A regulatory role for astrocytes in HIV-1 encephalitis. An overexpression of eicosanoids, platelet-activating factor, and tumor necrosis factor-alpha by activated HIV-1-infected monocytes is attenuated by primary human astrocytes.

HIV-1-infected brain macrophages participate in neurologic dysfunction through their continual secretion of neurotoxins. We previously demonstrated that astroglial cells activate HIV-1-infected monocytes to produce such neurotoxic activities. In this study, the mechanism underlying these monocyte secretory activities was unraveled and found dependent on HIV-1's ability to prime monocytes for activation. LPS stimulation of HIV-1-infected monocytes resulted in an overexpression of eicosanoids, platelet-activating factor (PAF), and TNF-alpha. This was dependent on the level of HIV-1 infection and monocyte stimulation. Cell to cell interactions between activated virus-infected monocytes and primary human astrocytes reduced monocyte secretions. The capacity of astrocytes to deactivate monocytes was, notably, TGF-beta independent. Although astrocytes constitutively produced latent TGF-beta 2, HIV-1-infected monocytes neither affected TGF-beta 2 production nor converted it into a bioactive molecule. Furthermore, addition of rTGF-beta 1 or rTGF-beta 2 or its Abs to LPS-stimulated monocyte-astrocyte mixtures had no effect on monokine production. In contrast, addition of rIL-10 to LPS-stimulated monocytes produced a dose-dependent decrease in TNF-alpha. IL-10 mRNAs were detected in monocytes, but not astrocytes, following LPS treatment. These results suggest that macrophage activation, a major component of HIV-1 infection in the brain, precipitates neuronal injury by causing virus-infected cells to synthesize neurotoxins. The neurotoxins produced by monocytes are then regulated by astrocytes. Astrocytes therefore, can play either positive or negative roles for disease depending on prior macrophage activation. These findings begin to unravel the cellular control mechanisms that influence cognitive and motor dysfunctions in HIV-1-infected individuals.

Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease.

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, > 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.

No direct neuronotoxicity by HIV-1 virions or culture fluids from HIV-1-infected T cells or monocytes.

Macrophages and microglia are the principal target cells for human immunodeficiency virus (HIV) in brain, and as such, are likely participants in the neuropathology of HIV infection. In a model system for this process, we found that fluids from human monocyte cultures enhanced survival and differentiation of the neurons in fetal rat brain explants. In contrast, fluids from HIV-infected monocyte cultures were strongly toxic to neurons and paradoxically enhanced the proliferation of glial cells. Further, neuronotoxic activity in these fluids was mediated through activation of NMDA binding receptors on the neurons and was inhibited by any of several different NMDA antagonists. Neuronotoxic activity was directly related to contamination of the HIV virus stock with Mycoplasma arginini and M. hominis. Pure cultures of mycoplasma, bacterial lipopolysaccharide (LPS), or murine recombinant tumor necrosis factor alpha (rTNF alpha) each induced neuronotoxicity which exactly mirrored that induced by the contaminated HIV stock. It is likely that mycoplasma or components of the mycoplasma plasma membrane stimulate TNF alpha production by the glial cells in the brain explants. Indeed, careful depletion of glial cells in these explants prevented mycoplasma or LPS-mediated neuronotoxicity. No neuronotoxicity was evident with HIV-1 virus stock, HIV-1 gp120, or culture fluids from HIV-infected T cells or monocytes when these preparations were free of contamination by mycoplasma and LPS. These findings suggest caution in interpretation of those experiments in which similar contamination has not been rigorously excluded.

Dehydroepiandrosterone antagonizes the suppressive effects of dexamethasone on lymphocyte proliferation.

Administration of dehydroepiandrosterone (DHEA) appears to have physiological effects opposing those of glucocorticoids in several animal models. Recently, immunomodulatory effects of treatment with DHEA have been described. This paper reports the effects of DHEA treatment on splenocyte blastogenic responses as well as thymic and spleen weights in C3H/HeN mice. Pretreatment of mice with sc DHEA (60 mg/kg.day) for 3 days in vivo antagonized the profound suppression of in vitro blastogenic responses seen in T- and B-lymphocytes after a single injection of dexamethasone (DEX; 60 mg/kg). Pretreatment with DHEA also significantly reduced dexamethasone-induced thymic and splenic atrophy. Splenic lymphocytes from DHEA-treated mice were markedly more resistant to in in vitro suppression of blastogenesis by DEX at 10(-6)-10(-8) M compared to lymphocytes from control mice. However, DHEA added to lymphocyte cultures in vitro over a concentration range from 10(-7)-10(-8) M failed to protect against suppression of mitogenic responses caused by addition of DEX to cultures. In summary, DHEA given in vivo antagonizes the suppressive actions of DEX on lymphoid target tissues in mice.

