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Deposition of antibodies to the collagen-like region of C1Q in renal glomeruli of patients with proliferative lupus glomerulonephritis.

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 8, August 1997, pp 1504-1511
0 1997, American College of Rheumatology
1504
DEPOSITION OF ANTIBODIES TO THE COLLAGEN-LIKE REGION OF
Clq IN RENAL GLOMERULI OF PATIENTS WITH
PROLIFERATIVE LUPUS GLOMERULONEPHRITIS
MART MA"1K and MARK H. WENER
Objective. To determine if antibodies to the
collagen-like region of Clq (Clq-CLR) are present in
the glomerular immune deposits of patients with systemic lupus erythematosus (SLE).
Methods. Kidney tissues were obtained at autopsy, glomeruli were isolated, and glomerular basement membrane fragments were prepared. Antibodies
were extracted with low pH or with DNase.
Results. The concentrations of antibodies to ClqCLR recovered from the glomeruli were 150-fold higher
per unit of IgG than that found in the serum or in the
serum and interstitial fluid entrained in glomeruli.
Antibodies to Clq-CLR were recovered from glomeruli
of 4 of 5 patients with proliferative glomerulonephritis
at autopsy.
Conclusion. This is the first demonstration that
antibodies to Clq-CLR are deposited and concentrated
in the renal glomeruli of patients with SLE. These
antibodies, thus, have the potential of contributing to
the pathogenesis of lupus glomerulonephritis.
In patients with systemic lupus erythematosus
(SLE), renal disease is a significant cause of morbidity
and mortality. Clinical evidence of kidney involvement is
found in about 50% of patients with this disease. Even in
SLE patients without proteinuria and with normal urine
sediment, however, renal biopsies have disclosed the
presence of immune deposits in glomeruli, particularly
in the mesangium (1,2). Immune complexes consisting of
Presented in part at the 60th National Scientific Meeting of
the American College of Rheumatology, Orlando, FL, October 1996.
Supported by grant R01-AR-11476 from NIAMS, and the
Lory Ford Fund.
Mart Mannik, MD, Mark H. Wener, MD: University of
Washington, Seattle.
Address reprint requests to Mart Mannik, MD, Division of
Rheumatology, Box 356428, University of Washington, Seattle, WA
98195-6428.
Submitted for publication February 19, 1997; accepted in
revised form April 4, 1997.
DNA and antibodies to double-stranded DNA (dsDNA)
play a pivotal role in the generation of proliferative
lupus glomerulonephritis. This concept evolved from
several observations. First, the prevalence of antibodies
to dsDNA is highest in patients with active SLE and
renal involvement (for review, see ref. 3). Second,
increasing levels of antibodies to dsDNA may herald
an exacerbation of the renal disease (4-6). Third, in
the pioneering studies of Koffler and colleagues and
of Krishnan and Kaplan, antibodies to dsDNA were
recovered from the glomeruli of SLE patients who had
died (7-9).
Studies that used Clq as a test for immune
complexes, and subsequent studies that used the
purified collagen-like region of Clq (Clq-CLR) as
antigen have provided definitive evidence for serum
antibodies to Clq-CLR in patients with proliferative
lupus glomerulonephritis (10-13). Several independent investigations have established the high prevalence of autoantibodies to Clq-CLR in patients with
proliferative lupus glomerulonephritis (14-17). In addition, increasing levels of these autoantibodies is
known to herald the flare of glomerulonephritis in
patients with SLE (18,19).
In view of these previous findings, the present
study was undertaken to determine if antibodies to
Clq-CLR are present in the glomeruli of patients with
SLE. The data presented herein demonstrate, for the
first time, the presence of antibodies to Clq-CLR in
extracts from the glomerular basement membrane
(GBM) fragments of patients with SLE.
PATIENTS AND METHODS
Preparation and extraction of GBM fragments. Kidney
tissues were obtained at autopsy, regardless of the cause of
death, from 13 patients (designated K1 through K13) with the
established diagnosis of SLE. These 13 specimens included 8
from the University of Washington Medical Center or from
Clq-CLR ANTIBODIES IN SLE GLOMERULI
affiliated hospitals, as approved by the Human Subjects Committee. Three samples were obtained through the Western
Division of the Cooperative Human Tissue Network, and 2
were from other medical centers. One specimen (K11) was not
studied, since the renal cortex was destroyed by multiple cysts.
