Cytokines and Disease

18 Pages·2000·0.215 MB·English

by  Feldmann M., Brennan F.M.

Cytokines and Disease
Marc Feldmann* and Fionula M. Brennan
Cytokine and Cellular Immunology Division, Kennedy Institute of Rheumatology, Imperial College
School of Medicine, 1 Aspenlea Road, Hammersmith, London, W6 8LH, UK
*corresponding author tel:[phone], fax:[phone],
e-mail: [email protected]
DOI: 10.1006/rwcy.2000.01003.
INTRODUCTION How can we establish whether a
cytokine is relevant to a disease
Whereas the health of an individual, organ or cell
process?
requires the precise maintenance of the function of
the product of the entire (cid:24)105 genes in the human
genome, pathology is potentially simpler. It could be There are multiple strategies for evaluating the roles
due to changes in a very small number of molecules, of cytokines in disease. A key distinction to make
initially possibly one. Subsequently there is usually concerns the role of a particular cytokine in animal
consequential damage to various cells and tissues, so models or in human diseases. The former is con-
that other genes become abnormally expressed as a siderably easier to establish, as an overexpression of
consequence,anditcanbedifficulttoassesswhatthe that cytokine in transgenic mice (e.g. Keffer et al.,
critical early events were. 1991), total abrogation in knockouts (Marino et al.,
With the involvement of cytokines in essentially all 1997), and infusion of large amounts of neutralizing
biological processes, such as in cell growth, differ- antibodies (Thorbecke et al., 1992) are all possible,
entiation, inflammation, immunity, repair, and fibro- admittedly with various degrees of time and effort.
sis (see the chapter on Introduction to the Role of This is not the case in humans, of course. As the
Cytokines in Innate Host Defense and Adaptive closeness of animal models to the corresponding
Immunity), it is apparent that cytokines will be human disease is highly variable, with only some
involved in a multitude of, and probably all patho- aspects of disease pathogenesis of complex human
logical processes. Their role in these processes will be diseases reflected faithfully in any of the animal
diverse,insomeinstancescytokinesbeingpathogenic, models, extrapolation from animal models is poten-
in others protective and probably most of the time, tially perilous.
innocent bystanders. This means that unraveling the So what is ‘important’ in an animal model only
role of cytokines in diseases is not as simple as some provides a working hypothesis for what is happening
might wish. The detection of a cytokine, upregulated in the related human disease. With authentic human
atthesiteofdiseaseordamageismerelythefirststep diseases,otherresearchstrategiesalsoyieldcluescon-
in that difficult path and in itself does not imply cerning the role of cytokines. These include genetic
pathogenic relevance. susceptibility associated with cytokine polymorph-
It is very difficult to generalize about the role of isms (e.g. Wilson et al., 1997), differential expression
cytokines in disease, notonly because of the diversity at sites of disease compared to uninvolved tissue (e.g.
(over 100 known cytokines), but also since diseases Feldmann et al., 1996), increased levels in the blood
are enormously heterogeneous. Nevertheless, this ofcytokines(e.g.Charlesetal.,1999)ortheirsoluble
chapter will attempt to provide an introduction to receptors (Cope et al., 1992). In vitro models of local
thisverycomplex,andrapidlygrowing,medicallyand humandiseases,whichinvolveculturingthetissuefrom
economically important field. the diseased site, can be very useful (Buchan et al.,
36 Marc Feldmann and Fionula M. Brennan
1988a, 1988b) if they continue to produce the same possibilities.OneisthatTNF(cid:11)isnotimportantinthe
spectrum of ‘pathological’ molecules considered or later stages of sepsis as it presents clinically, or that
known to be produced in vivo. These in vitro models the clinical trial design involving hundreds of trial
can be used for therapeutic tests with antibodies or centers makes it very difficult to discern the possible
other reagents to block cytokines that may influence therapeutic effect.
measurable indices of disease activity (Brennan et al., Whereas TNF(cid:11) is pathogenic in RA, there is
1989a). However, the only definitive proof of the evidence that the opposite may be true in systemic
importance of a cytokine in the pathogenesis of a lupus erythematosus (SLE). The clues come from
human disease is in vivo, in a clinical trial in the animal models of disease. (NZB(cid:2)NZW) F1 mice
affected population, using a cytokine inhibitor if the spontaneously develop, in an age- and sex-related
cytokine is considered detrimental or the cytokine fashion,IgMandthenIgGautoantibodiestodouble-
itself if it is considered beneficial. Only a limited stranded DNA (dsDNA), and subsequently other
number of such studies have been performed to date, manifestations of SLE, especially nephritis. In these
butitisinevitablethatmanymorewillbeexecutedin mice, treatment with anti-TNF(cid:11) antibody makes
the near future (Wendling et al., 1993; Paty et al., themworse(JacobandMcDevitt,1988)asdoestreat-
1993;Cooperetal.,1993;Peestetal.,1995;Bresnihan ment with IL-10 (Ishida et al., 1994), which would
et al., 1996; Keystone et al., 1998; Maini et al., 1998; also reduce TNF(cid:11) synthesis. Analysis of these mice
van den Bosch et al., 1998). indicated that they were genetically low producers of
TNF(cid:11).
What about human systemic lupus erythematosus?
Cytokines involved in a disease Theresultsarenotformallyproven.Thereisevidence
may be present at augmented, thatasubsetwithlupusnephritishasageneticallylow
productionofTNF(Jacobetal.,1990).Aspartofthe
normal or low levels
anti-TNF therapy clinical trials in rheumatoid
arthritis, two patients developed both clinical and
There is now definitive evidence that excess TNF(cid:11) is serological manifestations of SLE, one including
involved in the pathogenesis of two chronic inflam- lupus nephritis (Sander and Rau, 1997). Thus it is
matorydiseases,rheumatoidarthritis(RA)andCrohn’s very likely that low TNF(cid:11) production is one of the
disease(Elliottetal.,1993;VanDullemenetal.,1995; pathogenic steps in human SLE.
