CHARACTERISTICS OF ANTINUCLEAR ANTIBODIES

Transcription

1 528 CHARACTERISTICS OF ANTINUCLEAR ANTIBODIES IN RHEUMATOID ARTHRITIS Reactivity of Rheumatoid Factor with a -Dependent Nuclear Antigen CAROL T. AITCHESON, CAROL PEEBLES, FENNEKE JOSLIN, and ENG M. TAN The immunologic specificities of antinuclear antibodies (ANA) in sera from 97 patients with rheumatoid arthritis (RA) were studied. The frequency of ANA detected by immunofluorescence with mouse kidney sections as substrate was 6% (59/97) compared to 13% (7/ 55) in controls. IgM ANA was positive in 41% (4/97) of RA sera, IgG ANA in 4% (39/97), and IgA ANA in 33% (32/97). Specificities of antibodies in the ANA positive sera were examined by radioimmunoassay for antibodies to DNA and by immunodiffusion for antibodies to Sm antigen, nuclear ribonucleoprotein, and the SS-A and SS-B nuclear antigens. Antibodies to these nuclear antigens were found infrequently (1.7% to 6.8%). Sera were further investigated for antibodies to histones by an immunofluorescent method previously described using acid-extracted and histone reconstituted tissue sections. Twenty-four percent of the ANA positive sera (14/55) reacted with a histone-dependent nuclear antigen. The relationship of rheumatoid factor to ANA was studied by isolating rheumatoid factors (RF) from aggregated IgG absorbant columns. Ten of 19 isolated RF showed ANA activity, which did not result from complexes of rheumatoid factor with IgG ANA since IgM rheumatoid factor, dissociated from IgG under acid con- From the Division of Rheumatic Diseases, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado Carol T. Aitcheson, MD: Research Fellow; Carol Peebles, MS: Research Assistant; Fenneke Josh, BA: Research Assistant; Eng M. Tan, MD; Professor of Medicine, and Head, Division of Rheumatic Diseases. Address reprint requests to Carol Aitcheson, MD, University of Colorado Health Sciences Center, Division of Rheumatic Diseases, 42 East 9th Avenue, Box B 115, Denver, Colorado Submitted for publication November 2, 1979; accepted in revised form February 1, 198. ditions, continued to show ANA activity. In 8 of RA sera tested, both ANA and rheumatoid factor activity could be inhibited by aggregated IgG. Five of the crossreacting rheumatoid factors reacted specifically with a histone-dependent antigen in the reconstitution assay. These findings show that approximately WO of rheumatoid factors possess cross-reactivity with nuclear components, and histones are involved in a significant number of these reactions. The presence of antinuclear antibodies is an important feature of a variety of connective tissue diseases. In some diseases the specificity of the antibodies or the presence of certain antibodies in high titer helps characterize the disease. High titers of antibodies to nuclear ribonucleoprotein (n-rnp) in mixed connective tissue disease (1-3), anti-sm (43) and high titers of anti-native DNA (5-1) in systemic lupus erythematosus, anti-histone antibodies in drug-induced lupus (1 l), and anti- SS-A and SS-B antibodies in Sjogren s syndrome (12) are all examples of specific antibody profiles that can help define disease states. Patients with rheumatoid arthritis are also known to have antinuclear antibodies. The reported frequency of these antibodies as determined by indirect immunofluorescence varies from less than 1% to greater than 7% depending on the substrate used and the antibody titer considered significant (12-23). Antibodies to double-stranded DNA (dsdna) (5,9,2425), single-stranded DNA (ssdna) (26), soluble deoxyribonucleoprotein (snp) (9, n-rnp (5), SS-A, SS-B, and rheumatoid arthritis associated nuclear antigen (RANA) (12,27) have all been reported in patients with rheumatoid arthritis, but no characteristic antibody profile has been delineated. Antibodies to RANA have Arthritis and Rheumatism, Vol. 23, No. 5 (May 198)

