molecular targets in immune-mediated diseases: the case of tumour necrosis factor and rheumatoid...

Immunology and Cell Biology (2003) 81, 354–366

Special Feature

Molecular targets in immune-mediated diseases: the case of tumour necrosis factor and rheumatoid arthritis

I A N K C A M P B E L L , 1 L Y N D E N J R O B E R T S 2 a n d I A N P W I C K S 1,2

1Reid Rheumatology Laboratory, Walter & Eliza Hall Institute of Medical Research and 2Department of Rheumatology, Royal Melbourne Hospital, Melbourne, Victoria, Australia

Summary Rheumatoid arthritis is a common autoimmune condition in which, for unknown reasons, synovial jointsbecome the target of a sustained immune response. For many years, rheumatoid arthritis was in the ‘too hard basket’in terms of understanding disease mechanisms and providing rational therapy. This has changed dramatically overthe last 10 years and rheumatoid arthritis is now at the forefront of biotechnology. In this review, we outline one ofthe most exciting recent developments, namely antagonists of the cytokine TNF. The preclinical evaluation of TNFin animal models of rheumatoid arthritis, and subsequent clinical trials of TNF inhibitors in patients, provides insightinto the ‘bench to bedside’ paradigm. We therefore briefly review rheumatoid arthritis, animal models of rheumatoidarthritis, the biology of TNF, the pivotal clinical trials of TNF antagonists and the emerging data on side-effects.Tumour necrosis factor inhibitors in rheumatoid arthritis represent the first attempt to achieve sustained blockade ofa single cytokine in a human disease. Whilst this approach has been even more successful than might have beenpredicted, we suggest it is only the beginning of what has become a new therapeutic era.

Key words: animal models, autoimmunity, cytokine blockade, rheumatoid arthritis, tumour necrosis factor.

Epidemiology and clinical features of rheumatoid arthritis

The prevalence of rheumatoid arthritis (RA) is approximately0.5–1.0% and females are three times more commonlyaffected than males. Patients typically develop symptomsbetween the ages of 20–40, although RA can present at anyage. Rheumatoid arthritis is a systemic disease and symptomssuch as fatigue and weight loss are common, but inflamma-tion of synovial joints is the major clinical manifestation.Rheumatoid arthritis usually comes on insidiously and thenfollows a waxing and waning course over decades. Althoughthere is a spectrum of disease severity in RA, many patientshave persistent synovial inflammation, causing chronic pain,joint damage and progressive disability. Some patients alsohave extra-articular tissue involvement, such as vasculitis,scleritis and rheumatoid nodules. Rheumatoid arthritis isaccompanied by activation of the acute phase response, withelevation of serum C-reactive protein (CRP) and the erythro-cyte sedimentation rate (ESR). Disease activity can be relia-bly assessed by composite scores comprising the ESR orCRP, the number of tender and swollen joints, and globalmeasures of pain, disease activity and disability. Jointdestruction is currently quantified by serial radiologicalassessment of joint space (cartilage) width and erosions ofbone. Mortality is significantly increased in RA and the majorcause appears to be accelerated atherosclerosis.1 Rheumatoidarthritis also has major social consequences: 50% of patients

are unemployable from disability after 10 years of disease andin Australia, $3.2 billion per annum is spent on direct andindirect costs associated with RA.2

Pathogenesis of RA

The genetic susceptibility to RA consistently maps to theHLA-DRB1 region, the frequency of HLA-DRB1 *0401,*0101 and *0404 alleles is increased in more severe RA andthere is a gene dosage effect, all implying an important rolefor antigen presentation to CD4+ T cells.3 The above HLAalleles share a conserved sequence (QKRAA or QRRAA) inthe third hypervariable region (the ‘shared epitope’) of the βchain, but while many candidate foreign and self antigenshave been studied for over 30 years, there has been noconvincing demonstration of a consistent antigenic target forT cells in RA.4 The majority of RA patients have a character-istic autoantibody, rheumatoid factor (RF), which recognizesthe Fc region of IgG. Germline encoded IgM RF are producedby CD5+ B cells early in normal immune responses and mayplay a physiological role in accelerating immune complexformation. In RA, RF undergo isotype switching and affinitymaturation and are thought to be pathological,5 perhaps bydeposition of large immune complexes within joints. Somepatients also have anti-type II collagen antibodies6 and anti-bodies to molecules containing post-translationally modifiedamino acids.7 Local RF immune complexes activate comple-ment and Fc receptors and may thereby contribute to chronicsynovitis.8,9 Recently, serum autoantibodies to the ubiquitousmolecule glucose-6-phosphate isomerase were also found topassively transfer an RA-like synovitis in mice.10 However,not all RA patients have these HLA-DRB1 alleles, RF orother autoantibodies and RA may in fact represent a common

Correspondence: Professor Ian Wicks, Reid Rheumatology Labo-ratory, Walter & Eliza Hall Institute of Medical Research, 1G RoyalParade, Parkville, Vic. 3050, Australia. Email: [email protected]

Received 17 June 2003; accepted 17 June 2003.

Molecular targets in RA 355

phenotype resulting from a number of different immuno-pathogenetic mechanisms. Such a model would fit withclinical experience of the heterogeneity of RA, althoughreliable clinical classification of RA subtypes is still evolving.

