Spectrum of cdk-9 inhibitor activity against HIV-1 replication among various models of chronic and latent infection.

The cellular transcription elongation factor, P-TEFb, and its kinase component, cdk-9, have been implicated in the regulation of HIV-1 transactivation and transcription. We tested a panel of demonstrated cdk-9 kinase inhibitors for the ability to block HIV-1 expression in a variety of cell models representing chronic and latent/inducible infection. These agents induced cellular toxicity, in accordance with their potency for cdk-9 inhibition, with more pronounced toxicity in cultures of T-cell lineage. These agents also inhibited HIV-1 expression, in accordance with their potency for cdk-9 inhibition, in several latent models tested (representing T-lymphocytic, promonocytic and promyelocytic lineages) and using various extracellular stimuli that activate HIV-1 expression via distinct intracellular pathways. Such was the case even though some of these cell models of latent/inducible HIV-1 infection harbour viral defects in the HIV-1 transactivation mechanism. Two additional cell models of latent/inducible HIV-1 infection, both derived from Jurkat T-lymphocytes, were relatively resistant to inhibition of viral expression by these agents. This apparent lack of effect was most likely due to the narrow therapeutic range of these agents in T-cell cultures. Inhibition of HIV-1 replication by these agents was also observed in two cell models representing constitutive viral expression in cells of T-lymphocytic and promyelocytic lineages. Overall, the observed pattern of viral inhibition with these compounds suggests that cdk-9 enzymatic activity is important for HIV-1 expression irrespective of cell lineage or cellular pathway of viral activation. However, because of the non-selective nature of these inhibitors, other cellular pathways must also be considered. Agents that target cellular components essential for HIV-1 expression may provide new therapeutic approaches to limit viral replication, especially when combined with potent antiretroviral regimens.

Anatomically compartmentalized human immunodeficiency virus replication in HLA-DR+ cells and CD14+ macrophages at the site of pleural tuberculosis coinfection.

This study examined the impact of the host inflammatory microenvironment associated with localized tuberculosis (TB) on human immunodeficiency virus type 1 (HIV-1) replication within lymphocytes and macrophages in vivo. Paired plasma and pleural fluid samples from HIV-1-infected individuals with pleural TB (n=9) were analyzed. Detection of host proteins incorporated into the HIV-1 envelope by immunomagnetic capture analysis provided insight into the phenotype of cells supporting HIV-1 replication. The results indicated that the 4.0-fold greater median HIV-1 load in pleural fluid, compared with median load in plasma (P<.01), was derived in part from viral replication within HLA-DR+ cells, CD26+ lymphocytes, and, importantly, CD14+ macrophages. Greatly increased local concentrations of proinflammatory cytokines and immune activation markers in the pleural space correlated with the virologic findings. In summary, HIV-1 replication was increased at sites of Mycobacterium tuberculosis coinfection within activated cells, including lymphocytes and CD14+ macrophages.

Production of a novel viral suppressive activity associated with resistance to infection among female sex workers exposed to HIV type 1.

To investigate mechanisms of natural resistance to human immunodeficiency virus type 1 (HIV-1), we obtained blood samples from eight women who remained HIV-1 negative after > 3 years of high-risk sex work in Chiang Rai, Thailand. CD4+ T lymphocytes from these highly exposed, persistently seronegative (HEPS) women were readily infectable in vitro with HIV-1 subtypes B and E. Autologous CD8+ cell suppression of both HIV-1 subtypes was evident in HEPS infection cultures, but to an extent also observed in cultures from non-HIV-exposed individuals. Furthermore, production of beta-chemokines was not enhanced in HEPS cultures. However, HEPS cultures displayed significantly enhanced production of a soluble activity that suppressed postintegrated HIV-1 replication. This activity was the unique product of CD4+ T cell and monocyte cocultures. Therefore, although HEPS individuals are apparently susceptible to infection, the production of a postintegrated HIV-1 suppressive activity during monocyte-T cell interactions might protect against the establishment of infection by limiting viral dissemination.

The steroid receptor antagonists RU40555 and RU486 activate glucocorticoid receptor translocation and are not excreted by the steroid hormones transporter in L929 cells.

