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The clinical associations of antinuclear antibodies in juvenile rheumatoid arthritis
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
49, 19-27 (1988)
The Clinical Associations of Antinuclear Antibodies Rheumatoid Arthritis
in Juvenile
ALAN M. ROSENBERG Section of Rheumatology,
Department of Pediatrics, University of Saskatchewan, Saskatoon S7N 0X0, Canada
To clarify further the clinical correlates of antinuclear antibodies (ANA) in children with juvenile rheumatoid arthritis (JRA) this study compared the features of 60 ANA positive and 25 ANA negative children with JRA. ANA was more likely to be present in those with pauciarticular JRA than polyarticular JRA particularly if the ANA was of high titer. ANA positive subjects were more likely to have extraarticular manifestations, especially iridocyclitis. No significant differences were observed in onset ages, sex distribution, season of disease onset, family histories, or prognosis. There was no correlation between ANA titer and disease activity. Thus, while certain clinical features do correlate with ANA positivity in JRA, most clinical manifestations do not occur with distinctively different frequencies in the ANA negative and ANA positive groups. 0 1988 Academic
Press, Inc.
INTRODUCTION
Among children with juvenile rheumatoid arthritis (JRA), antinuclear antibodies (ANA) are found frequently enough that their possible pathogenic importance and their potential as serologic discriminators useful in the diagnosis and management of children with JRA ought to be considered. The substantially discrepant prevalences of ANA in JRA previously reported likely reflect, in part, variations in the assays employed for the detection of ANA (including substrates used) and inconsistencies in the criteria applied by different investigators for the establishment of the diagnosis of JRA (1-19). The availability now of improved and more standardized ANA detection techniques and the judicious application of more refined diagnostic criteria have helped foster continued enthusiasm for achieving an improved understanding of the significance of ANA in JRA. To clarify further the potential clinical utility of ANA in JRA we undertook to compare the features of ANA positive and ANA negative children with JRA. MATERIALS
AND METHODS
Subjects. The study group comprised 85 children younger than 17 years of age. All have attended the Pediatric Rheumatic Disease Clinic at the University Hospital, Saskatoon, Canada, for at least 1 year and all have had a disease which conformed to the American Rheumatism Association (ARA) criteria for the diagnosis of JRA (20). Not included in the study group were children with those diseases stipulated in the ARA criteria as diseases to be excluded; also, children with a previously described syndrome of seronegative enthesopathy and arthropathy (SEA Syndrome) (21) were excluded. Detection of ANA by immunojluorescence on Hep-2 cells. Sera were assayed 19 0090-1229/88 $1.50 Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
20
ALAN
M. ROSENBERG
for the presence of ANA by indirect immunofluorescence on Hep-2 cell substrate using reagents from a commercially available kit (Antibodies Incorporated, Davis, CA) as described previously (22). Assays yielding unequivocally positive nuclear fluorescence with clearly discernible patterns of fluorescence were considered positive. To determine the ANA titer, positive sera were serially diluted to a negative endpoint. Sera were considered negative if there was an unequivocal absence of nuclear fluorescence or if immunofluorescence was of insufficient intensity to clearly discern the pattern of fluorescence. Patients whose sera yielded equivocal results were excluded from the study. Patterns of nuclear fluorescence were characterized as: 1. Homogeneous/speckled: A fine, granular staining of the nucleus associated with an underlying, relatively fainter homogeneous fluorescent background staining (Fig. 1). 2. Homogeneous: A solid, diffuse, smooth staining of the interphase nucleus. Metaphase cells show chromosomal staining. 3. Coarse speckled: A course, granular staining of the nucleus without nucleolar staining and without chromosomal staining of the metaphase cell. 4. Discrete speckled (pseudocentromere): Tiny, discrete speckles generally distributed throughout the nucleus. In the interphase cell the appearance may be reminiscent of an anticentromeric pattern but metaphase cells do not demonstrate localization to the chromosomal centromere.
FIG. 1. A homogeneous/speckled --.l--..
fluorescent ANA staining pattern, t ,-I_.. _L..^ ^L__.. ing in additon to a tine residua
ANTINUCLEAR
ANTIBODIES
IN
JRA
21
5. Nucleolar: Large, pleomorphic, homogeneously stained bodies within the nucleus. The nucleoli may be apparent in interphase, anaphase, and telophase cells but not in metaphase. 6. Centromere: Tiny, discrete speckles throughout the interphase cell. In metaphase the speckles are localized to the chromosomal region. Detection of antibodies to specific nuclear antigens. Centers for Disease Control (CDC) reference sera were utilized for assays to native DNA (dsDNA), Sjogren’s syndrome-associated antigens A and B (SS-A and SS-B), extractable nuclear antigen (ENA), Smith antigen (Sm), and nuclear ribonucleoprotein (RNP). Detection of antibodies to dsDNA. Antibodies to dsDNA were detected by a commercially available Crithidia lucillae immunofluorescent assay (Kallestad Laboratories, Austin, TX) performed in accordance with the supplier’s prescribed methodology. The detection of antibodies to ENA. Antibodies to ENA (rabbit thymus, Pel Freeze, Rogers, AR) were detected by counterimmunoelectrophoresis (CIE) (23). ENA positive sera were tested for the presence of antibody to Sm and RNP by Ouchterlony double diffusions; lines of identity with CDC anti-RNP and anti-Sm reference sera were considered indicative of antibodies to the respective antigen. Detection of antibodies to SS-A and SS-B. Antibodies to SS-A and SS-B were detected by Ouchterlony double diffusion utilizing a commercial source of SS-A and SS-B (Zeus Laboratories, Raritan, NJ). Statistical analyses. x2 analyses with Yates continuity corrections were applied for comparison of frequency differences and Pearson’s product moment correlation was applied to determine significance of correlations. Student’s t test was applied for comparison of differences between means. RESULTS Among the 85 study group subjects 60 were ANA positive and 25 were ANA negative (Table 1). In the ANA positive group 41 (68.3%) had pauciarticular disease, 18 (30.0%) had polyarticular JRA, and 1 (1.7%) had systemic onset JRA. In the ANA negative group 10 (40.0%) had pauciarticular onset, 12 (48.0%) had polyarticular onset, and 3 (12.0%) had systemic onset disease. At a titer of >1:80 the frequency with which ANA occurred in the polyarticular and pauciarticular groups did not differ significantly (x2 = 3.006; P = 0.083). When considering only high titer ANA positive subjects (titer 21:320), 25 of the 51 patients with pauciarticular JRA (49%) had a high titer ANA but only 6 of 30 with polyarticular JRA (19.4%) had high titer ANA, a significant difference (x2 = 5.56; P = 0.018). Of the 60 ANA positive subjects 49 (81.7%) were female and 11 (18.3%) were male. The female predominance was maintained but was less striking in the ANA negative group; 18 of the 25 ANA negative subjects (72.0%) were female and 7 (28.0%) were male. The sex distributions in the ANA positive and negative groups did not differ significantly (x2 = 0.087; P = 0.769). ANA positive subjects had a somewhat younger mean onset age than did the ANA negative group but the difference was not significant (t = 0.88; P = 0.62).
