Ectopic Expression of Epidermal Renders the Lung a Target Organ in Paraneoplastic

This information is current as Tsuyoshi Hata, Shuhei Nishimoto, Keisuke Nagao, Hayato of September 29, 2021. Takahashi, Kazue Yoshida, Manabu Ohyama, Taketo Yamada, Koichiro Asano and Masayuki Amagai J Immunol 2013; 191:83-90; Prepublished online 31 May 2013;

doi: 10.4049/jimmunol.1203536 Downloaded from http://www.jimmunol.org/content/191/1/83

Supplementary http://www.jimmunol.org/content/suppl/2013/05/31/jimmunol.120353 Material 6.DC1 http://www.jimmunol.org/ References This article cites 42 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/191/1/83.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ectopic Expression of Epidermal Antigens Renders the Lung a Target Organ in Paraneoplastic Pemphigus

Tsuyoshi Hata,*,† Shuhei Nishimoto,* Keisuke Nagao,* Hayato Takahashi,* Kazue Yoshida,* Manabu Ohyama,* Taketo Yamada,‡ Koichiro Asano,x,{ and Masayuki Amagai*

Paraneoplastic pemphigus (PNP) is an of the skin and mucous membranes that can involve fatal lung com- plications. IgG target the cell adhesion molecules (Dsg)3 and plakins, but the nature and targets of infiltrating T cells are poorly characterized. Moreover, the lung involvement in this skin Ag-specific autoimmune condition rep- resents a paradox. To mimic autoimmunity in PNP, we grafted wild-type skin onto Dsg32/2 mice, which resulted in graft rejection and generation of anti-Dsg3 IgG and Dsg3-specific T cells. Transfer of splenocytes from these mice into Rag22/2 mice induced a combination of suprabasilar and interface , a histology unique to PNP. Furthermore, the recipient mice showed prominent bronchial inflammation of CD4+ and CD8+ T cells with high mortality. Intriguingly, ectopic Dsg3 expression Downloaded from was observed in the lungs of PNP mice, mirroring the observation that squamous metaplasia is often found in the lungs of PNP patients. Dsg3 and other epidermal Ags were ectopically expressed in the lungs after pulmonary injuries by naphthalene, which was sufficient for recruitment of Dsg3-specific CD4+ T cells. These findings demonstrate that squamous metaplasia after pulmo- nary epithelial injury may play a crucial role in redirecting the skin-specific autoimmune reaction to the lungs in PNP. The Journal of Immunology, 2013, 191: 83–90. http://www.jimmunol.org/ mong tissue-specific autoimmune diseases, pemphigus occurs in association with underlying , mainly of comprises a group of IgG -mediated blis- lymphoid origin, and has characteristic IgG autoantibodies A tering diseases of the skin and mucous membranes, against the plakin family (plectin, I and II, BP230, characterized histologically by intraepidermal blisters due to the loss envoplakin, and periplakin) (5, 6). Additionally, PNP patients of cell–cell adhesion, and immunopathologically by exhibit a cellular immune response in the skin and mucous mem- the in vivo finding of bound and circulating IgG autoantibodies branes. Histological examination shows not only blister formation directed against the surface of . Pemphigus has three caused by IgG autoantibodies against Dsg3 and/or Dsg1, but also major forms: (PF), (PV), interface dermatitis showing keratinocyte apoptosis and in- and paraneoplastic pemphigus (PNP) (1). The classic forms of filtration in the and mucosal epithelia. Furthermore, some by guest on September 29, 2021 pemphigus are PF and PV, which are mediated solely by humoral patients with PNP develop pulmonary involvement, such as bron- autoimmunity involving IgG autoantibodies against desmoglein chiolitis obliterans, which can cause fatal respiratory failure (7–11). (Dsg)1 and Dsg3, respectively, both of which are cadherin-type cell– Further investigation is required to determine the target Ag(s) of cell adhesion molecules in desmosomes (2, 3). T cells that infiltrate the skin and mucous membranes as well as the PNP shares the basic feature of pemphigus in that IgG auto- reason that such T cells also attack the lungs. The technical hurdles against Dsg3 or Dsg1 play a pathogenic role by me- to identification of these T cell Ags and the rarity of the disease diating blister formation in the mucous membranes and skin (4), hamper progress in solving these questions. but its phenotype is more complex than those of PV and PF. PNP An active disease mouse model for PV has been generated by adoptive transfer of T and B lymphocytes from Dsg32/2 mice immunized with recombinant mouse Dsg3 plus Freund’s adjuvant *Department of Dermatology, Keio University School of Medicine, Tokyo 160-8582, to Dsg3-expressing Rag22/2 mice (12, 13). PV model mice per- Japan; †Cutaneous Research Section, Fundamental Research Laboratories, KOSE´ Corporation, Tokyo 