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Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, > 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to- cell interactions between HIV-infected brain macrophages and astrocytes.

early after infection, either during the acute seroconversion reaction or during subclinical infection (11)(12)(13)(14)(15). However, exactly how HIV enters the brain and preferentially infects macrophages are areas of intense debate. Virus-infected brain macrophages may originate from an expansion of latently infected monocytes that carry HIV into the brain (the "Trojan horse" hypothesis) and later produce virus (16). Alternatively, virus may penetrate the brain through a disrupted blood-brain barrier by infected T cells or as free viral particles. In either case the results are identical: selective productive infection of brain macrophages and microglia. Whether these HIV-infected brain macrophages induce disease through metabolic, immune, and/or viral-induced mechanisms is critical to our understanding of HIV neuropathogenesis (17). These issues are made ever more apparent as several reports suggest that low-level infection of neurons and glia can produce neurological impairment during HIV infection (18)(19)(20). The ultimate approaches to therapy would vary dependent upon discovered pathogenic mechanisms for CNS injury.
Proposed theories of CNS dysfunction abound. These include: coexistence of opportunistic CNS infections (21,22), secretory toxic factors from infected monocytes, gpl20mediated neuronal growth factor blockade or killing, and neurotoxicity by HIV tat or other viral regulatory components. All or any of these mechanisms may result in cytotoxic effects in neurons and/or oligodendrocytes (23)(24)(25). For example, gpl20 may antagonize normal vasoactive intestinal peptide (VIP-ergic) function in brain (23) or be directly toxic to neurons. Studies show that gpl20 can induce neuronotoxicity by increasing free Ca 2+ levels in cultured neurons and is prevented by Ca z+ channel antagonists (24).
Recent reports suggest that brain dysfunction may be related to cell-encoded toxins generated from virus-infected macrophages (26,27). Secretory products from HIV-infected cells may alter neuronal viability, damage myelin, or stimulate neurotransmitters resulting in neuronal dysfunction. Indeed, macrophages play important roles in steady-state immune and tissue function. The regulatory role of macrophages occurs through the release of numerous secretory molecules made under a variety of physiologic conditions. Changes in the secretion or release of certain of these mediators may lead to disease. In support of this idea are recent studies demonstrating that disordered secretion of one or more cellular factors from HIV-infected macrophages produces neuronal death in vitro (26,27). In one report, HIV-infected U937 cells, a myelomonocytic cell line, released toxic factors that destroyed cultured chick and rat neurons (27). The monocyte-produced neurotoxin(s) were heat stable and protease resistant, and acted by way of N-methyl-o-aspartate (NMDA) receptors. The studies suggested that HIV-infected macrophages and microglia in brain continuously disrupt neurologic function leading to cognitive CNS dysfunction. However, two recent reports failed to confirm these observations (28,29). One study (29) demonstrated neurotoxicity only after cell-to-cell contact between HIV-infected monocytic and human neural cells. In this and a study from our own laboratory (28) investigators found no morphological alterations of neurons exposed to HIVol-infected monocyte fluids. Thus, if the macrophage plays a role in virus-induced neuropathology it may act through cellto-cell interactions with neurons and glia to produce CNS tissue damage (29). Perhaps this occurs through cytokine and/or other neuronotoxic factor release during cell-to-cell contact. Indeed, similar mechanisms are operative for cytokine induction in peripheral blood. For example, IFN-oe is induced during cell-to-ceU interactions with HIV-infected macrophages and PBMC (30). Similarly, neuronotoxins could be produced from glia during cell-to-cell interactions with infected brain macrophages. To investigate this possibility we recovered culture fluids from cell mixtures of virus-infected monocytes and astroglia. Assay of fluids after cocultivation showed high levels of arachidonic acid metabolites, cytokines, and neuronotoxic activity.

