Clin. exp. Immunol. (1985) 61, 496-502.

Human monoclonal antibodies from patients with rheumatoidarthritis: cross reactions against cellular constituents

0. 0. HASKARD, V. GUL, *ADRIENNE MORGAN, P. KATAAHA, *N. A.STAINES & J. R. ARCHER ARC Bone and Joint Research Unit, The London Hospital

Medical College, London El 2AD, UK and *Jmmunology Section, Chelsea College, University ofLondon, London SW3 6LX

(A ccepted for publication 3 January 1985)

SUMMARY

Hybridization of peripheral blood lymphocytes from patients with rheumatoid arthritishas yielded 14 monoclonal antibodies which react with cultured human epithelial cells.Immunofluorescence staining identifies at last five different types of antibody. Solid phaseimmunosorbent assays show a variety of cross-reaction patterns with nucleic acids,proteoglycan, cardiolipin and plastic, confirming that the various antibodies react withepitopes which are at least slightly different. These conclusions are confirmed by SDS gelelectrophoresis and immunoblotting on epithelial cell extracts. Similar antibodiespreviously found in association with lupus-like disease have been thought to berepresentative of the high antinuclear antibody response characteristic of lupus. Our dataare more consistent with the hypothesis that all or many of these antibodies are part of thenormal inflammatory response.

Keywords rheumatoid arthritis monoclonal autoantibodies intracellular antigens

INTRODUCTION

Recently we described a method for obtaining monoclonal antibodies by hybridization oflymphocytes from the peripheral blood of patients with rheumatoid arthritis. We found that asignificant proportion of them reacted with human cells (Haskard & Archer, 1984). This paperdescribes properties of these cell-reactive autoantibodies.

MATERIALS AND METHODS

Tissue culture. Human monoclonal antibodies were prepared by a modification of theEBV-hybridoma technique (Kozbor, Lagarde & Roder 1982, Haskard & Archer 1984). Hybrids,human laryngeal carcinoma cells (HEp 2) and human skin fibroblasts were grown in RPMI 1640medium (Gibco, Paisley) containing fetal calf serum (10%).

Sources ofpurified antigens. Native double strained DNA (dsDNA) and single stranded DNA(ssDNA) were prepared from calf thymus (Morgan et al.,1985). Yeast RNA, cardiolipin andcollagen types I and III were obtained from Sigma, Poole, Dorset. Gelatin was obtained fromHopkin & Williams, Chadwell Heath, Essex. Procedures described elsewhere were used for thepreparation of porcine proteoglycan monomer (PPGM) (Hardingham, Ewins, & Muir, 1976); typeII collagen (CII) (Staines et al., 1981); and fibronectin (Vuento & Vaheri, 1979).

Correspondence: Dr J. R. Archer, ARC Bone and Joint Research Unit, The London Hospital Med College,25-29 Ashfield Street, London El 2AD.

496

Monoclonal antibodies in rheumatoid arthritis 497Enzyme linked immunosorbent assay (ELISA) for 1gM. IgM immunoglobulin was detected by

ELISA (Haskard & Archer 1984) and quantified after titration in an ELISA in comparison with ahuman serum containing a known amount of IgM.

ELISA: Cellular constituents and gelatin. ELISAs to detect antigens in HEp 2 cells and gelatinwere as described by Haskard, Gul & Archer (1985).

ELISA: Nucleic Acids, connective tissue components and cardiolipin. Supernatants were dilutedin phosphate-buffered saline, pH 7-4 containing 0-05% Tween 20 and 1% bovine serum albumin,and were titrated against dsDNA, ssDNA, RNA and cardiolipin in an ELISA system described indetail elsewhere (Morgan et al., 1985; Staines et al., 1985); and against PPGM, CI, CIII andfibronectin in the ELISA described by Staines et al. (1981).

Competitive inhibition. Antibody at a concentration approximately 60% of the maximumpossible reaction were mixed with doubling dilutions of antigen and incubated at 40C for two hoursbefore 50 p1 samples were added to the appropriate antigen system. The ELISA was developed asdescribed above. Results are expressed as the amount of antigen required to inhibit 50% of thereaction that would have been obtained from antibody at 1 jg/ml.

Immunofluorescence. Immunofluorescent reactions ofsera and supernatants were tested on HEp2 cells and human skin fibroblasts, before and after the addition of colchicine (0 5 yg/ml for 16 h) tothe culture medium, as described by Haskard et al. (1985).

