Leukocyte rolling and emigration in response to inflammatory stimuli appears to involve both E-selectin- and P-selectin-dependent adhesion, which suggests that these molecules have overlapping functions. To clarify their relative contributions in chronic inflammation, we examined delayed-type contact hypersensitivity (DTH) responses in P-selectin, E-selectin, and E-/P-selectin-deficient mice. Oxazolone-induced increases in ear thickness and ear weight were equivalent in wild-type mice and in P-selectin and E-selectin mutants, but were significantly reduced in E-/P-selectin mutants. The number and area of microabscesses on the ears of E-/P-deficient mice were decreased by 72% and 93%, and the number of leukocytes invading the subdermal ear tissue was reduced. T cells from E-/P-deficient mice transferred oxazolone reactivity into naive wild-type mice. However, when donor T cells from wild-type mice were transferred into E-/P-selectin-deficient mice, the DTH response was significantly impaired. These results show that leukocyte recruitment into a subacute inflammatory reaction can occur when either P-selectin or E-selectin is present, but is significantly reduced when both selectins are absent. Both P- and E-selectin are likely to play important roles in the development and maintenance of inflammatory diseases.

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.