new cytokines and receptors make their debut in san antonio

in the treatment of tumors and infectious disease, apparently with- out the side effects of drugs, radioisotopes and toxins. In fact, considering the rate of progress in this field, many of the remaining problems with this approach may be

resolved by the time that Reinder Bolhuis reconvenes this group in the spring of 1992.

Michael Fanger is at the Dept of Microbiology, Dartmouth Medical School, Hanover, NH 03756, USA;

David Segal is at the Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA; and Jean-Loup Romet-Lemonne is at the Center National de Transfusion Sanguine, 3 avenue des Tropiques- BPI O0, 91943 Les Ulis Cedex, France.

New cytokines and receptors make their debut in San Antonio

Scott K. Durum, Daniel G. Quinn and

New cytokines A considerable number of new

cytokines were introduced at this meeting. The protooncogene c-kit was reported to encode a membrane tyrosine kinase. A ligand for kit has now been identified by groups at Immunex (Seattle) and Amgen (Thousand Oaks, California). This ligand, which is expressed by bone marrow stromal cells, is also a membrane protein, but it has a small intracellular tail. The extracellular portion binds to kit and acts as a mast cell growth factor. Natur~il~nu- tations in the ligand gene (the steel locus) produce a mouse with defec- tive erythropoiesis and mast cell production and with reproductive sterility.

M. Gately (Hoffman-La Roche, Nutley) described cloning of a new cytokine termed 'cytotoxic lympho- cyte maturation factor' (CLMF) that enhances proliferation and induces gamma-interferon (IFN-y) production in T cells and synergizes with interleukin 2 (IL-2) in natural killer (NK) cell and lymphokine-activated killer (LAK) cell activation. CLMF has two chains of 35 and 40kDa linked by disulphide bonds and is produced by B cells.

The name 'IL-11' has been suggested for a new cytokine that acts synergistically with IL-3 in stimulating megakaryocyte colony formation and is proposed to be an important inducer of mega- karyocytopoiesis (J. Gianotti, Gen- etics Institute, Boston). This molecule also stimulates proliferation of

©

Kathrin Muegge Two exciting developments were reported at the recent Seventh Inter- national Lymphokine Workshop*. First, a number of recently cloned cytokines and cytokine receptors were described, proving that there is still a great deal to be learned about the most fundamental aspects of cell-cell com- munication. Second, advances were reported in the understanding of post- receptor signalling at both cytoplasmic

and nuclear levels.

some, but not all, IL-6-dependent cell lines. The 24 kDa protein lacks disulfide bridges or N-linked gly- cosylation and is produced by bone marrow stromal cell lines.

New receptors An IL-1 receptor was cloned sev-

eral years ago and was termed the 'T cell' or 'type I' receptor. A second receptor, the 'B cell' or 'type II' receptor, has now been cloned by the same group (J. Sims, lmmunex, Seattle) from a human B-lympho- blastoid line. This 60-65 kDa protein has 28% homology to the type 1 receptor in the extracellular portion, and like the type I receptor it has three Ig-like extracellular domains. Unlike the type I receptor it has a very small (27 amino acid) cytoplasmic tail. The type 1I receptor is expressed in bone marrow s B cells and B-cell lines (such as Raji), macrophages, granulocytes, pla- centa, TH2 clones, activated normal T cells and activated keratinocytes;

"The Seventh International Lymphokine Workshop was held in San Antonio, USA on 1-5 October 1990. f99 I, Elsevier Science Publishers Ltd, UK. 0167--4919/9 i/$02.00

most of these cells express both receptors, but fibroblasts and smooth muscle cells exclusively express the type I receptor. The type II receptor is probably proteolytically cleaved to yield the soluble IL- 1-binding protein described by G. Duff (Univ. Sheffield). This molecule is released as a 47 kDa protein from Raji cells, and has high affinity for IL-113 (but does not bind IL-I~), which suggests that it is a naturally-occurring inhibitor for IL-l[3.

