819

QUANTITATION OF PRECIPITATING ANTIBODIES TO CERTAIN SOLUBLE NUCLEAR ANTIGENS IN SLE TH EI R CONTRIBUTION TO HY PERGAM MAG LOBULIN EM I A PETER J. MADDISON and MORRIS REICHLIN

Electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate was used to separate and quantitate the components of a washed immune precipitate. Serum was from patients with systemic lupus erythematosus known to have antibodies to soluble nuclear ribonucleoprotein (RNP) or to a soluble nuclear nonnucleic acid protein (Sm). Amounts of antibody that was predominantly IgG ranged from 0.2 to 8 mg/ml of patients’ serum, and in some cases accounted for over 20% of the total serum IgG. Results demonstrate that some patients respond to the disease by producing large amounts of a specific antibody, and that these antibodies can contribute significantly to hypergammaglobulinemia.

Patients with systemic lupus erythematosus (SLE) produce autoantibodies that react with many different nuclear and cytoplasmic constituents. In the only instance in which such autoantibodies have been quantitated in weight units ( l ) , the amount found was insufficient to contribute significantly t o the hypergammaglobulinemia commonly found in this disease. There is evidence for increased antibody titers to common

From the Departments of Medicine and Biochemistry, SUNY at Buffalo School of Medicine, and the Veterans Administration Hospital, Buffalo, New York. Supported by USPHS grant AM 10428 and by funds from the Veterans Administration. Peter J . Maddison. M.D.: Clinical Instructor in Medicine; Morris Reichlin, M.D.: Professor of Medicine and Biochemistry. Address reprint requests to Morris Reichlin, M.D., Department of Medicine, VA Hospital, 3495 Bailey Avenue, Buffalo, New York 14215. Submitted for publication September 8, 1976; accepted October 25, 1976. Arthritis and Rheumatism, Vol. 20, No. 3 (April 1977)

viruses, and such data have supported the opinion that the hyperglobulinemia is a manifestation of generalized hyperactivity of the humoral immune system (2). However the demonstration of high hemagglutination titers to an extractable nuclear antigen (ENA) in SLE (3) suggests the presence of large amounts of specific antibody in some patients’ sera. ENA is a mixture of soluble nuclear antigens, two of which .have been characterized and shown to be a ribonucleoprotein (RNP) antigen (Mo) (4), and a nonnucleic acid antigen (Sm) ( 5 ) . As this report will show, quantitative complement fixation studies using sera from patients with SLE reactive with one or both of these antigens also indicate large amounts of specific antibodies, in some cases of the order of several milligrams per milliliter of serum. Neither of these antigens has as yet been isolated in pure form, and thus direct methods for measuring the amount of these antibodies, such as quantitative precipitin analysis or immunoabsorbent methods, are impossible. This report describes a direct method for quantitating these antibodies by electrophoresis in polyacrylamide gels of solubilized washed immune precipitates in the presence of sodium dodecyl sulfate (SDS). This technique can be used to separate and quantitate the components of a washed immune precipitate. It makes possible the quantitation of precipitating antibodies to single antigenic components, even when the reactive antigen is a constituent of a crude extract of tissue. The technique also permits the description of the quantitative relationship between the content of precipitating antibody and the complement-fixing potency of individual human sera for specific antigen-antibody systems.