Materials and Methods
Isolation and Culture of Monocytes and Human Neural Cells. Monocytes were recovered from PBMC of HIV and hepatitis B-seronegative donors after leukapheresis and purified by countercurrent centrifugal elutriation. Cell suspensions were >98% monocytes by criteria of cell morphology in Wright-stained cytosmears, by granular peroxidase, and by nonspecific esterase. Monocytes were cultured as adherent monolayers (106 cells/ml in 24-mm plastic culture wells) in DMEM (Sigma Chemical Co., St. Louis, MO) with 10% heat-inactivated AB § human serum, 50/,g/ml gentamicin, and 1,000 U/ml highly purified (<0.01 ng/ml endotoxin) recombinant human macrophage MCSF (FAP-809; Cetus Corp., Emeryville, CA) (31). Human brain tumor-derived cell lines were obtained from the following sources: U251 MG from D. Bigner (32), U373 MG from B. Westermark (33), and SK-N-MC (34) and H4 (HTB 148) (35) from the from American Type Culture Collection (ATCC; Rockville, MD). The cells were grown as adherent monolayers in DMEM (Sigma Chemical Co.) with 10% heat-inactivated FCS and 50/zg/ml gentamicin. Human endothelial cells were a gift from P. I. Lelkes (36). Cell lines were fully characterized to their cell origins (37,38). Primary human astrocytes were prepared from second-trimester human fetal brain tissue obtained from elective abortions (performed in full compliance with both National Institutes of Health and University of Rochester guidelines). Brain tissue composed of telencephalon with both cortical and ventricular surfaces was dissected in cold HBSS with Hepes and 50/zg/ml gentamicin, then transferred to 20 ml ice-cold (4~ DMEM/F12 (Gibco Laboratories, Grand Island, NY) with 10% heat-inactivated FCS. The tissue was mechanically dissociated by teasing through a Nitex bag with a glass pestle. Cells were resuspended in media and filtered through a 230-and then 140-~m sieve. The cell suspension was centrifuged, washed twice in media, then plated in DMEM containing 10% FCS and 50/zg/ml gentamicin into 75-cm 2 tissue culture flasks (Coming, Coming, NY) at a cell density of 2 x 10 s cells/ml. Media was exchanged every 3 d. Nonadherent microglia and oligodendrocytes were removed by gentle agitation and circular shaking of cultured cell preparations 10 d after plating. The purity of the astrocyte cultures was ~95% by immunostaining for glial fibrillary acidic protein (GFAP) (39). Cells were cultured as adherent monolayers in DMEM (Sigma Chemical Co.) with 10% heat-inactivated FCS (Sterile Systems, Inc., Logan, UT), 20/*g/ml gentamicin, and 1% glutamine. All culture reagents were screened and found negative for endotoxin contamination.
Chemical Reagents. Dexamethasone was purchased from Sigma Chemical Co. and indomethacin and nordihydroguaiaretic acid (NDGA) from Cayman Chemical Co., Ann Arbor, MI. All reagents were dissolved in ethanol and diluted with complete macrophage media. Final ethanol concentration in cell cultures was ~<0.1%.
Quantitations of Cytokine Activity. Culture fluids from control and HIV-infected monocytes were analyzed by ELISA for the human cytokines TNF-~, IL-I~, IL-1/~, and IL-6 (Quantikine Immunoassay; Research and Diagnostics Systems, Minneapolis, MN). IFN activity in culture fluids was assayed by inhibition of cytopathic effects induced by vesicular stomatitis virus (VSV) in MDBK cells (42). TNF bioactivity was performed according to standard procedures (43). Briefly, the routine L929 cell line was propagated in DMEM (Sigma Chemical Co.), 5% FCS, 1% glutamine, and 20 ~tg/ml gentamicin. Cells were retrieved in log phase and placed (0.5 • 105/well) in 96-well plates (Costar) with actinomycin D. Culture fluids were inoculated into cell monolayers, and degree of cell lysis was determined by crystal violet staining after a 24-h incubation.
Coupled RT-PCR Detection of Cytokine and HIVspecific RNA. Levels of cytokine KNAs were estimated after KT with antisense primers and PCK amplification of the cDNA transcripts. The mKNA for the cellular enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), served as an internal control to allow analysis and comparison of KNA species between different samples. Briefly, 2.0/~g total cellular KNA in 0.025 ml was mixed with 0.3 #g of the antisense primers for GAPDH, TNF-c~, TNF-~/, IL-lc~, Ibl/~, II.-6, IFN-c~, IFN-% and IFN-/~ (44). Table 1 lists the primer sequences used in these studies. The mixture was heated at 70~ for 5 rain, cooled on ice, and treated with 500 U of Moloney mufine leukemia virus KT (Bethesda Research Laboratories, Bethesda, MD) and 0.5 mM each of all four deoxynucleotide triphosphates. ILT reactions were at 37~ for 15 rain, then stopped by heating at 95~ for 10 rain. For PCK amplification of the cDNA products, reaction mixtures were divided into equal aliquots and mixed with 0.5 #g sense and antisense primers, 0.5 mM deoxynucleotide triphosphates, and 2 U Amplitaq (Cetus Corp., Emeryville, CA). The products of 25 cycles (1.5 rain, 94~ 1.5 rain, 50~ and 2.0 min, 72~ were analyzed by Southern blot hybridization. The oligo-nucleotides were synthesized on DNA synthesizer (Applied Biosystems, Inc., Foster, City, CA) and checked for purity by polynucleotide kinase labeling and sequence gel analysis. Oligonucleotides were typically 95% pure.
