(CANCERRESEARCH52, 6129-6133, November1, 19921

Meeting Report

The Role of Cytokines in the Regulation of Cell Function and in the Pathogenesisof Disease: A Pathology A/Pathology B Study Section Workshop1

Working Report from the Division of Research Grants, National Institutes of Health

Cytokines are increasingly recognized as essential and powerful cell communication and regulatory molecules. They areimplicated in normal homeostasis and defense against manydiseases and are involved in the pathogenesis of a variety ofdisorders. As a result of their powerful and widespread regulatory functions, a number of cytokines are being tested as therapeutic agents. At the same time, many antagonistic factors arebeing developed for use in those diseases that may be initiatedor mediated by cytokines.

The field of cytokine biology is growing rapidly. Studies arebeing carried out that range from analysis of molecular regulation of cytokine expression through the use of cytokines astherapeutics. Diseases involved include cancer, infectious diseases, inflammatory processes, autoimmune disorders, developmental defects, and aging; the implications extend to everyorgan system of the body. One example of this pervasiveness isthe potential for use ofcytokines in cancer, where they are beingtested as direct antitumor agents, as enhancers of immunological reactivity against tumor cells, as potentiators of chemotherapy and radiotherapy, and as mitigators of the myelotoxicityassociated with current cancer treatment. Contrariwise somecancer cells produce cytokines, which may be involved in autocrine growth stimulation.

To explore these facets of cytokine biology, the NIH Pathology A and Pathology B Study Sections sponsored a workshopentitled, “TheRole of Cytokines in the Regulation of CellFunction and in the Pathogenesis of Disease.―The purpose ofthe workshop was to consider new findings in cytokine biology,and in particular questions concerning how the cytokines areregulated, how their effects on cells are mediated, how theyinteract with one another in complex regulatory networks, andhow such information can be used to understand and, ultimately, influence normal cell function and disease pathogenesis. A major objective was to bring together investigators fromwidely disparate areas on which cytokines impinge to examinecommon mechanisms and explore ways in which informationfrom one system could be brought to bear in gaining an understanding of cytokine effects on other processes.

Stephen J. Galli (Beth Israel Hospital and Harvard MedicalSchool, Boston, MA) discussed two areas of mast cell biologypertaining to cytokines: the cytokine-dependent regulation ofmast cell development and function; and the mast cell as asource of TNF-a2 and other cytokines which influence the expression of biological responses. Mast cell development is a

Received 5/27/92; accepted 8/21/92.I This workshop was held February 1, 1992, in Taos, NM. It was sponsored by

the PathologyA and Pathology B Study Sectionsandwassupported by the Divisionof Research Grants, NIH.

2 The abbreviations used are: TNF-a, tumor necrosis factor a; SCF, stem cellfactor, rrSCF, recombinant rat SCF; CSF, colony-stimulating factor, EC, endothehal cells hEC, human EC;IL-I, interleukin I; SMC, smooth musclecells SG-CSF,granulocyte-macrophagecolony stimulating factor, IL-6, interleukin 6; ANG-II,angiotensin II; TGF-il, transforming growth factor @;CNS, central nervous system;MS, multiple sclerosis; MIP-1, macrophage inflammatory protein 1; NK, naturalkiller, NKSF, natural killer cell-stimulatory factor, IL-12, interleukin 12; IFN-'y,7-interferon; FGF, fibroblast growth factor, LR, leukoregulin; HSV-1,

complex process that results in the appearance of phenotypically distinct populations of mast cells in different anatomicalsites. Mice with a double dose of mutations at the Wor S locusexhibit several phenotypic abnormalities, including a virtualabsence of mast cells in all organs and tissues. Recent workindicates that W encodes the c-kit tyrosine kinase receptor,whereas Si encodes a c-kit ligand that has been designated SCF,mast cell growth factor, kit ligand, and Steel factor. in vitro andin vivo studies in mice and in vivo studies in rats indicate thatrrSCF can induce the proliferation of both immature and matare mast cell populations and can induce the development ofboth connective tissue-type and mucosal mast cells. In addition,rrSCF can induce mouse mast cell maturation and heparmnsynthesis in vitro. rrSCF also can induce c-kit receptor-dependent mast cell degranulation and mediator release in vivo or invitro. This finding represents the first evidence that signalingthrough the c-kit receptor can induce expression of function bya cellular lineage bearing this receptor.