22
ALAN
CHARACTERISTICS
OF
M.
ROSENBERG
TABLE 1 ANA POSITIVE AND ANA NEGATIVE
JRA SUBJECTS
Numbers of patients Total
ANA Positive
Patients studied Females Pauciarticular JRA Polyarticular JRA Systemic JRA Extraarticular feature9
85 61 51 30 4 16
Functional class 1 II III
17 6 2
60 49 (82%) 41 18 I ‘P ‘--.-p 54 s I
Pauciarticular
JRA with ANA 2 1:320
ANA Negative 25 18 (72%) 10 I2 3 0 _ 0,016-.’ 23 I I
25 ___j P = 0.018
Polyarticular JRA with ANA 3 1:320 Fluorescent pattern distributionb Homogeneous Speckled Nucleolar Homogeneous Centromeric
61 36 14 4 3 I
a Number of patients without systemic JRA having extraarticular manifestations. b ANA patterns among the 58 patients whose patterns did not change during follow-up.
Significant differences were not apparent when onset subtypes were analyzed separately for age of onset (P 2 0.760). The highest ANA titers displayed by individual ANA positive subjects ranged from 1:80 to 1:10,240 with a median titer of 1:320 and a mode of 1:640. No significant differences were noted between those subjects with high-titer ANA (arbitrarily defined as 3 1:320) and low titer ANA (< 1: 320). However, when only patients with high-titer ANA were compared to ANA negative subjects the ANA positive patients had significantly fewer joints involved (mean number of joints 4.07 t 4.38) than did the ANA negative subjects (7.76 f 7.25) (f = 3.68; P =
0.010). Fifty-eight of the 60 ANA positive subjects (96.7%) retained the same ANA pattern throughout the follow-up period. The frequency distribution of patterns among these 58 ANA positive sera are shown in Table 1. Two of the 60 ANA positive subjects (3.3%) had an ANA pattern variably interpreted as homogeneous and homogeneous/speckled. In only three patients was reactivity to a specific nuclear antigen demonstrable. Two patients had antibodies to dsDNA. One patient was a male with deforming polyarthritis without additional clinical or serologic features of systemic lupus erythematosus (SLE). The second dsDNA-positive patient was the child with JRABLE overlap who had not had antibodies to dsDNA prior to the first clinical expression of her SLE 8 years after the onset of JRA. A female with seropositive,
ANTINUCLEAR
ANTIBODIES
IN JRA
23
nodular, erosive polyarticular JRA and recurrent parotitis had antibodies to RNP and to SS-A. Assays for specific nuclear antigen reactivity were negative in all other sera. When the family histories of ANA positive and ANA negative children were compared no significant differences were observed. Family histories of arthritis, allergy, cancer, diabetes, and thyroid disease did not occur with significantly different frequencies among first-, second-, or third-degree relatives in the two groups (x’ 2 0.685; P 3 0.408). ANA positive subjects were more likely to have extraarticular manifestations associated with their arthritis (x2 = 5.49; P = 0.016) (Table 1). The extraarticular manifestations included iridocyclitis in 10 patients; subcutaneous nodules in 3 patients (all of whom had the rheumatoid factor); splenomegaly and granulocytopenia associated with Felty’s syndrome in 1 patient; recurrent parotitis in a patient with Sjogren’s syndrome; and subcutaneous nodules, pleuritis, and alopecia in the patient with JRASLE overlap. Seventy-seven of the children were in functional class I (ARA criteria (24)) at their last evaluation (mean duration of follow-up in the ANA positive group was 2.91 & 2.23 years; mean duration of follow-up in the ANA negative group was 2.24 + 2.54 years). Of the 6 children with functional class II 5 were in the ANA positive group of 60 (8.3%) and 1 in the ANA negative group of 25 (4.0%). One patient in the ANA positive and 1 patient in the ANA negative groups were in functional class III. None of the patients were in functional class IV. At the time of last assessment 12 of the 60 ANA positive subjects and 5 of the 25 ANA negative subjects (20% in each group) were in clinical remission. To determine if ANA titer reflected disease activity the ANA titer of 11 ANA positive subjects with clinically apparent fluctuations in disease activity were evaluated at 1 year intervals for 5 consecutive years. ANA titers were correlated with white blood cell counts, polymorphonuclear leukocyte numbers, platelet counts, and erythrocyte sedimentation rates. No significant positive correlations were found with any of the four laboratory tests (Y d 0.27). Furthermore, there was no apparent correlation between ANA titers in individual patients and measures of acute phase reactivity. In the ANA positive group, rheumatoid factor titers correlated significantly with the titer of ANA (v = 0.87; P = 0.005). All but three ANA positive patients had persistently positive ANA tests; one ANA positive patient, a boy with systemic onset JRA, had had two determinations interpreted as negative and three as positive; the second patient, a girl with pauciarticular JRA and associated iridocyclitis, had one determination interpreted as negative and three as positive. The child with JRASLE overlap had a negative ANA test early in her course but became persistently ANA positive approximately 4 years prior to the clinical expression of her SLE. To explore possible epidemiological correlates with ANA we undertook to evaluate relationships of ANA status with certain environmental factors. Among the 85 subjects 72 families felt confident in identifying the month of the child’s onset of arthritis. Thirty of the 72 patients (41.7%) had the onset of their disease in the 3-month period of November to January. In comparison, only 13 patients had their onset in each of the two 3-month periods of February to April and August to October (x’= 8.49; P = 0.004) and 16 had their onset in the 3-month
24
ALAN
M.
