85 1
CHARACTERISTICS OF PATIENTS WITH SERUM ANTIBODIES TO EXTRACTABLE NUCLEAR ANTIGENS JAMES H. LEIBFARTH and ROBERT H. PERSELLIN A distinguishing feature of the mixed connective tissue disease (MCTD) syndrome is the presence in the serum of antibody in high titer to ribonucleoprotein (RNP). To determine whether this was an exclusive observation, a large rheumatic disease population was surveyed for the presence of antibody in high titer to extractable nuclear antigens (ENA) including RNP. Of 650 sera examined, 440 (from 240 patients) had antinuclear antibody. Only 39 patients had serum antibody to ENA in titers 2 1 :200 dilution. In 16 the anti-ENA was shown by RNAse digestion to be anti-RNP. Although many clinical and laboratory characteristics were similar in these two groups, the patients more closely resembled the previously described MCTD syndrome and, importantly, less often had severe renal and central nervous system disease manifestations. Thus the presence of serum antibodies to ENA that are predominantly RNAse-sensitive (anti-RNP) helps to identify a rheumatic disease syndrome and also appears to have prognostic value. From the Rheumatology Division, Department of Medicine, University of Texas Health Science Center at San Antonio. Research supported by grants from the South Central Texas Chapter of The Arthritis Foundation and from the Regional Medical Program of Texas. James H. Leibfarth, M.D.: Fellow in Rheumatology; Robert H . Persellin, M.D.: Professor of Medicine, and Head, Division of Rheumatology. Address reprint requests to Robert H. Persellin, M.D., Division of Rheumatology, Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284. Submitted for publication December 2, 1975; accepted April 14. 1976. Arthritis and Rheumatism, Vol. 19, No. 5 (September-October 1976)
The mixed connective tissue disease (MCTD) syndrome was suggested as a distinct rheumatic entity 5 years ago by Sharp and colleagues (1,2). Clinical features resembled systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), and dermatomyositis (DM), and, importantly, serious renal involvement was rare. Symptoms responded favorably to corticosteroid therapy. The unique feature of these patients was the presence of a serum antibody to an RNAse-sensitive extractable nuclear antigen (ENA), thought to be a ribonucleoprotein (RNP). Reichlin and Mattioli, studying SLE patients with serum antibodies against RNP, also noted that renal disease was less common, but no distinct clinical syndrome was apparent (3,4). The present authors have questioned whether the MCTD syndrome is a distinct entity and whether renal disease is less frequently found in patients with antibodies against RNP. To answer this question, a large rheumatic disease population was examined for the presence of this distinctive antibody. The clinical characteristics of patients with high titer antibody to ENA were compiled and evaluated.
MATERIALS AND METHODS Tanned Cell Hemagglutination for Anti-ENA. ENA was prepared from calf thymus nuclei by previously described methods (2), dialyzed against barbital buffer, 0.15 M ,pH 7.4, and stored at -2OOC until used. Sheep red blood cells (SRBC) (3 to 24 days old) in Alsever’s solution were washed in barbital buffer and tanned with a 1/25,000 solution of tannic acid for
LEIBFARTH AND PERSELLIN
852
10 minutes a t 37°C (5). Ten milligrams of ENA in 1 ml of barbital buffer were used to coat 1 ml of a 33% suspension of tanned SRBC at 37°C for 30 minutes. The tanned, coated SRBC were washed with barbital buffer containing 0.3% heatinactivated, SRBC-absorbed rabbit serum. SRBC absorbed sera were diluted in barbital buffer and 0.05 ml of each dilution was allowed to react overnight a t room temperature in microtiter V plates (Cooke Engineering Company, Alexandria, Virginia) with 0.025 ml of a 1% solution of tanned, coated SRBC. A hemagglutination titer 2 1 :200 was considered positive. Positive control serum was kindly evaluated by Dr. Gordon Sharp and Dr. Morris Reichlin, who confirmed the presence of antibody to ENA. Enzyme Digestion for Determination of Anti-RNP. The hemagglutination of tanned SRBC coated with ENA could be attributable to serum antibodies t o a variety of antigens present in the ENA preparation. To measure serum antibody to the RNP component of ENA, the coated SRBC were digested with RNAse (Ribonuclease A, 5139 U/mg, Worthington Biochemical Corp, Freehold, New Jersey) according t o the method of Holman: 37OC for I hour, 0.2 rng RNAse/ml in phosphate buffer, p H 6.8 (6). The RNAse-digested cells were then used to test for serum antibody. Other Antinuclear Antibody Determinations. Antinuclear antibody was determined by indirect immunofluores-
R N A s RESISTANT
.
BEFORE
cence using rat liver as substrate and a fluoresceinisothiocyanate-labeled polyvalent anti-human immunoglobulin that was raised in the rabbit. Antibody to native DNA was determined by the method of Kredich et al (7), with nitrocellulose filters and Hs-labeled E coli DNA. Complement (C3) was measured by radial imrnunodiffusion (Meloy Laboratory, Springfield, Virginia). Patients. Antibody to ENA was sought in 650 sera from 450 patients with rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis or polymyositis, and scleroderma. Antinuclear antibodies were found in 440 sera from 240 patients. Clinical records from patients with positive sera were reviewed and clinical and laboratory characteristics were compiled and evaluated. Statistical significance was determined by the x* test.
