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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. REFERENCES

Objective

Anti–citrullinated protein antibodies (ACPAs) are the serologic hallmark of rheumatoid arthritis. Functional studies on the role of ACPAs in experimental arthritis have yielded conflicting results, and therefore the present study was undertaken to assess systematically whether citrullinated proteins can really induce ACPAs and modulate arthritis in mice.

Methods

Balb/c, SJL, and DBA/1 mice were immunized with either native or citrullinated fibrinogen, myelin basic protein (MBP), and type II collagen (CII). ACPAs were detected with a peptide-based enzyme-linked immunosorbent assay (ELISA) and with Western blotting using fibrinogen as substrate. Arthritis was induced in mice by immunization with CII in Freund's complete adjuvant or by injection of anticollagen antibodies.

Results

Analysis of the sera of mice immunized with citrullinated proteins revealed false-positive results with the citrulline peptide–based ELISA. In contrast, Western blot analysis using either citrullinated or native fibrinogen as substrate reliably detected ACPAs in Balb/c mice immunized with citrullinated fibrinogen, MBP, and CII. However, these ACPAs failed to induce or aggravate disease in Balb/c mice in the anticollagen antibody–induced arthritis model. Immunization with citrullinated fibrinogen induced ACPAs but did not lead to arthritis development in SJL and DBA/1 mice. In contrast, immunization with citrullinated CII failed to induce ACPAs or enhance disease in these strains in the collagen-induced arthritis model.

Conclusion

Mice can develop genuine ACPAs, but detection of ACPAs is highly dependent on strain, immunogen, immunization protocol, and detection assay. Murine ACPAs are not overtly pathogenic, since neither preexisting ACPAs nor the use of citrullinated collagen as immunogen modulates the clinical course of arthritis.

Anti–citrullinated protein antibodies (ACPAs), the main serologic hallmark of rheumatoid arthritis (RA), are autoantibodies directed toward epitopes in which arginine amino acids have been posttranslationally modified toward citrulline by peptidyl arginine deiminases (1–3). ACPAs are intimately linked to the development of RA, as these antibodies are highly specific for RA (4), can appear years before the onset of clinical disease (5, 6), are associated with persistent and destructive disease (7, 8), and define a genetically and immunologically distinct set of RA patients (9, 10) who, despite having similar systemic and articular disease activity (11), show a better response to B cell depletion with rituximab (12).

Although these findings provide circumstantial evidence for the involvement of ACPA reactivity in the disease process, it remains to be determined in detail how, and at what stage and to what extent, ACPAs contribute to disease development. Ex vivo studies with human macrophages and mast cells have clearly indicated that ACPA-related immune complexes can trigger these cells to produce and release proinflammatory mediators (13, 14). However, it remains unknown whether ACPAs also play a role in the initiation phase of the disease, are just a mere bystander of the autoimmune process, or, on the contrary, even represent an abortive attempt to control and/or prevent the onset of RA (15).

The study of appropriate animal models may help us to gain a deeper insight into the potentially pathogenic role of ACPAs. Consistent with the findings from the above-mentioned studies with human macrophages and mast cells (13, 14), anti–citrullinated protein–specific antibodies have been reported to enhance tissue injury in experimental arthritis (16, 17). However, both the presence of genuine ACPAs and the arthritogenic role of these ACPAs in the initiation of experimental arthritis remain highly debated (18–23). The discrepancies between these reports may be due to the use of different citrullinated antigens for immunization, different mouse strains, and different detection methods. Therefore, in the present study, we aimed to revisit this issue by assessing, systematically, whether citrullinated proteins can induce a genuine anti–citrullinated protein response in mice and whether this response modulates clinical arthritis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. REFERENCES

Mice.

DBA/1 OlaHsd mice and Balb/c OlaHsd mice were purchased from Harlan. SJL/J mice were purchased from Charles River. Mice were housed in specific pathogen–free conditions at the University of Amsterdam Academic Medical Center, according to standard housing protocols. All experiments were approved by the animal ethics committee.

Citrullinated peptides and proteins.

