice as frequently (P < 0.0005) in infected mice as in uninfected controls. Moreover, in the absence of gross visual, motoric, or motivational impairment, LP‐BM5 MuLV‐infected animals exhibited neither the target directed search pattern nor the spatial preference characteristic of controls. The spatial learning and memory deficit described here is the first report of cognitive impairment accompanying viral‐induced immunosuppression in a nonprimate species.— Sei, Y., Arora, P. K., Skolnick, P., Paul, I. A. Spatial learning impairment in a murine model of AIDS. FASEB J. 6: 3008‐3013; 1992.

Morphine-induced immune alterations in vivo

The high incidence of human immunodeficiency virus (HIV) seropositivity among drug abusers prompted us to examine in an animal model the effects of morphine on aspects of the immune system that may be specifically related to HIV infection. We now report a robust, sustained elevation in the ratio of CD4+/CD8+ cells in the spleen and thymus of mice chronically treated with morphine. Since CD4+ cells have been reported to be target cells for HIV, these alterations, in concert with a marked cellular atrophy that appears to be restricted to organs of the immune system, suggest that opiates may serve as cofactors in altering the immune status of the host and thus contribute to the increased susceptibility to HIV infection and eventual development of AIDS in opiate abusers.

Quantitative analysis of calcium (Ca2+) mobilization after stimulation with mitogens or anti-CD3 antibodies: Simultaneous fluo-3 and immunofluorescence flow cytometry

A method is described for flow microfluorometric analysis of calcium mobilization in immune cells. This method is based on dual-color analysis of heterogeneous cell populations using fluo-3 and phycoerythrin (PE)-conjugated monoclonal antibody (mAb). This technique allows the detection of fluo-3 fluorescence as a measure of an increase in cytoplasmic free calcium ([Ca2+]i) while simultaneously discriminating PE-mAb-labeled cells without using wavelength ratio imaging. This method provides a unique way to distinguish patterns of calcium mobilization within a heterogeneous cell population.

Alterations in behavior, steroid hormones and natural killer cell activity in male transgenic TGFα mice

The expression of transforming growth factor alpha (TGF alpha) is widely distributed throughout many normal and neoplastic tissues, but its physiological significance remains unclear. We have utilized male transgenic mice overexpressing the gene encoding human TGF alpha in multiple tissues to further identify those functions which are influenced by this protein. Male TGF alpha mice develop hepatocellular carcinoma at the age of 10-15 months. At the age of 2-3 months these mice, compared to age matched CD-1 controls, spent significantly longer times immobile in Porsolts swim test, a model of stress and depressive behavior, and exhibiting aggressive behavior in the resident-intruder test. In contrast, the transgenic TGF alpha mice did not differ from the controls in either the plusmaze test of anxiety, or in their voluntary alcohol intake. Significantly, the TGF alpha mice exhibited a 25% lower Natural Killer (NK) cell activity and a four-fold increase in the plasma levels of 17-beta-estradiol (E2) than the controls. No significant changes in plasma testosterone or corticosterone levels were noted. The results indicate that transgenic male mice overexpressing TGF alpha exhibit behaviors characteristic of both an impaired ability to cope with stress and an increased aggressivity. The TGF alpha mice also show reduced NK cell activity and increased plasma estradiol concentrations. The present data suggest that TGF alpha may be important in influencing behavioral, immunological and hormonal systems prior to the onset of tumors. It remains to be determined whether hepatocarcinoma is associated with the direct proliferative and transforming effects of TGF alpha and/or indirect effects mediated through immune, hormonal and behavioral mechanisms.

Inhibition of calcium mobilization is an early event in opiate-induced immunosuppression.

Morphine administered as a subcutaneous implant inhibits the initial increase in cytoplasmic free‐calcium [Ca2+]i induced by mitogens in mouse splenocytes. This effect was not reproduced by incubation of splenocytes with morphine (10–8‐10–4 M). Analysis of splenocyte subpopulations demonstrates that this effect was manifest in both B and T cells. However, within T cell subpopulations, CD4+ but not CD8+ cells were affected. Adrenalectomy abolished this effect of morphine in CD4+ T but not CD4−, CD8− spleen cells (most likely Thy 1.2− B cells). Moreover, simultaneous administration of the opiate antagonist naltrexone blocked the effect of morphine in CD4−, CD8− spleen cells, but not in CD4+ T cells. These data indicate that the effects of morphine on mitogen‐stimulated increase in [Ca2+]i may be mediated through distinct glucocorticoid‐dependent and ‐independent mechanisms. The morphine‐induced inhibition of an increase in [Ca2+]; in immune cells reported here may be an early event mediating opiate‐induced immunosuppression.—Sei, Y.; McIntyre, T.; Fride, E.; Yoshimoto, K.; Skolnick, P.; Arora, P. K. Inhibition of calcium mobilization is an early event in opiate‐induced immunosuppression. FASEB J. 5: 2194–2199; 1991.

