EXTENDED REPORT

Tumour necrosis factor a independent disease mechanismsin rheumatoid arthritis: a histopathological study on theeffect of infliximab on rheumatoid nodulesD Baeten, F De Keyser, E M Veys, Y Theate, F A Houssiau, P Durez. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr D Baeten, Departmentof Rheumatology, 0K12IB,Ghent University Hospital,De Pintelaan 185, 9000Gent, Belgium; dominique.baeten@ugent.be

Accepted 29 July 2003. . . . . . . . . . . . . . . . . . . . . . .

Ann Rheum Dis 2004;63:489–493. doi: 10.1136/ard.2003.012302

Background: It has been suggested that the immunopathology of rheumatoid nodules parallels that ofinflamed synovium in rheumatoid arthritis (RA).Objective: To analyse the effect of infliximab on the immunopathology of rheumatoid nodules in order toprovide new insights into the relationship between synovial inflammation and rheumatoid nodules.Materials and methods: Nodules were present at baseline in six patients with RA and after infliximabtreatment in five patients, including paired nodules before and after treatment in three patients. In onepatient, the nodule appeared during treatment. Paraffin sections were used for histological analysis.Frozen sections were stained by immunohistochemistry for cellular markers (CD3, CD4, CD8, CD16,CD20, CD68), blood vessels (CD146, vWF, avb3), and adhesion molecules (E-selectin, VCAM-1, ICAM-1).Results: No manifest immunopathological differences were found between the nodules before and afterinfliximab treatment. All nodules depicted the classical structure with a central necrotic zone, surroundingthe palisade layer, and an outer connective tissue zone. Immunohistochemistry showed the presence ofCD68+ and CD16+ macrophages in the palisade and the connective tissue zone, as well as a smallnumber of CD3+, CD4+ T lymphocytes in the perivascular areas. Small vessels were seen in the connectivetissue and were sometimes positive for the neovascularisation marker avb3. They expressed no VCAM-1,E-selectin weakly, but ICAM-1 strongly. ICAM-1 was also strongly expressed on palisade cells.Conclusions: Despite an improvement of articular symptoms, infliximab treatment had no distinct effect onthe histopathology of rheumatoid nodules, suggesting that different pathogenetic mechanisms mediate thetwo disease manifestations in RA.

Rheumatoid nodules are the most characteristic extra-articular manifestation of rheumatoid arthritis (RA),occurring in about 25% of rheumatoid factor (RF)

positive patients with RA.1 They are typically located insubcutaneous tissues overlying bone or in other places ofmechanical stress (lungs, heart valves). Histologically, threecharacteristic zones can be recognised in the rheumatoidnodule. The inner zone of central necrosis contains fibrinoiddeposits and necrotic material, but no living cells. A palisadezone surrounds the central necrosis and is essentiallycomposed of radially arranged macrophages, but alsocontains some fibroblasts.2 Finally, the outer connectivetissue zone surrounds the palisade zone and is composed ofconnective tissue with macrophages and fibroblasts, a largenumber of small vessels, and a moderate number ofinfiltrating (mostly perivascular) lymphocytes. Clinically,there is an intriguing discrepancy between active jointinflammation and the extensive presence of nodules, notonly in the natural course of the disease but also in thefunction of disease modifying antirheumatic disease treat-ment, as illustrated by the worsening nodulosis in patientswith RA treated with methotrexate (MTX).3 Because thisobservation suggests that joint inflammation and nodulosisare not driven by the same pathogenetic mechanisms, itwould be of interest to analyse the effect of the newer, verypotent biological drugs for RA, on rheumatoid nodules.Despite the numerous studies and widespread use of tumournecrosis factor a (TNFa) blockers such as infliximab andetanercept, only one systematic prospective clinical study hasindicated that etanercept had neither an efficacious nor adeleterious effect on nodules.4 On the other hand, four cases

were described in which patients with RA developed nodulesduring etanercept treatment, even when they had cleararticular and inflammatory improvement.5 6 There are atpresent no studies comparing the histology of rheumatoidnodules before and after TNFa blockade or any data on theeffect of infliximab on nodules. The present study analysedhistologically and immunopathologically the effect of inflix-imab on rheumatoid nodules in order to evaluate theresponse and the pathogenic processes relevant to thissystemic feature of RA.