A filaggrin-derived citrullinated peptide, as is used in the first generation anti–cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) kits, was obtained from Eurodiagnostica, together with an arginine peptide as control. Human fibrinogen (Calbiochem-VWR), bovine type II collagen (CII) (Chondrex), chicken CII (Sigma-Aldrich), and bovine myelin basic protein (MBP) (Alexis Biochemicals–Enzo Life Sciences) were citrullinated (deiminated) in vitro as previously described (24). Proteins were dissolved in 10× deimination buffer (1M Tris HCl [pH 7.4], 100 mM CaCl2, 50 mM dithiothreitol) at a concentration of 1 mg/ml, along with rabbit skeletal peptidyl arginine deiminase type 2 (PAD-2; Sigma-Aldrich) at 10 units/ml. Citrullination was performed for 2 hours at 37°C. Nondeiminated proteins were treated identically, except that water was added instead of PAD enzyme. Samples were aliquoted and stored at −20°C until used.

Immunization protocols.

Balb/c, SJL, and DBA/1 mice (n = 6 per group) were immunized 5 times with native or citrullinated human fibrinogen (50 μg) in Freund's incomplete adjuvant at 14-day intervals. Mycobacterium tuberculosis was added to the mixture (2 mg/ml final concentration; Chondrex) in the first 2 immunizations. Balb/c mice (n = 10 per group) were immunized 3 times with native or citrullinated MBP (100 μg) in Freund's incomplete adjuvant at 14-day intervals, with the addition of M tuberculosis in the first 2 injections.

For experimental models of type II collagen–induced arthritis (CIA), SJL mice (n = 8 per group) and DBA/1 mice (n = 15 per group) were immunized on day 0 and day 21 with native or citrullinated bovine CII (50 μg) in Freund's complete adjuvant. A second cohort of SJL mice (n = 12 per group) and DBA/1 mice (n = 8 per group) was immunized with 100 μg of either native or citrullinated CII in Freund's complete adjuvant on day 0 and day 40. In Balb/c mice, immunization with 100 μg native or citrullinated CII was performed either 2 times (n = 8 per group) in Freund's complete adjuvant or 4 times (n = 10 per group) at 14-day intervals with the use of Freund's incomplete adjuvant in the last 2 immunizations. For experimental models of anticollagen antibody–induced arthritis (CAIA), Balb/c mice (n = 10 per group) were injected intraperitoneally with 6 mg anti-CII antibody cocktail (5 Clone Cocktail; Chondrex).

In all mice, the severity of arthritis was scored daily for each paw on a 4-point scale. The scores increased with increasing severity, as follows: 1 = mild swelling of the foot pad or redness; 2 = major swelling of the foot pad and ankle or toes; 3 = swelling of all joints; and 4 = dysfunction of the paw.

ELISA.

For detection of ACPAs, anti–PAD-2, or IgG anti-CII antibodies, Nunc MaxiSorp 96-well plates (eBioscience) were coated with 50 μl of 1 μg/ml citrulline or arginine control peptide (Eurodiagnostica), rabbit skeletal muscle PAD-2 (Sigma-Aldrich), or 100 μl of 5 μg/ml bovine CII (Chondrex) in 0.1M sodium carbonate buffer (pH 9). Plates were washed with phosphate buffered saline (PBS)–0.05% Tween, blocked with 1% bovine serum albumin (BSA), and incubated with mouse serum (dilutions of 1/20–1/200) and horseradish peroxidase–labeled goat anti-mouse IgG (Sigma-Aldrich). Color was developed using 3,3′,5,5′-tetramethylbenzidine (Merck) and measured at 450 nm using a Microplate Reader 680 (Bio-Rad). For inhibition ELISAs, serum was diluted 1/25 in PBS and preincubated for 1 hour with 8 μg/ml citrulline or arginine control peptide, followed by incubation on an ELISA plate.

Western blotting.

Citrullinated or native fibrinogen (10 μg/gel) was loaded on precast 12% NuPAGE 2-dimensional gels (Invitrogen). Gels were run in NuPAGE MES running buffer (Invitrogen). Protein transfer to Immobilon-FL transfer membranes (Millipore) was performed by tank blotting using the X cell Surelock system (Invitrogen) in NuPAGE blotting buffer. Membranes were blocked in 2% nonfat dry milk (Bio-Rad) and incubated overnight with serum diluted 1/250. After incubation with donkey anti-mouse IgG IRDye 800 (Li-Cor Bioscience), reactivity was visualized with an Odyssey infrared imaging system (Li-Cor Bioscience). Bands were quantified as the mean band intensity of the signal for immunoreactivity against the citrullinated γ-chain of fibrinogen minus the mean band intensity for the signal for immunoreactivity against the native γ-chain, after correction for background. These calculations were made using ImageJ image analysis software, with results expressed as the area under the curve of the band intensity peak (in arbitrary units [AU]).

