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Delayed lupus onset in NZB Ф NZWF1 mice expressing a human C-reactive protein transgene.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 6, June 2003, pp 1602–1611
DOI 10.1002/art.11026
© 2003, American College of Rheumatology
Delayed Lupus Onset in (NZB ⫻ NZW)F1 Mice Expressing a
Human C-Reactive Protein Transgene
Alexander J. Szalai,1 Casey T. Weaver,1 Mark A. McCrory,1 Frederik W. van Ginkel,1
Rachael M. Reiman,1 John F. Kearney,1 Tony N. Marion,2 and John E. Volanakis1
renal cortex was prevented. There was less glomerular
podocyte fusion, basement membrane thickening, mesangial cell proliferation, and occlusion of capillary
lumens in hCRPtg/BW mice, but dense deposits in the
mesangium were increased. With disease progression in
hCRPtg/BW mice, there was little rise in the plasma
CRP level, but CRP in the kidneys became increasingly
apparent due to local, disease-independent, age-related
expression of the transgene.
Conclusion. In hCRPtg/BW mice, CRP protects
against SLE by increasing blood and mesangial clearance of immune complexes and by preventing their
accumulation in the renal cortex.
Objective. Human C-reactive protein (CRP) binds
apoptotic cells and alters blood clearance of injected
chromatin in mice. To test whether CRP participates in
the pathogenesis of systemic lupus erythematosus
(SLE), we examined disease development in lupusprone (NZB ⴛ NZW)F1 (NZB/NZW) mice expressing a
human CRP transgene (hCRPtg/BW).
Methods. Mortality was monitored, proteinuria
was determined by dipstick, and serum levels of human
CRP and anti–double-stranded DNA (anti-dsDNA)
were determined by enzyme-linked immunosorbent assay in NZB/NZW and hCRPtg/BW mice. Thin sections
of kidneys were analyzed by immunofluorescence microscopy to compare deposition of IgG, IgM, C3, and
human CRP, and electron microscopy was used to
reveal differences in ultrastructure. In situ hybridization was performed to detect human CRP messenger
RNA expression.
Results. The hCRPtg/BW mice had less proteinuria and longer survival than NZB/NZW mice. They
also had lower IgM and higher IgG anti-dsDNA titers
than NZB/NZW mice, although the differences were
transient and small. In hCRPtg/BW mice, accumulation
of IgM and IgG in the renal glomeruli was delayed,
reduced, and more mesangial than in NZB/NZW mice,
while end-stage accumulation of IgG, IgM, and C3 in the
Systemic lupus erythematosus (SLE) is an autoimmune disease that can damage the skin, joints, lungs,
blood, blood vessels, heart, liver, brain, and especially,
the kidneys (1). Even with contemporary medical care,
renal involvement is a major cause of morbidity and
mortality in SLE patients (2). Much of our current
knowledge of the pathogenesis of SLE is based on
inference from murine models, the (NZB ⫻ NZW)F1
(NZB/NZW) hybrid strain being the oldest and best
characterized of these (3). The disease process in NZB/
NZW mice accurately reflects the process of SLE in
humans. Like humans with SLE, NZB/NZW mice display a pathognomonic antinuclear autoantibody response that includes anti–double-stranded DNA (antidsDNA), and they spontaneously develop a fatal
glomerulonephritis (4). Also similar to the characteristics in humans, the disease is most frequent in female
mice, susceptibility is influenced by the major histocompatibility complex (MHC; mouse H-2), and non–H-2 loci
are linked to the organ-specific symptoms (4,5).
In most diseases, the blood level of C-reactive
protein (CRP) accurately reflects the degree of underlying inflammation and tissue necrosis (6). In SLE
patients, however, there is often little or no increase in
Dr. Szalai’s work was supported in part by the National
Institute of Allergy and Infectious Diseases (grant AI-42183).
1
Alexander J. Szalai, PhD, Casey T. Weaver, MD, Mark A.
McCrory, BSc, Frederik W. van Ginkel, PhD, Rachael M. Reiman,
PhD, John F. Kearney, PhD, John E. Volanakis, MD (current address:
Biomedical Sciences Research Center “A. Fleming,” Vari, Greece):
The University of Alabama at Birmingham; 2Tony N. Marion, PhD:
The University of Tennessee at Memphis.
Address correspondence and reprint requests to Alexander J.
