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Decreased capacity to solubilize immune complexes in sera from patients with systemic lupus erythematosus.

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arthritis
and
rheumatism
Official Journal of the American Rheumatism Association Section of the Arthritis Foundation
DECREASED CAPACITY TO SOLUBILIZE IMMUNE
COMPLEXES IN SERA FROM PATIENTS WITH
SYSTEMIC LUPUS ERYTHEMATOSUS
M. TERESA AGUADO, L. H. PERRIN, P. A. MIESCHER, and P. H. LAMBERT
The capacity to solubilize immune complexes
formed in vitro is significantly decreased in patients with
systemic lupus erythematosus (SLE). This complement
function correlates significantly with serum C3 levels
and inversely with the presence of circulating immune
complexes. Clinically, patients with a decreased capacity to solubilize complexes show a worse evolution and
an increased incidence of renal involvement. In conclusion, the impaired solubilization capacity, related to
low complement activity, which is observed in patients
with SLE may favor the persistence of immune complexes in tissues.
The degree of lattice formation of immune complexes is dependent on the properties of both the antigen and the antibody, the size of the antigen, the number and type of antigenic determinants, the class of the
antibody, and the avidity of each antibody for its corresponding antigen (1,2).
In 1975, Miller and Nussenzweig demonstrated
that antigen-antibody aggregates formed in vitro can be
solubilized by addition of fresh serum. This phenomeFrom the WHO Immunology Research and Training Centre,
Department of Medicine, HBpital Cantonal, University of Geneva,
Switzerland.
This work was supported by the Swiss National Foundation
(grant no. 3.908.0.80), the Dubois-Femtre-Dinu-Lippatti Foundation, and the World Health Organization.
M. Teresa Aguado, PhD: Research Fellow; L. H. Pernn,
MD: Senior Research Associate; P. A. Miescher, MD: Professor of
Medicine; P. H. Lambert, MD: Head, WHO IRTC, WHO Immunology Research and Training Centre, Department of Medicine, HBpital
Cantonal, University of Geneva, Switzerland.
Address reprint requests to P. H. Lambert, WHO Research
and Training Centre, Centre de Transfusion, HBpital Cantonal, 121 1
Geneva 4, Switzerland.
Submitted for publication December 1, 1980; accepted in revised form May 6, 1981.
non depends essentially on the integrity of the complement alternative pathway (3). The solubihtion of
immune complexes requires the fixation of complement
components, particularly C3, which leads to the release
of smaller immune complexes from the lattice (4).
Our aim was to evaluate the in vivo significance
of this phenomenon. We measured the capacity to solubilize immune complexes in sera from patients suffering from systemic lupus erythematosus. In this disease,
immune complex deposits are observed in various tissues, and the plasma levels of components of classical
and alternative pathway are decreased (5-7). We have
related the solubilization capacity to the complement
activity, the presence of circulating immune complexes,
and the clinical expression of the disease.
MATERIALS AND METHODS
Patients and sera. In this study, we analyzed 32 sera
from 20 patients with systemic lupus erythematosus (SLE)
who had been hospitalized or seen as outpatients at the University Hospital of Geneva. Patients were diagnosed according to the criteria of the American Rheumatism Association
(8). Sera from 26 blood donors of the Blood Bank of Geneva
were used as controls. Human venous blood was allowed to
clot in glass tubes for 1 hour at room temperature and for 2
hours at 4°C. After centrifugation, the sera were frozen at
-7OOC. Normal serum pools were prepared with sera from 30
normal blood donors and were used either fresh, heated at
56OC for 30 minutes, or heated at 5OoC for 20 minutes.
Reagents and solutions. Bovine serum albumin (BSA)
(Calbiochem Behring Corporation, La Jolla, CA) was labeled
with '*'I by the method of chloramine T (9). Buffered saline
veronal supplemented with 0.15 m M Ca++ and 0.5 mM Mg++
(VBS") was used as diluent.
