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The Journal of Neuroscience, August 1, 1999, 19(15):6468-6474

Gp-41-Mediated Astrocyte Inducible Nitric Oxide Synthase mRNA Expression: Involvement of Interleukin-1beta Production by Microglia

Shuxian Hu, Humaira Ali, Wen S. Sheng, Laura C. Ehrlich, Phillip K. Peterson, and Chun C. Chao

Institute for Brain and Immune Disorders, Minneapolis Medical Research Foundation and the University of Minnesota Medical School, Minneapolis, Minnesota 55404


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanisms underlying human immunodeficiency virus-1 encephalopathy are not completely known; however, recent studies suggest that the viral protein gp41 may be neurotoxic via activation of inducible nitric oxide synthase (iNOS) in glial cells. In the present study, we investigated the NO-generating activity of primary human fetal astrocytes in response to gp41 and the relationship to microglial cell production of interleukin-1 (IL-1). Gp41 failed to trigger iNOS mRNA expression in highly enriched (>99%) astrocyte or microglial cell cultures. However, gp41-treated microglia released a factor(s) that triggered iNOS mRNA expression and NO production in astrocytes. Because IL-1 receptor antagonist protein blocked gp41-induced NO production, a pivotal role was suggested for microglial cell IL-1 production in astrocyte iNOS expression. Also, gp41 induced IL-1beta mRNA expression and IL-1 production in microglial cell but not astrocyte cultures. Using specific inhibitors, we found that gp41-induced IL-1beta production in microglia was mediated via a signaling pathway involving protein-tyrosine kinase. These data support the hypothesis that gp41 induces astrocyte NO production indirectly by triggering upregulation of microglial cell IL-1 expression.

Key words: astrocytes; cytokines; human immunodeficiency virus-1; interleukin-1; microglia; nitric oxide; nitric oxide synthase; protein-tyrosine kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human immunodeficiency virus-1 (HIV-1) encephalopathy is a devastating complication of CNS infection in patients with acquired immunodeficiency syndrome (AIDS), resulting in marked cognitive and motor abnormalities (Price et al., 1988; Janssen et al., 1991). The histopathological hallmarks of HIV-1 encephalopathy include HIV-1 infection of brain macrophages, multinucleated giant cell formation, astrogliosis, and neuronal cell loss in discrete regions of the brain (Navia et al., 1986; Ketzler et al., 1990; Wiley et al., 1991). For reasons that are not clear, this disease is most prevalent in HIV-1-infected children (Cohen et al., 1991). The precise cellular and molecular mechanisms underlying HIV-1 encephalopathy remain to be established; however, viral protein-induced generation of the neurotoxic free radical nitric oxide (NO) may be involved. For example, a recent study has suggested that inducible NO synthase (iNOS) is expressed in postmortem brains of patients with severe dementia and a corresponding elevation of the viral envelope protein gp41 (Adamson et al., 1996).

HIV-1 gp41, in concert with other viral proteins, has been reported to activate iNOS mRNA expression and NO production in mixed human microglia-astrocyte cocultures (Koka et al., 1995a). The sequence of events underlying gp41-induced NO production in mixed glial cell cocultures, however, is unknown. Mounting evidence has suggested that human microglia either are totally incapable of NO production (Lee et al., 1993) or are relatively weak producers of NO (Peterson et al., 1994; Ding et al., 1997). Instead, activated human astrocytes appear to be a major cellular source of inducible NO. Interleukin-1 (IL-1) appears to be the sole cytokine capable of triggering astrocyte iNOS mRNA expression and NO production, and this effect of IL-1 is potentiated by other cytokines, such as interferon-gamma (IFN-gamma ) and tumor necrosis factor-alpha (TNF-alpha ) (Lee et al., 1993; Chao et al., 1996c). In the present study, we investigated the hypothesis that gp41-induced NO production by glial cells would require a direct activation of microglial cells by the viral protein with production of IL-1, which in turn would trigger astrocyte iNOS mRNA expression.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. The following reagents were purchased from the indicated sources: antibodies to microglial cell CD68 antigen and astrocyte glial fibrillary acidic protein (GFAP) (Dako, Carpinteria, CA) and oligodendrocyte galactocerebroside (Polysciences, Warrington, PA); anti-digoxigenin-fluorescein antibody and propidium iodide (Boehringer Mannheim, Indianapolis, IN); biotinylated goat-anti rabbit IgG (Novacastra Laboratories, Burlingame, CA); cytokines (IL-1beta and IFN-gamma ), anti-IL-1beta antibodies, and IL-1 receptor antagonist protein (IRAP) (R & D Systems, Minneapolis, MN); anti-NOS2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT); viral envelope protein gp41 (Intracel, Inc., Issaquah, WA); oligo-dT12-18 primer and dNTP mixture (Pharmacia, Piscataway, NJ); reverse transcription (RT) buffers and SuperScript II RNase reverse transcriptase (Life Technologies, Gaithersburg, MD); Taq DNA polymerase (Promega, Madison, WI); and culture reagents, including DMEM, HBSS, protein kinase C (PKC) inhibitor H7, protein-tyrosine kinase (PTK) inhibitor G103, pyrrolidinedithiocarbamate (PDTC), polymyxin B, the iNOS inhibitor NG-monomethyl-L-arginine (NMMA), uridine, fluorodeoxyuridine, penicillin, and streptomycin (Sigma, St. Louis, MO). Cell culture medium containing 10% FBS was used under all experimental conditions.

