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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Objective

Blood C-reactive protein (CRP) is routinely measured to gauge inflammation. In rheumatoid arthritis (RA), a heightened CRP level is predictive of a poor outcome, while a lowered CRP level is indicative of a positive response to therapy. CRP interacts with the innate and adaptive immune systems in ways that suggest it may be causal in RA and, although this is not proven, it is widely assumed that CRP makes a detrimental contribution to the disease process. Paradoxically, results from animal studies have indicated that CRP might be beneficial in RA. This study was undertaken to study the role of CRP in a mouse model of RA, the collagen-induced arthritis (CIA) model.

Methods

We compared the impact of CRP deficiency with that of transgenic overexpression of CRP on inflammatory and immune responses in mice, using CRP-deficient (Crp−/−) and human CRP–transgenic (CRP-Tg) mice, respectively. Susceptibility to CIA, a disease that resembles RA in humans, was compared between wild-type, Crp−/−, and CRP-Tg mice.

Results

CRP deficiency significantly altered the inflammatory cytokine response evoked by challenge with endotoxin or anti-CD3 antibody, and heightened some immune responses. Compared to that in wild-type mice, CIA in Crp−/− mice progressed more rapidly and was more severe, whereas CIA in CRP-Tg mice was dramatically attenuated. Despite these disparate clinical outcomes, anticollagen autoantibody responses during CIA did not differ among the genotypes.

Conclusion

CRP exerts an early and beneficial effect in mice with CIA. The mechanism of this effect remains unknown but does not involve improvement of the autoantibody profile. In humans, the presumed detrimental role of a heightened blood CRP level during active RA might be balanced by a beneficial effect of the baseline CRP (i.e., levels manifest during the preclinical stages of disease).

Rheumatoid arthritis (RA) is a chronic, debilitating disease characterized by systemic inflammation and erosive destruction of the joints (1, 2). The hands and feet are the most commonly affected sites, but the disease can affect other joints, such as the elbow, shoulder, knee, and hip (3). Several theories have been proposed to explain the underlying mechanisms of RA, but none has been universally accepted or conclusively demonstrated. Since the discovery of rheumatoid factor (RF; antibodies against the Fc portion of IgG), it has been postulated that RA is an autoimmune disease (1). It is thought that RF interacting with the Fc portion of IgG promotes formation of immune complexes that activate the complement system and bind to various Fcγ receptors (FcγRs), thereby contributing to the inflammation associated with RA (1, 2, 4). In concert with the autoimmune model, various kinds of inflammatory cells (macrophages and dendritic cells, among others) infiltrate the synovium of patients with RA (1, 2) and are also thought to exert influence on the onset and clinical course of the disease. A critical role of T cells is postulated; their interaction with macrophages, fibroblasts, and other cells is thought to contribute to the production of deleterious cytokines (e.g., interleukin-2 [IL-2], IL-4, IL-10, and interferon-γ [IFNγ]) (1).

C-reactive protein (CRP) is a widely used blood marker of inflammation (5), and growing evidence indicates that it plays an active role in host defense (6) and certain cardiovascular diseases (7). It has long been recognized that in RA patients, the concentration of CRP in the blood correlates positively with disease severity and progression (8). Like RF, CRP can form immune complexes that activate complement (9, 10) and bind to FcγRs (11, 12). Therefore, it is not unreasonable to predict that CRP also participates in the RA disease process. Indeed, although many of the functions of CRP arguably are effected in the fluid phase (13), CRP is found within the arthritic joint (13, 14) and synovial fluid (15), and its presence in these sites can be used to differentiate inflammatory from noninflammatory arthritis (15). Measurement of the CRP blood level has also been incorporated into clinical algorithms used to determine the extent of RA disease activity (16).

Despite all of this “guilt by association,” very little is known about the biology of CRP in the context of arthritis. In fact, no human study to date has directly investigated the contribution of CRP to RA, and the animal studies performed so far have had mixed results. For instance, early studies of experimentally induced arthritis in rabbits established that the serum was the source of synovial CRP (17), and that intraarticular injection of (rabbit) CRP resulted in an elevation of the knee-joint temperature when arthritis was present, but not when the joint was healthy (18). These findings, pointing to CRP as a potentiator of already existing inflammation in RA, are in alignment with the clinical observations. In contrast, a more recent study of experimentally induced arthritis using rabbit CRP–transgenic mice (19) showed that (rabbit) CRP was protective, with the protective effect being exerted during a short time at the very beginning of disease initiation. The potential relevancy of this observation to the preclinical stages of RA has still not been investigated.

