Interleukin-6 and glucocorticoids synergistically induce human immunodeficiency virus type-1 expression in chronically infected U1 cells by a long terminal repeat independent post-transcriptional mechanism.

BACKGROUND: Glucocorticoids (GC) such as dexamethasone (Dex) can directly upregulate human immunodeficiency virus type-1 (HIV-1) replication in acutely infected cells and potentiate HIV expression from chronically infected promonocytic U1 cells stimulated with tumor necrosis factor-alpha (TNF-alpha). We have here investigated the potential effect of Dex in U1 cells stimulated with interleukin-6 (IL-6), a cytokine inducing virus expression by acting mostly at a post-transcriptional level on the virus life cycle. MATERIALS AND METHODS: Virus production in culture supernatants was evaluated by reverse transcriptase (RT) activity. GC receptor expression was tested by both binding of [3H]-Dexamethasone 21-mesylate and Northern blotting. Cell-associated HIV protein expression was analyzed by Western blotting, whereas both HIV and monocyte chemoattractant protein-1 (MCP-1) RNA accumulation were evaluated by Northern blotting. HIV transcription was tested by long terminal repeat (LTR) chloramphenicol acetyl transferase (CAT) assay after transient transfection of U1 or U937 cells. Formation of activating protein-1 (AP-1) DNA binding complex in nuclear cell extracts was visualized by electrophoretic mobility shift assay (EMSA), whereas ERK1/2 mitogen-activated protein kinase (MAPK) phosphorylation was studied by Western blotting. RESULTS: IL-6 and Dex synergistically induced HIV expression in U1 cells, and this effect was blocked by RU 486. No substantial HIV RNA accumulation was demonstrated in U1 cells co-stimulated with IL-6 and Dex, whereas IL-6 upregulated the expression of MCP-1 RNA, and this effect was inhibited by Dex. In contrast, Dex potentiated IL-6 induced activation of AP-1 and ERK1/2 MAPK phosphorylation, as revealed by EMSA. HIV-1 LTR driven transcription was observed in U1 cells stimulated with TNF-alpha and this effect was potentiated by Dex. In sharp contrast, no induction of LTR-directed CAT activity was observed in transfected U1 cells (or in their parental uninfected U937 cells) stimulated with IL-6 and Dex either alone or in combination. CONCLUSIONS: High levels of virion production can be induced in latently infected cells by stimulation with IL-6 and Dex in the absence of activation of the HIV LTR or viral transcription in spite of activation of both ERK1/2 MAPK and AP-1. These findings suggest the existence of LTR-independent pathways influenced by cytokine and GC through which HIV can maintain substantial levels of protein expression and virion production.

Effect of immunization with a common recall antigen on viral expression in patients infected with human immunodeficiency virus type 1.

BACKGROUND: Activation of the immune system is a normal response to antigenic stimulation, and such activation enhances the replication of human immunodeficiency virus type 1 (HIV-1). We studied the effect of immunization with a common recall antigen on viral expression in HIV-1-infected patients, on the ability to isolate virus, and on the susceptibility to HIV-1 infection of peripheral-blood mononuclear cells (PBMCs) from control subjects not infected with HIV-1. METHODS: Thirteen HIV-1-infected patients and 10 uninfected adults were given a 0.5-ml booster dose of tetanus toxoid. Studies were performed to evaluate changes in the degree of plasma viremia, proviral burden, the ability to isolate HIV-1, and the susceptibility of PBMCs to acute infection in vitro. Two patients underwent sequential lymph-node biopsies for the assessment of viral burden in these tissues. RESULTS: All 13 HIV-1-infected patients had transient increase in plasma viremia after immunization, and the proviral burden increased in 11. These changes did not correlate with the base-line CD4+ T-cell counts. The lymph-node tissue also had increases in the proviral burden and viral RNA after immunization. The virus was more easily isolated from PBMCs from nine of the patients after immunization than before immunization. Despite considerable variability in the results, PBMCs from 7 of the 10 normal subjects were more easily infected in vitro with HIV-1 after immunization than before immunization. CONCLUSIONS: Activation of the immune system by an ongoing antigen-specific immune response to an exogenous stimulus transiently increases the expression of HIV-1 and may enhance the susceptibility of uninfected subjects to HIV-1.

Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse.

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells.

Glucocorticoids synergize with tumor necrosis factor alpha in the induction of HIV expression from a chronically infected promonocytic cell line.

In this study we have investigated the effects of glucocorticoids (GCs) on the expression of human immunodeficiency virus (HIV) in a chronically infected promonocytic cell line, U1. Although no increase in virus production was observed in U1 cells stimulated with physiological concentrations of GC alone, costimulation with dexamethasone plus tumor necrosis factor alpha (TNF-alpha) synergistically enhanced TNF-alpha-dependent HIV expression. Molecular analysis demonstrated that GCs plus TNF-alpha resulted in an accumulation of steady state HIV RNA secondary to either an increase in transcription or an increase in message stability. These findings may be of physiological relevance because GCs are used in the treatment of certain disorders associated with HIV infection and TNF-alpha levels have been reported to be elevated in the plasma and cerebrospinal fluid of certain HIV-infected individuals.

Interferon gamma induces the expression of human immunodeficiency virus in persistently infected promonocytic cells (U1) and redirects the production of virions to intracytoplasmic vacuoles in phorbol myristate acetate-differentiated U1 cells.

Interferon gamma (IFN-gamma), a lymphokine that exerts multiple immunoregulatory effects, has been found to be elevated in the plasma, cerebrospinal fluid, and lymph nodes of human immunodeficiency virus (HIV)-infected individuals and has shown variable effects on HIV replication in acutely infected cells. In the present study, we have demonstrated that IFN-gamma is a potent modulator of HIV expression in persistently infected U1 promonocytic cells in which virus production is characterized by a constitutive state of relative latency. Direct stimulation of U1 cells with IFN-gamma (10-1,000 U/ml) activated HIV expression, as measured by reverse transcriptase (RT) activity in the culture supernatant and increased levels of cell-associated viral protein and mRNAs. These effects on virus expression were not accounted for by the induction of endogenous TNF-alpha secretion, as previously described in U1 cells stimulated with phorbol myristate acetate (PMA). At the ultrastructural level, the stimulatory activity of IFN-gamma was correlated with HIV particle production in intracytoplasmic vacuoles along with the differentiation of U1 into macrophage-like cells. Furthermore, costimulation of U1 cells with IFN-gamma and PMA significantly increased the accumulation of vacuole-associated HIV concomitant with decreasing membrane-associated particles and RT activity production, as compared with cells stimulated with PMA alone. No evidence of spontaneous secretion of intracellular vacuole-associated virus was obtained by kinetic analysis of the RT activity released in the supernatants throughout the culture period unless cells were deliberately disrupted. These findings suggest that vacuole-associated virions likely represent a relatively stable intracellular reservoir of HIV, as previously described in primary macrophages infected in vitro or in infected macrophages in the brains of patients with acquired immune deficiency syndrome. The reduced levels of RT activity observed in the culture supernatants of U1 cells stimulated with PMA in the presence of IFN-gamma were not indicative of a suppressive effect of IFN-gamma on PMA-induced expression of HIV proteins and mRNAs, either directly or mediated by the release of IFN-alpha/beta. This study suggests that IFN-gamma may play an important role as an inducer of HIV expression in infected mononuclear phagocytes.

CD34+ bone marrow cells are infected with HIV in a subset of seropositive individuals.

Individuals infected with HIV frequently develop cytopenias and suppressed hematopoiesis. The role of direct HIV infection of hematopoietic progenitor cells in this process has not been defined. In this study, purified CD34+ bone marrow progenitor cells from 74 Zairian and American patients were studied by both coculture viral isolation and polymerase chain reaction for evidence of HIV infection. A total of 36.5% of Zairian and 14% of American patients had HIV infection of the CD34+ cell subset, with as many as 1 in 500 CD34+ cells infected. Most of the Zairian patients in this study had advanced HIV infection and markedly decreased CD4/CD8 T lymphocyte ratios (mean 0.160 +/- 0.08), and no laboratory value predicted the presence of infection in the CD34+ subset of a given Zairian individual. In contrast, American patients with CD34+ cell infection had total CD4 cells less than 20/mm3 and a greater decrease of the CD4/CD8 T lymphocyte ratio compared to seropositive Americans without CD34+ cell infection (p = 0.003). Hematopoiesis, studied by methylcellulose colony assays, was depressed in all seropositive patients studied with no significant further suppression when CD34+ cells were infected. Thus, CD34+ bone marrow progenitor cells are infected in vivo in a subset of seropositive individuals and may serve as an additional reservoir of virus in HIV-infected individuals.

