Cytokines — soluble factors in immune responses

Cytokines

-

soluble

factors

in immune

responses

F. Balkwill Imperial

Cancer

Research

Current Opinion

Fund Laboratories,

in immunology

Introduction In the past a great deal of researchin immunology was based on the premise that antigens were solely responsible for intracellular events leading to proliferation and differentiation, and that cell contact, antigen receptor ideotypic network, suppressor T cells and antigen specific helper and suppressor factors were the regulatorsof the immune response.Our understandingof the role of antigen non-specitic soluble factors (variously called lymphokines, interleukins, cytokines,interferons, etc.) was as murky as the tissue culture supematants which contained them. Painstakingpurification aided by technological advances began to clarify the lymphokine ‘soup’, and the field was transformed as the genes were cloned. Purified recombinant products now becameavailablein milligram quantities as opposed to picogram quantities of the natural proteins.

Table 1 Other interleukin

names used for interleukin

IL-1 Endogenous Leucocyte

1 (IL-11 and

6 BL-6). IL-6 pyrogen

26 kD protein

endogenous

Hybridoma

growth

factor

mediator Mononuclear

cell factor

B-cell differentiation

factor,

BSF-2 B-ceil activating

factor

Hepatocyte

stimulating

factor Lymphocyte-activating

T-activating

factor

factor Catabolin Osteoclast

Interferon activating

Muscle proteolysis

gz

factor

factor

UK

1988, 1:241-249

tory molecules. Moreover, the system may well be numerically simpler than feared as we discover that many of the diverse factors delined by a particular activity are identical in their molecular structure (for example see Table 1). The cloning of the cytokine genes, and their widespread availability, has predictably led to an explosion of scientific information. In the past year, probably well over 1000 papers have been published on the (mainly in u&-o) properties of the interferons (IFNs), interleukins (IIs), colony stimulating factors and tumour necrosis factors (T’NFs).Unfortunately, instead of further clarifying the waters, the collective results of these papers have stirred up more mud from the bottom of the pool of immunological knowledge. We have moved from associating any one cytokine with just one major function to the realization that cytokines have, at least in Z&V, a great number of overlappingand equally important properties. Moreover, it is difiicult to attribute unequivocably any particular activity to any particular cytokine, as addition of any one of these molecules to an experimental system may stimulate the production of a ‘cascade’of other cytoklnes (Fig. 1) which can all change and modulate an observed biological effect (Balkwill. In Cytoklnes in Cancer Therapy, 1988, in press). It is an almost impossible task to select just a few papers from such an overwhelming barrageof information on the cytokines. What I have attempted to do in this article is to review the trends which have emerged and to select a few papers that may give us hints of a more rational understandingof the cytokine network, an understandlngwhich is so important to future progress in immunology and which has many practical applications. Of all the areasof immunology, cytokines have the most potential for therapy of human disease,and these recombinant proteins (or their antagonists) seem set to make a contribution to the physician’sarmoury in the next few decades.

The pleiotropic The murky cytokine waters have cleared sufIiciently for us to realize that intercellular communication in the immune system is largely driven by these protein regula-

London,

nature of cytokine activity

Cytokines comprise a broad class of protein cell regulators variously termed monokines, lymphokines, and In-

Abbreviations cDf4A-complementar-y Il-interleukin;

DNA; GVHD-graft versus host disease; IW-intetferon; IR-immunoglobulin; W-lymphokine activated killer; AUK-major histocompatibility complex; mRNA-messenger RNA; MS-multiple sclerosis; PRMC-peripheral blood mononuclear cells;

Pwlatelet

activating

factor; TGF-transforming

@ Current

growth factor; TNF-tumour

Science Ud ISSN 0952-7915

necrosis factor.

241

242

Immune

response

[c/

Activated

\

:F

Plasma cell

Fig. 1 Cytokines interact in a complex network to control immune defence cells when they respond to antigen. TNF, tumour factor; IL, interleukin; Ig, immunoglobulin. 0 , action of cytokines; I$ , cell division and differentiation.

tedeukins, which control the amplitude and duration of immune responses:(1) They are all of a low (less than 8OkD) molecular weight and am often glycosylated;(2) they generally act at short range in a paracrine or autocrlne (rather than endocrine) manner, and (3) they interact iirst with high aflinity cell surface receptors (distinct for each type or even subtype) and then regulate the transcription of a number of cellular genes by little understood second signals.This altered transcription (which can be an enhancementor an inhibition) results in changesin cell behaviour. There are over 20 proteins that could be considered as cytokines (Table 2). It is now obvious that the multiple, overlappingand sometimescontradictory functions of cytokines are dependent on their local concentration, the cell type they are acting on, and the other regulatoryinilu-

necrosis

ences to which the cell is exposed. This year has seen an absolute deluge of papers attributing a particular property to a particular cytokine and there is now an almost bewildering overlap of properties. A demonstration of this is seen in Table 3. Some light was shed upon this confusion by Spom and Roberts [ 11 in a well argued review. They discussthe peptides that are known as growth factors (and of course many of the cytokines come into this category as well). They ask what sensecan be made of the multiplicity of actions of so many of these regulatory proteins and suggestthat cytokines-growth factors form part of a complex cellular signalling languagewith the individual peptides equivalent to charactersin an alphabet or code. Thus the information which an individual cytokine conveysdepends on the pattern of regulatorsto which a cell is exposed, and not on one single cytokine.

