British Journal of Rheumatology 1992;31:185-188
ANTINUCLEAR AND ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA) IN THE SERA OF PATIENTS WITH FELTY'S SYNDROME BY A. JUBY, C. JOHNSTON, P. DAVIS AND A. S. RUSSELL Rheumatic Disease Unit, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
KEY WORDS:
Antinuclear antibodies, Antineutrophil cytoplasmic antibodies (ANCA), Felty's syndrome.
syndrome is a clinical variant of rheumatoid arthritis with a multifactorial and complex pathogenesis [1]. Understanding of this condition has been limited by the relatively few cases observed in any one centre although several extensive reviews with significant numbers of patients have been published [2, 3]. The major functional abnormality appears to result from a reduction in both neutrophil numbers and activity. Various contributing factors have been identified including cellular [4] and humoral mediated immune mechanisms [5], margination and sequestration of neutrophils [6] and defects in neutrophil metabolic activity [7]. In addition, a number of serological abnormalities have been reported in patients with Felty's syndrome. In particular, there is a high prevalence of both rheumatoid factors and fluorescent antinuclear antibodies although the specific reacting nuclear determinant has so far eluded definition [8, 9]. This study was undertaken to report the prevalence of autoantibodies in our patients with Felty's syndrome when compared to other subsets of patients with rheumatoid arthritis, and in particular to report our observations with antineutrophil cytoplasmic antibodies in this group of patients.
from Felty's syndrome [3]. Of these, 17 patients had classical Felty's syndrome manifest by features of rheumatoid disease with absolute neutropenia and splenomegaly. A further 15 patients with classical rheumatoid disease had neutropenia without evidence of a palpable spleen. In these patients their neutropenia could not be attributed to any other disease or drug therapy and marrow biopsies showed normal cellularity. Patients with Felty's syndrome had more severe disease and longer duration of disease than those patients with uncomplicated rheumatoid arthritis. However, the age and sex distribution was similar. The clinical features of our two subgroups of Felty's syndrome patients have previously been reported in more detail [10]. The methods utilized in our laboratory for the detection of autoantibodies have previously been described [11]. The techniques are briefly outlined as follows.
FELTY'S
Fluorescent antinuclear antibody An indirect immunofluorescent method for measuring antinuclear antibodies was used. Rat liver sections (4 um) prepared on the day of testing were used as the tissue substrate. Patient serum, diluted 1:15 in phosphate buffered saline (PBS), was placed on the sections and incubated for 30 min at room temperature in a moist chamber. Slides were then washed to remove excess serum for 10 min in PBS. Fluorescein conjugated antisera were then placed on the sections for a further 30-min incubation, followed by a 10-min wash in PBS. The slides were coverslipped with a glycerol mounting media and then read on a fluorescent microscope. Positive and negative controls were included in each run. Patient results were reported as homogenous, coarse orfinespeckled, peripheral, nucleolar or negative patterns.
PATIENTS AND METHODS Sera from 94 patients were studied utilizing the laboratory techniques outlined below. Patients were grouped as follows. Thirty-nine patients had classical rheumatoid arthritis manifest primarily as peripheral joint synovitis. These patients had no multisystem disease, had normal white counts, non-palpable spleens and serology revealed that their antinuclear antibody test was negative. Twenty-three patients with otherwise similar clinical features were segregated on the basis of a positive fluorescent antinuclear antibody test. Thirty-two patients were classified as suffering
Native DNA A millipore filter technique was used to quantitate the DNA binding. A synthetically prepared, radiolabelled DNA substrate was incubated with patient's
Submitted 2 January; revised version accepted 3 April 1991. Correspondence to Dr P. Davis, Division of Rheumatology, 562 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2. 185
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
SUMMARY The prevalence of antinuclear (ANA) and antineutrophil cytoplasmic antibodies (ANCA) has been studied in the sera of 62 patients with rheumatoid arthritis and 32 patients with Felty's syndrome. The presence of ANA was less in RA than Felty's syndrome (37% versus 69%). Specific autoantibody identification, where possible, was usually of SS-A or SS-B although two sera from patients with Felty's syndrome had low levels of DN A antibody. ANCA was detected in the sera of 33% of patients with Felty's syndrome and was absent in RA sera. The pattern of ANCA staining was either of a diffuse homogenous cytoplasmic or peripheral (pANCA) nature. Classical cytoplasmic granular staining (cANCA) was not identified.
