Transient modulation and internalization of T4 antigen induced by phorbol esters

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.     

Biological characterization of a simian immunodeficiency virus-like retrovirus (HTLV-IV): evidence for CD4-associated molecules required for infection

We have analyzed a number of biological features of HTLV-IV, a retrovirus indistinguishable from a macaque isolate of simian immunodeficiency virus (SIV), and compared this virus with several strains of human immunodeficiency virus type 1 (HIV-1). Although HTLV-IV was found to be similar to HIV-1 in its tropism for CD4+ lymphocytes, its effects on CD4 expression and the ability of its externalized envelope molecule to form a complex directly with the CD4 molecule, a number of striking differences were noted. Unlike with HIV-1, the range of cells susceptible to HTLV-IV infection and syncytia formation was restricted to a subset of CD4+ cell lines, particularly those that coexpressed CD4 with human leukocyte antigen (HLA) class II antigens. An analysis of the patterns of HTLV-IV infection with B x T somatic cell hybrids indicated that for this virus, molecules in addition to CD4 were probably required to facilitate infection and cell fusion. Additional studies of HTLV-IV infection of Sup-T1 cells, which are exquisitely sensitive to cytopathic effects induced by HIV-1, demonstrated that HTLV-IV infection could occur in the absence of cytopathic effects and, remarkably, with minimal or no downmodulation of the CD4 molecule from the cell surface. The failure of HTLV-IV infection to reduce the expression of several CD4 epitopes suggested that the HTLV-IV envelope produced by Sup-T1 cells was altered in its ability to interact with or bind to CD4. Additional differences were also noted in the size of the transmembrane envelope molecule of HTLV-IV produced by Sup-T1 cells, indicating that cell-specific alterations in processing of the HTLV-IV envelope occurred during the production of virus in this cell line. Understanding the basis for these biological differences between HTLV-IV and the HIV-1 viruses may help to elucidate more general mechanisms for pathogenesis of other members of the SIV and HIV families of retroviruses.     

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.     

Phorbol Esters and SDF-1 Induce Rapid Endocytosis and Down Modulation of the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time ~5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms.

SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1–mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

     

Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium

PMA causes rapid down-modulation of CD4 molecules on murine immature thymocytes, human PBL, and CD4-positive human tumor cell lines, but not on murine peripheral lymphocytes. The mechanisms of phorbol ester-induced down modulation of CD4 molecules, however, have not been elucidated. To determine how PMA down-modulates CD4 expression by T lymphocytes, we studied the ability of inhibitors of protein kinase C, calmodulin, actin, and tubulin to block PMA-induced modulation of CD4 in several murine and human cell types. We also tested the ability of intracellular and extracellular calcium chelators to block CD4 internalization. There was marked variability in the degree of PMA-induced down-modulation of CD4 among various cell types. The effects of PMA on CD4 expression were greater for murine thymocytes, for human PBL, and for the human lymphoblastic leukemia cell line, MOLT-3, than for any of the other cell types studied. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked phosphorylation but not internalization of CD4 molecules induced by PMA. Therefore, phosphorylation of CD4 molecules by protein kinase C is not required for the internalization of the molecules. Internalization was blocked by both inhibitors of calmodulin, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and trifluoperazine. PMA-induced internalization of CD4 was blocked by Quin-2 AM, which chelates intracellular calcium. EGTA, which chelates extracellular calcium, did not block internalization. Inhibitors of actin or tubulin did not block internalization. These results suggest that PMA-induced modulation of CD4 can occur in the absence of phosphorylation of the CD4 molecules and is calmodulin and intracellular calcium dependent.     

