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Monocyte fc receptor function in rheumatoid arthritis. enhanced cell-binding of igg induced by rheumatoid factors

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MONOCYTE Fc RECEPTOR FUNCTION IN
RHEUMATOID ARTHRITIS
Enhanced Cell-Binding of IgG Induced by Rheumatoid Factors
SUSAN K. MAcKINNON and GORDON STARKEBAUM
Monocytes from 11 patients with rheumatoid
arthritis and 10 control subjects were purified by
countercurrent elutriation. Rheumatoid arthritis monocytes had more cell-associated IgG ( P < 0.001) and
bound more 1251-labeledheat-aggregated IgG in vitro (P
< 0.02) than did monocytes from control subjects.
Interaction of rheumatoid factor (RF) with monocytes
was then investigated. Purified '251-labeled IgM-RF
and IgG-RF bound directly to monocytes from normal
individuals. Furthermore, preincubation of normal
monocytes with RF augmented subsequent binding of
aggregated IgG to the cells. We conclude that monocyteassociated RF can enhance binding of IgG-containing
immune complexes to the cells and can exaggerate the
measured number of Fc receptors. Such cell-bound RF
may affect clearance of immune complexes by the
reticuloendothelial system in vivo.
Previous studies have shown that circulating
monocytes from patients with rheumatoid arthritis
(RA) bind increased amounts of IgG, apparently reflecting increased numbers of Fc receptors (1-3). Such
elevated monocyte Fc receptor numbers may reflect
monocyte activation or up-regulation of Fc receptors
due to the presence of gamma interferon, immune
complexes, or other soluble factors in the serum of
patients with RA (4-7). Splenic Fc receptor function,
From the Department of Medicine, Veterans Administration Medical Center and the Division of Rheumatology, Department
of Medicine, University of Washington, Seattle, Washington.
Supported by the Veterans Administration Medical Research Funds.
Susan K. MacKinnon, MD; Gordon Starkebaurn, MD.
Address reprint requests to Gordon Starkebaurn, MD,
Chief, Arthritis Section, VA Medical Center ( l l l ) , 1660 South
Colurnbian Way, Seattle, WA 98108.
Submitted for publication August 18, 1986; accepted in
revised form December 15, 1986.
Arthritis and Rheumatism, Vol. 30, No. 5 (May 1987)
however, measured in vivo, using IgG-coated erythrocytes, is depressed in many patients with RA, which
suggests decreased monocytelmacrophage Fc receptor
function (8).
The apparent paradox in RA-depressed reticuloendothelial function despite increased monocyte Fc
receptors-led us to reexamine binding of IgG to RA
monocytes and to investigate the potential role of
rheumatoid factors (RF) in enhancing interaction of
IgG aggregates with the cells. The results indicate that
monocytes isolated from patients with RA by
countercurrent elutriation show increased binding of
labeled aggregated IgG (aIgG), as well as increased
cell-bound IgG, compared with normal monocytes.
Incubation of some RA sera with normal monocytes in
vitro led to increased binding of labeled algG. Finally,
isolated 1gG-RF and IgM-RF were found to bind
directly to normal monocytes and to enhance binding
of aIgG to the cells.
These findings suggest that some of the increased Fc receptor expression on RA monocytes may
reflect interaction of cell surface-bound rheumatoid
factors with monomeric or aIgG used to quantitate Fc
receptors. Such cell-associated R F may affect handling of immune complexes by monocytes and
macrophages in vivo.
PATIENTS AND METHODS
Patients and controls. Monocyte studies were performed on 1 1 patients with longstanding, classic seropositive
RA seen at the Seattle Veterans Administration Medical
Center. Their ages ranged from 27-87 years; 9 were men
and 8 had extraarticular manifestations. All patients had
stage I1 or stage I11 disease based on the criteria of
Steinbrocker et a1 (9). All patients but 1 were taking
nonsteroidal antiinflammatory agents, and 6 patients were
MONOCYTE-BINDING RF IN RA
taking low-dose prednisone. Four patients were taking second-line antirheumatic drugs. Serum alone was also obtained
from 5 additional patients with classic RA. Five patients who
had chronic inflammatory diseases (cellulitis of the legs
caused by diabetes mellitus, carcinoma of the lung,
seronegative polyarticular juvenile arthritis, Reiter's syndrome, and ankylosing spondylitis with peripheral arthritis)
were studied as inflammatory disease controls, and 10
healthy laboratory personnel at the Seattle Veterans Administration Medical Center served as normal controls.
