Infection Of Mononuclear Phagocytes Research Articles

Predictive biomarkers for cognitive decline during progressive HIV infection.

Predictive biomarkers for cognitive decline during progressive HIV infection.

Necroptosis of infiltrated macrophages drives Yersinia pestis dispersal within buboes.

When draining lymph nodes become infected by Yersinia pestis (Y. pestis), a massive influx of phagocytic cells occurs, resulting in distended and necrotic structures known as buboes. The bubonic stage of the Y. pestis life cycle precedes septicemia, which is facilitated by trafficking of infected mononuclear phagocytes through these buboes. However, how Y. pestis convert these immunocytes recruited by host to contain the pathogen into vehicles for bacterial dispersal and the role of immune cell death in this context are unknown. We show that the lymphatic spread requires Yersinia outer protein J (YopJ), which triggers death of infected macrophages by downregulating a suppressor of receptor-interacting protein kinase 1-mediated (RIPK1-mediated) cell death programs. The YopJ-triggered cell death was identified as necroptotic, which released intracellular bacteria, allowing them to infect new neighboring cell targets. Dying macrophages also produced chemotactic sphingosine 1-phosphate, enhancing cell-to-cell contact, further promoting infection. This necroptosis-driven expansion of infected macrophages in buboes maximized the number of bacteria-bearing macrophages reaching secondary lymph nodes, leading to sepsis. In support, necrostatins confined bacteria within macrophages and protected mice from lethal infection. These findings define necrotization of buboes as a mechanism for bacterial spread and a potential target for therapeutic intervention.

Chemokine CCL2 enhances NMDA receptor-mediated excitatory postsynaptic current in rat hippocampal slices-a potential mechanism for HIV-1-associated neuropathy?

Human immunodeficiency virus type 1 (HIV-1)-infected mononuclear phagocytes (brain macrophages and microglial cells) release proinflammatory cytokines and chemokines. Elevated levels of chemokine CC motif ligand 2 (CCL2, known previously as monocyte chemoattractant protein-1) have been detected in serum and cerebrospinal fluid (CSF) of HIV-1-infected individuals and the raised CCL2 in the CSF correlates with HIV-1-associated neurocognitive disorders. To understand how elevated CCL2 induces HIV-1-associated neuropathy, we studied effects of CCL2 on excitatory postsynaptic current (EPSCs) in the CA1 region of rat hippocampal brain slices using whole-cell patch recording techniques. The AMPA receptor (AMPAR)-mediated EPSC (EPSCAMPAR) and N-Methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated EPSCs (EPSCNMDAR) were isolated pharmacologically. Bath application of CCL2 produced a significant enhancement of the amplitudes of EPSCs, EPSCAMPAR and EPSCNMDAR. Further studies revealed that CCL2 potentiated NMDAR subtype NR2A-mediated EPSC (EPSCNR2AR) and NR2B-mediated EPSC (EPSCNR2BR). To determine the site of action, we recorded spontaneous mini EPSCs (mEPSC) before and during bath application of CCL2. Our results showed that CCL2 decreased inter event interval (IEI) and increased the frequency of mEPSCs without change on the amplitude, suggesting a presynaptic site of CCL2 action. CCL2 was also found to injure primary rat hippocampal neuronal cultures and neuronal dendrites in the CA1 region of hippocampal slices. The CCL2-associated neuronal and dendritic injuries were blocked by a specific NMDAR antagonist or by a CCR2 receptor antagonist, indicating that CCL2-associated neural injury was mediated via NMDARs and/or CCR2 receptors. Taken together, these results suggest a potential role CCL2 may play in HIV-1-associated neuropathology.