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 3
serum at 37°C for 15 min to allow complexes to form. At the end of this time, the reaction mixture was stopped by the addition of citrate buffer (pH 8.0). The mixture was then filtered onto a 0.45 urn nitrocellulose filter, placed in scintillation vials and allowed to dry overnight. The next day, scintillation cocktail was added and counts per minute (c.p.m.) determined. Patient DNA binding was quantitated by comparison against known controls.
Antineutrophil cytoplasmic antibodies The method recommended at the International Workshop on ANCA, 1988, was utilized [12]. Neutrophils were prepared from heparinized peripheral blood by Ficoll-Paque separation and purified by ammonium chloride lysis (0.83% NH4CI, 0.01 M KH2CO3 and 0.1 mM Na2EDTA). Cells were counted and spun in a Shandon cytospin centrifuge to obtain cell spots of 1.5 x ^cells/slide. Once air dried, they were fixed with cold 95% ethanol for 5-10 min. Slides were safely stored at —20°C, in air-tight containers for several months. Patient and control sera were diluted 1/20 in PBS. Previously prepared slides were removed from the freezer and allowed to come to room temperature in a moist chamber. Serum was applied to the cell spot and incubated for 30 min. After this incubation, the slides were washed in PBS for 10 min. A fluorescein conjugated anti-human IgG antisera was then placed on the slides for an additional 30 min after which a further 10min wash in PBS was performed. Slides were dried and coverslipped with a glycerol mounting media and read on afluorescentmicroscope. Positive and negative controls were included in every run. Readings were graded as to cytoplasmic, peripheral or nuclear staining pattern. The classic ANCA (cANCA) pattern was determined as a diffuse, cytoplasmic, granular staining with central accentuation of the nucleus. A distinct, homogenous, non-granular cytoplasmic staining pattern has also been identified. A perinuclear staining pattern was read as peripheral ANCA (pANCA). RESULTS The distribution of autoantibodies seen in our patients' sera are summarized in Table I. Of the 62 patients with classical rheumatoid arthritis, 23 (37%) had a positive fluorescent antinuclear antibody test. Of these, three had a positive precipitin line to SS-B on immunodiffusion. None of these patients had anti-
DISCUSSION Despite its relative rarity, numerous studies on patients with Felty's syndrome have shown that its pathogenesis is complex and certainly multifactorial [1]. One aspect of Felty's syndrome which has drawn attention but as yet has been unexplained has been the variable reports on the prevalence of antinuclear antibodies in patients' sera [2,3,8]. Our results of fluorescent antinuclear antibodies in two subsets of patients with Felty's syndrome showed a prevalence of 69% for ANA for both groups which is in accord with the prevalence previously reported. However, like other investigators we have been unable to elucidate the nature of the antigen despite testing sera against a
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
Extractable nuclear antigen The method employed screened and identified the EN A antibodies by the double diffusion, Ouchterlony technique. In brief, the patient's serum was screened against rabbit thymus extract (RTE) which was a source of RNP, Sm, Scl-70 and SS-B. Any precipitin lines that develop were further identified by reacting them against known prototype serum. Bovine spleen extract was the source for SS-A. Once identified, antibodies were titred.
