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Anti-CD45RO Suppresses Human Immunodeficiency Viru
http://www.100md.com 病菌学杂志 2006年第1期
     Department of Pathology, Albert Einstein College of Medicine, Bronx, New York

    Cardiology Division, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York

    ABSTRACT

    Macrophages and microglia are productively infected by HIV-1 and play a pivotal role in the pathogenesis of AIDS dementia. Although macrophages and microglia express CD45, a transmembrane protein tyrosine phosphatase, whether modulation of its activity affects human immunodeficiency virus type 1 (HIV-1) replication is unknown. Here, we report that of the five human CD45 isoforms, microglia express CD45RB and CD45RO (RB > RO) and treatment of microglia with a CD45 agonist antibody CD45RO (UCHL-1) inhibits HIV-1 replication. CD45RO prevented HIV-1 negative factor (Nef)-induced autophosphorylation of hematopoietic cell kinase (Hck), a myeloid lineage-specific Src kinase. Recombinant CD45 protein also inhibited HIV-1-induced Hck phosphorylation in microglia. Antennapedia-mediated delivery of Hck Src homology domain 3 (SH3), a domain that binds to the Nef PxxP motif with high affinity, reduced HIV-1-induced Hck phosphorylation and HIV-1 production in microglia. HIV-1-induced LTR transactivation was observed in U38 cells stably overexpressing wild-type Hck but not kinase-inactive Hck. In microglia, CD45RO reduced activation of transcription factors (NF-B and CCAAT enhancer binding protein) necessary for LTR transactivation in macrophages. These results establish that in myeloid lineage cells, Nef interacts with the Hck SH3 domain, resulting in autophosphorylation of Hck and an increase in HIV-1 transcription. CD45RO-mediated inhibition of HIV-1 replication in microglia identifies the CD45 protein tyrosine phosphatase as a potential therapeutic target for HIV-1 infection/AIDS dementia.

    INTRODUCTION

    CD45 (leukocyte common antigen) is a prototypic transmembrane protein tyrosine phosphatase (PTP) expressed in all hematopoietic cells except red blood cells (64). CD45 protein exists as multiple isoforms as a result of alternative splicing of variable exons (4/A, 5/B, and 6/C); the largest isoform (ABC) includes all three of these exons and the smallest isoform lacks all three exons (O). Five different isoforms of CD45 (ABC, AB, BC, B, and O) have been identified on human leukocytes, and these can be recognized by specific antibodies. All CD45 isoforms have a single transmembrane domain and a large cytoplasmic tail containing two tandemly duplicated PTP homology domains, D1 and D2; these are identical among different isoforms (64).

    CD45 can operate as a positive or negative regulator of Src family tyrosine kinases, depending in part on the tyrosine phosphate it dephosphorylates. Dephosphorylation of the tyrosine in the kinase domain (Y416 in c-Src) results in inactivation of Src kinases; this has been shown for hematopoietic cell kinase (Hck) and Lyn during integrin-mediated adhesion in macrophages, as well as Lck in developing thymocytes (17, 51). In contrast, CD45-dependent dephosphorylation of the negative regulatory tyrosine residue in the C-terminal tail (Y527 in c-Src) results in enhanced kinase activity; this has been shown for Lck during lymphocyte antigen receptor signaling (1). Furthermore, CD45-deficient mice demonstrate profound blocks in both T- and B-cell development and function (10, 31); in humans, mutations in the gene encoding CD45 can cause severe combined immunodeficiency (SCID) (11, 35). These findings reflect a critical role of CD45 in the positive regulation of lymphocyte development and signaling. In addition to Src, CD45 has been shown to dephosphorylate Janus kinases (Jak/Tyk), thereby modulating cytokine and interferon signaling (27).

    Microglia are resident central nervous system (CNS) macrophages critical to the maintenance of normal homeostasis, as well as innate and adaptive antimicrobial responses (15, 19). Microglia constitute the primary cell type productively infected by human immunodeficiency virus type 1 (HIV-1) in the CNS (12, 36); together with monocyte-derived macrophages, they are the prime instigators of the disease referred to as AIDS dementia/HIV encephalitis (HIVE) (49). Microglia and macrophages in AIDS dementia show diffuse activation and produce proinflammatory cytokines and neurotoxic molecules, ultimately leading to neuronal damage and CNS dysfunction (12, 16, 21). Previous studies have revealed that CD45 is expressed on resting microglia in normal brain tissue; CD45 expression is upregulated in diseases such as Alzheimer's disease and HIVE (12, 48). Furthermore, in murine microglia, Tan and colleagues have shown that CD45 negatively regulates -amyloid-induced microglial activation (61, 62) and that CD45RB inhibits microglial activation induced by microbial factors (63). In human microglia, activation of CD45 PTP by agonist antibody CD45RO (UCHL-1) inhibits granulocyte-macrophage colony-stimulating factor (granulocyte-M-CSF)-induced proliferation (59). Together, these studies demonstrate a potential role for CD45 PTP as a therapeutic target in regulating inflammatory and immune responses in the CNS.

