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Quantitation of warm reactive IgG antilymphocyte autoantibodies in systemic lupus erythematosus
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
17, 51.5-529 (1980)
Quantitation of Warm Reactive IgG Antilymphocyte Autoantibodies in Systemic Lupus Erythematosus S.H.
PACKER AND G.L.
Departments of Hematology and Medical Oncology, Veterans Administration and Duke University Medical
LOWE
and Departments Centers, Durham,
of Medicine, Durham North Carolina 27706
Received January 22, 1980 Warm-reacting lymphocyte-binding IgG was measured in normal subjects and patients with systemic lupus erythematosus (SLE). A quantitative antiglobulin assay was used to measure both the amount of IgG present on autologous lymphocytes (L-IgG) and lymphocyte-binding IgG in serum (IgG-LBA). L-IgG of 12.9 ? 8.6 x lo-l4 g IgG was detected on washed normal lymphocytes. Fifty-three percent of patients with SLE had L-IgG greater than two standard deviations above that of normal. IgG-LBA of normal subjects (the amount of IgG detected on lymphocytes after incubation in normal serum) was 9.0 2 4.7 x lo-l4 g IgGkell. This did not differ significantly from the amount of IgG detected on lymphocytes prior to reincubation in normal serum. Forty-seven percent of patients with SLE had serum IgG-LBA of greater than two standard deviations above normal. L-IgG was elevated in all patients with increased IgG-LBA in whom L-IgG was measured. Protein fractionation studies including Sephadex G-200 gel filtration, DEAE ion-exchange chromatography, and pepsin digestion to produce F(ab’), fragments were performed upon serum that had elevated IgG-LBA. The IgG-LBA activity appeared to represent binding of F(ab’), portions of monomeric IgG molecules to lymphocytes. The IgG antibody from one patient was able to produce human complement-dependent lymphocytotoxicity when present in high concentration (greater than 60 x lo-l4 g/cell). Thus, some patients with SLE have warm reactive antilymphocyte IgG present both in their serum and on their own lymphocytes. We are unable to define the clinical sequelae of this form of humoral autosensitization.
INTRODUCTION
Numerous abnormalities in immune function and regulation have been described in patients with systemic lupus erythematosus (SLE), including a variety of antibodies which react with lymphocytes (l-5). These antibodies may be either IgM or IgG, and optimally reactive at 4°C or at body temperature. Cold reactive complement-dependent IgM lymphocytoxins, detected by microcytotoxicity assays, appear to be most frequent and have been extensively characterized (6, 7). In contrast, human autoantibodies directed against red cells, platelets, and granulocytes are usually IgG and reactive at body temperatures (8- 11). Quantitative immunoassays of IgG binding to these other blood cells have proven useful in understanding the pathophysiology of humoral autosensitization, which frequently occurs in systemic lupus erythematosus (9- 11). Although warm reactive IgG antilymphocyte antibodies have been described in SLE, a number of questions regarding these antibodies remain. Using microcytotoxicity and immunofluorescent techniques, Winfield er al. found only an occasional SLE patient with IgG antilymphocyte antibodies (6). Steinberg and 515 0090-1229/80/120515-15$01.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
516
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co-workers were also unable to detect IgG antilymphocyte antibodies using flow microfluoremetry (4). In contrast, Williams reported that 80% of SLE patients’ sera contained IgG antilymphocyte antibodies when tested with lz51-labeled protein A (5). Some studies have demonstrated noncytotoxic IgG antibodies directed against the responding cells in MLC reactions (12), while other studies suggest that these IgG antibodies are lymphocytotoxic (1). In addition, the T or B specificity of these antilymphocyte antibodies has not been defined (5, 12, 13). Most important, whether these antibodies react with antigens on a patient’s own cells and are, therefore, true autoantibodies, or whether IgG binding to lymphocytes may involve binding of IgG containing immune complexes by lymphocyte Fc receptors demands clarification (4, 5). The present study was therefore undertaken to further investigate these warm reactive IgG antibodies. In this report, we describe the results of an antiglobulin consumption assay to quantitate the amount of IgG present on lymphocytes from normal controls and patients with SLE. We also studied the amount of IgG present in normal and SLE serum that would bind to lymphocytes. We found that some SLE patients have markedly increased amounts of IgG antibody on their own lymphocytes as well as increased quantities of lymphocyte-binding IgG antibody in their serum. Characterization of this antibody revealed it to be monomeric IgG which bound to lymphocytes by its F(ab’), portion. These antibodies, in high concentrations, can produce complement-mediated cytotoxicity of human lymphocytes. The possible significance of such autoantibodies is discussed. METHODS Patients. Eighteen patients with SLE were studied (Table 1). All met the American Rheumatic Association’s diagnostic criteria for SLE (14). Normal control sera and peripheral blood lymphocytes were obtained from healthy laboratory and hospital personnel. Preparation of lymphocytes. Forty milliliters of heparinized whole blood were obtained by routine venipuncture, mixed with a 1/5th volume of plasmagel (HTI Corp., Buffalo, N.Y.), and allowed to sediment for up to 3 hr. The resulting leukocyte-rich plasma was collected and incubated with carbonyl iron powder (GAF Corp., N.Y.) for 15 min in a 37°C shaking water bath. The phagocytic cells were then removed by passing the leukocyte-rich plasma-iron mixture through a magnetic field (15). The leukocyte-rich plasma was then layered on Ficoll - Hypaque (Winthrop Laboratories, New York, N. Y., specific gravity, 1.078) and centrifuged at 1OOOgfor 20 min to obtain a purified lymphocyte preparation (15). The interface, containing greater than 95% lymphocytes, was harvested, washed three times with veranol-buffered isotonic saline (VBS), pH 7.2, and resuspended to a concentration of 1 x 107/ml in VBS. Lymphocytes were fractionated into B- and T-cell subpopulations using techniques previously described (16, 17). B cells were obtained by incubating neuraminidase-treated sheep red blood cells with lymphocytes followed by centrifugation over a Ficoll-Hypaque gradient. E-rosetted cells separated into the pellet and the interface containing nonrosetted cells (T cell depleted) was collected. T cells were obtained by filtering lymphocytes over a nylon wool column.
IgG ANTILYMPHOCYTE
AIJTOANTIBODIES
IN SLE
517
The B- and T-cell subpopulations were washed three times in VBS and resuspended to the appropriate concentrations. The purity of the enriched B- and T-cell suspensions were determined by the percentage of cells bearing surface immunoglobulin and the percentage of cells which form rosettes with sheep erythrocytes. The percentage of lymphocytes with detectable surface immunoglobulin was measured by fluorescent microscopy using fluorescein-conjugated polyvalent goat anti-human immunoglobulin (18). The fraction of lymphocytes forming rosettes with sheep erythrocytes was determined by previously described methods (19). Preparation of immunologic reagents. Antibody to human IgG was made by injecting a mixture of purified IgG subclasses 2, 3, and 4 with Freund’s adjuvant into rabbit foot pads (9, 10). Serum was absorbed with sheep and human red blood cells as well as with K and A light chains. The absorbed serum did not react with human serum components other than IgG when tested by immunoelectrophoresis against whole human sera. Purified human IgG was obtained by DEAE ionexchange chromatography of serum from a patient with IgG subclasses Gl multiple myeloma. IgG-coated sheep erythrocytes were prepared by incubating 0.5 ml of sheep erythrocytes with 0.07 ml of 0.8% glutaraldehyde and 0.25 ml of purified IgG for 60 min at room temperature (10). The sheep red blood cells were then washed three times in VBS and adjusted to a concentration of 4.4 x 10Yml. Determination of lymphocyte-bound ZgG (L-ZgG): Direct assay. Lymphocytebound IgG was measured using a quantitative antiglobulin consumption technique similar to one previously described for platelets and granulocytes (9- 11). This assay and the assay for serum lymphocyte-binding antibody are outlined in Fig. 1. The surface IgG may be quantitated by the adsorption of a known amount of rabbit anti-human IgG during incubation with lymphocytes. The amount of antiIgG remaining is measured by lysis of IgG-coated sheep erythrocytes with an excess of guinea pig serum as a source of complement. One-tenth-milliliter duplicate serial twofold dilutions of lymphocytes or dilutions of known concentrations of human IgG were mixed with 0.1 ml of diluted rabbit anti-human IgG for 30 min at 37°C. One-tenth milliliter of IgG-coated sheep erythrocytes was then added to the mixture and allowed to react for 30 min at 37°C. This was followed by the addition of 0.2 ml of guinea pig serum diluted 1:20 with VBS. This final mixture was incubated for 30 min at 37°C following which 5.0 ml of VBS was added. The mixture was then centrifuged and the percentage of sheep cells lysed was measured spectrophotometrically at 412 nm. Standard curves were constructed by determining the percentage of inhibition of hemolysis by known amounts of IgG. When increasing concentrations of purified human IgG were assayed, progressive inhibition of lysis occurred. The result of such a standard curve is shown in Fig. 2. By referring to this curve, it is possible to ascribe an absolute IgG value to the amount of inhibition produced by an unknown quantity of IgG in a sample. In this experiment, 34 x IOeg g of IgG was required to produce 50% inhibition of hemolysis. Such standard curves were done with each assay. TO calculate the amount of IgG present on lymphocytes, inhibition curves which related lymphocyte number of percentage inhibition of hemolysis were constructed. Lymphocytes from a normal individual were always studied on the
518
PACKER AND LOGUE
4occ WHOLE BLOOD
!95% LYMPHOCYTES 1 x IO’ CELLS/ml
/ IgG-LEA
I
!LhPLELIENT
I
DUPLICATE SERIAL 2-FOLD DILUTIONS
HEMOLYSIS
FIG. 1. Schematic representation of the methods used to quantitate lymphocyte antibodies. Lymphocyte-bound IgG (L-&G) represents the amount of IgG detected on an individual’s own lymphocytes. Lymphocyte-binding antibody (IgG-LBA) represents the amount of antibody in serum which will bind to normal lymphocytes. Both the lymphocyte-bound IgG and the lymphocyte-binding antibody are determined by the inhibition of hemolysis of IgG-coated sheep red cells caused by anti-IgG and complement.
