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Antinuclear antibodies and nuclear antigens in rheumatoid synovial fluids
CLINKAL
IMMUNOLOGY
AND
IMMUNOPATHOLOCY
Antinuclear Antibodies in Rheumatoid PIERRE ROBITAILLE*, Division Scripps
1, 385-397
(19%)
and Nuclear Antigens Synovial Fluids’
NATHAN
J. ZVAIFLER,
AND ENG M. TANS
of Allergy und immunology und Department of Erperimentol Pathology, Clinic and Reseurch Foundation and from the Depurtment of Medicine. Unicersity of California at San Diego, La Jollu, California 92037 Receioed
Nowmber
6, 1972;
uccepted
December
26, 1972
Soluble nucleoprotein (sNP) antigen was extracted from calf thymus nuclei and labeled with Yodide. The labeled antigen (12SI-sNP) was used in a sensitive radioimmunoassay to detect either sNP antigen or sNP antibody. Synovial fluids from 54 patients with various rheumatic diseases were examined by this technique. arthritis (RA) patients and sNP antigen was detected in 18 of 31(58 Go / ) in rheumatoid 14 of 23 (61%) in non-R4 patients. The important finding was that 8 of 31 (26%) RA patients’ synovial fluids contained antibody whereas with only one exception, antibody could not be detected in 23 non-R4 patients. Presence of free sNP antigen or homologous antibody in synovial fluids of RA patients suggests that at certain times immune complexes might form and contribute to synovial injury.
Synovial fluid complement levels (CH,,) are generally significantly lower in rheumatoid arthritis (RA) than in other inflammatory joint diseases (l-3). In contrast, serum CH,, values are usually normal or slightly elevated in RA (2,4). Measurements of individual complement component activity in rheumatoid effusion have revealed diminished Cl, C4, C2, and C3 activities (3-5). The pattern of reduction in complement components (4), the presence of C3 convertase (6), and the utilization and consumption of the C3 activator system (7) are highly suggestive of immune activation of the complement sequence in RA synovitis (8). Although the exact mechanisms by which complement might be activated in RA synovial fluid remain unclear, the bulk of evidence supports the concept of complement depletion by immune complexes. Immunoglobulin complexes precipitable by IgM rheumatoid factor or Clq have been isolated from rheumatoid effusions (9,lO). The amount of such complexes was found to be inversely related to synovial fluid CH,, levels (10). Cryoprecipitable complexes containing immunoglobulins, deoxyribonucleic acid (DNA), and antinuclear antibodies have also been found in synovial fluids (11-13). Since DNA-anti-DNA complexes are thought to participate in the pathogenesis of systemic lupus erythematosus (SLE) nephritis (14,15), the possi’ This is publication Research Foundation, Health Service Grants tutes of Health. * Postdoctoral Fellow 3 Senior Investigator,
655 from the Department of Experimental Pathology, Scripps Clinic and La Jolla, California. This work was supported by United States Public AM 12198, AM 05694, AM 14916, and AI 00214 from the National lnstiof the Medical Research The Arthritis Foundation.
Council,
385 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
Canada.
386
KOBITAILLE,
ZVAIFLEH,
AND
7‘AN
bility that immune complexes containing nuclear antigens may be involved in RA synovitis has been raised (16,17). The present work was undertaken to determine whether such complexes involving nucleic acids could be found in R4 synovial fluids. Using a radioimmunoassay, synovial fluids from patients with RA and other rheumatic diseases were examined for presence of soluble nucleoprotein (sNP) antigen and its homologous antibody. METHODS Selection ofl~clticrrts. Patients were selected on the basis of availability of sufficient synovial fluid for arthrocentesis. Patients with R4 met the criteria of the American Rheumatism Association for classical or definite RA (18). The nonrheumatoid patients satisfied generally accepted diagnostic criteria. Sytwcicllfluicl crud ,set~nl s/)ecirrrc:rls. Synovial fluid was obtained by aseptic aspiration of an involved joint. The samples were immediately centrifuged at 4°C until the supematant liquid was cell-free and stored at -20°C. Although cryoprecipitable proteins were present in many of these samples, they usually formed after prolonged storage at 4°C and the centrifugation procedure did not result in removal of detectable amounts of cryoprotein. At the time of use, hyaluronidase (Wydase, Wyeth Laboratories, Philadelphia, 150 NF units per ml) was added to the synovial fluids (1 vol of enzyme for 50 vol of fluid) incubated at 37°C for 20 min, and further cleared by centrifugation at 2OOOgfor 15 min at 4°C. Venous blood was drawn from an antecubital vein and allowed to clot at room temperature. Serum was separated by centrifugation at 4°C and stored in aliquots at -20°C. Reference sera with precipitating antibody specific for native deoxyribonucleic acid (N-DNA) and soluble nucleoprotein (sNP) were obtained from patients with systemic lupus erythematosus (SLE) and were characterized according to methods previously described (19). Serum with precipitating antibody specific for single-stranded DNA (SS-DNA) was obtained from a rabbit immunized with SS-DNA complexed to methylated bovine serum albumin according to the method of Plescia ct (I/. (20). As reported previously, this serum reacted only with SS-DNA and did not crossreact with N-DNA (21). Stclrldnrdi=.atit)~l c!f‘ 111~:rc!dioil~zltlrl,lotr.~.vtrII. sNP was extracted from fresh calf thymus nuclei, purified, and labeled with ‘Yodide according to the method previously described (22). The percentage of uptake of radioactivity by sNP varied between 15 and 20% and specific activity of the labeled antigen averaged 2 $Zi per pg of protein, The ammonium sulfate method described by Minden and Farr was used to separate free antigen from antibody-bound antigen (23). This method could be applied since free lz5 I-sNP is soluble in 50% saturated ammonium sulfate (SAS), whereas antibodybound ‘251-sNP is precipitated. The assay was performed in 100 x 12-mm glass tubes and each sample was run in duplicate. All serum and synovial fluid samples were decomplemented by heating at 56°C for 30 min. Each tube contained 40 ng of ““I-sNP in 0.015 111NaCI, 0.0015 M sodium citrate (dilute sodium chloride-citrate:DSC) in a volume of 200 ~1 and gave between 10,000 and 20,000 cpm in a gamma spectrometer (Baird Atomic Nuclear Corp., Model 530). An SLE serum (JT) containing precipitating antibody to sNP was serially
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FLUIDS
diluted in pooled normal human serum diluent (diluted 1: 10 in DSC) and 2009.~1 aliquots added to the labeled antigen. After an overnight incubation at 4°C cold SAS was added to a final concentration of 50%. The samples were mixed, allowed to stand at 4°C for 30 min and then centrifuged at 2000g for 30 min. The supernatant fluids were decanted and precipitates washed with 3.0 ml of cold 50% SAS. The final precipitates were dissolved in 0.5 ml of 0.5 hr NaOH and radioactivity determined. The degree of nonspecific precipitation of 1251-sNP in 50% SAS in presence of 200 ~1 of pooled normal human serum diluent was approximately 10%. Specific binding in each sample due to antisNP antibody was calculated as follows: cpm precipitated
% Specific
binding
by JT serum - cpm precipitated = Total cpm added - cpm precipitated
by normal by normal
serum serum
Figure la illustrates the typical binding curve obtained with serial dilutions of JT serum. Test system for detecting antigen und antibody. A 1:20 dilution of JT serum, capable of binding 30-35% of labeled sNP was chosen as the fixed amount to use in a standardized radioimmunoassay. Two hundred pliters of test sample was added to 200 ~1 of JT serum diluted 1: 20 and incubated overnight at 4°C. Control tubes contained 200 ~1 of pooled normal human serum instead of test sample. The next morning 40 ng of ‘%I-sNP was added, the total reaction volume adjusted to 2.0 ml with addition of DSC, and the mixture in-
1.1. I/20 Uulabelltd all,
1.1. Sarum Oilutioas
pS Unlabclled
sNP Added
FIG. l(a). Antigen binding curve in the Farr-type radioimmunoassay was demonstrated by reaction of standard serum JT (containing antibody against sNP) and 40 ng of r2sI-labeled sNP. The curve represents percentage of radiolabeled antigen bound by various dilutions of serum. (b). Inhibition of standardized radioimmunoassay by unlabeled antigen (sNP). The standard system Adconsisted of JT serum at l/20 dilution and 40 ng L*SIsNP which bound 30% of total radiolabel. dition of unlabeled sNP to this system resulted in competition with isotope-labeled antigen and hence inhibition of bound radioactivity. This curve was used as reference to quantitate amount of sNP antigen present in synovial fluids.
