Clin. exp. Immunol. (1979) 35, 356-363.
DNA binding activity of serum from patients with systemic lupus erythematosus J. F. HALSEY, W. A. WOOLERY, S. OLEINICK, M. BASHAR KAHALEH & E. CARWILE LEROY Department of Microbiology and Department of Medicine, University of Oklahoma and the Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA
(Received 7 July 1978)
SUMMARY
The reaction conditions essential for reproducible use of the cellulose ester membrane filter assay for anti-DNA antibody levels in patients with systemic lupus erythematosus are described. A dependence of DNA-binding capacity on serum concentration was observed in the assay, requiring that serum concentrations be comparable in determinations of DNA-binding activity of different sera and when comparing different published studies. The DNA-binding capacity of serum from lupus patients was found to be significantly different from that of healthy controls. However, the binding capacities were not significantly different between lupus patients with and without nephritis. The relative avidity of the anti-DNA antibodies were estimated from plots of 1/DNA bound vs 1/DNA free and these data indicate that the avidities of the antibodies from the two groups of lupus patients are not significantly different. This observation suggests that the tightness of binding between the DNA and the serum anti-DNA antibodies cannot be used to predict immune complex-induced nephritis in lupus patients.
INTRODUCTION Antibodies reacting with a variety of nuclear and cytoplasmic antigens have been found in the serum of patients with systemic lupus erythematosus (SLE) (McDuffie & Bunch, 1977). There is considerable evidence suggesting that the DNA/anti-DNA system is important in the pathogenesis of lupus nephritis (Tan et al., 1966; Cochrane & Koffler, 1973). A positive correlation has been shown between antibody titres to double-stranded DNA and clinically active renal disease (Kredich, Skyler & Foote, 1973), decreased serum complement, and the number of SLE criteria present (Weitzman & Walker, 1977). Also, patients with persistently high titres of anti-DNA antibody usually have a clinical course characterized by progressive renal impairment (Adler et al., 1975). Similar results have been obtained in NZB/NZW mice where a correlation was found between a drug regimen associated with suppression of anti-DNA antibody titres and the lack of progression of glomerulonephritis (Hahn et al., 1973). However, anti-DNA antibodies are found in patients with and without lupus nephritis, and the relationship between the qualitative and quantitative features of the antibodies and their role in the pathogenesis of renal disease (Cohen et al., 197 lb; Pincus et al., 1969) is not clear. The properties of the anti-DNA antibodies that are important in the immunopathology are not known but several contrasting proposals have been made. Investigators have implicated non-precipitating serum DNA antibodies as causative in SLE nephritis (Alarcon-Segovia, Fishbein & Alcala, 1970), while others have emphasized precipitating serum DNA antibodies (Johnson, Edmonds & Holborow, 1973). Low avidity serum anti-DNA antibodies have been proposed by Soothill & Steward (1971) to be particularly important in the development of nephritis. It was suggested that these antibodies, similar to non-precipitating antibodies, are poorly cleared by Correspondence: Dr J. F. Halsey, Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103, USA. 0099-9104/79/0030-0356$02.00 (© 1979 Blackwell Scientific Publications
356
DNA binding activity in SLE serum
357
the reticuloendothelial system and remain in the circulation to initiate immune complex-mediated tissue damage. In contrast to this proposal, several laboratories have published apparent correlations of high avidity anti-DNA antibodies with the occurrence of lupus nephritis (Gershwin & Steinberg, 1974; Leon et al., 1977). Winfield, Faiferman & Koffler (1977), however, found the serum anti-DNA antibody avidity to be lower in patients with lupus nephritis. These divergent reports prompted the present investigation to determine with technically suitable procedures both the antigen-binding capacity and avidity of SLE sera from patients with and without active nephritis.
MATERIALS AND METHODS Patients were selected from the wards and clinics of the Medical University of South Carolina and affiliated hospitals and from the University Hospital of the University of Oklahoma. The clinical characteristics for the patients are shown in Table 1. TABLE 1. Clinical features of SLE patients
GB SHO OD Facial erythema Discoid lupus Raynaud's phenomenon..Alopecia Photosensitivity Oral ulceration Arthritis LE cells Proteinuria Cellular casts Serositis CNS disease Blood* DNA-binding capacity (.ug/
ml/) Active SLE; with nephritis Active SLE; no nephritis
+
-
-
JJj
AF
-
+
MC
BS
SHE RMt PK +
-
+
+
KN
CR
+ -+
+ -
-
+
-
-
+
+
+-+ + + + ± + + _ +
42
22
-
+ + + + + 4- + -+
+ +
+
+ +
-
-
22 +
-
-
+
22
-
+
9
-
+
+
+
41 +
+
+
-
-
+ + +
+ + + +
-
+
70 +
+ +
+ + +
+ + + +
-
+-
+
+
+
+ + -
+
+
+ + +
14 +
12 -
99 +
35 +
-
+
-
-
+
76
* Patient demonstrated any one of three blood abnormalities (haemolytic anaemia, leucopenia or thrombocytopenia). t This patient also had a chronic false positive serology for syphilis. jUrinary findings were 4 yr prior to study; at time of anti-DNA assay there was no evidence of nephritis.
Sera were studied from SLE patients; all satisfied the provisional ARA criteria (Cohen et al., 1971a) for classification of SLE. All SLE patients except one (see footnote, Table 3) were considered to have active disease, defined by the presence of active inflammatory involvement in three or more systems. Patients with active disease were further divided into two groups with and without active nephritis. Active lupus nephritis was defined as the presence of red cells (more than five per high-power field) or casts in the urine, proteinuria of greater than 400 mg/24 hr, and azotaemia or a positive renal biopsy. Patients with non-lupus rheumatic diseases and healthy controls were also studied. For several patients serum was taken on more than one occasion. The nine controls were five males and four females with no history ofrheumatic disease. All sera had been frozen prior to measurement. DNA. (Thymine-Methyl-"4C)-DNA (1-374 Curies/OD260 unit) was obtained from New England Nuclear. It was isolated from E. coli and had a reported molecular weight of 2 x 106 Daltons with less than 6% contamination with single-stranded DNA as estimated by hydroxylapatite chromatography. The 14C-DNA was pre-filtered through 0 45 p Millipore filters to remove single-stranded DNA (Ginsberg & Keiser, 1973). The buffer was 0-15 M NaCl, 0.01 M Tris, 0-015 M Citrate, pH 79. DNA-binding capacity. Twenty p1 of "4C-DNA (53 ng) and 30 p1 of buffer were added to 1-5 ml Brinkman tubes. Fifty ,1 of diluted serum (1/100 with buffer) were then added and the mixture incubated at 370C for 30 min. At that time 1-0 ml of ice-cold buffer was added, the tube was mixed and 1-0 ml was filtered with a pre-wetted 0 45 # Millipore filter. The filter
1. F. Halsey et al.
358
was washed with 1-0 ml of buffer and dissolved in a scintillation cocktail. Further washing had no significant effect on filter counts. For the filtration step a 30 sample Millipore manifold was used with the suction adjusted to about 4 ml per min. For each assay the amount of 14C-DNA bound to the filter in the absence of serum was subtracted from the "4C-DNA bound in the presence of serum. It was observed that DNA-binding capacity measurements were quite reproducible when duplicates of a patient sample were compared on different days, particularly if fresh dilutions of serum were used in the assay. Measurement of anti-D7VA avidity. The avidity of the DNA-binding activity in the sera of patients and controls was determined by analysing binding data with eight different antigen concentrations over a thirty-fold range using 0-3 ul of serum in the assay. Plots of antigen bound vs antigen unbound were examined to ensure that the antigen concentration range was sufficient to estimate avidities as accurately as possible. Plots of reciprocals of bound antigen vs free antigen (Soothill & Steward, 1971) were used to obtain the avidities reported. The avidities obtained with reciprocal plots were compared with bound vs free plots to verify that the linear least squares fit of the data did not introduce artifacts. Therefore, the reported avidities should be a reasonably accurate estimate of the average avidity for what is presumedly a heterogeneous population of anti-DNA antibody molecules.