RU40555 is a recently available glucocorticoid receptor (GR) antagonist that differs from RU486 by a methyl radical. We have used the mouse fibroblast cell line L929 to study the in vitro effects of RU40555 on GR translocation and function and on the membrane steroid hormones transporter. The results showed that: 1) RU40555 competed for the binding of labelled dexamethasone (Dex) with a K(i) of 2.4 nM; 2) both RU40555 and RU486 were equally potent inhibitors of Dex-induced GR-mediated gene transcription; 3) maximum GR translocation induced by micromolar concentrations of Dex and the GR antagonists was approximately 30-55% loss in the cytoplasmic GR and approximately 40-90% increase in the nuclear GR (assessed by GR immunostaining in cytoplasm and nucleus and western blots of immunoprecipitated GR protein in cytosolic and nuclear fractions) and was similar for the two antagonists; 4) at nanomolar concentrations, RU40555 and RU486 induced more GR translocation than Dex (assessed by [(3)H]Dex binding and western blot of immunoreactive GR in the same cytosolic homogenates); 5) blocking the steroids membrane transporter with verapamil (100 microM) in the presence of Dex (10 nM) increased GR translocation to levels similar to those induced by RU40555 (10 nM) and RU486 (10 nM) alone; 6) verapamil did not affect GR translocation in the presence of RU40555 or RU486. These data demonstrate similar quantitative effects on GR translocation by RU486 and the new GR antagonist, RU40555. Moreover, RU40555, like RU486, is an effective GR antagonist. Finally, there is no evidence that the intracellular concentrations of RU40555 or RU486 are regulated by the steroids membrane transporter in L929 cells.

Lymphocytic responses and the gradual hippocampal neuron loss following infection with lymphocytic choriomeningitis virus (LCMV).

Infection of rats with LCMV is known to cause a bi-phasic neurodegeneration characterized by acute T lymphocyte-mediated cerebellar damage, followed by gradual hippocampal neuron loss that occurs by an undefined mechanism. We found infiltration of CD8 + T-cells (but not CD4 + or NK cells) in the hippocampus which correlated with the acute phase, but not the chronic hippocampal degenerative phase. While immunosuppression of T lymphocytes protected the cerebellum and revealed the infection of corticohippocampal glia, the degeneration in the hippocampus was unabated. These data suggest that T lymphocytes control glial infection and mediate degeneration in the cerebellum but not the hippocampus.

The proinflammatory cytokine, interleukin-1alpha, reduces glucocorticoid receptor translocation and function.

Proinflammatory cytokines have been shown to influence the expression and function of the glucocorticoid receptor (GR). Specifically, several studies have found that cytokines induce a decrease in GR function, as evidenced by reduced sensitivity to glucocorticoid effects on functional end points. To investigate the potential mechanism(s) involved, we examined the impact of the proinflammatory cytokine, interleukin-1alpha (IL-1alpha), on 1) GR translocation from cytoplasm to nucleus using GR immunostaining, 2) cytosolic radioligand GR binding, and 3) GR-mediated gene transcription in L929 cells stably transfected with the mouse mammary tumor virus-cholamphenicol acetyltransferase reporter gene. L929 cells were treated with IL-1alpha (100 and 1000 U/ml) for 24 h in the presence or absence of dexamethasone (Dex; 10 nM to 1 microM). IL-1alpha inhibited Dex-induced GR translocation and alone induced GR up-regulation. Pretreatment with IL-1alpha followed by Dex treatment for 1.5 h led to about 20% inhibition of Dex-induced GR-mediated gene transcription, whereas coincubation of IL-1alpha plus Dex for 24 h inhibited Dex-induced GR-mediated gene activity up to 42%. The latter effect was reversed by the IL-1 receptor antagonist. These results suggest that cytokines produced during an inflammatory response may induce GR resistance in relevant cell types by direct effects on the GR, thereby providing an additional pathway by which the immune system can influence the hypothalamic-pituitary-adrenal axis.

Glucocorticoid receptors are differentially expressed in the cells and tissues of the immune system.