Cytokines -

Table 2 Human

cytokines

Zroup name

-

the current

list

Abbrev-

Alternative

Subtypes

iation

names

n = 22

IFN

nterferons

IFNa IFNB IFNy

fumour

necrosis

TNF

TNFa

tymphotoxin

LT

TNFB

interleukin-1

IL-1

lnterleukin-2

IL-2

lnterleukin-3

IL-3

Multi-CSF

lnterleukin-4

IL-4

BSF-1

nterleukin-5

IL-5

lnterleukin-6

IL-6

factor IL-la IL-l fl

IFNlh 26 kD protein

Probably 2 forms

BSF-2

of different

HSF

molecular

HCF

weight

TAF Colony stimulating

Multi-CSF

CSF

factors

CM-CSF M-CSF C-CSF

Transforming

growth

TCF-p

TCF-pl

factor-p

TGF-p2 TGF+3 TGF-01.2

Data taken from Balkwill, In Cytokines

in Cancer Therapy,

1988 fin press)

If one considers the number of words in the English languagewhich can be derived from an alphabet of 26 letters, or the number of proteins which can be made from a code of 21 amino acids, the response of a cell to the cytokine-growth factor ‘alphabet’could be expected to be extremely varied. In the next year or so, it is probable that further properties of the proteins known as growth factors will be uncovered that overlap with the known properties of cytokines and within 5 yearswe will see all these molecules as one broad class of agents interacting to regulate not only the immune system and inilammation, but also tissue repair, differentiation, and embryonic development. Papers that compare the mechanism of action of individual cytokines will help in our understanding of the complexity of the cytokine network One such paper was published by Beresini et al [2]. They studied cytokineinduced polypeptides in whole cell lysatesof human Iibroblasts by computer-basedanalysisof two-dimensional gels.

soluble factors in immune

response Balkwill

Interferons a and y (IFNa, IFNy) Induced 11 peptides in common; there was a further polypeptide induced solely by IFNa and 16 polypeptide specific for IFNy. Ratherpredictably (and mercifully!), TNF and IL-la (which have almost total identity in their biologic activity) induced sevenpolypeptides in common, although two additional polypeptideswere seen at higher concentrationsof TNF. Some of these polypeptides were shared by more than two cytokines: two were induced by TNF, IL-a , II-B and IFNa, and two were induced by TNF, IFNa and IFNy. These prelimimuy data thus con6rn-1the suspicion that, whatever the cytokine, the receptor to which it binds, or the signal it sends to the nucleus, the overlapping biologic activity is due, at least in part, to the induction or inhibition of a common set of cytokine-regulated genes and proteins. These cytokine-regulatedgenes may be controlled by common response sequencesin the DNA (Friedman and Stark, Nurure 1985,314:637439). At the moment, it therefore looks as if severalcytokines could do the same job, but there are some hints that there may be some more subtle differences between their actions, and moreover, that distinct subsets of immune cells may secretedifferent cytokines. For instance, Umetsu et al [3] examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretionand functional activity.In murine systems,other workers had found a dichotomy in the types of lymphokine secreted. One subset of T cell clones were shown to synthesizeL-2, IL-3 and IFNy while another subset synthesizedIL-4 and B-5. The latter subset (designated Th2) enhancedIgGr and IgE synthesis(Mosmann et aC,J Immunoll986, 136:2348-2351)(41. The situation does not appear to be so simple in humans, however. Out of three clones analyxd, one secreted IL-4, but not IL-2 or IFNy, while the other two secretedall three cytokines.The multiple lymphokine-secreting clones most likely represented the progeny of a single T cell becausethey were derived by lim iting dilution assays.The authors also describe another recently isolated T cell clone which secretesIL-2 and barely detectable amounts of B-4. It is encouraging that murine and human T lymphocyte clones do appear to secrete different spectra of cytokines. Maybe in time the pattern of cytokine secretion by T cell clones will provide a useful functional classi6cationof T cell subsets. Many different cytokines can be Involved in regulatingB cell proliferation and differentiation (Table 2). As IFNy and IL-4 are often’secreted by distinct T cell clones, a paper by Snapper and Paul [5] may throw some light on these functional dilferences. They found that IFNy and IL-4 (also known as BSF-1) reciprocally and independently regulatedimmunoglobulin isotype production. hxerferony stimulated the production of immunoglobulin (Ig) G2a by B cells but inhibited the production of IgG3, IgG2b and IgE. Interleukin-4, in contrast, promoted switching of B cells to produce IgGl and IgE and markedly inhibited the production of IgM, IgG3, IgG2a and IgG2b.

243

244

Immune

response

Table 3 Different

biological activities on human cells of various cytokines.

Cytotoxic for tumour cells Cytostatic for various cells Uitogenic for various cells Activate B cells Stimulate B-cell proliferation Stimulate B-cell differentiation Induce IgE receptor on B-cells Activate T cells Stimulate T cell proliferation Stimulate T cell differentiation Induce Class I MHC induce Class II MHC Activate macrophages Stimulate granulocyte activity Stimulate eosinophil activity Stimulate NK cell activity Stimulate LAK activity Stimulate osteoclastic bone resorption ’ Induce cellular cellular antiviral state Stimulate production of extracellular matrix proteins Induce chemotactic migration of cells

IFNa

IFNg

IFNy

TNF

LT

IL-1

J J

J J

J

J J J

J J J

J J

J J

J J J J J

J ?

no

J

IL-2

IL-4

J J J J

J J J

J J

J J J J

J

J J

J J

?