Kidney tissues were also obtained from 5 individuals (designated NKl through NK5) who had no evidence of SLE and no
known renal disease.
The kidney tissues were divided into quarters of a
kidney, and then frozen at -20°C while immersed in phosphate buffered saline (PBS; O.IM phosphate, 0.15M NaC1, pH
7.2) containing a mixture of protease inhibitors (PBS-I), as
defined below. Other tissues were frozen, shipped with dry ice,
divided into quarters of a kidney while frozen, and then
immersed in ice-cold PBS-I and frozen at -20°C.
When kidney tissue was processed for extraction, it was
thawed at room temperature. The cortex was harvested by
dissection and weighed and diced into -1-mm' pieces while in
petri dishes on ice. These pieces were forced with a glass pestle
through a 70-mesh (212-pm openings) stainless steel sieve and
collected in a pan on ice. The sieve was intermittently rinsed
with PBS-I, containing: 1 mM phenylmethylsulfonyl fluoride,
5 pA4 pepstatin A, 5 p'vl antipain, 5 f l leupeptin, 1 mM
benzamidine, 100 pA4 iodoacetamide, and 1 mM EDTA (all
purchased from Sigma, St. Louis, MO). The glomeruli were
allowed to settle in 50-ml conical centrifuge tubes for 20
minutes. Forty milliliters was aspirated from the top, and the
glomeruli were resuspended and settled 7 times.
After the final settle, the glomeruli in each tube were
suspended in 20 ml of PBS-I. In the same tube, the glomeruli
were disrupted with sonication using a Branson Sonifier 450
with a 1-cm diameter probe (Branson Ultrasonics Corporation,
Danbury, CT). To prevent heating, 1-minute pulses at 50%
duty cycle, 5.5 power level, were used with the tubes in an ice
bath. Between the usual 4 1-minute pulses, the tubes were
maintained on ice. Following sonication, the mixture was
passed through a 140-mesh sieve to remove the empty Bowman's capsules or undisrupted glomeruli. The sonicated and
sieved preparation was then pelleted in a 40-ml tube in an
Eppendorf 5403 centrifuge (Brinkmann Instruments, Westbury, NY) at 9600 revolutions per minute (10,OOOg) for 15
minutes. The pellet was resuspended in PBS-I and again
centrifuged. This was repeated 4 times, except that for the last
3 centrifugations, the pellet was suspended in PBS without the
protease inhibitors. The last suspension was divided into 2
equal aliquots so that the pellets could be submitted to
different extraction procedures.
Prior to sonication, a smear of the glomeruli was made
and examined to ensure the absence of tubules. After sonication and sieving, smears of the preparation were made, stained
with ethidium bromide, and examined for absence of nuclei
or DNA.
After the final wash, one-half of the pellet was extracted with low pH (0.1Mglycine HCI, 0.15M NaCI, pH 2.5) in
8-ml volume overnight at 4°C on a rotating mixer. The other
half of the pellet in PBS was extracted with DNase I (Sigma),
200 units per ml, and rendered 5 mM with MgSO,. The next
day, the GBM fragments were pelleted by centrifugation, and
the extracts were dialyzed against PBS-I, aliquoted, and frozen
at -20°C until analyzed further. A small amount of the pellets
was used, before and after extractions, to make smears on
1505
microscope slides. These slides were stained with fluoresceinated goat antibodies to human IgG, C3, or C l q (Organon
Teknika, Durham, NC).
Detection of antibodies in the extracts. IgG and human
serum albumin (HSA) were quantified in each supernatant of
settled glomeruli, in the supernatants of GBM fragments after
sonication, in the sequential washing of these fragments, and in
the low pH and DNase extracts of the GBM fragments, using
sensitive radioimmunoassays (20). Briefly, for detection of
IgG, microtiter wells were coated with antibodies to K and A
chains, and incubated with diluted or undiluted aliquots of the
supernatants or extracts. Bound IgG was detected with lZ5IF(ab'), fragments of purified goat antibodies to human IgG Fc
fragments (Jackson Laboratories, West Grove, PA). Standard
curves were constructed with each assay, using purified human
IgG. For detection of HSA, the wells were coated with purified
antibodies to HSA, and the bound HSA was detected with
'251-labeled antibodies to HSA. Standard curves were constructed with each assay using purified HSA.