same day as the patient’s lymphocytes. In Fig. 3, the results of such inhibition curves for a normal control, SLE patient K, and patient A with a marked increase in lymphocyte-bound IgG are shown. From these curves, the number of cells which produce 50% inhibition could be determined. By knowing the concentration of IgG required for 50% inhibition of lysis, the amount of lymphocyte-bound IgG could then be calculated. Determination of serum lymphocyte-binding antibody (ZgG-LBA): Indirect assay. The amount of antibody that would bind to normal lymphocytes was determined as outlined in Fig. 1. Three-tenths milliliter of serum was incubated with 0.3 ml of 1 x lO’/ml normal lymphocytes for 1 hr at 37°C. The mixture was washed three times with VBS at 37°C and the lymphocytes were counted by a Coulter counter (Coulter Diagnostics, Hialeah, Fla.). The amount of lymphocyte-bound IgG was determined as described above. In Fig. 4, the results of lymphocytebinding antibody at several serum dilutions is shown. At concentrations greater than 1:9, nonspecific binding of IgG from normal serum increased. All subsequent determinations were therefore performed at a 1:9 dilution. Serum fractionation. Sera from patients with SLE and normal controls were
IgG
ANTILYMPHOCYTE
AUTOANTIBODIES
100 I20 IgG (Gram x W9]
IN
I40
160
SLE
519
180
FIG. 2. Immunoglobulin G (IgG) calibration curve showing inhibition of anti-IgG-induced lysis of IgG-coated sheep erythrocytes when the anti-lgG is preincubated with increasing amounts of IgG.
fractionated using Sephadex G-200 gel filtration with 0.15 M saline at 4°C on a 3.0 x 90-cm column. Serum (2.5 ml) was passed over the column and representative fractions assayed for protein and lymphocyte-binding IgG. Normal and SLE sera were also fractionated by DEAE ion-exchange chromatography to obtain purified IgG. One-half milliliter of serum that had been dialyzed against 0.005 M phosphate buffer, pH 8.0, was applied to a 50 ml bed volume DEAE-cellulose column equili-
Lymphocytes
Added
per 0 I ml (x 10’)
3. Inhibition of anti-IgG-induced Iysis of IgG-coated sheep erythrocytes by lymphocytes from a normal subject and two patients with SLE. Those data can be used to calculate lymphocyte-bound IgG as described in the text. FIG.
520
PACKER
0 ’ ‘47
AND
‘4 SERUM
FIG. 4. Effect of dilution patients with SLE.
on lymphocyte-binding
LOGUE
‘4 DILUTION
antibody
of sera from
a normal
individual
and two
brated with the same buffer. IgG-containing fractions were then eluted with this buffer. To compare lymphocyte-binding antibody activity between samples from different patients, the IgG fractions from different patients were concentrated to equal protein concentrations as measured by the method of Lowery (20). Preparation of radiolabeled F(ab’), fragments. F(ab’), fragments were obtained by pepsin digestion of IgG-containing fractions from normal and SLE sera followed by gel filtration on Sephadex G-150 (21). The F(ab’)z fragments were then labeled with 1251by the Bolton-Hunter method (22). The radiolabeled F(ab’)2 fragments were then incubated with 0.1 ml of 3 x lO’/ml lymphocytes or control tubes without lymphocytes for 1 hr at 37°C. The tubes were washed three times with 37°C VBS and the pellets counted on a Beckman gamma counter. Complement-dependent lymphocytotoxicity method. The ability of these antibodies to lyse normal lymphocytes with human complement was investigated using a 51Cr release assay. One to two milliliters of 5 x lO’?ml lymphocytes was incubated with 100 to 300 &i Wr for 1 hr at 37°C and then washed three times in the McCoy’s 5A media (Grand Island Biological Co., Grand Island, N.Y.). Ten microliters of heat-inactivated test serum was incubated with 10 ~1 of 51Cr-labeled lymphocytes for 1 hr at 37°C in a microtiter plate (Limbro Scientific, Hamden, Conn.). One hundred microliters of dilutions of normal type compatible human serum as a source of complement was added and the mixture incubated for 4 hr at 37°C in a 5% CO, moist incubator. Following centrifugation of the microtiter plates at 800g for 10 min, 50 ~1 of supematant was aspirated and counted in a gamma counter. Percentage cytotoxicity was calculated by subtracting the supernatant counts in buffer controls from the test supernatant counts and dividing by
IgG ANTILYMPHOCYTE
AUTOANTIBODIES
IN SLE
521
the total releasable counts minus the buffer controls. Total releasable counts were determined by incubation of lymphocytes with 1% sodium deoxycholate followed by the addition of 100 ~1 of buffer and centrifugation. RESULTS Lymphocyte-Bound
ZgG (L-ZgG): Direct Assay
The amount of lymphocyte-bound IgG was determined on 17 normal controls and in 12 patients with SLE as shown in Fig. 5. Normal lymphocyte-bound IgG ranges from 2.3 to 35.4 x lo-l4 g/cell with a mean of 12.9 ? 8.6 x lo-l4 g/cell. Five of twelve SLE patients have levels of lymphocyte-bound antibody greater than two standard deviations above normal. In patient A, this represented three, and in patients C and E, two separate determinations on different days. Serum Lymphocyte-Binding
Antibody
(ZgG-LBA): Zndirect Assay
The results of the serum lymphocyte-binding antibody assay are shown in Fig. 6. IgG lymphocyte-binding antibody levels from 21 normal subjects ranges from 2.0 to 16.0 x lo-l4 g/cell with a mean of 9.0 ? 4.7 x lo-l4 g/cell. In sera of 8 of 15 patients with SLE, the lymphocyte-binding antibody level was greater than two standard deviations above normal. In patient A, the lymphocyte-binding antibody level was markedly elevated with a mean of 87.0 ? 42.6 x lo-l4 g/cell. These results represent 25 separate determinations on 15 different lymphocyte donors.
320 *A
:%
240 T 160 1 h i so: 0 x zD z 0 ,D
. B
.C
60.D .E
9 i3
g
40-
* c . E
? 2 t,
20-
t i T
0
NORMAL
SLE
5. Range of values for lymphocyte-bound IgG (L-IgG) in normal subjects and patients with SLE. Letters refer to separate determinations in patients with lymphocyte-bound IgG greater than two standard deviations above normal. The mean and standard deviations for normals are shown. FIG.
