Antagonistic effects of phorbol esters on lymphocyte activation. Evidence that protein kinase C provides an early signal associated with lytic function

There is increasing evidence that protein kinase C plays a role in the transduction of an activation signal in lymphocytes. The bulk of this evidence is based on pharmacological experiments involving the tumor promoter phorbol myristate acetate (PMA) as a protein kinase C agonist. However, in cytotoxic T lymphocytes, PMA has been shown to both stimulate and inhibit lytic function. By examining the effects of a series of phorbol esters on protein kinase C activity in lymphocytes, we will demonstrate that these antagonistic effects of PMA on cytotoxic T lymphocyte function are related to multiple effects of PMA on protein kinase C activity.     

Protein kinase C modulates actin conformation in human T lymphocytes

We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes.     

cAMP mediates IL-1-induced lymphocyte penetration through endothelial monolayers

Endothelial cell incubated with IL-1 have been shown adhere more lymphocytes than nontreated endothelial cells. Here we demonstrate that IL-1 can also increase lymphocyte penetration through endothelial monolayers in vitro. IL-1 induced a transient increase in the number of lymphocytes penetrated through the endothelial monolayer into a filter in a time- and dose-dependent manner. This effect could be mimicked by increasing the cytosolic cAMP levels in the endothelial cells either by forskolin or dibutyryl-cAMP. Concomitantly we were able to show that IL-1 increased the cytosolic cAMP levels in endothelial cells. An inhibitor of adenylate cyclase, ddAdo, decreased both the IL-1-induced cAMP elevation and lymphocyte penetration. A protein kinase A inhibitor HA 1004 could inhibit the IL-1-induced lymphocyte penetration, where as protein kinase C (N-(2-guamidino-ethyl)-5-isoquinolinesyl foamide hydrocloride) and calcium-calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalensulfanamide) inhibitors had no effect. Adding dibutyryl-cGMP or calcium ionophore to the endothelial cells could not mimic IL-1-induced penetration and finally IL-1 did not induce PKC translocation in endothelial cells. These data support the view that IL-1 acts via cAMP as a second messenger in regard to lymphocyte penetration through endothelial cells. The above data demonstrate that IL-1-induced lymphocyte penetration through endothelial cells and that this IL-1-induced signal is transduced via cAMP in endothelial cells.     

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

In vivo and in vitro modulation of HLA-DM and HLA-DO is induced by B lymphocyte activation.

Ag presentation via HLA class II molecules in B lymphocytes depends on the coordinated action of HLA-DM, the catalyst of class II-peptide loading, and HLA-DO, a pH-dependent modulator of DM, the expression of which is almost completely restricted to B lymphocytes. The relative expression levels of both class II modulators are critical for the composition of the HLA class II peptide repertoire. The data in this work demonstrate that DO and DM expression are both dependent on the cellular activation status in primary human B lymphocytes. In vivo low-density activated primary human B lymphocytes show a prominent reduction in DO and DM expression when compared with high-density resting primary B lymphocytes. In vitro, reduction of DO and DM expression can be induced by B lymphocyte activation via the B cell receptor or by use of the phorbol ester, PMA. Specific inhibition of protein kinase C resulted in a significant reduction of HLA-DO and is potentially due to protein degradation in lysosomal compartments as the phenomenon is reversed by chloroquine. Thus, the expression of the dedicated HLA class II chaperone DM and its pH-dependent modulator DO is regulated and tightly controlled by the activation status of the B lymphocyte.     

MAP kinase activity is downregulated by phorbol ester during mouse oocyte maturation and egg activation in vitro

The effects of protein kinase C (PKC) stimulator, phorbol 12-myriatate 13-acetate (PMA), on meiotic cell cycle regulation and mitogen-activated protein (MAP) kinase changes have been studied in mouse oocytes and eggs. The results showed that MAP kinase activation itself was not necessary for germinal vesicle breakdown (GVBD), but the ability of the ooplasm to phosphorylate MAP kinase was a prerequisite for this event. At concentrations of 1.6 nM, PMA effectively inhibited GVBD and MAP kinase activation, suggesting that PMA inhibits GVBD by inhibiting molecule(s) upstream to MAP kinase. At concentrations of 16.2 nM, PMA induced metaphase-interphase transition more effectively in eggs collected 19 hr after human chorionic gonadotropin (hCG) administration than in those collected 15 hr after hCG administration. The degree of MAP kinase activity decrease was well correlated with the time course and proportion of pronuclear formation. On the other hand, when the effect of PMA on cell cycle progression was abolished by protein phosphatase inhibitor, okadaic acid, MAP kinase was superactivated. The biologically inactive 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) had no evident effects on either GVBD and interphase transition or on MAP kinase activity. Furthermore, the effects of PMA on oocyte GVBD, egg activation, and MAP kinase activity could be overcome by the specific PKC inhibitor, calphostin C, suggesting the possible involvement of this enzyme in the regulation of MAP kinase activity. The results suggest that activation of PKC by PMA entrains a cascade of events that ultimately inhibits MAP kinase activation and GVBD in mouse oocytes and induces MAP kinase inactivation and metaphase-interphase transition in mouse eggs.     

Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. II. Interaction between phorbol ester- and cAMP-sensitive pathways.

Ag independent adhesion between lymphocytes and target cells is mediated in part by the interaction between lymphocyte function associated Ag-1 (LFA-1) and its coreceptor intercellular adhesion molecule-1 (ICAM-1). Within minutes, PMA treatment of JY cells, which express both LFA-1 and ICAM-1, induced capping of LFA-1 and augmentation of intercellular adhesion lasting for several hours. However, over the course of 15 to 30 min, both of these events were blocked by elevation of intracellular cAMP concentration ([cAMP]i) presumably via activation of protein kinase A. This short term inhibition of protein kinase C-induced adhesion was in contrast to the long term augmentation of adhesion caused by increased [cAMP]i as demonstrated in the companion article. Intercellular adhesion, due to LFA-1/ICAM-1 interactions, could also be induced by LPS treatment of JY cells. At submaximal concentrations, the extent of aggregation induced by LPS had two maxima, one at 30 to 60 min and the other with a plateau at 5 to 8 h. LPS is known to activate protein kinase C and we show that LPS treatment induced increased [cAMP]i. Using inhibitors of protein kinases C and A, possible mediators of the two components of adhesion induced by LPS could be identified. The early component was abrogated by inhibition of protein kinase C although the later component was unaffected. In contrast, an inhibitor of protein kinase A had no affect on the early component and attenuated, but did not entirely eliminate, the late component. These results suggest a model of sequential induction, inhibition, and reinduction of LFA-1/ICAM-1-mediated lymphocyte adhesion that is regulated by temporally ordered actions and interactions of protein kinases C and A.     

Modes of inhibitory action of 4 beta-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

The tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC50 vs thrombin = 0.5 nM, greater than 90% inhibition at 10 nM. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 nM PMA. A non-tumor-promoting phorbol ester, 4-O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated AA release, but not GTP gamma S- and AIF4(-)-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A2 by PMA pretreatment is discussed.     

Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.     

Phosphorylation of mouse thymocyte CD4 and CD8: regulation of surface expression.

Murine T cell surface antigens, CD4 and CD8 are phosphorylated in response to phorbol 12-myristate 13-acetate, a protein kinase C activator, but not phosphorylated after concanavalin A, Ca2+ ionophore or dibutyryl-cAMP treatment. We examined the cell surface expression of both antigens and show that surface CD4 on CD4+CD8+ and CD4+CD8- thymocytes is rapidly decreased after PMA treatment, while CD8 expression is unaffected. Prolonged PMA treatment, which down-regulates protein kinase C, allows CD4 reexpression only in the CD4+CD8- population, suggesting that different mechanisms of cell surface antigen expression are operating in the two thymocyte subpopulations.     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

B cell activation. IV. Induction of cell membrane depolarization and hyper-I-A expression by phorbol diesters suggests a role for protein kinase C in murine B lymphocyte activation

Analysis of the effects of phorbol diesters on mouse B lymphocyte kinase C activity, membrane potential, mI-A expression, and cell cycle state are reported. Results indicate that the phorbol diesters PMA and 4 beta-PDD, which are potent tumor promoters, activate partially purified B cell protein kinase C and stimulate B cell membrane depolarization and increased mI-A expression. The analog 4 alpha-PDD has none of these effects. Similarly, none of the phorbol diesters tested promoted G0 to G1 transition of B lymphocytes. Results are consistent with the possibility that the transmembrane signal transduction mediated by cell membrane immunoglobulin, which results in membrane depolarization and increased I-A antigen expression, operates via activation of protein kinase C.     

Modulation of the activity of the platelet-derived growth factor receptor by phorbol myristate acetate

Phorbol myristate acetate (PMA) weakly activates Na+/H+ exchange in NR-6 cells. Simultaneously, PMA blocks the activation of Na+/H+ exchange by platelet-derived growth factor or by serum. Phorbol esters that do not activate protein kinase C do not show this metabolic response. We conclude that activation of Na+/H+ exchange by platelet-derived growth factor or serum does not require the intermediate activation of protein kinase C. We postulate from this and previous observations that a major role of protein kinase C is to act as an inhibitor of the activity of cell surface receptors, in particular mitogen receptors.     

Enhanced gene expression of the murine ecotropic retroviral receptor and its human homolog in proliferating cells.

The receptor for gp70 envelope glycoprotein of murine ecotropic leukemia virus is essential for virus entry into the host cell and has been recently demonstrated to function as a cationic amino acid transporter. In the experiments reported herein, we compared the gene expression of the murine ecotropic retroviral receptor (ERR) and its human homolog (H13) in rapidly proliferating cells versus resting cells using four different systems. (i) The expression of ERR gene is enhanced during activation of T and B lymphocytes by concanavalin A and lipopolysaccharide, respectively. Similar enhancement is observed by adding phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). These phenomena appear to involve protein kinase C; two PMA analogs, 4 alpha-phorbol and 4 alpha-PMA, lacking the ability to activate protein kinase C fail to induce elevated levels of gene expression, and the protein kinase C inhibitor, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride[, inhibits the enhancement induced by PMA. (ii) Friend murine leukemia virus induces rapid splenomegaly, and acute erythroleukemia in sensitive mice. Concomitantly with splenomegaly, ERR gene expression in spleen cells increases dramatically. (iii) The level of expression of the ERR or H13 gene in a variety of tumor cells is highly elevated compared with the level in noncancerous cells. (iv) H13 gene expression decreases upon terminal differentiation of the human promyelocytic leukemia cell line HL-60 into granulocytes or macrophages by dimethyl sulfoxide or PMA, respectively. These results suggest that ERR and H13 genes play an important role in cellular proliferation.     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.