174-0051, Japan; ‡Department of Pathology, Keio University sistently produce anti-Dsg3 IgG and develop blisters on the skin School of Medicine, Tokyo 160-8582, Japan; xDepartment of Respiratory Medicine, and mucous membranes, as found in PV patients. Subsequently, { Keio University School of Medicine, Tokyo 160-8582, Japan; and Division of Dsg3-specific CD4+ T cell clones were isolated, and Dsg3-specific Pulmonary Medicine, Department of Medicine, Tokai University School of Medi- cine, Kanagawa 259-1193, Japan TCR transgenic (Dsg3H1) mice were generated (14, 15). Inter- + Received for publication December 27, 2012. Accepted for publication April 30, estingly, Dsg3-specific transgenic CD4 T cells, which developed 2/2 2013. in the absence of Dsg3 or in Dsg3 mice, induced not only the This work was supported by Grants-in-Aid for Scientific Research from the Ministry blisterformationcausedbyanti-Dsg3 IgG, but also interface of Education, Culture, Sports, Science and Technology of Japan, Health and Labor dermatitis with CD4+ T cell infiltration in the epidermis and Sciences Research Grants for Research on Measures for Intractable Diseases from the 2/2 Ministry of Health, Labor, and Welfare of Japan, and Keio Gijuku Academic Devel- keratinocyte upon adoptive transfer with Dsg3 B cells opment Funds. (15). This demonstrates that the cellular autoimmune response Address correspondence and reprint requests to Prof. Masayuki Amagai, Department against Dsg3 can lead to the formation of interface dermatitis, of Dermatology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, suggesting that Dsg3 may be a target autoantigen of the cellular Tokyo 160-8582, Japan. E-mail address: [email protected] immune reaction seen in PNP. The online version of this article contains supplemental material. In this study, by adoptive transfer of lymphocytes from Dsg32/2 Abbreviations used in this article: B6, C57BL/6J; Dsg, desmoglein; PF, pemphigus mice immunized with wild-type (WT) skin grafts, we generated foliaceus; PNP, paraneoplastic pemphigus; PV, pemphigus vulgaris. a PNP model mouse that showed both humoral and cellular im- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 mune reactions against Dsg3. PNP model mice showed supra- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203536 84 CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS basilar acantholysis caused by anti-Dsg3 IgG Abs as well as in- Immunization of Dsg32/2 mice via Dsg3+/+ skin grafts + + terface dermatitis with CD4 as well as CD8 T cell infiltration Dorsal skin from Dsg3+/+ WT mice (B6) was grafted onto the backs of sex- and keratinocyte apoptosis. Furthermore, these mice demonstrated matched Dsg32/2 or Dsg3+/2 mice, as described previously (17), and the bronchial inflammation with a high mortality rate. Surprisingly, grafted skin was biopsied at various times after transplantation. A second Dsg3 was ectopically expressed in the lungs of the PNP model skin graft was performed 28 d after the first graft for adoptive transfer mice, but not in the lungs of WT or PV model mice. Ectopic studies. Graft survival was determined in accordance with a method de- scribed previously (18). For adoptive transfer studies, splenocytes from expression of Dsg3 and other epidermal Ags in the lungs was mice that received a second skin graft for 14 d or more were transferred detected during epithelial remodeling after naphthalene-induced adoptively into Rag22/2 mice via the tail vein. In control PV model mice, 2 2 pulmonary injury. CD4+ T cells containing retrovirally transduced Dsg3 / mice were immunized with recombinant mouse Dsg3 in CFA, as Dsg3-specific TCR (15) preferentially infiltrated into bronchial described previously (12). epithelia in naphthalene-injected mice, but not in those that received Induction of acute lung injury by naphthalene the control corn oil injection. Squamous metaplasia in the lungs, To examine whether a skin-specific protein could be expressed ectopically which showed ectopic expression of various epidermal Ags, was in bronchial epithelium under certain conditions, we injected naphthalene indeed frequently identified in patients with PNP. These findings (Sigma-Aldrich, St. Louis, MO), which selectively injures Clara cells, and using PNP model mice provide valuable clues to solving a long- observed bronchial epithelium during the repair process. Naphthalene was standing mystery in PNP: why the lungs, which were thought to ex- dissolved in corn oil at 10 or 20 mg/ml and administered to mice (100 or 200 hibit no epidermal Ag expression, are involved in this skin-specific mg/kg) by i.p. injection weekly for 4 wk (19). Control mice received an identical volume of corn oil (Sigma-Aldrich). autoimmune condition.

