Signaling through T lymphocyte surface proteins, TCR/CD3 and CD28, activates the HIV-1 long terminal repeat

The state of T cell activation and proliferation controls HIV-1 replication and gene expression. Previously, we demonstrated that the administration of PHA and PMA to the human T cell line Jurkat activates the HIV-1 enhancer, which is composed of two nuclear factor kappa B (NF kappa B) binding sites. Here, we show that PMA alone is sufficient for this effect. In addition, activation of T cells through the surface proteins TCR/CD3 and CD28 increased gene expression directed by the HIV-1 long terminal repeat (LTR) to the same extent as PMA. Analysis of 5' deletions in the LTR revealed that the NF kappa B binding sites and sequences in the upstream U3 region are required for this response. Whereas cyclosporin A did not inhibit the effect of PMA, it reduced the effects of agonists to TCR/CD3 and CD28 on the LTR. H7, an inhibitor of protein kinase C (PKC), blocked the effects of all stimuli. Thus, PMA activates the NF kappa B sites through a PKC-dependent pathway while ligands to TCR/CD3 and CD28 activate the LTR through a cyclosporin A-sensitive, PKC-dependent pathway of T cell activation. We conclude that mechanisms involved in the expression of IL-2 and the alpha-chain of the IL-2R alpha genes also play a role in the regulation of HIV-1. Physiologic stimuli can activate HIV-1 gene expression; agents that block T cell activation also inhibit activation of the LTR. These observations might serve as a model for the regulation of HIV-1 gene expression in peripheral blood T cells.     

Suppression of interleukin-2 and interleukin-2 receptor expression in Jurkat cells stably expressing the human immunodeficiency virus Tat protein.

The Jurkat T cell line was stably transfected with an Epstein-Barr virus-based episomal replicon designed to express high levels of the HIV-1 Tat protein. After selection in hygromycin B, high-level Tat activity was detected in 3 of 18 transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor alpha chain (IL-2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL-2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-1 infection.     

A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction.

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.     

Differential signal requirements in T-cell activation by mitogen and superantigen

The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca²⁺ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca²⁺ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.     

Induction of nuclear factor-kappa B and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A

We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transferrin receptor antibody represents one signal for the induction of IL 2 production by a human T cell line

We previously demonstrated a two-signal requirement for the activation of the human T cell lines Jurkat and HUT 78. Interleukin 2 (IL 2) production by these lines can be induced by phytohemagglutinin (PHA), T3 antibodies, or calcium ionophores, but only in combination with phorbol myristate acetate (PMA). To obtain further information about surface structures involved in T cell activation, we produced a monoclonal antibody that could substitute for PMA in the activation of HUT 78. This antibody, designated J64, induced IL 2 secretion by HUT 78 in combination with PHA, T3 antibodies, or calcium ionophores, however not by itself. J64 also had other PMA-like effects on HUT 78, such as an increase in IL 2 receptor expression and an inhibition of cell growth. J64 was shown to immunoprecipitate the transferrin receptor (TfR). However, it bound to an epitope different from those recognized by other TfR antibodies and different from the transferrin-binding site. In addition, other previously described TfR antibodies did not, like J64, function as activating stimuli for HUT 78. Possible mechanisms for activation signaling in T cells involving the TfR are discussed.     

Substance P enhances IL-2 expression in activated human T cells.

Jurkat and HUT 78 T cell lines, as well as peripheral blood human T cells activated with PHA plus PMA were used to investigate the capacity of substance P (SP) neuropeptide to regulate IL-2 production. By using Northern blot analysis and dosage of the IL-2 release in cell supernatants, we show that SP can act as cosignal with PHA + PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. By using the N-terminal SP(1-4) or the C-terminal SP(4-11) fragments of the entire molecule, we show that the cosignal activity is carried by the C-terminal portion of SP. The SP and SP(4-11) optimal effects were observed at 10(-12) M and 10(-10) M when a broad range of concentrations from 10(-6) M to 10(-13) M was tested. The increase of IL-2 mRNA obtained with 10(-12) M of SP in the activated Jurkat cells was reduced by adding 10(-10) or 10(-9) M of the SP antagonist (D-Pro2,-D-Phe7,-D-Trp9)SP to the culture, indicating the specificity of SP action. The up-regulation observed when 10(-12) M of SP was applied together with the mitogens on Jurkat cells, persisted after a 16-h culture period, time at which the IL-2 mRNA signal is normally back to a minimum level when the mitogens are used alone. Furthermore, an induction of IL-2 mRNA accumulation, in a 2-h pulse, was obtained with 10(-12) M of SP on Jurkat cells previously activated with mitogens for 16 h.     

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.     

The effect of a T cell-specific NF-kappa B inhibitor on in vitro cytokine production and collagen-induced arthritis

NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors.     

Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells

In this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCalpha, a conventional Ca(2+)-dependent PKC, and to PKCtheta, a novel Ca(2+)-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCalpha activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.     

