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Quantitation of cells secreting rheumatoid factor of igg iga and igm class after elution from rheumatoid synovial tissue.

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1445
QUANTITATION O F CELLS SECRETING
RHEUMATOID FACTOR OF IgG, IgA, AND IgM
CLASS AFTER ELUTION FROM
RHEUMATOID SYNOVIAL TISSUE
TORSTEIN EGELAND, TOR LEA, GEORGE SAARI, OVE J. MELLBYE, and JACOB B. NATVIG
In an indirect hemolytic plaque assay that used
sheep erythrocytes coated with normal rabbit IgG or
reduced/alkylated IgG anti-sheep erythrocytes, mononuclear cells eluted from rheumatoid synovial tissue of 7
of 11 patients with seropositive rheumatoid arthritis
contained cells that secreted rheumatoid factor of IgG,
IgA, and IgM classes. The number of rheumatoid
factor-secreting cells varied from <1% to 53% of the
total number of eluted immunoglobulin-secreting cells.
In contrast, immunoglobulin-secreting cells and rheumatoid factor-secreting cells were scanty in blood compared with synovial tissue mononuclear cells.
the present study was to measure the number of
mononuclear cells (MNC) eluted from rheumatoid
synovial tissue that actively secreted RF of all the 3
main classes: IgG, IgA, and IgM. An indirect hemolytic plaque assay allows us to quantitate the cells that
actively secrete R F of these classes. In addition, the
results were compared with those of the total number
of Ig-secreting cells obtained from a reverse hemolytic
plaque assay ( I 1). This made it possible to estimate the
proportion of Ig-producing cells in synovial tissue
which is engaged in R F secretion.
Immunofluorescence studies of rheumatoid synovial tissue sections have revealed cells with rheumatoid factor (RF), primarily of IgG and IgM classes (14). Cells secreting IgM R F have been demonstrated in
eluates of cells from rheumatoid membranes ( 5 ) . Synovial fluid of rheumatoid arthritis patients and supernatants of cultured rheumatoid synovial membranes are
known to contain R F of IgG and IgM class ( 6 4 , and
there is also evidence of IgA R F (9).
Vaughan et a1 (10) first described a direct hemolytic plaque assay in which cells secreting complement-fixing IgM R F were demonstrated. The task of
Patients and synovial tissue. Materials from 13 patients, 9 females and 4 males with classic rheumatoid arthritis (12) who were undergoing synovectomy at Oslo Sanitetsforening Rheumatism Hospital and Oslo City Department of
Rheumatology and Rheumasurgery, Oslo, were used in the
study. The patients were receiving salicylates and related
drugs, chloroquines, D-penicillamine, gold sodium thiomalate, or glucocorticosteroids. The synovectomies were performed under general or nerve block anesthesia.
Elution of synovial tissue MNC. Elution of synovial
tissue MNC was performed according to Abrahamsen et al
(13). In short, the tissue samples were treated with collagenase and DNase during agitation, and the cells were filtered,
suspended in medium with 10% fetal calf serum, and incubated overnight in plastic culture flasks (Costar, USA). The
nonadherent cells were harvested and further purified by
Isopaque-Ficoll (Lymphoprep, Nygaard, Norway) gradient
centrifugation (14). The interphase cells were washed 3 times
at 37°C and resuspended in Hank’s balanced salt solution
(Flow Lab., UK). According to the trypan blue dye exclusion test, viability of the cells always exceeded 80%. The
cells were tested for RF-secreting cells and Ig-secreting
cells, and the results were related t o the number of MNC.
Separation of peripheral blood MNC. Peripheral
blood MNC from 6 of the patients were separated according
to B@yum(14). After being both washed and preincubated ( I
-
From the Institute of Immunology and Rheumatology,
Rikshospitalet, National Hospital, Oslo I , Norway.
Supported by the Norwegian Women’s Public Health Organization and the Norwegian Hydro Company.
Torstein Egeland, MD: Research Fellow; Tor Len, MSc:
Senior Biochemist; George Saari, MD: Research Fellow; Ove J .