L. Park (Immunex, Seattle) reported the cloning of an IL-7 receptor. Two alternatively spliced tran- scripts are made by human cells: one gives a membrane form and the other, which lacks a transmembrane portion, yields a soluble form that could, like the soluble tumor necrosis factor (TNF) receptors, act as a soluble inhibitor for the ligand. The currently identified structure has high affinity for IL-7 and is expressed on lymphoid and myeloid cells. There appears to be another IL-7 receptor, which has low affinity, but it has not yet been cloned.

A. Miyajima (DNAX, Palo Alto) reported cloning of a second chain of the granulocyte-macrophage colony- stimulating factor (GM-CSF) receptor. The previously described 80 kDa chain has low affinity while the new 120kDa chain has inter- mediate affinity. Both chains to- gether constitute a high-affinity receptor similar to the c~ and [3 chains of the IL-2 receptor.

S. Takaki (Kumamoto Univ.) reported cloning of an IL-5 receptor

Immunology Today 54 vo, 12 No. 2 1991

with a molecular mass of 60 kDa. Its intracellular domain is homologous to receptors for GM-CSF, prolactin and growth hormone. A form with- out the transmembrane region can be secreted. As the cloned IL-5 receptor has only low affinity, another chain providing high affinity still needs to be found.

All three of these newly cloned receptors (GM-CSF, IL-7 and IL-5) belong to the hematopoietin receptor superfamily. This family, charac- terized by cysteine repeats in the extracellular domain, includes several other cytokine receptors, such as the IL-2RI3, IL-3R, IL-4R and IL-6R.

A number of cytokine receptors have a two chain structure, including the newly discovered GM-CSF, IL-5 (and, presumably, the new B-cell IL-1 receptor, since it has such a small cytoplasmic domain) and the previously identified IL-2R and IL-6R. R. Klausner (NIH, Bethesda) pointed out that the ~ chain, first identified as part of the TCR-CD3 complex, is actually shared by several other receptors, including the Fce receptor and a receptor on NK cells. It is also possible that some chains of the cytokine receptors are shared: it was previously shown that IL-3 could compete for GM-CSF binding and T. Kishimoto (Osaka Univ.) reported that transfecting the p130 chain of the IL-6 receptor into IL-3-dependent cell lines increased their sensitivity to IL-3.

The TNF receptor consists of two TNF-binding proteins, p60 and p80. M. Kroenke (Univ. Goettingen) reported that monomeric p60 bound TNF but not lymphotoxin (LT), whereas p60 homodimers and p60- p80 heterodimers bind either TNF or LT.

Post-receptor events IL-2-receptor-associated tyrosine hinase activity

IL-2 has been shown to induce the phosphorylation of certain cellular proteins on serine and tyrosine resi- dues in responsive cells and it now appears that a number of distinct tyrosine kinases may be involved in signal transduction via this ligand- receptor system.

A new 64 kDa protein, tentatively termed the 'IL-2Ry' chain, co- precipitates with IL-2Rc~ (p55) and IL-2R[3 (p75) using a monoclonal

antibody to IL-2R{3 in the presence of IL-2 (K. Sugamura, Tohoku Univ., Sedai). This protein, which is expressed in lymphoid cells but not in fibroblastoid cells, is phosphoryl- ated on tyrosine in response to IL-2. The y chain was proposed to be a tyrosine kinase that associates with the IL-2R[3 chain upon binding the ligand (since in the absence of IL-2, the anti-IL-2R[3 precipitated the IL-2R[3 chain alone). D. Michiel (NCI, Frederick) reported that IL-2 induced tyrosine phosphorylation of two substrates, p58 and p97, detected by precipitation with anti- phosphotyrosine antibody or binding to an affinity column made of synthetic tyrosine kinase substrate. Both p58 and p97 appear to be associated with the IL-2 receptor and p97 showed tyrosine autophos- phorylation. Thus p97 is a good can- didate for a transducing structure. It was also reported that the lymphoid- specific tyrosine kinase p60 ;ck could be coprecipitated by an antibody to the ILT-2R[3 chain (in the absence of IL-2),Vbut not by an antibody to the IL-2R(x chain (T. Taniguchi, Osaka Univ.), suggesting that this kinase associates with the IL-2R[3 chain in the absence of ligand. Whether p60 ;ok plays a role in signal transduction via the IL-2R remains to be determined.