Electrophoresis and immunoblotting. Intermediate filament extracts were made by a modifica-tion of the method ofPruss et al. (198 1). Proteins were electrophoresed in a sodium dodecyl sulphate(SDS)/12-5% polyacrylamide gel in a discontinuous buffer using a 255% stacking gel (Laemmli,1970). Separated proteins were stained with Kenassie blue and reactive antigens were identified byimmunoblotting with the appropriate supernatant after electrophoretic transfer to nitrocellulose(Towbin, Staehelin & Gordon 1979). Controls included a culture supernatant containing IgMwhich did not react with intermediate filaments by immunofluorescence.

RESULTS

Fusion frequency. Nineteen fusions were carried out in which the cells survived more than 1 week.These used cells from 13 patients. Each fusion provided up to 136 primary clones (mean 30, median23). The clones described here were derived from 10 of our successful fusions on cells from sevenpatients with rheumatoid arthritis.

IgM secretion. Ninety percent of supernatants were shown to be secreting IgM. The quantity ofIgM secreted did not relate to positivity on any other assay. At the time of testing, IgM secretionranged from 19-8 to 0 9 yg/ml.

Anti-HEp 2 cell ELISA. Out of 425 supernatants tested (of which 383 were making IgMantibody), 18 reacted specifically with HEp 2 cells in the ELISA. A further supernatant, while givinga positive result, reacted more strongly with the control plate, suggesting that the HEp 2 cells werepartially blocking a reaction with the plastic. As reported elsewhere, four ofthe 18 supernatants thatreacted with HEp cells also reacted with Falcon 3912 Microtest III immunoassay plates (Haskard etal. (1985).

Immunofluorescence. Of the 18 supernatants selected by the HEp 2 ELISA, 12 gave positivestaining on immunofluorescence. The remaining six lost activity before their reaction patterns couldbe assessed. Ofa further 51 supernatants not originally tested on the ELISA, only two gave positivestaining (261 D12, 311 H10). Examples of the staining patterns of these 20 supernatants on HEp 2cells and fibroblasts are shown in Figs 1-6. Table 1 lists the number of each pattern observed.Supernatants were described as giving a vimentin-like reaction if they stained cytoplasmic filamentsin both HEp 2 cells and fibroblasts that collapsed on treatment with colchicine. Prekeratin-likestaining was a coarse filamentous pattern in HEp 2 cells that was unaffected by colchicine.'Ground-glass' staining was a fine background pattern in HEp 2 cells and fibroblasts that wasunaffected by colchicine.

Supernatants from clones 261 D12 and 473 E8 were the only ones giving positive immunofluor-escence that did not stain intermediate filaments. They both gave a similar coarse nuclear speckling

498 D. O. Haskard et al.

~~~~Fi. . 50D.nua ski firolat .._....,Fig..2. 50D5o co...in trae hua ski firbat x 5..

~~~~~~i.3 5B nclhc reae ~-2 cels (x 600).~~~~~~i.4. 42D on cocin treae H]- cel (x 600)

Fi.544G on ,ocin trate _p-2 (ucnetae suenaat ( x 0 :t~~~~~Fg 6 473E on!cociietetdHp2cls( 0)

Monoclonal antibodies in rheumatoid arthritis 499Table 1. Immunofluorescent staining pattern of hybrid supernatants on HEp 2 cells and human skin fibroblasts

Nuclear speckling 2 (473E8, 261D 12)Vimentin-like + /- 8 (402B6, 421 B 11, 452F8, 461C 11)cytoplasmic speckling (492D2, 501F7, 502C8, 503D1 1)Vimentin & prekeratin-like 2 (311H 10, 452B7)Vimentin & 'ground-glass' 1 (503D5)Vimentin & nucleolar 1 (442G3)

Total 14

(14-16 discrete immunofluorescent spots) on HEp 2 cells and fibroblasts. Supernatant from clone473 E8 did not react with Raji, Ramos or KR4 cells (all human B cell lines), and the precise stainingpattern on HEp 2 cells and fibroblasts varied with the cell cycle between nuclear speckled andnucleolar staining. This at first suggested it might be an anti-proliferating cell antibody similar tothat described by Miyachi, Fritzler & Tan (1978). However the Miyachi antibody reacts with anumber of EBV-transformed cell lines, and it seems likely that the antibodies are different.

The remaining antibodies all gave vimentin-like staining. They did, however, vary in theirstaining patterns and most also stained other structures. The most common additional appearancewas a speckled cytoplasmic staining. In one case (442 G3) the supernatant gave a speckledcytoplasmic staining if used unconcentrated, but was shown to stain vimentin-like filaments andnucleoli after five-fold concentration on a Minicon concentrator (Amicon, High Wycombe).