RESULTS Patients with Antibody to ENA. Thirty-nine patients had antibody to ENA in titers greater than or equal to 1 :200. Twenty-three patients had no significant change in antibody titer after digestion of ENA with RNAse (no more than a one-tube dilution decrease), 7 RNASC MARKEDLY
VnvE
.
AFTER
BEFORE
AFlcRBEFoFE
AFTER
RNAa DIGEmoN Fig 1. Anti-ENA titers before and afrer antigen digestion with RNAse. RNAse Resistant: fall in titer of one tube dilution or less; Moderately Sensitive: fall in titer of two to four tube dilutions; Markedly Sensitive:fall in titer greater than four tube dilutions.
SERUM ANTIBODIES TO ENA
853
Table 1. Diagnosisof 39 Patients with Serum Antibody to ENA at a Titer 2 I :200
Systemic lupus erythematosus Drug-induced SLE Progressive systemic sclerosis Dermatomyositis Overlap syndromes Vasculi tis Rheumatoid arthritis Osteoarthritis
Group I (23)*
G r o u p I1 (7)t
Group 111 (9)$
13 0
4 I 0 0 I 0 1 0
2 0 3
1
0 4 0 4
I
1
1 2 0 0
*Number of patients with anti-ENA not susceptible to RNAse: R NAse-resistant. t Number of patients with antibodies t o both R N P (titer decrease of two to four tube dilutions following RNAse) and to non-RNP ENA: RNAse moderately sensitive. $ N u m b e r of patients whose antibody t o ENA is mostly RNAsesensitive (titer decrease of greater than four tube dilutions): RNAse markedly sensitive.
patients showed a moderate fall in antibody titer (a twoto four-tube dilution decrease), and 9 patients showed a marked fall in antibody titer (a greater than four-tube dilution decrease). Figure 1 illustrates individual titer changes. The most frequent diagnosis that resulted from review of clinical records was SLE (Table 1). Of 19 SLE patients, 5 did not meet the diagnostic criteria established by the American Rheumatism Association (8). These 5 patients had multisystem disorders that fit no other clinical classification. Antibody to ENA was found in 1 patient with osteoarthritis but no systemic disease. One patient with dilantin-induced SLE had antibody directed against moderately sensitive ENA. Two patients with multisystem disorders and biopsy evidence of chronic vascular inflammation had antibody to RNP. Five patients with classic or definite rheumatoid arthritis (RA), all having characteristic erosive changes on x-ray, had antibodies to RNAse-insensitive antigen, and 1 also had antibody to RNP. In patients with antibody to both RNAse-sensitive and RNAse-insensitive antigen, overlap syndromes with manifestations of RA and SLE or manifestations of SLE, DM, as well as PSS were noted. Systemic Manifestations. Systemic manifestations with multiple organ involvement were apparent in each group of patients (Table 2). Tight, swollen fingers, Raynaud’s phenomenon, and leg ulcers were more common ( P < 0.05) in the markedly RNAse-sensitive group. Neurologic abnormalities were found in all groups, but severe central nervous system disease-an acute psychosis thought to be secondary to SLE-occurred in only 1
patient with antibody to RNP, and in 4 patients without antibody t o R N P (psychosis, seizure). Rash was less common ( P < 0.05) and the arthritis was nondeforming in the markedly RNAse-sensitive group, whereas other characteristics appeared in similar frequency in the two anti-RNP groups. Laboratory Characteristics. Laboratory characteristics are noted in Table 3. Thirty-eight of the 39 patients had positive antinuclear factor on routine screening of sera diluted 1 : 10. The one negative serum was positive when studied undiluted. The pattern was speckled in all patients. Two patients who had sera that gave homogenous patterns at a I : 10 dilution had speckled patterns at a 1 :20 sera dilution. Pulmonary abnormalities were seen more frequently ( P < 0.05) in the patients with predominantly antibody to RNP. No antibody to native DNA was detected in either group of patients having anti-RNP. Depressed complement and renal abnormalities were seen in all groups (Table 4). Azotemia was infrequent in all groups regardless of their antibody specificities. It is noteworthy that the patient with a diffuse proliferative glomerulonephritis in the mixed antibody group had completely normal renal function (creatinine clearance, 24-hour urine protein, and Addis count). Changing Antibody Titers. Changing titers of anti-ENA were found in 5 of 12 patients in whom simultaneous analysis of sequential serum samples were available (Figure 2). One of these patients (O--U), who had only antibody to RNP, had a fall in antibody titer from 1 : 1,600 to 1 :200 during a 4-year period. During this time the patient developed a peripheral neuropathy Table 2. Clinical Characteristics of 39 Patients with Serum Antibody to ENA at a Titer 2 1:ZOO
Weight loss Fever Fatigue Morning stiffness Rash Tight skin-swollen fingers Alopecia Raynaud’s phenomenon Leg ulcers Lymphadenopathy Pleurocarditis and/or pericarditis Arthritis-arthralgia Neurologic abnormalities *t$ See footnotes t o Table I .