bodies to native DNA nor did any of these patients have an antineutrophil cytoplasmic antibody. Our patients with rheumatoid arthritis who were ANA negative were uniformly negative for all the other autoantibodies tested. In contrast, the prevalence of fluorescent antinuclear antibodies in our two groups of patients with Felty's syndrome was significantly higher, being detectable in 12 of 17 (71%) patients with classical Felty's syndrome and 10 of our 15 (67%) patients with rheumatoid arthritis and neutropenia. In three ANA positive RA patients antibodies to SS-B were detected by immunodiffusion. In two of the patients with classical Felty's syndrome antibodies to native DNA were detectable at a level usually only observed in patients with systemic lupus erythematosus. These patients, however, had no clinical features of SLE and did not fulfil criteria for the classification of this disease. Antibodies to extractable nuclear antigens were also observed in these groups of patients with a prevalence somewhat higher than that observed in patients with uncomplicated rheumatoid arthritis. In two of these patients, however, it was not possible to identify the specific antigen producing the immunoprecipitin line despite testing against a batch of known prototype serum for specific antigens. Two patients in this group had antibodies to SS-A. The most striking difference between our patients with uncomplicated rheumatoid arthritis and those with Felty's syndrome was the high prevalence of positive fluorescent staining using the fluorescent antineutrophil cytoplasmic antibody technique. Two distinct patterns of immunofluorescent staining were observed. Four patients had a peripheral nuclear staining pattern (pANCA) while the remaining seven patients had cytoplasmic staining of a more diffuse nature but without the granular staining characteristically reported in patients with Wegener's granulomatosus. The presence or absence of antineutrophil cytoplasmic antibody correlated poorly with the presence or absence of fluorescent antinuclear antibody or any of the other autoantibodies detected in the sera. Nor was it possible to draw any correlation between the presence of ANCA and elevated levels of IgG polymorphonuclear leucocyte binding activity. No clinical differences were apparent between the ANCA positive and negative groups of patients.
JUBY ETAL.: AUTOANTIBODIES IN FELTY'S SYNDROME
187
TABLE I DISTRIBUTION OF AUTOANTIBODIES IN PATIENTS' SERA
Felty's
RA FANA neg (" = 39) ANA nDNA ENA* ANCA
0 0 0 0
FANA pos (« = 23) 23 0 3 0
RA + neutropenia (n = 15) 10 0 1 4
RA+ neutropenia + splenomegaly ( 17) 12 2 3 7
battery of specific antigens. In those few cases where a specific autoantibody was detected this was usually to antigens of the SS-A/SS-B group which has been previously reported in other groups of patients with uncomplicated rheumatoid arthritis. Although the numbers are small it is of note that two of our 17 patients with classical Felty's had levels of native DNA antibody in the range usually only observed in patients with systemic lupus erythematosus. This has been a feature of other studies of sera of patients with Felty's syndrome despite the relative specificity of native DNA antibodies for patients with SLE [2]. The role of these autoantibodies in the pathogenesis of Felty's syndrome and the relationship of patients with classical Felty's syndrome to SLE remain undefined. The most striking serological abnormality in our patients not previously reported was the high prevalence of ANCA in the sera of the patients with Felty's syndrome, being present in seven of 17 with classical Felty's syndrome and four of 15 with rheumatoid arthritis and neutropenia. There has been considerable interest in the role of ANCAs in the pathogenesis of connective tissue diseases and in particular the high specificity and sensitivity of cANCA in patients with Wegener's granulomatosus [13]. In our study none of our patients had cANCA lacking the diffuse granular cytoplasmic staining which is characteristic of this autoantibody. However, a number of our patients did have either a diffuse but more uniform cytoplasmic, nongranular staining pattern or peripheral staining (pANCA). These patterns of staining have been attributed to a variety of antibody systems including those to myeloperoxidase [14] or to granulocyte specific antinuclear antibodies [15]. They have been shown to have less disease specificity and sensitivity than cANCA, having been described in a variety of clinical conditions including rheumatoid vasculitis, polyarteritis nodosa and patients with idiopathic necrotizing and crescentic glomerulonephritis [16]. Our study therefore demonstrates another clinical subset of patients with Felty's syndrome associated with rheumatoid arthritis in whom ANCAs may be detected. However, as antinuclear antibodies and granulocyte specific ANA may complicate the evaluation of antineutrophil cytoplasmic antibodies, it is quite clear that caution and care must be taken in the interpretation of ANCA results. In this regard the use of different fixation techniques and more specific assays may be valuable in clarifying