Wor Si mutant mice also have been useful for the analysis ofmast cell function. W/Wv mice can be locally and selectivelyrepaired of the mast cell deficiency by the adoptive transfer ofin vitro-derived immature mast cells of congenic +1+ origin,providing a model for studying the expression of biologicalresponses in vivo in tissues which do or do not contain mastcells. Analysis of biological responses in this system indicatesthat the mast cells are essential for the expression of IgEdependent cutaneous responses and certain other cutaneousinflammatory responses and indicate that TNF-a of mast cellorigin represents an important mediator of the leukocyte infiltration which occurs during some ofthese reactions. In contrastto other cell types which can produce TNF-a, the mouse mastcell constitutively contains significant amounts of this cytokinein a preformed pool which can be released rapidly upon appropriate stimulation of the cell. Recent evidence indicates that therelease of preformed and newly synthesized TNF-a by mastcells may importantly contribute to the initiation of a variety ofexperimental and clinical allergic responses.

Cytokines are generally viewed as participants in inflammation, immunity, and the host response to infectious agents.Peter Libby (Brigham and Women's Hospital and HarvardMedical School) discussed the mechanisms by which these protein mediators may also contribute to deranged functions ofvessel wall cells during atherogenesis. The immunostimulatoryinterleukins and related leukocyte activating and chemoattractant proteins are one important class of cytokines. Other cytokines involved include TNF-a, CSFs, and the interferons. Cytokines can modulate the properties of EC that render theirsurface able to maintain blood in a liquid state. IL-i or TNF-acan alter the balance of anticoagulant, antithrombotic, and

herpes simplex virus type 1; ACV, acyclovir,HPV, human papilloma viruses;LAK, lymphokine activated killer, MMP, matrix-metalloproteinase;EGF, epidermal growth factor, AIDS, acquired immunodeficiency syndrome; HIV, humanimmunodeficiency virus.

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fibrinolytic mechanisms of EC in a fashion that reduces hemocompatibility. Adhesive interactions between leukocytes andendothelium contribute importantly to normal leukocyte trafficking and the mobilization of host defenses and in the pathogenesis of certain diseases. Early in the response to atherogenicdiets in many species, blood monocytes adhere to the endothehum of lesion-prone areas of arteries, enter the intima, andaccumulate lipids. IL-i, TNF-a, and ‘y-interferoncan modulatethe expression of leukocyte adhesion molecules on the endothelium and other cells that mediate such adhesive interactions.Recent studies indicate that in rabbits fed a cholesterol-supplemented diet for only 7 days, aortic endothelial cells expressregionally the cytokine-inducible endothelial-monocyte adhesion molecule, VCAM-I. Cytokines can also influence thegrowth ofEC and SMC by either direct or indirect mechanisms.TNF-a and IL-I tend to inhibit EC proliferation in vitro. IL-itreatment stimulates proliferation of SMC, perhaps by inducing autocrine growth factor production. IFN-'y limits the proliferation of both endothelial and SMC. IL-i or modified lipoproteins can modulate the expression of the hematopoieticgrowth factor granulocyte-monocyte CSF and monocyte-CSFin cultured human vascular cells. This is a potentially important

finding inasmuch as mononuclear phagocyte recruitment, infiltration, and even multiplication are characteristic of humanatheroma. In addition to effects on cell growth, cytokines canregulate the production of lipid mediators and other cytokinesby vascular wall cells. For example, IL-i and TNF-a potentlystimulate elaboration of prostaglandins and platelet-activatingfactor and augment IL-i and IL-6 production by vascular cells.

