Idiopathic Pulmonary Fibrosis Autoantigen in Acute Exacerbation of Identification of Annexin 1 as a Novel

Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary ﬁbrosis (IPF), we conducted serological identiﬁcation of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identiﬁed by SEREX analysis, ﬁve Abs were identiﬁed as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR V (cid:1) genes were identiﬁed in sequential materials obtained at 1–3 mo in all 10 acute exacerbation IPF cases, suggesting that some inﬁltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR V (cid:1) genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, displayed high titers of anti-annexin 1 Abs, and the CDR3 regions of iden- tical TCR V (cid:1) genes (clones 1-1, 3-1, and 5-1) from the ﬁrst BAL and second sequential biopsies from those cases showed high homology with the same portion (residues 18–26) of annexin 1 included in the epitopes of the autoantigens predicted by TEPITOPE analysis. In contrast, the other patients (cases 6, 7, and 9) were negative for anti-annexin 1 Abs. CD4-positive T cells were separated from BAL cells using anti-CD4 mAb- coated Dynabeads (Dynal Biotech). For puriﬁcation of alveolar macrophages, BAL cells were allowed to adhere to plastic plates for 3 h. Cells were recovered by gentle scraping with a rubber policeman. The alveolar macrophages thus obtained were 95% pure by morphology and nonspeciﬁc esterase staining. A total of 5 (cid:2) 10 4 CD4-positive T cells were cultured in 96-well U-bottom plates (Iwaki Glass) with the same number of alveolar macrophages for 6 days at 37°C with medium only, N-terminal annexin 1-positive peptide (20 (cid:2) g/ml, position 18–26: QEYVQTVKS), or a neg- ative peptide (20 (cid:2) g/ml, position 201–209: DARALYEAG) of annexin 1 predicted to display no binding to the HLA molecule. After triplicate cul- tures were maintained for 6 days, the BrdU assay procedure was conducted using a Frontier BrdU cell proliferation assay cell proliferation kit (Funa- koshi) according to the protocol of the manufacturer. The reaction was quantiﬁed by measuring the OD at a wavelength of 450 nm. Sequencing Core kit, full-length annexin 1 was expressed in E. coli (M15 cells) using TAGZyme (Qiagen). The induction of recombinant protein synthesis, subsequent puriﬁcation on Ni-NTA spin colums (Qiagen), and the complete removal of N-terminal His tags were conducted as instructed by the manufacture. Puriﬁed recombinant annexin 1 was incubated with human leukocyte elastase (Sigma-Aldrich) at a ﬁnal concntration of 0.3 U/ml in reaction buffer (30). After electrophoresis of full-length annexin 1 and elastase-cleaved annexin 1 (each 25 (cid:2) g protein), the proteins were electrotransferred to a nylon membrane and probed with sera of IPF pa- tients (15 acute exacerbation patients, 30 stable patients) and non-IPF patients (90 patients with interstitial pneumonia related to collagen vascular disease, 30 patients with pulmonary sarcoidosis, 10 patients with chronic eosinophilic pneumonia, 10 patients with HP, and 40 normal volunteers).