ROBITAILLE,
388
ZVAIFLER,
AND
TAX
cubated at 4°C for another overnight period. SAS was then added to a final concentration of 50%, precipitates sedimented by centrifugation, washed, and radioactivity determined as described above. When sNP antigen was present in synovial fluids, it competed with ‘251-sNP for the available anti-sNP antibody in the fixed amount of JT serum, thereby reducing or inhibiting the binding of labeled antigen. The percentage of inhibition was calculated as follows: % Inhibition
=
% binding
of JT - 70 binding of JT and test sample % binding of JT
x IO0
The percentage of inhibition could be interpreted in terms of amount of sNP antigen by reference to a standard curve (Fig. lb) in which known amounts of unlabeled sNP were added to the system and percentage of inhibition plotted against sNP added. Two tenths microgram sNP antigen could be detected with reproducibility in this system. When free antibody was present in synovial fluids, greater than 30-35s binding (enhancement) was noticed in the assay. Binding enhancement, indicative of antibody reacting with labeled sNP, was calculated as follows: % Enhancement
=
% binding
of JT and test sample - % binding % binding of JT
of JT x IO0
Enhancement of binding was not interpreted quantitatively into amount of antibody. Specificity of radioinznzunoclssays. The assay for sNP antigen reflected as inhibition of binding of standard serum JT was specific for sNP. Native calf thymus DNA, heat-denatured single-strand DNA, yeast RNA, and calf thymus total histones (all from Worthington Biochemical, Freehold, NJ) were tested as inhibitors in the system and amounts up to 20 pg of each of these materials did not inhibit JT binding of 1251-sNP. As reported above, reproducible inhibition could be detected by 0.2 pg of unlabeled sNP. Similarly, other nuclear antigens reactive with antinuclear antibodies, such as Sm and ribonucleoprotein antigens (24,25) were also tested as inhibitors. These antigens in amounts sufficient to produce precipitins with the respective antibodies were not capable of showing inhibition of JT binding. On the other hand, enhancement of binding of lz51-sNP by a test sample could be due to sNP antibody and/or DNA antibody. The latter antibody reacts with the DNA moiety of the DNA-histone complex which comprises the integral antigenic determinant molecule of sNP, whereas sNP antibody is reactive with the total complex (19). Differentiation between sNP antibody and DNA antibody was accomplished in the following manner. Test samples showing enhancement of binding were preincubated with 20 pg native calf thymus DNA (Worthington Biochemical) before the standard system of JT serum and 40 ng *z51-sNP was added. If binding enhancement was due to antibody reacting with DNA moiety of sNP, this was abolished by added calf thymus DNA whereas enhancement due to antibody to DNA-histone complex was not altered. Unlabeled DNA used for this procedure contained less than 1% protein, and the 20 pg DNA employed was shown in other studies to
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389
have no inhibition on JT serum which contains only specific antibody to DNA-histone. lmmunodiflusion. Double diffusion in 0.4% agarose was performed according to a method already described (24). Isolated calf thymus sNP was diluted in DSC and N-DNA and SS-DNA were diluted in phosphate-buffered saline (phosphate 0.01 M, 0.15 M NaCl-PBS), pH 7.4, at a final concentration of 500 pg per ml. Reference sera and synovial fluid samples were used undiluted. Enzyme digestions. Bovine pancreas deoxyribonuclease (DNAse) was a crystalline preparation obtained commercially (Worthington Biochemical, Freehold, NJ). DNAse was dissolved in PBS at a concentration of 200 pg per ml. Fifty microliters of DNAse solution (10 pg) was added to 1 ml of synovial fluid. For digestion of isolated calf thymus sNP, the antigen was dissolved in PBS at 500 pg per ml and 10 pg DNAse added to 1 ml of the antigen solution. In this instance, MgCl, was added to a final concentration of 0.003 M. The mixtures were incubated at 37°C for 1 hr. The reaction was stopped by addition of EDTA to a final concentration of 0.003 M and residual DNAse destroyed by heating at 56°C for 20 min (26). Occasionally a precipitate formed and was removed by centrifugation. This procedure destroyed reactivity of synovial fluid sNP in immunodiffusion, but more prolonged digestion was necessary to abolish its binding inhibition capacity in the radioimmunoassay. For more extensive digestion of sNP, the mixtures were incubated at 37°C for 18-24 hr followed by overnight dialysis against PBS. Appropriate controls containing DNAse + PBS or PBS + sNP were always performed simultaneously. Nucleur antigens. Calf thymus N-DNA, calf thymus total histones, and yeast RNA were obtained commercially (Worthington Biochemical, Freehold, NJ). SS-DNA was prepared by immersing a solution of N-DNA dissolved in PBS at a concentration of 500 kg per ml in a boiling water bath for 10 min followed by rapid chilling at 0°C. Previously described methods were utilized for the isolation of Sm (24) and nuclear ribonucleoprotein (RNP) (25) antigens from fresh calf thymus nuclei. Chemical determination of DNA was performed by a modification of the diphenylamine reaction (27). RESULTS Radioimmunoassay results obtained with 24 synovial fluids are presented in Table 1. These samples were also tested by double diffusion in agarose for presence of sNP, N-DNA, SS-DNA antigens. In addition, total DNA content was estimated by the diphenylamine reaction. sNP antigen was detected by radioimmunoassay in 17 of 24 samples; concentrations ranged from 5 to 60 pg per ml. Of the seven remaining samples, two gave neither inhibition nor enhancement of binding and five produced significant binding enhancement indicating presence of antibody to sNP and/or N-DNA. For the samples containing sNP antigen by radioimmunoassay, the diphenylamine reaction gave readings from 5 to 104 ,ug/ml of DNA-reacting material. sNP concentrations by radioimmunoassay were lower
Corrrsponding
sample
nu~nl~~t,
expressed
50 6 5 8 6 60 48 40 30 28 25 25 20 9 8 8 5 0 0 0 0 0 0 0
for sNP antigen
Infectious arthritis Infectious arthritis Infectious arthritis Palindromic rheumatism Osteoarthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rh eumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis
” These values of sNP. i. Synovial fluid
lb 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 are
number3
in weight
used
taken
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 60 75 108 118
in figures
from
the
and tlmmghout
text.
FLUIDS
inhibition
56 8 7 10 10 104 90 75 50 44 40 25 25 18 14 5 10 5 2 2 5 8 2 2
Diphenylamine reaction (bdnl)
IN JOINT
standard
TABLE 1 AND ANTIBODIES
sNP antibody (76 binding enhancement)
ANTIGENS
Radioimmunoassay
ACID
sNP antigen” (l%hl)
NUCLEIC
cuwc
and
-. -
--
.-
.t -
-
+ -
sNP
represent
activit!
