A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways

Interleukin-18 (IL-18) is a novel proinflammatory cytokine found in serum and joints of patients with rheumatoid arthritis (RA). We studied a novel role for IL-18 in mediating cell adhesion, a vital component of the inflammation found in RA and other inflammatory diseases. We examined the expression of cellular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and RA synovial fibroblasts using flow cytometry. Adhesion of the monocyte-like cell line HL-60 to endothelial cells was determined by immunofluorescence. IL-18 significantly enhanced ICAM-1 and VCAM-1 expression on endothelial cells and RA synovial fibroblasts. In addition, IL-18 induced E-selectin expression on endothelial cells and promoted the adhesion of HL-60 cells to IL-18-stimulated endothelial cells. Neutralizing anti-VCAM-1 and anti-E-selectin could completely inhibit HL-60 adherence to endothelial cells. IL-18-induced adhesion molecule expression appears to be mediated through nuclear factor kappa B (NF kappa B) and phosphatidyl-inositol 3 kinase (PI 3-kinase) since addition of inhibitors to either NF kappa B (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) or PI 3-kinase (LY294002) inhibited RA synovial fibroblast VCAM-1 expression by 50 to 60%. Addition of both inhibitors resulted in inhibition of VCAM-1 expression by 85%. In conclusion, the ability of IL-18 to induce adhesion molecule expression on endothelial cells and RA synovial fibroblasts indicates that IL-18 may contribute to RA joint inflammation by enhancing the recruitment of leukocytes into the joint. IL-18 requires NF kappa B as well as PI 3-kinase to induce VCAM-1 on RA synovial fibroblasts, suggesting that there may be two distinct pathways in IL-18-induced adhesion molecule expression.     

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.     

Activation of the selenoprotein SEPS1 gene expression by pro-inflammatory cytokines in HepG2 cells

SEPS1 (also called selenoprotein S, SelS) plays an important role in the production of inflammatory cytokines and its expression is activated by endoplasmic reticulum (ER) stress. In this report, we have identified two binding sites for the nuclear factor kappa B in the human SEPS1 promoter. SEPS1 gene expression, protein levels and promoter activity were all increased 2-3-fold by TNF-alpha and IL-1beta in HepG2 cells. We have also confirmed that the previously proposed ER stress response element GGATTTCTCCCCCGCCACG in the SEPS1 proximate promoter is fully functional and responsive to ER stress. However, concurrent treatment of HepG2 cells with IL-1beta and ER stress produced no additive effect on SEPS1 gene expression. We conclude that SEPS1 is a new target gene of NF-kappaB. Together with our previous findings that SEPS1 may regulate cytokine production in macrophage cells, we propose a regulatory loop between cytokines and SEPS1 that plays a key role in control of the inflammatory response.     

Human anti-human IL-18 antibody recognizing the IL-18-binding site 3 with IL-18 signaling blocking activity

IL-18 is an important regulator in both innate and acquired immune responses. The aberrant expression of IL-18 is associated with severe inflammatory conditions, such as autoimmune diseases and allergies. Thus, human antibodies with inhibitory activity on IL-18 signaling may be useful for therapeutic applications. We report here the first establishment of an antagonistic anti-IL-18 complete human antibody, h18-108, employing a human single chain antibody (scFv)-displaying phage library. The h18-108 scFv inhibited the IFN-gamma production of a human myelomonocytic cell line, KG-1. Flow cytometry analysis showed that h18-108 blocked the binding of IL-18 to KG-1 cells. Epitope mapping analysis using two kinds of random peptide-displaying phage libraries and an IL-18 alanine mutant (D98A) demonstrated that the h18-108 scFv binds to the site 3 of IL-18, which is suggested to be an association site with the IL-18 receptor beta. The complete human Fab and IgG forms of h18-108 have been successfully constructed to attain increases in both binding affinity and inhibitory activity.     

Effects of exercise on gene expression in human peripheral blood mononuclear cells.

Exercise leads to increases in circulating levels of peripheral blood mononuclear cells (PBMCs) and to a simultaneous, seemingly paradoxical increase in both pro- and anti-inflammatory mediators. Whether this is paralleled by changes in gene expression within the circulating population of PBMCs is not fully understood. Fifteen healthy men (18-30 yr old) performed 30 min of constant work rate cycle ergometry (approximately 80% peak O2 uptake). Blood samples were obtained preexercise (Pre), end-exercise (End-Ex), and 60 min into recovery (Recovery), and gene expression was measured using microarray analysis (Affymetrix GeneChips). Significant differential gene expression was defined with a posterior probability of differential expression of 0.99 and a Bayesian P value of 0.005. Significant changes were observed from Pre to End-Ex in 311 genes, from End-Ex to Recovery in 552 genes, and from Pre to Recovery in 293 genes. Pre to End-Ex upregulation of PBMC genes related to stress and inflammation [e.g., heat shock protein 70 (3.70-fold) and dual-specificity phosphatase-1 (4.45-fold)] was followed by a return of these genes to baseline by Recovery. The gene for interleukin-1 receptor antagonist (an anti-inflammatory mediator) increased between End-Ex and Recovery (1.52-fold). Chemokine genes associated with inflammatory diseases [macrophage inflammatory protein-1alpha (1.84-fold) and -1beta (2.88-fold), and regulation-on-activation, normal T cell expressed and secreted (1.34-fold)] were upregulated but returned to baseline by Recovery. Exercise also upregulated growth and repair genes such as epiregulin (3.50-fold), platelet-derived growth factor (1.55-fold), and hypoxia-inducible factor-I (2.40-fold). A single bout of heavy exercise substantially alters PBMC gene expression characterized in many cases by a brisk activation and deactivation of genes associated with stress, inflammation, and tissue repair.     

Differential effect of IL-4 and IL-13 on the expression of recombination-activating genes in mature B cells from human peripheral blood

We examined the expression of recombination-activating genes (RAG-1 and RAG-2) and activation-induced cytidine deaminase (AID) by mature human blood B cells stimulated with anti-CD40 in the presence of IL-4 or IL-13. IL-4 was an effective cofactor for RAG-1 and RAG-2 expression, whereas IL-13 was not. In addition, IL-4-dependent RAG expression combined with AID and IgE expression allowed predominant expression of newly rearranged lambda light chains on IgE+ cells generated from kappa+ cells. Although the magnitudes of IL-4- and IL-13-dependent AID and IgE expression were related to expression levels of binding subunits of the IL-4 and IL-13 receptors, IL-13 was ineffective for light chain replacement in the induced IgE+ cells due to the failure in RAG expression. Our studies using mature blood B cells indicate that IL-4-responsive cells, unlike IL-13-responsive cells, undergo lambda gene rearrangement leading to replacement in parallel with RAG expression and suggest that this replacement may contribute to the regulation of affinity maturation of IgE antibodies.