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Human IgG aggregates induce selective stimulation of IgM rheumatoid factor synthesis by rheumatoid blood mononuclear cells.

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502
HUMAN IgG AGGREGATES INDUCE SELECTIVE
STIMULATION OF IgM RHEUMATOID FACTOR
SYNTHESIS BY RHEUMATOID BLOOD
MONONUCLEAR CELLS
XUE LIAN TAO, NANCY OLSEN, MORRIS ZIFF, and HUGO E. JASIN
Peripheral blood mononuclear cells (PBM) from
patients with active rheumatoid arthritis (RA) contain
precursor B lymphocytes specific for IgM rheumatoid
factor (IgM-RF) synthesis. In this study, human IgG
aggregates (HaIgG) were used to stimulate monocytedepleted PBM from 46 patients with RA and 21 normal
controls. The cells were incubated with HaIgG and
pokeweed mitogen for 72 hours, washed, and then
cultured in microwells for an additional 11 days. HaIgG
induced an increase in IgM-RF synthesis by RA cells
with optimum response at 0.1 pg/ml ( P < 0.001). Total
IgM synthesis remained unchanged. In contrast, HaIgG
did not stimulate IgM-RF production by normal cells.
Specificity of the IgM-RF response was shown by the
concomitantly increased IgM-RF/IgM ratios, while IgM
anti-trinitrophenyl antibodies did not increase. Aggregation of the IgG was required for it to be an effective
stimulus. The findings suggest that circulating immune
complexes in RA patients may provide the stimulus for
sustained production of IgM-RF in vivo.
-~
From the Harold C. Simmons Arthritis Research Center
and the Department of Internal Medicine, University of Texas
Health Science Center at Dallas.
Supported by USPHS grants AM07055, AM16209, and
AM18505.
Xue Lian Tao, MD: Rheumatology Fellow (current address: Capital Hospital, Chinese Academy of Medical Sciences,
Beijing, China); Nancy Olsen. MD: Instructor, Department of
Internal Medicine, UTHSCD and recipient of a Postdoctoral Fellowship from the Arthritis Foundation; Morris Ziff, PhD, MD:
Professor of Internal Medicine and Director, Harold C. Simmons
Arthritis Research Center, UTHSCD; Hugo E. Jasin, MD: Professor of Internal Medicine, UTHSCD.
Address reprint requests to Dr. Nancy Olsen, Department
of Internal Medicine, The University of Texas Health Science
Center, 5323 Harry Hines Boulevard, Dallas, TX 75235.
Submitted for publication August 1, 1983; accepted in
revised form November 10. 1983.
Arthritis and Rheumatism, Vol. 27, No. 5 (May 1984)
The majority of adult patients with rheumatoid
arthritis (RA) have detectable levels of IgM rheumatoid factor (IgM-RF) in the blood. The presence of
high titers of this autoantibody is generally associated
with more severe disease manifestations (1). In addition, a majority of seropositive RA patients have been
shown to have circulating cells which synthesize IgMRF when cultured in vitro (2-4). In 40 to 50% of
rheumatoid patients, B cells synthesize IgM-RF spontaneously, suggesting in vivo activation. We have
previously shown that the presence of such circulating
cells is correlated with activity and severity of disease
(2). Additional precursor cells, which can be induced
by pokeweed mitogen (PWM) to synthesize and secrete IgM-RF, are present in most RA patients ( 2 ) and
in some normal controls (5). The aggregate of these
studies suggests that, in RA, there is a selective
expansion of the €3 cell population with specificity for
IgM-RF.
The antigenic stimulus responsible for IgM-RF
synthesis remains unknown. Soluble immune complexes and human aggregated IgG (HaIgG) have been
shown to have numerous nonspecific stimulatory effects on lymphocytes in vitro, including lymphocyte
transformation (6) and antibody synthesis (7,8). In a
recent study, normal human peripheral blood lymphocytes were induced to synthesize antibody in response
to HaIgG, and rheumatoid factor was produced as part
of the polyclonal response (9).
Since circulating immune complexes have been
shown to be present in patients with RA (lo), it is
possible that they could have a role in stimulating
antibody production in vivo. The present studies were
designed to investigate the possibility that such a
IgM-RF SYNTHESIS BY RA CELLS
503
stimulus might b c specific for IgM-RF production in
patients with RA. T h e results indicate that low concentrations of HaIgG increased IgM-RF synthesis by
rheumatoid, but not normal, lymphocytes. This response d o e s not occur in the presence of unaggregated
IgG. IgM-RF is selectively stimulated in this system
and it is not part of a polyclonal antibody response.