Synovial joints are specialized anatomical structureswhich allow for low friction movement between contiguousbone/cartilage surfaces (Fig. 1). Normal synovium is a thinlining of tissue comprising synovial fibroblasts that manu-facture components of lubricating synovial fluid, synovialmacrophages and a fenestrated capillary vascular network. InRA, the synovial compartment becomes the site of a sustainedinflammatory response, with the influx of neutrophils, mono-cytes, dendritic cells, T cells (mainly CD4+, memory pheno-type) and B lymphocytes (Fig. 1). The synovial liningbecomes hypertrophic, with expansion of the synovial fibrob-last and macrophage populations (Fig. 2) and angiogenesis.Lymphocytes can infiltrate the synovial membrane diffuselyor organize into perivascular or lymphoid follicles (Fig. 2).Neutrophils traffick through the synovium, accumulate in vastnumbers in the joint space and eventually die. Presumably,this creates an enormous load on the phagocytic capacity ofsynovial cells. There are profound changes in joint physiol-ogy, with increased synovial fluid volume and pressure,acidosis and hypoxia (Fig. 1). Inflamed, hypertrophic syno-vial tissue can become locally invasive (‘pannus’ tissue),destroying cartilage and eroding bone, through direct invasionand the combined effects of metalloproteinase and prostaglan-din production, as well as local osteoclast activation. Manystudies have documented intense local production of prosta-glandins, cytokines, chemokines, adhesion molecules andgrowth factors in RA synovial tissue, with a predominance ofmacrophage and fibroblast-derived mediators such as TNF,IL-1, IL-6, IL-12, IL-15, IL-18, GM-CSF, MIF andVEGF.8,11–13

This picture is in stark contrast to the selective destructionof particular cell types in organ specific autoimmune diseases

and the relative importance of innate compared to adaptiveimmune mechanisms in RA remains contentious.14 A numberof antigen-independent, self-sustaining mechanisms for RAhave been proposed, including dysregulation of cytokine geneexpression,11 pseudotransformation of synovial cells,8,14

impaired apoptosis,15 angiogenesis and sustained expressionof endothelial adhesion molecules,16 and persistence of acti-vated dendritic cells in the synovial compartment.17,18 Whilstmost investigators accept the concept of paracrine cytokinenetworks in RA, the relative importance of any singlecytokine in this complex scenario is also contentious.19,20

Given the important therapeutic implications, this debate is ofmore than academic interest.

Animal models of RA

The earliest preclinical stages of RA are not accessible toinvestigation and so animal models provide a very usefulmeans of assessing the importance of cytokines and othereffector pathways in contributing to disease pathogenesis.Table 1 lists some commonly used arthritis models and thecellular mediators involved. With the exception of adjuvantarthritis, all of the models can be performed in mice, whichalso allow transgenic and gene-knockout approaches. Of note,each model provides important information about particulareffector cells and no one model entirely mimics the compex-ity of human RA.

Collagen-induced arthritis (CIA) has become the industrystandard by which potential therapeutic targets are evaluated.Collagen-induced arthritis is elicited by the intradermal injec-tion of chick or bovine type II collagen (CII) in the presenceof Freund’s complete adjuvant. The mice develop an immuneresponse to foreign CII, which cross-reacts with self CIIresulting in chronic inflammation and tissue destruction in theperipheral joints. Type II collagen is a necessary componentfor the arthritic response, as mice do not develop disease with

Figure 1 Diagrammatic cross section of a normal synovial joint (a) and a rheumatoid arthritis (RA) joint. (b) ECM, extracellular matrix;HEV, high endothelial venule; JS, joint space; pO2, partial pressure of oxygen.

356 IK Campbell et al.

adjuvant alone. The appealing features of this RA model includeuse of a joint-specific antigen to elicit a chronic systemicautoimmune arthritis, similar histopathological features to RA(inflammatory cell infiltration, pannus formation, bone and

cartilage destruction), dependence on both T and B cellresponses to CII, and clinical as well as histological end-points. A potential limitation of this model is a reportedrestriction to mice of H-2q or H-2r haplotypes at the MHCclass II locus.

Basic biology of TNF and TNF receptors

Tumour necrosis factor is a pleiotropic cytokine which has abroad range of actions in inflammation, infection andimmunity.21 Tumour necrosis factor is produced predomi-nantly by activated macrophages but also by other immune(lymphocytes, natural killer cells, mast cells) as well asstromal (endothelial cells, fibroblasts, microglial cells) cells. Inturn, TNF can activate macrophages, endothelial cells, syno-vial cells, chondrocytes (cartilage cells) and bone cells.Tumour necrosis factor is first produced as a 26-kDa trans-membrane molecule (membrane-bound TNF) which is cleavedby TNF alpha converting enzyme (TACE) to generate asoluble molecule of 17 kDa (sTNF). memTNF is a type IImembrane protein and as such, lacks a cytoplasmic tail forsignal transduction. Both memTNF and sTNF are active ashomo-trimers that can bind to two cell surface receptors,TNF-R1 (p55) and TNF-R2 (p75) (Fig. 3). The receptorTNF-R1 preferentially binds sTNF and has a death domainmediating apoptosis.22 In contrast, TNF-R2 has greater affin-ity for memTNF and while it lacks a death domain, TNF-R2may also initiate cell death by enhancing TNF-R1 signal-ling.23,24 Both TNF receptors can activate the Rel/NF-κB andMAP kinase signal transduction pathways.22 These multipleintracellular signalling options endow TNF with the ability topromote a variety of cellular responses, including the clear-ance of intracellular bacterial infections, cell proliferation,apoptosis and regulation of leucocyte movement throughchemokine and adhesion molecule expression.21 The protein-ase TACE can also cleave TNF-R1 and TNF-R2 to yieldsoluble TNF receptors (sTNF-R) that act as competitivenon-signalling agonists for TNF.