All studies were approved by the Human Subjects
Review Committee of the University of Washington, Seattle.
Monocyte preparations. Monocytes were purified by
a modification of the method described by Fogelman et a1
(10). Mononuclear leukocytes were obtained from EDTAanticoagulated blood, following centrifugation over FicollHypaque gradients. The mononuclear cells were then
washed twice in phosphate buffered saline (PBS) containing
1 mM EDTA and were resuspended in 25 ml PBS-1 mM
EDTA. This volume (containing 1.5-2.5 x lo8 cells) was
then loaded into the reservoir of the countercurrent centrifugal elutriation apparatus, a J2-21 centrifuge and a JE-6
elutriator rotor fitted with a standard separation chamber
(Beckman Instruments, Palo Alto, CA). A peristaltic pump
(model 1203; Harvard Apparatus, Millis, MA) was used to
pump buffer and mononuclear cells into the chamber. The
temperature within the rotor and chamber was 4"C, and the
elution buffer (PBS-1 mM EDTA containing 1% autologous
platelet-free plasma) was kept on ice. The rotor speed was
2,020 revolutions per minute, and pressure within the system
was kept below 5 psi.
With the pump off and the rotor on, mononuclear
cells were loaded into the reservoir over a period of 5
minutes. The initial flow rate of 10.3 mlhinute was maintained for 50 minutes, during which most of the lymphocytes
and contaminating red blood cells were removed. The flow
rate was then increased to 15.8 m h i n u t e , and the monocyte
fraction was collected in a volume of 400 ml. The monocytes
were washed 4 times in PBS-EDTA and adjusted to a
concentration of 1 x 107/ml. Smears were obtained for
a-naphthyl esterase staining, and for Wright's staining to
determine neutrophil contamination. Viability was checked
by trypan blue exclusion.
Measurement of cell-associatedIgG. After purification
of monocytes by elutriation, the cells were washed 4 times,
transferred to clean tubes, and counted. An aliquot of cells
was lysed by rapid freezing and thawing. IgG in the lysate
was measured using the F(ab')2 anti-I2jI-Fab technique,
described previously (1 1). The results are expressed as ng of
IgG/lo6 monocytes.
Preparation of aIgG. Intermediate-sized soluble aggregates of human IgG were prepared as described by
Jimenez and Mannik (12). Briefly, human IgG was purified
from Cohn fraction I1 by DEAE-cellulose chromatography.
The purified IgG was aggregated by heating for 11 minutes at
63°C in O.IM Tris HCl-O.15M NaCl buffer, pH 7.55. Soluble
aggregates were obtained by sequential gel filtration on
Sepharose 4B and Sephadex G-200 (Pharmacia, Piscataway,
NJ) equilibrated with the same buffer. These stable aggregates were approximately 22-253 in size and contained
<lo% monomeric IgG as determined by sucrose density
499
gradient ultracentrifugation. The aggregates were labeled
with '251(New England Nuclear, Boston, MA) by the
chloramine T method (13), and the labeled aggregates were
stored at -70°C in 7 different concentrations, ranging from
0.5-100 pg/ml, in Tris buffer containing 1% 'rabbit serum
albumin. Unlabeled aggregates of human IgG were prepared
by heating Cohn fraction I1 for 11 minutes at 63°C at a
concentration of 20 mg/ml in Tris buffer and were stored at
-70°C. Immediately prior to use, all samples were spun in a
Beckman microcentrifuge for 30 seconds to remove any
precipitated material.
Binding assay. Siliconized 10 x 75-mm glass tubes
were prewashed with PBS-1% bovine serum albumin (BSA)
and allowed to dry. One million monocytes were mixed with
each concentration of labeled aIgG, in a final volume of 250
PI, containing I% BSA. Each concentration was run in
triplicate. To determine nonspecific protein binding to the
cells, at each concentration of '251-aIgG, excess cold aIgG
ranging from 3&6,000-fold was added in parallel.
The cells were incubated with aggregates at 0°C for
18 hours with gentle shaking. Earlier studies had shown that
maximum binding of '*'I-aIgG to monocytes was reached
after 18 hours. Furthermore, <7% of the radioactivity not
bound to the cells was precipitable by trichloroacetic acid,
suggesting minimal internalization and catabolism under
these conditions.
