Transient Exposure to HIV-1 Tat Protein Results in Cytokine Production in Macrophages and Astrocytes

Abstract

The pathological correlates of dementia due to human immunodeficiency virus (HIV) infection are glial cell activation and cytokine dysregulation. These findings occur in the setting of small numbers of productively infected cells within the brain. We determined whether exposure of susceptible cells to Tat protein of HIV could result in the production of select proinflammatory cytokines. In a dose-responsive manner, Tat induced interleukin (IL)-1β production in monocytic cells, while astrocytic cells showed an increase in mRNA for IL-1β, but had a translation block for IL-1β protein production. Conversely, IL-6 protein and mRNA productions were strongly induced in astrocytic cells and minimally in monocytic cells. IL-1β and IL-6 production were independent of tumor necrosis factor-α production. An exposure to Tat for a few minutes was sufficient for sustained releases of cytokines for several hours. This prolonged cytokine production is likely maintained by a positive feed back loop of Tat-induced nuclear factor κB activation and cytokine production that is independent of extracellular calcium. Thus a transient exposure may be sufficient to initiate a cascade of events resulting in cerebral dysfunction and a “hit and run” approach may be in effect. Hence cross-sectional measurement of viral load in the brain may not be a useful indicator of the role of viral products in the neuropathogenesis of HIV dementia. The pathological correlates of dementia due to human immunodeficiency virus (HIV) infection are glial cell activation and cytokine dysregulation. These findings occur in the setting of small numbers of productively infected cells within the brain. We determined whether exposure of susceptible cells to Tat protein of HIV could result in the production of select proinflammatory cytokines. In a dose-responsive manner, Tat induced interleukin (IL)-1β production in monocytic cells, while astrocytic cells showed an increase in mRNA for IL-1β, but had a translation block for IL-1β protein production. Conversely, IL-6 protein and mRNA productions were strongly induced in astrocytic cells and minimally in monocytic cells. IL-1β and IL-6 production were independent of tumor necrosis factor-α production. An exposure to Tat for a few minutes was sufficient for sustained releases of cytokines for several hours. This prolonged cytokine production is likely maintained by a positive feed back loop of Tat-induced nuclear factor κB activation and cytokine production that is independent of extracellular calcium. Thus a transient exposure may be sufficient to initiate a cascade of events resulting in cerebral dysfunction and a “hit and run” approach may be in effect. Hence cross-sectional measurement of viral load in the brain may not be a useful indicator of the role of viral products in the neuropathogenesis of HIV dementia. The pathogenesis of dementia associated with HIV 1The abbreviations used are: HIV, human immunodeficiency virus; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcriptase; PCR, polymerase chain reaction; IL, interleukin; TLCK, N-α-tosyl-l-lysine chloromethyl ketone; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; NFκB, nuclear factor κB infection involves the complex interactions of viral products and cytokines, which eventually result in neuronal dysfunction and cell loss. Several studies have shown that viral proteins such as Tat and gp120 can induce cytokine dysregulation in macrophages and glial cells as well as cause neurotoxicity (1Nath A. Geiger J.D. Prog. Neurobiol. 1998; 54: 19-33Crossref PubMed Scopus (215) Google Scholar). However, it remains uncertain as to why infection of the brain is limited in comparison with the severity of the clinical presentation. In fact, macrophage infiltration, glial cell activation and not viral load in the brain seem to correlate best with the severity of dementia (2Glass J.D. Wesselingh S.L. Selnes O.A. McArthur J.C. Neurology. 1993; 43: 2230-2237Crossref PubMed Google Scholar, 3Johnson R.T. Glass J.D. McArthur J.C. Chesebro B.W. Ann. Neurol. 1996; 39: 392-395Crossref PubMed Scopus (106) Google Scholar). In keeping with these observations cytokine levels in brain and cerebrospinal fluid are elevated in patients with HIV dementia (2Glass J.D. Wesselingh S.L. Selnes O.A. McArthur J.C. Neurology. 