CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 1994, p. 71-77 Vol. 1, No. 11071-412X/94/$04.00+0Copyright © 1994, American Society for Microbiology

A Confocal Microscopy Study of Anticytoskeletal AntibodyActivity in Patients with Connective Tissue Disease

PETER W. FRENCH,* RONALD PENNY, AND JIA-LIN YANGtCentre for Immunology, St. Vincent's Hospital, Victoria Street, Sydney 2010,

New South Wales, Australia

Received 29 July 1993/Returned for modification 27 August 1993/Accepted 10 September 1993

The significance of the presence of antibodies to cytoskeleton proteins in patients with connective tissuediseases is not clear, as there is a high level of these antibodies in healthy controls. In an attempt to improvethe visualization of the immunofluorescence binding pattern of autoantibodies to cytoskeletal structures incultured fibroblasts, we have used confocal microscopy. Of the 256 serum samples tested, 155 (61%) werereactive with cytoplasmic structures. These reactive samples could be divided into seven patterns of binding,as determined by double-blind examination of single-section confocal images. While confirming the results ofprevious immunofluorescence studies which have shown that autoantibodies that bind to filamentousstructures in the cytoplasm of cultured cells are common in patients with connective tissue diseases, we wereable to identify three patterns of cytoskeletal binding which may be useful as an adjunct to other tests for thediagnosis of some connective tissue diseases, in particular systemic sclerosis (scleroderma) and rheumatoidarthritis/Sjogren's syndrome. None of the seven patterns was exclusive to a particular disease. We concludethat confocal microscopy may be of limited use as an adjunct to other serological assays in the diagnosis ofsome forms of connective tissue disease.

The cytoskeleton plays a central role in a large number ofcellular processes, including muscle contraction, cell motility,and secretion (11, 26). Antibodies to cytoplasmic structures areoften associated with "organ-specific" autoimmune diseases.In 1973, Gabbiani et al. (5) demonstrated that some humansera reacted with microfilaments of smooth muscle tissue andof cultured cells. Since then, reactivity of human sera to theother major cytoskeletal systems, microtubules and intermedi-ate filaments, has been described for both pathological condi-tions and normal subjects (2, 7, 8, 20, 21, 25).

Natural autoantibodies present in the serum of healthyindividuals are polyreactive immunoglobulins predominantlyof the immunoglobulin M (IgM) isotype, although otherisotypes are increasingly recognized and are of low affinity (20).Pathologic autoantibodies are typically expressed in high con-centrations, are of the IgG isotype, are of high affinity, and aredefined as being associated with an organ-specific or systemicautoimmune disease (20). The finding of cytoskeleton autoan-tibodies has stimulated research into the function, biochemis-try, and pathology of the cytoskeleton (6, 16). Anticytoskeletonautoantibodies are thought to arise after tissue injury or cellturnover, leading to the release of cytoskeletal antigens intothe circulation (9, 10, 15, 23). It has been postulated that theseantibodies contribute to the chronicity of the inflammatoryprocess (13).The significance of elevated levels of antibodies to cytoskel-

eton proteins in patients with rheumatic disease remainscontroversial. Studies in which the cytoskeletal activity of serafrom patients with rheumatic disease has been described haveused indirect immunofluorescence (21), enzyme-linked immu-nosorbent assays (15), radioimmunoassays (24), and Westernblotting (immunoblotting) (17). There have been no reports of

* Corresponding author. Phone: 61-2-361-7700. Fax: 61-2-361-2391.t Present address: Oncology Research Centre, Prince of Wales

Hospital, Randwick 2031, NSW, Australia.

the use of confocal microscopy to study the pattern of cytoskel-eton binding by sera from patients with rheumatic diseases.Confocal microscopy offers the advantage of being able to takeoptical sections through cells in culture, thus obtaining anunobscured view of the localization of binding of fluorescentlylabeled antibodies to cellular structures. This feature is poten-tially useful for increasing the specificity of the autoantibodypattern of binding to cytoplasmic structures. Using a confocalmicroscope, we were able to define seven distinct patterns ofcytoplasmic cytoskeleton binding in a population of 256 pa-tients. None of these patterns was exclusive to a particulardisease, but combinations of two or three patterns were foundto account for the majority of antibody activity in rheumatoidarthritis and Sjogren's syndrome patients. We conclude thatimmunofluorescence confocal microscopy may be of limiteduse in diagnosis to define the cytoplasmic binding of humanautoantibodies in patients with some forms of connective tissuedisease.