the role of ANCAs in patients with Felty's syndrome and similar conditions. However, using a standard immunofluorescent technique, the presence of the diffuse staining pattern and pANCA did not correlate in any way with the presence of antinuclear antibodies. Nor was there any relationship to the presence of IgG polymorphonuclear leucocyte binding antibody. In conclusion, we have demonstrated a spectrum of autoantibodies including ANAs and particularly ANCAs in the serum of patients with Felty's syndrome. This study emphasizes the importance of care in the interpretation of ANCA results due to the high specificity of cANCA for Wegener's granulomatosus and the relatively low specificity of diffuse staining and pANCAs for other vasculitides. Our study would suggest that Felty's sera may be another source of value for the investigation of the pathogenic role of ANCAs. ACKNOWLEDGEMENTS
We would like to thank Dr H. Menard, Sherbrooke, Canada and Dr R. Dawkins, Perth, Australia for the donation of positive control sera for our assay. REFERENCES
1. Abdou NI. Heterogeneity of bone marrow-directed immune mechanisms in the pathogenesis of neutropenia in Felty' s syndrome. Arthritis Rheum 1983;26:947-53. 2. Campion G, Madison PJ, Goulding N, etal. The Felty syndrome: a case-matched study of clinical manifestations and outcome, serologic features and immunogenetic associations. Medicine 1990;69: 69-80. 3. Spivak JL. Felty's syndrome: an analytical review. John Hopkins MedJ 1977;141:152-62. 4. Abdou NI, NaPombejara C, Balentine L, Abdou NL. Suppressor cell-mediated neutropenia in Felty's syndrome. J Clin Invest 1978;61:738-43. 5. Starkebaum G, Arend WP, Nordella FA, Gavin SE. Characterizaton of immune complexes and immunoglobulin G antibodies reactive with neutrophils in the sera of patients with Felty's syndrome. J Lab Clin Med 1980;96:238-51. 6. Andrus M, Hurd EL, LoSpalloto J, Ziff M. Comparison of the presence of immune complexes in Felty's syndrome and rheumatoid arthritis. Arthritis Rheum 1978;21:310-15. 7. Chiu P, Davis P, Wong K, Dasgupta M. Superoxide production in neutrophils of patients with RA and Felty's syndrome. J Rheumatol 1983; 10:694-700. 8. Sienknecht CW, Urowitz MB, Pruzanski W, Stein
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
*RA, FANA pos, 3 patients SS-B; RA, neutropenia, 1 patient SS-A; RA, neutropenia + splenomegaly, 2 patients not identifiable, 1 itient SS-A SS-A patient
188
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 3 13. Nolle B, Specks V, Ludemann J, Rohrbach M, DeRemee R, Gross W. Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener's granulomatosis. Ann Intern Med 1989;lll:28-40. 14. Falk R, Jennette J. Antineutrophil cytoplasmic antibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988;318:1651-7. 15. Wiik A, Jensen E, Friis J. Granulocyte specific antinuclear factors in synovial fluids and sera from patients with rheumatoid arthritis. Ann Rheum Dis 1974;33:515-22. 16. Van Der Woude F, Daha M, Van Es L. The current status of neutrophil cytoplasmic antibodies. Clin Exp Immunol 1989;78:143-8.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
HB. Felty's syndrome: clinical and serological analysis of 34 cases. Ann Rheum Dis 1977;36:500-7. 9. Goldberg J, Pinals RS. Felty's syndrome. Semin Arthritis Rheum 1980;10:52-65. 10. Davis P, Johnston C, Bertouch J, Starkebaum G. Depressed superoxide radical generation by neutrophils from patients with rheumatoid arthritis and neutropenia: correlation with neutrophil reactive IgG. Ann Rheum Dis 1987;46:51-4. 11. Juby A, Johnston C, Davis P. Specificity, sensitivity and diagnostic predictive value of selected laboratory generated autoantibody profiles in patients with connective tissue diseases. J Rheumatol 1991; in press. 12. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS 1989; 97 (suppl 6):12-13.
