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Surface antigen specificity of cold-reactive IgM antilymphocyte antibodies in systemic lupus erythematosus.

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44
SURFACE ANTIGEN SPECIFICITY OF
COLD-REACTIVE IgM ANTILYMPHOCYTE
ANTIBODIES IN SYSTEMIC LUPUS
ERYTHEMATOSUS
AKIRA YAMADA, MELODY SHAW, and JOHN B. WINFIELD
Monoclonal antibodies to known surface antigens
on B cells and on resting and activated T cells of various
types were used in several approaches to examine the
specificity of IgM antilymphocyte antibodies in systemic
lupus erythematosus (SLE). Surface determinants that
were sought included: T3, T11, Leu-1, Leu-8 (pan-T);
T4, T8 (T subset); P2-microglobulin(Pzm); L243, Leu10 (DR and DS/DC framework, respectively); anti-Tac
(interleukin-2 receptor); 5E9 (transferrin receptor); and
4F2, AA1 (other activation antigens). The first strategy
was based on inhibition of rosette formation between
mouse monoclonal antibody-coated targets and antimouse IgG-coated erythrocytes by SLE sera, either
directly at 4°C or after modulation of IgM antilymphocyte antibody-reactive target cell antigen at 37°C. Significant rosette inhibition, defined as >2 standard deviations from the mean value for 10 control sera, was seen
only for P2m (13of 20 SLE sera were positive; inhibition
= 1548%). Next, relative fluorescence intensity of lymphocyte staining by monoclonal antibodies was assessed
by flow microfluorometry after preincubation of cells
with SLE serum at 4°C or after modulation of SLE
From the Division of Rheumatology and Immunology,
University of North Carolina at Chapel Hill, School of Medicine,
Chapel Hill, North Carolina.
Supported in part by NIH grant RO1 AM30863, a Multipurpose Arthritis Center grant, and an Arthritis Foundation Clinical
Research Center grant.
Akira Yamada, MD: Fellow in Rheumatology; Melody
Shaw, BS: Research Associate; John B. Winfield, MD: Professor of
Medicine.
Address reprint requests to John B. Winfield, MD, Division
of Rheumatology and Immunology, 932 FLOB, 231H, University of
North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC
27514.
Submitted for publication April 9, 1984; accepted in revised
form July 26, 1984.
Arthritis and Rheumatism, Vol. 28, No. 1 (January 1985)
antibody-reactive antigen. Modulation markedly reduced or eliminated SLE antilymphocyte antibody IgM
staining. Except for Pzm, neither cold nor warm temperature preincubations altered the relative fluorescence intensity for the known surface antigens. These
data confirm anti-P2m as a common antibody specificity
in SLE and suggest that antilymphocyte antibodies in
this disorder are not directed to Ia or to certain other
defined lymphocyte antigens of functional interest.
A variety of functional effects have been attributed to lymphocyte autoantibodies in systemic lupus
erythematosus (SLE). Examples of these include reduced concanavalin A-generated suppressor T cell
activity, reduced T cell proliferation to various specific and nonspecific stimuli, inhibition of natural killer
cell activity, suppression of antibody-dependent cellmediated cytotoxicity , inhibition of the generation of
alloreactive cytotoxic T cells, and enhancement of B
cell immunoglobulin secretion (reviewed in 1). Antilymphocyte antibodies also have been implicated in
lymphocyte depletion in vivo in this disorder, especially with respect to T cells and T cell subsets (1). For
these reasons, molecular characterization of the determinants on the cell membrane to which different types
of SLE antilymphocyte antibodies are directed has
been a pressing issue for some time. Despite the
considerable effort expended in various laboratories, a
remarkable lack of progress has been made in this
area. A principle obstacle to the characterization of
these determinants is the low avidity and low titer of
many of the most interesting antibodies, which greatly
limits their utility as probes for immunoprecipitation
and analysis of radiolabeled surface molecules. Indeed, with the exception of a single early effort in
ANTIBODIES IN SLE
whiclh the investigators identified a low molecular
weight glycoprotein on T cells involved in the allogeneic imixed leukocyte reaction (MLR) (2), this type of
approach has been unsuccessful.
