Clinical Atlergy, 1975, Volume 5, pages 165-174

Incidence of serum anti-DNA precipitins in patients with systemic lupus erythematosus by counterimmunoelectrophoresis

J. J. B I U N D O , J R , M . L O P E Z , P. F. K O H L E R ant/J. H. S A L V A G G I O Divisions of Clinical Immunology, Departmertt of Medicine, Louisiana State University Medical Center, New Orleans, Louisiana and the Department of Medicine, University of Colorado School of Medicine. Denver, Colorado Summary The technique of counterimmunoelectrophoresis (CIE) has been adapted for detection of serum precipitins to calf thymus (CT) DNA in patients with SLE. discoid LE, miscellaneous connective tissue and infectious diseases, and control populations. Of seventy-eight LE patients, 58";, demonstrated anti-ss DNA precipitins, and 20% exhibited anti-ds DNA precipitins. Good correlation was noted between the presence ofss DNA precipitins and ss DNA binding values determined by the more sensitive ammonium sulphate precipitation assay. Depressed total serum haemolytic complement activity in CH50 u/ml was noted in 64% of sera exhibiting ss DNA precipitins and 38*';, of those with negative ss DNA precipitins. There was a strong association, however, between ds DNA precipitins and depressed serum complement levels. Although less sensitive than primary binding assays, CIE can be used as a rapid and simple screening test for detection of circulating anti-native and denatured CT DNA precipitins. CT DNA serum precipitins are present in a significantly higher percentage of SLE patients when compared with other disease states and normal control populations. Introduction The disease activity of systemic lupus erythematosus has been associated with the presence of circulating anti-DNA antibodies, and considerable evidence supports the role of DNA-anti-DNA complexes in the pathogenesis of SLE nephritis. Immunoriuorescent studies have demonstrated DNA along glomerular basement membrane, unti-DNA antibodies have been eluted from post-mortem kidneys of SLE patients, and DNA-anti-DNA complexes have been detected in certain SLE sera (Andres et al., 1970: Koffler, Schur & Kunkei, 1967: Krishman & Kaplan, 1967: Harbeck et al.. 1973: Koffler et al., 1971: Tan et al.. 1966). The current methods used to measure anti-DNA antibodies such as complement iixation, tanned cell haemaggiutination, DNA binding by the ammonium sulphate technique, and Ouchteriony double diffusion are, however, either relatively insensitive. Correspondence: Dr. J. E. Salvaggio, Departmenl of Medicine. Louisiana Stale University, New Orleans, Louisiana.

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or complex and time consuming. Recent evidence from several laboratories has indicated that the technique of counterimmunoelectrophoresis (CIE) can be adapted for the rapid detection of circulating anti-DNA antibodies (Arquembourg et al., 1972: Arquembourg et al. (1974); Dorsch & Barnett, 1972: Davis, 1971: Klajman, Farkash & Meyers, 1973; Johnson, Edmonds & Holborow, 1973). This technique is simple, sufficiently sensitive, and can be performed rapidly. We have been able to analyse up to ninety sera simultaneously on two agar coated lantern slides in an approximate 30 min interval (Arquembourg et al., 1974). In the current study we report the incidence of precipitins to calf thymus (CT) DNA in groups of patients with SLE, miscellaneous connective tissue and infectious diseases and atopic diseases plus a normal control population. Materials and methods Antigen and antisera Calf thymus native double stranded (ds) and denatured single stranded (ss) DNA preparations (Worthington Biochemical Corp., Freehold, N.J.) were prepared in Tris bufTer as previously described {Arquembourg et al., 1974). Quantities of 5 0 fd containing 50 fig DNA were placed in cathodal wells and sera in anodal wells. Sera from seventy-eight patients with proven lupus erythematosus: seventy-one had SLE, and seven had discoid LE (Group I): 146 patients with miscellaneous connective tissue and infectious diseases {Group II); thirty-seven patients with atopic disorders (Group III) and thirty-four normal control subjects (Group IV) were obtained through the immunology service laboratories ofthe LSU Medical Center in New Orleans and the University of Colorado Medical School, Denver, Colorado. Sera had been previously frozen at - 7 0 X . Aliquots of 0-2 ml prepared from each sample were freshly thawed and used for duplicate analyses on different days. SLE sera were unselected and were chosen in the order in which the serum sample had been referred to the service laboratory for analysis. These sera were obtained from patients with SLE with or without nephritis who had been diagnosed on the basis of clinical findings plus a positive antinuclear antibody test by the indirect immunofluorescent technique and from patients with clinical evidence of discoid LE. Patients in the miscellaneous category included individuals with rheumatoid arthritis, juvenile rheumatoid arthritis, ulcerative colitis, myasthenia gravis, lung abscess, post hepatic cirrhosis, scleroderma, nocardiosis and brucellosis. Atopic patients were chosen on the basis of history of recently active allergic rhinitis and/or bronchial asthma plus broad patterns of immediate wheal and flare skin reactivity to common local inhalant allergens. Normal control sera were from laboratory technicians and staff personnel with no personal history of atopy or active disease.