Retinoic acid mimics transforming growth factor beta in the regulation of human immunodeficiency virus expression in monocytic cells.

Retinoic acid (RA) exerts potent suppressive and upregulatory effects on human immunodeficiency virus (HIV) expression in mononuclear phagocytes, strikingly similar to the effects of the cytokine transforming growth factor beta (TGF-beta). RA significantly inhibited phorbol ester-mediated, but not tumor necrosis factor alpha-mediated, induction of HIV transcription in the chronically infected promonocytic U1 cell line. RA and TGF-beta also completely suppressed the induction of virus production in U1 cells by interleukin 6 alone or in combination with glucocorticoids, which predominantly upregulate virus expression at the posttranscriptional level. Despite the close parallel to TGF-beta-induced effects, no evidence was obtained that RA mediated its effect by inducing secretion of active TGF-beta 1, -beta 2, or -beta 3. As with chronically infected U1 cells, similar inhibitory effects were also observed in primary monocyte-derived macrophages previously infected with HIV and then exposed to either RA or TGF-beta. In contrast, stimulation of monocyte-derived macrophages or U937 cells (the parental cell line of U1) with either RA or TGF-beta prior to in vitro infection resulted in the enhancement of virus production. Given the already successful use of retinoids in the treatment of several malignancies and the present demonstration of their capability of blocking the induction of HIV expression in infected mononuclear phagocytes, it would be of interest to pursue the potential role of this class of compounds in the development of strategies aimed at the pharmacologic regulation of HIV expression.

An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation.

A chronically HIV-1-infected T cell clone (J1.1) derived from Jurkat cells was developed that possesses defects in CD3 signaling. This clone was phenotypically determined to be CD4- and express a reduced surface density of CD3 as compared with a pool of uninfected Jurkat clones. Although J1.1 could be induced with TNF-alpha to produce HIV-1 particles, stimulation via the CD3 (T3-Ti) complex, using mAb cross-linking, had no effect on viral production. Further investigation revealed that J1.1 secreted approximately 20-fold less IL-2 than did uninfected Jurkat cells after anti-CD3 treatment. In addition, a separate defect in Ca2+ mobilization was noted in the HIV-1-infected J1.1 line when compared with uninfected Jurkat cells after anti-CD3 cross-linking. The cell line described offers a new model in which to study the mechanisms of several defects directly imposed by HIV-1 on CD3+ cells.

Transforming growth factor beta suppresses human immunodeficiency virus expression and replication in infected cells of the monocyte/macrophage lineage.

The pleiotropic immunoregulatory cytokine transforming growth factor beta (TGF-beta) potently suppresses production of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome, in the chronically infected promonocytic cell line U1. TGF-beta significantly (50-90%) inhibited HIV reverse transcriptase production and synthesis of viral proteins in U1 cells stimulated with phorbol myristate acetate (PMA) or interleukin 6 (IL-6). Furthermore, TGF-beta suppressed PMA induction of HIV transcription in U1 cells. In contrast, TGF-beta did not significantly affect the expression of HIV induced by tumor necrosis factor alpha (TNF-alpha). These suppressive effects were not mediated via the induction of interferon alpha (IFN-alpha). TGF-beta also suppressed HIV replication in primary monocyte-derived macrophages infected in vitro, both in the absence of exogenous cytokines and in IL-6-stimulated cultures. In contrast, no significant effects of TGF-beta were observed in either a chronically infected T cell line (ACH-2) or in primary T cell blasts infected in vitro. Therefore, TGF-beta may play a potentially important role as a negative regulator of HIV expression in infected monocytes or tissue macrophages in infected individuals.

Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals.