-. ._ ._ t i -
+ --
+ + + + -
-
-
-
SS-DNA
-. --
antigenic
analysis
-i t
+ + i -f
+ +
+
-k + -
+ + -
N-DNA
Precipitin
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FIG. 2. Immunodiffusion study illustrating presence of sNP antigen in synovial fluids. The center well contained an SLE serum with precipitating antibody to sNP antigen. Purified sNP antigen was in top and bottom wells. The four other peripheral wells contained synovial fluids. JT serum formed a line with synovial fluids 1 and 6 fusing in complete identity with that formed between JT serum and sNP. Samples 2 and 7 did not react.
than the values obtained by chemical measurement of DNA-reacting material. sNP antigen could be demonstrated by immunodiffusion in two samples (1 and 6) giving the greatest inhibition of binding. This finding is illustrated in Fig. 2 where serum JT containing precipitating antibody to sNP (center well) formed a precipitin line with synovial fluids 1 and 6 fusing in complete identity with each other and with lines formed between JT serum and purified calf thymus sNP. Synovial fluids 2 and 7 did not react. Synovial fluid 2 contained a very low concentration of sNP (6pg per ml, see Table l), but synovial fluid 7, although containing 48 pg of sNP per ml, did not precipitate with JT serum. Addition of isolated calf thymus sNP to normal human serum or to synovial fluid (devoid of inhibitory capacity by radioimmunoassay) indicated that the limit of sensitivity of standard anti-sNP (JT) used in the precipitin studies to detect sNP antigen was in the region of 40 pg per ml. Synovial fluid 7 could have been close to the limit of sensitivity of the system. Confirming the specificity of serum JT for nucleoprotein in radioimmunoassay studies, JT did not react with double-stranded or SS-DNA in immunodiffusion.
392
ROBITAILLE,
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AND
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FIG. 3. l’o),. Precipitin study illustrating presence of N-DNA-like material in synovial fluids. The center well contained an SLE serum (RM) with precipitating antibody to N-DNA. Isolated N-DNA was in top and bottom wells. The four other peripheral wells contained synovial fluids. RM serum formed a line with synovial fluids 25, and 10 fusing in complete identity with that formed between serum RM and N-DNA. Bottom. Demonstration of sNP antibody in synovial fluid by precipitin analysis. Anti-sNP serum (JT) in the center well precipitated with sNP antigen to form a line that identified completely with that formed between synovial fluid 24 and sNP antigen. Synovial fluids 2,5, and 10 were not reactive.
The results of immunodifhrsion tests for N-DNA and SS-DNA antigens are presented in the last two columns of Table 1. Material reacting like N-DNA was present in 16 synovial fluids and five fluids contained material reacting like SS-DNA. A representative study is illustrated in Fig. 3 (top) in which a serum containing precipitating antibody to N-DNA (RM) placed in the center well reacted with synovial fluids 2,5, and 10 to give precipitin lines fusing in identity with N-DNA. The serum used to detect SS-DNA was from a rabbit immunized with SS-DNA, and in separate studies this serum as well as serum
NUCLEIC
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RM (anti-N-DNA) were shown to form distinct precipitins with as little as 5 pg per ml of SS-DNA and N-DNA, respectively. Serum RM was one of the unusual SLE sera with antibodies reacting only with double-stranded DNA and not reactive with SS-DNA. Hence, where DNA antigen was concerned, some synovial fluids contained only double-stranded DNA, some only SS-DNA, and others both forms of DNA (see Table 1). Several samples containing antigen by precipitin analysis (two with sNP, five with SS-DNA, and 10 with N-DNA) were subjected to a 1-hr DNAse digestion. This short enzymatic treatment was sufficient to abolish precipitin formation in every instance between the digested synovial fluid and the appropriate serum containing precipitating antibody. More prolonged digestion was necessary to eliminate the sNP binding inhibition capacity in the radioimmunoassay; 24-hr DNAse digestion of four synovial fluids (1,6,7,8) giving the greatest degree of binding inhibition resulted in complete loss of their inhibitory capacity. Five synovial fluids from RA patients contained antibody activity (Table 1, 20-24). In two of them (21,22) free N-DNA antigen probably excluded the coexistance of free DNA antibody as the reason for1251-sNP binding enhancement, and the enhancement was probably due to sNP antibody. In the third fluid (24) which caused the greatest binding enhancement, specificity of the antibody to sNP was demonstrated by precipitin analysis. As illustrated in Fig. 3 (bottom) this sample precipitated with isolated calf thymus sNP forming a line of complete identity with specific anti-sNP serum JT. No line could be seen when this fluid was reacted against N-DNA. In addition, specificity of the antibody in these five samples was demonstrated by prior incubation of the fluids with either unlabeled N-DNA or sNP. Complete inhibition of binding of unlabeled N-DNA indicating presence of antibody to N-DNA occurred with only one sample (23). Partial inhibition by unlabeled N-DNA and complete inhibition by unlabeled sNP indicating presence of antibodies of both specificities was seen in two samples (20,24). In the fluids with free N-DNA (21,22), detectable by precipitin analysis, specificity of the antibody for sNP was confirmed since binding enhancement was abolished completely by unlabeled sNP but was uninfluenced by addition of unlabeled N-DNA. After our initial work on 24 synovial fluid samples, we expanded our study to include fluids from 54 patients. In this enlarged group, the proportion of samples from non-I&4 patients was purposely increased. Samples from these additional 30 patients were examined by radioimmunoassay and immunodiffusion but diphenylamine determinations were not performed. The incidence of sNP antigen or antibody found by radioimmunoassay in the fluids of the 54 patients studied is presented in Table 2. Of 31 samples from RA patients, 18 had sNP antigen (58%). The sNP concentration ranged from 2 to more than 250 pg per ml. Eight of these 31 RA patients (26%) had antibody as evidenced by binding enhancement. In samples from patients with infectious arthritis (IA), osteoarthritis (OA), SLE, and gout, 12 of 18 were positive for sNP antigen (67%), but none had antibody. In five samples from other inflammatory but non-RA joint diseases, two had sNP antigen, and antibody activity was de-
394
ROBITAILLE,
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AND
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2
INCIDENCE OF sNP ANTIGEN AND sNP AND/OR N-DNA IN SYNOVIAL FLUIDS
Number of patients studied
Diagnosis
AN-~IBODY
sNP antigenpositive samples
sNP and/or N-DNA antibodypositive samples
-___ Rheumatoid arthritis Infectious arthritis Osteoarthritis Systemic lupus erythematosus Gout OtheP Subtotal for nonrheumatoid arthritis patients
31 10 5 2 1 5 23
U One each: palindromic rheumatism, erythema nodosum, psoriatic arthritis. * Spondylitic arthritis, erythema nodosum. r Psoriatic arthritis.