ROSENBERG
period of May to July (x2= 5.40; P = 0.02). However, the frequencies with which onset occurred in each of the 3-month periods did not differ significantly between the ANA positive and ANA negative groups. At the time of onset 36 of the 60 ANA positive subjects (60%) lived in an urban area and 24 (40%) lived in a rural environment. These frequencies were not different from the number of ANA negative subjects residing in urban (13 patients) or rural (12 patients) areas. For administrative purposes the province of Saskatchewan is divided into 13 health regions. Two of these regions include a city in the southern half of the province (region A) and the immediately adjacent surrounding rural vicinity (region B). Eight of the 25 ANA negative subjects (32.0%) resided in area A or B at the time of diagnosis. In comparison, only 5 of the 60 ANA positive subjects (8.3%) resided in these areas significantly fewer than in the ANA negative group (x2 = 5.91; P = 0.015). No other geographical discrepancies were noted between the two groups. To strive to establish a more uniform ANA positive subgroup, only those patients with a homogeneous/speckled pattern were selected for separate analysis. Analyses of data derived from this subgroup, when compared to the ANA negative group, yielded no significantly different conclusions from those obtained when the entire ANA positive group was studied. In addition to excluding children with diseases stipulated in the ARA criteria we also excluded 12 children who, in addition to having arthritis for 6 weeks or longer, also had enthesitis and negative tests for ANA and rheumatoid factor and thus conformed to criteria for the SEA syndrome. None of these 12 children conformed to diagnostic or classification criteria for other diseases and thus conformed to the currently existing, unmodified criteria for the diagnosis of JRA. To determine if the exclusion of these subjects altered significantly certain of our statistical conclusions, data were subjected to statistical analyses again using these 12 patients. The group of 12 patients was comprised of 10 males and 2 females. All but one had oligoarthritis (fewer than five joints affected). The mean onset age of arthritis was 11 .O +- 2.19 years and the mean number ofjoints affected was 1.75 + 1.60. When statistical analyses were conducted using the expanded group of 97, including the 12 with the SEA syndrome, females were more likely to be ANA positive (x2= 7.21; P = 0.007). Those who were ANA positive had a significantly younger onset age (t = 2.41; P = 0.0085). The 12 patients showed no geographic or seasonal aggregations. When included in analysis of geographic distribution the statistically significant aggregation of ANA negative subjects in regions A and B was lost. Only 7 of 12 patients (58.7%) could identify the month of onset of their arthritis. Of these 7 none had the onset in the months of November, December, or January, the most common period for the onset of JRA. ,4nalysis of data including SEA syndrome patients eliminated the significant increase in disease onset during the November to January period. DISCUSSION
Antinuclear antibody positivity is a serologic hallmark of many rheumatic diseases. The specificity and the quantitation of such autoantibodies can provide useful guidance in the diagnosis and management of a variety of connective tissue
ANTINUCLEAR
ANTIBODIES
IN JRA
25
disorders as exemplified by the association of antibodies to dsDNA and Sm with SLE (25, 26), high titers of antibodies to RNP with mixed connective tissue disease (MCTD) (27), and the association of antibodies to SS-A and SS-B with Sjogren’s syndrome (28). Quantitative fluctuations of certain of these antinuclear antibodies with disease activity (such as that which occurs with antibodies to dsDNA in SLE) suggests the likelihood that certain antinuclear antibodies may be of pathogenic importance. A number of studies designed to determine the incidence of ANA in children with JRA have been reported (l-19) (Table 2). The reported frequencies with which antinuclear antibodies are found in children with JRA have varied substantially. This wide variability likely reflects, in part, variations in populations studied as a result of application of differing diagnostic criteria and inconsistencies in the methodology of ANA determination. The greater precision with which the diagnosis is being made and the emergence of indirect immunofluorescence on Hep-2 cells as a standardized method for the detection of ANA are factors contributing to the generation of frequencies of ANA in JRA that are acceptably consistent. In this present report ANA was more common in the pauciarticular subset than in the polyarticular subjects but only when high titer ANA was considered was a statistically significant difference demonstrated. The differences in onset ages remained insignificant even when only those patients with high titers of ANA were analyzed. The interpatient variability and intrapatient consistency in ANA patterns suggests that even within a relatively homogeneous clinical population the antigenic specificities of the ANA may differ and might suggest that different etiopathogenic mechanisms (reflected by different ANA patterns) may be associated with similar clinical expressions. A so-called homogeneous/speckled pattern, characterized by a tine granular appearance of the nucleus superimposed on a faintly homogeneous solid background was the most common pattern observed, appearing in almost half of the sera tested. Analyses of data comparing patients with a homogeneous/speckled pattern to those who were ANA negative did not yield conclusions different from those derived when analyzing the ANA positive group as a whole. Although we observed seasonal variations in the onset of JRA, differences in season of onset between ANA positive and ANA negative subjects were not observed. ANA positivity was not associated with different incidences of positive family histories. Apart from an apparent tendency for ANA negative subjects to reside in a particular combined urban and rural geographic region, demographic variables did not differ between the two groups. There was no difference in the frequency of the rheumatoid factor in the two groups. In the ANA positive group, however, the titer of nine positive tests for the rheumatoid factor displayed a significant positive correlation with the ANA titer. The association of ANA with uveitis has been well established previously and supported by the observation of ANA positivity in all 10 uveitic patients reported here and the absence of uveitis in any of the ANA negative subjects. All patients with uveitis had either a homogeneous/speckled or discretely speckled pattern suggesting the possibility of at least a partially shared (speckled) nuclear antigen.