2 ANA IN RHEUMATOID ARTHRITIS 529 been found in the greatest frequency (65%) (12), but these antibodies, along with antibodies to ssdna and SS-A, are not detected by immunofluorescence on organ sections which are often used as substrates, and thus do not account for the fluorescent ANA seen in RA. More extensive characterization of the antinuclear antibodies in rheumatoid arthritis was the purpose of this study. MATERIALS A METHODS Population studied. Sera were collected from 97 patients with definite or classic rheumatoid arthritis as defined by the American Rheumatism Association criteria (28). The ages of the patients ranged from 22 to 85 years with a mean age of 55 years. Male to female ratio was 51 :46. The control group consisted of 3 normal laboratory personnel, mean age 35, patients with degenerative joint disease (osteoarthritis) and no other known connective tissue disease, mean age 66, and 9 older normal controls, mean age 73. The mean age for the control group as a whole was 5 years with a male to female ratio of 26 : 29. Determination of antinuclear antibodies by indirect immunofluorescence. All sera were tested for the presence of antinuclear antibodies (ANA) by indirect immunofluorescence on two substrates, 4p cryostat sections of mouse kidney (29) and HEp-2 cells, a continuous cell line of human laryngeal carcinoma (Antibodies, Inc., Davis, California). Fluorescein-isothiocyanate (FITC) labeled goat anti-cohn Fraction 11, prepared in this laboratory (3), was the primary conjugate used. The patterns and intensity of fluorescence evaluations were read at 4~ with a Leitz Ortholux I1 incident light microscope with a 2W mercury light source, KP49 exciter lilter, and K53 barrier filter. On the HEp-2 substrate, sera were tested for ANA activity at dilutions 1 :4, 1:, and 1 :. Sera giving 1+ or greater fluorescence at a dilution of 1 : 4 were considered positive. Both dividing and nondividing cells were examined for patterns of staining. ANA on mouse kidney sections were determined by using serum dilutions of 1 :4, 1 :, 1 :, and 1 :. The intensity of the immunofluorescence at each dilution was graded as trace to 4+. Sera exhibiting 1+ or greater fluorescence at a dilution of 1 : were considered positive. The sera were also tested for ANA of specific IgG, IgA, and IgM immunoglobulin classes by using specific FITC-conjugated antisera (anti-igm and anti-igg, Calbiochem-Behring, La Jolla, California; anti-iga, Meloy, Springfield, Virginia). Sera were considered positive for IgA and IgG ANA if definite nuclear immunofluorescence was noted at 1 :4 serum dilution. IgM antibodies were considered positive only if present at 1+ or greater at a dilution of 1 :. The frequency in controls for IgG ANA and IgA ANA at serum dilution 1 : 4 was 11% and 13%, respectively. The frequency in controls for IgM ANA at serum dilution 1 : was 15%. The frequency of ANA of these classes in RA patients was significantly different (see Results). Detection of specific antibodies. Sera were heated for 3 minutes at 56 C to inactivate complement. They were then tested for precipitating antibodies by a modification of the Ouchterlony double-diffusion technique (3 1). Sera were investigated by immunodiffusion in agarose against rabbit thymus extract for antibodies to Sm and nuclear RNP (3,31), against soluble nucleoprotein for antibodies to snp (32), and against WiL-2 extract for antibodies to SS-A and SS-B (12). Antibodies to double- and single-stranded DNA were measured by the Millipore filter technique (Millipore Corp., Bedford, Massachusetts) (7,33). Antibodies to dsdna were considered significant if the binding was greater than 1% and antibodies to ssdna significant if the binding was greater than 2%. Antibodies to histones were determined by an immunofluorescent technique previously described (34) using acideluted and histone reconstituted mouse kidney sections as outlined below. The acid extraction removes Sm, n-rnp, SS-B, and histones as well as other unidentified nuclear antigens but does not remove DNA. Four micrometer cryostat sections of mouse kidney were fixed in acetone for 1 minutes at room temperature and air-dried. The mounted kidney sections were immersed in.1n HC1 and gently agitated for 3 minutes at room temperature. After rinsing in two changes of phosphate buffered salie (.15M NaC1;.1M phosphate buffer, ph 7.3), the sections were used as acid-extracted substrate for ANA detection or were reconstituted with purified histones. Lyophilized, acid-extracted calf thymus total histone (Worthington, Freehold, New Jersey) was used for reconstitution. Sera containing antibodies to histones have been shown to give a characteristic staining sequence when