RESULTS The study population consisted of control subjects, patients with rheumatic diseases other than SLE, and patients with active SLE (Table 1) with nephritis and without nephritis. In three patients with active SLE, serial determinations of DNA-binding capacity showed a positive correlation with disease activity. It has recently been reported that the molecular weight of the DNA can be an important parameter in an assay for anti-DNA antibody (Aarden, Lakmaker & Feltkamp, 1976a; Aarden, Lakmaker & deGroot, 1976b). Therefore, we chromatographed the Millipore filtered "4C-DNA on a Biogel A-150M column. As shown in Fig. 1, the "4C-DNA supplied by New England Nuclear appeared to be homogeneous and of 5000 A
4000 3000
A
-
.C
A
2000 l- A
2000
/;
_iR
10
20
HA
A ~
0
30 Fraction number
40
5C
FIG. 1. Fractionation of "4C-DNA on Bio-Gel A-150 M column. (0-0) Millipore filtered sonicated and filtered 14C-DNA; (A-A)22 Na marker; 0-85 ml per fraction.
14CDNA; (0 - -e)
high molecular weight. Several investigators have reported the use of sonicated DNA in these assays (Ginsberg & Keiser, 1973); therefore, an aliquot was sonicated for 15 sec on setting three of the Model 140W Branson sonifer (with microprobe) at ice-bath temperatures. This material was also chromatographed on the Bio-gel A-150M column; the profile (shown in Fig. 1) indicates a heterogeneous population of DNA molecules. Therefore all binding experiments were performed with unsonicated, filtered ' 4C-DNA. Effect of serum volume on DNA-binding capacity Four sera from lupus patients and two control sera were examined in the binding assay to assess the effect of serum volume. In all sera a striking increase in binding capacity was observed as lower serum volumes were used in the assay. It should be noted that this effect is not due to reductions in amount of available 14C-DNA at higher serum volumes since increases were observed when the percentage bound
DNA binding activity in SLE serum
359 was 9-15%. In addition, the same higher binding capacity is seen at low serum concentrations with twice the 14C-DNA concentration. In Fig. 2 the results of such an experiment with a lupus patient and a control are shown. Such an effect has been noted before (Kredich et al., 1973) and one investigator (Winfield et al., 1977) used 500 BSA to prevent this apparent increase in binding capacity at high dilutions. As shown in Fig. 2, dilution with 5%0 BSA did not alter this effect. There appears to be a factor in serum that inhibits the binding of DNA and this factor or property may explain the observations shown in Fig. 2. The effect of mixing normal human serum with patient 250r E 'o
200-
z 0
,
150
0
a 100 0
0'
'
50
z 0
0
0-25
050
Serum volume
0.75
1-0
(IL1)
FIG. 2. Effect of serum volume on DNA-binding capacity. (u-r) Lupus patient serum diluted with buffer; (A-A) lupus patient serum diluted with 5% bovine serum albumin; (0-.) control serum diluted with buffer.
serum is shown in Table 2. It can be seen that the addition of 15 P1 of normal human serum to 05 pl of patient serum resulted in a decrease in the percentage bound and in the binding capacity of the patient serum. As noted in Table 2, the normal serum which bound 31% of the DNA, in addition to being non-additive, actually reduced the total percentage bound. It should be observed that bovine serum albumin had no such effect, suggesting that the differences seen were not due to different protein concentrations in the assay. TABLE 2. Inhibition of DNA binding by a factor in normal serum
Percentage DNA-binding capacity bound (pugDNA/ml ofserum)* 0-5 ul lupus serum 0-5 pl lupus serum+ 15 p1 NHS f 15Ial NHS 0-5 jul lupus serum+ 15 p1 of 10 mg/ml BSA 15 pX1 of 10 mg/ml BSA
65 48 31
70 51 1
(± 2) (+ 1)+ (±0 1)
65 0
71 0
(±1)
* DNA binding capacity = (percentage bound) (0 053 pg DNA) (100) (ml of patient serum) The number in parenthesis is the standard error of the mean. t Abbreviations: NHS, normal human serum; BSA, bovine serum albumin. T The DNA-binding capacity in this instance was calculated using the volume of lupus patient serum (0.5 pl) to provide a comparison with the patient serum alone.
360
3. F. Halsey et al.
DNA binding capacity of SLE patient sera Using the standard assay conditions with 05 yl of serum, the DNA-binding capacity of two groups of patients and controls was determined. The results are shown in Table 3. Sera from controls had an average binding capacity of 4 jug DNA/ml serum with a range of 2-6 pg/ml. Eighteen sera from patients with SLE had an average binding capacity of 37 pg/ml. Control and patient-binding capacities were significantly different. When patients with nephritis were compared with the patients without nephritis, no significant differance in DNA-binding capacity was observed. TABLE 3. DNA-binding capacity of SLE patient sera
DNA-binding capacityt (jug of DNA/ml of serum)
Classification* SLE Patients with Nephritis (7) SLE Patients without Nephritis (11) Controls (9) Other rheumatic disease (5)+
44(± 11) 32(± 8 0) 4 0(± 0 4) 4-3(± 1-8)
* For some of the SLE patients sera were obtained a second time 4 months to 1 yr after the original sample. The number in parenthesis indicates the total number of serum samples that were analysed in duplicate or triplicate. t These values were obtained as described in the methods using 53 ng of 14C-DNA and 0 5 j1 of serum in buffer. The error shown is the standard error of the mean. lThis group includes one patient each with: inactive SLE, ankylosing spondylitis, undifferentiated connective tissue syndrome, fibrositis and rheumatoid arthritis.
c 600 C
0
400 _-
//
zA n10 / X
:'
200
0
50
100 150 200 I/Free DNA concentration
250
FIG. 3. Plot of reciprocal of bound DNA vs reciprocal of free DNA for three patients with SLE. The linear least squares fit for each patient is shown. (0) Patient SHE; (A) patient GB; (A) patient BS. The units for each axis are x 10-3 ml/ng.
