FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080511074> ?p ?o ?g. }

• W2080511074 endingPage "19369" @default.
• W2080511074 startingPage "19364" @default.
• W2080511074 abstract "Activation of CD4 positive T cells is a primary requirement for human immunodeficiency virus (HIV) entry, efficient HIV replication, and progression to AIDS. Utilizing CD4 positive T cell lines and purified T cells from normal individuals, we have demonstrated that native envelope glycoproteins of HIV, gp160, can induce activation of transcription factor, activated protein-1 (AP-1). The stimulatory effects of gp160 are mediated through the CD4 molecule, since treatment of gp160 with soluble CD4-IgG abrogates its activity, and CD4 negative T cell lines fail to be stimulated with gp160. Immunoprecipitation of the gp160-induced nuclear extracts with polyclonal antibodies to Fos and Jun proteins indicates that AP-1 complex is comprised of members of these family of proteins. The gp160-induced AP-1 complex is dependent upon protein tyrosine phosphorylation and is protein synthesis-independent. This stimulation can also be abolished by inhibitors of protein kinase C, but it is unaffected by calcium channel blocker or cyclosporine A. This gp160 treatment adversely affects the functional capabilities of T cells; pretreatment of CD4+ T cells with gp160 for 4 h at 37°C inhibited anti-CD3-induced interleukin-2 secretion. Effects similar to gp160 were seen with anti-CD4 mAb. The aberrant activation of AP-1 by gp160 in CD4 positive T cells could result in up-regulation of cytokines containing AP-1 sites, e.g. interleukin-3 and granulocyte macrophage colony-stimulating factor, and concurrently lead to T cell unresponsiveness by inhibiting interleukin-2 secretion. Activation of CD4 positive T cells is a primary requirement for human immunodeficiency virus (HIV) entry, efficient HIV replication, and progression to AIDS. Utilizing CD4 positive T cell lines and purified T cells from normal individuals, we have demonstrated that native envelope glycoproteins of HIV, gp160, can induce activation of transcription factor, activated protein-1 (AP-1). The stimulatory effects of gp160 are mediated through the CD4 molecule, since treatment of gp160 with soluble CD4-IgG abrogates its activity, and CD4 negative T cell lines fail to be stimulated with gp160. Immunoprecipitation of the gp160-induced nuclear extracts with polyclonal antibodies to Fos and Jun proteins indicates that AP-1 complex is comprised of members of these family of proteins. The gp160-induced AP-1 complex is dependent upon protein tyrosine phosphorylation and is protein synthesis-independent. This stimulation can also be abolished by inhibitors of protein kinase C, but it is unaffected by calcium channel blocker or cyclosporine A. This gp160 treatment adversely affects the functional capabilities of T cells; pretreatment of CD4+ T cells with gp160 for 4 h at 37°C inhibited anti-CD3-induced interleukin-2 secretion. Effects similar to gp160 were seen with anti-CD4 mAb. The aberrant activation of AP-1 by gp160 in CD4 positive T cells could result in up-regulation of cytokines containing AP-1 sites, e.g. interleukin-3 and granulocyte macrophage colony-stimulating factor, and concurrently lead to T cell unresponsiveness by inhibiting interleukin-2 secretion. INTRODUCTIONThe CD4 molecule is the binding site of the human immunodeficiency virus via the envelope glycoprotein, gp160/gp120(1Capon D. Ward R.H.R. Annu. Rev. Immunol. 1991; 9: 649-678Google Scholar). This interaction occurs at a specific region at the external domain of the CD4 molecule. There have been conflicting reports on the ability of gp160/gp120 to transduce biochemical signals through the CD4 molecule on T cells. While increase in intracellular calcium, hydrolysis of phosphatidyl inositol, and activation of tyrosine kinases have been demonstrated by some(2Kornfeld H. Cruickshank W.W. Pyle S. Berman J.S. Center D.M. Nature. 1988; 335: 445-454Google Scholar, 3Soula M. Fagard R. Fischer S. Int. Immunol. 1992; 4: 295-299Google Scholar, 4Hivroz C. Mazerolles F. Soula M. Fagard R. Graton S. Meloche S. Sekaly R.-P. Fischer A. Eur. J. Immunol. 1993; 23: 600-607Google Scholar, 5Cruickshank W.W. Center D.M. Pyle S.W. Kornfeld H. Biomed. Pharmacother. 