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Monoclonal rheumatoid factorigg immune complexes. poor fixation of opsonic c4 and c3 despite efficient complement activation

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Poor Fixation of Opsonic C4 and C3
Despite Efficient Complement Activation
Monoclonal IgM rheumatoid factor forms complexes with IgG in essential mixed cryoglobulinemia. We
demonstrate that such complexes fix C3 and C4 poorly,
although efficient fluid-phase C3 conversion can occur.
Fixation of small amounts of C4 may be sufficient to
generate a C3 convertase, but may prevent subsequent
fixation of C3 by competing for binding sites on the
complex. These complexes bind inefficiently to normal
erythrocyte complement receptor type 1 (CR1) in vitro,
and are undetectable on erythrocytes of patients with
essential mixed cryoglobulinemia in vivo. Clearance of
such phlogistic complexes from tissues by CR1-bearing
cells may be inefficient.
In essential mixed cryoglobulinernia (EMC),
monoclonal rheumatoid factor (mRF), usually IgM,
complexes with polyclonal IgG to form the characteristic cryoprecipitate (type 11 cryoglobulin) (1). The
clinical features consist of evidence of widespread
vasculitis (2) with purpura, arthralgias, glomerulonephritis, and neuropathy (2,3). Deposition of immune
complexes (IC) in tissues is a likely pathogenic mechFrom the Rheumatology Unit and MRC Clinical Immunology Research Group, Department of Medicine, Royal Postgraduate
Medical School, Hammersmith Hospital, London, UK.
Supported by a grant from the MRC. Dr. Ng is an MRC
Training Fellow.
Yin C. Ng, MA, MRCP: Research Fellow, Department of
Medicine, Hammersmith Hospital; D. Keith Peters, FRCP: Professor of Medicine, Hammersmith Hospital; Mark J. Walport, MA,
PhD, MRCP: Senior Lecturer in Medicine, Department of Medicine, Hammersmith Hospital.
Address reprint requests to Yin C. Ng, MA, MRCP, c/o 718
Wood Basic Science Building, Johns Hopkins University School of
Medicine, 725 N. Wolfe Street, Baltimore, MD 21205.
Submitted for publication March 3, 1987; accepted in revised form July 9, 1987.
Arthritis and Rheumatism, Vol. 31, No. 1 (January 1988)
anism, for the following reasons. 1) Immunoglobulins
and C3 have been demonstrated in vessel walls in
affected tissues such as skin (4), nerve ( S ) , and kidney
(6,7). The classes of immunoglobulin found in kidney
tissue were identical to those of the associated cryoglobulin (1 ,@.2) The cutaneous and renal lesions in
EMC are identical to those of acute serum sickness, an
IC disease (1). 3) Removal of cryoglobulin may be
associated with significant clinical improvement (5,9).
Tissue-bound mRF-IC probably accumulate under favorable local conditions, e.g., decreased temperature
at the peripheries (10) and raised protein concentration
at the end of glomerular capillary loops (11).
Recent evidence suggests that the erythrocyte
complement receptor (CR 1) in humans and other primates is important in the transport of circulating IC
that have fixed complement; these bind to CR1 via the
ligands C3b, iC3b, C4b, and iC4b. CR1 occurs on
phagocytic cells, but 90-95% of circulating CRl in
primates is found on erythrocytes (12). The role of
erythrocyte CR1 in IC transport was demonstrated in
the experiments of Cornacoff et a1 (13), who injected
'251-labeled IC into the aorta of baboons. Under circumstances in which the complexes failed to bind
efficiently to erythrocyte CR1, some IC were deposited in tissues outside the reticuloendothelial system
To investigate the manner in which the erythrocyte CR1 system handles IC formed by mRF and
IgG (mRF-IgG) in vivo and in vitro, we used patients
with EMC as a human model of IC disease. Patients
with EMC have no detectable erythrocyte-bound IC.
In vitro studies show that mRF-IgG IC do not bind
efficiently to normal erythrocyte CRl. Such com-
plexes fix C3 a n d C4 inefficiently, despite detectable
fluid-phase complement activation.