Avidity of anti-DNA antibody in serum Binding data at eight different DNA concentrations (range 10-324 ng) were examined to estimate avidities of the serum antibodies. A plot of the reciprocal of bound and free DNA for three SLE patients is shown in Fig. 3. In such plots the slope is equal to 1/(K.Abtotai), where K is the average avidity and Abtotal is the total antibody concentration, which is obtained from the y-intercept. The avidities estimated by this method of analysis are presented in Table 4 for the two groups of patients and controls. These data indicate that patients with nephritis do not have significantly different avidity than patients
DNA binding activity in SLE serum
361
TABLE 4. Avidity of anti-DNA antibody in serum
Classification
Avidity*
Range
SLE patients with nephritis (7) SLE patients without nephritis (8) Controls (6)
34(+± 1-1) 2 9(±0.7) 14(+± 03)
1-2-7-8 0 6-6-8 0 8-2-3
*Relative avidities are obtained from a 1/DNA bound vs 1/DNA free plot. The number in parenthesis is the standard error of the mean. The data include a second serum sample taken from three of the patients. The units for all values are x 10-3 ml/ng.
without nephritis. The differences in avidity between controls and patients are only marginally significant; when the data were analysed with Sips plots, no significant differences were observed between the two groups of patients. DISCUSSION A convenient and reliable method to detect and quantify the activity of anti-DNA antibodies might be of significant clinical use. Several assay methods for detecting and quantifying anti-DNA antibodies in sera of lupus patients have been reported. Assays based on the Farr ammonium sulphate technique have been used extensively; recently several reports using the cellulose ester filter assay have appeared. A filter assay for DNA-protein complexes was adapted for the direct measurement of serum antibodies to native DNA by Kredich et al. (1973) and Ginsberg & Keiser (1973). The success of the assay depends on the observation that native or double-stranded DNA, but not DNA-antibody complexes, can pass through the cellulose filters. Picazo & Tan (1975) successfully used this technique to study the specificities of antibodies to DNA. This method has advantages for the present study. The high salt concentrations (2 M) involved in the Farr method could drastically affect the measurement of the average avidity of the anti-DNA antibodies produced since low avidity interactions might be significantly altered (Davis, Russell & Percy, 1977). Thus, the use of a Farr assay might lead to significant error in estimating antibody avidity. The importance of controlling the physical and chemical properties of the DNA used in the binding assay has been discussed by a number of investigators. The effects of size and structure have been most carefully studied by Aarden et al. (1976a, b). They demonstrated that the size of the DNA was the most important parameter in binding studies and that differences in the binding properties of different DNA preparations can be largely ascribed to differences in the molecular weight of the DNA. These findings indicate that any DNA preparation used in a binding assay should be examined for the presence of low molecular weight fragments and the DNA should not be sonicated (Fig. 1). The other property of the DNA that may be important in evaluating patient sera is the purity in terms of the double-strandedness. This is a particularly troublesome problem since it is doubtful that absolutely pure double-stranded DNA of 106 Daltons can be prepared. Ifit were available it would very likely rapidly acquire some singlestranded regions during the assay procedures and during storage (Locker et al., 1977). It is possible that the use of synthetic DNA preparations (Steinman, Deesomchok & Spiera, 1976) or circular bacteriophage DNA (Aarden et al., 1976a) may have some advantages. The DNA-binding assay, done as described here, can be readily used to detect the increased levels of anti-DNA antibodies that appear to be specific for patients with active SLE. These studies have shown that the serum volume used in the assay must be standardized since the binding capacity varies when different quantities of serum are used. The cause of this effect is not known. Kredich et al. (1973) also noted the change in binding capacity with more dilute sera and suggested that it might be due to serum nucleases which at higher serum concentrations might degrade the '4C-DNA in the assay. If a nuclease were present, one would expect longer incubation times at a given serum concentration to result in lower binding; we found no such effect after several hours incubation (data not shown). A comparison of the DNA-binding capacity for the two groups of lupus patients indicated that the C
362
J. F. Halsey et (al.
serum from patients with nephritis bound slightly more DNA (44 pg/ml vs 32 pg/ml) than serum from patients without nephritis. Nevertheless these differences were not statistically significant. Gershwin & Steinberg (1974) also reported that the antigen binding capacities of sera from patients with and without lupus nephritis were similar. It appears unlikely that even with a larger patient population such differences would be useful in differentiating the txxo groups of active SLE patients. However, it is clear that measurements of DNA binding capacity can differentiate active SLE from controls and from other rheumatic diseases. The similarity in antigen binding capacity of sera from SLE patients with and without nephritis reported here differs from the findings in Winfield et al. (1977), who found significantly higher DNA binding capacity in patients with nephritis. In the Winfield study the amount of I)NA auras less (i.e. 25 ng) and the Farr assay was used to separate antibody-bound from free DNA. Most investigators who have compared DNA binding by normal and lupus patient sera have noted that there are factors in normal serum that bind DNA (Hasselbacher & LeRoy, 1974; Steinman et at/., 1976; Aarden et al., 1975). This binding has been attributed by some to an electrostatic interaction of the collagen like segment of Clq with nucleic acids. Since Clq is heat-labile and some of this binding activity in normal serum persists after heat treatment, other heat-stable basic proteins may be involved, as discussed by Izui, Lambert & Miescher (1976). Hasselbacher & LeRoy (1974) examined the nature of the molecules in normal sera that bind DNA and demonstrated that the DNA-binding component in normal sera was IgG. They suggested that the binding of DNA by antibody from normal and lupus sera w1as similar in kind, but not in degree, in that patient sera had much higher levels of anti-DNA antibody. Interestingly, the avidity of binding in control sera was similar to the avidity of antibody binding in patient sera (Table 4). Recently, Steward and his collaborators have proposed that susceptibility to immune complex disease may be a type of immunodeficiency with an inability to produce high-affinity antibody (Steward & Petty, 1976). It was suggested that low-affinity antibody would be unable to eliminate antigen with the resulting continued circulation and deposition of antigen excess complexes in filtering membrane tissues such as the glomerulus and the choroid plexus. This hypothesis was supported by the observation that the affinity of the antibodies produced in mice to a range of antigens appeared to be genetically controlled and related to a susceptibility to immune complex disease (Alpers, Steward & Soothill, 1972; Steward, Katz & West, 1975). The hypothesis that lowr-avidity antibody is of primary immunopathological significance is not supported by the present study, since