tested on untreated, acid-extracted, and histone-reconstituted sections (34). On control-untreated sections, sera containing antibodies to histones give a homogeneous or patchy pattern. On acid-extracted sections, these sera show either a complete loss of staining or a significant drop in titer. On histone-reconstituted sections, these sera show return of immunofluorescent staining, often in a more clumped pattern. (See Discussion.) All ANA positive sera were tested in the stated manner with both FITC-conjugated anti-cohn Fraction I1 and specific anti-igm. Isolation of rheumatoid factor. Cohn Fraction I1 at 2 mg/ml in PBS was aggregated by heating at 63 C for 15 minutes and was cleared of insoluble material by centrifugation at 1, rpm for 2 minutes. One gram of aggregated Fraction I1 was linked to 2 gm of cyanogen-bromide activated Sepharose 4B according to package instructions (Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey). The beads were stored at 4 C in PBS with.1% sodium azide. One volume of serum was mixed with two volumes of packed, coated beads and one volume of PBS and gently agitated on an Eberbach shaker overnight at room temperature. The mixture was poured into a small column (usually a 5 or 1 ml plastic pipette) and washed with at least 2 volumes PBS. Rheumatoid factor was eluted from the column with 2 volumes.1m glycine-acetate, ph 3.6, dialyzed against PBS, concentrated, and checked for latex activity using a slide test. Column chromatography of isolated rheumatoid factor. All column procedures were carried out at room temperature. Sephadex G2 was allowed to swell for 72 hours in.1m glycine-acetate buffer, ph 3.6. Excess buffer was removed and the gel was resuspended in fresh buffer and al-

3 53 AITCHESON ET AL lowed to settle for 15 minutes. This decanting procedure was repeated three times. The slurry was degassed for 18 hours, poured into a 2.3 X 35 cm column, and washed with 1 liter of degassed buffer at 1 cm pressure. The void volume of the column was determined by running I mg of Blue Dextran 2 through the column. All samples applied to the column had been dialyzed in glycine-acetate buffer and adjusted to 1% with sucrose before application. The volume of the samples was usually 1.25 ml. Pressure was 1 cm and the flow rate was approximately 6 ml/hour. Fractions were monitored on a Beckman spectrophotometer at 28 mp. Those showing absorbance were pooled, dialyzed against PBS, and concentrated. Ammonium sulfate precipitation. Serum was incubated for 1 hour at 4 C with an equal volume of saturated ammonium sulfate and centrifuged for 2 minutes at 15, rpm at 4 C. The precipitate was suspended in and dialyzed extensively against PBS. Inhibition of sera by aggregated fraction 11. For each serum to be absorbed, four aliquots were prepared. The control contained 2 p1 of a 1 :4 dilution of serum. The three other aliquots, also of 2 p1, additionally contained 2.,.5, or.5 mg heat-aggregated Cohn Fraction 11. The aliquots were left for 3 hours at room temperature with occasional shaking and then overnight at 4 C. They were centrifuged for 3 minutes in a Clay Adams serofuge. Supernatants were tested for ANA and latex activity at effective serum dilutions of 1 : 4, 1 :, 1 :, and 1 : to monitor changes in antibody titer. Anti-IgM conjugate was used for the ANA testing to avoid reaction with IgG complexes. Rheumatoid factor determination. Rheumatoid factor was quantitated in most studies by the 12-tube latex agglutination method (Behring, La Jolla, California) (35,36). Because the volumes of isolated rheumatoid factors were small, a micro-latex test was used on these samples. Twenty-five microliters of latex diluted 1 : 1 were added to 25 p1 of sample on a serologic ring slide (Scientific Products). The slide was shaken gently for 3 minutes and agglutination read over a dark background. This slide test is one-tenth as sensitive as the standard 12-tube method. Samples were considered positive for RF when 1 :4 dilution gave positive agglutination. Tests for determination of significance. Significance tests were based on the standard chi-square test for contingency tables. RESULTS Antinuclear antibodies by immunofluorescence. The frequency of antinuclear antibodies in sera of 97 patients with rheumatoid arthritis