    In spite of the enhanced expression of CD45 on microglia and macrophages in HIVE, whether CD45 can modulate viral production from these cells is unknown. In the current study, we addressed the role of CD45RO in HIV-1 production from microglia. Because of the well-documented interaction between HIV-1 negative factor (Nef) and Hck that results in activation of Hck in vitro (38, 54), we focused on the role of Hck as a potential substrate for CD45 PTP in HIV-1-infected cells. We hypothesized that in microglia Hck is phosphorylated following HIV-1 infection in a Nef-dependent manner and that CD45RO inhibits HIV-1 production by dephosphorylating Hck in the activating kinase domain. Here, we present evidence that this is indeed the case.

    (This work was presented in part at the annual meeting of the Society for Neuroscience, November 2002. This study was performed by M.-O. Kim in partial fulfillment of the requirements for a Ph.D. thesis of the Sue Golding Graduate Division of the Albert Einstein College of Medicine.)

    MATERIALS AND METHODS

    Antibodies. CD45RO (UCHL-1; immunoglobulin G 2a [IgG2a]) was obtained from DAKO (Carpinteria, CA) or Pharmingen (San Diego, CA), CD45RB was from Pharmingen (MT4; IgG1) or from Neomarkers (PD7/26; IgG1; Fremont, CA), CD45RA (HI100; IgG2b) was from Pharmingen, CD45RC (MT2; IgG1) was from BioGenex (San Ramon, CA), and anti-CD45 (HI30; IgG1) was from Pharmingen. All isotype controls were from Pharmingen.

    Cell culture. Primary human fetal microglial cell cultures were generated from mixed dissociated brain cell cultures of human fetal abortuses as previously described (40, 41). The protocols were reviewed and approved by the Albert Einstein College of Medicine Institutional Review Board. Briefly, floating cells in confluent monolayer cultures consisting of astrocytes, neurons, and microglia were pooled at 2 to 3 weeks in vitro and reseeded in 96-well plates at 40,000 cells per well or in 60-mm plastic culture dishes at 500,000 cells per dish. Microglial cultures (>98% CD68+) were fed with complete medium (Dulbecco's modified Eagle medium [DMEM] with 5% fetal calf serum and antibiotics). Highly pure primary human fetal astrocyte cultures were generated as previously described (41, 47). Jurkat and HUT78 cells (controls for CD45 expression) were obtained from the laboratories of H. Goldstein and M. Scharff, respectively, at the Albert Einstein College of Medicine.

    HIV-1. HIV-1ADA was obtained from the AIDS Research and Reference Reagent Program and propagated in peripheral blood mononuclear cells (PBMC) as previously described (55). Microglia were exposed to 2,000 50% tissue culture infective doses or 20- to 40-ng/ml p24 HIV-1ADA for 16 h at 37°C, washed, and incubated with fresh medium. Wild-type (WT) or Nef-deficient HIV-1ADA was generated by transfecting 293T cells with proviral plasmids with Effectene reagents (QIAGEN, Valencia, CA) as previously described (55). Vesicular stomatitis virus glycoprotein (VSV-G)/HIV env chimeric viruses were produced by cotransfecting 293T cells with pNL4.3 and pVSV-G env. The resulting viruses enter the cells primarily through the VSV-G env-mediated pathway. Mock supernatants were prepared by treating cells in the same manner without viral plasmids.

    Cell treatment with antibodies and virus. Microglial cell cultures were treated with CD45 or control IgG at 5 μg/ml 1 h prior to viral exposure. HIV-1 was washed out 16 to 24 h later, and then cultures were fed with fresh medium. Thereafter, medium was changed weekly. Antibodies were added back after each medium change.