serum antibody avidity in patients with nephritis is not significantly different from that in patients without nephritis. Measurements of avidity of anti-DNA antibodies have been made before (Tron & Bach, 1977; Gershwin & Steinberg, 1974; Winfield et al., 1977; Leon et at., 1977). Although the actual Scatchard and Sips plots were not published, it is clear (Fig. 3) that such data usually do not provide a very accurate or precise estimate of antibody avidity. Estimates of the number of binding sites using plots of 1/bound TVs 1/free are particularly troublesome (Fig. 3) where values at low free DNA concentratations greatly influence the linear regression line obtained. Attempts to improve the fit by invoking a higher order dependence, such as the Sips relationship, are not advised with multivalent antigens and heterogeneous antibodies. Therefore, it would appear that in such a complex system only large differences, of an order of magnitude or twoo, should be considered reliable and significant. Leon et at. (1977) reported avidity determinations for eight patients with SLE and observed highavidity antibody in patients with severe nephritis, It is difficult to compare our observations with this report because their patients were selected according to titre rather than clinical criteria and each patient was evaluated in the presence of variable amounts of normal and patient serum. Further, since they obtained their estimate of avidity from the steepest part of the Scatchard plot, the K values they report represent a variable and unknown fraction of the total antibody population. In the study reported by Winfield et al. (1977) the avidity of serum antibodies was found to be loxer in SLE patients with active glomerulonephritis. Nevertheless, the difference they observed was small (- 07 kcal/mol) and the biological significance of such differences in binding energy in terms of the percentage of antigen bound and the resulting immune complex size is of doubtful significance. It should be noted that in these measurements of antibody axidity, different serum concentrations were used for
DNA binding activity in SLE serum
363
each patient. It is possible that the variable serum concentrations used could produce an artifactual difference because of different amounts of the 'binding inhibitor' present in serum. In addition, the high concentrations of ammonium sulphate used in the Farr assay may have masked the binding of some very low-avidity molecules in the sera of patients without nephritis. The observation by Winfield et al. (1977) of high-avidity antibodies in glomerular eluates may be crucial and serves to weaken the argument for a preferential role for low-avidity antibodies proposed by Steward. The technical assistance of Sandar M. Halsey is greatly appreciated. This study was supported in part by a grant from the Research Council of the University of Oklahoma and by a South Carolina State Appropriation for Biomedical Research. REFERENCES AARDEN, L.A., LAKMAKER, F., DEGROOT, E., SWAAK, A. & FELTKAMP, T. (1975) Detection of antibodies to DNA by radioimmunoassay and immunofluorescence. Scand. j5. Rheumatol. suppl. 11, 12. AARDEN, L., LAKMAKER, F. & FELTKAMP, T. (1976a) Immunology of DNA. II. The effect of size and structure of the antigen on the Farr assay. _. Immunol. Methods. 10, 39. AARDEN, L.A., LAKMAKER, F. & DEGROOT, E. (1976b) Immunology of DNA. IV. quantitative aspects of the Farr assay. 3. Immunol. Methods 11, 153. ADLER, M.K., BAUMGARTEN, A., HECHT, B. & SIEGEL, N.J. (1975) Prognostic significance of DNA binding capacity patterns in patients with lupus nephritis. Ann. Rheum. Dis. 34, 444. ALARC6N-SEGOVIA, D., FISHBEIN, E. & ALCALA, H. (1970) The range and specificity of antinuclear antibodies in systemic lypus erythematosus. Clin. exp. Immunol. 6, 557. ALPERS, J., STEWARD, M. & SOOTHILL, J. (1972) Differences in immune elimination in inbred mice. The role of low affinity antibody. C/in. exp. Immunol. 12, 121. COCHRANE, C.G. & KOFFLER, D. (1973) Immune complex disease in experimental animals and man. Adv. Immunol. 16, 185. COHEN, A., REYNOLDS, W., FRANKLIN, E., KULKA, J., RoPES, M., SHULMAN, L. & WALLACE, S.) 1971a) Preliminary criteria for the classification of systemic lupus erythematosus Bull. Rheum. Dis. 21, 643. COHEN, S.A., HUGHES, G.R.V. & NOEL, G.L. (1971b) Character of anti-DNA antibodies in systemic lupus erythematosus. Clin. exp. Immunol. 8, 551. DAVIS, P., RUSSELL, A. & PERCY, J. (1977) A comparative study of technics for the detection of antibodies to native deoxyribonucleic acid. Amer. 7. clin. Path. 67, 374. GERSHWIN, M.E. & STEINBERG, A. (1974) Qualitative characteristics of anti DNA antibodies in lupus nephritis. Arthr. and Rheum. 17, 974. GINSBERG, B. & KEISER, H. (1973) A millipore filter assay for antibodies to native DNA in sera of patients with systemic lupus erythematosus. Arthr. and Rheum. 16, 199. HAHN, B.H., BAGBY, M.K., HAMILTON, T.R. & OSTERLAND, C.K. (1973) Comparison of therapeutic and immunosuppressive effects of azathioprine, prednisolone and combined therapy in NZB/NZW mice. Arthr. and Rheum. 16, 163. HASSELBACHER, P. & LEROY, E.C. (1974) Serum DNA binding activity in healthy subjects and in rheumatic disease. Arthr. and Rheum. 17, 63. IZUI, S., LAMBERT, P.H. & MIESCHER, P. (1976) In vitro demonstration of a particular affinity of glomerular basement mebrane and collagen for DNA. T. exp. Med. 144, 428.
JOHNSON, G.D., EDMONDS, J.P. & HOLBOROW, E.J. (1973) Precipitating antibody to DNA detected by two-stage electroimmunodiffusion. Study in SLE and in rheumatoid arthritis. Lancet, ii, 883. KREDICH, N.M., SKYLER, J. & ROOTE, L. (1973) Antibodies to native DNA in systemic lupus erythematosus. Arch. intern. Med. 131, 639. LEON, S.A., GREEN, A., EHRLICH, G.E., POLAND, M. & SHAPIRO, B. (1977) Avidity of antibodies in SLE. Arthr. and Rheum, 20, 23. LOCKER, J.D., MEDOF, M.E., BENNETT, R.M. & SUKHUPUNYARAKSA, S. (1977) Characterization of DNA used to assay sera for anti-DNA antibodies; determination of the specificities of anti-DNA antibodies in SLE and non-SLE rheumatic disease states. 5. Immunol. 118, 694. McDUFFIE, F.C. & BUNCH, T.W. (1977) Immunologic tests in the diagnosis of rheumatic diseases. Bull. Rheum. Dis. 27, 900. PICAZO, J. & TAN, E.M. (1975) Specificities of antibodies to native DNA. Scand. J. Rheumatol. Suppl. 11, 35. PINCUS, T., SCHUR, P.H., ROSE, J.A., DECKER, J.L. & TALAL, N. (1969) Measurement of serum DNA-binding activity in systemic lupus erythematosus. New Engl. 3. Med. 281, 701. SOOTHILL, J.F. & STEWARD, M.W. (1971) The immunopathological significance of the heterogeneity of antibody affinity. Clin. exp. Immunol. 9, 193. STEINMAN, C., DEESOMCHOK, U. & SPIERA, H. (1976) Detection of anti-DNA antibody using synthetic antigens. J. clin. Invest. 57, 1330. STEWARD, M., KATZ, F. & WEST, N. (1975) The role of low affinity antibody in immune complex disease. Clin. exp. Immunol. 21, 121. STEWARD, M. & PETTY, R. (1976) Evidence for the genetic control of antibody affinity from breeding studies. With inbred mouse strains producing high and low affinity antibody. Immunology, 30, 789. TAN, E.M., SCHUR, P.H., CARR, R. & KUNKEL, H. (1966) Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. 5. clin. Invest. 45, 1732. TRON, F. & BACH, J.F. (1977) Relationship between anti. bodies to native DNA and glomerulonephritis in systemic lupus erythematosus. Clin. exp. Immunol. 28, 426. WEITZMAN, R.J. & WALKER, S.E. (1977) Relation of titered peripheral pattern ANA to anti-DNA and disease actiN ity in systemic lupus erythematosus. Ann. Rheulm. Dis. 36, No. 1, 44. WINFIELD, J.B., FAIFERMAN, I. & KOFFLER, D. (1977) Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. 7. clin. Invest. 59, 90.
