Chronic inflammation of synovial joints with frequent extraarticular manifestations is an essential characteristic of rheumatoid arthritis (RA), the most common human autoimmune disease. The presence of autoantibodies to “citrullinated” (deiminated) proteins (anti–citrullinated protein antibodies [ACPAs]) in the serum of patients also constitutes a major feature of RA. These autoantibodies very probably play a significant role in the pathophysiology of RA. Indeed, ACPAs are most likely the most disease-specific of the RA-associated autoantibodies. They are detectable in the serum years before the onset of arthritis symptoms, and a significant positive correlation exists between the serum titer and clinical, biologic, and radiologic data related to RA activity and/or severity (for review, see ref.1). In addition, ACPAs are produced by plasma cells of rheumatoid synovial tissue (ST) and therein not only concentrate (2) but also probably interact with citrullinated proteins, among which citrullinated fibrin constitutes their major target (3).
Even if not sufficient, citrullyl residues are essential to the formation of ACPA epitopes on the target antigens (4–6). These epitopes result from the posttranslational transformation of the positively charged guanidino group of arginyl residues into the uncharged ureido group of citrullyl residues. A Ca2+-dependent enzyme activity, designated peptidyl arginine deiminase (PAD; also called protein-L-arginine iminohydrolase [EC 184.108.40.206]), catalyzes such conversion. Five PAD isotypes (PAD-1, PAD-2, PAD-3, PAD-4, and PAD-6), encoded by 5 paralogous genes (PADI1, PADI2, PADI3, PADI4, and PADI6, respectively) clustered on chromosome 1p35–36, have been described in humans (7). Their cellular expression pattern has not yet been extensively explored, particularly at the protein level. PAD-1 has been detected in the epidermis, in hair follicles, in arrector pili muscles, and in sweat glands (8–10). PAD-2 has been widely detected, notably in brain astrocytes (11, 12), sweat glands (9, 13), arrector pili muscles (9), macrophages (14), and epidermis (8, 15). To date, PAD-3 expression has been reported only in the upper layers of epidermis and in hair follicles (8–10, 16). PAD-4 expression has so far been described only in hematopoietic cells (14, 17–19). PAD-4 differs from other PAD isotypes by its capacity to undergo nuclear translocation (20). No data are available concerning expression of PAD-6 in human tissue, but PADI6 messenger RNA (mRNA) is present mainly in ovary, testis, and peripheral blood leukocytes (7).
Protein “citrullination” (deimination) leads to alterations in intramolecular and intermolecular interactions of the protein targets (21). It is implicated in terminal differentiation of epidermis (22) and in brain development (23). Nuclear PAD-4 could regulate gene expression via chromatin remodeling (24, 25). Diseases in which citrullination has been shown or suggested to play a role include not only RA but also multiple sclerosis, as well as psoriasis, Alzheimer's disease, primary open-angle glaucoma, and obstructive nephropathy (26).
One or several PADs are necessarily responsible for generation of the ACPA-targeted epitopes in rheumatoid ST, but therein the presence of the 5 PAD isotypes, including the most recently described PAD-6, has not yet been systematically explored. In the present study, we determined which of the 5 PADs are expressed in rheumatoid ST, at both the mRNA level and the protein level. After assessing the expression of PADs in human blood-derived mononuclear leukocytes, PADs were investigated in ST samples obtained from a large series of patients with RA, compared with patients with other arthritides or osteoarthritis (OA). Moreover, correlations were sought between the levels of PAD expression and the intensity of ST inflammation, detected histologically, both parameters being evaluated by semiquantitative methods. Finally, to identify the PAD(s) most likely to be involved in the generation of citrullinated fibrin, immunohistochemical analyses of ST samples from patients were RA were performed to analyze whether this major ACPA target is colocated with the ST-expressed PADs.
