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The Journal of Immunology, 2007, 178: 530-538.
Copyright © 2007 by The American Association of Immunologists, Inc.

C-Reactive Protein-Mediated Suppression of Nephrotoxic Nephritis: Role of Macrophages, Complement, and Fc{gamma} Receptors1

Wilfredo Rodriguez*, Carolyn Mold{dagger}, Milena Kataranovski{dagger}, Julie A. Hutt{ddagger}, Lorraine L. Marnell*, J. Sjef Verbeek§ and Terry W. Du Clos2,*,¶

* Department of Internal Medicine and {dagger} Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131; {ddagger} Experimental Toxicology Program, Lovelace Respiratory Research Institute, Albuquerque, NM 87108; § Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands; and Department of Veterans Affairs Medical Center, Albuquerque, NM 87108


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C-reactive protein (CRP) is a member of the pentraxin family of proteins and an acute phase reactant. CRP modulates the response to inflammatory stimuli including LPS and C5a. We recently demonstrated that CRP prevents and reverses proteinuria in accelerated nephrotoxic nephritis (NTN). NTN is a model of active inflammatory immune complex-mediated nephritis induced by injection of antiglomerular basement membrane. CRP treatment prevented the induction of NTN in C57BL/6 (B6) mice, increased survival, and reversed ongoing nephritis. Protection was associated with a decrease in IL-1beta and chemokines in the kidney and peritoneal cells as measured by quantitative RT-PCR. However, IL-10–/– mice were not protected by CRP either when given before disease onset or when disease activity was maximal. Fc{gamma}RI–/– mice developed NTN, but were only transiently protected by CRP treatment. This transient protection was abrogated by cobra venom factor depletion of complement from Fc{gamma}RI–/– mice. However, complement depletion did not prevent CRP-mediated protection in B6 mice, and CRP was protective in C3–/– mice. The role of macrophages in the protection provided by CRP was tested by treating B6 mice with liposomes containing clodronate. Clodronate-containing liposomes deplete mice of splenic and hepatic macrophages for 5–7 days. Pretreatment of NTN mice with clodronate but not control liposomes completely prevented CRP-mediated protection. These studies suggest that CRP mediates protection from NTN through the induction of IL-10 and that macrophages are required. In addition, Fc{gamma}RI plays an important role but is not the sole mediator of CRP-mediated protection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C-reactive protein (CRP)3 is a prototypic acute phase protein in humans, and levels increase rapidly from <1 µg/ml to as high as 1 mg/ml 24–48 h after an acute phase stimulus like inflammation, infection, or tissue damage (1, 2). CRP is produced primarily in the liver in response to IL-6 with other inflammatory cytokines like IL-1 playing a less prominent role (3). Therefore, CRP is present at very high concentration when acute inflammation is maximal.

CRP is a member of the pentraxin family of proteins. The pentraxins are characterized by cyclic pentameric or hexameric structure composed of five or six identical subunits (4). Each protomer contains two bound calcium ions on one face of the pentamer, which are involved in binding to its ligands. Binding sites for complement component C1q and Fc{gamma}R are present on the other face of the molecule.

CRP shares many properties in common with IgG, including the ability to activate the classical complement pathway (5), the capacity to interact with Fc{gamma}R (6, 7), and the ability to bind to ligands. The interaction of CRP with Fc{gamma}R mediates several functions that are analogous to those of IgG, including cytokine secretion and opsonization of bacteria and altered or exposed self-molecules on damaged cells (2).

The ability of CRP to suppress acute inflammation has been well documented in studies using mice transgenic for rabbit CRP developed by Samols and colleagues (8, 9, 10). Mice express only low levels of CRP (<5 µg/ml) and have been useful in models to test in vivo activities of transgenic or injected CRP. Human and rabbit CRP activate mouse complement and bind to receptors on mouse macrophages. Mice expressing high levels of CRP were protected from lethal shock induced by endotoxin, platelet-activating factor (PAF) or the combination of TNF-{alpha} and IL-1beta (9). These results suggest that CRP may regulate the inflammatory cytokine cascade or the response to it. Transgenic mice expressing high levels of CRP and challenged by intratracheal administration of chemotactic agents also showed decreased pulmonary inflammation compared to mice expressing low levels of CRP (8, 10). The mechanisms responsible for these effects are not well understood, although a direct inhibitory effect of CRP on neutrophil chemotaxis was proposed (8, 11, 12). CRP has also been shown to interact with PAF, and inhibition of PAF was proposed to account for its protective effect on PAF-induced shock (13). We have shown that CRP-mediated protection against endotoxin shock requires Fc{gamma}R and is associated with the induction of IL-10 and suppression of IL-12 synthesis (14). Mosser and colleagues (15, 16) have described similar regulatory effects of immune complexes (IC) on macrophage cytokine responses to LPS and other TLR ligands.