To gain new evidence to support the contributory role of CRP in RA, the present study examined the strength and direction of the contribution of CRP to inflammation, immunity, and development and progression of arthritis in a murine model of RA, the collagen-induced arthritis (CIA) model. In this study, we used, for the first time, human CRP–transgenic (CRP-Tg) mice (20, 21) in tandem with a newly engineered CRP-deficient (Crp−/−) mouse strain. We found that, compared to wild-type mice, Crp−/− mice 1) expressed less tumor necrosis factor α (TNFα) and IL-10 and more IL-6 in the blood following intraperitoneal (IP) endotoxin challenge, and also expressed less IFNγ, IL-2, and IL-4 in the blood after intravenous (IV) injection of anti-CD3 antibody, 2) had an enhanced antibody response to vaccination with the thymus-independent antigen trinitrophenyl (TNP)–Ficoll, and 3) exhibited more rapid clinical progression and more severe clinical symptoms of CIA. Conversely, CRP-Tg mice, which previously were shown to produce less IL-10 (22), had a weaker immune response to TNP-Ficoll, mounted a robust human CRP acute-phase response during the inductive phase of CIA, and had only mild symptoms of disease.

Surprisingly, the aggressiveness of CIA in Crp−/− mice and its mildness in CRP-Tg mice was not because of heightened autoantibody responses in the former or depressed autoantibody responses in the latter, since the anti–type II collagen (anti-CII) responses in Crp−/− and CRP-Tg mice were not different from those in wild-type mice. We do not dispute the notion that CRP might have a detrimental effect during active RA, but we caution that this can only be achieved in the context of already established disease. Our new observations reported herein, which align best with the observations made earlier by other investigators (19), indicate that CRP is probably beneficial during the early stages of experimentally induced arthritis in mice and during the preclinical stage of RA in humans.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Animals.

Transgenic mice carrying loxP-targeted Crp alleles (floxed Crp), wherein exon 2 of Crp was flanked by loxP sites (Figure 1A), were generated using a conditional targeting vector derived using the Lambda KOS system (Lexicon Pharmaceuticals). Floxed Crp–carrying mice were mated with protamine–Cre-recombinase–transgenic mice to generate hybrid offspring carrying one Cre-deleted Crp allele (Figure 1A). These mice were intercrossed to generate mice with CRP deficiency (Crp−/− mice) or were backcrossed to the C57BL/6 mouse strain (The Jackson Laboratory).

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Figure 1. Targeted deletion of the mouse Crp gene, and characterization of C-reactive protein (CRP) expression in mouse genotypes. A, CRP-deficient (Crp−/−) mice were generated by Cre-Lox recombination, wherein exon 2 (E2) of the Crp gene was flanked by loxP sites to direct Cre-recombinase–mediated deletion. Arrows indicate the positions and directions of elongation of the sense and antisense primers (BI.25-3, BI.25-33, and BI.25-27) used to discriminate Crp from floxed Crp and Cre-deleted Crp. B–D, Results of quantitative real-time polymerase chain reaction analysis of liver total RNA from Crp−/− and wild-type mice (B), Western blot analysis of plasma proteins from Crp−/− and wild-type mice (C), and enzyme-linked immunosorbent assay of sera from Crp+/+, Crp+/−, and Crp−/− mice (D) indicate that CRP is not expressed in the CRP-deficient strain. Arrow in C indicates the position of migration of the mouse CRP monomer. Bars in B are the mean ± SEM mRNA levels in 3 mice per genotype. Bars in D are the mean ± SEM serum levels in 5 mice per genotype.

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Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays, performed using standard methods, confirmed the absence of CRP messenger RNA (Figure 1B) and protein (Figure 1C) in Crp−/− mice. Results of an enzyme-linked immunosorbent assay (ELISA) performed on mouse sera at the time of acute-phase reaction (i.e., sera collected 24 hours after IP injection of endotoxin) confirmed the absence of blood CRP in Crp−/− mice and showed an ∼50% reduction in blood CRP in Crp+/− mice (Figure 1D).