Role of protease inhibitor 9 in survival and replication of Mycobacterium tuberculosis in mononuclear phagocytes from HIV-1-infected patients

Predisposition to opportunistic infections by Mycobacterium tuberculosis (MTB) is a concomitant of HIV-1 infection and occurrence of tuberculosis is independent of circulating CD4(+) T-cell count in HIV-1-infected patients. Infection of mononuclear phagocytes from healthy individuals by virulent MTB is associated with expression of the antiapoptotic molecule protease inhibitor 9 (PI-9), and PI-9 contributes to successful parasitism of macrophages by MTB. Here we studied the contribution of PI-9 to successful MTB infection of monocytes from HIV-1-infected patients. Blood monocytes obtained from HAART-treated HIV-1-infected patients (HIV+) and healthy controls were assessed for support of MTB H37Rv growth by assessment of MTB 16S ribosomal (r)RNA in cell lysates on day 1 and day 7 by real-time reverse transcription-PCR. PI-9 expression in monocyte cell lysates was assessed by ELISA and by reverse transcription-PCR. Inhibition of intracellular PI-9 was achieved by siRNA to PI-9 and compared to control constructs. Monocytes from HIV-infected patients supported higher MTB growth [MTB 16S rRNA (d7/d1)] as compared with monocytes from healthy controls. Both PI-9 protein and mRNA were significantly higher in monocytes from HIV-infected patients as compared with healthy controls. PI-9 protein levels prior to MTB infection correlated with MTB replication on day 7, and with plasma soluble CD14 levels. Silencing of PI-9 by transfection of monocytes from HIV-1-infected patients with PI-9-specific siRNA prior to infection improved intracellular containment of MTB. Increased intracellular PI-9 activity in mononuclear phagocytes from HIV-infected patients contributes to successful intracellular infection by virulent MTB.

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC50 of 11.26μg/ml against promastigotes and amastigotes of 0.27μg/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests.

Retrovirally induced CTL degranulation mediated by IL-15 expression and infection of mononuclear phagocytes in patients with HTLV-I–associated neurologic disease

AbstractCD8+ T cells contribute to central nervous system inflammation in human T-cell lymphotropic virus type I (HTLV-I)–associated myelopathy/tropical spastic paraparesis (HAM/TSP). We analyzed CD8+ T-cell dysfunction (degranulation and IFN-γ production) and have demonstrated that CD8+ T cells of patients with HAM/TSP (HAM/TSP patients) spontaneously degranulate and express IFN-γ in ex vivo unstimulated culture. CD8+ T cells of HTLV-I asymptomatic carriers and healthy donors did not. Spontaneous degranulation was detected in Tax11-19/HLA-A*201 tetramer+ cells, but not in CMV pp65 tetramer+ cells. Interestingly, degranulation and IFN-γ production in CD8+ T cells was induced by coculture with autologous CD14+ cells, but not CD4+ T cells, of HAM/TSP patients, which correlated with proviral DNA load in CD14+ cells of infected patients. Moreover, the expression of IL-15, which induced degranulation and IFN-γ production in infected patients, was enhanced on surface of CD14+ cells in HAM/TSP patients. Blockade of MHC class I and IL-15 confirmed these results. Thus, CD8+ T-cell dysregulation was mediated by both virus infection and enhanced IL-15 on CD14+ cells in HAM/TSP patients. Despite lower viral expression than in CD4+ T cells, HTLV-I–infected or –activated CD14+ cells may be a heretofore important but under recognized reservoir particularly in HAM/TSP patients.

Characterization of gene expression regulated by human OTK18 using Drosophila melanogaster as a model system for innate immunity

OTK18 is a human transcriptional suppressor implicated in the regulation of human immunodeficiency virus type-one infection of mononuclear phagocytes. It is ubiquitously expressed in all normal tissues, but its normal homeostatic function is yet to be characterized. One hypothesis is that OTK18 aids in the regulation of the innate immune system. To test this hypothesis, cDNA microarray analysis was performed on the total RNA extracted from Drosophila melanogaster embryonic Schneider 2 (S2) cells transfected with either pEGFP-OTK18 (enhanced green fluorescent protein) or empty vector controls (pEGFP-N3) for 6, 12 and 24 h. cDNA microarray analysis revealed differential expression of genes known to be important in regulation of Drosophila innate immunity. The expression levels of two genes, Metchnikowin and CG16708 were verified by quantitative real-time reverse transcription PCR. These results suggest a role for OTK18 in innate immunity.