PATIENTS AND METHODSPatientsEight patients fulfilling the American College ofRheumatology (ACR) criteria for RA and having nodules ofthe juxta-articular area were included in this study afterinformed consent was obtained. Five of these patients haderosive, refractory disease and received infliximab (3 mg/kg)at weeks 0, 2, 6, and subsequently every 8 weeks incombination with MTX (at least 15 mg/week) and low doseprednisone (,10 mg/day). Painful or disturbing rheumatoidnodules were removed from the olecranon area, the feet, orthe fingers under local anaesthesia. Table 1 shows that thestudy analysed nodules of six patients who had not receivedinfliximab and nodules of five patients after infliximab

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ACR, American College of Rheumatology; MTX,methotrexate; RA, rheumatoid arthritis; RF, rheumatoid factor; TNFa,tumour necrosis factor a; vWF, von Willebrand factor

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Figure 1 Immunohistochemical analysis of the effect of infliximab on cellular markers, blood vessels, and adhesion molecules in rheumatoid nodules.The nodules were obtained after 6–24 months of infliximab treatment. (A) Central necrotic zone with palisade layer (original magnification6160); (B)palisade layer with radially orientated mononuclear cells and occasional multinucleated giant cells (original magnification6160); (C) outer connectivetissue zone with small blood vessels and perivascular infiltration with lymphocyte-like mononuclear cells (original magnification 6160); (D) anti-CD3staining (original magnification 6320); (E) anti-CD68 staining (original magnification 6160); (F) anti-CD16 staining (original magnification 6160);(G) anti-vWF staining (original magnification 6160); (H) anti-aVb3 staining (original magnification 6160); (I) anti-ICAM-1 staining (originalmagnification 6160); (J) anti-ICAM-1 staining (original magnification 6320).

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treatment, with three paired observations before and afterinfliximab treatment. Nodules were obtained from threepatients independently of infliximab treatment, from threepatients both at baseline and after 2–12 months of inflix-imab, and from two patients after respectively 12 and30 months of infliximab.

Histopathological analysisThe 11 nodules were mounted in paraffin and sections werestained with haematoxylin and eosin for histological analysisby two ‘‘blinded’’ observers (DB and YT). In eight nodules(four baseline and four after infliximab treatment (see table 2for details)), tissue was also available for immunohistochem-istry. The nodules were mounted in freezing medium and5 mm sections were cut. The frozen sections were stained by aperoxidase technique, as extensively reported previously.7 Thesections were stained with mouse monoclonal antibodiesdirected against following cell surface markers and adhesionmolecules: CD3 (T cells, clone UCHT1; Dako, Glostrup,Denmark), CD4 (T helper cells, clone MT310; Dako), CD8(T suppressor/cytotoxic cells, clone DK25; Dako), CD16(FccIII receptor, expressed on a subset of neutrophils andmonocytes, clone 3G8; Immunotech, Marseille, France),CD20 (B cells, clone L26; Dako), CD54 (ICAM-1, clone6.5B5; Dako), CD62E (E-selectin, clone 1.2B6; Dako), CD68(macrophages, clone PG-M1; Dako), CD106 (VCAM-1, clone1.4C3; Dako), CD146 (endothelial cells, clone P1H12;Chemicon, Temecula, USA), von Willebrand factor (vWF;endothelial cells, clone F8/86; Dako), and the integrin aVb3(a marker of neovascularisation, clone 23C6; Pharmingen,San Diego, USA). Parallel sections were incubated withirrelevant isotype and concentration matched monoclonalantibodies as negative control. Stained sections were codedand scored on a semiquantitative four point scale.7 8

RESULTSClinical analysis of the five infl iximab treated patientsAs shown in (table 1, four of the five infliximab treatedpatients met the ACR 20% improvement criteria already at6 weeks of infliximab treatment and all five met the criteriaat 3 months (one patient met the ACR 50% improvementcriteria). Therefore these patients were classified as respon-ders to infliximab, although only three of the five patients(Nos 5, 7, and 8) had a clear decrease in swollen joint countbetween baseline and the time of nodule resection; in the twoother patients (Nos 4 and 6) with a more longstanding RA,the number of swollen joints remained stable. Similarly, Creactive protein serum levels were decreased in only threepatients, but this might be a biased reflection of clinicalresponse because they were assessed 8 weeks after the lastinfusion of infliximab. Interestingly, in four of the fivepatients, prednisone could be tapered while MTX remainedstable.