was 6% as determined by indirect immunofluorescence on mouse kidney sections by use of anti-cohn Fraction I1 conjugate. The frequency in a group of 55 controls of similar age and sex ratio was 13% (Table 1). The difference in frequency in rheumatoid arthritis patients and controls was significant (P c.1). Sera were considered positive if nuclear immunofluorescence of at least 1+ was present at a dilution of 1 :. Eighteen percent (17 of 97) of RA sera were still positive with an intensity of at least Table 1. Antinuclear antibodies in rheumatoid sera Frequency in Frequency in RA* controlst Fluorescein-conjugated reagent No. % No. % Anti-Cohn Fraction 11s Anti-IgG I1 Anti-IgMS Anti-IgA I 13 * Mean age 55; ma1e:female = 51:46; total number of patients was 97. t Mean age 5 makfemale = 26:29; total number of patients was 55. * Considered positive if I+ at 1:. 8 Considered positive if definite nuclear staining at 1:4. 1+ at a serum dilution of 1 :. Only 5.5% (3 of 55) of control sera were still positive at this dilution. IgG ANA was present in 4% of RA sera, IgM ANA in 4196, and IgA ANA in 33%. The frequency of ANA of each specific class was significant (P <.1) when compared to controls. Many rheumatoid sera contained antibodies of more than one immunoglobulin class. Most control sera, however, contained no detectable ANA or ANA of only one class (Table 2). It was noted that IgM ANA in low titer was the most frequent pattern (1 1%) of ANA in the control population (Table 2). Antinuclear antibody specificities. Those sera positive for ANA by immunofluorescence were tested for the presence of antibodies to known nuclear antigens (Table 3). Antibodies to double- and singlestranded DNA were found in low frequency, 3.5% and 6.8%, respectively. Antibodies to soluble nucleoprotein (snp), Sm antigen, and nuclear ribonucleoprotein (n- RNP) were found in one patient each. Antibodies to SS- A and SS-B nuclear antigens, seen commonly in patients with Sjogren s sicca complex, were present in 6.8% and 1.7%, respectively. Antibodies to histones. Antibodies to histones were seen in 23.7% of ANA positive sera, a strikingly Table 2. Immunoglobulin classes of antinuclear antibodies Classes of antibodies M+G-A- M-G+A- M-G-A+ M+G+A+ M+G+A- M+G-A+ M-G-A- M-G-A- RA sera Control sera No.* % No.? % * Total number of sera tested = 97. t Total number of sera tested =

4 ANA IN RHEUMATOID ARTHRITIS 53 1 Table 3. Specificities of antinuclear antibodies in rheumatoid sera Frequency of antibodies Nuclear antigen No.* 9% dsdna ssdna snp Sm n-rnp SS-A SS-B s * Total number of sera tested = higher frequency than for any of the other antibody specificities tested. Characteristics of the sera containing antibodies to histones are shown in Table 4. Most of these sera contained rheumatoid factor in high titer although this was not invariable (A-, B-l). The staining pattern on mouse kidney was diffuse or patchy homogeneous in all cases, sometimes associated with nucleolar or rim staining and was detected in a titer of at least 1 :. The titer of anti-histone antibodies de- Table 4. Serologic findings in sera with antibodies to histones Antibodies Patient RF Staining on to number latex titer mouse kidney histones* A-5 Horn? 1: 1: A- Neg Horn 1 : 1: B- 1 8 Horn + nucleo+ 1 :4 1: B-3 Horn 1: 1: B Horn + nucleo 1 :4 B : Horn 1 :4 B-12 1: Horn I : 1: B- 1,24 Horn 1: 1: c-2 I28 Horn + nucleo 1: c-11 1: Horn 1: D-4 1,24 1: Hom 1:4 1: D Horn 1: 1: D- Horn + rim 1: E-18 4,96 1: Horn 1: 1: * Determined by immunofluorescence as described in Materials and Methods. t Horn = homogeneous or patchy pattern. $ Nucleo = nucleolar pattern. tected on histone-reconstituted sections was often a few dilutions lower than the titer of ANA on untreated tissue sections. Sera were tested for anti-histone antibodies by use of fluorescein conjugated anti-cohn Fraction I1 and anti-igm. Three sera, A-, C-2, and D-4, were negative for anti-histone antibodies with fluorescein conjugated anti-cohn Fraction 11, but antibodies were detected with the anti-igm conjugate. All rheumatoid sera were stained for immunofluorescence on HEp-2 cells. The sera that contained antibodies to histone showed homogeneous staining on HEp-2 cells. These sera also stained metaphase chromosomes of dividing cells in a homogeneous