For comparison among specimens, the amounts of
recovered IgG and HSA were expressed in pdgm of wet
weight of renal cortex. The presence of HSA in the washes of
glomeruli, the sonication supernatants, the washes of GBM
fragments, and the extracts of GBM fragments was determined
as a measure of serum proteins in the blood of capillaries and
interstitial fluid, or serum proteins filtered through the GBM.
An increase in the molar ratio of IgG to HSA was used as
evidence for release of IgG bound to immune deposits in
glomeruli. The molar ratio of IgG to HSA was calculated for
each specimen, using molecular weights of 150,000 and 67,000,
respectively.
The presence of antibodies to Clq-CLR was detected
with an enzyme-linked immunosorbent assay (ELISA) system,
as previously described (13). Briefly, Clq was isolated from
outdated plasma and Clq-CLR was prepared with previously
described methods (21). The final step of purification of
Clq-CLR was removal of the small amounts of IgG using
affinity chromatography with an agarose-anti-human IgG column (22). The microtiter wells were coated with 500 ng of
Clq-CLR in 100-pl volume, with overnight incubation at 4°C.
After washing, the coated wells and the blank wells were
blocked with 5% bovine serum albumin (BSA) in PBS for 1
hour at room temperature. All washes were performed with
PBS, 0.05% Tween 20. One hundred microliters of undiluted
samples or samples diluted in 1% BSA in PBS were added to
wells, incubated overnight at 4"C, and washed. The presence of
IgG was detected with a 1:10,000 dilution of anti-human IgG
conjugated with horseradish peroxidase, and was subsequently
identified by color development. With each assay, standard
dilutions of a serum known to contain antibodies to Clq-CLR
were included. For each specimen, the optical absorbance of a
well without Clq-CLR was subtracted from the absorbance of
the Clq-CLR-coated well to yield the specific absorbance for
the specimen.
The specific absorbance was plotted against the IgG
concentration of serially diluted specimens to seek evidence
that antibodies to Clq-CLR were concentrated at the GBM.
Nonlinear regression of the absorbance and IgG concentration
data was performed with a modification of the logistic model
for immunoassays, to obtain a best-fit curve to the sigmoid,
log-linear data (23). Regression was performed using RS/1
MANNIK AND WENER
1506
Table 1. Yield of IgG per gm of cortex and the molar ratio of IgG to human serum albumin (HSA) for
1 systemic lupus erythematosus (SLE) kidney (K6) and 1 non-SLE kidney (NK-5)*
K6
First supernatant of glomeruli
Second supernatant of glomeruli
Third supernatant of glomeruli
Fourth supernatant of glomeruli
Fifth supernatant of glomeruli
Sixth supernatant of glomeruli
Seventh supernatant of glomeruli
Supernatant after sonication
First supernatant of GBM fragments
Second supernatant of GBM fragments
Third supernatant of GBM fragments
Fourth supernatant of GBM fragments
pH 2.5 extraction of GBM fragments
DNase extraction of GBM fragments
NK-5
IgG, Pdgm
cortex
1gG:HSA
IgG, PdP
cortex
1gG:HSA
3,785
588
97.0
12.8
2.35
1.11
1.20
12.5
0.672
0.015
0.008
0.001
0.013
0.096
0.249
0.183
0.193
0.347
0.529
1.13
3.03
2.48
3.20
No HSA
No HSA
No HSA
2.90
2.68
960
127
14.9
0.363
0
ND
ND
0.234
0.002
0
0
0
0.006
0.008
0.052
0.058
0.046
0.062
No HSA
ND
ND
0.055
0.012
No HSA
No HSA
No HSA
No HSA
No HSA
* Values are provided sequentially for the 7 supernatants of the settled glomeruli, the supernatant after
sonication, and the subsequent washes and extractions of glomerular basement membrane (GBM)
fragments. ND = not done.
software (BBN Software Products, Cambridge, MA) on a VAX
computer (Digital Equipment Corporation, Maynard, MA).
For tabulation of the data, the assay results were
expressed as (-), (+), (++), or (+++), on the following
basis: (-) when the several dilutions of a specimen showed no
optical absorbance at 405 nm in the assay for antibodies to
Clq-CLR; (+) when the midpoint of optical absorbance was
close to or higher than an IgG concentration of 100 pgiml;
(+ +) when the midpoint of the curve was closest to an IgG
concentration of 10 pg/ml; and (+++) when the midpoint
of the curve was closest to or below an IgG concentration
of 1 pg/ml.