1990; 44: 5-11Google Scholar), others have failed to observe these events(6Horak I.D. Popovic M. Horak E. Lucas P. Gress R.E. June C.H. Bolen J. Nature. 1990; 348: 557-560Google Scholar, 7Orloff G.M. Kennedy M.S. Dawson C. McDougal J.S. AIDS Res. Hum. Retroviruses. 1991; 7: 587-593Google Scholar).The interaction of the CD4 molecule with the nonpolymorphic β2 domain of the MHC 1The abbreviations used are: MHCmajor histocompatibility classHIVhuman immunodeficiency virusAP-1activated protein-1ILinterleukinPMAphorbol 12-myristate 13-acetateEMSAelectrophoretic mobility shift assayTCRT cell receptormAbmonoclonal antibody. class II molecule has been demonstrated to play a vital role in activation of mature T cells and in T cell development in the thymus(8Janeway C.A. Semin. Immunol. 1991; 3: 153-160Google Scholar). Several studies have now demonstrated that the CD4-MHC class II interaction is essential for effective signal transduction, at low antigen concentrations, to increase the avidity, in co-receptor-dependent systems(9Glainchenhaus N. Shastri N. Littman D.R. Turner J.M. Cell. 1991; 64: 511-520Google Scholar, 10Zamoyska R. Derham P. Gorman S.D. von Hoegden P. Bolen J.B. Veillette A. Parnes J.R. Nature. 1989; 342: 278-280Google Scholar). Studies demonstrating the association of the src homologous tyrosine kinase p56lck and the putative p32 G-protein with the cytoplasmic tail of the CD4 molecule have demonstrated that biochemical signals can be transduced through the CD4 molecule(11Veillette A. Abraham N. Caron L. Davidson D. Semin. Immunol. 1991; 3: 143-152Google Scholar, 12Telfer J.C. Rudd C.E. Science. 1991; 254: 439-441Google Scholar). In this respect, exposure of CD4+ T cells to anti-CD4 mAb or HIV gp120 has been shown to induce activation of the Raf-1-related 110-kDa polypeptide, and phosphatidylinositol 3- and phosphatidylinositol 4-kinases (13Prasad K.V.S. Keppler R. Janssen O. Repke H. Duke-Cohan J.S. Cantley L.C. Rudd C.E. Mol. Cell. Biol. 1993; 13: 7708-7717Google Scholar) and activation of NF-κB(14Chirmule N. Kalyanaraman V.S. Pahwa S. Biochem. Biophys. Res. Commun. 1994; 203: 498-505Google Scholar).Utilizing soluble envelope glycoproteins of HIV-1, gp160, we have previously demonstrated that CD4-mediated signals result in biological effects. These include up-regulation of CD40 ligand in CD4+ T cells, resulting in polyclonal B cell differentiation(15Chirmule N. Kalyanaraman V.S. Lederman S. Oyaizu N. Yagura H. Yellin M.J. Chess L. Pahwa S. J. Immunol. 1993; 150: 2478-2486Google Scholar); induction of IL-3, IL-6, and granulocyte macrophage colony-stimulating factor mRNA and cytokine secretion, which induce increased myelopoiesis(16Than S. Oyaizu N. Pahwa R.N. Kalyanaraman V.S. Pahwa S. Blood. 1994; 84: 184-189Google Scholar); and increased expression of Fas antigen on CD4+ T cells, resulting in accelerated apoptosis in peripheral blood mononuclear cells(17Oyaizu N. McCloskey T. Than S. Hu R. Kalyanaraman V.S. Pahwa S. Blood. 1994; 84: 2622-2631Google Scholar). To delineate the nature of the biochemical signals transduced through the CD4 molecule on T cells, we have investigated the ability of gp160 and anti-CD4 mAbs to induce activation of the transcription factor, activated protein 1 (AP-1).Physiological activation of T cells through the T cell receptor results in the activation of AP-1(18Jain J. Valge-Archer V.E. Rao A. J. Immunol. 1992; 148: 1240-1250Google Scholar). AP-1 is a collection of homodimeric and heterodimeric protein complexes of the c-fos and c-jun proto-oncogene products(19Curran T. Franza B.R. Cell. 1988; 55: 395-397Google Scholar). These proteins interact with a common DNA binding site, the TPA-responsive element (TGA(C/G)TCA) and activate gene transcription(20Lee W. Mitchell P. Tijan R. Cell. 1987; 49: 741-752Google Scholar). The binding of AP-1 to the TPA-responsive element has been attributed to post-translational modification of preexisting members of the Fos and Jun family of proteins, involving phosphorylation and dephosphorylation events(21Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Google Scholar). Our results have demonstrated that the CD4-induced signals transduced by gp160 or anti-CD4 mAb gp160 in T cells result in activation of AP-1 by a mechanism that involves post-translational activation of Fos and Jun family of proteins.Depression of antigen-specific T cell responses is a relatively early feature of HIV infection and precedes the quantitative decline of CD4+ T cells(22Fauci A. Science. 