Quantitations of Neuronal Cell Growth and Survival. The metabolic activity and number of viable cells/culture were assessed by conversion of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium (MTT) bromide, and color intensity was measured at OD490 om (47,48). Cell morphology in neuronal cultures depleted of glial cells was examined under phase-contrast microscopy or after fixation with 80% methanol and Wright-Giemsa stain. Morphologic changes in these neuron-enriched cell cultures correlated directly with MTT levels and were scored as 0 (no neuritic outgrowth), + (dendritic outgrowth 2-4 perikaryons distance in >/50% of cells/field), or + + (dendritic outgrowth 4-8 perikaryons distance in >~50% of ceils/field). Generally, 100 cells in four fields/culture were examined on successive days. In some studies, identical microscopic fields were photographed at serial intervals to decrease sampling variability.

Analysis of FH]Arachidonic Acid Metabolites Released by HIVinfected Monocytes and Cocultures of HIV-infected Monocytes and Astroglia.
HIV-infected or control uninfected monocytes (5 x 106 cells/ml) were cultured in 35-ram wells (Costar) in media containing 10% AB § human sera and [3H]arachidonic acid (1.0 mCi/ml; 1 Ci = 37 GBq; American Radiolabeled Chemicals Inc., St. Louis, MO) added to the cultures for 18 h. HPLC procedures followed previously published methods (49,50). A brief description of the protocol used in these experiments is outlined below.
The monocytes were washed three times with DMEM in 1% BSA (Sigma Chemical Co.) then incubated for 20 s to 180 rain with or without equal numbers of U251 MG astroglial cells. The reaction was stopped by the addition of 10 #l formic acid and 25 /zl butylated hydroxytoluene in methanol, and the samples were placed on dry ice. Supernatant fluids were removed and the cells scraped after the addition of 500/~1 of HPLC-grade water. The cell lysate was combined with the culture supernatants from each well and placed into 10-ml polypropylene centrifuge tubes. The fractured cell-supernatant mixtures were centrifuged at 400 g for 5 min and the clarified supernatants stored under argon at -70~ in 4-ml dram vials. Before analysis, a 2-ml thawed sample was adjusted to pH 4.0 with 22 M formic acid and microcentrifuged for 1 rain. The internal standard mix was added to the cell lysate. It contained hydroxyeicosadienoic acid (for spectrophotometric verification of elution position accuracy) and [14C]eicosatrienoic acid (for quantitation of sample recovery and inter/intrasample comparisons). DPMs of each sample were adjusted based on recovered [14C]eicostrienoic acid. A C18 Sep-Pak cartridge (Waters Associates, Milford, MA) was activated by placing 4 ml of HPLCgrade methanol through the cartridge. The C18 Sep-Pak was washed with 10 ml of HPLC-grade water. The sample was applied to the C18 Sep-Pack cartridge (Waters Associates) followed by a 2.5-ml wash, and arachidonic acid metabolites were quantitatively duted with a mixture of 85% acetonitrile and 15% methanol The ehant was concentrated and dried with a Speed-Vac concentrator (Savant Instruments, Inc., Farmingdale, NY). The arachidonic acid metabolites were then dissolved in methanol and transferred to HPLC vials (National Scientific Co., Lawrenceville, GA) for injection. The arachidonic acid metabolites extracted were then injected onto a reverse-phase C-18 column using an analytical HPLC system (Beckman Instruments, Inc., Palo Alto, CA). Spectraphotometric analyses were performed with a UV detector (166; Beckman Instruments, Inc.). l-rain fractions were collected during a 96-rain chromatography run, and total DPM was determined in each fraction. A second separately collected ehtion was performed on the Sep-Pak that quantitatively removed platelet-activating factor (PAF). The eluate was dried and analyzed using a quantitative RIA kit (Amersham Corp., Arlington Heights, IL). HIV-I~A at an MOI of 0.01. Culture fluids from the HIVinfected and control uninfected cells were half-exchanged at 2-3-d intervals then added, 14 d after plating and 7 d after infection, to rat brain explant and/or SK-N-MC human neuroblastoma cells. The composition of fetal rat brain cultures at the time of experimental inoculation (day 10 of culture) was 70-85% neurons (neurofilament and neuron-specific enolase-positive cells), 10-15% microglia (OX-6-positive cells that ingest latex beads), 5-10% astrocytes (GFAP-positive cells), and 0-3% fibroblasts (vimentin-positive cells [44]). Additions of fluids from HIV-infected and control uninfected monocytes to the rat brain explants showed neuronotrophic activity. Numbers of NSE + neurons treated with HIVinfected and control monocyte fluids for 5 d were two-to threefold higher than equal numbers of neurons treated with culture medium alone ( Table 2 and Fig. 1). Fetal rat brain cells inoculated with fluids from HIV-infccted or control uninfected U251 MG, U373 MG, or HTB 148 cells also showed no neuronotoxicity (Table 2).