How might cytokine gene expression be linked to factorsimplicated in the initiation of atherosclerosis? Ten weeks afterthe initiation of diets supplemented with saturated fat andgraded levels of cholesterol, aortic extracts do not contain augmented levels of IL-i or TNF-a mRNA. However, i.v. injectionof a classical cytokine inducer, bacterial endotoxin, elicitedgreater elevation of cytokine mRNA levels in animals fed dietscontaining 0.3 or 0.9% cholesterol in addition to saturated fat.This finding indicates that macrophage-derived foam cellsand/or other cell types in these rabbit atheromas retain theability to respond to exogenous stimuli, although they do notexhibit spontaneous elevation of cytokine gene expression. Ohservations on advanced human atherosclerotic lesions show noincrease in overall basal IL-i expression but do exhibit increased inducible IL-i activity, in agreement with the experimental results.

Eric G. Neilson (University of Pennsylvania, Philadelphia,PA) discussed evidence suggesting that tubular hypertrophymay be an important factor in the adaptive response of thekidney to various physiological and pathophysiological stimuliand that this could be a revealing model for in vivo regulationof cell and tissue growth. There is increasing evidence that atubular hypertrophy response may be at least one processinvolved in the progression of renal diseases. The importantobservations by others that the activation of the intrarenal renin-angiotensin axis is altered in situations associated with renal growth and the fact that angiotensin-converting enzymeinhibitors block compensatory renal hypertrophy in many models provided a basis to study extensively the influence ofANG-Il on the growth of various syngeneic renal cell lines. Theeffects of ANG-Il on growth are cell specific. In cultured mesangial cells it induces proliferation, while in syngeneic proximal tubular cells the peptide induces hypertrophy as deter

mined by increases in cell size, de novo protein synthesis, totalprotein content, and RNA without concomitant changes inDNA content.

ANG-Il-induced hypertrophy is paralleled by an increase intranscription and biosynthesis of type IV collagens, probablyreflecting the remodeling of the extracellular matrix associatedwith the enlargement ofthe cells. ANG-Il-induced hypertrophyin MCT cells is mediated by At1 receptors and a distinct signaltransduction pathway with a decrease of intracellular cyclicAMP as a second messenger. While some genes induced byANG-Il, like immediate early genes or homeobox genes, areengaged as part of a generalized activation of the nucleus, thehypertrophy effect also seems to be mediated by a set of novelgenes which may be part of an identifiable molecular programcausing or mediating enlargement. These genes have beenisolated by differential hybridization and the genes and thehypertrophy effect are sensitive to antisense block by phosophorothioate-capped oligonucleotides.

J. E. Merrill (Department of Neurology, UCLA School ofMedicine, Los Angeles, CA) discussed the role of cytokines inthe pathogenesis of neurological disorders, including MS andCNS AIDS. While certain cytokines such as IL-i, TNF-a, IL-6,and TGF-13 may be involved in pro- and antiinflammatoryevents in the CNS of patients with MS and CNS AIDS, there isno uniform consensus as to whether they are elevated or evendetectable in all compartments of the body such as serum, cerebrospinal fluid, and tissue. Furthermore, if they are elevated inthese diseases, there are no data as to whether they regulate thedisease process itself. Myelin damage in MS is punctate localdemyelination. In AIDS, white matter pathology is diffuse andmore global. Oligodendrocytes are destroyed in MS but notCNS AIDS, suggesting a different mechanism for myelin loss inthe two diseases. These different pathologies may provide cluesabout the role of macrophages, microglia, and/or the toxicproducts they produce in putatively giving rise to myelin damage. The stimuli that trigger such a destructive response bymacrophages or glial cells and/or the regulation of the toxicevents in the two diseases are likely to be different. In MS,effector cell-mediated lesion production and oligodendrocytecell destruction seems to occur. Merrill hypothesized that theeffector is the inflammatory blood macrophage and/or microglial cell induced and promoted to its cytotoxic activity by acollaboration of neurotransmitters and cytokines. In CNSAIDS, virus-induced toxic products of glia and their diffusionthrough white and gray matter areas of the brain have beensuggested. Such soluble mediators would then compromisemetabolic processes of neurons and glia without widespreadtarget cell loss.

