Extracellular HIV Tat and Tat cysteine rich peptide increase CCR5 expression in monocytes

Extracellular HIV Tat and Tat cysteine rich peptide increase CCR5 expression in monocytes 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yidayang@hotmail.com †E-mail:yidayang@hotmail.com yidayang@hotmail.com 1 2 Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Human Virology, University of Maryland Biotechnology Center, Baltimore, MD 21201, USA Institute of Human Virology, University of Maryland Biotechnology Center, Baltimore, MD 21201, USA In our previous work we reported that HIV Tat and 6 cysteine rich peptides of Tat induce tumor necrosis factor-related apoptosis-induced ligand (TRAIL) in human monocytes (Yang et al., 2003 2003 ). Here our results showed that HIV Tat and Tat cysteine rich peptide increase CCR5 expression in human monocytes, and this activity is inhibited by rabbit anti-Tat. Boiled Tat does not increase CCR5 expression in monocytes. These results provide insight into a new mechanism by which HIV Tat plays a key role in the pathogenesis of HIV-1 infection. HIV Tat Tat cysteine rich peptide CCR5 Monocytes INTRODUCTION 1 The human immunodeficiency virus-type 1 (HIV-1) Tat protein is essential for HIV-1 transcription and replication, and also modulates the expression of genes responsible for cell survival and proliferation (Jons, 1993 1993 ). Tat is also a secreted protein that can be detected in sera from HIV-infected individuals and that binds to cell surfaces through electrostatic interactions, chemokine receptors, or cell surface integrins and activates various interacellular signal transduction pathways (Barillari et al., 1993 1993 ). Cells treated with Tat showed increased expression of chemokine receptors CCR5 and CXCR4 (Huang et al., 1998 1998 ), overproduction of Interleukine-2 (IL-2) (Westendorp et al., 1994 1994 ), Interleukine-8 (IL-8) (Ott et al., 1998 1998 ) and enhanced expression of TNF-α related apoptosis induced ligand (TRAIL) (Yang et al., 2003 2003 ) on monocyte/macrophages that could cause cell death in cultured peripheral blood mononuclear cells (PBMC). Tat protein can also be taken up by infected and uninfected PBMC, thus inducing biological effects through an autocrine/paracrine mechanism (Zauli et al., 1995 1995 ). HIV Tat contains several functional subdomains, the first 13 amino acids (N-terminal) with amphipathic characteristics. Cysteines rich domain (aa 22–37), and the core region (1 to 48) together constitute the minimal activation domain for transcription in vitro. The cysteine rich subdomains are highly conserved between different isolates of HIV-1s (Jeang et al., 1999 1999 ). The cysteine rich domain mediates the pathogenesis of HIV infection in monocytes in vitro (Tikhonov et al., 2003 2003 ), and is also involved in monocyte chemotaxis (Boykins et al., 1999 1999 ; Albini et al., 1998 1998 ). The role of chemokines in regulating the progression of HIV infection has been identified by the ability of chemokines receptor of function as co-receptors that mediate HIV entry (Alkhatib et al., 1996 1996 ; Choe et al., 1996 1996 ). This study indicates that HIV Tat and cysteine rich peptide of Tat up-regulate CCR5 expression in monocytes, and that polyclonal rabbit anti-Tat inhibits CCR5 expression. MATERIALS AND METHODS 1 Reagents 2 HIV Tat protein, Tat-86, with a C-terminal truncation of 15 amino acids was obtained from the National Institute of Health AIDS Research and Reference Reagent Program (ARRRP). FITC-anti-CD4, phycoerythrin (PE)-anti-human CD8 + , FITC-anti-CD14, PE-anti-CCR5 (Pharmingen, San Diago, CA) were used for Flow Cytometry. The Tat peptides were as follows, with the included amino acid residues indicated in the subscript: Tat 1–20 (Peptide 1) is MEPVDPRLEPWKHPGSQPKT; Tat 16–35 (Peptide 8) is SQPKTACTNCYCKKCCFHCQ; Tat scramble (Peptide 12, a scramble of peptide 8) is TCCQKNKCPTKHQCCFSAYC; Tat 31–50 (Peptide 13) is CFHCQVCFMTKALGISYGRK; Tat 46–65 (Peptide 19) is SYGRKKRRQRRRAHQDSQTH; Tat 61–80 (Peptide 25) is NSQTHQASLSKQPTSQSR GD; Tat 76–95 (Peptide 31) is QSRGDPTGPKESKKK VERET. Cells and culture 2 Human monocytes were isolated from PBMC of healthy donors by adherence to plastic (i.e., they were incubated for 2 h in T125 flasks in RPMI 1640 medium containing 10% heat-inactivated AB serum). Fluorescence-activated cell sorter analysis demonstrated that more than 98% of the cells were CD14 + monocytes of high purity. CD4 + lymphocytes were purified by negative-selection on affinity columns (R & D systems, Minneapolis, MN). The purity of recovered cells was more than 90%, with no detectable crossing-contamination by CD8 + cells. CD4 + cells were incubated with 10% heat-inactivated FCS, 2 mmol/L L-glutamine, 100 U penicillin/ml, 100 g streptomycin/ml, and 50 IU interleukin-2/ml (RO 23-6019; Hoffmann-La Roche, Nutley, NJ). Flow Cytometry 2 Monocytes were detached with 0.5 mmol/L EDTA/PBS and washed with PBS. 5×10 5 monocytes were treated with HIV Tat or Tat peptide, then FITC-conjugated anti-CD14 (a monocyte marker) and PE-conjugated anti-CCR5 were added for 30 min at 4 °C. Cells were fixed in 2% paraformaldehyde and analyzed by Flow Cytometry. Gated acquisition of monocytes (10000 events) was performed based on forward- and side-scatter parameters. RESULTS 1 HIV Tat up-regulates CCR5 expression on monocytes 2 Human monocytes incubated with extracellular HIV Tat for 5 d express more cell-surface CCR5, as detected by Flow Cytometry, than monocytes incubated without Tat (Fig. 1 1 ). In a dose-dependent manner, HIV Tat stimulation caused a gradual increase in CCR5 expression that reached maximal levels by day 5. One hundred nmol/ml was the most appropriate concentration, as indicated in Fig. 2 2 . To further determine the specificity of HIV Tat activity, we mixed polyclonal rabbit anti-Tat with HIV Tat for 30 min, then added the mixture to monocytes, and observed that expression of CCR5 was inhibited by the rabbit anti-Tat. In order to exclude lipopolysacride (LPS) contamination, we also used boiled Tat (100 °C, 10 min) as a control, as indicated in Fig. 3 3 . Boiled Tat does not increase CCR5 expression in monocytes. Cysteine rich peptide of Tat is responsible for increasing CCR5 expression on monocytes 2 In order to determine which portion of Tat was responsible for up-regulation of CCR5 we incubated monocytes with different peptides covering six overlapping regions of the Tat molecule. Only peptide 8, the cysteine rich peptide, is responsible for CCR5 induction, the other peptides do not up-regulate CCR5 expression (Fig. 4 4 ). DISCUSSION 1 In this study, we demonstrated that extracelluar Tat and Tat cysteine rich peptide 8 dramatically increase HIV co-receptor CCR5 expression in monocytes. Tat and Tat peptide 8 increase CCR5 expression in monocytes. Polyclonal rabbit anti-Tat inhibits this activity. CCR5 is a key co-receptor in HIV infection, especially in macrophage tropic HIV infection. Our results indicated that Tat dramatically induced CCR5 expression in monocytes. These results are complementary to those reported by Boykins et al.( 1999 1999 ), who showed that HIV Tat and cyteine rich Tat peptide increase NF-kB and HIV-LTR transactivation in monocytes, corresponding to p24 levels in HIV infected monocytes. Detectable levels of Tat had been reported in HIV-infected patients (Westendorp et al., 1995 1995 ), which suggested the presence of extracellular HIV Tat protein in certain phases of HIV infection. It has also been shown that high levels of anti-Tat Abs are directly related to low viral load in seropositive nonprogression patients. Therefore, a strategy targeting required sites in Tat might provide a new therapeutic modality to reduce disease progression in HIV-infected patients. References A Albini , R Benelli , D Giunciuglio , T Cai , G Mariani , S Ferrini , DM Noonan . Identification of a novel domain of HIV Tat involved in monocyte chemotaxis . J Biol Chem , 1998 . 