Statistical analysis.

Normally distributed data are expressed as the mean ± SD, while non–normally distributed data are expressed as the median and interquartile range (IQR). Mann-Whitney tests were used for comparisons of 2 groups. Chi-square tests were used for comparisons of proportions. P values less than 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. REFERENCES

False-positive reactivity toward citrullinated peptides in experimental arthritis.

In mouse studies, the detection of genuine ACPAs that are specifically directed toward the citrullinated, but not native, epitopes remains a challenge, as the anti-CCP ELISA commonly used in humans has been reported to yield false-positive results in rodents (19, 25). To assess this issue, we developed a peptide-based ELISA using a filaggrin-derived citrullinated peptide as substrate (anti–CCP-1 ELISA). The assay was validated by our findings demonstrating that ACPA-positive human RA sera reacted strongly in the citrulline peptide–based ELISA, but not in the ELISA using the corresponding arginine peptide, and ACPA-negative human sera did not react in any of the ELISAs (results not shown). In addition, the reactivity of ACPA-positive human RA sera in the citrulline peptide–based ELISA was abrogated, in a dose-dependent manner, by preincubation with a citrullinated peptide, but not with the corresponding arginine peptide (Figure 1A).

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Figure 1. Immunoreactivity of human and mouse anti–citrullinated protein antibody (ACPA)–positive sera, as determined by peptide-based enzyme-linked immunosorbent assay (ELISA). A, Inhibition ELISAs were performed on 3 human ACPA-positive rheumatoid arthritis (RA) serum samples (p1, p2, and p3) to assess reactivity. Sera were preincubated at different concentrations with a citrullinated (citr) peptide, as compared to the corresponding arginine (arg) control peptide. B, A citrulline peptide–based ELISA was performed on serum samples from DBA/1 (n = 15), SJL (n = 12), and Balb/c (n = 6) mice immunized with native type II collagen (CII) or citrullinated CII. Results are expressed as the optical density (OD) at 450 nm. C, No residual signal that would indicate citrulline-specific reactivity was detected with the peptide-based ELISA in the sera of mice immunized with either native CII or citrullinated CII. Results are expressed as the difference in OD (delta OD), calculated as the immunoreactivity against citrulline peptide minus the immunoreactivity against arginine control peptide. D, Inhibition ELISAs were performed on the sera of mice immunized with either native CII or citrullinated CII, by preincubating the sera with citrulline peptide. Results are expressed as the percentage inhibition of citrulline-specific reactivity. Bars show the median and interquartile range.

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Since we found that the reactivity of human ACPA-positive sera could be inhibited with a minimal concentration of citrullinated peptide of 8 μg/ml, we analyzed the mouse serum using the same amount of peptide. Using these peptide-based ELISAs, we tested the sera of DBA/1, SJL, and Balb/c mice that were immunized twice with citrullinated bovine CII. All sera displayed modest immunoreactivity toward the citrullinated peptide (Figure 1B). However, this reactivity was similar between animals immunized with citrullinated CII and animals immunized with native CII (Figure 1B). Moreover, the reactivity toward the citrullinated peptide was similar to the reactivity toward the corresponding arginine peptide (Figure 1C), and the anticitrulline immunoreactivity could not be specifically inhibited by preincubation of the sera with citrullinated peptide or control native peptide (Figure 1D), not even when the concentration of the citrulline or arginine peptide was increased up to 100 μg/ml (results not shown).

To confirm and extend these findings, we hyperimmunized Balb/c mice with citrullinated human fibrinogen (5 immunizations at intervals of 14 days), since human fibrinogen contains multiple citrullination sites and was previously reported to induce ACPAs in Balb/c mice (26). Sera from these mice did not show any specific reactivity in the citrulline peptide–based ELISA as compared to the arginine peptide–based ELISA, nor did it show any reactivity in the inhibition assays (results not shown). Taken together, these results indicate that the anti-CCP–based ELISA used in our studies is sensitive to false-positive results, and therefore this peptide-based ELISA may not be an appropriate tool to detect genuine ACPAs in mice immunized with citrullinated proteins.