~- __
~--
__~
.-____-~-
Author(s) (reference)
i
i
+
+
Diagnostic criteria applied - .__.. -.___ AnselllBywater@ __ .__-____ +
..- __
Number of Percentage ANA -~patients positive Substrate used ARA” __ ~__-~~~__ .~~.__ __ -.~ __~.._ Weir et al. (1) 100 13 Thyroid Bamett et al. (2) 20 11 WBC, thyroid, heart Komreich ef al. (3) 85 19 WBC + Miller et al. (4) 42 16 WBC Bluestone et al. (5) 200 4 WBC Bianco et al. (6) 148 14 NS + Munthe et al. (7) 67 60 WBC i Petty et al. (8) 200 39 Mouse liver Schaller et al. (9) 113 30/88 Rat liver Rudnicki et a[. (10) 85 24 Mouse liver Moore et al. (11) 13 62 Mouse kidney Rosenberg et al. (12) 112 20 WBC Alspaugh et al. (13) 77 57 Mouse kidney + Permin et al. (14) 100 66 WBC, rat liver Pate1 cl a/. (15) 217 60 HEp-2 cells + Rosenberg et al. (16) 61 62 HEp-2 cells t ~.--__ - ~__--~ ~~ - .-._ Note. WBC, white blood cell count: NS, not stated. u Brewer, E. J.. et al., Arthritis Rheum. 20, 195, 1977. ’ Ansell, B. M.. and Bywaters. E. G. L.. Bull. Rheurn. nix. 9, 189. 1959. c 30% of the entire JRA group studied was ANA positive: 88% of girls with JR.4 and associated uveitis were ANA positive
__
TABLE 2 ANTINUCLEAR ANTIBODIES IN JRA ~~ __ ~- -.-.__ -~__--
+
NS
ANTINUCLEAR
ANTIBODIES
IN JR4
27
The ARA criteria applied to identify and classify children with JRA are criteria of exclusion and as such lack the precision of diagnostic criteria applied to most other rheumatic disorders. The potential exists, therefore, for study populations to become contaminated with children having disorders other than JRA but for whom other more appropriate diagnostic criteria are not available. The influence of such contamination is exemplified in this study by documenting significant changes in conclusions when patients with SEA syndrome are included in analyses. Thus, while certain clinical features do correlate with ANA positivity in JRA, most clinical manifestations do not occur with distinctively different frequencies in the ANA negative and ANA positive groups. Even so, the presence of ANA in a child suspected of having JRA would serve to support such a clinical impression. REFERENCES 1. Weir, D. M., Holborow, E. J., and Johnson, G. D., Brif. Med. J. 1, 933, 1961. 2. Bamett, E. V., North, A. F., Condemi, J. J., Jacox, R. F., and Vaughan, J. H., Ann. Intern. Med. 63, 100, 1965. 3. Komreich, H. K., Drexler, E., and Hanson, V., 1. Pediutr. 69, 1039, 1966. 4. Miller, J. J., Henrich, V. L., and Brandstrup, N. E., Pediutrics 38, 916, 1966. 5. Bluestone, R., Goldberg, L. S., Katz, R. M., Marchesano, J. M., and Calabro, J. J., J. Pediarr. 77, 98, 1970. 6. Bianco, N. E., Panush, R. S., Stillman, J. S., and Schur, P. H., Arthritis Rheum. 14, 685, 1971. 7. Munthe, E., &and. J. Rheum. 1, 161, 1972. 8. Petty, R. E., Cassidy, J. T., and Sullivan, D. B., J. Pediutr. 83, 386, 1973. 9. Schaller, J. G., Johnson, G. D., Holborow, E. J., Ansell. B. M., and Smiley, W. K., Arthritis Rheum. 17, 409, 1974. 10. Rudnicki, R. D., Ruder-man, M., Scull, E., Goldenberg, A., and Rothfield, N., Arthritis Rheum. 17, 1007, 1974. 11. Moore, T., Domer, R. W., and Zuckner, J., Ann. Rheum. Dis. 33, 255, 1974. 12. Rosenberg, J. N., Johnson, G. D., Holborow, E. J., and Bywaters, E. G. L., Ann. Rheum. Dis. 34, 350, 1974. 13. Alspaugh, M. A., and Miller, J. J., J. Pediutr. 90, 391, 1977. 14. Permin, H., Horbov, S., Wiik, A., and Knudson, J. V., Acta Puediatr. Sand. 67, 181, 1978. 15. Patel, N. J., Osbom, T. G., Moore, T. L., and Zuckner, J., Arthritis Rheum. 26, S57, 1983. 16. Rosenberg, A. M., Cordeiro, D. M., and Knaus, R. P., Arthritis Rheum. 26, S57, 1983. 17. Moore, T. L., Osbom, T. G., Weiss, T. D., Sheridan, P. W., Eisenwinter, R. K., Miller, A. V., Domer, R. W., and Zuckner, J., Semin. Arthritis Rheum. 13, 329, 1984. 18. Saulsbury, F. T., Clin. Res. 33, 512A, 1985. 19. Leak, A. M., Ansell, B. M., and Burman, S. J., Arch. Dis. Child. 61, 168, 1986. 20. Brewer, E. J., Bass, J., Baum, J., Cassidy, J. T., Fink, C., Jacobs, J., Hanson, V., Levinson, J. E., Schaller, J., and Stillman, J. S., Arthritis Rheum. 20, 195, 1977. 21. Rosenberg, A. M., and Petty, R. E., Arthritis Rheum. 25, 1041, 1972. 22. Rosenberg, A. M., and Prokopchuk, P. A., J. Rheumutol. 15, 148, 1988. 23. Bresnihan, B., Bunn, C., Snaith, M. L., Hughes, G. R., Brit. Med. J. 1, 610, 1977. 24. Steinbrocker, O., Traeger, C. H., and Batterman, R. C., JAMA 148, 659, 1949. 25. Tan, E. M., Carr, R. I., Schur, P. H., and Kunkel, H. G., J. Clin. Invest. 45, 1732, 1966. 26. Tan, E. M., and Kunkel, H. G., J. Immunol. 96, 464, 1966. 27. Sharp, G. C., Irvin, W. S., May, C. M., Holman, H. R., McDuffie, F. C., Hess, E. V., and Schmid, F. R., N. Engl. J. Med. 295, 1149, 1976. 28. Alspaugh, M. A., Talal, N., and Tan, E. M., Arthrifis Rheum. 19, 216, 1976. Received October 20, 1987; accepted with revision May 5, 1988
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
49, 19-27 (1988)
The Clinical Associations of Antinuclear Antibodies Rheumatoid Arthritis
in Juvenile
ALAN M. ROSENBERG Section of Rheumatology,
Department of Pediatrics, University of Saskatchewan, Saskatoon S7N 0X0, Canada
To clarify further the clinical correlates of antinuclear antibodies (ANA) in children with juvenile rheumatoid arthritis (JRA) this study compared the features of 60 ANA positive and 25 ANA negative children with JRA. ANA was more likely to be present in those with pauciarticular JRA than polyarticular JRA particularly if the ANA was of high titer. ANA positive subjects were more likely to have extraarticular manifestations, especially iridocyclitis. No significant differences were observed in onset ages, sex distribution, season of disease onset, family histories, or prognosis. There was no correlation between ANA titer and disease activity. Thus, while certain clinical features do correlate with ANA positivity in JRA, most clinical manifestations do not occur with distinctively different frequencies in the ANA negative and ANA positive groups. 0 1988 Academic
Press, Inc.
