SECOND INTERNATIONAL WORKSHOP ON CYTOKINES / 123

301

HISTAMINE SUPPRESSES THE SYNTHESIS OF TUMOR NECROSIS FACTOR-a IN VITRO VIA H2 RECEPTORS. E. Vannier, L.C. Miller, R. Schindler, B. Terlain,* and CA. Dinarellc Tufts Univ.Sch.Med./New Eneland Medical Center. Boston. MA 02111 and *Rhone-Poulenc~Sand, V&y/Seine, France.

Histamine is the main mediator released from mast cells and basophils during immediate-type hypersensitivity reactions. Histamine HI and HZ receptors have been characterized on human peripheral blood mononuclear cells (PBMC). We studied whether histamine could modulate the synthesis of various cytokines in vitro. Human PBMC were stimulated by LPS (E. coli 055:B5, 10 &ml) in the presence or absence of histamine (-10M to -4M). Cytokines (TNFa, ILla and p, IL6) were measured by specific radioimmunoassays, and expressed as total protein (cell-associated + secreted) 24 hrs after the addition of LPS. TNFcl synthesis was enhanced (114% of basal values, ~~0.05) by histamine at the lowest concentration (-lOM), but reduced at higher concentrations (from -7M to -4M) with an optimal effect at -5M (44% of basal values, pcO.01). Neither ILla, ILlp, nor IL6 synthesis was significantly modulated by histamine. Cimetidine, an HZ receptor antagonist, itself enhanced LPS-induced TNFa synthesis but prevented, in a dose-dependent manner, the histamine-mediated decrease in TNFa synthesis. In contrast, diphenhydramine, an Hl receptor antagonist, did not affect LPS-induced TNFa synthesis and did not reverse the histamine-mediated decrease in TNFa production. Neither antagonist modified ILlp synthesis. In conclusion, TNFa synthesis is regulated by histamine via HZ receptors. This pathway may contribute to immediate- type hypersensitivity reactions in vivo.

302

THE EFFECTS OF PSYCHOLOGICAL STRESS AND ANESTHESIA ON PLASMA INTERLEUKIN-6 (IL-6) ACTIVITY IN RAT. J in G I eMav, A. Van&r and M. Kluge~ Dept. of Physiology, Univ.‘Michigan Med. Sch., Ann Arbor, MI 48109. Exposure of male Sprague-Dawley rats to an “open-field” causes stress-induced hyperthermia. The purpose of this study was to determine whether such exposure also causes an increase in the plasma activity of the pyrogenic cytokine, IL-6. The effects of anesthesia on IL-6 was also tested. Body temperature (BT) was measured in unrestrained rats by biotelemetry, and plasma activity of IL-6 was measured using the IL-6 B-9 hybridoma cell line. Blood was collected following cervical dislocation. Control rats not exposed to open-field had a plasma IL-6 activity of 40.6 + 7.2 units/ml. Plasma IL-6 activity was 105 + 6.6 units/ml (P=O.O003) after 30 minutes of open-field exposure and 221 ;t 17 units/ml after 60 minutes (P=O.O003). Injection of the anesthetics ketamine hydrochloride (70 mg/kg) and xylazine (10 mg/kg), or inhalation of the anesthetic methoxyflurane, had no acute effect on plasma IL-6 activity. These data indicate that exposure to psychological stress can elevate the plasma concentration of a known mediator of the acute phase response. However, based on earlier data relating plasma IL-6 activity to BT changes following injection of LPS, we conclude that the plasma levels of IL-6 following exposure to an open-field are not high enough to account for the rise in BT observed in rats during this stress.

303

B. FERRUA. M. FAY. H. WAK&S!& CIPROFLOXACIN DIFFERENTLY MODULATES ILl& AND ILlBPRODUCTION. S. BAILLY, Y. MAHE. I T, TUBSZ and MA. GOUGEROT-POCIDALO

Ciprofloxa Fin (Cip), L CHU X. Bichat, Paris, France.

a quinoline derivative antibiotic juinolone) decreases the IL-I activity released by 5stimulated monocytes without affecting cell-associated .-1 activitv. ExtracellularIL-1 aCtivitV CorresDonds to

mature IL-l<and IL-l@molecules, wherea; cell-aisociated activity is mainly related to the IL-la precursor, the IL-l@percursor being virtually inactive. In order to analyse further the effects of Cip on LPS-induced synthesis of IL-lo( and IL-18, each species was measured in the extra cellular medium and in cell lysates of monocyte cultures over a 4-day period using an ELISA method. Cip delayed the production of IL-ldwith a peak occuring between 24 and 48 hrs compared to O-24 hrs in its absence. However, the total amount of IL-l&released during the culture period was not altered by Cip. The kinetics of cell-associated IL-lo( production showed a similar pattern, i.e. a delay but no decrease in synthesis. By contrast, Cip not only delayed but also decreased the production of both extracellular and cell-associated IL-~B

Cip did not decrease the accumulation of IL-lorahd IL-IamRNA in LPS-stimulated monocytes and led to an increase in cellular CAMP. These findings suggest that the differential action of Cip on the synthesis of IL-ldand IL-lk may occur at post-transcriptional level. Cip is, to our knowledge, the first pharmacological agent found to have a differential effect on the synthesis of IL-l&and IL-l!