Immune precipitates were prepared by incubating
rabbit antiserum to bovine serum albumin with Iz5I-labeled
BSA at 4 x antibody excess for 1 hour at 37OC and overnight
Arthritis and Rheumatism, Vol. 24, No. 10 (October 1981)
1225
AGUADO ET AL
1226
at 4°C (10). The resulting precipitates were washed two times
in cold VBS++,resuspended in VBS++,and thoroughly dispersed by passage through a syringe with a 30-gauge needle.
Measurement of the serum capacity to solubilize immune complexes. Twenty microliters of immune complex suspension containing approximately 3 pg of protein were added
to 250 pl of a 'h dilution of serum samples and incubated for 2
hours at 37°C. The reaction was stopped by addition of cold
saline or buffer. Insoluble and soluble complexes were separated by centrifugation (1,6OOg, 30 minutes). The precipitate
was washed once with 1 ml of cold saline or buffer, and the radioactivity was measured. Heat-treated serum (56"C, 30 minutes) and Factor B-inactivated serum (heated at 50"C, 20
minutes) were used as negative controls. The results are expressed as percent of solubilization of a normal serum pool by
the following formula:
ppt BSA with 56°C serum-ppt
BSA_
with
~_
- serum
_ _sample
_ppt BSA with 56°C serum-ppt BSA with serum pool
lM)
Complement studies and detection of circulating immune complexes. The hemolytic activities of the alternative
pathway and Factor B were studied using a 5'Cr release assay,
as previously described (7). In brief, the hemolytic activity of
the alternative pathway (APA) was measured by incubating
serial serum dilutions with 5'Cr-labeledrabbit red blood cells
in the presence of EGTA in order to block the classical pathway and then measuring the amount of radioactivity released
in the supernatant. The hemolytic activity of Factor B was
measured by mixing "Cr-labeled red blood cells with Factor
B-inactivated serum (heated at 50°C for 20 minutes) and serial dilutions as the source of Factor B. The amounts necessary for 50% lysis were calculated in both assays, and the results were expressed as percent of a standard normal serum
pool.
The hemolytic activity of complement was measured
with a semiautomatic continuous flow system ( I 1). Complement components (C3 and C4) and regulatory proteins of
the alternative pathway (JIH and C3b I NA) were measured
by single radial immunodiffusion using specific antisera. The
levels of circulating immune complexes were detected by I2'IClq binding assay, as described by Zubler et a1 (12).
Statistical analysis. A nonparametric test was used for
all calculations except for the clinical evaluation. In order to
compare the values, the Mann-Whitney ranking test and
Spearman rank test were used. To evaluate the frequency of
clinical events, we used the conventional Chi-square analysis
according to Woolf, except when the frequencies were 0. In
this case, the method of Haldane was used (13).
ground. The values of individual sera were then expressed as % in relation to the normal pool activity, after
correction for the heated serum background effect.
In 26 normal sera, the immune complex solubilization capacity (ICSC) was 107 f 36% (mean f 2 SD)
of a normal pool.
In sera from patients suffering from SLE, the capacity to solubilize immune complexes was lower than
in normal controls (Figure 1). Similarly, the function of
the total hemolytic complement (CH50), as well as the
levels of C3 and C4 components, was decreased in those
sera. Finally, the hemolytic activity of the alternative
pathway and hemolytic Factor B (hB) were decreased
(Figure 1). Levels of PIH and C3b INA were within
normal range.
As shown in Figure 2, there were significant correlations between the capacity to solubilize complexes
and the hemolytic activity of the alternative pathway
and Factor B. Similar correlations were also found with
CH50 (r = 0.71; P c 0.001), as well as C3 and C4 (Figure 2). In some cases, serum samples with decreased levels of C4 showed normal values of solubilization capacity. No correlation was found between the levels of the
regulatory proteins PIH and C3b INA and the capacity
to solubilize immune complexes.