Group I (23)*
G r o u p 11 (7)t
Group 111 (9)$
I2
2 6 7 3
1
9 18 11 11 4 8 7 2
4 0 0
9 8 3 I 6 2 8 5
6
3
2
5 16
4 6 3
4
5
5
0
6 4
LEIBFARTH A N D PERSELLIN
854
Table 3. Laboratory Characteristics of 39 Patients with Serum Antibody to ENA at a Titer 2 I :200
Anemia (Hct < 34%) Leukopenia (WBC < 4000mm3) Abnormal pulmonary findings (interstitial infiltrates or abnormal diffusion capacity) Abnormal esophageal motility Myositis (enzymes or biopsy) Hypergammaglobulinemia ( > 1.8g%)
Hypocomplementemia (C3) Rheumatoid factor Antinuclear antibody Antibodies to D N A Renal abnormalities (> 200 mg/24 hours protein, azotemia or abnormal biopsy)
10
cryptococcal meningitis, the second was a patient with RA and gram-negative bacterial pneumonia, and the third was a patient with SLE and pulmonary vasculitis. All had received long-term corticosteroid therapy and 2 had received immunosuppressive therapy. One of the 7 patients with antibody to RNAse-sensitive and RNAseinsensitive antigen died secondary to malignant hypertension and renal failure. One of the 9 patients in the RNAse-sensitive group died secondary to complications of multiple myeloma. Two patients with anti-RNP have not required steroids or immunosuppressive therapy to control symptoms. One patient with PSS has severe restrictive pulmonary disease that has severely limited his activities. The others have been generally well controlled on varying doses of prednisone.
I2 22 3
DISCUSSION
Group I (23)*
Group 11 (7 )t
Group 111 (9)$
10
2
2
8 4
2 I
0 6
5 8
4 4
10
10
*tS See footnotes to Table I . secondary to vasculitis and received an average of 30 mg of prednisone per day during the last year. Four patients in the RNAse-resistant group, all with the diagnosis of SLE, had a change in antibody titer of four or more tubes. Two patients had hemagglutination titers that fell from 1 : 12,800 to 1:200. One of these patients (0--0) required varying doses of prednisone (5-40 mg/day) to control her symptoms, while the other patient (.--a) also required varying doses of prednisone (10-30 mg/day) plus 9 months of therapy with chlorambucil(4 mg/day). The antibody titer of a third patient with SLE (A--A) fell from 1 :25,600 to 1 : 200 during a 12month period coincident with azathioprine (100 mg/day) and prednisone (75 mg/day) treatment for pulmonary vasculitis. The antibody titer of the fourth fell from 1 : 12,800 to 1 : 1,400 over a 4patient (W--H) year period associated with exacerbation of her multisystem disorder, the appearance of circulating antibody to native DNA, hypocomplementemia, and the development of a focal renal glomerulitis. She responded to corticosteroid therapy (7.5-40 mg/day) and her antiENA titer remained low. The relative amount of antibody to RNP remained constant in all patients’ sera that were serially studied. Prognosis. Three of the 23 patients with antiENA but without anti-RNP have died; one was an SLE patient with diffuse proliferative glomerulonephritis and
The MCTD syndrome has been previously described as an overlap syndrome with manifestations of SLE, DM, and PSS (1,2,9) and has been recently reviewed by Sharp (10). The presence of high titer antibody by hemagglutination assay against the RNP antigen in ENA is the distinctive laboratory characteristic Of this group Of patients. ENA has previously been shown to be comprised Of two major antigenic systems (10711) Plus several lesser antigenic systems (4). The major systems defined are the Sm and RNP antigens. The Sm antigen has been stated to be a marker for SLE (l2,13). Although specific studies to characterize antibodies in the present system other than RNP were not performed, several patients with diseases other than SLE had high titers of anti-
Table 4. Renal Abnormalities in 39 Patients with Serum
Antibody to ENA at a Titer 2 I :200
Proteinuria (> 200 mg/24 hours) Abnormal BUN-creatinine Biopsy Type 1 (focal glomerulitis) Type I 1 (membranous glomerulonephritis) Type I l l (diffuse proliferative glomerulonephritis)
*t$ See footnotes to Table I .
Group I (23)*
Group 11
10 2 6
3 I 4
0
2
2
I
2
I
1
2
I
0
(7)t
Group I11 (9)$ 2 2
SERUM ANTIBODIES TO ENA
Time in Months Fig 2. Changing antibody titers to ENA. SLE patient treated with steroids: .-4;SLE patient treated with steroids and azathioprine: A--k. uasculitis patient treated with steroids and cyclophosphamide: n--n: SLEpatient treated with steroids and chlorambucil: .--a; SLE patient treated with steroids: 0--0.
