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Ann Rheum Dis. Apr 2004; 63(4): 373–381.
PMCID: PMC1754951

Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages


Background: Antibodies directed to proteins containing the non-standard amino acid citrulline, are extremely specific for rheumatoid arthritis (RA). Peptidylcitrulline can be generated by post-translational conversion of arginine residues. This process, citrullination, is catalysed by a group of calcium dependent peptidylarginine deiminase (PAD) enzymes.

Objective: To investigate the expression and activity of four isotypes of PAD in peripheral blood and synovial fluid cells of patients with RA.

Results: The data presented here show that citrullination of proteins by PAD enzymes is a process regulated at three levels: transcription—in peripheral blood PAD2 and PAD4 mRNAs are expressed predominantly in monocytes; PAD4 mRNA is not detectable in macrophages, translation—translation of PAD2 mRNA is subject to differentiation stage-specific regulation by its 3' UTR, and activation—the PAD proteins are only activated when sufficient Ca2+ is available. Such high Ca2+ concentrations are normally not present in living cells. In macrophages, which are abundant in the inflamed RA synovium, vimentin is specifically citrullinated after Ca2+ influx.

Conclusion: PAD2 and PAD4 are the most likely candidate PAD isotypes for the citrullination of synovial proteins in RA. Our results indicate that citrullinated vimentin is a candidate autoantigen in RA.

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Figures and Tables

Figure 1
Messenger RNA expression of PAD isotypes in PBMCs. Total PBMCs from a healthy person were fractionated with MACS magnetic beads coated with either anti-CD3 (T cells), anti-CD14 (monocytes), anti-CD19 (B cells) or anti-CD56 (NK cells) for RNA isolation. ...
Figure 2
PAD4 mRNA is only expressed in monocytes, not in macrophages. RNA expression of PAD2 and PAD4 in freshly isolated monocytes (mono) from peripheral blood and in ex vivo differentiated macrophages (macro) of patients with RA (RA1–RA3) and healthy ...
Figure 3
PAD2 protein is only expressed in macrophages. Protein expression of PAD2 and PAD4 in freshly isolated monocytes (mono) from peripheral blood and in ex vivo differentiated macrophages (macro) of patients with RA (RA1–RA3) and healthy controls ...
Figure 4
PAD4 mRNA is only present in PBMCs, not in SFMCs. Mononuclear cells were isolated from PB and SF samples of patients with RA. RNA expression of PAD2 and PAD4 was analysed by RT-PCR. ß-Actin served as a control for mRNA input. Cloned cDNAs served ...
Figure 5
PAD2 protein is only expressed in SFMCs. Protein expression of PAD2 and PAD4 in mononuclear cells isolated from PB and SF samples of patients with RA was analysed by western blotting. Recombinant PAD2 and PAD4 proteins are included as controls. Molecular ...
Figure 6
Citrullinated proteins are only generated after calcium influx. Monocytes (A) and macrophages (B) from a healthy control (HC) and a patient with RA were treated for 0, 15 minutes, 60 minutes, or overnight (o/n) with 1 µM ionomycin in the presence ...
Figure 7
Vimentin is specifically citrullinated in calcium stimulated macrophages. Immunoprecipitated vimentin from monocytes (A) and macrophages (B) from a healthy control (HC) and a patient with RA, treated for 0, 15 minutes, 60 minutes, or overnight (o/n) ...
Figure 8
Model for synovial PAD infiltration. Many leucocytes infiltrate the inflamed synovium in RA. When monocytes from the PB enter the synovium, they will differentiate into macrophages. This differentiation process has several consequences for PAD expression: ...

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