The available information has been derived
from experimental strategies using antibody absorption or inhibition techniques, such as absorption of
SLE serum with purified antigen (3), inhibition of SLE
seruni cytotoxic activity by precoating target cells
with F(ab’)z antibody fragments (4), the lysostrip
and rosette inhibition using monoclonal
technique (3,
antibodies to known antigens (6).The latter method is
very sensitive and involves precoating target cells with
SLE antilymphocyte antibody, followed by enumeration of rosettes formed between the target cells in the
presence of monoclonal antibody of defined specificity
and erythrocytes coated with anti-mouse immunoglobulin.
In this investigation, we combined rosette inhibition, indirect immunofluorescence, and flow microfluorometry procedures in a series of experiments to
identify SLE serum antibodies directed to 13 surface
antigens defined by monoclonal antibodies. We took
special advantage of the capacity of SLE IgM antibody-reactive antigen to be modulated by brief incubation of target cells in SLE serum at 37°C.
MATERIALS AND METHODS
Patients and sera. Peripheral blood was obtained
from patients with SLE who met the revised American
Rheumatism Association diagnostic criteria (7) and from 10
norm,al subjects. After separation from whole blood, serum
was aliquoted and stored at -70°C. Each serum aliquot was
heated at 56°C for 30 minutes immediately before use. SLE
disease activity was assessed as described previously (8).
Lymphocytes. Normal human peripheral blood
mononuclear cells (PBMC) were separated from heparinized
venous blood by Ficoll-Hypaque flotation. T cells were
obtained by passage of PBMC over nylon wool columns
(Fenwall Laboratories, Berkeley, CA) and collecting the
nonatlherent population, according to the technique of Julius
et a1 (9). Nylon wool-adherent cells, containing enriched
numbers of B cells and monocytes, were removed by
wringing the nylon wool at cold temperatures. Markers used
to assess purity of these enriched cell populations included
esterase staining of monocytes and indirect immunofluorescence or complement-dependent cytotoxic reactivity with
monoclonal antibodies to Ia, IgM, and pan-T determinants.
Activated T cells were obtained by culturing PBMC
for 3 days in tissue culture flasks (Falcon, Oxnard, CA), at a
density of lo6 cells/ml, in RPMI 1640 medium (University of
North Carolina Cancer Center, Chapel Hill, NC) containing
1 w ‘ m l phytohemagglutinin (PHA; Burroughs Wellcome,
Research Triangle Park, NC), 2 mM glutamine, antibiotics,
and 10% fetal calf serum (FCS; Hyclone, Logan, UT) in a
45
humid 5% COz, 95% air incubator. Chronic lymphocytic
leukemia (CLL) cells were used in certain experiments.
Microcytotoxicity. Two-stage, complement-dependent microcytotoxicity assays using prescreened rabbit complement (C; Pel-Freez Biologicals, Rogers, AR) were performed as previously described (10). Assay temperatures of
15°C were used to detect cold-reactive antibodies, and
temperatures of 25°C were used to distinguish warm-reactive
anti-T blast antibodies (11). Cytotoxicity, expressed as the
percentage of nonviable cells failing to exclude eosin dye,
was assessed by inverted phase-contrast microscopy. Cytotoxicity with C alone or with C plus normal serum was
<lo%.
Special immunologic reagents. Monoclonal antibodies
used included the following: L243 (anti-DR framework;
American Type Culture Collection, Rockville, MD); antiTac (a generous gift from Dr. T. Waldmann, NIH, Bethesda,
MD); Leu-1, Leu-8, Leu-10, anti-b-microglobulin (antib m ; Becton-Dickinson, Sunnyvale, CA); OKT3, OKT4,
OKT8, OKTll (Ortho Diagnostics, Raritan, NJ); 5E9 (antibody to transferrin receptor; a generous gift from Dr. B.
Haynes, Duke University, Durham, NC); AA1 (anti-T blast;
a generous gift of Dr. N. Chiorazzi, Rockefeller University,
New York, NY); fluorescein isothiocyanate (FITCNonjugated F(ab’)2 goat anti-human IgM; and FITC-rabbit antimouse IgG (Cappel Laboratories, Cochranville, PA). Chicken and rabbit anti-human Ia antibodies were gifts of Dr. N
Bernard, University of North Carolina, Chapel Hill, NC.