1993; 43: 2230-2237Crossref PubMed Google Scholar, 4Merrill J.E. Chen I.S.Y. FASEB J. 1991; 5: 2391-2397Crossref PubMed Scopus (339) Google Scholar). The Tat protein of HIV is of particular interest, since it is released extracellularly from unruptured, HIV-infected lymphoid and microglial cells (5Ensoli B. Buonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R. Wingfield P. Gallo R. J. Virol. 1993; 67: 277-287Crossref PubMed Google Scholar, 6Tardieu M. Hery C. Peudenier S. Boespflug O. Montagnier L. Ann. Neurol. 1992; 32: 11-17Crossref PubMed Scopus (137) Google Scholar). Tat exits from cells via a leaderless secretory pathway in the absence of permeability changes (7Chang H.C. Samaniego F. Nair B.C. Buonaguro L. Ensoli B. AIDS. 1997; 11: 1421-1431Crossref PubMed Scopus (393) Google Scholar). This protein thus has the opportunity to interact with other uninfected cells. Further Tat can be detected in mononuclear cells within the brain of patients with HIV encephalitis (8Hofman F.M. Dohadwala M.M. Wright A.D. Hinton D.R. Walker S.M. J. Neuroimmunol. 1994; 54: 19-28Abstract Full Text PDF PubMed Scopus (92) Google Scholar) and in the sera of HIV-infected individuals (9Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (916) Google Scholar). Tat-mRNA levels are also elevated in the brains of patients with HIV dementia (10Wesselingh S.L. Power C. Glass J.D. Tyor W.R. McArthur J.C. Farber J.M. Griffin J.W. Griffin D.E. Ann. Neurol. 1993; 33: 576-582Crossref PubMed Scopus (403) Google Scholar, 11Wiley C.A. Baldwin M. Achim C.L. AIDS. 1996; 10: 943-947Crossref Scopus (119) Google Scholar). It has been shown previously that the Tat protein of HIV can induce macrophage infiltration in the brain (12Jones M. Olafson K. Del Bigio M.R. Peeling J. Nath A. J. Neuropathol. Exp. Neurol. 1998; 57: 563-570Crossref PubMed Scopus (143) Google Scholar) likely via production of monocyte chemoattractant protein-1 by astrocytes (13Conant K. Garzino-Demo A. Nath A. McArthur J.C. Halliday W. Power C. Gallo R.C. Major E.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3117-3121Crossref PubMed Scopus (506) Google Scholar). Tat also causes glial cell activation for several days post intracerebroventricular inoculation (12Jones M. Olafson K. Del Bigio M.R. Peeling J. Nath A. J. Neuropathol. Exp. Neurol. 1998; 57: 563-570Crossref PubMed Scopus (143) Google Scholar). Furthermore, a Tat-derived peptide when injected into the brain causes cytokine dysregulation (14Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-529Crossref PubMed Scopus (133) Google Scholar) similar to that observed in patients with HIV infection (10Wesselingh S.L. Power C. Glass J.D. Tyor W.R. McArthur J.C. Farber J.M. Griffin J.W. Griffin D.E. Ann. Neurol. 1993; 33: 576-582Crossref PubMed Scopus (403) Google Scholar, 15Vitkovic L. Geretta W.P. Major E.O. Fauci A.S. AIDS Res. Hum. Retroviruses. 1991; 7: 723-727Crossref PubMed Scopus (33) Google Scholar). In this study, we examine the possibility that a transient exposure of cells to Tat may be sufficient to induce a sustained production of cytokines and we determine the role of extracellular calcium and autoregulation in cytokine production. Peripheral blood monocytes were isolated by a Percoll gradient technique, and human fetal astrocyte cultures were prepared by differential adhesion as described previously (16Furer M. Hartloper V. Wilkins J. Nath A. Cell Adhes. Commun. 1993; 1: 223-237Crossref PubMed Scopus (21) Google Scholar). The human astrocytoma cell line U373 and monocytic cell line THP-1 were obtained from American Type Culture Collection (Rockville, MD). Human astrocytes and U373 cells were maintained in minimal essential medium with heat-inactivated 10% (v/v) fetal bovine serum and 1 mm sodium pyruvate. Monocytes were cultured in RPMI with 10% fetal bovine serum. All cell types were supplemented with 100 μg of streptomycin/ml and 0.25 μg amphotericin/ml. THP-1 cells were cultured in RPMI medium with 10% fetal bovine serum and 5.5 μm β-mercaptoethanol. The cells were cultured to approximately 95% confluence in 24-well plates (≈1 × 105 cells/ml). Highly purified (>95%) recombinant Tat protein was prepared from the tat gene encoding the first 72 amino acids as outlined previously (17Ma M. Nath A. J. Virol. 