Presentetal.,1999).Thecriticalevidencecomesfrom
double-blind, randomized, placebo-controlled trials
Cytokine equilibria
and has led to FDA approval of TNF(cid:11) inhibitors in
these diseases (an anti-TNF antibody Remicade,
inCrohn’sandRA,aTNFRfusionproteinEnbrelin AsdiscussedinthechapterontheIntroductiontothe
RA). For real certainty of the role of a cytokine in Role of Cytokines in Innate Host Defense and
disease more than one such trial and more than one AdaptiveImmunity, cells in vivoare neverexposed to
anticytokine agent would be useful and this has been a single cytokine. They are exposed to whole battery,
accomplishedinRA.TheroleofTNF(cid:11)inthesechro- simultaneously and sequentially, and at various con-
nic inflammatory diseases is discussed in more detail centrations. So cells must integrate the multiple sig-
in the chapter on TNF(cid:11). nals they receive, either individually or collectively,
This level of proof is difficult, expensive and time and translate them into biological effects. To help
consuming to acquire, but there is no alternative. understand this, we have formulated over the years a
Negativeclinicaltrialresults,however,aremorediffi- number of helpful oversimplifications, which can be
cult to interpret and do not necessarily invalidate the expressed in diagrams (Figure 1 and Figure 2).
hypothesis. Abundant animal experimentation was It may seem at first glance paradoxical that in
consistent with the importance of TNF(cid:11) in sepsis inflammatory diseases both the pro- and anti-inflam-
syndrome (Beutler et al., 1985; Van Zee et al., 1992). matorycytokinesmaybeupregulated.However,close
However, these studies clearly demonstrated benefit investigationhasrevealedthistobethecase,andone
only if a TNF(cid:11) inhibitor was used at, before, or very ofthemostimportantconceptsisthatofthecytokine
soon after the injection of LPS or bacteria. However, equilibrium, illustrated in Figure 1. This applies, for
humansepsissyndromeoccursrelativelylateafterthe example, to the ratios of proinflammatory to anti-
onset of bacterial infection, and despite thousands of inflammatory cytokine mediators, with the net effect
patients treated with TNF(cid:11) inhibitors, there have depending not on any one cytokine, but on the
been no successes (Wherry et al., 1993; Fisher et al., totality of expression, the equilibrium. Of course in
1996). What does this mean? There are at least two anyonetissuethereisacomplexmosaicofmanysuch
Cytokines and Disease 37
Figure 1 Cytokine disequilibrium in rheumatoid method increasing in popularity (because of its
arthritis. The cytokine profile at sites of inflammation simplicity) is PCR for cytokine mRNA. This is a
such as rheumatoid synovial tissue is characterized by relatively poor tool for understanding the role of
increased production of both pro- and anti-inflamma- cytokines in disease, since cytokines are also
tory molecules. Modified and reproduced with permis-
abundantly expressed in health, and it is the protein,
sion from Feldmann et al. (1996).
not the mRNA which signals to the cytokine
receptors.
TGF b IL-10 TIMP-1
MMP-3 sTNF-R IL-1ra TIMP-2
IL-1
TNF MMP-1 Receptor expression
Anti-inflammatory Becauseoftheiractiononveryhigh-affinityreceptors
cytokines are potent signaling molecules. This means
Proinflammatory
thatthebiologicaleffectofcytokinesalsodependson
thelevelsofthecorrespondingreceptorsexpressedon
Figure 2 Cytokine cascade in their neighboring target cells. While the levels of
rheumatoid arthritis. Proinflam- cytokine receptors are far less variable than those of
matory cytokines in rheumatoid cytokines, and most cytokine receptors are constitu-
arthritic synovium interact in a
tivelyexpressed,levelsofcytokinereceptorsareregu-
‘network’ or ‘cascade’. Modified
latable. The capacity to receive cytokine signals,
and reproduced with permission
especiallyformultichainreceptorcomplexes,depends
from Feldmann et al. (1996).
critically on receptor density, and so evaluating the
role of cytokines in disease also involves an under-
TIMP-1, TIMP-2 standing of receptor expression. The variable expres-
IL-10, IL-1ra, sTNF-R
sion of the IL-2 receptor (cid:11) chain (CD25) is the best
Anti-inflammatory
known example of variable cytokine receptor expres-
sion. Without CD25 the multifunction IL-2 receptor
Immune
TNF IL-1
system complex has a much lower affinity (Smith, 1988).
Proinflammatory
IL-6, IL-8, GM-CSF etc.
Cytokine inhibitors may reflect
MMP-1, MMP-3
cytokine expression
equilibria,indifferentspots;eachcellhastosummate
theeffectsofsignalsonitscellsurface,integratethem Like all powerful entities, cytokines have their inhib-
and then, if appropriate, produce secondary media- itors. Cytokines function physiologically as local
tors, move, commit suicide, etc. mediators and their inhibitors are involved in help-
Of much interest is the subdivision of CD4+ ing to localize their actions. As cytokine synthesis is
T lymphocytes into TH1 and TH2 cells. These cells sporadic, depending on local cell activation, but
are polarized into producing some but not all of the induced rapidly, it is consistent with the function of
repertoire of cytokines that T cells can produce cytokine inhibitors that, unlike cytokines, they are
(Romagnani,1991). TH1 cells, considered proinflam- present constitutively (e.g. Arend and Dayer, 1990),
matory, produce IFN(cid:13) and IL-2; these are not and are also inducible by the same type of stimuli as
produced by TH2 cells, which typically produce IL-4 induce the corresponding cytokine, and perhaps also
and IL-5. TH2 cells are involved in allergic diseases. bytherelevantcytokineitself.Hencethedefinitionof
In the same way that it is the balance between pro- a cytokine activity ‘profile’ needs to also take into
inflammatory/anti-inflammatory mediators that dic- account the presence and quantities of the inhibitors.