Following incubation overnight, the cell suspensions
were layered over 180 pl of a mixture of phthalate oils
(dibutyl phthalate: I -bis-2-ethylhexyl phthalate, at 0.7: 1 by
volume; Eastman Organic Chemicals, Rochester, NY) in
400-pl polypropylene microfuge tubes. Following 30 seconds
of centrifugation at 4"C, the tubes were cut below the
aqueous-organic interface above the pelleted cells, and the
tips containing monocytes and bound radioactivity were
counted in a standard gamma counter.
Calculations and Scatchard plots. The amount of
radioactivity bound per lo6 cells was determined by subtracting the counts that represented nonspecific binding from
the total counts at each concentration. The results are
expressed as ng of aIgG/106 monocytes. Scatchard plots of
the ratio of bound a1gG:free aIgG versus bound aIgG were
plotted for the cells of each donor (14). The line best fitting
the results was determined by the least-squares method.
Polymorphonuclear cell contamination was always
<I%. However, various numbers of lymphocytes were
present in each monocyte preparation, ranging from 5-20%.
In initial studies, the lymphocyte fraction obtained by
elutriation of cells from patients with RA and from normal
individuals was found to bind approximately 40% as much
aIgG as did monocytes. This value probably overestimated
specific binding of aIgG to lymphocytes, as it reflected, in
part, nonspecific binding to contaminating red cells and red
cell debris present in that fraction. Nevertheless, to facilitate
comparisons between donors, the amount of aIgG bound by
lo6 monocytes was calculated according to the following
formula, using the above estimate of lymphocyte binding:
aIgG boundllO6 monocytes
=
aIgG bound/106 cells
%
' monocytes
+ % lymphocytes
x 0.4
x 100
MacKINNON AND STARKEBAUM
Preincubation experiments. Sera or EDTA-treated
plasma from 9 normal donors, 16 patients with RA, and 5
patients with inflammatory diseases were used in preincubations with normal cells. Sera and EDTA-treated plasma were
shown to have similar effects on monocytes with regard to
subsequent binding of '251-aIgG(data not shown). Serum RF
was determined by tube dilution latex agglutination. Five
million normal monocytes in a volume of 200 pl of PBS-1
mM EDTA were incubated at 20°C for 10 minutes with 7.24
p M 2-deoxyglucose to minimize ingestion of immune complexes. Five hundred microliters of serum or plasma was
added prior to incubation for an additional 60 minutes at
20°C with gentle shaking. Each cell suspension was washed
twice in PBS-EDTA-1% glucose, and twice more in PBSEDTA. Triplicate samples of 1 x lo6 cells were then
incubated overnight at 0°C with 1 pg '2sI-aIgG. The cells
were then spun through oil and the binding of aIgG was
calculated as before.
Effect of RF on binding of aIgG to monocytes. IgM-RF
were purified from RA sera by adsorption to human IgG
linked to Sepharose 4B, as described (15). IgG-RF, F(ab')'RF, and IgM-RF (16) were kindly provided by Dr. Francis
Nardella (University of Washington, Seattle). Normal human IgG was purified from Cohn fraction I1 by sequential
chromatography over DEAE-cellulose, followed by
Sephadex G-200 to obtain the monomer. A non-RF
monoclonal IgMK paraprotein was also provided by Dr.
Nardella. All proteins gave a single band corresponding to
monomeric IgG or IgM when analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis.
Five million monocytes were preincubated with 100
pg of protein for 60 minutes at 20°C. After 4 washes,
triplicate samples of 1 x lo6 monocytes were incubated with
1 pg '*'I-aIgG overnight at 0°C before spinning through oil as
described above.
Direct binding of RF to monocytes. Purified IgG-RF,
F(ab')z-RF, and IgM-RF, normal IgG, and F(ab')2, aIgG, and
non-RF IgM were radiolabeled with "'I by the chloramine T
method. Normal monocytes (3.5 X lo6) were incubated with
15 pg of protein in 700 pl of PBS-BSA for 60 minutes at 20°C.
Triplicate samples of 1 x lo6 cells were spun through oil and
the monocyte-bound protein was determined.
Statistics. The significance of differences between
mean values was determined using the pooled 2-tailed t-test.