Agar, slide preparation^ and electrophoresis Preparation of agar and electrophoretic conditions were as previously described (Arquembourg ct al., 1974). Essentially, 0-82% ionagar No. 2 in Tris-HCL buffer, pH 7 0 was employed and electrophoresis was performed on constant voltage using a cell in which gel coated Kodak lantern slides were inverted over urethane foam wicks (Arquembourg, Bickers & Salvaggio, 1970). Conditions for electrophoresis were 5 4 V per linear cm of agar, and a 20 min migration interval.

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Serum complement and DNA antibody ammonium sulphate precipitation assay Serum complement assays in CH50 u/ml were determined by the method of Mayer (1961). Measurement of DNA antibody by the ammonium sulphate precipitation assay (Farr. 1958) was done through the courtesy of Drs R. Wold and R. Carr by means of a technique employing Bacillus subtilis tritiated DNA (H^ DNA), sera diluted 1:4 in borate buffer pH 8-4, overnight incubation at 4°C, and precipitation of the labelled DNA-anti-DNA complexes with 50% saturated ammonium sulphate (Wold et al., 1968). Results Figure 1 illustrates a typical washed and stained CIE pattern. Positive DNA-anti-DNA precipitin bands stained red with Pyronin Y alone or reddish purple after counterstaining with light green. Such precipitin bands have been shown to contain specific anti-DNA antibody by absorption studies, by the use of a goat anti-human IgG immunoabsorbent and by studies employing IgG rich fractions obtained from DEAE cellulose chromatography (Arquembourg et al., 1974). Table 1 lists results of serum precipitin analysis of the 295 patients and control sera tested for the presence of antibodies to ss and ds DNA using the CIE technique. Seventy-one Group I patients had SLE and seven discoid LE. Forty-four of the

Fig. 1. Typical washed and stained CIE pattern illustrating anti-ss DNA precipitin bands. Anodal wells (lefl) contain serum and cathodal wells contain ss DNA 50 /ig/well. Wells 3 and 4 illustrate positive precipitin bands.

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Table I. Percent positive anii-ss and ds DNA scrum precipitins by CIE in palients with SLE and discoid LE (Group 1), miscellaneous difl'use connective tissue and infectious diseases {Group ID, atopie diseases (Group III) and normal controls (Group IV) No. of positive anti-DNA precipitins

Group I SLE Discoid LE II Miscellaneous III Atopie IV Normal

No.

ssDNA

dsDNA

71 7 146 37 34

41 (58%) 3 (43";,) 19(13",») 3 (8%) 1 (3"J

14 (20%) 0 I (0 7%) 0 0

100

90 80

70

60

< z a 1/1

50 40 30 20

10

CIE positive

CIE negative

Fig. 2. Comparison between ammonium sulphate precipitation assay and CIE technique using ss CT DNA antigen in tweiKy-four selected CIE positive and twenty-four CIE negative sera. The horizontal line indicates the 20" „ binding capacity level accepted as the approximate cutoff between elevated and normal binding capacities using ss DNA antigen and the lechnique employed. SLE, • ; discoid LE, w; other, A.

