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Along with CD4+ T-lymphocytes, macrophage lineage cells serve as primary hosts for HIV replication in vivo. In some tissues such as brain, where T-cell infection is essentially absent, the development of HIV-associated disease is mediated through infection of macrophages. This fact underscores the importance of experimental methods that yield results and conclusions that accurately reflect the mechanisms operational in vivo. Unfortunately, our understanding of key aspects of HIV-macrophage interactions, most notably, features of viral entry, replication, latency and persistence, lags behind that of T-cell infection. While some questions are best approached by direct examination of patient specimens using methods such as immunohistochemistry and phylogenetics, experiments based on HIV infection of macrophages in vitro can, necessarily, identify and elucidate the events, molecular mechanisms, and pathological consequences associated with this infection. In addition, macrophage culture methods can provide for the isolation of infectious HIV from patient blood monocytes and tissue macrophages, as well as subsequent continued propagation of these isolates in their host cell of origin. Maintenance of the host cell pedigree limits the possibility of alteration of viral properties such as chemokine coreceptor usage that may then no longer reflect the situation in vivo. This chapter focuses on HIV infection of macrophages. We describe methods for the cultivation of human blood monocyte-derived macrophages, their infection with HIV and subsequent maintenance, and the isolation of infectious HIV from them. Also included is a protocol using accutase for macrophage detachment. Accutase is a relatively new dissociation medium, used primarily in stem cell research. In our laboratory, it has far out-performed all other methods by providing for the gentle, yet thorough, detachment of macrophages without the need for scraping, and without loss of surface antigens or viability.

Early after infection, large quantities of HIV are trapped on follicular dendritic cells (FDCs) thus establishing a potent reservoir of infectious virus adjacent to highly susceptible CD4-bearing T lymphocytes. Throughout much of the disease course, active HIV infection is largely confined to sites surrounding FDCs suggesting that this microenvironment is highly conducive to infection. FDCs maintain HIV infectivity and trapped virus can cause infection even in the presence of neutralizing antibody. FDCs also contribute signaling to the germinal center microenvironment that appears to increase HIV infection and replication. This article discusses these FDC contributions to HIV pathogenesis.

A population of circulating mononuclear cells from patients with AIDS was identified which expressed interleukin 2 receptors (IL-2R). By dual-fluorescence flow microfluorometry, the patients' IL-2R+ cells were further identified as Leu M3+ monocytes (29.4 +/- 5.2% of the Leu M3+ cells were IL-2R+, n = 15), whereas Leu M3+ monocytes from normal subjects were IL-2R negative (2.0 +/- 0.42%; P less than 0.001). By Northern analysis, monocytes from AIDS patients, but not control subjects, constitutively expressed steady-state levels of IL-2R mRNA. Functionally, the IL-2R+ monocytes were capable of depleting IL-2 from culture supernatants, suggesting a mechanism for the reduced IL-2 levels commonly seen in AIDS patients. IL-2R+ monocytes also expressed increased levels of surface HLA-DR which may favor monocyte T-cell interactions and the transmission of human immunodeficiency virus (HIV). In additional studies, normal monocytes were infected with a macrophage-tropic HIV isolate in vitro and monitored for IL-2R and HLA-DR expression. Within 24-48 h after exposure to HIV in vitro, but before evidence of productive infection, greater than 25% of the monocytes became IL-2R+ with increasing numbers of IL-2R+ cells and HLA-DR levels through day 6. These early signaling effects of HIV could be mimicked by adding purified HIV envelope glycoprotein gp120 to the monocytes. This stimulation of monocytes before or independent of productive infection of the cells by HIV is consistent with in vivo observations of activated and/or abnormal functions by monocytes that do not appear to be infected with HIV in AIDS patients.

Dipyridamole (DPM) is commonly used as a coronary vasodilator and inhibitor of platelet aggregation in the treatment of cardiovascular diseases. We report here that DPM potentiates the inhibitory effects of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine against human immunodeficiency virus type 1 (HIV-1) in human monocyte-macrophages. At the same concentrations, DPM does not potentiate the toxic effects of AZT on these cells or on human bone marrow (granulocyte-monocyte) progenitor cells. Since monocyte-macrophage lineage cells appear to be the major reservoir for HIV-1 in vivo, these findings suggest the possibility of using DPM or its analogues in combination chemotherapy of HIV infections.