CRP is a pattern recognition molecule that binds ligands on microbes and damaged tissue (1). The binding of CRP to nuclear Ags, including small nuclear ribonucleoproteins, histones, and chromatin, and to apoptotic and necrotic cells has led to investigations of its role in systemic autoimmunity (17, 18, 19). The effect of CRP on autoimmune disease has been examined in our laboratory and those of others. We first established that CRP could suppress autoantibodies and prolong survival of autoimmune (NZB x NZW)F1 female mice (NZB/NZW) treated with chromatin to accelerate disease (20). This work was extended by studies of Szalai et al. (21) who showed that transgenic NZB/NZW mice expressing human CRP from the human promoter had delayed proteinuria and prolonged survival. The transgenic NZB/NZW mice had decreased glomerular pathology with late IC accumulation in glomeruli and increased mesangial deposits. More recently, we determined that CRP given as a single injection to NZB/NZW mice either before disease onset or during active nephritis could rapidly and markedly decrease proteinuria (22). Survival of both groups of CRP-treated NZB/NZW mice was extended by 10–13 wk despite a lack of effect on anti-dsDNA Ab levels. In more recent studies using the more fulminant MRL/lpr model of systemic lupus erythematosus, we observed similar protective effects of CRP (23). Moreover, in the MRL/lpr model, CRP-treated mice showed a decrease in autoantibody levels to dsDNA and lymphadenopathy as well. IC deposition in glomeruli was not affected by CRP treatment, but there was greatly reduced glomerular as well as tubular and interstitial pathology.

A protective effect of transgenic CRP has also been demonstrated in a T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (24). CRP increased IL-10 synthesis in cultures of encephalitogenic T cells and APC. These studies suggested a possible common mechanism responsible for CRP-mediated immunomodulation involving the participation of IL-10. We hypothesized that the anti-inflammatory activity of CRP might be essential for its therapeutic effect on lupus nephritis, independent of any effect on the autoimmune response.

To investigate the protective effect of CRP in IC glomerulonephritis separately from the autoimmune response, we used an accelerated nephrotoxic nephritis model (NTN) (22). NTN is a model of acute glomerular inflammation, which is mediated by IC deposited on the glomerular basement membrane (GBM) (25). Disease is induced by immunizing animals with heterologous IgG followed by injection of anti-GBM Abs from the heterologous species. Our initial studies in this model suggested a requirement for IL-10 in the protective role of CRP (22). We now report on the cells and receptors responsible for the protective effect of CRP in NTN. A prominent role for macrophages in the induction and maintenance of suppression is seen. In addition, we determine that the high-affinity receptor for IgG and CRP, Fc{gamma}RI, is required for CRP-mediated protection. These studies suggest that CRP binding to Fc{gamma}RI on macrophages may provide a unique mechanism for immunoregulation by a member of the innate immune system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6) mice were from the National Cancer Institute. C3–/– mice and IL-10–/– mice on a B6 background were obtained from The Jackson Laboratory and maintained at the Department of Veterans Affairs Medical Center Animal Facility. IL-10–/– mice were further backcrossed onto B6 mice to 10 generations. Fc{gamma}RI–/– mice were developed as previously described and backcrossed onto B6 mice (26). Female mice at 6–12 wk of age were used in all experiments. All procedures involving animals were approved by the Institutional Review Board of the Department of Veterans Affairs Medical Center.

Reagents

CRP was purified from human pleural effusion fluid as described previously (27). All preparations were examined on overloaded SDS-PAGE gels to ensure purity. No additional bands other than the major band at ~23 kDa were seen. In addition, the preparations were examined for endotoxin by a quantitative chromogenic Limulus amebocyte lysate assay (BioWhittaker). All preparations used contained <3 endotoxin units (0.3 ng) of endotoxin per mg of protein. Recombinant CRP (Calbiochem) was dialyzed to remove sodium azide and treated for endotoxin contamination with an ActiClean Etox column (Sterogene Bioseparations) to <3 endotoxin units of endotoxin per mg of protein.

To prepare anti-GBM Ab, New Zealand White rabbits were immunized with purified mouse glomeruli. The purification of mouse GBM and immunization of rabbits were as described elsewhere (28, 29). Rabbit IgG and CFA were purchased from Sigma-Aldrich.

Induction of NTN and CRP treatment

Mice were injected i.p. with 0.5 mg of rabbit IgG in CFA. NTN was induced 5 days later (day 0) by injection of rabbit anti-GBM serum i.v. Mice received either a single injection of 100 µl of anti-GBM on day 0 or three injections of 100 µl of rabbit anti-GBM Ab on days 0, 1, and 2 as indicated in the figure legends. A single s.c. injection of 200 µg of human CRP was given for treatment. Control mice were treated with an equal volume of saline. CRP treatment was administered either at the same time as the first anti-GBM injection, or 9–10 days later, after the mice had developed 5+ proteinuria. Proteinuria was measured daily using Albustix (Bayer). Grades of proteinuria are expressed as 0, none; 1+, trace; 2+, 30 mg/dl; 3+, 100 mg/dl; 4+, 300 mg/dl; and 5+, >2000 mg/dl. Blood urea nitrogen (BUN) was measured using Azostix (Bayer).