seventy-eight patietits in this group or 58",, demonstrated positive precipitins to ss DNA.Threeof the forty-four positive sera were from patients wiih discoid LE and the remaining forty-one from SLE patients. Only fourteen (20%) demonstrated precipitins against ds DNA. Of the miscellaneous Group II sera 13",, (19/146) demonstrated positive ss DNA precipitins, arid one patient of this group had positive ds DNA

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precipitins. Three of the thirty-seven atopie subjects and one 'normal" control serum demonstrated positive ss DNA precipitins. It is of interest that of the fourteen SLE patients with positive ds DNA precipitins, five had associated diseases generally characterized by tissue destruction. These included pulmonary tuberculosis, polymyositis, myasthenia gravis. pulmonary infarction and acute bacteria! pneumonia. CIE is known to be at least ten times as sensitive as conventional Ouchterlony double diffusion for detection of serum precipitins in some antigen-antibody systems. We compared CIE with Ouchterlony double diffusion in twenty-four test sera using ss DNA antigen. Thirteen were positive by CIE and four were positive by double diffusion. None of the four positives by double diffusion were negative after CIE. A comparison was also made between CIE and the highly sensitive ammonium sulphate precipitation assay using selected sera from twenty-four LE patients with positive ss DNA precipitins and twenty-four negative sera. Results of this comparison are illustrated in Fig. 2. Twenty-two ofthe twenty-four CIE positive patients had SLE and two had discoid lupus. All twenty-four CIE positive sera exhibited ss DNA binding capacities greater than 30%. The mean binding value for SLE patients was 54% and for the two discoid LE patients 44%. The twenty-four patients with negative precipitins by CIE consisted of eleven SLE, two discoid LE and eleven miscellaneous connective tissue disease subjects. Eight of the eleven SLE patients with negative precipitins exhibited DNA binding above 30^^. The mean binding value for SLE patients with negative CIE precipitins was 28% and the miscellaneous group mean was 14%. Ofthe two discoid LE sera with negative ss DNA precipitins one demonstrated a very low binding value

•

60

• •

50 -

>

o

V

A V

20 -

1 - •

10 -

28/44decreosed 64%

CIE positive

•

11/29= decreaseC

•

38%

CIE Negative

Fig. 3. Comparison between total serum haemolylic complenieni activity in CHjo/inl and presence of anti-ss DNA precipitins by CIE. The horizontal lines indicate range of normal values of total haenioiytic complement activily for this laboratory using the technique employed. SLE, • ; discoid LE, ,

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70 60

50

40

20

10

t

s CIE positive

C l E negative

Fig. 4. Comparison between total serum haemolytic complement activity in CHso/ml and presence of anti-ds DNA precipitins by CrE. Horizontal lines delineate the range of normal values. SLE, • ; discoid LE, o.

while the other had a 44% value. These results indicated good correlation between the presence of CIE serum anti-ss DNA precipitins and the sensitive ss DNA binding ammonium sulphate precipitation assay. Comparison between the presence of anti-ss DNA serum precipitins by CIE and total serum haemolytic complement activity in CH50 u/ml is illustrated in Fig. 3. Complement activity was assayed in seventy-three LE sera. Forty-four demonstrated positive anti-ss DNA precipitins and twenty-nine were negative. Twenty-eight of the forty-four CIE positive sera (64%) demonstrated decreased serum haemoiytic complement activity is more apt to be present in patients with positive anti-ss DNA precipitins. With regard to ds DNA precipitins. however, there was a better correlation between their presence and depressed total serum haemolytic complement activity as illustrated in Fig. 4. While there were some patients with negative ds DNA precipitins who demonstrated decreased haemolytic complement titres. twelve of the thirteen sera with positive ds DNA precipitins also demonstrated decreased total haemolytic complement activity. This group of patients with positive ds DNA precipitins and low CH50 levels did not necessarily demonstrate evidence of acute renal disease with only four of twelve having known acute SLE nephritis at the time of the study. Discussion The primary findings of our survey are as follows, (a) CIE is a rapid, simple and sensitive screening test for detection of circulating anti-native and denatured CT DNA precipitins; (b) although CIE is less sensitive than primary binding assays, positive anti-ss DNA precipitins correlate well with elevated ss DNA binding values; (c) anti-ss and -ds DNA precipitins can be detected in patients with diseases other than SLE; (d) there is no clear relationship between anti-ss DNA precipitins and decreased CH50