Role of adrenal cortical activation in the immunosuppressive effects of chronic morphine treatment.

Implantation of a 75-mg morphine pellet in sham-adrenalectomized male C3H/HeN mice resulted in significant elevations of serum corticosterone levels within 6 h. Corticosterone levels remained elevated (3- to 4-fold) for 72 h and had returned to normal by 120 h postimplantation. Within 48 h of pellet implantation, morphine-pelleted mice exhibited marked reductions in spleen (35%) and thymus weight (56%) relative to values in placebo-pelleted controls. In addition, adrenal hypertrophy was observed in the morphine-pelleted shams (50% increase in adrenal weight relative to placebo. The magnitude of splenic and thymic atrophy was reduced by about 50% in adrenalectomized morphine-pelleted mice (17% and 22% reductions, respectively) compared to that in adrenalectomized mice implanted with placebo pellets. Lymphocyte proliferative responses to the T-cell mitogen Concanavalin-A and the B-cell mitogen bacterial lipopolysaccharide were also significantly reduced in the morphine-pelleted sham mice. Morphine-induced suppression of Concanavalin-A- or lipopolysaccharide-stimulated lymphocyte proliferation was absent in adrenalectomized mice. Effects similar to adrenalectomy (e.g. lessening of magnitude of morphine-induced suppression of lymphoid organ weight and lymphocyte proliferation) were found in morphine-pelleted mice given the glucocorticoid receptor antagonist RU-486 at a dose of 10 mg/kg, twice daily. These studies imply that morphine-induced immunosuppression is at least in part mediated by the increase in serum corticosterone levels after implantation of the morphine pellet.

Effects of soman on neuroendocrine and immune function.

We have previously reported that plasma growth hormone (GH) and prolactin levels were markedly decreased in rats two weeks following a single dose (100 micrograms/kg, SC) of soman. We have now conducted additional experiments to attempt to determine whether neuroendocrine responses to physiological or pharmacological challenge are altered in rat survivors of soman exposure, and whether immune function, which can be affected by circulating hormones, is altered in the soman-exposed rats. In the present study, basal prolactin levels were not significantly lower in the soman-treated rats although prolactin increases in response to physiological or pharmacological challenge were attenuated. Also, basal growth hormone levels in soman survivors were similar to control levels in 2 of 3 experiments in the present report. In the third experiment, growth hormone levels were lower in soman-treated animals. Endocrine abnormalities appeared to be related to the severity of soman insult as assessed by changes in body weight following exposure. Both ACTH and prolactin responses to stress were impaired in a severely affected subpopulation of soman survivors. The thymus, an important immune organ, was decreased in weight in severely affected soman survivors, but other tests of immune function did not show differences between control and soman-exposed rats.

Inhibition of lymphocyte proliferation by antibodies to prolactin.

Recent in vivo studies have shown that treatments that decrease circulating prolactin (PRL) in rodents result in significant immunosuppression. Our attempts to demonstrate corresponding direct stimulatory effects of PRL on cultured lymphocytes were unsuccessful. However, antibodies against pituitary PRL potently inhibited both murine and human lymphocyte proliferation in response to both T and B cell mitogens. Further studies using IL 2 and IL 4 responsive cell lines (CTLL-2 and HT-2) demonstrated that the same anti-PRL antibodies inhibited the proliferative response to these cytokine growth factors. Thus, antibodies to PRL appear to block an event occurring in the G1 to GS phase transition of these cell lines, which constitutively express growth factor receptors. The inhibitory activity of anti-PRL antibodies could be adsorbed by addition of purified human PRL or by immobilized PRL on an affinity column. Antibodies to other pituitary hormones were without inhibitory effect on CTLL-2 cell proliferation. Proliferation of lymphocytes in serum-free medium was also potently inhibited by anti-PRL antibodies, suggesting that antibody effects were not due to neutralization of PRL or other factors contained in culture serum supplements. We suggest from these data that a protein with homology to PRL and recognized by these anti-PRL antibodies is produced by lymphocytes and plays a critical role in their progression through the cell cycle.

Cysteamine produces dose-related bidirectional immunomodulatory effects in mice.