Tumour necrosis factor-knockout (TNF–/–) mice developnormally, but have disturbed lymphoid microarchitecture,with less demarcation between T and B cell regions andreduced follicular dendritic cells.25–27 We found these micehad defective isotype switching from IgM to IgG in responseto immunization with collagen.20 Genetically modified micethat express memTNF but not sTNF (memTNF∆/∆ mice) haverecently been generated through ‘knocking-in’ a TACE-resistant TNF allele.28 Compared to TNF–/– mice, thememTNF∆/∆ mice have partially restored lymphoid micro-architecture and, following immunization, improved germinalcentre formation and follicular dendritic cell networks. Inexperimental autoimmune encephalitis (EAE), disease sever-ity was reduced in the memTNF∆/∆ mice, suggesting the pro-inflammatory functions of TNF are largely mediated by sTNFin EAE. In support of this concept, a recent abstract reportedthat CIA is reduced in memTNF∆/∆ mice and restored bytreating these mice with neutralizing anti-TNF mAb.29 Insummary, these studies show that sTNF and memTNF mayhave quite distinct roles in lymphoid organ structure and moreimportantly, in tissue inflammation. Of clinical interest,TACE inhibitors have been developed and are currentlyundergoing clinical trials in RA.

Figure 2 Cellular features of rheumatoid arthritis (RA) synovium.(a) Immunohistology, stained for the activation/proliferationmarker, proliferation cell nuclear antigen-1 (PCNA-1) of RAsynovial tissue, showing hyperplasia of the synovial lining cells(SLC) adjacent to the joint space (JS). Smaller, lymphoid cellsinfiltrate the sub-synovial cell zone, some of which are alsoPCNA-1 positive. (b) RA synovial cells in culture. Rheumatoidsynovium was enzymatically digested and dissociated cells left inculture for 7 days. A cytospin shows heterogeneous cell popula-tions, with numerous macrophage polykaryons. (c) Phenotypicanalysis of SLC. The cells in (b) were stained with antibodies toCD68 and MHC Class II antibodies and analysed by flow cytom-etry. Two populations are evident, representing synovial macro-phages and fibroblasts. Dotted line represents staining withisotype matched control antibody.


Role of TNF in regulating the immune response

As with other cytokines, TNF and its receptors may have anumber of physiological and pathological roles. Administra-tion of TNF actually prolongs survival in lupus prone NZBmice and suppresses diabetes in the NOD mouse30,31 and inMOG-induced EAE, TNF appears to have an important anti-inflammatory role.32 In a model of infection with heat-killed

Corynebacterium parvum, TNF appears to play a pro-inflammatory role in the initial phase, but an anti-inflammatoryrole in the healing phase.26 A dual role has similarly beenproposed for TNF in myelin basic protein (MBP)-inducedEAE; an initial pro-inflammatory effect mediated throughTNF-R1 in the early disease phase, then a subsequentimmunosuppressive role that inhibits activated/memory T cellexpansion and thereby limits ongoing disease.33 Selective

Table 1 Cellular mediators in animal models of arthritis

Model Major effector cells involvedT cells B cells Mφ Other

Collagen-induced arthritis + + +Antigen-induced arthritis +mBSA/IL-1 acute arthritis + – +Adjuvant arthritis +Proteoglycan-induced arthritis +Streptococcal cell wall arthritis +Zymosan-induced arthritis +CpG-induced arthritis – – +Immune complex arthritis PMNAntibody-induced arthritis – – mast cells, PMNhuTNF transgenic mice synovial fibroblasts, chondrocytesK/Bxil transgenic mice + + mast cells, PMN

+, required; –, not required; blank, unknown. huTNF, human tumour necrosis factor; Mφ, macrophage; mBSA, methylated bovine serumalbumin; PMN, polymorphonuclear leucocytes.