Szalai, PhD, The University of Alabama at Birmingham, Department
of Medicine, Division of Clinical Immunology and Rheumatology,
THT 437B, 1530 Third Avenue South, Birmingham, AL 35294-0006.
E-mail: Alex.Szalai@ccc.uab.edu.
Submitted for publication September 26, 2002; accepted in
revised form February 19, 2003.
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DELAYED LUPUS ONSET IN MICE EXPRESSING HUMAN CRP
blood levels of CRP, even during disease flares (7,8).
Indeed, when CRP elevation is detected in an SLE
patient, it is indicative of intercurrent infection rather
than active lupus (8,9). There is additional clinical and
experimental evidence that together suggests a possible
participatory role of CRP in the pathogenesis of lupus.
For example, CRP binds to nuclear autoantigens of
relevance in SLE, such as chromatin, histones H1, H2A,
and H2B, and the 70-kd protein associated with U1
small nuclear RNP (10–13). Also, ligand-complexed
human CRP can interact with the complement system
(14) and Fc␥ receptors (Fc␥R) in mice as well as humans
(15–17). Furthermore, in SLE, and probably in other
diseases, CRP can potentially mediate an immunoregulatory effect by binding and opsonizing apoptotic cells
(18). Importantly in mice, where CRP is not an acutephase protein and is only a trace serum component (19),
injected human CRP alters the clearance of administered chromatin (20) and offers transient protection
against murine lupus in the NZB/NZW strain (21).
In the present study, we show that the development of autoimmune glomerulonephritis and death are
delayed in NZB/NZW mice carrying a human CRP
transgene (hCRPtg). Our data associate this protection
with the ability of CRP to limit renal damage, an effect
it achieves by preventing glomerular and extraglomerular deposition of immune complexes and/or enhancing
phagocytosis of immune complexes by mesangial cells.
MATERIALS AND METHODS
Animals. Our C57BL/6 congenic (H-2b/b) hCRPtg
mouse strain has been described in detail elsewhere (22).
These animals carry a 31-kb Cla I fragment of human genomic
DNA comprising of the CRP gene, 17 kb of the 5⬘-flanking
sequence, and 11.3 kb of the 3⬘-flanking sequence, and they
express high levels of human CRP in response to injection of
lipopolysaccharide (LPS) (23) or bacterial infection (22). We
crossed C57BL/6 hCRPtg mice with NZW mice (H-2z/z) obtained from The Jackson Laboratory (Bar Harbor, ME) to
generate hybrid pups (H-2b/z). Pups were screened for the
presence of the human CRP transgene by polymerase chain
reaction (PCR) (24), and hCRPtg F1 animals were backcrossed
with NZW mice. PCR amplification of an H-2z–linked polymorphism in the promoter of the tumor necrosis factor ␣ gene
(25) allowed us to identify F2 progeny that were H-2z/z, and
hCRPtg/H-2z/z mice were then backcrossed to the NZW strain
to fix the H-2 complex. Transgenic mice were backcrossed at
least 9 times prior to mating hCRPtg/NZW congenic males
with NZB females (obtained from The Jackson Laboratory) to
produce lupus-prone hybrids.
Disease development in female hCRPtg/BW and female NZB/NZW littermates was then monitored. All mice
eventually developed detectable levels of anti-dsDNA autoantibodies and exhibited severe anasarca and concomitant pro-
1603
teinuria, indicating that backcrossing transferred the NZW loci
responsible for lupus. Mice were housed in groups of 4, given
food and water ad libitum, and maintained according to
protocols approved by the Animal Resources Program of the
The University of Alabama at Birmingham.
Monitoring mortality, proteinuria, and serum proteins. The course of disease in hCRPtg/BW mice was compared with that in age-matched NZB/NZW mice. Urine and
blood were collected at biweekly intervals. Proteinuria was
assessed with Albustix test strips (Bayer, Elkhart, IN) and
scored as trace (⬍0.3 mg/ml), 1⫹ (0.3–0.9 mg/ml), 2⫹ (1–2.9
mg/ml), 3⫹ (3–20 mg/ml), or 4⫹ (⬎20 mg/ml), according to
the manufacturer’s scoring system. Blood was diluted immediately in ice-cold GVB–EDTA buffer (0.1% gelatin, 141 mM
NaCl, 1.8 mM sodium barbital, 3.1 mM barbituric acid, 10 mM
EDTA, pH 7.4), centrifuged at 1,000g, and the plasma was
collected and frozen at –70°C until used. Deaths of mice were
noted 4 times daily.
