(Received for publication, February 2, 1995; and in revised form, June 2, 1995)
From the
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.
The CD4 molecule is the binding site of the human immunodeficiency virus via the envelope glycoprotein, gp160/gp120(1) . 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(2, 3, 4, 5) , others have failed to observe these events(6, 7) .
The
interaction of the CD4 molecule with the nonpolymorphic 2 domain
of the MHC (
)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(8) . 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(9, 10) . Studies
demonstrating the association of the src homologous tyrosine
kinase p56
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(11, 12) . 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 (13) and activation of NF-
B(14) .
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(15) ; induction of IL-3, IL-6, and granulocyte macrophage colony-stimulating factor mRNA and cytokine secretion, which induce increased myelopoiesis(16) ; and increased expression of Fas antigen on CD4+ T cells, resulting in accelerated apoptosis in peripheral blood mononuclear cells(17) . 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(18) . AP-1 is a collection of homodimeric and heterodimeric protein complexes of the c-fos and c-jun proto-oncogene products(19) . These proteins interact with a common DNA binding site, the TPA-responsive element (TGA(C/G)TCA) and activate gene transcription(20) . 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(21) . 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(22) . Several investigators have clearly demonstrated the
inhibitory effects of gp120 on normal T cell functions (for review, see (23) ). 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
p56(24, 25, 26, 27, 28) .
The reduced proliferative responses were attributed to inhibition of
decreased IL-2 mRNA expression and IL-2 secretion(25) . 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, (18) ) and results in inhibition anti-CD3 mAb-induced IL-2
secretion.
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(15) .
Figure 1:
Stimulation of CD4 positive T cells
with gp160 induces AP-1 activation. Stimulation of E6-1 cells was
carried out by the addition of medium alone (lane1, unsti) or various concentrations of gp160 (lanes2, 3, and 4) and gp120 (lanes5, 6, and 7), or 1 µg/ml of
anti-CD4 mAb (Leu3a, lane8) or 50 ng/ml PMA (lane9), for 4 h at 37 °C. Lane10 comprised of competition of AP-1 binding by 10 cold AP-1
oligonucleotides corresponding to the IL-2 AP-1 site. The hollowarrow indicates mobility of free probe, and the solidarrow indicates the position of specific AP-1 binding.
The result is a representative of at least five separate
experiments.
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).
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.''
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.
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).
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.
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.''
We 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(8) . 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 (34) . 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(35) . In contrast, recent experiments have indicated that positive signals may be induced via the CD4 molecule, either by anti-CD4 mAbs (36) or by HIV envelope glycoproteins, gp160/gp120(2, 3, 4, 5) . Although the CD4-induced signals have been shown to synergize with anti-CD3 or anti-TCR mAb (37) the aberrant persistent activation through this molecule on T cells, may contribute to the pathogenesis of disease, e.g. in HIV infection(38) .
Cellular
activation plays a central role in HIV infection(22) . Virus
internalization, syncitium formation, and proviral replication have
been shown to require cellular activation(39, 40) .
Several investigators have demonstrated that binding of gp160/gp120 to
CD4 molecules on the cell surface results in activation of biochemical
signals(2, 3, 4, 5) . We have
demonstrated that binding of gp160, (at concentrations found in
vivo,(32) ) to CD4 molecules on T cells can induce
biological events, e.g. up-regulation of CD40
ligand(15) , secretion of IL-6, IL-3, granulocyte macrophage
colony-stimulating factor, interferon , tumor necrosis factor
(16, 17) , and up-regulation of Fas antigen (17) on CD4
T cells. These observations
unequivocally demonstrate that gp160 can transduce signals through the
CD4 molecule in T cells culminating into biological events.
In the present study, we have demonstrated that gp160 can induce activation of AP-1 in CD4+ T cells. The presence of Fos/Jun family of proteins in the gp160-induced AP-1 complex was confirmed by abrogation of AP-1 binding in immunoprecipitation experiments. Further studies are needed to determine the involvement and functional role of individual Fos/Jun components in the gp160-induced AP-1 complex. The stimulatory effects of gp160 are mediated through the CD4 molecule, since pretreatment of gp160 with soluble CD4- IgG abrogates its activity. Furthermore, cell lines expressing the CD4 molecule (H9, Molt4), but not CD4 negative cell line (JN), can be induced by gp160 to activate AP-1. gp160 can also stimulate peripheral blood CD4 positive cells, but not CD8 positive T cells to activate AP-1. Finally, the stimulatory effects of gp160 can be mimicked by anti-CD4 mAb. These results clearly demonstrate AP-1 activation by direct stimulation through the CD4 molecule.