522
PACKER
AND
LOGUE
,
i
Oi
NORUSS
SLE
pA’;EN’
FIG. 6. Range of values of lymphocyte-binding antibody (IgG-LBA) in normal subjects and patients with SLE. In patient A, values represent separate determinations on 15 different donor lymphocytes. The mean and standard deviations for normals are shown.
Patient A’s lymphocyte-binding antibody was elevated with all normal donor lymphocytes tested. Since this antibody reacted with all donor cells tested, it seemed unlikely that this antibody was directed against isologous antigens such as those of the HLA system. To further investigate the possibility that this antibody was directed against antigens of the HLA system, serum from patient A was absorbed extensively against pooled platelets from 150 different platelet donors (6). Following absorption, elevated levels of lymphocyte-binding antibody persisted, suggesting that this antibody was not directed against HLA antigenic determinants. To be certain that the increased amount of antilymphocyte antibody seen in these patients was not due solely to increased plasma concentrations of IgG, sera from two patients with multiple myeloma were studied. Despite markedly increased serum IgG concentrations, myeloma patients’ sera had normal levels of lymphocyte-binding antibody. In Table 1, the results of the lymphocyte-bound IgG and lymphocyte-binding antibody assays are tabulated for individual patients. Except for three patients with elevated lymphocyte-binding antibody whom we were unable to test, all patients with increased lymphocyte-binding antibody levels had elevated amounts of IgG present on their own washed lymphocytes. The reactivity of IgG-LBA with subpopulations of lymphocytes was studied. Sera from two SLE patients with elevated IgG-LBA and from a normal subject were tested with normal B- and T-lymphocyte preparations. The results of this study are shown in Table 2. The mean and range of duplicate determinations are shown. The IgG-LBA of both patients was detected on both B and T lymphocytes.
IgG
ANTILYMPHOCYTE
BLOOD
LEUKOCYTE
AUTOANTIBODIES
TABLE COUNTS
ANTIBODY
Patient
Total leukocyte count (cells/mm3)
A
2,400
B C D E F G
6.00’3
RESULTS
OF LYMPHOCYTE
IN SLE
QUANTITATION
PATIENTS
Lymphocyte-binding antibody (IgG-LBA x lo-” g/cell)
lymphocyte count (cells/mm3)
523
SLE
1
AND
Absolute
3,ooo 6,300 3,200 4,100 4,950 9,000 4,900 5,500 12,000 7,700 6,200 10,800 6,500 3,700 3,000
IN
336 800 780 630 1,376 920 1,100 810 931 2,255 840 1,070 800 3,780 1,200 1,295 990
81.9 k 42.4" 42.8 46.8 20.0 55.7 35.8 12.5 10.5 44.3 10.0 ND 11.4 13.9
Lymphocyte-bound antibody (L-IgG x lo-” g/cell)
+ 17.1b 130.2 53.5 55.8 41.0
262.6
ND’ 10.0 18.4
ND 14.3 15.5 12.0 9.0 3.0
ND 10.0
ND ND ND
5.0 20.4
u Results from 25 determinations. 0 Results from three determinations. c Not done.
The antibody of both patients reacted somewhat more strongly with B cells than with T cells as shown in this table. Serum Fractionation
Serum fractionations were done to determine whether the elevated levels of lymphocyte-binding antibodies were due to monomeric IgG, aggregated IgG, or immune complexes containing IgG. Serum from patient A was chromatographed on Sephadex G-200. In Fig. 7, the fraction number is plotted against the absorbance at 280 nm on the left ordinate and against the IgG-LBA on the right ordinate. Lymphocyte-binding antibody activity corresponded to monomeric IgG; there was no significant activity in void volume fractions where large molecular size immune complexes or aggregated IgG would be expected. To further confirm that the lymphocyte-binding antibody represented IgG alone, serum from patient
TABLE RESULTS
OF LYMPHOCYTE-BINDING
2
ANTIBODY
ON B AND
Lymphocyte
binding
(IgG-LBA,
Cells tested
Patient
B T n B- and T-enriched and range of duplicate
A
122.7 + 0.6 43.3 + 0.7 lymphocytes determinations.
were
prepared
x
IO-l4
Patient
E
57.0 27.0
as described
T LYMPHOCYTESO
antibody g/cell)
2 7.3 k 4.9 in the text.
Normal + 4.2 5.2 + 0.9
14.4
The results
are the mean
524
PACKER
AND
LOGUE
A and from a patient with normal amounts of lymphocyte-binding antibody (patient H) were fractionated by DEAE ion-exchange chromatography. The purified IgG was concentrated and confirmed by immunodiffusion against antisera to IgG, IgM, and whole serum. The protein concentrations of the samples from the two patients were equalized and assayed for lymphocyte-bound IgG as shown in Fig. 8. With equal amounts of IgG added, IgG-LBA of purified IgG from patient A was greater than that from patient H. Thus, results from G-200 gel and DEAE ionexchange chromatography suggested that this lymphocyte-binding antibody was monomeric IgG alone. To be certain that the lymphocyte-binding antibody activity was an antibody-antigen reaction rather than nonspecific binding of IgG by Fc receptors, we measured the binding of F(ab’)z fragments from SLE serum to lymphocytes. 1251-labeled F(ab’)z fragments from patient A and a patient with normal amounts of lymphocyte-binding antibody (patient H) were studied. As shown in Fig. 9, lymphocyte binding of patient A F(ab’)z was greater than for that of patient H. Thus, the serum from patient A appeared to contain antibodies directed against lymphocyte antigens. Human Complement-Dependent Lymphocytotoxicity These antibodies were tested for their ability to cause human complementdependent cytotoxicity. The results of the 51Cr release assay using various mixtures of serum is shown in Table 3. The mixture of heated patient A serum plus normal serum was lymphocytotoxic. When the normal serum was heated to remove its complement activity, lymphocytotoxicity was abolished. Thus, the antibody in this patient appeared to support human complement-dependent lymphocytotoxicity. The relationship between lymphocyte-binding antibody and lymphocytotoxicity was then examined. In Fig. 10, lymphocytotoxicity is plotted against lymphocyte-binding antibody. Approximately 65 x lo-l4 g/cell of IgG was re-
FRACTION
7. Results of Sephadex between protein concentration body is shown. FIG.
NUMBER
G-200 gel chromatography as estimated by absorbance
of serum from patient A. The relationship at 280 nm and lymphocyte-binding anti-
IgG ANTILYMPHOCYTE
AUTOANTIBODIES
IgG Concentrolton
Added
IN
SLE
525
jmg/ml)
8. Results of lymphocyte-binding antibody of DEAE-purified IgG. IgG was purified as described in the text. The results are shown for a patient with elevated whole serum IgG-LBA (patient A) and a patient with normal IgG-LBA (patient H). FIG.
quired to initiate lysis following which there was a dose-response between lymphocyte-binding antibody and lymphocytotoxicity.
relationship
DISCUSSION
Both IgG and IgM antibodies directed against lymphocytes are frequently present in patients with SLE. Cold reactive complement-dependent IgG antibodies
Flab):,
odded
(fig/ml)
FIG. 9. Results of binding of ‘251-labeled F(ab’), fragments to normal lymphocytes. Patient A had increased amounts and patient H normal amounts of lymphocyte-binding antibody as detected by the antiglobulin consumption assay.
