FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1781160907> ?p ?o ?g. }

• W1781160907 endingPage "408" @default.
• W1781160907 startingPage "399" @default.
• W1781160907 abstract "All plakin family proteins are known to be autoantigens in paraneoplastic pemphigus (PNP). In this study, we first examined whether PNP sera also react with epiplakin, another plakin protein, by various immunological methods using 48 Japanese PNP sera. Immunofluorescence confirmed that cultured keratinocytes expressed epiplakin. Epiplakin was detected by 72.9% of PNP sera by immunoprecipitation-immunoblotting with KU-8 cell extract, but not by immunoblotting of either normal human epidermal extract or KU-8 cell extract. Epiplakin was essentially not detected by 95 disease and normal control sera. Statistical analyses of various clinical and immunological findings revealed a significant correlation of the presence of anti-epiplakin antibodies with both bronchiolitis obliterans and mortality. No epiplakin-negative PNP case developed bronchiolitis obliterans. However, although 29.4% of European patients with PNP had bronchiolitis obliterans, significant correlation with anti-epiplakin autoantibodies was not observed. In further studies for lung, immunofluorescence showed the presence of epiplakin in normal human lung, particularly respiratory bronchiole, immunoprecipitation-immunoblotting showed that PNP sera reacted with epiplakin in cultured lung cells, and mice injected with polyclonal antibody specific to epiplakin histopathologically showed abnormal changes in small airway epithelia. These results indicated that epiplakin is one of the major PNP autoantigens and is related to PNP-related bronchiolitis obliterans. All plakin family proteins are known to be autoantigens in paraneoplastic pemphigus (PNP). In this study, we first examined whether PNP sera also react with epiplakin, another plakin protein, by various immunological methods using 48 Japanese PNP sera. Immunofluorescence confirmed that cultured keratinocytes expressed epiplakin. Epiplakin was detected by 72.9% of PNP sera by immunoprecipitation-immunoblotting with KU-8 cell extract, but not by immunoblotting of either normal human epidermal extract or KU-8 cell extract. Epiplakin was essentially not detected by 95 disease and normal control sera. Statistical analyses of various clinical and immunological findings revealed a significant correlation of the presence of anti-epiplakin antibodies with both bronchiolitis obliterans and mortality. No epiplakin-negative PNP case developed bronchiolitis obliterans. However, although 29.4% of European patients with PNP had bronchiolitis obliterans, significant correlation with anti-epiplakin autoantibodies was not observed. In further studies for lung, immunofluorescence showed the presence of epiplakin in normal human lung, particularly respiratory bronchiole, immunoprecipitation-immunoblotting showed that PNP sera reacted with epiplakin in cultured lung cells, and mice injected with polyclonal antibody specific to epiplakin histopathologically showed abnormal changes in small airway epithelia. These results indicated that epiplakin is one of the major PNP autoantigens and is related to PNP-related bronchiolitis obliterans. Paraneoplastic pemphigus (PNP), or paraneoplastic autoimmune multiorgan syndrome (Nguyen et al., 2001Nguyen V.T. Ndoye A. Bassler K.D. et al.Classification, clinical manifestations, and immunopathological mechanisms of the epithelial variant of paraneoplastic autoimmune multiorgan syndrome: a reappraisal of paraneoplastic pemphigus.Arch Dermatol. 2001; 137: 193-206PubMed Google Scholar), is an autoimmune blistering skin disease with severe mucocutaneous lesions. PNP is commonly associated with hematological malignancies (Anhalt, 1997Anhalt G.J. Paraneoplastic pemphigus.Adv Dermatol. 1997; 12: 77-96PubMed Google Scholar, Anhalt et al., 1990Anhalt G.J. Kim S.C. Stanley J.R. et al.Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.N Engl J Med. 1990; 323: 1729-1735Crossref PubMed Scopus (896) Google Scholar). Histopathology shows acantholysis and apoptotic cells in epidermis and interface changes (Anhalt, 1997Anhalt G.J. Paraneoplastic pemphigus.Adv Dermatol. 1997; 12: 77-96PubMed Google Scholar, Anhalt et al., 1990Anhalt G.J. Kim S.C. Stanley J.R. et al.Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.N Engl J Med. 1990; 323: 1729-1735Crossref PubMed Scopus (896) Google Scholar, Billet et al., 2006Billet S.E. Grando S.A. Pittelkow M.R. Paraneoplastic autoimmune multiorgan syndrome: review of the literature and support for a cytotoxic role in pathogenesis.Autoimmunity. 2006; 39: 617-630Crossref PubMed Scopus (69) Google Scholar, Sehgal and Srivastava, 2009Sehgal V.N. Srivastava G. Paraneoplastic pemphigus/paraneoplastic autoimmune multiorgan syndrome.Int J Dermatol. 2009; 48: 162-169Crossref PubMed Scopus (77) Google Scholar). Prognosis of PNP is poor with approximately 68% mortality (Leger et al., 2012Leger S. Picard D. Ingen-Housz-Oro S. et al.Prognostic factors of paraneoplastic pemphigus.Arch Dermatol. 2012; 148: 1165-1172Crossref PubMed Scopus (96) Google Scholar). Particularly, all PNP cases with chronic respiratory disease show fatal outcome (Nousari et al., 1999Nousari H.C. Deterding R. Wojtczack H. et al.The mechanism of respiratory failure in paraneoplastic pemphigus.N Engl J Med. 1999; 340: 1406-1410Crossref PubMed Scopus (204) Google Scholar, Takahashi et al., 2000Takahashi M. Shimatsu Y. Kazama T. Kimura K. Otsuka T. Hashimoto T. Paraneoplastic pemphigus associated with bronchiolitis obliterans.Chest. 2000; 117: 603-607Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Severe inflammation occurring in respiratory bronchioles leads to irreversible fibrotic reaction, resembling bronchiolitis obliterans (BO). In a previous study, BO occurred in 6% of 53 European patients with PNP (Leger et al., 2012Leger S. Picard D. Ingen-Housz-Oro S. et al.Prognostic factors of paraneoplastic pemphigus.Arch Dermatol. 2012; 148: 1165-1172Crossref PubMed Scopus (96) Google Scholar), whereas the prevalence was 25% in our recent study for 107 Japanese patients with PNP. However, pathogenesis in PNP-related BO is currently unknown. PNP develops autoantibodies to various antigens, mainly plakin family proteins. Immunoprecipitation (IP) first detected the 250-kDa desmoplakin I, the 230-kDa bullous pemphigoid 230 (BP230), the 210-kDa doublet of desmoplakin II and unknown protein, and the 190-kDa and 170-kDa unknown proteins (Anhalt et al., 1990Anhalt G.J. Kim S.C. Stanley J.R. et al.Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.N Engl J Med. 1990; 323: 1729-1735Crossref PubMed Scopus (896) Google Scholar). Then, immunoblotting (IB) of normal human epidermal extract showed that all PNP sera reacted with the 210-kDa and 190-kDa doublet proteins (Borradori et al., 1998Borradori L. Trueb R.M. Jaunin F. Limat A. Favre B. Saurat J.H. Autoantibodies from a patient with paraneoplastic pemphigus bind periplakin, a novel member of the plakin family.J Invest Dermatol. 1998; 111: 338-340Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Hashimoto, 2001Hashimoto T. Immunopathology of paraneoplastic pemphigus.Clin Dermatol. 2001; 19: 675-682Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, Hashimoto et al., 1995Hashimoto T. Amagai M. Watanabe K. et al.Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis.J Invest Dermatol. 1995; 104: 829-834Abstract Full Text PDF PubMed Scopus (167) Google Scholar). Thereafter, the 210-kDa and 190-kDa proteins were identified as envoplakin (EPL) and periplakin (PPL), respectively (Kiyokawa et al., 1998Kiyokawa C. Ruhrberg C. Nie Z. et al.Envoplakin and periplakin are components of the paraneoplastic pemphigus antigen complex.J Invest Dermatol. 1998; 111: 1236-1238Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This reactivity is very useful for diagnosis of PNP (Joly et al., 2000Joly P. Richard C. Gilbert D. et al.Sensitivity and specificity of clinical, histologic, and immunologic features in the diagnosis of paraneoplastic pemphigus.J Am Acad Dermatol. 2000; 43: 619-626Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, Mouquet et al., 2008Mouquet H. Drenovska K. Lartigue A. et al.Detection and characterization of anti-envoplakin linker autoantibodies in paraneoplastic pemphigus using specific bead-based assay.Clin Immunol. 2008; 129: 304-312Crossref PubMed Scopus (19) Google Scholar, Poot et al., 2013Poot A.M. Diercks G.F. Kramer D. et al.Laboratory diagnosis of paraneoplastic pemphigus.Br J Dermatol. 