Induction and reliable detection of ACPAs in Balb/c mice.

We next explored the use of in vitro citrullinated proteins such as fibrinogen as an alternative to citrullinated peptides as the substrate for detection of ACPAs in mice. However, since the PAD enzyme used for in vitro citrullination is difficult to separate completely from the citrullinated protein, immunization with in vitro citrullinated proteins led to a strong anti-PAD response in all tested mouse strains (Figure 2A), thus indicating a potential for false-positive results in ELISAs using PAD-containing in vitro citrullinated proteins as substrate. We therefore used citrullinated fibrinogen as the substrate in a Western blot assay, rather than in ELISA, as this would allow easy discrimination between reactivity toward the citrullinated γ-chain of fibrinogen at 47 kd and reactivity toward PAD around 60 kd (overlapping with reactivity toward the α- and β-chain of fibrinogen at 55–65 kd).

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Figure 2. Detection of antibodies to rabbit skeletal peptidyl arginine deiminase type 2 (anti-PAD) by enzyme-linked immunosorbent assay (ELISA) and anti–citrullinated protein antibodies (ACPAs) by Western blotting in mouse serum. A, An ELISA was used to detect anti-PAD antibodies in the serum of DBA (n = 15), SJL (n = 12), and Balb/c (n = 6) mice that had been immunized with either native type II collagen (CII) or citrullinated (Citr) CII. Bars show the median and interquartile range. ∗∗ = P < 0.01. B–D, Western blotting was used to detect ACPAs in the sera of Balb/c mice. The gels were loaded with native fibrinogen (fib) or citrullinated fibrinogen. After transfer, the blots were cut into strips and incubated with individual serum samples from Balb/c mice that had been immunized with either native fibrinogen or citrullinated fibrinogen (n = 6 mice per group) (B), native myelin basic protein (MBP) or citrullinated MBP (n = 10 mice per group) (C), or native CII or citrullinated CII (n = 10 mice per group) (D). OD = optical density.

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Analysis by this Western blot approach in the sera of Balb/c mice immunized with either native fibrinogen or citrullinated fibrinogen revealed clear reactivity toward both native and citrullinated fibrinogen in all animals (results not shown). Down-titration of the sera until reactivity toward native fibrinogen disappeared, however, revealed specific reactivity toward the citrullinated, but not native, γ-chain of fibrinogen in all 6 mice immunized with citrullinated fibrinogen, compared to 2 of 6 mice immunized with native fibrinogen (P = 0.014) (Figure 2B). Quantification of this reactivity confirmed that the mice displayed significantly higher anti–citrullinated protein immunoreactivity when immunized with citrullinated fibrinogen than when immunized with native fibrinogen (median 3,551 AU [IQR 1,698, 4316] versus 256 AU [IQR −53, 2,302]; P = 0.03).

Similar assays were performed with the sera of Balb/c mice immunized with either native or citrullinated MBP, since this protein can also be easily citrullinated on different positions. Nine of the 10 mice immunized with citrullinated MBP showed immunoreactivity against the γ-chain of citrullinated fibrinogen, as compared to none of the mice immunized with native MBP (P < 0.001) (Figure 2C). Identical results were obtained in Balb/c mice immunized with either native or citrullinated bovine CII, a substrate that is less susceptible to citrullination. Eight of 10 Balb/c mice immunized with citrullinated CII showed clear anti–citrullinated protein–specific immunoreactivity, in contrast to none of the 10 mice immunized with native CII (P < 0.001) (Figure 2D). This was confirmed by quantification of the band intensity (median 3,173 AU [IQR 1,165, 11,540] in those immunized with citrullinated CII versus 186 AU [IQR −50, 510] in those immunized with native CII; P = 0.004).

In addition, ACPAs could also be specifically detected by Western blot analysis in the serum of Balb/c mice immunized with citrullinated CII, but not in the serum of Balb/c mice immunized with native CII, when citrullinated BSA and citrullinated MBP were used as the substrate (results not shown). These findings thus indicate that Balb/c mice develop genuine ACPAs after immunization with different citrullinated proteins, and these ACPAs can be reliably detected by Western blot analysis using either citrullinated fibrinogen or other citrullinated proteins as substrate.

