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Activation of Human Microglial Cells by HIV-1 gp41 and Tat Proteins
Clinical Immunology Vol. 96, No. 3, September, pp. 243–251, 2000 doi:10.1006/clim.2000.4905, available online at http://www.idealibrary.com on
Activation of Human Microglial Cells by HIV-1 gp41 and Tat Proteins W. S. Sheng, S. Hu, C. C. Hegg,* S. A. Thayer,* and P. K. Peterson Institute for Brain and Immune Disorders, Minneapolis Medical Research Foundation, Department of Medicine, and *Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55404
The viral proteins, Tat (HIV-1 nuclear protein) and gp41 (HIV-1 coat protein), detected in the brains of HIV-1-infected patients have been shown to be neurotoxic. We investigated the effects of HIV-1 Tat and gp41 proteins on cytokine, chemokine, and superoxide anion (O 2ⴚ) production by microglia, the resident macrophages of the brain. Tat and gp41 dose-dependently stimulated cytokine and chemokine production by microglia. Peak production of these cytokines and chemokines differed in microglial cells treated with gp41 and Tat. Expression of cytokine and chemokine mRNA was also stimulated in gp41- and Tat-treated microglia. Neither gp41 nor Tat alone stimulated O 2ⴚ production by microglia. Treatment of microglial cells with Tat but not with gp41 evoked an increase in intracellular Ca 2ⴙ. The results of this study suggest that HIV-1 Tat and gp41 proteins impact several key functions of microglial cells which could contribute to the neuropathogenesis of HIV-1. © 2000 Academic Press Key Words: microglia; gp41; Tat; cytokines; chemokines.
INTRODUCTION
The pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) resulting in neuronal dysfunction and cell loss is incompletely understood. Microglia, the resident macrophages of the brain, are the main brain cell type to be productively infected by HIV-1. Upon activation, microglial cells produce many cytokines, chemokines, and other inflammatory mediators (e.g., reactive oxygen intermediates, [ROI]) (1). Such inflammatory mediators may have neuroprotective or neuropathological effects. For instance, some cytokines and chemokines can inhibit while others can enhance HIV infection of and replication in macrophages, including microglia (2–5). Proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣), interleukin-1 beta (IL-1), and IL-6 have been detected in the CNS of patients with HIV-1 encephalopathy, although no correlation between the levels of these cytokines and the severity of CNS disease has been found (6). Chemokines such as
regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-1 beta (MIP-1), and monocyte chemotactic protein-1 (MCP-1) are capable of directing the migration of lymphocytes and mononuclear phagocytes within infected/inflammatory sites, and these chemokines appear to participate in the host defense against HIV infection (7). However, these same chemokines also have been proposed to play a role in the neuropathogenesis of HIV-1 (8). The HIV-1 proteins Tat and gp41 have been detected in the brains of demented patients at postmortem examination. The levels of these viral proteins appear to correlate with the severity of CNS disease (9, 10). Both Tat and gp41 have been found to be neurotoxic, possibly via indirect mechanisms involving mediators released by activated microglia in the CNS. Tat has been reported to induce neuronal apoptosis mediated by proinflammatory cytokine release and activation of glutamate receptors (11). Gp41, on the other hand, was reported to stimulate inducible nitric oxide synthase (iNOS)-derived NO as a major mediator of neurotoxicity (12). Recently, work in our laboratory has shown that human microglial cells stimulated with gp41 release IL-1, which in turn stimulates astrocyte iNOS expression and NO production (13). Tat and gp41 have been shown by other investigators to alter many functions of monocytes, such as adhesion and chemotaxis (14), receptor expression (15, 16), and cytokine production (17–19). Also, Koka et al. have reported that human mixed glial cell cultures (consisting of microglia and astrocytes) stimulated with gp41 produce TNF-␣ and IL-1 (20); and recently, other investigators have reported that Tat induces chemokine (MIP-1␣, MIP-1, and MCP-1) production in human fetal microglial cell cultures (21). ROI, such as superoxide anion (O 2⫺), hydrogen peroxide, hydroxyl radical, and peroxynitrite, have both microbicidal and pathological properties at sites of infection and inflammation (22, 23). Increased O 2⫺ production has recently been reported in studies of HIV-1-infected macrophages (24). Microglia appear to be a major source of ROI in a variety of brain insults. Because little or nothing is known about the effects of
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gp41 and Tat on the generation of O 2⫺ by microglia and incomplete information is available on the production of cytokines and chemokines by purified human fetal microglial cells in response to gp41 and Tat, the present study was undertaken. MATERIALS AND METHODS
Reagents Cytokines (TNF-␣, IL-1, IL-6), chemokines (RANTES, MIP-1␣), and their antibodies were obtained from R&D Systems, Inc. (Minneapolis, MN). Antibodies to glial fibrillary acidic protein (GFAP) and CD 68 were purchased from DAKO (Carpinteria, CA). Other reagents were from the indicated sources: fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT); gp41 (Intracel Inc., Issaquah, WA); Tat (NIH AIDS Research and Reference Reagent Program, Rockville, MD); oligo (dT) 12–18 primer and dNTP mixture
(Pharmacia, Piscataway, NJ); reverse transcription (RT) buffers and SuperScript II RNase H ⫺ reverse transcriptase (Gibco BRL, Gaithersburg, MD); Taq DNA polymerase (Promega, Madison, WI); culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM), Hanks’ buffered salt solution, penicillin, streptomycin, ferricytochrome c, phorbol myristate acetate (PMA), and superoxide dismutase (Sigma Chemical Co., St. Louis, MO). Cell culture medium containing 10% FBS was used under all experimental conditions. Microglial 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. Briefly, brain tissues were dissociated after 30 min of trypsinization (0.25%) and plated in 75-cm 2
FIG. 1. Time course of cytokine and chemokine production by gp41-stimulated microglia. Microglial cell cultures were incubated with medium (control) and gp41 (100 nM) for the indicated periods of time. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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Falcon culture flasks in DMEM containing 10% heatinactivated FBS, penicillin (100 U/ml), and streptomycin (100 g/ml). The medium was replenished 4 days after plating in medium containing 10% FBS only. Microglia were harvested 10 –14 days later. Purified microglia were composed of a cell population of which ⬎99% stained with anti-CD 68 antibody (a human macrophage marker) and ⬍1% stained with anti-GFAP (an astrocyte marker).
IL-6, RANTES, or MIP-1␣ antibodies were used followed by the addition of supernatants and standards of TNF-␣, IL-1, IL-6, RANTES, or MIP-1␣. After incubation, goat anti-human TNF-␣, IL-1, IL-6, RANTES, or MIP-1␣ antibodies were added followed by donkey anti-goat IgG– horseradish peroxidase conjugate. A substrate buffer of K-blue (ELISA Technology, Lexington, KY) was used and the reaction was read at 450 nM.