Post-translational modifications upon T cell activation involve activation of pre-existing Fos/Jun by intracellular kinases and phosphatases(19) . The observation that protein synthesis inhibitor, CHX failed to abrogate (and in fact augments) the gp160-induced AP-1 binding at 4 h suggests that post-translational modification of preexisting Fos and/or Jun induces AP-1 binding at 4 h. Tyrosine phosphorylation inhibitor, herbimycin A, abrogated the gp160-induced AP-1 binding. In resting cells, the pre-existing Jun is phosphorylated on three sites at the C-terminal domain next to its DNA binding domains (21) ; phosphorylated states of these sites inhibits DNA binding(41) . The activation of Jun requires dephosphorylation of these sites, possibly by a protein kinase C-activated phosphatase(42) . Calcineurin, the phosphatase that modulates NFAT activity(43) , is not involved in c-Jun dephosphorylation, since AP-1 binding is unaffected by treatment of cells with cyclosporine A. While intracellular calcium channel blocker, verapamil, failed to block AP-1 binding, the requirement of protein kinase C activation for the gp160-mediated AP-1 binding was demonstrated by the addition of inhibitors, H7 and calphostin. Understanding the precise nature of the post-transcriptional modification involving activation of the Ha-Ras oncoprotein(44, 45) , mitogen-activated protein kinase pathway(46) , or the newly described JNK and SAPK pathways (47, 48) may give an insight into the regulatory role of CD4-mediated signals in T cell activation.
AP-1 has been
demonstrated to be target for T cell clonal anergy, as demonstrated by
down-modulation of AP-1 binding and transactivation in anergized T cell
clones(49) . We and others have previously demonstrated that
pretreatment of CD4+ T cells with envelope proteins of HIV-1 can
induce unresponsiveness of T cells upon stimulation through
TCRCD3
complex(23, 24, 25, 26) . It is
possible that the stimulatory effect of gp160 on AP-1 binding,
involving the repressive members of the Jun family, i.e. JunB(50, 51) , which may inhibit IL-2 gene
transcription. Our previous studies have demonstrated that pretreatment
of T cell clones with envelope glycoproteins or anti-CD4 mAb inhibited
IL-2 secretion at the transcriptional level(25, 26) .
In this study, we have shown that pretreatment of the CD4+ T cells
with gp160 or anti-CD4 mAb for 4 h at 37 °C (which results in
activation of AP-1 binding) inhibits anti-CD3 plus PMA-induced IL-2
secretion. Since the inhibition of IL-2 secretion by gp120 in these
experiments was independent of the presence of antigen presenting
cells, it can be hypothesized that signals mediated through the CD4
molecule in T cells, which results in activation of AP-1, may interfere
with signal transduction through the TCR
CD3 complex.
Given
that the promoters of IL-2, IL-3, IL-6, granulocyte macrophage
colony-stimulating factor, TCR, and the HIV long terminal repeat
contain AP-1 binding
sites(18, 52, 53, 54, 55, 56) ,
it is possible that the gp160-induced activation of AP-1 in T cells can
regulate the expression of these molecules. Since c-Jun and c-Fos have
also been implicated in the mechanism of
apoptosis(57, 58) , it is possible that gp160-induced
AP-1 activation may play a significant role in apoptosis mechanisms in
HIV infection. In conclusion, we have demonstrated that soluble
envelope glycoproteins of HIV-1, gp160, by binding to the CD4 molecule
on T cells, may transduce signals that result in aberrant activation of
AP-1. These effects by gp160, or by HIV itself in vivo, may
contribute to biological events, e.g. enhanced HIV
replication, hypergammaglobulinemia, increased cytokine secretion,
hypercellularity in bone marrow, apoptosis, and induction of T cell
unresponsiveness.