J

J

J

J

J

J

J

J J J J J

J J J

J

J J J

J J J

? J

?

J no no

J

J

J

J

J

J

J J t

IgE, immunoglobulin E; MHC, major histocompatibility complex; NK, natural killer; LAK, lymphokine activated killer; IFN, interferon; TNF, tumour necrosis factor; IL-interleukin; CSF colony stimulating factor; CM-CSF, CSF for granulocytes and macrophages; M-CSF, CSF for macrophages; C-CSF, CSF for granulocytes; TGF, transforming growth factor. Data from Balkwill Un Cytokines in Cancer Therapy 1988, in press).

lim iting and inhibitory cytokine network

members of the

The majority of the cytokine activities described in the litefature so far have been uprejylatoty (with the exception of cell growth inhibition and cytotoxicity), and there is a need to understand moleculesthat could potentially Iim it the immune response.One important contender for such a downregulatory protein has risen to prominence intheIastyearfromrheratherunIikeIybegmn@sasa growth factor that would reversibIystimulate the growth of fibrobIasts in soft agar [6]. It soon became apparent that this protein (or more correctly group of proteins), transforming growth factor (TGF)-p, had more potent and widespread activity as a growth inhibitor and stimuIator of extraceIIuiarmatrix formation. In the past two years a role for TGF-p in the reguIat.ionof immune responses has become evident. KeM et aL (I E @ Med 1!%6,163:1037-1049)found that not onIy did a subtype of TGF-p (TGF-fll) inhibit IL-2dependent T ceII proliferation without being cytotoxic, but after m itogenic stimulation, the level of TGF-gl receptors and TGF-j31messen-

ger RNA (mRNA) increased.However, TGF-pl was not releasedinto the culture medium for a further 2-4 days, a time at which the proliferative rate of the cells was slowing down. Thus TGF-pl could be invoked in autocrine growth control of activatedT Iymphocytes. A later paper by SmeIand et UC [7] has reported that TGF-j31inhibits DNA synthesisin normal and neoplastic B Iymphocytes stimulated to proliferate by anti-Ig or B ceII growth factors. InterestingIy,TGF-j31had little or no effect on severalearIy/intemrediateparametersof cell activation, e.g. inIlux of calcium or increasedc-myc expression, but did inhibit the induction of transferrin-receptor expression which occurs in the Iate Gl phase of the cell cycle. ke et al [8] showed that members of the TGF-j3family were potent inhibitors of acquisition of immunogIobuiin light chains during pre B cell maturation, and prevented the emergenceof surface x+ m itogen responsive B lymphocytes in v&o. Ongoing Ig RNA synthesis was not, however, sensitiveto TGF-pl or TGF-j32 and neither was classII major histocompatibiky complex (Ml-K) RNA Thus members of the TGF-g famiIy may se-

Cvtokines -

Table 3 (cont.) IL-5

MULTI CSF

IL-6

CM-CSF

M-CSF

CCSF

TGF-p

(IL-3)

J

J J J J

J

J

J

J J

J

J J

J

J

Abbreviations

-

J

J

reswmse Balkwill

interesting that the cytokine TNFa, if added along with TGF-p,was able to partiaUyreversethe latter’s inhibitory effect on irz vitm IAK-cell generation.The mechanismof this reversalis not understood, but could be due, for instance,to TGF-p receptor down modulation. The effect of TGF-j3described above could be primadly due to its antiproliferative action. One paper, however, has suggesteda way that TGF-g may modulate the immune response by inhibiting transcription of the classII MHC message[ 101. In contrast to the other cytokines, there is an additional level of control of the TGF-j3action, possibly reflectingits potency and importance. Many studies have shown that the majority of TGF-j3secretedis biologically inactk and fails to bind to cell surfacereceptors [6 1.This latent form can be activated in oirro by transient acidification or alkali, but in viva it is probably activated by exogenous proteases.Moreover, as with T cell activation, the TGF$ messagecan be present at high levels in some cells, but not secreteduntil a further stageof activationor differentiation is reached.Alveolar macrophagesexpress TGF-j3 mENA,but only releasethe protein upon activation [ll] . In fact, freshly isolated monocytes secrete an acid-labile binding protein that inhibits the action of TGF-j3.

J

J J

soluble factors in immune

J

see facing page

Thus, TGF-p secreted at an inkunmatory or immune site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-p is produced by various cancers,particularly glioblastomas,and therefore may contribute to immunosuppressionseen in some mal@ancies [12]. There may be other cytokines that exert an inhibitory/limiting influence on immune reactions.Two other obvious candidates would be IFNa and f!, but although

there have been

reports in the past on the ability of these two qtokines to exogenouslyinhibit some in vitro immune reactions, more information is now required on their endogenous production during such reactions. lectivelyinfluence gene expression at particular stagesof B lineagedifferentiation. More information on the interactions of TGF-j3with immune effector cells, and its reciprocal interaction with another cytokine was provided by Espevik et al [9]. They reported the TGF-j31inhibited IL-2 induced human lymphocyte activatedkiller (UK) cell activity in vitro against three different human and murine targets, but only if it was added early on during IL.-2stimulation of the effector cells. The TGF-j31had m inimal inhibitory activity if added directly into the w(-cell assay.Another subtype of TGF&, TGF-&I, had identical activity.The TGF-p did not only inhibit the developmentof cytolytic activityin a subsequent IAK assay,but also inhibited the production of three cytokines, IFNa, TNFa and TNFj3 (also called lymphotoxin) by the IL-~ stimulated lymphocytes,and the proliferative response of the peripheral blood mononuclear cells to B-2. As the data on the inhibition of the proliferative responsewere not given, it is difiicult to judge whether TGF-g acts in this system solely by inhibiting the initial proliferative response of the lymphocytes,or whether it has more complex effects. It was, however,

