Logo of annrheumdAnnals of the Rheumatic DiseasesVisit this articleSubmit a manuscriptReceive email alertsContact usBMJ
Ann Rheum Dis. Apr 2004; 63(4): 373–381.
PMCID: PMC1754951

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

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.

Full Text

The Full Text of this article is available as a PDF (534K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Lisse JR. Does rheumatoid factor always mean arthritis? Postgrad Med. 1993 Nov 1;94(6):133–139. [PubMed]
  • van Boekel Martinus A M, Vossenaar Erik R, van den Hoogen Frank H J, van Venrooij Walther J. Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res. 2002;4(2):87–93. [PMC free article] [PubMed]
  • NIENHUIS RL, MANDEMA E. A NEW SERUM FACTOR IN PATIENTS WITH RHEUMATOID ARTHRITIS; THE ANTIPERINUCLEAR FACTOR. Ann Rheum Dis. 1964 Jul;23:302–305. [PMC free article] [PubMed]
  • Young BJ, Mallya RK, Leslie RD, Clark CJ, Hamblin TJ. Anti-keratin antibodies in rheumatoid arthritis. Br Med J. 1979 Jul 14;2(6182):97–99. [PMC free article] [PubMed]
  • Simon M, Girbal E, Sebbag M, Gomès-Daudrix V, Vincent C, Salama G, Serre G. The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis. J Clin Invest. 1993 Sep;92(3):1387–1393. [PMC free article] [PubMed]
  • Sebbag M, Simon M, Vincent C, Masson-Bessière C, Girbal E, Durieux JJ, Serre G. The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. J Clin Invest. 1995 Jun;95(6):2672–2679. [PMC free article] [PubMed]
  • Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest. 1998 Jan 1;101(1):273–281. [PMC free article] [PubMed]
  • Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, van Venrooij WJ. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum. 2000 Jan;43(1):155–163. [PubMed]
  • van Venrooij WJ, Hazes JM, Visser H. Anticitrullinated protein/peptide antibody and its role in the diagnosis and prognosis of early rheumatoid arthritis. Neth J Med. 2002 Nov;60(10):383–388. [PubMed]
  • Vasishta Anil. Diagnosing early-onset rheumatoid arthritis: the role of anti-CCP antibodies. Am Clin Lab. 2002 Aug-Sep;21(7):34–36. [PubMed]
  • Bizzaro N, Mazzanti G, Tonutti E, Villalta D, Tozzoli R. Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis. Clin Chem. 2001 Jun;47(6):1089–1093. [PubMed]
  • Palosuo T, Tilvis R, Strandberg T, Aho K. Filaggrin related antibodies among the aged. Ann Rheum Dis. 2003 Mar;62(3):261–263. [PMC free article] [PubMed]
  • Visser Henk, le Cessie Saskia, Vos Koen, Breedveld Ferdinand C, Hazes Johanna M W. How to diagnose rheumatoid arthritis early: a prediction model for persistent (erosive) arthritis. Arthritis Rheum. 2002 Feb;46(2):357–365. [PubMed]
  • Kroot EJ, de Jong BA, van Leeuwen MA, Swinkels H, van den Hoogen FH, van't Hof M, van de Putte LB, van Rijswijk MH, van Venrooij WJ, van Riel PL. The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis. Arthritis Rheum. 2000 Aug;43(8):1831–1835. [PubMed]
  • Meyer O, Labarre C, Dougados M, Goupille Ph, Cantagrel A, Dubois A, Nicaise-Roland P, Sibilia J, Combe B. Anticitrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage. Ann Rheum Dis. 2003 Feb;62(2):120–126. [PMC free article] [PubMed]
  • Vencovský J, Machácek S, Sedová L, Kafková J, Gatterová J, Pesáková V, Růzicková S. Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis. Ann Rheum Dis. 2003 May;62(5):427–430. [PMC free article] [PubMed]