Monoclonal antibody rosettes and rosette inhibition.
The monoclonal antibody rosette assay was performed according to the method of Stocker et a1 (12), using goat antimouse IgG-coated bovine red blood cells (BRBC; Biological
Products, Winston-Salem, NC) as indicator cells. Two hundred microliters of 50% packed BRBC and 100 pl of affinitypurified goat anti-mouse IgG (800 pg/ml; a gift from Dr. R.
Eisenberg, University of North Carolina, Chapel Hill) were
mixed, and 200 pl of 2.5 mM chromic chloride was added
dropwise (13). After incubation of the reaction mixture for 5
minutes at room temperature, anti-IgG-coated BRBC were
washed thoroughly and resuspended at 1% in RPMI 1640
supplemented with 10% FCS.
Fifty microliters of a suspension of target lymphocytes (2.5 x 10’) was incubated with 50 pl of appropriate
dilutions of monoclonal antibody for 30 minutes at 4°C. After
washing, target cells were suspended in RPMI 1640, 10%
FCS, to a final density of 4 x lo6 ml, combined with an equal
volume (usually 50 pl) of a 1% suspension of anti-mouse
IgG-coated BRBC, and centrifuged for 10 minutes at 200g in
small tubes (Fisher Scientific, Pittsburgh, PA). After incubation in ice water for 30 minutes, the BRBC/lymphocyte
mixture was gently resuspended, stained with Giemsa, and
applied to glass microscope slides for microscopy to determine the percentage of rosette-forming cells. At least 200
cells were counted in each assay.
Antibodies to surface determinants recognized by
the various monoclonal antibodies were sought in the SLE
sera by rosette-inhibition assays. Target lymphocytes (1 x
lo6) were preincubated with 100 pl of SLE serum or normal
serum for 1 hour at 4”C, with frequent agitation. After
pelleting and aspiration of serum, the cells were resuspended
in the highest dilution of monoclonal antibody giving near-
YAMADA ET AL
46
maximal rosette formation, and rosettes were prepared and
enumerated as described above. Inhibition was calculated as
follows: % inhibition = [(a - b)hl x 100, where a = %
rosettes with normal serum preincubation, and b = %
rosettes with SLE serum preincubation.
The major surface antigen(s) on T cells with which
cold-reactive SLE antilymphocyte antibodies react can be
modulated (i.e., exhibit decreased fluorescence intensity for
SLE IgM staining) by brief preincubation of cells in SLE
serum at 37°C (14). Therefore, monoclonal antibody rosette
inhibition was also examined in experiments where the
target cells were preincubated with SLE serum at 37°C for 2
hours prior to rosette assay.
Indirect immunofluorescence and flow cytometry. IgM
and IgG antibody binding to T cells in SLE serum was
assessed by indirect immunofluorescence at 4°C using FITCconjugated rabbit F(ab‘)* antibodies to human p or y chains
as the second coat. Appropriate dilutions of mouse monoclonal antibodies and FITC-goat F(ab’)Z anti-mouse IgG were
used to determine known surface antigens. After staining,
cells were kept at 4°C and were examined immediately or
were fixed with paraformaldehyde. Analysis of fluorescence
intensity (log-integrated green fluorescence) and % positive
cells were determined by flow microfluorometry using an
Epics V instrument (Coulter Electronics, Hialeah, FL)
equipped with argon laser (488 nm), MDADS and EASY 1
computers, and appropriate software. Laser power, photomultiplier tube amplification, and gain settings were calibrated using fluorospheres (Coulter) and kept constant throughout the experimental run on a given lymphocyte preparation.
In some experiments, attempts were made to block or
modulate known determinants with SLE serum prior to
staining with mouse monoclonal antibodies.