MATERIALS AND METHODS

Serum samples. Serum samples were collected in the clinicaldiagnostic laboratories of St. Vincent's Hospital, Sydney, andSt. George Hospital, Sydney. The serum specimens werestored at - 70°C before use. In total, we studied 256 differentsamples from patients with rheumatic diseases, including rheu-matoid arthritis (44 cases), Sjogren's syndrome (53 cases),systemic lupus erythematosus (35 cases), systemic sclerosis (40cases), mixed connective tissue diseases (20 cases), and sys-temic vasculitis (25 cases), encompassing Wegener's granulo-matosis, polyarteritis, Behcet's disease, and giant cell arteritis;patients with tumors (5 cases) and hepatitis (2 cases); and 32healthy volunteers (controls).The cases of rheumatic disease that we selected all fulfilled

the international classification criteria. The criteria from theAmerican College of Rheumatology (formerly the AmericanRheumatism Association) were used (1, 14, 19, 22) for the

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diagnosis of rheumatoid arthritis, systemic lupus erythemato-sus, systemic sclerosis, and systemic vasculitis; for Sjogren'ssyndrome, the criteria of Fox et al. (4) were used.

Indirect immunofluorescence and confocal microscopy. Forthe immunofluorescence studies, fibroblast cells from humanforeskin tissue biopsy samples were cultured in sterile eight-well chamber slides (Nunc) at 5,000 cells per well for 24 h.Cells were fixed in 3.5% (vol/vol) formaldehyde in phosphate-buffered saline (PBS) containing 0.1% (vol/vol) Triton X-100for 1 h at room temperature (RT). The fixed cells were rinsedwith two changes of PBS, and nonspecific binding was blockedby using 5% (wt/vol) skim milk in PBS for 30 min at RT. Inorder to characterize the cytoskeletal patterns in these cells,monoclonal antibodies to 3-tubulin (Amersham), gelsolin,tropomyosin (Sigma), and vimentin (Serotec Ltd., Oxford,England) were added at appropriate dilutions. Anti-mouse Igconjugated with fluorescein isothiocyanate (FITC; SilenusLaboratories, Hawthorn, Australia) was used to visualize thebinding of these antibodies. To visualize F-actin, phalloidinlabeled with FITC (Sigma) was used. Patient sera were diluted1:20 in PBS and added to the cells for 1 h at RT. The cells werewashed in two changes of PBS containing 0.05% (vol/vol)Tween 20 (PBS-T). FITC-conjugated anti-human IgG, gammachain specific (Silenus), was diluted 1:40 in PBS-T and addedto the wells for 1 h at RT. After being washed in PBS-T asabove, the plastic chambers were removed and the slides weremounted in 90% (vol/vol) glycerol-10% (vol/vol) PBS (pH 8.0)containing 1 mg ofp-phenylenediamine per ml as an antifadingagent. Cells were examined under UV fluorescence micros-copy, with excitation at 488 nm.

In order to more closely examine the localization of thecytoskeleton binding, confocal laser scanning microscopy wasperformed with a Sarastro 2000 confocal laser scanning micro-scope (Molecular Dynamics, Sunnyvale, Calif.) with a planapochromat 60 x /1.40 numerical aperture (NA) oil immersionlens and an argon-ion class II laser. Cells were prepared forimmunofluorescence as described above. Optical sections(usually at 0.3-,um intervals) through FITC-labeled cells werecaptured with a 50-pLm fixed pinhole, with excitation at 488 nm,a 510-nm beam splitter, and a 510-nm barrier filter. Imageprocessing was performed with a Silicon Graphics Personal Iris4D 35 workstation.

Transparencies (35 mm) were made of all images obtainedand separated into common staining patterns by double-blindprocedures.

Statistical analysis. In order to test whether correlationsbetween disease and confocal fluorescence patterns could haveoccurred by chance, a chi-square test was performed on thedata.