18 (58%) 9 ”
0 2” 14 (61%)
multiforme,
spondylitic
8 (26%) 0 0 0 0 1” I (4%)
arthritis,
erythema
tected in one sample from a patient with psoriatic arthritis (PsA). Except for this single patient with PsA, the striking finding was that in eight of 31 synovial e&ions from R4 patients, antibody to sNP and/or N-DNA was found, whereas in 23 non-FM patients, none had antibody. By double diffusion, sNP antigen was detected in five of 54 samples (9%), N-DNA in 37 of 54 (69%), and SS-DNA in nine of 54 (17%). The incidence of samples containing any of the three nuclear antigens was comparable in the IL4 and non-IL4 groups. Except for the R4 synovial fluid samples (Table 1, 24 and Fig. 3) already mentioned, antibody precipitating with sNP was not found in the other fluids tested. None of the samples demonstrated precipitating antibody to N-DNA or SS-DNA antigen. Several samples were extensively digested with DNAse in an attempt to demonstrate presence of soluble antigen-antibody complexes. Fifteen randomly selected samples devoid of antibody activity were subjected to a 24-hr DNAse treatment as previously outlined. Enhancement of binding in the radioimmunoassay after DNAse treatment would have suggested the presence of DNA or sNP immune complexes if this treatment had released free antibody. However, all of the samples tested failed to show any significant enhanced binding after DNAse treatment. DISCUSSION A sensitive radioimmunoassay technique allowing quantitation of sNP antigen in biological fluids has been described, Inhibition of 1251-sNPbinding of a specific anti-sNP serum, indicated presence of sNP antigen. As little as 1 pg of sNP per ml could be detected by this method. This assay was shown to be specific for sNP antigen since a variety of other nuclear antigens failed to give any inhibition of binding even at high concentration. Additional evidence for
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specificity was obtained from DNAse digestion experiments. Since the antigenic specificity of sNP is dependent upon presence of both its DNA and protein moieties, DNAse treatment should destroy this antigenic determinant (19). Such a phenomenon was observed in the radioimmunoassay. Inhibition of binding by synovial fluids and by isolated calf thymus sNP was completely abolished after treatment with DNAse. Of the 54 synovial fluid samples tested, 60% had sNP antigen. In general, sNP concentrations correlated fairly well with the chemically determined DNA concentrations (diphenylamine reaction), The higher values obtained by the diphenylamine reaction could be partly accounted for by presence of either N-DNA or SS-DNA antigens which were not detected by radioimmunoassay. Furthermore, since radioimmunoassay measures the antigenic activity of sNP, partial denaturation of this antigen, causing loss of immunologic reactivity, may have reduced sNP values by radioimmunoassay without affecting the diphenylamine values. Finally, the possibility that other substances influencing the diphenylamine reaction (28) may have been present in some samples cannot be excluded. By immunodiffusion, five of 54 (9%) synovial fluids had sNP antigen, 37 of 54 (69%) had N-DNA antigen, and nine of 54 (17%) had SS-DNA antigen. Failure to identify sNP antigen in a greater number of samples by the precipitin technique might be due in part to the sensitivity of the antiserum, since it detected sNP antigen at concentrations in the region of 40 pg per ml. Another factor could be that a portion of the antigen was present in nonprecipitating form, perhaps as a result of partial in viva digestion by nucleases or proteases. Evidence drawn from the DNAse digestion experiments performed on two samples with sNP antigen detectable by precipitin analysis is in support of this possibility. Short enzymatic treatment prevented these fluids from precipitating with anti-sNP serum, whereas they still contained the same quantity of sNP antigen by radioimmunoassay since their inhibitory capacity remained unchanged. The availability of a strong monospecific antiserum to N-DNA, capable of detecting as little as 5 pg of antigen per ml might be partly responsible for the comparatively high incidence of N-DNA containing fluids by precipitin analysis. It must be stressed, however, that this antiserum can also react with the N-DNA moiety of sNP forming lines of complete identity with isolated N-DNA and making it impossible to differentiate these two antigens on the basis of this test alone. It is very likely that this factor might have elevated the incidence of N-DNA containing fluids since most of the fluids with N-DNA antigen by precipitin analysis had at least 5 pg of sNP antigen per ml by radioimmunoassay. The high incidence of N-DNA antigen found by immunodiffusion is in the range reported by others (29,30). SS-DNA antigen could not be detected by double diffusion in 63 synovial samples by one group of investigators (29), but has been identified by others (13,31). In our study, the presence of nuclear antigens could not be correlated with clinical diagnosis. This observation has previously been reported (29), and it would seem that presence of free nuclear antigens is a relatively nonspecific phenomenon, perhaps related to destruction of polymorphonuclear cells or local tissue breakdown. Such a possibility
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ROBITAILLE,
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is supported by the observation that lysosyme levels, generally associated with accelerated leukocyte turnover were found to parallel synovial fluid DNA concentrations (29). While appearance of free nuclear antigens in synovial fluids might be related to inflammation or tissue destruction and has been detected in many types of inflammatory arthritides, it is of interest that antinuclear antibodies were found almost exclusively in e&ions from patients with RA (17,32), However, the specificity of these antinuclear antibodies, except for anti-SSDNA (13,31), h as not been investigated. The striking finding in our study was that antibody to either sNP or N-DNA could be detected in 26% of the RA effusions examined. In contrast, with the exception of one sample from a patient with PsA, no antibody could be demonstrated in 23 non-RA samples. Although antibodies to sNP and N-DNA have not been reported in RA synovial fluids to our knowledge, the observation of it2 viva LE cell formation in synovial effusions from RA patients (33) supports our findings since antibody to nucleoprotein (19,34) and to a lesser extent N-DNA (19,35) are known to be responsible for the LE cell phenomenon. The demonstration of antibody in one quarter of the RA fluids tested and free antigen in more than half suggests that conditions are appropriate for immune complex formation. In this respect, the situation is probably analogous to what has been described in the sera of SLE patients where free DNA antigen and anti-DNA antibody have been found alternately (27). We were not able to demonstrate sNP immune complexes in synovial fluids. Failure to identify these complexes may have been technical. It is also possible that these soluble complexes are rapidly removed from the fluid phase by leukocytes and synovial cells. In this respect, leukocyte inclusions containing immunoglobulins, complement, nuclear antigen (17,36-39) and antinuclear antibody (32) are regularly demonstrated in the effilsions of patients with RA and could represent phagocytized antigen-antibody complexes. In this study, complement levels in synovial fluids could not be examined for a variety of reasons, and it was not possible to determine if a relationship existed with presence of antigen or antibody. Furthermore, paired sera were not available to determine if antibody in synovial fluid corresponded with any detectable antibody in blood. In the present work, indirect evidence is presented supporting the concept of sNP and DNA immune-complex formation in RA synovial fluids. The role of these complexes in the pathogenesis of RA synovitis remains unclear, but the ability of DNA and nucleoprotein antibodies to fix complement ill uilro (40) suggests that they may participate in synovial inflammation and local complement consumption. Valuable information on the potential pathogenic role of these complexes (DNA or sNP) might be obtained from sequential studies on RA synovial fluids in an attempt to correlate the presence of either free antibody or free antigen with disease activity and complement levels. Additional studies along these lines might provide further insights into the pathogenesis of synovial injury in rheumatoid arthritis.
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397
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HERDBERG, H., Actu Rheumutol. Scund. 9, 165, 1963. PEKIN, T. J., JR., AND ZVAIFLER, M. J.,J. Clin. Inoest. 43, 1372, 1964. SONOZAKI, H., AND TORISU, M., Ann. Rheum. Dis. 29,164,1970. RUDDY, S., AND AUSTEN, K. F., Arthritis Rheum. 13,713, 1970. FOSTIROPOULOS, G., AUSTEN, K. F., AND BLOCH, K. J., Arithritis Rheum. 8, 219, 1965. WARD, P. A., AND ZVAIFLER, N. J., J. Ckn. Inoest. 50,606, 1971. GOTZE, O., ZVAIFLER, N. J., AND Miiller-Eberhard, H. J., Arithritis Rheum. 15, 111, 1972 ZVAIFLER, N. J., Arthritis Rheum. 13,895, 1970. HANNESTAD, K., C/in. Exp. Immunol. 2, 511, 1967. WINCHESTER, R. J., AGNELLO, V, AND KUNKEL, H. G.,Ann. N. Y. Acad. Sci. 168, 195, 1969. MARCUS, R. L., AND TOWNES, A. S., Arthritis Rheom. 11,497, 1968. BARNETT, E. V., BLUESTONE, R., CRACCHIOLO, A., GOLDBERG, L. S., KANTOR, G. L., APED MCINTOSH, R., Ann. Intern. Med. 73,95, 1970. MARCUS, R. L., AND TOWNES, A. S., J. Immunof. 106, 1499, 1971. KOFFLER, D., SCHUR, P. H., AND KUNKEL, H. G., /. Erp. Med. 126,607, 1967. KRISHNAN, C., AND KAPLAN, M. H., I. Clin. Invest. 46,569, 1967. ZVAIFLER, N. J., Arthritis Rheum. 8,289, 1965. BARNETT, E. V., BIENENSTOCK, J., AND BLOCH, K. J., J. Amer. Med. Ass. 198, 143, 1966. ROPES, W. W., BENNETT, G. A., COBB, S., JACOX, R., AND JESSAR, R. A., Bull. Rheum. Dis. 9, 175, 1958. TAN, E. M., J. Clin. Invest. 46, 735, 1967. PLESCIA, 0. J., BRAUN, W., AND PALCZUK, W. C., PTOC. Nut. Acud. Sci. USA 52,279, 1964. TAN, E. M., AND NATALI, P. G., J. Immnnol. 104,902, 1970. ROBITAILLE, P., AND TAN, E. M.,f. Clin. fnvest., in press. MINDEN, P., AND FARR, R. S., in “Handbook of Experimental Immunology” (D. M. Weir, ed.), pp, 463-492, Blackwell, Oxford, 1967. TAN, E. M., AND KUNKEL, H. G., J. lmmunol. 96,464, 1966. NORTHWAY, J. D., AND TAN, E. M., Clin. Immunol. Immunoputhol., in press. STOLLAR, B. D., Biochim. Biophys. Actu 209,541, 1970. TAN, E. M., SCHUR, P. H., CARR, R. I., AND KUNKEL, H. G., J. Clin. Invest. 45, 1732, 1966. LEE, J. B., Nuture (London) 200,264, 1963. HUGHES, G. R. V., COHEN, S. A., LIGHTFOOT, R. W., MELTZER, J. I., AND CHRISTLAN, C. L,.. Arthritis Rheum. 14,259, 1971. CARR, R. I., Ph.D. Thesis, Faculty of Medicine, Rockefeller University, 1969. CRACCHIOLO, A., GOLDBERG, L. S., BARNETT, E. V., AND BLUESTONE, R., lmmunolog!l 20, 1067, 1971. ZVAIFLER, N. J., AND MARTINEZ, M. M., Clin. Exp. Immunol. 8, 271, 1971. HUNDER, G. G., AND PIERRE, R. V., Arthritis Rheum. 13,448, 1970. HOLMAN, H., AND DEICHER, H. R., J. C/in. Invest. 38,2059, 1959. SELIGMAN, M., in “Immunological Methods” (J. K. Ackroyd, ed.), pp. 417-432, Blackwell, Oxford, 1963. MALININ, T. I., PEKIN, T. J., ZVAIFLER, N. J., AND BAUER, H., Arthritis Rheum. 7,743, 1964. VAUGHAN, J. H., BARNETT, E. V., AND SOBEL, M. V., Arithritis Rheum. 11, 125, 1968. BRANDT, K. D., CATHCART, E. S., AND COHEN, A. S., J. Lab. Clin. Med. 72, 631, 1968. BRITTON, M. C., AND SCHUR, P. H., Arthritis Rheum. 14, 87, 1971. ROBBINS, W. C., HOLMAN, H. R., DEICHER, H., AND KUNKEL, H. G., PT~c. SOC. Exr,. pi&. Med. 96,575, 1957.
