Recombinant human interleukin-la (IL-la) and recombinant human IL-1P stimulate matrix proteoglycan degradation and inhibit glycosaminoglycan synthesis in bovine nasal cartilage explants. A 17-kd human recombinant IL-1 receptor antagonist protein (IRAP) caused a concentration-dependent (0.2-200 ng/ml) suppression of the effects of IL-la and IL-lP in cartilage organ cultures. IRAP inhibited the binding of radiolabeled IL-la to rabbit articular chondrocytes. Matrix metalloproteinase (collagenase, gelatinase, and stromelysin) and prostanoid production by IL-l-activated rabbit articular chondrocytes was also suppressed by IRAP. These results could have potential significance in the development of a new antiarthritis therapy based on an IRAP.Interleukin-1 (IL-1) is a 17-kd polypeptide that is produced by a variety of cells and is believed to play a major role in hoddefense and inflammation (1,2).Two forms of IL-1 ( a and p), which bind to the same receptor (2) and exert comparable effects on numerous target cells, have been identified (2). Although the role of IL-1 in physiologic events awaits clarification, the presence of IL-1 in arthritic joint fluid (1,2), together with the ability of this cytokine to induce cartilage degradation (2,3) and bone resorption (2), emphasizes the probable participation of IL-1 in the pathogenesis of rheumatic disease. Thus, inhibition of these and other deleterious activities of IL-1 might represent an effective therapy for inflammatory joint diseases.IL-1 receptor antagonists have been identified in the urine of patients with fever (4) and in culture media of growth factor-activated monocytes (5). An antagonist of IL-1 activity was purified from the culture supernatant of immune complex-activated monocytes (2). This protein inhibited IL-1 receptor binding and was termed IL-1 receptor antagonist (6,7). A human myelomonocytic cell line (U937)Aerived IL-1 receptor antagonist protein (IRAP) was recently purified, sequenced, cloned, and expressed (8). IRAP was shown to inhibit IL-I activity in vitro and in vivo (8).The purpose of the present study was to investigate the effects of IRAP on IL-l-elicited cartilage erosion and on matrix metalloproteinase (MMP) and prostaglandin E, (PGE,) production by IL-l-activated chondroc ytes. MATERIALS AND METHODSCartilage explant cultures. Methods for preparation of cartilage organ cultures and for quantitation of matrix degradation (glycosaminoglycan [GAG] release) and GAG synthesis (incorporation of 35S-sulfate into cartilage discs) have been described previously (3). Cartilage explants were cultured for 8 days in Dulbecco's modified Eagle's medium (DMEM) with 5% heat-inactivated fetal calf serum (FCS). Each experimental group was assayed in quintuplicate.Chondrocyte cultures. Confluent monolayer cultures of rabbit articular chondrocytes (RAC) were used throughout these studies. Monolayer cultures Arthritis and Rheumatism, Val. 34, No. 1 (January 1991)

ABSTRACT15(S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) exerted a time-and concentrationdependent inhibition of superoxide anion (O°) production and exocytosis of both azurophil and specific granule constituents from human polymorphonuclear neutrophils (PMN) stimulated with the receptor-specific agonists, N-formylmethionylleucylphenylalanine (FMLP), platelet-activating factor, and leukotriene B4, but not that elicited by phorbol 12-myristate 13-acetate. 15-HETE did not alter the binding of FMLP to its specific receptors on PMN but, rather, appeared to interfere with a subsequent process in signal transduction. Receptorcoupled production of inositol 1,4,5-trisphosphate (InsP3) and increases in cytosolic free calcium elicited with FMLP, plateletactivating factor, and leukotriene B4 were suppressed by 15-HETE. 15--HETE did not, however, inhibit the mobilization of45Ca from intracellular stores elicited by the addition ofInsP3 to permeabilized PMN. 15-HETE suppressed 0-production and increases in intracellular [Ca2+] induced when cell-surface receptors were bypassed and the PMN were activated directly by the guanine nucleotide-binding protein (G protein) activators aluminum fluoride (AIF ) and mastoparan. 15-HETE, however, did not perturb all G protein functions because cAMP production in FMLP-activated PMN was essentially unaffected by 15-HETE. These data support the proposition that 15-HETE modulates receptor-triggered activation of PMN either by uncoupling G protein stimulation of phospholipase C or by directly inhibiting phospholipase C, thus inhibiting the InsP3-dependent rise in intracellular [Ca2+] that is prerequisite for PMN responsiveness to receptor agonists.

