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. 2004 Apr;63(4):373–381. doi: 10.1136/ard.2003.012211

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

E Vossenaar 1, T Radstake 1, A van der Heijden 1, M A M van Mansum 1, C Dieteren 1, D-J de Rooij 1, P Barrera 1, A Zendman 1, W J van Venrooij 1
PMCID: PMC1754951  PMID: 15020330

Abstract

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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Figure 1 .

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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. Expression of PAD was analysed by RT-PCR. PAD1 and 3 were not detectable in any of the PBMC fractions. For PAD2 and PAD4, specific PCR products were produced in all fractions. In the case of CD3, CD19, and CD56, the cells that did not bind to the beads (CD3– CD19–, CD56–) showed higher expression than the cells that did bind (CD3+, CD19+, CD56+). In contrast, the cells that were bound by the anti-CD14 coated beads (CD14+, monocytes) showed higher PAD2 and PAD4 expression than the cells that did not bind the anti-CD14 coated beads (CD14–, all but the monocytes) as indicated by white squares. ß-Actin served as a control for mRNA input. Cloned cDNAs served as positive PCR controls, (no positive control included for ß-actin), PCR without template as negative control.

Figure 2 .

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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 controls (HC1–HC3) was analysed by RT-PCR. ß-Actin served as a control for mRNA input, α2-macroglobulin (α-2-m) expression was analysed as a control for macrophage differentiation. Cloned cDNAs served as positive PCR controls, (no positive control included for ß-actin), PCR without template as negative control.

Figure 3 .

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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 (HC1–HC3) was analysed by western blotting. Recombinant PAD2 or PAD4 proteins are included as controls. Molecular weight markers (kDa) are indicated on the left.

Figure 4 .

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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 as positive PCR controls, (no positive control included for ß-actin), PCR without template as negative control.

Figure 5 .

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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 weight markers (kDa) are indicated on the left.

Figure 6 .

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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 of 2 mM CaCl2. Extracts were analysed for the presence of citrullinated proteins by western blotting with anti-MC antibodies. In vitro citrullinated human fibrinogen (25 ng) is included as a positive control. Molecular weight markers (kDa) are indicated on the left.

Figure 7 .

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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) with 1 µM ionomycin in the presence of 2 mM CaCl2, was stained with anti-MC antibodies. In vitro citrullinated human fibrinogen (25 ng) is included on the western blot as a positive control. Molecular weight markers (kDa) are indicated on the left.

Figure 8 .

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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: the mRNA expression of PAD4 is lost during differentiation, but the PAD4 protein levels remain unchanged, indicating that the enzyme is not rapidly degraded. For PAD2, the situation is different: the mRNA levels remain more or less the same, but the mRNA is only translated into PAD2 protein in the macrophages. The patterns of PAD expression are identical in patients with RA and healthy controls, but because of the large numbers of macrophages that can be found in the RA (and not in healthy) synovium, the amount of PAD enzyme in the RA synovium will be much higher than in healthy synovium. During apoptosis or necrosis of the PAD-containing macrophages, free intracellular Ca2+ levels are increased, resulting in activation of the PAD enzymes and citrullination of cellular proteins. In the macrophages, vimentin is specifically citrullinated after increase of cytoplasmic Ca2+. Furthermore, PAD enzymes may leak out of necrotic cells, causing the citrullination of extracellular proteins (for example, fibrin21).

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