PATIENTS AND METHODS
Patients. Forty-six patients with definite or classic
RA, according to the American Rheumatism Association
diagnostic criteria (1 I ) , wcre selected for the present study.
All patients were seropositive for R F as determined by the
sensitized sheep cell agglutination assay. Sixteen patients
were treated with gold salt injections, and 7 were receiving
penicillamine in doses ranging from 250 to 1,000 mg daily.
The 21 normal controls were laboratory workers and students.
Cell separation. Peripheral blood mononuclear cells
(PBM) were obtained from heparinized blood by centrifugation on Ficoll-Hypaque cushions (12). The cells at the
interphase were washed 6 times with Hanks' solution (Grand
Island Biological Co., Grand Island, NY) and resuspended in
RPMI medium (Grand Island Biological Co.) with 10% fetal
calf serum (FCS) (Grand Island Biological Co.). The PBM
were partially depleted of monocytes by incubation on a
prewarmed glass Petri dish at 37°C for 1 hour. This was done
to minimize ingestion of the HaIgG by phagocytic cells, and
to avoid nonspecific suppression of the response by activated monocytes. Nonadherent cells (NAC) were removed by
gentle rinsing with medium and washed twice prior to
culture. The NAC contained 4-5% monocytes, as assessed
by ingestion of latex particles. The cells were suspended at
lo6 per milliliter in RPMI 1640 medium supplemented with
10% FCS, 0.002M glutamine. penicillin 10' unitsldl, and
gentamicin 6 mg/dl.
HaIgG. Human IgG was purified from Cohn fraction
I1 (Pentex, Miles Laboratory, Elkhart, IN) by DEAE cellulose ion exchange chromatography. IgG aggregates were
obtained by heating a solution of purified IgG (15 mg/ml) at
63°C for 30 minutes. Heavy aggregates were precipitated
with 4% polyethylene glycol, centrifuged at 3,000 revolutions per minute for 30 minutes at room temperature, and
dissolved in 0.15N NaCl solution.
Cell cultures. Twomilliliter volumes of the final
NAC suspension containing lo6 cells/ml were dispensed into
16 x 12.5-mm polystyrene tissue culture tubes (Falcon
#3033, Becton Dickinson and Co., Oxnard, CA). Varying
concentrations of HaIgG, in 0.2-ml volumes, were added to
each tube to yield final concentrations ranging from 0.001 to
100 Fglml. At each HalgG concentration, duplicate cultures
were made with and without 0.2 ml pokeweed mitogen
(PWM) (Grand Island Biological Co.), to yield a final dilution
of 1/500.
Cultures were incubated for 3 days at 37°C in air
containing 5% COz. The cells were then washed 3 times at
4°C with 3-5 ml culture medium per tube, counted, and
resuspended at a concentration of lo6 viable cells/ml. A
volume of 0.20 ml of this suspension was dispensed into
round-bottom microtiter plates (Dynatech Laboratories,
Inc., Alexandria, VA). PWM, in 2 0 - 4 volumes, was readded to those cells which had been cultured with PWM in
the initial 3-day period. The plates were incubated for 11
days, and the cultures were terminated by centrifugation at
500 rpm for 10 minutes. The cell-free supernatants from
quadruplicate cultures were pooled and stored at -20°C until
used.
Serologic reagents. Affinity purified rabbit anti-human IgM was prepared as previously described with some
modifications ( 2 ) . Anti-Fab antibodies were removed from
the rabbit anti-human IgM antisera by repeated absorption
over IgG-Sepharose columns. The globulin fraction was
obtained by precipitation with 50% saturated ammonium
sulfate solution. This fraction was dialyzed against 0.1M
acetic acid. The F(ab')* fragments were obtained by incubation with 2% crystalline pepsin for 18 hours at 37°C. After
dialysis against several changes of 0.15M NaCI, O.05M Tris,
pH 8.0 buEer, the F(ab')z fragments were absorbed with the
monoclonal IgM-Sepharose solid immunoabsorbents and
eluted with cold 0.1M glycine, 0.1M NaCI, pH 3.0 buffer.
The affinity purified F(ab')z anti-IgM was radiolabeled with
'"I as previously described (2).
Radioimmunoassays. Concentrations of IgM and
IgM-RF in culture supernatants and plasma were determined
by solid-phase radioimmunoassay as previously described
Table 1. IgM-RF and IgM synthesis by RA and normal NAC cultured without PWM*
HalgG (pg/ml)t
Source
of cells
Normal
(n = 21)
RA
(n = 46)
0
IgM-RF
ng anti-IgM
IgM
ndml
IgM-RF
ng anti-IgM
IgM
ng/ml
0.3
20.1
540
+- I50
1.?
