HIV-1 can invade the central nervous system early after infection and is detected in more than 80% of brains from individuals with AIDS [1–3]. Among HIV-1-infected children and adults, 20–30% develop neurological manifestations known as AIDS dementia complex during the course of their illness.
The transmembrane glycoprotein gp41 of HIV is known to play an important role in HIV pathogenesis and several biological and immunological functions are located in its extracellular domain [4,5]. Recent results implicate a role for gp41 in the altered cytokine expression pattern in serum which is related to HIV-induced immune dysfunction. Gp41 up-regulates the synthesis of interleukin (IL)-10 in human peripheral blood mononuclear cells (PBMC) and monocytes derived thereof while down-modulating the production of pro-inflammatory IL-2 and interferon-γ (IFN-γ) . Further experiments showed that cAMP increase and the p70S6 kinase activation are important steps in gp41-induced signal cascade leading to induction of IL-10 synthesis .
The role of gp41 in the development of AIDS dementia complex is under investigation. Gp41 is readily detected in brains of HIV-infected adult and paediatric patients [8–11]. It is reported to exert numerous effects on brain cells including induction of nitric oxide synthesis, inhibition of excitatory amino acid transport and up-regulation of the cytokines tumour necrosis factor-α and IL-1 [12–16]. Nothing is known about the role of gp41 in the induction of other cytokines such as IL-10 in the brain. Because IL-10 might alter the activity of circulating immune cells in the brain as well as brain cell function directly this question seemed to be of special importance.
HIV-1 entry into cells is dependent on the presence of cellular chemokine receptors which have been identified as coreceptors for the virus. CCR5, the main coreceptor on monocytes/macrophages is known to be also a coreceptor for HIV-infection of microglia . Microglia also express CXCR4, the main coreceptor for T-tropic HIV strains, but its usage is relatively inefficient . Astrocytes express both CCR5 and CXCR4 whereas on neurones high levels of CXCR4 and moderate levels of CCR5 are found [19–21].
Cellular expression of the chemokine receptors CXCR4 and CCR5 is not only essential for infectivity by HIV-1, the molecules are also involved in HIV-induced apoptosis [22,23]. In addition, their ligands SDF-1, MIP-1α, MIP-1β and RANTES behave as potent chemotactic factors for a variety of cell types thereby contributing to inflammation and antimicrobial response. Thus, chemokine receptor expression is a critical parameter for numerous processes. Synthesis of chemokine receptors like CXCR4 and CCR5 on monocytes is controlled by various cytokines including IL-10, IL-2 and IFN-γ[24–29], but the results are partly controversial.
In the present study we measured the production of IL-10 in different brain cell types after treatment with gp41. HIV gp41 was shown to markedly induce IL-10 synthesis in astrocytes and neurones in a time- and dose-dependent manner, to a similar degree as shown previously in monocytes. This up-regulation of IL-10 in astrocytes could be abrogated by specific inhibitors of cAMP synthesis and of p70S6 kinase activation. IL-10, induced by gp41, modulated the expression of the chemokine receptor CCR5 on gp41-treated monocytes, astrocytes and neurones, thus implicating a possible role of gp41 in cell migration and susceptibility to infection.
Materials and methods
Cell lines and monocyte isolation
The human astrocytic cell line U87-MG was purchased from MRC and cultured in Dulbecco's minimal essential medium (Life Technologies, Vienna, Austria) supplemented with 10% foetal calf serum (BioWhittaker, Verviers, Belgium), penicillin/streptomcycin, 0.1 mM non-essential amino acids and 2 mM l-glutamine (Life Technologies). The human neuroblastoma-derived cell line SK-N-SH (ATCC HTB-11) was a gift of from Dr I Blasko (University of Innsbruck, Austria) and was grown in minimal essential medium with the same supplements as described above plus 1 mM sodium pyruvate.
Human PBMC were isolated from buffy-coat blood fractions of healthy, HIV-seronegative donors by density gradient centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden). Monocytes were isolated from PBMC using gelatine/plasma-coated plates as described previously . Briefly, Petri dishes were coated with sterile 2% gelatine (Merck, Darmstadt, Germany) solution for 2 h at 37°C followed by incubation with autologous plasma for 30 min at 37°C. PBMC obtained from Ficoll separation were suspended in RPMI medium (Life Technologies) containing 10% foetal calf serum and 2 mM l-glutamine and loaded onto gelatine/plasma-coated plates at 4 × 106 cells/ml. After 40 min incubation at 37°C peripheral blood lymphocytes (PBL) were washed out with medium and the adherent monocytes were detached by 5 mM EDTA (Sigma, St. Louis, Missouri, USA) and further cultured in complete RPMI medium at 37°C in a humidified 5% CO2 atmosphere. Fluorescence-activated cell sorting (FACS) revealed that the cell population regularly consisted of 96–98% monocytes.