Retroviral transduction Downloaded from Materials and Methods Retroviral transduction of a Dsg3-specific TCR (Dsg3H1) to CD4+ T cells Mice from B6 mice was performed using a set of TCRs (Va8 and Vb6) of + 301–315 Dsg1tg/tgDsg32/2 mice with the C57BL/6J (B6) background, in which a CD4 T cell clone that recognizes the peptide Dsg3 , as described Dsg3-deficient phenotypes are rescued by ectopic Dsg1 expression (16), previously (14, 15, 20). were used as Dsg32/2 mice. In this study, “Dsg32/2 mice” refers to tg/tg 2/2 2/2 Histology and immunostaining in mice and humans

“Dsg1 Dsg3 mice” unless noted otherwise. These mice and Rag2 http://www.jimmunol.org/ mice in a B6 background (Central Institute for Experimental Animals, To quantify the induction of cellular immune responses, infiltrating CD4+ Tokyo, Japan) were maintained under specific pathogen-free conditions at and CD8+ cells were counted in frozen sections. The numbers of lymphocytes Keio University. All mouse studies were approved by the Animal Ethics and TUNEL+ cells in the oral epithelia (hard palate) and the peribronchial Review Board of Keio University. area (cells in the epithelia and within 100-mm depth from the basement by guest on September 29, 2021

FIGURE 1. WT skin grafted onto Dsg32/2 mice induced both humoral and cellular immune responses with resultant graft rejection. (A) Ex- perimental protocol for skin graft immunization. WT skin (Dsg3+/+) was grafted onto Dsg32/2 or Dsg3+/2 mice and was biopsied (yellow circles) weekly. (B) Histological changes on Dsg32/2 mice (top) and Dsg3+/2 littermates (bottom) on days 14, 21, and 28 after WT skin graft. Scale bar, 20 mm. (C) Skin graft survival rates after first (filled sym- bols) and second (open symbols) WT skin grafts on Dsg32/2 mice (diamonds) and Dsg3+/2 littermates (squares). On day 28 after the first skin graft, the second skin graft was applied. (D) Anti-Dsg3 IgG titers as measured by ELISA after first (day 0) and second (day 28) WT skin grafts on Dsg32/2 mice ()) and Dsg3+/2 littermates (N). (E) CD4+ (red) and CD8+ (red) T cell infiltrations, apoptotic cells as determined by TUNEL assay, and in vivo IgG deposition on keratinocyte surfaces in WT skin grafts on Dsg32/2 mice (top) and Dsg3+/2 litter- mates (bottom) on day 21. Scale bars, 20 mm. The Journal of Immunology 85 membrane) were calculated per 100-mm width of basement membrane in PNP model mice. The numbers of CD45.1+CD4+ T cells and CD45.12 CD4+ T cells infiltrating into bronchial epithelia (cells in the epithelia) were counted per 200-mm width of basement membrane as Dsg3-specific cells and Dsg3-nonspecific T cells, respectively. Anti-Dsg3 IgG production was monitored using a mouse Dsg3 ELISA (21), and and immunoblotting were performed to detect the production of anti- plakin family Abs (22). Statistical analyses were performed using an un- paired t test with Welch’s correction. Histological analyses and immunostaining of lungs were performed in a representative PNP patient, a 61-y-old female with diffuse large and positive for anti-Dsg3, anti-envoplakin, and anti- periplakin IgG autoantibodies. Deparaffinized sections were subjected to immunostaining after peroxidase deactivation by methanol containing 0.3% H2O2. Experiments with human samples were approved by Keio University Research Ethics Committee according to the Declaration of Helsinki.

Abs The following Abs were used: anti–E-tag mAb (GE Healthcare Bio- Sciences) conjugated with Alexa Fluor 488; anti-fluorescein/Oregon

Green goat IgG fraction conjugated with Alexa Fluor 488; goat anti-rat Downloaded from IgG conjugated with Alexa Fluor 568 (Invitrogen); AK18 mouse anti-Dsg3 mAb (23); anti-mouse CD4 (RM4-5, GK1.5), CD8 (53-6.7), and CD45.1 conjugated with FITC (A20; BioLegend, San Diego, CA); anti-human CD4 (1F6) and CD8 (4B11; Novocastra Laboratories, Newcastle, U.K.); anti-Dsg3 (3G133; Abcam, Cambridge, UK); HRP-conjugated anti-mouse IgG Ab (Medical and Biological Laboratories, Nagoya, Japan); and anti- envoplakin Ab (Santa Cruz Biotechnology). TUNEL+ cells were detected using the In Situ Cell Death Detection kit and TMR red (Roche, Mann- http://www.jimmunol.org/ heim, Germany).