2013; 169: 1016-1024Crossref PubMed Scopus (68) Google Scholar). Plectin, another plakin protein, was also found to be PNP autoantigen (Proby et al., 1999Proby C. Fujii Y. Owaribe K. Nishikawa T. Amagai M. Human autoantibodies against HD1/plectin in paraneoplastic pemphigus.J Invest Dermatol. 1999; 112: 153-156Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Thus, all known plakin proteins are PNP autoantigens. Non-plakin proteins are also detected by PNP sera. ELISAs detected antibodies to desmoglein 3 (Dsg3) and/or Dsg1 in PNP sera (Amagai et al., 1998Amagai M. Nishikawa T. Nousari H.C. Anhalt G.J. Hashimoto T. Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice.J Clin Invest. 1998; 102: 775-782Crossref PubMed Scopus (271) Google Scholar, Brandt et al., 2012Brandt O. Rafei D. Podstawa E. et al.Differential IgG recognition of desmoglein 3 by paraneoplastic pemphigus and pemphigus vulgaris sera.J Invest Dermatol. 2012; 132: 1738-1741Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). We also found that the unknown 170-kDa protein was alpha-2-macroglobulin-like-1 (Numata et al., 2013Numata S. Teye K. Tsuruta D. et al.Anti-α-2-macroglobulin-like-1 autoantibodies are detected frequently and may be pathogenic in paraneoplastic pemphigus.J Invest Dermatol. 2013; 133: 1785-1793Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, Schepens et al., 2010Schepens I. Jaunin F. Begre N. et al.The protease inhibitor alpha-2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human.PLoS One. 2010; 5: e12250Crossref PubMed Scopus (78) Google Scholar), and BP180 was frequently reacted by PNP sera (Tsuchisaka et al., 2014Tsuchisaka A. Kawano H. Yasukochi A. et al.Immunological and statistical studies of anti-BP180 antibodies in paraneoplastic pemphigus.J Invest Dermatol. 2014; 134: 2283-2287Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Epiplakin (EPPK) was originally identified as an autoantigen in a patient (Fujiwara et al., 1996Fujiwara S. Kohno K. Iwamatsu A. Naito I. Shinkai H. Identification of a 450kDa human epidermal autoantigen as a new member of the plectin family.J Invest Dermatol. 1996; 106: 1125-1130Abstract Full Text PDF PubMed Scopus (44) Google Scholar), and is expressed in entire epidermis, skin appendices, and other intestinal epithelia (Fujiwara et al., 2001Fujiwara S. Takeo N. Otani Y. et al.Epiplakin, a novel member of the plakin family originally identified as a 450-kDa human epidermal autoantigen.J Biol Chem. 2001; 276: 13340-13347Crossref PubMed Scopus (50) Google Scholar). Subsequent studies showed that EPPK is a plakin protein with many repeats of plakin-specific B-domain and linker-domain (Spazierer et al., 2003Spazierer D. Fuchs P. Proll V. et al.Epiplakin gene analysis in mouse reveals a single exon encoding a 725-kDa protein with expression restricted to epithelial tissues.J Biol Chem. 2003; 278: 31657-31666Crossref PubMed Scopus (31) Google Scholar, Takeo et al., 2003Takeo N. Wang W. Matsuo N. Sumiyoshi H. Yoshioka H. Fujiwara S. Structure and heterogeneity of the human gene for epiplakin (EPPK1).J Invest Dermatol. 2003; 121: 1224-1226Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). EPPK connects intermediate filaments (Spazierer et al., 2006Spazierer D. Fuchs P. Reipert S. et al.Epiplakin is dispensable for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia.Mol Cell Biol. 2006; 26: 559-568Crossref PubMed Scopus (24) Google Scholar, Spazierer et al., 2008Spazierer D. Raberger J. Gross K. Fuchs P. Wiche G. Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption.J Cell Sci. 2008; 121: 825-833Crossref PubMed Scopus (19) Google Scholar, Wang et al., 2006Wang W. Sumiyoshi H. Yoshioka H. Fujiwara S. Interactions between epiplakin and intermediate filaments.J Dermatol. 