Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A.

Human members of a family of structurally related cytokines, which play a role as effectors of inflammation, were analyzed for their expression and regulation in T lymphocytes. Members of this gene family include Platelet Basic Protein (PBP); Platelet Factor 4 (PF-4); IL-8/NAP-1; IP-10, a gamma interferon induced protein; GRO; pAT 464 and pAT 744. In resting T lymphocytes the RNAs of the individual genes could not be detected, but all genes were induced upon stimulation with PHA or with PHA/PMA. The induction of five genes was blocked by the immunosuppresive drug cyclosporin A (CSA), which appears to affect initial events in T cell activation. This expression in T lymphocytes, especially the sensitivity to CSA, indicates a common immunmodulatory role of these structural related proteins.     

Cyclosporin A blocks calcium-dependent pathways of gene activation.

We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A.     

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.     

Expression of a novel nuclear protein in activated and in tat-I expressing T cells

The intracellular events that occur in T lymphoid cells after activation or after infection with HIV-1 are not well defined. In the case of HIV-1 infection, it is unknown whether the tat-I gene, an essential gene for viral replication, affects host cell nuclear factors. Using two-dimensional PAGE, we have identified a novel nuclear protein, designated nuclear protein-28,000 (NP-28), which is induced in Jurkat T cells by stimulation with PMA and/or PHA or ionomycin. This nuclear protein has an apparent molecular mass of 28,000 Da and an isoelectric point of 4.6. Interestingly, Jurkat cells transfected with tat-I express higher levels of NP-28 constitutively, without added stimulation. Incubation of Jurkat cells expressing tat-I with PMA and/or PHA or ionomycin causes superinduction of NP-28. We have therefore identified a novel lymphoid nuclear protein induced by T cell activation that occurs in tat-I expressing cells in the absence of activating agents.     

Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line

Interleukin (IL)-32 was recently identified as a new cytokine which induces various proinflammatory cytokines in human monocytes and macrophages. Therefore, IL-32 has been primarily studied in inflammatory models such as rheumatoid arthritis and inflammatory bowel diseases. The regulation of endogenous IL-32 in other immune cells remains unknown. In the present study, we stimulated Jurkat T cells with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and examined IL-32 expression at both the mRNA and protein levels. All mRNAs of the four IL-32 isoforms and the 12-15 kDa IL-32 protein were independent of PHA and PMA stimulation, however a 9 kDa molecular weight IL-32 protein in the cell culture supernatant was induced by PHA and PMA after 16 h of stimulation. Compared to other human cell lines, the Jurkat cell line constitutively expressed a 12-15 kDa molecule of IL-32, which is smaller than the known IL-32 isoforms. We used IL-32 shRNA to examine the specificity of the 12-15 kDa molecule. Upon IL-32 shRNA transfection, the 12-15 kDa band was decreased specifically as compared to the control scrambled clone. Thus, the constitutive expression of IL-32 mRNA as well as the predominant production of a smaller sized IL-32 isoform in Jurkat cells may implicate a role for IL-32 in human T cell leukemia.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C

We recently reported that the myristoylated peptide N-myristoyl-Lys-Arg-Thr-Leu-Arg (N-m-KRTLR) is a novel protein kinase C inhibitor. In this study, we investigated the biological effects of N-m-KRTLR using as an in vitro model the induction of the IL-2 receptor and IL-2 secretion by Jurkat cells in response to stimulation with 12-O tetradecanoylphorbol-13-acetate (TPA) plus phytohemagglutinin (PHA) and TPA plus OKT3 mAb. N-m-KRTLR significantly suppressed induction of the IL-2 receptor on the surface of the Jurkat cells by TPA plus either PHA or OKT3 mAb. Furthermore, N-m-KRTLR inhibited the production and release of IL-2 from cultured Jurkat cells stimulated with TPA plus either PHA or OKT3 mAb. Similarly, this peptide significantly inhibited the IL-2 production in normal human peripheral blood mononuclear cells in response to stimulation by TPA and PHA. In contrast, this peptide did not affect expression of the CD3 complex on the surface of the Jurkat cells either alone or in the presence of TPA or PHA. Furthermore, N-m-KRTLR did not interfere with the spontaneous proliferation of the Jurkat cells, and its effects on IL-2 secretion and IL-2 receptor expression in the Jurkat cells were evident without loss of cell viability. These results suggest that the novel protein kinase C inhibitor N-m-KRTLR may selectively inhibit certain activation pathways of Jurkat cells and indicate the usefulness of N-m-KRTLR in the analysis of discrete events in T cell activation.     