CXCR4 function requires membrane cholesterol: implications for HIV infection

HIV requires cholesterol and lipid rafts on target cell membranes for infection. To elucidate a possible mechanism, we determined that cholesterol extraction by hydroxypropyl-beta-cyclodextrin (BCD) inhibits stromal cell-derived factor 1alpha (SDF-1alpha) binding to CXCR4 on T cell lines and PBMCs. Intracellular calcium responses to SDF-1alpha, as well as receptor internalization, were impaired in treated T cells. Loss in ligand binding is likely due to conformational changes in CXCR4 and not increased sensitivity to internalization. SDF-1alpha binding and calcium responses were effectively restored by reloading cholesterol. Immunofluorescence microscopy revealed that SDF-1alpha binding occurred in lipid raft microdomains that contained GM1. CXCR4 surface expression, on the other hand, only partially colocalized with GM1. HIV-1(IIIB) infection assays confirmed the functional loss of CXCR4 in the cell lines tested, Sup-T1 and CEM-NKR-CCR5. These data suggest that cholesterol is essential for CXCR4 conformation and function and that lipid rafts may play a regulatory role in SDF-1alpha signaling.     

T cell activation induced by anti-CD3 antibodies requires prolonged stimulation of protein kinase C

Accessory cell-depleted T cells required the presence of a protein kinase C (PKC) stimulating phorbol ester, such as phorbol 12,13-dibutyrate (PDB), to be activated by soluble antibodies to the CD3 molecular complex. To determine the duration of PDB costimulation necessary to induce a proliferative response, highly purified T cells were pulsed with anti-CD3, incubated with PDB for limited periods of time, and then washed and recultured in the absence of PDB. T cells stimulated with anti-CD3 and PDB for 2 hr were unable to proliferate unless IL-2 or PDB was added to the second culture. With more prolonged exposure to PDB (4-18 hr), anti-CD3-pulsed cells exhibited an increased capacity to proliferate in the absence of additional PDB. Proliferation could be augmented by exogenous IL-2, but remained submaximal. Optimal DNA synthetic responses required the presence of PDB throughout the entire culture. Despite this, costimulation with anti-CD3 and PDB induced a significant number of cells to express IL-2 receptors and enter the cell cycle after 18 hr of costimulation with PDB. Moreover, T cells costimulated by anti-CD3 and PDB produced IL-2 within 4 hr. However, T cells that were stimulated with anti-CD3 and PDB for 4 hr, washed, and recultured rapidly lost the ability to continue to produce IL-2, which reflected a decrease in the content of mRNA encoding IL-2. This loss of IL-2 production was prevented by reculturing the cells with PDB. These studies therefore indicate that after initial T cell activation by anti-CD3, continued stimulation of PKC is necessary for ongoing IL-2 production. These results suggest a model of T cell activation in which sustained stimulation of PKC after cell cycle entry is required to maintain growth factor production and continued proliferation.     

CD4 phosphorylation partially reverses Nef down-regulation of CD4

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (approximately 95%), whereas that induced by PMA was partial (approximately 40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.     

Induction of phosphorylation and intracellular association of CC chemokine receptor 5 and focal adhesion kinase in primary human CD4+ T cells by macrophage-tropic HIV envelope.

Binding of HIV-1 envelope glycoproteins to the surface of a CD4+ cell transduces intracellular signals through the primary envelope receptor, CD4, and/or the envelope coreceptor, a seven-transmembrane chemokine receptor. Macrophage-tropic strains of HIV-1 preferentially use CCR5 as an entry coreceptor, whereas T cell-tropic strains use CXC chemokine receptor-4 for entry. Intracellular signals transduced by HIV-1 envelope may have immunopathogenic consequences, including anergy, syncytium formation, apoptosis, and inappropriate cell trafficking. We demonstrate here that a recombinant envelope protein derived from an M-tropic isolate of HIV-1 can transduce CD4-dependent as well as CCR5-dependent intracellular signals in primary human CD4+ T cells. Novel HIV-induced intracellular signals that were identified include tyrosine phosphorylation of focal adhesion kinase (FAK) and CCR5, which are involved in cell adhesion and chemotaxis, respectively. HIV envelope-induced cellular association of FAK and CCR5 was also demonstrated, suggesting that ligation of CD4 and CCR5 leads to the formation of an activation complex composed of FAK and CCR5. Activation of this signaling pathway by HIV-1 envelope may be an important pathogenic mechanism of dysregulated cellular activation and trafficking during HIV infection.     