Results Grafting Dsg3-expressing skin onto Dsg32/2 mice elicits Dsg3-specific cellular and humoral immune responses A To generate both humoral and cellular immune responses against FIGURE 2. T cells infiltrating WT skin grafts are Dsg3-specific. ( ) Experimental protocol for determination of Dsg3-specific immune reac- Dsg3, we sought to use the skin graft technique as an immunization 2/2

2 2 tions caused by skin graft immunization. Dsg3 mice (n = 3) were first by guest on September 29, 2021 procedure instead of immunizing Dsg3 / mice with recombinant primed with WT (Dsg3+/+) skin grafts and challenged with second skin Dsg3 protein, because skin allografting is known to induce immune grafts side-by-side with WT skin and Dsg32/2 skin. were taken + + responses mediated by CD4 and CD8 T cells, as well as Abs from the grafted skin for histology and CD4/CD8 staining. (B) Histology, +/+ 2/2 (24–26). When Dsg3 WT skin was grafted onto Dsg3 mice CD4+ and CD8+ T cells (red) of WT skin grafts (left panel) and Dsg32/2 2 2 2 or Dsg3+/ littermates, the skin grafts on Dsg3 / mice (n = 8), but skin grafts (right panel) on Dsg32/2 mice after priming with WT skin 2 not on Dsg3+/ littermates (n = 4), were rejected on days 14–28 grafts. Scale bar, 20 mm. (Fig. 1A, 1C). Second, Dsg3+/+ skin grafts onto the same mice resulted in even more rapid rejection (days 7–10, n = 7). Histo- +/+ 2/2 logically, the first skin grafts on Dsg32/2 mice showed acanthosis Splenocytes from Dsg3 skin-grafted Dsg3 mice induce with mild lymphocytic infiltration on day 14, interface dermatitis characteristic features of PNP in recipient mice with significant lymphocytic infiltration and occasional keratino- Patients with PVand PNP present with mucocutaneous blisters and cyte apoptosis on day 21, and complete necrosis of the grafted skin erosions that histologically show suprabasilar acantholysis or the on day 28, in contrast to the grafts on Dsg3+/2 littermates, which loss of keratinocyte cell–cell adhesion just above the basal cell showed no apparent change (Fig. 1B). The infiltrating lymphocytes layer, which results from the binding of anti-Dsg3 IgG autoanti- included both CD4+ and CD8+ T cells, and the presence of apo- bodies to keratinocyte surfaces (Fig. 3A, 3B, Table I) (2, 4, 27). ptotic basal keratinocytes was confirmed via in situ TUNEL assay However, the oral lesions of PNP clinically appear more inflamed (Fig. 1E). In addition to the cellular immune response, Dsg32/2 with necrotic and lichenoid changes than do those of PV, and mice with WT skin grafts demonstrated circulating anti-Dsg3 IgG histological examination shows suprabasilar acantholysis with CD4+ on day 14 with continuous increases in their titers thereafter (Fig. and CD8+ T cell infiltration and keratinocyte apoptosis (6, 28). 1D), as well as in vivo IgG deposition on keratinocyte cell surfaces When lymphocytes from naive Dsg32/2 mice or recombinant of grafted skin (Fig. 1E, day 21). Dsg3-immunized Dsg32/2 mice were adoptively transferred to To determine whether the T cell infiltration in the grafted skin Rag22/2 mice, the recipient mice began to produce anti-Dsg3 was Dsg3-specific, WT skin and Dsg32/2 skin were grafted si- IgG, which mediates suprabasilar acantholysis. However, no ob- multaneously onto Dsg32/2 mice that had been primed with WT vious T cell infiltration was identified in the skin or mucous skin 28 d prior (Fig. 2A). The WT-grafted skin showed interface membranes (Fig. 3C, Supplemental Fig. 1) (12, 29). In contrast, dermatitis with marked CD4+ and CD8+ T cell infiltration and when splenocytes from Dsg32/2 mice immunized with Dsg3+/+ liquefaction degeneration, whereas the Dsg32/2 grafted skin skin grafts were transferred adoptively, the recipient mice devel- showed no apparent histological changes and no T cell infiltration oped oral lesions with combined suprabasilar acantholysis caused (Fig. 2B, n = 3). These findings indicate that grafting of Dsg3- by anti-Dsg3 IgG and interface dermatitis with CD4+ and CD8+ expressing WT skin onto Dsg32/2 mice induces both cellular and T cell infiltration and keratinocyte apoptosis (Fig. 3D). The numbers humoral Dsg3-specific immune responses. of infiltrating CD4+ and CD8+ T cells and TUNEL+ apoptotic cells 86 CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS Downloaded from http://www.jimmunol.org/