In MS, a demyelinating disease of the CNS, myelin sheathsare destroyed in the parenchyma of white matter, and oligodendrocytes, the myelin-producing cells, degenerate and disappearfrom the lesion where demyelination is occurring. The promflammatory cytokines IL-i and TNF-a from activated macrophages and brain microglia probably mediate loss of myelinand the myelin-producing cell, the oligodendrocyte. Macrophages have been shown to mediate a reduction in metabolic processes of and cytotoxicity against a variety of normal, nontransformed cells.

Merrill also suggested that Substance P, a neuropeptide released by nonmyelinated fibers innervating white matter bloodvessels, plays an important role in CNS inflammation by inducing or enhancing the production of IL-i, IL-6, and TNF-a.The function of these cytokines in lesion formation in MS is

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restricted to their expression on the surface of the macrophageor microglial cell (TNF-a) or at short distances only (IL-i andIL-6). These conclusions are based on (a) the identification ofTNF-a and IL-i within, but not outside, the gliotic plaques infrozen sections of MS tissue and (b) the inability of solubleTNF-a to lyse oligodendrocytes. Microglial lysis, however, isinhibited by anti-TNF-a suggesting a membrane-bound or associated form of TNF-or as the mediator.

Merrill's laboratory also previously demonstrated that HIV-1induces IL-i and TNF-a in cultures of normal peripheral bloodmacrophages. They demonstrated that cultures of glial cellsfrom rat or human brain can be induced to produce TNF-a andIL-i by HIV-i. The induction does not require productive viralreplication. Thus virus, even inactive virus, may participate in aparacrine loop of cytokine-HIV-1-cytokine which perpetratesvirus in inflammatory T-cells and macrophages in the CNS.

Barbara Sherry (Picower Institute for Medical Research,Manhasset, NY) discussed the biology of MIP-1, a member ofthe newly described chemokine family, which is made up of acollection of low molecular weight chemotactic and activatingpeptides of similar sequence and genomic structure. Other family members include PF4, IL-8, gro, MCP-1, and RANTES.The expression of MIP-i, which is isolated from culture supernatants as a doublet comprising two distinct peptides, MIP-laand MIP-i@, is markedly induced in macrophages in responseto inflammatory stimuli. It has been shown to elicit an inflammatory response consisting primarily of neutrophils when administered in vivo, and it has proved to be chemotactic andactivating for neutrophils when assayed in vitro. However, thework presented indicates that the role of MIP-i in inflammation is not limited to the modulation of neutrophil responses.MIP-1 peptides were shown to attract and activate macrophages as well. MIP-1-treated macrophages exhibited enhancedtumor cell killing in coculture assays and secreted the inflammatory cytokines, TNF-a, IL-i, and IL-6. The presence ofMIP-i was documented by Western analysis in sterile woundsites, and MIP-l was shown to induce the expression of cytokinc genes in vitro in fibroblasts cultured from wound fluids. Inaddition to these cellular activating effects, MIP-l is an endogenous pyrogen, eliciting a prostaglandin-independent feverwhen injected i.v. in rabbits or directly into the hypothalamus ofrats. In addition to its proinflammatory properties, work incollaboration with Hal Broxmeyer (Walther Oncology Center,Indianapolis, IN) indicates that MIP-l acts to enhance CSFdependent proliferation ofcommitted granulocyte-macrophageprogenitor cells. Additionally, MIP-i and recombinant MIPicr, but not recombinant MIP-1@9,selectively suppress less mature populations of early erythroid and multipotential progenitors which are dependent on multiple signals (erythropoietinplus interleukin 3) for proliferation.