1988; 239: 617-622Google Scholar). Several investigators have clearly demonstrated the inhibitory effects of gp120 on normal T cell functions (for review, see (23Habeshaw J.A. Dalgleish A.G. Bountiff L. Newell A.L. Wilks D. Walker L.C. Manca F. Immunol. Today. 1990; 11: 418-425Google Scholar)). The mechanism of gp120-mediated inhibition of T cell responses involves inhibition of intracellular calcium mobilization, hydrolysis of inositol phosphates, and activation of protein kinase C, and kinase activity of p56lck(24Chirmule N. Kalyanaraman V.S. Oyaizu N. Slade H. Pahwa S. Blood. 1990; 75: 152-159Google Scholar, 25Oyaizu N. Chirmule N. Kalyanaraman V.S. Hall W.W. Good R.A. Pahwa S. Proc. Natl. Acad. Sci. U. S. A. 1989; 87: 2379-2383Google Scholar, 26Oyaizu N. Chirmule N. Pahwa S. J. Clin. Invest. 1992; 89: 1807-1816Google Scholar, 27Goldman F. Jensen W.A. Johnson G.L. Heasley L. Cambier J. J. Immunol. 1994; 153: 2905-2917Google Scholar, 28Kalyanaraman V.S. Veronese F. Rahman R. Lusso P. Devico A.L. Copeland T. Oroszlan S. Gallo R.C. Sarngadharan M.G. AIDS Res. Hum. Retroviruses. 1990; 6: 371-380Google Scholar). The reduced proliferative responses were attributed to inhibition of decreased IL-2 mRNA expression and IL-2 secretion(25Oyaizu N. Chirmule N. Kalyanaraman V.S. Hall W.W. Good R.A. Pahwa S. Proc. Natl. Acad. Sci. U. S. A. 1989; 87: 2379-2383Google Scholar). In this study, we have suggested that binding of envelope glycoproteins of HIV to CD4+ T cells induces aberrant activation of the transcription factor AP-1 (which plays a critical role in IL-2 gene transcription, (18Jain J. Valge-Archer V.E. Rao A. J. Immunol. 1992; 148: 1240-1250Google Scholar)) and results in inhibition anti-CD3 mAb-induced IL-2 secretion.MATERIALS AND METHODSEnvelope Glycoproteinsgp160 and gp120 were purified from culture supernatants of a clone of HIV-infected Hut-78 cells, 6D4451, as described earlier(28Kalyanaraman V.S. Veronese F. Rahman R. Lusso P. Devico A.L. Copeland T. Oroszlan S. Gallo R.C. Sarngadharan M.G. AIDS Res. Hum. Retroviruses. 1990; 6: 371-380Google Scholar). Briefly, supernatant of cells grown in serum-free HB104 medium was concentrated and passed through a lentil-lectin Sepharose column. Glycoproteins were eluted with 400 mM α-methyl mannoside. gp160 was further purified by affinity chromatography over anti-HIV451 mAb-Sepharose 4B column. The envelope glycoprotein preparations were >95% pure and were not contaminated with endotoxins, as tested by the Limulus amoebocyte lysate assay (E-TOXATE, Sigma).Antibodies and ReagentsThe following reagents and resources were used: mAb to CD4 (Leu3a, IgG1; Becton Dickinson, Mountainview, CA); mAb to CD3 (mAb 454, IgG2a, gift from Dr. N. Chiorazzi, North Shore University Hospital, Manhasset, NY); nonimmune mouse Ig (mIg; Chrompure IgG, Jackson ImmunoResearch, West Grove, PA). Polyclonal antibodies to Fos and Jun proteins used were as follows; rabbit anti-c-Jun/AP1 and anti-c-Fos (Santa Cruz Biotechologies, Santa Crus, CA) recognize all of the members of the Fos/Jun family of proteins. Polyclonal rabbit anti-gp120451 was developed by Advanced BioScience Labs Inc., Kensington, MD; soluble CD4-IgG was a gift from Genentech, San Fransisco, CA. Herbimycin A (Life Technologies, Inc.), cyclosporine A (Sandoz, East Hanover, NJ), H-7, cycloheximide, 2-mercaptoethanol, verapamil, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma.CellsCD4 positive clone of Jurkat T cells, E6-1, obtained from the National Institutes of Health AIDS Reference Reagent Program, Bethesda, MD, (donated by Dr. A. Weiss, (29Weiss A.L. Skocil R.J. Stobo J.D. J. Immunol. 1984; 133: 123-128Google Scholar)) was maintained in RPMI 1640 medium (Whittaker) supplemented with pennicillin and streptomycin and 10% fetal calf serum. CD4 positive T cells H9, Molt4 were obtained from ATCC, Betheda, MD. CD4 negative Jurkat T cells (JN) were mutant CD4 negative (CD3+) cells by fluorescence-activated cell sorting analysis following staining with fluorescein isothiocyanate-conjugated anti-CD3 mAb and anti-CD4-conjugated with phycoerythrin (Becton Dickinson, Mountainview, CA).Peripheral blood lymphocytes were purified by Ficoll-Hypaque density gradient centrifugation. T cells were purified from peripheral blood lymphocytes by rosetting 2 times with neuraminidase-treated sheep red blood cells as described earlier(15Chirmule N. Kalyanaraman V.S. Lederman S. Oyaizu N. Yagura H. Yellin M.J. Chess L. Pahwa S. J. Immunol. 