ENA that were not directed against RNP. These patients had been variously diagnosed as having PSS, RA, and osteoarthritis. Therefore it is possible that in some cases antibody to Sm antigen may be present in sera of non-SLE patients. However further studies are needed to confirm whether the anti-ENA present in the serum of these individuals was directed against the Sm antigen. Patients with MCTD are clinically characterized by rarity of renal disease and by systemic symptoms that respond to steroids. Depressed complement was uncommon in Sharp’s series. Mattioli and Reichlin (3,4), using a precipitin reaction to determine an apparently similar RNAse-sensitive antigen, studied sera from SLE patients, and although renal disease was uncommon, those authors did not note a clinical syndrome distinct from SLE. In both series those patients with antibodies to RNP had a low frequency of antibodies to either native (2) or to single-stranded DNA (4). The current study was designed to evaluate the presence of ENA in a rheumatic disease population, to correlate the clinical and laboratory parameters of
855
patients with and without high titers of antibody to RNP, and to determine if the presence of anti-RNP is significant in defining a distinctive group of patients. The present patients with antibody to ENA had many common clinical and laboratory characteristics. However those patients with mainly anti-RNP appeared to have less serious renal and central nervous system disease, and although the numbers are small, those patients with essentially all antibody to R N P more often had tight, swollen fingers, Raynaud’s phenomenon, leg ulcers, and pulmonary abnormalities. Also, anti-DNA antibody was not found in patients with antiRNP. Thus in many characteristics these patients with anti-RNP resemble those with the previously described MCTD syndrome. Significant adenopathy or dermatitis were not noted in these anti-RNP patients. In contrast to Parker (14), who found that most patients with clinically defined MCTD had antibodies against RNP, clinical and laboratory parameters of the current study could not differentiate between the presence or absence of antibody to RNP. Titers of antibody to ENA have been reported to be relatively constant (1,lO) or variable (15) and to be unassociated with clinical course. Approximately half of the present patients with multiple antibody determinations showed a four tube dilution or greater change in antibody titers. In 2 patients a fall in titer was associated with an exacerbation of disease. In 2 patients the fall in antibody titer associated with cytotoxic therapy may reflect suppression of the immune response. Although titers were noted to fall during therapy, no increase in titer was noted during therapy. Whether antibodies to ENA or ENA itself inhibits the development of renal disease is uncertain. Nephritis was less severe in ENA-treated mice in a study performed by Morris et a1 ( 1 6). In addition Hamburger et a1 have suggested that ENA, by complexing with native DNA, could possibly inhibit DNA anti-DNA complex formation (17). Also, Morris and coworkers have recently demonstrated that ENA effectively inhibits the DNA anti-DNA hemagglutination reaction, but not DNA anti-DNA complexing (16). Further investigations along these intriguing pathways should help with the understanding and possibly the therapy of both renal and central nervous system complications of SLE. In summary, antibody to ENA can be detected in a number of rheumatic diseases. The presence or absence of MCTD cannot be determined by clinical parameters alone. However those patients with antibody t o RNP, especially in high titer, more closely resemble the MCTD syndrome of Sharp, and appear less likely to
LEIBFARTH AND PERSELLIN
856
h a v e severe renal and central nervous system involvement.
ACKNOWLEDGMENTS The authors are indebted to Dr. Nicholas M. Kredich for supplying labeled native DNA, to Dr. Gordon Sharp and Dr. Morris Reichlin for analyzing a serum, to Dr. William Arnold for referring 1 patient, and to Ms. Linda McChesney for technical assistance.
REFERENCES 1. Sharp GC, Irvin WS, LaRoque RL, et al: Association of
2.
3.
4.
5. 6.
7.
autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 50:350-359, 1971 Sharp GC, Irvin WS, Tan EM, et al: Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 52:148-159, I972 Mattioli M, Reichlin M: Characterization of a soluble nuclear ribonucleoprotein antigen reactive with SLE sera. J Immunol 107:1281-1290, 1971 Reichlin M, Mattioli M: Correlation of a precipitin reaction to an RNA protein antigen and a low prevalence of nephritis in patients with systemic lupus erythematosus. N Engl J Med 286:908-91 I , 1972 Stavitsky AB: Micromethods for the study of proteins and antibodies. J lmmunol 72:360-368, 1954 Holman HS: Partial purification and characterization of an extractable nuclear antigen which reacts with SLE sera. Ann N Y Acad Sci 124:800-806, 1965 Kredich NM. Skyler JS. Foote LJ: Antibodies to native
D N A in systemic lupus erythematosus. Arch Intern Med I31 :639-644, 1973 8. Cohen AS, Reynolds WE, Franklin EC, et al: Preliminary criteria for the classification of systemic lupus erythematosus. Bull Rheum Dis 21:643-648, 1971 9. Sharp GC, Irvin WC, May C, et al: Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and other rheumatic diseases. Clin Res 23:50A, 1975 10. Sharp GC: Mixed connective tissue disease. Bull Rheum Dis 25:828-831, 1975 11. Northway JD, Tan EM: Differentiation of antinuclear antibodies giving speckled staining patterns in immunofluorescence. Clin Immunol Immunopathol 1 :140-154, 1972 12. Tan EM, Kunkel HG: Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J lmmunol 96:464-471, 1966 13. Notman D D , Kurata N, Tan EM: Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann Intern Med 83:464-469, 1975 14. Parker MD: Ribonucleoprotein antibodies: frequency and clinical significance in systemic lupus erythematosus, scleroderma, and mixed connective tissue disease. J Lab Clin Med 82:769-775, 1973 15. Basch CM, Epstein WV: Prognostic implications of specificity and titer change in serum antibody to extractable nuclear antigen (ENA). Clin Res 23:132A, 1975 16. Morris AD, Littleton C, Corman LC, et al: Extractable nuclear antigen effect on the DNA anti-DNA reaction a n d N Z B / N Z W mouse nephritis. J Clin Invest 55:903-907, 1975 17. Hamburger M, Freidlander L, Barland P: Interactions of extractable nuclear antigen (ENA) and double stranded DNA. Arthritis Rheum 17:469-475, 1974