Arthritogenicity of ACPAs in Balb/c mice.

Despite the fact that immunization with citrullinated CII induced ACPAs in Balb/c mice, none of the animals developed clinical signs of arthritis (results not shown). Because Balb/c mice are not susceptible to CIA, we performed an additional experiment in which Balb/c mice were immunized 4 times, rather than 2 times, with either citrullinated CII or native CII (n = 12 per group). None of these animals developed arthritis (results not shown). Analysis of the sera of these mice showed a tendency toward a lower IgG anti-CII titer after 3 or 4 immunizations with citrullinated CII when compared to that after 3 or 4 immunizations with native CII (Figure 3A).

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Figure 3. Detection of antibodies to type II collagen (anti-CII) in mouse serum by enzyme-linked immunosorbent assay (ELISA), and incidence and severity of anticollagen antibody–induced arthritis in Balb/c mice. A, An ELISA was used to detect anti-CII in the sera of Balb/c mice that had been immunized with either native CII or citrullinated (Citr) CII in intervals of 14 days for several weeks. Bars show the mean ± SD optical density (OD) in sera from 12 mice per group. ∗ = P < 0.05. B and C, The incidence of arthritis (B) and clinical score of arthritis severity (C) were determined in Balb/c mice that had been preimmunized with myelin basic protein (MBP) or citrullinated MBP (n = 10 mice per group) before the induction of arthritis with anti-CII antibody cocktail. The clinical score was assessed for each paw on a 0–4-point scale and all of the scores were summed for each mouse (maximum [max] score of 16). Bars show the mean ± SD.

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As CII is not arthritogenic in Balb/c mice, even after citrullination, we assessed whether the ACPAs induced by immunization with citrullinated MBP had an effect on the subsequent development of arthritis or severity of arthritis produced by transfer of an anti-CII antibody cocktail (27). In this model, arthritis was observed to be present in the mice, but preimmunization with either citrullinated MBP or native MBP (n = 10 per group) did not influence either the incidence (Figure 3B) or the severity (Figure 3C) of the arthritis. These results indicate that there is no evidence for a pathogenic role of ACPAs in either CIA or CAIA in Balb/c mice.

Susceptibility of SJL and DBA/1 mice to ACPA development.

As ACPAs were observed to be inducible, but not arthritogenic, in Balb/c mice, we investigated whether the CIA-susceptible SJL and DBA/1 mouse strains could also develop ACPAs. Upon immunization of SJL mice with either native fibrinogen or citrullinated fibrinogen, specific immunoreactivity toward the citrullinated, but not native, γ-chain of fibrinogen was detected in 4 of the 5 SJL mice immunized with citrullinated fibrinogen, whereas none of the SJL mice immunized with native fibrinogen showed immunoreactivity (P = 0.04) (Figure 4A). Quantification of the band intensity confirmed that SJL mice displayed a significant increase in anti–citrullinated protein immunoreactivity when immunized with citrullinated fibrinogen as compared to that after immunization with native fibrinogen (median 9,097 AU [IQR 4,422, 13,695] versus 48 AU [IQR −1,294, 924]; P = 0.02). Immunization with citrullinated CII did not induce any detectable ACPAs in the SJL mice (n = 12), as no reactivity with the citrullinated γ-chain of fibrinogen was observed (results not shown).

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Figure 4. Detection of anti–citrullinated protein antibodies (ACPAs) in mouse serum by Western blotting. Gels were loaded with native fibrinogen (fib) or citrullinated (citr) fibrinogen. After transfer, the blots were cut into strips and incubated with individual serum samples from SJL mice that had been immunized with either native fibrinogen or citrullinated fibrinogen (n = 5 mice per group) (A), DBA/1 mice that had been immunized with either native fibrinogen or citrullinated fibrinogen (n = 6 mice per group) (B), or DBA/1 mice that had been immunized with either native type II collagen (CII) or citrullinated CII (n = 15 mice per group) (C).