    Immunoprecipitation and immunoblotting. Microglia, astrocytes, or Jurkat or HUT78 cells were grown in complete medium and harvested in lysis buffer (10 mM Tris-HCl [pH 8.8], 50 mM NaCl, 0.5 mM Na3VO4, 30 mM Na4P2O7, 50 mM NaF, 2 mM EDTA, 1% Triton X-100, and one tablet of the protease inhibitor cocktail [Boehringer-Mannheim, Indianapolis, IN] per 10 ml of lysis buffer). For immunoprecipitations, cell lysates were precipitated with 1.5 μg of anti-Hck (N-30; Santa Cruz Biotechnology) bound to protein A-agarose beads for 4 h at 4°C. The immunoprecipitates were probed with phospho-Src (Y416; Cell Signaling Technology, Beverly, MA) or total Hck (N-30; Santa Cruz) at a 1:1,000 dilution in 5% bovine serum albumin-Tris-buffered saline (TBS)-0.1% Tween-20 overnight at 4°C. Densitometry was performed using Scion Image software (Frederick, MD). For CD45 analysis, protein (70 μg) was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. The blots were blocked in TBS-0.1% Tween-20 containing 5% nonfat milk and then incubated with CD45 or CD45R (isoform-specific) antibodies, all at 1:1,000 dilutions at 4°C for 16 h. The secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG and was used at 1:2,000 for 1 h at room temperature. Total extracellular signal-regulated kinase (ERK) was used as a loading control, as previously described (59). Signals were developed using SuperSignal chemiluminescence reagents (West Pico or West Femto substrates; Pierce Biotechnology, Rockford, IL).

    FACS analysis. Microglial cell surface expression of CD45 isoforms was examined by fluorescence-activated cell sorter (FACS) analysis as previously described (42). Briefly, microglial cells (0.5 x 10 per sample) (6) were lifted from the culture dish by treatment with EDTA. Cells were incubated with 5% normal goat serum (NGS) in DMEM for 30 min at 4°C and then incubated with CD45R antibodies or isotype-matched control IgG at a 1:50 dilution in 5% NGS-DMEM for 1 h at 4°C. Secondary antibodies were fluorescein isothiocyanate-conjugated anti-mouse IgG at a 1:50 dilution for 1 h. Cells were fixed in 2% paraformaldehyde then analyzed with FACSCalibur.

    Immunocytochemistry. Microglial cultures were fixed in 100% ice-cold methanol and were then permeabilized with 0.1% Triton X-100 detergent in phosphate-buffered saline (PBS). Blocking for endogenous peroxidase and for nonspecific binding was performed with 3% H2O2, followed by 5% NGS in PBS, each for 30 min. Cultures were incubated with primary antibodies overnight at 4°C. Mouse anti-HIV-1 p24 monoclonal antibody (MAb; DAKO) was used at a concentration of 1:10. Alkaline phosphatase-labeled goat anti-mouse secondary (Biotechnology Associates, Inc.) was used at 1:200 in PBS, followed by 4-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium (blue chromogen; Sigma, St. Louis, MO).

    EMSA. Electrophoretic mobility shift assay (EMSA) was performed with HIV-1-infected microglial samples as previously described (29). Microglial cultures were treated with antibodies and/or virus as described above, and nuclear extract was harvested after 21 to 24 h. The NF-B consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGG-3') (Promega, Madison, WI) and the CCAAT enhancer binding protein (C/EBP) oligonucleotide (5'-ACTAGCATTTCATCACATGG-3') were derived from the HIV-1 long terminal repeat (LTR) sequences upstream of B sites. Supershift antibodies (p65, p50, c/EBP, c/EBP, and c/EBP) were purchased from Santa Cruz Biotechnology.

    Transient transfection of 293T cells. 293 cells were cotransfected overnight with WT or kinase-inactive (K269E) Hck plasmids obtained from Thomas Smithgall (University of Pittsburgh, Pittsburgh, PA) and the HIV-1 LTR-Luc reporter construct with Effectene reagents. Cultures were exposed to 90-ng/ml p24 of NL4.3/VSV-G env chimeric virus for 2 days, and luciferase activity was determined with a kit from Promega and expressed as relative light units per micrograms of total protein.

    U38 cells and the generation of Hck stable transfectants. WT and kinase-inactive (K269E) human Hck cells were subcloned into pcDNA3.1/Zeo expression vector (Invitrogen Life Technologies). The resulting constructs were used to transfect U38 cells by electroporation (program U-06; Amaxa, Gaithersburg, MD). The stable transfectants were selected in the presence of 50 μg of Zeocin/ml and then expanded. Hck transfectants were exposed to WT or Nef-deficient HIV-1ADA (100 ng of p24/ml) for 2 days, and chloramphenicol acetyl transferase (CAT) expression was determined by enzyme-linked immunosorbent assay (ELISA) with a kit from Roche Diagnostics (Indianapolis, IN).