526
PACKER AND LOGUE TABLE
3
COMPLEMENT-DEPENDENTLYMPHOCYTOTOXICITY~
Lymphocyte incubation mixture Heated patient A serum unheated normal serum Heated patient A serum + heated normal serum Heated normal serum + unheated normal serum
Lymphocytotoxicity (% Yr release) 23.0 zt 0.3 0.3 + 0.3 3.1 k 1.5
o Wr-labeled normal lymphocytes were incubated with mixtures of patient A and normal serum and lymphocytotoxicity determined as described in the text. The mean and standard deviation of triplicate determinations are shown.
have been extensively characterized. Data regarding IgG lymphocytic antibodies have been somewhat conflicting. Stastny and Ziff initially demonstrated a complement-dependent cytotoxic effect from serum of patients with SLE, for both ahogeneic and autologous lymphocytes (7, 23). They suggested that the antilymphocyte antibodies in SLE were IgG and maximally cytotoxic at physiologic temperatures. However, observations by Terasaki and later Winfield and co-workers demonstrated that the antilymphocyte antibodies detected by microcytotoxicity assays were, in fact, cold reactive and primarily IgM in class (2, 6). Other investigators have reported noncytotoxic antilymphocyte IgG that is active at physiologic temperatures and that suppresses mixed lymphocyte culture reactions (12). Williams and co-workers, using a 1251-labeled protein A, have further characterized warm reactive antilymphocyte IgG in SLE (5). They did not, however, investigate whether these patients’ own cells had elevated amounts of IgG. Glinski et al. have demonstrated anti-T-cell lymphocyte antibodies which appear to be of the IgG class (13). On the other hand, Steinberg and co-workers, using flow microfluorimetry, demonstrated anti-T-cell antibodies that were IgM in class, but were unable to demonstrate IgG antibodies (4). Results of this study clearly demonstrate the occurrence of warm reactive antilymphocytic IgG both in the serum and on the cells of some patients with SLE.
FIG. 10. The relationship between lymphocyte-binding lymphocytotoxicity is shown for patient A’s serum.
antibody and human complement-dependent
IgG
ANTILYMPHOCYTE
AUTOANTIBODIES
IN
SLE
527
Utilizing a quantitative antiglobulin consumption assay, we measured the amount of warm reactive lymphocyte-binding IgG present in the serum as well as the amount of IgG on the patient’s own lymphocytes. Fifty-three percent of patients had elevated serum levels of lymphocyte-binding IgG. In one patient, this represented 25 determinations performed on 15 different normal lymphocyte donors. In addition, IgG-LBA activity persisted after repeated absorption with pooled platelets from 150 different donors. This is an agreement with previous work indicating that antilymphocyte antibodies in SLE are directed against non-HLA determinants. We also investigated the subclass of lymphocytes against which these IgG antibodies were directed. Prior reports of either B- or T-cell specificity have been conflicting. In contrast to Glinski and co-workers, who reported IgG antibodies specific for T cells, Williams et al. found equal reactivity for both lymphocyte populations (5, 13). In our study, the IgG antibody bound to both B and T cells. In addition, we were able to directly measure IgG on these patients’ own lymphocytes. Forty-two percent of SLE patients tested had elevated amounts of lymphocyte-bound IgG. All the patients with elevated serum lymphocyte-binding IgG who were tested also had increased amounts of IgG present on their own lymphocytes. It would thus appear that the serum antibody reacted with the patients’ own lymphocytes. Other investigators, using other techniques, have also found antibodies on the lymphocytes of patients with SLE (7, 13). To confirm that this reactivity represented antibody-antigen interactions rather than nonspecific binding of IgG to lymphocyte Fc receptors, serum fractionation studies were performed. Serum was chromatographed on G-200 gel filtration and then assayed for lymphocyte-binding IgG. The IgG-LBA activity corresponded to monomeric IgG and there was no activity in the void fractions where immune complexes or aggregated IgG would be expected. Further confirmation that this activity was not due to immune complexes was obtained when purified IgG, prepared by DEAE ion-exchange chromatography, from a patient with elevated IgG-LBA was compared to that from a patient with normal IgG-LBA. Again, it appeared that the IgG-LBA activity resided in the IgG portion of serum. Finally, F(ab’), fragments were obtained by pepsin digestion of purified IgG. The lz51labeled F(ab’), fragments from the patient with elevated IgG-LBA bound to normal lymphocytes. It therefore appears that the reactivity seen in the antiglobulin consumption assay indeed represents an antibody-antigen reaction rather than nonspecific binding to lymphocyte Fc receptors. While warm reactive IgG autoantibodies directed against red cells, platelets, or granulocytes appear to cause destruction of these cells in some patients, we were unable to define the clinical consequences of this warm reactive antilymphocyte IgG. Although Winfield et al. and Butler et al. were able to correlate the level of antilymphocyte antibodies with lymphopenia, the microcytotoxicity assay they used detects predominantly cold reactive IgM antibodies (25,26). Statsny and Ziff initially suggested that there were warm reactive IgG antibodies in SLE capable of functioning in viva by direct complement-mediated lymphocytotoxicity (7). Since we are able to directly measure lymphocyte-bound antibody, we could define the absolute amount of IgG per lymphocyte necessary to cause human complement-
528
PACKER AND LOGUE
dependent cytotoxicity. A 65 x lo-l4 g quantity of IgG per lymphocyte was necessary to initiate lysis using human serum as a source of complement. Since only one patient bound antibody in this range, the ability of these antibodies to produce in viva complement-dependent cytotoxicity is questionable. It is of interest that the patient with lymphocyte-bound IgG in this range was severely lymphopenic (patient A). Otherwise, we are unable to show a correlation between IgG lymphocyte autoantibodies and lymphopenia in this group of SLE patients. This lack of correlation is not surprising since, as pointed out by others, a variety of different mechanisms may cause lymphopenia in SLE (25). The significance of such lymphocyte autoantibodies in the pathogenesis of SLE remains unclear. Lymphocytotoxins have been described in a number of diseases characterized by immune abnormalities suggesting that they may be secondary phenomena. In the rodent model of SLE, however, there is evidence that lymphocyte antibodies cause a loss of suppressor T cells, resulting in generalized B-cell hyperactivity (27, 28). Recent studies have shown that subsets of T cells, both T, and T,, can suppress differentiation of B lymphocytes (29, 30). In addition, Sagawa and Abdou have suggested that an IgG antilymphocyte antibody directed against suppressor cells is responsible for the B-cell hyperactivity in SLE (31). However, in this study, many patients with active SLE did not appear to have warm reactive IgG antilymphocyte antibodies. Patients who had neither increased IgG on their own lymphocytes nor increased lymphocyte-binding antibody in their serum had classic systemic lupus erythematosus with severe disease manifestations. Thus, it seems clear to us that the finding of IgG antilymphocyte autoantibodies in these patients is a manifestation rather than a cause of systemic lupus erythematosus. ACKNOWLEDGMENTS This work was supported by the Research and Education Branch of the Veterans Administration. The authors thank Ms. Barbara Connor for excellent technical assistance and Ms. LaVerne Johnson for help in preparation of the manuscript.
REFERENCES 1. Mittal, K. K., Rossen, R. D., Sharp, J. T., Lidsky, M. D., and Butler, W. T., Nature (London) 225, 1255, 1970. 2. Terasaki, P. I., Mottironi, V. D., and Barnett, E. V., N. Engl. J. Med. 283, 724, 1970. 3. Ooi, B. S., Orlina, A. R., Pesce, A. J., Mendoza, N., Masaitis, L., and Pollak, V. E., Clin. Exp. Immunol. 17, 237, 1974. 4. Steinberg, A. D., Klassen, L. W., Budman, D. R., and Williams, G. W., Arthritis Rheum. 22, 114, 1979. 5. Williams, R. C., Jr., Bankhurst, A. D., and Montano, J. D., Arthriris Rheum. 19, 1261, 1976. 6. Winfield, J. B., Winchester, R. J., Wemet, P., Fu, S. M., and Kunkel, H. G., Arthritis Rheum. 18, 1, 1975. 7. Stastny, P., and Ziff, M., Arthritis Rheum. 14, 733, 1971. 8. Rosse, W. F., .I. Clin. Invest. 50, 734, 1971. 9. Dixon, R., Rosse, W., and Ebbert, L., N. Engl. J. Med. 292, 230, 1975. 10. Logue, G., Ann. Intern. Med. 85, 437, 1976. 11. Logue, G. L., and Silberman, H. R., Amer. J. Med. 66, 703, 1979. 12. Wemet, P., and Kunkel, H. G., J. Exp. Med. 138, 1021, 1973. 13. Glinski, W., Gershwin, E., and Steinberg, A. D., J. C/in. Invest. 57, 604, 1976.
