Differential usage of NF-κB activating signals by IL-1β and TNF-α in pancreatic beta cells

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.     

IL-2 and IL-15 manifest opposing effects on activation of nuclear factor of activated T cells

IL-2 and IL-15 are cytokines involved in T cell activation and death. Their non-shared receptors, IL-2Ralpha and IL-15Ralpha, are important in the homeostasis of lymphocytes as evidenced by gene deletion studies. How these cytokine/receptor systems affect T cell antigen receptor signaling pathways is poorly understood. Here, we show that the IL-2 and IL-15 cytokine/receptor alpha systems regulate activation of nuclear factor of activated T cells (NF-AT) in opposing ways. IL-15Ralpha increased while IL-2Ralpha decreased basal NF-AT activation status in a Jurkat transient transfection model. The effect of each of the alpha chain receptors on NF-AT activation was further opposed by addition of the respective cytokine. These effects were inhibited by anti-cytokine and anti-cytokine receptor reagents as well as by inhibitors of TCR signaling. These results suggest a novel pathway of cytokine action to regulate T cell signaling, activation, death, and homeostasis.     

Double-stranded RNA regulates IL-4 expression

dsRNA, as genomic fragment, replicative intermediate, or stem and loop structure in cells infected by viruses, can act to signal the immune system of the presence of viral infections. Although most viral infections are associated with strong Th1 immune responses, Th2-type responses have also been observed. In this study, we characterize the effects of dsRNA on the induction of Th2 responses in human lymphocytes. We report that in addition to the well-known Th1-inducing capabilities of dsRNA, treatment of human lymphocytes with low concentrations of dsRNA (0.1-1 microg/ml) leads to the expression of the prototypic Th2 cytokine IL-4. This induction was accompanied with the concentration-dependent activation of NF-kappaB and NF-AT2 but not NF-AT1. In addition, dsRNA can directly activate an IL-4 promoter-driven chloramphenicol acetyltransferase reporter gene in transiently transfected Jurkat cells. These results are the first demonstration of a non-TCR-associated activator of NF-AT in human cells and suggest that dsRNA directly influences IL-4 gene expression through its effect on NF-AT activation. Our data provide support for the idea that dsRNA at low concentrations in vivo may induce a Th2-dominant response that is not optimal for protective immunity to the virus.     

NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation

NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-κB activation. However, the exact mechanism of its function has not been fully understood. Here, we report that knockdown of NEMO by RNA interference in Jurkat E6.1 cells enhanced TCR-induced NF-κB report gene activity and IL-2 production by promotion of IκBα degradation and p65 nuclear translocation, whereas inhibited TNF-α and LPS-induced IκBα degradation without influencing the phosphorylation of MAPKs. In human primary T and Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-κB activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-κB report gene activity was increased along with ascending doses of transfected siNEMO and reached the highest activity when knockdown about 70% of NEMO, then turned to decline and gradually be blocked once almost thoroughly knockdown of NEMO. Meanwhile, TNF-α induced NF-κB was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKKβ may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKKβ continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKKβ. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-κB activation and differentially regulates TNF-α and TCR-induced NF-κB pathways.     

Cyclosporin A sensitivity of the NF-kappa B site of the IL2R alpha promoter in untransformed murine T cells.

We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression.     

Cyclosporin A inhibits CD11a/CD18 adhesion molecules due to inhibition of TNF-α and IL-1β levels in the mouse model of pleurisy induced by carrageenan

The mouse model of pleurisy induced by carrageenan is characterized by a significant enhancement of cell migration due to neutrophils, 4 h after pleurisy induction. Forty-eight hours after pleurisy induction, a significant increase in cell migration due to mononuclear cells occurs. Recently, studies in our laboratory have demonstrated that cyclosporine A (CsA) inhibits leukocyte migration in the pleural cavity and lungs in the mouse model of pleurisy induced by carrageenan. In the present work we evaluated whether CsA was able to downregulate CD11a/CD18 adhesion molecule in the lungs, as well as TNF-α and IL-1β levels in the fluid leakage of the pleural cavity in this model. Our results showed that CsA significantly decreased CD11a/CD18 in the lungs, as well as TNF-α and IL-1β levels in the fluid leakage of the pleural cavity 4 h and 48 h after pleurisy induction. It is our hypothesis that the inhibitory effect elicited by CsA upon these adhesion molecules may be also be attributed to the downregulation of TNF-α and IL-1β cytokines.     