273 15895 - 15900 . . Identification of a novel domain of HIV Tat involved in monocyte chemotaxis G Alkhatib , C Combadiere , CC Broder , Y Feng , PE Kennedy , PM Murphy , EA Berger . CC Ck5: A RANTES, MIP-1 alpha, MIP-1 beta receptor as a fusion cofactor for macrophage-tropic HIV-1 . Science , 1996 . 272 1955 - 1958 . . CC Ck5: A RANTES, MIP-1 alpha, MIP-1 beta receptor as a fusion cofactor for macrophage-tropic HIV-1 G Barillari , R Gendelman , RC Gallo , B Ensoli . The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence . Proc Natl Acad Sci USA , 1993 . 90 7941 - 7945 . . The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence RA Boykins , R Mahieux , UT Shankavaram , YS Gho , SF Lee , IK Hewlett , LM Wahl , HK Kleinman , JN Brady , KM Yamada , 等 . A short polypeptide domain of HIV-1-Tat protein mediates pathogenesis . J Immunol , 1999 . 163 15 - 20 . . A short polypeptide domain of HIV-1-Tat protein mediates pathogenesis H Choe , M Farzan , Y Sun , N Sullivan , B Rollins , PD Ponath , L Wu , CR Mackay , G LaRosa , W Newman , 等 . The beta-chemokine receptor CCR3 and CCR5 facilitate infection by primary HIV-1 isolates . Cell , 1996 . 85 1135 - 1148 . . The beta-chemokine receptor CCR3 and CCR5 facilitate infection by primary HIV-1 isolates L Huang , I Bosch , W Hoffmann , J Sodroski , AB Pardee . Tat protein induces human immunodeficiency virus type 1 (HIV-1) coreceptors and promotes infection with both macrophage-tropic and T-lymphotropic HIV-1 strains . J Virol , 1998 . 72 8952 - 8960 . . Tat protein induces human immunodeficiency virus type 1 (HIV-1) coreceptors and promotes infection with both macrophage-tropic and T-lymphotropic HIV-1 strains KT Jeang , H Xiao , EA Rich . Multifaceted activities of the HIV-1 transactivator of transcription, Tat . J Biol Chem , 1999 . 27 28837 - 28840 . . Multifaceted activities of the HIV-1 transactivator of transcription, Tat KA Jons . Tat and HIV-1 promoter . Curr Opin Cell Biol , 1993 . 5 461 - 468 . . Tat and HIV-1 promoter M Ott , J Lee Lovett , L Mueller , E Verdin . Supperinduction of interleukine-8 in T cells by HIV Tat is mediated by NF-kB factors . J Immunol , 1998 . 160 2872 - 2880 . . Supperinduction of interleukine-8 in T cells by HIV Tat is mediated by NF-kB factors I Tikhonov , TJ Ruckwardt , GS Hatfield , CD Pauza . Tat-neutralizing antibodies in vaccinated macaques . J Virol , 2003 . 77 3157 - 3166 . . Tat-neutralizing antibodies in vaccinated macaques MO Westendorp , M Li-Weber , RW Frank , PH Krammer . Human immunodeficiency virus type 1 Tat up-regulates interleukin-2 secretion in activated T cells . J Virol , 1994 . 68 4177 - 4182 . . Human immunodeficiency virus type 1 Tat up-regulates interleukin-2 secretion in activated T cells MO Westendorp , R Frank , C Ochsenbauer , K Stricker , J Dhein , H Walczak , KM Debatin , PH Krammer . Sensitization of T cells to CD95-mediated apoptosis by HIV-Tat and gp120 . Nature , 1995 . 375 497 - 500 . . Sensitization of T cells to CD95-mediated apoptosis by HIV-Tat and gp120 Y Yang , I Tikhonov , T Ruckwardt , M Djavani , JC Zapata , CD Pauza , MS Salvato . Monocytes treated with human immunodeficiency virus Tat kill uninfected CD4 + cells by a tumor necrosis factor related apoptosis induced ligand mediated mechanism . J Virol , 2003 . 77 6700 - 6708 . . Monocytes treated with human immunodeficiency virus Tat kill uninfected CD4+ cells by a tumor necrosis factor related apoptosis induced ligand mediated mechanism G Zauli , M La Placa , M Vignoli , MC Re , D Gibellini , G Furlini , D Milani , M Marchisio , M Mazzoni , S Capitani . An autocrine loop of HIV Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal human immunodeficiency virus type 1 long terminal repeat transactivating activity . J Acquir Immune Defic Syndr , 1995 . 10 306 - 316 . . An autocrine loop of HIV Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal human immunodeficiency virus type 1 long terminal repeat transactivating activity 10.1631/jzus.2005.B0668 2004-06-01 2005-01-17 2005-06-23 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 6B