The human CRP transgene, its detection by PCR, and its human-like expression in CRP-Tg mice (also observed in the C57BL/6 mouse strain) have been fully described elsewhere (20, 21). Human CRP is present in the blood of CRP-Tg mice at concentrations relevant to those found in humans (5), i.e., low levels under steady-state conditions (<1–30 μg/ml) and high levels during an endotoxemia or infection-induced acute-phase response (100–500 μg/ml). Mouse CRP is still expressed in CRP-Tg mice, but mouse CRP is not a major acute-phase protein (23). For certain experiments, Crp−/− mice were reconstituted (by breeding) with CRP-Tg mice to generate mice that express only the human form of CRP.

Mice were housed in an atmosphere of constant humidity (mean ± SD 60 ± 5%) and constant temperature (mean ± SD 24 ± 1°C) with a 12-hour light cycle (6:00 AM to 6:00 PM), and were maintained ad libitum on sterile bottled water and regular chow (Harlan Teklad). Mice were 8–12 weeks old at the time of analysis, and both sexes were combined, unless specifically noted. All protocols for animal use were approved by the Institutional Animal Care and Use Committees at the University of Alabama at Birmingham and Boehringer Ingelheim Pharmaceuticals, and were consistent with the NIH Guide for the Care and Use of Laboratory Animals (1996 edition).

Measurement of inflammatory and immune responses.

To induce sterile peritonitis in the mice, 0.5 ml of zymosan A (2 mg/ml in sterile 0.9% NaCl; Sigma-Aldrich) was injected IP into each animal. Mice were killed 4 hours later and their peritoneal cavities were lavaged with ice-cold phosphate buffered saline. The total leukocyte count in the peritoneal exudates was determined, and differential cell counts were performed using Giemsa-stained cytospin preparations. Aseptic endotoxemia was induced by IV administration of 200 μl pyrogen-free 0.9% NaCl containing 200 ng Escherichia coli lipopolysaccharide serotype O55:B5 (Sigma) plus 1 mg N-acetyl-D-galactosamine (Sigma). One hour later, mice were anesthetized and blood samples were collected.

To activate T cells, mice received 10 μg of hamster anti-mouse CD3 antibody (eBioscience), administered IP, and blood was collected 3 hours later. To measure the delayed-type hypersensitivity (DTH) response, mice were immunized subcutaneously with 100 μg of ovalbumin (OVA; fraction V) emulsified with 50 μl Freund's complete adjuvant (both from Sigma) in 50 μl. Six days thereafter, each mouse was administered a challenge injection, into one ear pinnae, with 200 μg OVA in 10 μl of saline. The DTH response was assessed by measuring the thickness of the ear prior to and 24 hours after challenge, using an engineer's micrometer (Mitutoyo 2804F-10); the difference between the 2 measurements was calculated, to yield an index of ear swelling.

Carrageenan-induced paw edema was induced by administering an intraplantar injection of 0.3% carrageenan (Sigma) in a 20-μl volume into the left hind paw, using a 26-gauge needle. Paw volume was measured by determining fluid displacement upon immersion in water, and the difference in volume measured prior to and 3 hours following carrageenan administration was calculated.

To measure the antibody response, mice were immunized with 10 μg TNP-Ficoll (Biosearch Technologies), administered by IP injection. Pre- and postimmunization plasma samples were obtained to determine the titers of anti-TNP antibody.

In vitro proliferative responses and the IFNγ-producing ability of isolated mixed splenocytes were determined using the following methods. To elicit a contact hypersensitivity reaction, mice were painted on the shaved abdomen with 200 μl of 1% fluorescein isothiocyanate (FITC) or the same volume of 0.5% 2,4-dinitrofluorobenzene (DNFB) (both from Sigma) dissolved in acetone:dibutylphtalate (1:1). Six days later, a challenge with 10 μl of FITC or DNFB, painted on the dorsal side of one ear pinnae, was evoked. The contact hypersensitivity response was then determined by measuring ear thickness 24 hours after the challenge, similar to that described for DTH.