820

MADDISON AND REICHLIN

MATERIALS AND METHODS Preparation of Immune Precipitates for Electrophoresis Serum was from patients with SLE known to have precipitating antibodies t o either a nuclear R N P (Mo) (4), or a soluble nuclear nonnucleic acid protein (Sm) (5). There were 7 M o sera and 8 Sm sera. One serum contained antibodies to both antigens. Calf thymus extract (CTE), the source of both antigens, was prepared as described previously (4). Each serum was heated a t 56OC for 30 minutes t o minimize nonspecific precipitation. A specific immune precipitate was then prepared at equivalence proportions determined by supernatant testing of precipitin curves by using constant amounts of reactive serum and varying volumes of CTE. The tube with no detectable antigen or antibody in the supernatant was taken as the precipitate at equivalence. The precipitate was washed three times with cold 0.85% saline, then dissolved in 1% SDS at pH 7.1. Known concentrations of a commercial preparation of human Fraction I1 (Cohn Fraction 11, Sigma Chemical Company) were prepared in the same concentration of SDS and used as reference standards. . Fifty microliters of each sample were applied to the gels, and electrophoresis was performed in 7% polyacrylamide gel slabs containing 0.1 M phosphate and 0.1% SDS, at 300 volts for 4 hours. The migration of bromphenol blue was used as the reference point within each gel. The electrophoresis buffer was 0.1 M phosphate, p H 7.1, with 0.1% SDS. Each gel was fixed in 20% trichloracetic acid for 16 hours and stained with 0.25% Coomasie Blue R for 5 hours. After destaining, the gels were scanned on a transmission densitometer (E. C. Apparatus Corporation) fitted with a Coomasie blue filter. A tracing was made in which peaks corresponded to the density of the bands, and the area under each peak was measured. A graph of area versus the known concentration of the IgG reference standards was constructed for each gel, and from this the amount of antibody in each serum was calculated and expressed as milligrams per milliliter of serum. Two immune precipitates were prepared from each serum and treated in this way. In addition, for reasons to be described later, an immune precipitate prepared from serum Bo was dissolved in 1% 2-Mercaptoethanol to reduce the immunoglobulin component, yielding heavy and light chains. On another occasion it was dissolved in 1% SDS containing 0.05 M iodoacetamide to block sulfhydryl groups generated or exposed as a result of the effect on the protein by the SDS. In both cases the IgG reference standards were treated in the same way. The same procedure was repeated for eight of the other sera.

M phosphate-buffered saline (PBS), pH 7.3, double diffusion in 0.6% agarose was performed with immunoglobulin classspecific rabbit antisera. Wells 7 mm in diameter were used for the dissolved immune precipitate, and wells of 2 mm, spaced 3 mm apart, were used for the rabbit antisera. With this geometry, the sera could detect 15 pg/ml of each immunoglobulin class. The major immunoglobulin classes were quantitated in the sera by radial immunodiffusion using commercial Meloy AGM plates and W H O standards (Meloy). Gel filtration of serum Bo was performed on a Sephadex G-200 column equilibrated with PBS.

RESULTS Quantitation of AntjbFdies in the Immune Precipitate A n example of a polyacrylamide gel is shown in Figure 1, in which the bands to the right represent the IgG reference standards at three different concentrations. The other bands represent the separated components of four different immune precipitates. The slower band corresponds to the antibody, and the faster constant band is presumed to be the antigen. This assumption was supported by staining a gel, to which an immune precipitate composed of RNP anti-RNP had been applied, with 0.04% Methylene blue in 0.2 M sodium acetate, pH 4.7, after fixation in 5.6% acetic acid,

Immunologic Techniques Quantitative complement fixation tests were performed according to Mayer, with a modification described in previous reports (6). The classes of immunoglobulin found in the immune precipitate were determined in the following way: Immune precipitates were prepared, washed, then dissolved in 0.1 N acetic acid. Following neutralization by dialysis against 0.02

Fig 1. A polyacrylamidegel. with the origin at the top, in which the bands to the right represent IgG reJerence standards at three different concentrations (0.5, 17. and 1.0 mglml). The bands to the left represent the separated components of four different immune precipitates. The slower hands correspond to the antibody. and the faster bands are presumed to he the antigen.

QUANTITATION OF ANTIBODIES IN SLE

when a dense band, presumed to represent RNA, was demonstrated in the region of the antigen band shown in Figure 1. The rate of migration of the RNA band was, in fact, faster than the fast protein component, a fact indicating that the nucleic acid and protein components of this complex antigen were dissociated in SDS. Table 1 summarizes the results of the quantitative antibody determinations to Sm or nuclear RNP in each serum by this method. Each of the determinations is expressed as milligrams of antibody per milliliter of serum. Most of the sera contained large quantities of specific antibody, which in one case amounted to 8 mg/ml. Estimations on serial serum samples from patient Pe showed that anti-Sm diminished with time. An interesting but isolated phenomenon was seen with serum Bo. From complement fixation data (Figure 2) we would have predicted that this serum contained approximately 5 mg/ml of antinuclear RNP. This quantity could only be detected by electrophoresis when the precipitate was treated with either 2-mercaptoethanol or iodoacetamide. When dissolved in SDS alone, much less antibody was demonstrable. This effect was not seen with the other sera. Serum Bo was similar to the others in that there Table 1. Quantitation uf Antibodies in Immune Precipitate

