Annals ofthe Rheumatic Diseases 1991; 50: 653-657

REVIEW

Juvenile chronic arthritis, chronic iridocyclitis,and reactivity to histones

Alison M Leak, Patricia Woo

Autoimmune rheumatic diseases in childhoodpresent major differences in both clinical andlaboratory features compared with adult onsetdisease.' Although polyarthritis occurs in bothadults and children, the specific features ofseropositive erosive rheumatoid arthritis areonly seen in about 100/o of patients with juvenilechronic arthritis beginning under the age of 16years.' 2 Most have seronegative disease-thatis, without IgM rheumatoid factor, and sero-logically the more common finding is of anti-nuclear antibodies. These antibodies are detectedin 30-400/o of all children with juvenile chronicarthritis, but are most common in late onsetseropositive polyarthritis and in early onsetpauciarticular disease. 5 These latter childrenwith four or fewer joints affected in the firstthree months commonly have a limited arthritis,and with appropriate management usually havelittle long term disability. In antinuclear anti-body positive pauciarticular arthritis beginningunder the age of 5 years, however, 60-70% ofchildren develop chronic iridocyclitis, whichmay cause blindness,6 and around 8O0/o of thoseaffected are girls.7

It is not clear whether chronic anterior uveitisis an autoimmune disorder or an immunecomplex vasculitis.8 The initial cause of tissuedamage may be physical trauma, infection,chemical, or immunological injury. Uvealdamage caused by viruses or other microbialorganisms may expose antigens, normallycontained within cells, for subsequent auto-immune sensitisation.After an initial attack of uveitis it has been

postulated that the endothelial lining of theuveal blood vessels may become permanentlydamaged, thereby reducing the effectiveness, ofthe blood/aqueous barrier.9 This would allowdeposition of specific antinuclear antibody/antigen complexes or the later deposition ofunrelated immune complexes, which wouldthen cause a recrudescence and perpetuation ofinflammation.9-l" The rich blood supply of theuvea may trap immune complexes, and in non-granulomatous uveitis these are thought to beonly moderate in size. Evidence for immunecomplex formation in pauciarticular juvenilechronic arthritis is limited, however. 12-15 Lackof lymphatic drainage to the eye reduces seques-tration and processing of antigen by lymphoidtissue in peripheral nodes.'6 Primed lympho-cytes return to the uvea and production ofantibody occurs locally.9The antigenic stimulus to antinuclear anti-

body formation in both adult rheumatoid

arthritis and in juvenile chronic arthritis isunknown. Antinuclear antibodies are detectableon both HEp-2 cells and on rodent tissuesubstrate. The pattern of immunofluorescencemay be speckled,'7 but is usually homo-geneous,4 18 19 and suggests binding to thenucleosome, in which histones and DNA existin a highly organised structure.20 Many authorshave reported the uncommon existence of anti-bodies to double stranded DNA.2-26 Antibodiesto single stranded DNA, though detectable inactive juvenile chronic arthritis,'9 27 are alsofound in many other disorders and their patho-genic significance is uncertain.24 Also, none ofthe well characterised autoantibodies found inconnective tissue diseases (including antibodiesto Ro, La, RNP, Sm, PM-1, Scl-70) is presentwith any regularity. 17 21 25 2830

Attention recently has shifted towards furtherevaluation of antibodies to histones, and severalauthors have reported the presence of theseantibodies in juvenile chronic arthritis.3138

HistonesHistones are basic DNA binding proteins,which are arranged in a highly organised struc-ture in nucleosomal particles, subcomponentsof chromatin. A double loop of DNA (200 bplong) is wound round two H2A-H2B dimers oneither side of the (H3-H4)2 tetramer in the coreof the particle20 39 (figure). The association ofthe core histones with DNA causes a conden-sation of the double helix, which is furthercompacted by the binding of H1 in a secondorder supercoil.39 This organisation of thenucleosome allows separation of individual

H2ADH2B~H30H4

A single nucleosome showing the relation between the varioushistone molecules within two helical turns ofDNA (adaptedfrom ref39).