RESULTS

Comparison between standard fluorescence and confocalmicroscopy for the examination of anticytoskeletal autoanti-body patterns. Confocal microscopy was used in this study totake single optical sections midway through the fibroblasts.The fibroblasts are flat, well-spread cells, and consequently themain difference between confocal microscope images and thestandard immunofluorescence images was that the out-of-focus flaring was eliminated, resulting in clearer localization ofthe fluorescence staining without giving images substantiallydifferent from those derived by standard fluorescence. This wasmost useful in defining staining which appeared diffusethroughout the cytoplasm by standard fluorescence; with con-focal microscopy, fine filamentous staining could frequently beresolved (see, e.g., Fig. 2B). When major structures, such as

microfilaments and microtubules, were clearly visible by stan-dard fluorescence, confocal microscopy did not usually provideany advantage in interpretation of the pattern. In addition, inmost cases the localization of the nucleus was more obvious inthe confocal images than in standard fluorescence.

Defining confocal patterns of antiserum binding to fibro-blasts. Figure 1 shows the pattern of distribution of the maincytoskeletal elements in cultured human foreskin fibroblasts.These cells contain microfilaments, as visualized both byantitropomyosin antibody binding (Fig. la) and by phalloidin-FITC binding (Fig. lb); microtubules (Fig. ld); and interme-diate filaments, as visualized by antivimentin antibody binding(Fig. lc and f). Vimentin showed variable staining, in that mostcells showed a tight concentration perinuclearly, with fewerfilaments throughout the cytoplasm (Fig. lc), but some cellsshowed a more general distribution throughout the cytoplasm(Fig. lf). A cytoskeleton-associated protein, gelsolin, exhibiteda different distribution than these three major cytoskeletalfilament systems (Fig. le). These six patterns were used tocharacterize patient sera reactivities in these cells.Antiserum binding to cytoplasmic structures of fibroblasts.

Of the 256 serum samples tested, 155 (61%) were reactive withcytoplasmic structures in fixed fibroblast cells as detected byimmunofluorescence. These reactive samples were examinedby confocal microscopy and separated into seven patterns ofbinding, as determined by double-blind examination of trans-parencies of single-section confocal images. The seven patternswere defined as follows.

Microfilament pattern (pattern A, n = 14). Antibody stainsparallel filaments present throughout most of the cell (Fig.2A). This pattern corresponds to actin microfilaments andmultiple associated proteins, of which tropomyosin is one (Fig.la and b). The nucleus was variably reactive.Intermediate filament patterns. (i) Pattern B (n = 20).

Antibody staining is concentrated in the perinuclear region,where there are tight bundles of filaments, single filaments ofwhich are not distinguishable because of their high density.The more peripheral cytoplasmic areas contain a much lowerconcentration of filaments, which are diffuse. Filaments rarelyextend to the cell membrane (Fig. 2B). The nucleus was alwaysunstained. This pattern is similar to the vimentin staining inFig. ic.

(ii) Pattern C (n = 24). Antibody staining is similar topattern B except that there is less of a perinuclear concentra-tion and there are many more filaments throughout thecytoplasm, usually extending to the cell membrane (Fig. 2C).Filaments are not parallel but are arranged in whorls. Nuclearstaining varies from unstained to brightly homogeneous. Thispattern is similar to the vimentin staining shown in Fig. lc.

(iii) Pattern D (n = 35). Antibody staining is punctate toindividual filaments clearly observable throughout the cyto-plasm but not extending to the cell periphery (Fig. 2D). Thestaining in the nucleus is usually weakly diffuse. The punctatestaining of the filaments is similar to that of gelsolin (Fig. le)and suggests that the antigen is not a subunit protein but oneof many filament-associated proteins.

Microtubule staining (pattern G, n = 23). Antibody binds tocytoplasmic filaments which nucleate at the cell nucleus andradiate densely throughout the cytoplasm and to the cellperiphery (Fig. 2G). This pattern correlates with microtubuleand associated proteins (Fig. ld). The nucleus is variablystained.

