HIV-infected Macrophages Research Articles

IL-13 and IFN-γ Secretion by Activated T Cells in HIV-1 Infection Associated with Viral Suppression and a Lack of Disease Progression

AbstractThe immunopathology of HIV-1 infection includes immune defects in T cell cytokine secretion, resulting in decreased Ag-specific responses. In this report, IL-13 and IFN-γ were analyzed in progressive HIV-1 disease. Both cytokines exert positive effects on Ag presentation and inhibit HIV-1 infection of macrophages. Anti-CD3/anti-CD28-activated PBMC from HIV-1-infected individuals (n = 74) compared with uninfected subjects (n = 30) secreted significantly less IL-13 (median, 0.64 ng/ml vs 2.07 ng/ml; p < 0.001) and IFN-γ (median, 40.96 ng/ml vs 129.5 ng/ml; p < 0.005). Decreased IL-13 and IFN-γ secretion in HIV infection was present in sorted CD4+ and CD8+ T cell subsets, and additional analysis determined concurrent deficiency at the protein and transcriptional level. Longitudinal analysis showed that cytokine secretion levels correlated positively with CD4 count and negatively with plasma HIV-1 viral load. Patients changing to suppressive antiretroviral therapy during the study showed increases in IL-13 and IFN-γ secretion. Overall, results show a decline in IL-13 and IFN-γ secretion in progressive HIV-1 infection and suggest a role for both cytokines as part of T cell adaptive responses associated with a lack of disease progression.

Increased Peroxynitrite Activity in AIDS Dementia Complex: Implications for the Neuropathogenesis of HIV-1 Infection

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1beta, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.

Chemokine receptors and virus entry in the central nervous system

Several members of the chemokine receptor family are used as coreceptors together with CD4 for HIV and SIV entry in the central nervous system (CNS). CCR5 is the major coreceptor for HIV-1 infection of macrophages and microglia, the major target cells for HIV-1 infection in the CNS. CXCR4 and CCR3 are also expressed on microglia and can mediate infection by certain HIV-1 isolates but at lower efficiency than CCR5. Additional chemokine receptors that can function as HIV-1 and SIV coreceptors for a subset of viruses are expressed in the brain (i.e. Apj, CX3CR1, STRL33/BONZO, and gpr1), but their role in CNS infection has not been defined. The expression of CXCR4, and possibly other chemokine receptors, on subpopulations of neurons and glial cells may contribute to mechanisms of CNS injury that are independent of viral infection. Understanding the role of chemokine receptors and their chemokine ligands in HIV-1 and SIV infection of the CNS will elucidate mechanisms of viral tropism and pathogenesis and advance the development of new therapeutic strategies.

Activation of nuclear factor-kappaB in macrophages by mycoplasmal lipopeptides.

Mycoplasmas are potent macrophage stimulators. The active principle are lipopeptides or lipoproteins with a characteristic N-terminal S-[dihydroxypropyl]-cysteinyl group bearing two ester-bound fatty acids and lacking the amide-bound one common to other bacterial lipoproteins. Using synthetic analogues of mycoplasmal lipopeptides, we investigated activation of the transcription factor NF-kappaB in the C3H/HeJ mouse-derived DMBM-3 cell line. The lipopeptides activated NF-kappaB at below nanomolar concentrations. Activation in the murine system occurred distinctly earlier than TNF-alpha liberation, excluding autocrine stimulation by TNF-alpha. As determined from a supershift experiment, the active NF-kappaB complex consisted of the heterodimer p50/p65(RelA). The relevance of these findings for the inflammatory response to mycoplasmas and for mycoplasma-mediated effects on HIV-infected macrophages is discussed.

A murine model of HIV encephalitis: xenotransplantation of HIV-infected human neuroglia into SCID mouse brain.