1993; 150: 2478-2486Google Scholar).Immunomagnetic Separation of CD4+ and CD8+ T CellsPurified T cells stimulated with medium alone or with gp160 for 4 h were incubated with anti-CD8 mAb conjugated immunomagnetic beads (Dynal, Great Neck, NY) for 30 min at 4°C on a rotating shaker, as recommended by the manufacturer. The cells were subjected to a magnetic field, and the unbound cells (designated CD8 negative, CD8- T cells) were carefully aspirated. The cells bound to the beads were designated CD8 positive (CD8+) and were >90% CD4+ as determined by flow cytometry. Nuclear proteins were extracted as described below.Nuclear ExtractsSmall scale nuclear extracts were made from 2 × 107 unactivated or activated E6-1 cells as described previously(18Jain J. Valge-Archer V.E. Rao A. J. Immunol. 1992; 148: 1240-1250Google Scholar). Unless otherwise stated, cells were stimulated with medium alone or various stimuli for 4 h at 37°C. Cells were washed and resuspended in 10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride and lysed by the addition of Nonidet P-40 to a final concentration of 0.5%. Nuclei were pelleted and washed in the same buffer without Nonidet P-40, and nuclear proteins were extracted in buffer C (20 mM HEPES, pH 7.4, mM 0.42 NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 0.01% NaN3)(30Dignam J.D. Leibowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). After pelleting neclear debris, the supernatant was removed and diluted with an equal volume of buffer D (20 mM HEPES, pH 7.4, 50 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 0.01% NaN3). This extract was used directly in the electrophoretic mobility shift assay (EMSA). The equivalence of the extracts was verified by protein estimation using the BCA protein kit (Pierce).EMSADouble-stranded oligonucleotides corresponding to the consensus AP-1 binding sequence (5′-CGC TTG ATG AGT CAG CGC GAA-3′ (31Angel P. Imagawa M. Chiu R. Stein B. Imbra R.J. Rahmsdorf H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Google Scholar)) were obtained commercially (Promega, Madison, WI) and end-labeled with [γ32P]ATP and polynucleotide kinase. In some experiments, the binding site of AP-1 to the human IL-2 promoter (5′-AATTCCAAAGAGTCATCAGA-3′) was used as a competitor(18Jain J. Valge-Archer V.E. Rao A. J. Immunol. 1992; 148: 1240-1250Google Scholar). For each binding reaction, 10,000 cpm (0.2-0.5 ng) of end-labeled oligonucleotide was incubated for 30 min at room temperature with 5-8 μg of nuclear extract in the presence of 3 mg of sheared poly(dI-dC) (Pharmacia Biotech Inc.). The resulting DNA-protein complexes were analyzed by electrophoresis at 4°C of 4% polyacrylamide gels. Unlabeled oligonucleotides used for competitions were added to nuclear extracts and poly(dI-dC) prior to the addition of labeled probe. Gels were dried on a gel drier (Bio-Rad) and visualized by autoradiography. In some experiments, nuclear extracts were incubated with antibodies to c-Jun, c-Fos, or normal rabbit serum for 1 h on ice, followed by incubation with Protein A-conjugated Sepharose (Pharmacia) for 30 min at 4°C. After centrifugation at 5000 × g for 10 min, supernatants were analyzed for AP-1 binding in EMSA, as described above.IL-2 SecretionCD4+ E6-1 cells, CD4- JN cells, or purified CD4+ T cells were pretreated with medium, various concentrations of gp120, or anti-CD4 mAb (mAb Leu3a) followed by stimulation with anti-CD3 mAb (mAb 454) plus 10 ng/ml PMA for 24 h. Culture supernatants were collected and analyzed for the presence of IL-2 by commercial ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's protocol.RESULTSgp160-induced AP-1 Activation in CD4+ T CellsFig. 1shows that the basal level of AP-1 activation in the CD4+ E6-1 cells could be enhanced by stimulation with gp160 at a concentration as low as 0.01 mg/ml. This concentration of envelope proteins has been previously reported in the serum of HIV-infected individuals(32Oh S.