Localization of cytokine production

and action

One important facet of the cytokine systemis an understanding of the ways these potent molecules are generally contained in a localhed manner at sites of immunity and infknmation. In the past year two papers have made some contribution to this understanding.In an elegant series of experiments, Kriegler et al [131 proved that TNF could occur as a transmembraneprotein. When monocyteswere stimulated to produce this cytokine and subjected to Western analysiswith a polyclonal antibody tohumanTNF,a26kDproteinwasdetectedaswellas the commonly found 17 kD form of recombinant TNF that associatesin solution to form a biologically active trimer. The 26 kD protein is the size of the protein predicted by the TNF complementaryDNA (cDNA) if the putative signal sequenceis not removed,and further pulse chase experiments showed that the 26 kD molecule was a pre-

245

cursor of the 17 kD species. The primary sequence of the 26 kD TNF tevealed a hydrophobic domain of appmimady 20 amino acids danked by two hydrophilic domains characteristic of an integral transmembrane protein. Western blotting experiments of cell lysates confirmed that the majority of the 26 kD TNF protein was in the membrane fraction, and further studies showed that the amino terminus of the molecule was external to the Cell.

This membrane form of TNF was cytotoxic when inserted into a microsome. Kriegler et UC [13] suggested that this active tmnsmembrane molecule will act only locally because cell-cell contact ls required. In coniirmation of this are the observations of Decker et al [14] that pamformaldehyde treated activatedmacrophages are cytotoxic to TNF sensitive cells, but TNF cannot be detected in the supernatant fluids of activated macrophage cultures. All this fits in with the observations of the past twoyearsthatTNFlsdirectlyinvolvedinthekillingprocess of mactophages (Urban et a!, Proc Nutl Ad Sci USI 1986,83:52335237; Kombluth and Edgington, J Immund 1986,137:1585-2591) [ 141.There is a strong correlation between susceptibility of cell lines to TNF killing and monocyte-mediated cytotoxlcity, but more impor‘tantly, killing of tumour cells by activated macrophages can be inhibited by polyclonal and monoclonal antibodies to TNF. However, there is little evidence for extmcellular dease of TNF during such in vitn assays.A membrane-bound molecule would explain these contradicto~ data. It will be interesting to see, ln the next few years, how many other cytokines have membrane-bound forms and whether this correlates with their major biological activities. For instance, biologically active IL-1 has been detected on the surface of iibroblasts and monocytes [16,17]. . Even when cytokines are released they have to be localized, for instance, during antigen presentation. High local concentrations of cytokines could most efficiently be achieved, and their action directed against the appropriate cell, if they were actually synthesizedwhere the T cell ls in close contact with the antigen-presenting cell. A recent paper [ 181 has shown that cells of a cloned T helper llne exhibit directional exocytosis of the cytokine IL-4. An elegant culture systemwas devised in which T cells were forced into pores of 3 um nucleopore filters housed in sterile Swinnex chambers. When the membrane became hydrostatically sealed by the cells, antibodies to the T cell receptor which acted as an inducing stimulus for IL-4, were added to one side of the chamber. When the cytotoxin inducing stimulus was suboptimal, a strong polarity was observed ln IL-4 release to the side where the T-cell receptor antiwas present. Directional exocytosls occurs in non-polarized cells such as mast cells and T lymphocytes when they releasecytotoxlc storage granules, but the hding that the secretion of at least one cytoklnecanbepolarlzedisoneattract&waytoexplain the selectivity of cytokine action espe&lly at low antigen concentrations. When the inducer was increased equal cytokme production occurred on both sides of the membrane.

The potential of cytokines and their antagonists in disease therapy We are becoming increasingly aware that an imbalance in the cytokine-growth factor network contributes to many human diseases, and that restitution of that imbalance with cytokines or their specific antagonists offers a plausible therapeutic option. The cytokine system is a very potent force for good when activation of the network is local and transient, but when cytokine production is sustained and/or systemic there is no doubt that cytokines contribute to the signs, symptoms, and pathology of infectious, autoimmune, and malignant disease. The past year has seen some excellent papers describing examples of this, particularly in relation to the cytokine TNF which has a very destructive side to its character. There is now a strong case for the involvement of TNF in the pathogenesis of endotoxic shock. In rats, administration of suflicient puriiied recombinant TNF can reproduce the signs of shock and death follows rapidly (Tracey et al, Science 1986, 234:471-474). Moreover, antibodies to TNF will protect animals against endotoxic shock. If baboons were passivelyimmunized with murine monoclonal antibodies to human TNF, they could survive an LDloo dose of live Escheriichiacoli [19]. A third interesting link between TNF production and fatal outcome of infection was reported by Waage et al [20]. Using a highly sensitive biological assay for TNF, they found that 10 of 11 patients who died of meningococcal meningitis had detectable serum levels of TNF on admission to hospital, compared with only eight out of 68 who survived. Recently Camussi et al [21] have presented evidence that it is not TNF itself that is a major mediator of endotoxic shock, but platelet activating factor (PAF) which is induced by this cytokine. A potent and speciilc polyclonal antibody to murine TNF; which neutralized the production of TNF in vitro, proved a very useful tool for analysiingthe contribution of TNF to the pathogenesis of graft versus host disease(GVHD) and cerebral malaria [ 22,231.Mice with GVHD were injected weekly with 2 mg of rabbit antimouse recombinant TNF. This therapy largely prevented the acute manifestations of GVHD, skin epidemral cell necrosis, foci of lichenoid hyperplastic reactions, loss of hypodermic fat, gut dilation and crypt cell necrosis. The same antibody was given to mice who had been infected with Pkwnodium berg& 4-7 days previously.Just one injection of neutmlbing antibody to TNF completely protected mice from the cerebral complications of this parasite without modifyiq the ensuing parasitaemia These experiments reported over the last year suggest that TNF may be involved in the manifestations of some acute diseases. As IL-la and j3 and lymphotoxin (LT) have overlapping, if not identical, properties that these cytokine.5are also likely to be involved ln the manifestations of acute disease. Tumour necrosis factor and IL-1 appear to regulate the acute phase response to injury and infection, at least in