ANTINUCLEAR AND ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA) IN THE SERA OF PATIENTS WITH FELTY'S SYNDROME BY A. JUBY, C. JOHNSTON, P. DAVIS AND A. S. RUSSELL Rheumatic Disease Unit, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
KEY WORDS:
Antinuclear antibodies, Antineutrophil cytoplasmic antibodies (ANCA), Felty's syndrome.
syndrome is a clinical variant of rheumatoid arthritis with a multifactorial and complex pathogenesis [1]. Understanding of this condition has been limited by the relatively few cases observed in any one centre although several extensive reviews with significant numbers of patients have been published [2, 3]. The major functional abnormality appears to result from a reduction in both neutrophil numbers and activity. Various contributing factors have been identified including cellular [4] and humoral mediated immune mechanisms [5], margination and sequestration of neutrophils [6] and defects in neutrophil metabolic activity [7]. In addition, a number of serological abnormalities have been reported in patients with Felty's syndrome. In particular, there is a high prevalence of both rheumatoid factors and fluorescent antinuclear antibodies although the specific reacting nuclear determinant has so far eluded definition [8, 9]. This study was undertaken to report the prevalence of autoantibodies in our patients with Felty's syndrome when compared to other subsets of patients with rheumatoid arthritis, and in particular to report our observations with antineutrophil cytoplasmic antibodies in this group of patients.
from Felty's syndrome [3]. Of these, 17 patients had classical Felty's syndrome manifest by features of rheumatoid disease with absolute neutropenia and splenomegaly. A further 15 patients with classical rheumatoid disease had neutropenia without evidence of a palpable spleen. In these patients their neutropenia could not be attributed to any other disease or drug therapy and marrow biopsies showed normal cellularity. Patients with Felty's syndrome had more severe disease and longer duration of disease than those patients with uncomplicated rheumatoid arthritis. However, the age and sex distribution was similar. The clinical features of our two subgroups of Felty's syndrome patients have previously been reported in more detail [10]. The methods utilized in our laboratory for the detection of autoantibodies have previously been described [11]. The techniques are briefly outlined as follows.
FELTY'S
Fluorescent antinuclear antibody An indirect immunofluorescent method for measuring antinuclear antibodies was used. Rat liver sections (4 um) prepared on the day of testing were used as the tissue substrate. Patient serum, diluted 1:15 in phosphate buffered saline (PBS), was placed on the sections and incubated for 30 min at room temperature in a moist chamber. Slides were then washed to remove excess serum for 10 min in PBS. Fluorescein conjugated antisera were then placed on the sections for a further 30-min incubation, followed by a 10-min wash in PBS. The slides were coverslipped with a glycerol mounting media and then read on a fluorescent microscope. Positive and negative controls were included in each run. Patient results were reported as homogenous, coarse orfinespeckled, peripheral, nucleolar or negative patterns.
PATIENTS AND METHODS Sera from 94 patients were studied utilizing the laboratory techniques outlined below. Patients were grouped as follows. Thirty-nine patients had classical rheumatoid arthritis manifest primarily as peripheral joint synovitis. These patients had no multisystem disease, had normal white counts, non-palpable spleens and serology revealed that their antinuclear antibody test was negative. Twenty-three patients with otherwise similar clinical features were segregated on the basis of a positive fluorescent antinuclear antibody test. Thirty-two patients were classified as suffering
Native DNA A millipore filter technique was used to quantitate the DNA binding. A synthetically prepared, radiolabelled DNA substrate was incubated with patient's
Submitted 2 January; revised version accepted 3 April 1991. Correspondence to Dr P. Davis, Division of Rheumatology, 562 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2. 185
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
SUMMARY The prevalence of antinuclear (ANA) and antineutrophil cytoplasmic antibodies (ANCA) has been studied in the sera of 62 patients with rheumatoid arthritis and 32 patients with Felty's syndrome. The presence of ANA was less in RA than Felty's syndrome (37% versus 69%). Specific autoantibody identification, where possible, was usually of SS-A or SS-B although two sera from patients with Felty's syndrome had low levels of DN A antibody. ANCA was detected in the sera of 33% of patients with Felty's syndrome and was absent in RA sera. The pattern of ANCA staining was either of a diffuse homogenous cytoplasmic or peripheral (pANCA) nature. Classical cytoplasmic granular staining (cANCA) was not identified.