Mellbye, MD: Acting Head; Jacob B. Natvig, MD: Professor.
Address reprint requests to T. Egeland, MD, Institute of
Immunology and Rheumatology, Fr. Qvamsgt. 1, Oslo I , Norway.
Submitted for publication October 8, 1981; accepted in
revised form June I , 1982.
Arthritis and Rheumatism, Val. 25, No. 12 (December 1982)
MATERIALS AND METHODS
1446
hour) in Hank’s buffered salt solution at 3TC, MNC were
tested for RF- and Ig-secreting cells. Viability always exceeded 95%.
Preparation of antisera. The class-specific developing
antisera against human immunoglobulins were raised in
rabbits immunized with highly purified myeloma proteins.
The antisera were absorbed against relevant myeloma proteins and F(ab’)? fragments of normal human IgG. The
specificity of the antisera was tested in a sensitive, enzymelinked immunosorbent assay (ELISA) as well as in the
reverse hemolytic plaque assay. In the latter assay we added
the highly purified myeloma proteins of 1 Ig class instead of
MNC to the indicator erythrocytes and then added developing antiserum and complement.
In all instances, each antiserum showed exclusive
specificity for 1 Ig class, as previously documented ( 1 I) and
further described in the Results section. For all antisera, we
used IgG fractions after isolation by chromatography on
Sepharose 4B protein A columns (15) or by DE-52 ionexchange chromatography (16). The sensitivity of the assays
with the vanous developing antisera is comparable since the
sum of the number of IgG-, IgA-, and IgM-secreting cells
equaled the number of Ig-secreting cells developed by antiF(ab’)? (11).
F(ab’), fragments of human IgG that were used to
raise anti-F(ab’)*in rabbits, and those of isolated IgG from a
rabbit immunized with sheep erythrocytes (SRBC) (F(ab‘)2
anti-SRBC) were produced by pepsin digestion (17) of the
IgG fractions before gel filtration on calibrated Sephadex G200 columns.
Purification of myeloma proteins. IgM proteins were
isolated from sera of patients with Waldenstrom’s macroglobulinemia by euglobulin precipitation and gel filtration on
Sephadex (3-200 (17). IgA myeloma proteins were isolated
by chromatography on diethylaminoethyl (DEAE) cellulose
ion-exchange columns, and IgG proteins were obtained from
Kabi, Sweden, and further purified on DE-52 ion-exchange
columns (16). All proteins were further purified by affinity
chromatography on immunosorbent columns.
Indirect hemolytic plaque assay for RF-secreting cells.
This assay was done in 2 ways, and triplicate experiments
for each class of Ig were always made.
A . SRBC sensitized with subagglutinating dilutions
of reduced and alkylated rabbit IgG against SRBC (reduced
and alkylated IgG anti-SRBC) ( 5 ) were used as antigencoupled indicator cells. We prepared monolayers of indicator cells by adding sensitized SRBC to the bottom of plastic
culture cluster wells (Costar, USA). The wells had previously been incubated with F(ab‘)? anti-SRBC, which bound
SRBC in a monolayer (1 1). We added 0. I ml of test MNC,
1.106 MNC/ml, to the monolayer together with the classspecific, rabbit, developing antiserum for 1% hours at 37°C.
After addition of complement (guinea pig serum previously
absorbed against SRBC) and further incubation at 37°C for
one-half hour, concentric hemolytic plaques were seen in the
monolayer around nucleated test cells, which were interpreted as RF-secreting cells. The developing antisera and complement were used in the same dilutions as for the reverse
hemolytic plaque assay. We added phosphate buffered saline
(PBS) or normal rabbit IgG, instead of developing antisera,
for controls. In addition, control for tests were performed
EGELAND ET AL
with developing antisera and complement on uncoupled
SRBC.
B . IgG from the serum of an unimmunized rabbit was
separated through ion-exchange chromatography ( 16) and
absorbed 3 times, with SRBC. SRBC were then coated
through a chromium chloride method (18) with the rabbit
IgG, which served as antigen for RF. The test was otherwise
done as described above.