Fluids from HIV-infected Monocyte-Glial Cell Interactions Produce Neuronotoxic Factors,
The absence of neuronotoxic activity from HIV-infected monocytes or virus-infected neural cells led to assay of cell mixtures for neuronotoxic activities. Monocytes were infected with HIV-1AD^ for 7 d, harvested from teflon flasks, then placed onto equal numbers of neural cells. Fluids were harvested at 24 and 48 h after cocultivation then placed onto rat brain explant for assay of neuronotoxicity. In contrast to previous results, fluids from cocultures of HIV-infected monocytes with U251 MG, U373 MG, or HTB 148 astroglial cells were profoundly neuronotoxic ( Table  2 and Fig. 1). Within 2 d after fluid addition neurons were swollen and vacuolated (Fig. 1). These fluids were also toxic for SK-N-MC neuroblastoma cells. The neural cell toxic activity was only produced in mixtures of HIV-infected monocytes and glia (U251 MG, U373 MG, and HTB 148 cell lines) ( Table 2). Dose-response analysis of culture fluids from HIVinfected monocyte-astroglia (U251 MG) mixtures showed significant neuronotoxicity with dilutions of ~1:20 of the fluids. Indeed, a/>50% loss of viable neurons/well was evident when a 20-fold dilution of culture fluids of HIV-infected monocytes-astroglia was placed onto rat fetal neurons (Fig.  2). The toxic effects were cell specific. Fluids obtained from mixtures of HIV-infected monocytes and SK-N-MC (neuroblastoma)-or HIV-infected monocytes and endothelial cells showed no neuronotoxic activity ( Table 2). The HIV-infected monocytes and U251 glial cells fluids were not toxic for rat or human astrocytes and fibroblasts (data not shown).

Mechanisms for Neuronotoxicity Analysis of Cytokine Gene
Expression. A variety of cytokines may produce neurotoxicity and as such contribute to the pathogenesis of CNS disease. Two cytokines, IblB and TNF-ob, are associated with glial proliferation, neurotoxicity, and demydination. Interestingly, these cellular effects are all prominent features of HIVrelated encephalopathy. For example, TNF, at high concentrations, is a neurotoxin (28,51,52). Human astrocytes proliferate in response to TNF-c~ and IL-1B (53, 54), and conditioned medium from LPS-treated astrocytes stimulates HIV-1 gene expression in monocytic cells (55). These observations Initial experiments were performed to determine whether the addition of HIV-infected monocytes to astroglia resulted  in cytokine gene expression. We examined the levels of TNF-ce and I1:1fl by coupled RT-PCR in cell cocuhures. The mRNA for the constitutive cellular enzyme, GAPDH, was examined as the reference cellular transcript. GAPDH mRNA amplification products were present at equivalent levels in all cell lysates. IFN-7 mRNA was not detected in any cell lysate (data not shown), demonstrating that a mixed T cell reaction was not an explanation for any cytokine mRNA expression observed. The mRNAs for I1:1fl, IL-lc~, TNF-o~, and TNF-fl in cell lysates of uninfected and HIV-infected monocyte cultures were absent (Fig. 3, lanes 1 and 2; and Table  3). However, the predicted 237-and 179-bp amplification products of TNF-ot and IL-lfl mRNAs were readily seen in cell lysates of uninfected and HIV-infected monocytes and glia (U251 MG and HTB 148 cell lines) (Fig. 3, lanes 3 and 4, and 9 and 10). Interestingly, TNF-oe and IL-13 mRNAs were not detected in cell lysate and culture fluids of uninfected control or HIV-infected monocytes cocuhured with endothelial cells (Fig. 3, lanes 7 and 8) or neurohlastoma cells (SK-N-MC cells) (Fig. 3, lanes 11 and 12). Control uninfected and HIV-infected U251 MG cells also showed no TNF-ot and I1:1fl mRNAs (Fig. 3, lanes 5 and 6). Replicate experiments with primer pairs for three other cytokines (I1:1c~, TNF-fl, or I1:6) (data not shown) were below the limits of PCR detection (Fig. 3). These results, taken together, show a selective and cell-specific induction of TNF-ce and II:1/3 mRNA during the interaction between HIV-infected or control uninfected monocytes and glia. The high levels ofmRNAs were not always associated with  Table 1) similarly high levels of proteins (Table 3). TNF-ot and I1:lfl protein and biological activity were seen only in coculture fluids of HIV-l-infected monocytes and glia ( Table 3). The TNF-oe and I1:1fl proteins were observed during cocuhure of HIV-infected monocytes and astroglial (U251 MG). Maximum levels were present 12-48 h after cocuhivation. In a series of four replicate experiments maximum levels of TNF-o~ were 1,000-9,000 pg/ml (mean of 5,000), while I1:1fl levels ranged from 400 to 5,000 pg/ml (mean of 900) (Figs. 4 and 5). The results were confirmed by assays of TNF activity (Fig.  6). The underlying basis for this 10-fold difference was related to the levels of productive HIV infection. Peak cytokine levels occurred during the initial rise of RT activity, 3-5 d after virus infection, and diminished to baseline by day 10 (data not shown). TNF-cr and I1:1fl