Background/Purpose: The association of rheumatoid arthritis (RA) with smoking and silica exposure has led to the hypothesis that the lung is the site where RA autoimmunity is initiated.  In support of this, anti-citrullinated protein/peptide antibody (ACPA) positive RA is associated with a high prevalence of lung abnormalities at presentation, and airway changes have recently been reported in subjects with ACPA in the absence of joint disease. There are also reports of ACPA occurring in lung diseases in the absence of joint symptoms. Investigation of early lung associated citrullinated antigens may therefore give insight into RA pathogenesis.

Methods: Sera from 42 patients with idiopathic pulmonary fibrosis (IPF), but no arthritis, were tested for anti-CCP2 antibodies by commercial ELISA. Sera were further tested for antibodies to immunodominant peptides from 3 citrulllinated autoantigens in RA, α-enolase (KIHA-cit-EIFDS-cit-GNPTVE), vimentin (VYAT-cit-SSAV-cit-L-cit-SSVP) and fibrinogen (NEEGFFSA-cit-GHRPLDKK), as well as filaggrin (CCP1; SHQEST-cit-G-cit-SRGRSGRSGS) by in-house ELISA, with cysteines at the end of each peptide to facilitate cyclisation. Arginine-containing control peptides for all assays were run in parallel. A549 alveolar epithelial cells and HL-60 cells were cultured and lysates citrullinated in vitro with rabbit PAD2. Citrullinated and non-citrullinated lysates were separated by SDS-PAGE and probed with the anti-CCP2 positive sera.

Results: Six patients were anti-CCP2 positive (14%) with a mean antibody level of 44 AU/ml (range 29-61; normal <25).  None of these 6 sera were positive for antibodies to the α-enolase, vimentin and fibrinogen peptides and only one was positive for the CCP1 peptide. No reactivity of the anti-CCP2 positive sera against the in vitro citrullinated A549 cell lysates was observed, however with the HL-60 lysates,  reactive bands were identified in 3/6 sera (molecular weight approx. 52, 60 and 62 kDa) which were not citrulline-dependent. Sera were re-tested with the arginine-containing control peptide for CCP2, using a previously published method. This demonstrated that reactivity was not citrulline-dependent. Serum from a patient with RA-associated lung disease was used as a positive-control and had antibodies to CEP-1, cVim, cFib and CCP1, reacted predominantly with the citrullinated but not uncitrullinated A549 (bands at approx. 45, 50, 102 and 300 kDa) and HL-60 lysates, and showed citrulline-dependent specificity for the CCP2 peptides.

Conclusion: Our findings suggest that anti-CCP2 antibodies occurring in lung disease are not specific for citrullinated peptides. They may represent non-specific reactions, with correspondingly low antibody levels, as has previously been reported in autoimmune hepatitis. However, it remains a possibility that immunity to native antigens might breach tolerance to citrullinated proteins, as we have previously observed in DR4 transgenic mice immunised with α-enolase. Here we have identified candidate immunoreactive bands in the HL-60 lysates. In some individuals, this autoreactivity may be followed by epitope spreading to citrullinated protein, a corresponding rise in anti-CCP2 antibody levels, and the clinical onset of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-cyclic-citrullinated-peptide-antibodies-in-idiopathic-pulmonary-fibrosis-are-not-citrulline-specific-implications-for-the-pathogenesis-of-rheumatoid-arthritis/