Monocyte-bound IgG. Significantly more cellassociated IgG was present on RA monocytes, compared with normal monocytes: 45.0 2 14.2 ng IgG/106
monocytes versus 16.8 2 9.4 ng IgG/106 (P < 0.001)
(Figure 1). No correlation was found, however, between monocyte-bound IgG and maximum binding of
aIgG or RF titer (Table 1).
Monocyte binding of aIgG. After overnight incubation of monocytes with 1251-aIgG,maximum binding was calculated as noted in Patients and Methods.
Representative Scatchard plots for a normal subject
and an RA patient are shown in Figure 2. The results
indicate that monocytes from RA patients bound significantly more aIgG compared with that of controls:
51.7 -t 8.9 ng aIgG/106monocytes versus 40.0 2 11.1
ng aIgG/106 cells ( P < 0.02) (Figure 3). The slopes of
0
8
L
0
0
0
RESULTS
0
Monocyte preparation. Monocytes were purified by counterflow elutriation as described in Patients
and Methods. The yield from 100 ml of blood was
similar from patients with RA compared with that from
normal subjects: 32.1 ? 8.4 x lo6 cells versus 24.5 ?
11.3 X lo6(mean ? 1 SD). Likewise, the purity of the
monocytes, assessed by staining for nonspecific
esterase, was similar for RA patients and normal donors:
79 ? 7% versus 84 k 7%. The monocyte yield and
purity from control patients with inflammatory diseases were 51.6 k 9.4 x lo6 cells and 75 k 9%,
respectively.
A
0
w
0
m
0
Normals
n=8
RA
n=10
Figure 1. Monocyte-bound IgG in rheumatoid arthritis (RA)patients versus normal control subjects. The mean for the RA patients
was significantly greater than that for the normal subjects (horizontal bars) (P< 0.001).
MONOCYTE-BINDING R F IN RA
501
Table 1. Clinical and laboratory features of 11 rheumatoid arthritis patients
Patient
Erythrocyte
sedimentation
rate
(nmihour)
1
2
3
4
5
6
7
8
9
10
11
91
51
26
56
61
42
7
81
42
13
33
Rheumatoid
factor
(latex
fixation titer)
Maximum binding
of algC*
Cell-bound
IgGt
Effect of preincubation
with serum
on algG binding
(mean t I SD)S
48.6
52.2
48.8
55.1
52.1
74.9
52.2
53.2
42.1
45.9
42.8
69
ND
26
48
47
59
ND
45
58
ND
43
21.5 2 2.7
15.8 f 9.0
ND
20.7 t 2.9
13.6 f 0.6
15.0 ? 0.3
17.6 f 1.5
41.2 f 5.2
18.3 f 0.1
22.6 ? 4.1
32.4 7.0
1:640
1:640
1:320
1: 10,240
15,120
1:320
1:80
1:10,240
1:320
1:2,560
1 :20,480
~
~
~
* Nanograms of aggregated IgG (aIgC)/lO‘ monocytes, calculated from Scatchard plots after incubation of patients’ cells with aIgG overnight.
t Nanograms of IgG/lO‘ monocytes, measured on washed, purified patient monocytes. ND = not
done.
$ Nanograms of aIgG/106 monocytes, measured after preincubation of patient serum with normal
monocytes, followed by incubation with labeled aIgC (3 replicate determinations).
the binding curves, however, did not differ between
the 2 groups (data not shown). There was no correlation between maximum binding of aIgC and erythrocyte sedimentation rate or R F titer. Monocytes from 5
patients with inflammatory diseases bound 44.5 _t 7.9
ng aIgG/106 monocytes, which was not significantly
different from the binding by monocytes from normal
subjects. However, this value was also not significantly different from that found with monocytes from
RA patients ( P < 0.20), possibly because of the small
number of patients studied. These results indicate that
in addition to increased cell-bound IgC, monocytes
from patients with RA exhibited increased binding of
aIgG in vitro, compared with normal monocytes.
Effect of serum on binding of aIgG. We then
examined whether factors in RA serum could affect
binding of aIgG to normal monocytes in vitro. After
preincubation in normal serum for 1 hour at 20°C
followed by 4 washes, the binding of aIgG to normal
monocytes was slightly, but significantly, higher than
that seen with cells preincubated in buffer alone: 19.6
2 6.2 ng aIgG/106 monocytes (n = 11) versus 14.0 ?