Recombinant HIV gp41 protein
Recombinant (r) fusion protein of maltose binding protein (MBP) with an 82-amino acid-long extracellular region (aa565–647) of gp41 (Intracel, Cambridge, Massachusetts, USA) was produced in an Escherichia coli expression system. To rule out possible side-effects of MBP we used recombinant MBP (New England Biolabs, Beverly, USA) as a negative control. The presence of endotoxin in gp41–MBP and MBP preparations was less than 0.3 EU/μg of gp41–MBP or MBP protein, as measured by limulus amoebocyte lysate assay (Sigma).
Experimental settings and measurement of IL-10 by ELISA
Astrocytes or monocytes were seeded in 96-well plates at a concentration of 1 × 106 cells/ml. Depending on the experimental setting, cells were pre-incubated for 60 min at 37°C with the signal transduction inhibitors SQ 22536 or rapamycin (Calbiochem, La Jolla, California, USA), and for 4 h with Bordetella pertussis toxin (Calbiochem) at the concentrations indicated before addition of stimuli. Cells were stimulated with medium, gp41–MBP or MBP at various concentrations. Cell-free culture supernatants were collected at several time points and IL-10 levels were quantified by a cytokine-specific ELISA with matched antibody pairs (R&D Systems, Minneapolis, USA). The assay was performed according to manufacturer's instructions using ELISA plates coated with 4 μg/ml capture antibody in combination with the detection biotinylated antibody at a concentration of 500 ng/ml. An avidin–horseradish peroxidase conjugate (DAKO, Vienna, Austria) was used to detect the biotinylated antibody and was developed with tetramethylbenzidine dihydrochloride (Sigma). The detection sensitivity of the assay was ≤ 10 pg/ml.
FACS of CCR5 and CXCR4 expression
After the appropriate incubation time, cells were pre-treated with 0.5 mM EDTA for 10 min to detach adherent cells and washed twice with phosphate buffered saline/0.5% bovine serum albumin. To measure cell surface expression of the chemokine receptors cells were incubated with non-specific IgG as isotype control, anti-CCR5 or anti-CXCR4 antibodies (Pharmingen, San Diego, California, USA) for 30 min at 4°C, washed again in phosphate buffered saline/bovine serum albumin and then stained with a second FITC-labelled antibody (DAKO). Analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Diego, California, USA). The mean fluorescence intensity was determined by subtracting the mean fluorescence intensity of the isotype control from the mean fluorescence intensity of the CCR5 or CXCR4 signal. For blocking experiments the cells were pre-incubated for 60 min with 10 ng/ml α-IL-10 antibody (clone 23738.11) which is known to neutralize the bioactivity of IL-10; as a control an non-specific IgG was used for pre-incubation. Alternatively the cells were pre-treated for 60 min with the adenylate cyclase inhibitor SQ22536 before addition of the stimulus.
RNA preparation, reverse transcription and PCR
At different time points cells were lysed and RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. For quantification 2 μg of total RNA were reverse transcribed into cDNA using oligodT as a primer and Moloney murine leukaemia virus reverse transcriptase (Life Technologies) in a final volume of 25 μl. PCR was performed in a total volume of 50 μl containing 2 μl cDNA, 15 pmol each primer, 200 μM each deoxynucleotide and 2.5 U Taq polymerase (Promega, Mannheim, Germany) and buffer supplied with the polymerase. PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed with 30 cycles of 30 sec at 94°C, 1 min at 49°C and 2 min at 72°C. For CCR5 PCR the conditions were 1 cycle of 2 min at 95°C, followed by 30 cycles of 1 min at 94°C, 30 sec at 60°C and 2 min at 72°C. These conditions were determined to be optimal for quantitative studies. Half of the PCR reaction was separated by agarose gel electrophoresis and transferred to nylon membrane (BioRad, Munich, Germany) by alkaline capillary blotting. Filters were cross-linked by UV irradiation and hybridized with 32P-labelled probes under stringent conditions according to the method of Church and Gilbert . The radioactive signal obtained after hybridization was quantified using a Phosphoimager (Fuji) and expressed as photosensitive luminiscence.