Enzyme-Linked Immunosorbant Assay (ELISA)
RT-PCR Analysis
Supernatants of microglial cell cultures incubated with medium, gp41, or Tat at various concentrations and periods of time were harvested for ELISA analysis. Cytokine and chemokine levels in the microglial culture supernatants were measured by an ELISA developed in our laboratory as previously described (25). Briefly, purified mouse anti-human TNF-␣, IL-1,
Microglial cell cultures were incubated with medium, gp41 (100 nM), or Tat (100 ng/ml) for 3 h before harvesting RNA for RT-PCR analysis. Total RNA was isolated and reverse transcription of 1 g RNA was performed using an oligo-dT 12–18 primer followed by SuperScript II RT. Control reaction for RT had the SuperScript II RT omitted. Amplification of TNF-␣,
FIG. 2. Time course of cytokine and chemokine production by Tat-stimulated microglia. Microglial cell cultures were incubated with medium (control) and Tat (100 ng/ml) for the indicated periods of time. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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IL-1, IL-6, RANTES, MIP-1␣, or GAPDH (as a control) cDNA was performed with Taq DNA polymerase and specific primers. The control reaction for PCR contained no cDNA. The amplified DNA fragments were visualized on 2% agarose gel stained with ethidium bromide under UV light. The primer sequences and annealing temperatures were 5⬘-CAGAGGGAAGAGTTCCCCAG-3⬘ (sense) and 5⬘-CCTTGGTCTGGTAGGAGACG-3⬘ (antisense) for TNF-␣ at 60°C; 5⬘-AAACAGATGAAGTGCTCCTTCAGG-3⬘ (sense) and 5⬘-TGGAGAACACCACTTGTTGCTCCA-3⬘ (antisense) for IL-1 at 65°C; 5⬘-ATGAACTCCTTCTCCACAAGCGC-3⬘ (sense) and 5⬘-GAAGAGCCCTCAGGCTGGACTG-3⬘ (antisense) for IL-6 at 65°C; 5⬘-CCATGAAGGTCTCCGCGGCAC-3⬘ (sense) and 5⬘-CCTAGCTCATCTCCAAAGAG-3⬘ (antisense) for RANTES at 68°C; 5⬘-ACCAGTTCTCTGCATCACTTGCTG-3⬘ (sense) and 5⬘-AACAACCAGTCCATAGAAG AGGTAGCTG-3⬘ (antisense) for MIP-1␣ at 65°C; and 5⬘-CCACCCATGGCAAATTCCATGGCA-3⬘ (sense) and 5⬘-TCTAGACGGCAGGTCAGGTCCACC-3⬘ (anti-
sense) for GAPDH at 65°C. The sizes of DNA fragments for TNF-␣, IL-1, IL-6, RANTES, MIP-1␣, or GAPDH were 325, 391, 628, 162, 343, and 600 bp, respectively. Superoxide Production The production of O 2⫺ by microglial cells was measured through the superoxide-dismutase-inhibited reduction of ferricytochrome c, as described previously (26). Microglial cell cultures were incubated with HIV-1 gp41 (300 nM) or Tat (300 ng/ml) protein for 48 h followed by washing and stimulating with 50 nM PMA. After a 90-min incubation in a 5% CO 2 incubator at 37°C, plates were read at 550 nm. Measurement of [Ca 2⫹] i [Ca 2⫹] i was measured in single microglial cells using indo-1-based microfluorimetry as previously described
FIG. 3. Dose–response relationship of gp41-induced cytokine and chemokine production. Microglial cell cultures were incubated with the indicated concentrations of gp41 for the indicated times before harvesting supernatants for TNF-␣ (8 h), IL-1 (48 h), IL-6 (48 h), RANTES (48 h), and MIP-1␣ (48 h) assay. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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(27). After being loaded with dye, the microglial cell cultures were washed in Hanks’ solution and basal [Ca 2⫹] i was recorded for 10 min before treatment with gp41 (100 nM)) or Tat (50 ng/ml) for 2 min. A RANTESevoked (50 ng/ml, 1 min) increase in [Ca 2⫹] i served as a positive control in the gp41 experiments. RESULTS
Gp41 and Tat Protein-Induced Cytokine and Chemokine Production HIV-1 gp41 (100 nM) stimulated microglial cell production of cytokines (TNF-␣, IL-1, IL-6) and chemokines (RANTES and MIP-1␣) (Fig. 1). The production of TNF-␣ peaked at 8 h and diminished to minimal levels thereafter, while IL-1, IL-6, RANTES, and MIP-1␣ production rose slowly and peaked at 48 to 72 h. No detectable amounts of constitutive production of TNF-␣, IL-1, or RANTES were found, but low levels
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of IL-6 and MIP-1␣ were detectable in control microglial cell cultures (Fig. 1). HIV-1 Tat (100 ng/ml) also stimulated TNF-␣, IL-1, IL-6, RANTES, and MIP-1␣ production but with less potency than gp41 (Fig. 2). The time course of peak production of these cytokines and chemokines was similar to that stimulated by gp41. Production of TNF-␣, IL-1, IL-6, RANTES, and MIP-1␣ by microglial cells stimulated with gp41 (3-300 nM) (Fig. 3) or Tat (10-300 ng/ml) (Fig. 4) was found to be dose-dependent. At the doses tested, gp41 was more potent than Tat in stimulating TNF-␣, IL-1, RANTES, and MIP-1␣ production by microglia. Under no circumstances did the concentrations of gp41 and Tat used in this study induce cytotoxic effect on microglial cells as determined by trypan dye blue exclusion assay. Neither heat-inactivated (56°C, 45 min) nor trypsin-denatured (0.25%, 30 min) gp41 or Tat proteins induced production of any of the cytokines and chemokines studied.
FIG. 4. Dose–response relationship of Tat-induced cytokine and chemokine production. Microglial cell cultures were incubated with the indicated concentrations of Tat for the indicated times before harvesting supernatants for TNF-␣ (8 h), IL-1 (48 h), IL-6 (48 h), RANTES (48 h), and MIP-1␣ (48 h) assay. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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HIV-1 gp41 (100 nM) stimulated TNF-␣, IL-1, IL-6, RANTES, and MIP-1␣ mRNA expression in microglia (Fig. 5). With Tat (100 ng/ml) treatment, the induced mRNA expression of these cytokines and chemokines was less apparent. Expression of each cytokine and chemokine was observed at different PCR cycles: TNF-␣ and IL-1 at 30 cycles, IL-6 at 22 cycles, RANTES at 24 cycles, and MIP-1␣ at 18 cycles. No DNA contamination of the control samples lacking RT was observed at the above PCR cycles of each cytokine and chemokine (data not shown). Effects of gp41 and Tat Proteins on O 2⫺ Production Neither gp41 nor Tat by themselves stimulated O 2⫺ production by microglial cells (Fig. 6). When PMA alone was used to stimulate microglia, robust O 2⫺ production was observed. To determine whether gp41 or Tat would alter the generation of O 2⫺ by PMA-stimulated microglia, cells were pretreated for 48 h with varying concentrations of gp41 or Tat before stimulation with PMA. At the highest concentration tested (300 nM) gp41 modestly potentiated PMA-triggered O 2⫺ production (Fig. 6A), whereas Tat had no effect on PMA-triggered O 2⫺ production (Fig. 6B). Effects of gp41 and Tat Proteins on [Ca 2⫹] i Treatment of single microglia with gp41 (100 nM) failed to evoke an increase in [Ca 2⫹] i (Fig. 7A). Cells were confirmed responsive to chemokines, as indicated by an increase in [Ca 2⫹] i evoked by RANTES (50 ng/ ml), and then subsequently treated with gp41. In contrast, Tat evoked a rapid, transient increase in [Ca 2⫹] i in cultured microglia cells (Fig. 7B). DISCUSSION
In the present study, we found that primary human fetal microglial cells produced cytokines (TNF-␣, IL1, and IL-6) and chemokines (RANTES and MIP-1␣) in a dose- and time-dependent manner in response to the HIV-1 proteins, gp41 and Tat. Upregulation of mRNA expression of these cytokines and chemokines was also demonstrated after gp41 and Tat treatment of microglial cells. Although neither viral protein triggered O 2⫺ release, at the highest concentration tested, gp41 but not Tat amplified O 2⫺ production by PMAstimulated microglia. A robust [Ca 2⫹] i increase was seen in response to Tat, while gp41 did not affect [Ca 2⫹] i in microglia. Replication of HIV-1 in microglia in the CNS results in release not only of infectious HIV-1 virions but also of viral proteins, inflammatory mediators, and toxins, all of which may be neurotoxic. Thus, monocytes, the
FIG. 5. Gp41- and Tat-induced cytokine and chemokine mRNA expression. Microglial cell cultures were treated with medium (lane 1), gp41 (100 nM) (lane 2), or Tat (100 ng/ml) (lane 3) for 3 h before harvesting RNA for assaying for TNF-␣, IL-1, IL-6, RANTES, MIP1␣, and GAPDH mRNA expression by RT-PCR. Lane M is a DNA molecular weight marker.
