SECOND INTERNATIONAL WORKSHOP ON CYTOKINES / 89

91 100

REGULATION OF IMMUNE RESPONSES AND INFLAMMATION BY AN IL-l INHIBITOR V.Barak, D.Perritt, P.Yanai, M.Halperin, A.J.Treves. One. Dept., Hadassah Univ. Hospital, Jerusalem, Israel 91120.

ME INDUCTION IlF I VZlt IlF RFNAI CFI I CARCTNllMA CFII S RV RTSPFI-TFIC Y

We have described a factor with an IL-l Inhibitory activity secreted from a human myelo monocytic line developed in our lab. Biochemical characterization indicated that this activity resides in a protein with a molecular weight of 52KD, eluted at P.I. 4.1. The factor was found to inhibit IL-l induced proliferation of several types of cells, mouse thymocytes, human T cells, human fibroblasts as well as P6E2 secretion by IL-1 induced human fibroblasts. In in viva assays in experimental animals, the factor inhibited some inflammatory manifestations induced by or mediated by IL-l: We tested elevation of fever, lymph node enlargement and local acute inflammation [foot pad swelling). In addition, leukocytosis and levels of zinc and fibrinogen in plasma, were also changed by injected IL-l and the inhibitory factor reversed this effect. Similar responses which were induced by injection of TNF, were not affected by the IL-l Inhibitor. These results suggest that the IL-l Inhibitor has a strong anti inflammatory activity in viva, specific to IL-l.

_. _.___ _. .._...._ “--- _.” .--..-. “. I ---- -. ___. ___. __ ..DNDCLONAL ANTIBODIES. R.L.H. Bolhuis (11, G.J. Fleuren(Z), S. Warnaar (21, E. Braakman (1) and J. van DlJk (21 -* _ (I) Daniel d H d C c t Rtt d

i. (21 Stat: lJn~~ersi?yc?reid&i,efhe N%h~~l~r%.. The

Netnerland!

98

Endotoxin

MONOCLONAL ANTIBODY TO ENDOTCIXIN CORE PROTECTS MICE FROM

administratlon

GRAM NEGATIVE SEPSIS BY A MECHANISM TNDEPENDENT OF TUMOUR

reproduces the

NECROSIS TACTOR (TNF). K.Bayston*, A.T.Silva*, B.J.AppelmelkO,

features Of

W.Buurman ,

gram negative

J.Cohen*

septic

l RPMS,

shock

Hammersmith

in experimental animals. It

Hospital, London, UK: "Vrije Universiteit, Amsterdam, Netherlands;

is now evident that many of the actions of endotoxin are

+Rijks Universiteit Limburg, Maastricht, Netherlands.

mediated through endogenous factors. Of these TNF in parti- cular has been implicated. We have shown that clone 20, a murine TgM monoclonal antibody to S. minnesota Re 595, is protective in a murine model of gram negative sepsis. To investigate the mode of action of clone 20 we measured serial serum levels of TNF in male CD1 mice receiving an LD90 dose of E.coll 0111. Clone 20 or cell culture media (both contalnlng <lOOpg/ml endotoxin) were injected (i/v 1.5 hrs before admlnistration (i/v) of bacteria. Gentamicin 2mg/kg/day was given bd starting 2 hrs after infection. At 72 hrs survival of mice receiving clone 20 (n=36) was 97% versus 17% for mice given media (n=36), P<O.OOl. serum TNF measured at 0,15,50,90 and 300 mlns post challenge was similar in the 2 groups. Peak levels at 90 mins were 171.4 (clone 20) and 190.9 (media) "g/ml. In vitro experi- ments using human peripheral blood monocytes also demonstrated a lack of inhibition by clone 20 of endotoxln-induced TNF productlo". Clone 20, an antibody to endotoxin core, thus protects by a mechanism Independent of TNF.

99

AUTD-ARTIBDDIES TCI IL-Lx AND TNFu IN NORML INDM- DUALS ARD IN INFECTIOUS AND INRUNOIRFLARHA!L'DRY DISORDERS. K. Bendtzen. 13. Svenson, A. Fomsaaard, L.K. State Univ. Hosn., CODenhaaen, Denmark.