Cytokines -

m ice. Mortensen et al 1241showed that IL-la and g and TNF would induce the production of the mouse acute phasereactantsserum amyloid P-component,C3 and fibrinogen. One to 1Oug of IL-1 was sufilcient to induce a maximum in vivo response, but TNF was less effective. A combination of TNF and IL-l was additive or synergistic for the Induction of serum amyloid P-component and fibrinogen. The in vitm acute phase responseof hepatocytes to these cytokines was, Interestingly,reversed,with TNF being approximately 104 times more active than ILla or p. This could suggest that in vivo TNF is more active in a paracrine and IL-1 in an endocrine manner. The involvement of cytokines such as TNF in the pathogenesis of chronic disease is more controversial. Beutler et al OEcp Med 1985, 161:984-985)purl&d a protein they termed cachectin, from rabbits with wasting induced by Infection with Trypanasoma brucei. As this protein interfered with lipid metabolism they thought it m ight be responsible for the weight loss seen in these animals. They soon found that cachectln was identical to TNP (Beutler et al, 1985, Nature 316:552-554) and suggestedthat TNF production may contribute to the cachexiaseen in chronic infectious or malignantdisease. It has, however, proved difIicult to reproduce the syndrome of cachexia by administration of exogenous recombinant TNF. With repeated injections a tolerance occurs. However, by increasingtwice dally doses of recombinant TNF to rats, cachexia can be sustained,with the rats showing depletion of whole-body protein and lipid stores (251. A convincing experiment showing that endogenously produced TNF could cause cachexia came from Oliff et al [26l who transfected the cross-speciesreactive human TNF gene into CHO cells and isolated a cell line that constitutively secreted TNP. Nude m ice injected intramuscularlywith these cells developeda severecachetic syndrome even before the CHO cell grew Into tumours of appreciable size. Abnormal regulation of cytoldnes may be involved in the pathogenesisof autoimmune disease, and Bottazzo et al (Lmuet 1983, Ii:111~1119) advanced a theory for the generation of autolmmuneendocrine disorders.They suggestedthat viral infection or other factors would lead to activation of T cells and production of cytoldnessuch as IFNy. This would lead to inappropriate expression of ClassII MHC molecules on, for instance,thyroid epithellal cells, the presentation of self antigens and destruction of thyroid cells. Sarvetnick et al [27] have recently reported the production of transgenicmouse strains harbowing Class II MHC genes or IFNy genes which were linked to the human lnsulln promoter. The transgenes were only expressedin the pancreas.M ice containing either class II MHC or IFNy transgenesdeveloped insulindependent diabetes mellitus, but the histological picture differed. M ice expressing IFNy suffered an imlammatory destruction of the islets of Iangerhans,whereasthose expressing both chains of the MHC gene showed few inflammatory cells but cytotoxlclty of the B cells and progresslveatrophy of the islets. It is important that neither