Department ofRheumatology,Thanet District Hospital,St Peters Road, Margate,Kent CT9 4ANA M LeakMolecular RheumatologyDepartment,Clinical Research Centre,Watford Road, Harrow,Middlesex HAI 3UJP Woo

Correspondence to:Dr Leak.

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chromosomes at mitosis and contributes topolymorphisms of the DNA structure.39H3 and H4, forming the inner relatively

inaccessible core of nucleosomes, are evolution-arily highly conserved.' Differences betweencalf and pea plant histones H3 and H4 arelimited to four substitutions out of 135 aminoacid residues for H3, and two of 102 residues forH4.39 H2A and H2B also show major similaritiesbetween calf and human sources. This conser-vation of structure has been thought to beresponsible for the poor immunogenicity of corehistones.4' Only certain regions of each histonemolecule are exposed on the surface of theparticle, and it is thought that most antibodiesto histones in systemic rheumatic diseases aredirected to exposed parts of the histone mole-cule-that is, these antibodies are induced byepitopes on the intact nucleosomal particle.41-43

Techniques for the detection of antibodies tohistonesAs with any investigation of-antigen reactivity itis important to analyse the test systems used andto understand their limitations. Although anti-bodies reactive to histone were reported in1958," reinvestigation of their antigenicity didnot take place until 1976, when Tan andcolleagues introduced the acid elution/recon-stitution method for their detection.45 Serumsamples positive for antibodies to histones,which are antinuclear antibody positive byimmunofluorescence, will become negativeafter acid elution of the substrate, and recon-stitution with histones then reproduces thepositive fluorescence. Althoughinitiallyheraldedas a specific and reproducible assay, severalmethodological problems limit its use.4 His-tones H2A and H2B are most commonlydetected by this method, even though the sameserum samples may be positive for other his-tones by other tests.42 43 47 The reason may bethat H3 and H4 epitopes, which are prerequisitesfor binding of the H2A-H2B dimers, becomeconcealed by DNA binding to H2A/H2B in thereconstitution phase.42 Variability betweenlaboratories is also likely because immuno-fluorescence is a subjective assay open toobserver error.48A further technique for antigen detection and

analysis was described in 1979 by Towbin.49Immunoblotting or western blotting was origin-ally used to detect very small amounts ofribosomal protein after transference from thegel phase of electrophoresis to a solid phase forimmunoreaction with antisera. In general, blot-ting has been accepted as a useful technique fordetecting autoantibodies.50 The use of sodiumdodecyl sulphate in the gel electrophoresisbefore transference onto nitrocellulose maycause denaturation of histones and has been saidto interfere with histone-antibody complexformation.43

In rheumatoid arthritis and the equivalentdisease in children, seropositive juvenile ar-thritis, up to 300/o of patients may be antinuclearantibody positive. When investigating thesepatients by immunoblotting it is possible thatsome of the lines recognised may be false

positive reactions related to circulating immunecomplexes.48 Rheumatoid factors may bind tospecific IgG antihistone antibodies or directly tohistone,5' and Gripenberg reported that someantihistone activity is due to cross reactingrheumatoid factors.52 Analysis of reactivity tohistones in seropositive juvenile arthritis islimited, and no profile of reactivity to thedifferent histone classes has yet emerged.Development ofsolid phase assays was initially

difficult owing to problems of immobilisation ofcationic histone antigens.' A fluorimetric assaywas used by Hobbs et al to detect antibodies tohistones using purified histones,53 54 and foundto be more sensitive than the acid elution/reconstitution method. Non-specific antibodybinding is reduced by histone saturation of thetyrosine-glutamic acid copolymer coating thecuvettes.53 This copolymer, which increases thesensitivity of the assay, may affect the confor-mation of histone H4 and reduce detection ofanti-H4.51An enzyme linked immunosorbent assay

(ELISA) can offer a quantitative analysis andseems to be the method of choice for investigat-ing different conformational states of chroma-tin.55 As with blotting, considerable differencesare seen in the apparent antigenic activity ofdifferent histones depending on the assay.37Purified immunoreactive histones are essentialfor meaningful and reproducible results.48 Theheterogeneity of antihistone antibody reactivitymay reflect mixtures of antibodies to individualhistones, or antigenic cross reactivity.