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- AUTHOR CONTRIBUTIONS
In the first part of our study, the fact that none of the mononuclear leukocyte populations we examined expressed PADI1 and PADI3 fits with the reported absence of the corresponding mRNA transcripts in peripheral blood leukocytes (8, 31). It also was consistent with a recent study by Vossenaar et al, in which the authors reported the absence of mRNA for PADI1 and PADI3 in PB-derived T cells (CD3+ PBMCs), B cells (CD19+ PBMCs), monocytes (CD14+ PBMCs), and natural killer cells (CD56+ PBMCs) from 1 blood donor (14). Both studies are also in agreement concerning the presence of PADI2 mRNA in lymphocytes, monocytes, and monocyte-derived macrophages. However, at the protein level, we found PAD-2 in monocytes and macrophages, while it was detected in macrophages but not in monocytes by the other authors (14). This discrepancy may originate from differences in the methods used to obtain blood monocytes, because we purified monocytes using positive selection of CD14-positive PBMCs, while Vossenaar et al isolated monocytes from PBMCs by plastic adherence. Another discrepancy concerns PAD-4, which we could not detect in macrophages, while Vossenaar et al made an opposite observation. Again, this could originate from methodologic differences: to obtain monocyte-derived macrophages, we used a 7-day culture of the CD14-positive PBMCs in the presence of M-CSF and prevented adherence, while in the other study, macrophages were obtained by a 7-day culture of adherent PBMCs.
Concerning PAD-6, our analysis of its protein expression using specially developed and validated antibodies is original. The apparent molecular mass of the band that these antibodies detected in a protein extract of human adult ovary corresponds to that predicted for human PAD-6. Therefore, this protein is probably present at least in ovaries, similar to its mouse ortholog, ePAD (33, 34). In addition, we confirm previous reports mentioning detection of transcripts of PADI6 in peripheral blood leukocytes (7, 35) and, furthermore, specify their presence in several types of mononuclear leukocytes. However, real-time RT-PCR analysis showed that the expression level of PADI6 is very low, and this probably accounts for the fact that the corresponding protein could not be detected. Therefore, and on the whole, the results concerning expression of PAD in different mononuclear leukocyte populations indicate that only PAD-2 and PAD-4 reach detectable expression at the protein level in cells of lymphocyte and monocyte lineages.
The second part of our study constitutes the first systematic exploration of synovial expression of the 5 PAD isotypes in a large series of patients with RA, non-RA arthritides, or OA. We clearly demonstrate the absence of PAD-1 and PAD-3 in all disease types. Moreover, we show that PAD-2 is consistently expressed in the course of both arthritides and OA, while expression of PAD-4 is mainly associated with arthritides. Finally, no PAD-6 protein was found in any of the disease groups.
Concerning PAD-2 and PAD-4, our results can be compared with those of other less systematic studies analyzing their presence in human rheumatoid ST. The research group led by K. Yamamoto mentioned the presence of PAD-4 in the sublining region of ST from patients with RA (36, 37). However, in a much more detailed confocal microscopy analysis using the same anti–PAD-4 antibody, the presence of PAD-4 was detected in all areas of ST samples from patients with RA, in cells that were identified as T lymphocytes, B lymphocytes, macrophages, granulocytes, fibroblasts, and endothelial cells (19). The same group of investigators also reported immunohistologic detection of PAD-2 in the lining, sublining, and deep regions of 1 ST sample from a patient with RA (36).
In ST samples obtained from a series of patients including 19 patients with RA and 19 control patients with inflammatory or noninflammatory rheumatism, De Rycke et al detected PAD-2 in 59% of patients with RA but also in 17% of control patients (38). This is consistent with our observations, even though our immunoblot analysis with optimized antibody concentrations appears to be more sensitive (0.5–1 fmole), because it allowed us to detect the presence of PAD-2 in all RA and almost all control ST samples. Additionally, PAD-2 and PAD-4 have been explored in the ST of mice with collagen-induced arthritis (CIA) or streptococcal cell wall–induced arthritis, with identical results in both models (39). Transcripts for Padi2 were detected in naive and arthritic mice, while transcripts for Padi4 were detected only in arthritic mice. Both corresponding PAD-2 and PAD-4 proteins were absent in the ST of naive mice and, in the inflamed ST of arthritic mice, only PAD-4 was detected and reported to be expressed by neutrophils (39). PAD-4 has also been observed in the inflamed ST of dark agouti rats with CIA, but, in this case, mononuclear cells were found to be responsible for its expression (40).