soluble factors in immune

responseBalkwill 247

of these transgenescreated an exact pathological phenocopy of human insulin dependent diabetes,but they each showed some similar features,and their ability to obtain localizd production of a cytoldne transgeneopens avenues for exploring endogenouscytolcineproduction in health and disease. Some cytoklnes may lx Involved In the evolution of a malignancywhen they act as autocrine growth factors. Moreover, cytokines m ight also be found at a tumour site as part of a host response to the tumour [27]. A most compelling example of this was presented by Kawano et al [ 281, and concerned the role of the cytokine IL6 in . the development of human multiple myeloma. Interleuldn-6 is a potent m itogen for mouse plasmocytomas and hybridomas, and Kawano et al [28] found that freshly isolated human multiple myeloma cells produced IL-6 and exhibited IL-6 receptors. Moreover, antibodies to IL-~ inhibited the in vitro growth of myeloma cells, providing direct evidencefor an autocrine loop. As discussedpreviously, the TGF-ps, are potent growth inhibitors for many cell types. One step ln the process of malignant transformation may involve the inability to respond to negative growth regulatory signals. In this respect, Klmchi et al [29] have recently reported that, whereas fetal retinal cells were highly sensitive to the antimitogenic effects of TGF-PI, all retinoblastoma tumour cell lines were resistant to this factor and moreover, f&cl to express TGF-p receptor proteins. On the other side of the coin, there is now no doubt that cytolcinescan sometimes be useful in diseasetherapy. In the past year there have been severalpapers of interest in this respect.Talpaz et uL [30] reported a study of 51 patients with chronic myelogenousleukaemlawho received IPNa therapy; complete haematologicalremission was achievedln 71% of patients with a further 9% showing a partial response.Continuous but slow normalization of severalother blood and bone-marrow parameters, including platelet counts, B12 levels, marrow cellularity and marrow maturation index was seen during therapy. The most interesting linding, however, was that ln 20 of the patients who achieveda complete haematological remission, there was suppression of the Phlladelphla chromosome in the bone marrow. This was sometimes not evident for many months after therapy began. No other therapeutic option ls able to produce this &ect ln a diseasewhere progression to blastic crlsls and death almost invariably ensueswithin 3-4 years of diagnosis.If IPNa therapy can delay or inhibit the onset of blastic crlsis this would be a considerable advancein the therapy of this form of leukaemla. Interferon a is also effective against other haematologic malignanciessuch as hairy cell leukaemlaand lymphoma and some chronic virus d&as&. Adults with chronic hepatitis-Brespond to this cytoldne (Thomas and Scully, Br Med Bull 1985,41:374-386),but more recently those with non-A, non-B hepatitis showed a striking impmvement in serum aminotmnsferasesafter the start of therapy [31]. Regulardoses of Interferon were needed to maintain normal liver function tests.

Many of the clinical trails with cytokines reported so far have reported no clinical benefit, especially in advanced disease,but the deleterious effects of these agents have been transient. However, one small study using FNy in patients with multiple sclerosishas highlighted the potential hazards of manipulating the cytokine network by systemic administration of recombinant cytokine. The exacerbation rate of multiple sclerosis, compared retrospectively with the pretreatment and post-treatment rate, was significantly greater than expected [32]. Cytoklris are, therefore, regulatory proteins that have an impact that extends far beyond the field of immunology and are central to our understanding of cell biology, both in health and disease.

