846 ENII CONFERENCE

blocked function and, since C a 2+ inhibited Mg -'+-induced restoration of antibody 24 binding, this has led to a working hypothesis in which the type of cation bound to an integrin is proposed to have a critical role in determining the activation state of the receptor.

through the ability of D-containing peptide sequences to interact directly with cation bound in the special- ized EF-hand-type binding sites in integrin a subunits.

M. de Landazuri (Madrid)

M. Humphries (Manchester)

The final two presentations centred on the role of [31 integrins in lymphocyte adhesion and activa- tion. Humphries described work aimed at obtaining a molecular description of the interaction of adhe- sive ligands within their integrin receptors. As a first step, this had entailed identifying the minimal pep- tide sequences within ligands that retained adhesive activity. Using fibronectin as a prototype, two tripep- tide motifs had been characterized: RGD (recognized by a5131) and LDV (recognized by ~4131). Each se- quence appeared to be functionally equivalent in that each contained a critical D residue and both cross- inhibited each other's function. These findings were consistent with a recent hypothesis that adhesive specificity might be generated at the molecular level

Landazuri then continued the discussion of a4131 and described epitope mapping studies localizing functional sites on the a4 subunit through the use of antifunctional monoclonal antibodies. Four epitopes were identified - - occupancy of either A or B2 trig- gered homotypic cell aggregation, occupancy of B I perturbed adhesion to fibronectin and VCAM-1, while occupancy of C had no effect on adhesion. These data were felt to be consistent with the presence of three ligands for a4[~l ; fibronectin, VCAM-I and an as yet unidentified molecule mediating cell aggre- gation. Interestingly, the recognition of fibronectin and VCAM-1 appeared to be independently and differentially regulatable - - treatment of cells with activators of protein kinase C or Ca 2÷ fluxes acti- vated adhesion to fibronectin and VCAM-1, while activators of protein kinase A and intracellular cAMP only enhm~ced adhesion to VCAM-1. The mechanism of this difference is currently unclear.

Cytokines and their receptors

Chai r : W. Haas (Basel)

Z. Dembic (BaseO

Interferons are a large family of cytokines which include 24 IFNa, 1 IFN'r and 1 IFN[3. Many of these type I IFN have been shown to compete for one receptor. IFN-f (type II IFN) shows no significant se- quence similarity to type I IFN and binds to a differ- ent receptor, but X-ray structure analysis revealed a striking similarity between the 3-D structures of !FN~ and IFN~, (Falick eta:. (1991), Science, 252, 698). Moreover, the recently published sequences of recep- tors for human IFNaB and human and murine IFN~, also suggest an evolutionary resemblance between the two receptors (Bazan (1990), Cell, 61,753). Murine cells transfected with the human IFN~B receptor cDNA were 100-fold less sensitive to human IFNaB than human cells and, in addition, were poorly sen-

sitive to type I subspecies like IFN~A and IFN~3. The most likely explanation for these findings is that the interaction of the human receptor polypeptide with putative mouse accessory proteins is suboptimal. Different accessory molecules might associate with the same type I IFN binding polypeptide to form specific high affinity receptors for individual type I IFN. The IFN-f-binding polypeptide requires acces- sory :m__o!ecu!es for signal transduction rather than for the generation of a high affinity IFNT-binding site. The genes encoding the IFNT-binding protein and the accessory protein have been located at human chro- mosome 6q and 21q, respectively. Transfection of cDNA constructs encoding human-murine IFN 7 receptor hybrid molecules in murine cells have shown that the intracellular domain of the murine IFN~, receptor is not sufficient for the interaction with the

I M M U N E F U N C T I O N S A T THE M O L E C U L A R LEVE L 847

murine accessory protein that is required for signal transduction. Z. Dembic also reviewed the various IFN-inducible transcription factors with particular emphasis on ISGF3. IFN~, induces the synthesis of a latent form of a subunit of this transcription fac- tor which is then activated in response to IFN0c (Levy et al. (1990), E M B O J., 9, 1105). This may explain the synergy between type I and type II IFN that has been observed in various functional assays.

A. Rubartelli (Genoa)

Most cytokines are secreted via the classical secre- tory pathway of eukaryotic cells which is associated with the formation of disulphide bridges in the ox- idizing milieu of the ER and with glycosylation in the ER and the Golgi apparatus. The translocation of secretory proteins across the ER membrane depends on hydrophobic signal sequences that are usually lo- cated at the N-terminus of the precursor peptides and cleaved off during the transfer across the membrane. Several secretory proteins such as ILl and the dis- tantly related fibroblast growth factors which lack precursor peptides with signal sequences appear to be exported from the cell via a novel secretory path- way (for review, see Muesch et al. (1990), Trends in biol. Sci., 15, 86). Immune electron microscopy and subcellular fractionation studies failed to demon- strate ILl in the ER and the Golgi apparatus. ILl[3 is normally not glycosylated, but glycosylation oc- curs if it is directed to the classical secretory path- way by the artificial attachment of a cleavable signal sequence. Secretion of IL 1 is blocked by methylamine and enhanced by brefeldin A and monensin, which do inhibit secretion via the classical secretory path- way (Rubartelli et aL (1990), E M B O J., 9, 1503). The alternative pathway of secretion remains poorly de- fined. One possibility is that the cells are lysed. A more interesting possibility is that special transport proteins are involved (McGrath and Varshavsky (1989), Nature (Lond.), 340, 400).

