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.     

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.     

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.     

Protein Kinase C�� Modulates Ligand-induced Cell Surface Death Receptor Accumulation: A MECHANISTIC BASIS FOR ENZASTAURIN-DEATH LIGAND SYNERGY*

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.     

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.     

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.     

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.     

人IL-3基因原核表达载体的构建及表达

目的:构建人IL-3基因原核表达载体pET32a-IL-3,并在大肠杆菌中诱导表达。方法:通过佛波酯(TPA)和植物球血凝素(PHA)刺激人T淋巴细胞系Jurkat细胞,增加IL-3mRNA表达水平,提取mRNA,逆转录-聚合酶链反应(RT-PCR)获得cDNA,以Jurkat细胞cDNA为模板,通过PCR方法扩增得到人IL-3基因,将其克隆入原核表达载体pET32a(+)中,将重组质粒转化入大肠杆菌宿主菌株BL21中,以异丙基硫代半乳糖苷(IPTG)诱导融合蛋白表达,并通过改变IPTG浓度,诱导时间,诱导温度等条件最终实现蛋白的可溶性表达。表达产物用SDS-PAGE检测表达情况。结果:酶切鉴定和测序结果证明成功构建了原核表达载体pET32a-IL-3。SDS-PAGE检测结果证明实现了人IL-3基因在大肠杆菌中的可溶性表达。结论:成功构建了人IL-3基因的原核表达载体并在大肠杆菌中获得了良好的表达。     

Rainbow trout (Oncorhynchus mykiss) sIgM- leucocytes secrete an interleukin-2 like growth factor after mitogenic stimulation in vitro

Two secreted proteins were detected in culture supernatants of PHA or PMA stimulated, immunomagnetically separated, sIgM(-) leucocytes of rainbow trout (Oncorhynchus mykiss) with 60kDa and with 12-15kDa (multiple bands). So called conditioned media (CM), containing these proteins, induced significant activation of blood and head kidney leucocytes. Immunomagnetically separated, naive as well as PHA activated sIgM(-) T lymphocytes and LPS prestimulated sIgM(+) B lymphocytes could be identified to be responding to these secreted proteins. Using a monoclonal antibody specific for mouse IL-2 (clone JES6-1A12), one of the multiple 12-15kDa proteins could be stained in Western blots. It was also shown that the induced proliferation was due to this protein in the CM, as the same anti-IL-2 mab was able to block the CM induced proliferation. Furthermore, survival of the IL-2 dependent mouse cell line HT-2 was enhanced after addition of various concentrations of CM. The data presented show, for the first time, that mitogen stimulated trout sIgM(-) leucocytes secrete a cytokine like growth factor sharing functional and structural similarities with mammalian IL-2.     

Regulation of galectin-9 expression and release in Jurkat T cell line cells

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.     

Suppressing IL-32 in monocytes impairs the induction of the proinflammatory cytokines TNFα and IL-1β

Targeting major proinflammatory cytokines such as IL-1β and TNFα is of great interest in patients with chronic inflammatory diseases, including rheumatoid arthritis, colitis, and psoriasis. The cytokine Interleukin (IL)-32 induces proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines. We previously used an IL-32 ligand-affinity column to purify proteinase 3, which is abundantly expressed in neutrophil and monocytic leukocytes but not in other cell types, and found that IL-32 is mainly produced by monocytic leukocytes. This evidence suggested that silencing endogenous IL-32 by short hairpin RNA (shRNA) in monocytic cells might reveal the precise function of endogenous IL-32. Indeed, lipopolysaccharide (LPS)- or phorbol myristate acetate (PMA)-induced proinflammatory cytokine production was significantly inhibited in shRNA/IL-32 stable clones as compared to control clones. Furthermore, macrophages in PMA-differentiated shRNA/IL-32 stable clones displayed remarkably impaired LPS- and IL-1β-induced proinflammatory cytokine production. These data suggest that IL-32 is not only involved in host defense against pathogens, but also might play a role in chronic inflammatory diseases. IL-32 production leads to major proinflammatory cytokine production during the initial immune response.     