FIGURE 3. Generation and characterization of a mouse model with immunological features of PNP. Clinical and histological images stained for CD4+ and CD8+ T cells in a representative PV patient (A) and PNP patient (B). (C) PV model mice were generated by adoptive transfer of splenocytes from Dsg32/2 mice immunized with recombinant Dsg3 into Rag22/2 mice. The recipient mice stably produced anti-Dsg3 IgG and developed the characteristic PV phenotype, including suprabasilar acantholysis in the hard palate and in vivo IgG deposition (green) on keratinocyte surfaces with no obvious infiltration of CD4+ and CD8+ T cells (red). Scale bars, 40 mm. (D) PNP model mice were generated by adoptive transfer of splenocytes from Dsg32/2 mice im- by guest on September 29, 2021 munized twice with WT skin grafts into Rag22/2 mice. The recipient mice showed marked CD4+ and CD8+ T cell infiltration (red) with keratinocyte apoptosis (TUNEL+ in red) in addition to anti-Dsg3 IgG production (green) and suprabasilar acantholysis. (E) Infiltrating CD4+ T cells, CD8+ T cells, and apoptotic cells were counted per 100 mm basement membrane (BM) in the oral mucosa. Error bars indicate SD. (F) Anti-envoplakin (top panel, arrow; molecular mass, 210 kDa) or anti-Dsg3 (bottom panel, arrowhead; molecular mass, 130 kDa) IgG production was determined by immunoprecip- itation and immunoblotting. in the oral epithelia were significantly higher in these mice than in receiving splenocytes from Dsg3+/+ skin graft–immunized Dsg32/2 PV model mice (Fig. 3E). Furthermore, by day 30, 4 of 12 mice mice developed characteristic histological and immunological fea- produced anti-envoplakin IgG, a characteristic autoantibody in PNP, tures seen in PNP. Consequently, these mice constitute a model for whereas none of the 40 PV model mice did so (Fig. 3F). Thus, mice analyzing the immunological aspects of PNP without neoplastic complications, and are referred to as PNP mice in this study.

Table I. Summary of the phenotypes of PV and PNP in humans and High mortality rate and lung involvement in PNP mice mice PNP mice had significantly higher mortality than did PV mice (Fig. 4A, survival rate 46 [n = 41] versus 81% [n = 16] at day 49, p = Human Mouse 0.02 [Wilcoxon test]). To investigate the cause of this difference, PV PNP PV Model PNP Model we performed whole-body histological analyses and found intense Clinical phenotype mononuclear cell infiltrates in the peribronchial and perivascular Blisters and erosions + +a ++ spaces in the lungs of PNP mice, but not in those of PV mice (Fig. Histology 4B). Most of the infiltrating cells were CD4+or CD8+ T cells, Acantholysis + + + + which were significantly more plentiful in PNP mice than in PV 2 2 Interface dermatitis + + mice (Fig. 4C, 4D). The degree of inflammation was profound in Humoral autoimmunity Anti-Dsg3 IgG + + + + some animals, and robust infiltration of neutrophils and mono- Anti-plakin IgG 2 + 2 + nuclear cells was observed in the alveolar spaces; other organs, in- Cellular autoimmunity cluding the liver, kidney, heart, and small intestine, showed no such 2 2 T cell–mediated cytotoxicity + + findings (Supplemental Fig. 2). Dsg3-specific T cell attack 2 N.D. 2 + Lung involvement 2 +b 2 + To determine why skin Ag-specific T cells were found in lungs, we examined the lungs of PNP and PV mice for Dsg3 expression. aOral PNP lesions showed more necrosis and lichenoid changes than did PV lesions. Surprisingly, the PNP mice expressed various levels of Dsg3, as bHuman PNP shows bronchiolitis obliterans. shown by RT-PCR, whereas no Dsg3 was detected in PV mice (Fig. The Journal of Immunology 87

