SECOND INTERNATIONAL WORKSHOP ON CYTOKINES / 87

85

TUMOR NECROSIS FACTOR SIGNAL TRANSDUCTION: CELL-TYPE-SPECIFIC ACTIVATION AND TRANSLOCATION OF PROTEIN KINASE C. s. Schiitze, S. Nottrott, K. Pfizenmaier and M. Krtinke. Clinical Research Group of the Max-Planck-Society, University of Giittingen, D-

3400 Gcttingen, F.R. Germany We have investigated the changes in protein kinase C

(PKC) activity following treatment of several cell lines with tumor necrosis factor (TNF). Binding-studies with t3H)-PBt on whole cells revealed rapid and transient activation of PK s I" Jurkat, K562, and U937 cells with a maximum of phorbol ester- binding at 6 min. after TNF-administration. Upon subfractiona- tion of TNF-treated U937 cells, binding in the membrane

a transient increase of PBt2- fraction was accompanied by a long-

term loss of PBt2-Pinding in the cytosol, indicating a TNF- induced translocatlon of PKC from the cytosol to the cell membrane. Determination of the specific PKC-activity, using histone III-S as substrate, revealed similar kinetics of PKC translocation in U937 cells. TNF also induced PKC-transloca- tion in K562 and Jurkat cells. However, while TNF caused long- -term downregulation of cytosolic PKC-activity in U937 cells, the cytosolic PKC-activity only transiently decreased in both Jurkat and K562 cells and then recovered to near basal levels. In the human nonmalignant fibroblast cell line CCD18, PKC was not activated by TNF. Together our data suggest that PKC- activation may play a major role in TNF signal transduction in some, but not all target cells.

86

INTERFERON INHIBITION OF ENVELOPED VIRUSES: ROLE OF INTRACELLULAR pH.G. Sidhu, D. Bhartiya, R. Friedman and R. Maheshwari. Uniformed Services University of the Health Sciences, Bethesda, MD 20814

Interferon (IFNs) have a" unusual inhibitory effect on some membrane-associated viruses. The inhibitory effects of IFN can be expressed in two major ways: inhibition of virus assembly, release and budding as see" in most murine leukemia (MLV's), mnuse mammary tumor, parainfluenza, herpes simplex-l

and human-immnnodeficiency (HIV) viruses; or, the combination of a small decrease in the production of virus particles and a much greater reduction in the infectivity of released virions as see" in some infections with MLV, vesicular stomatitis (VSV) and vaccina viruses. These particles have been shown to

be deficient in glycoproteins and could therefore be of reduced infectivity. We have show" that IFN inhibited the transport of VSV glycoprotein (G) to the plasma membrane. Primary amines added one to two hour post-infection significantly enhanced (5-250 fold) the antiviral activity against VSV and inhibited the transport of G protein to the cell surface. Since primary amines are known to raise the intracellular pH, we were interested whether IFNs had any effect on the intracellular pH. Using SNARF-1 carboxyacetate we found that IFN raised the intracellular pH significantly. This rise in intracellular pH by IFN may play a role in the inhibition of some membrane viruses, possibly including HIV.

87

THE LPS- AND PMA-INDUCED EXPRESSION OF INTERLEUKIN-la AND 0 mRNAs ARE CONTROLLED BY MULTIPLE MECHANISMS. M.F.. Smith. Temple Univ. Sch. Med. and Smith Kline and French Labs., Phila., PA.

The molecular mechanisms involved in regulating the expression of IL-lo! and IL-la mRNAs in human monocytes have been examined in order to determine if the expression of the two genes are coordinately regulated. We have previously shown that in response to LPS, PBM express approximately lo-fold more IL-1s mP.NA than IL-lo mRNA. We now report that in response to treatment with the PKc activators PMA, PdBu, PDA, and mezerein, PBM express exclusively IL-lp mRNA. Furthermore, under conditions where PKc was rendered inactive, at least part of the LPS-induced expression of both IL-lo and /3 mRNA was PKc independent. Calcium ionophore could not alone induce or in combination with LPS or PMA enhance the expression of either form of IL-1 suggesting that increases in intracellular calcium do not play a major role in the regulation of IL-1 mRNA exnression. Additionallv. we oresent evidence that-a pertussis toxin-sen%<ive G protein may play a role in regulating LPS-induced IL-1 mFtNA expression. Together these results demonstrate that there are multiple mechanisms involved in regulating IL-1 mRNA expression and that the two forms of IL-1 can be independently controlled.

88

NCNOKINE INwCl'IoNBY FcY TYPE I REcEPToRCROSSLINKIffi. G. Szabo, C.L. Miller, U.Mass.Msd.Center, Worcester, MA 01655.

