THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 499

295

INTERLEUKIN I-STIMlJL.ATED CARTILAGE PROTEOGLYCAN DEGRADATION IS INHIBITED BY SOME CYSTEINE ENDOPEPTIDASE INACTIVATORS

David 3. Buttle, Jeremy Saklatvala, Alan J. Barrett Demrtments of Biochemistry and Cytokine St>angeways Research Labora tory, Cambridge, Ii. K.

Biochemistry,

IL1 damages cartilage by stimulating chondrocytes to degrade their proteoglycan matrix. This well-known phenomenon is thought to be a primary event in damage to cartilage in RA, but the proteolytic enzymes responsible have not been identified. We tested the ability of inactivators of cysteine endopeptidases to inhibit release of proteoglycan from explants of bovine nasal septum cartilage stimulated by 1Lla. Some lipophilic inactivators of lysosomal cysteine endopeptidases inhibited ILl-induced degradation at concentrations as low as 10 or 1 pM. These agents were not cytotoxic, as judged by reversibility, rates of glycolysis, protein and proteoglycan synthesis. Nor did they inhibit other responses to ILl, including EGF receptor transmodulation and prostaglandin synthesis. We conclude that the lysosomal cyst&w endopeptidases are involved in ILl-stimulated cartilage breakdown, by acting in a membrane- restricted environment.

296

PRODUCTION OF IL-1 RECEPTOR ANTAGONIST (IL-IRA) AND IL-lb IN WHOLE BLOOD. Judith L. Nerad, Debra D. Poutsiaka, Charles A. Dinarello and Joseuh G. Cannon. Dept. of Med., N. Engl. Med. Ctr. and USDA Human Nutr. Res. Ctr, Tufts Univ, Boston, MA 02111.

We have studied the production of IL-18 and IL-IRA in whole blood using a method we have developed recently as an alterna- tive to mononuclear cell cultures. Blood was collected in stan- dard 3 cc vacuum tubes containing EDTA and stimulated by adding a 100 ul solution of LPS and PHA via tuberculin syringe through the rubber stopper of the tube (final cont., 10 rig/ml and 30 ug/ml, respectively). Whole blood from 5 donors (8 tubes each) was stimulated. The plasma was separated and frozen after periods of incubation at 37’C ranging from 1 to 24 hrs, then assayed for IL-1RA and IL-18. IL-IRA was detectable in the plas- ma after 3 hrs and reached maximal levels of 11.8 f 1.4 rig/ml at 10 hrs; the magnitude was similar to IL-IRA production by LI’S- stimulated cells in vifro, but the peak occurred sooner (24 hrs, see accompanying abstract by Poutsiaka et al.). IL-18 reached a maximum of 5.8 + 2.3 rig/ml at 8 hrs. These results indicate that the whole blood method is a suitable alternative to mononuclear cell cultures for simultaneous assessment of both IL-18 and IL-IRA production that requires less equipment and labor and is less likely to become contaminated. Therefore it is of parti- cular value in field studies in developing countries.

291

IN VIVO ASSESSMENT OF IL-18 AND IL-1 RECEPTOR ANTAGONIST (IL-IRA) IN PHYSICAL TRAUMA. loseuh G. Cannon, Roger A. FieldinP. Debra D. Poutsiaka. Maria A. Fiatarone, William 1. Evans, Charles A. Dinarello. USDA Human Nutr. Res. Ctr., Tufts Univ. and Dept. of Medicine, N. Engl. Medical Ctr. Boston, MA 02111.

Forced lengthening of muscle during tension development, such as occurs in downhill running, causes myofibrillar damage, leuko- cyte infiltration, muscle soreness and proteolysis. We studied the induction of IL-18 and IL-IRA in 9 subjects who ran downhill on a treadmill for 45 minutes. IL-18 content in biopsies taken from the vastus lateralis before and after exercise was detected immunohis- tochemically and quantified by computer image analysis. Mean IL- 18 staining increased 144% by 1 hr after exercise, well in advance of peak plasma levels of IL-18 (49 * 13 pg/ml at 6 hr) and peak cellular production in vitro (63% increase at 24 hr). At 5 days, staining was increased 272%. In this model of trauma, plasma IL-18 typically reaches 70% of the levels observed in humans receiving 4 rig/kg LPS, but we have found no evidence of circulating IL-1RA in any subject. This is in sharp contrast to plasma IL-1RA levels observed after giving LPS (-7,000 pg/ml, see abstract by Granowitz et al). We conclude that tissue levels of IL-18 may rise faster and remain ele- vated longer than indicated by plasma or in oitro analyses. In addi- tion, IL-19 may play a significant role in host responses to trauma not associated with infection because it is not opposed by IL-IRA.

298

MOLECULAR CLONING OF RABBIT, IGl-RECEPTO; $T;ZO”yT (ILARA) GENE. F. Cpmmell~. M.T. Brewer. an

se be e, Division of GI and Liver Diseases, University of Southern California, School of Medicine, Los Angeles, CA; Synergen Inc., Boulder, CO.