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values, but precipitins against ds DNA do correlate with decreased total serum haemolytic complement activity. In assessing the practical value of CIE as opposed to other methods of detecting anti-DNA or antinuclear antibodies, it seems evident that it offers a less cutnbersome and more rapid method than tanned cell passive haemagglutination or complement fixation, and a more sensitive tnethod than conventional Ouchterlony double dilTusion. Among its advantages over the indirect fluorescent antibody technique are its lack of fluorochrome labelling requirements and tissue substrates plus its ability lo detect specifically anti-DNA as opposed to antinuclear antibody. When compared with the highly sensitive primary binding assays CIE oflFers the advantage of simplicity of performance. Although CIE is not as sensitive as primary binding assays, its lack of requirement for complex radiolabelling procedures, and monitoring equipment, and its rapidity of performance, suggest that it would be more easily used as a rapid screening procedure in clinics and small hospitals where more sophisticated equipment and techniques are generally not in use. Indeed, in a large SLE clinic population we have found it advantageous to draw serum samples on patients before they are seen by the attending physician, perform CIE during clinic hours and have results available immediately after clinic. While the detection of DNA antibodies by CIE appears promising, there are a variety of problems regarding technique which warrant further investigation. Among these problems are the following. Several methods employing different types of agar. buffers and electrophoretic conditions are currently being used in preliminary CIE surveys for anti-DNA precipitins (Arquembourg et al., 1972; Arquemboutg et al., 1974; Dorsch & Barnett, 1972; Davis, 1971; Klajman, Farkash & Myers, 1973). Purified deproteinized CT DNA preparations have not yet been employed for precipitin detection, and some false positive or equivocally positive reactions may be noted using commercial CT DNA preparations. It would seem that future surveys using deproteinized DNA antigen and a standard methodology would aid in perfecting a simple generally accepted procedure for widespread use. The use of deproteinized CT DNA preparations (Harbeck et al., 1973) may be of considerable practical importance in determining whether or not serum precipitins represent specific DNA antibodies. The CT DNA antigens employed in our CIE survey and in other current anti-DNA CIE studies (Arquembourg et al., 1974; Dorsch & Barnett, 1972) might have contained histone and non-histone protein and it is possible that precipitins detected in some sera, particularly these among our miscellaneous group, might not represent specific anti-DNA antibody. In this regard, it is well known that antibodies to many nuclear constituents including ss DNA, ds DNA, soluble nucleoprotein, nuclear RNA protein, ribosomes and other components have been detected in SLE sera {Arquembourg c/a/.. 1972; Wold et al., 1968; Reichlin & Mattioli. 1972;Schur, Moroz & Kunkel, 1968; Komer et at., 1973; Kunkel & Tan, 1964; Holman & Diecher, 1959). Recent evidence using a primary binding assay has indicated that while most SLE patients have antibody to both soluble nucleoprotein and native DNA. antibody to soluble nucleoprotein parallels SLE renal disease activity (Tan, 1967). With the availability of different purified nuclear antigen preparations it would be of interest to perform CIE surveys in SLE with a battery of antigens in order to compare the incidence of precipitins against the various antigens with such variables as SLE activity and presence of renal disease. The commercial CT DNA