CHARACTERISTICS OF PATIENTS WITH SERUM ANTIBODIES TO EXTRACTABLE NUCLEAR ANTIGENS JAMES H. LEIBFARTH and ROBERT H. PERSELLIN A distinguishing feature of the mixed connective tissue disease (MCTD) syndrome is the presence in the serum of antibody in high titer to ribonucleoprotein (RNP). To determine whether this was an exclusive observation, a large rheumatic disease population was surveyed for the presence of antibody in high titer to extractable nuclear antigens (ENA) including RNP. Of 650 sera examined, 440 (from 240 patients) had antinuclear antibody. Only 39 patients had serum antibody to ENA in titers 2 1 :200 dilution. In 16 the anti-ENA was shown by RNAse digestion to be anti-RNP. Although many clinical and laboratory characteristics were similar in these two groups, the patients more closely resembled the previously described MCTD syndrome and, importantly, less often had severe renal and central nervous system disease manifestations. Thus the presence of serum antibodies to ENA that are predominantly RNAse-sensitive (anti-RNP) helps to identify a rheumatic disease syndrome and also appears to have prognostic value. From the Rheumatology Division, Department of Medicine, University of Texas Health Science Center at San Antonio. Research supported by grants from the South Central Texas Chapter of The Arthritis Foundation and from the Regional Medical Program of Texas. James H. Leibfarth, M.D.: Fellow in Rheumatology; Robert H . Persellin, M.D.: Professor of Medicine, and Head, Division of Rheumatology. Address reprint requests to Robert H. Persellin, M.D., Division of Rheumatology, Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284. Submitted for publication December 2, 1975; accepted April 14. 1976. Arthritis and Rheumatism, Vol. 19, No. 5 (September-October 1976)
The mixed connective tissue disease (MCTD) syndrome was suggested as a distinct rheumatic entity 5 years ago by Sharp and colleagues (1,2). Clinical features resembled systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), and dermatomyositis (DM), and, importantly, serious renal involvement was rare. Symptoms responded favorably to corticosteroid therapy. The unique feature of these patients was the presence of a serum antibody to an RNAse-sensitive extractable nuclear antigen (ENA), thought to be a ribonucleoprotein (RNP). Reichlin and Mattioli, studying SLE patients with serum antibodies against RNP, also noted that renal disease was less common, but no distinct clinical syndrome was apparent (3,4). The present authors have questioned whether the MCTD syndrome is a distinct entity and whether renal disease is less frequently found in patients with antibodies against RNP. To answer this question, a large rheumatic disease population was examined for the presence of this distinctive antibody. The clinical characteristics of patients with high titer antibody to ENA were compiled and evaluated.
MATERIALS AND METHODS Tanned Cell Hemagglutination for Anti-ENA. ENA was prepared from calf thymus nuclei by previously described methods (2), dialyzed against barbital buffer, 0.15 M ,pH 7.4, and stored at -2OOC until used. Sheep red blood cells (SRBC) (3 to 24 days old) in Alsever’s solution were washed in barbital buffer and tanned with a 1/25,000 solution of tannic acid for
LEIBFARTH AND PERSELLIN
852
10 minutes a t 37°C (5). Ten milligrams of ENA in 1 ml of barbital buffer were used to coat 1 ml of a 33% suspension of tanned SRBC at 37°C for 30 minutes. The tanned, coated SRBC were washed with barbital buffer containing 0.3% heatinactivated, SRBC-absorbed rabbit serum. SRBC absorbed sera were diluted in barbital buffer and 0.05 ml of each dilution was allowed to react overnight a t room temperature in microtiter V plates (Cooke Engineering Company, Alexandria, Virginia) with 0.025 ml of a 1% solution of tanned, coated SRBC. A hemagglutination titer 2 1 :200 was considered positive. Positive control serum was kindly evaluated by Dr. Gordon Sharp and Dr. Morris Reichlin, who confirmed the presence of antibody to ENA. Enzyme Digestion for Determination of Anti-RNP. The hemagglutination of tanned SRBC coated with ENA could be attributable to serum antibodies t o a variety of antigens present in the ENA preparation. To measure serum antibody to the RNP component of ENA, the coated SRBC were digested with RNAse (Ribonuclease A, 5139 U/mg, Worthington Biochemical Corp, Freehold, New Jersey) according t o the method of Holman: 37OC for I hour, 0.2 rng RNAse/ml in phosphate buffer, p H 6.8 (6). The RNAse-digested cells were then used to test for serum antibody. Other Antinuclear Antibody Determinations. Antinuclear antibody was determined by indirect immunofluores-
R N A s RESISTANT
.
BEFORE
cence using rat liver as substrate and a fluoresceinisothiocyanate-labeled polyvalent anti-human immunoglobulin that was raised in the rabbit. Antibody to native DNA was determined by the method of Kredich et al (7), with nitrocellulose filters and Hs-labeled E coli DNA. Complement (C3) was measured by radial imrnunodiffusion (Meloy Laboratory, Springfield, Virginia). Patients. Antibody to ENA was sought in 650 sera from 450 patients with rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis or polymyositis, and scleroderma. Antinuclear antibodies were found in 440 sera from 240 patients. Clinical records from patients with positive sera were reviewed and clinical and laboratory characteristics were compiled and evaluated. Statistical significance was determined by the x* test.