The degree of neuronal damage in HIV encephalitis (HIVE) and the mechanisms leading to it are not known. Post-mortem human studies provide limited information concerning pathogenesis, and there are few animal models of HIVE. We have developed a murine model of HIVE based on HIV-infected human brain tissues grafted within the host CNS milieu. HIV-infected blood-derived macrophages were cocultured with and incorporated within second trimester human fetal brain neuroglia. Mixed neuronal-glial aggregates were injected into the striatum of SCID mice where they survived for more than 6 months. Cellular proliferation and differentiation were determined by immunohistochemical staining for proliferating cell nuclear antigen and cellular markers. Synapse formation was seen by immunocytochemistry for synapsin and by electron microscopy. Virus was detected by immunohistochemical staining for HIV gp41. Based on the long-term survival of human neuroglial xenografts containing HIV infected macrophages, we believe that this model will support the study of chronic HIV-associated neurodegeneration and the testing of various CNS targeted therapeutic interventions.

Death via gp120

When HIV-infected patients progress to AIDS, there is not only a drop in CD4 T-cell numbers but also an appreciable decline in CD8 T-cell populations. To determine if the depletion of CD8 T cells can be mediated directly by HIV, Herbein et al., 1998xApoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. Herbein, G. et al. Nature. 1998; 395: 189–194Crossref | PubMed | Scopus (325)See all ReferencesHerbein et al., 1998analyzed the rate of CD8 T cell apoptosis in vitro in the presence of infectious HIV or recombinant HIV gp120. They observed that recombinant gp120 alone was sufficient to induce CD8 T cell apoptosis, but that the addition of macrophages to the cell culture was required for apoptosis to occur. Macrophage/gp120- induced apoptosis of CD8 T cells was mediated through the chemokine receptor CXCR4 but did not require Fas–Fas-ligand interactions. Instead, apoptosis occurred through the cognate interaction of membrane-bound tumor necrosis factor α (TNF-α) (expressed on HIV-infected or gp120-treated macrophages) binding to the TNF-receptor II molecule on the CD8 T cells. As CD8 T cells play an important role in controlling HIV infection, Herbein et al., 1998xApoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. Herbein, G. et al. Nature. 1998; 395: 189–194Crossref | PubMed | Scopus (325)See all ReferencesHerbein et al., 1998speculate that the apoptotic effect of gp120 on this T-cell subset might contribute to the development of AIDS.

Neurotrophic and chemokine dysfunction of HIV infected human brain macrophages

Neurotrophic and chemokine dysfunction of HIV infected human brain macrophages

HIV-ASSOCIATED NEOPLASTIC DEMENTIA: A MODEL IMPLICATING A ROLE FOR CLONAL MACROPHAGES.

91 Objective: The clonality of HIV within a given tissue can be determined using an inverse PCR (IPCR) technique. Clonal HIV has been observed in a T-cell lymphoma, several polyclonal lymphomas, and in early Kaposi's sarcoma. Recent cell separation studies have identified tumor-associated macrophages that contain clonal HIV. Integration site mapping studies have identified a common integration region just 5′ to the c-fes oncogene in 6 HIV-associated tumors. One characteristic of this class of neoplasia is the presence of clonal macrophages that express high levels of HIV proteins. Another major HIV-associated disease process associated with these HIV expressing macrophages is AIDS-related dementia. The purpose of the current study was to determine whether HIV-infected macrophages associated with AIDS dementia were clonal, and if clonal, to map integration sites to determine whether a similar insertional mutagenesis process as that observed in the AIDS-related lymphomas might be operative in AIDS-related dementia. Methods: Brains from patients with AIDS dementia and controls were obtained at autopsy. Specimens from cortical and subcortical regions were obtained for analysis. IPCR, immunohistochemistry, Southern blot, and DNA sequencing were performed on each specimen as required. Results: Using IPCR, clonal forms of HIV were found in 8 of 10 AIDS dementia brain specimens, and 5 of 5 AIDS dementia CSF specimens analyzed. In 4 of 5 CSF specimens the only cellular components identified were macrophages. DNA sequencing and RFLP mapping studies identified the platelet-derived growth factor-B (PDGF-B) gene locus as the integration region in 4 specimens containing clonal IPCR products. Immunohistochemistry studies identified areas of astrogliosis that contained HIV-infected perivascular macrophages that expressed HIV, PDGF-B, and the proliferating cell marker, PCNA. Conclusions: We postulate that this macrophage population, with a clonal form of HIV integrated near the PDGF locus, may contribute to brain abnormalities through constitutive production of PDGF, causing secondary astrocyte proliferation. Because of the clonal nature of the HIV infected macrophage involved in these brain/CSF specimens, we term this process HIV-associated neoplastic dementia. This new category of disease could define certain forms of AIDS dementia as paraneoplastic processes.