1997; 71: 2495-2499Crossref PubMed Google Scholar). The functional properties of this protein were confirmed using a transactivation assay in HL3T1 cells containing the HIV-1 long terminal repeat, chloramphenicol acetyltransferase construct (17Ma M. Nath A. J. Virol. 1997; 71: 2495-2499Crossref PubMed Google Scholar). For experiments designed to determine the effect of a transient exposure to Tat, cells were treated with 100 ng/ml Tat for either 5, 30, or 60 min, following which the cells were washed five times and then reincubated in culture media without Tat protein. In each case, culture supernatants were collected at 30, 90, and 180 min following reincubation without Tat and analyzed for the presence of IL-1β, IL-6, and TNF-α. For experiments designed to determine the role of TNF-α in IL-1 and IL-6 production, the monocytes and astrocytes were preincubated with antisera to TNF-α (0.5 μg/ml; R&D Systems) for 30 min followed by incubation with 100 ng/ml Tat for either 5, 30, or 60 min. The cells were then washed and reincubated with culture media containing antisera to TNF-α. Culture supernatants were then analyzed after another 90 and 180 min for the presence of IL-1, IL-6, or TNF-α. Cells from two different donors were analyzed in triplicates. Data from a representative experiment are shown. To establish dose profiles for each cell type, Tat was used at 0, 10, 100, and 1000 ng/ml concentrations for 4 h with THP-1 cells for IL-1β and IL-6 mRNA detection in THP-1 cells and for 6 h with U373 cells. IL-1β and IL-6 proteins were analyzed in culture supernatants, 16 h post-Tat treatment. Since IL-1β could not be detected in cell culture supernatants of U373 cells, cell extracts of U373 cells were also measured at 1, 3, 6, 12, 24, and 48 h following treatment with 1 μg/ml Tat for IL-1β by ELISA. Cells were stimulated with lipopolysaccharide from Escherichia colitype 055:B5 (Sigma) 1.0 μg/ml as positive controls. Negative controls included mock (PBS)-treated cells and cells that were treated with solutions from which Tat had been immunoadsorbed as described previously (18Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Cell culture supernatants were analyzed for IL-1β, IL-6, or TNF-α. Additionally, cell extracts of U373 cells were analyzed for IL-1β. In each case, ELISA kits were used from R&D systems, and the procedure was followed as per the manufacturer's instructions. Briefly, 200 μl of standard or sample was added to each well, which had been precoated with a murine monoclonal antibody (2 μg/ml) against the appropriate cytokine. Following a 2-h incubation with the test sample and three washes, 200 μl of a rabbit polyclonal antibody (diluted 1:1000) directed against the appropriate cytokine was added. One hour later, the plates were washed, color developed, and analyzed using a microtiter plate reader. A standard curve was generated on each microtiter plate, which was used for quantitating the amount of cytokine in each sample. The sensitivity of detection for IL-1β was 4 pg/ml, while that of IL-6 and TNF-α was 5 pg/ml. First strand cDNA was prepared from total cellular RNA as per the manufacturer's protocol (Amersham Pharmacia Biotech). PCR was conducted using published primers for IL-1β, IL-6, and β-actin (19Yamamura M. Uyemura K. Deans R.J. Weinberg K. Rea T.H. Bloom B.R. Modlin R.L. Science. 1991; 254: 277-279Crossref PubMed Scopus (1065) Google Scholar). β-Actin primers served as internal controls in each reaction. PCR products were resolved in a 1.5% agarose gel and transferred to a nylon membrane and probed with [32P]ATP end-labeled oligonucleotide probes. IL-β, IL-6, and β-actin oligonucleotide probes were designed based on products amplified using the above primers (IL-1β, 5′-CTG CAC GCT CCG GGA CTC ACA CCA)-3′; IL-6, 5′AAT CGG GTA CAT CCT CGA CGG CAT CT-3′; β-actin, 5′-GAG ACC TTC AAC ACC CCA GCC ATG T-3′). U373 or THP-1 cells were washed in PBS, treated with EGTA (0.5 mm) or BAPTA (200 μm) for 10 min, followed by incubation with 100 ng/ml Tat in a calcium-free buffer for 1 h. Alternatively, the cells were pretreated with 100 μm TLCK (Sigma) for 30 min followed by treatment with 100 ng/ml Tat for 6 h in serum-containing medium. mRNA expression in each case was analyzed by RT-PCR and Southern blot analysis. To determine the effect of a transient exposure of Tat on cytokine production, we treated monocytes and astrocytes with concentrations of Tat (100 ng/ml) previously shown to induce cytokine production in these cells (18Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 20Rautonen N. Rautonen J. Martin N.L. Wara D.W. AIDS. 1994; 8: 1504-1506Crossref PubMed Scopus (24) Google Scholar, 21New D.R. Maggirwar S.B. Epstein L.G. Dewhurst S. Gelbard H.A. J. Biol. Chem. 1998; 273: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 22Lafrenie R.M. Wahl L.M. Epstein J.S. Yamada K.M. Dhawan S. J. Immunol. 