6.1 ng aIgG/106 monocytes (n = 29) (mean 2 1 SD) (P
< 0.05). However, preincubation of normal monocytes with 2 of the 10 sera from the RA patients
previously studied (patients 8 and 11, Table 1) consistently resulted in increased binding of aIgG, which was
greater than that seen with 10 of 11 normal sera (Figure
4). Of 6 additional RA sera studied, 3 also induced
elevated binding of aIgG to monocytes; sera from the
5 patients with inflammatory diseases did not increase
binding of aIgC to monocytes (Figure 4).
Effect of RF. T o determine whether R F in RA
serum could augment binding of aIgG to normal
monocytes, IgG-RF and IgM-RF were purified from
sera as outlined in the Patients and Methods. Of the 6
IgM-RF tested, 4 increased binding of aIgG to
monocytes, compared with binding in buffer controls
or compared with a non-RF monoclonal IgM (Table 2).
Two IgG-RFs also increased binding of aIgG, whereas
normal IgG had no effect (Table 2). Similarly,
endotoxin did not increase binding of aIgG to
monocytes. These results indicate that some RFs can
increase binding of aIgG to normal monocytes.
In additional studies, binding of 12sI-labeledR F
to normal monocytes was measured. Both IgM-RFs
and 1 of 2 IgG-RFs tested were found to bind directly
to normal monocytes in greater amounts than did
monoclonal IgM or normal IgG (Table 3). Furthermore, F(ab’)z from 2 of 3 IgG-RFs bound to
monocytes in amounts that were more than twofold
higher than that seen with normal F(ab’)z (Table 3),
indicating that the IgG-RFs were not binding to the
cells by way of their Fc portion.
DISCUSSION
Countercurrent centrifugal elutriation proved to
be a practical method for isolating monocytes without
the need for adherence to surfaces or exposure to
hyperosmolar Percoll. The purity obtained by this
502
MacKINNON AND STARKEBAUM
.lo.
Our finding that monocytes from patients with
RA have increased binding of aIgG, which suggests
increased Fc receptor expression, is consistent with
findings by other investigators who have used different
monocyte purification methods, incubation times, and
IgG preparations (1-3). We found no correlation between maximum binding of aIgG and erythrocyte
sedimentation rate or RF titer. In contrast, Katayama
et a1 noted a correlation with R F titer ( l ) , and other
researchers have found a correlation with levels of
circulating immune complexes (2,3).
Recent studies indicate that human monocytes
possess 2 types of Fc receptors: a low-affinity receptor
for IgG aggregates or immune complexes (MW 72 kd)
and a high-affinity receptor for monomeric IgG (MW
40 kd) (21,22). Since the latter Fc receptor would also
be expected to bind multimeric IgG complexes, it is
.09
.08.
-07.
.06
Q)
2
*
2
a
n
.05
.04
8ol
-03
.02
70
.o 1
60
0
I
lb
I
20
I
30
-I
1
40
A
50
ng algG bound/ 1O6 cells
Figure 2. Representative Scatchard plots of monocyte binding data
from a rheumatoid arthritis patient (closed circles) and a normal
control (open circles). Each point represents a mean of triplicate
samples at each of 7 concentrations of aggregated IgG (aIgG).
method (6692%) compares favorably with that obtained by Percoll gradient centrifugation (2,3). Two
functionally different subsets of monocytes have been
obtained by other investigators by the elutriation
method: a larger, F c receptor-positive fraction and a
smaller, Fc receptor-negative fraction (17-19). It is
likely that we examined only the larger monocytes,
since the smaller monocytes were probably in the
discarded lymphocyte fraction. The main type of cells
contaminating our monocyte preparation were lymphocytes. We did not examine the Fc receptor function of these larger lymphocytes, although Carter et a1
found that the lymphocyte fraction obtained by Percoll
gradients bound only 5% of labeled IgG compared with
that bound by monocytes (20).
me
&
Normals
n=lC
8
RA
n=ll
Figure 3. Maximum binding of aggregated IgG (aIgG) to purified
monocytes from rheumatoid arthritis (RA) patients versus normal
controls. The mean for the RA patients was significantly greater
than that for the normal subjects (horizontal bars) (P< 0.02).