Clone 442 E7 which secretes an IgM rheumatoid factor (Haskard & Archer 1984) gave noimmunofluorescent staining.

Binding to purified antigens. Twelve supernatants found to give positive immunofluorescentstaining, together with 10 negative control supernatants containing IgM of unidentified specificity,were tested for reactivity against ssDNA, dsDNA, RNA, cardiolipin and a purified porcineproteoglycan monomer (Table 2). The table also indicates supernatants previously shown to react

Table 2. Antigen binding of supernatants showing cross-reactivity between cytoskeletal structures and otherantigens*

Proteoglycan FalconClone ssDNA dsDNA RNA cardiolipin monomer plastic

31lH10 - - - - +/-t +402B6 0 30 - - - 4.57 +421BI I - - - - - _442G3 1 71 17 1 2-63 2 63 492 +452B7 33 0 - - 490 - -452F8 025 - - - 293 +461C Il - - - - - -

473E8 - - - - - -

492D2 - - - - -

501F7 0 81 - - - - -503D5 - - - - - +503Dl1 - I _ _

Results expressed as yg/ml IgM required to give an O.D. of 0-35.* Ten control supernatants which gave negative immunofluorescent

staining were negative on all other assays. Clone 442E7 which hasrheumatoid factor activity specific for human yl, y2 and y4 was alsonegative on immunofluorescence and other assays apart from weakreactivity with Falcon plastic.

t May be caused by direct binding to plastic.

Table 3. Competitive inhibition of binding of antibodies to HEp 2 cells and ssDNA by purified antigens

Concentration of inhibitorcausing 50% inhibition of

Concentration IgM at 1 pg/ml (pg/ml)of IgM used Antigen

Antibody (pg/ml) on plate ssDNA dsDNA RNA Cardiolipin PPGMM

311H10 62-5 HEp 2 nd nd nd 1 4 ndssDNA nd nd nd nd nd

402B6 2 6 HEp 2 0 8 nd nd nd 28 8ssDNA 8-1 nd nd nd 8-8

442G3 5-0 HEp 2 1-8 104 > 200 0 3 12-4ssDNA 52-0 144 > 200 1-2 9 8

452B7 90 HEp 2 > 111 nd nd 6-3 ndssDNA 13 9 nd nd < 0-2 nd

452F8 7 0 HEp 2 7-0 nd nd nd ndssDNA 8 9 nd nd nd nd

501F7 8 0 HEp 2 15 0 nd nd nd ndssDNA 2-2 nd nd nd nd

nd = Not done.

with Falcon polyvinyl chloride immunoassay plates (Haskard et al. 1985). Supernatant from therheumatoid factor secreting clone 442 E7 (see above) was also tested on these assays and gaveuniformly negative results.

The pattern ofcross-reactivity found amongst the anti-vimentin antibodies correlated fairly wellwith the observed immunofluorescence pattern. Antibodies 402 B6, 452 F8, and 501 F7, all causinga 'pure' + vimentin staining, reacted well with ssDNA and, in two cases, with higher concentrationsof PPGM. Antibody 442 G3, the only antibody which in addition caused nucleolar staining, wasalso the only antibody to react with every tested variety of nucleic acid, cardiolipin and PPGM.Antibody 452 B7, which in common with 31 1 H 10 caused a pre-keratin like staining pattern, reactedwith ssDNA and cardiolipin only at very high concentrations. It is possible that 311 H10 failed toreact only because it was too dilute. No evidence ofcross-reactivity was found for 421 BI 1, 461 C1 1,473 E8, 492 D2, and 503 D lI.

Competitive inhibition. Inhibition experiments were carried out on selected supernatants whichhas shown evidence of cross-reactivity. The inhibition results (Table 3) were in substantialagreement with the direct tests and confirmed that the various antibodies are not identical inspecificity. However, some extra reactions were observed and occasionally a reaction which clearlyoccurred on the solid-phase could not be seen in the liquid phase. Cardiolipin, for example,inhibited in every system in which it was tested, and particularly inhibited 452 B7, with which it didnot apparently react except at high concentration in the direct test. In most cases the HEp 2 cellELISA reaction was inhibited as expected by purified antigens. The unexplained exception was thefailure of inhibition by ssDNA of the reaction with 452 B7.

Tests of supernatants from the cross-reactive clones 311 H 10, 402 B6, 442 G3 and 503 D5, andthe rheumatoid factor 442 E7 against gelatin, collagen types I, II and III, actin and fibronectin wereall negative.