precursor cells of microglia, and microglial cells have been proposed to play a pivotal role in the neuropathogenesis of HIV-1 (28). Results of the present study indicate that activation of microglial cells does not require HIV-1 replication since the viral proteins gp41 and Tat were both found to be capable of stimulating cytokines and chemokines and Tat induced a robust [Ca 2⫹] i. Proinflammatory cytokines have been implicated in the pathogenesis of many CNS infections and in certain dementias, such as that seen in AIDS patients. Microglia are likely to be a major source of cytokines in the CNS (26, 29, 30), although astrocytes are also capable of producing cytokines (31–33). Expression of certain cytokines (e.g., TNF-␣, IL-1, and IL-6) in the CNS could be neurotoxic (34, 35), although neuroprotective activity has been reported under certain circumstances. Also, in an in vitro study, IL-1 and IL-6 have been shown to inhibit HIV-1 replication in acutely infected human fetal microglial cultures (5), whereas such proinflammatory cytokines have been shown to upregulate HIV-1 expression in chronically infected cells (36 –38). Activated microglia and astrocytes are capable of secreting a number of chemokines, such as, MIP-1␣, MIP-1, MCP-1, IL-8, and RANTES (25, 39 – 41). These inflammatory mediators are thought to direct migration of blood monocytes and lymphocytes into the CNS. Chemokines could also direct migration of microglia to sites of inflammation and injury in the brain. The consequences of such cell trafficking could be beneficial or harmful to the host. MIP-1␣, MIP-1, and RANTES have been found to inhibit HIV-1 repli-
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cultures (20). Also, iNOS and gp41 positive staining in microglia of brains from HIV-infected patients has been reported (46). Superoxide production by PMAstimulated microglial cells is well documented (47, 48). In the present study, gp41 and Tat treatments by themselves did not stimulate O 2⫺ production in microglia. However, a 55% increase in PMA-stimulated O 2⫺ production was observed with pretreatment of microglia with gp41, but only at the highest concentration of gp41 tested. Other investigators have reported that Tat increases oxidative stress in HeLa cells, Jurkat cells, and human neurons (49, 50). Gp41 and Tat proteins are detectable in the brains and Tat in the serum of HIV patients (51). The concentrations of gp41 and Tat used in the present study were within the physiological range and were similar to those used by other investigators. Changes in intracellular Ca 2⫹ levels regulate a variety of cell functions. Human monocytes stimulated with gp41 (0.1–100 nM) showed no induction of Ca 2⫹ mobilization (15), whereas Tat was found to increase [Ca 2⫹] i in a dose-dependent manner in cultured human fetal neurons and astrocytes (52) and in monocytes and macrophages (53). The findings in the present study
FIG. 6. Effects of gp41 and Tat on microglial cell O 2⫺ production. Microglial cell cultures were treated with (A) medium (c) or gp41 (0.3 to 300 nM) or (B) medium (c) or Tat (0.3 to 300 ng/ml) for 48 h either alone (left side of A and B) or prior to stimulation with PMA (50 nM) for 90 min (right side of A and B). Data are means ⫾ SE of triplicates and are representative of three separate experiments. *P ⬍ 0.05 versus PMA alone.
cation (7), suggesting a beneficial role of these chemokines. IL-1 can trigger MIP-1␣, MIP-1, MCP-1, and RANTES production by human microglial cells (25, 41), suggesting that, in addition to inhibiting HIV-1 in acutely infected microglia, this cytokine may also indirectly suppress HIV-1 infection of microglia. Therefore, in addition to their role in neuropathogenesis, cytokines and chemokines released from microglia could contribute to the host defense of the brain against HIV-1. Oxidative stress has been implicated in a number of neurodegenerative diseases, including AIDS dementia (42), and ROI as well as NO appear to be involved in the defense and injury of the brain (43, 44). Previously, we found that IL-1 released from gp41-treated microglia stimulated iNOS and NO production in human astrocyte cultures (13). Other investigators have reported iNOS and NO production in human microglial cell cultures (45) and in gp41-treated mixed glial cell
FIG. 7. Effects of gp41 and Tat on [Ca 2⫹] i. [Ca 2⫹] i was recorded from single microglia with indo-1-based microfluorimetry and peptides were applied by bath superfusion at the times indicated by the horizontal bars. (A) In a cell that responded to RANTES (50 ng/ml) a subsequent application of gp41 (100 nM) failed to elicit a response (n ⫽ 3). (B) Tat evoked a robust [Ca 2⫹] i response in chemokineresponsive microglial cells (n ⫽ 6).
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support the view that gp41 has no direct effect on Ca 2⫹ mobilization in mononuclear phagocytes and add microglia to the types of brain cells in which Tat elicits an increase in [Ca 2⫹] i. Based upon recent findings (27), it appears that the Tat-induced increase in [Ca 2⫹] i results from a Ca 2⫹ influx subsequent to microglial cell CCR3 activation. Many anti-HIV-1 agents including vaccines against Tat and gp120 have been used in an attempt to combat the devastating consequences of infection by this retrovirus. The efficacy of antiviral drugs varies but eradication of HIV-1 may not be achievable. Understanding the cellular responses to gp41 and Tat in the CNS may lead to the development of new approaches to the prevention and treatment of HIV-1-related brain disease. ACKNOWLEDGMENT This study was supported by U.S. Public Health Service Grants DA09924, DA04381, DA07304*, and T32-DA07239. REFERENCES 1. Peterson, P. K., Gekker, G., Hu, S., and Chao, C. C., Microglia: A “double-edged sword.” In “In Defense of the Brain: Current Concepts in the Immunopathogenesis and Clinical Aspects of CNS Infections” (P. K. Peterson and J. S. Remington, Eds.), pp. 31–55, Blackwell Sci., Malden, MA, 1997. 2. Koyanagi, Y., O’Brien, W. A., Zaho, J. Q., Golde, D. W., Gasson, J. C., and Chen, Y. S., Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241, 1673–1675, 1988. 3. Saville, M. W., Taga, K., Foli, A., Broder, S., Tosato, G., Yarchoan, R., Interleukin-10 suppresses human immunodeficiency virus-1 replication in vitro in cells of the monocyte/macrophage lineage. Blood 83, 3591–3599, 1994. 4. Poli, G., and Fuci, A. S., The effect of cytokines and pharmacologic agents on chronic HIV infection. AIDS Res. Hum. Retroviruses 8, 191–197, 1992. 5. Lokensgard, J. R., Gekker, G., Ehrlich, L. C., Hu, S., Chao, C. C., and Peterson, P. K., Proinflammatory cytokines inhibit HIV-1 SF162 expression in acutely infected human brain cell cultures. J. Immunol. 158, 2449 –2455, 1997. 6. Tyor, W. R., Glass, J. D., Griffin, J. W., Becker, P. S., McArthur, J. C., Bezman, L., and Griffin, D. E., Cytokine expression in the brain during the acquired immunodeficiency syndrome. Ann. Neurol. 31, 349 –360, 1992. 7. Cocchi, F., Devico, A. L., Garzinodemo, A., Arya, S. K., Gallo, R. C., and Lusso, P., Identification of RANTES, MIP1-alpha, and MIP1-beta as the major HIV-suppressive factors produced by CD8(⫹) T-cells. Science 270, 1811–1815, 1995. 8. Schmidtmayerova, H., Nottet, H. S., Nuovo, G., Raabe, T., Flanagan, C. R., Dubrovsky, L., Gendelman, H. E., Cerami, A., Burkrinsky, M., and Sherry, B., Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: Implications for recruitment of leukocytes into brain and lymph nodes. Proc. Natl. Acad. Sci. USA 93, 700 –704, 1996. 9. Kure, K., Lyman, W. D., Weidenheim, K. M., and Dickson, D. W., Cellular localization of an HIV-1 antigen in subacute AIDS encephalitis using an improved double-labelling immunohistochemistry method. Am. J. Pathol. 136, 1085–1092, 1990.