  • Masson-Bessière C, Sebbag M, Durieux JJ, Nogueira L, Vincent C, Girbal-Neuhauser E, Durroux R, Cantagrel A, Serre G. In the rheumatoid pannus, anti-filaggrin autoantibodies are produced by local plasma cells and constitute a higher proportion of IgG than in synovial fluid and serum. Clin Exp Immunol. 2000 Mar;119(3):544–552. [PMC free article] [PubMed]
  • Reparon-Schuijt CC, van Esch WJ, van Kooten C, Schellekens GA, de Jong BA, van Venrooij WJ, Breedveld FC, Verweij CL. Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis. Arthritis Rheum. 2001 Jan;44(1):41–47. [PubMed]
  • Masson-Bessière C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C, Senshu T, Serre G. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin. J Immunol. 2001 Mar 15;166(6):4177–4184. [PubMed]
  • van Venrooij WJ, Pruijn GJ. Citrullination: a small change for a protein with great consequences for rheumatoid arthritis. Arthritis Res. 2000;2(4):249–251. [PMC free article] [PubMed]
  • Vossenaar Erik R, Zendman Albert J W, van Venrooij Walther J, Pruijn Ger J M. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays. 2003 Nov;25(11):1106–1118. [PubMed]
  • Rus'd AA, Ikejiri Y, Ono H, Yonekawa T, Shiraiwa M, Kawada A, Takahara H. Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I, type III and type IV, and the expression pattern of type I in mouse. Eur J Biochem. 1999 Feb;259(3):660–669. [PubMed]
  • Rogers G, Winter B, McLaughlan C, Powell B, Nesci A. Hair follicle peptidylarginine deiminase. Exp Dermatol. 1999 Aug;8(4):362–363. [PubMed]
  • Kanno T, Kawada A, Yamanouchi J, Yosida-Noro C, Yoshiki A, Shiraiwa M, Kusakabe M, Manabe M, Tezuka T, Takahara H. Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin. J Invest Dermatol. 2000 Nov;115(5):813–823. [PubMed]
  • Guerrin Marina, Ishigami Akihito, Méchin Marie-Claire, Nachat Rachida, Valmary Séverine, Sebbag Mireille, Simon Michel, Senshu Tatsuo, Serre Guy. cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I. Biochem J. 2003 Feb 15;370(Pt 1):167–174. [PMC free article] [PubMed]
  • Watanabe K, Akiyama K, Hikichi K, Ohtsuka R, Okuyama A, Senshu T. Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues. Biochim Biophys Acta. 1988 Sep 8;966(3):375–383. [PubMed]
  • Nagata S, Senshu T. Peptidylarginine deiminase in rat and mouse hemopoietic cells. Experientia. 1990 Jan 15;46(1):72–74. [PubMed]
  • Ishigami Akihito, Ohsawa Takako, Asaga Hiroaki, Akiyama Kyoichi, Kuramoto Masashi, Maruyama Naoki. Human peptidylarginine deiminase type II: molecular cloning, gene organization, and expression in human skin. Arch Biochem Biophys. 2002 Nov 1;407(1):25–31. [PubMed]
  • Nakashima K, Hagiwara T, Ishigami A, Nagata S, Asaga H, Kuramoto M, Senshu T, Yamada M. Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3). J Biol Chem. 1999 Sep 24;274(39):27786–27792. [PubMed]
  • Asaga H, Nakashima K, Senshu T, Ishigami A, Yamada M. Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils. J Leukoc Biol. 2001 Jul;70(1):46–51. [PubMed]
  • Nakashima Katsuhiko, Hagiwara Teruki, Yamada Michiyuki. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem. 2002 Dec 20;277(51):49562–49568. [PubMed]
  • Cutolo M, Sulli A, Barone A, Seriolo B, Accardo S. Macrophages, synovial tissue and rheumatoid arthritis. Clin Exp Rheumatol. 1993 May-Jun;11(3):331–339. [PubMed]
  • Asaga H, Yamada M, Senshu T. Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun. 1998 Feb 24;243(3):641–646. [PubMed]
  • Mizoguchi M, Manabe M, Kawamura Y, Kondo Y, Ishidoh K, Kominami E, Watanabe K, Asaga H, Senshu T, Ogawa H. Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro. J Histochem Cytochem. 1998 Nov;46(11):1303–1309. [PubMed]
  • Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988 Mar;31(3):315–324. [PubMed]
  • Rodenburg RJ, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Peripheral blood monocytes of rheumatoid arthritis patients do not express elevated TNF alpha, IL-1beta, and IL-8 mRNA levels. A comparison of monocyte isolation procedures. J Immunol Methods. 1998 Dec 1;221(1-2):169–175. [PubMed]
  • Senshu T, Sato T, Inoue T, Akiyama K, Asaga H. Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. Anal Biochem. 1992 May 15;203(1):94–100. [PubMed]
  • Kikuno Reiko, Nagase Takahiro, Waki Mina, Ohara Osamu. HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project. Nucleic Acids Res. 2002 Jan 1;30(1):166–168. [PMC free article] [PubMed]
  • Watanabe K, Senshu T. Isolation and characterization of cDNA clones encoding rat skeletal muscle peptidylarginine deiminase. J Biol Chem. 1989 Sep 15;264(26):15255–15260. [PubMed]
  • Tsuchida M, Takahara H, Minami N, Arai T, Kobayashi Y, Tsujimoto H, Fukazawa C, Sugawara K. cDNA nucleotide sequence and primary structure of mouse uterine peptidylarginine deiminase. Detection of a 3'-untranslated nucleotide sequence common to the mRNA of transiently expressed genes and rapid turnover of this enzyme's mRNA in the estrous cycle. Eur J Biochem. 1993 Aug 1;215(3):677–685. [PubMed]
  • Senshu T, Akiyama K, Kan S, Asaga H, Ishigami A, Manabe M. Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues. J Invest Dermatol. 1995 Aug;105(2):163–169. [PubMed]
  • Asaga H, Senshu T. Combined biochemical and immunocytochemical analyses of postmortem protein deimination in the rat spinal cord. Cell Biol Int. 1993 May;17(5):525–532. [PubMed]
  • Treiman M, Caspersen C, Christensen SB. A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPases. Trends Pharmacol Sci. 1998 Apr;19(4):131–135. [PubMed]
  • Urano Y, Watanabe K, Sakaki A, Arase S, Watanabe Y, Shigemi F, Takeda K, Akiyama K, Senshu T. Immunohistochemical demonstration of peptidylarginine deiminase in human sweat glands. Am J Dermatopathol. 1990 Jun;12(3):249–255. [PubMed]
  • Standart N, Jackson RJ. Regulation of translation by specific protein/mRNA interactions. Biochimie. 1994;76(9):867–879. [PubMed]
  • Steger K. Haploid spermatids exhibit translationally repressed mRNAs. Anat Embryol (Berl) 2001 May;203(5):323–334. [PubMed]
  • Ostareck DH, Ostareck-Lederer A, Wilm M, Thiele BJ, Mann M, Hentze MW. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end. Cell. 1997 May 16;89(4):597–606. [PubMed]
  • Ostareck DH, Ostareck-Lederer A, Shatsky IN, Hentze MW. Lipoxygenase mRNA silencing in erythroid differentiation: The 3'UTR regulatory complex controls 60S ribosomal subunit joining. Cell. 2001 Jan 26;104(2):281–290. [PubMed]
  • Mazumder Barsanjit, Seshadri Vasudevan, Fox Paul L. Translational control by the 3'-UTR: the ends specify the means. Trends Biochem Sci. 2003 Feb;28(2):91–98. [PubMed]
  • Hagiwara Teruki, Nakashima Katsuhiko, Hirano Hisashi, Senshu Tatsuo, Yamada Michiyuki. Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes. Biochem Biophys Res Commun. 2002 Jan 25;290(3):979–983. [PubMed]
  • Tombal B, Denmeade SR, Gillis J-M, Isaacs JT. A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis. Cell Death Differ. 2002 May;9(5):561–573. [PubMed]
