Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

Phosphorylation and down-regulation of CD4 and CD8 in human CTLs and mouse L cells

The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4⁺ or CD8⁺ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4⁺ or CD8⁺ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4⁺ and CD8⁺ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4⁺ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8⁺ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.     

Induction of phosphorylation of human immunodeficiency virus type 1 Nef and enhancement of CD4 downregulation by phorbol myristate acetate.

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) encodes a 27 to 34 kDa myristoylated protein that induces downregulation of CD4 from the cell surface and enhances virus infectivity. As shown by experiments on SIV-infected adult macaques, Nef is important in pathogenesis and disease progression. In vitro, protein kinase C (PKC) phosphorylates Nef, but the role of phosphorylation in the function and expression of this protein has not yet been determined. Here we show that in HIV type 1-infected cells, phosphorylation of Nef increased 8- to 12-fold after treatment with phorbol myristate acetate and phytohemagglutinin (PMA/PHA). Basal and PMA/PHA-induced phosphorylation occurred on serine residues of Nef and was independent of other HIV proteins. The PMA/PHA-induced phosphorylation of Nef was inhibited by bisindolylmaleimide I, a potent and specific inhibitor of PKC, but was unaffected by H89, an inhibitor of protein kinase A. In contrast, treatment with bisindolylmaleimide I did not affect the basal level of Nef phosphorylation, suggesting two different phosphorylation pathways. A PMA-insensitive CD4 mutant in which three serine residues in the cytoplasmic domain have been replaced by alanines was used to determine whether PMA-induced phosphorylation affects Nef-induced CD4 downregulation. In Nef-expressing cells, treatment with PMA enhanced downregulation of the CD4 serine triple mutant from the cell surface, suggesting that phosphorylation is important for Nef function.     

Murine T-cell differentiation antigen CD8 is a direct substrate of protein kinase C

Murine T cell differentiation antigen CD8 alpha (Lyt-2) is phosphorylated in vivo after phorbol 12-myristate 13-acetate (PMA) treatment of cells. Concanavalin A,dibutyryl cAMP and calcium ionophore are unable to stimulate phosphate incorporation into CD8 alpha. Depletion of cellular protein kinase C (PKC) by prolonged PMA treatment abolished this phosphorylation, suggesting that PKC is required for this effect. Using the amino acid sequence derived from cloning CD8 alpha, peptides encompassing both possible intracellular phosphorylation sites were made and used to test the ability of various kinases to phosphorylate CD8 alpha sequences. Only the proximal serine peptide was a kinase substrate, and of PKC, cAMP-dependent kinase and the multifunctional calcium/calmodulin-dependent kinase, only PKC was able to phosphorylate this peptide. These studies provide the first definitive evidence that CD8 alpha is a direct substrate of PKC.     

Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and class I MHC-encoded proteins on T cells. Role of intracytoplasmic domains analyzed with hybrid CD8/class I molecules

We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.     

Modulation of CD40L antigen expression in Jurkat cells: involvement of protein kinase C activity

The CD40L expressed on activated CD4+ T cells delivers contact-dependent proliferative and anti-apoptotic signals to B lymphocytes. Little is known about molecular mechanisms of constitutive expression of CD40L on some non-Hodgkin's lymphomas, especially about involvement of two signal pathways regulating its expression in normal cells; one involving calcineurin, and the other protein kinase C. We analyzed by flow cytometry the effects of 6-hour stimulation of both pathways (stimuli: PMA and ionomycin) and their inhibitors: cyclosporin A and chelerythrine, on CD40L expression. Two Jurkat clones differing in CD40L surface expression: clone 217.6 (CD40L-) and 217.7 (CD40L+) were studied. Our experiments showed that high level of CD40L expression on the surface of 217.7 cells was reduced after stimulation with PMA. The same effect was observed for combination of PMA and chelerythrine or for PKC inhibitor alone. In 217.6 cells, only chelerythrine used alone induced low level of CD40L expression, while PMA and ionomycin were without effect. These results suggest that CD40L surface expression is mainly dependent on protein kinase C activity. By using PepTag Assay we have confirmed that in both Jurkat clones PKC activity is higher than in normal blood lymphocytes.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

Signal transduction via leukocyte antigen CD43 (sialophorin). Feedback regulation by protein kinase C