tatesthe‘outcome’,ratherthanlevelsofeither,soitis Thiscanbedonefunctionallybyusingbioassaysthat
thebalancebetweenTH1andTH2andtheirproducts ‘automatically’ take endogenous inhibitors into
that dictates the effects. The concept of equilibrium account (rather than the easy and hence popular
emphasizes the need for accurate quantitation of ELISA assays). In some circumstances there are a
cytokines in vitro, in animal models and in human variety of inhibitors, for example for IL-1, soluble
pathology, if greater insight is to be gained. Poorly type I and type II IL-1 receptor, and IL-1 receptor
quantitative and indirect methods for measuring antagonist,andtwosolubleTNFreceptorsforTNF(cid:11)
cytokinelevels are oflimited value.For example, one and LT(cid:11).
38 Marc Feldmann and Fionula M. Brennan
The role of cytokines in a variety level, rather than the abrogation of cytokine syn-
thesis, which occurs with a targeted mutation. In this
of biological, pathological processes
context, total lack of important immune mediators
and diseases such as IL-2 and IL-10 lead to major immune and
inflammatory disorders (Schorle et al., 1991; Kuhn
et al., 1993). An excess of TNF(cid:11) in transgenic mice
While it is clearly not possible in an introductory
also produces a spectrum of pathology, with a
chapter of reasonable size to discuss the role of cyto-
destructiverheumatoidtypeofarthritisandaCrohn’s
kines in disease in any depth, we will attempt to
like inflammatory bowel disease the most common
illustratethediversityofeffectsofcytokinesindisease
manifestations(Kefferetal.,1991).Incontrast,alack
processes. In only a small number of these diseases is
of proinflammatory mediators, e.g. TNF(cid:11), produces
there sufficient information to definitely argue that
a much more subtle phenotype, discernible upon
cytokinesplayakeyroleinthediseaseprocess.Thisis
stressing the animals (Marino et al., 1997).
only true so far for rheumatoid arthritis and Crohn’s
There is a diverse spectrum of immune and inflam-
disease, where the blockade of TNF(cid:11) has reprodu-
matory diseases, with differences in the phenotypes
cibly led to clear clinical benefit. However, these are
displayed. The most salient features are described
unlikelytoremaintheonlyexamplesinthiscategory.
below.
Because of the greater depth of knowledge in these
diseases, we will start the more detailed discussion of
cytokines in disease with these examples.
Rheumatoid arthritis
The role of cytokines in the pathogenesis of
THE ROLE OF CYTOKINES IN rheumatoid arthritis (RA) has been the most exten-
INFLAMMATORY AND IMMUNE sively investigated of the human diseases since the
mid-1980s(reviewedbyFeldmannetal.,1996).RAis
DISEASES
a chronic inflammatory disease that affects the peri-
pheral synovial joints, resulting in inflammation and
Theevidentroleofcytokinesinimmunityandinflam- swelling, and eventually leading to destruction of the
mationhasledtoawidespreadexplorationoftherole underlying connective tissue. The synovial membrane
of cytokines in this group of diseases. Over the years liningthejointcapsuleinRAbecomesinfiltratedwith
the understanding of the cytokine network in these cells from the blood, mainly activated monocytes,
diseases has evolved dramatically. In the late 1980s T and B cells, while the tissue-resident cells, such as
and early 1990s it was widely believed that because a fibroblasts and endothelium, become activated and
number of proinflammatory cytokines with closely increase in number. The eventual destruction of the
overlapping properties (e.g. IL-1, TNF(cid:11), GM-CSF) underlyingcartilageandboneisnowthoughttoresult
were expressed in inflammatory sites, blocking a from the activities of these cells in the synovium and
single cytokine was unlikely to be clinically useful as the cytokine and enzyme products that they release.
the proinflammatory features would still be main- Inparticularthe‘pannus’tissue,whichmigratesfrom
tained by the remaining cytokines. Studies with anti- the synovium into the cartilage, is thought to
TNF(cid:11)antibody in rheumatoid joint cell cultures first represent the ‘moving edge’ of inflammation and, as
demonstratedthattheexpressionofproinflammatory such, is an important source of proinflammatory
cytokines were coordinated (Brennan et al., 1989b), cytokines and other mediators.