IgG ANTILYMPHOCYTE
AUTOANTIBODIES
IN SLE
529
14. Cohen, A. S., Reynolds, W. E., Franklin, E. C., Kulka, J. P., Ropes, M. W., Shulman, L. E., and Wallace, S. L., Bull. Rheum. Dis. 21, 643, 1971. 15. Logue, G. L., and Cohen, H. J., J. Clin. Invest. 60, 1159, 1977. 16. Jarvis, S. C., Snyderman, R., and Cohen, H. J., Blood 48, 717, 1976. 17. Aisenberg, A. C., Long, J. C., and Wilkes, B., J. Nat. Cancer Inst. 52, 13, 1974. 18. Cohen, H. J., J. Clin. Invest. 55, 84, 1975. 19. Jondal, M., Holn, G., and Wigzell, H., J. Exp. Med. 136, 207, 1972. 20. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193,265, 1951. 21. Edelman, G. M., and Marchalonis, J. J., In “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase Eds.), Vol. 1, pp. 422-423, Academic Press, New York, 1967. 22. Bolton, A. E., and Hunter, W. M., B&hem. J. 133, 529, 1973. 23. Stastny, P., and Ziff, M., Clin. Exp. Immunol. 8, 543, 1971. 24. Williams, R. C., Jr., Lies, R. B., and Messner, R. P., Arthritis Rheum. 16, 597, 1973. 25. Winfield, J. B., Winchester, R. J., and Kunkel, H. G., Arthritis Rheum. 18, 587, 1975. 26. Butler, W. T., Sharp, J. T., Rossen, R. D., Lidsky, M. D., Mittal, K. K., and Gard, D. A., Arthritis Rheum. 15, 231, 1972. 27. Krakauer, R. S., Waldmann, T. A., and Strober, W., J. Exp. Med. 144, 663, 1976. 28. Steinberg, A. D., Gerber, N. L., Gershwin, M. E., Morton, R., Goodman, D., Chused, T. M., Hardin, J. A., and Barthold, D. R., In “Suppressor Cells in Immunity,” Proceedings of the First International Symposium on Suppressor Cells (S. K. Singhal N. R. St. C. Sinclair, Eds.), p. 174, Univ. of Western Ontario Press, London, 1975. 29. Moretta, L., Webb, S. R., Grossi, C. E., Lydyard, P. M., and Cooper, M. D., J. Exp. Med. 146, 184, 1977. 36. Hayward, A. R., Layward, L., Lydyard, P. M., Moretta, L., Dagg, M., and Lawton, A. R., J. Immunol. 121, 1, 1978. 31. Sagawa, A., and Abdou, N. I., J. Clin. Invest. 63, 536, 1979.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
17, 51.5-529 (1980)
Quantitation of Warm Reactive IgG Antilymphocyte Autoantibodies in Systemic Lupus Erythematosus S.H.
PACKER AND G.L.
Departments of Hematology and Medical Oncology, Veterans Administration and Duke University Medical
LOWE
and Departments Centers, Durham,
of Medicine, Durham North Carolina 27706
Received January 22, 1980 Warm-reacting lymphocyte-binding IgG was measured in normal subjects and patients with systemic lupus erythematosus (SLE). A quantitative antiglobulin assay was used to measure both the amount of IgG present on autologous lymphocytes (L-IgG) and lymphocyte-binding IgG in serum (IgG-LBA). L-IgG of 12.9 ? 8.6 x lo-l4 g IgG was detected on washed normal lymphocytes. Fifty-three percent of patients with SLE had L-IgG greater than two standard deviations above that of normal. IgG-LBA of normal subjects (the amount of IgG detected on lymphocytes after incubation in normal serum) was 9.0 2 4.7 x lo-l4 g IgGkell. This did not differ significantly from the amount of IgG detected on lymphocytes prior to reincubation in normal serum. Forty-seven percent of patients with SLE had serum IgG-LBA of greater than two standard deviations above normal. L-IgG was elevated in all patients with increased IgG-LBA in whom L-IgG was measured. Protein fractionation studies including Sephadex G-200 gel filtration, DEAE ion-exchange chromatography, and pepsin digestion to produce F(ab’), fragments were performed upon serum that had elevated IgG-LBA. The IgG-LBA activity appeared to represent binding of F(ab’), portions of monomeric IgG molecules to lymphocytes. The IgG antibody from one patient was able to produce human complement-dependent lymphocytotoxicity when present in high concentration (greater than 60 x lo-l4 g/cell). Thus, some patients with SLE have warm reactive antilymphocyte IgG present both in their serum and on their own lymphocytes. We are unable to define the clinical sequelae of this form of humoral autosensitization.
INTRODUCTION
Numerous abnormalities in immune function and regulation have been described in patients with systemic lupus erythematosus (SLE), including a variety of antibodies which react with lymphocytes (l-5). These antibodies may be either IgM or IgG, and optimally reactive at 4°C or at body temperature. Cold reactive complement-dependent IgM lymphocytoxins, detected by microcytotoxicity assays, appear to be most frequent and have been extensively characterized (6, 7). In contrast, human autoantibodies directed against red cells, platelets, and granulocytes are usually IgG and reactive at body temperatures (8- 11). Quantitative immunoassays of IgG binding to these other blood cells have proven useful in understanding the pathophysiology of humoral autosensitization, which frequently occurs in systemic lupus erythematosus (9- 11). Although warm reactive IgG antilymphocyte antibodies have been described in SLE, a number of questions regarding these antibodies remain. Using microcytotoxicity and immunofluorescent techniques, Winfield er al. found only an occasional SLE patient with IgG antilymphocyte antibodies (6). Steinberg and 515 0090-1229/80/120515-15$01.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
516
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co-workers were also unable to detect IgG antilymphocyte antibodies using flow microfluoremetry (4). In contrast, Williams reported that 80% of SLE patients’ sera contained IgG antilymphocyte antibodies when tested with lz51-labeled protein A (5). Some studies have demonstrated noncytotoxic IgG antibodies directed against the responding cells in MLC reactions (12), while other studies suggest that these IgG antibodies are lymphocytotoxic (1). In addition, the T or B specificity of these antilymphocyte antibodies has not been defined (5, 12, 13). Most important, whether these antibodies react with antigens on a patient’s own cells and are, therefore, true autoantibodies, or whether IgG binding to lymphocytes may involve binding of IgG containing immune complexes by lymphocyte Fc receptors demands clarification (4, 5). The present study was therefore undertaken to further investigate these warm reactive IgG antibodies. In this report, we describe the results of an antiglobulin consumption assay to quantitate the amount of IgG present on lymphocytes from normal controls and patients with SLE. We also studied the amount of IgG present in normal and SLE serum that would bind to lymphocytes. We found that some SLE patients have markedly increased amounts of IgG antibody on their own lymphocytes as well as increased quantities of lymphocyte-binding IgG antibody in their serum. Characterization of this antibody revealed it to be monomeric IgG which bound to lymphocytes by its F(ab’), portion. These antibodies, in high concentrations, can produce complement-mediated cytotoxicity of human lymphocytes. The possible significance of such autoantibodies is discussed. METHODS Patients. Eighteen patients with SLE were studied (Table 1). All met the American Rheumatic Association’s diagnostic criteria for SLE (14). Normal control sera and peripheral blood lymphocytes were obtained from healthy laboratory and hospital personnel. Preparation of lymphocytes. Forty milliliters of heparinized whole blood were obtained by routine venipuncture, mixed with a 1/5th volume of plasmagel (HTI Corp., Buffalo, N.Y.), and allowed to sediment for up to 3 hr. The resulting leukocyte-rich plasma was collected and incubated with carbonyl iron powder (GAF Corp., N.Y.) for 15 min in a 37°C shaking water bath. The phagocytic cells were then removed by passing the leukocyte-rich plasma-iron mixture through a magnetic field (15). The leukocyte-rich plasma was then layered on Ficoll - Hypaque (Winthrop Laboratories, New York, N. Y., specific gravity, 1.078) and centrifuged at 1OOOgfor 20 min to obtain a purified lymphocyte preparation (15). The interface, containing greater than 95% lymphocytes, was harvested, washed three times with veranol-buffered isotonic saline (VBS), pH 7.2, and resuspended to a concentration of 1 x 107/ml in VBS. Lymphocytes were fractionated into B- and T-cell subpopulations using techniques previously described (16, 17). B cells were obtained by incubating neuraminidase-treated sheep red blood cells with lymphocytes followed by centrifugation over a Ficoll-Hypaque gradient. E-rosetted cells separated into the pellet and the interface containing nonrosetted cells (T cell depleted) was collected. T cells were obtained by filtering lymphocytes over a nylon wool column.