IMMUNOLOGY
AND
IMMUNOPATHOLOCY
Antinuclear Antibodies in Rheumatoid PIERRE ROBITAILLE*, Division Scripps
1, 385-397
(19%)
and Nuclear Antigens Synovial Fluids’
NATHAN
J. ZVAIFLER,
AND ENG M. TANS
of Allergy und immunology und Department of Erperimentol Pathology, Clinic and Reseurch Foundation and from the Depurtment of Medicine. Unicersity of California at San Diego, La Jollu, California 92037 Receioed
Nowmber
6, 1972;
uccepted
December
26, 1972
Soluble nucleoprotein (sNP) antigen was extracted from calf thymus nuclei and labeled with Yodide. The labeled antigen (12SI-sNP) was used in a sensitive radioimmunoassay to detect either sNP antigen or sNP antibody. Synovial fluids from 54 patients with various rheumatic diseases were examined by this technique. arthritis (RA) patients and sNP antigen was detected in 18 of 31(58 Go / ) in rheumatoid 14 of 23 (61%) in non-R4 patients. The important finding was that 8 of 31 (26%) RA patients’ synovial fluids contained antibody whereas with only one exception, antibody could not be detected in 23 non-R4 patients. Presence of free sNP antigen or homologous antibody in synovial fluids of RA patients suggests that at certain times immune complexes might form and contribute to synovial injury.
Synovial fluid complement levels (CH,,) are generally significantly lower in rheumatoid arthritis (RA) than in other inflammatory joint diseases (l-3). In contrast, serum CH,, values are usually normal or slightly elevated in RA (2,4). Measurements of individual complement component activity in rheumatoid effusion have revealed diminished Cl, C4, C2, and C3 activities (3-5). The pattern of reduction in complement components (4), the presence of C3 convertase (6), and the utilization and consumption of the C3 activator system (7) are highly suggestive of immune activation of the complement sequence in RA synovitis (8). Although the exact mechanisms by which complement might be activated in RA synovial fluid remain unclear, the bulk of evidence supports the concept of complement depletion by immune complexes. Immunoglobulin complexes precipitable by IgM rheumatoid factor or Clq have been isolated from rheumatoid effusions (9,lO). The amount of such complexes was found to be inversely related to synovial fluid CH,, levels (10). Cryoprecipitable complexes containing immunoglobulins, deoxyribonucleic acid (DNA), and antinuclear antibodies have also been found in synovial fluids (11-13). Since DNA-anti-DNA complexes are thought to participate in the pathogenesis of systemic lupus erythematosus (SLE) nephritis (14,15), the possi’ This is publication Research Foundation, Health Service Grants tutes of Health. * Postdoctoral Fellow 3 Senior Investigator,
655 from the Department of Experimental Pathology, Scripps Clinic and La Jolla, California. This work was supported by United States Public AM 12198, AM 05694, AM 14916, and AI 00214 from the National lnstiof the Medical Research The Arthritis Foundation.
Council,
385 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
Canada.
386
KOBITAILLE,
ZVAIFLEH,
AND
7‘AN
bility that immune complexes containing nuclear antigens may be involved in RA synovitis has been raised (16,17). The present work was undertaken to determine whether such complexes involving nucleic acids could be found in R4 synovial fluids. Using a radioimmunoassay, synovial fluids from patients with RA and other rheumatic diseases were examined for presence of soluble nucleoprotein (sNP) antigen and its homologous antibody. METHODS Selection ofl~clticrrts. Patients were selected on the basis of availability of sufficient synovial fluid for arthrocentesis. Patients with R4 met the criteria of the American Rheumatism Association for classical or definite RA (18). The nonrheumatoid patients satisfied generally accepted diagnostic criteria. Sytwcicllfluicl crud ,set~nl s/)ecirrrc:rls. Synovial fluid was obtained by aseptic aspiration of an involved joint. The samples were immediately centrifuged at 4°C until the supematant liquid was cell-free and stored at -20°C. Although cryoprecipitable proteins were present in many of these samples, they usually formed after prolonged storage at 4°C and the centrifugation procedure did not result in removal of detectable amounts of cryoprotein. At the time of use, hyaluronidase (Wydase, Wyeth Laboratories, Philadelphia, 150 NF units per ml) was added to the synovial fluids (1 vol of enzyme for 50 vol of fluid) incubated at 37°C for 20 min, and further cleared by centrifugation at 2OOOgfor 15 min at 4°C. Venous blood was drawn from an antecubital vein and allowed to clot at room temperature. Serum was separated by centrifugation at 4°C and stored in aliquots at -20°C. Reference sera with precipitating antibody specific for native deoxyribonucleic acid (N-DNA) and soluble nucleoprotein (sNP) were obtained from patients with systemic lupus erythematosus (SLE) and were characterized according to methods previously described (19). Serum with precipitating antibody specific for single-stranded DNA (SS-DNA) was obtained from a rabbit immunized with SS-DNA complexed to methylated bovine serum albumin according to the method of Plescia ct (I/. (20). As reported previously, this serum reacted only with SS-DNA and did not crossreact with N-DNA (21). Stclrldnrdi=.atit)~l c!f‘ 111~:rc!dioil~zltlrl,lotr.~.vtrII. sNP was extracted from fresh calf thymus nuclei, purified, and labeled with ‘Yodide according to the method previously described (22). The percentage of uptake of radioactivity by sNP varied between 15 and 20% and specific activity of the labeled antigen averaged 2 $Zi per pg of protein, The ammonium sulfate method described by Minden and Farr was used to separate free antigen from antibody-bound antigen (23). This method could be applied since free lz5 I-sNP is soluble in 50% saturated ammonium sulfate (SAS), whereas antibodybound ‘251-sNP is precipitated. The assay was performed in 100 x 12-mm glass tubes and each sample was run in duplicate. All serum and synovial fluid samples were decomplemented by heating at 56°C for 30 min. Each tube contained 40 ng of ““I-sNP in 0.015 111NaCI, 0.0015 M sodium citrate (dilute sodium chloride-citrate:DSC) in a volume of 200 ~1 and gave between 10,000 and 20,000 cpm in a gamma spectrometer (Baird Atomic Nuclear Corp., Model 530). An SLE serum (JT) containing precipitating antibody to sNP was serially
NUCLEIC
ACID
ANTIBODIES
IN
JOINT
387
FLUIDS
diluted in pooled normal human serum diluent (diluted 1: 10 in DSC) and 2009.~1 aliquots added to the labeled antigen. After an overnight incubation at 4°C cold SAS was added to a final concentration of 50%. The samples were mixed, allowed to stand at 4°C for 30 min and then centrifuged at 2000g for 30 min. The supernatant fluids were decanted and precipitates washed with 3.0 ml of cold 50% SAS. The final precipitates were dissolved in 0.5 ml of 0.5 hr NaOH and radioactivity determined. The degree of nonspecific precipitation of 1251-sNP in 50% SAS in presence of 200 ~1 of pooled normal human serum diluent was approximately 10%. Specific binding in each sample due to antisNP antibody was calculated as follows: cpm precipitated
% Specific
binding
by JT serum - cpm precipitated = Total cpm added - cpm precipitated
by normal by normal
serum serum
Figure la illustrates the typical binding curve obtained with serial dilutions of JT serum. Test system for detecting antigen und antibody. A 1:20 dilution of JT serum, capable of binding 30-35% of labeled sNP was chosen as the fixed amount to use in a standardized radioimmunoassay. Two hundred pliters of test sample was added to 200 ~1 of JT serum diluted 1: 20 and incubated overnight at 4°C. Control tubes contained 200 ~1 of pooled normal human serum instead of test sample. The next morning 40 ng of ‘%I-sNP was added, the total reaction volume adjusted to 2.0 ml with addition of DSC, and the mixture in-