RESULTS
Recovery of IgG from glomeruli. As expected,
large amounts of IgG and HSA were present in the first
supernatants after the glomeruli were allowed to settle,
reflecting serum proteins in blood vessels and interstitial
fluid. With repeated settling of glomeruli, the amount of
IgG in the supernatants declined. The amount of released IgG increased in the supernatants after sonication of the glomeruli from the kidneys of SLE patients,
and then decreased with sequential washes of the GBM
fragments produced by sonication. Less IgG was recovered in each supernatant from normal kidneys than in
the corresponding supernatant from SLE kidneys. Furthermore, in most SLE specimens, the 1gG:HSA molar
ratio increased in the washes as well as in the sonication
supernatant and in the low pH and DNase extracts.
Table 1 shows the data for 1 SLE kidney and 1 normal
kidney.
Sonication of the glomeruli from SLE specimens
increased the release of IgG, as indicated by the increased pg of IgG per gm of cortex in the supernatant
after sonication, as well as by the finding that the
1gG:HSA molar ratio was always higher in the sonication
supernatant than in the first supernatant after settling of
glomeruli. Indeed, in 6 of the 12 SLE kidneys, this ratio
in the sonication supernatants exceeded 1.0. These
findings suggest that IgG was preferentially released
from the glomeruli fragmented by sonication. In contrast, in the 5 normal kidneys, the 1gG:HSA ratio did not
increase in the sonication supernatant, and remained
below 0.1 (Table 2).
The amount of IgG recovered by extraction from
the GBM fragments using low pH or DNase was relatively small, ranging from 0.002 to 2.97 pg of IgG per gm
of cortex in the low pH extracts and from 0.002 to 1.48
pg of IgG per gm of cortex in the DNase extracts. In
these extracts, however, the 1gG:HSA ratio exceeded 1.0
in 7 of the 12 low pH extracts and in 6 of the 12 DNase
extracts (Table 2). In all extracts except 2, the 1gG:HSA
ratio was higher than in the initial supernatant of
glomeruli. In contrast, in normal kidneys, the 1gG:HSA
molar ratio in the extracts from GBM fragments was
lower than in the initial supernatants (Table 2).
Small aliquots of the GBM fragments pelleted by
centrifugation were used to make smears on microscope
slides. Before extraction, the GBM fragments were
stained with antibodies to IgG, and this staining was
Clq-CLR ANTIBODIES IN SLE GLOMERULI
Table 2. The 1gG:HSA molar ratios for the 12 SLE kidneys studied
(Kl-K13) and the range for 5 normal kidneys (NK) in the first
supernatant of settled glomeruli, the supernatant after sonication, and
the low pH and DNase extracts of the GBM fragments*
K1
K2
K3
K4
K5
K6
K7
K8
K9
K10
K12
KI 3
NK (n
=
5)
GBM fragments
First supernatant
of glomeruli
Sonication
supernatant
Low pH
DNase
0.357
0.200
0.149
0.222
0.103
0.436
0.522
0.105
0.113
0.201
0.450
0.050
0.024-0.052
1.66
0.893
0.523
5.08
1.40
4.73
2.88
0.353
0.349
1.02
0.543
0.336
0.006-0.055
1.14
0.330
0.226
1.72
3.37
2.49
1.70
4.07
0.859
0.411
1.11
0.037
0-0.026
20.6
0.506
0.111
1.38
1.39
2.62
3.00
0.651
0.620
0.596
5.02
0.079
0-0.015
* K11 was not studied because the renal cortex was destroyed by
multiple cysts. See Table 1 for definitions.
blocked by a large excess of IgG added to the anti-IgG
reagent. In the smears made of GBM fragments after
extraction with low pH or DNase, the staining for IgG
was reduced, but was not absent (data not shown). These
findings indicate that the extraction was incomplete.