    In vitro phosphatase assay. Hck immunoprecipitates from HIV-1-infected microglia were used to perform an in vitro phosphatase assay as previously described (27). Microglia (5 x 105) infected with HIV-1ADA for 7 days were harvested in phosphatase buffer. Recombinant CD45 (Calbiochem: San Diego, CA) was then added at 0.01 to 50 U in the presence or absence of 5 μM potassium bisperoxo(1,10-phenanthroline)oxovanadate (phen), a tyrosine phosphatase inhibitor (Calbiochem), for 20 min at 30°C. The lysates were then immunoblotted with anti-phospho-Src (Y416) antibody.

    Preparation of cell permeable Hck SH3 protein. Antennapedia (AP)-conjugated Hck Src homology domain 3 (SH3) was generated essentially as previously described (58). PCRs were performed to generate a chimeric DNA containing Hck SH3, S-protein (marker protein), and antennapedia (RQIKIWFQNRRMKWKK) sequences, and DNA was cloned into the pGEX4.1 vector (Amersham). PCR sequences were as follows (Hck SH3 sequences are underlined): 5'-AAA GGA TCC GAC ATC ATC GTG GTT GCC CTG TAT (sense), 5'-TTT AGC AGC AGC GGT TTC TTT TGT CTC CAG AGA GTC AAC GCG (first-round antisense), 5'-GCT GTC CAT GTG CTG GCG TTC GAA TTT AGC AGC AGC GGT TTC (second-round antisense), 5'-GTT CTG GAA CCA GAT CTT GAT CTG TCT GCT GTC CAT GTG CTG GCG TTC (third-round antisense), and 5'-AAA GTC GAC TCA CTT CTT CCA CTT CAT CCT CCT GTT CTG GAA CCA GAT CTT GAT (fourth-round antisense). A control Nck-SH2 AP protein was generated as previously described (58). The resulting plasmids were transformed into BL21-Gold(DE3) Escherichia coli (Stratagene). Protein expression was induced with IPTG (isopropyl--D-thiogalactopyranoside), and recombinant proteins were purified with glutathione-Sepharose, followed by dialysis against TBS and treatment with thrombin as previously described (58). Transduction efficiency was determined by staining microglia with S-protein-fluorescein isothiocyanate conjugate (1:500; Novagen, Madison, WI).

    Hck SH3-Nef peptide binding assay. Biotinylated Nef peptides (WT, amino acids 65 to 82 of HIV-1 NL4-3 and a mutant Nef peptide; P72, 75A) were generated and incubated with NeutrAvidin-coupled agarose beads (Pierce, Rockford, IL). Following the addition of Hck SH3 protein, samples were separated on 4 to 20% linear gradient gels and Western blotting was performed with S-protein-horseradish peroxidase conjugate.

    Statistical analysis. One-way analysis of variance, followed by Scheffer's multiple comparison procedure, was used. In certain experiments, Student's t test was performed to compare the difference between two treatment groups. Differences between treatments were considered statistically significant when P values were <0.05.

    RESULTS

    Microglia express CD45RB and CD45RO. We first determined the expression of CD45 isoforms in primary human microglial cells by Western blot analysis and FACS analysis using CD45 (reacts with all isoforms) or CD45R (reacts with specific isoforms). Jurkat and HUT78 T cell lines and human astrocytes were used as positive and negative controls, respectively. As shown in Fig. 1A, microglia were reactive with CD45RB and CD45RO, but not CD45RA. CD45RC reactivity was not seen in any of the cells (not shown). A longer exposure with a sensitive substrate (SuperSignal West Femto) was necessary to detect CD45RO, indicating a very low level of expression. Cell surface expression by FACS analysis showed similar results, demonstrating abundant RB, barely detectable RO, and no RA expression (Fig. 1B). From these results we conclude that of the five human CD45 isoforms (ABC, BC, AB, B, and O), microglia express CD45RB and CD45RO.