503
MONOCYTE-BINDING RF Ir\t RA
Table 2. Binding of aggregated IgG (aIgG) after preincubation of
normal monocytes with purified rheumatoid factors (RFs)
70
g
-
60-
r,
V
e
E
50a
z 40ID
0
\
0
3
0
30-
P
20
10
0 '
BSS with 1% BSA
Monomeric IgG
aIgG
IgG-RF Le
IgG-RF Ga
IgMK Gi
IgM-RF Ga
IgM-RF MO
IgM-RF HO
IgM-RF St
IgM-RF Li
IgM-RF Fu
Endotoxin, 10 pglml
14.0 t 6.1
13.2 2 6.2
3.7 ? 2.4
22.1 2 4.3*
58.6 2 5.9*
17.2 2 0.6
49.1 2 17.8*t
57.4 ? 22.Q*t
28.7 t 8.0*t
28.8 2 10.4*
22.9 t 3.8*t
20.6 2 4.0*
18.8 2 0.6
Number of
replicates
29
18
9
12
5
3
11
6
16
15
9
8
3
* Significantly increased compared with balanced salt solution
(BSS) containing 1% bovine serum albumin (BSA) (P< 0.01).
I' Significantly increased compared with IgMK (P< 0.05).
a
80
-
Preincbbation
condition
Binding of aIgG
(ng of aIgG/106
monocytes,
mean 2 1 SD)
Normals
ID
RA
n=9
n=5
n=16
Figure 4. Binding of aggregated IgG (aIgG) to normal monocytes
after preincubation in sera of normal controls, patients with inflammatory diseases (ID), and patients with rheumatoid arthritis (RA).
Five RA sera consistently augmented binding of aIgG over levels
seen in 10 of 11 normal sera.
likely that our IgG aggregates could bind td both
receptors. Likewise, the results of earlier studies using
aIgG (1,2) or monomeric IgG (3) are consistent either
with an itlcrease of the monomeric IgG receptors alone
or with an increase of both types of receptors. Studies
using the recently described anti-p40 monotlonal antibody (21) might resolve this question.
Previous studies have shown that RA monocytes are activated, with elevated synthesis of complement components (23), prostaglandin El (24), increased phagocytic activity (25,26), and increased
antibody-dependent cell-mddiated cytotoxity (ADCC)
(27). Increased Fc receptor numbers in these monocytes could reflect such activation, possibly resulting
from elevated levels of gamma interferon, seen in
some patients with RA (4-7). Alternatively, upregulation of Fc receptor numbers could result from
prolonged exposure to circulating immune complexes
(28), which are also present in many RA patients (29).
The finding of increased Fc receptors on ciraulating nlonocytes is in apparent conflict with the impaired splenic Fc receptor function seen in many
patients with RA (8). It is possible, however, that the
peripheral blood monocyte is an invalid model of the
fixed macrophage of the reticuloendothelial system
(RES). Furthermore, splenic clearance of IgG-coated
particles is not indicative of the Fc receptor functidn of
the entire RES, since soluble immune complexes are
predominantly taken up by the Kupffer cells of the
liver (12). Another explanation for the discrepancy
Table 3. Direct binding of IgG, F(ab'),, ahd IgM rheumatoid factors (RFs) to normal monocytes
Condition
IgMK Gi
IgM-RF HO
IgM-RF St
Normal IgG
Normal aggregated IgG
IgG-RF Ga
IgG-RF Le
Normal F(ab'),
F(ab'),-RF Ga
F(ab'),-RF Le
F(ab'),-RF Jj
Amount bound
(ng/1O6 cells,
mean ? 1 SD)
7.1
28.4
26.1
20.0
50.4
31.1
21.0
8.9
22.7
11.8
20.8
Number of
replicates
* 3.4
t 16.0*
2 7.1*
2 7.5
2 18.8t
2 10.8t
t 7.2
2 1.7
2 1.6$
2 1.6$
5 3.3$
12
8
8
18
12
9
9
6
6
6
6
* Significantly increased compared with IgMK (P < 0.001).
t Significantly increased compared with normal igG ( P < 0.005).
Significantly increased compared with normal F(ab')? ( P < 0.02).
t
504
may be that increased Fc receptor expression is only
apparent, due to the IgG probe binding to the
monocyte cell surface, through RFs. Rheumatoid factors might, in addition, affect clearance of immune
complexes by the RES.
Our results indicate that RA monocytes have
elevated levels of cell-associated IgG, compared with
levels in normal monocytes. The nature of the IgG
detected in this assay was not identified in these
studies, nor was the relative contribution of cell surface versus ingested IgG determined. However, immune complexes in RA sera have been shown to bind
to neutrophil Fc receptors, and it seems likely that
such immune complexes, some of which contain RFs
(13, might also bind to monocyte Fc receptors.