CD43 is a constitutively phosphorylated 115-kDa sialoglycoprotein expressed on a variety of blood cells including lymphocytes and monocytes. L10, a mAb directed against CD43, triggers T cell activation and enhances hydrogen peroxide production in monocytes. Activation of mononuclear cells by L10 initiates phosphoinositides hydrolysis, C2+ mobilization, and protein kinase C (PKC) activation. In turn, activated PKC hyperphosphorylates CD43, suggesting a potential role for PKC in the regulation of signaling via CD43. To address this issue, we have analyzed the effect of PKC activation by the tumor promoter PMA on L10-triggered rise in intracellular free Ca2+ concentrations ([Ca2+]i). Treatment of mononuclear cells with PMA profoundly inhibited the increase in [Ca2+]i induced by L10. The inhibition of CD43-mediated signaling by PMA was due, in part, to uncoupling of CD43 from the signal-transducing G protein. This was evidenced by the comparatively modest inhibition by PMA of the increase in [Ca2+]i induced by the direct G protein activator AlF4-. PMA treatment did not affect the surface expression of CD43. However, it induced the hyperphosphorylation of CD43, the extent of which correlated with the inhibition of CD43-mediated increase in [Ca2+]i. Staurosporine, a potent inhibitor of PKC, abrogated the hyperphosphorylation of CD43 and normalized CD43-mediated signaling in PMA-treated cells. Significantly, in the absence of PMA, staurosporine enhanced the rise in [Ca2+]i triggered by L10, suggesting that engagement of CD43 by activating ligands results in feedback inhibition by PKC. It is concluded that activation of PKC inhibits signaling via CD43 by mechanisms involving phosphorylation and uncoupling of CD43 from the signal-transducing apparatus and by distal, post-receptor events.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.     

Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Activation of CD3/TCR negative human thymocytes via CD28 molecule.

We have observed that the CD28 molecule was present on the cell surface of a large fraction of resting CD3- thymocytes (40 to 100%). Interestingly, the majority (greater than 90%) of surface CD3-CD28-cells reacted in the cytoplasm with anti-CD28 (CK248, 9.3) and anti-CD3 epsilon chain mAbs (Leu4, OKT3). Along this line, we found that CD28 surface expression could be induced within 18 hr on CD3-CD28- thymocytes using very low doses of phorbol-13-myristate-12-acetate (PMA). This event was accompanied by the appearance of CD25 and CD69 activation antigens but not of CD3/TCR complex. These results were further confirmed by immunoprecipitation studies. It is noteworthy that the T-cell activation pathway initiated via the CD28 molecule is functional in resting CD3- thymocytes in the presence of PMA and/or IL2. Finally, stimulation of CD3- immature thymocytes via CD28 gave rise to a large fraction (about one-third) of CD3-CD8+ cells.     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.     

Cell surface comodulation of CD4 and T cell receptor by anti-CD4 monoclonal antibody

In our study we have used anti-CD4 mAb to investigate the cell surface association between CD4 and the Ag-specific TCR complex on mature peripheral T cells. Anti-CD4 mAb was administered in vivo and in vitro and its effects on CD4 and CD3 cell surface expression were determined. In vivo, anti-CD4 mAb reduced cell surface expression of its ligand, CD4, and secondarily also reduced cell surface expression of CD3/TCR on CD4+ splenic T cells. In vitro, multivalent cross-linking of CD4 by anti-CD4 mAb and either FcR+ cells or anti-Ig mAb also resulted in decreased surface expression of CD4 and specific comodulation of CD3/TCR. The secondary reduction in cell surface CD3/TCR expression induced by CD4 cross-linking could be pharmacologically disrupted by high doses of PMA, indicating that the comodulation of CD3 with CD4 was dependent upon intracellular mediators, possibly including protein kinase C. These results demonstrate that, in the presence of anti-CD4 mAb, CD4 is functionally associated with the CD3/TCR complex, and that this association is dependent upon the activity of intracellular mediators. Such intracellular mediators might induce the coordinate down-modulation of physically unassociated CD4 and CD3/TCR molecules, or, alternatively, might promote a physical interaction between CD4 and CD3/TCR molecules.     

Phorbol ester induces both gene expression and phosphorylation of the plasma membrane Ca2+ pump.

Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.     

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.