Neuronal injury associated with HIV-1: approaches to treatment.

Mounting evidence suggests that cognitive dysfunction developing as a result of HIV-1 infection is mediated at least in part by generation of excitotoxins and free radicals in the brain. This syndrome is currently designated HIV-1-associated cognitive/motor complex, was originally termed the AIDS Dementia Complex, and for simplicity, is called AIDS dementia in this review. Recently, brains of patients with AIDS have been shown to manifest neuronal injury and apoptotic-like cell death. How can HIV-1 result in neuronal damage if neurons themselves are only rarely, if ever, infected by the virus? Experiments from several different laboratories have lent support to the existence of HIV- and immune-related toxins in a variety of in vitro and in vivo paradigms. In one recently defined pathway to neuronal injury, HIV-infected macrophages and microglia, or immune-activated macrophages and astrocytes (activated by the shed HIV-1 envelope protein, gp120, or other viral proteins and cytokines), appear to secrete excitants and neurotoxins. These substances may include arachidonic acid, platelet-activating factor, free radicals (NO. and O2.-), glutamate, quinolinate, cysteine, amines, and as yet unidentified factors emanating from stimulated macrophages and reactive astrocytes. A final common pathway for neuronal susceptibility is operative, similar to that observed in stroke and several neurodegenerative diseases. This mechanism involves excessive activation of N-methyl-D-aspartate (NMDA) receptor-operated channels, with resultant excessive influx of Ca2+ and the generation of free radicals, leading to neuronal damage. With the very recent development of clinically tolerated NMDA antagonists, there is hope for future pharmacological intervention.

HIV decreases glutamate transport in SK-N-MC neuroblastoma cells.

Autopsy studies of patients with AIDS dementia have shown neuronal loss consistent with a neurotoxic component of this disease. In vitro studies suggest that viral products or cytokines from HIV-infected macrophages (Mphi) may modulate or directly mediate excitotoxic cell death of neurons. Mphi differentiated from peripheral mononuclear blood cultures were infected with HIV, and conditioned media (CM) were harvested from these cultures. Exposure of SK-N-MC (neuroblastoma) cells to CM from HIV-infected Mphi for 4, 24 or > or = 48 h resulted in a mean suppression of 12-34% of the glutamate transport Vmax with no appreciable change in transport Km. An astrocytoma tumor cell, U373MG, showed similar CM-mediated glutamate uptake suppression. Changes were evident in total and Na+-dependent glutamate uptake, with significantly more suppression of Na+-dependent uptake. Similar effects were seen with the nonmetabolizable transporter agonist D-aspartate, indicating that the effect was on transport and not metabolism. No suppression was seen with CM from uninfected Mphi or Mphi infected with heat-inactivated HIV. The magnitude of uptake suppression was not correlated with CM p24 values, and removal of CM virions by ultracentrifugation and immunoprecipitation did not alter the uptake-suppressive properties of infected Mphi CM. Uptake suppression was seen when Mphi were infected with Mphi-tropic strains HIV(SF162), HIV(JR-CSF), HIV(NFN-SX) and a Mphi-tropic patient isolate, but not the lymphotropic strain HIV(LAI). HIV-infected Mphi may produce substances which suppress neuronal and glial glutamate neurotransmitter uptake, resulting in higher extracellular glutamate levels and leading possibly to deficits in cell signaling and neurotoxicity.