Auto-antibodies against IL-la was initially de- tected in a pool of sera from healthy blood donors. IgG (particularly IgGI) dominated the rIL-la-bin- ding capacity. The frequency of detectable anti-rIL- la IgG in sera from 32 male and 32 female donors was 25% and 22%, respectively. However, the capacity and avidity of binding were greater in the male *era (mean capacity to bind =‘=J-rIL-la = 10 (0.7-27) v. 3.3 (0.5-7.3) rig/ml and mean Kd = 5.5 (5-7) v.11 (4- 16) PM; n=5). There was a correlation between anti- body titres and inhibition of binding of 13xJ-rIL-la to the murine cell line EL4 (r = 0.86; P < 0.01). Auto-antibodies to TNFa (IgG and IgM) were detected by a semi-quantitative immunoblotting technique using SDS-PAGE'd human rTNFa. Strong antibody reac- tions were seen in 8% (weak in 40%) of normal sera. Increased prevalence of antibodies to both cytokines was seen in patients with Gram-negative bacterial diseases and various rheumatic diseases, and antibo- dies to TNFa were induced in early stages of septic endotoxic shock conditions. Auto-antibodies to IL- la, TNFa and, possibly, other cytokines may serve as specific and native cytokine inhibitors.

Bispecific mAb recognize one renal cell carcinoma (RCC) associated antigen on the one hand CD3 (OKT3) and the effector cell on the other. The dual specificity of these quadromas was analyzed by immunohistochemistry and in cytotoxicity assays. The quadromas induced TCRCXB'/CD~~ and TCRy6+/CD3+ cloned lymphocytes to lyse RCC but not other tumor target cells. Contamination of bispecific mAb preparations by paren- tal OKT3 mAb does not significantly affect induction of lysis by uptill a 10 fold excess present. Several IgG subclass switch variants of the G250 hybridoma were used to produce quadromas. the OKT3 hybridoma. Except for IgE, all IgG sub- class (IgG1,2a,2bl combinations in the quadroma equally induced cytolysis.

101 IUPNS'TFICA'IION 4ND CHARACTERIZATION OF A PLASMA PROTEIN GROUP, THE THIOL ESTER PLASMA PROTEINS (TEPP) WHICH BIND IL-l&. 'W.Borth, -A.Urbanski, "T.A.Luger, "L.Sottrup-Jensen; 'NY Hosp.Cornell Univ.,

Thiol ester plasma proteines (TEPP) appear to have a com-

NY 10021; ‘Dept.Derm.11 and LBI-DVS, Lab.Cellbiol., Univ.Vienna; "Dept.Mol.Biol.,

mon evolutionary origin and share an unique peptide bond

Univ.Aarh"s, Denmark

known as internal thiol ester. The human proteinase inhibi- tors a2-macroglobulin (a2M), pregnancy zone protein (PZP) as well as rat alM and a2M are able to bind IL-113 upon activa- tion of the thiol ester. Binding of IL-15 to activated aI4s is dependent on the presence of suitable transition metal ions, which mediate complex formation involving free thiol groups and histidyl residues. Small molecular mass proteins related or unrelated to the interleukin family could not block binding of IL-1B to aMs. Moreover, activation of fresh serum mediates also binding of IL-15 to the third complement component, which also belongs to the TEPP. TEPP IL-la com- plexes retained IL-1 like activity in two independent bioas- says. TEPP IL-lfl complex formation could be modulated by some antirheumatic drugs. TEPP IL-15 complex formation in viva is indicated since reductive treatment of inflarmnatory synovial fluids increased IL-15 levels as measured by ELISA and bioassay. The results suggest that activated TEPP exert a modulatory role for IL-18. Supported by a grant from the "Biirgermeister der Bundes- hauptstadt Wien" (to W.B.).

102

DEMONSTRATION OF THE EFFECT OF HUMAN INTERLEUKIN-1 INHIBITOR (IL-li) IN CULTURED BOVINE NASAL CARTILAGE EXPLANT. David F. Carmichael. Christopher G. Smith. and Robert C. Thomuson. Synergen Inc., Boulder, Colorado USA

We have used the cartilage explant model to demonstrate the mitigating effect of a human IL-li on human IL-l/3 mediated cartilage breakdown. Bovine nasal septum was used as a source of cartilage tissue. Cartilage degradation was monitored by the release of proteoglycans into the culture medium. (J. Steinberg, et al., 1979 Biochem. J. 180:403-412). Interleukin-1 was found to increase the proteoglycan release in a dose dependent fashion. The inhibitor alone had no effect on the cartilage. The effect of high dose (5.0 "g/ml) of IL-1 was found to be antagonized in a dose dependent manner by the inhibitor (5-50 "g/ml). The highest dose of IL-li returned the proteoglycan release rate to the basal level. The effect of IL-11 was reversible since its removal from the culture made the tissue susceptible to the action of IL-l.