Giorgio Trinchieri (Wistar Institute, Philadelphia, PA) described the structure and function ofa novel cytokine, NKSF orIL-12. NKSF/IL-12 was originally identified in the supernatantfluid ofseveral human Epstein-Barr virus-transformed cell linesfrom which it was purified. NKSF/IL-12 have a variety of biological functions on T- and NK cells, including: (a) the ability toinduce, alone or in synergism with other stimuli (e.g., IL-l2,phorbol diesters, mitogens, antigens, or anti-CD3 stimulation)the production of IFN-'y and other cytokines; (b) an enhancingeffect on the cytotoxic activity of NK and T cells; and (c) complex modulatory effects, mostly but not uniquely stimulatory,on the proliferation of T- and NK cells.

The ability of IL-l2 to induce IFN-7 was used by MichikoKobayashi in Trinchieri's laboratory for monitoring the purification ofnatural NKSF/IL-12 from the supernatant fluid of thehuman B-lymphoblastoid cell line RPMI 8866. IL-i 2 wasfound to have a structure different from that ofother cytokines,being a heterodimer, formed from two covalently linked glycosylated Mr 40,000 and 35,000 chains. In collaboration withSteven Clark and Stan Wolf (Genetics Institute, Inc., Cambridge, MA), the two genes encoding IL-12 were cloned on thebasis of primary internal and amino-terminal sequences derivedfrom the purified natural NKSFIIL-1 2 and biologically activerecombinant IL-i 2 was produced. The p40 gene encodes a protein with characteristics of the hematopoietin or cytokine receptor family, with over 20% homology in primary amino acidsequence with the extracellular portion of the IL-6 receptor.The p35 gene encodes an ct-helix-rich polypeptide with somehomology with IL-6 and granulocyte-colony stimulating factor.Kishimoto and colleagues showed that IL-6 can bind in solutionwith the soluble extracellular portion of the a chain of the IL-6receptor which is homologous to IL-12 p40. The complex ofIL-6 and IL-6R can then bind to the@ chain of IL-6R (orgpl3O) and mediate IL-6 biological functions on differentiationand proliferation of the target cells. It is therefore possible thatIL-12 p35 is a primitive cytokine which during evolution became covalently linked to the secreted extracellular portion ofits receptor (p40); the complex would then be able to bind to asecond transmembrane chain of the receptor, similarly to whatis observed for IL-6. The covalent linkage between the twochains may represent an efficient way to increase receptor affinity, and it is striking that several biological functions ofIL-1 2are mediated at concentrations well below 1 pM.

Preliminary evidence indicates that natural NKSF/IL-i2 isproduced by normal B-cells and monocytes. Fixed Staphylococcus aureus Cowan I strain is a powerful inducer of IL-i2 production both from monocytes and B-cells, suggesting that IL-l2might be produced in vivo during bacterial infection. Because ofthe ability ofNKSF/IL-12 to modulate many important cellularfunctions of T- and NK cells, this new cytokine is likely to playimportant roles in the regulation of both adaptive and naturalimmunity. In particular, the powerful ability of NKSF/IL-l2 toinduce the production of IFN-'y and to synergize in this effectwith many other T- and NK cell stimuli (e.g., IL-2, TCR-CD3or CD16 cross-linking) that are specifically activated during animmune response suggest that NKSF/IL-l 2 when producedduring infection or immunization might preferentially inducethe expansion of the Thi subset of helper T-cells, with possiblyprofound effects on the type of immune response generated andits efficiency to protect against infection.