Monoclonal RF-IgM. Blood specimens were obtained
from EMC patients who had mRF that was IgMK. Serum and
plasma EDTA (10 mM, pH 7.4) samples were separated
within 1 hour of collection and stored at -70°C until
Cryoglobulins. Blood taken from patients with EMC
was allowed to clot at 37°C. Serum was separated, 10 mM
azide was added, and the mixture was stored at 4°C for 5
days. The cryoprecipitate was washed 5 times in phosphate
buffered saline (PBS; pH 7.2)/azide (10 mM) by centrifugation at 1,8008 for 25 minutes, resuspended in PBS/azide, and
stored at 4°C.
Monoclonal RF-IgM was purified from the cryoprecipitates of 2 patients with EMC. IgM was separated by
fractionation on Sephacryl S300 (Pharmacia, Uppsala, Sweden) in 0.6M sodium acetate buffer, pH 4.4, and dialyzed
extensively against PBS before use. IgM preparations retained rheumatoid factor activity with latex titers of 1 :1,280
and 1540, respectively. Evidence in support of the monoclonality of these RF is as follows. First, the RF was
restricted to the K light chain type. Second, using monoclonal antibodies raised against private idiotypes on these RF,
idiotype-positive B cells were detectable in peripheral blood
from these patients. Furthermore, these cells were all shown
by 2-color immunofluorescence to be K- and p-positive
(Grennan D: unpublished observations). Third, clonal rearrangement of Ig genes was demonstrated in peripheral blood
lymphocytes from these patients, using a genomic probe for
the JH region of the heavy chain (Foroni L: unpublished
Normal human IgG was prepared by fractionation of
normal human serum (NHS) on DEAE Sephacel (Pharmacia) in 15 mM phosphate buffer, pH 7.6. The eluate was
pooled, concentrated, and dialyzed against PBS before use.
On immunoelectrophoresis, this preparation produced a
single line against anti-whole human serum. Heat-aggregated IgG (HAG) was prepared by heating normal human
IgG (50 mg/ml) at 63°C for 20 minutes. After centrifugation at
143,OOOg for 90 minutes, the pellet was resuspended in PBS
and the HAG was stored at -70°C. Clq was prepared from
NHS by the method of Reid (16). Bovine serum albumin
(BSA) and ovalbumin (OVA) were purchased from Sigma
(Poole, UK).
Antibodies. The antibodies used in this investigation
were obtained from the following sources. Anti-CRl was a
murine monoclonal antibody, El I , which was a gift from Dr.
N. Hogg (Lincolns Inn Fields, UK). Anti-C3d was a rat
monoclonal antibody, clone 3, a gift from Professor P. J.
Lachmann (Cambridge, UK). Anti-C4d was a rat monoclonal antibody, D5.87.5, a gift from Dr. R. A. Harrison (Cambridge, UK). Anti-human IgG was a murine monoclonal
antibody, 1410KG7, a gift from Dr. G. D. Ross (Chapel Hill,
NC). In 2 patients, an affinity-purified goat-anti-human IgG
was used. AntLC3 and anti-C4 were rabbit IgG from Dako
(Copenhagen, Denmark). F(ab'), preparations of the antLC3
and anti-C4 were prepared in our laboratory according to the
method described by Lachmann ( 1 7). Rabbit-anti-rat IgG
was purchased from Sigma, and was affinity-purified.
Normal human erythrocytes were obtained from
healthy laboratory workers. Heparinized blood (1 0 unitdml)
was separated by Ficoll-Hypaque (Pharmacia) density centrifugation at 800g for 15 minutes at room temperature. The
erythrocyte pellet was washed 3 times in PBS and resuspended in an equal volume of 0.2% OVAIPBS.
Immune complexes. '251-labeled BSA-anti-BSA IC
were prepared according to the method described by Medof et
a1 (I@, with minor modifications. 1251-labeledBSA (specific
activity 5.25 pCUpg) was incubated with heat-inactivated
rabbit anti-BSA, which was diluted 200-fold in 0.2% OVA/complement fixation diluent (CFD; Oxoid, Basingstoke,
UK). In a total volume of 250 pl, 124 ng of '2SI-labeled BSA
was incubated with a fourfold excess of antibody at 37°C for
30 minutes, and the IC mixture was diluted fourfold in 0.2%
OVNCFD before use. The IC were non-precipitating.