IL-2 stimulation of T cells results in increased incorporation of phos- phate into the 3 position of phos- phatidylinositol (PI). This was shown to be due to the activation, via tyrosine phosphorylation, of the 86kDa PI-3 kinase (Augustine, Mayo Clinic), an enzyme of unknown function. Immunoprecipitation ex- periments demonstrated the association of PI-3 kinase with the tyrosine kinase p59[y n in T cells.

Signalling from IL-2R~ D. Jankovic (Institut Pasteur)

suggested that the IL-2Rc~ chain is required for growth signalling. A cell line was developed that had only p70 and lacked p55; this line showed a response to IL-2 by producing myc and myb. Adding p55 to this cell line using transfection permitted proliferation to IL-2.

IL-1 and cAMP IL-1 synergizes with phorbol myr-

istate acetate (PMA) to induce the

expression of the DNA-binding factor AP-1 in the mouse EL4 T-cell line, but has no effect on its own (S. Mizel, Wake Forest Univ.). The fol- lowing pathway was proposed: the IL-1 effect depends on constitutive intracellular cAMP levels, which would continually activate PK-A; this may downregulate phosphatase (PPase) activity (possibly PPase 1 or PPase 2a), and these PPases could constitutively inactivate the AP-1 complex.

Colony-stimulating factor 1 (CSF-1) receptor

Signal transduction by the CSF-1 receptor, which unlike most cytokine receptors is itself a tyrosine kinase, was discussed by C. Sherr (St Judes, Memphis). Binding of CSF-1 to its receptor results in tyrosine phosphorylation of the receptor at three sites: 699,708 and 809. Using specific mutations of these tyrosines, it was shown that the critical site for mitogenesis is 809, whereas the association of PIG kinase with the receptor depends on sites 699 and 708. As in the case of the IL-2 receptor, the possible significance of ligand- induced PI-3 kinase association with the CSF-1 receptor is unclear, but it does not appear to be required for mitogenesis.

TNF receptor There are two TNF-binding recep-

tors (p60 and p80), as noted above (M. Kroenke, Univ. Goettingen). TNF stimulation of Jurkat, K562 and U937 cells results in a decrease in phosphocholine (PC), an increase in diacylglycerol, and a subsequent activation of protein kinase C. An antibody against p60 blocks these effects, which suggests that this receptor interacts with a PC-specific phospholipase.

Nuclear events Efforts continue to be made to

understand how cytokines are induced (and act) at the gene level. One highlight of the meeting was M. Karin's (Univ. California, San Diego) finding of a connection between glucocorticoid and the AP-1 transcription factor. Glucocor- ticoids were previously thought to mediate their actions via the glucocorticoid responsive element (GRE); but this would not explain

Immunology Today 5 5 Vol. 12 No. 2 1991

the inhibition of many genes, including tL-2, that lack a GRE. Karin's new finding is that the glucocorticoid-receptor complex di- rectly binds to the jun component of AP-1, blocking DNA binding. Since the AP-1 factor appears to activate many genes, including the IL-2 gene, many of the anti-inflammatory and immunosuppressive activities of glucocorticoids could occur via the blocking of the AP-1 factor. The recently cloned transcription factor NF-IL-6 has been suggested to trans- duce the IL-6 signal on a number of genes (such as haptoglobin) during the acute phase response (T. Kishimoto). Perhaps NF-IL-6, which has a leucine-zipper motif, can also interact with jun or the glucocorticoid receptor, since the zipper is the basis of these associations.

Examination of cytokine promoter regions reveals common DNA-binding motifs. Thus an NF-KB-like binding site has been de- fined in functionally important regions of the GM-CSF and the IL-3 promoter (Arai, DNAX, Palo Alto) as well as in the 1L-8 promoter (K. Matsushima, NCI, Frederick). The NF-KB motif of the IL-2R~x chain promoter binds four different proteins. One of them (p85) probably participates in negative regulation of the gene and was suggested to be identical with c-rel (W. Greene, Duke Univ.). V-rel was shown to inhibit tax-induced activation of the IL-2 receptor gene. ADF, an enzyme with reducing capacity in human T-cell leukemia virus 1 (HTLV-1)- infected cells, has been known to activate the IL-2RoL chain. W. Leonard (NIH, Bethesda) reported that oxidation (or alkylation) de- creased NF-KB DNA-binding activity which could be restored by reduction. Free sulfhydril groups ap- peared to be required for NF-KB activity.