Molecular mechanism(s) involved in the synergistic induction of CXCL10 by human immunodeficiency virus type 1 Tat and interferon-γ in macrophages

Synergistic interactions between viral proteins and soluble host factors released from infected mononuclear phagocytes play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia (HAD). The chemokine CXCL10 has been found to be closely associated with the progression of HIV-1-related central nervous system (CNS) disease and its related neuropsychiatric impairment. In this report the authors demonstrate that the HIV-1 protein Tat can interact with the proinflammatory cytokine interferon (IFN)-gamma to dramatically induce the expression of CXCL10 in macrophages. Synergistic induction of CXCL10 by both Tat and IFN-gamma was susceptible to inhibition by the MEK1/2 inhibitor U0126 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. In addition, JAK/STAT pathway plays a major role in Tat/gamma-mediated CXCL10 induction in macrophages because pretreatment of stimulated macrophages with JAK inhibitor completely abrogated the synergistic induction of the chemokine. Functionality of the synergistically induced CXCL10 was further demonstrated by its chemotactic activity for peripheral blood lymphocytes. Taken together, these findings demonstrate that the cooperative interaction of Tat and IFN-gamma results in enhanced chemokine expression, which in turn can amplify the inflammatory responses within the CNS of HAD patients by recruiting more lymphocytes in the brain.

Animal models of highly pathogenic RNA viral infections: Hemorrhagic fever viruses

A diverse group of highly pathogenic RNA viruses cause a severe multisystemic illness in humans commonly referred to as viral hemorrhagic fever (VHF). Although they can vary widely in clinical presentation, all VHFs share certain features that include intense fever, malaise, bleeding and shock. Effective antiviral therapies for most of the VHFs are lacking. Complicating development of intervention strategies is the relative infrequency and unpredictability of VHF outbreaks making human clinical trials extremely challenging or unfeasible. Therefore, animal models that can recapitulate human disease are essential to the development of effective antivirals and vaccines. In general, a good animal model of VHF will demonstrate systemic dispersion of the virus through infection of mononuclear phagocytes and dendritic cells, which induces the release of inflammatory mediators that increase vascular permeability and facilitate coagulation. The culmination of this process leads to significant loss of plasma volume and terminal hypovolemic shock. Although it is clear that nonhuman primate models are the most faithful to human disease, the more accessible and less costly rodent models, including those based on infection with related surrogate viruses, can reproduce certain components of VHF and can serve as suitable preclinical models for initial development of effective countermeasures. Such models are sufficient for testing of drugs that directly block viral replication, but may be inadequate for evaluating therapies that depend for their success on the activation or inhibition of host responses.

Inhibition of long-term potentiation by interleukin-8: implications for human immunodeficiency virus-1-associated dementia.

Human immunodeficiency virus type 1 (HIV-1)-infected mononuclear phagocytes (MP; brain macrophages and microglia) secrete a number of toxic factors that affect the pathogenesis of HIV-1-associated dementia (HAD). The identification and relative role of each MP toxin for neuronal dysfunction during HAD are not well understood. Interleukin-8 (IL-8), a CXC chemokine involved in leukocyte activation and chemotaxis, is constitutively produced by MP, and elevated levels of IL-8 mRNA were detected in the brains of patients with HIV-1 encephalitis (HIVE) by both ribonuclease protection assays and real-time PCR. To determine the role that IL-8 might play in the neuronal dysfunction in HAD, we studied its effect on synaptic transmission and plasticity in the CA1 region of hippocampus, the seat of learning and memory. Bath application of IL-8 (50 ng/ml) to rat hippocampal slices had no effect on basal synaptic transmission. However, IL-8 was shown to inhibit long-term potentiation (LTP) in a concentration-dependent manner. In control and IL-8-treated slices, the LTP magnitudes were 167.8% +/- 11.9% (mean +/- SE; n = 17) and 122.2% +/- 16.2% of basal levels (n = 13), respectively. These differences were statistically significant (P < 0.05). Preincubation of hippocampal slices with a monoclonal CXCR2 antibody (2 microg/ml) but not control IgG (2 microg/ml) blocked IL-8-induced inhibition of LTP. The expression of CXCR2 receptors in the CA1 region was shown by Western blot assays. The induction of IL-8 in HAD, its inhibition of LTP, and the expression of its receptor, CXCR2, in the hippocampus all suggest that it plays a role in the cognitive dysfunction associated with HAD.