The following primers were used for PCR: GAPDH, 5′-GTGAAGGTCGGAGTCAACG-3′ and 5′-GG TGAAGACGCCAGTGGACTC-3′; for CCR5, 5′-GAACAAGATGGAT TATCAAGTGTCAAGTCC-3′ and 5′-GTCACAAGCCCACAGATATTTCCTG C-3′. Hybridization probes were generated with the same primers.
Statistical analysis (Student's t test) was performed using the Origin 4.1 software (serial No: 6014906).
Gp41 up-regulates IL-10 production in the astrocytic cell line U87
To mimic the three-dimensional structure of gp41 better than is possible with small peptides we used a fusion construct between the extracellular part of gp41 and maltose binding protein (MBP) which enhanced gp41 solubility (gp41–MBP). For comparison, MBP protein was used to control for any possible side-effects.
Whereas incubation of astrocytes with MBP alone did not alter IL-10 production, addition of gp41–MBP highly up-regulated IL-10 production in U87 cells (Fig. 1a). This effect was maximal after 60 h with > 12 fold increased IL-10 levels in the medium of gp41-treated cells compared with control cells (6.4 ng/ml versus 0.5 ng/ml).
To evaluate the dose-dependence of IL-10 induction in astrocytes U87 cells were incubated with increasing concentrations of gp41–MBP, ranging from 1 nM to 100 nM (Fig. 1b). A low but statistically significant increase (P < 0.05) of IL-10 was observed even at 1 nM after 48 h of incubation. The highest increase was achieved by the addition of 100 nM gp41–MBP. Higher concentrations were not tested due to limited gp41 material. Incubation with control protein MBP did not alter the cytokine expression.
Expression of chemokine receptors on gp41-treated monocytes and astrocytes
Further experiments aimed to study the physiological consequence of IL-10 up-regulation by incubation with gp41 in different cell types. Both monocytes and astrocytes were treated with MBP or gp41–MBP followed by quantification of chemokine receptor expression by FACS.
Expression of the chemokine receptor CCR5 on monocytes is highly up-regulated by incubation with gp41 after 120 h of culture (Fig. 2a). The MBP-treated monocytes showed low expression of CCR5, only slightly above the FACS signal for non-specific antibody binding as measured with isotype control antibody. After incubation with gp41–MBP there was a significant shift of the CCR5 FACS expression signal with an increase of CCR5-positive cells within the gate from 1% to 100%. Depending on the blood donor mean fluorescence intensity increased up to 10 fold. A significant shift was already visible after 90 h with a maximal response after 120 h (data not shown). A similar effect could also be measured on mRNA level (Fig. 2b). During incubation with medium (data not shown) or MBP an increase of CCR5 mRNA level was detected, probably due to differentiation. Incubation of the monocytes with gp41 enhanced CCR5 mRNA expression up to fivefold above the expression in MBP-treated cells. Expression of the chemokine receptor CXCR4 was not altered by incubation with gp41–MBP (Fig. 2c). For both MBP-treated and gp41–MBP-treated monocytes approximately 27% of the cells were positive for CXCR4 within the gate.
Similarly, the expression of CCR5 was significantly enhanced on the astrocytic cell line U87 by incubation with gp41–MBP. The percentage of CCR5-positive cells within the gate increased from 8.6% of the MBP-treated control cells to 85.4% of the cells incubated with 20 nM gp41–MBP for 120 h (data not shown). In contrast with the situation on monocytes, CXCR4 expression was also slightly up-regulated by gp41–MBP (7.6% positive cells within the gate for MBP-treated cells versus 42% after incubation with gp41–MBP).
Evaluation of the gp41-induced signal transduction pathway in astrocytes
As previous results showed that the effect of gp41 on IL-10 production is mediated by cAMP elevation and activation of the serine/threonine kinase p70S6-kinase we investigated the signal transduction pathway in astrocytes. In fact, 50 nM rapamycin, an efficient and specific inhibitor of p70S6-kinase activation by the mTOR (mammalian target of rapamycin) kinase, suppressed gp41-induced IL-10 production in astrocytes to the background level of medium-treated or MBP-treated cells (Fig. 3a). Furthermore the adenylate cyclase inhibitor SQ22536, which had been shown to abolish gp41-induced IL-10 synthesis in monocytes, also abolished the effect of gp41 on U87 astrocytes. These results indicate that, like monocytes, the effect of gp41 is transduced by cAMP elevation and activation of p70S6-kinase.