10. Dickson, D. W., Lee, S. C., Mattiace, L. A., Yen, S. C., and Brosnan, C., Microglia and cytokines in neurologic disease, with special reference to AIDS and Alzheimer’s disease. Glia 7, 75– 83, 1993. 11. New, D. R., Maggirwar, S. B., Epstein, L. G., Dewhurst, S., and Gelbard, H. A., HIV-1 Tat induces neuronal death via tumor necrosis factor-␣ and activation of non-N-methyl-D-aspartate receptors by a NFB-independent mechanism. J. Biol. Chem. 273, 17852–17858, 1998. 12. Adamson, D. C., Kopnisky, K. L., Dawson, T. M., and Dawson, V. L., Mechanisms and structural determinants of HIV-1 coat protein, gp41-induced neurotoxicity. J. Neurosci. 19, 64 –71, 1999. 13. Hu, S., Ali, H., Sheng, W. S., Ehrlich, L. C., Peterson, P. K., and Chao, C. C., Gp-41-mediated astrocyte inducible nitric oxide synthase mRNA expression: Involvement of interleukin-1 production by microglia. J. Neurosci. 19, 6468 – 6474, 1999. 14. Lafrenie, R. M., Wahl, L. M., Epstein, J. S., Hewlett, I. K., Yamada, K. M., and Dhawan, S., HIV-1-Tat protein promotes chemotaxis and invasive behavior by monocytes. J. Immunol. 157, 974 –977, 1996. 15. Ueda, H., Howard, O. M. Z., Grimm, M. C., Su, S. B., Gong, W. H., Evans, G., Ruscetti, F. W., Oppenheim, J. J., and Wang, J. M., HIV-1 envelope gp41 is a potent inhibitor of chemoattractant receptor expression and function in monocytes. J. Clin. Invest. 102, 804 – 812, 1998. 16. Weiss, J. M., Nath, A., Major, E. O., and Berman, J. W., HIV-1 Tat induces monocyte chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood– brain barrier and up-regulates CCR5 expression on human monocytes. J. Immunol. 163, 2953–2959, 1999. 17. Barcova, M., Kacani, L., Speth, C., and Dierich, M. P., Gp41 envelope protein of human immunodeficiency virus induces interleukin (IL)-10 in monocytes, but not in B, T, or NK cells, leading to reduced IL-2 and interferon-␥ production. J. Infect. Dis. 177, 905–913, 1998. 18. Lafrenie, R. M., Wahl, L. M., Epstein, J. S., Yamada, K. M., and Dhawan, S., Activation of monocytes by HIV-Tat treatment is mediated by cytokine expression. J. Immunol. 159, 4077– 4083, 1997. 19. Nath, A., Conant, K., Chen, P., Scott, C., and Major, E. O., Transient exposure to HIV-1 Tat protein results in cytokine production in macrophages and astrocytes. J. Biol. Chem. 274, 17098 –17102, 1999. 20. Koka, P., He, K., Zack, J. A., Kitchen, S., Peacock, W., Fried, I., Tran, T., Yashar, S. S., and Merrill, J. E., Human immunodeficiency virus 1 envelope proteins induce interleukin 1, tumor necrosis factor ␣, and nitric oxide in glial cultures derived from fetal, neonatal, and adult human brain. J. Exp. Med. 182, 941– 952, 1995. 21. McManus, C. M., Weidenheim, K., Woodman, S. E., Nunez, J., Hesselgesser, J. H., Nath, A., and Berman, J. W., Chemokine and chemokine-receptor expression in human glial elements. Induction by the HIV protein, Tat, and chemokine autoregulation. Am. J. Pathol. 156, 1441–1453, 2000. 22. Babior, B. M., Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298, 659 – 668, 721–725, 1978. 23. Chao, C. C., Hu, S., and Peterson, P. K., Glia: The not so innocent bystanders. J. Neurovirol. 2, 234 –239, 1996. 24. Boven, L. A., Gomes, L., Hery, C., Gray, F., Verhoef, J., Portegies, P., Tardieu, M., and Nottet, H. S. L. M., Increased peroxynitrite activity in AIDS dementia complex: Implications for the neuropathogenesis of HIV-1 infection. J. Immunol. 162, 4319 – 4327, 1999.