INTRODUCTION
Among children with juvenile rheumatoid arthritis (JRA), antinuclear antibodies (ANA) are found frequently enough that their possible pathogenic importance and their potential as serologic discriminators useful in the diagnosis and management of children with JRA ought to be considered. The substantially discrepant prevalences of ANA in JRA previously reported likely reflect, in part, variations in the assays employed for the detection of ANA (including substrates used) and inconsistencies in the criteria applied by different investigators for the establishment of the diagnosis of JRA (1-19). The availability now of improved and more standardized ANA detection techniques and the judicious application of more refined diagnostic criteria have helped foster continued enthusiasm for achieving an improved understanding of the significance of ANA in JRA. To clarify further the potential clinical utility of ANA in JRA we undertook to compare the features of ANA positive and ANA negative children with JRA. MATERIALS
AND METHODS
Subjects. The study group comprised 85 children younger than 17 years of age. All have attended the Pediatric Rheumatic Disease Clinic at the University Hospital, Saskatoon, Canada, for at least 1 year and all have had a disease which conformed to the American Rheumatism Association (ARA) criteria for the diagnosis of JRA (20). Not included in the study group were children with those diseases stipulated in the ARA criteria as diseases to be excluded; also, children with a previously described syndrome of seronegative enthesopathy and arthropathy (SEA Syndrome) (21) were excluded. Detection of ANA by immunojluorescence on Hep-2 cells. Sera were assayed 19 0090-1229/88 $1.50 Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
20
ALAN
M. ROSENBERG
for the presence of ANA by indirect immunofluorescence on Hep-2 cell substrate using reagents from a commercially available kit (Antibodies Incorporated, Davis, CA) as described previously (22). Assays yielding unequivocally positive nuclear fluorescence with clearly discernible patterns of fluorescence were considered positive. To determine the ANA titer, positive sera were serially diluted to a negative endpoint. Sera were considered negative if there was an unequivocal absence of nuclear fluorescence or if immunofluorescence was of insufficient intensity to clearly discern the pattern of fluorescence. Patients whose sera yielded equivocal results were excluded from the study. Patterns of nuclear fluorescence were characterized as: 1. Homogeneous/speckled: A fine, granular staining of the nucleus associated with an underlying, relatively fainter homogeneous fluorescent background staining (Fig. 1). 2. Homogeneous: A solid, diffuse, smooth staining of the interphase nucleus. Metaphase cells show chromosomal staining. 3. Coarse speckled: A course, granular staining of the nucleus without nucleolar staining and without chromosomal staining of the metaphase cell. 4. Discrete speckled (pseudocentromere): Tiny, discrete speckles generally distributed throughout the nucleus. In the interphase cell the appearance may be reminiscent of an anticentromeric pattern but metaphase cells do not demonstrate localization to the chromosomal centromere.
FIG. 1. A homogeneous/speckled --.l--..
fluorescent ANA staining pattern, t ,-I_.. _L..^ ^L__.. ing in additon to a tine residua
ANTINUCLEAR
ANTIBODIES
IN
JRA
21
5. Nucleolar: Large, pleomorphic, homogeneously stained bodies within the nucleus. The nucleoli may be apparent in interphase, anaphase, and telophase cells but not in metaphase. 6. Centromere: Tiny, discrete speckles throughout the interphase cell. In metaphase the speckles are localized to the chromosomal region. Detection of antibodies to specific nuclear antigens. Centers for Disease Control (CDC) reference sera were utilized for assays to native DNA (dsDNA), Sjogren’s syndrome-associated antigens A and B (SS-A and SS-B), extractable nuclear antigen (ENA), Smith antigen (Sm), and nuclear ribonucleoprotein (RNP). Detection of antibodies to dsDNA. Antibodies to dsDNA were detected by a commercially available Crithidia lucillae immunofluorescent assay (Kallestad Laboratories, Austin, TX) performed in accordance with the supplier’s prescribed methodology. The detection of antibodies to ENA. Antibodies to ENA (rabbit thymus, Pel Freeze, Rogers, AR) were detected by counterimmunoelectrophoresis (CIE) (23). ENA positive sera were tested for the presence of antibody to Sm and RNP by Ouchterlony double diffusions; lines of identity with CDC anti-RNP and anti-Sm reference sera were considered indicative of antibodies to the respective antigen. Detection of antibodies to SS-A and SS-B. Antibodies to SS-A and SS-B were detected by Ouchterlony double diffusion utilizing a commercial source of SS-A and SS-B (Zeus Laboratories, Raritan, NJ). Statistical analyses. x2 analyses with Yates continuity corrections were applied for comparison of frequency differences and Pearson’s product moment correlation was applied to determine significance of correlations. Student’s t test was applied for comparison of differences between means. RESULTS Among the 85 study group subjects 60 were ANA positive and 25 were ANA negative (Table 1). In the ANA positive group 41 (68.3%) had pauciarticular disease, 18 (30.0%) had polyarticular JRA, and 1 (1.7%) had systemic onset JRA. In the ANA negative group 10 (40.0%) had pauciarticular onset, 12 (48.0%) had polyarticular onset, and 3 (12.0%) had systemic onset disease. At a titer of >1:80 the frequency with which ANA occurred in the polyarticular and pauciarticular groups did not differ significantly (x2 = 3.006; P = 0.083). When considering only high titer ANA positive subjects (titer 21:320), 25 of the 51 patients with pauciarticular JRA (49%) had a high titer ANA but only 6 of 30 with polyarticular JRA (19.4%) had high titer ANA, a significant difference (x2 = 5.56; P = 0.018). Of the 60 ANA positive subjects 49 (81.7%) were female and 11 (18.3%) were male. The female predominance was maintained but was less striking in the ANA negative group; 18 of the 25 ANA negative subjects (72.0%) were female and 7 (28.0%) were male. The sex distributions in the ANA positive and negative groups did not differ significantly (x2 = 0.087; P = 0.769). ANA positive subjects had a somewhat younger mean onset age than did the ANA negative group but the difference was not significant (t = 0.88; P = 0.62).