The rabbit has been used to study the involvement of IL-1 in the pathogenesis of colitis, arthritis, septic shock, ocular inflammation and atherosclerosis. It is of interest to study the regulation of endogenous IL-lra gene expression in these disease models. In addition, the use of homologous recombinant protein will allow repeated administrations without generation of neutralizing antibodies in the experimental animals. We therefore initiated a project to clone and express rabbit IL- Ii-a. We first generated a rabbit IL-ha-specific probe by PCR amplification of rabbit genomic DNA. The PCR primers were degenerate oligonucleotides that correspond to sequences flanking the fourth exon of the gene for human, mouse, and rat IL-lra. The probe was then used to screen a rabbit genomic library, from which several clones were isolated. The sequence of the coding region of the IL-lra gene from one of the clones was then determined. By sequence comparison, we found that rabbit IL-lra shows a high degree of homology to the IL-lra proteins from human, mouse and rat. Based on the sequence of the protein, a synthetic gene has been constructed and recombinant protein has been expressed in E. coli. Specific nucleic acid probes and antibodies against rabbit IL-lra generated by these studies will allow the investigation of the role of IL-l and IL-lra in inflammatory disease models induced in the rabbit.

299

PREVENTION AND TREATMENT OF RABBIT IMMUNE COLITIS BY HUMAN RECOMBINANT IGl RECEPTOR ANTAGONIST @rIGlra). F. Cominelli. Division of GI and Liver Diseases, University of Southern California, School of Medicine, Los Angeles, CA.

To determine that IL-l is the key mediator of rabbit formalin-immune complex colitis we have examined the effects of hrll-lra on inflammation, tissue damage and production of inflammatory mediators in this model. Treatment of rabbits with intravenous administration of IL-lra before and during the first 43 hours after the induction of colitis suppressed inflammation and tissue damage in a dose-related manner. Maximum inhibition of inflammatory index (by 73%), edema (by 74%), percent of mucosai necrosis (by 84%) and MPO activity (by 80%) occurred with the dose of 5 mg/kg every 8 hours. Colonic PGE2 and LTB4 production, measured in rectal dialysates by specific radioimmunoassays, was also dose-dependently suppressed (84% and 83% inhibition respectively at 5 mglkg). In contrast, colonic IL-la tissue levels, measured by a specific radioimmunoassay after tissue extraction, were similar in all groups. When only 2 doses of IL-lra, 10 minutes before and 3 hr after the induction of colitis were given, there was no longer an inhibitory effect on inilammation or production of inflammatory mediators. Delaying the IL-lra treatment 3 hours after the induction of colitis was still effective in reducing inflammatory index (by 60%), MPO activity (by 79%), PGE2 (by 62%) and LTB4 (by 72%) whereas colonic IL-lo levels were unchanged compared to vehicle-treated animals. These studies demonstrate the ability of hrll-lra to suppress the proinflammatory activity of IL-1 produced in the colon during the induction and progression of rabbit immune complex colitis. Thus, the use of the IL-lra may be beneficial in inflammatory diseases characterized by overproduction of IL-l, including rheumatoid arthritis and inflammatory bowel disease.

300

INHIBITION OF LTB4 MONOCYTE PRODUCTION BY INTERLEUKIN-1 RECEPTOR ANTAGONIST (IL-1RA). Pio Conti, “Maria R. Pnnara, Rennto C. Barbacane, Marcella Reale and ‘Matno Bongrszio and meehnris C. The&arid- Immunology Division. Insrirute of Experimenfal Medicine, University of Chieti Medico/ School, Chiefi. Iloly and “Institute of Normal and Porhologic Cytomorphology, CNR. Universiry of Chieri Medical School, Chieti. Italy, PDepartment of Phormocology. Tufts Universiry. Boslon. MA. USA.

The effect of human recombinant interlenkin-I receptor antagonist &IL-lra) on lenkntriene B4 (LTB4) release was investigated in activated human menocyte cultures. To stimnlate LTB4 Prcnhtetion twe agonists were used: calcium ionepbore A23187 and PMLP. The detection was performed by employing the highly sensitive radioimmnneassay method. Preincnbation of monocytes with cytechalasin B (CB) (5 pe/ml), for 15 mitt, augmented the release of LTB4 when PMLP, net calcium ionophore A23187, was nsed. The cells were treated with various concentrations of A23187 (0.05 - 50 )IM) and PMLF’ (5 x 10-s - 5 x IO-5 M) for different periods of time. The greater LTB4 stimulation was found at A23187 (5 uM) and PMLP (5 x 10-6 M) for 10 mln incubation time. when human monocytes were pretreated for I hr with MIL-lra at different cencentmtions (0.25 - 250 @ml) and then heated with A23187 (5nM) or CB + FMLP (5 x lo6M) for IO mln, a dose-dependent inhibition wns found. The maximum inhibition was observed at the hiehest concentration of hrIL-lra (250 me/ml). when n relatively selective 5-lipoxygenase-inhibitor, nordihydroguaiaretic ncid (N-DCA), used at 10 nM, was added 15 min before A23187, a dose-dependent inhibition of LTB4 was found. Cells pretreated with amchldonic acid, at various concentrations (10-o - 10-5 M), for 10 mln and then treated with A23187 (5 pM ) for 10 ml”, were also inhibited in a dose-dependent tnnnner by hrILlra in their production of LTB4.

The inhibition of LTB4 release by hrILlra, in stimulated human monocytes, may suggests an important modulatory role for this new monokine in inflammation and imnnmity and may hold future therapeutic implications in diseases involving LIB4 as B mediator.