Histological studies

Kidneys were removed and fixed for 2 h in Bouin’s solution and then transferred to 70% ethanol. They were then embedded in paraffin and 2-µm sections were cut and stained with H&E or with periodic acid-Schiff reagent. The sections were examined by one of us (J.A.H.) in a blinded manner and scored for glomerular and other renal changes. Histopathology scores were assigned on a 4-point scale based on the number of glomeruli involved and the severity of the lesions (1, <10%, minimal; 2, 10–25%, mild; 3, 25–50%, moderate; and 4, >50%, marked). The glomerular lesion score was determined by averaging scores for glomerular hypertrophy, protein rich fluid, or basement membrane disruption; fibrin thrombi; necrotic debris; periglomerular fibrosis, crescents, or bridging; and presence of neutrophils (>2/glomerulus). Thirty glomeruli in each kidney were examined.

To measure the presence of macrophages in the glomeruli, kidneys were fixed in acetone for 10 min at 4°C and then air dried. The slides were washed in PBS and blocked with 10% normal rabbit serum for 30 min. For the staining, a primary rat anti-mouse CD68 mAb (Serotec) was used. Slides were washed and incubated with biotinylated rabbit anti-rat IgG secondary Ab for 30 min and washed. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in PBS for 30 min. The sections were developed with a Vectastain ABC kit with 3,3'-diaminobenzidine stain and hematoxylin counterstain. Slides stained with a rat IgG2a isotype control Ab were used as negative controls.

Chemokine and cytokine mRNA measurement in kidneys

NTN was induced and mice were injected s.c. with 200 µg of CRP or saline on day 0 (early treatment) or day 10 (late treatment). Mice were killed on days 1 and 3 after early CRP treatment or day 11 after late CRP treatment. One kidney from each mouse analyzed was placed in RNAlater (Ambion). RNA was extracted using RNA-STAT60 (Tel-Test) as described by the manufacturer. The integrity of the RNA was confirmed by agarose gel electrophoresis and fluorescent staining with GelStar (Cambrex). The High Capacity cDNA Archive Kit (Applied Biosystems) was used to make cDNA, and mRNA levels were determined by quantitative RT-PCR (qRT-PCR) using TaqMan Gene Expression assays in the Applied Biosystems 7500 Real-Time PCR System. The RNA was normalized to ribosomal protein 13 and compared with levels in untreated B6 mouse kidney.

Chemokine and cytokine mRNA measurement in peritoneal cells

NTN was induced as described above and mice were injected i.p. with 200 µg of CRP or saline on day 8. After 8 h, mice were killed and peritoneal cells were collected into RNAlater and the RNA was extracted with RNAqueous (Ambion). 32P-radiolabeled cDNA probes were made from the RNA and hybridized to a Mouse Inflammatory Cytokines and Receptors Gene Array (SuperArray Biosciences). The arrays were washed and scanned on a Storm 860 phosphor imager (Amersham Biosciences) and analyzed with ImageQuant TL software. Results are expressed as the fold increase or decrease in CRP-treated compared with control mice.

Macrophage depletion

Liposomes containing dichloromethylene bisphosphonate (clodronate) were prepared as previously described (30). Clodronate was provided by Boehringer Mannheim. Phosphatidylcholine and cholesterol were purchased from Sigma-Aldrich. Mice were injected i.v. with 0.2 ml of clodronate liposomes. We previously documented that this treatment depletes 99% of Kupffer cells and 95% of splenic macrophages (31). Control liposomes were prepared with PBS in place of clodronate.

Complement depletion

Mice were depleted of complement by treatment with cobra venom factor (CVF, Naja naja kaouthia; Quidel) as previously described (32). Complement depletion was induced by three i.p. injections of 4 U of CVF at 12-h intervals with the last injection 12 h before injection of anti-GBM. This treatment decreases serum C3 levels to <10% of normal (32).

Statistical analysis

Survival curves were plotted by the method of Kaplan and Meier and compared by the log-rank test (Mantel-Haenszel test). This analysis takes into account the time of death as well as the absolute numbers of mice surviving. Other comparisons were made between CRP-treated and saline-treated mice using either the two-tailed Student’s t test or the Mann- Whitney U test for nonparametric data. Graphical and statistical analyses were performed using GraphPad Prism software version 4.0 (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CRP treatment prevents and reverses proteinuria in B6 mice with NTN

We previously demonstrated that CRP treatment prevents and reverses NTN in B6 mice (23). These experiments were repeated here to define the conditions required for the analysis of mechanisms involved in CRP-mediated protection. NTN was induced in B6 mice as described in Materials and Methods using three daily doses of anti-GBM serum on days 0, 1, and 2. Mice were treated with 200 µg of CRP or saline on day 0 (early CRP treatment). Some of the saline-treated mice (n = 6) were injected with 200 µg of CRP on day 9 (late CRP treatment). Proteinuria was measured daily using Albustix on a 0–5+ scale. The mice that were treated with CRP on day 0 were largely protected from the onset of severe proteinuria for the duration of the experiment, although mild proteinuria developed slowly over time in some mice. Mice that were treated with CRP on day 9 showed a rapid reversal of proteinuria (Fig. 1A). To measure renal function, BUN was measured on day 29. Both the early and late CRP treatments reduced the level of BUN, which is considered a more reliable measurement of renal function than proteinuria (Fig. 1B). The survival of mice treated with CRP on day 0 was also significantly improved over saline-treated mice, Fig. 1C. To determine the dose requirements for CRP and to ensure that the effect of treatment was not due to any possible minor contaminant of the purified CRP, rCRP was tested. Mice were treated on day 0 with different concentrations of purified human CRP or with 200 µg of rCRP that had been dialyzed and depleted of endotoxin. As seen in Fig. 1D, full protection required 200 µg of CRP and rCRP was similarly effective as native purified human CRP in preventing proteinuria.