RESULTS Patients with Antibody to ENA. Thirty-nine patients had antibody to ENA in titers greater than or equal to 1 :200. Twenty-three patients had no significant change in antibody titer after digestion of ENA with RNAse (no more than a one-tube dilution decrease), 7 RNASC MARKEDLY
VnvE
.
AFTER
BEFORE
AFlcRBEFoFE
AFTER
RNAa DIGEmoN Fig 1. Anti-ENA titers before and afrer antigen digestion with RNAse. RNAse Resistant: fall in titer of one tube dilution or less; Moderately Sensitive: fall in titer of two to four tube dilutions; Markedly Sensitive:fall in titer greater than four tube dilutions.
SERUM ANTIBODIES TO ENA
853
Table 1. Diagnosisof 39 Patients with Serum Antibody to ENA at a Titer 2 I :200
Systemic lupus erythematosus Drug-induced SLE Progressive systemic sclerosis Dermatomyositis Overlap syndromes Vasculi tis Rheumatoid arthritis Osteoarthritis
Group I (23)*
G r o u p I1 (7)t
Group 111 (9)$
13 0
4 I 0 0 I 0 1 0
2 0 3
1
0 4 0 4
I
1
1 2 0 0
*Number of patients with anti-ENA not susceptible to RNAse: R NAse-resistant. t Number of patients with antibodies t o both R N P (titer decrease of two to four tube dilutions following RNAse) and to non-RNP ENA: RNAse moderately sensitive. $ N u m b e r of patients whose antibody t o ENA is mostly RNAsesensitive (titer decrease of greater than four tube dilutions): RNAse markedly sensitive.
patients showed a moderate fall in antibody titer (a twoto four-tube dilution decrease), and 9 patients showed a marked fall in antibody titer (a greater than four-tube dilution decrease). Figure 1 illustrates individual titer changes. The most frequent diagnosis that resulted from review of clinical records was SLE (Table 1). Of 19 SLE patients, 5 did not meet the diagnostic criteria established by the American Rheumatism Association (8). These 5 patients had multisystem disorders that fit no other clinical classification. Antibody to ENA was found in 1 patient with osteoarthritis but no systemic disease. One patient with dilantin-induced SLE had antibody directed against moderately sensitive ENA. Two patients with multisystem disorders and biopsy evidence of chronic vascular inflammation had antibody to RNP. Five patients with classic or definite rheumatoid arthritis (RA), all having characteristic erosive changes on x-ray, had antibodies to RNAse-insensitive antigen, and 1 also had antibody to RNP. In patients with antibody to both RNAse-sensitive and RNAse-insensitive antigen, overlap syndromes with manifestations of RA and SLE or manifestations of SLE, DM, as well as PSS were noted. Systemic Manifestations. Systemic manifestations with multiple organ involvement were apparent in each group of patients (Table 2). Tight, swollen fingers, Raynaud’s phenomenon, and leg ulcers were more common ( P < 0.05) in the markedly RNAse-sensitive group. Neurologic abnormalities were found in all groups, but severe central nervous system disease-an acute psychosis thought to be secondary to SLE-occurred in only 1
patient with antibody to RNP, and in 4 patients without antibody t o R N P (psychosis, seizure). Rash was less common ( P < 0.05) and the arthritis was nondeforming in the markedly RNAse-sensitive group, whereas other characteristics appeared in similar frequency in the two anti-RNP groups. Laboratory Characteristics. Laboratory characteristics are noted in Table 3. Thirty-eight of the 39 patients had positive antinuclear factor on routine screening of sera diluted 1 : 10. The one negative serum was positive when studied undiluted. The pattern was speckled in all patients. Two patients who had sera that gave homogenous patterns at a I : 10 dilution had speckled patterns at a 1 :20 sera dilution. Pulmonary abnormalities were seen more frequently ( P < 0.05) in the patients with predominantly antibody to RNP. No antibody to native DNA was detected in either group of patients having anti-RNP. Depressed complement and renal abnormalities were seen in all groups (Table 4). Azotemia was infrequent in all groups regardless of their antibody specificities. It is noteworthy that the patient with a diffuse proliferative glomerulonephritis in the mixed antibody group had completely normal renal function (creatinine clearance, 24-hour urine protein, and Addis count). Changing Antibody Titers. Changing titers of anti-ENA were found in 5 of 12 patients in whom simultaneous analysis of sequential serum samples were available (Figure 2). One of these patients (O--U), who had only antibody to RNP, had a fall in antibody titer from 1 : 1,600 to 1 :200 during a 4-year period. During this time the patient developed a peripheral neuropathy Table 2. Clinical Characteristics of 39 Patients with Serum Antibody to ENA at a Titer 2 1:ZOO
Weight loss Fever Fatigue Morning stiffness Rash Tight skin-swollen fingers Alopecia Raynaud’s phenomenon Leg ulcers Lymphadenopathy Pleurocarditis and/or pericarditis Arthritis-arthralgia Neurologic abnormalities *t$ See footnotes t o Table I .