LEE G, EIUNGSWORTHLR, GOUS S, WAU. R, K~NCADEPW: j3 transforming factors are potential regulators of B lym phopoiesis. J w Med 1987, 166:129&-1299. TGF-gs are potent inhibitors of x iight chain expression on cloned B cells and their maturation to mitogen responsiveness.This is the first natural substance found to be a negative regulator of B iymphopoiesis. ESPY T, FIGARIIS, RANGESGE, PAUM)IM MA JR: Transform9. 00 ing growth factor-& (TGF-PI) and recombinant human tumor necrosis factor-a reciprocally regulate the generation of lymphokine-activated killer ceU activity: comparison between natural porcine platelet-derived TGF-Ut and TGF-gz and recombinant human TGF-8,. J Immunol 1988, 140:23132316. TGF-8 inhibits IL-2 dependent L4K cell proliferation and development of cytotoxicity. This effect is rewsed by TNF. This shows reciprocal reguiation of IAK ceU activity by 2 cytokines. TGF-8 could be a negative regulator of IAK celi activity. 10. Cwwacm 07, Cm HH, WONGGHW, MCCABESM, Pwuio 00 MA Transforming growth factor-81 modulates the expression of ciass B histocompatibiity antigens on human cells. J Immunol 1988, 140:4217-5223. Annotated references and recommended TGF-gl partiaiiy inhibits the induction of IFNy of ciass II MHC mRNA and protein, another means by which TGF-g could downreguiate imreading mune responses. 11. ASSOIANRK. FLEURDELYS BE, SlFVENSONHC, MIUER PJ, MADTES 0 Of interest DK, m EW, Ross R, SPORNMB: Expression and secretion 0 00 Of ouwanding interest of type U transforming growth factor by activated human macrophages. Proc Nat1 Acud Sci USA 1987, 84:602&6024. 1. SPORNMB, RoBEal’s AB: Peptide growth lhctors are muhiExpression of TGF-f? mRNA is unrelated to macrophage actiwtion, but .a IimcdonaL N&we 1988, 332:217-219. production of the protein oniy occurs on activation. TGF-U production A good discusion of why protein ceil rquiators may have overlapping may enable macrophages to participate in tissue remcdeiiing and repair. function is the iirst attempt to address this complex issue. 12. W~ANNM, BODMERS, DE M.urn~ R, SSPL C, HOFER-WAI~BLNEK 2. BeREsINlMH, LEMpm MJ, Ersn~t~ LB: Overlapping polypepR, Ftw K, HOFER E, FONT- A T ceU suppressor fatl a. tide induction in human ftbroblasts in response to treattor from human glioblastoma ceils is a 12.5kd protein ment with interferon-q interferon-y, interleultin-a, interdoseIy related to transforming growth factor-g. Embo J leddn lb, and tumor necrosis factor. J fmmunoi 1988, 1987, 6:1633-1636. 140485493. A T cell suppressor factor produced by a giiobiastoma ceU line is a 2-D @ anatysh of changes in protein synthesis in cytokine treated celis member of the TGF-g family. If giioblastoma ceiis produced this factor shows that cytokines with overlapping biologic functions induce a simin vitq it may contribute to the immunodeficiency seen in this disease. ilar range of cellular proteins, and presumabiy regulate the same genes. KIUEG~ER M, PERU C, DEFAYK, ALBERTI, Lu SD: A novel form 13. UMElXJ DT, JIBARAHH, DEKRuws RH, ABBhs AK, AnRAMsJS, 3. of THF/cachectin is a ceU surface cytotoxic transmembrane a* 0 Gnu. RS: Punctional heterogeneity among human inducer protein ramifications for the complex physiology of TNF. T cell clones. / Immunol 1988, 140:4211-4216. cdl 1988, 5345-53. Different human helper/inducer T ceii clones secrete different cytokines A good series of experiments proved the existence of membrane or cytokine combinations. Tbe secretion of different T ceil clones may bound molecules of TNF. The ceU membrane form of TNF may be be.dated to their functional differences. an important mediator of directed inilammatoty responses. 4. KnuRL,M~cDo~G,W~srJ,WooosA,Bcn-rohtL~K 14. DECKERT, Lo~hwwMA?-r~ss ML, GIFFORDGE: Cell-associated l cloned, la-restricted T cells that do not produce intertumor necrosis factor (THF) as a killing mechanisms of actil leukin d(IL-4) B ceU stimulatory factor @SF-l) fail to help vated cytotoxic macrophages. J Immunoll987,138:957-%2. antigen-specific B cells. J lmmunoll987, 138~1674-1679. TNF dependent lysii of tumor ceiis could be performed by pamformalCloned mouse l3T4+ T celis have 3 functional subtypes that can be dehyde iixed activated macrophages. CeU membrane TNF is biologicaUy further chamctetiz.ed by their cytokine production. IL.4 is a necessaty active. but not sufiicient signai for diiferentiation into antibody forming ceils. 15. finuhw~ R, DaSntrANo DH, Tsu~l~o~o M, V~L~EKJ: Tumor SNAPPER CM, PAULWE: Interleron-y and B ceU stimulatory 5. necrosis factor is an important mediator of tumor cell *a *a factor-1 reciprocal& regulam Ig isotype production. Science killing by human monocytes. J lmmund 1987, 138:63%40. 1987,23@44+47. Correlation is shown between in vitro sensitivity of ceiis to TNF iysis In the mouse, lFN-y stimulates B celi production of IgGh and IL-4 (BSFand monocyte kiiling. Also inhibition of monocyte idiling by a mono1) stimulate I&X and IgE. Different cytokines regulate dilTerent Ig isocionai antibody to TNF. The latter is involved in some forms of monotypes. cyte killing, at least in vfha 6. SPORNMB, ROBERTSAB, WAKEFIEU)IN, DE C~~~MBRUGGHE 16. Iz J, WE~NS?EWD, GUB~ER U, Vncuc J: Induction of 0 B: Some recent advances in the chemistry and biolmembrane-associated interleuldn-1 by tumor necrosis fatl ogy of transforming growth factor&m. / Ceu Bid 1987, tor in human libroblas& J Immunol 1987, 1%2137-2142. 105:183~1045. TNF induces membrane associatedIL-la and fl in fibrobiasts. IL.-la and AnuptodatereviewofTGF-g. j3 also has a membrane bound form. 0, BROWNDC, IAci-th&w LB: Subcellular locaiiaa7. Shuutm EB, B~MHOPF HK, HOLE H, Ruuo E, Buse K, 17. BA~~OUCHE l tion of human monocyte interleukin 1: evidence for an FUNDERUD S, GoDAL T, &llSSON R Transtbrming growth facl inactive precursor molecule and a possible mechanism for tor type WGP @) inhibits G1 to S transition, but not IL 1 release. J Immund 1987, 138~4249-4255. activadon of human B lymphocytes. IT@ CeN Res 1987, 171:213-222. IL-1 activity is shown in membrane and iysosome fractions of monocyte TGF-f3 inhibits B celi DNA synthesis by an effect on iate Gt phase. It is iysates but not in endopiasmic reticulum. IL-1 is not a conventionaUy a gtowtb inhibitor of immune ceils. secreted protein. 8. l

Cytokines Poe WJ, CONRADI, JANEWAYCA JR (letter to the Editor) Receptor-directed focusing of lymphokine release by helper T cells. Nature 1988, 332:378-380. Some elegant experiments showed that when the stimulus is suboptional, cytokines are releasedfrom the cell membrane in a directional manner. This mechanismwould allow IocaExedcytokine releaseifgenerally applicable. 18.

l e

TRACEYKJ, FONG Y, HENCEDG, MANOGUEKR, IRE AT, KUO GC, lowRy SF. CERAM~ A (Letter to the Editor) Anticachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia Nature 1987, 338662664. Baboons passivelyimmunixed againstendogenous TNF with a neutralizing monoclonal antibody are protected from a subsequentLDt~ dose of live EC& TNF is a mediator of fatal bacteraemic shock 19. 00

20.

A EsptrvtxT: Association between tuW M G E A HN~IIXNSEN

mour necrosis iactor in serum and fatal outcome in patients with meningococcal disease. Lancet1987, i:355-357. Serum TNF was detected in 10/l 1 patients who died of meningococcal meningitis but only S/68 survivors. This is further evidence that TNF may be invohed in the pathogenesisof shock l

21.