  • Sasamura Satoshi, Furukawa Ken-Ichi, Shiratori Miwa, Motomura Shigeru, Ohizumi Yasushi. Antisense-inhibition of plasma membrane Ca2+ pump induces apoptosis in vascular smooth muscle cells. Jpn J Pharmacol. 2002 Oct;90(2):164–172. [PubMed]
  • Schwab BL, Guerini D, Didszun C, Bano D, Ferrando-May E, Fava E, Tam J, Xu D, Xanthoudakis S, Nicholson DW, et al. Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis. Cell Death Differ. 2002 Aug;9(8):818–831. [PubMed]
  • Senshu T, Kan S, Ogawa H, Manabe M, Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis. Biochem Biophys Res Commun. 1996 Aug 23;225(3):712–719. [PubMed]
  • Senshu T, Akiyama K, Nomura K. Identification of citrulline residues in the V subdomains of keratin K1 derived from the cornified layer of newborn mouse epidermis. Exp Dermatol. 1999 Oct;8(5):392–401. [PubMed]
  • Inagaki M, Nishi Y, Nishizawa K, Matsuyama M, Sato C. Site-specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature. 1987 Aug 13;328(6131):649–652. [PubMed]
  • Inagaki M, Takahara H, Nishi Y, Sugawara K, Sato C. Ca2+-dependent deimination-induced disassembly of intermediate filaments involves specific modification of the amino-terminal head domain. J Biol Chem. 1989 Oct 25;264(30):18119–18127. [PubMed]
  • Müller K, Dulku S, Hardwick SJ, Skepper JN, Mitchinson MJ. Changes in vimentin in human macrophages during apoptosis induced by oxidised low density lipoprotein. Atherosclerosis. 2001 May;156(1):133–144. [PubMed]
  • Byun Y, Chen F, Chang R, Trivedi M, Green KJ, Cryns VL. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ. 2001 May;8(5):443–450. [PubMed]
  • Morishima N. Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action. Genes Cells. 1999 Jul;4(7):401–414. [PubMed]
  • Cao L, Goodin R, Wood D, Moscarello MA, Whitaker JN. Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein. Biochemistry. 1999 May 11;38(19):6157–6163. [PubMed]
  • Resing KA, al-Alawi N, Blomquist C, Fleckman P, Dale BA. Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage. J Biol Chem. 1993 Nov 25;268(33):25139–25145. [PubMed]
  • Tarcsa E, Marekov LN, Mei G, Melino G, Lee SC, Steinert PM. Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin. J Biol Chem. 1996 Nov 29;271(48):30709–30716. [PubMed]
  • Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell. 1996 May 3;85(3):307–310. [PubMed]
  • Rodenburg RJ, Ganga A, van Lent PL, van de Putte LB, van Venrooij WJ. The antiinflammatory drug sulfasalazine inhibits tumor necrosis factor alpha expression in macrophages by inducing apoptosis. Arthritis Rheum. 2000 Sep;43(9):1941–1950. [PubMed]
  • Vossenaar Erik R, Nijenhuis Suzanne, Helsen Monique M A, van der Heijden Annemarie, Senshu Tatsuo, van den Berg Wim B, van Venrooij Walther J, Joosten Leo A B. Citrullination of synovial proteins in murine models of rheumatoid arthritis. Arthritis Rheum. 2003 Sep;48(9):2489–2500. [PubMed]
  • Ménard HA, Lapointe E, Rochdi MD, Zhou ZJ. Insights into rheumatoid arthritis derived from the Sa immune system. Arthritis Res. 2000;2(6):429–432. [PMC free article] [PubMed]
  • Després N, Boire G, Lopez-Longo FJ, Ménard HA. The Sa system: a novel antigen-antibody system specific for rheumatoid arthritis. J Rheumatol. 1994 Jun;21(6):1027–1033. [PubMed]

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: ...

Articles from Annals of the Rheumatic Diseases are provided here courtesy of BMJ Group

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • BioAssay
    BioAssay
    PubChem BioAssay links
  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...