Glial cell cultures. Human fetal brain tissue was obtained from 16- to 22-week-old aborted fetuses under a protocol approved by the Human Subjects Research Committee at our institution. The procedure for isolating highly enriched primary human fetal microglial cells has been previously described (Chao et al., 1996a). Briefly, brain tissues were dissociated after 30 min trypsinization (0.25%) and plated in 75 cm2 Falcon culture flasks in DMEM containing 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). The medium was replenished 4 d after plating in medium containing 10% FBS only. Microglia were harvested 10-14 d later. Purified microglia were composed of a cell population of which >99% stained with anti-CD68 antibody (a human macrophage marker) and <1% stained with anti-GFAP and anti-galactocerebroside antibodies (astrocyte and oligodendrocyte markers, respectively).

Enriched astrocyte cultures were prepared as previously described with minor modifications (Chao et al., 1996c). In brief, after removing microglia as described above, flasks were incubated with Ca2+- and Mg2+-free HBSS containing 0.125% trypsin for 20 min at 37°C, which was followed by addition of 10% FBS-containing medium. After centrifugation, the cell suspension was seeded into new flasks with medium containing 10% FBS, and this culture medium was changed 24 hr later. This subculture procedure was repeated three times at a weekly interval. Finally, highly enriched (99% positive by anti-GFAP staining) astrocytes were seeded into 96-well plates at a density of 105 cells per well for NO and IL-1 production and into 12-well plates at 2 × 106 cells per well for RNA analysis. At the end of cell culture, in all experiments, >99% of cells remained GFAP-positive, and <1% stained positively with anti-CD68 antibodies.

Experimental protocols. Glial cell cultures were treated with gp41 (20 µM) for 8 hr before harvesting total RNA for evaluating iNOS, glyceraldehyde 3-phosphate dehydrogenase (GADPH), or IL-1beta mRNA expression, for 48 hr for IL-1 bioassay (Chao et al., 1992a), and for 5 d for assaying nitrite levels. The optimal timing for harvesting supernatants or cells for mRNA expression and NO and IL-1 assays has been reported previously (Chao et al., 1996c). The concentration (20 µM) of gp41 selected was based on a previous study demonstrating that lower concentrations of gp41 failed to trigger iNOS mRNA expression in rat glial cell cultures (Adamson et al., 1996). To evaluate the possibility that a soluble factor(s) was released from gp41-treated microglia, microglial cell cultures were first treated with gp41 for 48 hr, and supernatants were transferred to highly enriched astrocyte cultures in the absence or presence of 200 U/ml IFN-gamma for 8 hr for assessing iNOS mRNA expression and for an additional 5 d for assaying nitrite levels. Glial cells were cultured in glass chambers for staining with the indicated reagents. Polymyxin B (20 µg/ml) was used in one experiment to block endotoxin as a potential contaminant in gp41-induced cytokine expression. Polymyxin B had been shown previously to reduce >97% of lipopolysaccharide (LPS)-induced TNF-alpha production by microglia (Peterson et al., 1995b). For studies of signaling transduction pathways, microglial cell cultures were incubated with gp41 in the absence or presence of the inhibitors H7 or G103 (30 µM), as previously described (Chao et al., 1996c). To evaluate the involvement of nuclear factor-kappa B (NF-kappa B) activation in gp41-induced IL-1 production, microglial cells were pretreated with PDTC (30 µM) for 2 hr, followed by extensive washing as previously described (Ehrlich et al., 1998a) and then replacement with gp41 (20 µM) for an additional 48 hr. These concentrations of inhibitors have been shown to effectively block the signaling pathways in other systems (Chao et al., 1996c; Ehrlich et al., 1998a).