12%labeled mRF at 500 pg/ml (specific activity 7
pCi/mg) was added to NHS to form 12SI-labeledmRF-IgG
IC. '2SI-labeled mRF-IgG IC were formed in vitro by incubating 50 pg mRF with 134 pg normal IgG (equivalence as
determined by precipitin curve), in a total volume of 20 pl,at
37°C for I hour and then at 4°C overnight. The IC were
centrifuged at 2,OOOg for 15 minutes and diluted fourfold in
OVA/CFD before use. 1251-labeledcryoglobulin (10 mg/ml)
from patient 4 was labeled to specific activity 2.7 pCi/mg.
Tetanus toxoid (TT)-anti-TT IC were a gift from Dr.
J. A. Schifferli (Clinique Medicale, HBpital Cantonale, Geneva, Switzerland). These were non-precipitating and prepared at twentyfold antibody excess from TT (gift from Dr.
H. Furer, Berne, Switzerland) and anti-TT antiserum (human
IgG; purchased from Berna, Berne, Switzerland).
Radiolabeling of cryoglobulin with 1251was performed using chloramine T (19). Staphylococcus protein A
(Pharmacia) was labeled by the method of Bolton and
Hunter (20). All other proteins were labeled by the iodogen
method (21).
Binding of IC to normal erythrocytes in vitro. 12%
labeled IC were incubated at 37°C with an equal volume of
serum or, as a negative control, lOmM EDTA serum. At
intervals, they were transferred to ice and 650 p1 of normal
erythrocytes (-2 x lo9) was added. They were washed 3
times in 0.2% OVAIPBS by centrifugation at 400g for 3
minutes, and bound counts were then measured. Specific
binding of IC was measured by subtracting the counts
obtained in EDTA serum.
Measurement of IC-associated C3 and C4. Solidphase
C l 9 binding assay. Plastic plates (Dynatech, Chantilly, VA)
were coated with 1 pg C Iq/well and stored overnight at 4°C.
These were washed 5 times in PBS/IO mM azide/0.2%
OVNO.O5% Tween 2040 mM EDTA, pH 7.4. Fifty microliters of the sample was added and incubated overnight at 4°C.
After 5 washes, this was probed with '2SI-labeled protein A
(specific activity 2.6 pCilpg) or 12sI-labeled clone 3 (specific
activity 0.34 pCilpg) or monoclonal anti-C4d followed by
1251-labeledrabbit-anti-rat IgG (specific activity 2.8 pCiIpg)
yielding 40,000-50,000 counts per minute/well. After 3 hours
at room temperature, 5 washes were performed, and the
radioactivity in the wells was counted.
Coprecipitation with anti-C3 and anti-C4. Radiola:
beled IC were prepared as previously described and were
incubated with NHS at 37°C for 5 minutes, then transferred
to ice. Conditions for precipitation of 0.4 pg '2SI-labeled
BSA-anti-BSA IC were optimized using different volumes of
the F(ab'), preparations. Precipitation of RF-containing IC
formed from 0.3 pg 12'I-labeled mRF in 5 pl NHS was not
significantly altered by varying the amounts of F(ab'),
added. The '251-labeled IC were incubated with F(ab'),
antLC3 or anti-C4 in the presence of 10 mM EDTA, pH 7.4,
and incubated at 4°C overnight. The IC were centrifuged at
1,200g for 30 minutes and washed twice in PBS, and the
radioactivity in the pellet was counted.
Detection of erythrocyte-bound IC in vivo. Erythrocyte-associated IgG was measured using 12sI-labeledantihuman IgG. Preliminary experiments were performed to
distinguish between IgG bound to erythrocytes as autoantibodies and IgG bound to erythrocyte CRI in the form of IC.
Factor I releases IC from erythrocyte CRI by cleaving iC3b
to C3dg, which is no longer a ligand for CR1. Erythrocytes
bearing '2'I-labeled BSA-anti-BSA IC were incubated with
varying dilutions of factor I-containing plasma, to establish
conditions for decreasing the rate of release of bound IC. It
was demonstrated that plasma diluted a hundredfold released 22% bound IC after 1 hour at 4"C, compared with
plasma diluted twofold, which released 79% of bound IC.
To measure CR1-bound IC, an aliquot of freshly
drawn blood was immediately diluted a hundredfold in icecold PBS, and a second aliquot was collected in 10 mM
EDTA, pH 7.4, at room temperature, to allow factor I to
release bound IC. Within 20 minutes of collection, the cells
were washed twice, the buffy coat discarded, and the cells
resuspended to 5 x 108/ml,as determined by Coulter counter.