For both setups, control studies were performed by
substituting MNC with heat-inactivated serum containing
RF (titer 1,024) diluted in PBS. Inhibition studies were done
with heat-aggregated (19), reduced, and alkylated human
IgG (Kabi, Sweden) (20); human IgG; heat-aggregated human IgG; and normal rabbit IgG. All the reagents had
previously been absorbed against SRBC.
Reverse hemolytic plaque assay for Ig-secreting cells.
For this assay, SRBC were coated through the chromium
chloride method (18) with isolated IgG fraction of antiF(ab’)z antiserum; the monolayers of indicator SRBC were
prepared by binding through F(ab’)z anti-SRBC (1 1). Test
MNC and developing antisera were layered on the monolayer for 1% hours before guinea pig serum as complement
source was added. After one-half hour, hemolytic plaques
with a central Ig-secreting cell were counted. Standardization experiments had been performed earlier on peripheral
blood MNC stimulated with pokeweed mitogen for 7 days.
We used the highest dilutions of developing antisera and
guinea pig serum that resulted in maximal numbers of Igsecreting cells and in total hemolysis of the monolayer
preincubated with myeloma proteins. Control experiments
were undertaken with uncoated, chromium chloride-treated
SRBC, or by substituting the developing antisera with PBS
or normal rabbit IgG. All gave negative results.
Counting of RF- or Ig-secreting cells. Both RF- and
Ig-secreting cells were counted in an inverted microscope
( l l ) , and each result was calculated as the mean of the
plaque count of 3 wells of RF- or Ig-secreting cells, with
corrections for possible direct plaques, e.g., cells secreting
IgM able to form plaques, but without a developing antiserum. During calculations, values of <20 were recorded as
10.
RF in serum. The routine Waaler-Rose test was
performed to detect RF, using group 0 human erythrocytes
sensitized with rabbit antibodies.
RESULTS
Cells secreting RF and immunoglobulins of IgG,
IgA, and IgM classes. Synovial tissue MNC samples
varied in their RF-secreting cell activity. As Table 1
shows, RF-secreting cells were detected in 7 of I 1
(64%) samples from seropositive patients (serum RF
titer 232), and not in 2 samples from seronegative
patients. IgG and IgA RF-secreting cells were always
present together, except in Patient OB, in whom only
IgA RF-secreting cells were found. IgM RF-secreting
cells were less frequently observed. Table 1 also
shows the high numbers of the total Ig-secreting cells
RF-SECRETING CELLS
1447
Table 1. Number of rheumatoid factor-secreting cells (RF-SC) and Ig-secreting cells (ISC) in eluted synovial tissue mononuclear cells versus
serum titer for R F , and the number of RF-SC versus the number of ISC (%)*
Serum
IgG
IgA
IgM
Total
PatiRF
ents
titer
RF-SC
ISC
%
RF-SC
ISC
%
RF-SC
ISC
9%
RF-SC
I sc
%
~~
JK
<I6
<I6
<20
<20
1,660
3,920
32
32
<20
GAT
760
NT
NT
RKat
RSt
64
64
520
140
4,900
6,120
128
I28
128
<20
<20
<20
700
I , 160
10,260
256
256
256
256
I40
480
<20
NT
1,540
18,360
8,060
2,280
TA
OBt
TS
ER
WN
RKr
AS
GL
ss
Mean
5,360
<I
<I
<20
<20
360
6,460
NC
NC
40
80
3,280
2.120
1
4
11
180
2
I40
1,320
2.420
<20
<20
<20
5,500
620
12,340
1,100
1,220
3,180
9,320
2,000
<I
<I
<I
9
3
<I
NC
I80
<20
I .320
3,857
<20
120
1,440
140
<I
(7
<20
<20
3,460
10,520
<20
<20
2,480
2,660
<I
(1
40
840
25,760
24,780
13
120
6
<20
1,600
720
7
<I
820
280
7,820
9,260
<20
<20
<20
1,380
240
5,080
<I
<4
<I
<20
<20
<20
7,580
2,020
27.680
680
120
<20
660
840
2,980
4,820
1,420
80
<I
<I
46
1,920
660
<20
>1,980
3,600
24,520
22,200
5,700
<3
<I
90
6
<I
66
1,985
<1
<I
53
3
<1
234
210,377
* NT = not tested; NC = not calculable. The figures for RF-SC and ISC are given as RF-SC/lO' and ISCilO' mononuclear cells, respectively.