A common factor regulates both Th1- and Th2-specific cytokine gene expression.

Murine T helper cell clones are classified into two distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), on the basis of cytokine secretion patterns. Th1 clones produce interleukin-2 (IL-2), tumor necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), while Th2 clones produce IL-4, IL-5, IL-6 and IL-10. These subsets differentially promote delayed-type hypersensitivity or antibody responses, respectively. The nuclear factor NF-AT is induced in Th1 clones stimulated through the T cell receptor-CD3 complex, and is required for IL-2 gene induction. The NF-AT complex consists of two components: NF-ATp, which pre-exists in the cytosol and whose appearance in the nucleus is induced by an increase of intracellular calcium, and a nuclear AP-1 component whose induction is dependent upon activation of protein kinase C (PKC). Here we report that the induction of the Th2-specific IL-4 gene in an activated Th2 clone involves an NF-AT complex that consists only of NF-ATp, and not the AP-1 component. On the basis of binding experiments we show that this 'AP-1-less' NF-AT complex is specific for the IL-4 promoter and does not reflect the inability of activated Th2 cells to induce the AP-1 component. We propose that NF-ATp is a common regulatory factor for both Th1 and Th2 cytokine genes, and that the involvement of PKC-dependent factors, such as AP-1, may help determine Th1-/Th2-specific patterns of gene expression.     

Cyclosporin A inhibits initiation but not progression of human T cell proliferation triggered by phorbol esters and calcium ionophores

Cyclosporin A (CsA) is a potent inhibitor of T lymphocyte proliferation induced by Ag and mitogens. In an attempt to further delineate the mechanism of action of CsA, we have examined its effects on T cell proliferation induced by the combination of the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin. T cells were rendered competent as the result of a 30-min initial incubation with both drugs, after which the drugs were washed out. Competence is defined as the ability to subsequently proliferate in response to exogenously added IL-2 or PDB in the second phase of the culture, but not to synthesize IL-2 or proliferate without these additions. Addition of CsA (1 microgram/ml) to the cells in the initial, competence-inducing 30-min incubation with PDB/ionomycin abrogated their subsequent response to IL-2 or PDB. In contrast, addition of CsA to cells after they had been treated for 30 min with PDB/ionomycin and then washed did not affect their responses to subsequent addition of either IL-2 or PDB. Treatment with CsA during induction of competence prevented the expression of the 55-kDa IL-2R gene during competence induction and inhibited IL-2 gene expression and IL-2 production in response to PDB in the second phase. These results indicate that the effects of CsA are limited to the initiation (competence induction) period of T cell activation, that CsA apparently affects expression of more than one gene, and in competent cells, CsA does not affect their ability to progress to DNA synthesis.     

Regulation of the human interleukin-2 receptor alpha chain promoter: activation of a nonfunctional promoter by the transactivator gene of HTLV-I

We have characterized regulatory regions of the human IL-2 receptor alpha chain (IL2R alpha) promoter. 5' deletion constructs extending to -327 directed CAT expression in HTLV-I-infected T cells, which express IL2R alpha constitutively, and in Jurkat cells, which express IL2R alpha only after induction. Deletions to -267 and -265 were active only in HTLV-I-transformed T cells, but their activity in Jurkat cells was restored by cotransfection of a construct expressing the HTLV-I transactivator protein (tat-I). However, HTLV-I-infected human osteosarcoma cells do not express IL2R alpha-CAT constructs. Thus cell-type-specific factors are required for IL2R alpha expression, and direct or indirect interaction(s) between tat-I and a specific region of the IL2R alpha promoter may cause altered regulation. Tat-I also augments IL2-CAT expression under some conditions, suggesting possible autocrine or paracrine mechanisms for HTLV-I-induced leukemogenesis.