22
ALAN
CHARACTERISTICS
OF
M.
ROSENBERG
TABLE 1 ANA POSITIVE AND ANA NEGATIVE
JRA SUBJECTS
Numbers of patients Total
ANA Positive
Patients studied Females Pauciarticular JRA Polyarticular JRA Systemic JRA Extraarticular feature9
85 61 51 30 4 16
Functional class 1 II III
17 6 2
60 49 (82%) 41 18 I ‘P ‘--.-p 54 s I
Pauciarticular
JRA with ANA 2 1:320
ANA Negative 25 18 (72%) 10 I2 3 0 _ 0,016-.’ 23 I I
25 ___j P = 0.018
Polyarticular JRA with ANA 3 1:320 Fluorescent pattern distributionb Homogeneous Speckled Nucleolar Homogeneous Centromeric
61 36 14 4 3 I
a Number of patients without systemic JRA having extraarticular manifestations. b ANA patterns among the 58 patients whose patterns did not change during follow-up.
Significant differences were not apparent when onset subtypes were analyzed separately for age of onset (P 2 0.760). The highest ANA titers displayed by individual ANA positive subjects ranged from 1:80 to 1:10,240 with a median titer of 1:320 and a mode of 1:640. No significant differences were noted between those subjects with high-titer ANA (arbitrarily defined as 3 1:320) and low titer ANA (< 1: 320). However, when only patients with high-titer ANA were compared to ANA negative subjects the ANA positive patients had significantly fewer joints involved (mean number of joints 4.07 t 4.38) than did the ANA negative subjects (7.76 f 7.25) (f = 3.68; P =
0.010). Fifty-eight of the 60 ANA positive subjects (96.7%) retained the same ANA pattern throughout the follow-up period. The frequency distribution of patterns among these 58 ANA positive sera are shown in Table 1. Two of the 60 ANA positive subjects (3.3%) had an ANA pattern variably interpreted as homogeneous and homogeneous/speckled. In only three patients was reactivity to a specific nuclear antigen demonstrable. Two patients had antibodies to dsDNA. One patient was a male with deforming polyarthritis without additional clinical or serologic features of systemic lupus erythematosus (SLE). The second dsDNA-positive patient was the child with JRABLE overlap who had not had antibodies to dsDNA prior to the first clinical expression of her SLE 8 years after the onset of JRA. A female with seropositive,
ANTINUCLEAR
ANTIBODIES
IN JRA
23
nodular, erosive polyarticular JRA and recurrent parotitis had antibodies to RNP and to SS-A. Assays for specific nuclear antigen reactivity were negative in all other sera. When the family histories of ANA positive and ANA negative children were compared no significant differences were observed. Family histories of arthritis, allergy, cancer, diabetes, and thyroid disease did not occur with significantly different frequencies among first-, second-, or third-degree relatives in the two groups (x’ 2 0.685; P 3 0.408). ANA positive subjects were more likely to have extraarticular manifestations associated with their arthritis (x2 = 5.49; P = 0.016) (Table 1). The extraarticular manifestations included iridocyclitis in 10 patients; subcutaneous nodules in 3 patients (all of whom had the rheumatoid factor); splenomegaly and granulocytopenia associated with Felty’s syndrome in 1 patient; recurrent parotitis in a patient with Sjogren’s syndrome; and subcutaneous nodules, pleuritis, and alopecia in the patient with JRASLE overlap. Seventy-seven of the children were in functional class I (ARA criteria (24)) at their last evaluation (mean duration of follow-up in the ANA positive group was 2.91 & 2.23 years; mean duration of follow-up in the ANA negative group was 2.24 + 2.54 years). Of the 6 children with functional class II 5 were in the ANA positive group of 60 (8.3%) and 1 in the ANA negative group of 25 (4.0%). One patient in the ANA positive and 1 patient in the ANA negative groups were in functional class III. None of the patients were in functional class IV. At the time of last assessment 12 of the 60 ANA positive subjects and 5 of the 25 ANA negative subjects (20% in each group) were in clinical remission. To determine if ANA titer reflected disease activity the ANA titer of 11 ANA positive subjects with clinically apparent fluctuations in disease activity were evaluated at 1 year intervals for 5 consecutive years. ANA titers were correlated with white blood cell counts, polymorphonuclear leukocyte numbers, platelet counts, and erythrocyte sedimentation rates. No significant positive correlations were found with any of the four laboratory tests (Y d 0.27). Furthermore, there was no apparent correlation between ANA titers in individual patients and measures of acute phase reactivity. In the ANA positive group, rheumatoid factor titers correlated significantly with the titer of ANA (v = 0.87; P = 0.005). All but three ANA positive patients had persistently positive ANA tests; one ANA positive patient, a boy with systemic onset JRA, had had two determinations interpreted as negative and three as positive; the second patient, a girl with pauciarticular JRA and associated iridocyclitis, had one determination interpreted as negative and three as positive. The child with JRASLE overlap had a negative ANA test early in her course but became persistently ANA positive approximately 4 years prior to the clinical expression of her SLE. To explore possible epidemiological correlates with ANA we undertook to evaluate relationships of ANA status with certain environmental factors. Among the 85 subjects 72 families felt confident in identifying the month of the child’s onset of arthritis. Thirty of the 72 patients (41.7%) had the onset of their disease in the 3-month period of November to January. In comparison, only 13 patients had their onset in each of the two 3-month periods of February to April and August to October (x’= 8.49; P = 0.004) and 16 had their onset in the 3-month
24
ALAN
M.