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All of the DBA/1 mice immunized with citrullinated fibrinogen developed ACPAs, in comparison with 4 of the 6 DBA/1 mice immunized with native fibrinogen (Figure 4B). Quantification of the band intensity showed larger numeric values in those immunized with citrullinated fibrinogen compared to those immunized with native fibrinogen (median 5,312 AU [IQR 1,660, 127,090] versus median −11 AU [IQR −2,208, 7,881]), but these differences failed to reach statistical significance. Immunization of DBA/1 mice with citrullinated CII induced immunoreactivity toward the citrullinated γ-chain of fibrinogen in only 1 of the 15 animals, compared to none of the mice immunized with native CII (Figure 4C). Results of a second independent experiment in 8 animals (not shown) revealed no immunoreactivity, which confirmed the low ACPA response in DBA/1 mice immunized with citrullinated CII. These results indicate that ACPAs can be induced by citrullinated fibrinogen, but rarely by citrullinated CII, in SJL and DBA/1 mice, demonstrating that these strains are less susceptible to ACPA development than are Balb/c mice.

Disease induction with citrullinated CII versus native CII in SJL and DBA/1 mice.

We assessed whether the immunization with citrullinated substrates could induce and/or aggravate the clinical manifestations of disease in the arthritis-susceptible SJL and DBA/1 mouse strains. Using either citrullinated fibrinogen or native fibrinogen as substrate, we did not observe any clinical evidence of arthritis induction in either the SJL mouse strain (n = 6 per group) or the DBA/1 mouse strain (n = 6 per group), not even after 5 injections. In contrast, after immunization with either native CII or citrullinated CII, 50% of the DBA/1 mice developed arthritis 5–7 days after the booster injection, reaching a maximum mean clinical arthritis score of 5.2 (±SD 3.2) (Figures 5C and D). Similar results were obtained in SJL mice, with an incidence of arthritis of 66% and a mean severity score of 4.6 (±SD 3.6). In the SJL mice, there were no significant differences in the incidence or severity of CIA between those immunized with citrullinated CII and those immunized with native CII (n = 8 per group) (Figures 5A and B). We did not observe any influence on IgG anti-CII antibody levels when either citrullinated or native CII was used for immunization in the SJL and DBA/1 mouse strains (Figure 5E).

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Figure 5. Effects of immunization with either native type II collagen (CII) or citrullinated (citr) CII on arthritis incidence and severity in SJL and DBA/1 mice. AD, The incidence of arthritis (A and C) and severity of arthritis (B and D) were assessed on day 0 (not shown) and day 21 (arrow) in SJL mice (n = 8 mice per group) (A and B) and DBA/1 mice (n = 15 mice per group) (C and D) that had been immunized with either native CII or citrullinated CII. The clinical score was assessed for each paw on a 0–4-point scale and all of the scores were summed for each mouse. E, An enzyme-linked immunosorbent assay was used to detect anti-CII antibodies in the sera of SJL and DBA/l mice that had been immunized with either native CII or citrullinated CII. F, Experiments were performed in which SJL and DBA/1 mice (n = 12 mice per group) received a booster injection of native CII or citrullinated CII at a later time point (day 40 [arrow] rather than day 21) to detect differences in the day of arthritis onset. The severity of arthritis before and after the booster injection was assessed as the summed clinical score. Bars show the mean ± SD. OD = optical density.

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As SJL mice showed a slightly more rapid onset of arthritis upon immunization with citrullinated CII (median onset day 20 [IQR 15, 22] versus day 26 [IQR 25, 33] in DBA/1 mice; P = 0.03), we performed experiments in which SJL and DBA/1 mice received a booster injection at a later time point (day 40 rather than day 21) in order to detect differences in the day of onset. Most of the SJL mice developed arthritis before the booster immunization, but there was no difference in the clinical arthritis severity score between those immunized with citrullinated CII and those immunized with native CII (n = 12 per group) (Figure 5F). Similarly, in DBA/1 mice, there was no difference in the day of onset, incidence, or severity of arthritis between those immunized with citrullinated CII and those immunized with native CII on day 0 and day 40 (n = 8 per group) (results not shown). These data indicate that when disease is induced with citrullinated CII rather than native CII, this does not significantly accelerate and/or aggravate CIA in SJL and DBA/1 mice.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. REFERENCES

ACPAs are important diagnostic and prognostic biomarkers in human RA, but the pathologic relevance of these antibodies remains largely unknown. Studies with human macrophages and mast cells have clearly indicated that the immune complexes that are formed by ACPAs can trigger these cells to produce and/or release proinflammatory mediators (13, 14). Interpreting the observations from experimental models, however, remains challenging, because the data on the presence and arthritogenicity of ACPAs in murine arthritis have been conflicting (16–23).