Clinical associations of antibodies to histonesIn adult rheumatoid arthritis, using the tech-nique of acid elution/reconstitution, Aitchesonet al demonstrated antibodies to histones in 24%of antinuclear antibody positive patients." In astudy by Epstein et al, using unfractionatedhistones in an ELISA,4 antibodies to histoneswere detected in 100%/o of patients with druginduced systemic lupus erythematosus, in 42%with idiopathic systemic lupus erythematosus,but in only 15% of adults with rheumatoidarthritis, and at very low concentrations, com-pared with one of 24 healthy control subjects.With a more sensitive ELISA (Nucleoscreenkit-see below), however, Muzellec et al foundthat 80% of antinuclear antibody positivepatients with rheumatoid arthritis had antibodiesto histones, of which antibodies to HI weremost common, being significantly more pre-valent than in controls.57 Patients treated withD-penicillamine had a significantly higher con-centration of a'ntibodies to histone HI thanthose not so treated.With acid elution/reconstitution we were

only able to detect antibodies to histones in twoof 15 patients with antinuclear antibodypositive juvenile chronic arthritis. Both weregirls with extended pauciarticular arthritiscomplicated by severe chronic iridocyclitis(Leak A M, MD thesis, London, 1991).Similarly, Rosenberg,25 Saulsbury,' andHaynes19 found only 0-100/o of children withantinuclear antibody positive juvenile chronicarthritis possessed antibodies to histones by this

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Juvenile chronic arthritis, chronic iridocyclitis, and reactivity to histones

method. Job-Deslandre et al, however, used atotal histone ELISA (calf thymus) to. test aspectrum of rheumatic diseases in children anddetected antibodies to histones in 23 of 39 (59%)children with juvenile chronic arthritis, ofwhom most of the children with antibodies tohistones had a pauciarticular onset.35 Antibodiesto histones were also found in a significantnumber of children with other inflammatoryconditions (42%), including infections, and inbetween 14 and 20% of children not usuallypossessing autoantibodies-for example,patients with irritable hip and mechanicalmusculoskeletal problems. In the subgroup ofpatients with juvenile chronic arthritis with anage of onset of under 5 years antihistoneantibody concentrations were significantlyhigher than in control groups.Four groups reported the use of immuno-

blotting to characterise the antinuclear anti-bodies found in juvenile chronic arthritis. -All the groups studied mixed seronegative typesof juvenile chronic arthritis, about half withpolyarticularand halfwith pauciarticular disease.The children chosen for analysis had a highprevalence of positive antinuclear antibodies byimmunofluorescence of HEp-2 cells (70-1000/opositive at 1:20 dilution). A preliminary studyby Brunet in 1986 suggested that six of ninechildren tested had antibodies to histonesthought to be directed against H2B.31 Morerecently, Malleson,32 Pauls,33 and Rosenberg34have detected bands by immunoblotting whichwere thought to represent histone HI. Thesewere found in 42%, 35%, and 37% of theirpatients respectively. In addition to HI,Malleson's group reported that 12% of patientshad a band identified at 45 kD which wasspecifically not associated with iridodcyclitis.32Pauls noted that many patients with HI also hadantibodies to H5, a variant of HI, whereas theintensity of reaction with core histones wasmuch weaker, and Rosenberg reported thatalthough many bands were seen on immuno-blotting as well as the 33 kD band thought to beHI, a similar spectrum of bands was also seen inpatients without juvenile chronic arthritis whowere analysed as controls.3Newer ELISA techniques have been de-

veloped by Muller and colleagues in whichantibodies to each of the histone classes can bedetected independently using purified histonesfrom chicken erythrocytes for the four corehistones and calf thymus for HI (Nucleoscreenkit, Neosystem, Strasbourg57).Three studies have been performed in children