304

INTERLEUKIN 1 INDUCES INTERLmTKIN 2 GENE EXPRESSION VIA A SITE HOMOLOGOUS TO THE AP-1 RECOGNITION S1TE.K. Mueeee'. T, Williams'. J. Kant'. H. You"e3. A. Schmidt4. U. Siebe"list4 and S. Dam', 'BCDP, PRI, NCI-FCRF, Frederick, MD, 'Univ. of Pen". Philadelphia, PA, 3Lab. of Exp. Inrmunol., BRMP DCT. NCI-FCRF, Frederick, MD, %IH, NIAID, Bethesda, MD, and 'Lab. of Molec. Immunoregulation, BRMP, DCT, NCI-FCRF, Frederick, MD.

We examined the IL 1 responsive element on the human IL 2 gene in the mouse T cell lymphoma LBRM 33 lA5. This cell line responds to exogenous IL 1 along with PHA to stimulate a" increase in IL 2 protein and mRNA expression (up to 50 fold). In transient transfections we used a construct containing a 548 bp upstream enhancer region of the human IL 2 gene linked to the chloramphenicol acetyl transferase gene (ILPCAT). We observed that IL 1 addition yielded a 10 fold increase in activity of ILPCAT, whereas no IL 1 effect could be observed in control plasmids. Deletion of the upstream region -218-176 of IL2CAT removed the enhancing effect of IL 1. This region contained a" Bbp homologue to the binding site of the transcription factor AP-1. I" gel mobility shift assays, IL 1 stimulation enhanced a nuclear factor binding to a" oligonucleotide harboring the AP-1 homologue site. The use of a CAT construct containing a trimer of AP-1 sites confirmed that IL 1 can act on gene expression via a" AP-1 site. In Northern analysis IL 1 augmented mRNA for the AP-1 factor (encoded by the jun-oncogene). The finding that IL 1 acts via an AP-1 homologue site suggests a general mechanism for multiple IL 1 effects operating at the nuclear level.

305

INTERLEXJKIN 1 INCREXES GTP BINDING AND HYDRDLYSIS IN MEMBRANESOFA- CELL LINE (EL4 NOB.l). L.A.J. O'Neill, T.A. Bird & J. Saklatvala, Strangeways Research Lab., Warts museway, -ridge, CBl 4RN, U.K.

The events which DCCUT following the binding of interleukin 1 to its receptor are unclear. Here we describe a" investigation into the involvement of guanine nucleotide binding proteins IG proteins) in IL1 signal transduction. The results show that in membranes prepared from an ILl- receptor rich strain of the EL4 marine thymoma line, IL1 increased the binding of GTPjS and hydrolysis of GTP, suggesting G protein involvement. in IL1 receptor signaIling. ILla caused a time dependent increase in GTpy35S binding to cell membranes at 37'C which was detectable from 1 minute and was dose dewndent (0.1-1000 rig/ml). Varying the concentration of GTP+jS revealed that the enhanced binding was due to an increase in affinity of available sites. ILla also caused an increase in [y 3*-PIGTP hydrolysis with a time course and concentration dependency similar to ahanced GTpy jjS binding. IL113 produced similar results to ILla but was 10 times less potent which ag'rees with receptor binding studies for ILlo and ILlI? on these cells. Neutralising antisera to ILla and IL113 abolished the response and minted to specificity. Half-maxim1 enhwcement of GTPfsS binding by ILla, occurred at 5% IL1 receptor occu~cy. We conclude that the IL1 receptor activates a GTP binding protein in a responsive cell line.

306

EVIDENCE FOR THE EXISTENCE OF MORE THAN ONE SECOND NESSENGER IN INTERLEUKIN 1 ACTION. S.B.Mizel, M.Chedid, F.Shirakawa Wake Forest University Medical Center, Winston-Salem, NC 27103.

In a series of recent studies, we demonstrated that CAMP serves as an intracellular second messenger for IL 1 in murine thymocytes, human rheumatoid synovial cells, the pre-B cell line, 702/3, and the human natural killer-like cell line, YT. The IL 1 receptor (IL 1R) is coupled to adenylate cyclase in these cell types via a pertussis toxin-sensitive G-binding protein. In the present study, we analyzed a variety of IL l-responsive fibroblast and T cell lines for the presence of one or more components of this pathway. IL 1 stimulated the production of PGE, by several fibroblast cell lines (Swiss 3T3, NIH 3T3, FS-4, MRC-5, and human dermal fibroblasts). Pertussis toxin (PT) markedly inhibited IL l-induced PGE, production. PT also inhibited CAMP production in these cell lines. IL l-induced IL 2 production by EL 4 6.1 Cl0 cells was also inhibited by PT. However, IL 1 did not induce CAMP production in EL 4 cells. Furthermore, IL 1 failed to induce CAMP production in three other IL l-responsive T cell lines: DlO, Jurkat 9.9, and LBRM 33. These results suggest that the IL 1R is coupled to a PT-sensitive G protein in fibroblasts and T cell lines. However, in T cells this G protein interacts with a different second messenger generating system than in fibroblasts.