To investigate whether CCR5 up-regulation is induced directly by gp41 or mediated by gp41-induced IL-10 we performed inhibitor experiments using an α-IL-10 antibody with the ability to neutralize the bioactivity of human IL-10. In addition, we tested the effect of SQ22536.
After stimulation of U87 astrocytes the percentage of CCR5-positive cells within the gate increased from 10.1% for MBP-treated cells to 77% for the cells treated with gp41–MBP (Fig. 3b). The use of both inhibitors abrogated the gp41-induced enhancement of CCR5 surface expression to a level corresponding to that achieved by the addition of MBP. Pre-incubation with α-IL-10 antibody decreased the percentage of CCR5-positive cells after gp41-stimulation to 37%. Similarly, pre-incubation with SQ22536 decreased the percentage of CCR5-positive cells to 34%. The same results were found for monocytes where pre-incubation of the cells with inhibitors prior to stimulation with gp41 reduced the percentage of CCR5-positive cells from 95% to 12% for α-IL-10 antibody and to 45% for the adenylate cyclase inhibitor SQ22536 (data not shown). These inhibitory effects indicate that CCR5 up-regulation by gp41 is mediated by IL-10.
Expression of IL-10 and CCR5 in neurones
To evaluate whether astrocytes are the only cell line in the brain to be sensitive to gp41 we treated the neuronal cell line SK-N-SH with gp41–MBP. Production of IL-10 was monitored by ELISA and surface expression of CCR5 was analysed by FACS.
Incubation of SK-N-SH neuronal cells with gp41–MBP resulted in increased expression and secretion of IL-10 (Fig. 4a). Compared to the medium and the MBP control the level of IL-10 in the medium increased almost threefold. A maximum was reached at 24 h then the amount of IL-10 in the supernatant began to decrease again. In parallel, the surface expression of CCR5 increased significantly (Fig. 4b). Whereas in MBP-treated cells the percentage of CCR5-positive cells within the gate is 19.8% this percentage increases to 94.7% after incubation with gp41–MBP.
The results presented above clearly demonstrate that the HIV transmembrane protein gp41 up-regulated IL-10 production and secretion in astrocytes and neurones. Previous results had shown a similar effect on monocytes, whereas no increase was observed in B cells, T cells or natural killer cells . In contrast to the situation in neurones and monocytes where IL-10 enhancement by gp41 reaches a plateau after 24–28 h, the gp41-induced up-regulation in astrocytes continues at least for 60 h.
Whereas there is good evidence for a relationship between enhanced IL-10 expression in the blood and immunological abnormalities found in HIV-positive patients, nothing is known about the role of gp41-induced IL-10 in the generation of AIDS dementia complex. As a hypothesis, gp41 – by induction of IL-10 in various brain cell types – could modulate activity of brain-circulating monocytes and T cells, thus inhibiting effective host defence against HIV-infection of the brain. From blood it is known that IL-10 inhibits T helper cell response to antigens which can be restored by antibodies against IL-10 [31,32]. Enhanced IL-10 expression has also been related to monocyte/macrophage functional impairment, polyclonal B cell proliferation and hypergammaglobulinaemia [33–35]. It is not only the blood-derived immune cells that have been shown to be regulated in their activity by IL-10. Treatment of brain-resident microglia with IL-10 down-modulated the expression of the costimulatory molecules B7–1 and B7–2 which are essential for the presentation of antigens . Furthermore, IL-10 inhibits microglia proliferation in concentrations that are achieved in our experiments after stimulation by gp41 .
IL-10 could also regulate immune activation by modulating production of pro-inflammatory cytokines both from infiltrating immune cells and from brain cells directly; it is known from studies on blood to inhibit markedly the synthesis of IFN-γ, IL-1, tumour necrosis factor-α, IL-6, IL-8 and IL-12 . Interestingly, production of IL-1 and tumour necrosis factor-α was described to be up-regulated by gp41 in human glial cultures implying that cytokine interaction in the brain differs from that in the blood . A similar co-expression pattern of pro-inflammatory and anti- inflammatory cytokines was also shown for systemic inflammation of the central nervous system . The effect of gp41 on production of pro-inflammatory cytokines needs further elucidation.