Milligram of Antibody per Milliliter of Serum Sera containing Anti-Sm I . Pe (7-27-70) 2. Pe ( 1 1-2-70) 3. Pe (7-22-71) 4 . Pe (4-18-73) 5 . Pe (6-16-75) 6. Do 7. Ki 8. Le

3.3; 3.6 3.1; 2.8 2.0; 2.5 0.4; 0.4 Not measurable 5.2; 5.5; 6.1* 0.7; 0.6; 0.6; 6.7; 7.1'

Sera containing antinuclear RNP I . Ba 2. Br 3. Sa 4. Ku 5. Bo 6. Mo 7. Ro

8 . 6 7.9; 7.5,* 8.2* 4.0; 4.0 4.0; 4.7; 4.9t 0.6; 0.8 1.2; 2.0; 4.5t; 5.0,* 5.2* 0.2; 0.25 2.5; 2.0; 2.3*

Sera containing Anti-Sm and antinuclear RNP 1 . Wa

4.3; 4.7; 5.2"

Each value represents a separate experiment.

* Immune precipitate dissolved in 1% SDS and 0.05 M iodoacetamide. t Immune precipitate dissolved in 1% SDS and 1% 2-mercaptoethanol.

82 1

25

x

E E

15

L

al YI L 0

-

10

> 0

5

0

1

2

3

4 mg

5

6

7

8

o f a n t i b o d y p e r ml

9

10

o f serum

Fig 2 . Relationship hetween the volume of serum t o j i x 50 units of complement and milligrams of antibody per milliliter of serum measured hy electrophoresis. A-immune precipitate prepared from serum Bo, dissolved in I % SDS alone: A-immune precipitate prepared from serum Bo. dissolved in I% S DS and 0.05 M iodoacetamide.

was no rheumatoid factor activity and the immune precipitate contained no detectable IgM. It did differ, however, in being the only serum to contain antibodies reactive with the soluble cytoplasmic nonnucleic acid antigen (Ro) (6). We hypothesized that reduction to heavy and light chains by mercaptoethanol permitted entry of immunoglobulin material which otherwise could not penetrate the gel because of molecular aggregation. T o see if the aggregates were present in the serum before immune precipitation formation, serum Bo was subjected to gel filtration on Sephadex G-200. No evidence of the high molecular weight form of IgG in serum Bo was found by gel filtration on a Sephadex (3-200 column, where there was good resolution of the 19s and 7s peaks. N o immunologically detectable IgG was found in any fraction from tubes that eluted before the 7s fraction. Such experiments suggest that aggregation of the immunoglobulin molecules involving covalent bonding between sulfydrul groups occurs during SDS solubilization of the precipitate. This isolated effect implies that the antinuclear RNP IgG in serum Bo is more prone to an aggregation phenomenon requiring disulfide interchange which is enhanced by the addition of SDS. Such effects are prevented by iodoacetamide or reversed by 2-mercaptoethanol.

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MADDISON AND REICHLIN

Comparison with Complement Fixation The amount of specific antibody measured by electrophoresis was correlated with the quantitative complement fixation results with the same sera. Figure 2 shows the inverse relationship between the volume of serum required to fix 50 CH,, units of complement, and the quantity of antibody measured directly by electrophoresis. A very close relationship is shown, except when electrophoresis was performed on the immune precipitate from serum Bo dissolved in SDS alone. The efficiency of complement fixation per unit antibody weight was calculated as the amount of antibody required to fix 50 units of complement. The values ranged from 0.007 to 0.0367 mg antibody with a mean of 0.021 f 0.0088 mg required to fix 50 units of complement. This range is nearly the same as that of complementfixing efficiencies for rabbit antibodies to ovalbumin in which a range of 0.0063 to 0.0300 mg was required with different rabbit antisera to fix 50 units of complement (7).