Of the two distinct I$-Fc receptors on human monocytes(IqZ) only the type I, 72 kW receptor (FCRI) has high affinity for monomeric human IgGl/IgG3 and murine IgG2a. Anti-I& coated human erythrocytes preferentially bind to w FcRI, resulting in EA rosette formation and FcRI crosslinking. Here we show that this FcRI crosslinking induces w TNF production without additional stimulation(measured in LEI bioassay range:O.l-6.7 median:2.3ng/ml). The non-rosetting rgr produced no lWF even though some binding of the FcRII must have occurred. Most of the FcRI crosslinking induced lNF was cell-associated lNF in cell-lysates. Even after IFNy+NDP stimulation, the FcRI cosetting I$&' TNF levels were still greater than those of the non-rosetting w (p<O.OOl), suggesting that only stimulation through the FcRI receptor can augment IFNy+MDP induced v 'INF production. FcRI crosslinking (rosetted M@) also significantly augmented IFNwMDP induced PGE, (p<O.OOl) and IL-6 (p<O.OOl). Even after blocking @Z ?NF activity with neutralizing &iF Ab, the FcRI rosetted m population had greater IL-6 and PGE, levels, than the non-rosetting w, indicating that FCRI stimulation of IL-6 and PGEz was not solely a secondary result of lWF induction. Soluble Fc fragments did not induce q 'INF (O+o~g/Inl) or IL4(262+217Um) production, but moderately increased M$3 PGEl levels(8.1+9.8ng/ml.). Neither lNF, IL-6 nor FGE, was induced or augmented by soluble FC fragments in the non-FcRI rosetting &Jo. These data suggest, only stimulation through FcRI crosslinking can induce mpr PGE,! 'ItiF and IL-6 production, and occurs Only in the FcRI ixarrng w population.

89

TUMOR NECROSIS FACTOR ALPHA STIMULATES THE GROWTH OF MYELOIO LEUKAEMIA CELLS

BY A GM-CSF MEDIATED PATHWAY.

Iabilio A.,Higliorati G.,Falretti F.,Velley C.,Pebusque H.J.,Gabert

J.,Riccardi C.,Martelli M.f.,Hannoni P. Dept. Intern. Med. I and Inst.

Pharmacology, University of Perugia, Italy and INSERH U 119,Regional Cancer Center.Harreille.France. Tumor Necrosis Factord (INFJ.) h as been reported to inhibit normal myeloid

cell proliferation and induce production GM-CSF by accessory cells. In previous studies ye showed GM-CSF induced proliferation of blast cells from patients with Acute Hyeloyd Leukaemias (AMLs)and these leukaemic cells

synthesized some of the growth factors to which they respond,lainly GM-CSF, It-land IL-S. The existence of the autocrine loops,responsible for the growth of ine leukaemic clone was thus demonstrated. By testing the antiproliferative activity of TNFd on the in vitro proliferation of the leukaenic cells.we demonstrated: (1) INFd induced proliferation of AML cells in 30% of the cases;

(2) in a few cases a slight inhibition of the spontaneous in vitro growth uas observed;(3) a strong synergisni between TNFd and GM-CSF was observed in selected cases. resulting in It in vitro I1 AML cell proliferation; (4) this synergism is mediated by GH-CSF, since monoclonal antibodies against GM-CSF inhibited poliferation. Furthermore. 1NFd also induced mRNA-GM-CSF

expression in selected cases. Thus INFd ,a~ uell 3% other lynphokines. might be involved in the arxplification of the leukaenic clone.

90

IL-2 INDUCES T-CELL RECEPTOR GAMMA CHAIN mRNA EXPRESSION IN IMMATURE GAMMA/DELTA X-NMOCYTES IN VITR0.L. Takacs'. W, Gotlieb'. and S. Durum'. 'Laboratory of Molecular Immunoregulation, BRMP, DCT, NCI-FCRF, Frederick, MD 21701- 1013, USA and 'BCDP, Program Resources, Inc., NCI-FCRF, Frederick, MD 21701-1013, USA.

One of the most important differentiation events in intrathymic T-cell development is the expression of TcR genes. Ontogenetic analysis of TcR gene expression suggests that expression of each chain of the TcR complex (gamma/delta and alpha/beta) are regulated independently in immature thymocytes. Here we describe a" in vitro model of gamma/delta thymocyte development. CD4-/CD8-/It-2r- precursor T-cells cultured in the presence of IL-1 plus ConA give rise to a novel intermediate cell type that expresses IL-2r, high levels of TcR delta mRNA but low levels of TcR gamma mRNA and no detectable TcR alpha/ beta. IL-2 upregulates TcR gamma mRNA expression in these cells. The effect of IL-2 is detectable as early as 2 hours and peaks at eight hours. Thus we were able t" separate proliferation of precursor cells from a differentiation event by sequential cytokine treatment. This is the first demonstration of lymphokine regulation of TcR gene expression. Our observation parallel the late steps of B-cell development, and the involvement of cytokines in that process suggests that cytokines might have an important role in the regulation of late differentiation events associated with Ig or TcR gene expression, in both lymphoid lineages.