Nonfilamentous staining. (i) Pattern E (n = 10). Antibodystaining has two definite characteristics, diffuse filamentouscytoplasmic staining and brightly speckled nuclei (Fig. 2E).There appear to be two reactivities in these sera, one to a

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ANTICYTOSKELETAL AUTOANTIBODIES 73

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FIG. 1. Single confocal microscopy sections of cytoskeletal structures in human foreskin fibroblasts. (A) Antitropomyosin antibody binding; (B)actin filaments visualized with phalloidin-FITC; (C and F) antivimentin antibody binding; (D) antitubulin binding; (E) antigelsolin binding. Bars,5 ,um.

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TABLE 1. Observed and expected pattern frequencies

No. of sera giving pattern":Sera" (n)

A B C D E F G

Controls (9)Observed 4 3 2 0 () 0 0Expected t).81 1.16 1.39 2.03 0.58 1.68 1.34

RA (4t))Observed 2 4 8 9 0 5 12Expected 3.61 5.16 6.19 9.03 2.58 7.48 5.94

SS (33)Observed 1 1 5 14 0 4 8Expected 2.98 4.26 5.11 7.45 2.13 6.17 4.9

SLE (27)Observed 1 1 4 10 3 7 1Expected 2.44 3.48 4.18 6.1 1.74 5.05 4.01

SSc (27)Observed 3 11 5 2 5 0 1Expected 2.44 3.48 4.18 6.1 1.74 5.05 4.01

Other (19)Observed 3 () 0 0 2 13 1Expected 1.72 2.45 2.94 4.29 1.23 3.55 2.82

Totals (155) 14 20 24 35 10 29 23

"For definitions, see the legend to Fig. 3."Chi-square, 125.945; P = (.00t)1 for the difference between observed and

expected values. This table gives the chi-square analysis for the 155/256 reactivesamples. The remainder (101) were negative and not included.

nuclear antigen, and the other to an intermediate filamentprotein, similar to the pattern of vimentin staining in Fig. If.

(ii) Pattern F (n = 29). Antibody staining is diffuse through-out the cytoplasm, with an increased concentration around thenucleus. The stain binds not to distinguishable filaments but toa less defined cytoplasmic meshwork (Fig. 2F). This is not aclassical cytoskeletal pattern and was not seen in the standardantiserum staining.

Association of confocal fluorescence patterns with disease:statistical analysis. Chi-square test analysis was performed onthe data in Table 1. The difference between the observedvalues and the expected values was significant at P = 0.0001.However, Fig. 3 shows that no pattern is exclusively diagnosticof a single disease. Nevertheless, some correlations are observ-able.

Pattern A is the least specific, containing representativesfrom every disease tested and from the healthy controls.

Patterns B and C have a similar broad distribution, althoughthe majority of samples with pattern B reactivity are fromscleroderma patients.

Pattern D appears to be specific for connective tissuediseases, with no healthy controls or other disease groupsfalling into this pattern.

Pattern E was rare in this group of samples and includes afew antisera from patients with scleroderma and systemic lupuserythematosus.

Pattern F included serum samples from all the diseasesstudied in this trial but not those from healthy controls. It is thepattern against which most of the other disease groups reacted,in particular, antisera from patients with vasculitis.

Pattern G is predominantly positive for rheumatoid arthritisand Sjogren's syndrome.

DISCUSSION

Anticytoskeletal antibodies have been reported to occur invarious human sera, both in patients with certain diseases andin normal subjects. In some cases, these antibodies are diag-nostic of particular diseases, particularly chronic active hepa-titis (12). In other cases, they may contribute to the pathogen-esis of specific diseases (13). Many studies have attempted todetermine whether these antibodies have diagnostic signifi-cance. However, the significance of these antibodies is unclearbecause of the often high frequencies detected in normalhuman sera (18). Despite the use of a variety of different assaysin an attempt to increase the specificity of detecting anticy-toskeletal antibodies, there has as yet been no clearly definedpattern of binding which is diagnostic for a specific disease.We report here the results of a study with confocal micros-

copy used to examine the pattern of cytoskeletal binding ofantisera from patients with connective tissue diseases. Confo-cal microscopy offers the advantage of being able to visualizeonly those cellular structures which are in the plane of focus,thus eliminating out-of-focus information which can make fora confusing image in standard immunofluorescence tech-niques. It is theoretically possible that an improvement indetecting the structures to which human autoantibodies in serabind may clarify what to now has been an apparent lack of clearcorrelation between the specificity of cytoskeletal autoantibod-ies and rheumatic disease. In addition, it may be possible toidentify two or more antibody specificities in sera by using thistechnology.