t Results from the test performed on the monolayer of sheep erythrocytes sensitized with subagglutinating dilutions of reduced and alkylated
rabbit IgG against sheep erythrocytes.
of IgG, IgA, and IgM classes, mean 210,377 Igsecreting ~ e l l s / l MNC.
0~
For Patients RKr and SS, both with serum R F
titers of 256, the proportion of RF-secreting cells to the
total number of Ig-secreting cells was considerable:
53% and 234%, respectively (Table 1). For the other 5
samples positive for RF-secreting cells, such cells
constituted 5 1-18% of the total number of lg-secreting
cells (Table 1). Statistical analysis showed that the
number of RF-secreting cells was not correlated to the
total number of Ig-secreting cells (r = -0.478, P >
0.05).
In 1 sample (RS), only IgG and IgA RF-secreting cells, but not IgM RF-secreting cells were countable (Table I ) . However, the total number of Igsecreting cells of IgM class in this sample was also
very low. In other samples (OB, GA, RKa, and AS) in
which the numbers of IgA- and IgM-secreting cells
were comparable, the numbers of IgM RF-secreting
cells were still lower than the numbers of IgA RFsecreting cells (Table 1). The cells from Patient RKr
revealed a pattern different from the other cell samples: these cells had very high numbers of cells secreting IgA and IgM RF, but low numbers of IgG RFsecreting cells (Table 1).
Blood MNC were distinctly lower in number of
Ig-secreting cells (mean 910/106MNC) than synovial
tissue MNC. The difference was highly significant ( P
< 0.001). No RF-secreting cells were observed in the
blood in any of the cases (<20/I06MNC).
Comparison of the assay for RF-secreting cells
with various coupled antigens. In 4 of the samples, tests
were made both with SRBC sensitized with reduced
and alkylated IgG anti-SRBC and SRBC coated with
normal rabbit IgG by the chromium chloride method.
It was a consistent finding that a few more RFsecreting cells were recovered with the former type of
SRBC in the monolayer, but the differences were
small. In addition, the few direct plaques (21) formed
by IgM RF-secreting cells could be seen only in
monolayers of SRBC sensitized with reduced and
alkylated IgG anti-SRBC. The use of antiIgM-developing antiserum made more IgM RF-secreting cells
appear, however. Direct plaques were never observed
in monolayers of SRBC coated with normal rabbit TgG
or anti-F(ab')2.
In 2 cases studied, we also counted the number
of RF-secreting cells using human IgG or reduced and
alkylated, heat-aggregated human IgG for coating by
chromium chloride. This did not result in higher numbers of RF-secreting cells than coating with normal
rabbit IgG did. In addition, IgG RF-secreting cells
could not be counted since antilgG-developing antiserum reacted directly with the coupled IgG or the heat-
EGELAND ET AL
1448
aggregated, reduced and alkylated human 1gG. Hemolytic plaques were never seen in monolayers of
uncoated, chromium chloride-treated SRBC o r of unsensitized SRBC.
Control for the specificity and reactivity of the
assays for RF-secreting cells. A series of control experiments was performed to assure the specificity of the
indirect plaque assay. Except for the few directly
developed IgM RF-secreting cells in monolayer of
SRBC sensitized with reduced and alkylated IgG antiSRBC, no plaques were formed when normal rabbit
IgG was used instead of the developing antisera in the
same concentrations. This clearly demonstrated that
normal rabbit IgG did not bind to R F in a way that
activated complement and induced lysis with plaque
formation.