Patients with decreased ICSC had higher levels
of circulating immune complexes as assessed by the C 1q
binding assay than patients with normal ICSC (P c
0.01) (Figure 3). The clinical data from these patients
were evaluated in relation to the solubilization capacity
and levels of C3. The results are shown in Table 1. It
appears that there is a tendency to greater renal inICSC
-
hB
1
.A.
+.;.
*.
aR
2o
4
!
oJ
P.0
001
C3
P<O.OOI
C4
..
...
...
;
;
..
-+a .
.......
CH 50
I
lj
...
..
....*
RESULTS
After 2 hours of incubation, a normal serum pool
solubilized approximately 80% of the amount of radiolabeled immune complexes added (without serum:
100% radiolabeled BSA is precipitated; with serum:
-20% radiolabeled BSA is precipitated). Solubilization
values of heat-incubated serum (at 56"C, 30 minutes) or
Factor B-inactivated serum (at 50°C, 20 minutes) were
about lo%, and these data were considered the back-
APA
P<0.005
p<O.OOI
P*0.001
+....
.".
P<O.WI
Figure 1. Immune complex solubilization capacity (ICSC), alternative pathway (APA), Factor B (hB) and CH50 hemolytic activities,
and levels of C3 and C4 in 32 sera from patients with systemic lupus
erythematosus. Closed areas represent the normal range (mean f 2
SD of 26 normal sera). Points represent individual values of patients.
SOLUBILIZATION OF IMMUNE COMPLEXES IN SLE
c3 %
c4 %
= 0.70
P- 0.001
I
12OT
+
R.0.71
p.o.001
r: 0.67
P- 0 001
/
60
*
\
-.. 1.
0 1
b 6
't20'100'8b'6b'Lb'~O'
APA %
. .
R: 0.68
p'.O.OOl
A...
...
1227
lence point, which normally precipitate due to lattice
formation, may be solubilized by the addition of fresh
serum. The solubilization appears to be the result of a
particular dissociation and subsequent decrease in the
size of complexes after fixation of C3. The integrity of
the alternative pathway is required for the production
of this phenomenon (14,15). The in vivo significance of
this phenomenon is not well known, but there is evidence that immune complexes play an important role in
the development of tissue lesions in animal and human
diseases (16-1 8). The mechanisms governing the deposition and the removal of immune complexes are not
completely defined, but it is likely that a balance exists
between complexes that remain in either intravascular
or extravascular spaces and those that are eliminated by
the phagocytic system.
We have considered the possibility that the solubilization capacity of complement may have important
biologic significance in vivo in relation to the immunopathology of various diseases, particularly in patients
suffering from systemic lupus erythematosus, an im-
ICSC
norrna
dQCrUaSQd
' & ' L b ' 6 b 'sb'l~0'1;O
hB %
Figure 2. Relationship between immune complex solubilization capacity and alternative pathway hemolytic activity (APA), Factor B
hemolytic activity (hB), C3 and C4 levels in 32 patients with systemic
lupus erythematosus.R = Spearman's rank correlation coefficient, r =
linear correlation coefficient.
volvement in patients with decreased capacity to solubilize complexes as judged by proteinuria and renal
biopsy. In this study, patients with greater renal involvement were also remarkable for their low serum C3
values. No correlation has been found between the frequency of articular, dermatologic, or neurologic manifestations of the disease and the capacity to solubilize
complexes or the C3 levels. In addition, patients with a
normal solubilization capacity or normal C3 level appear to have a better evolution than patients with decreased values of those parameters (Table 1).
***
p<o.o1
DISCUSSION
Miller and Nussenzweig (3) have demonstrated
that in vitro complexes formed near or at the equiva-
Figure 3. Levels of circulating immune complexes in patients' sera in
relation to normal decreased values of immune complex solubilization capacity (ICSC). Shaded area represents the normal range. P =
significance of Mann-Whitney ranking test.