DNA binding activity of serum from patients with systemic lupus erythematosus J. F. HALSEY, W. A. WOOLERY, S. OLEINICK, M. BASHAR KAHALEH & E. CARWILE LEROY Department of Microbiology and Department of Medicine, University of Oklahoma and the Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA
(Received 7 July 1978)
SUMMARY
The reaction conditions essential for reproducible use of the cellulose ester membrane filter assay for anti-DNA antibody levels in patients with systemic lupus erythematosus are described. A dependence of DNA-binding capacity on serum concentration was observed in the assay, requiring that serum concentrations be comparable in determinations of DNA-binding activity of different sera and when comparing different published studies. The DNA-binding capacity of serum from lupus patients was found to be significantly different from that of healthy controls. However, the binding capacities were not significantly different between lupus patients with and without nephritis. The relative avidity of the anti-DNA antibodies were estimated from plots of 1/DNA bound vs 1/DNA free and these data indicate that the avidities of the antibodies from the two groups of lupus patients are not significantly different. This observation suggests that the tightness of binding between the DNA and the serum anti-DNA antibodies cannot be used to predict immune complex-induced nephritis in lupus patients.
INTRODUCTION Antibodies reacting with a variety of nuclear and cytoplasmic antigens have been found in the serum of patients with systemic lupus erythematosus (SLE) (McDuffie & Bunch, 1977). There is considerable evidence suggesting that the DNA/anti-DNA system is important in the pathogenesis of lupus nephritis (Tan et al., 1966; Cochrane & Koffler, 1973). A positive correlation has been shown between antibody titres to double-stranded DNA and clinically active renal disease (Kredich, Skyler & Foote, 1973), decreased serum complement, and the number of SLE criteria present (Weitzman & Walker, 1977). Also, patients with persistently high titres of anti-DNA antibody usually have a clinical course characterized by progressive renal impairment (Adler et al., 1975). Similar results have been obtained in NZB/NZW mice where a correlation was found between a drug regimen associated with suppression of anti-DNA antibody titres and the lack of progression of glomerulonephritis (Hahn et al., 1973). However, anti-DNA antibodies are found in patients with and without lupus nephritis, and the relationship between the qualitative and quantitative features of the antibodies and their role in the pathogenesis of renal disease (Cohen et al., 197 lb; Pincus et al., 1969) is not clear. The properties of the anti-DNA antibodies that are important in the immunopathology are not known but several contrasting proposals have been made. Investigators have implicated non-precipitating serum DNA antibodies as causative in SLE nephritis (Alarcon-Segovia, Fishbein & Alcala, 1970), while others have emphasized precipitating serum DNA antibodies (Johnson, Edmonds & Holborow, 1973). Low avidity serum anti-DNA antibodies have been proposed by Soothill & Steward (1971) to be particularly important in the development of nephritis. It was suggested that these antibodies, similar to non-precipitating antibodies, are poorly cleared by Correspondence: Dr J. F. Halsey, Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103, USA. 0099-9104/79/0030-0356$02.00 (© 1979 Blackwell Scientific Publications
356
DNA binding activity in SLE serum
357
the reticuloendothelial system and remain in the circulation to initiate immune complex-mediated tissue damage. In contrast to this proposal, several laboratories have published apparent correlations of high avidity anti-DNA antibodies with the occurrence of lupus nephritis (Gershwin & Steinberg, 1974; Leon et al., 1977). Winfield, Faiferman & Koffler (1977), however, found the serum anti-DNA antibody avidity to be lower in patients with lupus nephritis. These divergent reports prompted the present investigation to determine with technically suitable procedures both the antigen-binding capacity and avidity of SLE sera from patients with and without active nephritis.
MATERIALS AND METHODS Patients were selected from the wards and clinics of the Medical University of South Carolina and affiliated hospitals and from the University Hospital of the University of Oklahoma. The clinical characteristics for the patients are shown in Table 1. TABLE 1. Clinical features of SLE patients
GB SHO OD Facial erythema Discoid lupus Raynaud's phenomenon..Alopecia Photosensitivity Oral ulceration Arthritis LE cells Proteinuria Cellular casts Serositis CNS disease Blood* DNA-binding capacity (.ug/
ml/) Active SLE; with nephritis Active SLE; no nephritis
+
-
-
JJj
AF
-
+
MC
BS
SHE RMt PK +
-
+
+
KN
CR
+ -+
+ -
-
+
-
-
+
+
+-+ + + + ± + + _ +
42
22
-
+ + + + + 4- + -+
+ +
+
+ +
-
-
22 +
-
-
+
22
-
+
9
-
+
+
+
41 +
+
+
-
-
+ + +
+ + + +
-
+
70 +
+ +
+ + +
+ + + +
-
+-
+
+
+
+ + -
+
+
+ + +
14 +
12 -
99 +
35 +
-
+
-
-
+
76
* Patient demonstrated any one of three blood abnormalities (haemolytic anaemia, leucopenia or thrombocytopenia). t This patient also had a chronic false positive serology for syphilis. jUrinary findings were 4 yr prior to study; at time of anti-DNA assay there was no evidence of nephritis.
Sera were studied from SLE patients; all satisfied the provisional ARA criteria (Cohen et al., 1971a) for classification of SLE. All SLE patients except one (see footnote, Table 3) were considered to have active disease, defined by the presence of active inflammatory involvement in three or more systems. Patients with active disease were further divided into two groups with and without active nephritis. Active lupus nephritis was defined as the presence of red cells (more than five per high-power field) or casts in the urine, proteinuria of greater than 400 mg/24 hr, and azotaemia or a positive renal biopsy. Patients with non-lupus rheumatic diseases and healthy controls were also studied. For several patients serum was taken on more than one occasion. The nine controls were five males and four females with no history ofrheumatic disease. All sera had been frozen prior to measurement. DNA. (Thymine-Methyl-"4C)-DNA (1-374 Curies/OD260 unit) was obtained from New England Nuclear. It was isolated from E. coli and had a reported molecular weight of 2 x 106 Daltons with less than 6% contamination with single-stranded DNA as estimated by hydroxylapatite chromatography. The 14C-DNA was pre-filtered through 0 45 p Millipore filters to remove single-stranded DNA (Ginsberg & Keiser, 1973). The buffer was 0-15 M NaCl, 0.01 M Tris, 0-015 M Citrate, pH 79. DNA-binding capacity. Twenty p1 of "4C-DNA (53 ng) and 30 p1 of buffer were added to 1-5 ml Brinkman tubes. Fifty ,1 of diluted serum (1/100 with buffer) were then added and the mixture incubated at 370C for 30 min. At that time 1-0 ml of ice-cold buffer was added, the tube was mixed and 1-0 ml was filtered with a pre-wetted 0 45 # Millipore filter. The filter
1. F. Halsey et al.
358
was washed with 1-0 ml of buffer and dissolved in a scintillation cocktail. Further washing had no significant effect on filter counts. For the filtration step a 30 sample Millipore manifold was used with the suction adjusted to about 4 ml per min. For each assay the amount of 14C-DNA bound to the filter in the absence of serum was subtracted from the "4C-DNA bound in the presence of serum. It was observed that DNA-binding capacity measurements were quite reproducible when duplicates of a patient sample were compared on different days, particularly if fresh dilutions of serum were used in the assay. Measurement of anti-D7VA avidity. The avidity of the DNA-binding activity in the sera of patients and controls was determined by analysing binding data with eight different antigen concentrations over a thirty-fold range using 0-3 ul of serum in the assay. Plots of antigen bound vs antigen unbound were examined to ensure that the antigen concentration range was sufficient to estimate avidities as accurately as possible. Plots of reciprocals of bound antigen vs free antigen (Soothill & Steward, 1971) were used to obtain the avidities reported. The avidities obtained with reciprocal plots were compared with bound vs free plots to verify that the linear least squares fit of the data did not introduce artifacts. Therefore, the reported avidities should be a reasonably accurate estimate of the average avidity for what is presumedly a heterogeneous population of anti-DNA antibody molecules.