2006; 33: 518-527Crossref PubMed Google Scholar). Despite abundant expression of EPPK in stratified and simple epithelia, gene-targeted mice showed no abnormality in either phenotype or keratin filament organization, except for mild delay of wound healing (Goto et al., 2006Goto M. Sumiyoshi H. Sakai T. et al.Elimination of epiplakin by gene targeting results in acceleration of keratinocyte migration in mice.Mol Cell Biol. 2006; 26: 548-558Crossref PubMed Scopus (32) Google Scholar, Ishikawa et al., 2010Ishikawa K. Sumiyoshi H. Matsuo N. et al.Epiplakin accelerates the lateral organization of keratin filaments during wound healing.J Dermatol Sci. 2010; 60: 95-104Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Because EPPK is a plakin protein, we speculated that EPPK might also be a PNP autoantigen. In this study, we investigated whether anti-EPPK antibodies were present in PNP sera by various analyses. Immunoprecipitation-immunoblotting (IP-IB) detected anti-EPPK autoantibodies in 35 of 48 PNP sera. Statistical analysis indicated correlations of anti-EPPK antibodies with PNP-related BO and mortality. Additional studies indicated that EPPK is expressed in respiratory cells and tissues and was reacted by PNP sera, suggesting that anti-EPPK autoantibodies may develop BO. Clinical and immunological parameters for all 48 Japanese patients with PNP are summarized in Supplementary Table S1 online. By IHC and IF of normal human skin, rabbit polyclonal antibody (pAb) for human EPPK (anti-EPPK pAb) (Figure 1a and c), but not normal rabbit IgG (Figure 1b and d), showed cytoplasmic staining in entire epidermis. By IF, anti-EPPK pAb (Figure 1e), but not normal rabbit IgG (Figure 1f), showed perinuclear cytoplasmic staining in cultured KU-8 cells. Finally, by IF using the rat bladder, anti-EPPK pAb (Figure 1g), but not normal rabbit IgG (Figure 1h), showed cell surface and cytoplasmic reactivity. We first attempted to detect anti-EPPK antibodies in 48 Japanese PNP sera by IB of normal human epidermal extract. Anti-EPPK pAb detected approximately 500-kDa EPPK (Supplementary Figure S1 online). However, whereas all PNP sera detected the 210-kDa EPL and 190-kDa PPL, neither PNP nor normal sera reacted with EPPK. In IB of normal human epidermal extract, anti-EPPK pAb also reacted with many lower molecular weight protein bands, which may be other molecules or degraded EPPK fragments. To confirm the specificity of anti-EPPK pAb, we performed absorption analysis using human EPPK RP (Supplementary Figure S2a online). Both intact EPPK and additional lower protein bands seen before absorption were significantly reduced by absorption with 0.3 μg EPPK RP, and completely disappeared with 0.6 and 1.2 μg EPPK RP. The reduction of EPPK reactivity was not seen by absorption with 5 and 10 μg of total protein in lysate of Escherichia coli transformed with the pGEX empty vector. Because we confirmed strong expression of EPPK in KU-8 cells by IF, we then tried to detect anti-EPPK antibodies in PNP sera by IB using KU-8 cell extract. Anti-EPPK pAb strongly reacted with the approximately 500-kDa EPPK, as well as additional lower bands (Supplementary Figure S3 online). However, none of the representative PNP sera reacted with EPPK, although all sera reacted with EPL and PPL. No normal sera reacted with any plakin proteins. To overcome the difficulty in detection of EPPK by IB analyses, we performed IP-IB of KU-8 cell extract (Figure 1i for representative sera, and Supplementary Figure S4 online for all sera). Nineteen PNP sera and anti-EPPK pAb reacted strongly and exclusively with the approximately 500-kDa EPPK, 16 PNP sera showed relatively weak but clear reactivity with EPPK, and 13 PNP sera showed no reactivity. Thus, 35 (72.9%) of 48 PNP sera reacted with EPPK. This EPPK band was not detected by 20 pemphigus vulgaris and 20 pemphigus foliaceus sera, but was very weakly detected by one of 20 BP and two of 35 normal sera (Supplementary Figure S4). We also performed this IP-IB for additional 22 European PNP sera. Eight sera reacted strongly and exclusively with EPPK, and four sera reacted relatively weakly with EPPK (Supplementary Figure S5 and Table S2 online). Thus, European PNP sera also clearly and frequently reacted with EPPK. To further confirm the specific reactivity of PNP sera with EPPK, we performed IP-IB using KU-8 cell extract, which was preabsorbed with anti EPPK pAb. In this IP-IB, two selected PNP sera reacted with EPPK in non-preabsorbed KU-8 cell extract, but not in KU-8 cell extract preabsorbed with anti-EPPK pAb (Supplementary Figure S6 online). To further confirm the lack of cross-reactivity with the anti-EPPK pAb with other plakin proteins, after IP of KU-8 extract with anti-EPPK pAb, immunoprecipitated proteins were immunoblotted with antibodies specific to various plakin proteins. As expected, EPPK was detected strongly