Positive controls. Erythrocytes bearing opsonized
TT-anti-TT IC. Four hundred microliters of IC was incubated with 400 pl of NHS, in a total volume of 3 ml at 37°C
for 2 minutes, and was added to lo' normal erythrocytes on
ice. After I wash by centrifugation at 400g for 5 minutes,
these were divided into 2 aliquots. One aliquot was immediately washed in the volume of PBS x 100, and the other was
incubated with an equal volume of autologous plasma EDTA
(10 mM, pH 7.4) at room temperature for 30 minutes to allow
C3b cleavage by factor I.
Erythrocytes bearing opsonized HAG. Five microliters of HAG (2.5 mg/ml) was incubated with 5 p1 of NHS at
37°C for 5 minutes, added to 4 x 10' normal erythrocytes on
ice, and washed 3 times in PBS by centrifugation at 400g for
3 minutes.
Quantitation of erythrocyte-associated antigens. E 1 1
was labeled to a specific activity of 0.6 pCi/pg and used at 2
pg/ml concentration. Clone 3 was labeled to specific activity
3 pCi/pg and used at 2 pg/ml concentration. Goat antihuman IgG was labeled to specific activity 3 pCi/pg and used
at 0.3 pg/ml concentration. 1410KG7 was labeled to specific
activity 95 pCilmg and used at a concentration of 28.7 pg/ml.
Erythrocyte-associated antigens were enumerated
by the method of Walport et a1 (22). Briefly, erythrocytes
were washed in 1% BSA/PBS. Then, 1.5 x 10' erythrocytes
were incubated with the appropriate radiolabeled antibody in
a final volume of 375 pI at 37°C for 30 minutes. Aliquots (125
pl) were layered onto oil (dibutylpthalate [BDH, Poole, UK]
and dinonylpthalate, 4: 1 volume/volume), microfuged at
10,000g for 1 minute, and frozen at - 70°C. The erythrocyte
pellet was clipped off and bound counts were measured.
C3 conversion of NHS incubated with various 1C at
37°C for 30 minutes was measured by crossed electrophoresis (23).
Complement components. Serum concentrations of
C3 and C4 were measured by single radial immunodiffusion.
Total hemolytic complement (CH50) and factor B were
measured by a hemolytic plate technique (24). Plasma C3d
was measured by the method of Perrin et a1 (25), using single
radial immunodiffusion and a rabbit anti-C3d (Dako).
Rheumatoid factor activity was measured by agglutination of IgG-coated latex particles (Ortho, Beerse, Belgium). All assays were performed in duplicate.
We studied the binding to erythrocyte CR1 of
IC that contained mRF derived from patients with
EMC. IC containing mRF were prepared in 3 different
ways, then incubated with NHS and their binding to
normal erythrocytes was measured.
First, '251-labeled mRF was added to NHS,
incubated at 37"C, and binding to erythrocytes was
measured. As shown in Figure lA, no binding occurred. In contrast, '251-labeled BSA-anti-BSA IC
bound readily to erythrocytes after 5 minutes. No
significant fluid-phase C3 conversion of NHS occurred
with added mRF or with BSA-anti-BSA IC (Table 1).
The fixation of C3 and C4 to IC was measured using a
coprecipitation assay with F(ab'), anti-C3 and F(ab'),
anti-C4; whereas BSA-anti-BSA IC fixed C3 and C4,
mRF-IgG IC fixed neither of these complement components (Table 1).
Second, because it was possible that complement activation by such IC would be more efficient
(26), '251-labeledmRF-IgG IC were preformed in vitro.
Figure 1B shows that preformed mRF-IgG binds very
poorly to normal erythrocytes in vitro, compared with
BSA-anti-BSA IC. However, incubation of preformed
mRF-IgG IC with NHS gave 80% conversion of C3
(Table 1).
Third, we have previously noted that cryoglobulins derived from patients' serum could activate
complement in the fluid phase. Therefore, we tested
'251-labeled cryoglobulin for its ability to bind to
erythrocytes following complement activation. As
shown in Figure lC, such IC did not bind significantly
to normal erythrocytes. However, cryoglobulin incubated in NHS gave 50% conversion of C3 (Table 1).