Thomas Maciag (American Red Cross, Rockville, MD) discussed regulation of endothelial cell function by signal-lessgrowth factors and cytokines. FGF-l and IL-la are positive andnegative regulators of hEC growth in vitro. An interesting feature of these polypeptides is the absence of a signal peptidesequence for directed secretion. While recent studies withFGF-l have suggested that it contains a nuclear translocationsequence, the nuclear translocation sequence does not appear tobe functional when FGF-1 is expressed as an endogenous cytosolic protein. In contrast, exogenous FGF-i is rapidly importedinto the nucleus and further studies have suggested that thisoccurs continuously throughout the G0 to G1 transition periodof the BALB/c 3T3 cell type. In addition, the commitment toinitiate DNA synthesis requires a continuous 12-h exposure toFGF-1. Interestingly, there appears to be a low steady state level

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of FGF receptors on the cell surface that initiates a continuousseries ofdifferential FGF-1-induced phosphotyrosyl-containingproteins that are present during the entire G0 to G1 transitionperiod. Although the mechanism utilized by FGF-l for secretion is not established, recent experiments argue that heatshock may be involved in the regulation of this process.

During these studies with FGF-1, Maciag and colleaguesdetermined whether intracellular IL-la can function as a repressor of hEC growth in vitro. They observed that as hECsenter into their senescence pathway, the mRNA for plasminogen activator inhibitor I and cyclooxygenase, two hEC IL-laresponse genes, are exaggerated in the senescent hEC population. To determine the role of IL-la as a potential regulator ofhEC senescence, young hEC were transfected with an antisenseAUG oligomer to IL-la. While they observed that the repression of IL-la translation significantly extended the life span ofthe hEC population, these conditions did not result in the formation of an immortal hEC phenotype. Thus, the repression ofhEC growth by the process ofcellular senescence is regulated bythe potential intracellular function of Il-la and studies are inprogress to determine whether this function involves the nuclear translocation of endogenous IL-la.

Cytokines as destructive or protective agents in viral diseasewere discussed by Charles H. Evans (National Cancer Institute,Bethesda, MD). An increasing number of cytokines are beingrecognized as able to inhibit as well as stimulate virus geneexpression and replication after virus infection in addition tothe widely recognized ability of interferons to induce a cellularstate of resistance to subsequent virus infection. A model ofcytokine intervention in acute human herpesvirus infection andanother model of chronic human papillomavirus infection developed at the NIH are proving useful for investigation of cytokine modulation of virus infection, virus gene expression,virus replication, and the development ofneoplasia. In the acuteinfection model, Evans' laboratory in collaboration with JohnHooks (National Eye Institute, Bethesda, MD) and BarbaraDetrick (George Washington University, Washington, DC)have demonstrated that LR is able to prevent the secretion ofinfectious virus when applied to cells within hours after infection with human herpes simplex virus. LR is a naturally occurring M@ 50,000 cytokine secreted by lymphocytes which increases membrane permeability and drug uptake in tumor butnot in normal cells. LR also increases membrane permeabilityof HSV-l-infected cells. More importantly, LR significantlyenhances the ability of ACV to inhibit the cellular release ofinfectious HSV-l. The ability of 1—100@iMACV to inhibitinfectious HSV-l production is increased up to 100-fold whenHSV-l-infected human amnion (WISH) cells are treated with 5units LR/ml and ACY 3 h after virus infection. Under theseconditions LR alone is unable to inhibit HSV-l infectivity. Inaddition, three unrelated cytokines, IL-Ia, IFN-a, and IFN-'y,lack the ability to enhance the anti-HSV actions of ACV whentheir treatment is initiated after HSV-l infection. These findings demonstrate that a combination of immunotherapy andchemotherapy can produce a substantial inhibition of herpesvin's replication and provide a rationale for the application of thisapproach to the interventive treatment of virus infection.