Proinflammatory response during Ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily

Ebola virus (EBOV) infections are characterized by dysregulation of normal host immune responses. Insight into the mechanism came from recent studies in nonhuman primates, which showed that EBOV infects cells of the mononuclear phagocyte system (MPS), resulting in apoptosis of bystander lymphocytes. In this study, we evaluated serum levels of cytokines/chemokines in EBOV-infected nonhuman primates, as possible correlates of this bystander apoptosis. Increased levels of interferon (IFN)-α, IFN-β, interleukin (IL)-6, IL-18, MIP-1α, and MIP-1β were observed in all EBOV-infected monkeys, indicating the occurrence of a strong proinflammatory response. To investigate the mechanism(s) involved in lymphoid apoptosis, soluble Fas (sFas) and nitrate accumulation were measured. sFas was detected in 4/9 animals, while, elevations of nitrate accumulation occurred in 3/3 animals. To further evaluate the potential role of these factors in the observed bystander apoptosis and intact animals, in vitro cultures were prepared of adherent human monocytes/macrophages (PHM), and monocytes differentiated into immature dendritic cells (DC). These cultures were infected with EBOV and analyzed for cytokine/chemokine induction and expression of apoptosis-related genes. In addition, the in vitro EBOV infection of peripheral blood mononuclear cells (PBMC) resulted in strong cytokine/chemokine induction, a marked increase in lactate dehydrogenase (LDH) activity, and an increase in the number of apoptotic lymphocytes examined by electron microscopy. Increased levels of sFAS were detected in PHM cultures, although, <10% of the cells were positive by immunohistochemistry. In contrast, >90% of EBOV-infected PHM were positive for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by immunohistochemistry, RNA analysis, and flow cytometry. Inactivated EBOV also effected increased TRAIL expression in PHM, suggesting that the TNF receptor superfamily may be involved in apoptosis of the host lymphoid cells, and that induction may occur independent of viral replication. In further studies with infected PHM, expression of MHC II was remarkably suppressed after 6 days, an additional correlate of immunological dysregulation. In conclusion, our findings suggest that infection of mononuclear phagocytes is critical, triggering a cascade of events involving cytokines/chemokines and oxygen free radicals. It is the consequence of these events rather than direct viral infection that results in much of the observed pathology. Identification of cytokine/chemokine, nitric oxide, and reactive oxygen species involvement in the observed filoviral pathogenesis may lend insight into the rational design of therapeutic countermeasures of filoviral pathogenesis.

Neurotrophins prevent HIV Tat-induced neuronal apoptosis via a nuclear factor-kappaB (NF-kappaB)-dependent mechanism.

HIV-1 associated dementia is thought to be caused by neuronal damage and death in response to the production of soluble neurotoxic factors by virally infected mononuclear phagocytes. These neurotoxins include HIV-1 Tat. The ability of neurotrophins to promote cell survival prompted us to examine whether neurotrophins might also be capable of opposing the pro-apoptotic effects of Tat. Here, we show that Tat-induced neuronal apoptosis in primary cultures of rat cerebellar granule cells and in neuronally differentiated human SK-N-MC cells is profoundly inhibited by brain-derived neurotrophic factor, nerve growth factor and activity-dependent neurotrophic factor nonamer peptide. These neurotrophins activated the transcription factor NF-kappaB, and inhibition of NF-kappaB activation using a super-repressor IkappaB-alpha mutant was found to block the survival-promoting activity of the neurotrophins. Reporter gene assays and immunoblot experiments revealed that the neurotrophins also up-regulated the expression of Bcl-2, at both the transcriptional and protein levels. Overexpression of the super-repressor IkappaB-alpha mutant prevented this induction of Bcl-2 expression. Moreover, overexpression of either Bcl-2, alone, or the RelA subunit of NF-kappaB, alone, protected neurons from Tat-induced apoptosis. These findings suggest that the activation of NF-kappaB by neurotrophic factors may promote survival of neurons exposed to Tat, via regulation of anti-apoptotic genes including Bcl-2.