gp41 AND Tat ACTIVATION OF MICROGLIAL CELLS 25. Peterson, P. K., Hu, S., Gekker, G., Molitor, T. W., and Chao, C. C., Differential production of and migratory response to  chemokines by human microglia and astrocytes. J. Infect. Dis. 175, 478 – 481, 1997. 26. Chao, C. C., Gekker, G., Sheng, W. S., Hu, S., Tsang, M., and Peterson, P. K., Priming effect of morphine on the production of tumor necrosis factor-␣ by microglia: Implications in respiratory burst activity and human immunodeficiency virus-1 expression. J. Pharmacol. Exp. Ther. 269, 198 –203, 1994. 27. Hegg, C. C., Hu, S., Peterson, P. K., and Thayer, S. A., -Chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia. Neuroscience 98, 191–199, 2000. 28. Gendelman, H. E., Ghorpade, A., and Persidsky, Y., The neuropathogenesis of HIV-1-associated dementia. In “In Defense of the Brain: Current Concepts in the Immunopathogenesis and Clinical Aspects of CNS Infections” (P. K. Peterson and J. S. Remington, Eds.), pp. 291–304, Blackwell Sci., Malden, MA, 1997. 29. Giulian, D., Baker, T. J., Shih, L. N., and Lachman, L. B., Interleukin-1 of the central nervous system is produced by ameboid microglia. J. Exp. Med. 164, 594 – 604, 1986. 30. Sebire, G., Emilie, D., Wallon, C., Hery, C., Devergne, O., Delfraissy, J-F., Galanaud, P., and Tardieu, M., In vitro production of IL-6, IL-1, and tumor necrosis factor-␣ by human embryonic microglial and neural cells. J. Immunol. 150, 1517–1523, 1993. 31. Lee, S., Liu, W., Dickson, D. W., Brosnan, C. F., and Berman, J. W., Cytokine production by human fetal microglia and astrocytes. J. Immunol. 150, 2659 –2667, 1993. 32. Aloisi, F., Care, A., Borsellino, G., Gallo, P., Rosa, S., Bassani, A., Cabibbo, A., Testa, U., Levi, G., and Peschle, C., Production of hemalymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 and tumor necrosis factor-␣. J. Immunol. 149, 2358 –2366, 1992. 33. Bethea, J. R., Chung, I. Y., Sparacio, S. M., Gillespie, G. Y., and Benveniste, E. N., Interleukin-1 induction of tumor necrosis factor-␣ gene expression in human astroglioma cells. J. Neuroimmunol. 36, 179 –191, 1992. 34. Chao, C. C., and Hu, S., Tumor Necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures. Dev. Neurosci. 16, 172–179, 1994. 35. Hu, S., Peterson, P. K., and Chao, C. C., Cytokine-mediated neuronal apoptosis. Neurochem. Int. 30, 427– 431, 1997. 36. Schuitemaker, H., Kootstra, N. A., Koppelman, M. H., Bruisten, S. M., Huisman, H. G., Tersmette, M., and Miedema, F., Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages. J. Clin. Invest. 89, 1154 –1160, 1992. 37. Poli, G., Bressler, P., Kinter, A., Duh, E., Timmer, W. C., Rabson, A., Justement, J. S., Stanley, S., and Fauci, A. S., Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and post-transcriptional mechanisms. J. Exp. Med. 172, 151–158, 1990. 38. Poli, G., Kinter, A. L., and Fauci, A. S., Interleukin 1 induces expression of the human immunodeficiency virus alone and in synergy with interleukin 6 in chronically infected U1 cells: Inhibition of inductive effects by the interleukin 1 receptor antagonist. Proc. Natl. Acad. Sci. USA 91, 108 –112, 1994. 39. Ehrlich, L. C., Hu, S., Sheng, W. S., Sutton, R. L., Rockswold, G. L., Peterson, P. K., and Chao, C. C., Cytokine regulation of human microglial cell IL-8 production. J. Immunol. 160, 1944 – 1948, 1998. Received April 6, 2000; accepted with revision June 15, 2000
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40. McManus, C. M., Brosnan, C. F., and Berman, J. W., Cytokine induction of MIP-1␣ and MIP-1 in human fetal microglia. J. Immunol. 160, 1449 –1455, 1998. 41. Hu, S., Chao, C. C., Ehrlich, L. C., Sheng, W. S., Sutton, R. L., Rockswold, G. L., and Peterson, P. K., Inhibition of microglial cell RANTES production by IL-10 and TGF-. J. Leukocyte Biol. 65, 815– 821, 1999. 42. Kimura, T., Kameoka, M., and Ikuta, K., Amplification of superoxide anion generation in phagocytic cells by HIV-1 infection. FEBS Lett. 326, 232–236, 1993. 43. Peterson, P. K., Hu, S., Anderson, W. R., and Chao, C. C., Nitric oxide production and neurotoxicity mediated by activated microglia from human versus mouse brain. J. Infect. Dis. 170, 457– 460, 1994. 44. Chao, C. C., Anderson, W. R., Hu, S., Gekker, G., Martella, A., and Peterson, P. K., Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism. Clin. Immunol. Immunopathol. 67, 178 –183, 1993. 45. Ding, M., St. Pierre, B. A., Parkinson, J. F., Medberry, P., Wong, J. L., Rogers, N. E., Ignarro, L. J., and Merrill, J. E., Inducible nitric-oxide synthase and nitric oxide production in human fetal astrocytes and microglia. J. Biol. Chem. 272, 11327–11335, 1997. 46. Rostasy, K., Monti, L., Yiannoutsos, C., Kneissl, M., Bell, J., Kemper, T. L., Hedreen, J. C., and Navia, B. A., Human immunodeficiency virus infection, inducible nitric oxide synthase expression, and microglial activation: Pathogenetic relationship to the acquired immunodeficiency syndrome dementia complex. Ann. Neurol. 46, 207–216, 1999. 47. Colton, C., Wilt, S., Gilbert, D., Chernyshev, O., Snell, J., and Dubois-Dalcq, M., Species differences in the generation of reactive oxygen species by microglia. Mol. Chem. Neuropathol. 28, 15–20, 1996. 48. Chao, C. C., Hu, S., and Peterson, P. K., Modulation of human microglial cell superoxide production by cytokines. J. Leukocyte Biol. 58, 65–70, 1995. 49. Westendorp, M. O., Shatrov, V. A., Schulze-Osthoff, K., Frank, R., Kraft, M., Los, M., Krammer, P. H., Dro¨ge, W., and Lehmann, V., HIV-1 Tat potentiates TNF-induced NF-B activation and cytotoxicity by altering the cellular redox state. EMBO J. 14, 546 –554, 1995. 50. Shi, B., Raina, J., Lorenzo, A., Busciglio, J., and Gabuzda, D., Neuronal apoptosis induced by HIV-1 Tat protein and TNF-␣: Potentiation of neurotoxicity mediated by oxidative stress and implications for HIV-1 dementia. J. NeuroVirol. 4, 281–290, 1998. 51. Westendorp, M. O., Frank, R., Ochsenbauer, C., Stricker, K., Dhein, J., Walczak, H., Debatin, K-M., and Krammer, P. H., Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature 375, 497–500, 1995. 52. Haughey, N. J., Holden, C. P., Nath, A., and Geiger, J. D., Involvement of inositol 1,4,5-trisphosphate-regulated stores of intracellular calcium in calcium dysregulation and neuron cell death caused by HIV-1 protein tat. J. Neurochem. 73, 1363– 1374, 1999. 53. Albini, A., Ferrini, S., Benelli, R., Sforzini, S., Giunciuglio, D., Aluigi, M. G., Proudfoot, A. E. I., Alouani, S., Wells, T. N. C., Mariani, G., Rabin, R. L., Farber, J. M., and Noonan, D. M., HIV-1 Tat protein mimicry of chemokines. Proc. Natl. Acad. Sci. USA 95, 13153–13158, 1998.
Activation of Human Microglial Cells by HIV-1 gp41 and Tat Proteins W. S. Sheng, S. Hu, C. C. Hegg,* S. A. Thayer,* and P. K. Peterson Institute for Brain and Immune Disorders, Minneapolis Medical Research Foundation, Department of Medicine, and *Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55404
The viral proteins, Tat (HIV-1 nuclear protein) and gp41 (HIV-1 coat protein), detected in the brains of HIV-1-infected patients have been shown to be neurotoxic. We investigated the effects of HIV-1 Tat and gp41 proteins on cytokine, chemokine, and superoxide anion (O 2ⴚ) production by microglia, the resident macrophages of the brain. Tat and gp41 dose-dependently stimulated cytokine and chemokine production by microglia. Peak production of these cytokines and chemokines differed in microglial cells treated with gp41 and Tat. Expression of cytokine and chemokine mRNA was also stimulated in gp41- and Tat-treated microglia. Neither gp41 nor Tat alone stimulated O 2ⴚ production by microglia. Treatment of microglial cells with Tat but not with gp41 evoked an increase in intracellular Ca 2ⴙ. The results of this study suggest that HIV-1 Tat and gp41 proteins impact several key functions of microglial cells which could contribute to the neuropathogenesis of HIV-1. © 2000 Academic Press Key Words: microglia; gp41; Tat; cytokines; chemokines.