20.4
235
? 280
0.01
0.5
20.2
554
i248
I .5
t0.5
287
2388
0. I
0.4
20.2
573
172
1.3
'0.4
266
2308
*
I .o
10
0.4
20.1
507
2115
0.3
to.I
390
1111
1.1
0.6
20.2
I68
t I46
20.3
23 1
2 207
* RF = rheumatoid factor; RA = rheumatoid arthritis; NAC = nonadherent cells; PWM
mitogen.
t Results expressed as mean ? SEM. HaIgG = human IgG aggregates.
=
pokeweed
TAO ET AL
504
Table 2.
IgM-RF and IgM synthesis by RA and normal NAC cultured with PWM'
HaIgG (pg/ml)t
Source
of cells
Normal
(n = 21)
RA
(n = 46)
0
IgM-RF
ng anti-IgM
IgM
nghl
IgM-RF
ng anti-lgM
Ig1M
ng/ml
1.0
?0.3
1,848
? 640
2.9
L0.6
2,097
2419
0.001
0.01
0. I
1 .o
10
I00
ND
0.8
20.2
3.241
21154
4.8$
1.5
?0.5
1,825
+497
4.9$
1 .2
1.2
20.6
1.242
t325
3.7
0.9
20.5
1,088
2425
1.4
20.4
1316
-555
ND
2.1
20.9
1,888
2749
20.8
k0.8
2,009
~485
1.624
2280
+O.S
1,212
5454
3.39
20.9
1,540
5417
~
* R F = rheumatoid factor; RA - rheumatoid arthritis; NAC
rnitogen.
t Results expressed as mean
$ P < 0.001.
8P<OO5.
2
SEM. Hal&
-
=
( 2 ) . Briefly, affinity-purified anti-IgM or HaIgG was bound
to wells of flexible polyvinyl microtiter plates (Dynatech
Laboratories, Inc.) by a 4-hour incubation at room temperature. The plates were washed with phosphate buffered saline
(PBS) and PBS with 10% newborn calf serum. Volumes of
50 pl of culture supernatants or suitable dilutions of plasma
were added to duplicate wells, and the plates were incubated
at room temperature overnight. After washing, 50 pI of
i251-labeledF(ab')? anti-human IgM containing approximately 2 pdml of the radiolabeled antibody was added to each
well. Plates were again incubated overnight and washed.
Individual wells were cut and counted in a Packard gamma
scintillation counter. Standard curves were constructed and
results expressed as ng/ml for total IgM, and nanograms of
F(ab')2 anti-IgM bound per well for IgM-RF.
IgM anti-triiitrophenyl (anti-TNP) antibodies were
measured in selected culture supernatants by a similar solidphase radioimmunoassay. In this assay, the only difference
consisted of the addition to the wells of 50 pi of a I-mg/rnl
solution of TNPlo-human serum albumin (TNPlo-HSA),
which was the kind gift of Dr. Randolph Noelle. Control
plates were coated with HSA alone. Plates were washed and
incubated with the supernatants as described above. The
'*'I-labeled anti-IgM F(ab')* was again added in the final
step. Results are expressed as nanograms of Frab')? anti-IgM
bound per well.
Statistical analysis. Results are expressed as mean
values 2 standard error of the mean. Significance levels
were calculated using a paired Student's t-test.
RESULTS
When NAC from normal controls and RA patients were cultured with varying concentrations of
HaIgG in the absence of PWM, there was no significant stimulation of IgM-RF or total IgM (Table 1). At
the highest concentration of aggregates tested, 10 pg/
ml, total IgM synthesis was lower than the corresponding baselines for both groups, but these differences were not significant.
In the presence of PWM and HaIgG, normal
NAC did not synthesize increased amounts of IgM-RF
=
21.8
1.182
+I50
~
~
nonadherent cells; PWM
human IgG aggregates; N D
=
~~
=
pokeweed
not done.
(Table 2). Total IgM synthesis was increased at 0.01
pglml HaIgG, but this was not significantly greater
than the corresponding baseline ( P > 0.3). In contrast,
RA NAC cultured at HaIgG concentrations of 0.01 and
505
IgM-RF SYNTHESIS BY RA CELLS
These data suggested that HaIgG-induced stimulation of IgM-RF by RA NAC was selective and not
part of a polyclonal response. Further support for this
observation was obtained by expressing the results as
the ratio of IgM-RF to total IgM synthesized and
secreted by each individual donor. The individual
ratios were then averaged, and the corresponding
mean values for the normal and rheumatoid groups are
shown in Figure 2. It can be seen that IgM-RF/IgM is
increased in rheumatoid cells, but not in normal cells.