1997; 159: 4077-4083PubMed Google Scholar). We found that a 5-min exposure to Tat was sufficient to induce maximal amounts of proinflammatory cytokines IL-1β, IL-6, or TNF-α in the monocytes (Fig.1, A–C) and IL-6 in the astrocytes (Fig. 1 D). IL-1β or TNF-α could not be detected in the astrocyte culture supernatants at all time points tested. To determine whether the IL-1β or IL-6 production in these cells was regulated via TNF-α, we analyzed the ability of TNF antisera to inhibit IL-1β or IL-6 production. IL-1β and IL-6 induction by Tat was independent of TNF-α production (Fig. 1,A, B, and D). Even though we used highly purified cultures of monocytes and astrocytes, we could not exclude the possibility of small amounts of other contaminating cell types in these cultures. Hence we used a human monocytoid (THP-1) and astrocytic (U373) cells for further experiments. We have previously characterized Tat-induced TNF-α in these cells (18Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Hence, to determine the effect of Tat on the production of cytokines IL-1β and IL-6, we treated THP-1 and U373 cells with Tat and measured IL-1β and IL-6 in the culture supernatants. Increasing levels of IL-1β were produced by THP-1 cells in a dose-dependent manner. Significant increases were noted with concentrations of 100 ng/ml Tat (Fig.2 A). To determine whether the induction of IL-1β occurred at the level of transcription or translation, we estimated IL-1β mRNA levels in THP-1 cells by RT-PCR. Tat induced IL-1β mRNA expression in a dose-dependent manner. The RT-PCR was much more sensitive and was able to detect IL-1β mRNA induction with 10 ng/ml Tat (Fig. 2 D). The U373 cells did not produce detectable levels of IL-1β in the culture supernatants, even with 1 μg/ml Tat. Since stimuli such as IL-1α and IL-2 result in production of cell associated IL-1β (23Dinarello C.A. Immunol. Lett. 1987; 16: 227-231Crossref PubMed Scopus (110) Google Scholar,24Numerof R.P. Kotik A.N. Dinarello C.A. Mier J.W. Cell Immunol. 1990; 130: PubMed Scopus Google we cell extracts for the presence of IL-1β but were to detect IL-1β protein not To determine whether the block in IL-1β production was at the level of transcription or translation, we measured mRNA levels in Tat treated U373 cells. IL-1β mRNA levels in U373 cells were with that of THP-1 cells (Fig. Hence was a translation block in the U373 cells. Significant amounts of IL-6 were produced in the culture supernatants of THP-1 cells and the U373 cells (Fig. the U373 cells produced more IL-6 the THP-1 cells at 100 ng/ml and 1 μg/ml concentrations of Tat. IL-6 mRNA production the production of IL-6 protein in U373 cells (Fig. and THP-1 cells not We conducted experiments to determine whether a transient exposure to Tat be sufficient to induce cytokine production in U373 and THP-1 cells. A 5-min incubation was for TNF-α or IL-1β in THP-1 cells, but was sufficient to induce IL-6 production in U373 cells at 180 min post Tat exposure Following a or exposure to Tat, all three cytokines could be A exposure to levels and of cytokines (Fig. We used highly purified Tat protein in all experiments and have previously shown the of Tat (18Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, Geiger J.D. R.M. Nath A. Ann. Neurol. 1995; PubMed Scopus Google Scholar). However, to further determine whether were contaminating that may result in IL-1β or IL-6 production, we immunoadsorbed Tat and used the for the cells. The cell extracts were analyzed for IL-1β and IL-6 mRNA since this was a much more sensitive for detection of Tat as with cytokine detection in culture supernatants. A block in IL-1β and IL-6 mRNA production was noted as shown in the U373 cells (Fig. that Tat are of other studies have shown that Tat activation K. M. Nath A. Major E.O. J. Virol. 