G. Kroemer (Madrid)

The role of IL2 in the pathogenesis of autoim- mune diseases is not clear. On the one hand, T cells from patients or animals with autoimmune diseases were found to be defective in IL2 production in vitro (Kroemer and Wick (1989), Immunol. Today, 10, 246) and in vivo IL2 could prevent or delay the ap- pearance of autoimmune symptoms in MLR/Ipr mice (Guitierrez et al. (1990), Nature (Lond.), 346, 271). On the other hand, elevated IL2 levels were observed in the sera of animals before the appearance of au- toimmune symptoms (Kroemer and Wick, op. cit.) and some IL2-treated cancer patients developed au-

toimmune lesions (Atkins et al. (1988), New Engl. J. Med., 318, 1157). Normally, autoaggressive T-cell clones are deleted or inactivated. G. Kroemer showed that IL2 administration in the form of a recombinant vaccinia virus did not prevent the deletion of self- specific T cells in TCR transgenic mice, but induced expression of CD8 in peripheral T cells e×pressing the transgenic TCR. Self-specific T cells expressing V[311 that were anergic in thymectomized mice be- came responsive to anti-TCR antibodies, and autoim- mune symptoms were induced in thyrnectomized mice and in nude mice when the animals were injected with the IL2 recombinant virus (A. Aznchez et al. (1991), J. exp. Med., 173, 1323).

M. Brockhaus (Basel)

TNFa is a product of monocytes/macrophages, granulocytes, T cells, mast cells and keratinocytes; TNF[3 is mainly produced by T cells. In vitro, they induce expression of adhesion molecules in endotheli- al cells, granulocytes and keratinocytes. Moreover, both forms of TNF stimulate B and T lymphocytes, but only when combined with other lymphokines. In vivo TNF may play a role in the immediate defence of infection at the site of entry as well as in im- muneregulation. Two types of TNF receptors (TNFR), the 75-kDa type A and the 55-kDa type B TNFR, can be distinguished biochemically and by specific monoclonal antibodies (Brockhaus et al. (1990), Proc. nat. Acad. Sci. (Wash.), 87, 3127). The cDNA ~ . . . . . . . . . . . nav~ oc t , ~u:,~u ,t,u ~- I o r both r e t : c p t u ~ s L . . . . t _ , _ _ _ . a _ _ . ~ h A

quenced. Both TNFR belong to a family of cysteine- rich cell surface molecules which includes the recep- tor for nerve growth factor, the Ox40, CD40 and CD27 antigens (Smith et ai. (1990), Science, 248, 1019; Loetscher et al. (1990), Cell, 61,351). The two types of TNFR show a,most identical binding curves for TNFa and also for TNF[3 when tested in a solid phase assay. On the cell surface, TNFRA has a 5-times higher affinity for I'NF0t than TNFRB. An- tibodies to TNFRB have TNF-agonistic properties. Translocation of NFxB is seen already at very low receptor occupancy. This response occurs within minutes and is cycloheximide-insensitive. Most cell types express both receptor types" TNFRB predominates on the surface of T cells. Fibroblasts appear to express only TNFRB. In section~ of ton- sils, TNFRB expression is confined to germinal centres (probably reticular dendritic ce:.ls) and TNFRA-expression to T-cell areas. Soluole TNFR are found at 2-3 ng/ml in sera of healthy individu- als and are increased up to 10-fold in patients with inflammatory and malignant diseases. A transient in- duction of TNFR release into the serum is seen in volunteers treated with a low dose of lipopolysac- charide.

848 ENII CONFERENCE

T. van der Pouw Kraan (Amsterdam)

An important aspect of current lymphokine research is the elucidation of the requirements for the induction of the production of individual lym- phokines. IL4 is of particular interest in this regard. IL4 is a growth factor for lymphocytes and mast cell and plays a key role in the induction of IgE produc- tion by B cells. A sandwich ELISA was used to de- tcrmine IL4 production of human peripheral blood T cells in response to various stimuli. IL4 produc- tion was observed after stimulation with combina- tions of CD2 mAb and CD28 mAb or CD2 mAb and IL2. Interestingly, PMA inhibited the IL4 produc- tion but enhanced the production of IFN'r and of IL2. Only memory (CD45RO) T cells produced IL4, while only naive (CD45RA) T cells proliferated in response to anti-CD3 and IL4.