Plasma levels of human CRP were measured by
enzyme-linked immunosorbent assay (ELISA) (22). The assay
has a lower limit of detection of 20 ng of human CRP/ml and
does not detect mouse CRP. To induce acute-phase expression
of human CRP, mice received an intraperitoneal injection of
25 ␮g of LPS (Sigma-Aldrich, St. Louis, MO).
Anti-dsDNA antibody titers were measured by ELISA
(26) using proteinase/ribonuclease-treated and sonicated calf
thymus DNA (Sigma-Aldrich) as immobilized antigen and
horseradish peroxidase–conjugated goat anti-mouse immunoglobulins specific for IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3
(Southern Biotechnology, Birmingham, AL) as reporters.
Plasma was diluted serially in GVB–EDTA, and ABTS
(Sigma-Aldrich) was used as chromogenic substrate. The antidsDNA end-point titer for each sample corresponds to the
highest dilution of plasma that resulted in an absorbance at 405
nm that was more than twice the level achieved using undiluted
plasma from a 6-week-old C57BL/6 mouse. As positive controls for IgG and IgM anti-dsDNA, we used DNA6 and 25-12
monoclonal antibodies, respectively (26).
Histologic analysis. Kidneys harvested from randomly
selected age-matched hCRPtg/BW and NZB/NZW mice were
embedded in OCT compound (Sakura, Torrance, CA) and
stored (–70°C) until they were processed for light and immunofluorescence microscopy, or they were fixed in glutaraldehyde and processed for electron microscopy. For light and
immunofluorescence microscopy, serial sections (4 ␮m thick)
were cut on a cryostat microtome at –20°C, mounted on glass
slides, air-dried, acetone-fixed, and stained with hematoxylin
and eosin to allow comparison of gross pathology.
To detect immunoglobulins and C3, sections were
blocked with normal horse serum and stained with tetramethylrhodamine isothiocyanate (TRITC)–labeled goat F(ab⬘)2 antimouse IgG or anti-mouse IgM (␮ chain) and fluorescein
isothiocyanate (FITC)–labeled goat F(ab⬘)2 anti-mouse C3
(ICN Pharmaceuticals, Aurora, OH). To detect human CRP,
we used biotinylated HD2-4 monoclonal antibody (27) in
conjunction with R-phycoerythrin (PE)–streptavidin (Southern Biotechnology). Sections were washed and mounted in
Fluormount G (Southern Biotechnology) and viewed with a
Leica DMRB fluorescence microscope (Bannockburn, IL)
equipped with appropriate filter cubes (Chromatechnology,
Battleboro, VT). Images were acquired with a Hamamatsu
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Figure 1. Reduced proteinuria and increased survival in (NZB ⫻
NZW)F1 (BW) mice made transgenic for human C-reactive protein
(hCRPtg/BW). A, Kaplan-Meier cumulative survival is plotted for 25
mice per group. Longevity of hCRPtg/BW mice (290 ⫾ 12 days) is
significantly increased compared with BW mice (235 ⫾ 9 days) (P ⫽
0.004, by Student’s t-test). B, Incidence of proteinuria is plotted for
25–30 mice per group. Severe proteinuria (⬎0.9 mg of protein/ml) is
detected 4 weeks later, with a 2–5-fold reduced incidence in
hCRPtg/BW mice compared with BW mice. ⴱ ⫽ P ⫽ 0.016 for the
incidence of severe proteinuria at 30 weeks, by chi-square test.
SZALAI ET AL
Photonic System digital color camera (Hamamatsu, Bridgewater, NJ) and processed with Adobe Photo Shop (Adobe
Systems, San Jose, CA) and IP LAB Spectrum software (Signal
Analytics Software, Vienna, VA). Images of TRITC and/or
R-PE fluorescence (red) superimposed on images of FITC
fluorescence (green) produced a yellow-orange color when the
2 fluorochromes coincided.
Glutaraldehyde-fixed tissue was processed for electron
microscopy according to a standard procedure that included
postfixation in osmium tetroxide, dehydration with acetone,
and embedding in Spurr’s resin. Ultrathin sections were cut on
an LKB Ultratome III (Stockholm, Sweden), poststained with
aqueous uranyl acetate and Reynold’s lead citrate, and examined with a Phillips model TEM 301 transmission electron
microscope at an accelerating voltage of 60 kV.