proteins were also detected at low levels (<100 pg/ml) in cocultures of uninfected monocytes and astroglia (U251 MG and human astrocytes), The latter results suggest that the interactions seen between HIVinfected monocytes and glia are an extension of a normal physiological response. In all experiments assayed, the cytokine response mirrored the neuronotoxic response (data not shown). TNF-a mRNA and protein in cell lysates and culture fluids of uninfected control, HIV-l~^-infected monocytes, and/or U251 MG astroglial cells after 24-h incubations with the treatments listed. Monocytes cultured 7 d as adherent monolayers were exposed to HIV at a MOI of 0.01.1 wk after infection virus-infected monocytes were cocultured with media or equal numbers of: endothelial cells, SK-N-MC (neuroblastoma) cells, HTB 148 glial cells, U251 MG astroglial cells, cycloheximide then U251 MG astroglial cells, and cydoheximide then U251 astroglial cells. The U251 MG astroglial cells were incubated with: uninfected monocytes, HIV-I^D^, mycoplasma-infected monocytes, and freeze-thawed or par^formaldehyde-fixed HIV-infected monocytes. Cytokine mKNA levels were detected by coupled KT-PCK amplifications from cell lysates. The extracted KNAs were mixed with antisense primers and, after KT, eDNA was amplified by PCK. The products of 25 cycles were analyzed by Southern btot hybridization. KNA was not detected (0), detected at low levels (+), was readily detected (+ + ), or detected at high levels ( + + + ). Cytokine levels in culture fluids were determined by ELISA.

Analysis of the Cytokine-producing Cell in Monocyte.Astroglia Cocultures. Cytokines produced during the interactions between HIV-infected monocytes and glia required viable mix-
tures of both call types. When U251 MG or HTB 148 were mixed with 4% par^formaldehyde-fixed or freeze-thawed HIVinfected monocytes, cytokines were not detected (Tables 3). Pretreatment of HIV-infected monocytes with cydoheximide or actinomycin D before coculture was used to determine if the infected monocytes were primary cytokine producers. Cycloheximide and actinomycin D were titrated to inhibit HIV mRNA and protein synthesis (positive controls for this assay system) and used at concentrations of 30 and 5/~g/ml, respectively. In these assays, cycloheximide-or actinomycin D-treated monocytes were washed extensively before addition ofU251 MG astroglial cells at equal cell concentrations. Analysis of TNF-c~ mRNA levels from cell lysates prepared from cycloheximide-or actinomycin D-treated cocultures showed increased and decreased levels of TNF-c~ mRNA, respectively (Fig. 7). However, both additions resulted in significant reductions in both TNF-c~ and II,1B proteins (Table  3; and unpublished observations). The higher levels of TNF-a protein seen after cycloheximide compared with actinomycin D treatment likely reflect the reversibility of cycloheximide after a 24-h wash out. That these inhibitors of RNA and protein synthesis had opposite effects on levels of TNF-ol mRNA was not surprising. Indeed, previous reports show an upregulation of TNF-c~ mRNA in monocytes after both LPS and cycloheximide treatment (56,57). These results coupled with the recent demonstration of TNF-c~ and IblB protein in HIV-infected brain macrophages (58) support the notion that the macrophage is the primary cytokine producer. Furthermore, reproduction of these experimental findings with cocukures of H1V-infected monocytes and normal human fetal astrocytes (data not shown) lends support for the biological relevance of the described experimental system. The role of TNF-(x and IL-1/3 in the observed neuronotoxicity was next explored. Toxicity of recombinant human (rh)TNF-ol (Amgen Biologicals, Thousand Oaks, CA) and rhll.-1/3 (Collaborative Research, Bedford, MA) was tested on rat fetal neuronal cultures as described above. Replicate experiments were performed with recombinant murine (rm)TNF-ol (Genzyme, Boston, MA) and rmlblB (Hoffman-La Roche, Inc., Nutley, NJ). The cytokine concentrations used in these assays were extrapolated from data from cocultures of HIV-infected monocytes and astroglia (1-10 ng/ml of recombinant protein). Inoculation of TNF-o~ and IL-I~ alone or in combination to fetal neuronal cultures produced no neuronotoxicity. Moreover, mycoplasma and endotoxin contaminations were ruled out as the neuronotoxin in the cell and viral preparations. Mycoplasma was not detected by hybridization assays in any of 10 randomly selected culture fluids. The levels of endotoxin contamination as detected by the Limulus amebocyte lysate assay were <50 pg/ml. The addition of polymyxin B at 15/~g/ml, a concentration known to inhibit the LPS-induced cytokine production, had no significant effect on cytokine levels observed in HIV-infected monocyte-astroglia cocultures. These results, taken together, demonstrate a clear association of cytokine and neuronotoxicity in cocultures of HIV-infected monocytes and astroglia.