The clinical nodulosis was stable in three of the fivepatients (Nos 4, 5, and 8), despite the clear improvement ofthe swollen joint count in two of them. New nodules as wellas mild nailfold vasculitis appeared during infliximabtreatment in one patient (No 6). The fifth patient (No 7),who was treated with a high dose of MTX (20 mg/week), hadno rheumatoid nodules at baseline but developed a uniquenodule of the olecranon region after 9 months of infliximabtreatment, whereas there was a clear decrease of both theswollen joint count and the C reactive protein.

Histological analysis of the nodulesThe global histological structure of the 11 nodules wassimilar, independently of the time of sampling and the factthat they developed before or during infliximab treatment(table 2). All nodules had clear regions of central necrosis,

surrounded by a palisade layer of radially orientated mono-nuclear cells and, occasionally, multinucleated giant cells(figs 1A and B). These characteristic structures wereembedded in loose connective tissue containing a smallnumber of mononuclear cells. In some regions, mostlylocated at the periphery of the nodules, a moderate numberof small blood vessels were observed; some of these vesselswere surrounded by perivascular infiltrates of lymphocyte-like mononuclear cells (fig 1C). There were no manifesthistological differences between the nodules formed beforeand after infliximab treatment.

Immunohistochemical analysisFour baseline and four nodules after infliximab treatmentwere assessed by immunohistochemistry. Globally, all eightnodules had a similar staining pattern for the differentcellular markers and adhesion molecules (table 2). Thecellular infiltration was characterised by a small to moderatenumber of CD3+ T lymphocytes, mostly localised in thevicinity of small blood vessels in the surrounding connectivetissue zone (fig 1D). Almost all T lymphocytes were CD4+,whereas no CD8+ cells were detected. The number of CD20+B cells was extremely low (score zero for all nodules). As formacrophages, CD68+ macrophages were found in thepalisade layer as well as in the surrounding connective tissuezone (fig 1E). Although less numerous than the CD68+macrophages, CD16+ mononuclear cells were found through-out the nodules but especially in the vicinity of the palisadelayer (fig 1F). Small vessels were found exclusively in theperiphery of the surrounding connective tissue zone. Theywere consistently positive for the endothelial cell markersvWF and CD146 (fig 1G). In two nodules obtained afterrespectively 1 and 2 years of infliximab treatment, some ofthese small vessels expressed the aVb3 integrin, pointing to acertain degree of neovascularisation (fig 1H). As to theadhesion molecules, VCAM-1 expression was absent fromblood vessels and palisade layer. E-selectin expression wasnegative in the nodules before infliximab treatment and intwo nodules after infliximab treatment; in the two othernodules, a faint expression was noticed on the endothelium.In contrast, ICAM-1 was strongly expressed in all samplesboth on endothelium and on cells of the palisade layer (figs 1Iand J).

DISCUSSIONThree studies have previously compared the immunopathol-ogy of the rheumatoid nodule and the synovial membrane inRA in order to analyse putative common pathogenicmechanisms.2 8 9 They indicated a number of strikingsimilarities between synovium and nodules: similar expres-sion of adhesion molecules (except for E-selectin expression,which was higher in nodules), palisade formation mimickingthe proliferation of the synovial lining, predominant infiltra-tion with CD68+ macrophages which produce proinflamma-tory cytokines such as TNFa and interleukin 1b, andperivascular CD3+ T cells. These findings suggested thatinflammation mediated by macrophages, amplified by adhe-sion molecule expression and continuous influx of additionalinflammatory cells, might be similar in the synovium andnodule. However, there were also important histopathologicaldifferences between both disease manifestations: the absenceof necrosis in the synovial membrane, the absence of B cells,plasma cells, and lymphoid follicles in the nodules, and thedifferential expression of VCAM-1. This study confirms theglobal baseline histopathological structure described in thepreviously mentioned reports, but challenges the concept ofsimilar disease mechanisms in inflamed synovium andrheumatoid nodules by indicating no detectable effect ofTNFa blockade by infliximab on the clinical presentation and