Cytokines versus HIV A. Fauci and co-workers (NIH,

Bethesda) have found that different cytokines can induce or inhibit viral expression. For example, in infected monocytes, TNF, IL-6 or GM-CSF induce the production of reverse transcriptase. The mechanisms are different: TNF induces transcription of the gene (probably via NF-KB), whereas IL-6 activates post-tran-

scriptionally. Cytokines can also inhibit human immunodeficiency virus (HIV): transforming growth factor [3 (TGF-[3) blocks induction of reverse transcn ,tase by some stimuli (PMA) but not others (TNF). IFN-~ inhibits post-translational budding.

T cells K. Smith (Dartmouth Univ.) and

S. Swain (Univ. California, San Diego) both reported that different repertoires of cytokines were produced by naive T cells and memory T cells. For naive cells, Smith used neo- natal CD4 cells from human cord blood, whereas Swain purified CD4 cells from adult spleen cells. Naive cells could be stimulated by anti- CD3 antibody to produce IL-2, but not other cytokines (except slow production of GM-CSF); however, once naive cells had been stimulated for a few days, they could also produce IL-3, IL-4, IL-5 and IFN-y.

Inhibitors Berger (UpJohn, Kalamazoo) re-

viewed findings on the IL-1 inhibitor, 'IRAP', which is an IL-1 homolog. It is an effective inhibitor: a ten-fold excess in vitro gives 50% inhibition, and in mice 30 ~g blocks IL-1 induction of corticosteroids. Confusingly, IRAP blocks both type I and II receptors of human origin, but in mice the human IRAP blocks only type I receptors. C. Dinarello (Tufts Univ., Boston) reported that in rabbits IRAP is very effective in vivo in two different systemic inflammatory reactions. In immune complex colitis IRAP blocks inflam- mation, edema and necrosis, and in endotoxic shock IRAP blocks hypotension and leukopenia and prevents death. Moreover, IRAP was able to inhibit proliferation and spon- taneous GM-CSF production in myeloid leukemia. These findings implicate ILd in the pathogenesis of these disorders and offer therapeutic promise. IRAP was not found to be toxic by either group.

Other inhibitors include a new compound, CPD 406, reported by H-D. Flad (Borstel), that blocks lipopolysaccharide (LPS) induction of IL-1 in macrophages. IL-4 was shown to be an effective inhibitor of monocyte production of IL-1, TNF and IL-6 (M. Fenton, Boston Univ.); this was due, at least in part, to an

effect on stability of the mRNA for these cytokines.

IL-10 (M. Howard, DNAX, Palo Alto) has been cloned, based on the inhibition of cytokine production by TH1 cells. The current thinking on IL-10 is that it stimulates T cells, but inhibits the antigen-presenting cell (APC) function of macrophages (including inhibition of IL-1, IL-6 and TNF production); however, B-cell APC function is not inhibited by IL- 10. A remarkable feature of IL-t 0 is its high homology to a product of Epstein-Barr virus (EBV), which is termed 'BCRF-I'. This molecule even shares some of the biological activities of IL- 10, and it is likely that EBV has appropriated this gene from the human genome.

Notes on cancer and cytokines J. Yang (NIH, Bethesda) described

exciting plans to study tumor- infiltrating lymphocytes (TILs) transfected with TNF in a retroviral vec- tor. The reasoning here is that TNF has anti-tumor activity in mice, which can tolerate high doses of the cytokine; but humans cannot be given enough TNF to be effective, because they are far more susceptible than mice to systemic toxicity. By giving the patient TNF via T1Ls, it is hoped that enough TNF will be produced locally in the tumor to have an effect, but not so much that systemic toxicity is a problem.

W. Peters (Duke Univ.) described studies using GM-CSF to promote the recovery of neutrophils after radiation and autologous bone marrow transplantation for breast cancer. Patients were first treated with GM-CSF or G-CSF to induce more progenitors for granulopoiesis. After this treatment, bone marrow was taken out and reserved for grafting, and this bone marrow contained increased granulopoietic precursors. After treatment, the bone marrow was replaced, resulting in granulopoietic reconstitution that was much faster than normal.

