Eugene Mochan

Manipulation of Distinct NFκB Proteins Alters Interleukin-1β-induced Human Rheumatoid Synovial Fibroblast Prostaglandin E2 Formation

Interleukin 1β (IL-1β) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the “classic” NFκB consensus site. Immunoblot analysis identified NFκB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1β-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1β-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFκB consensus motif. An NFκB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = ∼2 μM), indicating a role of NFκB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1β-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFκB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the “alternative” IL-8 κB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFκB protein dimers in RSF prostanoid and IL-8 formation.

INTERLEUKIN-4 SUPPRESSION OF INTERLEUKIN-1–INDUCED TRANSCRIPTION OF COLLAGENASE (MMP-1) AND STROMELYSIN 1 (MMP-3) IN HUMAN SYNOVIAL FIBROBLASTS

OBJECTIVE To determine the effects of interleukin-4 (IL-4) on IL-1 induction of collagenase (matrix metalloproteinase 1 [MMP-1]) and stromelysin-1 (MMP-3) in human synovial fibroblasts. METHODS Northern blot analysis was performed to determine the effects of IL-4 on IL-1 induction of MMP messenger RNA (mRNA). MMP protein levels were determined by enzyme-linked immunosorbent assay, and prostaglandin E2 (PGE2) levels were measured by enzyme immunoassay. Run-on transcription assays and transient transfection experiments were performed to determine whether the effects of IL-4 occur at the level of transcription. Activator protein 1 (AP-1) binding was assessed by electrophoretic mobility shift assay. RESULTS Northern blot analysis revealed that coincubation of synovial fibroblasts with IL-1 and IL-4 resulted in a significant decrease in both collagenase and stromelysin mRNA levels compared with IL-1 alone, with a concomitant decrease in MMP protein levels. This inhibition is dose dependent, with an IC50 (50% inhibition concentration) for both MMPs of approximately 0.3 ng of IL-4 per ml, and is at least somewhat selective, since IL-1 induction of c-fos mRNA is not affected. Nuclear run-on experiments and transient transfection studies demonstrated that the suppression of IL-1-induced collagenase and stromelysin expression by IL-4 occurs at least in part at the transcriptional level, and that binding of transcription factor AP-1 is not affected. Although IL-1-induced levels of PGE2 are reduced by IL-4, exogenous addition of PGE2 does not abrogate the inhibitory effects of IL-4 on MMP expression. CONCLUSION IL-4 inhibits IL-1 induction of both collagenase and stromelysin, as well as PGE2 production, in human synovial fibroblasts. The inhibition occurs at least in part at the level of transcription, does not affect binding of transcription factor AP-1, and appears to involve a mechanism that is independent of the ability of IL-4 to inhibit production of PGE2.

Mechanistic features associated with induction of metalloproteinases in human gingival fibroblasts by interleukin-1

Human gingival fibroblasts were treated with recombinant interleukin-1 (IL-1) to determine the effect of this stimulus on the relative expression of collagenase (MMP-1), stromelysin (MMP-3) and plasminogen activator (PA) mRNA. The steady-state mRNA levels for these genes were determined on Northern blots. IL-1 induced steady-state levels of these mRNAs to different extents. Nuclear run-on transcription studies showed that IL-1 induction of neutral metalloproteinase may be transcriptionally regulated. Actinomycin D and protein kinase inhibitors decreased the mRNA production for all three metalloproteinases, whereas cycloheximide decreased the production of collagenase and stromelysin mRNA. Protein kinase inhibitors (H7/H8) decreased production of the three mRNAs to different extents. This study demonstrates a potentially important role for IL-1 in the regulation of metalloproteinase expression in human gingival fibroblasts. The ability of IL-1 to induce the expression of stromelysin, collagenase and PA may define a pivotal role for this cytokine in the pathogenesis of periodontitis.

Molecular cloning and sequencing of a human cDNA encoding ornithine decarboxylase antizyme

We report the cloning of a cDNA encoding the human homolog of ornithine decarboxylase antizyme from a human gingival fibroblast cDNA library. The human antizyme is 84% identical to the rat sequence and shows almost no homology to the E. coli antizyme. Northern analysis studies show that this gene is expressed in both human gingival and synovial fibroblasts.

Interleukin-1 selectively potentiates bradykinin-stimulated arachidonic acid release from human synovial fibroblasts.