Group I (23)*
G r o u p 11 (7)t
Group 111 (9)$
I2
2 6 7 3
1
9 18 11 11 4 8 7 2
4 0 0
9 8 3 I 6 2 8 5
6
3
2
5 16
4 6 3
4
5
5
0
6 4
LEIBFARTH A N D PERSELLIN
854
Table 3. Laboratory Characteristics of 39 Patients with Serum Antibody to ENA at a Titer 2 I :200
Anemia (Hct < 34%) Leukopenia (WBC < 4000mm3) Abnormal pulmonary findings (interstitial infiltrates or abnormal diffusion capacity) Abnormal esophageal motility Myositis (enzymes or biopsy) Hypergammaglobulinemia ( > 1.8g%)
Hypocomplementemia (C3) Rheumatoid factor Antinuclear antibody Antibodies to D N A Renal abnormalities (> 200 mg/24 hours protein, azotemia or abnormal biopsy)
10
cryptococcal meningitis, the second was a patient with RA and gram-negative bacterial pneumonia, and the third was a patient with SLE and pulmonary vasculitis. All had received long-term corticosteroid therapy and 2 had received immunosuppressive therapy. One of the 7 patients with antibody to RNAse-sensitive and RNAseinsensitive antigen died secondary to malignant hypertension and renal failure. One of the 9 patients in the RNAse-sensitive group died secondary to complications of multiple myeloma. Two patients with anti-RNP have not required steroids or immunosuppressive therapy to control symptoms. One patient with PSS has severe restrictive pulmonary disease that has severely limited his activities. The others have been generally well controlled on varying doses of prednisone.
I2 22 3
DISCUSSION
Group I (23)*
Group 11 (7 )t
Group 111 (9)$
10
2
2
8 4
2 I
0 6
5 8
4 4
10
10
*tS See footnotes to Table I . secondary to vasculitis and received an average of 30 mg of prednisone per day during the last year. Four patients in the RNAse-resistant group, all with the diagnosis of SLE, had a change in antibody titer of four or more tubes. Two patients had hemagglutination titers that fell from 1 : 12,800 to 1:200. One of these patients (0--0) required varying doses of prednisone (5-40 mg/day) to control her symptoms, while the other patient (.--a) also required varying doses of prednisone (10-30 mg/day) plus 9 months of therapy with chlorambucil(4 mg/day). The antibody titer of a third patient with SLE (A--A) fell from 1 :25,600 to 1 : 200 during a 12month period coincident with azathioprine (100 mg/day) and prednisone (75 mg/day) treatment for pulmonary vasculitis. The antibody titer of the fourth fell from 1 : 12,800 to 1 : 1,400 over a 4patient (W--H) year period associated with exacerbation of her multisystem disorder, the appearance of circulating antibody to native DNA, hypocomplementemia, and the development of a focal renal glomerulitis. She responded to corticosteroid therapy (7.5-40 mg/day) and her antiENA titer remained low. The relative amount of antibody to RNP remained constant in all patients’ sera that were serially studied. Prognosis. Three of the 23 patients with antiENA but without anti-RNP have died; one was an SLE patient with diffuse proliferative glomerulonephritis and
The MCTD syndrome has been previously described as an overlap syndrome with manifestations of SLE, DM, and PSS (1,2,9) and has been recently reviewed by Sharp (10). The presence of high titer antibody by hemagglutination assay against the RNP antigen in ENA is the distinctive laboratory characteristic Of this group Of patients. ENA has previously been shown to be comprised Of two major antigenic systems (10711) Plus several lesser antigenic systems (4). The major systems defined are the Sm and RNP antigens. The Sm antigen has been stated to be a marker for SLE (l2,13). Although specific studies to characterize antibodies in the present system other than RNP were not performed, several patients with diseases other than SLE had high titers of anti-
Table 4. Renal Abnormalities in 39 Patients with Serum
Antibody to ENA at a Titer 2 I :200
Proteinuria (> 200 mg/24 hours) Abnormal BUN-creatinine Biopsy Type 1 (focal glomerulitis) Type I 1 (membranous glomerulonephritis) Type I l l (diffuse proliferative glomerulonephritis)
*t$ See footnotes to Table I .
Group I (23)*
Group 11
10 2 6
3 I 4
0
2
2
I
2
I
1
2
I
0
(7)t
Group I11 (9)$ 2 2
SERUM ANTIBODIES TO ENA
Time in Months Fig 2. Changing antibody titers to ENA. SLE patient treated with steroids: .-4;SLE patient treated with steroids and azathioprine: A--k. uasculitis patient treated with steroids and cyclophosphamide: n--n: SLEpatient treated with steroids and chlorambucil: .--a; SLE patient treated with steroids: 0--0.