Primary cultures from a brain biopsy specimen of a human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) seropositive patient with progressive dementia contained small numbers of monocytoid cells and showed reverse transcriptase activity that persisted for as long as 100 days. Electron microscopy of these cells revealed the presence of HTLV-III/LAV virions. Subcultured cells removed from primary cultures by trypsinization were nonspecific esterase negative and did not express virus or show evidence of HTLV-III/LAV proviral sequences, while those remaining in the original flasks were nonspecific esterase positive and continued to produce virus. Virus from primary cultures was transmitted to peripheral blood-derived monocyte-macrophages and T cells. Virus production in T-cell cultures was transient while the monocyte-macrophages, like the primary cultures, produced virus for at least 120 days. Infection of several brain-derived cells with this and another HTLV-III/LAV isolate failed to demonstrate virus replication. These results indicate that the HTLV-III/LAV-infected cells recovered from the brain of this patient are cells of the mononuclear phagocyte series.

Because of the probable role of HIV-infected monocyte/macrophages in the pathogenesis and progression of AIDS, it is essential that antiretroviral therapy address viral replication in cells of this lineage. Several dideoxynucleosides have been shown to have potent in vitro and, in the case of 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), in vivo activity against HIV. However, because these compounds must be phosphorylated (activated) in target cells, and because monocyte/macrophages may have levels of kinases that differ from those in lymphocytes, we investigated the capacity of these drugs to suppress HIV replication in monocyte/macrophages using HIV-1/HTLV-IIIBa-L (a monocytotropic isolate). In the present study, we observed that HTLV-IIIBa-L replication in fresh human peripheral blood monocyte/macrophages was suppressed by each of three dideoxynucleosides: 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyadenosine (ddA). Similar results were observed in 5-d-cultured monocyte/macrophages, although higher concentrations of the drugs were required. We then studied the metabolism of AZT and ddC in such cells. The phosphorylation of ddC to a triphosphate moiety was somewhat decreased in monocyte/macrophages as compared with H9 T cells. On the other hand, the phosphorylation of AZT in monocyte/macrophages was markedly decreased to 25% or less of the level in T cells. However, when we examined the level of the normal endogenous 2'-deoxynucleoside triphosphate pools, which compete with 2',3'-dideoxynucleoside triphosphate for viral reverse transcriptase, we found that the level of 2'-deoxycytidine-triphosphate (dCTP) was six- to eightfold reduced, and that of 2'-deoxythymidine-triphosphate (dTTP) was only a small fraction of that found in T cell lines. These results suggest that the ratio of dideoxynucleoside triphosphate to normal deoxynucleoside triphosphate is a crucial factor in determining the antiviral activity of dideoxynucleosides in HIV target cells, and that the lower levels of dTTP may account for the antiretroviral activity of AZT in the face of inefficient phosphorylation of this compound.

We describe an acute human immunodeficiency virus (HIV) infection in 16 homosexual men who presented with painful swallowing (odynophagia). Eleven men had a maculopapular rash and 3 had palatal ulcers. At esophagogastroduodenoscopy (endoscopy), multiple discrete esophageal ulcers measuring 0.3 to 1.5 cm in diameter were observed. Electron microscopy of biopsy specimens taken from the ulcer margins in 8 men revealed viral particles 120 to 160 nm in diameter whose morphologic characteristics were those of retroviruses. Human immunodeficiency virus seroconversion was documented in 15 men by Western blot analysis. In 3 men, HIV-1 was isolated from peripheral blood mononuclear cells, in 2 men HIV-1 was isolated from peripheral blood monocytes, and in 1 man HIV-1 was isolated from tissue taken from the margins of the esophageal ulcers. These observations extend our knowledge of the clinical spectrum of acute HIV infection syndromes and suggest that cells in the esophagus are a target for HIV-1 infection. [ABSTRACT FROM AUTHOR]

Decline in blood CD4+ lymphocytes during primary symptomatic infections with HIV is usually attributed to viral killing, and has not been considered in terms of altered lymphocyte migration and sequestration. We therefore sought to examine whether CD4+ cell loss from blood of macaques undergoing an acute primary SIV infection might be due to increased synthesis of cytokines, known to profoundly affect lymphocyte trafficking, rather than to direct lymphocyte destruction by virus. The findings indicate that rapid lymphocyte depletion following acute infection is not selective for CD4+ cells, correlates precisely with increased plasma IFN-gamma and tumor necrosis factor-alpha levels, and is reversible. CD4/CD8 ratios in lymph nodes with high viral burdens remain relatively unchanged despite lymphocyte loss from blood. Levels of cytokine mRNA measured in lymphoid organs reflect neither cytokine plasma levels nor their potential to induce sequestration. These results support a model of cytokine-induced lymphocyte extravasation to account for the acute HIV/SIV-induced CD4+ cell lymphopenia and raise questions regarding the extent to which altered lymphocyte migration plays a role in the gradual CD4+ cell depletion throughout infection. [ABSTRACT FROM AUTHOR]