ROSENBERG
period of May to July (x2= 5.40; P = 0.02). However, the frequencies with which onset occurred in each of the 3-month periods did not differ significantly between the ANA positive and ANA negative groups. At the time of onset 36 of the 60 ANA positive subjects (60%) lived in an urban area and 24 (40%) lived in a rural environment. These frequencies were not different from the number of ANA negative subjects residing in urban (13 patients) or rural (12 patients) areas. For administrative purposes the province of Saskatchewan is divided into 13 health regions. Two of these regions include a city in the southern half of the province (region A) and the immediately adjacent surrounding rural vicinity (region B). Eight of the 25 ANA negative subjects (32.0%) resided in area A or B at the time of diagnosis. In comparison, only 5 of the 60 ANA positive subjects (8.3%) resided in these areas significantly fewer than in the ANA negative group (x2 = 5.91; P = 0.015). No other geographical discrepancies were noted between the two groups. To strive to establish a more uniform ANA positive subgroup, only those patients with a homogeneous/speckled pattern were selected for separate analysis. Analyses of data derived from this subgroup, when compared to the ANA negative group, yielded no significantly different conclusions from those obtained when the entire ANA positive group was studied. In addition to excluding children with diseases stipulated in the ARA criteria we also excluded 12 children who, in addition to having arthritis for 6 weeks or longer, also had enthesitis and negative tests for ANA and rheumatoid factor and thus conformed to criteria for the SEA syndrome. None of these 12 children conformed to diagnostic or classification criteria for other diseases and thus conformed to the currently existing, unmodified criteria for the diagnosis of JRA. To determine if the exclusion of these subjects altered significantly certain of our statistical conclusions, data were subjected to statistical analyses again using these 12 patients. The group of 12 patients was comprised of 10 males and 2 females. All but one had oligoarthritis (fewer than five joints affected). The mean onset age of arthritis was 11 .O +- 2.19 years and the mean number ofjoints affected was 1.75 + 1.60. When statistical analyses were conducted using the expanded group of 97, including the 12 with the SEA syndrome, females were more likely to be ANA positive (x2= 7.21; P = 0.007). Those who were ANA positive had a significantly younger onset age (t = 2.41; P = 0.0085). The 12 patients showed no geographic or seasonal aggregations. When included in analysis of geographic distribution the statistically significant aggregation of ANA negative subjects in regions A and B was lost. Only 7 of 12 patients (58.7%) could identify the month of onset of their arthritis. Of these 7 none had the onset in the months of November, December, or January, the most common period for the onset of JRA. ,4nalysis of data including SEA syndrome patients eliminated the significant increase in disease onset during the November to January period. DISCUSSION
Antinuclear antibody positivity is a serologic hallmark of many rheumatic diseases. The specificity and the quantitation of such autoantibodies can provide useful guidance in the diagnosis and management of a variety of connective tissue
ANTINUCLEAR
ANTIBODIES
IN JRA
25
disorders as exemplified by the association of antibodies to dsDNA and Sm with SLE (25, 26), high titers of antibodies to RNP with mixed connective tissue disease (MCTD) (27), and the association of antibodies to SS-A and SS-B with Sjogren’s syndrome (28). Quantitative fluctuations of certain of these antinuclear antibodies with disease activity (such as that which occurs with antibodies to dsDNA in SLE) suggests the likelihood that certain antinuclear antibodies may be of pathogenic importance. A number of studies designed to determine the incidence of ANA in children with JRA have been reported (l-19) (Table 2). The reported frequencies with which antinuclear antibodies are found in children with JRA have varied substantially. This wide variability likely reflects, in part, variations in populations studied as a result of application of differing diagnostic criteria and inconsistencies in the methodology of ANA determination. The greater precision with which the diagnosis is being made and the emergence of indirect immunofluorescence on Hep-2 cells as a standardized method for the detection of ANA are factors contributing to the generation of frequencies of ANA in JRA that are acceptably consistent. In this present report ANA was more common in the pauciarticular subset than in the polyarticular subjects but only when high titer ANA was considered was a statistically significant difference demonstrated. The differences in onset ages remained insignificant even when only those patients with high titers of ANA were analyzed. The interpatient variability and intrapatient consistency in ANA patterns suggests that even within a relatively homogeneous clinical population the antigenic specificities of the ANA may differ and might suggest that different etiopathogenic mechanisms (reflected by different ANA patterns) may be associated with similar clinical expressions. A so-called homogeneous/speckled pattern, characterized by a tine granular appearance of the nucleus superimposed on a faintly homogeneous solid background was the most common pattern observed, appearing in almost half of the sera tested. Analyses of data comparing patients with a homogeneous/speckled pattern to those who were ANA negative did not yield conclusions different from those derived when analyzing the ANA positive group as a whole. Although we observed seasonal variations in the onset of JRA, differences in season of onset between ANA positive and ANA negative subjects were not observed. ANA positivity was not associated with different incidences of positive family histories. Apart from an apparent tendency for ANA negative subjects to reside in a particular combined urban and rural geographic region, demographic variables did not differ between the two groups. There was no difference in the frequency of the rheumatoid factor in the two groups. In the ANA positive group, however, the titer of nine positive tests for the rheumatoid factor displayed a significant positive correlation with the ANA titer. The association of ANA with uveitis has been well established previously and supported by the observation of ANA positivity in all 10 uveitic patients reported here and the absence of uveitis in any of the ANA negative subjects. All patients with uveitis had either a homogeneous/speckled or discretely speckled pattern suggesting the possibility of at least a partially shared (speckled) nuclear antigen.