RESULTS
In initial experiments, sera from patients with
SLE were screened for antibodies to different types of
lymphocytes using C-dependent microcytotoxicity
and indirect immunofluorescence assays. Of 20 sera
with antilymphocyte antibody activity, 13 contained
cold-reactive IgM cytotoxic antibodies against resting
T cells (mean % SD dead cells = 56 & 24). Sixteen
sera were cytotoxic for B cells (mean % t SD dead
cells = 47 ? 23). Relatively warm-reactive IgM antibodies to 3-day PHA-activated T blasts were detected
in 7 sera (mean % ‘r_ SD dead cells = 55
26) in
cytotoxicity assays performed at 25°C. None of these
sera gave detectable IgG staining of the different
targets. This panel of 20 sera was then used in subsequent experiments to identify IgM antibody reactivity
against a series of known cell surface determinants.
Direct rosette inhibition. The monoclonal antibody specificities and the different types of target cells
used in rosette-inhibition assays are given in Table 1 .
Also shown is the mean percent of rosette-forming
cells observed in the presence of standard normal AB
serum, as well as the variation about this mean when
sera from 10 normal individuals were substituted for
the standard AB serum in the assay. Figure 1A illustrates the relationship between rosette formation and
monoclonal antibody dilution. From preliminary titrations of this sort, a working dilution for each monoclonal antibody was chosen to maximize inhibitability of
rosette formation by test sera. Figure 1B shows a
typical direct rosette inhibition curve. In this experiment, target cells were preincubated with varying
dilutions of rabbit anti-human Ia antiserum before
addition of L243, a monoclonal antibody to human Ia.
Thirteen specificities identified by monoclonal
*
*
Table 1. Monoclonal antibody specificities and target cells studied
% rosette-forming
cells*
Monoclonal
antibody
Specificity (ref.)
Target
cell
Mean
L243
Leu-10
aP2m
OKT4
OKT8
OKT3
OKTl1
Leu-1
Leu-8
5E9
4F2
Tac
AAl
DR framework (28)
DSlDC framework
&microglobulin
HLA class I1 receptor (29)
HLA class I receptor (30)
T cell antigen receptor (31)
E receptor (18)
Pan-T
Pan-T
Transfemn receptor (32)
Activation antigen (33)
Interleukin-2 receptor (34)
Activation antigen (35)
CLLt
CLL
CLL
Resting T
Resting T
Resting T
Resting T
Resting T
Resting T
T blast
T blast
T blast
T blast
13
52
I1
42
20
72
64
53
53
72
88
63
30
?
2 SD
61-85
42-62
61-81
34-50
16-24
60-84
52-76
43-63
44-62
61-83
63-95
54-12
23-31
* Mean % rosette-forming cells in the presence of standard normal AB serum, and the variation about
this mean when substituting 10 normal sera in the rosette inhibition assay (determined as % of total
lymphocytes).
t CLL = chronic lymphocytic leukemia.
47
ANTIBODIES IN SLE
t
I
I
Po-2
I
I
10-3
I
I
10-4
I
I
1
L
10-5
Dilution of L243
10-6
"
15
110
150
11001200
Dilution of Robbit Anti-DR
Figure 1. Monoclonal antibody rosette formation and rosette inhibition. A, Chronic lymphocytic leukemia (CLL) cells were incubated with various dilutions of L243, a monoclonal anti-DR framework
antibody, washed, and then combined with anti-mouse IgGxoated
bovine erythrocytes. The arrow indicates the dilution of L243
chosen for use in rosette inhibition assays. B, A typical rosette
inhibition assay. Here the CLL targets were preincubated with
rabbit anti-DR framework antiserum prior to addition of L243 and
antimouse IgG-coated erythrocytes.
antibodies were sought in direct rosette inhibition
assays performed at 4°C with the 20 SLE sera (Figure
2). Thirteen of 20 sera inhibited anti-p2m rosettes
(range 15-58%). Rosette inhibition was rarely observed with the other monoclonal antibodies. This was
true even when the procedure was modified to allow
continuous presence of SLE serum (data not shown).
The failure to detect rosette inhibition with L243
rosettes was unexpected since, with this technique,
others have identified anti-Ia antibodies directed to the
L243 DR framework epitope (6).