The amount of C3 and C4 fixed to cryoglobulin that
activated complement in the fluid phase was mea-
M i n u t e s a t 3loC
Figure 1. Binding to normal erythrocytes (- 2 x 109 of monoclonal rheumatoid factor (mRF)-lgG immune complexes (IC) (O),compared with
1.6 pg of bovine serum albumin (BSA)-anti-BSA IC (+) incubated with 10 p1 of normal human serum (NHS). A, 'Z51-labeledmRF (0.5pg)
incubated with NHS; B, '*'I-labeled mRF-lgG IC formed in vitro (2 pg); C, '251-labeled cryoglobulin (1 pg).
sured, using a solid-phase Clq binding assay. HAG
and BSA-anti-BSA IC opsonized in NHS had detectable IgG, C4d, and C3d; in contrast, only IgG was
detected in the cryoglobulin (Figure 2).
Thus, mRF-lgG IC generated in each of 3
different ways were inefficient at fixing C4 and C3 and
binding to erythrocytes. The possibility that mRF
directly inhibits the fixation of C3 and C4 to IC was
investigated by the addition of mRF to BSA-anti-BSA
IC before incubation with NHS. Monoclonal RF had
Table I .
Complement activation by various immune complexes
C3 conversion of NHS, %
Binding to normal
erythrocytes, % 1C
C3 fixed to IC, %
C4 fixed to IC, %
* One hundred percent C3 or C4 fixation to IC was defined as the
amount of fixation by bovine serum albumin (BSAkanti-BSA. The
amount (by weight) of various rheumatoid factor-containing ICs
studied was comparable with that of BSA-anti-BSA IC, and did not
exceed a 2.5-fold excess. mRF = monoclonal rheumatoid factor;
NHS = normal human serum; ND = not done.
no effect on fluid-phase C3 conversion of NHS by
BSA-anti-BSA IC (Table 2). In contrast, the binding
of BSA-anti-BSA IC to erythrocytes and the amount
of C3 fixed to these IC, as measured by coprecipitation
with F(ab'), anti-C3, were both significantly reduced.
Addition of mRF to BSA-anti-BSA IC that had been
preopsonized with NHS had no effect on their binding
to erythrocytes (data not shown).
Further experiments were performed to investigate aspects of processing of 1C in patients with
EMC. To examine the binding of IC to erythrocyte
CRl in vivo, we measured the amount of IgG bound to
erythrocytes in the presence or absence of factor I,
using a monoclonal anti-human IgG. Normal erythrocytes bearing TT-anti-TT IC, when exposed to factor
I, showed a marked reduction in erythrocyteassociated IC (Figure 3). In contrast, 3 patients with
EMC had no significant erythrocyte-associated IgG, in
the presence or in the absence of factor I. Erythrocytes from 2 additional patients with EMC were studied using erythrocytes bearing HAG as positive controls and a goat-anti-human IgG. There was no
increase in erythrocyte-associated IgG compared with
that of normal controls in the presence or in the
absence of factor I (data not shown). Fourteen healthy
subjects had an average of 220 k 37 (SEM) molecules
Normal E
Factor I'absent
Normal E
1 + IC
1 ;resent
Figure 3. Erythrocyte (Etassociated IgG in the presence or the
absence of factor I. Freshly drawn erythrocytes from 3 patients with
essential mixed cryoglobulinemia (EMC), compared with normal
erythrocyte bearing opsonized tetanus toxoid-anti-tetanus toxoid
immune complexes (IC). Values shown are the mean & SEM.
Figure 2. Measurement of A, C3d and B, C4d. C3d associated with
1C bound to the Clq plate. Unlabeled 1C were incubated with NHS
at 37°C for 30 minutes. The mixture was diluted before use. IC used
were 2.5 pg heat-aggregated IgG (HAG), 5 pg BSA-anti-BSA, and
12.5 pg cryoglobulin (cryo). EDTANHS = EDTA-treated normal
human serum. See Figure I for other definitions.
IgGferythrocyte in the absence, and 222 +- 37 in the
presence, of factor 1.
There was no increase in erythrocyte-associated C3d in the patients (Table 3). Their erythrocyte
CR1 numbers were also within the normal range (data
Table 2. Effect of mRF (0.3 mg/ml) on C3 conversion and
opsonization in NHS of BSA-anti-BSA IC*
Control mRF
C3 conversion, % total C3
Binding to normal erythrocytes, % yielded counts
Anti-C3 coprecipitation, % yielded counts
not shown). In contrast, levels of plasma C3d were
raised in 4 patients (Table 3). Together with the
decreased concentrations of C4, these results suggest
complement activation in vivo; together with normal
factor B concentrations, they suggest activation by the
classical pathway.