Arachidonic Acid Metabolites Induced during Cocultures of HIVinfected Mono~tes and Astroglia: Implications for Cytokine Regulation and Neuronotoxicity. Viable HIV-infected monocytes
are required for the generation ofcytokines and neuronotoxicity. Supernatant fluids from astroglial ceils (U251 MG) mixed with 4% par^formaldehyde-fixed or freeze-thawed HIV- infected monocytes or HIV-I^D^ viral stock (>10 s total particles/ml) each failed to produce neuronotoxicity. These results suggested yet another component for both cytokine and neuronotoxic responses. For several reasons, arachidonic acid metabolites were pursued as this possible missing component. First, arachidonic acid metabolites are upregulated in monocytes after incubation with the viral envelope glycoprotein, gp120 (50). Second, these metabolic products can regulate TNF-cz and IL-13 production in macrophages. Indeed, TNF causes amplification of arachidonic acid metabolites in response to IL-1, while PAF enhances TNF production (59)(60)(61). An autocrine/paracrine loop between arachidonic acid metabolites and cytokines and vice versa could explain the need for viable cell-to-cell interactions. Third, arachidonic acid metabolites play important roles in development neurobiology, neuronal function, and were reported as neuronotoxins (62)(63)(64). For these reasons, we determined whether arachidonate metabolites were produced during HIV-infected monocytes-astroglia interactions and whether they played a physiologically important role in this experimental system. HPLC separation of arachidonic acid metabolic products released from uninfected control monocytes, HIV-infected monocytes, uninfected monocytes and U251 MG astroglial cells, and HIV-infected monocytes and U251 MG astroglial cells were evaluated. Cells were radiolabeled with [3H]arachidonic acid for 18 h before coculture. The arachidonic metabolites were identified based on elution position standards ( Table 4) and use of increasing polar solvents. The dution profiles of uninfected control (Fig. 8, broken lines) and HIV-1711 Genis et al.    Monocytes cultured 7 d as adherent monolayers were exposed to HIV at an MOI of 0.01. 7 d after infection HIV-infected or control uninfected monocytes were treated with cycloheximide (30 #g/ml) or actinomycin D (5/~g/ml) for 1 h. After 3 h IkNA was extracted from cell lysates and then mixed with antisense primers. After RT, cDNAs were amplified by PCR and the products of 25 cycles analyzed by Southern blot hybridization with a TNF-c~-specific probe (see Table I). Coupled RT-PCR amplification products from cell lysates of uninfected and HIV-infected monocytes cocultured with U251 MG astroglial cells and treated with or without cycloheximide, actinomycin D, or media (control) are shown.