The sulfhydryl reducing agent, cysteamine, is known to functionally inactive prolactin and other neurohormones that have been recently shown to play a role as immunomodulators. Cysteamine was administered to mice to evaluate its effects upon immune organ size and mitogen-induced lymphocyte proliferative responses in relation to corresponding effects on the immunomodulatory hormones, prolactin and corticosterone. The lowest dose of cysteamine, 12.5 mg/kg given once per day for 3 consecutive days, produced significant elevations of both concanavalin A-(Con A) and lipopolysaccharide-induced blastogenesis. Serum prolactin levels were also significantly elevated with 12.5 mg/kg cysteamine. By contrast, at 300 and 400 mg/kg, cysteamine significantly reduced Con A and lipopolysaccharide-induced proliferation. This suppression of mitogen-induced proliferative responses was accompanied by marked atrophy of the thymus. Levels of both prolactin and corticosterone in the serum were significantly reduced at 400 mg/kg cysteamine. A positive correlation was observed between serum prolactin levels and Con A-induced proliferation as well as between serum prolactin and corticosterone levels in cysteamine-treated mice. The immunomodulatory effects of cysteamine were not limited to correlative effects on neuroendocrine parameters. A similar pattern of effects was observed following in vitro administration of cysteamine. Low concentrations in vitro (0.1 mM) stimulated Con A-induced proliferation of normal mouse splenocytes, and higher concentrations in vitro (2.0 mM) suppressed proliferation. These studies indicate that, depending upon the dose, cysteamine has bidirectional effects on mitogen-induced proliferation of lymphocytes; these effects are correlated with cysteamine-related alterations in the neuroendocrine status of the animal but may also be observed with direct addition of the drug to stimulated lymphocytes in culture.

Rapid release of multiple hormones from rat pituitaries perifused with recombinant interleukin-1.

Previous studies demonstrated a direct action of interleukin-1 (IL-1) on release of hormones from rat anterior pituitary cells in monolayer culture. To rule out any possibility of a paracrine effect from the elevated hormones in the static monolayer system, and to examine further the dynamics of hormone release elicited by IL-1, studies were conducted with rat anterior pituitary tissue in a computer-controlled automated perifusion system. In experiments performed on the same day as sacrifice, IL-1 stimulated the release of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), growth hormone (GH) and prolactin (PRL) in a dose-related manner. Peak levels were achieved within 6 minutes of exposure to IL-1. However, PRL was not increased over the baseline fluctuations when pituitaries were perifused with IL-1 after 72 hours of incubation. Hormone release did not appear to undergo desensitization after multiple short pulses of IL-1. Heat-denatured IL-1 had no effect on hormone release. The rapid response suggests that IL-1 acts acutely to release preformed hormone stores.

Morphine pellet-induced immunomodulation in mice: temporal relationships.

Morphine, an alkaloid known for its potent analgetic action, also affects a variety of immunologic functions. In the experiments reported here, the time course for the effect of 75-mg morphine pellet implants on spleen and thymus size and cellularity and in vitro proliferative responses of lymphocytes from male C3H/HeN mice is described. T lymphocyte proliferation in response to concanavalin A (Con A) was not significantly affected at 6 or 24 hr after morphine pellet implantation but was reduced at 48 and 72 hr. B lymphocyte proliferation in response to lipopolysaccharide was more sensitive to morphinization, as the response was reduced at the 24, 48 and 72 hr intervals after implantation of the morphine pellet. No differences in Con A- or lipopolysaccharide-induced proliferation were observed 96 hr after pellet implantation. Interestingly, a slight elevation of Con A-induced proliferation was observed 120 hr after morphine pellet implantation. By contrast with Con A proliferative data, lipopolysaccharide-induced proliferation of lymphocytes from morphine-treated mice was not different from placebo-pelleted mice at 120 hr. A marked atrophy of the spleen and thymus accompanied the reduced splenocyte proliferative responses of morphine-treated mice and was greatest at the 48 to 72 hr postimplantation interval. The attenuation of mitogen-induced proliferative responses and atrophy of immune organs was accompanied by hypothermia and a marked tolerance to the antinociceptive effect of morphine in morphine-pelleted mice at all of the time points that were monitored.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphine-induced immunomodulation is not related to serum morphine concentrations.

Morphine pellets produced atrophy of the spleen and thymus and affected mitogen-induced lymphocyte proliferation in mice as characterized by marked suppression of concanavalin A-induced blastogenesis at 48 h, and mild stimulation at 120 h. Morphine blood levels in these animals indicated that changes in the immunomodulatory effects of morphine over time were not related to dramatic shifts in circulating morphine. Enclosure of the pellet in nylon mesh did not alter blood levels or morphine-induced immunomodulation.

Suppression of macrophage activation and T-lymphocyte function in hypoprolactinemic mice.

The effects of prolactin on lactation and reproductive organs are well known. However, the other possible target organs and physiological consequences of altered levels of circulating prolactin remain poorly understood. In this study, mice were treated with bromocryptine, a dopamine receptor agonist that inhibits pituitary prolactin secretion. Bromocryptine treatment prevented T-cell-dependent induction of macrophage tumoricidal activity after the intraperitoneal injection of Listeria monocytogenes or Mycobacterium bovis. Coincident treatment with ovine prolactin reversed this effect. Of the multiple events leading to macrophage activation in vivo, the production by T-lymphocytes of gamma-interferon was the most impaired in bromocryptine-treated mice. Lymphocyte proliferation after stimulation with mitogens in vitro was also depressed in spleens of bromocryptine-treated mice, and coadministration of prolactin also reversed this effect. Bromocryptine treatment also reduced the number of deaths resulting from inoculation of mice with Listeria; exogenous prolactin significantly reversed this effect. The critical influence of pituitary prolactin release on maintenance of lymphocyte function and on lymphokine-dependent macrophage activation suggests that, in mice, lymphocytes are an important target tissue for circulating prolactin.