Regulation of anti-HIV-1 activity of RANTES by heparan sulfate proteoglycans.

The role of cell surface proteoglycans in CC chemokine-mediated anti-HIV-1 activity in T cells and macrophages was investigated. Enzyme digestion of heparan sulfate (HS), but not chondroitin sulfate, from the surface of PM1(CD26H) cells (a human T cell line selected for high CD26 expression) rendered them resistant to the antiviral effects of RANTES and macrophage-inflammatory protein-1beta at otherwise inhibitory chemokine concentrations. HIV-1 infection of macrophages, however, was inhibited only partially, even at high concentrations of RANTES, and this inhibition was not prevented by HS removal. Flow cytometry revealed that digestion of cell surface proteoglycans, including HS, prevented the binding of RANTES at 10 to 100 nM concentrations to PM1(CD26H) cells. However, the binding of RANTES to activated macrophages occurred only at higher concentrations (100-300 nM) and was mostly chondroitin sulfate, and not HS, dependent. These results support a role for HS in facilitating the interaction of CC chemokines with the cell surface and the consequent inhibition of HIV-1 infection. The absence of HS-dependent binding of RANTES at lower concentrations to macrophages is consistent with the resistance of these cells to the antiviral effects of chemokines.

Apoptosis of cd4+ t cells induced after contact with hiv1-infected or non-infected macrophages

The hallmark of human immunodeficiency virus type 1 (HIV1) infection is the relentless destruction of CD4+ T lymphocytes. Indirect cell killing mechanisms are thought to play an outstanding role in lymphocyte depletion. One such proposed mechanism is the induction of apoptosis through cross-linking of the CD4 receptor by anti-CD4 antibodies or by the HIV1 envelope protein expressed at the surface of infected cells. Here we provide evidence that apoptosis is triggered in CD4+ lymphoblastoid cells (MT4) following cocultivation with monocyte-derived macrophages (MDMs) productively infected with the monocytotropic isolate HIV1 PAR. Blocking virus replication by AZT abrogates apoptosis of MT4 cells. Infected MDMs do not transmit virus infection to target cells. DNA nucleosomal fragmentation occurs at 46-66 h after starting cocultures. It is inhibited by the addition of neutralizing anti-gp120 monoclonal antibody (mAb), implying that the gp120/CD4 interaction triggers the apoptotic process. Cocultivating with MDMs, either infected or not, in the presence of anti-CD4 mAb Leu-3a, also leads to MT4 cell death, with DNA fragmentation being detected at 24-40 h. Leu-3a induced apoptosis does not require cross-linking of CD4 by anti-immunoglobulin, showing that MDMs provide an alternative to conventional cross-linking. Both the infected and the non-infected MDMs were found to stimulate 3H-thymidine incorporation in cocultivated MT4 cells.

Cellular aspects of HIV-1 infection of macrophages leading to neuronal dysfunction in in vitro models for HIV-1 encephalitis.

HIV-1 is a hematogenously spread virus that most likely gains entry into the brain within blood-derived macrophages. Indeed, productive viral replication selectively occurs within perivascular and parenchymal blood-derived macrophages and microglia and HIV-infected macrophages have increased potential to bind and transmigrate through the blood-brain barrier. Once inside the brain, HIV-infected macrophages secrete a variety of pro-inflammatory mediators that display neuromodulatory and neurotoxic activities in several in vitro models for HIV-1 encephalitis. The final outcome regarding neuronal function and cell loss is regulated through intercellular interactions between these virus-infected cells and astrocytes. In this regard, both HIV-induced intracellular events in macrophages and interactions between HIV-infected macrophages and brain cells are reviewed as factors that might lead to neuronal injury in in vitro model systems for HIV-1 encephalitis.

MCP-1 and CCR2 in HIV infection: regulation of agonist and receptor expression.