186
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 3
serum at 37°C for 15 min to allow complexes to form. At the end of this time, the reaction mixture was stopped by the addition of citrate buffer (pH 8.0). The mixture was then filtered onto a 0.45 urn nitrocellulose filter, placed in scintillation vials and allowed to dry overnight. The next day, scintillation cocktail was added and counts per minute (c.p.m.) determined. Patient DNA binding was quantitated by comparison against known controls.
Antineutrophil cytoplasmic antibodies The method recommended at the International Workshop on ANCA, 1988, was utilized [12]. Neutrophils were prepared from heparinized peripheral blood by Ficoll-Paque separation and purified by ammonium chloride lysis (0.83% NH4CI, 0.01 M KH2CO3 and 0.1 mM Na2EDTA). Cells were counted and spun in a Shandon cytospin centrifuge to obtain cell spots of 1.5 x ^cells/slide. Once air dried, they were fixed with cold 95% ethanol for 5-10 min. Slides were safely stored at —20°C, in air-tight containers for several months. Patient and control sera were diluted 1/20 in PBS. Previously prepared slides were removed from the freezer and allowed to come to room temperature in a moist chamber. Serum was applied to the cell spot and incubated for 30 min. After this incubation, the slides were washed in PBS for 10 min. A fluorescein conjugated anti-human IgG antisera was then placed on the slides for an additional 30 min after which a further 10min wash in PBS was performed. Slides were dried and coverslipped with a glycerol mounting media and read on afluorescentmicroscope. Positive and negative controls were included in every run. Readings were graded as to cytoplasmic, peripheral or nuclear staining pattern. The classic ANCA (cANCA) pattern was determined as a diffuse, cytoplasmic, granular staining with central accentuation of the nucleus. A distinct, homogenous, non-granular cytoplasmic staining pattern has also been identified. A perinuclear staining pattern was read as peripheral ANCA (pANCA). RESULTS The distribution of autoantibodies seen in our patients' sera are summarized in Table I. Of the 62 patients with classical rheumatoid arthritis, 23 (37%) had a positive fluorescent antinuclear antibody test. Of these, three had a positive precipitin line to SS-B on immunodiffusion. None of these patients had anti-
DISCUSSION Despite its relative rarity, numerous studies on patients with Felty's syndrome have shown that its pathogenesis is complex and certainly multifactorial [1]. One aspect of Felty's syndrome which has drawn attention but as yet has been unexplained has been the variable reports on the prevalence of antinuclear antibodies in patients' sera [2,3,8]. Our results of fluorescent antinuclear antibodies in two subsets of patients with Felty's syndrome showed a prevalence of 69% for ANA for both groups which is in accord with the prevalence previously reported. However, like other investigators we have been unable to elucidate the nature of the antigen despite testing sera against a
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
Extractable nuclear antigen The method employed screened and identified the EN A antibodies by the double diffusion, Ouchterlony technique. In brief, the patient's serum was screened against rabbit thymus extract (RTE) which was a source of RNP, Sm, Scl-70 and SS-B. Any precipitin lines that develop were further identified by reacting them against known prototype serum. Bovine spleen extract was the source for SS-A. Once identified, antibodies were titred.