The capacity of arachidonic acid (AA) to stimulate granule exocytosis from human polymorphonuclear neutrophils (PMNs) was investigated. AA induced the selected extracellular release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12 binding protein) granule constituents from human PMNs in a time‐and concentration‐dependent manner. Cytochalasin B (CB) enhanced but was not required for PMN activation with AA. Although extracellular calcium had no effect on granule exocytosis, AA did stimulate the mobilization of intracellular sequestered Ca2+ which resulted in an increase in cytosolic‐free Ca2+ ([Ca2+]i) as reflected by increased fluorescence of Fura‐2‐treated cells. AA stimulated Ca2+/phospholipid‐dependent protein kinase C (PK‐C) activity in PMNs. 4,4′‐Diisothiocyano‐2,2′‐disulphonic acid stilbene (DIDS), an anion channel blocker, caused a concentration‐dependent inhibition of granule enzyme release. Activation of PMNs with AA was unaffected by the lipoxygenase/cyclo‐oxygenase inhibitors, 5,8,11, 14‐eicosatetraynoic acid (ETYA) and benoxaprofen, a lipoxygenase inhibitor, 6, 9, deepoxy‐6,9‐(phenylimino)‐Δ6,8‐prostaglandin 11 (piriprost potassium) or a pure cyclo‐oxygenase inhibitor, flurbiprofen. These data define the properties of AA as a secretory stimulus for human PMNs.

Recombinant human monocyte-derived interleukin-8 (IL-8M), recombinant human endothelium-derived IL-8 (IL-8E), and a recombinant human truncated form of IL-8 (IL-8T) stimulated a time-dependent (t 1/2 approximately 2-3 s) and concentration-dependent (0.1-100 nM) release of azurophil (myeloperoxidase) and specific (vitamin B12 binding protein, gelatinase) granule constituents from cytochalasin B-treated human neutrophils (HNs) wherein IL-8T = IL-8M greater than IL-8E. An increase in the cytosolic free calcium concentration ([Ca2+]i) was greater in IL-8T- than in IL-8M- or IL-8E-activated HNs, and IL-8T was more potent than either IL-8M or IL-8E in sequentially desensitizing the HNs to the effects of the other IL-8 forms. IL-8 induced a time- and concentration-dependent (0.1-100 nM) increase in the production of inositol 1,4,5-trisphosphate (IP3) in HNs. U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), a potent inhibitor of phospholipase C-catalyzed events in HNs, suppressed IL-8-triggered IP3 production, increased [Ca2+]i and granule exocytosis in HNs. The membrane-associated activity of the alpha and beta subtypes of protein kinase C was significantly enhanced in IL-8-activated cells.

Recombinant human interleukin-la (IL-la) induced a time-dependent (0-72 hours) and concentration-dependent (0.01-10 ng/ml) production of metalloproteinases (collagenase, gelatinase, stromelysin) and prostaglandin E, (PGE,) in rabbit articular chondrocytes (RAC). Exposure of RAC to recombinant human plateletderived growth factor homodimer BB (PDGF-BB; 2-200 ng/ml) in the presence of stimulatory and substimulatory concentrations of IL-la resulted in a marked augmentation of metalloproteinase and PGE, production. PDGF-BB exerted no agonist effects on RAC responsiveness. PDGF-BB up-regulated the number of IL-1 receptors per chondrocyte but had no effect on receptor affinity. Cycloheximide and actinomycin D caused a concentrationdependent suppression of the PDGF-BB-mediated potentiation of radiolabeled IL-la binding to RAC and cell responsiveness to IL-la. Similarly, IL-1 increased the number of PDGF receptors on RAC without changing receptor affiity. These data are discussed within the context of cytokine-growth factor interactions as components of the pathogenesis of arthritic diseases.Articular cartilage erosion and resorption of subchondral bone in inflammatory joint disease is caused by matrix metalloproteinases (MMP) and prostanoids released from the proliferative synovial lesion that is in contact with the cartilage surface (1,2). The inflamed synovium is composed of heterogenous cell populations which produce a broad spectrum of cytokines and growth factors, which, in turn, stimulate MMP and prostaglandin E, (PGE,) production by synovial fibroblasts (3-5). The occurrence of interleukin-1 (IL-1) (6), tumor necrosis factor a (TNFa) (7), platelet-derived growth factor (PDGF) (8), and IL-6 (9) in arthritic synovial fluid implies a pathogenic role(s) for these factors.IL-1 is a 17-kd polypeptide that is produced by rheumatoid synovial tissue (10,ll) and chondrocytes (12). Synovial cells (13) and chondrocytes (14,15) express IL-1 receptors and produce MMP and PGE, in response to 4,[15][16][17][18][19]. A major role of IL-I in the pathogenesis of arthritic diseases is supported by reports of IL-I stimulating B and T lymphocyte proliferation, a febrile response, bone resorption, and the production of acute-phase reactants (20). PDGF is produced by macrophages, platelets, and endothelial cells (21), which are components of the inflamed synovium. PDGF is a chemoattractant for monocytes, fibroblasts, and neutrophils (22) and is mitogenic for fibroblasts, vascular smooth muscle cells, and chondrocytes (21).Since it is unlikely that tissue destruction in rheumatoid arthritis is mediated by any one cytokine and/or growth factor, synergistic interactions between MATERIALS AND METHODSChondrocyte culture. Confluent monolayer cultures of rabbit articular chondrocytes (RAC) were used throughout this study. The monolayer cultures were obtained from single-cell suspensions of chondrocytes isolated from the articular cartilage of the knee joints of New Zealand white rabbits, as described by Chin and Lin (18). Briefly, cartilage t...