Immunoglobulin Classes in the Immune Precipitate The classes of immunoglobulin found in the immune precipitates are summarized in Table 2. All the precipitates contained predominantly IgG. Four conTable 2. Qualiiaiive Assessment of Immunoglobulin Classes in Immune Precipitate IgG Sera containing Anti-Sm I . Pe (7-27-70) 2. Pe (7-22-71) 3. Do 4. Ki 5. Le Sera containing antinuclear R N P 1. Ba 2. Br 3. Sa 4. Ku 5. Bo 6. Mo 7. Ro Sera containing Anti-Sm and antinuclear R N P I . Wa

-

+

+ +

+ +

+ + + + + + +

+

+

Table 3. Quantitation of Total Imnninoglobulins in the Sera

_-

Percent IgG IgA IgM IgG as (mg/100 (mn/l00 (mn/100 Specific ml) . &I) Antibody ml) 83 160 I10 92 25 180 200 52

20.1 9.7 8.2 2.6

0 0 0 0 0 0 0

Sera containing antinuclear R N P I . Ba 2. Br 3. Sa 4. Ku 5. Bo 6. Mo 7. Ro

3150 3100 1350 2200 3200 1180 2100

100 270 142 275 214 480 274

124 600 36 124 63 70 240

26.0 12.9 33.0 3.2 15.6 2. I 16.9

0

Sera containing anti-Sm and antinuclear R N P I . Wa

2800

310

240

16.1

760-1400

65-300

40-150

-

0 0 0 0 0

0 0 0 0

-

240 480 405 142 170 580 360 350

0

+ 0 +

Table 3 shows the results of quantitating the total IgG, IgA, and IgM in each serum by using commercial immunodiffusion plates. The serum levels of IgG were elevated in most cases, and in some the elevation was marked. This finding is common in this disease, but three sera and notably serum Sa had normal levels. IgA was less elevated, and although there was a wide scatter of values for IgM, most values were close to the normal range. The last column of Table 3 lists the amount of specific antibody in the serum as a percentage of the total IgG. With one exception, when the amount of

1730 3200 2800 1500 1350 2500 2500 3700

IgM

0 0 0

Quantitation of Immunoglobulins in the Sera

Sera containing Anti-Sm I . Pe (7-27-70) 2. Pe ( I 1-2-70) 3. Pe (7-22-71) 4. Pe (4-18-73) 5. Pe (6-16-75) 6. Do 7. Ki 8. Le

IgA

+

tained small amounts of IgA, but IgM was not detected in any. The limit of sensitivity for the geometry in the gels employed with known standards of purified immunoglobulins for each of the immunoglobulin class-specific antisera was 15 pg/ml. Autoantibodies belonging to all the major classes of immunoglobulin have been found in SLE sera (8), but our results reflect the common knowledge that they are predominantly IgG.

Sensitivity: the lowest concentration of IgG, IgA. or IgM detected was 15 pg/ml of antisera.

Normal values

-

21.6 2.8 18.6

QUANTITATION OF ANTIBODIES IN SLE

antibody was too small to be measured, the values range from 2% to 33%, and they show that these antibodies contribute significantly to the hypergammaglobulinemia. One case, serum Sa, is particularly interesting in that the antibody amounted to 33% of the total IgG, and this patient did not have elevated levels of immunoglobulin.

DISCUSSION Large amounts of specific precipitating antibodies reactive with nuclear R N P antigen (Mo) or a soluble nuclear nonnucleic acid protein (Sm) were demonstrated in the sera of patients with SLE by electrophoresis of washed immune precipitates in polyacrylamide gels in the presence of SDS. In one serum (Ba) as many as 8 mg/ml of antinuclear RNP were detected. In one study (1) antibodies to DNA-histone were isolated by binding and elution from DNA-histone. These eluted antibodies were then measured by a quantitative complement fixation test, but they invariably amounted to less than 0.5 mg/ml, even in active cases of SLE. Their average value was in the range of 0.1-0.2 mg/ml. Clearly, in this study specific antibodies are present at a higher order of magnitude. The specific antibody was predominantly IgG, and when the amount is expressed as a percentage of the total serum IgG, values in four cases exceeded 20%. Therefore antibodies to Sm and nuclear R N P can contribute significantly to the hypergammaglobulinemia. However raised serum levels of immunoglobulin were not invariably present, and in one of these cases (serum Sa) we could still demonstrate large amounts of antinuclear RNP which amounted to 33% of the total IgG. None of the sera demonstrated an abnormal homogeneous protein spike in the gamma region on paper electrophoresis. The demonstration of autoantibodies in SLE to an ever widening'spectrum of antigens has pointed to a generalized hyperactivity of the humoral immune system. However it appears from this study that some patients d o indeed respond to the disease by producing large amounts of a specific antibody to a single definable antigen. Different patterns of specific autoantibody formation have been associated with the pattern of clinical disease. Antibodies to DNA are frequently associated with acute, severe SLE (9), and there is strong evidence for a major role of DNA anti-DNA complexes in the development of lupus nephritis (10-12). The presence of antibodies to the nuclear R N P antigen (Mo) in SLE, on the other hand, is associated with a more benign course,