An important point of our study is that it clearly shows that PAD expression in the ST is not specific for RA, which is consistent with our finding that citrullination of fibrin in the ST occurs during a variety of synovitides (41), and that citrullinated proteins are present in several other inflamed tissues, such as the muscle, the colon, or the lung of patients with polymyositis, inflammatory bowel disease, or interstitial pneumonia, respectively (42, 43). Similarly, in patients with RA, we could not demonstrate a relationship between the serum ACPA titer and the level of PAD-2 or PAD-4 in the ST, which is consistent with the absence of correlation between the serum ACPA titer and the amount of citrullinated fibrin in the ST (41). This emphasizes that, in the ST, PAD expression and citrullination of target proteins are not specifically associated with RA, in contrast to our findings with ACPAs.
We actually show that PAD expression in the ST is an inflammation-dependent phenomenon. A close correlation is observed in arthritides and OA between the level of PAD-2 and the degree of infiltration by inflammatory cells, suggesting basal expression of this isotype by these cells in these 2 disease types. Because PAD-4 is also correlated with the degree of infiltration, inflammatory cells also constitute a source for this isotype. However, PAD-4 is confined to the arthritides. Differences in the composition of the infiltrate, but also in the state of differentiation and/or activation of infiltrating cells, probably influenced by differences in their cytokine environment, may account for PAD-4 expression in arthritides but not OA. Moreover, PAD-4 is frequently observed in the lining layer, and its expression level is particularly correlated with the synovial lining thickness. This suggests that the hyperplastic cells of the synovial lining constitute another important source of PAD-4 in these diseases. It would be interesting to evaluate whether the proportion of PAD-4–positive cells exhibiting nuclear staining is related to the concentration of tumor necrosis factor α in the ST, because it was recently shown that this cytokine induces nuclear translocation of this PAD isotype in murine and human oligodendroglial cell lines (44).
Studies of PAD polymorphisms have shown that a haplotype of PADI4 was associated with RA in Japanese (37, 45) and Korean populations (46). These results were confirmed in a Caucasian population from North America (47) but were not reproduced in various European Caucasian populations (47–51). In the initial study by Suzuki et al (37), in vitro results indicated that the presence of this haplotype could lead to more stable mRNA, suggesting that PAD-4 expression was increased in the ST of patients with RA, leading to enhanced levels of citrullinated proteins. However, so far the question of whether the levels of PAD-4 expression are related to the PADI4 haplotype has not been explored. Given the association between PAD-4 expression and ST inflammation, it would be interesting to evaluate the influence of PADI4 haplotypes in groups of patients stratified according to the degree of ST inflammation.
Owing to the use of anti-PAD antibodies with equalized sensitivities of detection, we could observe that, in the ST of patients with RA, the levels of PAD-4 detected by immunoblotting tended to be higher than those of PAD-2. This is corroborated by the observation of higher numbers of PAD-4–positive cells than PAD-2–positive cells by immunohistochemical analysis. However, our results strongly indicate that both PAD-2 and PAD-4 are involved in the process of fibrin citrullination, because even if their simultaneous detection in the same area was quite rare, both were observed directly or in the close vicinity of fibrin deposits in the ST. This is compatible with the observation that, in vitro, recombinant forms of both human PAD-2 and human PAD-4 are able to citrullinate human fibrinogen (36). Moreover, the presence of citrullinated fibrin in samples of inflamed ST from patients with OA (41), in which PAD-4 is rarely seen (ref.19, and the present study), is necessarily attributable to a PAD-2 activity.
In conclusion, the present study has provided the first extensive exploration of all existing PAD enzymes in the ST of patients with RA. Even if it remains to be precisely determined which factors induce their production and how/why they are released and activated to be able to target extracellular proteins such as fibrin, most probably both PAD-2 and PAD-4 play a role in the generation of synovial ACPA targets. In this respect, inhibition of the activity or expression of PAD-2 and/or PAD-4 may constitute a valuable therapeutic strategy for RA.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Serre 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 design. Sebbag, Serre.
Acquisition of data. Foulquier, Sebbag, Clavel, Chapuy-Regaud, Al Badine.
Analysis and interpretation of data. Foulquier, Sebbag, Chapuy-Regaud, Al Badine, Guerrin.
Manuscript preparation. Foulquier, Sebbag, Chapuy-Regaud, Méchin, Simon, Guerrin, Serre.
Statistical analysis. Vincent.
Provision of indispensable reagents. Clavel, Méchin, Nachat, Yamada, Takahara.