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the histopathology of the rheumatoid nodules. This is to ourknowledge the first systematic comparison of rheumatoidnodules before and after treatment with infliximab, whichextends histopathologically previous case reports withanother TNFa blocker, etanercept.6 7 The absence of adetectable histopathological effect of infliximab on therheumatoid nodule contrasts with the improvement ofperipheral joint and global inflammation in these patients(all patients met the ACR 20% improvement criteria at3 months despite tapering prednisone in four of the fivepatients and stable MTX in all patients). Moreover, whereasthe limited number of observations (due to the ethical andclinical difficulty of performing sequential surgical interven-tions for a rather mild disease manifestation), the semi-quantitative approach (which might be relatively insensitiveto small changes), and the variable timing of biopsy sampling(between 1 and 7 weeks after infliximab infusion) suggeststhat some caution is needed in the interpretation of thepresent results, the absence of any detectable effect onrheumatoid nodules remains in sharp contrast with thepreviously documented histopathological effect on autoim-mune synovitis observations using an identical approach.7 10

Different explanations can be envisaged for the absence ofa detectable effect of infliximab on rheumatoid nodules.Firstly, it may be that the drug cannot effectively penetratethe nodules owing to the specific granuloma-like architec-ture. Although this hypothesis cannot be formally excluded,it is in contradiction with the clear presence of blood vesselsin the outer connective tissue zone and with the effect ofinfliximab on other granuloma-like structures such as intuberculosis.11 Secondly, longstanding rheumatoid nodulesmay have to be considered as residual lesions that becomeinsensitive to anti-inflammatory treatment once the activeinflammatory process has terminated. This theory, however,cannot explain the appearance of new nodules in two of thepatients or the presence of nodules after infliximab treatmentof features of active inflammation such as aVb3 positivevessels, ICAM-1, and E-selectin expression, and perivascularlymphocyte infiltration. It is interesting to note that inhibi-tion of vascular activation by TNFa and subsequent infiltra-tion with inflammatory cells was proposed as one of themajor modes of action of infliximab in peripheral jointinflammation.7 10 The third and most likely explanation isthat, although TNFa may have some role in nodulosis, thepivotal mechanisms of these lesions are largely TNFaindependent. Such alternative mechanisms might includeadenosine release by monocytes induced by MTX12 and/or thestimulation of specific macrophage subsets expressing theFccIIIa receptor (CD16) by small immune complexes asproposed by Edwards and coworkers.13 Self associateddimeric complexes of IgG RF could evade clearance bycomplement receptors and diffuse into the extravascularspace, especially at sites of mechanical stress and repetitivemicrotrauma. When binding to macrophages through theFccIIIa receptor, this could lead to stimulation of these cells,production and release of cytokines and growth factors suchas transforming growth factor b, and, ultimately, noduleformation. This hypothesis is supported by the fact thatnodules are almost exclusively found in RF positive patients,that FccIIIa positive macrophages are preferentially found atsites of biomechanical stress such as the dermis on extensorsites, which are the typical localisation for rheumatoidnodules,14 and that FccIIIa polymorphism is associated withnodular disease.15 Accordingly, the present study indicatesthat a subset of the CD68+ macrophages, present in nodulesboth before and after infliximab treatment, expresses theFccIIIa receptor, especially in the palisade layer. These

observations warrant further research on the role of smallimmune complexes and their receptors in the pathogenesis ofrheumatoid nodules as well as other extra-articular manifes-tations in RA.

In conclusion, this study demonstrated no distinct effect ofinfliximab treatment on the clinical and histopathologicalpresentation of rheumatoid nodules, despite an improvementof joint inflammation. These data support the concept thatsynovial inflammation and nodule formation in RA are, atleast partially, mediated by different mechanisms. Althoughimmune complex mediated activation of specific macrophagesubsets is likely to play a part at both disease sites, wepropose that this results in TNFa dependent mechanisms ofinflammation in synovitis but leads to a largely TNFaindependent proliferative connective tissue lesion in thenodule.