To further examine the question of anti-Ia
specificities in the SLE sera, an additional series of
experiments was performed. First, T cell blasts were
used in place of CLL cells as targets in the rosette
inhibition assay, but no inhibition was observed (mean
% inhibition k SD = 3 ? 8; % control L243 rosetteforming cells = 14). Second, the anti-Ia rosette assay
with CLL targets was modified for use with rabbit
anti-DR heteroantiserum by substituting goat antirabbit IgG Fc for anti-mouse IgG as the coating for the
BRBC indicator cells. In this system, L243 at a
1 : 1,000 dilution inhibited rosette formation -40%
(85% + 51%), but inhibition by the SLE sera was
insignificant (mean ? SD = 1 2 5%). Third, the
presence of anti-Ia antibody was sought in an assay
based upon inhibition of C-mediated cytotoxicity using
chicken anti-Ia antibody, which does not fix C. Preincubation of nylon wool-purified non-T cells (85% Ia+)
with chicken anti-Ia completely prevented killing by
L243, but had no effect on cytotoxicity of the SLE sera
.
0
c
..-
t
n
Y
r
-c
401
..
...
.
-
L243
a&m
OKT8
OKTll
Leu 8
4F2
AA1
Leu 10
OKT4
OKT3
Leu 1
5E9
Tac
Monoclonal Antibodies
Figure 2. Rosette inhibition by systemic lupus erythematosus (SLE) sera. Target cells were preincubated with SLE serum before
performance of the monoclonal antibody rosette assay. Monoclonal antibody specificities and target cells used are shown in Table
1. The horizontal bars represent 2 SD above the mean % inhibition observed with a panel of 10 control sera (within normal limits).
With rare exceptions, significant inhibition was found only for p2-microglobulin (&m).
YAMADA ET AL
48
(Table 2). This latter experiment does not exclude the
presence of anti-Ia antibodies in SLE sera, but indicates that the predominant anti-B cell specificity is not
directed to Ia.
Finally, attempts were made to block indirect
immunofluorescent staining by L243 by preincubating
T cell blasts with SLE serum at 4°C. Neither the
percentage of positively stained cells, nor the relative
fluorescence intensity of L243 staining was decreased
relative to control preparations preincubated with normal AB serum (data not shown). Inhibition of monoclonal antibody immunofluorescence staining for Tac,
OKT3,OKT4, and OKT8 specificities was studied in a
similar manner with 10 SLE sera. In no case was
monoclonal antibody staining altered by preincubation
with the SLE sera.
Modulation studies. In studies to be reported in
detail separately (14), it was observed that brief preincubation of lymphocytes with SLE serum at 37°C
resulted in loss of IgM-reactive determinant(s) from
the cell surface. When cells treated in this manner
were incubated at 4°C with a fresh aliquot of the same
SLE serum used in the 37°C preincubation, both the
percent of IgM+ cells and the relative fluorescence
intensity for IgM staining fell sharply. Since SLE IgM
antibody-reactive antigen(s) is modulated under these
conditions, one would predict that if the IgM antibody
were directed to known determinants identified by
Table 2. Inhibition studies of the cytotoxicity of systemic lupus
erythematosus (SLE) sera for peripheral non-T cells, using non-complement-fixing chicken anti-Ia*
Preincubation of target cells
Normal
chicken
serum.
1:2
1:2
1:8
80
65
60
80
65
45
ND
50
45
75
ND
65
50
70
75
35
35
50
10
80
70
50
30
40
35
80
80
65
50
80
70
40
ND
50
40
70
L243
90
90
SLE
serum
None
1
90
2
3
4
5
6
7
8
9
60
60
Chicken anti-Ia
<I0
40
70
<I0
* Nylon wool-adherent cell targets were 85% I a + , 70% sIgM+.
Target cells were incubated with indicated dilutions of normal
chicken serum or chicken anti-la for 30 minutes at 4°C prior to
assay. Values expressed as % dead cells, determined in a 2-stage
complement-dependent microcytotoxicity assay performed at 15°C.
ND = not determined.
monoclonal antibodies, these would be modulated as
well.
This hypothesis was tested with 10 SLE sera for
the monoclonal antibody specificities listed in Table 1.
Despite a substantial reduction in IgM staining of T
cells with 8 of 10 sera following modulation (60 -+ 25%
IgM+ cells 3 26 rt 22%), the percentage of rosettes
was unchanged, relative to control values, for all
monoclonal antibodies except anti-&m. Slight, but
significant (>12%) reduction in anti-p2m rosettes was
observed following modulation with 4 of 10 sera.