Chemokines induced by infection of mononuclear phagocytes with mycobacteria and present in lung alveoli during active pulmonary tuberculosis.

The capacity of Mycobacterium tuberculosis (MTB) to induce production of chemokines with known chemotactic activity for monocytes and lymphocytes, the cellular building blocks of granulomas, was investigated. These chemokines included regulated upon activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). MTB stimulated production of MCP-1 and MIP-1alpha by blood monocytes (MN) and alveolar macrophages (AM). MTB infection of MN and AM stimulated release but not production of RANTES. AM produced or released significantly higher levels than MN of RANTES (by 2.1-fold), MCP-1 (by 6.9-fold), and MIP-1alpha (by 5. 5-fold) (P < 0.05 for each). This study also confirmed that MTB-infected AM produce the chemokine interleukin (IL)-8. MTB infection of AM resulted in increased steady-state expression of messenger RNA (mRNA) for MCP-1 and MIP-1alpha and minimal increased expression of RANTES mRNA. Both an avirulent (H37Ra) and a virulent (H37Rv) strain of MTB and purified protein derivative of H37Rv but not latex beads induced production of chemokines. Supernatants of MTB-infected cells demonstrated chemotactic activity for both monocytes and lymphocytes partially inhibitable by neutralizing antibodies against the chemokines studied. Bronchoalveolar lavage fluid from patients with active pulmonary tuberculosis as compared with healthy control subjects contained increased levels of RANTES (by 8-fold), MCP-1 (by 2.7-fold), and IL-8 (by 8.9-fold) (P < 0.05), but not MIP-1alpha, as compared with healthy control subjects. Thus, multiple chemokines may be involved in recruitment of cells for granuloma formation in tuberculosis.

Listeria monocytogenes infects human endothelial cells by two distinct mechanisms

Infection of endothelial cells by bacteria may be an important component of the bacteria's ability to escape host defenses and cause disease. Listeria monocytogenes cause sepsis and central nervous system infection in domesticated animals and immunocompromised humans, suggesting that this bacterium interacts with endothelial cells in a significant fashion. The experiments presented here tested the hypothesis that L. monocytogenes can invade and replicate within human endothelial cells. We found that L. monocytogenes grows readily in umbilical vein endothelial cells and that its intracellular life cycle involves phagosomal escape, F-actin-based motility, and cell-to-cell spread. We found that L. monocytogenes invaded endothelial cells by cell-to-cell spread from adherent mononuclear phagocytes which were previously infected by this bacterium. Interestingly, L. monocytogenes mutants lacking the invasion protein, internalin, bound less well to endothelial cells than did wild-type bacteria in the absence, but not the presence, of serum, and their invasion of endothelial cells was diminished under both conditions. Thus, endothelial cell infection by L. monocytogenes can occur by two distinct mechanisms: direct bacterial invasion of the endothelial cells in an internalin-mediated fashion or cell-to-cell spread from adherent, infected mononuclear phagocytes. These data support the idea that endothelial cell infection by L. monocytogenes is an important event in the pathogenesis of listeriosis.

Cellular CD44S as a determinant of human immunodeficiency virus type 1 infection and cellular tropism.