Figure 1
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  		FIGURE 1. CRP treatment prevents and reverses NTN in B6 mice. NTN was induced in B6 mice using three daily doses of anti-GBM serum on days 0, 1, and 2. Mice were treated with 200 µg of CRP or saline on day 0 (early CRP). Half of the saline-treated mice were injected with 200 µg of CRP on day 9 (late CRP). A, Proteinuria was measured daily using Albustix. B, BUN was measured on day 29 using Azostix. C, Survival of saline and early CRP groups. D, Mice were treated on day 0 with different concentrations of purified human CRP (injected dose in µg) or with 200 µg of rCRP. Proteinuria values are means ± SEM using Albustix. The scale is 0, none; 1+, trace; 2+, 30 mg/dl; 3+, 100 mg/dl; 4+, 300 mg/dl; and 5+, >2000 mg/dl (n = 6 for all groups).

 
CRP did not affect the Ab response to rabbit IgG as determined by ELISA on day 6 (data not shown). There were also no reproducible differences between CRP-treated and control mice in IC or C3 deposition in glomeruli. Kidney sections were analyzed for glomerular deposition of rabbit and mouse IgG and mouse C3 by immunofluorescence on days 1, 3, 6, and 11 (data not shown). These results suggest that CRP is primarily affecting the inflammatory response to IC rather than the generation or deposition of these complexes.

Macrophage depletion prevents the protective effect of CRP on NTN

NTN was induced in B6 mice using a single dose of anti-GBM serum on day 0. On day 6, mice were injected with either clodronate or control liposomes using a dose and route designed to deplete splenic macrophages and Kupffer cells (30). Both groups of mice had a modest and transient decrease in proteinuria that may have been due to either paralysis of macrophages by liposome ingestion or consumption of complement following liposome injection. On day 9, mice were treated with 200 µg of CRP or saline. Mice that were treated with control liposomes and then with CRP showed a reversal of proteinuria to baseline levels by day 16 (Fig. 2A). If, however, the mice had been treated with clodronate liposomes, subsequent treatment with CRP had no effect on proteinuria. Mice treated with saline on day 9 had no response whether they had been pretreated with clodronate or control liposomes (Fig. 2B). Thus, in the absence of macrophages, CRP is unable to reverse proteinuria.


Figure 2
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  		FIGURE 2. Macrophage depletion prevents the protective effect of late CRP treatment. NTN was induced in B6 mice using a single dose of anti-GBM serum at day 0. On day 6, mice were injected with either clodronate liposomes (Clod-L) or control liposomes (PBS-L). On day 9, mice were treated with 200 µg of CRP (A) or saline (B). Proteinuria values are means ± SEM using Albustix. (n = 4–6/group).

 
In a second experiment (Fig. 3), the effect of macrophage depletion was tested on mice treated at day 0 with CRP to prevent the onset of NTN. Mice were treated with either 200 µg of CRP (Fig. 3A) or saline (Fig. 3B) on day 0. Mice treated with CRP were completely protected from the onset of proteinuria. The CRP-treated mice were then exposed to clodronate or control liposomes on day 3. Mice that were treated with control liposomes were not affected and remained proteinuria-free. However, clodronate treatment rapidly reversed the effect of CRP and mice uniformly developed high-grade proteinuria. Proteinuria persisted until 6 days following clodronate treatment, at which time the proteinuria dropped to 0, suggesting that the effect of CRP was recalled after the return of macrophages, which is expected on days 5–7 after depletion (Fig. 3A) (33). NTN was induced in control mice, which were then treated with clodronate or control liposomes on day 3 (Fig. 3B). Clodronate liposome treatment resulted in decreased proteinuria for the period of macrophage depletion (6 days), indicating that effector macrophages in this disease model are accessible to depletion by i.v. administered clodronate liposomes.


Figure 3
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  		FIGURE 3. Macrophage depletion reverses the protective effect of early CRP treatment. A, NTN was induced in B6 mice using a single injection of anti-GBM on day 0. Mice were treated with CRP or saline on day 0. A, CRP-treated mice were injected i.v. with clodronate liposomes (Clod-L) or control liposomes (PBS-L) on day 3. The decreased proteinuria seen with CRP injection was temporarily reversed by clodronate liposome injection. B, Saline-treated mice with NTN were injected with clodronate liposomes (Clod-L) or control liposomes (PBS-L) on day 4. Proteinuria was temporarily decreased by clodronate liposome injection. Proteinuria values are means ± SEM using Albustix (n = 4/group).