Figure 3 Interaction of soluble TNF (sTNF) and membrane-bound TNF (memTNF) with TNF receptors (TNF-R1 and TNF-R2) anddown-stream signalling pathways. Arrow (in red) shows cleavage sites for TACE.


inhibition of TNF-R1 may thus have advantages over totalTNF blockade. Along with other members of the TNF super-family, such as Fas and TRAIL, TNF may therefore play arole in terminating T cell responses. Mutations in Fas andFasL cause the lpr and gld phenotypes, respectively, in miceand autoimmune lymphadenopathy in humans. Exposure toTNF or FasL induces apoptosis in activated mature T cells,34

although the relative contributions of IL-2 withdrawal, TNFand FasL to death of CD4 and CD8 T cells in differentcircumstances remains controversial.35 Lethal disruption ofMycobacterium avium infected granulomas occurred in TNF-R1–/– mice and was mediated in part by excessive accumula-tion of activated T cells.36

Role of TNF in animal models of RA

The role of TNF has been extensively evaluated in severalarthritis models, including CIA (Table 2). Injection of TNFeither systemically (intraperitoneal in mice)37 or locally (intra-articular in rats),38 prior to disease onset, exacerbated CIA.More importantly, inhibition of TNF with neutralizingmonoclonal37,39,40 or polyclonal antibodies,41 or with TNF-Rconstructs,40,42 ameliorated but did not completely block CIA.

The incomplete inhibition of disease using TNF blockerscould be explained by an insufficient dose, inappropriatedosing schedule, or inaccessibility of TNF to the blockingagents. To overcome these possible shortcomings, we used

TNF–/– mice to evaluate the effect of complete TNF depletionon CIA. This was made possible by use of our modifiedimmunization procedure, which elicits CIA in mice ofC57BL/6 (H-2b) background.43 Surprisingly, these studiesshowed that disease was only slightly reduced in the absenceof TNF while, in individual mice, severe disease coulddevelop that exhibited all aspects of the arthritic response.20

The persistence of CIA in TNF-deficient mice provides auseful working model for further studies into TNF-independentmechanisms of disease progression.

We extended these studies to two other arthritis models: (i)acute IL-1-dependent methylated bovine serum albuminarthritis (mBSA/IL-1)44 and (ii) antigen-induced arthritis(AIA). Tumour necrosis factor–/– mice showed a smallreduction in disease severity in the acute model,20 but anormal AIA response (unpubl. data). With the exception ofIFN-γ, the profile of cytokines produced in TNF–/– synoviumfrom each of these models was similar to that of wild-typecells (Fig. 4).20 This finding implies a non-redundant role forTNF in the regulation of IFN-γ production. It has beenproposed that TNF is at the apex of a cytokine cascadeleading to the production of inflammatory mediators andarthritis.45 However, taken together, our findings question theconcept of TNF as an obligatory cytokine in experimentalmodels of acute and chronic inflammatory arthritis.

Overall, the data in Table 2 are supportive of a role forTNF in several aspects of arthritis pathogenesis. However, it

Table 2 The role of TNF in animal models of arthritis

Model Animal Test Outcome

Collagen-induced arthritisCIA mouse TNF–/– minor ↓20

TNF-R1–/– ↓42 and →92

TNF-R1/IgG1 i.p.; TNFbp i.p. ↓40,42

AdTNF-R1/IgG1 i.v. ↓ then ↑48

anti-TNF mAb i.p. ↓37,39,40

endogenous anti-TNF pAb ↓41

rmTNF i.p. ↑37

CIA rat rhTNF i.a. ↑38

Antibody-induced arthritisCAbIA mouse rmTNF s.c. ↑55

anti-TNF mAb i.p. ↓55

TNF–/– ↓55

K/BxN AbIA mouse TNF–/– ↓56

TNF-R-/- → but delayed93

Other modelsmBSA/IL-1 mouse TNF–/– ↓20

AIA mouse TNF–/– → (unpubl. data)mouse anti-TNF mAb i.v. → PG synthesis94

rabbit anti-TNF mAb i.v. ↓ joint swelling only49

AA rat anti-TNF pAb i.p. ↓95

PEG rhTNF-R1 s.c. ↓96

ICA mouse anti-TNF mAb i.v. →97

ZIA mouse anti-TNF mAb i.v. → PG synthesis93

SCW mouse anti-TNF mAb i.p.; TNFbp i.p. ↓50

CpG mouse TNF–/– ↓54

spontaneous mouse huTNF transgene ↑98,99

K/Bx transgenic mouse anti-TNF-mAb i.p. →93

AA, adjuvant arthritis; AbIA, antibody-induced arthritis; AdTNF-R1/IgG1, adenoviral gene transfer of TNF-R1/IgG1; AIA, antigen-inducedarthritis; CAbIA, collagen AbIA; CIA, collagen-induced arthritis; huTNF, human TNF; i.a., intra-articular; ICA, immune complex arthritis; i.p.,intraperitoneal; i.v., intravenous; mAb, monoclonal antibody; mBSA, methylated bovine serum albumin; pAb, polyclonal antiserum; PEG, poly-ethylene glycol; PG, proteoglycan; rh, recombinant human; rm, recombinant murine; SCW, Streptococcal cell wall arthritis; s.c., subcutaneous;TNFbp, TNF binding protein; TNF-R1, TNF receptor-1; TNF-R1/IgG1, TNF-R1 IgG1 fusion protein; ZIA, zymosan-induced arthritis; ↑ increased;↓ decreased; → unchanged.