    CD45RO (UCHL-1) inhibits HIV-1 production from microglia. We next tested several CD45R antibodies for their effect on microglial HIV-1 production. Microglia were exposed to HIV-1ADA, and HIV-1 production was determined as previously described (32, 55, 56). CD45RO (UCHL-1), which we have previously shown to enhance microglial tyrosine phosphatase activity (59), inhibited HIV-1 production from microglia (Fig. 2A). HIV-1 p24 release was inhibited during the entire 4-week culture period; intracellular p24 expression and syncytium formation were also reduced (Fig. 2B). Furthermore, when the timing of CD45RO addition was changed with respect to HIV-1 exposure, antibody addition up to 1 day after viral exposure still showed a similar degree of inhibition (Fig. 2C). While CD45RB expression was abundant on microglia, two different CD45RB antibodies tested (PD7/26 and MT4) did not alter viral production from microglia (Fig. 2D). Together, the results show that although microglia express a low level of CD45RO, CD45RO (UCHL-1) is effective in stimulating microglial CD45 PTPase activity and inhibiting HIV-1 production. They further show that CD45RO inhibits HIV-1 replication at some point after viral entry. The results with CD45RB antibodies suggest that they are ineffective antibodies (see Discussion).

    Anti-CD45RO or rCD45 prevent phosphorylation of the Hck tyrosine kinase in microglia. Because Hck has been shown to interact with the HIV-1 Nef PxxP motif with high affinity, resulting in autophosphorylation of Hck (8, 54), we asked whether HIV-1 induces phospho-Hck and whether CD45RO (or rCD45; see below) dephosphorylates Hck tyrosine kinase in microglia. HIV-1-infected microglial lysates were immunoprecipitated with -total Hck and then immunoblotted with -phospho-Src (Y416) that cross-reacts with several Src kinases (Lyn, Fyn, Lck, Yes, and Hck) that are phosphorylated at the corresponding tyrosine residue (Fig. 3A and C). The results show that p-Hck is induced in HIV-1-infected microglia and that CD45RO inhibits p-Hck. In some experiments, total microglial lysates were immunoblotted with -p-Src antibody without immunoprecipitation with -Hck (Fig. 3B). These experiments yielded positive bands corresponding to p-Hck (59 to 61 kDa). The results show that the level of HIV-1-induced p-Src is reduced by CD45RO in microglia. Densitometric ratios (p-Src to T-Hck) from pooled data are shown in Fig. 3C.

    We next tested the effect of rCD45 protein (D1 and D2 domains) on HIV-1-induced phosphorylation of Hck by incubating HIV-1-infected microglial cell lysates with various concentrations of rCD45 protein. Immunoprecipitation and immunoblotting were performed for p-Hck as above. As shown in Fig. 3D, rCD45 at 1 to 50 U inhibited p-Hck induced by HIV-1 infection. PHEN, a phosphatase inhibitor, reversed the effect of rCD45. Densitometric analysis of this blot is shown in Fig. 3E. Additional experiments using lower concentrations of rCD45 (1, 0.1, 0.01, and 0 U) demonstrate a dose-dependent inhibition of p-Src (two experiments). Densitometric analysis of one blot is shown on the right in Fig. 3E. These results directly demonstrate that HIV-1-induced p-Hck is a substrate for CD45 tyrosine phosphatase and indicate that the effect of CD45RO on microglial HIV-1 production is likely due to its stimulation of CD45 tyrosine phosphatase activity.

    The role of Nef. Since CD45 has an effect on Hck phosphorylation and since Hck has been shown to interact with Nef, we wanted to examine the role of Nef in CD45RO-mediated effect. Microglial cultures were infected with Nef+ or Nef– HIVADA, and the pHck induction was compared. Results shown in Fig. 3F and G show that p-Hck was induced in Nef+ (WT) but not in Nef– cultures, demonstrating that Nef was necessary in the induction of p-Hck. In an effort to establish the role of Nef, we next compared the effect of CD45RO on p24 production in microglial cultures infected with Nef+ (WT) or Nef– HIV-1ADA. CD45RO's inhibitory effect was observed in all cultures infected with Nef+ virus. However, the replication of Nef– virus in microglial cultures was consistently poor (not shown). Therefore, we could not establish a role of Nef in CD45RO-mediated suppression of p24 release (see Discussion).