In situ hybridization. Plasmids (pcDNA3.1; Promega,
Madison, WI) carrying CRP complementary DNA inserts
derived from clone HLCRP-23 (28) were used as templates for
the synthesis of digoxigenin-labeled riboprobes. One plasmid
carrying a 1.5-kb fragment that spans the entire CRP gene,
including 0.76 kb of 3⬘-untranslated sequence, was used to
make the sense probe; and a second plasmid carrying the
protein coding sequence only (0.74 kb) was used to make the
antisense probe. Plasmids were linearized by digestion with
Hind III (Promega), and in vitro transcription was performed
using an RNA transcription kit containing T7 RNA polymerase (Promega).
Kidneys were harvested from mice, frozen, and sectioned as described above for light microscopy. Serial sections
were mounted on glass slides and fixed in 3% paraformaldehyde, pH 7.4. These were hybridized for 16 hours at 50°C with
the riboprobes diluted 1:10 in hybridization buffer (Fisher
Scientific, Fair Lawn, NJ). After hybridization, 3 washes were
performed at 50°C. To reveal the location of RNA/RNA
duplexes, slides were incubated with alkaline phosphatase–
conjugated anti-digoxigenin F(ab⬘)2 (Boehringer Manheim,
Indianapolis, IN), followed by reaction with BCIP/nitroblue
tetrazolium substrate (Sigma-Aldrich).
Figure 2. Reduced IgM and elevated IgG anti–double-stranded DNA (anti-dsDNA) responses in (NZB ⫻ NZW)F1 (BW) mice made transgenic
for human C-reactive protein (hCRPtg/BW). Enzyme-linked immunosorbent assay was used to measure IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3
anti-dsDNA antibodies. Values are the mean ⫾ SEM of 10 randomly selected mice per time point. ⴱⴱ ⫽ P ⬍ 0.05 for anti-dsDNA titers in
age-matched groups, by Student’s unpaired t-test. Arrows indicate ages at which a difference between the 2 groups of mice is apparent but could
not be tested for statistical significance because of zero variance in one or both groups.
DELAYED LUPUS ONSET IN MICE EXPRESSING HUMAN CRP
1605
considered significant in all analyses. Data are reported as the
mean ⫾ SEM.
RESULTS
On average, hCRPtg/BW mice lived 8 weeks
longer than NZB/NZW mice (mean ⫾ SEM 290 ⫾ 12
days versus 235 ⫾ 9 days; P ⫽ 0.004, by Student’s t-test)
(Figure 1A), and 85% of the hCRPtg/BW mice were still
alive when 50% of the NZB/NZW mice had already
succumbed to lupus (33 weeks). The onset of severe
proteinuria was also delayed by at least 4 weeks in
hCRPtg/BW mice compared with NZB/NZW mice, and
the incidence of severe proteinuria in hCRPtg/BW mice
was 2–5 times lower (Figure 1B). The difference in the
incidence of severe proteinuria between NZB/NZW and
Figure 3. Reduction of IgG and C3 levels in the kidneys of (NZB ⫻
NZW)F1 (BW) mice made transgenic for human C-reactive protein
(hCRPtg/BW). Thin sections of kidneys from BW mice and agematched hCRPtg/BW mice were stained with antibodies specific for
mouse IgG (red) and mouse C3 (green). In 5-week-old mice, C3 is
detected in the epithelia, and no IgG is present. By the age of 26 weeks,
virtually all the renal glomeruli of the BW mice contain both IgG and
C3 deposited in a mesangial and capillary pattern. The yellow-orange
color resulting from coincident fluorescence suggests that IgG and C3
are colocalized. Compared with age-matched BW mice, the kidneys of
26-week-old hCRPtg/BW mice contain less mesangial IgG, and less of
it is colocalized with C3. By the age of 34 weeks, IgG and C3 are
present in the extraglomerular region in BW mice but not in
hCRPtg/BW mice. Each image is representative of the results obtained
with multiple tissue sections from both kidneys of ⱖ4 mice per group.
(Original magnification ⫻ 200.)