RT-PCR analysis. Total RNA was isolated as previously described (Ehrlich et al., 1998b). Reverse transcription of 1 µg of RNA was performed using an oligo-dT12-18 primer. Briefly, 1 µg of RNA was incubated with 1 µl of 0.5 µg/µl oligo-dT12-18 primer for 10 min at 70°C. The RT reaction was performed in a final volume of 20 µl containing 4 µl of 5× first-strand buffer (in mM: 250 Tris-HCl, pH 8.3, 375 KCl, and 15 MgCl2), 2 µl of 0.1 M DTT, 1 µl of dNTP mixture (10 mM dATP, dTTP, dGTP, and dCTP), and 1 µl (200 U) of SuperScript II reverse transcriptase. Control reaction for RT had the SuperScript II reverse transcriptase enzyme omitted. The reaction mixture was incubated at 42°C for 1 hr followed by termination at 95°C for 5 min in a programmable Tempcycler (Coy Corp., Ann Arbor, MI). The cDNA was stored at -80°C before amplification.

Amplification of iNOS, IL-1beta , or GADPH (as a control) cDNA was performed in a final reaction volume of 50 µl consisting of 5 µl of 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0 at 25°C, and 1% Triton X-100), 3 µl of 25 mM MgCl2, 1 µl of dNTP mixture, 2 U of Taq DNA polymerase, 1 µl of each (sense and antisense) primer (from a 25 µM stock), 2 µl of cDNA, and H2O. Control reaction for PCR contained no cDNA. The mixture was subjected to amplification cycles with each cycle as follows: 94°C for 45 sec, 65°C for 45 sec, and 72°C for 90 sec. The amplification for iNOS in highly enriched microglia or astrocytes was 40 cycles. For IL-1beta and GADPH, the amplification cycles were 26 and 22, respectively. A 10 µl aliquot of PCR product was loaded on a 2% agarose gel for electrophoresis, and the amplified DNA fragments were visualized with ethidium bromide stain under ultraviolet light.

The iNOS primer sets were 5'-TCAGAAGCAGAATGTGACCA-3' (sense) and 5'-TACATGCTGGAGCCGAGGCCAAA-3' (antisense). This iNOS primer set was designed as previously described (Chao et al., 1996c). The IL-1beta and GADPH (control) primer sets were obtained from Perkin-Elmer (Foster City, CA) and Stratagene (La Jolla, CA), respectively. The sizes of the DNA fragments for iNOS, IL-1beta , and GADPH were 615, 391, and 600 bp, respectively.

In situ hybridization for iNOS mRNA expression. Microglia or astrocytes were treated with or without gp41 (20 µM) for 36 hr before fixation at room temperature for 30 min in a solution of 4% (w/v) formaldehyde, 5% (v/v) acetic acid, and 0.9% (w/v) NaCl. After washing with PBS, fixed cells were treated with 0.1% pepsin in 0.1N HCl for 1 min to increase permeability and then post-fixed with 1% formaldehyde for 10 min.

For hybridization, the digoxigenin-labeled human iNOS probe (R & D Systems) was denatured at 80°C shortly before use and diluted to the concentration of 5 ng/µl with the hybridization solution containing 60% formamide, 300 mM NaCl, 30 mM sodium citrate, 10 mM EDTA, 25 mM NaH2PO4, pH 7.4, 5% dextran sulfate, and 250 ng/µl sheared salmon sperm DNA. After hybridization at 37°C for 16 hr and washing with 60% formamide, 300 mM NaCl, and 30 mM sodium citrate, cells were incubated with anti-digoxigenin-fluorescein antibody followed by washing with 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 and dehydration in series of ethanol solutions. Finally, cell samples were embedded with DNA counterstain propidium iodide (50 ng/ml) and visualized by fluorescence microscopy. After staining, nuclei appear red-orange and the target iNOS mRNA appears green under fluorescence microscopy.