FIGURE 4. PNP model mice showed higher mortality and CD4+ and CD8+ T cell infiltration in the lungs. (A) Survival rates of PNP model mice ()) and PV model mice (N) throughout the course after adoptive transfer. (B) Histology of the lungs of PV model mice (left panel) and PNP model mice (right panel) under various magnifications. Scale bars, 100 mm. (C) Stain- ing of CD4+ and CD8+ T cells in the intra- epithelial and peribronchial areas in the lungs of PV model mice (left) and PNP model mice (right). Scale bar, 20 mm. (D) The numbers (6 SD) of intraepithelial and peribronchial CD4+ and CD8+ T cells in PV model mice (filled bars) Downloaded from and PNP model mice (open bars) per 100 mm basement membrane (BM). (E) RT-PCR of lung tissues for Dsg3 expression (arrow) in PV model mice and PNP model mice on days 12–14 after adoptive transfer (negative control [NC] using WT lung). http://www.jimmunol.org/

4E). Dsg3 in the lungs of PNP mice could not be detected at Naphthalene is concentrated in Clara cells that are enriched in p450 by guest on September 29, 2021 protein level using either various mAbs (23) or sera from patients enzymes, and its metabolism generates toxic metabolites that result with PV, probably due to its low expression or poor stability on in selective injury to Clara cells (37). On day 1, the bronchiolar cell membranes that lacked proper formation of desmosomes at surface appeared to be denuded, and exfoliated cells were evident the time points examined (up to day 49). in the bronchiolar lumen. On day 3, squamous cells were lining the These findings suggest that ectopic pulmonary Dsg3 expression injured bronchioles, which exhibited a relatively homogeneous cu- might redirect Dsg3-specific CD4+ and CD8+ T cells to the lungs boidal cell appearance (Fig. 6A), as described previously (19, 37). and there induce inflammation. Ectopic expression of Dsg3 was transiently found on days 1–3, which declined after day 4, as determined by RT-PCR (Fig. 6B). To Squamous metaplasia is frequently found in the lungs of examine the effects of naphthalene during the chronic phase, a lower patients with PNP dose (100 mg/kg) was administered once per week for 4 wk and the It is well established that Dsg3 is not expressed in the normal lungs of expression of epidermal Ags was examined. Interestingly, not only mice and humans (8, 30). However, the lungs of patients with PNP Dsg3, but also other epidermal Ags such as keratin 5 and keratin 14 frequently show squamous metaplasia at autopsy or in bronchial were detected by RT-PCR throughout the course (Fig. 6C); however, specimens, which is often accompanied by CD4+ and CD8+ non-epidermal peripheral Ags, such as a-fibrinogen (liver-specific T cell infiltration (Fig. 5A) (8, 10, 31). Squamous metaplasia Ag), were not detected. Thus, these results demonstrate that Dsg3 develops in response to acute or chronic inflammation caused by and other epidermal Ags may be ectopically expressed during epi- smoking (32) or viral infection (33–35), and it shows striking thelial remodeling or squamous metaplasia as part of the wound similarities to the skin and mucosa not only in terms of morphology, healing process after tissue injury. but also gene expression (36). Immunohistochemical analysis of Next, we determined whether Dsg3-reactive CD4+ T cells are human lungs with squamous metaplasia confirmed ectopic Dsg3 preferentially recruited to lung in which Dsg3 was ectopically expression in 18 of 19 samples (representative data in Fig. 5B). expressed. CD4+ T cells from CD45.1+ congenic B6 mice and B6 mice (CD45.2+) were retrovirally transduced with Dsg3-specific Ectopic Dsg3 expression during the tissue repair process in the + TCR (Dsg3H1) and mock T cells, respectively. Equal numbers lungs is sufficient to recruit Dsg3-specific CD4 T cells (1 3 106) of Dsg3H1 (CD45.1+) and mock T cells (CD45.2+) were The above findings indicate that the stratified squamous epithelia– adoptively transferred into Rag22/2 mice (n = 5), followed by specific adhesion molecule Dsg3 may be ectopically expressed weekly naphthalene administration (100 mg/kg) (Fig. 6D). Dsg3H1 under certain conditions. To further explore a setting in which ec- T cells infiltrated to the skin and induced interface dermatitis, with topic expression of Dsg3 is induced in bronchial epithelium, a single the gross phenotype becoming apparent ∼4wkafteradoptive dose of naphthalene (200 mg/kg), which selectively injures non- transfer (15). We therefore examined the lungs of naphthalene- ciliated Clara respiratory epithelial cells, was administered i.p. treated mice at the time points after adoptive transfer of T cells 88 CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS

Dsg3 in Freund’s adjuvant or naive Dsg32/2 mice into Rag22/2 mice (12, 29). These PV model mice showed only a humoral im- mune reaction as a form of anti-Dsg3 IgG production, but no ap- parent cellular immune reaction to Dsg3 (Table I). When Dsg3- specfic T cell clones isolated from Dsg32/2 mice were adoptively transferred together with Dsg32/2 B cells, the recipient mice exhibited only humoral responses (14). However, Dsg3H1 trans- genic CD4+ T cells, which developed in the absence of Dsg3 or in Dsg32/2 mice, were capable of inducing not only humoral but also cellular responses to Dsg3 in the form of interface derma- titis (15). In contrast, Dsg3H1-transgenic CD4+ T cells, which developed in the presence of Dsg3 or in WT mice, induced only cellular responses (15). Taken together, these findings indicate that CD4+ T cells with the same TCR specificity for Dsg3 may be involved in induction of both pemphigus and interface der- matitis. To induce polyclonal Dsg3-specific CD4+ and CD8+ T cells, we modified the immunization step and immunized Dsg32/2 mice 2 2 with WT skin grafts and transferred splenocytes to Rag2 / mice. Downloaded from The phenotype of the skin and oral mucosa in recipient mice showed both acantholysis and interface dermatitis with CD4+ and CD8+ T cell infiltration and keratinocyte apoptosis, which is a histological finding unique to PNP (Fig. 3, Table I). These findings suggest that Dsg3 is a target Ag of autoimmune T cells in

PNP. The identification of target Ags is an important step in un- http://www.jimmunol.org/ derstanding the pathophysiology of autoimmune diseases. How- ever, it is more difficult to identify a T cell–targeted Ag than the target of IgG, because TCRs recognize antigenic peptides pre- FIGURE 5. Squamous metaplasia in the lungs of PNP patients. (A) sented in the context of MHC molecules. Additionally, direct Lung histology in a PNP patient. Transition of squamous metaplasia with multilayered epithelial cells (left square in the top panel) from columnar evidence for autoimmune diseases requires transmissibility of the ciliated respiratory epithelial cells (right square; squares were enlarged in characteristic lesions from humans to animals, which is difficult to the columns below) are shown. CD4+ and CD8+ cells were stained in the do with T cells, primarily because of MHC mismatches. Currently, area of squamous metaplasia. Scale bars, 100 mm. (B) Squamous meta- it is not feasible to identify the target Ag of infiltrating individual plasia (top panel) in patients with chronic bronchitis (left) and lung T cells in the skin or mucosal lesions of PNP. by guest on September 29, 2021 (right) was stained for Dsg3 (bottom panel). Scale bars, 100 mm. When PVand PNP model mice were compared, the latter showed significantly higher mortality, and intense CD4+ and CD8+ Tcell at which the skin phenotype became apparent. Compared with infiltrations were found in the peribronchial area in the lungs of corn oil–treated control mice, naphthalene-treated mice showed PNP model mice, but not in PV model mice (Fig. 4). It is not certain greater T cell infiltration in the lungs. The lungs of naphthalene- whether the lung involvement is the direct cause of mortality in treated mice showed significantly higher numbers of infiltrating PNP model mice, but it seems to be at least associated. It has been CD45.1+CD4+ T cells in bronchial epithelia with squamous meta- long considered that the lung expresses Dsg2, but not Dsg3 (8, 30). plasia than did mock CD45.2+CD4+ T cells (Fig. 6E, 6F). These However, the expression profile had been examined only under findings indicate that ectopic Dsg3 expression by bronchial epithelial normal conditions. Our unexpected finding of Dsg3 expression in cells is sufficient to recruit Dsg3-specific CD4+ Tcells. the lungs of PNP mice suggests that Dsg3 and other epidermal Ags Taken together, these findings indicate that Dsg3 and other are expressed in the lungs of mice during squamous metaplasia after epidermal Ags can be expressed ectopically in the lungs during naphthalene-induced pulmonary epithelial injury (Fig. 5A, 5C). tissue repair after acute inflammation, as well as in tissues showing Dsg3H1 CD4+ T cells transduced using a retrovirus did not infiltrate squamous metaplasia, rendering the bronchial epithelium prone to in the lungs in the absence of ectopic Dsg3 expression; however, Dsg3-specific or epidermal Ag-specific cellular autoimmune attack they preferentially infiltrated into the pulmonary epithelia with in mice and humans. squamous metaplasia, in which Dsg3 was expressed ectopically, in naphthalene-treated mice (Fig. 5D, 5E). These findings indicate that Discussion the ectopic expression of Dsg3 in the pulmonary epithelial cells was The autoimmune reaction in PNP is unique among the pemphigus sufficient to recruit Dsg3-specific T cells. group in at least two immunological aspects (Table I): 1) patients It is unclear how the ectopic Dsg3 expression is induced in the with PNP show cellular autoimmune attack of stratified squamous first place in the lung of PNP mice. One speculation is that epithelia in the skin and oral mucosa in addition to humoral au- bronchial epithelia may be constantly remodeled after minor toimmune attack by anti-Dsg IgG autoantibodies; and 2) a subset physiological insults, followed by transient ectopic expression of of patients with PNP develops fatal pulmonary involvement as epidermal Ags, including Dsg3 (19). Another speculation is that a form of bronchiolitis obliterans, although no epidermal Ags are the inflammatory processes in the skin may produce certain known to be expressed in the lungs. In this study, we attempted to cytokines, such as IL-22, which are able to affect epithelial pro- clarify the mechanisms underlying these unique features of PNP liferation (38, 39). These humoral factors may get into circulation using an experimental pemphigus mouse model. and remotely affect bronchial epithelia to induce ectopic Dsg3 PV model mice were originally generated by adoptive transfer expression. Epidermal Ags, including Dsg3, keratin 14, and 2 2 of lymphocytes from Dsg3 / mice immunized with recombinant involucrin, are able to be upregulated by TGF-b in human bron- The Journal of Immunology 89