INTRODUCTION
The pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) resulting in neuronal dysfunction and cell loss is incompletely understood. Microglia, the resident macrophages of the brain, are the main brain cell type to be productively infected by HIV-1. Upon activation, microglial cells produce many cytokines, chemokines, and other inflammatory mediators (e.g., reactive oxygen intermediates, [ROI]) (1). Such inflammatory mediators may have neuroprotective or neuropathological effects. For instance, some cytokines and chemokines can inhibit while others can enhance HIV infection of and replication in macrophages, including microglia (2–5). Proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣), interleukin-1 beta (IL-1), and IL-6 have been detected in the CNS of patients with HIV-1 encephalopathy, although no correlation between the levels of these cytokines and the severity of CNS disease has been found (6). Chemokines such as
regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-1 beta (MIP-1), and monocyte chemotactic protein-1 (MCP-1) are capable of directing the migration of lymphocytes and mononuclear phagocytes within infected/inflammatory sites, and these chemokines appear to participate in the host defense against HIV infection (7). However, these same chemokines also have been proposed to play a role in the neuropathogenesis of HIV-1 (8). The HIV-1 proteins Tat and gp41 have been detected in the brains of demented patients at postmortem examination. The levels of these viral proteins appear to correlate with the severity of CNS disease (9, 10). Both Tat and gp41 have been found to be neurotoxic, possibly via indirect mechanisms involving mediators released by activated microglia in the CNS. Tat has been reported to induce neuronal apoptosis mediated by proinflammatory cytokine release and activation of glutamate receptors (11). Gp41, on the other hand, was reported to stimulate inducible nitric oxide synthase (iNOS)-derived NO as a major mediator of neurotoxicity (12). Recently, work in our laboratory has shown that human microglial cells stimulated with gp41 release IL-1, which in turn stimulates astrocyte iNOS expression and NO production (13). Tat and gp41 have been shown by other investigators to alter many functions of monocytes, such as adhesion and chemotaxis (14), receptor expression (15, 16), and cytokine production (17–19). Also, Koka et al. have reported that human mixed glial cell cultures (consisting of microglia and astrocytes) stimulated with gp41 produce TNF-␣ and IL-1 (20); and recently, other investigators have reported that Tat induces chemokine (MIP-1␣, MIP-1, and MCP-1) production in human fetal microglial cell cultures (21). ROI, such as superoxide anion (O 2⫺), hydrogen peroxide, hydroxyl radical, and peroxynitrite, have both microbicidal and pathological properties at sites of infection and inflammation (22, 23). Increased O 2⫺ production has recently been reported in studies of HIV-1-infected macrophages (24). Microglia appear to be a major source of ROI in a variety of brain insults. Because little or nothing is known about the effects of
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gp41 and Tat on the generation of O 2⫺ by microglia and incomplete information is available on the production of cytokines and chemokines by purified human fetal microglial cells in response to gp41 and Tat, the present study was undertaken. MATERIALS AND METHODS
Reagents Cytokines (TNF-␣, IL-1, IL-6), chemokines (RANTES, MIP-1␣), and their antibodies were obtained from R&D Systems, Inc. (Minneapolis, MN). Antibodies to glial fibrillary acidic protein (GFAP) and CD 68 were purchased from DAKO (Carpinteria, CA). Other reagents were from the indicated sources: fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT); gp41 (Intracel Inc., Issaquah, WA); Tat (NIH AIDS Research and Reference Reagent Program, Rockville, MD); oligo (dT) 12–18 primer and dNTP mixture
(Pharmacia, Piscataway, NJ); reverse transcription (RT) buffers and SuperScript II RNase H ⫺ reverse transcriptase (Gibco BRL, Gaithersburg, MD); Taq DNA polymerase (Promega, Madison, WI); culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM), Hanks’ buffered salt solution, penicillin, streptomycin, ferricytochrome c, phorbol myristate acetate (PMA), and superoxide dismutase (Sigma Chemical Co., St. Louis, MO). Cell culture medium containing 10% FBS was used under all experimental conditions. Microglial 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. Briefly, brain tissues were dissociated after 30 min of trypsinization (0.25%) and plated in 75-cm 2
FIG. 1. Time course of cytokine and chemokine production by gp41-stimulated microglia. Microglial cell cultures were incubated with medium (control) and gp41 (100 nM) for the indicated periods of time. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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Falcon culture flasks in DMEM containing 10% heatinactivated FBS, penicillin (100 U/ml), and streptomycin (100 g/ml). The medium was replenished 4 days after plating in medium containing 10% FBS only. Microglia were harvested 10 –14 days later. Purified microglia were composed of a cell population of which ⬎99% stained with anti-CD 68 antibody (a human macrophage marker) and ⬍1% stained with anti-GFAP (an astrocyte marker).
IL-6, RANTES, or MIP-1␣ antibodies were used followed by the addition of supernatants and standards of TNF-␣, IL-1, IL-6, RANTES, or MIP-1␣. After incubation, goat anti-human TNF-␣, IL-1, IL-6, RANTES, or MIP-1␣ antibodies were added followed by donkey anti-goat IgG– horseradish peroxidase conjugate. A substrate buffer of K-blue (ELISA Technology, Lexington, KY) was used and the reaction was read at 450 nM.