ENA that were not directed against RNP. These patients had been variously diagnosed as having PSS, RA, and osteoarthritis. Therefore it is possible that in some cases antibody to Sm antigen may be present in sera of non-SLE patients. However further studies are needed to confirm whether the anti-ENA present in the serum of these individuals was directed against the Sm antigen. Patients with MCTD are clinically characterized by rarity of renal disease and by systemic symptoms that respond to steroids. Depressed complement was uncommon in Sharp’s series. Mattioli and Reichlin (3,4), using a precipitin reaction to determine an apparently similar RNAse-sensitive antigen, studied sera from SLE patients, and although renal disease was uncommon, those authors did not note a clinical syndrome distinct from SLE. In both series those patients with antibodies to RNP had a low frequency of antibodies to either native (2) or to single-stranded DNA (4). The current study was designed to evaluate the presence of ENA in a rheumatic disease population, to correlate the clinical and laboratory parameters of
855
patients with and without high titers of antibody to RNP, and to determine if the presence of anti-RNP is significant in defining a distinctive group of patients. The present patients with antibody to ENA had many common clinical and laboratory characteristics. However those patients with mainly anti-RNP appeared to have less serious renal and central nervous system disease, and although the numbers are small, those patients with essentially all antibody to R N P more often had tight, swollen fingers, Raynaud’s phenomenon, leg ulcers, and pulmonary abnormalities. Also, anti-DNA antibody was not found in patients with antiRNP. Thus in many characteristics these patients with anti-RNP resemble those with the previously described MCTD syndrome. Significant adenopathy or dermatitis were not noted in these anti-RNP patients. In contrast to Parker (14), who found that most patients with clinically defined MCTD had antibodies against RNP, clinical and laboratory parameters of the current study could not differentiate between the presence or absence of antibody to RNP. Titers of antibody to ENA have been reported to be relatively constant (1,lO) or variable (15) and to be unassociated with clinical course. Approximately half of the present patients with multiple antibody determinations showed a four tube dilution or greater change in antibody titers. In 2 patients a fall in titer was associated with an exacerbation of disease. In 2 patients the fall in antibody titer associated with cytotoxic therapy may reflect suppression of the immune response. Although titers were noted to fall during therapy, no increase in titer was noted during therapy. Whether antibodies to ENA or ENA itself inhibits the development of renal disease is uncertain. Nephritis was less severe in ENA-treated mice in a study performed by Morris et a1 ( 1 6). In addition Hamburger et a1 have suggested that ENA, by complexing with native DNA, could possibly inhibit DNA anti-DNA complex formation (17). Also, Morris and coworkers have recently demonstrated that ENA effectively inhibits the DNA anti-DNA hemagglutination reaction, but not DNA anti-DNA complexing (16). Further investigations along these intriguing pathways should help with the understanding and possibly the therapy of both renal and central nervous system complications of SLE. In summary, antibody to ENA can be detected in a number of rheumatic diseases. The presence or absence of MCTD cannot be determined by clinical parameters alone. However those patients with antibody t o RNP, especially in high titer, more closely resemble the MCTD syndrome of Sharp, and appear less likely to
LEIBFARTH AND PERSELLIN
856
h a v e severe renal and central nervous system involvement.
ACKNOWLEDGMENTS The authors are indebted to Dr. Nicholas M. Kredich for supplying labeled native DNA, to Dr. Gordon Sharp and Dr. Morris Reichlin for analyzing a serum, to Dr. William Arnold for referring 1 patient, and to Ms. Linda McChesney for technical assistance.
REFERENCES 1. Sharp GC, Irvin WS, LaRoque RL, et al: Association of
2.
3.
4.
5. 6.
7.
autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 50:350-359, 1971 Sharp GC, Irvin WS, Tan EM, et al: Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 52:148-159, I972 Mattioli M, Reichlin M: Characterization of a soluble nuclear ribonucleoprotein antigen reactive with SLE sera. J Immunol 107:1281-1290, 1971 Reichlin M, Mattioli M: Correlation of a precipitin reaction to an RNA protein antigen and a low prevalence of nephritis in patients with systemic lupus erythematosus. N Engl J Med 286:908-91 I , 1972 Stavitsky AB: Micromethods for the study of proteins and antibodies. J lmmunol 72:360-368, 1954 Holman HS: Partial purification and characterization of an extractable nuclear antigen which reacts with SLE sera. Ann N Y Acad Sci 124:800-806, 1965 Kredich NM. Skyler JS. Foote LJ: Antibodies to native
D N A in systemic lupus erythematosus. Arch Intern Med I31 :639-644, 1973 8. Cohen AS, Reynolds WE, Franklin EC, et al: Preliminary criteria for the classification of systemic lupus erythematosus. Bull Rheum Dis 21:643-648, 1971 9. Sharp GC, Irvin WC, May C, et al: Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and other rheumatic diseases. Clin Res 23:50A, 1975 10. Sharp GC: Mixed connective tissue disease. Bull Rheum Dis 25:828-831, 1975 11. Northway JD, Tan EM: Differentiation of antinuclear antibodies giving speckled staining patterns in immunofluorescence. Clin Immunol Immunopathol 1 :140-154, 1972 12. Tan EM, Kunkel HG: Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J lmmunol 96:464-471, 1966 13. Notman D D , Kurata N, Tan EM: Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann Intern Med 83:464-469, 1975 14. Parker MD: Ribonucleoprotein antibodies: frequency and clinical significance in systemic lupus erythematosus, scleroderma, and mixed connective tissue disease. J Lab Clin Med 82:769-775, 1973 15. Basch CM, Epstein WV: Prognostic implications of specificity and titer change in serum antibody to extractable nuclear antigen (ENA). Clin Res 23:132A, 1975 16. Morris AD, Littleton C, Corman LC, et al: Extractable nuclear antigen effect on the DNA anti-DNA reaction a n d N Z B / N Z W mouse nephritis. J Clin Invest 55:903-907, 1975 17. Hamburger M, Freidlander L, Barland P: Interactions of extractable nuclear antigen (ENA) and double stranded DNA. Arthritis Rheum 17:469-475, 1974