RESULTS The study population consisted of control subjects, patients with rheumatic diseases other than SLE, and patients with active SLE (Table 1) with nephritis and without nephritis. In three patients with active SLE, serial determinations of DNA-binding capacity showed a positive correlation with disease activity. It has recently been reported that the molecular weight of the DNA can be an important parameter in an assay for anti-DNA antibody (Aarden, Lakmaker & Feltkamp, 1976a; Aarden, Lakmaker & deGroot, 1976b). Therefore, we chromatographed the Millipore filtered "4C-DNA on a Biogel A-150M column. As shown in Fig. 1, the "4C-DNA supplied by New England Nuclear appeared to be homogeneous and of 5000 A
4000 3000
A
-
.C
A
2000 l- A
2000
/;
_iR
10
20
HA
A ~
0
30 Fraction number
40
5C
FIG. 1. Fractionation of "4C-DNA on Bio-Gel A-150 M column. (0-0) Millipore filtered sonicated and filtered 14C-DNA; (A-A)22 Na marker; 0-85 ml per fraction.
14CDNA; (0 - -e)
high molecular weight. Several investigators have reported the use of sonicated DNA in these assays (Ginsberg & Keiser, 1973); therefore, an aliquot was sonicated for 15 sec on setting three of the Model 140W Branson sonifer (with microprobe) at ice-bath temperatures. This material was also chromatographed on the Bio-gel A-150M column; the profile (shown in Fig. 1) indicates a heterogeneous population of DNA molecules. Therefore all binding experiments were performed with unsonicated, filtered ' 4C-DNA. Effect of serum volume on DNA-binding capacity Four sera from lupus patients and two control sera were examined in the binding assay to assess the effect of serum volume. In all sera a striking increase in binding capacity was observed as lower serum volumes were used in the assay. It should be noted that this effect is not due to reductions in amount of available 14C-DNA at higher serum volumes since increases were observed when the percentage bound
DNA binding activity in SLE serum
359 was 9-15%. In addition, the same higher binding capacity is seen at low serum concentrations with twice the 14C-DNA concentration. In Fig. 2 the results of such an experiment with a lupus patient and a control are shown. Such an effect has been noted before (Kredich et al., 1973) and one investigator (Winfield et al., 1977) used 500 BSA to prevent this apparent increase in binding capacity at high dilutions. As shown in Fig. 2, dilution with 5%0 BSA did not alter this effect. There appears to be a factor in serum that inhibits the binding of DNA and this factor or property may explain the observations shown in Fig. 2. The effect of mixing normal human serum with patient 250r E 'o
200-
z 0
,
150
0
a 100 0
0'
'
50
z 0
0
0-25
050
Serum volume
0.75
1-0
(IL1)
FIG. 2. Effect of serum volume on DNA-binding capacity. (u-r) Lupus patient serum diluted with buffer; (A-A) lupus patient serum diluted with 5% bovine serum albumin; (0-.) control serum diluted with buffer.
serum is shown in Table 2. It can be seen that the addition of 15 P1 of normal human serum to 05 pl of patient serum resulted in a decrease in the percentage bound and in the binding capacity of the patient serum. As noted in Table 2, the normal serum which bound 31% of the DNA, in addition to being non-additive, actually reduced the total percentage bound. It should be observed that bovine serum albumin had no such effect, suggesting that the differences seen were not due to different protein concentrations in the assay. TABLE 2. Inhibition of DNA binding by a factor in normal serum
Percentage DNA-binding capacity bound (pugDNA/ml ofserum)* 0-5 ul lupus serum 0-5 pl lupus serum+ 15 p1 NHS f 15Ial NHS 0-5 jul lupus serum+ 15 p1 of 10 mg/ml BSA 15 pX1 of 10 mg/ml BSA
65 48 31
70 51 1
(± 2) (+ 1)+ (±0 1)
65 0
71 0
(±1)
* DNA binding capacity = (percentage bound) (0 053 pg DNA) (100) (ml of patient serum) The number in parenthesis is the standard error of the mean. t Abbreviations: NHS, normal human serum; BSA, bovine serum albumin. T The DNA-binding capacity in this instance was calculated using the volume of lupus patient serum (0.5 pl) to provide a comparison with the patient serum alone.
360
3. F. Halsey et al.
DNA binding capacity of SLE patient sera Using the standard assay conditions with 05 yl of serum, the DNA-binding capacity of two groups of patients and controls was determined. The results are shown in Table 3. Sera from controls had an average binding capacity of 4 jug DNA/ml serum with a range of 2-6 pg/ml. Eighteen sera from patients with SLE had an average binding capacity of 37 pg/ml. Control and patient-binding capacities were significantly different. When patients with nephritis were compared with the patients without nephritis, no significant differance in DNA-binding capacity was observed. TABLE 3. DNA-binding capacity of SLE patient sera
DNA-binding capacityt (jug of DNA/ml of serum)
Classification* SLE Patients with Nephritis (7) SLE Patients without Nephritis (11) Controls (9) Other rheumatic disease (5)+
44(± 11) 32(± 8 0) 4 0(± 0 4) 4-3(± 1-8)
* For some of the SLE patients sera were obtained a second time 4 months to 1 yr after the original sample. The number in parenthesis indicates the total number of serum samples that were analysed in duplicate or triplicate. t These values were obtained as described in the methods using 53 ng of 14C-DNA and 0 5 j1 of serum in buffer. The error shown is the standard error of the mean. lThis group includes one patient each with: inactive SLE, ankylosing spondylitis, undifferentiated connective tissue syndrome, fibrositis and rheumatoid arthritis.
c 600 C
0
400 _-
//
zA n10 / X
:'
200
0
50
100 150 200 I/Free DNA concentration
250
FIG. 3. Plot of reciprocal of bound DNA vs reciprocal of free DNA for three patients with SLE. The linear least squares fit for each patient is shown. (0) Patient SHE; (A) patient GB; (A) patient BS. The units for each axis are x 10-3 ml/ng.
