Human astrocytes inhibit HIV-1 expression in monocyte-derived macrophages by secreted factors.

OBJECTIVE: To determine the effects of primary human fetal and adult astrocytes on HIV-1 replication in monocyte-derived macrophages (MDM). DESIGN: HIV-1 can infect the brain in the early stage of systemic infection. The HIV-1-associated cognitive/motor complex develops later in the course of the disease, suggesting that brain cells may inhibit the early productive infection and the development of neurological disease. In this study, we established an in-vitro coculture system to determine whether astrocytes can modulate HIV-1 replication in MDM. METHODS: Elutriated human monocytes were differentiated in culture, then infected with monocyte tropic HIV-1. One day after infection, MDM were co-cultured with primary astrocytes. Reverse transcriptase (RT) activity was used to monitor virus replication. RT-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and bioassay were used to assess cytokine production. RESULTS: Primary human astrocytes suppressed HIV-1 replication in MDM via the production of soluble factors. Cytokine inhibitors of HIV-1, such as IFN-gamma, IL-4, IL-10 and IL-13, were not detectable, whereas transforming growth factor beta (TGF-beta) was constitutively produced only in its latent form. Paraformaldehyde-fixed astrocytes, unable to secrete cytokines, failed to inhibit HIV-1. These cells caused enhanced virus replication, however, which correlated with an increase in macrophage colony stimulating factor (M-CSF) production. CONCLUSIONS: Human astrocytes can increase and decrease HIV-1 expression in MDM. An imbalance between the positive and negative effects of astrocytes may contribute to the expression of virus in the brain, and the development of HIV-1-associated cognitive/motor complex.

Human immunodeficiency virus-1-infected macrophages induce inducible nitric oxide synthase and nitric oxide (NO) production in astrocytes: astrocytic NO as a possible mediator of neural damage in acquired immunodeficiency syndrome.

Nitric oxide (NO) plays an important role in normal neural cell function. Dysregulated or overexpression of NO contributes to neurologic damage associated with various pathologies, including human immunodeficiency virus (HIV)-associated neurological disease. Previous studies suggest that HIV-infected monocyte-derived macrophages (MDM) produce low levels of NO in vitro and that inducible nitric oxide synthase (iNOS) is expressed in the brain of patients with neurologic disease. However, the levels of NO could not account for the degree of neural toxicity observed. In this study, we found that induction of iNOS with concomitant production of NO occurred in primary human astrocytes, but not in MDM, when astrocytes were cocultured with HIV-1-infected MDM. This coincided with decreased HIV replication in infected MDM. Supernatants from cocultures of infected MDM and astrocytes also stimulated iNOS/NO expression in astrocytes, but cytokines known to induce iNOS expression (interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha) were not detected. In addition, the recombinant HIV-1 envelope protein gp41, but not rgp120, induced iNOS in cocultures of uninfected MDM and astrocytes. This suggests that astrocytes may be an important source of NO production due to dysregulated iNOS expression and may constitute one arm of the host response resulting in suppression of HIV-1 replication in the brain. It also leads us to speculate that neurologic damage observed in HIV disease may ensue from prolonged, high level production of NO.

Endogenous macrophage CSF production is associated with viral replication in HIV-1-infected human monocyte-derived macrophages.

Human monocyte-derived macrophages (MDM) cultured in medium containing macrophage (M) CSF are more susceptible to infection with HIV-1. M-CSF increases the frequency with which MDM become infected, the level of HIV mRNA expressed per infected cell, and the level of proviral DNA expressed per infected culture. Because of these effects of M-CSF on HIV-1 replication and the reported function of this factor as a survival and differentiation factor for human monocytes, we investigated whether HIV-1 could induce endogenous M-CSF production by MDM and the potential role of endogenous M-CSF on HIV-1 infection in these cells. MDM infected with HIV and maintained in the absence of exogenous M-CSF produced this cytokine endogenously at levels 5- to 24-fold higher than uninfected cells. In contrast, the proinflammatory cytokines IL-1, IL-6, and TNF-alpha and the growth factor granulocyte-macrophage CSF were not detected. The kinetics of M-CSF production following infection paralleled the kinetics of virus replication. Furthermore, enhanced production of M-CSF was dependent on viral entry and active replication of HIV-1. Thus, endogenous M-CSF production may contribute to the survival of HIV-infected MDM, enable them to function as a reservoir for HIV, and facilitate the spread of virus in vivo.

Potent stimulation of monocytic endothelin-1 production by HIV-1 glycoprotein 120.

Monocytes/macrophages play a critical role in the pathogenesis of HIV infection, both as targets for virus replication and as sources of production of multifunctional cytokines. Endothelins, peptides with potent vasoconstricting activities originally isolated from endothelial cells, are also produced and secreted by macrophages in a manner similar to that of other cytokines. In an attempt to explore the potential role of endothelins in HIV-infection, we investigated the effect of the HIV-1 envelope glycoprotein, glycoprotein 120, on monocytic endothelin-1 production. This glycoprotein has been identified as a potent stimulator of monokines such as TNF-alpha and IL-6, which have been implicated as potential mediators of HIV-encephalopathy. We found that glycoprotein 120, similar to LPS, stimulates the secretion of endothelin-1, as well as TNF-alpha, from macrophages in a concentration-dependent manner. Using reverse transcriptase polymerase chain reaction, we found that circulating monocytes in HIV-infected individuals show a distinct expression of the endothelin-1 gene that is not detectable in healthy controls, indicating chronic activation of this gene in HIV-infection. In addition, cerebral macrophages in patients with HIV-encephalopathy were strongly positive for endothelin. Thus, monocytic endothelins appear to be stimulated during HIV infection. Their potent vasoactive properties render them potential candidates for mediating alterations in the cerebral perfusion pattern associated with the AIDS dementia complex.

The HIV-1 gp120 envelope protein has the intrinsic capacity to stimulate monokine secretion.

Results and conclusions concerning the ability of HIV glycoprotein (gp) 120 to stimulate monokine secretion have been equivocal, based on observations using natural gp120 derived from infected human cells and a Chinese hamster ovary (CHO) cell-derived recombinant fusion protein. Current studies were designed to determine whether differences in recombinant gp120 proteins could result in failure to trigger monokine production. We found that natural gp120 could stimulate monocytes to release TNF-alpha, IL-1 beta, IL-6, and granulocyte-macrophage-CSF, and this effect could be blocked with soluble CD4. Full-length rgp120 either expressed from an adenovirus vector and purified from infected human cells, or derived from CHO cells, could function similarly. In contrast, full-length recombinant envelope protein expressed in a baculovirus system and a CHO cell-derived recombinant fusion protein tested previously, consistently failed to stimulate monokine production. The stimulatory capacity of both natural and full-length CHO cell-derived gp120 was eliminated by heating at 100 degrees C, and could be blocked with excess CHO cell-derived gp120 fusion protein. Inasmuch as the baculovirus-expressed gp120 and the CHO cell-derived recombinant fusion protein can bind to CD4, these results suggest that HIV gp120 binding to CD4 on the monocyte surface may of itself be insufficient for stimulation of monokine secretion. Therefore, primary protein structure, as well as posttranslational protein modifications, may determine this activity.