CD4+ T cells of patients with AIDS exhibit a qualitative defect in their ability to respond to soluble antigen while their responses to mitogens remain normal. CD4+ T cells can be broadly divided phenotypically into "naive" [CD45RA+ (2H4+)] and "memory" [CD29+ (4B4+) or CD45RO+ (UCHL1+)] cell subpopulations, which represent distinct maturation stages. To determine the human immunodeficiency virus type 1 (HIV-1) infectability of memory and naive CD4+ T-cell subsets in vitro and to determine the in vivo preference of HIV-1 in these subpopulations, we obtained highly purified CD4+ T-cell subsets from normal and HIV-1-infected individuals and studied them by viral cultivation, quantitative polymerase chain reaction, and functional assays. Polymerase chain reaction studies demonstrated that the memory cell subset of CD4+ T cells is preferentially infected (4- to 10-fold more than naive T cells) by HIV-1 in vitro, and these memory cells are the principal reservoir for HIV-1 within CD4+ T cells obtained from infected individuals. Functional abnormalities attributable to CD4+ T cells in HIV-infected individuals (failure to respond in vitro to soluble antigen or to anti-CD3 monoclonal antibodies) were shown to reside primarily within these memory cells. Thus, the present study suggests that the selective functional defects present in the memory CD4+ T-cell subset of HIV-infected individuals may be a direct result of the preferential infection and consequently greater viral burden within these cells.

Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and post-transcriptional mechanisms.

The immunoregulatory cytokine interleukin 6 (IL-6) directly upregulates production of human immunodeficiency virus (HIV) in acutely as well as in chronically infected cells of monocytic lineage. In addition, IL-6 synergizes with tumor necrosis factor alpha (TNF-alpha) in the induction of latent HIV expression. Unlike TNF-alpha, upregulation of viral expression induced by IL-6 alone does not occur at the transcriptional level and it is not associated with accumulation of HIV RNA. However, when IL-6 and TNF-alpha synergistically stimulate HIV production, accumulation of HIV RNA and increased transcription are observed, indicating that IL-6 affects HIV expression at multiple (transcriptional and post-transcriptional) levels.

Tumor necrosis factor alpha functions in an autocrine manner in the induction of human immunodeficiency virus expression.

Tumor necrosis factor alpha (TNF-alpha) is an immunoregulatory cytokine capable of inducing viral expression in cells chronically infected with the human immunodeficiency virus (HIV), such as the promonocytic line U1 and the T-lymphocytic line ACH-2. In the present study, we demonstrate an autocrine mechanism of TNF-alpha-mediated HIV induction. Stimulation of U1 and ACH-2 cells with phorbol 12-myristate 13-acetate (PMA) resulted in the induction of TNF-alpha mRNA and the secretion of TNF-alpha. Of note is the fact that anti-TNF-alpha antibodies significantly suppressed the expression of HIV in PMA-stimulated U1 and ACH-2 cells. Furthermore, anti-TNF-alpha antibodies also suppressed both the constitutive and inducible levels of viral expression in the chronically infected promonocytic clone U33.3. This study illustrates the interrelationship between the regulation of HIV expression and normal immunoregulatory mechanisms in that virus expression, both constitutive and induced, can be modulated by an autocrine pathway involving TNF-alpha, a cytokine involved in the complex network of regulation of the normal human immune response.

Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow.

Myeloid progenitor cells were highly purified from normal human bone marrow by positive immunoselection with high-affinity monoclonal antibodies linked to magnetic beads and were successfully infected in vitro with the human immunodeficiency virus type 1 (HIV-1). From 99 to 100 percent pure bone marrow cells expressing the CD34 phenotypic marker were obtained. These cells were devoid of mature myeloid or T cell surface and intracellular markers as analyzed by immunohistochemical staining and flow cytometry. HIV-1 particles were detected by supernatant reverse transcriptase activity and transmission electron microscopy 40 to 60 days after infection. Viral particles were predominantly observed assembling and accumulating from within intracellular membranes, while phenotypically the cells were observed to have differentiated into CD4+ monocytes. These studies have important implications in understanding the pathogenesis of HIV-1 as well as the possible cause of certain of the observed hematologic abnormalities in HIV-1 infection. They also indicate that the bone marrow may serve as a potentially important reservoir of HIV-1 in the body.