Statistical analysis. Survival curves were generated
using 25 hCRPtg/BW mice and 25 NZB/NZW mice, and
kidneys were obtained from 2 contemporaneous but separate
groups of 18 hCRPtg/BW mice and 20 NZB/NZW mice
housed in the same facility. The deaths of the mice had a
normal distribution, so an unpaired Student’s t-test was used to
evaluate differences in lifespan. The chi-square test with
Fisher’s correction was used to evaluate differences in the
incidence of severe (ⱖ2⫹) proteinuria. Anti-dsDNA titers
were log-transformed to achieve normality and then compared
using Student’s t-tests; in some cases, this analysis could not be
applied due to zero variance. P values less than 0.05 were
Figure 4. Reduction of IgM levels in the kidneys of (NZB ⫻ NZW)F1
(BW) mice made transgenic for human C-reactive protein (hCRPtg/
BW). Thin sections of kidneys from BW mice and age-matched
hCRPtg/BW mice were stained with antibodies specific for mouse IgM
(red) and mouse C3 (green). In 5-week-old mice, no IgM is detected in
either strain. In the glomeruli at 26 weeks of age, the IgM signal is
strong in BW mice but weak in hCRPtg/BW mice. At 34 weeks of age,
IgM is present in the glomeruli in both strains at more comparable
levels. Each image is representative of the results obtained with
multiple tissue sections from both kidneys of ⱖ4 mice per group.
(Original magnification ⫻ 400.)
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SZALAI ET AL
Figure 5. Electron micrographs of glomerular capillaries from 35-week-old (NZB ⫻ NZW)F1 (BW) mice
and in BW mice made transgenic for human C-reactive protein (hCRPtg/BW). In BW mice (top), the
healthiest-looking capillary loops (A) already have extensive subepithelial, subendothelial, and intramembranous dense deposits (arrows), a thickened glomerular basement membrane (bm) (inset), and extensive
fusion of podocyte (p) foot processes (ⴱ) (inset). The majority of capillary loops in BW mice were almost
completely occluded (B and C) due to proliferation and invasion of the lumen (l) by mesangial (m) and
endothelial (e) cells, and some dense deposits were observed in the mesangium (thick arrow) (C). In
contrast, in the glomeruli of hCRPtg/BW mice (bottom), accumulation of dense deposits was more
pronounced in the mesangial region (F) (thick arrows). There was little or no basement membrane
thickening or foot process fusion (D and inset), and the capillary loops remained open (E and F). Each
image is representative of the results obtained with multiple tissue sections from 1 kidney of 3 mice per
group. ec ⫽ erythrocyte; us ⫽ urinary space. (Original magnification ⫻ 5,300 in A–F; ⫻ 16,000 in insets.)
hCRPtg/BW mice achieved statistical significance at 30
weeks (␹2 ⫽ 5.788, P ⫽ 0.016).
To decipher the basis for CRP protection, plasma
was collected from mice and assayed for the presence of
circulating anti-dsDNA antibodies. There were marked
age-related variations in the titers of the different classes
and subclasses of anti-dsDNA in NZB/NZW mice (Figure 2). This finding confirms those of previous investigators who studied the characteristics of anti-dsDNA
responses in NZB/NZW mice (29). In general, in young
NZB/NZW mice, the predominant anti-dsDNA class
was IgM. As the animals aged, there was increased
production of IgG. Anti-dsDNA of all isotypes peaked
at ⱖ30 weeks.
The anti-dsDNA response of the hCRPtg/BW
mice was equally variable and also peaked at ⱖ30 weeks,
but had a slightly different tempo (Figure 2). For
example, in NZB/NZW mice, there was early and transient rise in IgM anti-dsDNA, but this did not occur in
hCRPtg/BW mice. Consequently, in 16- and 20-weekold hCRPtg/BW mice the IgM anti-dsDNA titer was
6–7-fold lower than that in age-matched NZB/NZW
mice (P ⬍ 0.05, by Student’s t-test). In contrast to IgM,
the IgG anti-dsDNA titer was 3–5-fold higher in 18- and
24-week-old hCRPtg/BW mice than in age-matched
NZB/NZW mice (P ⬍ 0.05, by Student’s t-test). The
elevation in IgG antibody was a consequence of the
increased production of IgG1, IgG2a, and IgG2b subclasses.