1.1. I/20 Uulabelltd all,
1.1. Sarum Oilutioas
pS Unlabclled
sNP Added
FIG. l(a). Antigen binding curve in the Farr-type radioimmunoassay was demonstrated by reaction of standard serum JT (containing antibody against sNP) and 40 ng of r2sI-labeled sNP. The curve represents percentage of radiolabeled antigen bound by various dilutions of serum. (b). Inhibition of standardized radioimmunoassay by unlabeled antigen (sNP). The standard system Adconsisted of JT serum at l/20 dilution and 40 ng L*SIsNP which bound 30% of total radiolabel. dition of unlabeled sNP to this system resulted in competition with isotope-labeled antigen and hence inhibition of bound radioactivity. This curve was used as reference to quantitate amount of sNP antigen present in synovial fluids.
ROBITAILLE,
388
ZVAIFLER,
AND
TAX
cubated at 4°C for another overnight period. SAS was then added to a final concentration of 50%, precipitates sedimented by centrifugation, washed, and radioactivity determined as described above. When sNP antigen was present in synovial fluids, it competed with ‘251-sNP for the available anti-sNP antibody in the fixed amount of JT serum, thereby reducing or inhibiting the binding of labeled antigen. The percentage of inhibition was calculated as follows: % Inhibition
=
% binding
of JT - 70 binding of JT and test sample % binding of JT
x IO0
The percentage of inhibition could be interpreted in terms of amount of sNP antigen by reference to a standard curve (Fig. lb) in which known amounts of unlabeled sNP were added to the system and percentage of inhibition plotted against sNP added. Two tenths microgram sNP antigen could be detected with reproducibility in this system. When free antibody was present in synovial fluids, greater than 30-35s binding (enhancement) was noticed in the assay. Binding enhancement, indicative of antibody reacting with labeled sNP, was calculated as follows: % Enhancement
=
% binding
of JT and test sample - % binding % binding of JT
of JT x IO0
Enhancement of binding was not interpreted quantitatively into amount of antibody. Specificity of radioinznzunoclssays. The assay for sNP antigen reflected as inhibition of binding of standard serum JT was specific for sNP. Native calf thymus DNA, heat-denatured single-strand DNA, yeast RNA, and calf thymus total histones (all from Worthington Biochemical, Freehold, NJ) were tested as inhibitors in the system and amounts up to 20 pg of each of these materials did not inhibit JT binding of 1251-sNP. As reported above, reproducible inhibition could be detected by 0.2 pg of unlabeled sNP. Similarly, other nuclear antigens reactive with antinuclear antibodies, such as Sm and ribonucleoprotein antigens (24,25) were also tested as inhibitors. These antigens in amounts sufficient to produce precipitins with the respective antibodies were not capable of showing inhibition of JT binding. On the other hand, enhancement of binding of lz51-sNP by a test sample could be due to sNP antibody and/or DNA antibody. The latter antibody reacts with the DNA moiety of the DNA-histone complex which comprises the integral antigenic determinant molecule of sNP, whereas sNP antibody is reactive with the total complex (19). Differentiation between sNP antibody and DNA antibody was accomplished in the following manner. Test samples showing enhancement of binding were preincubated with 20 pg native calf thymus DNA (Worthington Biochemical) before the standard system of JT serum and 40 ng *z51-sNP was added. If binding enhancement was due to antibody reacting with DNA moiety of sNP, this was abolished by added calf thymus DNA whereas enhancement due to antibody to DNA-histone complex was not altered. Unlabeled DNA used for this procedure contained less than 1% protein, and the 20 pg DNA employed was shown in other studies to
NUCLEIC
ACID
ANTIBODIES
IN JOINT
FLUIDS
389
have no inhibition on JT serum which contains only specific antibody to DNA-histone. lmmunodiflusion. Double diffusion in 0.4% agarose was performed according to a method already described (24). Isolated calf thymus sNP was diluted in DSC and N-DNA and SS-DNA were diluted in phosphate-buffered saline (phosphate 0.01 M, 0.15 M NaCl-PBS), pH 7.4, at a final concentration of 500 pg per ml. Reference sera and synovial fluid samples were used undiluted. Enzyme digestions. Bovine pancreas deoxyribonuclease (DNAse) was a crystalline preparation obtained commercially (Worthington Biochemical, Freehold, NJ). DNAse was dissolved in PBS at a concentration of 200 pg per ml. Fifty microliters of DNAse solution (10 pg) was added to 1 ml of synovial fluid. For digestion of isolated calf thymus sNP, the antigen was dissolved in PBS at 500 pg per ml and 10 pg DNAse added to 1 ml of the antigen solution. In this instance, MgCl, was added to a final concentration of 0.003 M. The mixtures were incubated at 37°C for 1 hr. The reaction was stopped by addition of EDTA to a final concentration of 0.003 M and residual DNAse destroyed by heating at 56°C for 20 min (26). Occasionally a precipitate formed and was removed by centrifugation. This procedure destroyed reactivity of synovial fluid sNP in immunodiffusion, but more prolonged digestion was necessary to abolish its binding inhibition capacity in the radioimmunoassay. For more extensive digestion of sNP, the mixtures were incubated at 37°C for 18-24 hr followed by overnight dialysis against PBS. Appropriate controls containing DNAse + PBS or PBS + sNP were always performed simultaneously. Nucleur antigens. Calf thymus N-DNA, calf thymus total histones, and yeast RNA were obtained commercially (Worthington Biochemical, Freehold, NJ). SS-DNA was prepared by immersing a solution of N-DNA dissolved in PBS at a concentration of 500 kg per ml in a boiling water bath for 10 min followed by rapid chilling at 0°C. Previously described methods were utilized for the isolation of Sm (24) and nuclear ribonucleoprotein (RNP) (25) antigens from fresh calf thymus nuclei. Chemical determination of DNA was performed by a modification of the diphenylamine reaction (27). RESULTS Radioimmunoassay results obtained with 24 synovial fluids are presented in Table 1. These samples were also tested by double diffusion in agarose for presence of sNP, N-DNA, SS-DNA antigens. In addition, total DNA content was estimated by the diphenylamine reaction. sNP antigen was detected by radioimmunoassay in 17 of 24 samples; concentrations ranged from 5 to 60 pg per ml. Of the seven remaining samples, two gave neither inhibition nor enhancement of binding and five produced significant binding enhancement indicating presence of antibody to sNP and/or N-DNA. For the samples containing sNP antigen by radioimmunoassay, the diphenylamine reaction gave readings from 5 to 104 ,ug/ml of DNA-reacting material. sNP concentrations by radioimmunoassay were lower
Corrrsponding
sample
nu~nl~~t,
expressed
50 6 5 8 6 60 48 40 30 28 25 25 20 9 8 8 5 0 0 0 0 0 0 0
for sNP antigen
Infectious arthritis Infectious arthritis Infectious arthritis Palindromic rheumatism Osteoarthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rh eumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis
” These values of sNP. i. Synovial fluid
lb 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 are
number3
in weight
used
taken
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 60 75 108 118
in figures
from
the
and tlmmghout
text.