in both KU-8 extracts and immunoprecipitates (Supplementary Figure S7a online). EPL and PPL were detected in KU-8 extracts but not in immunoprecipitates. Plectin and desmoplakin were not detected in both extracts of KU-8 cells and immunoprecipitated samples, suggesting that antibodies to these plakins were not suitable for IB. Nevertheless, the results for EPL and PPL strongly indicated that anti-EPPK pAb had no cross-reactivity with other plakins. To further demonstrate the specificity of anti-EPPK pAb and anti–mouse-EPPK pAb, we performed IF of skin and lung tissues of wild-type and Eppk-knockout mice using these pAbs. Both pAbs reacted with wild-type mouse tissues, but no positive reactivity was observed in Eppk-knockout mouse tissues (Supplementary Figure S7b and c). These results indicated that both pAbs were specific to EPPK in mouse tissues, and had no cross-reactivity with other plakins. We also performed IB of EPPK RP for 48 PNP sera. Both anti-GST monoclonal antibody and anti-EPPK pAb reacted strongly with the 80-kDa EPPK RP, as well as additional lower bands that were considered degradation products (Supplementary Figure S8 online). The EPPK RP was also recognized weakly by almost all PNP sera, including sera negative for EPPK in IP-IB. None of control pemphigus vulgaris, pemphigus foliaceus, BP, and normal sera reacted with the RP. We designated 35 EPPK-positive Japanese PNP cases and 13 EPPK-negative PNP cases in IP-IB as EPPK(+) PNP and EPPK(−) PNP, respectively. Relevant results in statistical analyses are graphically shown (Figure 1j–l). EPPK(+) PNP showed significantly higher frequency of BO than EPPK(–) PNP (P = 0.0303), and none of the 13 patients with EPPK(−) PNP had BO (Figure 1j). Mortality was significantly higher in EPPK(+) PNP (P = 0.0341) (Figure 1k). Dsg3 indices in EPPK(+) PNP (71.47 ± 66.93) were significantly lower than EPPK(−) PNP (116.57 ± 90.80) (P = 0.0333) (Figure 1l). The results of other parameters showed no statistical significance between EPPK(+) PNP and EPPK(−) PNP (Table 1).Table 1Results of clinical and immunological features in 48 patients with PNP and differences between EPPK(+) PNP and EPPK(−) PNP, which were not statistically significant (P > 0.05), except for BO (P = 0.0303)Parameters (n = cases with description)EPPK(+) PNPEPPK(−) PNPTotalAge (n = 46)59.09 ± 11.9753.77 ± 13.4857.59 ± 12.49Gender (n = 47) Females24/34 (70.6%)9/13 (69.2%)33/47 Males10/34 (29.4%)4/13 (30.8%)14/47Cutaneous lesions Trunk (n = 44)20/33 (60.6%)9/11 (81.8%)29/44 Extremities (n = 44)18/33 (54.5%)5/11 (45.5%)23/44Mucous lesions Oral (n = 44)33/33 (100.0%)11/11 (100.0%)44/44 Ocular (n = 39)16/28 (57.1%)8/11 (72.7%)24/39 Nasal (n = 39)8/28 (28.6%)2/11 (18.2%)10/39 Genital (n = 37)10/27 (37.0%)6/10 (60.0%)16/37BO (n = 48)10/35 (28.6%)0/13 (0%)10/48Acantholysis (n = 39)14/29 (48.3%)8/10 (80.0%)22/39Necrotic cells (n = 39)9/29 (31.0%)5/10 (50.0%)14/39Liquefaction degeneration (n = 39)5/29 (17.2%)1/10 (10.0%)6/39Reactivity for therapy (n = 43)13/31 (41.9%)9/12 (75.0%)22/43Rat bladder indirect IF (n = 48)27/35 (77.1%)9/13 (69.2%)36/48Human skin indirect IF (n = 48)22/35 (62.9%)11/13 (84.6%)33/48Dsg1 (n = 48)23.94 ± 40.2814.98 ± 20.3821.51 ± 36.00Abbreviations: BO, bronchiolitis obliterans; Dsg, desmoglein; EPPK, epiplakin; IF, immunofluorescence; PNP, paraneoplastic pemphigus. Open table in a new tab Abbreviations: BO, bronchiolitis obliterans; Dsg, desmoglein; EPPK, epiplakin; IF, immunofluorescence; PNP, paraneoplastic pemphigus. We also obtained information of BO in 17 of 22 European patients with PNP (Supplementary Table S2).However, there was no significant relationship between anti-EPPK antibodies and BO in European patients with PNP (Mann-Whitney U-test; P = 0.297). Because statistical analyses showed a clear relationship between EPPK(+) PNP and BO, we then examined whether EPPK is present in lung tissues and respiratory cells. By IHC and IF of normal human lung samples obtained from three unrelated subjects, anti-EPPK pAb reacted relatively weakly with alveolus cells (Figure 2a and c) and strongly with bronchiole epithelia (Figure 2a, b, d, and e). Particularly, granular staining was observed inside bronchiole epithelial cells (Figure 2e, arrows). Normal rabbit IgG did not react with these cells (Figure 2g, h, i, j, and