Kidneys harvested at different time points and
DELAYED LUPUS ONSET IN MICE EXPRESSING HUMAN CRP
1607
Electron microscopy (Figure 5) confirmed the
presence of dense deposits in the glomeruli of both
NZB/NZW and hCRPtg/BW mice in a pattern consistent with the presence of immune complexes. In the
glomeruli of NZB/NZW mice, there was extensive fusion of podocyte foot processes, thickening of the glomerular basement membrane, and proliferation of mesangial cells with invasion and occlusion of the capillary
lumens (Figure 5, top panels). In contrast, in the glomeruli of hCRPtg/BW mice, neither obvious foot process fusion nor any obvious basement membrane thickFigure 6. Plasma levels of human C-reactive protein (CRP) in
(NZB ⫻ NZW)F1 (BW) mice made transgenic for human CRP
(hCRPtg/BW). Plasma levels of human CRP were not substantially
elevated in plasma from untreated hCRPtg/BW mice (E), but they
rose in response to lipopolysaccharide (LPS) treatment in agematched hCRPtg/BW mice that had received an intraperitoneal injection of LPS 24 hours previously (F). Values are the mean ⫾ SEM of
2–7 mice per time point.
stained with hematoxylin and eosin revealed progression
of glomerular disease typical of murine lupus in both the
hCRPtg/BW and the NZB/NZW groups, with mesangial
thickening and hypercellularity evolving into crescent
formation and end-stage sclerosis (data not shown).
Kidneys were then analyzed more extensively by immunofluorescence (Figures 3 and 4) and electron microscopy (Figure 5).
In the kidneys of healthy 5-week-old mice, no
IgG or IgM was detected in either strain, but C3 was
detected in both (Figures 3 and 4). Importantly, in young
mice, immunoreactive C3 was detected mainly in the
epithelial layer, with the strongest signal in the Bowman’s capsule, most likely because of local production of
C3 rather than the accumulation of blood-borne C3. By
26 weeks, heavy accumulations of immunoglobulin and
C3 were seen inside the glomeruli of NZB/NZW mice
(Figures 3 and 4), with a fluorescence pattern suggesting
the presence of immune complexes in both the capillary
loops and the mesangium. In comparison, the glomeruli
of age-matched hCRPtg/BW mice had less accumulation
of IgG and IgM, with a more mesangial pattern but a
similar distribution of C3. By 34 weeks, accumulation of
immunoglobulins and C3 in the kidneys of NZB/NZW
mice had expanded beyond the glomeruli to the cortex
(Figures 3 and 4). In contrast, in age-matched
hCRPtg/BW mice, there was no accumulation of IgM,
IgG, or C3 in the cortex. Again, there was no apparent
difference in the amount of C3 present within glomeruli
in the two strains.
Figure 7. Accumulation of human C-reactive protein (CRP) in the
kidneys of (NZB ⫻ NZW)F1 (BW) mice made transgenic for human
C-reactive protein (hCRPtg/BW). Thin sections of kidneys from BW
mice and age-matched hCRPtg/BW mice were stained with antibodies
specific for mouse C3 (green) and human CRP (red). In the
hCRPtg/BW groups, little or no human CRP is present in the kidney
of 5-week-old mice, but the amount of human CRP increases in
26-week-old and 34-week-old mice. Note that human CRP is present in
the cortical (tubular) region and is not accompanied by C3. The
comparatively weak anti-human CRP signal seen in the kidneys of
26-week-old BW mice was unexpected because HD2-4 does not
cross-react with mouse CRP; nevertheless, this staining might reflect
nonspecific binding of the biotinylated monoclonal antibody and/or
cross-reactivity with mouse CRP. Each image is representative of the
results obtained with multiple tissue sections from 1 kidney of 3 mice
per group. (Original magnification ⫻ 200.)
1608
SZALAI ET AL
As in female hCRPtg mice with non-autoimmune
genetic backgrounds (22,24), blood levels of CRP in the
hCRPtg/BW mice were low. However, relative to baseline values determined at age 6 weeks, there was a
50–100-fold increase observed with aging (Figure 6).
Since hCRPtg/BW mice did not secrete human CRP into
their urine (as determined by ELISA; data not shown)
and the transgene maintained full acute-phase inducibility, as judged by LPS responsiveness (Figure 6), we
examined whether human CRP might be present in the
kidneys. Human CRP–specific immunofluorescence was
detected in the kidneys of older hCRPtg/BW mice
(Figure 7). Parallel analysis of kidneys obtained from
older, non–lupus-prone hCRPtg/C57BL/6 mice (22) also
revealed human CRP in the kidneys (data not shown),
suggesting that the appearance of CRP in the kidneys of
hCRPtg/BW mice is age- rather than disease-related.