Monocyte chemotactic protein-1 (MCP-1) interacts with the chemokine receptor CCR2. Two CCR2 cDNAs have been described. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated natural killer (NK) cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. We found that bacterial products and cytokines affect CCR2 expression. Interleukin-2 (IL-2) augmented CCR2 mRNA in monocytes and NK cells. The augmented migratory capacity of IL-2-activated versus resting NK cells was associated with increased CCR2 transcript levels. Lipopolysaccharide (LPS) and other microbial agents caused a rapid and drastic reduction of CCR2 mRNA levels. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced. These results suggest that regulation of receptor expression, in addition to agonist production, is probably a crucial point in the regulation of the chemokine system. Down-regulation of chemokine receptor expression may play a role in the modulation of HIV infection in macrophages by LPS. Levels of MCP-1 were markedly elevated in the cerebrospinal fluid (CSF) but not in blood of HIV-infected patients with cytomegalovirus (CMV) encephalitis. The CSF levels of MCP-1 in CMV encephalitis were markedly higher than those found in the CSF of HIV-infected patients with or without unrelated neurological diseases. IL-8, the prototype of C-X-C chemokines and RANTES and macrophage inflammatory protein-1 alpha (C-C chemokines) were not substantially increased in the liquor of CMV encephalitis patients. High levels of MCP-1 may underlie monocyte recruitment and tissue damage in CMV encephalitis and may represent a rapid and useful tool in the diagnostic armamentarium for neurological disorders associated with HIV.

HIV infection of macrophages and pathogenesis of AIDS dementia complex: interaction of the host cell and viral genotype.

AIDS dementia complex (ADC) develops in only a third of HIV-infected patients who progress to AIDS. Macrophages and microglial cells are the major cellular sites of productive HIV replication in brain. Using 11 blood isolates of HIV from asymptomatic patients there was marked variation in tropism and the level of productive infection in recently adherent monocytes and monocyte-derived macrophages cultured in vitro. However, less variation was seen with 19 blood isolates from advanced HIV infection and 11 postmortem tissue isolates from brain, cerebrospinal fluid, spleen, and lung. Newly adherent monocytes expressed CCR5 in all seven patients tested, consistent with their susceptibility to infection but not explaining the above variability. There is, also marked regional variability in neuropathology in the brain of patients with ADC. We have demonstrated that there was marked variation in the V3 sequences of HIV clones from different regions of the cortex of a patient with ADC, suggesting independent evolution of HIV replication in brain. Furthermore, production of the neurotoxin quinolinic acid from HIV-infected macrophages varied, depending on the host and source of HIV isolate. Hence variations in viral genotype, production by infected macrophages, and subsequent toxin production may contribute to the variability in neuropathology between individuals and between different regions of the brain in the same individual.

SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1.

Tyrosine kinases of the Src family are regulated via their Src homology 2 (SH2) and SH3 domains. The Nef protein of human immunodeficiency virus-1 (HIV-1) has previously been shown to bind with high affinity and specificity in vitro to the SH3 domain of Hck, a Src family member expressed primarily in myeloid cells. However, the effect of Nef on Hck activity in living cells is unknown. Here we show that Rat-2 fibroblasts co-expressing Hck and Nef rapidly developed transformed foci, whereas control cells expressing either protein alone did not. Nef formed a stable complex with Hck and stimulated its tyrosine kinase activity in vivo. Mutagenesis of the Nef proline-rich motif essential for SH3 binding completely blocked complex formation, kinase activation, and transformation, indicating that the Nef SH3-binding function is required for its effects on Hck. These results provide direct evidence that SH3 engagement is sufficient to activate a Src family kinase in vivo and suggest that Hck may be activated by Nef in HIV-infected macrophages.

