SECOND INTERNATIONAL WORKSHOP ON CYTOKINES / 113

241

STIMULATION OF CYTOKINE PRODUCTION BY THE BACTERIALLY DERIVED BIOLOGICAL RESPONSE MODIFIER IMUVERT. C. McCall, L. Weimer, S. Baldwin. Cell Technology, Inc. Boulder, CO 80301

ImuVert is a biological response modifier prepared from the bacterium Serratia marcescens, consisting of 0.1 - 0.2 urn vesicles derived from the inner and outer bacterial membranes, and ribosomes. It is currently in Phase 2 clirical trials for recurrent primary brain cancer. ImuVert is a potent macro- phage activator and also augments the activity of NK cells and cytotoxic T lymphocytes. When human peripheral blood mononuclear cells are cultured with ImuVert (50 ug/ml), Inter- leukin-1 (IL-l>. Tumor Necrosis Factor (TNF). Interleukin-2 (IL-2), I&rf&n-gamma, and Granulocy;e m&ophage colony stimulating factor (GM-CSF) are released into the culture supernatant, measurable by ELISA or RIA. The addition of Polymixin B to the cultures has no effect on the production of IL-1 and TNF, indicating that endotoxin is not the stimulus causing release of these cytokines. A time course of cytokine release in vitro shows characteristic kinetics, with early -__ (2 hours culture time) release of IL-l, TNF and IL-Z. G%STlUl~ interferon and GM-CSF show biphasic release kinetics, with peaks at E-10 hours and 3-5 days culture time, presumably re- flecting release first by the monocyte, and subsequently by T lymphocyte populations.

242

INMUNOMODULATING EFFECTS OF PLATELET ACTIVATING FACTOR (PAF): STIMULATION OF IN VITRO LYMPHOCYTE MIGRATION. R G McFadden, M, Bishop, K E Vickers, L J Fraher. Lawson Research Inst., St Joseph's Hospital, London, Ontario, CANADA. N6A 4V2

PAF has been reported to be present at sites of inflammation and to enhance vascular permeability, as well as augment the migration of neutrophils, eosinophils and monocytes. PAF has also been shown to down-regulate mitogen -induced lymphocyte proliferation, but its effect on lymphocyte migration (LyM) is not known. We examined the effect of PAF in a nitrocellulose filter-microchemotaxis chamber assay employing freshly harvested human peripheral blood lymphocytes as the target cell population. PAF stimulated LyM in a dose-dependent fashion to levels of 200-355X of control at 10 9 M while augmenting polymorpho- nuclear leucocyte (PMNL) migration to 294 * 45% of control. The chemokinetic response to PAF was completely blocked by the specific PAF antagonists, CV 3988 10 7 M and SRI 63072 10 6 M. Although PAF has been reported to induce the release of other immunomodulatory autacoids such as histamine, serotonin and substance P, none of these compounds are chemokinetic in our LyM assay. The chemokinetic effec_t of PAF on both lymphocytes and PMNLs is blocked by 10 s M 1-(5-isoquinoline sulphonyl)-2-methyl piperazine (H7), a specific protein kinase C inhibitor. These data suggest that PAF may function to recruit nonsensitized lymphocytes to inflansnatory foci, and may act through stimulation of protein kinase C.

243

ALTERED SYNTHESIS AND EXPRESSION OF GANGLIOSIDES OF LEUKEMIC T-LYMPHOBLASTS BY PHORBOL ESTER, INTERLEUKIN 2, AND INTERFERON W.D. Merritt, C. Kueter, M.B. Sztein and G.H. Reaman. Children's Hospital Natl. Med. Ctr., Washington, DC 20010, and The George Washington Univ. Med. Ctr., Washington, DC 20037.

The major gangliosides of blasts of human primary T-cell acute lymphoblastic leukemia (T-ALL), GM3 (hematoside) and GD3 (disialolactosylceramide), are also synthesized in certain T- ALL cell lines. Cm-2 cells were cultured for 3 days with 14C- galactose and I+-glucosamine and in the presence or absenceof phorbol myristate acetate (PMA), interleukin 2 (IL-2)andinter- ferons (IF), either alone or in combination. Gangliosides were isolated from harvested cells, and synthesis wasquantitated by scintillation counting and densitometryof TLC autoradiograms. PMA increased total ganglioside synthesis 2.5 times relative to controls,and PMA combined with either IL-2 or IF-a enhanced synthesis 3-4 times. IL-2 and IF-a together with PMA resulted in additive effects. IF-Y could not replace IF-a, and lL-2 or IF-o did not increase total synthesis without PMA. PMA in the presence or absence of cytokinesincreased absoluteamounts of GD3 and the proportion of GM3 synthesized. IL-2 andIF-atogether increased the proportion of GD3. Results of indirect inmuno- fluorescence flow cytometrystudieswithanti-GD3 showed that PMA also enhanced cell susfaceGD3 2-2.5-fold. The results indicate that ganglioside expression by T-ALL blasts can be modulated with differentiation agents and/or cytokines, implicating their potential use in altering glycolipid surface expression for therapeutic applications of anti-ganglioside antibodies.

244

LTB. NEGATIVELYAND POSITIVELYMODULRTES ~TNFaPKODUCTION. C.Mlller,G.Szabo L K.Kodys,U.Mass.Med.C., !&rcester, MA 01655. prig LPS, 1FNy plus mummy1 dipeptide (MDP),and IgC-antigen coa@&es can in&ce w ‘INF~ production.

We have shown that crosslinking the w FcRI receptor during rosetting with anti+& coated erythrocytes also stimulates TNF by itself and synerqizes with MDP in inducing M0 WF. Since l&kotrine B, CL;ra, ); another w product, aug&nts LPS induced MO TkiF levels. here the LTS. effect on induction of H0 lNF bv I’&+MDP or 67 crosslinkingqof the FcRI receptor are ‘assesseh in the LM bioassay. Total TNF, secreted and cell associated (cell lysates), was augmented in whole M@ populations stimulated with IFN~+MDP(X-Z~.~% increase). In contrast, rosette selected, FcRI crosslink-activated f$0 showed depressed total MF levels when LTS, was added with MDP or with IFNy+MDP as a stimuli (X=31.6% decrease p>O.Ol). LPS or MDP induced prostaglandin E, depresses 'INFO at the mRNA level. Consequently, increased or decreased levels of ‘INF after LTB, addition might result from increased or decreased PGE, levels. However, concomitantly measured q FGE levels were essentially unchanged (X increase-0.9%jin both the whole q population stimlated with IFNy+MDP or in the FcRI selected and crosslink activated KQ. This suggests that the LTSl effect is not secondary to a PGE, effect. Current experiments are assessing the LTS, effect at the level of ‘INF message. These data further support both the suggestion that stimulation of w TNF production by FcRI crosslinking represents a distinct pathway from IFNy+MDP, or LPS induction and the concept that @Z produced LTS, has a regulatory role in induction of M0 ‘INF.

245

INTEFERON-y ACTIVATES ALVEOLAR MACROPHAGES FOR ANT- CRYPTOCOCCAL ACTNITY. CH MODY. CL TYLER CA JACKSON. GB TOEWS, DN OF PULMONARY MED. UNN OF MICH. ANN ARBOR MI.

The Alveolar Macrophage (AMI Is a resident cell within the a&pace of the lung, and thus is ideally situated to defend the lung from invading microbial pathogens. Cyptococcus neoformans is a pathogenic yeast which is acquired by the respiratory route, and produces pulmonary infections. Previous studies have demonstrated that resting rat AM had minimal ant&cfyptococcal activity, but that activity was present when AM were incubated with supematants of Concanavalin A stimulated syngeneic splenocyte cultures [Con A Media]. The purpose of the present study was to determine which cytokine was responsible for the activity. AM were activated overnight in Con A Media with a neutralizing anti- IFN-7 antibody, or with a control antibody. In other experiments AM were incubated in the presence of recombinant rIFN, or with media alone. AM were infectedwith C. neoformans C3D atid at various intervals the % inhibition in cryptococcal growth determined. AM stimulated with Con A Media resulted in 98% Inhibition in cryptococcalgrowth.but this was reduced to 78% followirx addition of 1000 neutralizine units ofanti- IFN-‘y Ab, and in a separate experiment, the inhibiton was reduced from 70% to 6% @EM of quadmplicate determinants <2%). Thus, y-IFN is necessarv for oDtIma1 anti-crvntococcal activitv bv Con A Media. AM __ I - incubateh in media with Rat IFN-y 2000u/mlresulted in 84% inhibition in cryptococcal growth at 6 hours, and 98% inhibition at 48 hours. 20,00Ou/ml resulted In 100% inhlbltion at 6 hours and 98 % inhibition at 48 hours. Therefore, IFN-ywas sufficient to activate AM for anti- cryptococcal activity. Infections may occur in patients with defects in cell mediated immunity because of the inability to produce y-IFN and thus activate AM, and furthermore this deficiency may be amenable to exogenous immunomodulation.

246

OPIATE-DEPENDENT SUPPRESSION OF SWINE PERIPHERAL BLOOD LEUKOCYTE FUNCTION. T. Molitor’ M.Murtauah’. S. Chinsakchai’, G. Gekkef. and P. Peterson*. Departments of lACS and Vet. Pathobiol, CVM, Univ of Mn. St. Paul, Mn.’ and Hennepin County Medical Center, Mpls, Mn.’

Two recent developments have increased interest in the interaction of opiates and the immune system. First, endogenous opiates, i.e., met-enkephalin and Bendorphin modulate immune functions. Second, clinical observation is that administration of exogenous opiates increases risk of infection. To determine if the pig will serve as a suitable model for examining the interactions of opiates and the immune system we evaluated the effect of exogenous opiates on immune cell function and cytokine expression. We present here results from in vitro investigations of chronic methadone exposure on swine PBL function. PBL were exposed to methadone at concentrations ranging from 1O’6 to IO M for 24-48 hrs and then examined for the following: NK activity, lymphocyte blastogenesis, IL- 2 production, phagocytosis, phagolysosome formation, superoxide formation and interferon production. Methadone exposure at IO” and IO “M concentrations resulted in suppression ofselected macrophage functions such as phagolysosome and superoxide formation. NK activity and IL-2 synthesis was not affected. The immunosuppression was naloxone reversible but not associated with a decrease in cell viability or normal cell metabolism. The macrophage suppression was mediated by soluble factors released from opiate-exposed cells.