For induction of passive cutaneous anaphylaxis, a single ear of each mouse was injected with 10 μl monoclonal antidinitrophenyl (anti-DNP) clone SPE-1 (a mouse IgE produced on location at Boehringer Ingelheim). In this case, ear thickness was measured before and 24 hours after injection of anti-DNP, to ascertain the background swelling reaction. To stimulate the passive cutaneous anaphylaxis reaction, mice were then injected intravenously with 100 μl of 0.3 mg/ml DNP–human serum albumin (A-6661; Sigma) in PBS, and 10 minutes later, ear swelling was measured a final time. The resulting change in ear thickness is proportional to the mast cell–mediated inflammatory response.

For isolation and stimulation of splenocytes, spleens were isolated from wild-type and Crp−/− mice, and single-cell suspensions were prepared by crushing the tissue through a 40-μm cell strainer (BD PharMingen). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA, pH 7.4), and the remaining cells were washed twice with RPMI 1640 (BioWhittaker Europe). Splenocytes were suspended in fresh medium (RPMI 1640 with L-glutamine, 5% autologous serum, 5% antibiotic–antimycotic [Invitrogen Life Technologies]) and then seeded into 96-well flat-bottomed culture plates at a cell density of 1 × 106 cells/well, each in triplicate, incubated at 37°C in 5% CO2, and stimulated with 6.25 ng/ml phorbol myristate acetate in combination with 625 ng/ml ionomycin (calcium salt from Streptomyces conglobatus; Sigma), 1.25 μg/ml T cell mitogen concanavalin A (ConA; Sigma), 6.25 μg/ml staphylococcal enterotoxin B (SEB; Sigma), or 1.25 μg/ml anti–T cell receptor anti-mouse CD3ϵ (BD PharMingen), all in an end volume of 200 μl. Anti-CD3 was presented either in solution or as a plate-bound version.

Supernatants were harvested after 48 hours, and cytokine levels were assessed by ELISA (R&D Systems). Separate plates were used to measure cell proliferation, and in this case, 16 hours prior to harvesting, the cells were pulsed with 0.5 μCi 3H-thymidine. Incorporation of 3H-thymidine was determined using a liquid scintillation counter.

For histologic assessment of the mouse joint tissue, the fore and hind limbs were removed from the mice after they had been killed humanely. The soft tissue was removed, and the articulated bones were fixed in 10% formalin. Decalcified bones were embedded in paraffin and sectioned (5 μm), and serial sections were stained with hematoxylin and eosin for histology.

To assess bone density and volume, we performed microfocal computed tomography (micro-CT) scans of the mouse joints, as described previously (1), using a high-resolution micro-CT imaging system (μCT40; Scanco Medical). The regions of interest were the tarsals and metatarsals of the paw. Histologic analyses and micro-CT scans both confirmed the development of typical CIA in each genotype and verified the validity of our clinical scoring system (results available from the corresponding author upon request).

CIA induction protocol.

CIA was elicited in mice using a previously described protocol (24). Briefly, Freund's complete adjuvant containing 4 mg/ml Mycobacterium tuberculosis was emulsified 1:1 with a 4-mg/ml solution of chicken CII (both from Chondrex). At the start of each experiment (day 0), 100 μl of a freshly prepared emulsion was injected intradermally using a 23-gauge needle at a site toward one side of the base of the tail. On day 21, a booster injection (100 μl of CII emulsified in Freund's incomplete adjuvant) was administered at a site contralateral to the primary injection site. Three times per week thereafter, and until day 50, the clinical signs of arthritis were recorded for each paw. The clinical scoring system used was described by Brand et al (25), where 0 = no evidence of erythema and swelling, 1 = erythema and mild swelling confined to the tarsals or ankle joint, 2 = erythema and mild swelling extending from the ankle to the tarsals, 3 = erythema and moderate swelling extending from the ankle to the metatarsal joints, and 4 = erythema and severe swelling encompassing the ankle, foot, and digits, or ankylosis of the limb. Several individuals (NRJ, MAP, and AJS) scored the mice for arthritis, and we verified the accuracy of our scoring by micro-CT and histologic analyses (26) of representative arthritic limbs (results available from the corresponding author upon request).