One of the major reasons for the discrepancies in the published reports may be the different methods used for the detection of ACPAs in mice. In contrast to the findings in human serum, in mouse serum, the antibodies binding the CCPs used in the commercially available ELISAs were reported to bind with similar avidity and affinity to the arginine counterpart, indicating that these antibodies are not specific for the citrulline residue, but rather are specific for the surrounding amino acids making up the immunogenic epitope (19). Our findings extend this concept by demonstrating, in 3 different mouse strains, that reactivity to the citrullinated peptide was clearly detected in CIA, but the immunoreactivity was similar upon immunization with citrullinated CII and that upon immunization with native CII, was not specific for the citrullinated epitope, as similar reactivity was observed with the arginine epitope, and, in contrast to experiments with human serum, could not be inhibited by preincubation with citrullinated epitopes. These results confirm that the commercially available anti-CCP assays are not reliable in the detection of ACPAs in mice. The possibility that other citrullinated peptides, such as epitopes from vimentin or α-enolase, might be more reliable substrates in rodents remains to be investigated.

To avoid this potential for false-positivity, ELISA methods using whole citrullinated proteins as coating antigen have been developed by several groups (18, 20–22, 26). However, one caveat, which has not always been taken into account, is the contamination of the in vitro citrullinated protein with PAD in the protein mixture used for immunization and/or in the substrate used for detection of ACPAs. Herein, we clearly showed that all investigated mouse strains developed a strong immunoreactivity against the rabbit PAD-2 enzyme used to citrullinate the antigens in vitro, leading to false-positive results in the ELISAs using in vitro citrullinated proteins as substrate. Moreover, the PAD protein itself can become citrullinated (results not shown) (28), which further complicates the interpretation of these data.

We circumvented this issue by developing a Western blot assay in which we utilized citrullinated fibrinogen as the substrate, since this substrate contains a wide variety of epitopes that can be citrullinated and specifically recognized by human ACPAs (29–31), and since the γ-chain can clearly be distinguished from contaminating PAD based on the molecular weight. Whereas the immunoreactivity of human ACPAs is largely restricted to 3 dominant epitopes of the α- and β-chain (32), we demonstrated specific immunoreactivity toward the citrullinated, but not native, γ-chain of fibrinogen in Balb/c mice that were hyperimmunized not only with citrullinated fibrinogen but also with other citrullinated proteins, such as citrullinated MBP or CII.

Using this robust and reliable detection method, we investigated the immunogenicity of different citrullinated proteins in 3 different mouse strains: Balb/c mice, which are well known for strong humoral responses but are not susceptible to CIA, SJL mice, which show a mild form of arthritis after CII injection, and DBA/1 mice, which are the most susceptible to CIA. In all 3 strains, ∼80% of the animals developed ACPAs after multiple immunizations with citrullinated fibrinogen in Freund's complete adjuvant/incomplete adjuvant, which is consistent with previous observations in experiments with Balb/c mice, DR4-IE–transgenic B6 mice, and Lewis and Brown-Norway rats (21, 22, 26). Interestingly, 2 of 6 Balb/c mice and 4 of 6 DBA/1 mice immunized multiple times with native fibrinogen also developed some ACPA reactivity, suggesting that the native protein injected for immunization either 1) already contains some citrullinated epitopes (23) or 2) can be citrullinated in vivo by mouse PAD. The former hypothesis is unlikely, as blotted human native fibrinogen is not recognized by the Senshu antibody (results not shown) (33). The latter concept, however, fits with the results reported in several studies indicating that ACPAs can develop upon immunization of mice with other native proteins (16, 17), and that, when endogenous PAD enzymes are chemically blocked, citrullinated synovial protein levels and severity of CIA are reduced (34).

Balb/c mice also developed ACPAs when immunized with citrullinated MBP or with citrullinated collagen, a substrate which is pathogenetically relevant but contains less epitopes that can be citrullinated (17). In SJL mice, ACPA reactivity was detected to some extent upon immunization with citrullinated MBP (results not shown), but not upon immunization with citrullinated CII.