~- __
~--
__~
.-____-~-
Author(s) (reference)
i
i
+
+
Diagnostic criteria applied - .__.. -.___ AnselllBywater@ __ .__-____ +
..- __
Number of Percentage ANA -~patients positive Substrate used ARA” __ ~__-~~~__ .~~.__ __ -.~ __~.._ Weir et al. (1) 100 13 Thyroid Bamett et al. (2) 20 11 WBC, thyroid, heart Komreich ef al. (3) 85 19 WBC + Miller et al. (4) 42 16 WBC Bluestone et al. (5) 200 4 WBC Bianco et al. (6) 148 14 NS + Munthe et al. (7) 67 60 WBC i Petty et al. (8) 200 39 Mouse liver Schaller et al. (9) 113 30/88 Rat liver Rudnicki et a[. (10) 85 24 Mouse liver Moore et al. (11) 13 62 Mouse kidney Rosenberg et al. (12) 112 20 WBC Alspaugh et al. (13) 77 57 Mouse kidney + Permin et al. (14) 100 66 WBC, rat liver Pate1 cl a/. (15) 217 60 HEp-2 cells + Rosenberg et al. (16) 61 62 HEp-2 cells t ~.--__ - ~__--~ ~~ - .-._ Note. WBC, white blood cell count: NS, not stated. u Brewer, E. J.. et al., Arthritis Rheum. 20, 195, 1977. ’ Ansell, B. M.. and Bywaters. E. G. L.. Bull. Rheurn. nix. 9, 189. 1959. c 30% of the entire JRA group studied was ANA positive: 88% of girls with JR.4 and associated uveitis were ANA positive
__
TABLE 2 ANTINUCLEAR ANTIBODIES IN JRA ~~ __ ~- -.-.__ -~__--
+
NS
ANTINUCLEAR
ANTIBODIES
IN JR4
27
The ARA criteria applied to identify and classify children with JRA are criteria of exclusion and as such lack the precision of diagnostic criteria applied to most other rheumatic disorders. The potential exists, therefore, for study populations to become contaminated with children having disorders other than JRA but for whom other more appropriate diagnostic criteria are not available. The influence of such contamination is exemplified in this study by documenting significant changes in conclusions when patients with SEA syndrome are included in analyses. Thus, while certain clinical features do correlate with ANA positivity in JRA, most clinical manifestations do not occur with distinctively different frequencies in the ANA negative and ANA positive groups. Even so, the presence of ANA in a child suspected of having JRA would serve to support such a clinical impression. REFERENCES 1. Weir, D. M., Holborow, E. J., and Johnson, G. D., Brif. Med. J. 1, 933, 1961. 2. Bamett, E. V., North, A. F., Condemi, J. J., Jacox, R. F., and Vaughan, J. H., Ann. Intern. Med. 63, 100, 1965. 3. Komreich, H. K., Drexler, E., and Hanson, V., 1. Pediutr. 69, 1039, 1966. 4. Miller, J. J., Henrich, V. L., and Brandstrup, N. E., Pediutrics 38, 916, 1966. 5. Bluestone, R., Goldberg, L. S., Katz, R. M., Marchesano, J. M., and Calabro, J. J., J. Pediarr. 77, 98, 1970. 6. Bianco, N. E., Panush, R. S., Stillman, J. S., and Schur, P. H., Arthritis Rheum. 14, 685, 1971. 7. Munthe, E., &and. J. Rheum. 1, 161, 1972. 8. Petty, R. E., Cassidy, J. T., and Sullivan, D. B., J. Pediutr. 83, 386, 1973. 9. Schaller, J. G., Johnson, G. D., Holborow, E. J., Ansell. B. M., and Smiley, W. K., Arthritis Rheum. 17, 409, 1974. 10. Rudnicki, R. D., Ruder-man, M., Scull, E., Goldenberg, A., and Rothfield, N., Arthritis Rheum. 17, 1007, 1974. 11. Moore, T., Domer, R. W., and Zuckner, J., Ann. Rheum. Dis. 33, 255, 1974. 12. Rosenberg, J. N., Johnson, G. D., Holborow, E. J., and Bywaters, E. G. L., Ann. Rheum. Dis. 34, 350, 1974. 13. Alspaugh, M. A., and Miller, J. J., J. Pediutr. 90, 391, 1977. 14. Permin, H., Horbov, S., Wiik, A., and Knudson, J. V., Acta Puediatr. Sand. 67, 181, 1978. 15. Patel, N. J., Osbom, T. G., Moore, T. L., and Zuckner, J., Arthritis Rheum. 26, S57, 1983. 16. Rosenberg, A. M., Cordeiro, D. M., and Knaus, R. P., Arthritis Rheum. 26, S57, 1983. 17. Moore, T. L., Osbom, T. G., Weiss, T. D., Sheridan, P. W., Eisenwinter, R. K., Miller, A. V., Domer, R. W., and Zuckner, J., Semin. Arthritis Rheum. 13, 329, 1984. 18. Saulsbury, F. T., Clin. Res. 33, 512A, 1985. 19. Leak, A. M., Ansell, B. M., and Burman, S. J., Arch. Dis. Child. 61, 168, 1986. 20. Brewer, E. J., Bass, J., Baum, J., Cassidy, J. T., Fink, C., Jacobs, J., Hanson, V., Levinson, J. E., Schaller, J., and Stillman, J. S., Arthritis Rheum. 20, 195, 1977. 21. Rosenberg, A. M., and Petty, R. E., Arthritis Rheum. 25, 1041, 1972. 22. Rosenberg, A. M., and Prokopchuk, P. A., J. Rheumutol. 15, 148, 1988. 23. Bresnihan, B., Bunn, C., Snaith, M. L., Hughes, G. R., Brit. Med. J. 1, 610, 1977. 24. Steinbrocker, O., Traeger, C. H., and Batterman, R. C., JAMA 148, 659, 1949. 25. Tan, E. M., Carr, R. I., Schur, P. H., and Kunkel, H. G., J. Clin. Invest. 45, 1732, 1966. 26. Tan, E. M., and Kunkel, H. G., J. Immunol. 96, 464, 1966. 27. Sharp, G. C., Irvin, W. S., May, C. M., Holman, H. R., McDuffie, F. C., Hess, E. V., and Schmid, F. R., N. Engl. J. Med. 295, 1149, 1976. 28. Alspaugh, M. A., Talal, N., and Tan, E. M., Arthrifis Rheum. 19, 216, 1976. Received October 20, 1987; accepted with revision May 5, 1988