is noteworthy that the level of inhibition attained with TNFblockade is often less than what can be achieved throughinhibition of other cytokines/targets in these models. Forexample, we reported more profound effects on CIA whenmice were lacking GM-CSF46 or the NF-κB subunits, c-Rel orp50,47 while antibody blockade of IL-1 in CIA had a moremarked effect than anti-TNF, particularly when given later indisease.40 Tumour necrosis factor may only be driving certainaspects, or stages of disease.40,48 In this regard, several animalstudies have suggested that TNF is more important in mediat-ing the inflammatory, rather than destructive, aspects ofarthritis.49–52 Nevertheless, emerging clinical data tend tosupport anti-TNF therapy as being protective against jointdestruction in RA patients.53

That TNF is involved early on in the innate immuneresponse was shown by the weaker responses observed in theStreptococcal cell wall (SCW) and CpG arthritis modelsfollowing TNF blockade.50,54 Macrophages are major produc-ers of TNF and orchestrate these disease models. Antibody-induced arthritis (AbIA), whether elicited using antibodiesreactive to CII55 or those derived from spontaneously arthriticK/BxN mice,56 also requires TNF for full expression. Thisantibody transfer model is dependent on neutrophils and mastcells, rather than lymphocytes.57,58 Since different cell types

may predominate at particular stages of human disease, thiscould influence whether anti-TNF therapy works for anygiven individual.

Tumour necrosis factor in Mycobacterium tuberculosis infection and the granulomatous response

Our studies with TNF–/– mice also supported the concept thatTNF has an important role in regulating the adaptive immuneresponse. Tumour necrosis factor–/– mice failed to produceanti-CII IgG and developed lymphadenopathy followingimmunization with CII, the latter associated with the accumu-lation of memory CD4+ T cells.20 As stated earlier, theaccumulation of activated/memory CD4+ T cells has alsobeen reported in TNF–/– mice immunized for EAE.33 Wefound that there was no correlation between the incidence ofarthritis and the presence of lymphadenopathy in TNF–/–mice, suggesting that the accumulating memory T cells maybe reactive to non-arthritogenic antigens. Complete Freund’sadjuvant is common to both the CIA and EAE models, withheat-killed Mycobacterium tuberculosis being the major reac-tive component. Tumour necrosis factor is produced bymacrophages in vitro in response to M. tuberculosis-derivedantigens,59 and it is required for the formation and main-tenance of granulomas resulting from infection of mice withM. tuberculosis.60

Assessing the effect of TNF blockade in human RA

A number of TNF inhibitors have now been studied in RA(Table 3). The evaluation of new treatments for RA requirestools that measure disease activity in randomized clinicaltrials. There are currently two complementary measures ofRA disease activity: the American College of Rheumatology(ACR) response and serial X-ray examinations. Assessing theACR response involves counting the number of tender andswollen joints, subjective global assessments of pain, diseaseactivity and disability, and the ESR or CRP. This assessmentis performed at baseline and then repeated serially afterthe therapeutic intervention to calculate ACR 20, 50 or 70%improvements. The ACR50 represents a greater than 50%reduction in the composite criteria mentioned above and isclinically meaningful.61 The ACR50 has been used to comparetreatments in Table 4. A complementary measure of disease

Figure 4 Synovial tissue cytokine expression is not reduced inthe absence of TNF. Total RNA was extracted from synovialtissue from wild-type (WT) or Tnf–/– mice with antigen-inducedarthritis and analysed by RNASe protection analysis. L32 wasused as a loading control. Arrows show increased RNA for IFN-γ.

Table 3 Human TNF inhibitors*

Structure Target Components

Infliximab mAb TNF mouse and human

Adalimumab mAb TNF humanEtanercept p75 TNF-R dimer TNF, lymphotoxin α humanLenercept p55 TNF-R dimer TNF, lymphotoxin α human

*There have been four different TNF inhibitors used in humans.Infliximab is a chimeric IgG1 mAb comprising a human Fc portionand murine Fv. Adalimumab is a fully humanized IgG1 mAb made byphage display from human components. Etanercept is a dimer of thep75 component of the human TNF-R fused to human IgG Fc.Lenercept is a dimer of the p55 component of the human TNF-R fusedto human IgG Fc. mAb, monoclonal antibody; TNF-R, TNF receptor.


progression employs serial X-ray examinations. As RAprogresses, bony erosions and cartilage destruction developand correlate with disability. Using serial X-ray examination,drugs used to treat RA can be assessed by the ability to slowthe rate of joint damage. Drugs that do so are called diseasemodifying antirheumatic drugs (DMARD). To eliminate thepotential for bias, the randomized double-blinded placebocontrolled trial design using intention to treat analysis is used.In these trials, patients with RA are randomly allocated toreceive the active agent, for example an Infliximab infusion,or a placebo infusion and all other treatments are heldconstant during the study period. ‘Double-blinding’ ofphysician and patient reduces subjective human biases, andmeasures the placebo response, which is significant in studiesof RA (Table 4). Intention to treat analysis requires thatpatients who drop out prior to the conclusion of the study areincluded in the final analysis, ensuring that drop-out as wellas response rates to the active agent are included.

An inhibitor of dihydrofolate reductase (purine synthesis),Methotrexate, is currently the most widely used DMARD forRA. Methotrexate slows the rate of progression of bonyerosions on serial X-rays and achieves ACR50 response ratesof around 30%.62 Other DMARD are in general use for RA –as single agents and in combination – including the inhibitorof dihydroorotate dehydrogenase (pyrimidine synthesis)Leflunomide, Sulfasalazine, Hydroxychloroquine, gold injec-tions and Cyclosporine.