k). By IF of cultured normal human small airway epithelial (NHSAE) cells, anti-EPPK pAb showed perinuclear cytoplasmic staining (Figure 2f), similar to that seen in KU-8 cells (Figure 1e). Normal rabbit IgG showed no staining (Figure 2l). By IB of NHSAE cell extract, anti-EPPK pAb reacted strongly with the approximately 500-kDa EPPK, as well as smear-like additional lower bands (Figure 3a). However, none of representative PNP sera with or without BO showed the reactivity with EPPK (Figure 3a). PNP sera reacted with PPL but not EPL. Normal sera reacted with no plakin protein in NHSAE cell extract. Interestingly, whereas KU-8 cell extract showed only one EPPK protein band (K-EPPK for keratinocyte-EPPK), NHSAE cell extract showed two different EPPK bands, K-EPPK and L-EPPK (for lung-EPPK), with weaker reactivity with L-EPPK (Figure 3b). We next performed IP-IB of NHSAE cell extract for representative BO(+) and BO(−) PNP sera (Figure 3c). Anti-EPPK pAb reacted with doublet bands of the approximately 500-kDa EPPK, with stronger reactivity with the lower band (K-EPPK) and weaker reactivity with the upper band (L-EPPK). Some of both BO(+) and BO(−) PNP sera reacted with the doublet EPPK bands in the same pattern. No normal sera showed this reactivity. To further investigate whether EPPK relates to BO, we performed injection experiments of anti–mouse-EPPK pAb into mice. In our preliminary study, the anti–mouse-EPPK pAb reacted with the approximately 700-kDa mouse EPPK by IB of mouse skin extract, and the reactivity reduced significantly by preabsorption with mouse EPPK RP (Supplementary Figure S2b), confirming the specific reactivity with EPPK. In injection experiments, to exclude the nonspecific effect of normal IgG injection, we first injected normal rabbit IgG at doses of 0 (control), 1, 2, 4, and 6 mg (n = 3) into BALB/c mice, and bred for 2 weeks. Histopathologically, no abnormal morphological changes were found in both skin and lung tissues taken from the mice (Supplementary Figure S9 online). In preliminary IF of skin and lung tissues from noninjected mice, anti–mouse-EPPK pAb clearly stained entire epidermis and bronchiole epithelia, respectively (Figure 4a). In mice injected with anti–mouse-EPPK pAb, direct IF of skin and lung tissues using FITC-conjugated anti-rabbit IgG showed positive staining of entire epidermis and bronchiole epithelia, respectively (Figure 4b), indicating that injected anti–mouse-EPPK pAb actually penetrated into skin and lung cells. In mice injected with anti–mouse-EPPK pAb, the histopathological study showed no obvious abnormality in skin (Figure 4c). In contrast, lung showed mononuclear cell infiltration and extracellular matrix deposition in peribronchial areas, as well as detached epithelial cells, suggesting loss of cell-cell adhesion (Figure 4c). No significant change was observed in lung tissue from mice injected with normal rabbit IgG. In this study, we first showed that none of 48 Japanese PNP sera reacted with EPPK by IB of both normal human epidermal extract and cultured KU-8 cell extract, probably due to loss of conformation of EPPK during IB procedure. Then, we performed IP-IB of KU-8 cell extract, which successfully detected anti-EPPK antibodies in 35 (72.9%) of 48 PNP sera. By IHC and IF, anti-EPPK pAb showed clearly positive reactivity in both normal human skin and rat bladder, suggesting high and wide expression of EPPK in various epithelia. Our statistical analysis revealed that BO was significantly more prevalent in EPPK(+) PNP than EPPK(−) PNP. Several mechanisms for PNP-related BO were proposed (Nousari et al., 1999Nousari H.C. Deterding R. Wojtczack H. et al.The mechanism of respiratory failure in paraneoplastic pemphigus.N Engl J Med. 1999; 340: 1406-1410Crossref PubMed Scopus (204) Google Scholar, Takahashi et al., 2000Takahashi M. Shimatsu Y. Kazama T. Kimura K. Otsuka T. Hashimoto T. Paraneoplastic pemphigus associated with bronchiolitis obliterans.Chest. 