Electrophoresis and immunoblotting. The results obtained from electrophoresis and immunob-lotting are summarized in Table 4. A set of seven proteins in the molecular weight range14,000-66,000 was used for standards in calculating the molecular weights of antigens detected bythe human antibodies. We were unable to find material reactive with two of the clones (421 B 1 and473 E8). Each of the remainder gave one of two patterns: most stained a double band in the

D. 0. Haskard et al.500

Monoclonal antibodies in rheumatoid arthritisTable 4. Molecular weights of antigens stained by monoclonal antibodies

Band molecular weight (x 103)

Clone 46 48 53 56 58 70

311H10 + + + + + - + -402B6 ++++ - - - -421 B11 - - - - -442G3 ++ ++ + +452B7 + + + + + + -452F8 + + + + + + -473E8 - - - - -501F7 ++++ - - - -503D5 ++ - ++ ++ ++ +

501

46-48Kd region. Antibody 503 D5, which recognised only a single band in this region, also gave adistinctive immunofluorescence pattern and clearly belongs to a different group. All of the positiveantibodies reacted with several bands. This is characteristic of serum antibodies to intermediatefilaments and also of mouse monoclonal antibodies to these antigens (Lane, 1982). It was no morepossible to define these antibodies in terms of conventional specificity by immunoblotting than itwas by immunofluorescence or ELISA.

DISCUSSION

We have no doubt that the supernatants tested in this paper contained antibodies that wereeffectively monoclonal, even though our initial work made it clear that many were derived fromwells which originally contained more than one immunoglobulin producing clone. As the incidenceof a positive result on our HEp 2 ELISA was 0-047 (the number of positive clones divided by thetotal number of IgM secreting clones tested), the probability of two positive clones growing in thesame well is only 0-002. Hybrids that have been subcloned (402 B6, 442 E7, 503 D5) have retainedtheir initial antigen specificities. It should be noted, however, that estimates of IgM in culturesupernatants may overestimate antibody concentrations (and therefore underestimate theiractivity) because of the presence of irrelevant IgM in the supernatants.

The antibodies we have found can roughly be classified into a minority that give circumscribedreactions and the rest that react with intermediate filaments with or without other cross-reactions.The former group is confined to 261 D12 and 473 E8, which give similar nuclear staining onimmunofluorescence; 442 E7, which is a restricted rheumatoid factor, and 503 D5. These antibodiesshow few cross-reactions with the other antigens against which they have been tested. The fact thatour HEp 2 ELISA readily detected 473 E8 suggests that the small number of clones found to besecreting antibody of circumscribed specificity is not due to assay insensitivity.

The second and larger group of antibodies all gave characteristic vimentin staining. Howeverthere was variation in the other structures with which the antibodies cross-reacted and therefore intheir fine specificity. This was demonstrated not only by the immunofluorescent reactions but with aseries of solid phase assays against nucleic acids, cardiolipin, proteoglycan and immunoassay platesthemselves. Such variation would have been difficult to prove using sera, as it would probably havebeen dismissed as an artefact caused by mixtures of autoreactive antibodies. Attempts to obtain'pure' reactions by dilution or absorption would have led to misleading results.

Similar clones secreting antibodies showing cross-reactions between nucleic acids, proteogly-cans and other cellular constituents have been described by a number of groups (Shoenfeld et al.,1983, Faaber et al., 1984, Andre-Schwartz et al., 1984, Rubin et al., 1984, Rauch et al., 1984, Jacobet al., 1984). Many of these were derived from patients with systemic lupus erythematosus (SLE) or

502 D. 0. Haskard et al.mice with an SLE like disease, and were initially selected for their reactivity with nucleic acids orimmunoglobulin. It has been implicit in most discussions relating to these antibodies that they are ofdirect relevance to this specific disease.

Our own cross-reactive antibodies were initially identified on an ELISA that could be expectedto screen for a wider range of autoantibodies against cellular constituents and were derived frompatients with rheumatoid arthritis, in whom antinuclear antibodies are not thought to be animportant feature.

Anti-vimentin antibodies are extremely common in a large number of infections andinflammatory diseases and are frequently found in low titres in 'normal' individuals (Kurki &Virtanen 1984). The cross-reactive antibodies that we describe may be part of this antibodypopulation, and be a general manifestation of inflammation in the same manner as acute phasereactants.

We thank Dr J. C. Roder (Queens University, Kingston, Ontario) for providing the KR4 cell line; Dr 1. Olsen(Kennedy Institute) for providing the DNA; and Mr R. A. Lake (Chelsea College) for providing the fibronectin.This work was supported by the Arthritis and Rheumatism Council for Research. DOH was in receipt of anArthritis and Rheumatism Council Research Fellowship.

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