Another test was aimed at showing that RF was
detected in the system. For this reason, various experiments were made in which test MNC were substituted with serum that was positive or negative for R F
activity in the Waaler-Rose test. When complement
and an RF-positive serum were added to a monolayer
of sensitized SRBC, a diffuse hemolysis of the monolayer was seen (Table 2). In addition, when the developing antiserum specific for human IgM was also
added, more hemolysis of the monolayer was seen. In
a monolayer of SRBC coated with normal rabbit IgG,
no hemolysis was seen when antilgM was substituted
with PBS or normal rabbit IgG in the same dilutions as
the developing antisera. RF-negative sera did not give
hemolysis.
When the RF-positive serum was preincubated
with heat-aggregated, reduced and alkylated human
IgG, the above-mentioned hemolysis of both kinds of
monolayers was totally inhibited (Table 2 ) . Preincubation with native human or rabbit IgG did not inhibit the
lysis. Lysis of the monolayer was also seen after the
addition of complement together only with heat-aggregated human IgG that had not been reduced or alkylated. The control experiments with an RF-positive serum show that the plaque assay has a distinct
specificity for R F and thus will also specifically detect
RF-secreting cells.
To demonstrate the class specificity of the
developing antisera, various experiments were made.
Table 3 shows a typical example for antiIgA-developing antiserum. It has been demonstrated by blocking
and myeloma protein experiments that antiIgA binds
IgA only in a sensitive ELISA system and in the
plaque assay used. A comparable specificity was obtained with antiIgG- and antiIgM-developing antisera.
Further specificity tests are described elsewhere (1 1).
DISCUSSION
IgM RF-secreting cells have earlier been detected by direct plaque assays (5,lO). It is believed,
however, that some IgM R F clones are not able to fix
complement in this assay (22), and in such cases direct
plaques (21) cannot be formed. In accordance with
these statements, we found that the addition of devel-
Table 2. Demonstration of the specificity and reactivity of the indirect plaque assay for rheumatoid factor-secreting cells*
Experiments performed on monolayers of
indicator SRBC coated with
Developing
antiserum
Complement
R/A IgG anti-SRBC
Normal rabbit IgG
Hemolysis experiments
RF-positive serum
RF-positive serum
RF-negative serum
RF-positive serum + RIA HAGG
RF-positive serum + IgGl
HAGG
PBS
AntiIgM
AntiIgM
AntilgM
AntilgM
PBS
+
+
+
+
+
+
10% hemolysist
40% hemolysis
Nil
Nil
40% hemolysis
>YO% hemolysis
Nil
40% hemolysis
Nil
Nil
40% hemolysis
>90% hemolysis
Plaque experiments
Synovial tissue MNC
Synovial tissue MNC
Synovial tissue MNC
Synovial tissue MNC
AntiIgG
AntilgA
AntiIgM
PBS
+
+
+
+
760 RF-SCB
180 RF-SC
380 RF-SC
<20 RF-SC
260 RF-SC
940 RF-SC
1 2 0 RF-SC
<20 RF-SC
* Abbreviations used in this table are: RF-SC (rheumatoid factor-secreting cells); SRBC (sheep erythrocytes); RIA (reduced and alkylated);
HAGG (heat-aggregated human IgG); PBS (phosphate buffered saline); MNC (mononuclear cells).
f' Judged by visual impression of the hemolytic disintegration of the monolayer.
$- Native human or rabbit IgG.
4 RF-SC/106 MNC.
RF-SECRETING CELLS
1449
Table 3. Demonstration of the class specificity for antiIgA-developing antiserum in a reverse
hemolytic plaque assay
MNC or
myeloma protein*
MNC
MNC
MNC
IgA
IgA
IgA
Developing
antiserum
AntiIgA
AntiIgA
AntiIgA
AntiIgA
AntiIgA
AntiIgA
Immunoglobulinst
Complementt
-
+
+
+
+
+
+
IgA
IgG or IgM
-
IgA
IgG or IgM
* MNC = mononuclear cells.
t -, without; +, with. IgA inhibits in concentration down to <1
Results
1,480 IgA-SCt
Nil
1,46011,520 IgA-SC
>90% hemolysis
Nil
>9Wc hemolysis
~ g / m lIgG
; and IgM do not inhibit in
excess amounts (400 Fg/ml).