 
IL-10 is required for CRP-mediated protection in NTN

We have previously demonstrated that CRP fails to provide protection from NTN in IL-10–/– mice (23). These results were repeated and expanded here. NTN was induced as described in Fig. 1 using IL-10–/– mice (Fig. 4). The IL-10–/– mice had decreased survival as compared with B6 mice due to NTN. CRP treatment given either early or late failed to decrease proteinuria (Fig. 4A) or mortality (Fig. 4B) in IL10–/– mice. Thus, the ability to produce IL-10 is required for CRP-mediated protection.


Figure 4
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  		FIGURE 4. IL-10 is required for CRP-mediated protection in NTN. NTN was induced in IL-10–/– mice using three daily doses of anti-GBM serum on days 0, 1, and 2. A, Proteinuria was measured daily by Albustix. B, Survival of saline and early CRP groups. Proteinuria values are means ± SEM using Albustix. Results from two experiments are combined (n = 13–15/group).

 
CRP treatment decreases glomerular pathology and macrophage infiltration in B6, but not in IL-10–/– mice

Mice with NTN were killed on day 11 after CRP treatment on day 0 (early) or day 9 (late) and renal pathology was analyzed. Glomerular lesion scores are average glomerular pathology scores determined from H&E sections on a 0–4+ scale. The glomerular lesion scores were markedly decreased in late or early CRP-treated B6 mice as compared to saline-treated NTN mice (Fig. 5A). Two of six mice in the early CRP-treated group failed to respond and were excluded from analysis.


Figure 5
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  		FIGURE 5. CRP treatment decreases glomerular pathology and macrophage infiltration in B6, but not IL-10–/– mice. NTN was induced using three daily doses of anti-GBM serum on days 0, 1, and 2. Mice with NTN were killed on day 11 after CRP treatment on day 0 (early) or day 9 (late). A, Glomerular lesion scores. Mean ± SD on a 4-point scale. *, p < 0.05 vs saline. B, Kidney sections were stained for CD68+ macrophages using immunoperoxidase. Mean ± SEM of CD68+ cells per glomerulus are shown. *, p < 0.05 vs saline; **, p < 0.01 vs saline. C, Renal pathology in saline-treated IL-10–/– mice (day 7). D, Renal pathology in CRP-treated IL-10–/– mice (day 7). Large arrows show dilated tubules filled with protein-rich fluid. Small arrows show intraglomerular fibrin thrombi.

 
Renal pathology in CRP-treated IL-10–/– mice did not differ significantly from saline-treated mice, suggesting that IL-10 is important for CRP’s effect on renal disease (Fig. 5A). As shown in Figs. 1C and 4B, there was greater mortality in IL-10–/– mice compared with B6 mice and many of the mice died before the analysis on day 11. To confirm the pathology findings using a larger number of IL-10–/– mice, an additional experiment was done in which renal pathology was analyzed on day 7. The glomerular lesion scores were 3.76 ± 0.04 for saline-treated mice and 3.6 ± 0.14 for CRP-treated mice, again showing no effect of CRP treatment. The increased pathology in IL-10–/– mice suggests that endogenous IL-10 may ameliorate renal disease in this model and is consistent with earlier studies showing an important effect of IL-10 in a crescentic glomerulonephritis model (34). Representative H&E-stained sections are shown from a saline-treated IL-10–/– (Fig. 5C) and an early CRP-treated IL-10–/– mouse (Fig. 5D). Both sections show glomerular hypertrophy, cellular infiltration, fibrin thrombi, and basement membrane disruption. The improved histopathology in CRP-treated B6 mice was previously published (22).

Frozen sections of kidneys were stained for infiltrating macrophages using anti-CD68. The average number of CD68+ cells per glomerulus is shown (Fig. 5B). The number of CD68+ cells correlated well (r = 0.95) with the glomerular lesion score in saline-treated B6 mice. In the B6 mice, early and late CRP treatment decreased the numbers of macrophages in glomeruli nearly to control levels. In IL-10–/– mice, CRP treatment had no effect on CD68+ cell infiltration of the kidneys (Fig. 5B).

CRP treatment of Fc{gamma}RI–/– mice provides early protection from NTN, but protection is not sustained

NTN was induced in Fc{gamma}RI–/– mice and the mice were treated with CRP in an identical manner to B6 mice. Similar to the control mice, Fc{gamma}RI–/– mice initially were protected from proteinuria by CRP. However, this protection was short-lived and proteinuria recurred at days 5–7 and became severe by days 9 and 10 (Figs. 6 and 7A). Thus, CRP is able to protect mice from NTN in an Fc{gamma}RI-independent manner, but only briefly. Long-term protection was dependent on Fc{gamma}RI. If mice were given CRP at day 9, CRP again provided moderate transient protection from proteinuria. Unlike the results in B6 mice, this protection was incomplete and of short duration, indicating an important role of Fc{gamma}RI for long-lasting protection. CRP had no significant effect on the survival of Fc{gamma}RI–/– mice (data not shown).