NO determination. Nitrite levels were measured using the Griess reagent, as previously described (Chao et al., 1996c), as a reflection of NO production. A standard curve was established using nitrite levels in a range between 1 and 125 µM. The Griess reagent consists of equal volumes of 0.1% naphthylethylene diamine dihydrochloride in distilled water and a mixture of 1% sulfanilamide plus 5% H3PO4. After a 10 min reaction at room temperature, a mixture of equal volumes of standard or supernatant samples and Griess reagent was read on a spectrophotometer at 550 nm.

Immunocytochemical staining. Glial cell cultures were stained with primary antibodies (anti-NOS2 and anti-IL-1beta ) followed by secondary antibodies labeled with avidin-biotin reagents as previously described (Ehrlich et al., 1998a). Briefly, glial cells were fixed with 4% paraformaldehyde for 20 min followed by washing with PBS and incubated with 10% normal rabbit or goat serum in PBS for 1 hr. Primary antibody (rabbit anti-human NOS2 at 1:500) in the presence or absence of specific blocking peptide (2 µg) in PBS was added and incubated overnight at 4°C. After washing, biotinylated goat anti-rabbit IgG (1:200 in PBS) was added for 1 hr at room temperature followed by the avidin-biotinylated enzyme complex and 3,3'-diaminobenzidine for color development. Normal rabbit IgG was used as control antibody. Goat anti-human IL-1beta at 1:1000 as primary antibody and goat IgG isotype as control antibody were used, followed by peroxidase conjugate secondary antibody for IL-1beta staining in glial cells.

Statistical analysis. All experiments were repeated at least three times using glial cells that were obtained from different fetal brain tissue specimens. Where appropriate, data were expressed as mean ± SEM of triplicate samples. To compare means of two groups, Student's t test was used. For comparison of means of multiple groups, ANOVA was performed, followed by Scheffe's F test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gp41-induced iNOS mRNA expression

Gp41 in the absence or presence of IFN-gamma did not induce iNOS mRNA expression in highly enriched astrocyte or microglial cell cultures (Fig. 1). However, iNOS mRNA expression was induced in IFN-gamma -treated astrocyte cultures incubated with supernatants from gp41-treated microglial cell cultures (Fig. 1). Results from controls (RT and cDNA omitted) were negative (data not shown). These findings suggest that gp41-treated microglial cell cultures release a soluble factor(s), which is transferable and activates astrocyte iNOS mRNA expression.



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  		Figure 1.   Gp41-induced iNOS mRNA expression. Highly enriched astrocytes (left) or microglia (right) were incubated with medium (lane 1) or medium containing 20 µM gp41 (lane 2), 200 U/ml IFN-gamma (lane 3), or gp41 plus IFN-gamma (lane 4) for 8 hr before harvesting total RNA for assaying iNOS and GAPDH mRNA expression by RT-PCR analysis. In a separate group (middle), astrocyte cultures were incubated with supernatants derived from microglial cell cultures treated as above (lanes 1-4).

Also, in situ hybridization studies provided further support for iNOS mRNA expression at a single-cell level. Astrocytes exposed to supernatants derived from gp41-treated microglial cell cultures expressed iNOS mRNA (green) in perinuclei (Fig. 2B). Neither highly enriched microglia (data not shown) nor astrocytes (Fig. 2A) alone expressed iNOS mRNA in response to gp41.



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  		Figure 2.   In situ hybridization of iNOS expression. Astrocytes were exposed to gp41 (20 µM) (A) or supernatants derived from gp41-treated microglial cell cultures (B) for 36 hr before fixation and hybridization with the iNOS oligonucleotide probe. With this technique, the cell nucleus appears red-orange, and iNOS mRNA, in the perinuclear area, appears green. Data are representative of three separate experiments.

The effect of gp41 on NO production was elucidated further by assaying supernatants derived from glial cell cultures for nitrite levels after 5 d of incubation. Again, gp41 did not have a direct effect on astrocyte or microglial cell NO production (Table 1). Instead, a factor(s) released by gp41-treated microglial cell cultures stimulated astrocyte NO production. Also, gp41-induced NO production was markedly enhanced when IFN-gamma was included in astrocyte cultures (Table 1). These findings strongly support the hypothesis that microglia play a primary role in activating the NO pathway in human astrocytes.