Detection of antibodies to Clq-CLR in extracts of
glomeruli. Initially, the first supernatant after settling of
the sieved glomeruli and the supernatant after sonication of the glomeruli were examined for the presence of
antibodies to Clq-CLR. The first supernatants after
settling of glomeruli were chosen, since these preparations contained the highest amount of IgG and represented the serum proteins present in blood vessels and
interstitial fluid in the renal cortex. In 4 of the 12 first
supernatants of glomeruli, and in the corresponding
sonication supernatants, antibodies to Clq-CLR were
identified by ELISA. In 2 of these 4 specimens (K6 and
K7), antibodies to Clq-CLR were also present in the low
pH extracts and in the DNase extracts of the GBM
fragments. Adsorption of the sonication supernatant of
K7 with wells coated with dsDNA, an adsorption
method previously employed by Burlingame et a1 (24),
did not remove the antibodies to Clq-CLR. In addition,
the antibodies to Clq-CLR present in the sonication
supernatant of the K7 glomeruli cosedimented with
monomeric IgG on sucrose density gradient ultracentrifugation (data not shown), which was performed by
previously described methods (25). In the low pH and
DNase extracts of the GBM fragments of K2 and K3,
the volumes were small and the IgG concentrations
were so low that the presence or absence of antibodies
1507
to Clq-CLR could not be determined with certainty
(Table 3).
The antibody activity against Clq-CLR in the
recovered IgG, as determined by ELISA, was plotted
against the IgG concentration of the serially diluted
samples, This analysis showed that the antibody activity
to Clq-CLR in the IgG recovered by sonication, by low
pH extraction, or by DNase digestion was 250-fold
more concentrated than in the initial supernatant of
settled glomeruli (Figures 1A and B). The latter supernatant was considered to represent the serum proteins
entrained in blood vessels and interstitial fluid of the
renal cortex. This assumption was supported by the
finding that the curves of antibody activity plotted
against IgG concentration for antemortern serum and
for the first supernatant of glomeruli were essentially
superimposable for the 1 specimen for which both were
available (Figure 1A).
Antibodies to Clq-CLR were present in the
glomeruli of 4 (K2, K3, K6, and K7) of 5 specimens with
active proliferative or inflammatory glomerular lesions.
Diffuse proliferative glomerulonephritis was present at
autopsy in K10, together with thrombi in glomerular
capillaries, but antibodies to Clq-CLR were not detected. In K9, diffuse proliferative glomerulonephritis
had been present 5 years prior to death, but the patient
Table 3. Summary of the results of tests for antibodies to Clq-CLR
for the 12 SLE kidneys in the first supernatant of settled glomeruli, the
supernatant after sonication, and the low pH and DNase extracts of
the GBM fragments*
First supernatant
of glomeruli
K1
K2
K3
K4
K5
K6
K7
K8
K9
K10
K12
K13
-
+
+
Sonication
supernatant
-
+++
++
-
-
-
-
+
+
+++
+++
GBM fragments
Low pH
DNase
ND
?
?
ND
ND
?
?
++
+++
-
ND
+++
+++
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
* Antibody activity in relation to IgG concentration is indicated as
follows: - = no optical absorbance at 405 nm; + = midpoint of optical
absorbance is close to or higher than an IgG Concentration of 100
pgiml; + + = midpoint of the curve is closest to an IgG concentration
of 10 pgiml; + + + = midpoint of the curve is closest to or below an
IgG concentration of 1 pgiml. The question mark indicates that the
presence or absence of the antibodies could not be determined for
these samples, due to a low IgG concentration in the extracts. See
Table 1 for definitions.
MANNIK AND WENER
1508
0.I
1
10
100
1000
IgG c~dml
Figure 1. Antibody activity against Clq-CLR in extracts of the glomeruli from specimens K6 (A) and K7 (B). Absorbance at 405 nm in the
enzyme-linked immunosorbent assays (ELISA) is plotted against the IgG concentration in the test sample and in dilutions of the sample. 0 = first
supernatant of settled glomeruli; X = dilutions of antemortem serum (available for K6 only); 0 = supernatant after sonication; V = low pH extract
of glomerular basement membrane (GBM) fragments; = DNase extract of GBM fragments. The midpoint of the ELISA absorbance curves
corresponded to the following IgG concentrations (95% confidence intervals): in K6, serum and first supernatant of glomeruli 177 pg/ml (133-223):
low pH extract of GBM fragments 35 pdml (uncertainty very high); sonication supernatant and DNase extract of GBM fragments 4.2 pgml
(3.2-5.1); in K7, first supernatant of glomeruli 142 pdml (133-150); sonicalion supernatant 1.9 pdml (0-5.0); low pH extract of GBM fragments
1.5 pg/ml (0-5.0); DNase extract of GBM fragments 0.2 pgiml (0.006-0.38).
had been on dialysis for 3 years and global glomerulosclerosis was found on autopsy; antibodies to Clq-CLR
were not recovered from this specimen (Table 4).