with TNF(cid:11) blockade leading to marked diminution
ofIL-1bioactivity.Thesestudiesledtotheconceptof
Cytokine Expression in RA Synovial Tissue
the cytokine cascade, as described in detail in the
section on Rheumatoid arthritis. Using a combination of immunohistological, mRNA
A wide spectrum of cytokines is involved in both studies, and protein assays, studies from our own
the normal immune and inflammatory responses. group and others have documented that many
Hence it is anticipated that in diseases of these sys- cytokines are produced in RA synovium. These
tems, alterationsin thelevels ofcytokinesmayoccur, investigations have indicated the abundance of
and could be important in the abnormal outcomes. proinflammatory cytokines (Fontana et al., 1982;
There are clues from cytokine ‘knockouts’ that Buchan et al., 1988a, 1988b; Di Giovine et al., 1988;
lowered levels of certain mediators may be important Eastgate et al., 1988; Hirano et al., 1988; Hopkins
incertaindiseases.However,inthehumandiseases,it et al., 1988; Hopkins and Meager, 1988; Houssiau
is much more likely that there is a reduced cytokine et al., 1988; Saxne et al., 1988; Brennan et al., 1989b;
Cytokines and Disease 39
Chu et al., 1991; Deleuran et al., 1992; Wood et al., Table 1 Cytokine expression in rheumatoid arthritis
1992), hematopoietic growth factors (Firestein et al.,
1988; Xu et al., 1989; Alvaro-Garcia et al., 1989; Cytokine Expression
Haworth et al., 1991), chemokines (Brennan et al.,
mRNA Protein
1990a; Koch et al., 1991, 1992, 1993, 1994a, 1994b;
Akahoshi et al., 1993; Hosaka et al., 1994), and Proinflammatory
fibrotic growth factors. These are summarized in
IL-1(cid:11), IL-1(cid:12) + +
Table 1 and reviewed by Feldmann et al. (1996). In
TNF + +
the main, activated macrophages, fibroblasts, and
endothelium produce the most abundant cytokines. LT + (cid:6)
In contrast there is a paucity of T cell-derived IL-6 + +
cytokines (Firestein et al., 1988; Saxne et al., 1988;
GM-CSF + +
Buchanetal.,1988a; Brennanet al., 1989a;Xu et al.,
M-CSF + +
1989), leading to an ongoing debate regarding the
importance of T cells in RA, especially in its late LIF + +
stages (Firestein and Zvaifler, 1990; Panayi et al., Oncostatin M + +
1992). Recently, however, using more sensitive
IL-2 + +
techniques, the presence of T cell-derived cytokines,
IL-3 (cid:255) (cid:255)
IFN(cid:13), IL-2, and lymphotoxin, which are character-
istic of TH1 cells, has been described in RA tissue IL-7 (cid:6) (cid:6)
(Ulfgren et al., 1995a, 1995b; Steiner et al., 1999). IL-9 (cid:6) (cid:6)
IL-12 + +
Cytokine Networks in RA Synovial Tissue IL-15 + +
While the studies described above document the IFN(cid:11),(cid:12) + +
spectrum of cytokines produced in an inflammatory IFN(cid:13) +
tissue such as that in RA, these observations do not
IL-17 + +
indicate which are of major importance with respect
IL-18 + +
todiseasepathology,nordotheyaddressthecomplex
interactions between the cytokines. These issues have
Immunoregulatory
been addressed using an ex vivo system in which dis-
IL-4 (cid:6) (cid:255)
sociated rheumatoid synovial tissue cells are cultured
for short periods without exogenous stimulation, and IL-10 + +
the spontaneous production of cytokines detected IL-11 + +
in the presence or absence of cytokine antagonists
IL-13 + +
(reviewed by Feldmann et al., 1996). Prolonged cyto-
TGF(cid:12) + +
kineexpressionwasdetected,atboththemRNAlevel
whichwasnotduetoincreasedhalf-life(Buchanetal.,
Chemokines
1988b) and the protein level, mimicking what was
IL-8 + +
presumed to occur in vivo. As it had been reported
that TNF(cid:11) was a strong inducer of IL-1 production GRO(cid:11) + +
(Nawroth et al., 1986) we investigated the effect of MIP-1 + +
neutralizingTNFonIL-1synthesis.Itwasfoundthat
MCP-1 + +
blockade of TNF(cid:11) but not TNF(cid:12) (LT) in these RA
ENA-78 + +
synovialculturessignificantlyinhibitedIL-1bioactiv-
ity (Brennan, 1989). As both IL-1 and TNF induce RANTES + +
the resorption of cartilage and bone (Gowen et al.,
Mitogens
1983; Saklatvala et al., 1985; Dayer et al., 1985;
FGF + +
Thomas et al., 1987) this suggested that TNF
blockade could have potent effects upon the disease PDGF + +
process (Figure 2). VEGF + +
Since there are many other possible inducers of
IL-1, such as GM-CSF, IFN(cid:13), and immune com-
plexes, these results demonstrated the predominance
of TNF, and led us to investigate whether other
40 Marc Feldmann and Fionula M. Brennan
cytokines produced in the RA synovial cultures may unclear whether a genesuch as IL-15, which contains
alsobeTNF?dependent.ItwasfoundthatGM-CSF, multiple start codons, is translated efficiently into
IL-6, and IL-8 (Haworth et al., 1991; Butler et al., protein (Tagaya et al., 1996) in rheumatoid tissue.
1995) were also downregulated by anti-TNF in these We have focused upon the hypothesis that TNF
ex vivo cultures. In order to evaluate the role of IL-1 synthesis in RA tissue is T cell dependent, based
in the synovial cytokine network, IL-1 receptor upon the observation that removal of T cells from
antagonistwasused,anditwasfoundthatIL-6,IL-8 the monocytic/macrophage population markedly
were downregulated, but not TNF(cid:11) (Butler et al., reducesthecapacityofthelattertomakeTNF(cid:11).Sur-
1995). These results clearly pointed to the possibility prisingly, the few monocytic cells left in the T cell-
that blocking TNF(cid:11) may be useful therapeutically enriched fraction, together with the T cells, release
and it also highlighted the central or ‘pivotal’ role of more TNF(cid:11) than T cell-depleted synovial macro-
TNF(cid:11) in this inflammatory disease (Brennan et al., phages (Brennan, unpublished; Figure 3). Direct cell
1992). contactbetweenmonocytesandTcellsprovidesfora
Mostofthestudiesdescribedabovehavebeenper- potent stimulus for TNF(cid:11) and IL-1 production (Isler
formed upon tissue from late-stage disease. Thus it is et al., 1993; Sebbag et al., 1997). The nature of the
unclear whether or what cytokines are involved in signalsfromTcellstomacrophagesisnotwellunder-
promoting the earlier events, such as the TH1/pro- stood but has been studied. Using normal T cells/cell
inflammatory immune response, which may involve lines, several groups have investigated and proposed
IL-12 or IL-18, the most potent inducers of IFN(cid:13). some possibilities, e.g. CD69 and LFA-1 (Isler et al.,
The role of IL-12 has been investigated in murine 1993;Manieetal., 1993),VLA4(Laffonet al.,1991),
collagen-inducedarthritisinDBA/1mice.Thus,IL-12 (Alderson et al., 1993; Wagner et al., 1994; Kiener
coadministered with collagen type II boosts arthritis et al., 1995; Shu et al., 1995) as well as unidentified
in these animals, and disease is ameliorated if IL-12 proteins in the range 25–35kDa (Isler et al., 1993).