pattern as shown in Figure 1. This pattern of staining was seen in all 14 sera that contained antibodies to histone. Rheumatoid factor with antinuclear antibody activity. The relationship of rheumatoid factor (RF) to antinuclear antibodies was studied. Fifteen rheumatoid sera were selected for ANA positivity and high rheumatoid factor latex titer. Rheumatoid factors were isolated by absorption onto aggregated Cohn Fraction I1 affinity columns followed by thorough washing with saline and elution with dilute acid. Table 5 shows that 8 of the 15 isolated rheumatoid factors had antinuclear antibody activity by immunofluorescence. Results on 4 additional sera with no ANA detected in sera but with high titers of rheumatoid factor are also shown. Two of these rheumatoid factor gave positive ANA. This finding suggests that antinuclear antibodies were present in low levels in these sera and were concentrated by isolating rheumatoid factor. Eight of the 19 sera tested contained antibodies to histones. Of these eight, the isolated rheumatoid factors in five also had anti-histone activity. The immunofluorescent ANA in each of these cases was completely negative on acid-extracted sections and was reconstituted by histone addition. The reconstituted titer in three cases was lower than the original ANA titer. (Specific titers are given in Table 5.) One rheumatoid factor had ANA activity but was not anti-histone (D-4), and the other two did not contain ANA activity (A-5, B-3). The 3 sera with antibodies to SS-B, n-rnp, and Sm (A- 8, C-13, C-15, respectively) demonstrated no ANA activity of the isolated rheumatoid factor. Relationship of RF and ANA activities. The possibility existed that antinuclear antibodies were bound to rheumatoid factor in the form of complexes of IgG antibody and antigen, and that these complexes, not the rheumatoid factor itself, were responsible for the positive ANA. Further studies were carried out in an at-

5 532 AITCHESON ET AL Figure 1. Fluorescent staining on HEp-2 cells of a rheumatoid serum containing antibodies to histones. The homogeneous pattern of staining on nondividing cells and cells in metaphase is characteristic. Table 5. Antinuclear antibody activity of isolated rheumatoid factor Antinuclear antibody in Serum RF Serum Isolated RF latex Patient titer Titer Specificity Titer Specificity (titer) B-9 B-I6 c-2 c-11 B-8 D-4 B-18 D- 1 A-5 B-3 A-8 C-13 C-15 A-3 B , , Not histone, unknown Not histone, unknown SS-B n-rnp Sm Unknown Unknown 4 () () () (4) (4) Not histone, unknown Not tested Not tested c-9 D-12 D-11 D Unknown Unknown

6 ANA IN RHEUMATOID ARTHRITIS 533 tempt to separate any such complexes from the RF. Isolated RF with ANA activity was chromatographed on a G2 column at acid ph. The results of one such chromatography are shown in Figure 2. The fractions were pooled and concentrated. The void volume fraction, which by immunoelectrophoresis showed IgM but no IgG, had high rheumatoid factor titer and IgM-ANA activity but no IgG-ANA activity. The second peak showed IgG but no IgM, had IgG-ANA, and had only negligible rheumatoid factor activity. This IgG-ANA may represent IgG complexes attached to the RF or may have been IgG-RF which also had cross-reacting ANA activity. In either case, IgG was separated from IgM rheumatoid factor, but the IgM-RF continued to demonstrate ANA activity. This chromatographic separation was performed on two additional isolated rheumatoid factors, C-1 1 and D-4, which by immunofluorescence had demonstrated ANA activity. In each OD28 Antinuclear Antibody Activity OD28 Latex Titer Anti-lgM Anti-lgG Isolated RF Void Volume s Figure 2. Rheumatoid factor was isolated from serum B- by chromatography using Sepharose beads coated with heat-aggregated Cohn Fraction 11. The adsorbed RF was eluted with dilute acid. Isolated rheumatoid factor was then chromatographed on a G2 column at ph 3.6. Fractions were dialyzed against PBS. The void volume fraction, which by immunoelectrophoresis contained IgM and no detectable IgG, had high RF titer and ANA activity. The 7s peak showed IgG ANA and negligible RF. case, the results were similar; i.e., IgM rheumatoid factor continued to have ANA activity after separation of IgG from IgM under dissociating conditions at acid ph. Another attempt to separate