Cell Proliferation Is Not Required for Productive HIV-1 Infection of Macrophages

Lentiviruses, including HIV-1, are considered a rare example of retroviruses which do not require cell proliferation for their replication. However, this notion was questioned in several publications where productive HIV-1 infection was found to be restricted to a small fraction of macrophages with proliferative capacity. Since the mode of HIV-1 replication in macrophages is of great clinical relevance, we performed a single-cell analysis of HIV-1 replication and [3H]thymidine incorporation. Our results indicate that while 17% macrophages were detected as HIV-1 DNA-positive 12 hr after infection, only 2% of those cells had incorporated tritium, about the same percentage as in the uninfected cell population. Forty-eight hours after infection, 38% macrophages were HIV-1 DNA-positive and 47% of those had incorporated tritium, while the percentage of tritium-positive uninfected cells did not change (1%). These results demonstrate directly that HIV-1 DNA does not colocalize with [3H]thymidine and support the notion that cell proliferation is not required for HIV-1 infection of macrophages.

Expression of pro‐ and anti‐apoptosis gene products in brains from paediatric patients with HIV‐1 encephalitis

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV-1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene-directed neural cell death, we studied the in-situ expression of the Bcl-2 family of proteins, including the pro-apoptosis gene product Bax, the anti-apoptosis gene product Bcl-2, and Bcl-x. We demonstrate significantly elevated numbers of Bax-positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV-1 infected children without encephalitis or children who were seronegative for HIV-1. In contrast, patients with HIVE, but not HIV-1 without encephalitis, or seronegative controls, had increased expression of Bcl-2 and Bcl-x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up-regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl-2 in HIV-1 infected macrophages, and in LPS-activated macrophages, relative to levels in virus-negative unstimulated macrophages. These results suggest that productive HIV-1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain-resident macrophages and microglia in patients with HIV-1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV-1 infection in the CNS of children with HIV-1 encephalitis.

Dexamethasone therapy worsens the neuropathology of human immunodeficiency virus type 1 encephalitis in SCID mice.

Human immunodeficiency virus (HIV) dementia is a late complication of viral infection. Cognitive dysfunction revolves around the secretion of neurotoxins from immunologically competent virus-infected brain macrophages and microglia. Such macrophage neurotoxins are inflammatory factors that produce selective neuronal dysfunction and ultimately cell death. To evaluate the potential efficacy of antiinflammatory therapy for HIV dementia, dexamethasone was administered to severe combined immunodeficient mice with HIV-1 encephalitis. Mice were given therapeutic doses of dexamethasone before intracerebral inoculation with HIV-1-infected human monocytes. Histochemical evaluation showed a worsening of neuropathology after treatment, with astrogliosis and increased apoptosis of neurons. Laboratory investigation of the mechanisms for the dexamethasone effects revealed increased viability of HIV-infected macrophages and incomplete suppression of neurotoxic inflammatory secretions. The results suggest the need for caution in administering glucocorticoids for treatment of HIV encephalitis in humans.

Functionally relevant changes occur in HIV-infected individuals' alveolar macrophages prior to the onset of respiratory disease.

We have compared the phenotypic and functional changes found in alveolar macrophages recovered from the lungs of 39 HIV-positive individuals with no respiratory disease with those from 33 HIV-positive individuals with pneumonitis and 31 healthy controls. Bronchoalveolar lavage (BAL) cell cytospin preparations were stained using monoclonal antibody immunoperoxidase and double immunofluorescence techniques. Cytokine levels within supernatant BAL were determined using enzyme immunoassay. There were marked differences in alveolar macrophage phenotype between the three groups. In particular, the relative proportion of cells staining RFD1+RFD7- (inducer cells) was reduced in the HIV-positive individuals without respiratory disease. This was correlated with measures of declining systemic immunity. Patients with pneumonitis had the highest levels of measured cytokines [interleukin-1 beta, tumour necrosis factor-alpha and transforming growth factor (TGF)-beta 2], followed by the HIV-positive individuals without respiratory disease. In this latter population a negative correlation was found between active (non acid dissociated) TGF-beta 2 and blood CD4 cell count. The differences between the three groups suggest that alterations of potential relevance to the pulmonary immune response are occurring in alveolar macrophages prior to the onset of respiratory disease. This study confirms the importance of investigating asymptomatic HIV-positive individuals.