Avidity of anti-DNA antibody in serum Binding data at eight different DNA concentrations (range 10-324 ng) were examined to estimate avidities of the serum antibodies. A plot of the reciprocal of bound and free DNA for three SLE patients is shown in Fig. 3. In such plots the slope is equal to 1/(K.Abtotai), where K is the average avidity and Abtotal is the total antibody concentration, which is obtained from the y-intercept. The avidities estimated by this method of analysis are presented in Table 4 for the two groups of patients and controls. These data indicate that patients with nephritis do not have significantly different avidity than patients
DNA binding activity in SLE serum
361
TABLE 4. Avidity of anti-DNA antibody in serum
Classification
Avidity*
Range
SLE patients with nephritis (7) SLE patients without nephritis (8) Controls (6)
34(+± 1-1) 2 9(±0.7) 14(+± 03)
1-2-7-8 0 6-6-8 0 8-2-3
*Relative avidities are obtained from a 1/DNA bound vs 1/DNA free plot. The number in parenthesis is the standard error of the mean. The data include a second serum sample taken from three of the patients. The units for all values are x 10-3 ml/ng.
without nephritis. The differences in avidity between controls and patients are only marginally significant; when the data were analysed with Sips plots, no significant differences were observed between the two groups of patients. DISCUSSION A convenient and reliable method to detect and quantify the activity of anti-DNA antibodies might be of significant clinical use. Several assay methods for detecting and quantifying anti-DNA antibodies in sera of lupus patients have been reported. Assays based on the Farr ammonium sulphate technique have been used extensively; recently several reports using the cellulose ester filter assay have appeared. A filter assay for DNA-protein complexes was adapted for the direct measurement of serum antibodies to native DNA by Kredich et al. (1973) and Ginsberg & Keiser (1973). The success of the assay depends on the observation that native or double-stranded DNA, but not DNA-antibody complexes, can pass through the cellulose filters. Picazo & Tan (1975) successfully used this technique to study the specificities of antibodies to DNA. This method has advantages for the present study. The high salt concentrations (2 M) involved in the Farr method could drastically affect the measurement of the average avidity of the anti-DNA antibodies produced since low avidity interactions might be significantly altered (Davis, Russell & Percy, 1977). Thus, the use of a Farr assay might lead to significant error in estimating antibody avidity. The importance of controlling the physical and chemical properties of the DNA used in the binding assay has been discussed by a number of investigators. The effects of size and structure have been most carefully studied by Aarden et al. (1976a, b). They demonstrated that the size of the DNA was the most important parameter in binding studies and that differences in the binding properties of different DNA preparations can be largely ascribed to differences in the molecular weight of the DNA. These findings indicate that any DNA preparation used in a binding assay should be examined for the presence of low molecular weight fragments and the DNA should not be sonicated (Fig. 1). The other property of the DNA that may be important in evaluating patient sera is the purity in terms of the double-strandedness. This is a particularly troublesome problem since it is doubtful that absolutely pure double-stranded DNA of 106 Daltons can be prepared. Ifit were available it would very likely rapidly acquire some singlestranded regions during the assay procedures and during storage (Locker et al., 1977). It is possible that the use of synthetic DNA preparations (Steinman, Deesomchok & Spiera, 1976) or circular bacteriophage DNA (Aarden et al., 1976a) may have some advantages. The DNA-binding assay, done as described here, can be readily used to detect the increased levels of anti-DNA antibodies that appear to be specific for patients with active SLE. These studies have shown that the serum volume used in the assay must be standardized since the binding capacity varies when different quantities of serum are used. The cause of this effect is not known. Kredich et al. (1973) also noted the change in binding capacity with more dilute sera and suggested that it might be due to serum nucleases which at higher serum concentrations might degrade the '4C-DNA in the assay. If a nuclease were present, one would expect longer incubation times at a given serum concentration to result in lower binding; we found no such effect after several hours incubation (data not shown). A comparison of the DNA-binding capacity for the two groups of lupus patients indicated that the C
362
J. F. Halsey et (al.
serum from patients with nephritis bound slightly more DNA (44 pg/ml vs 32 pg/ml) than serum from patients without nephritis. Nevertheless these differences were not statistically significant. Gershwin & Steinberg (1974) also reported that the antigen binding capacities of sera from patients with and without lupus nephritis were similar. It appears unlikely that even with a larger patient population such differences would be useful in differentiating the txxo groups of active SLE patients. However, it is clear that measurements of DNA binding capacity can differentiate active SLE from controls and from other rheumatic diseases. The similarity in antigen binding capacity of sera from SLE patients with and without nephritis reported here differs from the findings in Winfield et al. (1977), who found significantly higher DNA binding capacity in patients with nephritis. In the Winfield study the amount of I)NA auras less (i.e. 25 ng) and the Farr assay was used to separate antibody-bound from free DNA. Most investigators who have compared DNA binding by normal and lupus patient sera have noted that there are factors in normal serum that bind DNA (Hasselbacher & LeRoy, 1974; Steinman et at/., 1976; Aarden et al., 1975). This binding has been attributed by some to an electrostatic interaction of the collagen like segment of Clq with nucleic acids. Since Clq is heat-labile and some of this binding activity in normal serum persists after heat treatment, other heat-stable basic proteins may be involved, as discussed by Izui, Lambert & Miescher (1976). Hasselbacher & LeRoy (1974) examined the nature of the molecules in normal sera that bind DNA and demonstrated that the DNA-binding component in normal sera was IgG. They suggested that the binding of DNA by antibody from normal and lupus sera w1as similar in kind, but not in degree, in that patient sera had much higher levels of anti-DNA antibody. Interestingly, the avidity of binding in control sera was similar to the avidity of antibody binding in patient sera (Table 4). Recently, Steward and his collaborators have proposed that susceptibility to immune complex disease may be a type of immunodeficiency with an inability to produce high-affinity antibody (Steward & Petty, 1976). It was suggested that low-affinity antibody would be unable to eliminate antigen with the resulting continued circulation and deposition of antigen excess complexes in filtering membrane tissues such as the glomerulus and the choroid plexus. This hypothesis was supported by the observation that the affinity of the antibodies produced in mice to a range of antigens appeared to be genetically controlled and related to a susceptibility to immune complex disease (Alpers, Steward & Soothill, 1972; Steward, Katz & West, 1975). The hypothesis that lowr-avidity antibody is of primary immunopathological significance is not supported by the present study, since