-Y. Cruickshank W.W. Raina J. Blanchard G.C. Adler W.H. Walker J. Kornfeld H. J. AIDS. 1992; 5: 251-256Google Scholar). Soluble gp120, anti-CD4 mAb (Leu3a) and PMA alone could also induce activation of AP-1 in the CD4+ E6-1 cells. Specific binding was demonstrated by abrogation of the AP-1 binding in the presence of excess unlabeled oligonucleotides corresponding to the AP-1 site in the IL-2 promoter (competitor, lane10). The kinetics of the induction of the proteins was examined by using nuclear extracts from E6-1 cells stimulated for increasing amounts of time. The gp160-induced AP-1 binding was observed within 30 min, peaked at 4 h, persisted for 24 h of stimulation (Fig. 2). The gp160-induced AP-1 binding was specific, since it could be abrogated by pretreatment of gp160 with goat anti-gp120 polyclonal antibody (Fig. 3, compare lane2 with lanes6 and 7); normal goat serum had no significant effect on the stimulatory effect of gp160 (lane8). That the constituents of the gp160-induced AP-1 complex comprises Jun and Fos components was confirmed by abrogation of AP-1 binding by immunoprecipitation of the gp160-stimulated nuclear extracts with polyclonal antibodies to c-Jun and c-Fos (Fig. 4); even the basal level binding of AP-1 was inhibited by the addition of these antibodies. Normal rabbit serum had no significant effect on the gp160-induced AP-1 binding. These results indicate that gp160 can induce activation of AP-1 in CD4+ T cells and that the AP-1 complex consists of Fos/Jun family of proteins.Figure 2:Kinetics of the gp160-induced AP-1 activation. E6-1 cells were stimulated with 1 mg/ml gp160 for various time intervals indicated. AP-1 binding was analyzed by EMSA. The lowerband shows nonspecific binding (ns).View Large Image Figure ViewerDownload (PPT)Figure 3:Pretreatment of gp160 with soluble CD4-IgG or anti-gp160 antibodies abrogates its activity. E6-1 cells were stimulated with medium alone (lane1, unsti) or 1 μg/ml gp160 (lanes2-8) in the presence of 10 or 1 μg/ml soluble CD4-IgG (Genentech, CA) (lanes3 and 4) or 1 μg/ml bovine serum albumin, (lane5), 1:1000, 1:3000 dilution of polyclonal goat anti-gp160 antibodies (lanes6 and 7), or 1:1000 dilution of normal goat serum (lane8). EMSA were performed as described under “Materials and Methods.”View Large Image Figure ViewerDownload (PPT)Figure 4:The gp160-induced AP-1 complex contains Fos and Jun family of proteins. Nuclear extracts were generated from E6-1 cells either unstimulated (lane1) or stimulated with 1 μg/ml gp160 for 4 h at 37°C (lanes2-8). The gp160-induced nuclear extracts were incubated with medium (lanes1 and 2), 1 μg (in 1 μl), or 0.1 μg (in 0.1 μl) of antibodies to Fos and Jun proteins (which recognize all of the members of the Fos/Jun family of proteins) or normal rabbit serum for 1 h at 4°C. Immune complexes were immunoprecipitated with Protein A-Sepharose beads (Pharmacia), and supernatants were analyzed for AP-1 binding. Results are representative of five separate experiments.View Large Image Figure ViewerDownload (PPT)The gp160-induced Activation of AP-1 Was Mediated through the CD4 MoleculeIn order to demonstrate that the cell surface molecule involved in the gp160-mediated stimulatory effects was CD4, gp160 was first preincubated with soluble CD4- IgG for 30 min at 4°C, prior to addition to the cell cultures. Fig. 3shows that the stimulatory activity of gp160 on E6-1 cells could be abrogated by pretreatment of gp160 with soluble CD4-IgG (compare lane2 with lanes3 and 4). An irrelevant protein, bovine serum albumin, did not have any effect on the gp160-induced activation of AP-1 (lane5).In order to further demonstrate that gp160-induced activation of AP-1 was mediated through the CD4 molecule, CD4+ and CD4 negative T cell lines were analyzed. Fig. 5shows that gp160 could stimulate AP-1 activation in CD4+ H9 cells, Molt4 cells, but not in CD4 negative mutant Jurkat T cells (JN). Here again, pretreatment of gp160 with soluble CD4 abrogated AP-1 activation in CD4+ T cells. All of these cells could be effectively induce AP-1 activation upon stimulation with PMA. These results demonstrate that the stimulatory activity of gp160 on AP-1 activation is mediated through the CD4 molecule.Figure 5:CD4 positive T cell