CD4 is the predominant cell membrane protein that binds human immunodeficiency virus type 1 (HIV-1) gp120 and facilitates HIV-1 infection, but other membrane-associated molecules may be involved in determining HIV-1 cellular infection. Our prior work had suggested that CD44, the transmembrane receptor for hyaluronan, might play a role in the infection of mononuclear phagocytes with HIV-1. In the present work, we have used cells of the CD4-positive, CD44-negative human T-lymphoblast cell line Jurkat to study the role of CD44 in HIV-1 infection and tropism. Cells were transfected with cDNA for the standard (S, or hematopoietic) CD44 isoform CD44S or the epithelial isoform CD44E. The resultant lines expressed appropriate CD44S or CD44E mRNA and protein. While the parent Jurkat cells, those transfected with vector alone, and those transfected with CD44E could be productively infected with only the lymphocytotropic strain HIV-1-LAI, cells transfected with CD44S were rendered susceptible to productive infection with the monocytotropic strains HIV-1-BaL and HIV-1-ADA. Also, CD44S-transfected cells displayed higher levels of infection with HIV-1-LAI than did the other transfected Jurkat cells. The transfected cell line cells all had comparable growth rates and expressed similar levels of the membrane antigens CD4, CD7, major histocompatibility complex (MHC) class I, MHC class II, and CD11a, while levels of CD3 were slightly higher in cells transfected with vector alone and in one of the clones transfected with CD44S. Hyaluronan binding was increased in cells transfected with either CD44S or CD44E. Mouse NIH 3T3 fibroblasts transfected with human CD4, human CD44S, or both human CD4 and CD44S displayed the appropriate antigens, but they could not be productively infected with lymphocytotropic or monocytotropic strains of HIV-1. The results indicate that in human leukocytes, CD44S is an important determinant of HIV-1 productive infection and may be involved in viral cellular tropism.

Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type-1 in Mononuclear Phagocytes

HIV-1 vpr encodes a 96-amino acid, nuclear protein whose function is not well understood. Unlike the other lentivirus regulatory proteins, Vpr is present in virions at relatively high copy number. In cells, Vpr is localized to the nucleus. Possible functions for vpr consistent with these findings include the nuclear import of preintegration complexes, transactivation of cellular genes, or induction of cellular differentiation. We show here, using both replication competent, macrophage-tropic virus and a sensitive, single-cycle luciferase HIV-1 reporter vector, that vpr is important for efficient viral replication in primary monocyte/macrophages, but appears to play no role in activated or resting T cell infection. The block to infection in monocytes was localized by PCR analysis of newly synthesized viral DNA and with the luciferase reporter vector to a stage in the viral life cycle after entry and reverse transcription, yet prior to, or at the time of, proviral transcription. In addition, infection of mononuclear phagocytes with virions that had been loaded with Vpr molecules in the producer cells by trans-complementation still showed a vpr-phenotype. These data suggest a role for vpr molecules produced in newly infected cells, in addition to its presumed function in the virion.

Kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors.

Human immunodeficiency virus type 1 infection of mononuclear phagocytes has been implicated in disease manifestations, but postentry viral replication events in these cells have not been well characterized. Productive infection of activated T cells is associated with cell proliferation and accumulation of full-length viral DNA within 6 h. In infected, nondividing quiescent peripheral blood lymphocytes, reverse transcription is aborted prior to full-length viral DNA formation. For nondividing, cultured mononuclear phagocytes, we now report a third pattern of reverse transcription with relatively slow kinetics, in which full-length viral DNA did not accumulate until 36 to 48 h. The reverse transcription rate in mononuclear phagocytes could be accelerated by addition of exogenous nucleotide precursors, but still not to the rate seen in activated T cells. These results indicate that substrate limitations in mononuclear phagocytes slow but do not arrest human immunodeficiency virus type 1 reverse transcription.

Human immunodeficiency virus type 1 infection of human monocytes and macrophages does not alter their ability to generate an oxidative burst.

Human immunodeficiency virus type 1 (HIV-1) infects mononuclear phagocytes, cells that may serve as a reservoir for viral persistence. Infection with HIV-1 leads to progressive compromise of the immune system, resulting in infections with opportunistic pathogens and eventual death. Experiments were designed to determine if in vitro HIV-1 infection of mononuclear phagocytes would diminish their oxidative capabilities, thus decreasing their antimicrobial effectiveness. Blood monocytes and peritoneal macrophages were obtained from uninfected donors and inoculated with a monocytotropic strain of HIV-1. Hydrogen peroxide production and reduction of nitroblue tetrazolium were measured after acute stimulation of cells with PMA or a phagocytic stimulus. Despite vigorous virus production, no difference was seen in oxidative burst between uninfected cells and infected cells or between monocyte-derived and peritoneal macrophages. In conclusion, reduced antimicrobial activity of HIV-infected mononuclear phagocytes is probably not secondary to decreased ability to generate reactive oxygen species.

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.

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.