Cytosolic glucocorticoid receptor (GR) binding studies on immune tissues demonstrate that the thymus exhibits three to four times higher levels of GR protein than the spleen. High levels of GR are consistent with the exquisite sensitivity of the thymus to glucocorticoid exposure. Nevertheless, whole cell binding studies reveal similar levels of GR in immature thymic T lymphocytes and more mature, splenic T lymphocytes. Moreover, whole cell binding techniques indicate that neutrophils (which represent roughly 30% of splenic leukocytes) exhibit higher GR than both T and B lymphocytes, further contradicting results from cytosolic binding studies. To address these inconsistencies, GR protein was assessed in immune cells and tissues using cytosolic radioligand binding. Western blot analysis, and immunocytochemistry. Consistent with previous cytosolic receptor binding studies on immune tissue homogenates, thymic T cells were found to have higher levels of GR than T cells isolated from the spleen. In addition, neutrophils were found to have fewer GR than lymphocytes and monocytes. These results indicate a meaningful relationship between receptor expression and known sensitivity to glucocorticoids.

Steroid-independent translocation of the glucocorticoid receptor by the antidepressant desipramine.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that in its unactivated form resides primarily in the cytoplasm. After being bound by steroid, the GR undergoes a conformational change and translocates to the nucleus, where it influences gene transcription. Because the GR mediates negative feedback exerted by circulating glucocorticoid hormones on the hypothalamic-pituitary-adrenal (HPA) axis, it has been hypothesized that abnormalities in GR expression and/or function may underlie the HPA axis hyperactivity described in patients with major depression. In further support of this hypothesis, animal studies have shown that long term in vivo treatment with antidepressants enhances glucocorticoid feedback inhibition, possibly through a direct effect on the GR. To examine this latter possibility, we evaluated translocation of the GR from the cytoplasm to the nucleus after 24-hr in vitro treatment of L929 cells (mouse fibroblasts) with the tricyclic antidepressant desipramine (0.1-10 microM) in the presence or absence of the synthetic steroid dexamethasone. In addition, GR-mediated gene transcription was measured with the use of L929 cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene. Desipramine was found to (i) induce GR translocation from the cytoplasm to the nucleus in the absence of steroids (with no effect alone on GR-mediated gene transcription) and (ii) potentiate dexamethasone-induced GR translocation and dexamethasone-induced GR-mediated gene transcription. Treatment with desipramine for 24-96 hr had no effect on the expression of GR protein as measured by cytosolic radioligand receptor binding. We suggest that one important aspect of the effects of antidepressants in vivo may be to facilitate GR-mediated feedback inhibition on the HPA axis, by facilitating GR translocation and function, and thereby reverse glucocorticoid hypersecretion in depression.

1996 Curt P. Richter Award. Effects of viral infection on corticosterone secretion and glucocorticoid receptor binding in immune tissues.

During an immune challenge it has been suggested that responding cells secrete cytokines which then stimulate the release of glucocorticoids. Glucocorticoids, in turn, are believed to bind to their receptors in target immune tissues and provide feedback inhibition on evolving immune responses. The foundations for this hypothesis have been drawn primarily from studies on animal models of autoimmune and/or inflammatory processes, and the relevance of these glucocorticoid-immune interactions to viral infections has not been extensively examined. Accordingly, we infected mice with lymphocytic choriomeningitis virus (LCMV) and measured plasma corticosterone and cytosolic glucocorticoid receptor (GR) binding at multiple time points throughout the day and throughout infection (days 3, 5, 7 and 10 post infection). Despite a vigorous immune response to this virus, LCMV infection was associated with minimal and transient increases in corticosterone secretion. Interestingly, however, significant decreases in cytosolic GR were found in immune tissues. Receptor decreases were characterized by a significant decrease in GR binding during the diurnal rise in corticosterone in the spleen and thymus of infected but not uninfected animals on days 5-10 post infection. In addition, in the morning on these days, GR binding in the spleen of infected mice was decreased compared to uninfected control mice. Following an acute injection of corticosterone on day 7 post infection, LCMV-infected animals exhibited a significantly greater decrease in splenic GR binding than uninfected control mice, suggesting an increased sensitivity to corticosterone in infected animals. No changes were found in the affinity (Kd) of the GR during infection, nor was there evidence of an infection-associated decrease in plasma corticosteroid binding globulin. The appearance of significant GR changes in the spleen and thymus, in the absence of significant elevations in corticosterone or decreases in its binding protein, suggests that cytokines and/or other factors produced within the immune tissues during infection either directly influenced GR number and/or function or influenced the local availability of corticosterone. Taken together, the results indicate that interactions between the neuroendocrine and immune systems can be modified at the level of the GR in the context of an ongoing immune response such as during a viral infection.