lines, but not CD4 negative T cell lines could be induced by gp160 to increase AP-1 activation. H9 cells were stimulated with medium alone (lane1, unsti), or with PMA, gp160, or gp120 (lanes2, 3, and 4) in the presence of soluble CD4 IgG (lanes5, 6, and 7); E6-1 cells were stimulated with medium alone (lane8), PMA and gp160 (lanes9 and 10) as positive controls. CD4 positive Molt4 and CD4 negative JN (mutant CD4 negative Jurkat cells) were stimulated with medium alone (lanes1 and 7, unsti), gp160, gp120 (lanes2 and 3 and lanes8 and 9) in the presence of soluble CD4-IgG (lanes4 and 5 and lanes10 and 11) or PMA alone (lanes6 and 12).View Large Image Figure ViewerDownload (PPT)Gp160 Induced AP-1 Activation in Peripheral Blood CD4+ T CellsIn order to determine the ability of gp160 to activate physiological cells and peripheral blood T cells, purified T cells were first stimulated with gp160 for 4 h at 37°C. CD4+ T cells were separated from CD8+ T cells by negative selection using anti-CD8 mAb-conjugated magnetic beads; cells bound to the beads were designated CD8+, and unbound T cells designated CD8-. Fig. 6shows that stimulation of CD8- T cells (>90% CD4+ by flow cytometry) with gp160 induced AP-1 activation (upperrightpanel) no activation of AP-1 occurred in the CD8+ T cells (upperleftpanel). The stimulatory effects of gp160 on CD8- T cells could be abrogated by pretreatment of gp160 with soluble CD4- IgG (lowerpanel). Soluble CD4- IgG itself did not induce activation of AP-1 (data not shown).Figure 6:gp160 can induce CD8- but not CD8+ peripheral blood T cells to induce AP-1 binding. Upper panel, purified T cells were stimulated with medium alone (lane1, unsti), 1 μg/ml gp160 (lanes2 and 3), or gp120 (lanes4 and 5). CD4 and CD8 positive T cells were separated by anti-CD8 mAb-conjugated magnetic beads (Dynal, Great Neck, NY). Adherent cells were denoted CD8 positive and nonadherent cells as CD4 positive. Nuclear extracts were assessed for AP-1 binding by EMSA. The arrow indicates specific binding for AP-1, and the lower band indicates nonspecific binding (ns). Lower panel, gp160 induced AP-1 binding in CD4+ peripheral blood T cells can be abrogated by soluble CD4- IgG. Purified T cells were stimulated with medium alone (lane1, unsti) or 1 μg/ml gp160 (lanes2 and 3) or gp120 (lanes4 and 5) in the presence of soluble CD4-IgG (lanes3 and 5); 50 ng/ml PMA (lane6). Nuclear extracts of CD4+ T cells were analyzed for AP-1 binding by EMSA.View Large Image Figure ViewerDownload (PPT)Signal Requirements for the gp160-induced AP-1 ActivationIn order to investigate the nature of the signals involved in the activation of AP-1 by gp160, several pharmacological inhibitors were utilized. Fig. 7shows that addition of herbimycin A (HA, inhibitor of tyrosine phosphorylation), and H7 (inhibitor of protein kinase C) abrogated gp160-induced AP-1 activation. A more specific inhibitor of protein kinase C activity, calphostin (33Gublins H. Coggeshall K.M. Baier G. Telford D. Langlet C. Baier-Bitterlich G. Berrard-Bonnefoy N. Burn P. Wittinghofer A. Altman A. Mol. Cell. Biol. 1994; 14: 4749-4758Google Scholar) also inhibited the gp160-induced AP-1 binding (data not shown). Cyclosporine A (CsA) and the protein synthesis inhibitor, cycloheximide (CHX) had no significant effect on activation of AP-1, as did the calcium channel blocker, verapamil (ver). These studies indicate that tyrosine phosphorylation and activation of protein kinase C were involved in the mechanism of the gp160-induced activation of AP-1 binding.Figure 7:The gp160-mediated AP-1 binding is dependent on tyrosine phosphorylation, activation of protein kinase C, but not on protein synthesis or increase in intracellular calcium or CsA. E6- 1 cells were pretreated with various concentrations of cyclosporine A (CsA), herbimycin A (HA), verapamil (ver), cycloheximide (CHX), or H7 and stimulated with gp160 for 4 h at 37°C. Electromobility shift assays were performed as described under “Materials and Methods.”View Large Image Figure ViewerDownload (PPT)gp120 Inhibits IL-2 Secretion by CD4+ T CellsE6-1 cells or purified peripheral blood CD4+ T cells were pretreated with 1 μg/ml gp120 or anti-CD4 mAb for 4 h at 37°C (which induced AP-1 binding) and stimulated with anti-CD3 mAb plus PMA for an additional 