action is blocked with anti-IL-12 antibody (Malfait We have observed additionally, that the manner in
et al., 1998), which is particularly effective in con- which the T cell is stimulated influences which cyto-
junctionwithanti-TNF(cid:11)(Butleretal.,1999).Littleis kine are produced by the monocyte. Thus T cells
yet known about IL-18 expression in rheumatoid activated with anti-CD3 antibodies mimicking an
arthritis, but it is expected to be present at the early antigensignalinduceabundantTNF(cid:11)andIL-10syn-
stages (Yamamura et al., 1997). thesis in monocytes when co-cultured (Parry et al.,
1997). In contrast, T cells activated for 8 days with a
cocktail of cytokines (IL-2, TNF, and IL-6) when
Regulation of TNF(cid:11) in Rheumatoid Arthritis
fixed and co-cultured with monocytes induce TNF(cid:11)
TNF(cid:11)productionisahighlyregulatedprocesswhich but not IL-10 synthesis (Sebbag et al., 1997)
includes involvement ofboth the 50 promoter and the (Figure 4). This raises the interesting possibility that
30 untranslated region of the gene (Beutler and chronic inflammation can be maintained as a con-
Cerami, 1989). The 30 UTR contains an AU-rich sequence of proinflammatory cytokine production
motif, which regulates translation of the mRNA and
tissue expression. Indeed replacement of this AU Figure3 TNFproductioninRAsynovialjoint
region in the human TNF gene expressed in trans- cell cultures is T cell dependent. Rheumatoid
genic mice results in the spontaneous development of synovial cells were cultured either as a total
arthritis (Keffer et al., 1991). In rheumatoid joints, population or without CD3+T cells (removed
the TNF(cid:11) is produced predominantly from CD68+ bymagneticbeadenrichment).TNFproduction
macrophage-like cells. The factor(s) that induce TNF was measured by ELISA.
synthesis in RA synovial joints have obviously been
of much interest. Blockade of one likely candidate, 750
IL-1, with the IL-1 receptor antagonist had no effect
on TNF production (Butler et al., 1995). Others have
suggested that the IL-2-like cytokine IL-15, which is ml) 500
g/
found in RA synovium, may be of importance (
aF
(McInnes et al., 1997). Thus, IL-15 was observed to N
T 250
activateT cells, whichtheninducedTNFsynthesis in
monocytes in a cognate-dependent manner, and
in vivo, arthritis disease in rodents was prevented 0
Total T cell Total T cell
with a soluble IL-15 receptor protein (McInnes et al., depleted depleted
1996; Ruchatz et al., 1998). However, it remains Day 2 Day 5
Cytokines and Disease 41
Figure 4 T cell activation modulates the cytokine Figure 5 Blockade of IL-11 activity enhances
profile produced by monocytes following T cell TNF(cid:11) production: synergy with IL-10. Rheuma-
monocyte cognate interaction. toid arthritis synovial membrane cells (pooled
data from five experiments) were cultured in tri-
plicate, treated for 48 hours with neutralizing
TNFa + IL-10
Macrophage anti-IL-11mAb(10(cid:22)g/mL),neutralizinganti-IL-
10 mAb (10(cid:22)g/mL) or both antibodies together,
andsupernatantstestedforTNF(cid:11)byELISAand
TNFa illustratedas%levelofTNF(cid:11)control.TNF(cid:11)pro-
ductionwassignificantlyincreasedincellcultures
treated with anti-IL-11 mAb (2-fold), anti-IL-10
mAb (3-fold) or both antibodies (22-fold) com-
Cytokine stimulus Antigen stimulus pared with basal production, anti-IL-11 mAb-,
‘bystander activation’ ‘specific activation’
and anti-IL-10 mAb-treated synovial membrane
cell cultures, respectively. Modified and repro-
ducedwithpermissionfromHermannetal.(1998).
from macrophages, induced by ‘bystander’ T cells,
which themselves are activated by the cytokine prod-
ucts of the macrophage. In the absence of insuffi-
cient immunoregulation by cytokines such as IL-10, a IL-11 + a IL-10 *
chronic inflammation ensues.
a IL-10 (10 m g/ml) **
* p <0.05
Anti-inflammatory Cytokines a IL-11 (10 m g/ml) ** **p <0.01
The evaluation of cytokine expression in rheumatoid
synovium revealed that some cytokines with chiefly Control
anti-inflammatorypropertieswerealsoresentinabun-
dantquantitiesinrheumatoidsynovium.Theseinclude 0 500 1000 1500 2000 2500 3000
TGF(cid:12) and IL-10 (Brennan et al., 1990a; Fava et al., % inhibition of TNFa control
1991;Katsikisetal.,1994).Incontrast,IL-4production
hasnotusuallybeenidentified,anditslackmaybeone that in RA synovial tissue the system is not in
of the factors accounting for the relative TH1 T cell homeostasis.
preponderance in rheumatoid joints. There are con- Recently,weinvestigatedwhetherIL-11,amember
flicting data concerning IL-13 (Ulfgren et al., 1995b; of the IL-6 gene superfamily displayed anti-inflam-
Isomaki et al., 1996) and the expression and role of matory effects similar to those of IL-10. It had been
IFN(cid:11)/(cid:12) is not well defined (Hopkins and Meager, described that IL-11 inhibited LPS-induced TNF in
1988).ThetypeIinterferonshavebothpro-andanti- mouse macrophages (Trepicchio et al., 1996). How-
inflammatory activities and it is not clear which, if ever, IL-11 alone in RA cultures had no effect on
either, predominates in RA. However, the recent evi- TNF synthesis, unless the soluble IL-11 receptor was
dence that IFN(cid:11) induces STAT4 phosphorylation in added together with IL-11 (Hermann et al., 1998).