The lack of erythrocyte-associated IC in patients with EMC was consistent with the poor C3
fixation by RF-IgG IC demonstrated in vitro. Two
additional factors that could contribute to failure of IC
binding to erythrocytes in these patients were hypocomplementemia and the presence of RF (Table 3).
Therefore, the ability of sera derived from patients
with EMC to opsonize '251-labeledBSA-anti-BSA IC
for binding to normal erythrocyte CRl was examined.
Three of these sera had a reduced capacity to bind
BSA-anti-BSA IC to erythrocytes, but surprisingly, 2
sera exhibited normal binding (Table 3). This normal
binding is apparently in conflict with the finding that
purified mRF inhibits the binding of BSA-anti-BSA IC
to erythrocytes (Table 2 and Figure 4). This inconsistency may be due to the different affinity of RF for
rabbit IgG (27).
* BSA-anti-BSA IC (4 mg/ml) were prepared at fourfold antibody
excess. Results are the average of 2 experiments. See Table 1 for
We have demonstrated that IC of mRF with IgG
do not bind to normal erythrocyte CRI . This is associ-
Table 3. Serum rheumatoid factor (RF), complement, and ability to bind immune complexes (IC) to normal erythrocytes (E) in vitro, in
patients with essential mixed cryoblobuEnemia*
1 540
(60-1 35)
Factor B
(50-1 25)
1C binding
to E
1 I5
* Results were measured as follows. RF: latex titer in serum stored at -70°C; C3, C4, CHSO, and factor B: % pooled normal human serum;
plasma C3d: % of standard pool of zymosan-treated plasma; IC binding to E: 'Z51-labeledbovine serum albumin-anti-bovine serum albumin IC
binding, expressed as % yielded counts; E-C3d: molecules/cell. Values in parentheses are normal range.
ated with limited fixation of C4 and C3 to the IC. Under
some circumstances, however, there is evidence of
significant fluid-phase complement activation by such
IC. This contrasts with BSA-anti-BSA IC, in which
efficient opsonization by NHS is associated with weak
C3 conversion. The poor opsonization of mRF-IgC IC
in vitro is consistent with the absence of detectable IC
on erythrocytes from patients with EMC.
The poor C3 fixation by RF-IgG IC observed in
our experiments has been shown previously by other
workers. Balestrieri et al (28) demonstrated that IC
formed by the addition of monoclonal RF to NHS
failed to bind to conglutinin, which binds iC3b. Druet
et a1 (6)described 2 patients with EMC whose IgM-IgG
cryoglobulins contained no C3. This was in contrast to
patients in the same series with acute endocapillary
proliferative glomerulonephritis and membranoproliferative glomerulonephritis who had cryoglobulins containing C3. Similarly, Balazs and Frohlich (29) did not
detect C3 in RF-containing IgM-IgG cryoglobulins,
although these were capable of reducing the hemolytic
titer of fresh complement.
In contrast, other work has suggested that
complement fixation by RF-IgG IC does occur. There
are 2 points, however, to be considered in the interpretation of these studies. First, some studies demonstrated complement activation in the fluid phase only,
and the investigators did not pursue fixation of complement by the IC (30). Second, complement activation by IgM may result in C4 being deposited on
adjacent surfaces, and not on the IgM molecule itself.
Circolo and Borsos (31) eluted IgM from its antigen
following complement activation. Whereas residual C4
molecules that were hemolytically active were detectable on the antigen, no C4 was associated with the
eluted IgM. Several investigators demonstrated complement fixation in association with RF-IgG IC, but do
not distinguish complement deposited on adjacent
surfaces from complement that is fixed to IgM itself
(32-34). Others reported the demonstration of C3containing RF-IgG IC in patients' sera, but their
studies were performed under conditions that did not
exclude in vitro binding of RF to complement-fixing
IgG 1C (35,36).
Reports of IgM-IgG cryoglobulins containing
Dilution of It!!
Figure 4. Effect of monoclonal rheumatoid factor (MRF) on binding
of '251-labeledBSA-anti-BSA 1C to normal erythrocytes by NHS.