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preparation employed in our current survey has been shown to exhibit stable serum DNA binding values after absorption with anti BGG immunosorbents (Robitailie & Tan, 1973). It is, however, theoretically possible that some bovine thymus DNA preparations might result in false positive precipitin tests due to contaminating BGG since antibody to BGG has been detected in over 50",, of SLE sera using sensitive primary binding methods Carr. Wold & Farr (1972) and anti-BGG precipitins are present in approximately 15/., of such sera by conventional Ouchteriony double diffusion analysis (R. I. Carr, personal communication). Our finding of a 58% incidence of anti ss DNA precipitins versus a 20";, incidence of ds DNA precipitins in SLE patients is also of interest and poses some problems in interpretation of results. Although ds DNA has been found in high levels in patients with diseases other than SLE (Carr, 1969), high levels of antibody to ds DNA and depressed serum complement activity have traditionally been associated with active SLE (Carr, 1969; Schur & Sandson, 1968; Friou, 1967). Our comparatively low incidence of anti-ds DNA precipitins suggests that CIE might not be a sufficiently sensitive technique for their detection under the etectrophoretic and technical conditions employed in our survey. Granted that this is so, our high incidence of anti-ss DNA precipitins would still likely be of significance since recent evidence indicates that ss DNA is present in higher concentrations in the serum of patients with SLE than in patients with other diseases (Schur et al., 1968). Other evidence that ss DNA may be an important immunogen in SLE has been provided by the finding that it can form complexes in vivo with anti-ss and anti-ds DNA (Schur et al., 1968). The relationship between anti-ss and ds DNA precipitins and SLE disease activity is also not clear from our current survey. It has been established that high levels of free circulating ss DNA, anti-ss DNA anlibody and anti-ds DNA antibody (Tan et al., 1966: Schur et al., 1968; Carr, 1969) plus decreased total serum baemolytic complement activity and other complement components, notably Clq. C3 and C4 (Carr, 1969; Townes, Stewart & Osier. 1963; Morse, Muller-Eberhard & Kunkel, 1962) are characteristic of active SLE. Although 62% of our anti-ss DNA positive sera demonstrated decreased serum haemolytic complement activity, 39% of our negative sera also demonstrated decreased activity suggesting no clear relationship between positive anti-ss DNA precipitins and SLE disease activity as determined by decreased CH50 values. Since virtually all of our patients who exhibited positive ss DNA precipitins and normal serum haemolytic complement levels had inactive renal disease or nonrenal manifestations of SLE, it is possible that decreased serum complement levels rather than anti-ss DNA precipitins per .se were associated with SLE disease activity. Although high levels of anti-ds DNA antibody have been noted in diseases other than SLE. the frequently observed findings of ds DNA antibody and depressed serum complement levels in patients with active SLE is consistent with our current observations in that virtually all of our SLE patients with anti-ds DNA precipitins detnonstrated decreased serum haemolytic complement levels and clinical evidence of other active diseases associated with tissue necrosis. In spite ofthe above described limitations with regard to antigen purity, technique sensitivity, antibody specificity, and interpretation of positive anti-DNA precipitin tests, our current results indicate that CIE can be used as a rapid screening procedure for the detection of circulating anti-CT DNA precipitins, and that these precipitins are present in a significantly higher percentage of SLE patients wheti compared with other disease states and normal control populations.

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In considering disease pathogenesis rather than diagnosis, it is obvious that our finding of anti-ss and ds DNA precipitins in some patients with miscellaneous connective tissue and infectious diseases, the well known occurrence of circulating ss and ds DNA antibodies plus free DNA (Kohler & ten Bensel, 1969; Davis & Davis, 1973) in a variety of non-SLE disease states, and the finding of free DNA in many postoperative situations, indicates a role for additional host response factors in SLE pathogenesis. it also follows from our current study and other reports using CIE that neither the detection of free circulating DNA or anti-DNA precipitins by this procedure in any given patient is diagnostic of SLE.

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