TH1CD4+Tcells(Roggeetal.,1997)incommonwith Blockade of IL-11 enhanced TNF levels by 2-fold.
IL-12, may suggest that increased IFN(cid:11) in RA may However,ifIL-10andIL-11werebothblocked,TNF
predisposetoTH1ratherthanTH2predominance. levels increased 22-fold (Figure 5). This suggests that
Our own interest has focused on the inhibitory both IL-10 and IL-11 are important endogenous
cytokines, which are present in abundance in RA immunoregulators in inflammatory tissue. IL-11
synovial tissue, and include TGF(cid:12), IL-10, and IL-11. also inhibited the release of MMP-1 and MMP-3
However, while TGF(cid:12) displays several anti-inflam- whilst increasing TIMP levels in these cultures, sug-
matory effects, the addition to RA joint cell cultures gesting that there is a direct action of IL-11 on
had little effect on the spontaneous production of fibroblastic cells.
proinflammatory cytokines (Brennan et al., 1990a),
Targeting TNF(cid:11) in Disease In Vivo
suggesting that the quantities already present were
probably at maximal effective concentration. In con- The definition that TNF(cid:11) was a therapeutic target,
trast,theadditionofIL-10diminishedtheproduction based on the in vitro studies described above, was
of IL-1 and TNF(cid:11) furthermore, blockade of endo- confirmed with anti-TNF(cid:11) antibody in animal
genous IL-10 enhanced TNF levels by 3–4-fold models of arthritis (Williams et al., 1992, 1995;
(Katsikis et al., 1994). This suggests that IL-10 is an Piguetetal.,1992;Thorbeckeetal.,1992),andhelped
important immunoregulator of inflammation, and provide the rationale for anti-TNF(cid:11) antibody
42 Marc Feldmann and Fionula M. Brennan
therapyinpatientswithrheumatoidarthritis(Elliottet Figure 6 Effect of cA2 on circulating IL-1Ra.
al., 1993, 1994). The clinical trials of anti-TNF(cid:11) Rheumatoid arthritis patients were treated with
antibody, using cA2, a high-affinity neutralizing either placebo (circles); 1mg/kg cA2 (triangles);
chimeric antibody produced by Centocor, Inc., or 10mg/kg cA2 (filled squares). *p<0.05,
**p<0.01, ***p<0.001 compared with placebo.
began in May 1992, and were successful in rapidly
Reproduced with permission from Charles et al.
diminishing many aspects of disease activity, includ-
(1999).
ing symptoms (e.g. pain, joint stiffness), signs (joint
swelling and tenderness) as well as biochemical 50
markers of disease (e.g. C-reactive protein, CRP,
and erythrocyte sedimentation rate, ESR). It was ml) 0
g/ Placebo
striking that this happened even in longstanding p
active patients who were resistant to existing therapy y 0; –50
a
d
(Elliott et al., 1993). These results were confirmed in m 1 mg/kg cA2
o –100 *
a randomized double-blind, placebo-controlled trial e fr
owfithcAd2iff(eErellniotttanetti-TalN.,F1(cid:11)99a4n)t,ibaonddiebsy(Roatnhkeringertouapl.s, chang –150 ** *** *** 10 mg/kg cA2
t1h9e94;pS7a5lfieTldNFet arle.,ce1p9t9o8r), faunsdionmorperorteecienntlEynwbirtehl IL-1ra ( –200 *** * *****p* pp < <<0.000..000151
(Moreland et al., 1997). –250
01 3 7 14 28
The cA2 trial demonstrated that in addition to a
rapid fall in the acute phase response, serum levels of Day
IL-6 were reduced within the first 24 hours of treat-
Blockade of Other Cytokines in RA
ment, and reached normal levels in the majority of
patients (Charles et al., 1999). Not all cytokines are In view of the many actions of TNF(cid:11) mediated by
detectable in serum samples, so it is not yet known if IL-1, and the synergy between these two cytokines, it
allthecytokinesshowntobeTNF(cid:11)dependentinthe would be predicted that IL-1 blockade might be just
rheumatoid joint tissue ex vivo are in fact reduced as effective as that of TNF(cid:11). In murine collagen-
invivo.SofaritisclearthatIL-1,IL-6,IL-8,MCP-1, inducedarthritis(CIA)thatpossibilityhasbeentested
and VEGF are diminished by anti-TNF(cid:11) therapy and is the case, provided that therapy is performed
in vivo. Furthermore, the trial demonstrated that when these cytokines are present (Wooley et al.,
endothelium was deactivated following anti-TNF 1993). If therapy is delayed until TNF(cid:11) is no longer
therapy, with a marked reduction in circulating solu- expressed in mouse joints, then it is not surprising
ble E-selectin and ICAM-1, reduced trafficking of that it is not effective (Joosten et al., 1996).
leukocytes to the inflamed synovium, associated with IL-1blockadeinhumanRAhasinvolvedtheuseof
reduced expression of adhesion molecules on the subcutaneous IL-1Ra, which is cleared rapidly, and
blood vessels in synovium. the doses used probably do not block IL-1 receptors
One of the possible limitations of the effectiveness efficiently. This may account for the marginal results
of anti-TNF therapy given as a short course may be reported so far, with little reduction in inflammatory
the observation that cytokine inhibitors are also symptomsandsigns(Campionetal.,1996;Bresnihan
reducedafterthistherapy.Thus,serumIL-1Ralevels et al., 1996). IL-6 blockade in RA has been reported
are diminished by about 50% and enter the normal tobeeffectivefor2–3weeks,usingamurineanti-IL-6
range (Figure 6). The rate of change is rapid, just as antibody (Wendling et al., 1993; Wijdenes et al.,
rapid as changes in acute phase proteins, and it has 1994). Professor T. Kishimoto has reported at meet-
beenreportedrecentlythatIL-1Raisproducedinthe ings that anti-IL-6 receptor antibody is also effective
liver by hepatocytes (Gabay et al., 1997) just like in RA. Both therapies would block IL-6 signaling.