Decreasing amounts of MRF were added to the 1C before opsonization at 37°C for 5 minutes. Neat MRF represented a latex titer of
1: 1,280. Values shown are the mean of 2 experiments. See Figure 1
for other definitions.
C3 are apparently incompatible with our observations.
With one possible exception (37), however, none of
these reported C3-containing cryoglobulins were obtained from patients with EMC, but came from patients with systemic lupus erythematosus (38), Sjogrens syndrome (39), and various glomerulonephritides (6,40); indeed, many of these cryoglobulins did not contain RF.
Significant fluid-phase C3 conversion was detected using preformed mRF-IgG IC, a finding that is
consistent with other reports (41); however, C3 fixation by these complement-activating IC was poor.
Similarly, when mRF was added to BSA-anti-BSA IC,
there was decreased opsonization of these IC, but no
decrease in fluid-phase C3 conversion of NHS. The
effect of mRF on complement activation by IC is
complicated, because it depends on the balance between 2 opposing effects. Monoclonal RF binds to the
Fc portion of IgG (42) and decreases complement
activation, probably by interfering with the C1 binding
site (43,44). It also increases lattice size of IC (26),
which favors complement activation (45) and will itself
activate complement in the staple configuration (46).
Thus, rnRF inhibits complement activation by IC in
some circumstances (47,48), but increases complement activation in others (49,50).
The effect of rnRF on complement activation by
BSA-anti-BSA IC shown in Table 2 may be interpreted as follows: mRF inhibited complement activation by BSA-anti-BSA 1C. It was itself an activator of
complement (as shown by C3 conversion of NHS), but
unlike IgG, it was a poor acceptor of C4 (31), so there
was no fixation of C4 or C3 to the IC.
These results show that preformed RF-IgG IC
activate complement but do not fix C3 or C4 efficiently. While it is possible that failure of RF to bind
C4 and C3 is secondary to denaturation of the RF
during purification, this does not appear to explain the
identical findings with whole cryoprecipitate. Further
findings were consistent with poor binding of C3 and
C4 to mRF-IgG IC in vivo: No IC were detected bound
to erythrocytes freshly drawn from patients with
EMC. The significance of these findings is unclear,
however, because even in diseases with circulating
C3-opsonized IC, such as systemic lupus erythematosus, erythrocyte-associated IC have been demonstrated in only a small proportion of patients (51).
Further evidence for absence of erythrocyte-bound IC
in EMC is found in the normal amounts of erythrocyteassociated C3d in these patients: Binding of IC to
erythrocytes in vitro is accompanied by bystander
deposition of C3dg on the cells, which persists even
after the IC has been released (52).
The mechanisms for the poor incorporation of
C4 and C3 into RF-containing ICs in the presence of
efficient fluid-phase complement activation are unclear. Poor binding of C4 and C3 may be a general
property of IgM-containing IC and is consistent with
the relative insensitivity of the Raji cell assay for
IgM-containing IC (53); this assay detects primarily
iC3b and C3dg (45). The numbers of acceptor sites for
C3 and C4 on these RF-IgG IC may be enough to fix
C4 in small amounts, which may be sufficient to
generate a C3 convertase (5433, but subsequent
fixation of significant amounts of C3 may be prevented
by competition for the same type of hydroxyl- or
amino-binding site (56).
The poor fixation of C4 and C3 by mRF-IgG IC
shown in these studies would result not only in failure
of transport of circulating IC by erythrocyte CR1 to
the reticuloendothelial system, but also in inefficient
removal of IC from tissues by phagocytic cells, because Fc receptors would be acting alone without the
synergy provided by ligand-occupied CR1 (57,58).
Similar mechanisms may contribute to inefficient removal of ICs of polyclonal RF and IgG from joints of
patients with rheumatoid arthritis. The persistence of
IC in tissues has 2 potential consequences. First, the
multivalency of IgM may contribute to enlargement of
these IC in situ by successive trapping of antigen and
antibody molecules (59). The higher affinity of RF for
IgG that is surface-bound rather than monomeric
would favor IC formation in these circumstances (60).
Second, although there is no opsonization of the IC,
complement activation results in bystander fixation of
C4 and C3 to surrounding tissues, and this results in
tissue injury.
We thank Dr. C. G. Winearls for helpful discussions.
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