with juvenile chronic arthritis with the Nucleo-screen ELISA. Ostensen reported results on agroup of 121 patients with juvenile chronicarthritis, 72 pauciarticular and 49 either poly-articular or systemic onset.36 A total of 58patients had antibodies to histones, including 21with antibodies to HI. Among the children withantibodies to histones there was a strong associ-ation with antibodies to H3 in 26 of 28 (93%)patients who had chronic iridocyclitis comparedwith 14 of 30 (47%) anti-H3 positive patientswithout eye disease. Children without eye diseasehad a more homogeneous response to all histonesrather than a peak for H3 seen in children with

uveitis. No associations were seen in either this,or the other reported studies using immuno-blotting,32 33 between antibodies to histonesand clinical disease activity, median antinuclearantibody titres, or drug treatment. Recentanalysis by Tuaillon et al of a further group ofunselected children with juvenile chronicarthritis (14 pauciarticular onset, 18 poly-articular or systemic) again shows reactivity tohistones ofall types, withantibodies toHI andH3being most often detected.37 Insufficient clinicaldetails were available to test for associationswith disease activity or with chronic iridocyclitis.We recently analysed antibodies to histones

by an ELISA in collaboration with Muller in 44patients selected because of early onset (meanthree years, all less than eight years) pauci-articular juvenile chronic arthritis.38 Previouswork suggested that the highest antinuclearantibody titres seen in this subgroup of diseasesare found in the first few months of the disease'8and, therefore, particular attention was paid tocollecting early serum samples. In addition,serial samples were collected from 32 children.Thirty five of 44 were girls, and 23 haddeveloped chronic iridocyclitis. IgM antihistoneantibodies were found in 75% of the patientsand antibodies to H3 were the most common (in56%) followed by HI (490/%), H2A (490/%), H4(46%), and anti-H2B (37-5%).Assessment of clinical variables showed an

association of antibodies to histone HI withjoint score, erythrocyte sedimentation rate, andIgG (p=005) and with antinuclear antibodytitres (p=0001). Regression of disease activityfrom year 1 to years 2 and 3 was paralleled by asignificant fall in antibodies to HI and H2A,while anti-H3 remained high. There was noassociation of antibodies to histones witheither the presence or degree of activity ofchronic anterior uveitis, and no clinical associa-tions with antibodies to H3 were found despitetheir frequent occurrence. The disparity betweenthese results and Ostensen's study may be dueto differences in the duration of disease. Moreof our patients, still early in the disease course,may develop uveitis over the next two to threeyears, though after that additional numbers arelikely to be small. Will raised anti-H3 concen-trations be a marker for eye disease?

Further analysis of reactivity to 32 synthetichistone peptides was made according to thetechnique described by Tuaillon et al.37 Themost commonly recognised peptides were to theN-terminal regions of H2A and H3 and the C-terminal peptides of HI and H3. Thus 29 of our44 patients had IgG antibodies to either one orother of the H3 peptides (amino acid residues1-21, 130-135). When anti-H3 peptide positiveserum samples were compared with H3 peptidenegative samples the former were significantlymore likely to be associated with positiveantinuclear antibodies (58% v 7%, p=0-001) orwith chronic iridocyclitis (690% v 290%, p0= 008).

Tuaillon et al used the same technique ofpeptide analysis in juvenile chronic arthritis andagain detected significant reactivity to H3 pep-tides, but also to the N-terminal peptide ofH4.37 The authors noted a poor correlationbetween H3 histone and H3 peptide anti-

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bodies,37 and our results corroborate this finding.

Antibodies to histones and chronicindocyclitisThe association of antinuclear antibodies withchronic anterior uveitis has been clinicallyreported for nearly 20 years, but a pathogenicrole for the antinuclear antibody remains elusive.Antibodies to histones have now been shownmost commonly in the early onset pauciarticularsubgroup of juvenile chronic arthritis, who aresusceptible to uveitis. Clinical associations witheye disease seem to be strongest with antibodiesto H3,36 38 though it is not known whetherantibodies to intact H3 molecules are the sameas those reacting with synthetic H3 peptides.The chronic iridocyclitis of juvenile chronic

arthritis is unique and there is no exact experi-mental model of this non-granulomatousanterior uveitis, especially in relation to thechronic relapsing course. Petty studied theuveitis of adjuvant induced arthritis in rats, thenearest animal model. Antibodies to bacterialcomponents, ocular and articular antigens, didnot correlate with uveitis."