In 3 patients (K5, K8, and Kl3), the cause of
death was antiphospholipid syndrome. In these 3 patients, variable amounts of IgG were recovered from the
GBM fragments using low p H extraction. In K13, the
Table 4.
Kidney histology on biopsy and/or autopsy*
K1
K2
K3
K4
Membranous glomerulonephritis on autopsy.
Focal inflammation and wire loop lesions on autopsy.
Diffuse proliferative glomerulonephritis on autopsy.
Focal hypercellularity of glomeruli and GBM thickening on
autopsy.
KS Patient died of antiphospholipid syndrome. By light
microscopy, no glomerulonephritis on autopsy.
K6 Diffuse proliferative glomerulonephritis on biopsy and
autopsy.
K7 Clinical flare of SLE, pneumococcal sepsis. Mesangial
proliferation on autopsy.
K8 Patient died of antiphospholipid syndrome. Kidney histology
on autopsy was not available to us.
K9 Diffuse proliferative glomerulonephritis on biopsy 5 years ago;
3 years on dialysis. Global glomerulosclerosis on autopsy.
K10 Diffuse proliferative glomerulonephritis with intracapillary
thrombi on autopsy.
K12 Trophoblast pulmonary emboli and nephritis with wire loop
lesions on autopsy.
K13 Patient died of antiphospholipid syndrome. By light
microscopy, no glomerulonephritis on autopsy.
* GBM
= glomerular basement membrane; SLE = systemic lupus
erythematosus.
yield of IgG was 0.002 p d g m of renal cortex at this step;
this value is within the range of IgG recovery from
normal kidneys. In K5, however, the molar ratio of
1gG:HSA was high both in the low pH extract and in the
DNase extract, in the absence of glomerulonephritis on
autopsy. These ratios were also high in K8, but renal
histology results on autopsy were not available. The
reasons for the recovery of increased amounts of IgG
from the GBM fragments in these 2 patients is not known.
DISCUSSION
This is the first study to demonstrate the presence of
antibodies to Clq-CLR in the glomeruli of some patients with SLE. In previous studies on the recovery of
antibodies to DNA or other nuclear antigens from
immune deposits in the glomeruli of patients with SLE,
these molecules were extracted from intact glomeruli or
fragmented glomeruli. In this study, preparations of
GBM fragments were used for the extraction of antibodies. This approach has been validated in experimental
systems by demonstrating that immune deposits persist
in GBM fragments, as determined by electron microscopy (26) or by the use of radiolabeled antigen or
antibody molecules (27).
For the present study, kidneys were sought and
obtained at autopsy, regardless of the cause of death,
from patients who had the established diagnosis of SLE.
Clq-CLR ANTIBODIES IN SLE GLOMERULI
Among the 12 specimens studied, antibodies to ClqCLR were found in 4 of the initial supernatants of
glomeruli, which were believed to represent serum proteins. For comparison, in unselected series of patients
with SLE, the prevalence of IgG antibodies to Clq-CLR
has varied from 17% to 46% (13-17).
Several points of interest emerged from the attempts to recover IgG from the GBM fragments. First,
IgG continued to be released by serial washing of the
glomeruli or GBM fragments, and the 1gG:HSA ratio
increased. This suggests that the IgG was released from
immune deposits. Such release by washing can be expected, owing to the nature of the equilibrium reaction
between antigens and antibodies, and the amount and
rate of release would depend on the association constant of these reactants. Continued release would not
be expected from molecules entrained passively and
nonselectively in tissues, but could occur if IgG is
otherwise retained nonspecifically by noncovalent
bonds. In the latter mechanism, however, enrichment
of specific antibodies would not be expected. Second,
a substantial release of IgG occurred with sonication
of the glomeruli to produce GBM fragments. This was
reflected by the increased 1gG:HSA ratio and the
concentration of antibody activity. Third, the recovery
of IgG from GBM fragments was incomplete, as
determined by immunofluorescence microscopy. The
difficulty of extracting antibodies from the immune
deposits in renal glomeruli was previously documented with studies in mice (27).