                              
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  		Table 1.   Gp41-induced NO production

Next, the expression of iNOS protein was visualized using antibodies specific to human iNOS protein. Exposure of highly enriched astrocytes (Fig. 3A) to gp41 plus IFN-gamma failed to induce iNOS protein, suggesting a lack of a direct effect on iNOS protein production. Only IFN-gamma -treated astrocytes exposed to supernatants derived from gp41-treated microglial cell cultures stained positively with anti-iNOS antibody (Fig. 3B). When astrocytes were treated with microglial cell supernatants and then stained with iNOS antibodies plus iNOS peptides, no positive staining of iNOS was observed (Fig. 3C), supporting the specificity of iNOS staining. As can be seen, astrocytes exposed to microglial cell supernatants assume a marked morphological alteration similar to that described with IL-1beta (Lee et al., 1993). When microglial cells were treated with IFN-gamma plus gp41 for 48 hr, no evidence of iNOS protein production was found with the same staining technique (data not shown). These data collectively support the notion that gp41 indirectly triggers astrocyte NO production by stimulating a soluble factor(s) from microglial cell cultures.



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  		Figure 3.   Gp41-induced iNOS protein expression. Highly enriched astrocyte cultures were treated with IFN-gamma (200 U/ml) and exposed to either gp41 (20 µM) (A) or supernatants from gp41-treated microglial cell cultures (B, C) for 48 hr. After 5 d cells were then fixed and stained with anti-human NOS2 antibodies alone (A, B) or anti-human NOS2 antibodies plus iNOS peptides (C), demonstrating specificity of the antibody immunoreactivity.

Role of microglial cell IL-1beta  production

Because human astrocytes have been shown to respond only to IL-1 for iNOS mRNA expression and NO production (Lee et al., 1993; Chao et al., 1996c), we investigated the potential involvement of gp41-induced microglial cell IL-1 production in iNOS activation in astrocytes. Treatment of astrocyte cultures with 200 ng/ml IRAP, a dose previously shown to block the effect of IL-1beta on astrocytes (Hu et al., 1995; Liu et al., 1996; Chao et al., 1996c), totally blocked iNOS mRNA expression (Fig. 4) and NO production (Table 2) induced by microglial cells that had been treated with gp41 alone or gp41 plus IFN-gamma . Results from controls (RT and cDNA omitted) were negative (data not shown). This finding supports a pivotal role for gp41-induced microglial cell IL-1 production in the subsequent induction of NO release by astrocytes. Treatment of cell cultures with NMMA (500 µM) suppressed gp41-induced NO production (Table 2). Similar results were obtained when anti-IL-1beta antibodies were used (Table 2), confirming the essential role of IL-1beta in this phenomenon.



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  		Figure 4.   IRAP blockade of gp41-induced iNOS mRNA expression. Astrocyte cultures were incubated with supernatants derived from highly enriched microglial cell cultures incubated with medium (lanes 1, 4) or medium containing 20 µM gp41 (lanes 2, 5) and gp41 plus 100 ng/ml IRAP (lanes 3, 6) in the absence (lanes 1-3) or presence of 200 U/ml IFN-gamma (lanes 4-6) for 8 hr before harvesting RNA for assaying mRNA expression by RT-PCR. Data are representative of three separate experiments.


                              
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  		Table 2.   IRAP and NMMA blockade of gp41-induced NO production

Stimulation of human microglial cell IL-1beta production by gp41

We next evaluated whether gp41 would stimulate IL-1beta production by microglia. Figure 5 reveals that gp41 directly augmented expression of IL-1beta mRNA in microglia but not in astrocytes. Results from controls (RT and cDAN omitted) were negative (data not shown). Furthermore, we measured IL-1 protein levels in microglial cell cultures and found that gp41 stimulated IL-1 production in a dose- and time-dependent manner (Fig. 6). To test whether gp41-induced IL-1beta expression was attributable to potential endotoxin contamination of the gp41 preparation, polymyxin B was used. Treatment of microglial cell cultures with polymyxin B (20 µM) blocked LPS (100 ng/ml)-induced IL-1beta production by 75% (LPS, 32.85 ± 1.69 pg of IL-1/ml vs LPS and polymyxin B, 8.34 ± 0.73 pg of IL-1/ml; n = 3) but had little effect on gp41 (20 µM)-induced IL-1beta production (control, 0.08 ± 0.02 pg of IL-1/ml; gp41, 26.4 ± 0.9 pg of IL-1/ml; gp41 and polymyxin B, 24.9 ± 1.9 pg of IL-1/ml), suggesting that gp41-induced IL-1beta production is not caused by endotoxin contamination. By immunocytochemical staining, the stimulatory effect of gp41 on IL-1beta production was observed in microglia (Fig. 7B) but not in control microglia (Fig. 7A) or gp41-stimulated astrocytes (Fig. 7C).