Inhibitors of TNF were initially developed as a treatment forseptic shock,63 an acute, life-threatening condition caused bydisseminated bacterial infection. Many of the clinical mani-festations of septic shock are thought to be mediated by TNF64

and in animal models, TNF inhibition was protective. However,a randomized controlled trial of a neutralizing mAb Infliximab toTNF in humans with septic shock suggested that TNF inhibitionactually increased mortality.63 It was fortuitous that at about thistime, human RA synovial cell cultures were found to havesustained overproduction of TNF65 and subsequent studies inCIA showed a beneficial effect from TNF inhibition.39 In 1994,the first randomized trial of a single infusion of Infliximabdemonstrated that TNF blockade could produce substantial,short-term improvement in patients with chronic RA.66

Subsequent clinical trials of TNF inhibitors in humanshave demonstrated effectiveness in decreasing the severity of

RA and in slowing the progression of bony erosions(Table 4). These trials have demonstrated benefits comparedwith placebo in patients who are on no other DMARDtreatment (two trials), or who are on stable doses of Meth-otrexate (two trials). Etanercept is the only TNF inhibitordirectly tested against Methotrexate; it was equally effectiveand less toxic.62 Of note, combination therapy with Meth-otrexate, Sulfasalazine, and Hydoxychloroquine is superior tosingle drug therapy,67 but has not yet been compared to TNFinhibitors.

The effect of TNF inhibitors in RA can be dramatic andmay be seen in patients who have been unresponsive tostandard treatments. Approximately 40% of patients withlongstanding and treatment-resistant RA achieved an ACR50

response (Table 4). Tumour necrosis factor inhibitors are theonly agents besides corticosteroids that improve the disablingfatigue that can accompany RA. The beneficial effect of TNFinhibition only persists while treatment continues and relapseof RA to pretreatment levels occurs rapidly when treatmentstops. There is great interest in whether more durable diseaseremission can be induced by TNF blockade during the earliestphase of the disease and studies are underway to address thisquestion.62

The success of TNF blockade in RA has prompted clinicaltrials in other inflammatory diseases. There is randomizedcontrolled trial evidence for the effectiveness of TNF inhibi-tors in Crohn’s disease,68,69 psoriatic arthritis,70 psoriasis,70

juvenile inflammatory arthritis,71 and ankylosing spondylitis.72,73

Small uncontrolled case series have suggested TNF inhibitorsmay also be effective in adult onset Still’s disease,74 temporalarteritis,75 Wegener’s granulomatosis76 and Behcet’s syndrome.77

The down side

Lack of efficacy

Lack of efficacy, tolerability and toxicity are frequent prob-lems with all DMARD in RA. On average, more than 50% ofthe RA patients in TNF inhibitor trials fail to achieve anACR50 response. Furthermore, an ACR50 only reflects a betterthan 50% improvement, rather than complete disease remis-sion. Although most trial patients were selected as poorresponders to other treatments, TNF blockade is clearly noteffective in all patients with RA. This incomplete response is

Table 4 Randomized controlled trials of TNF inhibitors in rheumatoid arthritis

Patient characteristics Drug Year n = Design %ACR50 (placebo)

Trial length (mths)

long-standing active disease despite DMARD

Infliximab 10 mg/kg 199466 73 one infusion, ITT, RCT, no DMARD 58 (8) 1

long-standing active disease despite ≥ 1 DMARD

Etanercept 16 mg/m2 1997100 180 ITT, RCT, no DMARD 57 (7) 3

long-standing active disease despite MTX

Etanercept 25 mg 1999101 89 oral MTX in all, ITT, RCT 39 (3) 6

long-standing active disease despite MTX

Infliximab 10 mg/kg 200053 428 oral MTX in all, ITT, RCT 40 (9) 12

early active disease Etanercept 25 mg 200062 632 ITT, RCT, with MTX comparator 45 (40)* 12long-standing active disease despite MTX

Adalimumab 40 mg 2003102 271 oral MTX in all, ITT, RCT 55 (8) 6

DMARD, disease modifying antirheumatic drug; ITT, intention to treat; MTX, Methotrexate; RCT, randomized control trial; *, comparison iswith MTX.


consistent with data from animal studies and suggests thatnon-TNF-cytokine pathways are important in many patientswith RA.

Toxicity

Overall, TNF inhibitors have been remarkably well toleratedby humans. By August 2002, over 340 000 and 150 000patients had used Infliximab or Etanercept (respectively)worldwide. The commonest problems are related to theparenteral administration of the drugs, with one third ofEtanercept patients reporting injection site reactions, and10% of Infliximab patients developing infusion reactions(Wyeth and Immunex surveillance data). Serious side-effectsare rare but include bacterial sepsis, tuberculosis and otheropportunistic infections, multiple sclerosis and drug inducedlupus (Table 5). Infliximab and Etanercept show similar ratesof these serious side-effects, suggesting that they are directlyrelated to TNF inhibition.