RF and ANA in sera in which both activities were present is demonstrated in Figure 3. Immunoglobulins were precipitated by ammonium sulfate from serum B-9, a serum which, by previous studies, had demonstrated cross-reacting RF. This precipitate was chromatographed on a G2 column at acid ph. The void volume pool had both RF and IgM ANA activity. The 7s pool from the column had RF and IgG ANA activity. The material from each peak was passed over an affinity column of heat-aggregated IgG in order to isolate rheumatoid factor. The eluate obtained from the void volume pool showed both IgM ANA activity and RF activity. The eluate from the 7s pool had neither ANA nor RF activity. Figure 4 demonstrates that in another serum, A- 5, rheumatoid factor and ANA activity were clearly separable from each other by the same procedure. IgM ANA activity was not absorbed to aggregated IgG and was recovered in the effluent, whereas IgM rheumatoid factor was adsorbed and recovered in the eluate. The data from these experiments suggest that certain rheumatoid factors cross-react with nuclear antigens. This could be further tested by showing that crossreacting ANA could be inhibited by IgG aggregates. Sera positive for both ANA and rheumatoid factor were incubated with varying amounts of heat-aggregated Cohn Fraction I1 and titers were measured to determine residual immunofluorescent ANA. Table 6 shows the ANA results on two such absorbed sera. The ANA reaction of serum B-9 is clearly inhibited by IgG aggregates; that of A-5 is not. Eight of rheumatoid sera thus tested were inhibited (Table 7). Sera from 9 patients with SLE, MCTD, and Sjogren s syndrome containing antibodies to a variety of nuclear antigens including SS- B, n-rnp, Sm, DNA, snp, and histones were also tested in this manner. None showed inhibition of ANA activity with aggregated IgG. In addition to the inhibition results, Table 7 summarizes the results of all isolated rheumatoid factors studied, showing that of 19 rheumatoid factors tested, 1 had ANA activity. Nine isolated rheumatoid factors did not have ANA, although whole sera showed this activity. In these cases, aggregated IgG did not remove ANA activity from serum (A-5, B-3, C-13, C-15, A-8). Table 7 shows that a total of 25 RA patients sera were tested for cross-reacting RF and ANA, either by isolation of RF or inhibition with aggregated IgG. Thirteen of the 25 showed this cross-reaction.

7 534 AITCHESON ET AL Serum 9-9 I Saturated Ammonium Sulfate Protein Precipitate Containing Immunoglobulins Void Volume RF IgM ANA 1 : IgG ANA Sepharose Linked to Heat -aggregated IgG 7s Fraction RF 8 IgM ANA IgG ANA 31 : Sepharose Linked to Heat -aggregated IgG Effluent Eluate Effluent - Eluate RF RF RF IgM ANA IgM ANA IgG ANA IgG ANA IgG ANA t IgG ANA Figure 3. Immunoglobulins were precipitated by ammonium sulfate from serum B-9, a rheumatoid serum which by previous studies had demonstrated cross-reacting rheumatoid factor. This precipitate was chromatographed on a G2 column at acid ph. The void volume had both RF and IgM ANA activity. The 7s peak had RF activity and IgG ANA. The material obtained from each peak was passed over an affinity column of heat-aggregated IgG. Adsorbed IgM was eluted from the affinity column and continued to show RF and ANA activity. The 7s fraction did not bind to the affinity column, and the effluent contained ANA activity but no RF activity. Inhibition by aggregated IgG was also used to determine if this removed antibody to histones. Three sera containing anti-histone antibodies (B-3, B-9, B- ) were tested for inhibition of specific anti-histone activity by soluble IgG aggregates. Sera B-9 and B- showed inhibition of anti-histone activity, consistent with the previous finding that the isolated rheumatoid factors from these sera had anti-histone activity (Table 5). Serum B-3 showed no inhibition. DISCUSSION In this study, antinuclear antibodies in the sera of patients with RA were studied for frequency, immu- noglobulin class, specificity, and cross-reactivity with aggregated IgG. Antibodies of the IgG, IgM, and IgA immunoglobulin classes were all seen in both the RA and control sera, but in a higher percentage in the former. Only 3 of 55 or 6% of the controls had antibodies of more than one immunoglobulin class but 36 of 97 or 37% of RA patients had ANA of more than one class. The control group most often had antibodies of the IgM class alone and these were present in low titer. No immunoglobulin class or combination of classes of ANA antibodies was notably more prevalent than any other in the RA population. Antibodies to specific nuclear antigens Sm, n-