FLUIDS
inhibition
56 8 7 10 10 104 90 75 50 44 40 25 25 18 14 5 10 5 2 2 5 8 2 2
Diphenylamine reaction (bdnl)
IN JOINT
standard
TABLE 1 AND ANTIBODIES
sNP antibody (76 binding enhancement)
ANTIGENS
Radioimmunoassay
ACID
sNP antigen” (l%hl)
NUCLEIC
cuwc
and
-. -
--
.-
.t -
-
+ -
sNP
represent
activit!
-. ._ ._ t i -
+ --
+ + + + -
-
-
-
SS-DNA
-. --
antigenic
analysis
-i t
+ + i -f
+ +
+
-k + -
+ + -
N-DNA
Precipitin
NUCLEIC
ACID
ANTIBODIES
IN
JOINT
FLUIDS
391
FIG. 2. Immunodiffusion study illustrating presence of sNP antigen in synovial fluids. The center well contained an SLE serum with precipitating antibody to sNP antigen. Purified sNP antigen was in top and bottom wells. The four other peripheral wells contained synovial fluids. JT serum formed a line with synovial fluids 1 and 6 fusing in complete identity with that formed between JT serum and sNP. Samples 2 and 7 did not react.
than the values obtained by chemical measurement of DNA-reacting material. sNP antigen could be demonstrated by immunodiffusion in two samples (1 and 6) giving the greatest inhibition of binding. This finding is illustrated in Fig. 2 where serum JT containing precipitating antibody to sNP (center well) formed a precipitin line with synovial fluids 1 and 6 fusing in complete identity with each other and with lines formed between JT serum and purified calf thymus sNP. Synovial fluids 2 and 7 did not react. Synovial fluid 2 contained a very low concentration of sNP (6pg per ml, see Table l), but synovial fluid 7, although containing 48 pg of sNP per ml, did not precipitate with JT serum. Addition of isolated calf thymus sNP to normal human serum or to synovial fluid (devoid of inhibitory capacity by radioimmunoassay) indicated that the limit of sensitivity of standard anti-sNP (JT) used in the precipitin studies to detect sNP antigen was in the region of 40 pg per ml. Synovial fluid 7 could have been close to the limit of sensitivity of the system. Confirming the specificity of serum JT for nucleoprotein in radioimmunoassay studies, JT did not react with double-stranded or SS-DNA in immunodiffusion.
392
ROBITAILLE,
ZVAIFLER,
AND
TAN
FIG. 3. l’o),. Precipitin study illustrating presence of N-DNA-like material in synovial fluids. The center well contained an SLE serum (RM) with precipitating antibody to N-DNA. Isolated N-DNA was in top and bottom wells. The four other peripheral wells contained synovial fluids. RM serum formed a line with synovial fluids 25, and 10 fusing in complete identity with that formed between serum RM and N-DNA. Bottom. Demonstration of sNP antibody in synovial fluid by precipitin analysis. Anti-sNP serum (JT) in the center well precipitated with sNP antigen to form a line that identified completely with that formed between synovial fluid 24 and sNP antigen. Synovial fluids 2,5, and 10 were not reactive.
The results of immunodifhrsion tests for N-DNA and SS-DNA antigens are presented in the last two columns of Table 1. Material reacting like N-DNA was present in 16 synovial fluids and five fluids contained material reacting like SS-DNA. A representative study is illustrated in Fig. 3 (top) in which a serum containing precipitating antibody to N-DNA (RM) placed in the center well reacted with synovial fluids 2,5, and 10 to give precipitin lines fusing in identity with N-DNA. The serum used to detect SS-DNA was from a rabbit immunized with SS-DNA, and in separate studies this serum as well as serum
NUCLEIC
ACID
ANTIBODIES
IN
JOINT
FLUIDS
393
RM (anti-N-DNA) were shown to form distinct precipitins with as little as 5 pg per ml of SS-DNA and N-DNA, respectively. Serum RM was one of the unusual SLE sera with antibodies reacting only with double-stranded DNA and not reactive with SS-DNA. Hence, where DNA antigen was concerned, some synovial fluids contained only double-stranded DNA, some only SS-DNA, and others both forms of DNA (see Table 1). Several samples containing antigen by precipitin analysis (two with sNP, five with SS-DNA, and 10 with N-DNA) were subjected to a 1-hr DNAse digestion. This short enzymatic treatment was sufficient to abolish precipitin formation in every instance between the digested synovial fluid and the appropriate serum containing precipitating antibody. More prolonged digestion was necessary to eliminate the sNP binding inhibition capacity in the radioimmunoassay; 24-hr DNAse digestion of four synovial fluids (1,6,7,8) giving the greatest degree of binding inhibition resulted in complete loss of their inhibitory capacity. Five synovial fluids from RA patients contained antibody activity (Table 1, 20-24). In two of them (21,22) free N-DNA antigen probably excluded the coexistance of free DNA antibody as the reason for1251-sNP binding enhancement, and the enhancement was probably due to sNP antibody. In the third fluid (24) which caused the greatest binding enhancement, specificity of the antibody to sNP was demonstrated by precipitin analysis. As illustrated in Fig. 3 (bottom) this sample precipitated with isolated calf thymus sNP forming a line of complete identity with specific anti-sNP serum JT. No line could be seen when this fluid was reacted against N-DNA. In addition, specificity of the antibody in these five samples was demonstrated by prior incubation of the fluids with either unlabeled N-DNA or sNP. Complete inhibition of binding of unlabeled N-DNA indicating presence of antibody to N-DNA occurred with only one sample (23). Partial inhibition by unlabeled N-DNA and complete inhibition by unlabeled sNP indicating presence of antibodies of both specificities was seen in two samples (20,24). In the fluids with free N-DNA (21,22), detectable by precipitin analysis, specificity of the antibody for sNP was confirmed since binding enhancement was abolished completely by unlabeled sNP but was uninfluenced by addition of unlabeled N-DNA. After our initial work on 24 synovial fluid samples, we expanded our study to include fluids from 54 patients. In this enlarged group, the proportion of samples from non-I&4 patients was purposely increased. Samples from these additional 30 patients were examined by radioimmunoassay and immunodiffusion but diphenylamine determinations were not performed. The incidence of sNP antigen or antibody found by radioimmunoassay in the fluids of the 54 patients studied is presented in Table 2. Of 31 samples from RA patients, 18 had sNP antigen (58%). The sNP concentration ranged from 2 to more than 250 pg per ml. Eight of these 31 RA patients (26%) had antibody as evidenced by binding enhancement. In samples from patients with infectious arthritis (IA), osteoarthritis (OA), SLE, and gout, 12 of 18 were positive for sNP antigen (67%), but none had antibody. In five samples from other inflammatory but non-RA joint diseases, two had sNP antigen, and antibody activity was de-
394
ROBITAILLE,
ZVAIFLER, TABLE
AND
TAN
2
INCIDENCE OF sNP ANTIGEN AND sNP AND/OR N-DNA IN SYNOVIAL FLUIDS
Number of patients studied
Diagnosis
AN-~IBODY
sNP antigenpositive samples
sNP and/or N-DNA antibodypositive samples
-___ Rheumatoid arthritis Infectious arthritis Osteoarthritis Systemic lupus erythematosus Gout OtheP Subtotal for nonrheumatoid arthritis patients
31 10 5 2 1 5 23
U One each: palindromic rheumatism, erythema nodosum, psoriatic arthritis. * Spondylitic arthritis, erythema nodosum. r Psoriatic arthritis.