24 h at 37°C, and culture supernatants were analyzed for IL-2 secretion. Table I shows that gp120- or anti-CD4 mAb-treated E6-1 cells and CD4+ peripheral blood T cells were markedly inhibited in their ability to secrete IL-2 in response to anti-CD3 mAb. Pretreatment of the CD4 negative T cells (JN) cells with gp120 or anti-CD4 mAb, however, had no effect on anti-CD3 mAb plus PMA-induced IL-2 secretion.DISCUSSIONWe have demonstrated that the addition of gp160 to CD4+ T cells induces activation of transcription factor AP-1 by signals transduced directly through the CD4 molecule.Signals transduced through the CD4 molecule on T cells has been shown to play an important role in regulating T cell functional responses mediated through the T cell receptor(8Janeway C.A. Semin. Immunol. 1991; 3: 153-160Google Scholar). Earlier studies had implicated that the binding (adhesion) of the CD4 molecule with its natural ligand, MHC class II molecule, participated in T cell activation by stabilizing the T cell receptor (TCR)-MHC interactions (34Biddison W.E. Rao P.E. Talle M.A. Goldstein G. Shaw S. J. Immunol. 1983; 131: 152-187Google Scholar). In addition, inhibition of T cell activation by anti-CD4 mAbs in MHC class II independent systems suggested that inhibitory signals were transduced through the CD4 molecule(35Bank I. Chess L. J. Exp Med. 1985; 162: 1294-1303Google Scholar). In contrast, recent experiments have indicated that positive signals may be induced via the CD4 molecule, either by anti-CD4 mAbs (36Carrel S. Salvi S. Gallay P. Rapin C. Sekaly R.P. Res.Immunol. 1991; 142: 97-108Google Scholar) or by HIV envelope glycoproteins, gp160/gp120(2Kornfeld H. Cruickshank W.W. Pyle S. Berman J.S. Center D.M. Nature. 1988; 335: 445-454Google Scholar, 3Soula M. Fagard R. Fischer S. Int. Immunol. 1992; 4: 295-299Google Scholar, 4Hivroz C. Mazerolles F. Soula M. Fagard R. Graton S. Meloche S. Sekaly R.-P. Fischer A. Eur. J. Immunol. 1993; 23: 600-607Google Scholar, 5Cruickshank W.W. Center D.M. Pyle S.W. Kornfeld H. Biomed. Pharmacother. 1990; 44: 5-11Google Scholar). Although the CD4-induced signals have been shown to synergize with anti-CD3 or anti-TCR mAb (37Ledbetter J.A. Deans J.P. Aruffo A. Grsomarie L.S. Kanner S.B. Bolen J.B. Schieven G.L. Curr. Opin. Immunol. 1993; 5: 334-343Google Scholar) the aberrant persistent activation through this molecule on T cells, may contribute to the pathogenesis of disease, e.g. in HIV infection(38Ascher M.S. Sheppard H.W. J. AIDS. 1990; 3: 177-191Google Scholar).Cellular activation plays a central role in HIV infection(22Fauci A. Science. 1988; 239: 617-622Google Scholar). Virus internalization, syncitium formation, and proviral replication have been shown to require cellular activation(39Gowda S.D. Stein B.S. Mohagheghpour N. Benike C.J. Engleman E.G. J. Immunol. 1989; 142: 773-780Google Scholar, 40McDougal J.S. Mawle A. Cort S.P. Nicholson J.K.A. Cross G.D. Scheppler-Campbell J.A. Hicks D. Sligh J. J. Immunol. 1985; 135: 3151-3162Google Scholar). Several investigators have demonstrated that binding of gp160/gp120 to CD4 molecules on the cell surface results in activation of biochemical signals(2Kornfeld H. Cruickshank W.W. Pyle S. Berman J.S. Center D.M. Nature. 1988; 335: 445-454Google Scholar, 3Soula M. Fagard R. Fischer S. Int. Immunol. 1992; 4: 295-299Google Scholar, 4Hivroz C. Mazerolles F. Soula M. Fagard R. Graton S. Meloche S. Sekaly R.-P. Fischer A. Eur. J. Immunol. 1993; 23: 600-607Google Scholar, 5Cruickshank W.W. Center D.M. Pyle S.W. Kornfeld H. Biomed. Pharmacother. 1990; 44: 5-11Google Scholar). We have demonstrated that binding of gp160, (at concentrations found in vivo,(32Oh S.-Y. Cruickshank W.W. Raina J. Blanchard G.C. Adler W.H" @default.
• W2080511074 created "2016-06-24" @default.
• W2080511074 creator A5002278563 @default.
• W2080511074 creator A5014900807 @default.
• W2080511074 creator A5033173015 @default.
• W2080511074 creator A5051571388 @default.
• W2080511074 creator A5065426964 @default.
• W2080511074 creator A5070314915 @default.
• W2080511074 date "1995-08-01" @default.
• W2080511074 modified "2023-09-27" @default.
• W2080511074 title "HIV-1 Envelope Glycoproteins Induce Activation of Activated Protein-1 in CD4+ T Cells" @default.
• W2080511074 cites W1487277447 @default.