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  		Figure 5.   Gp41-induced IL-1beta mRNA expression. Highly enriched astrocyte or microglial cell cultures were treated with medium (lanes 1, 3) or 20 µM gp41 (lanes 2, 4) for 8 hr before harvesting RNA for assaying IL-1beta and GADPH mRNA expression. Data are representative of three separate experiments.



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  		Figure 6.   Gp41 induction of IL-1 production. Microglial cell cultures were incubated with 20 µM gp41 (A) for the indicated periods or with the indicated concentrations of gp41 (B) for 48 hr before harvesting supernatants for IL-1 assay. Data are mean ± SEM of triplicates and are representative of three separate experiments.



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  		Figure 7.   Gp41-induced IL-1beta protein. Control microglia (A), gp41 (20 µM)-treated microglia (B), or gp41-treated astrocytes (B) for 48 hr were fixed and stained with antibodies specific to human IL-1beta . Data are representative of three separate experiments.

We next delineated the signaling pathways underlying gp41-induced microglial cell IL-1beta production using inhibitors of PKC (H-7), PTK (G103), or NF-kappa B activation (PDTC). Gp41-induced IL-1beta production was markedly blocked by G103 (~80%) and to a lesser extent (~20%) by H7 or PDTC (Fig. 8), suggesting that postreceptor binding of gp41 involves predominantly activation of a PTK signaling pathway.



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  		Figure 8.   Signal transduction pathways involved in gp41-induced IL-1beta production. Microglial cell cultures were incubated with medium or medium containing 30 µM H7, G103, or PDTC followed by extensive washing and stimulation with 20 µM gp41 for 48 hr before assaying for IL-1. Unstimulated cells produced a nondetectable amount of IL-1. Data are mean ± SEM of triplicates. **p < 0.01 versus gp41 group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates, for the first time, that the HIV-1 envelope protein gp41 triggers expression of IL-1beta in human microglia but not in astrocytes. Although gp41 failed to activate iNOS directly in astrocytes, the IL-1beta produced by gp41-stimulated microglial cells did upregulate astrocyte iNOS mRNA expression and NO production. The finding that IRAP and anti-IL-1beta antibodies abolished the iNOS mRNA expression and NO production by astrocytes that had been treated with supernatant from gp41-stimulated microglia supports a primary role of microglial cell release of IL-1beta in NO production by astrocytes. It should be pointed out that our studies were performed exclusively with glial cells obtained from fetal brain tissue. Fetal glial cells appear to be functionally similar to adult glial cells (Liu et al., 1998; Zhao et al., 1998). Nonetheless, HIV-1 encephalopathy occurs predominantly in children and adults. Thus, it would be of interest to extend these studies to glial cells of adult origin.

The potential role of gp41 in HIV-1 encephalopathy has been proposed recently on finding that gp41 is capable of inducing the neurotoxin NO in mixed rodent glial and neuronal cell cultures (Adamson et al., 1996). Results of previous studies with a human glial cell culture system led other investigators to suggest that release of NO from astrocytes in response to microglial cell IL-1 production could be involved in HIV-1 encephalopathy (Lee et al., 1995). Autopsy evidence of brains from patients with AIDS dementia supports the hypothesis that NO production in the brain is related to viral envelope gp41 but not to gp120 (Adamson et al., 1996). It also has been noted that gp41-induced neurotoxicity in the rodent brain culture system requires the presence of glia (Adamson et al., 1996), suggesting that glia are the source of neurotoxins, although the particular glial cell type (i.e., microglia vs astrocytes) was not identified. Cytokine-induced NO has been shown to be neurotoxic in primary animal (Boje and Arora, 1992; Chao et al., 1992b) and human (Chao et al., 1996c) neuronal cell cultures. Although the precise mechanism whereby NO damages neurons remains to be determined, a common pathway involving activation of NMDA receptors has been proposed in neurodegenerative diseases, such as AIDS dementia (Chao et al., 1996b; Lipton, 1996), and this hypothesis has been supported by in vitro studies using human brain cell cultures (Chao et al., 1995).