Figure 6
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  		FIGURE 6. CRP treatment of Fc{gamma}RI–/– mice provides early protection from NTN, but protection is not sustained. NTN was induced in Fc{gamma}RI–/– mice with three injections of anti-GBM serum on days 0, 1, and 2. CRP-treated mice received 200 µg of CRP on day 0 (early CRP treatment) or on day 9 (late CRP treatment). Proteinuria was measured daily by Albustix. Proteinuria values are means ± SEM (n = 6–7/group).

 

Figure 7
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  		FIGURE 7. Effect of complement depletion on CRP-mediated protection from NTN in B6 and Fc{gamma}RI–/– mice. NTN was induced in B6 mice and Fc{gamma}RI–/– mice with three injections of anti-GBM serum on days 0, 1, and 2. C3–/– mice received one injection of 20 µl of anti-GBM on day 0. Mice were treated on day 0 with 200 µg of CRP or saline. To deplete circulating complement, mice were pretreated with CVF as described in Materials and Methods. Proteinuria was measured daily by Albustix. A, Fc{gamma}RI–/– mice. B, B6 mice. C, C3–/– mice. Proteinuria values are means ± SEM using Albustix (n = 8/group).

 
Effect of complement depletion on CRP-mediated protection from NTN in B6 and Fc{gamma}RI–/– mice

Although CRP activates the classical complement pathway, this activation differs from IC activation of complement in that it is limited to the early steps of the pathway. CRP binds the regulatory protein factor H, thereby preventing C3 and C5 convertase amplification and preventing generation of C5a and the membrane attack complex (35). Complement activation contributes to the early inflammatory response in NTN primarily through the actions of C5a and the membrane attack complex, and we hypothesized that the early transient suppression of proteinuria by CRP in Fc{gamma}RI–/– mice might be complement dependent. Complement was depleted in Fc{gamma}RI–/– (Fig. 7A) and B6 (Fig. 7B) mice by CVF treatment before induction of NTN. Mice were treated with 200 µg of CRP at day 0 or with saline and proteinuria was measured daily. CVF treatment eliminated the transient protection from NTN afforded by CRP in Fc{gamma}RI–/– mice (Fig. 7A). BUN levels determined in these mice on day 4 reflected the proteinuria values. The BUN values were 70 ± 6.5 and 75 ± 5.0 in the saline-treated and CVF-treated controls, respectively. The BUN values were 17.8 ± 1.8 in the CRP-treated mice without complement depletion and 75.0 ± 5 in the CRP-treated mice with complement depletion (p < 0.001). In B6 mice, no effect of CVF was seen and CRP was fully protective, suggesting that the major effect of CRP is not mediated by complement in normal mice (Fig. 7B). These results were supported by experiments using C3–/– mice. In these mice, early CRP treatment provided full protection from proteinuria and mortality in NTN (Fig. 7C).

Effect of CRP on chemokine mRNA expression in renal tissue of mice with NTN

As CRP treatment decreased the accumulation of macrophages in NTN, we measured the effect of CRP treatment on IL-1beta and chemokine production in the kidney. Previous studies have demonstrated the importance of IL-1beta production by inflammatory macrophages in the induction of chemokine production by intrinsic renal cells, such as tubular epithelial cells (36, 37). These locally produced chemokines then induce further cellular infiltration and activation, resulting in renal pathology. NTN was induced in B6 mice with a single injection of anti-GBM. Mice were either treated on day 0 with 200 µg of CRP or saline and killed at day 1 or 3, or treated with CRP on day 10 and killed on day 11. The kidneys were removed and mRNA was extracted and analyzed by qRT-PCR for IL-1beta, CCL2, CCL3, and CXCL2. Results are presented as the mRNA expression relative to kidneys from untreated B6 mice. As shown in Fig. 8, CRP treatment decreased the production of IL-1beta, CCL2, CCL3, and CXCL2 on days 1 and 3 after early CRP treatment. All decreases were significant on day 3. Lesser decreases in chemokine production were seen after late CRP treatment. Since chemokines are produced by tubular epithelial cells in the kidney in response to IL-1beta stimulation, we also looked at the effect of CRP on IL-1beta production. As seen in Fig. 8A, IL-1beta production was decreased at days 1, 3, and 11 following CRP treatment. These results were statistically significant on days 3 and 11, 1 day following late CRP treatment. The rapid down-regulation of IL-1beta following CRP treatment could account for the decreases in local chemokine production.


Figure 8
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  		FIGURE 8. Effect of CRP on chemokine mRNA expression in renal tissue of mice with NTN. NTN was induced in B6 mice using a single injection of anti-GBM on day 0. Mice were treated on day 0 with 200 µg of CRP or saline and killed on day 1 or 3, or treated with CRP or saline on day 10 and killed on day 11 (late CRP). The kidneys were removed and mRNA was extracted and analyzed by qRT-PCR for IL-1beta (A), CCL2 (B), CCL3 (C), or CXCL2 (D). Results are expressed as the ratio of mRNA in each sample compared with a normal, nondiseased B6 mouse kidney. Individual values and group means ± SEM are shown.