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a low prevalence of the occurrence of antibodies to DNA, and a much lower incidence of renal disease (3,13). A sustained favorable course is associated with a sustained high titer of antibody to nuclear RNP. Therefore it is clear that the qualitative nature of the immune response is important in determining the type of disease. In our experience, a change in the titer of antibodies to Sm and nuclear RNP is accompanied by an alteration in the clinical status. We have observed in 2 patients, as have others (14,15), that a reduction of antinuclear RNP accompanied the appearance of anti-DNA and the subsequent development of renal disease. Conversely, the reduction in the amount of anti-Sm in serial serum samples from patient Pe, included in this study, accompanied clinical remission of his renal disease, manifested by the nephrotic syndrome, while he was on corticosteroids. These clinical observations suggest that a sustained vigorous immune response to the nuclear RNP antigen is closely linked to the failure of such patients to make antibodies to DNA. The mechanism of such a linkage has been speculatively explored previously (13,16). What has now b.een shown is that the vigor of this immune response to nuclear R N P and Sm is such that in some patients this response contributes significantly to the hypergammaglobulinemia.

REFERENCES I . Townes AS, Stewart CR Jr, Osler AG: Immunological studies of systemic lupus erythematosus. 1. Quantitative estimations of nucleoprotein-reactive gamma-globulin in systemic lupus erythematosus and other diseases. Bull Johns Hopkins Hosp 112:202-219, 1962 2. Phillips PE, Christian CL: Virus antibody studies in the connective tissue diseases. Arthritis Rheum 14:180-181, I97 I 3. Sharp G C , Irvin WS, LaRoque RL, e t al: Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 50350-359, 1971 4. Mattioli M, Reichlin M: Characterization of a soluble nuclear ribonucleoprotein antigen reactive with SLE sera. J Immunol 107:1281-1290, 1971 5. Tan EM, Kunkel HG: Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J lmmunol 9:464-471, 1966 6. Clark G M , Reichlin M, Tomasi TB: Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic lupus erythematosus. J Immunol 102:117-122, 1968

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7. Wallace AL, Osler A G , Mayer MM: Quantitative studies of complement-fixing potency of immune sera and its relation to antibody-nitrogen content. J Immunol 65:661-673, 1950 8. Barnett EV, Condemi JJ, Leddy JP, et al: Gamma 2, gamma IA, and gamma I M antinuclear factor in human sera. J Clin Invest 43:l104-11l5, 1964 9. Schur PH, Sandson J: Immunologic factors and clinical activity in systemic lupus erythematosus. N Engl J Med 278:533-538, 1968 10. Tan EM, Schur PH, Carr RI: Deoxyribonucleic acid (DNA) and antibodies to D N A in the serum of patients with systemic lupus erythematosus. J Clin Invest 45:1732-1740, 1966 11. Koffler D, Schur PH, Kunkel H G : Immunological studies concerning the nephritis of systemic lupus erythematosus. J Exp Med 126:607-623, 1967 12. Cochran CG, Koffler D: Immune complex disease in ex-

13.

14.

15.

16.

perimental animals and man. Adv Immunol 16:185-264, 1973 Reichlin M, Mattioli M: Correlation of a precipitin reaction to an R N A protein antigen and a low prevalence of nephritis in patients with systemic lupus erythematosus. N Engl J Med 286:908-911, 1972 Maddison PJ, Reichlin M: Association of the disappearance of precipitating antibodies to certain soluble nuclear and cytoplasmic antigens and disease exacerbation in SLE. Arthritis Rheum 18:808, 1976 (abstr) Sharp G C , Irvin WS, Tan EM, et al: Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 52:148-159, 1972 Reichlin M: Mixed connective tissue disease, Modern Topics in Rheumatology. London, Heinneman Publishers, in press

Quantitation of precipitating antibodies to certain soluble nuclear antigens in SLE.

819 QUANTITATION OF PRECIPITATING ANTIBODIES TO CERTAIN SOLUBLE NUCLEAR ANTIGENS IN SLE TH EI R CONTRIBUTION TO HY PERGAM MAG LOBULIN EM I A PETER J...
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