Human T lymphotropic virus I infection deregulates surface expression of the transferrin receptor

Human T-lymphotropic virus I (HTLV-I) is an etiologic agent in adult T cell leukemia. In an effort to understand the relationship between HTLV-I infection and malignant transformation, we have examined transferrin receptor expression in HTLV-I-infected cells. Transferrin receptor expression in normal T cells is tightly regulated and essential for cell proliferation. We have used matched T cell sets originating from a normal donor, consisting of tetanus toxoid-specific normal T cell clones (TM3 and TM5) and their in vitro HTLV-I-infected counterparts (TM3H and TM5H). Using these matched sets of virus-infected and normal T cells, we have determined that HTLV-I infection leads to hyperexpression of surface transferrin receptors (five- to six-fold higher than normal counterparts). Although the growth rates of the virus-infected cells did not differ significantly from their normal controls, HTLV-I-infected cells constitutively hyperexpressed surface transferrin receptors, whereas the level of surface receptor expression of normal counterpart cells varied during the cycle of antigenic stimulation. Immunoprecipitation of total (surface plus cytoplasmic) transferrin expression showed that the HTLV-I-infected cells did not possess a greater total number of transferrin receptors than their normal counterparts. This data was supported by Northern blot analysis, which showed equivalent transferrin receptor mRNA expression in HTLV-I-infected and uninfected cells. Functional analysis revealed a marked defect in 59Fe-transferrin internalization in the HTLV-I-infected cells. Furthermore, the HTLV-I-infected cells showed markedly decreased transferrin receptor phosphorylation and internalization in response to active phorbol ester. Thus the data demonstrate that in peripheral blood T cells, HTLV-I infection is accompanied by surface transferrin receptor overexpression secondary to subcellular redistribution and defective internalization.     

Human immunodeficiency virus type 1 Nef-induced CD4 cell surface downregulation is inhibited by ikarugamycin

One well-characterized in vitro function of Nef is its ability to remove CD4, the human immunodeficiency virus (HIV) receptor, from the cell surface. Nef accomplishes this by accelerating the internalization and degradation of CD4. Current models propose that Nef promotes CD4 internalization via an increased association of CD4 with clathrin-coated pits (CCP). Here, we investigated the effect of a naturally occurring antiprotozoan antibiotic, ikarugamycin (IKA), on CD4 cell surface expression in human monocytic cells stably expressing HIV type 1 SF2 Nef. IKA was able to efficiently restore CD4 cell surface expression in Nef-expressing cells without affecting either CD4 synthesis or Nef expression. In addition, we demonstrate that IKA is also capable of efficiently blocking CD4 down-modulation in response to phorbol myristate acetate. Our data suggest that IKA may be an efficient and useful inhibitor of CCP-dependent endocytosis.     

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4⁺ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.     

HIV-1 envelope glycoproteins-mediated apoptosis is regulated by CD4 dependent and independent mechanisms

The progressive loss of CD4 T lymphocytes is one of the hallmarks of HIV infection. The reverse correlation observed in vivo, between plasmatic HIV levels and CD4 T lymphocyte counts, supports the concept that direct HIV-mediated cell death contributes to this depletion. Previously, we and others have demonstrated, in vitro, that interactions between membrane-expressed HIV-envelope glycoprotein complexes and CD4 ecto-molecules are critical to cell killing which occurs mainly by apoptosis. Here, by the use of a co-culture model, in which chronically HIV-1 infected cells trigger apoptosis in uninfected CD4+ target cells, we have investigated the role of different CD4 domains in HIV envelope-mediated apoptosis. Target cells were A201 lymphoblastoid cell lines expressing wild-type CD4 or mutant forms of CD4. We show that the cytoplasmic domain of CD4 was not required for apoptosis induction. In contrast, the HIV permissive cell line expressing a CD4/CD8 chimeric molecule which contains only the first 171 amino acids of CD4, appeared to be resistant to HIV-induced apoptosis; thus suggesting that the D3-D4 CD4 module plays somewhat a regulatory role. Pre-treatment of wild-type CD4 expressing target cells by the phorbol ester PMA which leads to down-regulation of CD4, completely abolished apoptosis. Interestingly, in cells expressing CD4 devoid of its cytoplasmic domain, PMA blocked partially cell death without affecting, as expected, the CD4 expression. Taken together, these results demonstrate that although CD4 expression is essential for HIV envelope induced apoptosis, the apoptotic signal could be delivered in the absence of its cytoplasmic domain. Consistent with this, we suggest that other membrane associated molecule(s) are recruited for the signalling to initiate apoptosis.     