• W2080511074 cites W1589819858 @default.
• W2080511074 cites W1593158365 @default.
• W2080511074 cites W1966815515 @default.
• W2080511074 cites W1967004332 @default.
• W2080511074 cites W1975791322 @default.
• W2080511074 cites W1977302628 @default.
• W2080511074 cites W1980692649 @default.
• W2080511074 cites W1993243592 @default.
• W2080511074 cites W1997901301 @default.
• W2080511074 cites W1999747291 @default.
• W2080511074 cites W2002588802 @default.
• W2080511074 cites W2003058837 @default.
• W2080511074 cites W2007096062 @default.
• W2080511074 cites W2024395223 @default.
• W2080511074 cites W2025227540 @default.
• W2080511074 cites W2025701387 @default.
• W2080511074 cites W2026951979 @default.
• W2080511074 cites W2032856364 @default.
• W2080511074 cites W2044457945 @default.
• W2080511074 cites W2052977964 @default.
• W2080511074 cites W2053132428 @default.
• W2080511074 cites W2055456747 @default.
• W2080511074 cites W2064022513 @default.
• W2080511074 cites W2065114401 @default.
• W2080511074 cites W2077309544 @default.
• W2080511074 cites W2077880395 @default.
• W2080511074 cites W2078676274 @default.
• W2080511074 cites W2081135713 @default.
• W2080511074 cites W2084223366 @default.
• W2080511074 cites W2087586960 @default.
• W2080511074 cites W2118477707 @default.
• W2080511074 cites W2126440278 @default.
• W2080511074 cites W2129190720 @default.
• W2080511074 cites W2134352843 @default.
• W2080511074 cites W2148370330 @default.
• W2080511074 cites W2178145019 @default.
• W2080511074 cites W2240730501 @default.
• W2080511074 cites W252613826 @default.
• W2080511074 cites W99188018 @default.
• W2080511074 doi "https://doi.org/10.1074/jbc.270.33.19364" @default.
• W2080511074 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7642615" @default.
• W2080511074 hasPublicationYear "1995" @default.
• W2080511074 type Work @default.
• W2080511074 sameAs 2080511074 @default.
• W2080511074 citedByCount "50" @default.
• W2080511074 countsByYear W20805110742013 @default.
• W2080511074 countsByYear W20805110742015 @default.
• W2080511074 countsByYear W20805110742018 @default.
• W2080511074 countsByYear W20805110742019 @default.
• W2080511074 crossrefType "journal-article" @default.
• W2080511074 hasAuthorship W2080511074A5002278563 @default.
• W2080511074 hasAuthorship W2080511074A5014900807 @default.
• W2080511074 hasAuthorship W2080511074A5033173015 @default.
• W2080511074 hasAuthorship W2080511074A5051571388 @default.
• W2080511074 hasAuthorship W2080511074A5065426964 @default.
• W2080511074 hasAuthorship W2080511074A5070314915 @default.
• W2080511074 hasBestOaLocation W20805110741 @default.
• W2080511074 hasConcept C108625454 @default.
• W2080511074 hasConcept C12554922 @default.
• W2080511074 hasConcept C159047783 @default.
• W2080511074 hasConcept C185592680 @default.
• W2080511074 hasConcept C3013748606 @default.
• W2080511074 hasConcept C41008148 @default.
• W2080511074 hasConcept C554190296 @default.
• W2080511074 hasConcept C55493867 @default.
• W2080511074 hasConcept C65155139 @default.
• W2080511074 hasConcept C76155785 @default.
• W2080511074 hasConcept C86803240 @default.
• W2080511074 hasConcept C95444343 @default.
• W2080511074 hasConceptScore W2080511074C108625454 @default.
• W2080511074 hasConceptScore W2080511074C12554922 @default.
• W2080511074 hasConceptScore W2080511074C159047783 @default.
• W2080511074 hasConceptScore W2080511074C185592680 @default.
• W2080511074 hasConceptScore W2080511074C3013748606 @default.
• W2080511074 hasConceptScore W2080511074C41008148 @default.
• W2080511074 hasConceptScore W2080511074C554190296 @default.
• W2080511074 hasConceptScore W2080511074C55493867 @default.
• W2080511074 hasConceptScore W2080511074C65155139 @default.
• W2080511074 hasConceptScore W2080511074C76155785 @default.
• W2080511074 hasConceptScore W2080511074C86803240 @default.
• W2080511074 hasConceptScore W2080511074C95444343 @default.
• W2080511074 hasIssue "33" @default.
• W2080511074 hasLocation W20805110741 @default.
• W2080511074 hasOpenAccess W2080511074 @default.
• W2080511074 hasPrimaryLocation W20805110741 @default.
• W2080511074 hasRelatedWork W1516164909 @default.

• next