Based on elution times of 3H standard (Table 4), the major products of the lipoxygenase pathway were LTB4, LTD4, and lipoxin A4 (Fig. 8, a, c, and e). Although increased amounts of 5-HETE methyl ester and lactone were in HIVinfected cells the levels of these metabolites were indistinguishable between infected and uninfected ( Fig. 8, a, c, and e, block broken lines) cell cocultures. Major increases were also seen in levels of 5-HETEs, methyl ester, and ~-lactone. Interestingly, LTB4 levels were transient, produced at 90 s, but not sustained. This is in keeping with its known cyclical production (65). Moreover, cyclooxygenase products were identified only at low levels. Uninfected monocytes cocultured with astroglia produced 15-HETE, a metabolite found in low quantities in HIV-infected cells (Fig. 8, a, c, and e). The differences in the metabolic arachidonate profiles persisted through 90 min. At that time HIV-infected monocyteastroglia cocultures showed a >20-fold increase in LTD4 and uninfected cultures showed a >14-fold increase in 5-HPETE. LTB4 oxidation products were cyclic but were always greater in HIV-infected cocultures. By 180 rain, LTD, levels remained elevated in HIV-infected cocultures and increased greater than fourfold from cocultures of uninfected monocytes and astroglia. The levels of PAF were evaluated by a quantitative klA. Increased levels of PAF in cocultures of HIV-infected monocytes and astroglia were strongly associated with TNF-ot  (Table 5). To investigate a possible casual relationship between arachidonic acid metabolites and TNF-ot, we added dexamethasone, an inhibitor of phospholipase A; indomethacin, a cyclooxygenase inhibitor; or NDGA, a lipoxygenase inhibitor (at concentration used), to cocultures of HIV-infected monocytes and astroglia, and measured TNF-a production ( Table 6). Monolayers were infected with HIV at an MOI of 0.01 for 7 d before addition of U251 MG astroglial cells. The compounds were added together with equal numbers of astroglia. Both dexamethasone and NDGA markedly reduced the levels of TNF-ot in supernatant fluids of these cocultured cells. Interestingly, indomethacin increased TNF-ot levels. This likely reflected shunting of the arachidonate metabolites into the lipoxygenase pathway. The failure of NDGA to completely abrogate the TNF-et response suggests that PAF also participates in this cytokine response. Indeed, TNF-ot is reprodudbly detected after addition of PAF to cocultures of uninfected monocytes and astroglia (P. Genis, unpublished observations). Thus, arachidonic acid metabolites and PAF both likely participate in the TNF-ct induction demonstrated in this experimental system.

Discussion
In the present study we demonstrated that the interactions between HIV-infected human monocytes and astroglia pro-   duced high levels of IL-13 and TNF-ol. The Ib13 and TNF-ot responses were cytokine specific, correlated with neuronotoxicity, and occurred only during coculture of HIV-infected monocytes and astroglia. TNF-/L Iblot, IL-6, and IFN-q/were not produced. Mixtures of neuronal or endothelial cells and HIV-infected monocytes failed to elicit cytokines or neuronotoxins. Interestingly, the addition of TNF-ot and Ib13 alone or in combination to neurons at the concentrations found in HIV-infected monocyte-astroglia culture fluids did not produce neuronotoxicity. Viable glial cell-to-cell interactions were required. Moreover, HIV-infected monocyte culture fluids, sucrose gradient-concentrated viral partides, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocyte cell membranes failed to produce cytokine or neuronotoxic activity when placed on astrocytes. These data, taken together, suggested that other factor(s) were required for the observed responses. Two of these factors were identified as lipidic compounds derived from membrane phospholipids, including products of the 5-1ipoxygenase pathway and PAE These products, potent low molecular weight mediators of immune activation, were secreted within 90 s of the mixture between HIV-infected monocytes and astroglia. The large numbers of HIV-infected macrophages and astrogliosis in virus-infected brain tissue coupled with histological evidence of neuronotoxicity support the biological relevance of these observations. Pathological outcomes of HIV-1 infection in brain tissue include neuronal loss, reactive astrogliosis, and myelin damage (66). Neuronal loss (67) is strongly associated with axonal and dendritic damage in the cortex and subcortex of affected individuals (68,69). The paucity of productively infected neurons supports indirect mechanisms for neuronal damage seen during HIV disease (6)(7)(8)(9)(10). Indeed, viral antigens are found predominantly, if not exclusively, in cells of macrophage lineage (microglia, macrophages, and multinucleated giant cells).
It is tempting to attribute the pathogenesis of HIV encephalopathy to secretory products produced from HIVinfected macrophages or through the interactions between HIV-l-infected macrophages and astrocytes. Perhaps interplay, or "synergy" occurs between a number of toxic factors, including cytokines, arachidonic acid metabolites, and viral structural and/or regulatory proteins (gp120, tat, nef, etc.). Our results support such a notion and suggest that cytokines, viral proteins, and lipidic compounds produced during glial cellular interactions all play roles in the neuropathogenesis of HIV disease. Arachidonate metabolites and PAF may contribute to neuronal injury while stimulating TNF-ot and Ib13 production, resulting in astroglial proliferation (60,61).
Macrophages play a pivotal role in the generation of the described neuronotoxin response. This result is not surprising. Indeed, macrophages can contribute to disease progression by several mechanisms. At sites of infection macrophages secrete scores of toxic effector molecules that damage tissue. Proliferation of autoreactive T cells through indigenous antigen presentation also leads to tissue damage and occurs in multiple sclerosis and rheumatoid arthritis (70,71). In multiple sclerosis, cytokines produced from macrophages and T cells produce myelin damage. In rheumatoid arthritis, the secretion of monokines leads to alterations in endothelial cell adhesion and neutrophil influx. Ultimately, alterations in macrophage secretory and antigen-presenting functions leads to brain inflammation in multiple sclerosis and synovial hyperplasia of cartilage, tendons, and subchondral bone in rheumatoid arthritis.