Activation of protein kinase C accelerates internalization of transferrin receptor but not of major histocompatibility complex class I, independent of their phosphorylation status.

Phosphorylation of membrane glycoproteins has often been invoked as a determinant of receptor internalization and receptor trafficking in a more general sense. Here we have studied the trafficking of major histocompatibility complex (MHC) Class I molecules and transferrin receptor (Tfr) related to their phosphorylation status in the human lymphoblastoid cell line JY. High resolution isoelectric focusing (IEF) allows the visualization of phosphorylated and non-phosphorylated protein species simultaneously, using protein backbone-labeling. Analysis on IEF was combined with a neuraminidase protection assay, in which sialic acid modification of the N-linked glycans present on Tfr and Class I molecules is used as a reporter group for cell surface expression. Phosphorylation of Class I heavy chains and Tfr was induced by exposure of cells to the phorbol ester tetradecanoyl phorbol acetate. We show that 1) phosphorylation of MHC Class I molecules is restricted to the cell surface fraction, 2) phosphorylation of MHC Class I molecules by protein kinase C (PKC) is not correlated with their internalization, as no internalization of Class I molecules, phosphorylated or non-phosphorylated, could be detected, 3) the initial rate, but not the final extent of the internalization of Tfr is affected by activation of PKC, and 4) phosphorylated Tfr behaves in a manner identical to non-phosphorylated Tfr in terms of internalization. The effect of activation of PKC on internalization of Tfr therefore most likely takes place at the level of the internalization machinery. Our data concerning the internalization of MHC Class I molecules contrast with earlier studies describing constitutive internalization in the B lymphoblastoid cell line A 46 and in HPB-ALL cells.     

Inhibition of T cell antigen receptor-dependent phosphorylation of CD4 in human immunodeficiency virus type 1 infected cells.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.     

Infection of monocytic cells by HIV1: combined role of FcR and CD4

Human immunodeficiency virus (HIV) complexed with human anti-HIV IgG can attach to Fcγ receptors (Fch) of mononuclear phagocytes. To determine whether the FcR-mediated infection that results also requires interaction between HIV gp 120 and cell membrane CD4, monocytic cells of the U937 line were transiently treated with phorbol 12, 13-dibutyrate (PDB) so that they temporarily presented a CD4⁻FcR⁺ phenotype at the time of HIV infection. HIV production was not abolished, but only significantly delayed after infection of these cells with free virus. Leu3a monoclonal antibody or soluble recombinant CD4 completely blocked this delayed infection. This indicates that enough CD4 still remained at the membrane to allow infection of a reduced cell number. Infection of PDB-treated cells with virus preincubated with high anti-HIV IgG concentrations was inhibited, contrasting with what was observed with control cells infected under the same conditions. Inhibition of infection was also observed when HIV became attached to untreated U937 cells through the binding of CD4-IgG hybrid molecules to FcR. Thus, the binding of IgG-coated virus to FcR is not sufficient in itself to elicit productive infection of monocytic cells, which still requires the interaction of viral gp120 and membrane CD4.     

Failure of human immunodeficiency virus entry and infection in CD4-positive human brain and skin cells.