other acute phase proteins. With respect to soluble TNF blockade diminishes the production of natu-
TNF receptors (sTNFR), there is also a rapid dimi- ral cytokine inhibitors such as IL-1Ra and soluble
nution in serum levels, which lasts for many weeks. TNFR(Charlesetal.,1999).Thismaybeanimportant
The effect is statistically significant for p75 sTNFR, part of why TNF(cid:11) blockade alone does not, in the
but not for the less abundant p55 sTNFR (Charles late-stageRApatientsusedforclinicaltrials,leadtoa
et al., 1999). There are no data available yet con- cure, as might have been considered possible on the
cerning other cytokine inhibitors, e.g. soluble IL-1 basis of the equilibrium concept (Figure 1). One con-
receptor. In contrast there is an abstract indicating sequence of the anti-TNF(cid:11) antibody-induced reduc-
that IL-10 levels may be increased (Ohshima et al., tion of natural cytokine inhibitors is that it would
1996). make sense to consider restoring them. The one that
Cytokines and Disease 43
drops the most is IL-1Ra, which is currently in thaninRA(reviewedbyWestacottandSharif,1996).
clinical trials, with marginal efficacy if used alone CytokinesdetectableincludeTNF(cid:11),IL-1,IL-6,IL-8,
(Bresnihan et al., 1998). It may be more useful to use LIF, and oncostatin M (Smith et al., 1997). These
it in combination with anti-TNF(cid:11) antibody. Simi- have been detected in synovial fluid, and in the
larly, IL-10 may be more useful in conjunction with supernatant of osteoarthritis synovial cell cultures.
anti-TNF(cid:11), as a maintenance therapy. Combination Inhibitors of cytokine action such as IL-1Ra, soluble
therapies of multiple types are very effective in colla- TNFR, and TGF(cid:12) are also present in appreciable
gen type II-induced arthritis (reviewed by Williams quantities in synovial fluid (SF) and synovial mem-
et al., 1998). brane (SM) cultures. Anabolic cytokines involved in
The reduction in serum cytokines provides a useful maintaining cartilage function, such as TGF(cid:12), FGF,
laboratory test of whether therapy is effective, and and IGF1, are also found (e.g. Brennan et al., 1990a;
may provide a useful surrogate marker for clinical Martel-Pelletier et al., 1990; Denko et al., 1990;
efficacy. Serum IL-6 changes after anti-TNF(cid:11) anti- Westacott and Sharif, 1996). Which angiogenic
bodytherapyarerapid,andreachnormallevels.This cytokines or chemokines are of major importance in
assay could be a useful tool for ascertaining whether the angiogenesis and cell recruitment that take place
other antirheumatic therapies act by influencing the iscurrentlynotclear,althougharoleformacrophage
cytokinesystem.WhethertheIL-6assayisbetterthan inflammatory protein has been proposed by Koch
the conventional assays of CRP and ESR used by et al. (1995).
rheumatologists remains to be ascertained, especially
as CRP and ESR are chiefly regulated by IL-6
(Andusetal.,1988;Geigeretal.,1988;Morroneetal., Cytokine expression in
1988) – CRP directly, and ESR by IL-6-induced
Crohn’s disease
elevations of fibrinogen.
While there is strong evidence for a genetic asso-
Osteoarthritis (OA)
ciation in Crohn’s disease, the important genes have
not yet been identified. All the classic autoimmune
Osteoarthritis is the commonest joint disease and, diseaseshaveassociationswithcertainHLAantigens.
unlike rheumatoid arthritis, its pathogenesis is poorly For example, rheumatoid arthritis is associated with
understood. It is not clear which type of pathology a HLA-DR4 and DR1, insulin-dependent diabetes
‘degenerative’ disease of this type belongs to, and so mellitus with HLA-DR3 and DR4 and more closely
thereisnoclearreferenceframework,ascomparedto with HLA-DQ8. Systemic lupus erythematosus (SLE)
autoimmunity or cancer. It is considered to be a is associated with HLA B2 DR3, but in fact the
heterogeneous disease, with a variety of predisposing strongest association within the HLA gene cluster is
risk factors, including genetic predisposition, obesity, with the complement component C4. However, in
and trauma (Cooper, 1994; Bullough, 1994). Crohn’sdiseasetherehasbeenmuchcontroversy,and
It is believed that there are a number of stages in reported associations are variable. Sometimes the
the pathogenesis of human OA, which are recapitu- disease is associated with DR1 or D95, but in more
latedintheSTR/ortmousewhichdevelopspathology recent studies it is not associated with HLA at all,
with similar histology (Chambers et al., 1997). First even within afflicted families (Satsangi et al., 1996).
there is a cartilage-specific stage, in which matrix This suggests that the immunological aspects of
turnover is increased and chondrocytes divide, then a Crohn’s will differ from those of other diseases
stagewithhypertrophyofbonewiththeformationof considered autoimmune. There has been a lot of
osteophytesattheedgeofjointsandlastofall,astage interest in the relevance of mycobacteria and other
with marked synovial membrane enlargement, leuko- bacteria to Crohn’s disease, but there is no convin-
cyte infiltration, inflammation, cytokine production cing link.