Enzyme-Linked Immunosorbant Assay (ELISA)
RT-PCR Analysis
Supernatants of microglial cell cultures incubated with medium, gp41, or Tat at various concentrations and periods of time were harvested for ELISA analysis. Cytokine and chemokine levels in the microglial culture supernatants were measured by an ELISA developed in our laboratory as previously described (25). Briefly, purified mouse anti-human TNF-␣, IL-1,
Microglial cell cultures were incubated with medium, gp41 (100 nM), or Tat (100 ng/ml) for 3 h before harvesting RNA for RT-PCR analysis. Total RNA was isolated and reverse transcription of 1 g RNA was performed using an oligo-dT 12–18 primer followed by SuperScript II RT. Control reaction for RT had the SuperScript II RT omitted. Amplification of TNF-␣,
FIG. 2. Time course of cytokine and chemokine production by Tat-stimulated microglia. Microglial cell cultures were incubated with medium (control) and Tat (100 ng/ml) for the indicated periods of time. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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IL-1, IL-6, RANTES, MIP-1␣, or GAPDH (as a control) cDNA was performed with Taq DNA polymerase and specific primers. The control reaction for PCR contained no cDNA. The amplified DNA fragments were visualized on 2% agarose gel stained with ethidium bromide under UV light. The primer sequences and annealing temperatures were 5⬘-CAGAGGGAAGAGTTCCCCAG-3⬘ (sense) and 5⬘-CCTTGGTCTGGTAGGAGACG-3⬘ (antisense) for TNF-␣ at 60°C; 5⬘-AAACAGATGAAGTGCTCCTTCAGG-3⬘ (sense) and 5⬘-TGGAGAACACCACTTGTTGCTCCA-3⬘ (antisense) for IL-1 at 65°C; 5⬘-ATGAACTCCTTCTCCACAAGCGC-3⬘ (sense) and 5⬘-GAAGAGCCCTCAGGCTGGACTG-3⬘ (antisense) for IL-6 at 65°C; 5⬘-CCATGAAGGTCTCCGCGGCAC-3⬘ (sense) and 5⬘-CCTAGCTCATCTCCAAAGAG-3⬘ (antisense) for RANTES at 68°C; 5⬘-ACCAGTTCTCTGCATCACTTGCTG-3⬘ (sense) and 5⬘-AACAACCAGTCCATAGAAG AGGTAGCTG-3⬘ (antisense) for MIP-1␣ at 65°C; and 5⬘-CCACCCATGGCAAATTCCATGGCA-3⬘ (sense) and 5⬘-TCTAGACGGCAGGTCAGGTCCACC-3⬘ (anti-
sense) for GAPDH at 65°C. The sizes of DNA fragments for TNF-␣, IL-1, IL-6, RANTES, MIP-1␣, or GAPDH were 325, 391, 628, 162, 343, and 600 bp, respectively. Superoxide Production The production of O 2⫺ by microglial cells was measured through the superoxide-dismutase-inhibited reduction of ferricytochrome c, as described previously (26). Microglial cell cultures were incubated with HIV-1 gp41 (300 nM) or Tat (300 ng/ml) protein for 48 h followed by washing and stimulating with 50 nM PMA. After a 90-min incubation in a 5% CO 2 incubator at 37°C, plates were read at 550 nm. Measurement of [Ca 2⫹] i [Ca 2⫹] i was measured in single microglial cells using indo-1-based microfluorimetry as previously described
FIG. 3. Dose–response relationship of gp41-induced cytokine and chemokine production. Microglial cell cultures were incubated with the indicated concentrations of gp41 for the indicated times before harvesting supernatants for TNF-␣ (8 h), IL-1 (48 h), IL-6 (48 h), RANTES (48 h), and MIP-1␣ (48 h) assay. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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(27). After being loaded with dye, the microglial cell cultures were washed in Hanks’ solution and basal [Ca 2⫹] i was recorded for 10 min before treatment with gp41 (100 nM)) or Tat (50 ng/ml) for 2 min. A RANTESevoked (50 ng/ml, 1 min) increase in [Ca 2⫹] i served as a positive control in the gp41 experiments. RESULTS
Gp41 and Tat Protein-Induced Cytokine and Chemokine Production HIV-1 gp41 (100 nM) stimulated microglial cell production of cytokines (TNF-␣, IL-1, IL-6) and chemokines (RANTES and MIP-1␣) (Fig. 1). The production of TNF-␣ peaked at 8 h and diminished to minimal levels thereafter, while IL-1, IL-6, RANTES, and MIP-1␣ production rose slowly and peaked at 48 to 72 h. No detectable amounts of constitutive production of TNF-␣, IL-1, or RANTES were found, but low levels
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of IL-6 and MIP-1␣ were detectable in control microglial cell cultures (Fig. 1). HIV-1 Tat (100 ng/ml) also stimulated TNF-␣, IL-1, IL-6, RANTES, and MIP-1␣ production but with less potency than gp41 (Fig. 2). The time course of peak production of these cytokines and chemokines was similar to that stimulated by gp41. Production of TNF-␣, IL-1, IL-6, RANTES, and MIP-1␣ by microglial cells stimulated with gp41 (3-300 nM) (Fig. 3) or Tat (10-300 ng/ml) (Fig. 4) was found to be dose-dependent. At the doses tested, gp41 was more potent than Tat in stimulating TNF-␣, IL-1, RANTES, and MIP-1␣ production by microglia. Under no circumstances did the concentrations of gp41 and Tat used in this study induce cytotoxic effect on microglial cells as determined by trypan dye blue exclusion assay. Neither heat-inactivated (56°C, 45 min) nor trypsin-denatured (0.25%, 30 min) gp41 or Tat proteins induced production of any of the cytokines and chemokines studied.
FIG. 4. Dose–response relationship of Tat-induced cytokine and chemokine production. Microglial cell cultures were incubated with the indicated concentrations of Tat for the indicated times before harvesting supernatants for TNF-␣ (8 h), IL-1 (48 h), IL-6 (48 h), RANTES (48 h), and MIP-1␣ (48 h) assay. Data are means ⫾ SE of triplicates and are representative of three separate experiments.
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HIV-1 gp41 (100 nM) stimulated TNF-␣, IL-1, IL-6, RANTES, and MIP-1␣ mRNA expression in microglia (Fig. 5). With Tat (100 ng/ml) treatment, the induced mRNA expression of these cytokines and chemokines was less apparent. Expression of each cytokine and chemokine was observed at different PCR cycles: TNF-␣ and IL-1 at 30 cycles, IL-6 at 22 cycles, RANTES at 24 cycles, and MIP-1␣ at 18 cycles. No DNA contamination of the control samples lacking RT was observed at the above PCR cycles of each cytokine and chemokine (data not shown). Effects of gp41 and Tat Proteins on O 2⫺ Production Neither gp41 nor Tat by themselves stimulated O 2⫺ production by microglial cells (Fig. 6). When PMA alone was used to stimulate microglia, robust O 2⫺ production was observed. To determine whether gp41 or Tat would alter the generation of O 2⫺ by PMA-stimulated microglia, cells were pretreated for 48 h with varying concentrations of gp41 or Tat before stimulation with PMA. At the highest concentration tested (300 nM) gp41 modestly potentiated PMA-triggered O 2⫺ production (Fig. 6A), whereas Tat had no effect on PMA-triggered O 2⫺ production (Fig. 6B). Effects of gp41 and Tat Proteins on [Ca 2⫹] i Treatment of single microglia with gp41 (100 nM) failed to evoke an increase in [Ca 2⫹] i (Fig. 7A). Cells were confirmed responsive to chemokines, as indicated by an increase in [Ca 2⫹] i evoked by RANTES (50 ng/ ml), and then subsequently treated with gp41. In contrast, Tat evoked a rapid, transient increase in [Ca 2⫹] i in cultured microglia cells (Fig. 7B). DISCUSSION
In the present study, we found that primary human fetal microglial cells produced cytokines (TNF-␣, IL1, and IL-6) and chemokines (RANTES and MIP-1␣) in a dose- and time-dependent manner in response to the HIV-1 proteins, gp41 and Tat. Upregulation of mRNA expression of these cytokines and chemokines was also demonstrated after gp41 and Tat treatment of microglial cells. Although neither viral protein triggered O 2⫺ release, at the highest concentration tested, gp41 but not Tat amplified O 2⫺ production by PMAstimulated microglia. A robust [Ca 2⫹] i increase was seen in response to Tat, while gp41 did not affect [Ca 2⫹] i in microglia. Replication of HIV-1 in microglia in the CNS results in release not only of infectious HIV-1 virions but also of viral proteins, inflammatory mediators, and toxins, all of which may be neurotoxic. Thus, monocytes, the
FIG. 5. Gp41- and Tat-induced cytokine and chemokine mRNA expression. Microglial cell cultures were treated with medium (lane 1), gp41 (100 nM) (lane 2), or Tat (100 ng/ml) (lane 3) for 3 h before harvesting RNA for assaying for TNF-␣, IL-1, IL-6, RANTES, MIP1␣, and GAPDH mRNA expression by RT-PCR. Lane M is a DNA molecular weight marker.