    Role of Hck-SH3 domain. To directly demonstrate a role of Nef in activating Hck in microglia, we generated a cell-permeable form of Hck SH3 protein to transduce microglia. We surmised that adding excessive amounts of Hck-SH3 protein (the domain that binds to Nef PxxP motifs with high affinity) would inhibit the interaction between endogenous Hck protein and Nef in HIV-1-infected microglia. Antennapedia-mediated delivery of the Hck SH3 protein to microglia was efficient, as demonstrated by immunofluorescence for the marker protein S-protein (Fig. 4A). The specific in vitro binding between Hck SH3 protein and the Nef peptide bearing the PxxP motif (WT), but not mutated (p72, 75A) Nef, (54) was also demonstrated (Fig. 4B). We determined the effect of overexpression of Hck SH3 proteins on HIV-1 p24 production and Hck phosphorylation following HIV-1 infection of microglia. As shown, treatment of microglia with cell-permeable Hck SH3 protein inhibited p24 production, whereas the control Nck protein did not (Fig. 4C). Furthermore, inhibition of Hck phosphorylation was observed in an immunoprecipitation assay of Hck SH3-treated, HIV-1-infected microglial cell cultures but not control (Nck-treated) cultures (Fig. 4D). Together, these results support that in primary microglia, Nef is involved in phosphorylation of Hck tyrosine kinase and that p-Hck positively regulates HIV-1 production.

    Role of Hck in LTR transactivation in cell lines. Although our results support a positive role for Hck tyrosine kinase in HIV-1 replication, conflicting reports exist with respect to the role of Hck in HIV-1 replication (24, 34). Therefore, we next tested the effect of overexpression of kinase-inactive (K269E) Hck protein in HIV-1 LTR transactivation (1). First, 293T cells were transiently transfected with WT or (K269E) Hck and HIV LTR-Luc constructs and were subsequently infected with NL4.3/VSV-G env chimeric virus. As shown in Fig. 5A, HIV-1-induced LTR transactivation was induced in WT but not in kinase-inactive Hck-expressing cells, supporting a positive role for Hck (2). Second, we generated a stable transfectant of WT or kinase-inactive Hck in a macrophage cell line containing two copies of stably integrated HIV-1 LTR (U38 cells) (18). The cells were then infected with Nef+ or Nef– HIV-1ADA and the reporter gene (CAT) expression was determined. As shown in Fig. 5B, LTR transactivation was seen in WT but not in kinase-inactive Hck-expressing cells; furthermore, Nef+ (but not Nef–) virus was able to induce LTR transactivation. Western blot analysis confirmed overexpression of Hck protein in both WT and KE transfectants compared to parental cells (Fig. 5C). Together, these results establish a positive role of Hck in HIV-1 transcription and the involvement of Nef in the observed effect.

    CD45RO effect on LTR transactivation. The results with kinase-inactive Hck suggest that the effects of CD45RO in microglia and macrophages should converge at the level of LTR activity. We directly examined this first in U38 cells. In U38 cells, CD45RO treatment inhibited HIV-1ADA-induced LTR activity (Fig. 6A). In microglia, we examined the activation of transcription factors required for HIV-1 transcription, i.e., NF-B and C/EBP proteins (25, 28), by EMSA. In HIV-1-infected microglia, p50/p50 and p65/p50 NF-B complexes were formed and CD45RO decreased the amount of these complexes (Fig. 6B). Densitometric analysis was performed and pooled data are shown in Fig. 6C: they show that HIV-1-induced NF-B activation is significantly inhibited by CD45RO in microglia. Because the C/EBP family transcription factors are important for HIV transcription in macrophages, we analyzed the formation of nuclear C/EBP complexes in microglia. We show that HIV-1-infected microglia exhibit C/EBP complexes composed of C/EBP and C/EBP, but not C/EBP (Fig. 6D). Treatment of microglia with CD45RO reduced the amount of C/EBP complexes. Together, our results demonstrate formation of NF-B and C/EBP complexes following HIV-1 infection of primary microglia. They suggest that CD45RO inhibits HIV-1 production from microglia, in part by preventing activation of transcription factors required for HIV-1 transcription in macrophages.

    DISCUSSION

    In the present study, we demonstrate that treatment of microglia with CD45RO antibody inhibited HIV-1 production. The inhibition was strong and sustained through the entire period of culture (4 weeks) in a spreading infection model of primary human fetal microglial culture when antibody was administered weekly. Furthermore, antibody administered after the viral exposure still inhibited viral production from microglia. Our results are the first to demonstrate an antiviral activity of CD45 MAb in macrophages and in combination with the reported anti-inflammatory properties of CD45 suggest the possibility for a novel CD45-based therapy to treat HIV-1 infection.