For statistical analysis, a mouse was considered to have developed CIA on the day that the clinical score reached 2, and a mouse was considered to have full-blown CIA only if the symptoms were sustained thereafter. The rate of progression of disease was estimated by calculating the slope of the linear regression of days since clinical presentation (abscissa) versus clinical score (ordinate). This regression was done on data collected for the first week following disease onset.

Measurement of CRP, cytokines, and antibodies.

An anti-TNP ELISA was performed using TNP–bovine serum albumin–coated plates (Biosearch Technologies), biotinylated goat anti-mouse IgM (Caltag), and streptavidin–horseradish peroxidase (Caltag). Plasma cytokines were measured using specific ELISA kits (R&D Systems). Serum mouse CRP was measured using a Life Diagnostics mouse CRP kit, according to the manufacturer's instructions, and human CRP was measured using an ELISA developed in our laboratory (21). The latter does not detect mouse CRP and has a lower limit of detection, defined as a level of ∼20 ng of human CRP per ml of mouse serum. Anti-CII IgG was measured using ELISA-grade CII and mouse anti-CII IgG standards (both from Chondrex).

Statistical analysis.

All pooled data are expressed as the mean ± SEM, without transformation, and the sample size is given. Group comparisons were done using Student's unpaired t-tests or one-way analysis of variance, followed by protected least significant difference post hoc pairwise tests or Dunnett's test for multiple comparisons. Differences were considered significant when the P value was less than 0.05. Statistical analyses were performed using GraphPad Prism (version 3.02) or StatView (version 5.0.1) software.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Alteration of inflammatory and immune responsiveness by CRP deficiency.

Deletion of Crp altered the inflammatory response to endotoxin challenge in mice. Crp−/− mice displayed a significantly weakened serum IL-10 and TNFα response compared to that in wild-type mice (for IL-10, mean ± SEM 71 ± 11 pg/ml versus 179 ± 17 pg/ml, respectively; for TNFα, 1,498 ± 201 pg/ml versus 2,786 ± 395 pg/ml, respectively) and had a significantly strengthened IL-6 response (mean ± SEM 4,007 ± 117 pg/ml versus 3,193 ± 109 pg/ml, respectively) (Figure 2A). Carrageenan-induced paw inflammation was also affected, being significantly worsened in Crp−/− mice compared to wild-type mice (mean ± SEM 0.54 ± 0.05 ml of swelling versus 0.40 ± 0.03 ml of swelling, respectively) (Figure 2B).

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Figure 2. Targeted deletion of mouse Crp alters the inflammatory response. Compared to wild-type mice, Crp−/− mice had significantly lower serum concentrations of interleukin-10 (IL-10) and tumor necrosis factor α (TNFα) and significantly higher serum concentrations of IL-6 after endotoxin challenge (A), and had a significantly greater inflammatory response to carageenan injection, as measured by change in paw volume (B). In contrast, recruitment of peritoneal exudate cells (PECs) into the peritoneal cavity, assessed as the proportion of neutrophils, monocytes, mast cells, and eosinophils, during zymosan-induced peritonitis was unaffected (C). Bars show the mean ± SEM results in 23 mice per group in A (note break in y-axis), 13 Crp−/− mice and 20 wild-type mice in B, and 12 mice per group in C, from 2 separate experiments. In A, ∗ = P < 0.006; ∗∗ = P < 0.0001 versus wild-type mice, by protected least significant difference post hoc pairwise test. In B, ∗ = P = 0.01 versus wild-type mice, by Student's t-test.

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In contrast, Crp deletion had no effect on the total number of inflammatory cells recruited to the body cavity during zymosan-induced peritonitis (results not shown), and also had no effect on the proportion of each type of inflammatory cell (neutrophils, monocytes, mast cells, and eosinophils) recruited to the body cavity (Figure 2C). Likewise, deletion of Crp had no effect on FITC- or DNFB-induced contact hypersensitivity or OVA-induced active cutaneous anaphylaxis (results available from the corresponding author upon request). These results suggest that, under certain circumstances, mouse CRP dampens the inflammatory response.