Resting T cells and T cell blasts modulated with SLE
serum were also examined by indirect immunofluorescence to detect any subtle change in staining characteristics for the L243, OKT3, OKT4, OKT8, Pzm, and
Tac specificities. Anti-am staining was shifted to
lower levels of fluorescence intensity by 37°C pretreatment with 7 of 10 sera in the same manner as that
observed for SLE IgM (Figure 3). Staining by the
other monoclonal antibodies was not altered.
DISCUSSION
This investigation utilized sensitive rosetting
and immunofluorescence techniques to define cell
surface determinants reactive with IgM antilymphocyte antibodies in SLE. Specificities for epitopes
identified by the monoclonal antibodies L243 (DR
framework), Leu-10 (DSDC framework), OKT4,
OKT8, OKT3, OKTll (E receptor), Leu-I, Leu-8,
5E9 (transferrin receptor), 4F2 (activation neoantigen), Tac (interleukin-2 receptor), and AA1 (activation
neoantigen) were not demonstrable in direct rosette
inhibition assays. Additional evidence against the
presence of antibodies to the molecules expressing
these epitopes was obtained in a series of modulation
experiments. Thus, preincubation of target cells with
SLE serum at 37°C caused a reduction in cold-reactive
IgM staining, but (except for anti-p2m) did not alter
monoclonal antibody rosette formation or relative
fluorescence intensity of staining. While it is possible
that the known antigens would not be modulated under
the conditions used even if specifically bound by SLE
antibodies, the dramatic loss of IgM-reactive antigen(s) following modulation indicates that the latter
are largely distinct from the molecules recognized by
the monoclonal antibodies. It should be emphasized,
however, that our approaches to defining the surface
antigen specificity of antilymphocyte antibodies in
SLE are indirect and subject to certain caveats. For
example, the failure of SLE serum to inhibit monoclonal antibody rosette formation could reflect a relative-
ANTIBODIES IN SLE
49
chains of HLA-A, B, or C antigens (3), or absorption
with purified p2-microglobulin (4) both inhibit the
cytotoxic activity of antilymphocyte antibodies in
SLE sera. Antibody to &-microglobulin has been
found in other diseases, such as Felty’s syndrome, as
well (16).
The existence of antibodies with Ia specificity is
more controversial. In early experiments, F(ab’)2fragments of operationally-specific xenoantisera to HLADR antigens reduced the cytotoxicity of SLE sera for
B lymphoblastoid cell lines, but not for peripheral
lymphocytes (4). The SLE patients had been pregnant,
however. More recently, Okudaira et a1 (6) reported
inhibition of anti-DR framework antibody-mediated
rosette formation by serum from patients with active
SLE. We could not confirm their findings despite our
use of the same monoclonal antibody, L243, and both
T blasts and CLL cells (-75% Ia+) as targets. Anti-Ia
antibodies also were not detected in a series of modulation, indirect immunofluorescence, and inhibition of
C-dependent cytotoxicity experiments.
These data may provide some additional insight
into conceptualization of the mechanisms by which
antilymphocyte antibodies in SLE influence cell function. As an example, there is strong evidence that
antilymphocyte antibodies inhibit T cell proliferation
to soluble antigen or to allogeneic non-T cells through
effects at the level of the responding T cell (2,17). The
failure to demonstrate specificities to Ia, Tac, T3, T8,
or T4 argues against an inhibitory mechanism involving specific blockade or modulation of, respectively,
DR antigens, interleukin-2 receptors, or antigen, or
classes I and I1 molecule-recognition structures. Similarly, the failure of other investigators to inhibit Erosette formation, and our data against antibodies to
the OKT11 epitope suggest that an “off-signal” effect
(18) does not occur. It should be emphasized, however, that such arguments represent aids in conceptualization, rather than proofs, since the available data are
both limited and indirect. More conclusive answers to
questions concerning structure/function relations of
antilymphocyte antibodies in SLE will require systematic analyses of the entire array of antibody specificities, using more sophisticated approaches, e.g.,
cloned cells as targets and human monoclonal antilymphocyte autoantibodies derived from patients.