CAMUSSIG, Bussott~o F, SAL~~DIOG, BAGLIONIC: TUmor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neuuophiks, and vascuku endothelial cells to synthesize and release platelet-activating factor. / E @ Med 1987, 166:139&1404. TNF stimulates transient PAF production by macrophages,endothelial cells, and polymorphs. PAF may be involved in the toxicity and intlammatory effects of TNF. me

PIGURTP-F, C&AU GE, ALUXTB, VASSAUP: Tumor necrosis factor/cachetin is an effector of skin and gut lesions of the acute phase of the graft-vs.-host disease. J ELI Med 1987, 166:1280-1289. Passiveimmunization of mice with antibodies to TNF prevented the development of the acute gut and skin lesions of GVHD and decreased mortality. This shows that activation of grafted lymphocytes leads to local releaseof TNF which is involved in the pathology of this disease. 22.

a.

GRAU GE, FAJARDOLF, PIQLET P-F, ALET B, IMIBERT PH, VASSW P: Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 1987, 237:121&1212. Passiveimmunization of mice with antibodies to TNF prevents the Deb velopment of cerebral malaria, showing that TNF plays a role in the pathogenesisof cerebral malaria. 23. l *

MOR’~RNSEN RF, SHAPIRO J, LENBF, DOUCHES S, NETAR: Interaction of recombinant IL-1 and recombinant tumor necrosis factor in the induction of mouse acute phase proteins. J Immunol 1988, 140:226&2266. IL-la and g are more active than TNF in inducing acute phase proteins in viuo in mice but their combination may be additive or synergistic. The combined effect of TNF and B-la and g accounts for a substantial portion of acute phase protein synthesis. 24. l e

25.

TRACEYKJ, WEI H, MANO~XEE, FONGY, HES.SE DG, NGWEN

l

HT, Kuo GC, BEUIIJXRB, COTRANIIS, CERAM~ A, LOWRYSF: Cachectidtumor necrosis factor induced cachexia, anemia, and ingammation. J .?Zr@ Med 1988, 167:1211-1227.

soluble factors in immune

response Balkwill

New data on the role of TNF in the development of cachexia reports that animals Injected chronicaUy with TN-F develop weight loss aasociated with depletion of whole-body protein and lipid stores. This is some evidence for the involvement of TNF in cachexia. 26. Otw A, DEFEO-JONES D, BOYRRM, MMTINEZ D, KtRFRRD, l e VUCXOLOG, WOLFEA SCItXRRSH: Tumors secreting human TNF/cachectin induce cachexia in mice. Call 1987, 50:555-563.

CHO J cells transfectedwith the TNF gene and spontaneously secreting this cytokine cause severe cachexia and weight loss when growing intramuscukuly in nude mice. Thus endogenously produced TNF can induce a syndrome resembling cachexia 27. S~XTNICK N, Ltocx’rr D, Prrn SL, HANSENSE, S’rxwARTTA: l e Insulin-dependent diabetes meflints induced in transgenic mice by ectopic expression of class II MHC and interferongamma Cell 1988, 52~773-782. Transgenic mice harbouring MHC Class 11or lFNa genes linked to the human insulin promoter develop insulin dependent diabetes mellitus, lDDM.This work shows site-specilicactivation of a cytokine transgene and some insiit into the pathogenesisof IDDM. 28. KAWANOM, HIRANOT, MAWJDA T, TA~A T, HORRY, ~WATO em K, As~oxu H, TANG B, TANABE0, TANAEIE0, TANARAH, KURAMATO A Ktsttworo T: Autocrine gesrat.ion and requirement of BSF/IL-6 for human multiple myelomas. N& ture 1988, 332:8385. Fresh human myeloma cells are stimulated to grow by B-6 and to express B-6 receptors. Their spontaneous in &ro proliferation is inhibited by antibodies to E-6, which may be an autocrine growth factor for multiple myeloma. 29.

KIMCHI4 W A N G XF, WEINBERGm

CHEIFE’IZS, I~ASAGIJE J: Ab-

sence of TGF-g receptors and growth inhibitory responses in retinoblastoma cells. Science 1988, 240:1+199. 8etinoblastoma cell lines are not growth inhibited by TGF-gt and lack all 3 species of TGF-g receptor. Loss of TGF-g receptors may reptesent one mechanism through which cells escape negative control and become malignant. 30. T.UPAZM, KANTARRAN HM, MCCRED~R KB, Kwrtu~ MJ, TRt8nt.o invesdgation of human alpha inl e J, Gunx~wu J: Chid terferon in chronic myelogenous leukemia B&d 1987, 69:lS1288. Detailed analysis of the responses of 51 chronic myelogenous leukaemic patients to long term IFNa therapy. 71% achieve complete haematologic remission. A trend towards bone marrow normalltywas seen in 56% of patients examined. No other thempy suppressesthe Philadelphia chromosome in the bone marrow. ‘l-HOhlPSON BJ, DORANM, IJXVRR AMt, WRRstRRADB tipha31. interferon therapy for non-& non-B hepatitis transmitted by l gammaglobulin replacement therapy. Lancet 1987, E539-541. 3 of 3 patients with non-A, non-B hepatitis responded to IFNa therapy. Poor outcome of this diseasecould be improved with IFNa therapy. PAN~CHHS, H~L~CHru, HARRYAS, JOHNSONKP: Exacetba32. tions of multiple sclerosis in patients treated with gamma l htcrferon. Luncet 1987, i893-894. 7 of 18 patients receiving lFN7 therapy for muhiple sclerosis (MS) had exacerbations during therapy, a statisticaUysignificant worsening of their condition. fFN7 is unsuitable for MS therapy and may cause l e

exacerbations.

249