Exposure of human synovial fibroblasts prelabelled with [3H]arachidonic acid to bradykinin causes a rapid and sustained increase in arachidonic acid release, a transient increase in cytosolic calcium and an increase in radiolabelled diacylglycerol. Activation of arachidonic acid release by bradykinin was potentiated by interleukin-1 added either simultaneously with bradykinin or to cultures 24 h before addition of bradykinin. In contrast, interleukin-1 did not modify bradykinin-induced increases in cytosolic calcium or diacylglycerol. The stimulation of arachidonic acid release in response to bradykinin, in the absence or presence of interleukin-1, was not affected by RHC-80267, an inhibitor of diacylglycerol kinase, suggesting that deacylation of diacylglycerol was not an important pathway of arachidonic acid production in cultures exposed to bradykinin. This conclusion is supported by the observation that increased release of arachidonic acid was not accompanied by increased release of [14C]stearic acid in cultures labelled with both isotopes. Bradykinin-stimulated release of arachidonic acid was prevented by down-regulating protein kinase C by pretreatment with phorbol 12-myristate 13-acetate and was unaffected by inhibitors of protein synthesis actinomycin D or cycloheximide. On the other hand, interleukin-1 amplification of bradykinin-stimulated release of arachidonic acid was blocked by actinomycin D and cycloheximide. The results from this study point to activation of phospholipase A2 as the source of arachidonic acid in response to bradykinin. Our data further indicate that interleukin-1 selectively potentiates bradykinin activation of a phospholipase A2 by a mechanism requiring protein synthesis, but has no effect on bradykinin activation of phospholipase C.

Evidence that suppression of IL-1 induced collagenase mRNA expression by dihomo-γ-linolenic acid (DGLA) involves inhibition of NFκB binding

Joint destruction in rheumatoid arthritis is thought to be mediated by increased production of metalloproteinases (e.g. collagenase, stromelysin) by synovial fibroblasts. Evidence suggests that the cytokine interleukin-1 (IL-1) plays an important role in stimulating transcription of these genes. However, the mechanisms involved in the IL-1 induction of MMP have not yet been fully elucidated. Studies have suggested that diets supplemented with g-linolenic acid (GLA; 18:3q-6) or its botanical lipid precursors, may be a safe and effective anti-inflammatory treatment for rheumatoid arthritis, reducing symptoms such as joint pain and morning stiffness in humans [1, 2] and tissue injury in animal models [3, 4]. We have shown [5] that incorporation of dihomo-g-linolenic acid (DGLA, a precursor of GLA) into the membranes of synovial fibroblasts suppresses the IL-1 induction of collagenase mRNA, but has no effect on induction of stromelysin. Here we present evidence that this discoordinate regulation of MMP genes by DGLA may be mediated by inhibiting binding of transcription factor NF kB to the collagenase promoter.

Concanavalin a stimulation of plasminogen activator production in pig kidney cells in culture

Addition of subtoxic doses of the lectin concanavalin A to growing subconfluent monolayer cultures of pig kidney cells causes an increase in extra- and intracellular plasminogen activator activity which is reversibly inhibited by actinomycin D, cycloheximide and alpha-methyl-D-mannoside. These results suggest that cell surface events may play an important modulatory role in plasminogen activator production.

DGLA discoordinately suppresses IL-1 induced metalloproteinase mRNA levels in human synovial fibroblasts

Joint destruction in rheumatoid arthritis (RA) is thought to be mediated by increased synovial fibroblast production of metalloproteinases (MMP e.g., collagenase, stromelysin) in response to cytokines such as interleukin-1 (IL-1). Studies [1, 2] in animal models and human trials have suggested that diets supplemented with omega-6 fatty acids ( glinolenic acid (GLA: 18:3), dihomogammalinolenic acid (DGLA: 20:3)) may be safe and effective anti-inflammatory treatments for rheumatoid arthritis (RA). In the present study, we examined whether DGLA affects the IL-1 induction of MMP mRNA expression in human synovial fibroblasts in culture.

Loss of body cell mass in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease of unknown etiology. It is characterized by an abnormal immune response involving antigen-specific T and B lymphocyte hyperactivity [1]. Systemic manifestations of SLE include arthritis, skin rash, fatigue, fever, and weight loss. Decreased body cell mass (BCM) without a decrease in fat mass (FM) has been reported to occur in AIDS [2], cancer [3], rheumatoid arthritis (RA) [4] and normal aging [5]. It has been suggested that BCM is an influential factor in energy metabolism and a good indicator of health status in inflammatory diseases [4]. The present study was designed to determine whether a loss of BCM occurs in another chronic autoimmmune disease such as SLE. BCM levels were measured in SLE patients by bioelectrical impedance analysis (BIA) to determine whether there was a difference in BCM levels between SLE patients and controls. Determining BCM by BIA may be useful in monitoring the health status of SLE patients.

Identification of IL-1 regulated genes in human synovial and gingival fibroblasts by differential display

Interleukin-1 (IL-1) is a pro-inflammatory, multifunctional cytokine, which affects nearly every cell type, often acting in concert with other cytokines or small mediator molecules. In rheumatoid arthritis, IL-1 and tumor necrosis factor-alpha (TNF-a) not only promote the inflammatory response but also induce cartilage degradation. It has also been shown that fibroblasts derived from inflammatory synovitis are one of the major sources of damaging mediators in this disorder [1]. Elevated levels of cytokines such as IL-1, IL-2, IL-6, TNF-a, TGFb have been detected in these cells as well as in the synovial fluid. Abnormal gene programs are activated, leading to enhanced inflammatory response and cell proliferation [1]. Differential display analysis of mRNA was utilized to identify genes that are regulated by IL-1 in human synovial fibroblasts.