For micetreated with a T cell–targeting anti-CD3 antibody, the plasma IL-2 and IFNγ responses in Crp−/− mice were significantly impaired compared to those in wild-type mice (for IL-2, mean ± SEM 666 ± 63 pg/ml versus 1,267 ± 134 pg/ml, respectively; for IFNγ, 293 ± 28 pg/ml versus 538 ± 16 pg/ml, respectively), whereas the IL-4 response was not significantly different between the 2 strains (Figure 3A). The impaired production of these cytokines in Crp−/− mice treated with anti-CD3 antibody was likely attributable to a trans-effect of FcγR-bearing cells, and not due to an intrinsic defect in T cells, since stimulation of mixed splenocytes from Crp−/− mice with anti-CD3 in vitro induced less proliferation and less IFNγ production than that seen in wild-type mouse splenocytes, only when the anti-CD3 was presented in soluble form. Furthermore, T cell proliferation and cytokine production in Crp−/− mouse splenocytes did not differ from that in wild-type mouse splenocytes when the splenocytes were directly stimulated with the superantigen SEB or the mitogen ConA (results available from the corresponding author upon request).

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Figure 3. Targeted deletion of Crp alters the immune response. The serum cytokine (interleukin-2 [IL-2], IL-4, and interferon-γ [IFNγ]) response to intraperitoneally administered anti-CD3 antibody was reduced (A), whereas the ovalbumin-induced delayed-type hypersensitivity response, measured as the change in ear thickness (B), and the trinitrophenyl (TNP)–Ficoll–elicited anti-TNP antibody response, measured as IgM antibody levels on day 7 after immunization (C), were significantly enhanced in Crp−/− mice compared to wild-type mice. Bars show the mean ± SEM results in 21 mice per group in A, 10 mice per group in B, and 4 female and 5 male mice per group in C, from 2 separate experiments. ∗∗ = P < 0.002 versus wild-type mice, by protected least significant difference post hoc pairwise test (A and C) or Student's t-test (B).

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In accordance with this interpretation, deletion of Crp significantly strengthened the DTH response to OVA, as measured by change in ear thickness (Figure 3B), a response thought to be T cell–initiated but macrophage-driven (27). Interestingly, the antibody response of Crp−/− mice immunized with the thymus-independent antigen TNP-Ficoll was significantly stronger than that of wild-type mice (Figure 3C), whereas that of CRP-Tg mice was weaker (results available from the corresponding author upon request). These results suggest that under certain circumstances, mouse CRP can influence both T cell and B cell responses, albeit indirectly.

Exacerbation of CIA by CRP deficiency.

Given the observed pleiotropic effects of mouse CRP deficiency and human CRP expression on inflammatory and immune responses in mice, the clinical data linking the rise and fall of blood CRP levels to worsened and improved RA severity, respectively, and the paradoxical reports of CRP-mediated benefits in mouse models of RA, we investigated the impact that mouse CRP deficiency and human CRP excess might each have on the incidence, onset, and progression of CIA. Micro-CT and histopathologic analyses of tissue sections revealed gross pathologic features that are typical of mouse CIA, with no readily observable differences among the genotypes (results available from the corresponding author upon request). Likewise, the incidence of CIA was similar among the 3 genotypes, and its clinical onset was uniformly manifest among the genotypes at ∼4 weeks after induction (Table 1).

Table 1. Clinical traits of collagen-induced arthritis in human C-reactive protein–deficient (Crp−/−) mice compared with wild-type mice and human CRP–transgenic (CRP-Tg) mice
 No. of miceOnset of arthritis, mean ± SEM daysIncidence of arthritis, %Progression, slope/r2*
  • *

    Values are the slope and r2 values calculated from linear regression analysis of days since presentation with arthritis versus clinical score, for days 1 through 7 (see Figure 4A).