Mannel (DKFZ, Heidelberg) showed that several tumor cell types (including Jurkat and K562) can induce macrophages to produce IL-1 and TNF. The inducing principle seems to be on the tumor cell membrane and it appears to be a carbohydrate.

G. Forni (Univ. Torino) has been

Immunology Today 56 VoL 12 No. 2 1991

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treating tumors with local adminis- tration of cytokines. He found that IL-1 plus IL-4 given into draining lymphatics resulted in both tumor regression and immunological memory.

Rearrangement of the gene for the low molecular weight BCGF was detected in 50% of Burkitt's lymph- omas by R. Ford (M.D. Anderson, Houston), and reported to have autocrine growth factor activity in these cells.

IL-1 phase I trials (W. Urba, NCI, Frederick) showed considerable toxicity, with hypotension being the

dose-limiting side effect. IL-4 was transfected into an IL-4-

addicted line and IL-5 was transfected into an IL-5-addicted line (T. Blankenstein, Univ. Berlin). Both types of cell line became independent of their respective growth factors. But, interestingly, only the IL-5 line was tumorigemc in vivo. It was suggested that the IL-4 line induced immunity, leading to its own rejection.

The authors thank W. Farrar, J.J. Oppenheim and D. Longo for their comments on this manuscript. Daniel

Quinn is partially funded by the Psoriasis Association.

Scott Durum is at the Laboratory of Molecular Immunoregulation, Bio- logical Response Modifiers Program, National Cancer Institute, Frederick, MD 21702-1201, USA; Daniel Quinn is at the Institute of Dermatology, St Thomas' Hospital, Lambeth Place Rd, London SE1 7EH, UK; Kathrin Muegge is at the Biological Carcino- genesis and Development Program, Program Resources Inc., National Cancer Institute, Frederick, MD 21702-1201, USA.

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Genetic variation in the stress response: susceptibility to experimental allergic

encephalomyelitis and implications for human inflammatory disease

Don Mason

Glucocorticoids that are released from the adrenal glands in response to stress can have profound effects on the immune system. Here, Don Mason illustrates how genetic variation in the magnitude of such a response can determine susceptibility to an experimental autoimmune disease in rats and discusses the implications for susceptibility to inflammatory diseases in humans. He also addresses the possible long-term effects of glucocorticoids on the balance between the cell-mediated and the humoral aspects of immunity and how this

balance may influence the temporal development of an immune reaction.

In the evolution of the immune system, a number of potent bactericidal and cytocidal mechanisms have developed. These reactions are potentially damaging to the host and a variety of immunoregulatory mechanisms may have evolved in parallel to limit them. Since virtually all biological parameters show genetic variation, the level to which an immune response may develop before being checked by these immunoregulatory mechanisms will also be expected to vary from one individual to another. In this article the possible consequences of such variation are examined and illustrated by the effect of genetically determined variation in the glucocorticoid stress response during inflammatory immune reactions.

Glucocorticoids and the immune response In all mammalian species studied, a major response to

stress is the secretion of glucocorticoids from the adrenal

gland. This stress response can be induced by a wide range of stimuli such as heat, cold, fear, hunger and physical injury and it produces an equally broad diversity of physiological effects, not only on metabolism but also on the immune system 1. There is evidence that the magnitude of an immune response may be limited by glucocorticoids that are released as a consequence of the immune response itself 2-4 and it has been proposed that such a neuroendocrine feedback loop prevents the immune response from reaching a level that is potentially damaging to the host s . This idea is strongly supported by the finding that adrenalectomized rats, which are otherwise apparently well, die within 24--48 hours of being immunized with Freund's Complete Adjuvant (FCA) but can be protected by corticosterone replacement therapy 6. It is probable that the lethal effects of FCA are mediated by interleukin 1 and tumour necrosis factor as it has been

© 1991, Elsevier Science Publishers Ltd, UK. 0167--4919191/$02.00

Immunology Today 57 Vol. 12 No. 2 1991

new cytokines and receptors make their debut in san antonio

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