The finding that antibodies to Clq-CLR were
concentrated in IgG extracted from the GBM fragments
indicates that these antibodies were present in the
immune deposits in the glomeruli. These molecules were
released, at least in part, by extraction with pH 2.5, a
condition known to disrupt antigen-antibody bonds. The
unexpected finding was that treatment with DNase also
released antibodies to Clq-CLR. Adsorption with
dsDNA did not remove the IgG binding to Clq-CLR. In
previous investigations, antibodies to Clq-CLR and
antibodies to dsDNA have been observed to coexist in
the sera of patients with SLE (14). Therefore, the
release of antibodies to Clq-CLR by DNase suggests
that the immune deposits contained immune complexes
composed of DNA and antibodies to DNA, which then
bound Clq and, in turn, antibodies to Clq-CLR. This
sequence of events is also supported by the preliminary
finding that antibodies to dsDNA were present in the
first supernatant of glomeruli and were concentrated in
the low pH and DNase extracts of K6 and K7 (Wener
MH, Mannik M: unpublished studies). Extracts of both
1509
of these kidney specimens also contained antibodies to
Clq-CLR. A similar sequence of reactions has been
demonstrated in mice by first forming glomerular immune deposits consisting of cationized HSA and rabbit
antibodies to HSA, followed by human Clq and then
human antibodies to Clq-CLR (28).
Antibodies to Clq-CLR do not bind to native,
circulating Clq in the trimolecular complex of C1 that
contains Clr and Cls bound to the collagen-like region
of Clq. Antibodies to Clq-CLR readily interact with
Clq bound to immune complexes (29), and with Clq
bound to plastic wells used in ELISA systems (12). If
antibodies to Clq-CLR were contained only in antigenantibody complexes of Clq and antibodies to Clq-CLR,
DNase should not release antibodies to Clq-CLR from
such complexes. An alternative possibility, however,
needs to be considered. Clq binds to dsDNA and to
single-stranded DNA at physiologic salt concentration
(30). DNA in immune complexes or nucleosomal DNA
at the GBM could bind free Clq. If this interaction
changes the conformation of the CLR of Clq to permit
binding of antibodies to Clq-CLR, then DNase could
release the antibodies to Clq-CLR. This potential mechanism for the binding of antibodies to Clq-CLR to
glomeruli also could enhance inflammation in the
glomeruli.
Several studies have indicated that the highest
prevalence of antibodies to Clq-CLR in patients with
SLE is found among patients with proliferative lupus
glomerulonephritis (ll,18,19). Furthermore, the appearance of circulating antibodies to Clq-CLR has served as
a harbinger for flares of lupus glomerulonephritis
(18,19). In the present study, antibodies to Clq-CLR
were recovered from the glomeruli of 4 of 5 patients with
proliferative or inflammatory lesions at autopsy. In 2 of
these 4 patients, antibodies to Clq-CLR were also
recovered from the GBM fragments. Collectively, these
observations strongly argue that antibodies to Clq-CLR
contribute to the pathogenesis of proliferative glomerulonephritis in patients with SLE. This pathogenic role
may come about by Clq binding to immune deposits,
consisting of DNA and antibodies to DNA or other
antigen-antibody complexes, in the subendothelial or
mesangial areas of glomeruli, and then followed by the
interaction of antibodies to Clq-CLR with the bound
Clq. These events would increase the cross-linking of
the immune deposits and prolong their persistence in
the subendothelial area. In turn, the persistence of
immune deposits in the subendothelial area would promote the inflammatory responses that result from immune deposits in this area of the glomeruli.
MA" 1 K AND WENER
1510
A large body of information supports the concept
that the autoimmune responses to nuclear components
in patients with SLE are driven by specific antigens (24).
Furthermore, several lines of evidence indicate that
subcellular molecular assemblies, rather than single molecules, play a pivotal role in the development of the
autoimmune response in these patients, as reviewed by
Tan et a1 (31,32). In view of the accumulating data
indicating a pathogenic role for antibodies to Clq-CLR
in proliferative glornerulonephritis of SLE, the presence
of antibodies to an extracellular and circulating protein
must be added to the paradigms intended to explain the
autoimmunity in patients with SLE.
ACKNOWLEDGMENTS
The authors thank Ronald D. Krofft, Judy Huang,
Judy B. Anderson, and Richard E. Person for their technical
assistance in the conduct of this work, Corinne L. Fligner, MD
(Department of Pathology, University of Washington) for
her help in obtaining the kidney specimens, Dennis W.
Boulware, MD (University of Alabama) and Herbert B.
Lindsley, MD (University of Kansas) for providing kidney
tissues for the study, and the Cooperative Human Tissue
Network, which is funded by the National Cancer Institute.
Other investigators may have received samples from these
same tissues.
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