As in the case of the IgM-RF values, the ratios are
significantly increased in the rheumatoid group at
HaIgG concentrations of 0.01 and 0.1 pg/ml ( P <
0.00 1) .
Additional evidence to support the selectivity
of the IgM-RF response was sought by measuring IgM
anti-TNP antibodies in supernatants showing increased IgM-RF synthesis. Fifteen RA supernatants
which had shown increased IgM-RF in response to
HaIgG were selected. As shown in Figure 3, anti-TNP
antibody levels in these samples were not increased
over the baseline, while IgM-RF levels doubled. These
additional data indicate that the observed increase in
IgM-RF synthesis was specific and not part of a
polyclonal activation.
The next series of experiments was carried out
to investigate whether aggregation of IgG was required
for it to be an effective stimulus. IgM-RF synthesis
I
I
I
I
I
0 0.001 0.01 0.1 1.0
I
10
1*O
I
100
HalgG p g / m l
Figure 2. IgM rheumatoid factor to IgM (IgM-RF/lgM) ratios in
cultures stimulated with human IgG aggregates (HalgG) and pokeweed mitogen. The IgM-RF/IgM ratio increased in rheumatoid
arthritis patients (0-0) at HaIgG concentrations of 0.01 and
0.1 Kg/ml ("P < 0.001); there was no increase in normal controls
(0-0,.
0.1 pg/ml synthesized almost twice as much IgM-RF
as the corresponding baseline with PWM alone ( P <
0.001) (Table 2). A smaller, but still significant increase
was also observed with HaIgG at 1 .0 pg/ml ( P < 0.05).
IgM-RF synthesis appeared to be inhibited by the
highest concentration of HaIgG used, 100 pg/ml ( P =
0.20). Total IgM synthesis was not increased at any
HaIgG concentration. The results for IgM-RF in the 2
groups are also shown in Figure I . Increased synthesis
of IgM-RF occurred only with the rheumatoid cells.
with the peak response at 0.01 and 0.1 pg/ml of HaIgG.
0
0.01
0.1
I
HalgG pg/ml
Figure 3. Effect of human IgG aggregates (HaIgG) o n anti-trinitrophenyl (anti-TNP) antibody production by rheumatoid arthritis
nonadherent cells. Levels of IgM rheumatoid factor (IgM-RF)
increased. while levels of anti-TNP were unchanged. All cultures
were done in the presence of pokeweed mitogen.
506
TAO ET AL
60
40
Q)
0
20
c
0
+
C
Q)
E
a"
0
- 20
0
0.01
0.1
I
HalgG or IgG pg/ml
Figure 4. Comparison of the effects of human IgG aggregates
(HaIgG) (0-0)
and unaggregated IgG (0-0) on IgM rheumatoid
factor (IgM-RF) production by rheumatoid arthritis nonadherent
cells. Increased IgM-RF synthesis was observed only with HalgG.
All cultures were done in the presence of pokeweed mitogen.
was measured in experiments from RA cells incubated
with aggregated or unaggregated IgG. The results,
shown in Figure 4, indicate that unaggregated IgG did
not stimulate IgM-RF synthesis over baseline levels,
while HaIgG, at equivalent protein concentrations,
stimulated IgM-RF synthesis up to 50% greater than
baseline in this group of RA cell donors.
DISCUSSION
The studies reported here indicate that HaIgG
can specifically stimulate IgM-RF secretion by RA
NAC in the presence of PWM. Normal cells did not
show increased IgM-RF production in response to
HaIgG. The specificity of the response is shown by 2
sets of data. First, IgM-RF synthesis was significantly
increased in the supernatants obtained from RA lymphocytes incubated with 0.01 to 1 pg/ml HaIgG, while
total IgM synthesis remained unchanged. Therefore,
the ratio of IgM-RF to total IgM increased as the IgMRF production increased. Second, measurement of an
unrelated IgM antibody, anti-TNP, did not show an
increase. Thus, increased IgM-RF levels are not a
result of polyclonal stimulation. The stimulating IgG
must be aggregated to induce the response; unaggregated IgG tested at the same concentrations had no
effect.