serum antibody avidity in patients with nephritis is not significantly different from that in patients without nephritis. Measurements of avidity of anti-DNA antibodies have been made before (Tron & Bach, 1977; Gershwin & Steinberg, 1974; Winfield et al., 1977; Leon et at., 1977). Although the actual Scatchard and Sips plots were not published, it is clear (Fig. 3) that such data usually do not provide a very accurate or precise estimate of antibody avidity. Estimates of the number of binding sites using plots of 1/bound TVs 1/free are particularly troublesome (Fig. 3) where values at low free DNA concentratations greatly influence the linear regression line obtained. Attempts to improve the fit by invoking a higher order dependence, such as the Sips relationship, are not advised with multivalent antigens and heterogeneous antibodies. Therefore, it would appear that in such a complex system only large differences, of an order of magnitude or twoo, should be considered reliable and significant. Leon et at. (1977) reported avidity determinations for eight patients with SLE and observed highavidity antibody in patients with severe nephritis, It is difficult to compare our observations with this report because their patients were selected according to titre rather than clinical criteria and each patient was evaluated in the presence of variable amounts of normal and patient serum. Further, since they obtained their estimate of avidity from the steepest part of the Scatchard plot, the K values they report represent a variable and unknown fraction of the total antibody population. In the study reported by Winfield et al. (1977) the avidity of serum antibodies was found to be loxer in SLE patients with active glomerulonephritis. Nevertheless, the difference they observed was small (- 07 kcal/mol) and the biological significance of such differences in binding energy in terms of the percentage of antigen bound and the resulting immune complex size is of doubtful significance. It should be noted that in these measurements of antibody axidity, different serum concentrations were used for
DNA binding activity in SLE serum
363
each patient. It is possible that the variable serum concentrations used could produce an artifactual difference because of different amounts of the 'binding inhibitor' present in serum. In addition, the high concentrations of ammonium sulphate used in the Farr assay may have masked the binding of some very low-avidity molecules in the sera of patients without nephritis. The observation by Winfield et al. (1977) of high-avidity antibodies in glomerular eluates may be crucial and serves to weaken the argument for a preferential role for low-avidity antibodies proposed by Steward. The technical assistance of Sandar M. Halsey is greatly appreciated. This study was supported in part by a grant from the Research Council of the University of Oklahoma and by a South Carolina State Appropriation for Biomedical Research. REFERENCES AARDEN, L.A., LAKMAKER, F., DEGROOT, E., SWAAK, A. & FELTKAMP, T. (1975) Detection of antibodies to DNA by radioimmunoassay and immunofluorescence. Scand. j5. Rheumatol. suppl. 11, 12. AARDEN, L., LAKMAKER, F. & FELTKAMP, T. (1976a) Immunology of DNA. II. The effect of size and structure of the antigen on the Farr assay. _. Immunol. Methods. 10, 39. AARDEN, L.A., LAKMAKER, F. & DEGROOT, E. (1976b) Immunology of DNA. IV. quantitative aspects of the Farr assay. 3. Immunol. Methods 11, 153. ADLER, M.K., BAUMGARTEN, A., HECHT, B. & SIEGEL, N.J. (1975) Prognostic significance of DNA binding capacity patterns in patients with lupus nephritis. Ann. Rheum. Dis. 34, 444. ALARC6N-SEGOVIA, D., FISHBEIN, E. & ALCALA, H. (1970) The range and specificity of antinuclear antibodies in systemic lypus erythematosus. Clin. exp. Immunol. 6, 557. ALPERS, J., STEWARD, M. & SOOTHILL, J. (1972) Differences in immune elimination in inbred mice. The role of low affinity antibody. C/in. exp. Immunol. 12, 121. COCHRANE, C.G. & KOFFLER, D. (1973) Immune complex disease in experimental animals and man. Adv. Immunol. 16, 185. COHEN, A., REYNOLDS, W., FRANKLIN, E., KULKA, J., RoPES, M., SHULMAN, L. & WALLACE, S.) 1971a) Preliminary criteria for the classification of systemic lupus erythematosus Bull. Rheum. Dis. 21, 643. COHEN, S.A., HUGHES, G.R.V. & NOEL, G.L. (1971b) Character of anti-DNA antibodies in systemic lupus erythematosus. Clin. exp. Immunol. 8, 551. DAVIS, P., RUSSELL, A. & PERCY, J. (1977) A comparative study of technics for the detection of antibodies to native deoxyribonucleic acid. Amer. 7. clin. Path. 67, 374. GERSHWIN, M.E. & STEINBERG, A. (1974) Qualitative characteristics of anti DNA antibodies in lupus nephritis. Arthr. and Rheum. 17, 974. GINSBERG, B. & KEISER, H. (1973) A millipore filter assay for antibodies to native DNA in sera of patients with systemic lupus erythematosus. Arthr. and Rheum. 16, 199. HAHN, B.H., BAGBY, M.K., HAMILTON, T.R. & OSTERLAND, C.K. (1973) Comparison of therapeutic and immunosuppressive effects of azathioprine, prednisolone and combined therapy in NZB/NZW mice. Arthr. and Rheum. 16, 163. HASSELBACHER, P. & LEROY, E.C. (1974) Serum DNA binding activity in healthy subjects and in rheumatic disease. Arthr. and Rheum. 17, 63. IZUI, S., LAMBERT, P.H. & MIESCHER, P. (1976) In vitro demonstration of a particular affinity of glomerular basement mebrane and collagen for DNA. T. exp. Med. 144, 428.
JOHNSON, G.D., EDMONDS, J.P. & HOLBOROW, E.J. (1973) Precipitating antibody to DNA detected by two-stage electroimmunodiffusion. Study in SLE and in rheumatoid arthritis. Lancet, ii, 883. KREDICH, N.M., SKYLER, J. & ROOTE, L. (1973) Antibodies to native DNA in systemic lupus erythematosus. Arch. intern. Med. 131, 639. LEON, S.A., GREEN, A., EHRLICH, G.E., POLAND, M. & SHAPIRO, B. (1977) Avidity of antibodies in SLE. Arthr. and Rheum, 20, 23. LOCKER, J.D., MEDOF, M.E., BENNETT, R.M. & SUKHUPUNYARAKSA, S. (1977) Characterization of DNA used to assay sera for anti-DNA antibodies; determination of the specificities of anti-DNA antibodies in SLE and non-SLE rheumatic disease states. 5. Immunol. 118, 694. McDUFFIE, F.C. & BUNCH, T.W. (1977) Immunologic tests in the diagnosis of rheumatic diseases. Bull. Rheum. Dis. 27, 900. PICAZO, J. & TAN, E.M. (1975) Specificities of antibodies to native DNA. Scand. J. Rheumatol. Suppl. 11, 35. PINCUS, T., SCHUR, P.H., ROSE, J.A., DECKER, J.L. & TALAL, N. (1969) Measurement of serum DNA-binding activity in systemic lupus erythematosus. New Engl. 3. Med. 281, 701. SOOTHILL, J.F. & STEWARD, M.W. (1971) The immunopathological significance of the heterogeneity of antibody affinity. Clin. exp. Immunol. 9, 193. STEINMAN, C., DEESOMCHOK, U. & SPIERA, H. (1976) Detection of anti-DNA antibody using synthetic antigens. J. clin. Invest. 57, 1330. STEWARD, M., KATZ, F. & WEST, N. (1975) The role of low affinity antibody in immune complex disease. Clin. exp. Immunol. 21, 121. STEWARD, M. & PETTY, R. (1976) Evidence for the genetic control of antibody affinity from breeding studies. With inbred mouse strains producing high and low affinity antibody. Immunology, 30, 789. TAN, E.M., SCHUR, P.H., CARR, R. & KUNKEL, H. (1966) Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. 5. clin. Invest. 45, 1732. TRON, F. & BACH, J.F. (1977) Relationship between anti. bodies to native DNA and glomerulonephritis in systemic lupus erythematosus. Clin. exp. Immunol. 28, 426. WEITZMAN, R.J. & WALKER, S.E. (1977) Relation of titered peripheral pattern ANA to anti-DNA and disease actiN ity in systemic lupus erythematosus. Ann. Rheulm. Dis. 36, No. 1, 44. WINFIELD, J.B., FAIFERMAN, I. & KOFFLER, D. (1977) Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. 7. clin. Invest. 59, 90.