2000; 117: 603-607Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Airway mucosa in patients with PNP with BO showed acantholysis and IgG deposition in bronchial epithelium, suggesting that autoantibody-mediated acantholytic process occurred in respiratory tissues. However, because bronchial epithelia express only Dsg2, anti-Dsg1 and Dsg3 autoantibodies in PNP sera should not contribute to this change. In contrast, respiratory epithelium expresses various plakin proteins including EPPK. Therefore, we speculated that anti-EPPK antibodies in PNP sera may react with EPPK in lung tissues and cause BO. To confirm this speculation, we first showed that EPPK was present in normal lung tissue and NHSAE cells by IHC and IF using anti-EPPK pAb. EPPK expressed strongly in bronchiole epithelia and weakly in alveolar cells. IB also confirmed expression of EPPK in NHSAE cells. Similar to IB of epidermal extract or KU-8 cell extract, no PNP sera recognized EPPK in IB of NHSAE cell extract. In contrast, in IP-IB of NHSAE cell extract, most sera from patients with BO(+) PNP and patients with BO(−) PNP reacted with both L-EPPK (weakly) and K-EPPK (strongly). This positive reactivity of PNP sera with EPPK in lung cells further suggested that anti-EPPK autoantibodies cause BO. However, the result that both BO(+) PNP and BO(−) PNP reacted with EPPK in NHSAE cells made the pathogenic role of EPPK in the development of BO obscure. To further unravel the pathogenic role of EPPK in BO development, we injected anti–mouse-EPPK pAb into mice. IF indicated that the injected anti–mouse-EPPK pAb could penetrate into the skin and lung cells of mice. Histopathology revealed abnormal changes in lung tissue, but not in skin. These results further indicated that anti-EPPK antibodies can develop BO. Similar abnormal histopathological features were also shown in CXCL10 transgenic mice with BO-like inflammation (Jiang et al., 2012Jiang D. Liang J. Guo R. et al.Long-term exposure of chemokine CXCL10 cause bronchiolitis-like inflammation.Am J Respir Cell Mol Biol. 2012; 46: 592-598Crossref PubMed Scopus (11) Google Scholar). However, other mechanisms in the development of BO were also proposed. Proinflammatory cytokines and chemokines and growth factors were secreted from epithelial cells or T cells during BO development (Boehler and Estenne, 2003Boehler A. Estenne M. Post-transplant bronchiolitis obliterans.Eur Respir J. 2003; 22: 1007-1018Crossref PubMed Scopus (172) Google Scholar). In a recent study, Dsg3 expression in lung was observed in a PNP mouse model using Dsg3-target mice and naphthalene treatment (Hata et al., 2013Hata T. Nishimoto S. Nagao K. et al.Ectopic expression of epidermal antigens renders the lung a target organ in paraneoplastic pemphigus.J Immunol. 2013; 191: 83-90Crossref PubMed Scopus (34) Google Scholar). Pten knockout mice showed lung fibrosis with upregulation of snail and downregulation of E-cadherin and tight junction proteins (Miyoshi et al., 2013Miyoshi K. Yanagi S. Kawahara K. et al.Epithelial Pten controls acute lung injury and fibrosis by regulating alveolar epithelial cell integrity.Am J Respir Crit Care Med. 2013; 187: 262-275Crossref PubMed Scopus (63) Google Scholar). Therefore, these other factors and anti-EPPK antibodies may coordinately develop BO. The mechanism by which injected pAb accessed intracytoplasmic EPPK in skin and lung was currently unclear. However, the penetration of autoantibodies into living cells was reported for antinuclear antibodies in systemic lupus erythematosus (Rekvig et al., 2012Rekvig O.P. Putterman C. Casu C. et al.Autoantibodies in lupus: culprits or passive bystanders?.Autoimmun Rev. 2012; 11: 596-603Crossref PubMed Scopus (86) Google Scholar, Toubi and Shoenfeld, 2007Toubi E. Shoenfeld Y. Clinical and biological aspects of anti-P-ribosomal protein autoantibodies.Autoimmun Rev. 2007; 6: 119-125Crossref PubMed Scopus (91) Google Scholar) and" @default.
• W1781160907 created "2016-06-24" @default.
• W1781160907 creator A5001915636 @default.
• W1781160907 creator A5020447788 @default.
• W1781160907 creator A5031138415 @default.
• W1781160907 creator A5032167452 @default.
• W1781160907 creator A5033451411 @default.
• W1781160907 creator A5045431309 @default.
• W1781160907 creator A5045491062 @default.
• W1781160907 creator A5050422951 @default.
• W1781160907 creator A5050841942 @default.
• W1781160907 creator A5059334726 @default.
• W1781160907 creator A5064765702 @default.
• W1781160907 creator A5075201374 @default.
• W1781160907 creator A5076387135 @default.
• W1781160907 creator A5080015762 @default.
• W1781160907 creator A5085348329 @default.