precursor cells of microglia, and microglial cells have been proposed to play a pivotal role in the neuropathogenesis of HIV-1 (28). Results of the present study indicate that activation of microglial cells does not require HIV-1 replication since the viral proteins gp41 and Tat were both found to be capable of stimulating cytokines and chemokines and Tat induced a robust [Ca 2⫹] i. Proinflammatory cytokines have been implicated in the pathogenesis of many CNS infections and in certain dementias, such as that seen in AIDS patients. Microglia are likely to be a major source of cytokines in the CNS (26, 29, 30), although astrocytes are also capable of producing cytokines (31–33). Expression of certain cytokines (e.g., TNF-␣, IL-1, and IL-6) in the CNS could be neurotoxic (34, 35), although neuroprotective activity has been reported under certain circumstances. Also, in an in vitro study, IL-1 and IL-6 have been shown to inhibit HIV-1 replication in acutely infected human fetal microglial cultures (5), whereas such proinflammatory cytokines have been shown to upregulate HIV-1 expression in chronically infected cells (36 –38). Activated microglia and astrocytes are capable of secreting a number of chemokines, such as, MIP-1␣, MIP-1, MCP-1, IL-8, and RANTES (25, 39 – 41). These inflammatory mediators are thought to direct migration of blood monocytes and lymphocytes into the CNS. Chemokines could also direct migration of microglia to sites of inflammation and injury in the brain. The consequences of such cell trafficking could be beneficial or harmful to the host. MIP-1␣, MIP-1, and RANTES have been found to inhibit HIV-1 repli-
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cultures (20). Also, iNOS and gp41 positive staining in microglia of brains from HIV-infected patients has been reported (46). Superoxide production by PMAstimulated microglial cells is well documented (47, 48). In the present study, gp41 and Tat treatments by themselves did not stimulate O 2⫺ production in microglia. However, a 55% increase in PMA-stimulated O 2⫺ production was observed with pretreatment of microglia with gp41, but only at the highest concentration of gp41 tested. Other investigators have reported that Tat increases oxidative stress in HeLa cells, Jurkat cells, and human neurons (49, 50). Gp41 and Tat proteins are detectable in the brains and Tat in the serum of HIV patients (51). The concentrations of gp41 and Tat used in the present study were within the physiological range and were similar to those used by other investigators. Changes in intracellular Ca 2⫹ levels regulate a variety of cell functions. Human monocytes stimulated with gp41 (0.1–100 nM) showed no induction of Ca 2⫹ mobilization (15), whereas Tat was found to increase [Ca 2⫹] i in a dose-dependent manner in cultured human fetal neurons and astrocytes (52) and in monocytes and macrophages (53). The findings in the present study
FIG. 6. Effects of gp41 and Tat on microglial cell O 2⫺ production. Microglial cell cultures were treated with (A) medium (c) or gp41 (0.3 to 300 nM) or (B) medium (c) or Tat (0.3 to 300 ng/ml) for 48 h either alone (left side of A and B) or prior to stimulation with PMA (50 nM) for 90 min (right side of A and B). Data are means ⫾ SE of triplicates and are representative of three separate experiments. *P ⬍ 0.05 versus PMA alone.
cation (7), suggesting a beneficial role of these chemokines. IL-1 can trigger MIP-1␣, MIP-1, MCP-1, and RANTES production by human microglial cells (25, 41), suggesting that, in addition to inhibiting HIV-1 in acutely infected microglia, this cytokine may also indirectly suppress HIV-1 infection of microglia. Therefore, in addition to their role in neuropathogenesis, cytokines and chemokines released from microglia could contribute to the host defense of the brain against HIV-1. Oxidative stress has been implicated in a number of neurodegenerative diseases, including AIDS dementia (42), and ROI as well as NO appear to be involved in the defense and injury of the brain (43, 44). Previously, we found that IL-1 released from gp41-treated microglia stimulated iNOS and NO production in human astrocyte cultures (13). Other investigators have reported iNOS and NO production in human microglial cell cultures (45) and in gp41-treated mixed glial cell
FIG. 7. Effects of gp41 and Tat on [Ca 2⫹] i. [Ca 2⫹] i was recorded from single microglia with indo-1-based microfluorimetry and peptides were applied by bath superfusion at the times indicated by the horizontal bars. (A) In a cell that responded to RANTES (50 ng/ml) a subsequent application of gp41 (100 nM) failed to elicit a response (n ⫽ 3). (B) Tat evoked a robust [Ca 2⫹] i response in chemokineresponsive microglial cells (n ⫽ 6).
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support the view that gp41 has no direct effect on Ca 2⫹ mobilization in mononuclear phagocytes and add microglia to the types of brain cells in which Tat elicits an increase in [Ca 2⫹] i. Based upon recent findings (27), it appears that the Tat-induced increase in [Ca 2⫹] i results from a Ca 2⫹ influx subsequent to microglial cell CCR3 activation. Many anti-HIV-1 agents including vaccines against Tat and gp120 have been used in an attempt to combat the devastating consequences of infection by this retrovirus. The efficacy of antiviral drugs varies but eradication of HIV-1 may not be achievable. Understanding the cellular responses to gp41 and Tat in the CNS may lead to the development of new approaches to the prevention and treatment of HIV-1-related brain disease. ACKNOWLEDGMENT This study was supported by U.S. Public Health Service Grants DA09924, DA04381, DA07304*, and T32-DA07239. REFERENCES 1. Peterson, P. K., Gekker, G., Hu, S., and Chao, C. C., Microglia: A “double-edged sword.” In “In Defense of the Brain: Current Concepts in the Immunopathogenesis and Clinical Aspects of CNS Infections” (P. K. Peterson and J. S. Remington, Eds.), pp. 31–55, Blackwell Sci., Malden, MA, 1997. 2. Koyanagi, Y., O’Brien, W. A., Zaho, J. Q., Golde, D. W., Gasson, J. C., and Chen, Y. S., Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241, 1673–1675, 1988. 3. Saville, M. W., Taga, K., Foli, A., Broder, S., Tosato, G., Yarchoan, R., Interleukin-10 suppresses human immunodeficiency virus-1 replication in vitro in cells of the monocyte/macrophage lineage. Blood 83, 3591–3599, 1994. 4. Poli, G., and Fuci, A. S., The effect of cytokines and pharmacologic agents on chronic HIV infection. AIDS Res. Hum. Retroviruses 8, 191–197, 1992. 5. Lokensgard, J. R., Gekker, G., Ehrlich, L. C., Hu, S., Chao, C. C., and Peterson, P. K., Proinflammatory cytokines inhibit HIV-1 SF162 expression in acutely infected human brain cell cultures. J. Immunol. 158, 2449 –2455, 1997. 6. Tyor, W. R., Glass, J. D., Griffin, J. W., Becker, P. S., McArthur, J. C., Bezman, L., and Griffin, D. E., Cytokine expression in the brain during the acquired immunodeficiency syndrome. Ann. Neurol. 31, 349 –360, 1992. 7. Cocchi, F., Devico, A. L., Garzinodemo, A., Arya, S. K., Gallo, R. C., and Lusso, P., Identification of RANTES, MIP1-alpha, and MIP1-beta as the major HIV-suppressive factors produced by CD8(⫹) T-cells. Science 270, 1811–1815, 1995. 8. Schmidtmayerova, H., Nottet, H. S., Nuovo, G., Raabe, T., Flanagan, C. R., Dubrovsky, L., Gendelman, H. E., Cerami, A., Burkrinsky, M., and Sherry, B., Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: Implications for recruitment of leukocytes into brain and lymph nodes. Proc. Natl. Acad. Sci. USA 93, 700 –704, 1996. 9. Kure, K., Lyman, W. D., Weidenheim, K. M., and Dickson, D. W., Cellular localization of an HIV-1 antigen in subacute AIDS encephalitis using an improved double-labelling immunohistochemistry method. Am. J. Pathol. 136, 1085–1092, 1990.
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