Cyclosporin A-mediated inhibition of mitogen-induced gene transcription is specific for the mitogenic stimulus and cell type

We have examined the effect of cyclosporin A (CSA) on the mitogen-induced expression of 11 genes previously cloned from mitogen-activated T lymphocytes. Levels of induced gene expression in the human T cell line Jurkat were determined by mRNA blotting and nuclear run-on assay, after stimulation with one or combinations of the mitogens PMA, PHA, and the ionophore A23187. In the presence of CSA, gene expression induced with PMA alone was not inhibited, whereas PHA-induced increases in gene expression were inhibited by CSA. For one group of genes, including IL-2 and two novel genes with sequences suggestive of lymphokines, A23187 plus PMA-induced gene expression was inhibited by CSA. In contrast, another group of induced genes was unaffected by CSA after A23187 and PMA induction. This finding implies that A23187 and PMA stimulate gene induction by more than one mechanism, and that not all activation signals mediated through calcium fluxes are sensitive to CSA. In addition, 8 of the 11 genes were expressed in the fibroblast cell line Mrc 5 after stimulation with PMA, A23187, or serum; CSA had no effect on genes induced with these agents in Mrc 5 cells in both mRNA blotting and run-on experiments, although 5 of these genes were markedly inhibited by CSA in Jurkat after PMA/PHA induction. These data indicate that separate pathways for induction of identical genes exist, and that the inciting stimulus and cell type are determining factors in the ability of CSA to inhibit gene expression.     

The generation of oxygen radicals: a positive signal for lymphocyte activation

The induction of ornithine decarboxylase (ODC) activity in lymphocytes is associated with activation and the initiation of cellular proliferation. ODC is also an essential component in tumor promotion. Phorbol myristic acetate (PMA) is a mitogen for lymphocytes, but can also promote tumor formation. Tumor promotion is linked to the generation of free radicals induced by PMA. Modulation of intracellular glutathione is associated lymphocyte activation and in protection of cells from damage due to oxygen radicals. We examined the interaction between ODC activity and intracellular glutathione concentrations in EL4 murine lymphoblastoid cells. The intracellular glutathione concentration could be augmented in EL4 cells when cultured with the cysteine delivery agents 2-oxothiazolidine 4-carboxylate (OTC) and 2-mercaptoethanol (2-ME) and suppressed with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO). OTC and 2-ME suppressed ODC activity in fresh serum and PMA-activated EL4 cells. BSO had no effect on ODC activity of EL4 cells cultured in the presence of PMA. While both OTC and 2-ME augmented the total intracellular glutathione concentration, PMA enhanced only the level of oxidized glutathione. To determine if the mechanism by which PMA or fresh serum altered intracellular glutathione and ODC activity was through the generation of oxygen radicals, EL4 cells were cultured with free radical scavengers. The nonpermeant electron acceptor potassium ferricyanide, and the H2O2 scavenger catalase, lowered ODC activity in both serum-stimulated and PMA-activated EL4 cells. Similarly, incubation of EL4 cells with either potassium ferricyanide or catalase elevated intracellular glutathione concentrations. These data suggest that (a) modulation of intracellular glutathione in the EL4 lymphoblastoid cell line alters ODC activity induced by fresh serum and by the mitogen PMA; (b) activation of EL4 cells by PMA alone alters intracellular glutathione metabolism, which may be associated with its role as a mitogen in lymphocyte activation; and (c) the generation of free radicals in EL4 cells may play a positive role in cellular activation.     

Expression and regulation of phospholipase D isoforms in mammalian cell lines

Phospholipase D (PLD) is activated in mammalian cells in response to diverse stimuli that include growth factors, activators of protein kinase C, and agonists binding to G-protein-coupled receptors. Two forms of mammalian PLD, PLD1 and PLD2, have been identified. Expression of mRNA and protein for PLD1 and PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle); EL4 (mouse thymoma); HL-60 (human myeloid leukemia); Jurkat (human leukemia); PC-3 (human prostate adenocarcinoma); PC-12K (rat phaeochromocytoma); and Rat-1 HIR (rat fibroblast). All, with the exception of EL4, express agonist-activated PLD activity. PLD1 is expressed in A7r5, HL-60, PC-3, and Rat-1, while PLD2 is expressed in A7r5, Jurkat, PC12K, PC-3, and Rat-1. Neither isoform is expressed in EL4. Guanine nucleotide-independent PLD activity is present in membranes from all cells expressing PLD2. In PC12K cells, which express only PLD2, treatment with nerve growth factor causes neurite outgrowth and increases expression of PLD2 mRNA and protein within 6-12 h. A corresponding increase is observed in membrane PLD activity and in phorbol-12-myristate-13-acetate (PMA)-stimulated PLD activity in intact cells. These results show that PLD2 can be regulated both pretranslationally and posttranslationally by agonists.     

Comparison of the capacity of murine and human class I MHC molecules to stimulate T cell activation.

The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.