Taken together, an effective immune response against the virus and virus-infected cells might be disturbed by the IL-10-mediated action of gp41 on immune cell activation and production of pro-inflammatory cytokines. Therefore up-regulation of IL-10 by gp41 could favour viral spread in the brain. However, IL-10 synthesis might not have only detrimental effects on the brain. Anti-inflammatory IL-10 may also help to prevent the immunopathological effects of an uncontrolled immune response and immune-mediated injury of brain tissue. Furthermore IL-10 induces a dose-dependent increase of nerve growth factor secretion by astrocytes and may thus provide a neurotrophic support to injured neurones . In addition, because IL-10 had been shown to inhibit viral replication in monocytes, gp41 via its effect on IL-10 production of brain cells could contribute to the establishment of viral latency in the brain and suppress extensive virus synthesis. Further experiments are necessary to study viral replication under the influence of gp41 and IL-10 in different brain cells.
Production of IL-10 by astrocytes, neurones and microglial cells has been described previously [41–43] but only few facts are known about its regulation. Our results demonstrate that the signal transduction cascade between cellular binding of gp41 and up-regulation of IL-10 involves generation of cAMP by adenylate cyclase and activation of the serine/threonin kinase p70S6. Previous results showed the same signal transduction cascade for the gp41-induced up-modulation of IL-10 in freshly isolated human monocytes , implicating that gp41 binds to the same receptor in both cell types. The identification of the gp41 surface receptor is presently under investigation. The dose-dependence of IL-10 enhancement on astrocytes indicates that these cells express a rather high level of gp41 receptor on the cell surface as IL-10 up-regulation did not reach a plateau up to a concentration of 100 nM gp41.
The hypothesis that gp41 modulates chemokine receptor synthesis is intriguing as several cytokines and molecules which are altered in their synthesis by gp41 are described to influence chemokine receptor production. Nevertheless, the results are controversial – making it unclear how gp41 might alter chemokine receptor expression. For example IL-10 is described to both up-regulate and down-modulate CCR5 expression [24,25]. On the other hand, IL-2 and IFN-γ which are described to be down-modulated by gp41  can up-regulate CCR5 expression [26,28,29] making it more likely that CCR5 production might decrease after incubation with gp41. In addition cAMP which is highly increased after treatment with gp41  down-regulates CCR5 synthesis on macrophages . Our results show that gp41 enhances the expression of the chemokine receptor and HIV coreceptor CCR5. Thus gp41 could contribute to the increase of CCR5 expression found in vivo. CCR5 synthesis on CD4 T cells from HIV-infected patients is reported to be enhanced in AIDS disease progression . According to our results this effect might not be a direct one but could be mediated by gp41-induced IL-10. This hypothesis is supported by our inhibitor experiments with a neutralizing α-IL-10 antibody and also by the fact that up-regulation of CCR5 (after 90–120 h) is delayed relative to the up-regulation of IL-10 (after 6–12 h). CCR5 up-regulation might be dependent on a threshold level of IL-10 in the medium.
Upregulation of chemokine receptor expression in monocytes and brain cells might have tremendous consequences for virus spreading and cell function. It has been shown that up-regulation of CCR5 on monocytes is associated with enhancement of viral entry and CCR5 levels correlate with infectability by macrophage-tropic HIV-1 [24,46]. Enhanced CCR5 levels might also result in a shift of virus tropism from T-cell tropic to macrophage tropic strains. In addition, a higher expression of chemokine receptors on monocytes and microglia might implicate increased sensitivity to the chemotactic activity of their ligands, thus modulating host defence against pathogens including HIV itself. Furthermore, the CCR5-dependent increase of migration of infected monocytes into organs might contribute to spread of the virus throughout the body. In accordance with that hypothesis the overall frequency of CCR5-positive mononuclear cells was increased in the brains of patients with severe HIV-1 encephalitis compared with non-AIDS control patients . Since SDF-1, the ligand of CXCR4, has been shown to induce apoptosis in neurones and lymphocytes, enhanced chemokine receptor expression could be involved in generation of HIV-induced lesions in the brain [23,48]. This hypothesis is especially intriguing as high levels of chemokine receptors are detected in neocortex and hippocampus, areas of HIV-induced pathology and clinical impairment . Chemokine receptors are also found to be associated with pathological changes in Alzheimer's disease. Further investigation will aim to clarify the role of gp41, gp41-induced IL-10 and modulated chemokine receptor expression in the development of AIDS dementia complex.
The authors thank I. Blasko for providing the SK-N-SH cell line and C. Nolf for technical assistance.
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