Release of multiple hormones by a direct action of interleukin-1 on pituitary cells.

Exposure to bacterial endotoxins has long been known to stimulate the release of anterior pituitary hormones; administration of endotoxin was at one time a common clinical test of anterior pituitary function. Endotoxin is a potent stimulus for production of the endogenous pyrogenic protein, interleukin-1 (IL-1), by macrophages and monocytes. The possibility that IL-1 has a direct effect on the secretion of hormones by rat pituitary cells in a monolayer culture was investigated. Recombinant human IL-1 beta stimulated the secretion of adrenocorticotropic hormone, luteinizing hormone, growth hormone, and thyroid-stimulating hormone. Increased hormone secretion into culture supernatants was found with IL-1 concentrations ranging from 10(-9) M to 10(-12) M. Prolactin secretion by the monolayers was inhibited by similar doses. These concentrations of IL-1 are within the range reported for IL-1 in serum, suggesting that IL-1 generated peripherally by mononuclear immune cells may act directly on anterior pituitary cells to modulate hormone secretion in vivo. Incubation of IL-1 solutions with antibody to IL-1 neutralized these actions. These pituitary effects of IL-1 suggest that this monokine may be an important regulator of the metabolic adaptations to infectious stressors.

Immunosuppressive effects of chronic morphine treatment in mice.

In this report we describe the immunomodulatory effects of subcutaneous morphine pellets in mice, a model commonly used in the study of opiate tolerance and dependence. Mice given a single 75 mg morphine pellet displayed marked atrophy and reduced cellularity of the spleen and thymus, and an attenuated lymphocyte proliferative response to T- and B-cell mitogens (concanavalin A and bacterial lipopolysaccharide, respectively). These immunosuppressive effects were observed 72 hr following implantation of the pellet, a time point by which the mice also had developed tolerance to the antinociceptive effect of the pellet. Splenic and thymic atrophy with reduced mitogen-induced lymphocyte proliferative responses and opiate tolerance were also apparent in mice subjected to a multiple pellet implantation schedule. However, implantation of a pellet containing 37.5 mg morphine did not suppress mitogen-stimulated lymphocyte proliferation, which was slightly elevated in this group. These findings concur with other observations suggesting immunosuppression with morphine tolerance. Furthermore, we suggest that chronic morphine treatment acts as a pharmacologic stressor that mimics behavioral stress.

Naloxone and TRH in the treatment of shock and trauma: what future roles?

Endogenous opioid peptides are released in response to stressful situations, such as circulatory shock, both as hormones and as central and peripheral neurotransmitters. Naloxone, an opiate antagonist, improves cardiovascular function in a variety of animal models of shock caused by endotoxemia, hemorrhage, anaphylaxis, or spinal trauma. Administration of thyrotropin-releasing hormone (TRH) in supraphysiologic doses also has pressor effects in these shock models. Given acutely after injury, TRH improves recovery in models of spinal trauma; however, the experimental effects of TRH do not involve action at the opiate receptor. Clinical evaluation of the use of naloxone in patients with shock has been largely limited to treatment of sepsis. The paucity of prospective, randomized trials makes these clinical data difficult to evaluate, but in septic patients the use of naloxone does not seem to improve survival. The use of naloxone in shock of other etiologies has not been clinically investigated, and may hold greater promise. Acute-phase treatment of spinal trauma victims with TRH is currently undergoing clinical trials.

Opioids and neuropeptides: mechanisms in circulatory shock.

Endogenous opioid systems are activated in stressful situations such as circulatory shock. The opiate antagonist naloxone improves cardiovascular function in several models of shock caused by endotoxemia, hypovolemia, anaphylaxis, and spinal trauma. The ergotropic neuropeptide, thyrotropin-releasing hormone, in supraphysiological doses, also improves cardiovascular function in these shock models, but this effect does not result from action at the opiate receptor. For both these agents a central nervous system (CNS) site of action has been partially characterized. A variety of neuropeptides, including the opioids, seem capable of modulating autonomic function through their CNS actions. In addition, they may play a role in peripheral integration and transmission of autonomic nervous activity by actions at the ganglia and/or at nerve endings. Some neuropeptides also have direct autacoid effects on cells, including those of the microvasculature. This raises new questions concerning possible peripheral functions of neuropeptides during circulatory shock, and the nature of their interactions with other potential shock mediators such as monokines and arachidonic acid derivatives.