Identification of a soluble IL-2 receptor beta-chain from human lymphoid cell line cells

A clone was isolated from the human lymphoid cell line YT that displayed IL-2R beta, and was found to express much higher levels of IL-2R beta than the original cells. Combining cell surface iodination, affinity labeling of the released soluble protein, and fluorescence sandwich-ELISA for both IL-2 and IL-2.(soluble)(s)IL-2R beta reactants revealed the presence of IL-2-binding protein in the culture supernatant as soluble forms of IL-2R beta. By using the fluorescence sandwich-ELISA elevated levels of sIL-2R beta were measured in culture supernatants of human T cell leukemia virus I positive T cell lines. In addition to this constitutive production of sIL-2R beta, normal PBMC could release low levels of IL-2R beta by stimulation with PHA. In contrast, this was not found in certain human T cell leukemia virus I negative T cell, B cell and macrophage lines. Immunoprecipitation of the soluble protein with IL-2R beta-specific mAb characterized it as an apparent 50- to 55-kDa molecule that is distinct from the 45-kDa soluble IL-2R alpha. Moreover, 10 to 15% of the total cell surface molecules were released into culture supernatants. These results suggest that the released IL-2R beta might serve as an immunoregulatory function in IL-2 dependent both normal and abnormal immune responses.     

人IL-3基因原核表达载体的构建及表达

目的：构建人IL-3基因原核表达载体pET32a-IL-3,并在大肠杆菌中诱导表达。方法：通过佛波酯（TPA）和植物球血凝素（PHA）刺激人T淋巴细胞系Jurkat细胞,增加IL-3mRNA表达水平,提取mRNA,逆转录-聚合酶链反应（RT-PCR）获得cDNA,以Jurkat细胞cDNA为模板,通过PCR方法扩增得到人IL-3基因,将其克隆入原核表达载体pET32a（＋）中,将重组质粒转化入大肠杆菌宿主菌株BL21中,以异丙基硫代半乳糖苷（IPTG）诱导融合蛋白表达,并通过改变IPTG浓度,诱导时间,诱导温度等条件最终实现蛋白的可溶性表达。表达产物用SDS-PAGE检测表达情况。结果：酶切鉴定和测序结果证明成功构建了原核表达载体pET32a．IL-3。SDS-PAGE检测结果证明实现了人IL-3基因在大肠杆菌中的可溶性表达。结论：成功构建了人IL-3基因的原核表达载体并在大肠杆菌中获得了良好的表达。     

用于靶向于NFAT的免疫抑制类小分子化合物筛选的细胞模型的建立

建立IL-2启动子以及NFAT-AP1增强子调控报告基因包括D2egfp或Luciferase在Jurkat细胞中瞬时表达的细胞模型。首先,利用三步连接将IL-2-promoter -255～+285处的序列插入到Pnf-Κb-D2egfp表达载体启动子上游,形成3个拷贝的前后串联的增强子序列;然后从人外周血钓取两条IL-2-promoter序列,包括-326～＋46以及-89～＋46两段基因组序列,分别替代上述重组表达载体中的TK minimal promoter, 构建所需的IL2 promoter以及NFAT-AP1 enhancer调控的报告基因表达质粒：3×NFAT-AP1-IL-2P/D2egfp和3×NFAT-AP1-TATA/D2egfp; 最后,分别将3×NFAT-AP1-IL-2P以及3×NFAT-AP1-TATA融合基因克隆到Pgl3-basic载体中,构建相应的Luciferase报告基因表达质粒。利用电转染方法将重组的报告基因表达载体瞬时转入Jurkat细胞后发现,未刺激以及PMA、离子霉素（Ionomycin）单刺激均不能激活下游报告基因的表达,只有PMA和离子霉素联合刺激才能启动D2egfp以及Luciferase的转录表达,并且5μg/Ml FK506预先作用1h能几乎完全阻断刺激剂诱导的无论是IL-2 promoter还是NFAT-AP1enhancer调控的报告基因的表达。实验结果提示,所构建的Jurkat细胞瞬时表达模型可用于靶向于NFAT信号通路的FK506类免疫抑制小分子化合物的初步筛选。