This study has confirmed the results of previous studieswhich have shown that autoantibodies that bind to filamentousstructures in the cytoplasm of fibroblasts are common inpatients with connective tissue diseases. Using the technique ofconfocal microscopy, we have found that there is considerableoverlap between the different patterns of binding when com-paring various connective tissue diseases. This may be theresult of multiple cytoskeletal autoantibody specificities withinthe same antiserum.

However, we have identified three patterns of cytoskeletalbinding which may be useful as an adjunct to other tests, suchas those for antinuclear and extractable nuclear antibodies (3)in the evaluation of scleroderma (pattern B), connective tissuediseases generally (pattern D), and rheumatoid arthritis/Sjog-ren's syndrome (pattern G). We can also conclude from thesedata that visualization of microfilament binding (pattern A)appears to be of no use for diagnostic purposes.There are several possibilities to explain why confocal

immunofluorescence microscopy may not provide diagnosticspecificity for the connective tissue diseases. First, the anticy-toskeletal reactivity may arise indirectly as a result of inflam-matory conditions, and therefore this activity on its own is notspecific to a particular disease. Second, there have beenreports of more than one anticytoskeleton antibody specificitybeing present in the same serum sample (21). This maytherefore give rise to the lack of fluorescence pattern specific-

FIG. 2. Single confocal microscopy sections of the binding of human serum samples to human foreskin fibroblasts. One example of each of theseven patterns of binding is shown. (A) Pattern A, serum from a healthy control; (B) pattern B, serum from a patient with systemic scleroderma;(C) pattern C, serum from a patient with systemic scleroderma; (D) pattern D, serum from a patient with rheumatoid arthritis; (E) pattern E,serum from a patient with systemic lupus erythematosus; (F) pattern F, serum from a patient with Sjogren's syndrome; (G) pattern G, serum froma patient with rheumatoid arthritis. Bars, 10 l.m (A and C) or 5 ,um (B, D, E, F, and G).

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76 FRENCH ET AL.

Pattern A Pattern B Pattern C

Other

SSc

SE

SS

RA

Control

3

3

4

0 5 10 15No.

Pattern D

Other 0

SSc 2

SE 10

SS a

RA / 9

Control 0

5 10

Other

SSc

SE

SS

RA

Control

0

11

1

1

4

3

0 5No.

Pattern E

Other

SSc

SE

SS

RA

Control

1 5

No.

0 5 10

No.

10 15

Other lO

Skc

SE

SS

RA

Control

5

5

8

2l l

0 5 1 0 1 5

No.

Pattern F

1 5

Other

SSc

51E

SS

RA

Control

0 5 10 15

No.

Pattern G

Other 1

SSc 1

SE 1

Ss 8

RA 11

Control 0

0 5 10 15

No.

ity associated with connective tissue disease. Third, the methodof cell fixation that we have used (Formalin with Triton X-100)has been shown in this and other studies (4a) to leave themicrofilament, microtubule, and intermediate filament systems

FIG. 3. Association of confocal fluorescence patterns with diseasestate. The diagnosis associated with each antiserum giving one of theseven confocal fluorescence antibody binding patterns A through G(Fig. 2) was tallied in order to determine whether any of the confocalpatterns correlated with specific disease states. The x axis representsthe absolute number of patients, and they axis represents the differentdisease conditions. Control, healthy controls; RA, rheumatoid arthri-tis; SS, Sjogren's syndrome; SLE, systemic lupus erythematosus; SSc,systemic scleroderma; Other, non-connective tissue diseases.

intact and clearly observable, as determined by antibody andphalloidin binding (Fig. 1). The fixation method, however, maybe critical for certain epitopes on associated proteins (21). It ispossible, therefore, that a different fixation protocol may result

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ANTICYTOSKELETAL AUTOANTIBODIES 77

in different patterns of cytoskeleton binding. This will be afuture direction of this work.

ACKNOWLEDGMENTS

We thank Alan Sturgess, St. George Hospital, for provision of serumsamples and Helen Blake, Centre for Immunology, for collection ofserum samples.

Dr. Yang was supported by a research grant from the ArthritisAssociation of Australia.

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