Finally, in DBA/1 mice, ACPAs did develop upon immunization with citrullinated collagen, but this was observed in only a small minority of the animals. However, the use of citrullinated fibrinogen as the detection substrate in Western blot analysis may not detect all ACPAs, and antibodies against other citrullinated epitopes that are not present in citrullinated fibrinogen could be present in the sera of SJL and DBA/1 mice.

These results thus indicate that all 3 different strains can develop ACPAs, but that this humoral response depends on the strain (with Balb/c mice being most susceptible), the immunogen (with collagen being poorly immunogenic in comparison with fibrinogen or MBP), and, most probably, also the immunization protocol. Indeed, in Balb/c mice, the appearance of ACPAs was most pronounced after multiple immunizations with citrullinated CII; a similar protocol could not be used in DBA/1 and SJL mice due to development of severe arthritis after 2 injections with CII. In combination with the use of different detection methods, the variability in ACPA development related to these different factors could possibly explain many of the discrepancies reported in the literature.

The ability to induce and detect murine ACPAs also allowed us to reassess the pathogenic role of these antibodies in these models. First, we investigated whether the presence of ACPAs can aggravate experimental arthritis. It was previously shown that transfer of monoclonal ACPAs was insufficient to induce arthritis in DBA/1 mice, but did aggravate disease activity in mice with CAIA (16). In contrast, ACPAs raised by immunization with citrullinated fibrinogen did not modulate the course of subsequent adjuvant-induced arthritis in Lewis rats (21). Similarly, in our experiments, the presence of ACPAs induced by active immunization of Balb/c mice failed to affect the incidence or severity of CAIA. Although this discrepancy between the results of the former study (16) and the results of the latter study (21) and our own study may be attributed to the specific features of specific monoclonal ACPAs (17), overall these findings indicate that naturally raised ACPAs do not consistently affect the course of experimental arthritis.

Second, we assessed whether the response raised by immunization with native or citrullinated fibrinogen was arthritogenic, as has been reported previously not only in DR4-IE–transgenic B6 mice but also in wild-type SJL mice (22, 23). In contrast with the previous findings in SJL mice in the study by Ho et al (23), our study found that none of the Balb/c, SJL, or DBA/1 mice immunized up to 5 times with either native or citrullinated fibrinogen developed arthritis. This could be due to differences in the immunization protocol used, the mildness of the arthritis observed in the study by Ho et al (mean arthritis score of 4, maximum score of 12), or the sample size in those experiments (n = 10 mice). Our observations are in accordance with those from previous studies in rats (21) and mice (18), and further confirm that the presence of circulating ACPAs alone is not sufficient to induce arthritis in rodents.

Finally, even if we were not able to detect robust ACPAs during the course of CIA in SJL and DBA/1 mice, we assessed whether the use of citrullinated CII, rather than native CII, for immunization modulated the clinical course of CIA, since a previous report indicated that this immunization protocol had a modest effect on arthritis incidence and day of onset of disease in rats (20). We did not observe any difference in incidence or severity of arthritis in both SJL and DBA/1 mice. In one experiment in SJL mice, we observed a slightly accelerated onset of disease when citrullinated CII was used, but this effect was not reproducible in subsequent experiments. Moreover, the anti-CII antibody levels were not augmented by citrullination of CII; on the contrary, similar experiments in Balb/c mice even showed lower IgG anti-CII levels when citrullinated CII was used for immunization.

Two caveats should be considered when interpreting these data. First, the disease severity scores were relatively low in these experiments in mice with CIA, which may be explained by partial denaturation of CII during the 2 hours of incubation at 37°C needed for the in vitro citrullination reaction. Second, it needs to be emphasized that all of our experiments were performed with heterologous proteins (either human or bovine). Whether immunization with citrullinated autologous proteins, as was performed by Duplan and colleagues in a previous study (21), yields similar results needs to be further assessed in comparative studies.

Collectively, our findings indicate that mice can develop genuine ACPAs, but the detection of ACPAs is highly dependent on the strain, immunogen, immunization protocol, and, most importantly, the use of a reliable detection method. However, these ACPAs were not overly pathogenic, since neither the presence of preexisting ACPAs nor the use of citrullinated collagen could modulate the clinical course of arthritis.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. 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. Baeten 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. Cantaert, Tak, Baeten.

Acquisition of data. Cantaert, Teitsma, Baeten.

Analysis and interpretation of data. Cantaert, Teitsma, Baeten.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. REFERENCES