Cytokine interaction in chronic virus infection is being studied by Evans in collaboration with Joseph DiPaolo and CraigWoodworth at the National Cancer Institute, Bethesda, MD,using the system of human papillomavirus transformation ofcervical epithelial cells developed in DiPaolo's laboratory forthe study ofcervical carcinogenesis. HPV, in particular type 16,

are major factors in the etiology of cervical cancer. The HPVtransforming genes E6 and E7 are retained and expressed in themajority of cervical cancers implying an important role forthese proteins in maintenance of the malignant phenotype. LRand IFN-'y but not IFN-a inhibit transcription of E6 and E7genes in several human epithelial cervical cell lines immortalized by recombinant HPV-16, 18-, and -33 DNAS. LR andIFN--y enhance transcription of class 1 cell surface HLA andIFN-'y additionally induces HLA class 2 expression. HPV-immortalized cells develop partial resistance to the growth-inhibitory effects of the cytokine after malignant transformation orextended propagation in culture. This is the first demonstrationthat LR and IFN-'y selectively inhibit transcription of HPVtransforming genes and suggests molecular mechanisms bywhich these cytokines participate in regression of premalignantcells. NK lymphocytes, an important defense against viral diseases, are present in most HPV-associated lesions and in manycases of cervical intraepithelial neoplasia. Current investigations, however, indicate that HPV-16-positive cervical cancercells and HPV-l6 immortalized human cervical epithelial cellswhich possess properties similar to those of cells from cervicaldysplasia are resistant to lysis by NK lymphocytes. HPV-positive immortalized and carcinoma cells are sensitive to LAKlymphocyte lysis. Their sensitivity can be further enhanced bytreatment with LR. Combination treatment with LR and achemotherapeutic drug, e.g., cisplatin, further enhances the sensitivity of HPV-16-infected cells to LAK lymphocyte lysis. Incontrast, combined treatment with IFN—ycan result in decreased sensitivity of HPV-infected cells to destruction by LAKlymphocytes.

Lynn M. Matrisian (Vanderbilt University, Nashville, TN)discussed the regulation of the MMP gene stromelysin bygrowth factors and oncogenes. EGF stimulation of rat stromelysin gene expression in cultured rat fibroblasts occurs at thetranscriptional level through a pathway that requires the induction of the protooncogenes c-los and c-jun and the activation ofprotein kinase C. The AP-i site located at position —71in therat stromelysin promoter is required but is not sufficient formaximal activation of stromelysin gene expression. Repressionof the rat stromelysin gene by TGF-@ is mediated through adistinct element, the TGF-fl-inhibitory element, at position—709in the rat stromelysin promoter. Interestingly, TGF-@repression of stromelysin gene expression also requires the induction of the c-los protooncogene, and Fos is a component ofthe TGF-fi-mduced protein complex that binds to the TGF-$inhibitory element sequence. The apparent paradox of Fos involvement in both positive and negative regulation of stromelysin gene expression suggests that very subtle differences in thegrowth factor regulation of protooncogenes can dramaticallyalter the subsequent effects of these protooncogene transcription factors on the regulation of cellular genes.

Growth factor and oncogene regulation of MMP gene expression is believed to impact on normal processes and contribute to pathological conditions. For example, EGF treatment ofdeveloping mouse lungs in an organ culture system causes adramatic inhibition in the degree of branching and results inbroad, dilated end buds after 3 days in culture. These effects canbe completely reversed by simultaneous treatment with the tissue inhibitor of metalloproteinases, a specific inhibitor of matrix metalloproteinase activity. These results suggest that EGFcan effect normal branching morphogenesis through a mechanism that involves the regulation of matrix-degrading metalloproteinases. In addition, the expression of several members of