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.     

人淋巴毒素基因5′端上游NF－KB类因子特异性结合位点的研究

淋巴毒素（Ｌｙｍｐｈｏｔｏｘｉｎ,ＬＴ）是由活化的Ｔ淋巴细胞分泌的一种糖蛋白。ＬＴ基因的表达调控主要是在转录水平上。ＰＨＡ＋ＰＭＡ诱导的Ｊｕｒｋａｔ细胞总ＲＮＡ点渍杂交发现,Ｊｕｒｋａｔ细胞的内源性ＬＴｍＲＮＡ的数量随诱导时间的延长而增加。凝胶迟滞电泳分析发现,ＰＨＡ＋ＰＭＡ诱导４小时的Ｊｕｒｋａｔ细胞核抽提物形成多种复合物,且在诱导不同时间后有明显变化。系列缺失片段的竞争反应表明,这些复合物的形成与ＬＴ基因５＇端上游──１２８ｂｐ──９３ｂｐ左右的序列相关。ＤＮａｓｅｌ足迹法及其竞争实验发现,ＬＴ基因５＇端上游─—１０４ｂｐ──８３ｂｐ（共２２ｂｐ）序列具有明显的蛋白质保护足迹。同源性分析发现此２２ｂｐ的序列中存在一个ＫＢ样结合位点：─１００ｂｐ──９０ｂｐ（５’－ＧＧＧＧＧＣＴＴＣＣＣ－３’）。ＮＰ－ＫＢ类蛋白质因子参与了此序列的ＤＮＡ－蛋白质的特异性结合,可能存在多种类型的ＮＰ－ＫＢ类蛋白因子参与了ＬＴ基因的表达调控。     

Bovine papilloma virus encoded E2 protein activates lymphokine genes through DNA elements, distinct from the consensus motif, in the long control region of its own genome.

Activation of T cells by antigen, lectin or a combination of phorbol ester (PMA) and calcium ionophore (A23187) leads to the induction of a set of lymphokine genes. Transfection of a human T cell leukemia cell line, Jurkat, or an African green monkey kidney cell line, CV1, with a cDNA encoding E2 protein, a trans-activator of bovine papilloma virus type 1, results in activation of interleukin 2 (IL-2), interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) genes in a transient transfection assay. 5' deletion and mutation analyses showed that the sequence between positions -60 and a TATA-like sequence is required for basic promotor function and that the sequence between positions -95 and -73 containing conserved lymphokine element 2 (CLE2) and a GC box (CLE2/GC box) mediates the positive response to E2 protein. The latter has been previously shown to respond to PMA/A23187 stimulation or to p40tax, a trans-activator encoded by human T cell leukemia virus type 1 (HTLV-I). The sequence located between -108 and -99 (CLE1) is inhibitory to E2 protein or PMA/A23187 stimulation. The combination of E2 protein and PMA/A23187 appears to eliminate an inhibitory effect of the upstream region. However, E2 protein, like p40tax, mediates a positive response through CLE1 alone linked to the basic promoter sequence. The level of activation of the long control region (LCR) by E2 protein is unaffected by the number of CLE2/GC box sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines

A new family of human genes xiap, hiap-1 and hiap-2, which are homologous to the baculovirus iap (inhibitor of apoptosis) genes cp-iap and op-iap, has been recently cloned and shown to suppress apoptosis after serum withdrawal or exposure to a free radical inducer. In order to examine the role of one of these human genes, namely xiap, in lymphoid cells, we studied XIAP expression, after PHA stimulation in three different human T cell lines. We report here that stimulation with PHA resulted in the human T cell lines undergoing apoptosis, as assessed by DNA fragmentation and by propidiumiodide (PI) staining and flow cytometry. When XIAP protein expression was evaluated by Western blot, we observed that the induction of apoptosis by PHA was associated with a parallel decrease of XIAP expression. We also provide evidence that stably transfected Jurkat cells containing the xiap open reading frame became resistant to PHA-induced apoptosis. These data suggest a role for XIAP protein in the regulation of apoptosis in lymphoid cells. This revised version was published online in June 2006 with corrections to the Cover Date.