Given the myriad functional effects attributed
to antilymphocyte antibodies in SLE and the range of
reactivities seen with cell targets of different types, it
is generally assumed that the antibody specificities
must reflect this heterogeneity. This presumed diver-
!
modulated cells
OKT3 Staining
Anti-&m
Staihing
Figure 3. Modulation of systemic lupus erythematosus (SLE) IgMreaciive surface antigen(s) and &-microglobulin (p2m). Nylon woolpurified T cells were modulated by incubation with SLE serum for 2
hours at 37°C. Modulated cells and fresh, untreated T cells were
then incubated at 4°C with a fresh aliquot of SLE serum (upper
panel), OKT3 (middle panel), or anti-&m (lower panel) followed by
staining with fluorescein isothiocyanate-conjugated antiimmunoglobulin reagents. Modulation reduced the fluorescence intensity
(log-integrated green fluorescence) of SLE IgM and anti-p2m staining, but did not alter that of OKT3.
ly lower avidity of the autoantibodies. More precise
delineation of autoantibody specificities will require
imrriunoprecipitation and immunoblotting procedures.
Each of the experimental approaches identified
Pzm as a major specificity of cold-reactive IgM antilymphocyte antibodies in SLE. The data here are
consistent with those obtained by previous investigators using different techniques (3,4,15). For example,
preincubation of target cells with F(ab’)2fragments of
xenoantisera against P2-microglobulin and heavy
50
sity contrasts sharply with the unambiguous molecular
data in this regard, i.e., p2m (3,4,15) and a surface
determinant cross-reactive with core mononucleosomes (19). Is it possible that cold lymphocytotoxins
in this disorder, and in a variety of other disorders,
actually are rather limited in specificity in a manner
analogous to IgM rheumatoid factor and cold agglutinins? If such were the case, the differential reactivity
with various cells and the multiple functional effects
might derive from variation in cell density and/or
membrane stability of a small number of functionally
relevant antigens.
Regardless of the exact antibody specificity, the
observation that the predominant antigen(s) with
which IgM antibodies react can be easily modulated at
physiologic temperatures is of special interest because
of the potential for important in vivo effects (20).
Shedding of surface antigens in association with antilymphocyte antibody was first postulated by the recovery of allogeneic MLR responsiveness by T cells
exposed to MLR-blocking antibodies from SLE serum
after short-term culture (2). Further evidence for the
existence of circulating complexes of shed surface
antigen and antilymphocyte antibody is the demonstration of specific enrichment of antilymphocyte antibody in purified cryoprecipitates from patients with
SLE (21,22) and chronic lymphocytic leukemia (23),
and the development of antibodies to human lymphocytes in animals immunized with SLE serum cryoproteins (24). Both p2m and anti-p2m antibody have been
demonstrated in immune complexes isolated from the
serum of patients with Felty’s syndrome (16).
Although it remains to be determined whether
shedding or capping and internalization of antigen is
occurring in our experiments, in vivo antigenic modulation has been demonstrated directly in other situations. Thus, treatment of leukemia patients with antibodies to leukemic cells results in the disappearance of
reactive membrane antigens (25). Similarly, administration of the monoclonal antibody OKT3 to renal
allograft recipients causes a decrease in OKT3+ T
cells and the appearance of OKT4+/T3 and OKT8+/
T3 cells in peripheral blood followed by re-expression
of the OKT3 determinant after overnight incubation in
culture (26). Work is in progress to determine whether
such phenomena occur spontaneously in SLE, as is
perhaps suggested by the observation of Steinberg and
colleagues that brightly IgM-staining cells are selectively “lost” in patients with active disease (27). If
such proves to be the case, in vivo surface receptor
modulation by autoantibodies may be an important
YAMADA ET AL
mechanism for immune system functional abnormalities in SLE and related disorders.
ACKNOWLEDGMENTS
The technical assistance of Rekha Shah is gratefully
acknowledged. We also thank Dr. Philip Cohen for helpful
discussions and Cathy Miller-Wells for typing the manuscript.
Winfield JB: Anti-lymphocyte antibodies in SLE: specificity and relationship to abnormal cellular function,
Systemic Lupus Erythematosus. Edited by R Lahita.
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