J. van Damme (Louvain)

IL8 was isolated on the basis of its neutrophil at- tracting/activating activity (hence the name NAP-1) and its cDNA cloned and sequenced by several differ- ent groups. It is produced by ieukocytes, endotheli- al cells, fibroblasts, keratinacytes, synovia! cells, chondrocytes and some carcinoma cells in response to stimuli such as LPS, ConA, ILl, TNF, poly I/C, measles virus or rubella virus. Multiple forms of IL8 are generated by proteolytic truncations at the N-terminal end. X-ray crystallography indicated that, in solution, IL8 occurs as a dimer (Baldwin et al. ( I Q Q l l P r n r n a t 4 t a d .~r i I ' ~ d ' ~ c h ~ RR g O g l I1

belongs to a large family of cytokines which can be divided into CXC and CC subfamilies according to the spacing of four cysteines which form two disul- phide bridges. Platelet basic protein (PBP), which is - - like IL8/NAP - - a member of the CXC subfami- ly, also occurs with various N-terminal truncations. IL8/NAP-1 and PBP show only sequence similarity starting at the N-terminal residues of their ultimately processed forms (van Damme (1989), Europ. J. Bin- chem., 181,337). The shortest version of PBP (NAP-2) and its second shortest version [3-thromboglobulin ([3TG) also activate neutrophils. Intravenous injec- tion of IL8/NAP-I into rats leads to a doubling of the number of circulating granulocytes within 1 hour. [3TG has a similar effect but is less potent. Intrader- mal injection of IL8/NAP-I, [3TG or NAP-2 leads to an accumulation of indium-labelled neutrophils at the injection site. IL8/NAP-1 has been identified in psoriatic skin lesions and in synovial fluid from rheu- matoid arthritis patients. A novel chemoattractant protein (MCP-I) which attracts monocytes but not neutrophils could be separated from IL8/NAP-1 by chromatographic procedures (van Damme et al. (1989), Europ. J. Immur, ol., 19, 2367). This factor belongs to the CC subfamily. The MCP gene is prob-

ably the human homologue of the murine JE gene that is transcribed in PDGF-treated fibroblasts.

J.C. Renauld (Brussels)

IL9 was purified and cloned by different groups on the basis of its growth-promoting activity on cer- tain T-cell lines, mast cell lines and a megakaryocyt- ic cell line. More recently, it was found to enhance the formation of BFU-E in the presence of erythropoietin (Donahue et al. (1990), Blood, 75, 2271), but the true physiological function of IL9 re- mains to be determined, lLq-cDNA-transfected, |L9-dependent T-cell lines grow autonomously and form tumours when injected into syngeneic mice (Uyttenhove et al. (1991), J. exp. Med., 173, 519). J.C. Renauld reported that tumours, mostly of thym- ic origin, developed in about 5 % of IL9 transgenic mice, which express the transgene under the control of a pim-1 promoter combined with an E~ enhancer and a Moloney-MuLv LTR. The tumours were trans- plantable to syngeneic mice with the exception of one, which had lost the transgene. Two of four methylnitrosourea-induced thymomas were also found to be IL9-responsive.

T.R. Mosmaun (Edmonton)

ILl0 is a polypeptide of 16, 18 or 20 kDa (depend- ing on the extent of glycosylation) which forms dimers. It is produced by TH2 clones. The follow- I I J L ~ I L 4 1 1 ~ , L I I t . J I I L . ~ I I L L V q w g ~ r ~ , F l l I ~ , ~ S Q , . 7 ~ l l l d r ~ t t ~ 4 L L.11kJ l ~ l , l . I ~ I L l l l - -

hibits the antigen-induced synthesis of cy-tokines by TH 1 clones and CTL clones, probably via an effect on antigen-presenting cells; 2) it inhibits the synthe- sis of ILl, IL6 and TNF0c by LPS-stimulated mac- rophages; 3) it inhibits the synthesis of IFN-f by NK cells; 4/i t enhances mast cell survival and promotes the growth of these cells in synergy with IL3 and IL4; 5) like IL4, it induces class II MHC antigen expres- sion by small B cells, but in contrast to IL4, it does not induce CD23 expression by these cells; 6) it main- tains B-cell viability in culture; and 7) it enhances thymoc~te growth in response to IL2 and IL4. Elevat- ed levels of ILl0 are produced by cells from Nippostrongylus-infected mice or by cells that were taken from mice 7 weeks after infection with schisto- somes. The in vivo conditions which lead to the de- velopment of THI ceils that produce IFN~, and of TH2 cells which produce IL4, IL3, IL5 and ILl0 re- main to be elucidated. It appears that the ILl0 gene was captured in the EBV genome. EBV-infected cells produce a polypeptide (BCRF1) with high homolo- gy to ILl0 and the same biological effects as ILl0. The viral factor could prevent the killing of virus- infected cells by IFN~, and TNF, since it inhibits the production of these cytokines.