bodies to native DNA nor did any of these patients have an antineutrophil cytoplasmic antibody. Our patients with rheumatoid arthritis who were ANA negative were uniformly negative for all the other autoantibodies tested. In contrast, the prevalence of fluorescent antinuclear antibodies in our two groups of patients with Felty's syndrome was significantly higher, being detectable in 12 of 17 (71%) patients with classical Felty's syndrome and 10 of our 15 (67%) patients with rheumatoid arthritis and neutropenia. In three ANA positive RA patients antibodies to SS-B were detected by immunodiffusion. In two of the patients with classical Felty's syndrome antibodies to native DNA were detectable at a level usually only observed in patients with systemic lupus erythematosus. These patients, however, had no clinical features of SLE and did not fulfil criteria for the classification of this disease. Antibodies to extractable nuclear antigens were also observed in these groups of patients with a prevalence somewhat higher than that observed in patients with uncomplicated rheumatoid arthritis. In two of these patients, however, it was not possible to identify the specific antigen producing the immunoprecipitin line despite testing against a batch of known prototype serum for specific antigens. Two patients in this group had antibodies to SS-A. The most striking difference between our patients with uncomplicated rheumatoid arthritis and those with Felty's syndrome was the high prevalence of positive fluorescent staining using the fluorescent antineutrophil cytoplasmic antibody technique. Two distinct patterns of immunofluorescent staining were observed. Four patients had a peripheral nuclear staining pattern (pANCA) while the remaining seven patients had cytoplasmic staining of a more diffuse nature but without the granular staining characteristically reported in patients with Wegener's granulomatosus. The presence or absence of antineutrophil cytoplasmic antibody correlated poorly with the presence or absence of fluorescent antinuclear antibody or any of the other autoantibodies detected in the sera. Nor was it possible to draw any correlation between the presence of ANCA and elevated levels of IgG polymorphonuclear leucocyte binding activity. No clinical differences were apparent between the ANCA positive and negative groups of patients.
JUBY ETAL.: AUTOANTIBODIES IN FELTY'S SYNDROME
187
TABLE I DISTRIBUTION OF AUTOANTIBODIES IN PATIENTS' SERA
Felty's
RA FANA neg (" = 39) ANA nDNA ENA* ANCA
0 0 0 0
FANA pos (« = 23) 23 0 3 0
RA + neutropenia (n = 15) 10 0 1 4
RA+ neutropenia + splenomegaly ( 17) 12 2 3 7
battery of specific antigens. In those few cases where a specific autoantibody was detected this was usually to antigens of the SS-A/SS-B group which has been previously reported in other groups of patients with uncomplicated rheumatoid arthritis. Although the numbers are small it is of note that two of our 17 patients with classical Felty's had levels of native DNA antibody in the range usually only observed in patients with systemic lupus erythematosus. This has been a feature of other studies of sera of patients with Felty's syndrome despite the relative specificity of native DNA antibodies for patients with SLE [2]. The role of these autoantibodies in the pathogenesis of Felty's syndrome and the relationship of patients with classical Felty's syndrome to SLE remain undefined. The most striking serological abnormality in our patients not previously reported was the high prevalence of ANCA in the sera of the patients with Felty's syndrome, being present in seven of 17 with classical Felty's syndrome and four of 15 with rheumatoid arthritis and neutropenia. There has been considerable interest in the role of ANCAs in the pathogenesis of connective tissue diseases and in particular the high specificity and sensitivity of cANCA in patients with Wegener's granulomatosus [13]. In our study none of our patients had cANCA lacking the diffuse granular cytoplasmic staining which is characteristic of this autoantibody. However, a number of our patients did have either a diffuse but more uniform cytoplasmic, nongranular staining pattern or peripheral staining (pANCA). These patterns of staining have been attributed to a variety of antibody systems including those to myeloperoxidase [14] or to granulocyte specific antinuclear antibodies [15]. They have been shown to have less disease specificity and sensitivity than cANCA, having been described in a variety of clinical conditions including rheumatoid vasculitis, polyarteritis nodosa and patients with idiopathic necrotizing and crescentic glomerulonephritis [16]. Our study therefore demonstrates another clinical subset of patients with Felty's syndrome associated with rheumatoid arthritis in whom ANCAs may be detected. However, as antinuclear antibodies and granulocyte specific ANA may complicate the evaluation of antineutrophil cytoplasmic antibodies, it is quite clear that caution and care must be taken in the interpretation of ANCA results. In this regard the use of different fixation techniques and more specific assays may be valuable in clarifying