IgG ANTILYMPHOCYTE
AIJTOANTIBODIES
IN SLE
517
The B- and T-cell subpopulations were washed three times in VBS and resuspended to the appropriate concentrations. The purity of the enriched B- and T-cell suspensions were determined by the percentage of cells bearing surface immunoglobulin and the percentage of cells which form rosettes with sheep erythrocytes. The percentage of lymphocytes with detectable surface immunoglobulin was measured by fluorescent microscopy using fluorescein-conjugated polyvalent goat anti-human immunoglobulin (18). The fraction of lymphocytes forming rosettes with sheep erythrocytes was determined by previously described methods (19). Preparation of immunologic reagents. Antibody to human IgG was made by injecting a mixture of purified IgG subclasses 2, 3, and 4 with Freund’s adjuvant into rabbit foot pads (9, 10). Serum was absorbed with sheep and human red blood cells as well as with K and A light chains. The absorbed serum did not react with human serum components other than IgG when tested by immunoelectrophoresis against whole human sera. Purified human IgG was obtained by DEAE ionexchange chromatography of serum from a patient with IgG subclasses Gl multiple myeloma. IgG-coated sheep erythrocytes were prepared by incubating 0.5 ml of sheep erythrocytes with 0.07 ml of 0.8% glutaraldehyde and 0.25 ml of purified IgG for 60 min at room temperature (10). The sheep red blood cells were then washed three times in VBS and adjusted to a concentration of 4.4 x 10Yml. Determination of lymphocyte-bound ZgG (L-ZgG): Direct assay. Lymphocytebound IgG was measured using a quantitative antiglobulin consumption technique similar to one previously described for platelets and granulocytes (9- 11). This assay and the assay for serum lymphocyte-binding antibody are outlined in Fig. 1. The surface IgG may be quantitated by the adsorption of a known amount of rabbit anti-human IgG during incubation with lymphocytes. The amount of antiIgG remaining is measured by lysis of IgG-coated sheep erythrocytes with an excess of guinea pig serum as a source of complement. One-tenth-milliliter duplicate serial twofold dilutions of lymphocytes or dilutions of known concentrations of human IgG were mixed with 0.1 ml of diluted rabbit anti-human IgG for 30 min at 37°C. One-tenth milliliter of IgG-coated sheep erythrocytes was then added to the mixture and allowed to react for 30 min at 37°C. This was followed by the addition of 0.2 ml of guinea pig serum diluted 1:20 with VBS. This final mixture was incubated for 30 min at 37°C following which 5.0 ml of VBS was added. The mixture was then centrifuged and the percentage of sheep cells lysed was measured spectrophotometrically at 412 nm. Standard curves were constructed by determining the percentage of inhibition of hemolysis by known amounts of IgG. When increasing concentrations of purified human IgG were assayed, progressive inhibition of lysis occurred. The result of such a standard curve is shown in Fig. 2. By referring to this curve, it is possible to ascribe an absolute IgG value to the amount of inhibition produced by an unknown quantity of IgG in a sample. In this experiment, 34 x IOeg g of IgG was required to produce 50% inhibition of hemolysis. Such standard curves were done with each assay. TO calculate the amount of IgG present on lymphocytes, inhibition curves which related lymphocyte number of percentage inhibition of hemolysis were constructed. Lymphocytes from a normal individual were always studied on the
518
PACKER AND LOGUE
4occ WHOLE BLOOD
!95% LYMPHOCYTES 1 x IO’ CELLS/ml
/ IgG-LEA
I
!LhPLELIENT
I
DUPLICATE SERIAL 2-FOLD DILUTIONS
HEMOLYSIS
FIG. 1. Schematic representation of the methods used to quantitate lymphocyte antibodies. Lymphocyte-bound IgG (L-&G) represents the amount of IgG detected on an individual’s own lymphocytes. Lymphocyte-binding antibody (IgG-LBA) represents the amount of antibody in serum which will bind to normal lymphocytes. Both the lymphocyte-bound IgG and the lymphocyte-binding antibody are determined by the inhibition of hemolysis of IgG-coated sheep red cells caused by anti-IgG and complement.
same day as the patient’s lymphocytes. In Fig. 3, the results of such inhibition curves for a normal control, SLE patient K, and patient A with a marked increase in lymphocyte-bound IgG are shown. From these curves, the number of cells which produce 50% inhibition could be determined. By knowing the concentration of IgG required for 50% inhibition of lysis, the amount of lymphocyte-bound IgG could then be calculated. Determination of serum lymphocyte-binding antibody (ZgG-LBA): Indirect assay. The amount of antibody that would bind to normal lymphocytes was determined as outlined in Fig. 1. Three-tenths milliliter of serum was incubated with 0.3 ml of 1 x lO’/ml normal lymphocytes for 1 hr at 37°C. The mixture was washed three times with VBS at 37°C and the lymphocytes were counted by a Coulter counter (Coulter Diagnostics, Hialeah, Fla.). The amount of lymphocyte-bound IgG was determined as described above. In Fig. 4, the results of lymphocytebinding antibody at several serum dilutions is shown. At concentrations greater than 1:9, nonspecific binding of IgG from normal serum increased. All subsequent determinations were therefore performed at a 1:9 dilution. Serum fractionation. Sera from patients with SLE and normal controls were
IgG
ANTILYMPHOCYTE
AUTOANTIBODIES
100 I20 IgG (Gram x W9]
IN
I40
160
SLE
519
180
FIG. 2. Immunoglobulin G (IgG) calibration curve showing inhibition of anti-IgG-induced lysis of IgG-coated sheep erythrocytes when the anti-lgG is preincubated with increasing amounts of IgG.
fractionated using Sephadex G-200 gel filtration with 0.15 M saline at 4°C on a 3.0 x 90-cm column. Serum (2.5 ml) was passed over the column and representative fractions assayed for protein and lymphocyte-binding IgG. Normal and SLE sera were also fractionated by DEAE ion-exchange chromatography to obtain purified IgG. One-half milliliter of serum that had been dialyzed against 0.005 M phosphate buffer, pH 8.0, was applied to a 50 ml bed volume DEAE-cellulose column equili-
Lymphocytes
Added
per 0 I ml (x 10’)
3. Inhibition of anti-IgG-induced Iysis of IgG-coated sheep erythrocytes by lymphocytes from a normal subject and two patients with SLE. Those data can be used to calculate lymphocyte-bound IgG as described in the text. FIG.
520
PACKER
0 ’ ‘47
AND
‘4 SERUM
FIG. 4. Effect of dilution patients with SLE.
on lymphocyte-binding
LOGUE
‘4 DILUTION
antibody
of sera from
a normal
individual
and two
brated with the same buffer. IgG-containing fractions were then eluted with this buffer. To compare lymphocyte-binding antibody activity between samples from different patients, the IgG fractions from different patients were concentrated to equal protein concentrations as measured by the method of Lowery (20). Preparation of radiolabeled F(ab’), fragments. F(ab’), fragments were obtained by pepsin digestion of IgG-containing fractions from normal and SLE sera followed by gel filtration on Sephadex G-150 (21). The F(ab’)z fragments were then labeled with 1251by the Bolton-Hunter method (22). The radiolabeled F(ab’)2 fragments were then incubated with 0.1 ml of 3 x lO’/ml lymphocytes or control tubes without lymphocytes for 1 hr at 37°C. The tubes were washed three times with 37°C VBS and the pellets counted on a Beckman gamma counter. Complement-dependent lymphocytotoxicity method. The ability of these antibodies to lyse normal lymphocytes with human complement was investigated using a 51Cr release assay. One to two milliliters of 5 x lO’?ml lymphocytes was incubated with 100 to 300 &i Wr for 1 hr at 37°C and then washed three times in the McCoy’s 5A media (Grand Island Biological Co., Grand Island, N.Y.). Ten microliters of heat-inactivated test serum was incubated with 10 ~1 of 51Cr-labeled lymphocytes for 1 hr at 37°C in a microtiter plate (Limbro Scientific, Hamden, Conn.). One hundred microliters of dilutions of normal type compatible human serum as a source of complement was added and the mixture incubated for 4 hr at 37°C in a 5% CO, moist incubator. Following centrifugation of the microtiter plates at 800g for 10 min, 50 ~1 of supematant was aspirated and counted in a gamma counter. Percentage cytotoxicity was calculated by subtracting the supernatant counts in buffer controls from the test supernatant counts and dividing by
IgG ANTILYMPHOCYTE
AUTOANTIBODIES
IN SLE
521
the total releasable counts minus the buffer controls. Total releasable counts were determined by incubation of lymphocytes with 1% sodium deoxycholate followed by the addition of 100 ~1 of buffer and centrifugation. RESULTS Lymphocyte-Bound
ZgG (L-ZgG): Direct Assay
The amount of lymphocyte-bound IgG was determined on 17 normal controls and in 12 patients with SLE as shown in Fig. 5. Normal lymphocyte-bound IgG ranges from 2.3 to 35.4 x lo-l4 g/cell with a mean of 12.9 ? 8.6 x lo-l4 g/cell. Five of twelve SLE patients have levels of lymphocyte-bound antibody greater than two standard deviations above normal. In patient A, this represented three, and in patients C and E, two separate determinations on different days. Serum Lymphocyte-Binding
Antibody
(ZgG-LBA): Zndirect Assay
The results of the serum lymphocyte-binding antibody assay are shown in Fig. 6. IgG lymphocyte-binding antibody levels from 21 normal subjects ranges from 2.0 to 16.0 x lo-l4 g/cell with a mean of 9.0 ? 4.7 x lo-l4 g/cell. In sera of 8 of 15 patients with SLE, the lymphocyte-binding antibody level was greater than two standard deviations above normal. In patient A, the lymphocyte-binding antibody level was markedly elevated with a mean of 87.0 ? 42.6 x lo-l4 g/cell. These results represent 25 separate determinations on 15 different lymphocyte donors.