In situ hybridization experiments showed that in
the kidneys of hCRPtg/BW mice, human CRP messenger RNA was produced locally and was up-regulated
with age (Figure 8), and thus appears there coincident
with the progression of glomerular disease. In concordance with the staining pattern seen with immunofluorescence (Figure 7), the staining pattern seen with in situ
hybridization (Figure 8) suggests that cells in the cortical
tubular area are the main source of the renal human
CRP.
DISCUSSION
Figure 8. Expression of the human C-reactive protein (CRP) transgene in the kidneys of (NZB ⫻ NZW)F1 (BW) mice made transgenic
for human C-reactive protein (hCRPtg/BW). Thin sections of kidneys
from 9-, 23-, and 35-week-old mice were incubated with a digoxigeninlabeled antisense probe to reveal human CRP mRNA. Note the
increased CRP signal in the cortical tubular area of 23- and 35-weekold hCRPtg/BW mice. Only background staining is seen in kidneys
from 35-week-old hCRPtg/BW mice hybridized with the sense probe,
and no CRP signal is detected in kidneys from 35-week-old BW mice
hybridized with the antisense probe. Each image is representative of
the results obtained with multiple tissue sections from 1 kidney of 2
mice per group. (Original magnification ⫻ 200.)
ening was seen, and the capillary lumens largely
remained more open (Figure 5; bottom panels). Importantly, in accordance with the increased mesangial pattern of fluorescence seen when hCRPtg/BW glomeruli
were stained with anti-Ig (Figures 3 and 4), the number
and size of dense deposits seen in the mesangium of
hCRPtg/BW mice (Figure 5F) were increased compared
with that in NZB/NZW mice.
We show here that endogenously expressed human CRP is protective in a mouse model of SLE, that is,
the spontaneous development of lethal glomerulonephritis was delayed and the lifespan was extended in
NZB/NZW mice carrying a human CRP transgene. This
finding confirms and extends earlier observations made
by Du Clos et al (21), who showed that 26-week-old
NZB/NZW mice injected repeatedly with chromatincoated latex beads pretreated with human CRP lived
longer than age-matched animals injected with untreated chromatin-coated beads. In those experiments,
CRP did not alter the IgM and IgG anti-DNA response,
the beneficial effect of CRP was short-lived, and protection was not observed in 19-week-old mice. The transient nature of the CRP protective effect was attributed
to development of anti-CRP responses in mice given
human CRP (21). In comparison, we found that in our
transgenic NZB/NZW mice, expression of human CRP
was associated with slight and transient, but significant,
lowering of IgM anti-dsDNA autoantibody titers, and
slight and transient elevation of IgG1, IgG2a, and IgG2b
DELAYED LUPUS ONSET IN MICE EXPRESSING HUMAN CRP
anti-dsDNA titers. Precisely how human CRP changes
the tempo and repertoire of the anti-dsDNA response in
NZB/NZW mice is not known with certainty, but the
data from both our study and that by Du Clos et al are
consistent with the proposal by Du Clos and colleagues
(21) that this is a consequence of altered clearance
and/or processing of autoantigens by circulating CRP.
Such a process could hasten the isotype switch and might
also explain the apparent discordance in the IgM and
IgG results. Presumably, renal CRP could have similar
contributions.
As shown in Figure 6, although CRP was elevated
up to 100-fold during disease development, its absolute
level remained low compared with that in the LPSinduced acute phase. We found also that human CRP
was locally produced in the kidneys of hCRPtg/BW
mice, and like the blood-borne (hepatically synthesized)
protein, its expression there increased with age. The
finding that CRP is expressed in the kidney is not
surprising, since in a previous study of mice carrying the
same CRP transgene, Klein et al (30) detected (by
nested PCR) the expression of human CRP in the
kidneys. The levels they detected, however, were scant,
and they failed then to recognize that renal expression of
the CRP transgene increased with age; this presumably
occurred because only the kidneys of young adult mice
were examined (30). Recent clinical evidence suggests
that our observation of CRP expression in the kidneys is
an accurate reflection of the situation found in SLE in
humans. For example, in SLE patients, CRP is often not
elevated in the blood even during disease flares (5–8)
and is rarely lost into the urine (31), yet it is frequently
found in the kidneys of patients with glomerular disease
(32). These clinical observations together with our observations of hCRPtg/BW mice suggest that locally
expressed CRP, and perhaps its regulation, might be
more important in the pathogenesis of SLE than is
blood-borne CRP and its hepatic expression.