$ IgA-SC/106 MNC. SC = secreting cells.
oping antisera developed more RF-secreting cells than
occurred when no such sera were added.
RF-secreting cells were detected in 7 of I 1
(64%) synovial tissue MNC samples from seropositive
patients. No RF-secreting cells were seen in 2 samples
from seronegative patients. A considerable number of
Ig-secreting cells of all classes were present in the
samples, whereas Ig-secreting cells were scanty and
RF-secreting cells not recovered in blood MNC. The
number of RF-secreting cells was not related to the
total number of Ig-secreting cells. Except for 1 patient,
secreted R F was always of IgG and IgA classes. IgM
RF-secreting cells were observed in fewer samples. In
some samples this could be a reflection of the very low
number of IgM-secreting cells, but in other samples it
was caused by an absolute low number of IgM RFsecreting cells, despite many IgM-secreting cells. Because of lack of eluted cells, we evaluated only optimal
dilutions of the developing antisera while testing peripheral blood MNC in the reverse assay. It is therefore a possibility that the dilutions might be suboptimal
in the indirect assay for RF-secreting cells. This would
explain some of the low numbers of IgM RF-secreting
cells.
RF-secreting cells were seen in synovial tissue
MNC from only seropositive patients. There was no
other correlation between the serum titer of R F and
the number of RF-secreting cells. However, some of
our synovial tissue MNC samples could have been
taken from joints that happened to have low or no RF
production despite a high serum R F titer and possibly
high RF production in other joints.
In most systems that detect IgG RF, it is
possible that the figures may represent some
understimulation because of the self-association of
IgG RF (23). R F activity may therefore be blocked
intracellularly or extracellularly ( 2 , 3 , 8 ) ,and the figures
on IgG RF-secreting cells are therefore minimum
figures. This phenomenon is not seen with the 2 other
classes of RF. This adds further evidence that IgG RF
is the major type of R F produced by synovial tissue
plasma cells. In addition, R F could have inhibited
complement fixation (24), but we always added excess
of complement to assure that this would not be a
limiting factor.
The specificity of the assays for RF-secreting
cells seems to be adequate since reduced and alkylated, heat-aggregated human IgG, but not native human
or rabbit IgG ( 2 5 ) , inhibited completely the hemolysis
of the indicator SRBC monolayer, which was otherwise seen after incubation with RF-positive serum,
antiIgM, and complement. Heat-aggregated human
IgG activated complement by itself and set off a
hemolysis. It is important to note that, except for the
few directly developed IgM RF-secreting cells in
monolayers of SRBC sensitized with reduced and
alkylated IgG anti-SRBC, no plaques were formed
with PBS o r normal rabbit IgG instead of the developing antisera. Hence, secreted R F did not bind the Fc
part of IgG in these antisera in a way that activated
complement to lyse SRBC and form plaques. The
plaques are therefore entirely defined according to the
specificity of the developing antisera.
In conclusion, the rheumatoid synovial membrane is, compared with blood, an active site for the
production of R F of IgG and IgA as well as IgM
classes. In addition, anti-rubella antibodies (18) and
other immunoglobulins of hitherto-unknown specificities are also produced in even amounts in affected
joints. Further, others (8) and ourselves (19) have
shown that synovial tissue MNC also secrete lymphokines in vitro without antigen and mitogen stimulation.
Many possible factors, including antigens, mitogenlike stimulation, o r coactivation (for instance by IgG-
EGELAND ET AL
1450
IgG RF complexes)(18), could have caused the stimulation of this local production of RF, other immunoglobulins, and lymphokines.
ACKNOWLEDGMENTS
Thanks are due to Professor E. Kiss and Dr. J. A.
Pahle, Oslo Sanitetsforening Rheumatism Hospital, and to
Dr. T. Ottesen, Det Norske Diakonhjem Hospital, for providing tissue for the investigation. The assistance by Mrs.
Margaretha W. Kabbe, Mrs. Anne-Marie Rasmussen, and
Mrs. Sissel Bergersen is gratefully acknowledged.
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