Crp−/−2728.18 ± 2.5340.70.757/0.945
Wild-type3628.18 ± 1.8730.80.365/0.924
CRP-Tg2527.50 ± 2.3532.00.083/0.570
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Figure 4. Collagen-induced arthritis (CIA) is exacerbated by targeted deletion of the mouse Crp gene and dampened by transgene-expressed human C-reactive protein (CRP). CIA was induced in wild-type mice (n = 36), Crp−/− mice (n = 27), and human CRP–transgenic (CRP-Tg) mice (n = 25), and arthritis symptoms were monitored for 50 days using a clinical scoring system. Following the initial clinical presentation, Crp−/− mice had the most rapid progression of disease, whereas disease was nearly static in CRP-Tg mice, as indicated by the clinical score over time (see also Table 1) (A). Furthermore, compared to wild-type mice, disease symptoms were significantly worse in Crp−/− mice and significantly better in CRP-Tg mice, as measured by the cumulative disease index (B) and the clinical score (C) for each group. Bars show the mean ± SEM results in mice from 3 separate experiments. ∗ = P < 0.0001, by analysis of variance; ∗∗ = P < 0.0001 versus wild-type mice, by protected least significant difference post hoc pairwise test.

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Importantly, however, following its clinical presentation, the progression of CIA was fastest (Table 1 and Figure 4A) and its severity was greatest (Figures 4B and C) in Crp−/−mice. Conversely, in CRP-Tg mice, in which an elevation in the levels of human CRP well above baseline values was observed (Figure 5A), the tempo of CIA was slowest (Table 1 and Figure 4A) and its severity was the lowest among the 3 genotypes (Table 1 and Figures 4A–C).

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Figure 5. Deletion of mouse Crp and expression of human C-reactive protein (CRP) do not alter the anti–type II collagen (anti-CII) IgG response during collagen-induced arthritis. A, In human CRP–transgenic (CRP-Tg) mice, a robust elevation of serum human CRP levels was observed 1 day after initial immunization with CII and Freund's complete adjuvant (30-fold increase above baseline) and again 1 day following the booster injection (3.6-fold increase), and levels of human CRP slowly returned to normal during the symptomatic phase, whereas, in the same animals, the levels of mouse CRP were only modestly elevated. B, Serum anti-CII IgG levels did not differ between Crp−/− mice, wild-type mice, and CRP-Tg mice. Bars show the mean ± SEM results in 27 Crp−/− mice, 36 wild-type mice, and 25 CRP-Tg mice.

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Furthermore, an experiment using Crp−/− mice reconstituted (by breeding) with the human Crp transgene showed that replenishment with human CRP restores resistance to CIA (results available from the corresponding author upon request). Despite the disparate effects, the anti-CII IgG autoantibody responses of wild-type, Crp−/−, and CRP-Tg mice were not different (Figure 5B). These findings show that CRP exerts a significant and beneficial effect on the development of arthritis in mice, an effect that was seemingly unrelated to the autoantibody response.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Our comprehensive comparison of wild-type, Crp−/−, and CRP-Tg mice reveals that CRP exerts a significant influence on both the inflammatory and immune responses. At least in mice, CRP actively participates in these processes, as opposed to being merely associated with these processes. CRP has been shown to have numerous effects in vitro that indicate that it should be able to interact with the inflammatory and immune systems on multiple levels in vivo. For example, the interaction of the protein with phosphocholine (9, 28) on biologically relevant ligands (6, 7, 29) could allow CRP to target the sources of inflammation, whereas its interaction with the complement proteins C1q and factor H (30) could allow it to influence immune complex formation, and its interaction with various FcγRs (11, 12, 21) could allow it to influence antigen presentation, and thereby affect cellular and humoral immunity.

Considering these known abilities of CRP, it is perhaps not so surprising that, in the present mouse study, Crp deletion influenced the cytokine response evoked by challenge with both endotoxin and anti-CD3 antibody, altered the DTH response to OVA, and strengthened the antibody response to immunization with the thymus-independent antigen TNP-Ficoll. In fact, in earlier studies, we showed that transgenic expression of human CRP had the opposite effects on some cytokine and immune responses; for example, our results indicated that transgenic expression of human CRP could lead to increased production of IL-10 (22) and decreased production of IgM autoantibodies (31). Similarly, we showed herein that the immune response to TNP-Ficoll was weaker in CRP-Tg mice than in wild-type mice (results available from the corresponding author upon request).