AGUADO ET AL
1228
Table 1. Correlation of the capacity to solubilize immune complexes and C3 level with clinical manifestations in systemic lupus erythematosus
Patients with*
Patients with'
Clinical manifestations
Proteinuria (200 mg/24 hours)
Renal biopsy
Minimal changes or
rnesangial lesions
Intra- and extracapillary or
focal segmentary lesions
Articular involvement
Dermatologic involvement
Evolution8
Normal
ICSC
Decreased
ICSC
2
fi
Normal
c3
Decreased
c3
2
3/10
9/10
5.83
t0.02
3/10
9/10
5.83
<0.02
6
0
5
I
9.67
<0.005
6.54
t0.02
0.84
0.20
8.69
NS
NS
<0.005
0
10/10
4/10
2/10
4
9/10
7/10
8/10
0.84
1.75
6.14
NS
NS
t 0 .0 2
0
10/10
5/10
1/10
4
9/ 10
6/10
9/10
P
71% (Mean f 2 SD of control group).
t NS = not significant.
$68% (Mean f 2 SD of control group).
8 Normalization of the erythrocyte sedimentation rate and proteinuria after treatment without clinical reactivation evaluated after more than 12
months of followup.
mune complex disease associated with immune complex
deposition in various tissues (6,19). In patient sera, we
have found a significant decrease in the capacity to solubilize complexes, as well as an overall decrease in
complement activity. We have observed a very significant correlation between this capacity and the levels of
C3. Similar results have been shown previously by
Krettli, Nussenzweig, and Nussenzweig (20) using an
animal model of malarial infection. The solubilization
capacity also correlated significantly with the alternative and classical pathway hemolytic activities, Factor B
and C4. These results are consistent with the observation that both complement pathways are involved in
systemic lupus erythematosus. The observation that, in
some cases, the levels of C4 were decreased while the
solubilization capacity of these sera remained within
normal limits is in agreement with the nonessential role
of the classical pathway in the solubilization phenomenon (14,21).
The importance of these results should also be
considered in relation to in vivo distribution of immune
complexes. Indeed, it can be expected that the binding
of complement to immune complexes formed or localized in the extravascular compartment should lead to
some solubilization of these complexes. This would favor their subsequent diffusion to lymph and blood and
their removal in the reticuloendothelial system. Along
this line, a decreased complement activity that would
impair the capacity to solubilize complexes should lead
to the persistence of complexes at the tissue level and favor the development of tissue damage.
In this study, we found that lupus patients with
an increased level of circulating immune complexes had
a decreased capacity to solubilize complexes. This may
be interpreted in two ways: first, if the level of complexes is increased, C3 can be fixed by complexes, and
subsequently solubilization capacity will be diminished.
Alternatively, if the C3 level is diminished, the capacity
to solubilize complexes will be decreased, favoring the
persistence of immune complexes.
We also observed that those patients with decreased solubilization capacity showed more renal involvement and a worse evolution. In lupus, this relationship may be caused by the close correlation between
the decrease of complement activity and the reduced capacity to solubilize complexes. However, one cannot exclude other factors that may interfere with the solubilization phenomenon, such as rheumatoid factor or
immunoconglutinin.
In recent studies, we have found that patients
with rheumatoid arthritis may have a decreased serum
capacity to solubilize complexes that is not associated
with a decreased complement hemolytic activity. The
decreased serum capacity to solubilize complexes in patients with SLE may also influence the size of circulating complexes and their relative pathogenicity. This
hypothesis is now under investigation using ultracentrifugation analysis.
ACKNOWLEDGMENTS
The authors are grateful to Dr. U. E. Nydegger for
providing the anti-PIH and anti-C3b INA antisera. We thank
Mr. Brandon for his help in programming the statistical analysis of the results and Mr. M. Rychner for his help in the
preparation of the iconographic material. We would also like
to thank Dr. A. Celada for his assistance in the clinical investigation.
SOLUBILIZATION OF IMMUNE COMPLEXES IN SLE
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