 
CRP treatment affects the cytokine and chemokine profile of cells in the peritoneal cavity of mice with NTN

Because CRP produced decreased changes in renal cytokines and chemokines, it was important to determine whether the effects were restricted to the kidney. Since mice are immunized i.p. with rabbit IgG in CFA for the induction of accelerated NTN, we decided to determine whether peritoneal cell production of cytokines and chemokines induced by CFA were altered by CRP treatment. NTN was induced as described in Fig. 2, and mice were injected i.p. with 200 µg of CRP or saline on day 8. After 8 h, mice were killed and peritoneal cells were collected. The RNA extracted from these cells was used to produce 32P-labeled cDNA probes that were hybridized to mouse inflammatory cytokines and receptors gene array. Representative array data are shown in Fig. 9A. The results shown in Fig. 9B are expressed as the fold increase or decrease in CRP-treated compared with control mice. CRP treatment decreased the production of IL-1beta similar to results seen in the kidney. There were also substantial decreases in CC and CXC chemokines and their receptors. However, in contrast to the kidneys, CCL2 mRNA was increased in peritoneal cells from CRP-treated mice. Similar results were seen in peritoneal cells collected 2 h after CRP treatment. These results suggest that there is a systemic effect of CRP on inflammatory chemokines and cytokines. In particular, a marked and rapid decrease in IL-1beta, which is produced primarily by macrophages, was observed in both the kidneys and peritoneal cells following CRP treatment of mice with NTN.


Figure 9
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  		FIGURE 9. CRP treatment affects the cytokine and chemokine profile of cells in the peritoneal cavity of mice with NTN. NTN was induced in B6 mice using a single injection of anti-GBM on day 0 and mice were injected i.p. with 200 µg of CRP or saline on day 8. After 8 h, mice were killed and peritoneal cells were collected and RNA extracted. A, 32P-radiolabelled cDNA probes were made and hybridized to a Mouse Inflammatory Cytokines and Receptors Gene Array (Superarray Biosciences) and scanned. B, Results are expressed as the fold increase or decrease in CRP-treated mice compared with control mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NTN is an IC-mediated disease that is initiated by heterologous Abs to GBM deposited in the kidney and is enhanced by innate immune signals (25, 38). In preliminary studies, we described the marked suppression of proteinuria and glomerular pathology in mice with NTN following CRP treatment (22). The major findings reported here concern the mechanisms by which CRP modulates renal disease in NTN. We first demonstrate that prior depletion of clodronate-sensitive macrophages completely prevents the suppression of NTN by CRP. Intravenously injected clodronate liposomes specifically eliminate macrophages in the spleen and liver (30). These findings suggest that macrophages are crucial to the effector phase of CRP-mediated suppression. However, this experiment does not indicate whether the initiator cell is a macrophage or another cell with which CRP may interact directly. In mice in which proteinuria had been suppressed by early CRP treatment, depletion of macrophages by treatment with clodronate liposomes reversed suppression. Disease suppression was restored 6 days later without further CRP treatment, suggesting two possibilities. One possibility is that a suppressed population of monocyte/macrophage precursors exists in another compartment like the bone marrow that are programmed by CRP treatment to become suppressive effector cells as they mature. Another possibility is that an initiator cell is generated that can induce the suppressive phenotype in macrophages regenerated from the bone marrow. The findings are most consistent with the latter possibility as the return of suppression corresponds with the time period when macrophages repopulate the liver and spleen after clodronate liposome treatment (5–7 days).

The role of IL-10 in CRP-mediated immunomodulation was indicated in a previous publication from our laboratory, but effects on pathology and the role of macrophage infiltration or glomerular pathology were uncertain due to the high level of mortality in IL-10–/– mice, which are known to be highly sensitive to NTN. These studies confirmed the requirement for IL-10 in CRP treatment of NTN. IL-10–/– mice developed more pathology and higher mortality than B6 mice. CRP treatment had no measurable effect on proteinuria, BUN, survival, renal pathology, or macrophage infiltration of glomeruli in IL-10–/– mice. CRP has been shown to induce IL-10 in macrophages and IL-10 has been suggested to provide an important role in immunosuppression in the experimental allergic encephalomyelitis model (24). We have previously shown that CRP increases IL-10 synthesis by bone marrow macrophages stimulated with low doses of LPS and that CRP pretreatment of mice increases serum IL-10 levels following LPS injection (14). This CRP-mediated enhancement of IL-10 production required the FcR {gamma}-chain. Similar findings have been reported by Mosser et al. (15, 16) using IC to enhance IL-10 synthesis in macrophages stimulated with LPS. More recently, we repeated the in vivo endotoxin challenge experiments using mice deficient in the {alpha}-chain of Fc{gamma}RI and confirmed that CRP induction of IL-10 requires Fc{gamma}RI (our unpublished results). Thus, the experiments reported here suggest that one of the main mechanisms by which CRP may regulate inflammation is through IL-10 production. The results further suggest that Fc{gamma}RI may be the receptor responsible for IL-10 production induced by CRP.