1996; PubMed Google Scholar) and that Tat-induced TNF-α production is (18Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Hence, we pretreated the cells with TLCK, an of activation followed by incubation with Tat. A block in IL-1β and IL-6 production was noted in the U373 cells A and and the THP-1 cells not We the role of extracellular calcium in Tat-induced cytokine production. EGTA BAPTA was able to block cytokine production. the effect on IL-1β production in U373 cells. A similar of was for IL-6 production in U373 cells and IL-1β and IL-6 production in THP-1 cells not studies that cytokine expression in monocytes and astrocytes are regulated by Tat. monocytes could be induced to produce all three cytokines, IL-1β, IL-6, and TNF-α, astrocytes produced The levels of IL-6 produced by astrocytic cells were produced by monocytic cells. The astrocytic cells did not produce amounts of IL-1β, even though mRNA for IL-1β could be we have shown previously that Tat could induce small amounts of TNF-α in the astrocytic cells (18Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Furthermore, we were to inhibit the production of IL-6 with antisera to TNF-α in astrocytes or This is an since IL-1β and TNF-α have been shown to induce IL-6 gene expression in astrocytes L. S.M. J. Immunol. 1994; Google Scholar). This that the effect of Tat on IL-6 production is the IL-1β is produced by D.W. W. J. Neuroimmunol. 1993; Full Text PDF PubMed Scopus Google Scholar). HIV-infected macrophages also levels of IL-1β P. M. Gelbard H.A. K. L. Epstein L. Gendelman H. J. Exp. 1992; PubMed Scopus Google Scholar). studies that Tat protein at in to the of IL-1β levels in the brain of patients with HIV dementia. Tat-induced IL-1β is independent of TNF-α production. The increase in IL-1β may induce A. Tyor W.R. Glass J.D. L. Res. 1993; PubMed Scopus Google HIV-1 G. Fauci A.S. 1992; PubMed Scopus Google and induce other cytokines such as TNF-α D.W. W. J. Neuroimmunol. 1993; Full Text PDF PubMed Scopus Google resulting in further brain astrocytic cells not produced amounts of IL-6, but the elevated levels were for prolonged Several studies have shown that astrocytes are an of IL-6 S.M. J. Neuroimmunol. 1990; Full Text PDF PubMed Scopus Google Scholar). IL-6 has on the which activation of the and neuronal S. Immunol. 1992; Scholar). Furthermore, IL-6 has been in neuronal with IL-6 with and P. J.D. L. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). IL-6 has also been in pathogenesis of neuronal in M. K. S. B. J. Neurobiol. 1996; PubMed Scopus Google Scholar). Tat has been shown to induce changes in calcium in an of extracellular calcium Nath A. Geiger J.D. J. 1998; Scholar). We the role of extracellular calcium in Tat-induced cytokine production. of extracellular calcium had effect on cytokine production. These observations are since a showed that calcium not the of HIV dementia U. D. McArthur J.C. C. L. Neurology. 1998; PubMed Scopus Google Scholar). studies have shown activation in the brains of patients with HIV infection Epstein L.G. Gelbard H.A. Dewhurst S. Neuropathol. Neurobiol. 1995; PubMed Scopus Google Scholar). Further Tat activation in glial cells K. M. Nath A. Major E.O. J. Virol. 1996; PubMed Google Scholar). We that Tat-induced cytokine production is likely the cytokines have also been shown to J.W. D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Thus cytokine production by Tat could result in a positive loop and cytokine production. This may to an of cytokine production without the presence of Tat. in this we that exposure to Tat for even a few minutes is sufficient to induce cytokine production in monocytes and astrocytes for prolonged of These findings are with observations following a of Tat in glial activation and macrophage infiltration could be for several even though Tat could not be detected in the brain after a few (12Jones M. Olafson K. Del Bigio M.R. Peeling J. Nath A. J. Neuropathol. Exp. Neurol. 1998; 57: 563-570Crossref PubMed Scopus (143) Google Scholar). Furthermore, an exposure of Tat in the of is sufficient to induce prolonged in Geiger J.D. R.M. Nath A. Ann. Neurol. 1995; PubMed Scopus Google Scholar, J. Nath A. B. S. Geiger J.D. M. 1998; PubMed Scopus Google Scholar). these studies that a transient exposure to protein in a cascade of events to glial cell activation and neuronal We thus that a “hit and run” may be in neuropathogenesis of HIV which may also why cross-sectional of viral load in the brain at not correlate with neuronal and dementia. We and for and Geiger and for