To examine the possibility that factors in rheumatoid serum could increase the binding of aIgG to the
cells, normal monocytes were preincubated in sera
from RA patients or normal controls prior to overnight
incubation with aIgG. Five of 16 RA sera consistently
caused increased binding of aIgG to normal monocytes, compared with sera from normal individuals or
patients with inflammatory diseases. The effect of RA
sera on monocyte Fc receptor function has also been
investigated by other researchers. Katayama (1) found
that sera had no effect on subsequent binding and
metabolism of aIgG by normal monocytes. In contrast,
Hoch and Schur, using as their probe of Fc receptor
expression the bihding and phagocytosis of IgG-coated
red cells by monocyte monolayers, found decreased
Fc receptor expression after preincubation with RA
sera (30). Using a similar assay, Temple and Loewi
(31) found no change in 6 of 8 sera, although 1 of the 8
did increase binding. Finally, Bar-Eli et a1 (32) found
that preincubation of adherent monocytes with RA
sera decreased subsequent ADCC for target tumor
cells. Since these investigators used different probes of
Fc receptor expression (aggregates, IgG-coated red
cells. ADCC), different methods of monocyte purification (adherence, thrombin clot), different concentrations and types of RA sera (5%, lo%, 50%; fresh,
heat-inactivated), and different incubation times abd
temperatures, their results are not directly comparable
with ours.
To determine whether R F in sera could be
responsible for increased binding of aIgG to the cells,
we preincubated purified R F with monocytes. Both
IgG-KF and IgM-RF caused increased binding of aIgG
to the cells. Endotoxin did not increase binding of
aIgG, suggesting that short-term nonspecific activation
of the monocytes was not responsible for elevated
MacKINNON AND STARKEBAUM
binding of aIgG. Not all of the purified rheumatoid
factors induced increased binding of aIgG, however.
This may reflect heterogeneity of RF (33) and could
explain the lack of correlation between R F titer and
amount of algG bound to the cells, noted by us and by
other investigators (2,3).
In further studies, labeled IgG-RF, F(ab’),-RF,
and IgM-RF were found to bind directly to normal
monocytes. Rheumatoid factors could bind to the
monocyte cell surface through several possible mechanisms. Nonspecific binding, or binding to a putative
IgM receptor on monocytes (34), appear unlikely as
explanations, since R F binding was in excess of that
seen that with non-RF IgG or IgM. Since the majority
of washed blood monocytes have detectable surfacebound IgG ( 3 9 , rheumatoid factors could bind to the
Fc region of such IgG. Alternatively, complementfixing immune complexes containing IgG may adhere
to the cell surface via Fc or C3b receptors and, in turn,
bind RF, or RF-containing immune complexes themselves might attach to these monocyte receptors. The
finding of increased cell-associated JgG in RA monocytes is consistent with such possibilities.
Rheumatoid factors with antinuclear reactivity
(36,37) could also react with monocyte surface-bound
nuclear antigens (38,39). Finally, IgG-RF and IgM-RF
from patients with RA have been shown to be
hyposialylated (40). Removal of sialic acid may allow
interaction of R F with lectin-like receptors on
monocytes (4 1,42) or may increase interaction with the
monocyte cell surface by decreasing the negative
charge on the rheumatoid factor molecule (43). After
binding to the cell surface through any of these possible avenues, R F could then bind labeled aIgG. Of
note, reactivity of rheumatoid factors with aIgG appears to be optimal with aggregates of molecular size
lo6 kd (44), similar to those used in our studies.
In vivo, the effect of interaction between rheumatoid factors and immune complexes at the
monocyte cell surface might be to increase the size of
the immune complexes, and thereby trigger ingestion
(45). In addition, activation of complement might be
facilitated, promoting ingestion by way of complement
receptors. Determination of the actual in vivo effects
of cell-bound rheumatoid factors in rheumatoid arthritis, however, will require further studies.
ACKNOWLEDGMENTS
We thank Dr. Francis Nardella for providing purified
rheumatoid factors and for reviewing the manuscript,
MONOCYTE-BINDING RF IN RA
Douglas Webster for his excellent technical assistance,
and Ronald Phillips and Donella Sawin for manuscript
preparation.
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