If, as we suspect, the presence of human CRP in
the kidneys of hCRPtg/BW mice is the key to understanding its protective effect, then exactly how does CRP
retard the development of lupus nephritis? The ultimate
fate of CRP produced locally in the kidney remains
elusive. We could not detect (by ELISA) the protein in
the urine, and it is still not known if the presence of CRP
in the cortical tubular area has any impact on renal
physiology per se. However, we do know that in
hCRPtg/BW mice, there is a virtual absence of immune
complex deposition in the renal cortex and an increased
accumulation of dense deposits in the glomerular mesangium, and we think this is informative. As we demon-
1609
strated here for C3, other complement proteins are also
expressed by the renal epithelium in murine lupus
nephritis (33–35). CRP is known to activate complement
via the classical pathway (14), and the classical pathway
plays an important physiologic role by preventing the
formation of immune complexes (36). Thus, one possibility is that by activating the classical pathway of
complement, CRP inhibits the formation of immune
complexes in the cortex, thereby reducing the local
inflammatory response and delaying the onset of glomerulonephritis. This scenario requires that immune
complex formation occur in the cortex, but what if
immune complexes are preformed distally and then
deposited locally?
In contrast to its effect on the classical pathway,
CRP inhibits the alternative pathway (37), and the
alternative pathway affects the solubilization of large
insoluble preformed immune complexes (38). Recent
evidence shows that activation of the alternative pathway
has a prominent detrimental effect in the lupus kidney
(39). In a second scenario, by inhibiting the alternative
pathway, CRP could still reduce local inflammation and
support the improved clearance of deposited immune
complexes by resident phagocytes.
In a third scenario, since CRP is known to bind
Fc␥R (15–17) and since occupation of Fc␥R is known to
modulate the complement-mediated inflammatory response in the kidney (40), the protective effect might
involve the interaction of CRP with cells bearing Fc␥R.
Importantly, mesangial cells do express Fc␥R and have a
phagocytic clearance function (ref. 41 and for review, see
ref. 42). Perhaps CRP enhancement of the mesangial
cell clearance of immune complexes is responsible for
the protection observed and the increase in dense deposits in the mesangium of hCRPtg/BW mice. It is
important to point out that these 3 scenarios are not
mutually exclusive, and that in all instances, the ability of
blood CRP to clear apoptotic and necrotic cells (18,43)
would further enhance the antiinflammatory effect.
SAP is very closely related to CRP and supports
many of the same effector functions (44). In a recent
report (45), Bickerstaff et al showed that in an otherwise
healthy strain of mice, targeted deletion of the SAP gene
led to the development of antinuclear autoimmunity and
severe glomerulonephritis. Like CRP, SAP binds and
can solubilize chromatin (46), and it binds to apoptotic
and necrotic cells (47,48). Thus, the SLE-like phenotype
of SAP-deficient mice was linked to their impaired
handling and degradation of chromatin, which allowed
for increased anti-DNA responses (45). These results
from SAP-deficient mice and our findings from
1610
SZALAI ET AL
hCRPtg/BW mice strongly support the notion that CRP
and SAP provide an important physiologic function.
CRP-deficient NZB/NZW mice are not available to
confirm this, but presumably, mouse CRP, which is also
expressed in the kidney (30), is protective as well.
Although the additional local expression of human CRP
in hCRPtg/BW mice does not prevent glomerular injury,
it clearly delays disease development above and beyond
that achieved by endogenous mouse CRP and SAP.
Recognizing that generalizing the results of
mouse studies to humans must be done with caution, our
data suggest that the beneficial role of CRP likely is
2-fold in SLE and in other diseases in which the kidney
is a major target organ. On the one hand, local and
systemic production of CRP delays the onset of renal
involvement, and on the other hand, circulating CRP has
significant antimicrobial properties (for review, see ref.
49) and reduces the likelihood of intercurrent infection.
12.
13.
14.
15.
16.
17.
18.
ACKNOWLEDGMENTS
The authors thank Nuzhat Iqbal (Department of Medicine, The University of Alabama at Birmingham) for assistance and Dr. A. Agrawal (Department of Pharmacology, East
Tennessee State University, Johnson City, TN) for helpful
advice.
19.
20.
21.
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