8 ANA IN RHEUMATOID ARTHRITIS 535 Serum A-5 Saturated Ammonium 1 Sulfate Protein Precipitate Containing Immunoglobulins n G2 Column ph 3.6 Void Volume : RF 128 IgM ANA 1 : IgG ANA / U 1 Sepharose Linked to Heat -aggregated IgG 7s Fraction RF 2 IgM ANA IgG ANA 1 : Sepharose Linked to Heat-aggregated IgG Effluent Eluate (Isolated RF) Effluent RF Wl RF RF - Eluate I IgM ANA 1 : 1 IgM ANA IgM ANA.IgM ANA IgG ANA IgG ANA IgG ANA 1 : IgG ANA Figure 4. Immunoglobulins were precipitated by ammonium sulfate from serum A-5, shown to have unrelated ANA and RF activities. The same chromatography was performed as in Figure 3. When the 19s fraction from the G2 column was passed through the affinity column, the IgM ANA did not bind to aggregated IgG, and the RF and ANA activities were separated. Table 6. Inhibition of IgM antinuclear antibody activity by aggregated IgG Mg aggregated Cohn Unab- II/ml serum Serum sorbed 1 mg/ml 1 mg/ml 4 mg/ml B-9 1:4* 1: 1:84 1: A-5 1:4* 1: 1: 1: Tr 1+ I+ Tr 2 Trt I+ Tr 2 1+ Tr * Sera were absorbed at 1:4 dilution only and titers were then determined. t Tr = trace RNP, snp, SS-A, SS-B, and ss- and dsdna were each found in only a small percentage of the rheumatoid patients. The frequency of antibodies to n-rnp was somewhat lower than the frequency of 1% found by Notman (5). In the present study, antibodies to n-rnp were investigated only by immunodiffusion. It is possible that the hemagglutination method for detecting antibodies to n-rnp (2) used in Notman s study picked up low levels of anti-rnp, not detected by the less sensitive immunodiffusion method used in this study. The RA sera were tested for antibodies to histones by an immunofluorescent technique using acidextracted and histone-reconstituted tissue sections. Previous work with this technique (1 1,34) has shown the need for DNA in the acid-extracted tissue sections for reconstitution of the histones. Since histones have a

9 ~~ 536 AITCHESON ET AL Table 7. Summary of experimental data on the relationship of rheumatoid factor to antinuclear activity Inhibition RF ANA of of IgM ANA in Patient Serum (latex rheumatoid serum by number ANA titer) factor aggregated IgG B-8 B-9 B- c-2 c-1 I A-5 B-3 c-13 C-15 A-8 C-6 D-5 B- 1 D-15 B-12 D- A-3 B-6 B-18 c-9 D- 1 D-4 D-11 D-12 D , , strong tendency to self-aggregate (37), the requirement for DNA may be related to the fact that histone antigenic sites are made available for reaction with antibody by unfolding of histones on some form of framework like that provided by DNA in the acid-extracted sections. It is also possible that histones complex with DNA to form a new junctional antigenic determinant. We have not determined which of these two possibilities is correct, and it is advisable to consider that the RA antibodies are reactive with a histone-dependent nuclear antigen. The frequency of antibodies to histones in the RA sera as determined by the reconstitution technique was much higher than antibodies to any of the other antigens. The titer of anti-histone antibodies detected on histone-reconstituted sections was often a few dilutions lower than the titer of ANA detected on untreated tissue sections. This observation could be accounted for by at least two possible explanations: 1) more than one antinuclear antibody may be present in certain sera, and anti-histone antibodies may account for only a portion of the nuclear immunofluorescence seen on untreated sections; or 2) certain classes of histones may not reconstitute on the acid-extracted sections in adequate quantity or in such a way that the antigenic sites are exposed. In addition, several sera (not considered positive in the final tally) gave very weak staining at low serum dilutions when tested for antibodies to histones. This finding suggests that some RA sera contain low titer anti-histone antibodies not reproducibly detected by the currently used technique. It is possible that with more sensitive techniques a