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the MMP family has been implicated in tumor invasion andmetastasis. Recent studies in collaboration with Rudy Pozzatti(American Red Cross, Rockville, MD) suggest that the regulation of stromelysin gene expression by oncogenes influencessubsequent tumor metastasis in a rat fibroblast model system.Transfection of rat fibroblasts with activated Ha-ras results in adramatic increase in the ability of these cells to metastasizefollowing injection into nude mice. Coexpression of the adenovirus Ela gene product, however, completely represses themetastatic ability of these cells. The metastatic activity of thecells correlates with the expression of enzymatic activity capable of degrading type IV basement membrane collagen. Anexamination of the expression of several type IV collagen-degrading metalloproteinases revealed a striking correlation between metastatic activity and the expression of stromelysin 1and the closely related stromelysin 2 at the protein and mRNAlevels. The Ela gene product repressed Ha-ras-induced stromelysin expression at the transcriptional level.

Judah Folkman (Children's Hospital and Harvard MedicalSchool, Boston, MA) discussed the biology of angiogenesis.While there is now substantial evidence that tumor growth andmetastasis are angiogenesis dependent, a central question intumor biology is “Howand when do tumors switch to theangiogenic phenotype during tumorigenesis?― Subsidiary questions are: “Howdoes one detect an angiogenic phenotype? Areall tumor cellsin avascularizedtumor, angiogenic?―Datawerepresented to suggest that in a highly vascularized tumor, only aportion of tumor cells are angiogenic. In the early stages oftumorigenesis before a tumor is vascularized, almost no tumorcells are angiogenic. While it is difficult to see the early prevascular stage in transplantable tumors, the prevascular and thevascular stage can be detected in transgenic mice where tumorsarise de novo and then switch to the vascularized phase. Inhuman cancer, a distinct separable prevascular and vascularstage can also be seen, especially in breast cancer, where in agiven specimen it is possible to detect microscopically vascularized and nonvascularized carcinomas in situ. Studies in collaboration with Noel Weidner showed that the intensity of neovascularization in breast biopsies was highly correlated withmetastatic outcome. Furthermore, recent unpublished studiescarried out in collaboration with Weidner and Giampietro Gasparini show that neovascularization may be a more accuratepredictor of tumor recurrence, metastasis, or death in breastcancer than any other conventional marker.

Of the seven angiogenic factors which have been sequencedand cloned to date, basic FGF is among the most potent and isubiquitous in a variety of human tumors. It has recently beenmeasured in the blood and urine of cancer patients and appearsto correlate with metastatic burden. A newly discovered property of a-interferon is its antiangiogenic activity. While a-interferon is a relatively weak angiogenesis inhibitor, it has been veryeffective in the treatment of life-threatening hemangiomas inbabies and infants. These results indicate that the field of angiogenesis research studied for the past 20 years in the laboratory is now beginning to yield both diagnostic and therapeuticapplications which are being transferred to the clinic.

It was apparent from the studies presented at the workshopthat while cytokine biology is becoming even more complex,with the identification of additional effect on molecules, moreactivities, and complex interactive regulatory networks, enormous progress is being made in understanding the role andmechanisms of a chain of this diverse and potent class of molecules. Several are nearing clinical use and a number of othersare in trials for a broad range of diseases, alone and in concertwith other therapeutic agents. There is no doubt that as thisresearch progresses, many of the cytokines will become significant components of a new armamentarium against cancer anda variety of other diseases.

Philip FurmanskiDepartment of BiologyNew York UniversityNew York, New York 10003

Eric G. NeilsonRenal Electrolyte DivisionSchool of MedicineUniversity of PennsylvaniaPhiladelphia, Pennsylvania

Jaswant S. Bhorjee

Martin@Division ofResearch GrantsNIHBethesda, Maryland 20892

3 To whom requests for reprints should be addressed.

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1992;52:6129-6133. Cancer Res   Philip Furmanski, Eric G. Neilson and Jaswant S. Bhorjee  Research Grants, National Institutes of HealthSection Workshop: Working Report from the Division ofthe Pathogenesis of Disease: A Pathology A/Pathology B Study The Role of Cytokines in the Regulation of Cell Function and in

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