Tuberculosis

That TNF might be important in controlling M. tuberculosishad been suggested by animal studies. Tuberculoid granu-loma formation is dependent on TNF, and TNF-deficientmice are highly susceptible to tuberculosis.60 Active tubercu-losis is uncommon in the first world countries currently usingTNF inhibitors. However, latent tuberculosis is commonenough to be a concern. Latent tuberculous infection remainsdormant unless the host is compromised, because of immuno-suppressive drugs, AIDS, or old age. Demonstration that TNFblockade reactivates tuberculosis in humans required carefulpost marketing surveillance.78 Most of the reactivation infec-tions occurred within months of TNF inhibition, and dis-seminated tuberculosis occurred in many of the patients.Disseminated infection is normally a rare manifestation oftuberculosis, suggesting an important role for TNF in limitingdissemination. All patients are now screened for latent tuber-culosis prior to the use of TNF inhibitors.

Multiple sclerosis

Multiple sclerosis (MS) is thought to be an autoimmuneinflammatory disease and causes progressive damage to thecentral nervous system (CNS) through the demyelination ofneurones. Tumour necrosis factor is thought to mediate tissueinjury in MS.79 Therefore, TNF blockade was investigated asa treatment for multiple sclerosis using the TNF inhibitorLenercept. The trial was prematurely stopped because ofworsening of disease in some of the Lenercept treatedpatients.80 There have also been case reports of worsening ofmultiple sclerosis with the other TNF inhibitors, Infliximaband Etanercept. Furthermore, in patients without previousmultiple sclerosis, there are reports of new CNS demyelina-tion that resolves upon cessation of the drug (Table 5).Although the rate of new MS in patients on TNF inhibitors islow (31 per 100 000 patients per year of exposure), it ishigher than the background rate of MS (5 per 100 000 patientsper year of exposure). The mechanism of TNF inhibitor-induced MS is unclear. It is unlikely that a TNF inhibitor drugis able to enter the CNS because of protein blockade at theblood–brain barrier. It has been suggested that TNF blockadeallows activation and survival of peripheral autoreactivemyelin-specific T cells. These could enter the CNS andprovoke demyelination.79

Lymphoma

Lymphoma is a known complication of immunosuppressivemedications such as Cyclophosphamide. Methotrexate canalso cause a pseudolymphoma which remits on withdrawal.The odds ratio (observed compared with expected number ofcases) for lymphoma in patients with long-standing active RAis 26.81 There is circumstantial evidence that TNF may beimportant in the control of malignancy. In vitro, TNF caninhibit tumour growth.64,82 In the 5 years that TNF inhibitorshave been in widespread human use, there has been noapparent increase in the rate of true lymphoma in treatedpatients.83 However, definitive proof of no effect awaits long-term follow up.

Table 5 Side-effects of TNF inhibitors

Agent Event n =

Infliximab87 Serious adverse events (fever, pneumonia, dyspnoea, rash) 4.4% with Infliximab vs. 1.8% with placebo 700 trial patientsEtanercept87 Rates of serious infection (4%/patient year), and cancer (n = 41) identical to control group 4794 patient-yearsInfliximab103 Aseptic meningitis following 2nd infusion 1 caseInfliximab78 70 patients with tuberculosis, 4 times higher than expected rate, high incidence of extrapulmonary disease

(57% vs. 18% usually)147 000 exposed

Infliximab104 14 patients with Listeria monocytogenes septicaemia or meningitis; 6 deaths 186 500 exposedInfliximab105 9 patients with pulmonary Histoplasma capsulatum; 1death 150 000 exposedInfliximab106 Invasive Aspergillus fumigatus 1 deathLenercept80 Increased exacerbations of multiple sclerosis in randomized trial 168 trial patientsInfliximab107 Worsening of multiple sclerosis 2 casesEtanercept108 Precipitating multiple sclerosis 1 caseEtanercept109 Reversible central nervous system demyelination 17 casesInfliximab109 Reversible central nervous system demyelination 2 casesEtanercept110 Drug induced systemic lupus erythematosus 4 casesInfliximab* Reversible drug induced systemic lupus erythematosus 301 casesEtanercept* Injection site reactions 35% 764 trial patients

*Wyeth and Immunex company data on file.


Induction of antinuclear antibodies and systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune diseasecharacterized by the production of antinuclear autoantibodies(ANA). In contrast to RA, SLE has been associated with lowlevels of TNF.84–86 It is therefore intriguing that over 50% ofRA patients treated with Infliximab developed a positiveANA during treatment.53 In addition, 13% developed anti-bodies to double stranded DNA (dsDNA Abs). With Etaner-cept, 11% of treated RA patients developed a new ANA and2% developed dsDNA Abs, suggesting that ANA develop-ment is at least partly an effect of TNF inhibition. Despite thehigh rates of autoantibodies in these patients, however, veryfew patients developed clinical manifestations of SLE.87

These clinical observations raise intriguing possibilitiesregarding TNF and Th1/Th2 bias in the expression of autoim-mune disease. Interestingly, TNF inhibition in RA patients isassociated with elevated IFN-γ levels.88