Strain-dependent association between immune function and paw preference in mice.

The relationship between immune function and the preferred direction of behavioral asymmetry was examined in several mouse strains. Mixed leukocyte reaction, natural killer cell activity, cytotoxic T lymphocyte response and lymphoproliferation in response to mitogens were investigated in animals with left or right paw preference. From the 7 strains and substrains examined, it appeared that differences in immune function between left and right pawed mice, when present, vary in directionality. Thus, in C3H/HeJ and 129/J, left pawed mice had higher immune responses than right pawed mice, whereas in C3H/HeNCr MTV- and BALB/cJ animals, the reverse was found. In C3H/HeNCr MTV+, C57BL/6J and Collins heterogenous control population for the high/low asymmetry lines, no differences between animals with left or right paw preference were found. The statistical significance of these differences were not uniform for all the immune parameters studied. These data indicate that the association between immune function and preference for using the left versus right paw is a strain-dependent phenomenon and may suggest that the inconsistent evidence for an association between immune deficiency and left-handedness could be due to genetic heterogeneity among subpopulations.

Central nervous system infection in a murine retrovirus-induced immunodeficiency syndrome

Astrocyte-enriched primary glial cultures (AGC) from C57BL/6 mice were found to be highly susceptible to infection with the replication competent components of LP-BM5, consisting of the ecotropic and mink cell focus-inducing (MCF) helper murine leukemia viruses (MuLVs). The presence in infected AGC of defective LP-BM5 MuLV genome, a critical component for induction of the disease referred to as murine AIDS, was confirmed by Southern blot hybridization using a probe reactive with the p12 gag sequence of the 4.9 kb defective genome. Electron microscopic studies demonstrated C-type retrovirus particles in both astrocytes and microglial cells. In vivo studies demonstrated that the ecotropic MuLVs and the defective genome could be detected within AGC obtained form either 14-day-old mice following intraperitoneal inoculation or 7-day-old mice following intracranial inoculation. These findings suggest that: (1) the central nervous system (CNS) infection is present at an early stage in murine AIDS, (2) both astrocytes and microglial cells are possible CNS targets in which helper MuLVs replicate, and (3) these cells can harbor the defective genome that is a critical component for disease induction.

Opposing behavioural alterations in male and female transgenic TGFα mice: association with tumour susceptibility

Psychosocial factors are thought to influence risk and survival from cancer. We have previously studied specific behaviours in transgenic male CD-1 MT42 mice, which overexpress the gene encoding human transforming growth factor alpha (TGF alpha) in multiple tissues, and which develop a high incidence of spontaneous hepatocellular carcinoma. The male TGF alpha mice spent a lengthened time immobile in the swim test, were highly aggressive, had increased plasma levels of 17 beta-estradiol (E2), and reduced natural killer (NK) cell activity. The female transgenic MT42 TGF alpha mice do not develop an increased rate of tumours at any site. We hypothesised that if the alterations in male TGF alpha mice are associated with their development of hepatocellular carcinomas, female TGF alpha should not show these alterations. The data in the present study indicate that female TGF alpha mice display shortened immobility in the swim test, suggesting an improved ability to cope with stress, and appear less aggressive in the resident-intruder test than non-transgenic female CD-1 mice. The female TGF alpha mice also exhibit a 3-fold increase in the plasma levels of E2, and a 3-fold increase in NK cell activity. These findings suggest that the elevated expression of TGF alpha in the transgenic mice is associated with gender-specific behavioural alterations, and the development of spontaneous hepatocellular tumours in the males. Furthermore, TGF alpha alters hormonal and immune parameters similarly in both sexes. It remains to be determined whether the development of hepatocarcinoma in the male TGF alpha animals is associated with an impaired ability to cope with stress and elevated aggressive tendencies and/or whether manipulations leading to an impaired ability to cope with stress will promote tumourigenesis in female TGF alpha mice.

Immune function in lines of mice selected for high or low degrees of behavioral asymmetry

Cerebral lateralization has been suggested to play a regulatory role in immune function. In this study, several measures of immune function were evaluated in mice selectively bred for either a strong (HI) or weak (LO) degree of behavioral asymmetry (paw preference) and compared to an unselected control population (HET). Both HI and LO animals had fewer spleen cells but higher degrees of [3H]thymidine incorporation into DNA (on a per cell basis) than HET mice. However, only HI mice had lower immune functions compared to HET controls manifest as reduced mixed leukocyte reaction (MLR), cytotoxic T lymphocyte (CTL) activity, and natural killer (NK) cell activity. These findings indicate that although both extremes in the degree of paw preference may be associated with deviations from the norm, a high degree of behavioral lateralization is associated with decreased immune responsiveness in this animal model.