and joint destruction, often leading to joint replace- At the cytokine level, the abnormalities in Crohn’s
ment surgery. It is only from this last stage that there appear to be closely related to those of RA. The
is considerable information about cytokine expres- strongest evidence for this is that both diseases have
sion, as it is the time that joint samples are obtained responded very well to therapy with an anti-TNF(cid:11)
at surgery. antibody (Remicade) (Van Dullemen et al., 1995;
The cytokine profile in late OA is typical of what Targan et al., 1997; Present et al., 1999). Consistent
would be anticipated from an activated cell popula- improvement is seen in the majority of patients,
tion composed chiefly of macrophages, fibroblasts, lasting 8 or more weeks from a single infusion. Biop-
and endothelial cells, with considerably less T cells sies of affected sites via an endoscope have added
44 Marc Feldmann and Fionula M. Brennan
considerablytoourknowledgeofthepathogenesisof Atherosclerosis
Crohn’s disease. There is a considerable infiltrate in
the lamina propia of T cells and macrophages. There
Currentconceptsin thisdiseasearechanging rapidly.
is evidence that these cells are activated, as judged by
Whileitisstillclearthatabnormalitiesinlipidmetab-
their surface markers and the production of cyto-
olism are of importance, it is now acknowledged that
kines. Cytokines of the proinflammatory TH1 type,
this does not explain all the phenomenology. Several
such as IFN(cid:13) and IL-2, have been detected, whereas
studies, initially that of Russell Ross, have high-
those of the proallergic TH2 type, such as IL-4 and
lighted that atherosclerosis has the features of an
IL-5, are difficult to find. There is evidence that the
inflammatory disease (Ross, 1989, 1999). More
antigen-presenting function is upregulated; for exam-
recently, autoimmunity and infection have been pro-
ple, high levels of HLA class II, extending to
posed (e.g. Wick et al., 1997; Danesh et al., 1997,
epithelium have been observed. This is likely to be a
Caligiuri et al., 1999). The cellular composition is
reflection of bioactive levels of IFN(cid:13) in the tissues.
analogous to that in other inflammatory diseases,
Macrophage proinflammatory cytokines, such as
with macrophages (often lipid laden, hence called
TNF(cid:11), IL-1, IL-6, are also found (Radford-Smith
‘foam cells’) and smooth muscle being the most
and Jewell, 1996; Fiocchi, 1998). Anti-inflammatory
abundant cells, with T lymphocytes and even some
mediators such as IL-10 are present. IL-4 levels are
mastcells present. Earlylesions termed‘fatty streaks’
reduced compared to those in normal gut or asso-
only contain macrophages and T cells (Hansson and
ciated with ulcerative colitis. TGF(cid:12) is present at
Libby, 1996). Adhesion molecules, such as ICAM-1
elevated levels (Beck and Podolsky, 1999).
andVCAM-1arealsoupregulated.Theexpressionof
The signals that drive the presence of these cyto-
cytokines in atherosclerotic plaques resembles that of
kines are not known. In animal models the Ox40
other inflammatory sites. Cytokines expressed in the
pathway has been implicated in therapeutic studies
local lesions, and hence believed to be important in
(Higgins et al., 1999). Chemokines expressed include
thepathogenesis,areTNF(cid:11),MCP-1,IL-1,GM-CSF,
IL-8, GRO, MCP-1, and more work needs to be
IL-8, VEGF, MCP-4, BMP-6, CD40/CD40L, IFN(cid:13),
performed to define the others. The relatively scarce
IL-12, and IL-6 (Libby and Galis, 1995; Libby et al.,
results obtained with human tissue have been corro-
1995; Raines and Ross, 1996). This profile of cyto-
borated in a variety of animal models that resemble
kines is very similar to that seen at other chronic
Crohn’s disease, such as IL-10 knockouts, or the
inflammatorysites,suchasinRA,anditisofinterest
transfer of CD4+CD45RB+T cells.
that there is accelerated onset of atherosclerosis in
RApatients.PDGFisalsoexpressedinatherosclero-
Ulcerative colitis (UC)
tic plaques, and has been implicated in the migration
ofsmoothmusclecellsintotheplaques.Expressionof
The etiology and pathogenesis of ulcerative colitis is, some of the receptors for the proinflammatory cyto-
like that of most diseases, incompletely understood. kines have been noted, such as TNFR, and pre-
Therearegeneticassociations(Fiocchi,1998),includ- sumably the others are also present, as would be
ing TNF(cid:11) 308 allele 2. Antibodies to neutrophils expected on activated cells.
(pANCA) are very prevalent. Also present in atherosclerotic plaques are some
Unlike Crohn’s disease, where the inflammation is cytokines that are believed to be protective. These
transmural, in UC the inflammation is more super- include IL-10, which reduces the production of a
ficial. The cytokine profile has been studied. Chemo- wholegamutofproinflammatorycytokines,including
kines such as MCP-1, IL-8, RANTES, ENA-28, and most of those listed above. TGF(cid:12) has been reported
proinflammatory cytokines such as IL-1, TNF, IL-6, widely to be present, and because it has deactivating
IL-15 have been described (Radford-Smith and effects on endothelium, macrophages, and smooth
Jewell, 1996; Fiocchi, 1998). muscle, and it is abundant in sites of the arterial tree
TheTcellcytokineprofileisvaried,butthereisnot most prone to atherosclerosis, it is probable pro-
theTH1 preponderanceseenin Crohn’sdisease.Thus tective (Borkowski et al., 1995). Confirmatory evi-
lamina propria T cells do not make large amounts dence of the protective role of TGF(cid:12) comes from
ofIFN(cid:13).TH2cytokinessuchasIL-5arepresent,but findings of genomic instability (deletions) in replica-
IL-4isnotelevatedcomparedtolevelsinnormalgut; tion-prone microsatellites in the type II TGF(cid:12) recep-
IL-10ispresent.UnlikeCrohn’sdisease,theresponse tor with consequent loss of function in cells from
to anti-TNF(cid:11) antibody was not dramatic, emphasi- plaques but not in surrounding tissue (McCaffrey
zing that at the cytokine level, UC is different from etal.,1997).Whilethepathogenesisofatherosclerosis
Crohn’s disease. is becoming clearer, the current state of knowledge