Experimental autoimmune uveitis (an acuteanterior and posterior uveitis) without arthritiscan be induced in laboratory animals by retinalS antigen,59 and one of the sequences of thisprotein, known to be uveito-pathogenic (aminoacids 303-320), has a similar sequence to avariety of proteins.' The best match is withamino acids 106-121 from baker's yeast histoneH3. Singh et al were able to induce experimentalautoimmune uveitis in Lewis rats by injection ofthis histone peptide.'" We were unable to findany patients with antibodies to H3 peptide98-112, which has the closest homology to theS antigen peptide 303-320. This synthetic H3peptide has also been tested in an experimentaluveitis system with negative results (Graham E,personal communication).The pathogenetic mechanism proposed by

Singhet al was one of molecular mimicry, whichmight explain the development of autoimmunechronic uveitis, whereby immune response isinitially directed against the antigen in the yeasthistone, and persists because of shared deter-minants with host retinal S antigen.60 Molecularmimicry has been proposed for other sequencehomologies linking the retrovirus, visna, withexperimental allergic encephalitis (the animalmodel of multiple sclerosis), a human adeno-virus with a-gliadin in the production of coeliacdisease, and shared epitopes between HLA-B27and Klebsiella pneumoniae nitrogenase.Y Induc-tion of any of these diseases by specific micro-bial agents may occur in a susceptible subject.In the uveitis model yeast histones are implicatedas the possible initiators of the autoimmuneinflammation.One significant aspect of chronic iridocyclitis

in juvenile chronic arthritis is that it is mostcommon in girls and in children with an age ofonset between the first and third birthday (42 of73 (58%) affected in one study) (Leak A M,MD thesis, London, 1991). Eye disease is veryuncommon if juvenile chronic arthritis beginsafter the age of 5 years (7% of cases) (Leak A M,MD thesis, London, 1991), though the uveitis

itself may not become manifest until many yearslater. The eye is, in fact, relatively fullydeveloped at birth and three quarters of all sub-sequent development takes place in the first threeyears of life. Perhaps it is only at this stage thatan initiating event may occur which subsequentlyin a susceptible subject could lead directly toperpetuation of the inflammatory response.There is strong evidence for genetic association

with this form of juvenile chronic arthritis,6163predominantly with the HLA class II genes.Production of antinuclear antibodies and anti-bodies to histones may be a further characteristicof this impaired or altered immune response.The question of whether these antibodies aredirectly concerned with the pathogenesis of theeye disease remains to be tested.

1 Leak A M, Isenberg D A. Autoimmune rheumatic disordersin childhood-a comparison with adult onset disease. Q 7Med 1989; 73: 875-993.

2 Ansell B M. Juvenile chronic arthritis with persistentlypositive tests for rheumatoid factor. Ann Pediatr (Paris)1983; 30: 545-50.

3 Petty R E, Cassidy J T, Sullivan D B. Clinical correlates ofantinuclear antibodies in juvenile rheumatoid arthritis.J Pediair 1973; 83: 386-9.

4 Kornreich H K, Drexler E, Hanson V. Antinuclear factors inchildhood rheumatic diseases. J Pediatr 1966; 69: 1039-45.

S Schaller J G, Johnson G D, Holborow E J, et al. Theassociation of antinuclear antibodies with the chroniciridocyclitis of juvenile rheumatoid arthritis (Still's disease).Arthritis Rheum 1974; 17: 409-16.

6 Kanski J J. Anterior uveitis in juvenile rheumatoid arthritis.Arch Ophthalmol 1977; 95: 1794-7.

7 Rosenberg A M. Uveitis associated with juvenile rheumatoidarthritis. Semin Arthritis Rheun 1987; 16: 158-73.

8 Merriam J C, Chylack L T, Albert D M. Early-onsetpauciarticular juvenile rheumatoid arthritis. A histopatho-logical study. Arch Ophthalmol 1983; 101: 1085-92.