    We show that Nef was necessary in the activation of Hck kinase in HIV-1-infected microglia and that CD45RO inhibited HIV-1 production from microglia by suppressing Hck phosphorylation. Nef is an early regulatory protein known to play a crucial role in HIV-macrophage interactions. Nef protein from HIV-1 and simian immunodeficiency virus is a membrane-associated myristoylated protein that is critical for high levels of viremia and the progression to AIDS in infected humans and monkeys (14, 30). Nef is known to trigger multiple signaling cascades in infected cells (50, 53). In macrophages, Nef activates CD40-like signaling that renders T cells permissive to HIV-1 infection (60). Nef also modulates survival of HIV-1-infected and bystander cells, contributing to viral persistence (7, 20). These findings together indicate that Nef functions as the master regulator of viral and cellular activation and that inactivating Nef could lead to the suppression of a number of downstream effects, resulting in the prevention or halting of AIDS. In vitro, Nef has an especially high affinity for Hck (38, 54). Binding between the proline-rich motif of Nef [(P69xxP)4] and the SH3 domain of Hck has been shown to cause autophosphorylation and activation of Hck and to result in oncogenic transformation of certain cells (8). However, the biological consequences of the Nef-Hck interaction in HIV-1-infected cells are unknown, as HIV-1-infected monocyte-derived macrophages reportedly show no detectable p-Hck (9). To determine the mechanism by which CD45RO inhibits HIV-1 production from microglia, we first examined whether Hck phosphorylation occurs in HIV-1-infected microglia. Our results demonstrate that Hck phosphorylation (corresponding to Y416 in pSrc) occurs following HIV-1 infection in a Nef-dependent manner. These differing results in monocytes and microglia suggest that the growth conditions of microglia are more permissive to Hck activation. For example, microglia in vivo and in vitro differentiate in the presence of astrocytes that are known producers of M-CSF (23, 41, 42); in monocyte-derived macrophages, M-CSF has been shown to increase HIV-1 production by modulating the amount of Hck protein expression (34).

    Conflicting data exist with regard to the role of Hck kinase in HIV-1 replication, since both positive and negative effects have been reported. While Komuro et al. showed a positive role in macrophages by use of antisense Hck treatment (34), another study reported Hck as an inhibitor of HIV-1 replication counteracted by the Vif protein (24). In our study, several lines of evidence indicate that Hck positively contributed to HIV-1 replication. First, HIV-1 LTR transactivation did not occur in cells (U38 and 293T) overexpressing the kinase-inactive Hck mutant (43). Second, in microglia, transfer of the Hck SH3 domain using the antennapedia-Hck fusion protein also suppressed HIV-1-dependent activation of Hck and suppressed HIV-1 replication. In our microglial cultures, we saw changes in Hck phosphorylation, but we did not see the changes in total Hck protein expression (this report and data not shown), indicating that posttranslational modification rather than transcription (34) is the main mechanism of Hck regulation. These results demonstrate the positive role of Hck tyrosine kinase in HIV-1 replication and provide a physiologically relevant context for the high-affinity interaction between Nef and Hck. Moreover, our results confirm others by Hiipakka et al., who showed that cellular functions of HIV-1 Nef can be inhibited by artificial domains of Hck SH3 (26) and further suggest that the SH3 strategy can be utilized for therapy.

    The downstream pathway(s) of pHck that leads to enhanced HIV-1 expression is unclear, but we observe that CD45RO inhibits virus-induced NF-B and C/EBP activation in microglia, suggesting them as possible downstream targets of Hck. The HIV-1 LTR has two B sites near the transcriptional initiation site; occupancy of the enhancer by the NF-B p65/p50 heterodimer is required for ongoing viral transcription in monocyte-lineage cells (28). HIV-1-infected microglia show both p65/p50 and p50/p50 dimers, and CD45RO reduces the amounts of both. Because NF-B also activates a host of macrophage genes involved in inflammation and tissue damage, these results suggest that CD45RO could block the generation of macrophage activation products implicated in the pathogenesis of AIDS dementia (33, 39, 46). C/EBPs have also been shown to be crucial in HIV-1 LTR transactivation in macrophages (25). We show for the first time that C/EBP complexes are induced in HIV-1-infected primary microglia and inhibited by CD45RO and that the C/EBP complexes are composed of C/EBP and C/EBP, but not C/EBP. Similar to NF-B, the C/EBP proteins are involved in transactivation of macrophage inflammatory genes, in addition to viral promoters (65). Therefore, our results show that in vivo, CD45RO could potentially affect different signaling pathways, resulting in synergistic inhibition of HIV-1 transcription and microglial activation.