320 *A
:%
240 T 160 1 h i so: 0 x zD z 0 ,D
. B
.C
60.D .E
9 i3
g
40-
* c . E
? 2 t,
20-
t i T
0
NORMAL
SLE
5. Range of values for lymphocyte-bound IgG (L-IgG) in normal subjects and patients with SLE. Letters refer to separate determinations in patients with lymphocyte-bound IgG greater than two standard deviations above normal. The mean and standard deviations for normals are shown. FIG.
522
PACKER
AND
LOGUE
,
i
Oi
NORUSS
SLE
pA’;EN’
FIG. 6. Range of values of lymphocyte-binding antibody (IgG-LBA) in normal subjects and patients with SLE. In patient A, values represent separate determinations on 15 different donor lymphocytes. The mean and standard deviations for normals are shown.
Patient A’s lymphocyte-binding antibody was elevated with all normal donor lymphocytes tested. Since this antibody reacted with all donor cells tested, it seemed unlikely that this antibody was directed against isologous antigens such as those of the HLA system. To further investigate the possibility that this antibody was directed against antigens of the HLA system, serum from patient A was absorbed extensively against pooled platelets from 150 different platelet donors (6). Following absorption, elevated levels of lymphocyte-binding antibody persisted, suggesting that this antibody was not directed against HLA antigenic determinants. To be certain that the increased amount of antilymphocyte antibody seen in these patients was not due solely to increased plasma concentrations of IgG, sera from two patients with multiple myeloma were studied. Despite markedly increased serum IgG concentrations, myeloma patients’ sera had normal levels of lymphocyte-binding antibody. In Table 1, the results of the lymphocyte-bound IgG and lymphocyte-binding antibody assays are tabulated for individual patients. Except for three patients with elevated lymphocyte-binding antibody whom we were unable to test, all patients with increased lymphocyte-binding antibody levels had elevated amounts of IgG present on their own washed lymphocytes. The reactivity of IgG-LBA with subpopulations of lymphocytes was studied. Sera from two SLE patients with elevated IgG-LBA and from a normal subject were tested with normal B- and T-lymphocyte preparations. The results of this study are shown in Table 2. The mean and range of duplicate determinations are shown. The IgG-LBA of both patients was detected on both B and T lymphocytes.
IgG
ANTILYMPHOCYTE
BLOOD
LEUKOCYTE
AUTOANTIBODIES
TABLE COUNTS
ANTIBODY
Patient
Total leukocyte count (cells/mm3)
A
2,400
B C D E F G
6.00’3
RESULTS
OF LYMPHOCYTE
IN SLE
QUANTITATION
PATIENTS
Lymphocyte-binding antibody (IgG-LBA x lo-” g/cell)
lymphocyte count (cells/mm3)
523
SLE
1
AND
Absolute
3,ooo 6,300 3,200 4,100 4,950 9,000 4,900 5,500 12,000 7,700 6,200 10,800 6,500 3,700 3,000
IN
336 800 780 630 1,376 920 1,100 810 931 2,255 840 1,070 800 3,780 1,200 1,295 990
81.9 k 42.4" 42.8 46.8 20.0 55.7 35.8 12.5 10.5 44.3 10.0 ND 11.4 13.9
Lymphocyte-bound antibody (L-IgG x lo-” g/cell)
+ 17.1b 130.2 53.5 55.8 41.0
262.6
ND’ 10.0 18.4
ND 14.3 15.5 12.0 9.0 3.0
ND 10.0
ND ND ND
5.0 20.4
u Results from 25 determinations. 0 Results from three determinations. c Not done.
The antibody of both patients reacted somewhat more strongly with B cells than with T cells as shown in this table. Serum Fractionation
Serum fractionations were done to determine whether the elevated levels of lymphocyte-binding antibodies were due to monomeric IgG, aggregated IgG, or immune complexes containing IgG. Serum from patient A was chromatographed on Sephadex G-200. In Fig. 7, the fraction number is plotted against the absorbance at 280 nm on the left ordinate and against the IgG-LBA on the right ordinate. Lymphocyte-binding antibody activity corresponded to monomeric IgG; there was no significant activity in void volume fractions where large molecular size immune complexes or aggregated IgG would be expected. To further confirm that the lymphocyte-binding antibody represented IgG alone, serum from patient
TABLE RESULTS
OF LYMPHOCYTE-BINDING
2
ANTIBODY
ON B AND
Lymphocyte
binding
(IgG-LBA,
Cells tested
Patient
B T n B- and T-enriched and range of duplicate
A
122.7 + 0.6 43.3 + 0.7 lymphocytes determinations.
were
prepared
x
IO-l4
Patient
E
57.0 27.0
as described
T LYMPHOCYTESO
antibody g/cell)
2 7.3 k 4.9 in the text.
Normal + 4.2 5.2 + 0.9
14.4
The results
are the mean
524
PACKER
AND
LOGUE
A and from a patient with normal amounts of lymphocyte-binding antibody (patient H) were fractionated by DEAE ion-exchange chromatography. The purified IgG was concentrated and confirmed by immunodiffusion against antisera to IgG, IgM, and whole serum. The protein concentrations of the samples from the two patients were equalized and assayed for lymphocyte-bound IgG as shown in Fig. 8. With equal amounts of IgG added, IgG-LBA of purified IgG from patient A was greater than that from patient H. Thus, results from G-200 gel and DEAE ionexchange chromatography suggested that this lymphocyte-binding antibody was monomeric IgG alone. To be certain that the lymphocyte-binding antibody activity was an antibody-antigen reaction rather than nonspecific binding of IgG by Fc receptors, we measured the binding of F(ab’)z fragments from SLE serum to lymphocytes. 1251-labeled F(ab’)z fragments from patient A and a patient with normal amounts of lymphocyte-binding antibody (patient H) were studied. As shown in Fig. 9, lymphocyte binding of patient A F(ab’)z was greater than for that of patient H. Thus, the serum from patient A appeared to contain antibodies directed against lymphocyte antigens. Human Complement-Dependent Lymphocytotoxicity These antibodies were tested for their ability to cause human complementdependent cytotoxicity. The results of the 51Cr release assay using various mixtures of serum is shown in Table 3. The mixture of heated patient A serum plus normal serum was lymphocytotoxic. When the normal serum was heated to remove its complement activity, lymphocytotoxicity was abolished. Thus, the antibody in this patient appeared to support human complement-dependent lymphocytotoxicity. The relationship between lymphocyte-binding antibody and lymphocytotoxicity was then examined. In Fig. 10, lymphocytotoxicity is plotted against lymphocyte-binding antibody. Approximately 65 x lo-l4 g/cell of IgG was re-
FRACTION
7. Results of Sephadex between protein concentration body is shown. FIG.
NUMBER
G-200 gel chromatography as estimated by absorbance
of serum from patient A. The relationship at 280 nm and lymphocyte-binding anti-
IgG ANTILYMPHOCYTE
AUTOANTIBODIES
IgG Concentrolton
Added
IN
SLE
525
jmg/ml)
8. Results of lymphocyte-binding antibody of DEAE-purified IgG. IgG was purified as described in the text. The results are shown for a patient with elevated whole serum IgG-LBA (patient A) and a patient with normal IgG-LBA (patient H). FIG.
quired to initiate lysis following which there was a dose-response between lymphocyte-binding antibody and lymphocytotoxicity.
relationship
DISCUSSION
Both IgG and IgM antibodies directed against lymphocytes are frequently present in patients with SLE. Cold reactive complement-dependent IgG antibodies
Flab):,
odded
(fig/ml)
FIG. 9. Results of binding of ‘251-labeled F(ab’), fragments to normal lymphocytes. Patient A had increased amounts and patient H normal amounts of lymphocyte-binding antibody as detected by the antiglobulin consumption assay.