Animal species differences in the cellular source of NO within the brain have been reported. For example, human microglial cells cocultured with human neurons are not as toxic as murine microglia when stimulated with lipopolysaccharide and IFN-gamma , potent inducers of iNOS in murine but not in human microglia (Peterson et al., 1994). Human mononuclear phagocytes appear to have the potential to express iNOS mRNA under certain circumstances (Nathan, 1997); however, only a low output of NO is detected in primary human microglial cell (Peterson et al., 1994; Koka et al., 1995a) and HIV-1-infected monocyte (Bukrinsky et al., 1995) cultures. Experimentally, it appears that high-output NO is necessary to elicit neuronal damage.

Gp41 has previously been found to trigger NO production in mixed human glial cell cultures (Koka et al., 1995a). In human brain cell cultures, astrocytes appear to be the main source of inducible NO (Lee et al., 1993), and the results of the present study suggest that astrocytes are the source of NO in mixed glial cell preparations treated with gp41. However, we clearly demonstrated that human astrocytes are not the direct target of gp41. Instead, an indirect mechanism was discovered involving gp41-elicited microglial cell production of IL-1beta (the sole cytokine currently known to trigger iNOS mRNA expression in human astrocytes). In the presence of IFN-gamma or TNF-alpha , a high output of the neurotoxic free radical NO by astrocytes is attainable (Chao et al., 1996c; Hu et al., 1997).

Although IFN-gamma is not required for iNOS expression in astrocytes, this cytokine is known to potentiate the effect of IL-1beta (Lee et al., 1993), as was seen in the present study. There is little evidence that IFN-gamma is produced in the brain itself; however, in certain infections of the CNS, such as HIV encephalitis (Griffin et al., 1991; Tyor et al., 1992), this cytokine may be present.

The finding that gp41 induces microglial cell but not astrocyte IL-1beta mRNA expression, as determined by RT-PCR and in situ hybridization analyses, suggests that IL-1beta production by microglia is a primary event in gp41-induced NO production and subsequent neurotoxicity. It has been found that gp41 induces IL-1 production in rat glial cells (Koka et al., 1995b) and human peripheral blood mononuclear cell (Tyring et al., 1991) cultures. Gp41 also has been shown to upregulate IL-6 and IL-10 mRNA expression and protein production in monocyte but not in lymphocyte cultures (Takeshita et al., 1995; Koutsonikolis et al., 1997; Barcova et al., 1998), suggesting that binding sites (receptors) for gp41 exist in human mononuclear phagocytes. From flow cytometry analysis, gp41-binding sites appear to be constitutively expressed in both lymphocytes and monocytes (Chen et al., 1993). To further investigate whether gp41-induced IL-1beta production is a receptor-mediated event, we evaluated the potential involvement of several signaling transduction pathways in human microglia stimulated by gp41. Our study supports an intracellular signaling cascade involving PTK in gp41-induced IL-1beta production after binding of the viral protein to microglial cell membrane receptors. Additional studies are necessary, however, to characterize gp41 receptors on microglia.

In summary, our data support the notion that gp41 is potentially neurotoxic via induction of NO production in the brain. Because there could be an age-related effect on the activity of gp41, it would be of interest to evaluate the response of glial cells obtained from adult human brain specimens. Our findings demonstrate that upregulation of microglial cell IL-1beta production is a primary event in gp41-induced iNOS mRNA expression and stimulation of high-output NO production by astrocytes. The signaling pathway associated with gp41-induced microglial cell IL-1beta production involves PTK. Thus, therapeutic maneuvers aimed at minimizing gp41-induced NO production could target microglial cell IL-1beta production as well as astrocyte iNOS activation. Although such an approach could be beneficial in terms of protecting neurons from the toxic properties of NO, it must be remembered that this free radical also has beneficial antimicrobial properties (Nathan, 1997), and thus this approach could interfere with astrocyte defense against opportunistic CNS pathogens (Peterson et al., 1995a).


    FOOTNOTES

Received Sept. 28, 1998; revised April 15, 1999; accepted May 13, 1999.

This study was supported in part by United States Public Health Service Grants DA09924, DA04381, and T32-DA07239 from the National Institute on Drug Abuse. We are grateful to Dr. Fred Kravitz for invaluable technical assistance.

Correspondence should be addressed to Dr. Shuxian Hu, Minneapolis Medical Research Foundation, 914 South Eighth Street, D3, Minneapolis, MN 55404.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Copyright © 1999 Society for Neuroscience  0270-6474/99/19156468-07$05.00/0


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