As expected based on its effect on inflammatory and immune responsiveness, CRP deletion did influence the course of CIA. Consistent with the findings from an earlier study showing that elevation of rabbit CRP during induction of antigen-induced arthritis suppressed disease in rabbit CRP–transgenic mice (19), our data from Crp−/− and CRP-Tg mice together suggest an early and beneficial effect of CRP in CIA. Notably, compared to wild-type mice, Crp−/− mice showed more rapid progression of disease with more severe symptoms, whereas CRP-Tg mice showed slower progression of disease with milder symptoms. As expected, replenishment of Crp−/− mice with the human Crp transgene restored their resistance to CIA. In summary, these findings strongly suggest that CRP confers benefit during the early inductive phase of CIA.

We believe that this beneficial effect of CRP requires accessory cells. Indeed, in preliminary experiments wherein we induced arthritis by direct injection of arthritogenic monoclonal antibodies, thereby bypassing the need for accessory cells and T cells, we have observed no difference in onset, progression, or severity of arthritis in wild-type mice compared with Crp−/− mice (results not shown).

At first glance, our findings might seem to be in conflict with the large body of evidence showing that a higher blood CRP level is positively associated with worsening of symptoms in RA patients. With one important caveat, i.e., that our observations in mice with CIA might not be extrapolated to humans with RA, we believe this apparent paradox can be resolved. We propose that during health, the baseline blood CRP level is sufficient to enable tonic suppression of inflammation that would otherwise predispose to autoimmunity. Thus, in mice, deletion of Crp renders animals prone to a more rapidly evolving and more severe form of CIA, because the inflammatory response is desuppressed, whereas transgenic overexpression of Crp has the opposite effect. It is important to understand that in the mouse model that was utilized in the present study, the influence of CRP (or lack of CRP) was exerted (or not) already prior to disease induction. In humans, this beneficial, tonic-suppressive effect of CRP would go unrecognized, as it would be manifest during the preclinical stage of RA. In the context of clinically diagnosed, active arthritis, on the other hand, levels of blood CRP could be raised either in response to worsening of the associated inflammation, as most assume, or in an effort to dampen it, as we propose. This model fully accounts for our findings and the positive association of elevated blood CRP level with symptoms of ongoing RA.

In this study, we provide the first direct evidence that CRP deficiency alters the course of experimentally induced arthritis in mice. Other investigators recently described a separately generated CRP-deficient C57BL/6 mouse strain (32), but whether that strain is, like ours, susceptible to CIA remains to be tested. Regardless, our observation of worsened CIA in the CRP-deficient mouse suggests that the baseline CRP maintains health rather than promotes disease.

Further study is needed to identify the means by which baseline CRP provides tonic suppression of inflammation and autoimmunity in CIA, but based on observations previously made in another model of experimentally induced autoimmunity (22, 33), we believe that the beneficial effect of CRP is likely mediated by FcγRIIB-expressing cells. The disruption of a protective CRP–FcγRIIB pathway may be one reason that FcγRIIB-deficient mice also show increased susceptibility to CIA (34). Our findings suggest that we may have to rethink CRP epidemiology as it relates to CRP biology in the context of arthritis.

Since patients seldom visit rheumatologists before symptoms of RA manifest, much of the data pointing to a positive correlation of higher blood CRP level with worsening disease have been obtained in patients with active RA rather than in those with preclinical RA. Our model predicts that in healthy people, the baseline blood CRP level should be inversely correlated with future RA risk. In people whose baseline level of CRP is insufficient to maintain the tonic-suppressive effect, there is progression of disease. Ultimately, an observational study will be needed to validate these predictions. In the future, we hope to illuminate the mechanisms of action of CRP in arthritis and point the way toward novel therapies.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Szalai had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Jones, Kerr, Sellati, Berger, Madwed, Szalai.

Acquisition of data. Jones, Pegues, McCrory, Kerr, Jiang, Villalona, Parikh, McFarland, Pantages, Szalai.

Analysis and interpretation of data. Jones, Kerr, Madwed, Szalai.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Boehringer Ingelheim assisted in the development of the CRP-knockout mouse but had no role in the CIA study design or in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. Publication of this article was not contingent upon approval by Boehringer Ingelheim.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

The authors would like to thank members of the University of Alabama Birmingham Comparative Pathology Laboratory for their help with histology and tissue analysis, and the University of Alabama Birmingham Small Animal Phenotyping Core for assisting with micro-CT analysis.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES