Protein kinase C (PKC) inhibitors inhibit primary (1-0) but not secondary (1-1) or cloned cytotoxic T lymphocytes

The isoquinolinesulfonamide PKC inhibitors H-7 and H-8 inhibit primary, in vivo generated cytotoxic T lymphocyte (CTL) activity by 50% at concentrations approximating their reported Ki values for PKC, 6 uM and 15 uM respectively. However, a greater than ten-fold higher concentration of H-7 (100 uM) is required to reduce secondary or clone 8F CTL-mediated cytotoxicity by 50%. At this concentration H-7 is also reported to inhibit calmodulin (CaM)-dependent enzymes. To distinguish between the effect of 100 uM H-7 on PKC versus CaM the napthalenesulfonamide CaM antagonist W-7 was investigated. W-7 inhibited primary, secondary and clone 8F CTL-mediated cytolysis by 50% near its reported Ki value for CaM-dependent kinase activity, 12 uM. We conclude that W-7 and 100 uM H-7 reduce cytolysis by inhibiting CaM-dependent reactions and not PKC. Thus, these findings indicate that primary killers require both PKC- and CaM-dependent activation pathways for lethal hit delivery, whereas highly lytic cultured CTL use only one pathway dependent upon CaM.     

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Killing of mouse blastocyst stage embryos by cytotoxic T lymphocytes directed to major histocompatibility complex antigens

A new assay, mixed embryo leukocyte interaction assay, in which the ability of cytotoxic T lymphocytes (CTL) to kill preimplantation mouse embryos could be investigated, is described. CTL were generated both in vitro and in vivo to the H-2b and H-2d haplotypes. The specificity of the CTL was verified by using EL-4 (H-2b) and P815 (H-2d) target cells in a 51Cr-release assay. The cytolytic effect of the CTL on mouse blastocysts was measured by assessing blastocoel retention and inhibition of [3H]thymidine incorporation by the embryos. It was shown that CTL kill blastocyst stage embryos from C57BL/6J (H-2b) and B10.D2 (H-2d) mice with the zona pellucida removed, but not with the zona pellucida intact. These results demonstrate that the H-2 antigens present on mouse blastocysts can be recognized by CTL. It is suggested that one biologic role for the zona pellucida is the prevention of cell-mediated destruction of preimplantation embryos in utero.     

Inhibition of cytotoxic T lymphocyte and natural killer cell-mediated lysis by O,S,S,-trimethyl phosphorodithioate is at an early postrecognition step

O,S,S,-Trimethyl phosphorodithioate (OSS-TMP), an organophosphate esterase inhibitor, has been shown to block the effector phase of the cytolytic reaction mediated by murine and human cytotoxic T lymphocytes (CTL) and human natural killer cells. The murine interleukin 2-dependent CTLL-1 (anti-Iad) clone was used to determine the phase of the cytolytic pathway inhibited by OSS-TMP. Pretreatment of the CTL or target cell with OSS-TMP was not effective at blocking lysis; however, inhibition of lysis was achieved if the reaction was carried out in the continuous presence of OSS-TMP (IC50 = 55 microM) or when CTL-target conjugates were performed and incubated with OSS-TMP (IC50 = 640 microM). Two structural analogues of OSS-TMP were unable to inhibit CTL-mediated lysis. In contrast to OSS-TMP, N-alpha-p-tosyl-L-lysine chloromethylketone required only a 5-min preincubation with the CTL to inhibit lysis. OSS-TMP did not block recognition-adhesion step(s) of the reaction since the ability to form conjugates was not impaired; however, the lytic efficiency of individual CTL-target pairs were blocked. OSS-TMP did not appear to be an inhibitor of the major granule-associated protease that cleaves the substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzylester. Ca2+ pulse and kinetic experiments indicated that the OSS-TMP-sensitive site was at a pre-Ca2+-dependent phase but after recognition-adhesion. Human CTL and natural killer cell activity was also inhibited by OSS-TMP, suggesting the presence of a common site of action among these cytolytic systems. The results indicate that OSS-TMP may be a useful reagent in characterizing the early post-recognition events in the cytolytic pathway of CTL and natural killer effector cells.     

Features of target cell lysis by class I and class II MHC-restricted cytolytic T lymphocytes

The lytic activity of influenza virus-specific murine cytolytic T lymphocyte (CTL) clones that are restricted by either H-2K/D (class I) or H-2I (class II) major histocompatibility (MHC) locus products was compared on an influenza virus-infected target cell expressing both K/D and I locus products. With the use of two in vitro measurements of cytotoxicity, conventional 51Cr release, and detergent-releasable radiolabeled DNA (as a measure of nuclear disintegration in the early post-lethal hit period), we found no difference between class I and class II MHC-restricted CTL in the kinetics of target cell destruction. In addition, class II MHC-restricted antiviral CTL failed to show any lysis of radiolabeled bystander cells. Killing of labeled specific targets by these class II MHC-restricted CTL was also efficiently inhibited by unlabeled specific competitor cells in a cold target inhibition assay. In sum, these data suggest that class I and class II MHC-restricted CTL mediate target cell destruction by an essentially similar direct mechanism.     

H-2D(b-/-) mice are susceptible to persistent infection by Theiler's virus

H-2(b) mice are resistant to persistent infection of the central nervous system by Theiler's virus. They clear the infection 7 to 10 days after intracranial inoculation. Resistance maps to the H-2D gene and not to the H-2K gene and is associated with a potent antiviral cytotoxic T-lymphocyte (CTL) response. We used H-2(b) mice in which the H-2D or the H-2K gene had been inactivated to dissect the respective roles of these genes in resistance. We report that H-2D(-/-) but not H-2K(-/-) mice were susceptible to persistent infection. Furthermore, whereas H-2K(-/-) mice mounted a vigorous virus-specific CTL response, similar to that of control C57BL/6 mice, the CTL response of H-2D(-/-) mice was nil or minimal. Using target cells transfected with the H-2D(b) or the H-2K(b) gene, we showed that the H-2K-restricted CTL response against the virus was minimal in H-2D(-/-) mice. These results demonstrate that the H-2D(b) and H-2K(b) genes play nonredundant roles in the resistance to this persistent infection.     

Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine

L-Ornithine was shown to inhibit the development of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Lymphokines were unable to reverse the suppressive effect, and cytotoxic activity was not revealed by coupling ornithine-inhibited MLC cells to target cells with phytohemagglutinin (PHA). If addition of ornithine to MLC were delayed, sensitivity of CTL to inhibition was reduced after 24 hr and lost by 48 hr. Suppression of CTL development was not due to a toxic effect. MLC washed free of ornithine after 3 days produced detectable cytolytic activity within 24 hr of secondary culture, and to the same degree as the uninhibited MLC control within 48 hr. Cytotoxic cells generated in secondary cultures were Lyt-2+, did not kill the natural killer-sensitive YAC-1 cell line, and were shown to be antigen-specific by virtue of the findings that cytolysis and cold target inhibition were observed only with cells carrying the original, inducing H-2 haplotype. Cytolysis of target cells by normal CTL effector cells was not inhibited by L-ornithine. MLC depleted of accessory cells so that CTL activation was dependent upon addition of lymphokines remained susceptible to inhibition by ornithine. Our findings indicate that in the ornithine-inhibited MLC, CTL precursors undergo clonal expansion, but their maturation is arrested at a precytolytic stage. L-Arginine and putrescine also suppressed generation of CTL in primary MLC, and cells recovered from arginine- and putrescine-inhibited MLC developed control levels of CTL within 48 hr of secondary culture. Inhibition by putrescine was observed in tissue culture medium supplemented with human serum but not with fetal calf serum, presumably due to the presence of diamine oxidase activity in fetal calf serum. Similar to ornithine, the suppressive effects of arginine and putrescine on T lymphocytes were apparently selective for CTL because they did not inhibit mitogen activation with concanavalin A or the production of interleukin 2 and interleukin 3. These findings are consistent with a hypothesis that the inhibitory effects of ornithine, arginine, and putrescine are mediated by polyamines, and exerted on the differentiative stage of CTL development.     

Inhibition of cytolytic T lymphocyte clones reactive with Moloney leukemia virus-associated antigens by monoclonal antibodies: a direct approach to the study of H-2 restriction

H-2 restriction in cytolytic T lymphocyte (CTL)-mediated lysis of syngeneic murine Moloney leukemia virus (MoLV)-induced tumor cells was studied at the clonal level by testing the inhibitory effect of monoclonal anti-H-2 antibodies on the lytic interaction between CTL clones and target cells. Large numbers of MoLV-specific CTL clones were generated by placing limiting numbers of C57BL/6 regressor (responder) spleen cells into micro-mixed leukocyte-tumor cell cultures. The clonal CTL populations thus obtained were split into 5 aliquots and tested for lytic activity in the presence (or absence) of 1 of 3 monoclonal antibodies or of an anti-whole H-2b haplotype antiserum. Two of the monoclonal antibodies were directed against H-2Db and one against H-2Kb determinants. Specificity of these reagents had been verified by demonstrating inhibition of lysis by CTL populations directed against H-2Db and H-2Kb alloantigens. In 44 of a total of 51 clones tested, results showed selective inhibition by the anti-H-2Db (and the anti-whole haplotype) reagents, and lack of inhibition by the anti-H-2Kb antibody., Of the remaining 7 clones, none was inhibited by the anti-H-2Db antibody, and 3 were inhibited by the anti-whole haplotype antiserum. These studies show that the recognition of MoLV-associated antigens by the majority of CTL clones was restricted to the H-2Db region, and that there exists limited heterogeneity in the H-2 restriction of such clones.     

Herpes simplex virus glycoprotein D is recognized as antigen by CD4+ and CD8+ T lymphocytes from infected mice. Characterization of T cell clones

Several previous reports have described the surprising inability to detect murine CTL specific for glycoprotein D (gD), one of the important protective immunogens of HSV. Using slight variations of published procedures, we were able to show that the immune response to HSV in infected mice includes the generation of CTL specific for gD. C3H/OuJ (H-2k) mice were infected by injection in the hind footpads with purified HSV-1. Lymphocytes from draining lymph nodes were then isolated and shown to proliferate in response to, and to kill, transformed fibroblasts (H-2k) expressing HSV-1 gD. Two gD-specific T cell clones were isolated. One clone, designated CGD1, was shwon to be CD8+. This clone recognizes HSV-1 gD, but not HSV-2 gD, in the context of class I MHC molecules and kills the appropriate MHC-matched fibroblasts expressing HSV-1 gD. Unusual features of this cytolytic clone include augmentation by IL-4 of proliferative responses to Ag, inhibition of its lytic activity by a mAb specific for Thy-1 and recognition of infected fibroblasts in preference to infected lymphoblasts. The other clone, designated CGD3, was shown to be CD4+. This clone recognizes both HSV-1 gD and HSV-2 gD in the context of class II MHC molecules and has cytolytic potential.     

Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism.

Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.     

In vitro anti-parasitic activity of Cyclosporin A analogs on Trypanosoma cruzi

Cyclosporin A (CsA) nonimmunosuppressive analogs were evaluated against Trypanosoma cruzi and on TcCyP19, a cyclophilin of 19 kDa. Two out of eight CsA analogs, H-7-94 and F-7-62 showed the best anti-parasitic effects on all in vitro assays. Their IC(50) values were 0.82 and 3.41 microM, respectively, compared to CsA IC(50) value 5.39 microM on epimastigote proliferation; and on trypomastigote lysis their IC(50) values were 0.97 and 2.66 microM compared to CsA IC(50) value 7.19 microM. H-7-94 and F-7-62 were also more effective than CsA in inhibiting trypomastigote infection. The enzymatic activity of TcCyP19 was inhibited by all CsA derivatives, suggesting this target is involved in the trypanocidal effects observed.     

Dissociation of H-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2b/H-2b). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (all H-2b/H-2b), FY7 failed to induce the primary in vitro generation of anti-H-2b CTL by (B10.A x A)F1 (H-2a/H-2a) or B10.D2 x BALB/c)F1 (H-2d/H-2d) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2b CTL. Quantitative absorption tests with H-2Kb and H-2Db antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2Kb product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2Kb CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2Kb-gene product expressed by FY7.     

Lymphokine-mediated induction of cytolytic activity in a T cell hybridoma

Functionally inducible CTL hybridomas were constructed by fusing alloantigen-specific T cells (C57BL/6 alpha-DBA/2) with cells from the rat thymoma line W/FU (C58NT)D. A cloned hybridoma line (KSH.4.13.6) that was specifically cytolytic in the presence of activated rat spleen cell supernatant fluid (rat Con A SN) lost activity when transferred to normal medium. However, a cytolytic activity could be reinduced by culturing KSH.4.13.6 cells in medium containing rat Con A SN or secondary mixed leukocyte culture SN. By using various sources of SN, it was found that cytolytic induction required two different factors. PMA-induced EL-4 SN and SN from antigen-activated cloned T cells, neither of which were capable of inducing cytolytic activity alone, were able to synergize in the cytolytic induction of KSH.4.13.6 IFN-gamma and IL 1 failed to induce cytolytic activity even in the presence of EL-4 SN. Furthermore, this hybridoma produced macrophage activating factor (MAF) upon culture in rat Con A SN, although MAF production could not be induced by either specific antigen or lectins. The kinetics of induction and loss of cytolytic activity mediated by rat Con A SN were similar to those of the induction of MAF production. However, EL-4 SN, which by itself was incapable of inducing cytolytic activity, was able to induce MAF production in the KSH.4.13.6 hybrid to an extent similar to that induced by rat Con A SN. These results suggest that the induction of cytolytic activity and of MAF production in this cloned hybridoma cell line are regulated by different mechanisms. Such a functionally inducible T cell hybrid may provide a tool for biochemical and molecular analysis of T cell function and regulation, and of the characterization of cytokines required for CTL differentiation.     

Synthesis, cytotoxicity, and anti-inflammatory evaluation of 2-(furan-2-yl)-4-(phenoxy)quinoline derivatives. Part 4

A number of 2-(furan-2-yl)-4-phenoxyquinoline derivatives have been synthesized and evaluated for anti-inflammatory evaluation. 4-[(2-Furan-2-yl)quinolin-4-yloxy]benzaldehyde (8), with an IC(50) value of 5.0 microM against beta-glucuronidase release, was more potent than its tricyclic furo[2,3-b]quinoline isomer 3a (>30 microM), its 4'-COMe counterpart 7 (7.5 microM), and its oxime derivative 13a (11.4 microM) and methyloxime derivative 13b (>30 microM). For the inhibition of lysozyme release, however, oxime derivative 12a (8.9 microM) and methyloxime derivative 12b (10.4 microM) are more potent than their ketone precursor 7 and their respective tricyclic furo[2,3-b]quinoline counterparts 4a and 4b. Among them, 4-[4-[(2-furan-2-yl)-quinolin-4-yloxy]phenyl]but-3-en-2-one (10) is the most active against lysozyme release with an IC(50) value of 4.6 microM, while 8 is the most active against beta-glucuronidase release with an IC(50) value of 5.0 microM. (E)-1-[3-[(2-Furan-2-yl)quinolin-4-yloxy]phenyl] ethanone oxime (11a) is capable of inhibiting both lysozyme and beta-glucuronidase release with IC(50) values of 7.1 and 9.5 microM, respectively. For the inhibition of TNF-alpha formation, 1-[3-[(2-furan-2-yl)quinolin-4-yloxy]phenyl]ethanone (6) is the most potent with an IC(50) value of 2.3 microM which is more potent than genistein (9.1 microM). For the inhibitory activity of fMLP-induced superoxide anion generation, 11a (2.7 microM), 11b (2.8 microM), and 13b (2.2 microM) are three of the most active. None of above compounds exhibited significant cytotoxicity.     

Synthesis and antioxidant properties of novel N-methyl-1,3,4-thiadiazol-2-amine and 4-methyl-2H-1,2,4-triazole-3(4H)-thione derivatives of benzimidazole class

Some novel 1-methyl-4-(2-(2-substitutedphenyl-1H-benzimidazol-1-yl)acetyl)thiosemicarbazides (16a-20a), 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl]-N-methyl-1,3,4-thiadiazol-2-amines (17b-20b), and 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl-4-methyl-2H-1,2,4-triazole-3(4H)-thiones (16c-20c) were synthesized and tested for antioxidant properties by using various in vitro systems. Compounds 16a-20a were found to be a good scavenger of DPPH radical (IC(50), 26 microM; IC(50), 30 microM; IC(50), 43 microM; IC(50), 55 microM; IC(50), 74 microM, respectively) when compared to BHT (IC(50), 54 microM). Noteworthy results could not be found on superoxide radical. Compound 19b, which is the most active derivative inhibited slightly lipid peroxidation (28%) at 10(-3)M concentration. Compound 17c inhibited the microsomal ethoxyresorufin O-deethylase (EROD) activity with an IC(50)=4.5 x 10(-4)M which is similarly better than the specific inhibitor caffeine IC(50)=5.2 x 10(-4)M.     

Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. II. Development of syngeneic cytotoxicity in the absence of specific antigenic stimulation

Two out of four long-term murine allospecific cytotoxic T lymphocyte (CTL) clones tested could develop high levels of cytotoxicity against syngeneic target cells when cultured under appropriate conditions. All CTL clones maintained strict allospecificity so long as they were cultured with both appropriate allogeneic stimulator cells and growth factor (supernatant from secondary mixed lymphocyte cultures). In two of the clones, syngeneic reactivity rapidly developed when the allogeneic stimulator cells were replaced with syngeneic or third party stimulator cells, and when the supernatant from EL4 thymoma cells stimulated with phorbol ester was used as growth factor. In addition to killing the appropriate allogeneic target, clones with syngeneic reactivity could kill both syngeneic C57BL/6 targets and H-2-congenic BALB.B targets but not third party unrelated targets, suggesting that the self structure recognized was coded for within the major histocompatibility complex. Such clones did not kill the natural killer (NK) target YAC. The results obtained from cold target inhibition and from subcloning at limiting dilution of clones with syngeneic reactivity suggested that both allogeneic and syngeneic reactivity could be expressed by the same individual cell in the CTL clone. The specificity for syngeneic H-2 as opposed to third party H-2 and NK-sensitive target cells, and the observation that both allospecific and syngeneic killing could be partially blocked by anti-Lyt-2 antibody treatment of the CTL, strongly suggested that different recognition structures are involved in CTL-mediated syngeneic cytotoxicity and NK cytotoxicity.     

Regulation of phospholipase D2 activity by protein kinase C alpha

It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, G? 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.     

Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)