higher frequency of antibodies to histone can be detected in rheumatoid arthritis sera. Of special interest was the relationship of rheumatoid factors with antinuclear antibody activity. Hannestad and Johannessen first reported this type of reaction in 1976 (38). They described a serum that contained antibodies to IgG which had antinuclear antibody activity. These antibodies were isolated on an IgG-agarose immunosorbent column. The column was able to absorb 1% of the RF activity and 99% of the ANA activity from the immunoglobulin fraction of the serum. The polyclonal nature of these cross-reacting antibodies was demonstrated by the presence of K and h light chains and y and p heavy chains. Further work by Hannestad showed that a cross-reacting RF bound to nucleosomes, the repeating histone-dna subunit of chromatin (39). It did not bind to native DNA alone but still bound to nucleosomes after histone fractions H1 and H5 had been removed. Agnello and associates have also done work in this area, demonstrating by gel diffusion that cross-re-

10 ANA IN RHEUMATOID ARTHRITIS 537 acting rheumatoid factors form precipitating lines of identity with aggregated IgG and a nuclear antigen which they describe as a DNA protein (4). They found this type of antibody in 21 of 56 RA patients, 4 of 1 RA overlap syndromes, and 3 of 5 patients with mixed connective tissue disease. In this study, 25 RA sera were tested for crossreacting rheumatoid factor either by isolating rheumatoid factor and testing for ANA activity or by inhibiting the ANA reaction in the serum by adding IgG aggregates. Thirteen of the 25, or one-half of the sera tested, showed rheumatoid factors cross-reacting with nuclear components. In 5 of 13 positive sera, the rheumatoid factor reacted specifically with a histone-dependent nuclear antigen. This anti-histone activity demonstrated by immunofluorescence is especially interesting in light of the report by Hannestad who, using a completely different technique, demonstrated the cross-reaction of one of his isolated rheumatoid factors with nucleosomes, which contain histone. Thus, studies from several laboratories report the occurrence of RFs which cross-react with nuclear antigens. In Hannestad s and our studies, there is evidence that at least one of the nuclear antigens involved is histone or a histone-dna complex. It is not yet known whether the dual reactivity of the antibodies involved is a true cross-reaction in the sense that both antigens share a common structural component or is an example of multispecificity of antibodies for different antigens that are structurally unrelated. The latter concept was discussed in detail by Richards et a1 (41). They studied protein 46, a mouse IgA myeloma protein which bound the haptens 2- DNP-lys and 2 methyl-1, 4 naphthoquinone (menadione). The binding for menadione could be inhibited by either 2,4 dinitrophenol or treatment with 2% dimethyl sulfoxide without impairing the binding to Z- DNP-lys. The binding to Z-DNP-lys but not to menadione could be inhibited by denaturation and partial unfolding. These data suggested spatially separated binding sites for the two haptens. Further studies showed the distance between these binding sites to be approximately 14 A and nonoverlapping, thus demonstrating an antibody with at least two separate binding potentials. Further identification of the nuclear antigen involved in the cross-reacting of RF and ANA should help determine which of the explanations is operative in this case. If the observed phenomenon is a true cross-reaction, an implication is that not only IgG but also nuclear antigens are stimuli for the production of a certain percentage of the antibody we call rheumatoid factor. There may be other as yet undiscovered primary anti- gens and the reaction of RF with both IgG and histones may be secondary. Further studies in this area are of importance since more complete characterization of antinuclear antibodies, rheumatoid factor, and their interrelationships may well lead to further knowledge about the etiology and pathogenesis of rheumatoid arthritis. REFERENCES 1. Sharp GC, Irvin WD, LaRoque RL, Velez C, Daly V, Kaiser AD, Holman HR: Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. 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