526
PACKER AND LOGUE TABLE
3
COMPLEMENT-DEPENDENTLYMPHOCYTOTOXICITY~
Lymphocyte incubation mixture Heated patient A serum unheated normal serum Heated patient A serum + heated normal serum Heated normal serum + unheated normal serum
Lymphocytotoxicity (% Yr release) 23.0 zt 0.3 0.3 + 0.3 3.1 k 1.5
o Wr-labeled normal lymphocytes were incubated with mixtures of patient A and normal serum and lymphocytotoxicity determined as described in the text. The mean and standard deviation of triplicate determinations are shown.
have been extensively characterized. Data regarding IgG lymphocytic antibodies have been somewhat conflicting. Stastny and Ziff initially demonstrated a complement-dependent cytotoxic effect from serum of patients with SLE, for both ahogeneic and autologous lymphocytes (7, 23). They suggested that the antilymphocyte antibodies in SLE were IgG and maximally cytotoxic at physiologic temperatures. However, observations by Terasaki and later Winfield and co-workers demonstrated that the antilymphocyte antibodies detected by microcytotoxicity assays were, in fact, cold reactive and primarily IgM in class (2, 6). Other investigators have reported noncytotoxic antilymphocyte IgG that is active at physiologic temperatures and that suppresses mixed lymphocyte culture reactions (12). Williams and co-workers, using a 1251-labeled protein A, have further characterized warm reactive antilymphocyte IgG in SLE (5). They did not, however, investigate whether these patients’ own cells had elevated amounts of IgG. Glinski et al. have demonstrated anti-T-cell lymphocyte antibodies which appear to be of the IgG class (13). On the other hand, Steinberg and co-workers, using flow microfluorimetry, demonstrated anti-T-cell antibodies that were IgM in class, but were unable to demonstrate IgG antibodies (4). Results of this study clearly demonstrate the occurrence of warm reactive antilymphocytic IgG both in the serum and on the cells of some patients with SLE.
FIG. 10. The relationship between lymphocyte-binding lymphocytotoxicity is shown for patient A’s serum.
antibody and human complement-dependent
IgG
ANTILYMPHOCYTE
AUTOANTIBODIES
IN
SLE
527
Utilizing a quantitative antiglobulin consumption assay, we measured the amount of warm reactive lymphocyte-binding IgG present in the serum as well as the amount of IgG on the patient’s own lymphocytes. Fifty-three percent of patients had elevated serum levels of lymphocyte-binding IgG. In one patient, this represented 25 determinations performed on 15 different normal lymphocyte donors. In addition, IgG-LBA activity persisted after repeated absorption with pooled platelets from 150 different donors. This is an agreement with previous work indicating that antilymphocyte antibodies in SLE are directed against non-HLA determinants. We also investigated the subclass of lymphocytes against which these IgG antibodies were directed. Prior reports of either B- or T-cell specificity have been conflicting. In contrast to Glinski and co-workers, who reported IgG antibodies specific for T cells, Williams et al. found equal reactivity for both lymphocyte populations (5, 13). In our study, the IgG antibody bound to both B and T cells. In addition, we were able to directly measure IgG on these patients’ own lymphocytes. Forty-two percent of SLE patients tested had elevated amounts of lymphocyte-bound IgG. All the patients with elevated serum lymphocyte-binding IgG who were tested also had increased amounts of IgG present on their own lymphocytes. It would thus appear that the serum antibody reacted with the patients’ own lymphocytes. Other investigators, using other techniques, have also found antibodies on the lymphocytes of patients with SLE (7, 13). To confirm that this reactivity represented antibody-antigen interactions rather than nonspecific binding of IgG to lymphocyte Fc receptors, serum fractionation studies were performed. Serum was chromatographed on G-200 gel filtration and then assayed for lymphocyte-binding IgG. The IgG-LBA activity corresponded to monomeric IgG and there was no activity in the void fractions where immune complexes or aggregated IgG would be expected. Further confirmation that this activity was not due to immune complexes was obtained when purified IgG, prepared by DEAE ion-exchange chromatography, from a patient with elevated IgG-LBA was compared to that from a patient with normal IgG-LBA. Again, it appeared that the IgG-LBA activity resided in the IgG portion of serum. Finally, F(ab’), fragments were obtained by pepsin digestion of purified IgG. The lz51labeled F(ab’), fragments from the patient with elevated IgG-LBA bound to normal lymphocytes. It therefore appears that the reactivity seen in the antiglobulin consumption assay indeed represents an antibody-antigen reaction rather than nonspecific binding to lymphocyte Fc receptors. While warm reactive IgG autoantibodies directed against red cells, platelets, or granulocytes appear to cause destruction of these cells in some patients, we were unable to define the clinical consequences of this warm reactive antilymphocyte IgG. Although Winfield et al. and Butler et al. were able to correlate the level of antilymphocyte antibodies with lymphopenia, the microcytotoxicity assay they used detects predominantly cold reactive IgM antibodies (25,26). Statsny and Ziff initially suggested that there were warm reactive IgG antibodies in SLE capable of functioning in viva by direct complement-mediated lymphocytotoxicity (7). Since we are able to directly measure lymphocyte-bound antibody, we could define the absolute amount of IgG per lymphocyte necessary to cause human complement-
528
PACKER AND LOGUE
dependent cytotoxicity. A 65 x lo-l4 g quantity of IgG per lymphocyte was necessary to initiate lysis using human serum as a source of complement. Since only one patient bound antibody in this range, the ability of these antibodies to produce in viva complement-dependent cytotoxicity is questionable. It is of interest that the patient with lymphocyte-bound IgG in this range was severely lymphopenic (patient A). Otherwise, we are unable to show a correlation between IgG lymphocyte autoantibodies and lymphopenia in this group of SLE patients. This lack of correlation is not surprising since, as pointed out by others, a variety of different mechanisms may cause lymphopenia in SLE (25). The significance of such lymphocyte autoantibodies in the pathogenesis of SLE remains unclear. Lymphocytotoxins have been described in a number of diseases characterized by immune abnormalities suggesting that they may be secondary phenomena. In the rodent model of SLE, however, there is evidence that lymphocyte antibodies cause a loss of suppressor T cells, resulting in generalized B-cell hyperactivity (27, 28). Recent studies have shown that subsets of T cells, both T, and T,, can suppress differentiation of B lymphocytes (29, 30). In addition, Sagawa and Abdou have suggested that an IgG antilymphocyte antibody directed against suppressor cells is responsible for the B-cell hyperactivity in SLE (31). However, in this study, many patients with active SLE did not appear to have warm reactive IgG antilymphocyte antibodies. Patients who had neither increased IgG on their own lymphocytes nor increased lymphocyte-binding antibody in their serum had classic systemic lupus erythematosus with severe disease manifestations. Thus, it seems clear to us that the finding of IgG antilymphocyte autoantibodies in these patients is a manifestation rather than a cause of systemic lupus erythematosus. ACKNOWLEDGMENTS This work was supported by the Research and Education Branch of the Veterans Administration. The authors thank Ms. Barbara Connor for excellent technical assistance and Ms. LaVerne Johnson for help in preparation of the manuscript.
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