    Although we demonstrated that CD45RO inhibited Hck autophosphorylation, our results do not exclude the possibility that CD45 targeted additional signaling pathways induced by HIV-1 in microglia. For example, because of the poor replication of Nef– virus in microglia, contribution of viral factors other than Nef could not be definitely excluded. Furthermore, in various cell contexts, Nef has been shown to alter cell signaling directly or indirectly by interacting with various kinases such as a serine/threonine kinase, p21-activated kinase mitogen-activated protein kinases, c-Raf, and protein kinase C (50, 53). In macrophages, Nef activates NF-B (60), as well as activating protein-1 and ERK mitogen-activated protein kinase (5). p21-activated kinase is known to play a positive regulatory role in HIV expression (45) and is also expressed in macrophages (9), and it is thus another potential target of CD45 in microglia. Furthermore, other myeloid Src kinases such as Fgr (44) could have been an additional target of CD45 in microglia.

    Our study is not the first to demonstrate a role of MAbs directed against CD45 in the regulation of microglial cell activity, as previous studies with a mouse model reported suppression of microglial activation by CD45 MAb, particularly by CD45RB (63). This clone of CD45RB (MB23G2) is also effective in the treatment of renal and islet cell transplantation rejection in the mouse, showing in vivo efficacy as a therapeutic tool (3, 37). However, not all CD45RB MAbs are effective in the mouse (3); our experiments indicated that CD45RB did not work effectively in human microglia. Because of the lack of response to CD45RB MAb, we wondered whether human microglial cells do not express CD45RB. Our investigation of CD45 isoform expression in human microglia in vitro and in vivo demonstrated that microglia express CD45RB and CD45RO. The level of microglial CD45RO was low, while CD45RB was abundant. In vivo, CD45RB and CD45RO are also expressed in microglia, but a sensitive technique is required for detection of CD45RO. CD45RO is upregulated in HIVE (M. Cosenza et al., submitted for publication). The abundance of CD45RB isoform is also reported for murine microglia in vitro (63), and thus the difference in the antibody activity is unlikely due to the difference in CD45 isoform expression but rather related to the fine specificity of each antibody. The UCHL-1 clone of CD45RO appears to be particularly effective in human cells, including lymphoid cells (6, 59), whereas antihuman CD45RB clones (MT4 and PD7/26) are shown to be ineffective in our study. Importantly, CD45RB did not induce tyrosine phosphatase activity in human microglia (not shown). Together, our data suggest that CD45RO could efficiently target CD45RO, which is upregulated in HIV encephalitis.

    In our experiments with human microglia and mouse antibodies, we observed that normal mouse IgG2a often showed a partial effect compared to no antibody controls (Fig. 3 and 6). These results culd indicate that some of the antibody effect was mediated by the Fc portion of the immunoglobulin. Mouse IgG2a binds to human Fc receptors with high affinity and can trigger cell activation (13, 57), an effect that could be overcome by isotope switching or by humanizing the therapeutic antibody (22).

    In addition to macrophages, T cells are productively infected with HIV-1; therefore, it would be important to determine the effect of CD45 MAb on T cell HIV-1 production. Earlier studies by Baur et al. demonstrated that an antibody to CD45 indeed inhibited HIV production from human PBMC (4), suggesting that anti-CD45 strategy might lower the systemic viral load as well. The specific effect of CD45RO on viral replication in PBMC has not yet been tested, but in Jurkat T cells HIV-1 has been shown to replicate preferentially in CD45RO+ cells (2, 52). Our results hint at the possibility of targeting peripheral CD45RO+ memory T cells in HIV-positive individuals with an agonist antibody to maximize virus eradication in vivo. Further studies are necessary to elucidate how CD45RO may alter virological, immunological, and clinical parameters of HIV-1-infected individuals.

    ACKNOWLEDGMENTS

    This study was supported by NIH RO1 MH55477 and AI44641 to S.C.L., RO1 CA086289 to B.I.T., and Einstein CFAR AI051519. Q.S. was supported by NIH training grant NS07098.

    We thank Wa Shen for microglial cultures, the Einstein Human Fetal Tissue Repository for tissue, and the Einstein Laboratory for Macromolecular Analysis and Proteomics (LMAP) for peptide synthesis. The members of the Lee laboratory are acknowledged for their helpful comments and technical assistance. Melissa Cosenza provided editorial assistance. We thank Thomas Smithgall for his generous gift of Hck plasmids and Jin S. Im for her help for the generation of Hck stable transfectants. Mario Stevenson provided the Nef+ and Nef– HIV-1ADA plasmids and NL4-3 HIV provirus. Other viruses were provided by the NIH AIDS Research and Reference Reagent Program.

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