the role of ANCAs in patients with Felty's syndrome and similar conditions. However, using a standard immunofluorescent technique, the presence of the diffuse staining pattern and pANCA did not correlate in any way with the presence of antinuclear antibodies. Nor was there any relationship to the presence of IgG polymorphonuclear leucocyte binding antibody. In conclusion, we have demonstrated a spectrum of autoantibodies including ANAs and particularly ANCAs in the serum of patients with Felty's syndrome. This study emphasizes the importance of care in the interpretation of ANCA results due to the high specificity of cANCA for Wegener's granulomatosus and the relatively low specificity of diffuse staining and pANCAs for other vasculitides. Our study would suggest that Felty's sera may be another source of value for the investigation of the pathogenic role of ANCAs. ACKNOWLEDGEMENTS
We would like to thank Dr H. Menard, Sherbrooke, Canada and Dr R. Dawkins, Perth, Australia for the donation of positive control sera for our assay. REFERENCES
1. Abdou NI. Heterogeneity of bone marrow-directed immune mechanisms in the pathogenesis of neutropenia in Felty' s syndrome. Arthritis Rheum 1983;26:947-53. 2. Campion G, Madison PJ, Goulding N, etal. The Felty syndrome: a case-matched study of clinical manifestations and outcome, serologic features and immunogenetic associations. Medicine 1990;69: 69-80. 3. Spivak JL. Felty's syndrome: an analytical review. John Hopkins MedJ 1977;141:152-62. 4. Abdou NI, NaPombejara C, Balentine L, Abdou NL. Suppressor cell-mediated neutropenia in Felty's syndrome. J Clin Invest 1978;61:738-43. 5. Starkebaum G, Arend WP, Nordella FA, Gavin SE. Characterizaton of immune complexes and immunoglobulin G antibodies reactive with neutrophils in the sera of patients with Felty's syndrome. J Lab Clin Med 1980;96:238-51. 6. Andrus M, Hurd EL, LoSpalloto J, Ziff M. Comparison of the presence of immune complexes in Felty's syndrome and rheumatoid arthritis. Arthritis Rheum 1978;21:310-15. 7. Chiu P, Davis P, Wong K, Dasgupta M. Superoxide production in neutrophils of patients with RA and Felty's syndrome. J Rheumatol 1983; 10:694-700. 8. Sienknecht CW, Urowitz MB, Pruzanski W, Stein
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
*RA, FANA pos, 3 patients SS-B; RA, neutropenia, 1 patient SS-A; RA, neutropenia + splenomegaly, 2 patients not identifiable, 1 itient SS-A SS-A patient
188
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 3 13. Nolle B, Specks V, Ludemann J, Rohrbach M, DeRemee R, Gross W. Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener's granulomatosis. Ann Intern Med 1989;lll:28-40. 14. Falk R, Jennette J. Antineutrophil cytoplasmic antibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988;318:1651-7. 15. Wiik A, Jensen E, Friis J. Granulocyte specific antinuclear factors in synovial fluids and sera from patients with rheumatoid arthritis. Ann Rheum Dis 1974;33:515-22. 16. Van Der Woude F, Daha M, Van Es L. The current status of neutrophil cytoplasmic antibodies. Clin Exp Immunol 1989;78:143-8.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 28, 2015
HB. Felty's syndrome: clinical and serological analysis of 34 cases. Ann Rheum Dis 1977;36:500-7. 9. Goldberg J, Pinals RS. Felty's syndrome. Semin Arthritis Rheum 1980;10:52-65. 10. Davis P, Johnston C, Bertouch J, Starkebaum G. Depressed superoxide radical generation by neutrophils from patients with rheumatoid arthritis and neutropenia: correlation with neutrophil reactive IgG. Ann Rheum Dis 1987;46:51-4. 11. Juby A, Johnston C, Davis P. Specificity, sensitivity and diagnostic predictive value of selected laboratory generated autoantibody profiles in patients with connective tissue diseases. J Rheumatol 1991; in press. 12. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS 1989; 97 (suppl 6):12-13.