The absence of critical regulatory signals between macrophages, microglia, and astrocytes may also contribute to the CNS disease process. Macrophages play important roles in cell differentiation and tissue repair, and together with astrocytes regulate steady-state homeostatic CNS function. These functions provide an additional means for how HIV might disrupt neural function. For example, a critical homeostatic factor required for neuronal function might be lost because of HIV macrophage infection. However, the lack of supporting experimental evidence for this mechanism, the focality of tissue pathology and disease progression seen without large numbers of virus-infected cells, suggests that HIV-associated neuropathology occurs through the daboration of toxic factors.
The astrocyte functions, in this experimental system, in a supportive role for cytokines and neuronotoxin induction. Although astrocytes can produce toxins (e.g., quinolinate) (72,73) and secrete cytokines after appropriate stimulation, (e.g., IL-1, IL-3, TNF, and II.,6) (74), there is no evidence that the astrocyte produces any such factors in vivo (75). Moreover, cycloheximide and actinomycin D added to HIVinfected monocytes significantly reduced or diminated cytokines within culture fluids of HIV-infected monocytes and astroglia. Nonetheless, astrocytes are required for cytokine and neurotoxin production. This suggests that they play an important role in the generation of CNS injury during HIV encephalopathy.
TNF and Ibl likely contribute to the in vivo neuropathology of HIV infection. TNF regulates class I and II MHC antigens and induces proliferation of astrocytes. Additionally, TNF can cause myelin damage and lysis in oligodendrocytes and upregulates HIV gene expression in monocytic cells (51)(52)(53)(54)76). During HIV infection in brain, increased levels of TNF and/or IL-1 can induce intracellular adhesion molecule 1 expression (77) in endothelial cells and/or astrocytes. Thus, TNF can facilitate the production of inflammatory infiltrates in brain parenchyma and permit the penetration of virus-infected monocytes through the blood-brain barrier.
Several lines of evidence demonstrate that the regulation of TNF and II.-1 revolves around both the production of arachidonic acid metabolites and PAE First, the addition of dexamethasone, a potent inhibitor of phospholipase A, to cocultures of HIV-infected monocytes and astroglia abrogates this cytokine response. Second, downregulation in TNF-ce was seen with NDGA, an inhibitor of the lipoxygenase pathway, while indomethacin, a cyclooxygenase inhibitor, increased TNF-ot production. Third, a temporal relationship between production of LTB~, LTD4, lipoxin A4, and PAF and TNF was shown. PAF enhances TNF production by inducing 5-1ipoxygenase activity in macrophages (60,61). PAF also causes the release of LTB4 suggesting an interrelationship between the stimulation of TNF by PAF and endogenous lipoxygenase activity (61). TNF and IL-1 can also induce LTB4 in human macrophages. The addition of PAF alone or in combination with LTB4 into uninfected monocytes and astroglia cocultures induced TNF activity (P. Genis, unpublished observation). Last, the HIV glycoprotein (gp120) strongly upregulates (up to 40-fold) arachidonic acid metabolites and 1L,1 activity in human monocytes (50). In this fashion HIV-1 gp120 may be inducing TNF-c~ and IblB in primary brain cultures (78).
There is precedent for the ability of HIV-l-infected monocytes to trigger cytokine production from calls during cellto-cell contact (e.g., IFN-ot from PBMC and TGF-~8 from astrocytes) (30,79). PBMC cocultured with HIV-infected monocytes release high levels of IFN-a activity. Levels of this IFN are associated with the interstitial pneumonitis seen in ungulate lentiviral infections and in children infected with HIV (80). The release of TGFo~, a potent chemotactic factor, is initiated from the interactions between HIV-infected macrophages and astrocytes. The production of TGF-~ in brain likely permits recruitment of HIV-infected monocytes into brain, providing a mechanism for efficient viral spread and disease (79). The exact secretory factors that regulate neural injury in HIV disease likely revolve around combinations of factors including arachidonic acid metabolites, cytokines, and other toxins. Nonetheless, the mechanism of cytokine and neuronotoxic factor induction described in this report coupled with identifiable compounds that augment the neurological response should prove helpful in understanding the basic mechanisms underlying HIV-induced CNS injury and for providing therapeutic strategies for AIDS-associated encephalopathy.
We thank Ms. Victoria Hunter and Debra Joyne for excellent graphics, and Christopher Welch, Victoria Lancaster, and Michael Zurer for excellent technical assistance and Dr. Mark DeCoster for skillful assistance with the rat neuronal culture system.