The importance of the Fc{gamma}RI/IL-10 pathway is supported by the requirement for Fc{gamma}RI in the suppression of NTN by CRP. The critical requirement for Fc{gamma}RI in the mediation of suppression may be unique to CRP. Enhancement of IL-10 synthesis by IC requires the FcR {gamma}-chain, but does not require either Fc{gamma}RI or Fc{gamma}RIII (26), indicating either redundant roles or involvement of Fc{gamma}RIV. Other models of suppression of NTN and other IC-mediated diseases by high-dose i.v. Ig (IVIG) in mice have shown a crucial role for the regulatory receptor Fc{gamma}RIIb (39, 40). More recently, an important role for the activating receptors has been shown in a mouse model of immune thrombocytopenic purpura (ITP) (41). In this model, it was demonstrated that the initiator cell responding to IVIG was a splenic dendritic cell (DC), that it was present in Fc{gamma}RIIb–/– mice, and that these DC could transfer suppression to naive mice with ITP.

CRP activation of the human classical complement pathway through the binding of C1q has been well described (1), although recent data suggest that CRP may activate complement in the mouse through a C1q-independent pathway (42). CRP activation of complement is limited to the early steps in the pathway (C1, C4, C2, and C3) with little activation of C5b-C9 (43). CRP regulates the generation of late inflammatory complement components C5a and the membrane attack complex by recruiting the regulatory protein factor H (35). Our results show that CRP’s effect on NTN may involve a contribution of the complement pathway, although it did not provide a dominant role in normal B6 mice. The effect of CVF depletion of complement was only seen in Fc{gamma}RI–/– mice, suggesting that interaction with Fc{gamma}RI is the major mechanism by which inflammation is suppressed. The early protection from proteinuria during the first week of the treatment may be related to the early phase of NTN, which is more dependent on complement activation than the later phase, which is dominated by Fc{gamma}R and macrophages.

CRP induced a marked reduction in IL-1beta and chemokines that are important in attracting macrophages to the kidney in NTN. This decrease in message was seen as early as 1 day after CRP injection but was more evident at day 3. Late injection of CRP was less effective in decreasing chemokine mRNA despite the observed decrease in renal pathology. The decrease in IL-1beta was most marked at day 3, and this cytokine is known to be an important stimulus to chemokine secretion by tubular epithelial cells (37). The source of IL-1beta in this model is reported to be the infiltrating macrophages (36). Results of chemokine and cytokine expression in the peritoneal cavity suggest that the effect of CRP is rapid and not restricted to the kidney. Marked decreases in the level of IL-1beta message in the peritoneum 2 and 8 h after injection suggest that CRP may provide systemic immunomodulation based on the down-regulation of this proximal activator of the inflammatory cascade.

Several questions remain unanswered. One is whether the initiator cell involved in CRP-mediated suppression of inflammation is of the monocyte/macrophage lineage or another cell type. Our experiments indicate that the suppressive cell itself is a macrophage that may be eliminated by clodronate liposome treatment. However, it is unclear whether CRP acts directly on the macrophage or whether CRP may induce suppression through lymphocytes, DC, or T cells. It has recently been demonstrated that the initiator cell in IVIG-induced suppression of ITP is of the DC lineage and that this cell requires the presence of a {gamma}-chain-associated receptor, although the effector cell required expression of the suppressive Fc{gamma}RIIb in the recipient (41). The possibility that CRP acts through such an initiator cell is being examined currently in our laboratory. Another question raised by these studies is the source and role of IL-10. We did not find evidence by qRT-PCR for IL-10 synthesis by either renal cells or peritoneal cells in CRP-treated mice. This could indicate a very transient induction of IL-10 as has been described for macrophages stimulated with LPS in the presence of IC (44). Alternatively, IL-10 produced centrally in the liver or spleen may be responsible for the suppression of macrophage cytokine and chemokine expression in the kidney and peritoneal cavity. These questions are currently being addressed by additional studies.

In summary, CRP inhibits NTN, a nonautoimmune IC-mediated glomerulonephritis, supporting previous experiments that showed that inhibition of autoantibody formation is not solely responsible for CRP suppression of nephritis in systemic lupus erythematosus mice. These experiments further implicate an important role for Fc{gamma}RI in CRP-mediated immune modulation. The ability of CRP to regulate ongoing active nephritis suggests that CRP may potentially have a role in the clinical treatment of patients with inflammatory disease.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
T. W. Du Clos and C. Mold have a pending patent on the use of CRP to treat IC-mediated glomerulonephritis.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Department of Veterans Affairs and by National Institutes of Health Grant AI-28358. Back

2 Address correspondence and reprint requests to Dr. Terry W. Du Clos, Veterans Affairs Medical Center, Research Service 151, 1501 San Pedro Southeast, Albuquerque, NM 87108. E-mail address: tduclos{at}unm.edu Back

3 Abbreviations used in this paper: CRP, C-reactive protein; PAF, platelet-activating factor; IC, immune complex; NTN, nephrotoxic nephritis; GBM, glomerular basement membrane; BUN, blood urea nitrogen; qRT-PCR, quantitative RT-PCR; CVF, cobra venom factor; IVIG, intravenous Ig; ITP, immune thrombocytopenic purpura; DC, dendritic cell. Back

Received for publication August 28, 2006. Accepted for publication October 24, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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