9 Rahi A H S, Kanski J J, Fielder A. Immunoglobulins andantinuclear antibodies in aqueous humour from patientswith juvenile "rheumatoid" arthritis (Still's disease). TransOphthalmol Soc UK 1977; 97: 217-22.

10 Petty R E. Current knowledge of the aetiology and patho-genesis ofchronic uveitis accompanying juvenile rheumatoidarthritis. Rhewn Dis Clin North Am 1987; 13: 19-36.

11 Char D H, Stein P, Masi R, Christensen M. Immunecomplexes in uveitis. AmJ Ophthalmol 1979; 87: 678-81.

12 Rossen R D, Brewer E J, Person D A, et al. Circulatingimmune complexes and antinuclear antibodies in juvenilerheumatoid arthritis. Arthritis Rheun 1977; 20: 1485-90.

13 Miller J J, Osborne C L, Hsu Y P. Clq binding in serum injuvenile rheumatoid arthritis. J Rheumatol 1980; 7:665-70.

14 Balogh Z, Meretey K, Falus A, Bozsoky S. Serologicabnormalities in juvenile chronic arthritis: a review of 46cases. Ann Rhewn Dis 1980; 39: 129-34.

15 Egeskjold E M, Johansen A, Permin H, Hoyeraal H M,S0rensen T. The significance of antinuclear antibodies injuvenile rheumatoid arthritis associated with chronicbilateral iridocyclitis. ActaPaediatrScand 1982; 71:615-20.

16 Chader G. Biochemistry of the eye. In: MillerS, ed. Clinicalophthalmology. Bristol: Wright, 1987: 24-45.

17 Osborn T G, Patel N J, Moore T L, Zuckner J. Use of theHEp2 cell substrate in the detection of antinuclear anti-bodies in juvenile rheumatoid arthritis. Arthritis Rheum1984; 27: 1286-9.

18 Leak A M, Burman S J, Ansell B M. Antinuclear antibodystudies in juvenile chronic arthritis. Arch Dis Child 1986;61: 168-72.

19 Haynes DC, Gershwin M E, Robbins D L, Miller J J, CoscaD. Autoantibody profile in juvenile arthritis. J Rheunatol1986; 13: 358-63.

20 Richmond T J, Finch J T, Rushton B, Rhodes D, Klug A.Structure of the nucleosome core particle at 7 A resolution.Nature 1984; 311: 532-7.

21 Alspaugh M A, Miller J J. A study of specificities ofantinuclear antibodies in juvenile rheumatoid arthritis.J Pediatr 1977; 90: 391-5.

22 Rudnicki R D, Ruderman M, Scull E, et al. Clinical featuresand serological abnormalities in juvenile rheumatoidarthritis. Arthritis Rheum 1974; 17: 1007-15.

23 Permin H, Halberg P, Christiansen E. Antibodies againstdsDNA in patients with connective tissue disorders. ActaMed Scand 1978; 203: 61-5.

24 CassidyJ T, Walker S E, Soderstrom S J, Petty RE, SullivanD B. Diagnostic significance of antibody to native DNA inchildren with juvenile chronic arthritis.J7Iediatr 1978; 93:416-20.

25 RQsenberg A M, Cordeiro D M, Knause R P. Studies on thespecificity of antinuclear antibodies (ANA) in juvenilerheumatoid arthritis [abstract]. Arthritis Rheum 1983; 26:S57.

26 Bell C, Talal N, Schur P. Antibodies to DNA in patients withrheumatoid arthritis and juvenile rheumatoid arthritis.Arthritis Rheum 1975; 18: 535-40.

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27 Leak A M, Colaco C B, Isenberg D A, Dudeney C, Burman SJ, Ansell B M. Anti-cardiolipin and anti-ssDNA antibodiesin ANA positive juvenile arthritis and other rheumaticdiseases [abstract]. Clin Exp Rheumatol 1987; 5 (suppl 2):18.

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nn Rheum

Dis: first published as 10.1136/ard.50.9.653 on 1 S

eptember 1991. D

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