CD4 molecules on human cells function as a major receptor for human immunodeficiency virus (HIV); however, certain CD4-negative cell types may also be susceptible to infection. Therefore, we attempted to quantitate the relationship between HIV infection and CD4 expression on human cell lines before and after introduction of the CD4 gene by using a retrovirus vector. Prior to introduction of the CD4 expression vector, low levels of HIV infection were detected by a sensitive focal immunoassay on all three cell types studied. With several HIV strains in clones of human cervical carcinoma (HeLa) cells expressing different levels of CD4, HIV titer increased with increasing CD4 expression. In contrast, in squamous cell carcinoma cells (SCL1) and astroglial cells (U87MG), even high levels of CD4 expression failed to augment HIV infection. The CD4 protein expressed in these two cell lines had the expected molecular weight and was capable of binding HIV virions. However, in contrast to CD4-positive HeLa cells, CD4-positive U87MG and SCL1 cells were unable to form syncytia when cultured with cells expressing HIV envelope protein. Thus, the inability of HIV to infect these cells appeared to be due to lack of fusion between HIV virion envelope proteins and CD4-positive cell membranes. This block is infectivity was overcome when cells were infected with HIV which was pseudotyped with the envelope protein of amphotropic murine leukemia virus. Thus, in addition to CD4, other cell surface molecules appear to be required for successful HIV entry into and infection of these two human cell lines.     

Rapid and reversible modulation of T4 (CD4) on monocytoid cells by phorbol myristate acetate: effect on HIV susceptibility

The effect of phorbol myristate acetate (PMA) on T4 (CD4) expression by monocytoid cells was studied. Greater than 99% of untreated U937 and HL-60 cells expressed surface T4 as measured with a fluorescence-activated cell sorter. The percentage of T4 positive cells decreased to less than 20% after incubation with PMA (10(-8) M). A decrease was observed within 15 min of PMA exposure, was maximal within 1 hr, and persisted for at least 3 days in the continuous presence of PMA. The susceptibility of untreated and PMA-treated U937 cells to human immunodeficiency virus (HIV) was also studied. Pretreatment of cells with PMA for 18 hr decreased the production of viral RNA and p24 antigen 24 hr after infection. The dose of PMA resulted in a parallel reduction of both T4 expression and infection by HIV. When PMA was washed from cultures and replaced with fresh medium for 48 hr, then T4 expression and the production viral RNA and p24 antigen following infection were restored. These data suggest that pharmacologic manipulation of surface T4 expression may have a potential role in the prevention or treatment of HIV infection.     

Structural features of the cytoplasmic region of CD4 required for internalization.

CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.     

The tumor promoter teleocidin induces IL 2 receptor expression and IL 2-independent proliferation of human peripheral blood T cells

In testing the recently discovered tumor promoter teleocidin (TCD), we found that like phorbol esters, TCD was mitogenic to human peripheral blood lymphocytes (PBL) and preferentially stimulated sheep erythrocyte-rosetted (ER) T cell-enriched populations. Stimulation of PBL with TCD induced synthesis and expression of receptors for interleukin 2 (IL 2), as shown by dot-blot analysis with the use of a synthetic oligonucleotide probe, cell surface staining with anti-Tac antibody followed by fluorescence-activated cell sorter analysis, and a functional proliferation assay in which TCD-stimulated cells were washed free of TCD and were recultured with human recombinant IL 2 (rIL 2). Increased expression of cell surface markers after TCD stimulation of PBL is not general, because TCD did not affect the expression of Leu-2a antigen, and it also reduced the density of Leu-3a and Leu-4 antigens. Stimulation of cultured, IL 2 receptor-positive PBL with rIL 2, but not TCD, was blocked by anti-rIL 2 antibodies. Furthermore, IL 2-specific mRNA was not detected in TCD-stimulated PBL, demonstrating that IL 2 was not required for TCD-induced T cell proliferation. In addition, TCD replaced IL 2 in inducing short-term proliferation of IL 2-dependent murine cytotoxic T cell lines. The findings that TCD induced IL 2-independent proliferation of T cells, and TCD and IL 2 synergized in inducing T cell proliferation, suggest that they initiate T cell proliferation via different mechanisms. The IL 2-independent activation of T cells, and the induction of IL 2 receptor expression by TCD, may be related to its ability to activate protein kinase C in cell membrane.