Soluble immune complexes and aggregated human IgG have previously been shown to stimulate
lymphocyte transformation (6) and antibody synthesis
(7-9). Yamasaki and Ziff reported that a mixture of
heat-aggregated human IgG, IgM-RF, and complement enhanced the in vitro immunoglobulin synthesis
by lymphocytes from RA patients, but not from normal controls or patients with miscellaneous seronegative arthritides (7). Morgan and Weigle demonstrated
stimulation of a polyclonal antibody response by murine spleen cells exposed to HaIgG (8). I n a more
recent study, Pisko et a1 described polyclonal stimulation of normal human peripheral blood lymphocytes
exposed to HaIgG with RF production as part of the
polyclonal response (9).
In our system, stimulation of IgM-RF by HaIgG
was selective and not part of a polyclonal response.
One possible explanation for the difference between
our results and those reported previously is the concentration range of HaIgG used. The concentrations of
HaIgG which induced IgM-RF in our system, 0.01 and
0.1 pg/ml, were in the range previously shown to be
effective in exogenous antigen-specific stimulation
(13). In contrast, Pisko et a1 used concentrations from
100 to 5,000 pglml, and Morgan and Weigle used 300 to
500 pglml. It is possible that at these high concentrations, the mechanism of B cell activation differs,
resulting in nonspecific stimulation and a polyclonal
response. This dose-dependent qualitative difference
in antibody responses has been described for human B
cells by Volkman and coworkers, who used tetanus
toxoid (13). Low concentrations of tetanus toxoid
( 4 0 ng/ml) stimulated a specific anti-tetanus response, while high concentrations (>100 ng/ml) stimulated increases in total Ig synthesis, which suggested a
nonspecific response.
Another important methodologic difference
which may have played a role in the results obtained
was the culture conditions. Morgan and Weigle (8)
used flat-bottom wells, while our cultures were done in
round-bottom wells. Such geometric differences have
been shown to be critical in determining the specificity
of in vitro antigen-induced antibody responses (l4),
507
IgM-RF SYNTHESIS BY RA CELLS
and this may partly explain the different results obtained.
Our data provide evidence that aggregated IgG
may trigger activation of IgM-RF-specific B cells in
RA. While we (2) and others (3,4) have previously
reported that peripheral blood lymphocytes from RA
patients synthesize IgM-RF spontaneously or in response to PWM, prior to this report, there has been no
evidence for specific IgM-RF synthesis by these cells
that had been induced by a defined stimulus.
This type of antigen-specific activation has
been studied in human systems using well-defined
stimuli. Studies by other investigators have led to a
postulated pathway for B cell activation and antibody
formation (15-18). According to this scheme, small
resting B cells can undergo activation in vivo by
exposure to antigen. These cells then increase in size
and become responsive t o proliferative signals, resulting in expansion of the antigen-specific B cell population. In the final step, the cells become sensitive to
differentiation factors, resulting in antibody synthesis.
Depending on exposure to antigen and other factors in
vivo, circulating B cells may be in various stages of
activation. It is likely that completion of this sequence
can occur in vivo, since spontaneous antibody-secrcting cells can be demonstrated in circulating lymphocyte populations of recently immunized donors
(18,19).
In our studies, PWM-induced B cell maturation
was required to demonstrate the specific increase in
IgM-RF synthesis. It has recently been shown that
PWM-responsive B cells constitute a small, distinct
subset of the total human B cell population (20). These
large B cells are characterized by the presence of
surface IgG, but the predominant antibody class secreted in response t o PWM is IgM. The requirement
for PWM in our system suggests that this subpopulation may be responsible for the induced IgM-RF
synthesis. HaIgG may stimulate a small subpopulation
of specific precursor B cells from a PWM-nonresponsive to a PWM-responsive state.
Also pertinent to our studies is the recent
demonstration that mitogen-activated B cells can function as antigen-presenting cells (21). The requirement
for PWM in our system might indicate such a role for
activated B cells in the HaIgG-specific response. Studies on the lymphocyte subpopulations responsible for
the HaIgG-specific response are in progress in our
laboratory.
In RA, we (2) and others (3.4) have reported
that a significant percentage of circulating B cells
secrete IgM-RF spontaneously. These data, and observation of large blastoid-appearing circulating cells
in RA (22,23), suggest that in vivo activation occurs.
Our present findings suggest that circulating immune
complexes present in low concentrations in rheumatoid patients may provide the stimulus for rheumatoid
B cells to produce IgM-RF in vivo. This autoantibody
could, in turn, form complexes with immunoglobulins
which would then stimulate additional production of
IgM-RF. Such a positive feedback mechanism could
explain the long-term sustained synthesis of R F observed in the majority of patients with active disease.
ACKNOWLEDGMENTS
We wish to thank Ms Barbara Strother for skilled
technical assistance and Ms Debbie McInnis for expert
secretarial assistance.
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