18 (58%) 9 ”
0 2” 14 (61%)
multiforme,
spondylitic
8 (26%) 0 0 0 0 1” I (4%)
arthritis,
erythema
tected in one sample from a patient with psoriatic arthritis (PsA). Except for this single patient with PsA, the striking finding was that in eight of 31 synovial e&ions from R4 patients, antibody to sNP and/or N-DNA was found, whereas in 23 non-FM patients, none had antibody. By double diffusion, sNP antigen was detected in five of 54 samples (9%), N-DNA in 37 of 54 (69%), and SS-DNA in nine of 54 (17%). The incidence of samples containing any of the three nuclear antigens was comparable in the IL4 and non-IL4 groups. Except for the R4 synovial fluid samples (Table 1, 24 and Fig. 3) already mentioned, antibody precipitating with sNP was not found in the other fluids tested. None of the samples demonstrated precipitating antibody to N-DNA or SS-DNA antigen. Several samples were extensively digested with DNAse in an attempt to demonstrate presence of soluble antigen-antibody complexes. Fifteen randomly selected samples devoid of antibody activity were subjected to a 24-hr DNAse treatment as previously outlined. Enhancement of binding in the radioimmunoassay after DNAse treatment would have suggested the presence of DNA or sNP immune complexes if this treatment had released free antibody. However, all of the samples tested failed to show any significant enhanced binding after DNAse treatment. DISCUSSION A sensitive radioimmunoassay technique allowing quantitation of sNP antigen in biological fluids has been described, Inhibition of 1251-sNPbinding of a specific anti-sNP serum, indicated presence of sNP antigen. As little as 1 pg of sNP per ml could be detected by this method. This assay was shown to be specific for sNP antigen since a variety of other nuclear antigens failed to give any inhibition of binding even at high concentration. Additional evidence for
NUCLEIC
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ANTIBODIES
IN
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specificity was obtained from DNAse digestion experiments. Since the antigenic specificity of sNP is dependent upon presence of both its DNA and protein moieties, DNAse treatment should destroy this antigenic determinant (19). Such a phenomenon was observed in the radioimmunoassay. Inhibition of binding by synovial fluids and by isolated calf thymus sNP was completely abolished after treatment with DNAse. Of the 54 synovial fluid samples tested, 60% had sNP antigen. In general, sNP concentrations correlated fairly well with the chemically determined DNA concentrations (diphenylamine reaction), The higher values obtained by the diphenylamine reaction could be partly accounted for by presence of either N-DNA or SS-DNA antigens which were not detected by radioimmunoassay. Furthermore, since radioimmunoassay measures the antigenic activity of sNP, partial denaturation of this antigen, causing loss of immunologic reactivity, may have reduced sNP values by radioimmunoassay without affecting the diphenylamine values. Finally, the possibility that other substances influencing the diphenylamine reaction (28) may have been present in some samples cannot be excluded. By immunodiffusion, five of 54 (9%) synovial fluids had sNP antigen, 37 of 54 (69%) had N-DNA antigen, and nine of 54 (17%) had SS-DNA antigen. Failure to identify sNP antigen in a greater number of samples by the precipitin technique might be due in part to the sensitivity of the antiserum, since it detected sNP antigen at concentrations in the region of 40 pg per ml. Another factor could be that a portion of the antigen was present in nonprecipitating form, perhaps as a result of partial in viva digestion by nucleases or proteases. Evidence drawn from the DNAse digestion experiments performed on two samples with sNP antigen detectable by precipitin analysis is in support of this possibility. Short enzymatic treatment prevented these fluids from precipitating with anti-sNP serum, whereas they still contained the same quantity of sNP antigen by radioimmunoassay since their inhibitory capacity remained unchanged. The availability of a strong monospecific antiserum to N-DNA, capable of detecting as little as 5 pg of antigen per ml might be partly responsible for the comparatively high incidence of N-DNA containing fluids by precipitin analysis. It must be stressed, however, that this antiserum can also react with the N-DNA moiety of sNP forming lines of complete identity with isolated N-DNA and making it impossible to differentiate these two antigens on the basis of this test alone. It is very likely that this factor might have elevated the incidence of N-DNA containing fluids since most of the fluids with N-DNA antigen by precipitin analysis had at least 5 pg of sNP antigen per ml by radioimmunoassay. The high incidence of N-DNA antigen found by immunodiffusion is in the range reported by others (29,30). SS-DNA antigen could not be detected by double diffusion in 63 synovial samples by one group of investigators (29), but has been identified by others (13,31). In our study, the presence of nuclear antigens could not be correlated with clinical diagnosis. This observation has previously been reported (29), and it would seem that presence of free nuclear antigens is a relatively nonspecific phenomenon, perhaps related to destruction of polymorphonuclear cells or local tissue breakdown. Such a possibility
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is supported by the observation that lysosyme levels, generally associated with accelerated leukocyte turnover were found to parallel synovial fluid DNA concentrations (29). While appearance of free nuclear antigens in synovial fluids might be related to inflammation or tissue destruction and has been detected in many types of inflammatory arthritides, it is of interest that antinuclear antibodies were found almost exclusively in e&ions from patients with RA (17,32), However, the specificity of these antinuclear antibodies, except for anti-SSDNA (13,31), h as not been investigated. The striking finding in our study was that antibody to either sNP or N-DNA could be detected in 26% of the RA effusions examined. In contrast, with the exception of one sample from a patient with PsA, no antibody could be demonstrated in 23 non-RA samples. Although antibodies to sNP and N-DNA have not been reported in RA synovial fluids to our knowledge, the observation of it2 viva LE cell formation in synovial effusions from RA patients (33) supports our findings since antibody to nucleoprotein (19,34) and to a lesser extent N-DNA (19,35) are known to be responsible for the LE cell phenomenon. The demonstration of antibody in one quarter of the RA fluids tested and free antigen in more than half suggests that conditions are appropriate for immune complex formation. In this respect, the situation is probably analogous to what has been described in the sera of SLE patients where free DNA antigen and anti-DNA antibody have been found alternately (27). We were not able to demonstrate sNP immune complexes in synovial fluids. Failure to identify these complexes may have been technical. It is also possible that these soluble complexes are rapidly removed from the fluid phase by leukocytes and synovial cells. In this respect, leukocyte inclusions containing immunoglobulins, complement, nuclear antigen (17,36-39) and antinuclear antibody (32) are regularly demonstrated in the effilsions of patients with RA and could represent phagocytized antigen-antibody complexes. In this study, complement levels in synovial fluids could not be examined for a variety of reasons, and it was not possible to determine if a relationship existed with presence of antigen or antibody. Furthermore, paired sera were not available to determine if antibody in synovial fluid corresponded with any detectable antibody in blood. In the present work, indirect evidence is presented supporting the concept of sNP and DNA immune-complex formation in RA synovial fluids. The role of these complexes in the pathogenesis of RA synovitis remains unclear, but the ability of DNA and nucleoprotein antibodies to fix complement ill uilro (40) suggests that they may participate in synovial inflammation and local complement consumption. Valuable information on the potential pathogenic role of these complexes (DNA or sNP) might be obtained from sequential studies on RA synovial fluids in an attempt to correlate the presence of either free antibody or free antigen with disease activity and complement levels. Additional studies along these lines might provide further insights into the pathogenesis of synovial injury in rheumatoid arthritis.
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