Authors’ affiliations. . . . . . . . . . . . . . . . . . . . .

D Baeten, F De Keyser, E M Veys, Department of Rheumatology, GhentUniversity Hospital, BelgiumY Theate, Department of Pathology, Cliniques Universitaires St Luc,Universite Catholique de Louvain, Brussels, BelgiumF A Houssiau, P Durez, Department of Rheumatology, CliniquesUniversitaires St Luc, Universite Catholique de Louvain, Brussels, Belgium

D Baeten is a senior clinical investigator of the Fund for ScientificResearch (FWO)–Vlaanderen.

REFERENCES1 Veys EM, De Keyser F. Rheumatoid nodules: differential diagnosis and

immunohistological findings. Ann Rheum Dis 1993;52:625–6.2 Edwards JCW, Wilkinson LS, Pitsillides AA. Palisading cells of rheumatoid

nodules: comparison with synovial intimal cells. Ann Rheum Dis1993;52:801–5.

3 Kerstens PJ, Boerbooms AM, Jeurissen ME, Fast JH, Assmann KJ, van dePutte LB. Accelerated nodulosis during low dose MTX therapy for rheumatoidarthritis: an analysis of ten cases. J Rheumatol 1992;19:867–71.

4 Kaiser MJ, Bozonnat MC, Jorgensen C, Daures JP, Sany J. Effect of etanercepton tenosynovitis and nodules in rheumatoid arthritis. Arthritis Rheum2002;46:559–60.

5 Cunnane G, Warnock M, Fye KH, Daikh DI. Accelerated nodulosis andvacsulitis following etanercept therapy for rheumatoid arthritis. Arthritis Rheum2002;47:445–9.

6 Kekow J, Welte T, Kellner U, Pap T. Development of rheumatoid nodulesduring anti-tumor necrosis factor alpha therapy with etanercept. ArthritisRheum 2002;46:843–4.

7 Baeten D, Kruithof E, Van Den Bosch F, Demetter P, Van Damme N,Cuvelier C, et al. Immunomodulatory effects of anti-TNF-a therapy onsynovium in spondyloarthropathy: histologic findings in eight patients from anopen-label pilot study. Arthritis Rheum 2001;44:186–95.

8 Elewaut D, de Keyser F, De Wever N, Baeten D, van Damme N,Verbruggen G, et al. A comparative phenotypical analysis of rheumatoidnodules and rheumatoid synovium with special reference to adhesionmolecules and activation markers. Ann Rheum Dis 1998;57:480–6.

9 Wikaningrum R, Highton J, Parker A, Coleman M, Hessian PA, Roberts-Thomson PJ, et al. Pathogenic mechanisms in the rheumatoid nodule:comparison of proinflammatory cytokine production and cell adhesionmolecule expression in rheumatoid nodules and synovial membranes from thesame patients. Arthritis Rheum 1998;41:1783–97.

10 Tak PP, Taylor PC, Breedveld FC, Smeets TJM, Daha ME, Kluin PM, et al.Decrease in cellularity and expression of adhesion molecules by anti-tumornecrosis factor a monocloncal antibody treatment in patients with rheumatoidarthritis. Arthritis Rheum 1996;39:1077–81.

11 Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J,Schwieterman WD, et al. Tuberculosis associated with infliximab, a tumornecrosis factor alpha-neutralizing agent. N Engl J Med 2001;345:1098–104.

12 Merrill JT, Shen C, Schreibman D, Coffey D, Zakharenko O, Fisher R, et al.Adenosine A1 receptor promotion of multinucleated giant cell formation byhuman monocytes: a mechanism for methotrexate-induced nodulosis inrheumatoid arthritis. Arthritis Rheum 1997;40:1308–15.

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14 Edwards JCW, Blades S, Cambridge G. Restricted expression of FccRIII(CD16) in synovium and dermis: implications for tissue targeting inrheumatoid arthritis. Clin Exp Immunol 1997;108:401–6.

15 Morgan AW, Griffiths B, Ponchel F, Montague BMN, Ali M, Gardner PP, et al.Fcc receptor type IIIA is associated with rheumatoid arthritis in two distinctethnic groups. Arthritis Rheum 2000;23:28–34.

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