• W1781160907 creator A5087453757 @default.
• W1781160907 creator A5091560416 @default.
• W1781160907 creator A5091888563 @default.
• W1781160907 date "2016-02-01" @default.
• W1781160907 modified "2023-10-11" @default.
• W1781160907 title "Epiplakin Is a Paraneoplastic Pemphigus Autoantigen and Related to Bronchiolitis Obliterans in Japanese Patients" @default.
• W1781160907 cites W1838672982 @default.
• W1781160907 cites W1968500406 @default.
• W1781160907 cites W1971260998 @default.
• W1781160907 cites W1973473575 @default.
• W1781160907 cites W1973497455 @default.
• W1781160907 cites W1987053226 @default.
• W1781160907 cites W1989256666 @default.
• W1781160907 cites W1993966098 @default.
• W1781160907 cites W1994489029 @default.
• W1781160907 cites W1995133400 @default.
• W1781160907 cites W2009741791 @default.
• W1781160907 cites W2009881053 @default.
• W1781160907 cites W2011427670 @default.
• W1781160907 cites W2016303365 @default.
• W1781160907 cites W2019045924 @default.
• W1781160907 cites W2031145183 @default.
• W1781160907 cites W2040020320 @default.
• W1781160907 cites W2044124136 @default.
• W1781160907 cites W2044837409 @default.
• W1781160907 cites W2045875442 @default.
• W1781160907 cites W2060034838 @default.
• W1781160907 cites W2064326890 @default.
• W1781160907 cites W2068972095 @default.
• W1781160907 cites W2072041604 @default.
• W1781160907 cites W2074052453 @default.
• W1781160907 cites W2076203837 @default.
• W1781160907 cites W2077069867 @default.
• W1781160907 cites W2081323987 @default.
• W1781160907 cites W2091243777 @default.
• W1781160907 cites W2091408822 @default.
• W1781160907 cites W2094800200 @default.
• W1781160907 cites W2102838769 @default.
• W1781160907 cites W2104726963 @default.
• W1781160907 cites W2105937700 @default.
• W1781160907 cites W2120192832 @default.
• W1781160907 cites W2120356806 @default.
• W1781160907 cites W2129944058 @default.
• W1781160907 cites W2137996367 @default.
• W1781160907 cites W2140677259 @default.
• W1781160907 cites W2171919496 @default.
• W1781160907 cites W2328517306 @default.
• W1781160907 doi "https://doi.org/10.1038/jid.2015.408" @default.
• W1781160907 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26802236" @default.
• W1781160907 hasPublicationYear "2016" @default.
• W1781160907 type Work @default.
• W1781160907 sameAs 1781160907 @default.
• W1781160907 citedByCount "48" @default.
• W1781160907 countsByYear W17811609072016 @default.
• W1781160907 countsByYear W17811609072017 @default.
• W1781160907 countsByYear W17811609072018 @default.
• W1781160907 countsByYear W17811609072019 @default.
• W1781160907 countsByYear W17811609072020 @default.
• W1781160907 countsByYear W17811609072021 @default.
• W1781160907 countsByYear W17811609072022 @default.
• W1781160907 countsByYear W17811609072023 @default.
• W1781160907 crossrefType "journal-article" @default.
• W1781160907 hasAuthorship W1781160907A5001915636 @default.
• W1781160907 hasAuthorship W1781160907A5020447788 @default.
• W1781160907 hasAuthorship W1781160907A5031138415 @default.
• W1781160907 hasAuthorship W1781160907A5032167452 @default.
• W1781160907 hasAuthorship W1781160907A5033451411 @default.
• W1781160907 hasAuthorship W1781160907A5045431309 @default.
• W1781160907 hasAuthorship W1781160907A5045491062 @default.
• W1781160907 hasAuthorship W1781160907A5050422951 @default.
• W1781160907 hasAuthorship W1781160907A5050841942 @default.
• W1781160907 hasAuthorship W1781160907A5059334726 @default.
• W1781160907 hasAuthorship W1781160907A5064765702 @default.
• W1781160907 hasAuthorship W1781160907A5075201374 @default.
• W1781160907 hasAuthorship W1781160907A5076387135 @default.
• W1781160907 hasAuthorship W1781160907A5080015762 @default.
• W1781160907 hasAuthorship W1781160907A5085348329 @default.
• W1781160907 hasAuthorship W1781160907A5087453757 @default.
• W1781160907 hasAuthorship W1781160907A5091560416 @default.
• W1781160907 hasAuthorship W1781160907A5091888563 @default.
• W1781160907 hasBestOaLocation W17811609071 @default.

• next