Human anti-chimaeric molecule antibodies

Infliximab is 25% murine derived. Human anti-chimaericmolecule antibodies (HACA) to the murine Fv componentdevelop in 40% of RA patients and 61% of Crohn’s diseasepatients.66,89 It is a clinically relevant phenomenon. Infusionreactions occur predominantly in patients with HACA and aremore likely in patients with high titre HACA, suggesting animmune-mediated effect.53,89 In Crohn’s disease, the averagelength of the clinical response was reduced by 50%, and thehalf-life of Infliximab was dramatically shortened in patientswith HACA.89 In RA however, HACA was not associatedwith reduced efficacy.53,66 Human anti-chimaeric moleculeantibodies can be largely prevented by the concomitantadministration of Methotrexate,90 and this is now the recom-mended treatment approach when using Infliximab. In aseparate study in RA, the development of HACA could beprevented by giving higher doses of Infliximab at eachinfusion. Higher rates of HACA are seen in Infliximab treatedCrohn’s disease patients (61%) compared to treated RApatients (40%).89 This may be due to the sporadic administra-tion of Infliximab in Crohn’s disease, where Infliximab isused to heal fistulae.

Cost

Huge costs are incurred in development of all new drugs,particularly the costs of multicentre clinical trials, which canrun into hundreds of millions of dollars. Production costs ofbiologic agents are high compared with traditional medicinalchemistry. At a non-subsidized average current drug cost perpatient per year of $A15 000,91 in a disease with a 1%prevalence, clearly TNF inhibitors cannot be used for every-one with RA. In Australia alone, this would cost $3 billioneach year. In contrast, the non-subsidized cost of Metho-trexate is less than a few hundred dollars per patient per year.Clinical trial evidence confirms the benefit of TNF inhibitorsin patients who have persistently active RA despite optimalDMARD therapy. Although this probably represents about10–20% of patients with RA, the overall cost to the healthcare budget could still be substantial. Against this must be

weighed the fact that RA causes substantial personal andsocietal costs.2 Patients in the US and in Europe have hadrelatively open access to the TNF inhibitors for several years.In Australia, where pharmaceuticals are heavily subsidized bygovernment, regulatory agencies have insisted on rigorouscost-benefit analysis and have to date been very cautiousabout how TNF inhibitors will be made available to clinicianstreating RA.

Discussion

Rheumatoid arthritis has always been one of the most difficultautoimmune diseases to understand and to treat. For manyyears, the focus has been on identifying genetic susceptibili-ties and abnormalities in the adaptive immune response.Clearly, this approach will continue and is likely to yieldimportant insights, especially relevant to the earliest phases ofdisease. More rapid progress has been made in understandingthe molecular mechanisms underlying established disease insynovial joints and a great deal is now known about the localinflammatory environment in RA. Persistent overproductionof an array of inflammatory mediators is the cardinal feature,converting the normally quiescent synovial compartment intoa self-sustaining, chronic inflammatory site. The synovium inRA seems to provide functional aspects of secondary lym-phoid tissue, which attract and sustain dendritic cells, T and Bcells, an activating trap for myeloid cells (particularly macro-phages and neutrophils) and immune complexes, and thestimuli for the induction of tissue remodelling programs insynovial fibroblasts and osteoclasts.

The discovery that there may be key molecular contribu-tors to this complex disease has both conceptual and thera-peutic significance. Although appealing in some respects, aTNF-driven cytokine heirarchy45 seems untenable, in view of(i) clinical benefit with TNF inhibitors in some but not all RApatients (ii) relative lack of side-effects with TNF inhibitorsand (iii) animal model evidence. Perhaps individual cytokinessuch as TNF do predominate in some patients with RA andthe responsiveness of individual RA patients to cytokineinhibitors may allow clinical definition of RA subtypes.Whilst not advocating a ‘cytokine chaos’ model of RA, wesuggest a pluralist model is a better explanation and allowsfor independent overproduction of multiple key cytokines andsome degree of redundancy. The corollary of this model is thepossibility that blockade of one or several key cytokines mayrestore homeostasis in the majority of RA patients.

The story of TNF in RA also shows the benefits of a‘paradigm shift’. Acute septicaemic shock was the originalbiotechnology target for a chimaeric anti-TNF mAb, butfailed. Early success with the same anti-TNF mAb inpatients with chronic, treatment resistant RA was the begin-ning of a new chapter in RA research and renewed interestin chronic inflammatory diseases. Although parenteraldelivery remains a disadvantage compared with the oralintake of small chemical entities, there is also renewedenthusiasm for wider therapeutic applications of humanizedmAbs and soluble receptors. Many biological inhibitors arenow in development or clinical trials, including mAbs toC5a, GM-CSF, IL-6, CD20 and VLA-4, and soluble inhibi-tors such as IL-1R, CTLA4-Ig and osteoprotegerin. Thesestrategies will greatly expand the range of therapeutic options


beyond inhibition of TNF and may offer the prospect ofdisease control for the vast majority of patients with RA.

Acknowledgements

We gratefully acknowledge the support of the the ReidCharitable Trusts, the National Health & Medical ResearchCouncil of Australia and the Arthritis Foundation of Aus-tralia, for the work of the Reid laboratory at WEHI. We thankKristy O’Donnell for technical assistance with RNASe pro-tection analysis and Suthep Sachthep for immunohistochem-istry. Ian Wicks is an NHMRC Clinical Practitioner Fellow.

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