FIGURE 6. Naphthalene-induced ectopic Dsg3 expression in the lungs is sufficient to recruit Dsg3-specific CD4+ T cells. (A) Histo- logical changes in the bronchial epithelia after a single dose of naphthalene (200 mg/kg) or control corn oil. Scale bar, 50 mm. (B) RT-PCR for Dsg3 in lung tissue after a single dose of naphthalene (200 mg/kg) or control corn oil. (C) RT-PCR for Dsg3, keratin 5 (K5), keratin 14 (K14), and a-fibrinogen (a-Fib, liver-specific Ag) in lung tissue of mice that received weekly naphthalene injections (100 mg/kg) for 4 wk. As positive controls (P), skin was subjected to

RT-PCR for Dsg3, K5, and K15, and liver tissue Downloaded from for a-Fib. (D) Experimental protocol for deter- mination of recruitment of Dsg3-specific CD4+ T cells in the lungs. CD4+ T cells from CD45.1+ mice and CD45.2+ mice were retrovirally transduced with Dsg3-specific TCR (Dsg3H1) and mock, respectively, and equal numbers 6 + + (1 3 10 ) of CD45.1 and CD45.2 T cells were http://www.jimmunol.org/ adoptively transferred into Rag22/2 mice, fol- lowed by weekly naphthalene administration (100 mg/kg). (E) Lung tissue was stained with anti-CD4 (red), anti-CD45.1 (green) Abs, and Hoechst (blue). CD45.1+CD4+ T cells (arrow- heads) were found in the intraepithelial area in naphthalene-treated mice, but not in corn oil- treated mice. Scale bar, 50 mm. (F) The num- bers (6SD) of CD45.1+CD4+ T cells and by guest on September 29, 2021 CD45.2+CD4+ T cells infiltrating the intra- epithelial area in the lungs of mice treated with naphthalene or corn oil were counted per 200 mm basement membrane (BM).

chial epithelial cells in vitro (40). Further intensive investigations surfaces and acantholysis of epithelial cells were found in the are necessary to clarify these issues. lungs of PNP patients (8). However, at the time of the previous T cell infiltration observed in the lungs of PNP model mice was study, no target Ag was known. It is tempting to speculate that the a mixture of CD4+ and CD8+ cells, and it remains to be elucidated autoantibodies deposited in pulmonary lesions with squamous whether both populations are necessary or whether one population metaplasia are anti-Dsg3 IgG that recognize ectopic Dsg3, thereby is more important than the other for the lung injury. Based on the inducing acantholysis in bronchial epithelia. observation that Dsg3H1 CD4+ T cells alone caused interface der- Respiratory epithelia are constantly exposed to hazardous matitis (15), it is speculated that CD4+ T cells are sufficient to induce conditions, including viral or bacterial infection, chemical in- tissue injury. However, mice receiving Dsg3H1 CD4+ T cells from halation, and smoking. Repair and maintenance of lung functions Dsg3H1 TCR transgenic mice by adoptive transfer did not neces- under such hazardous conditions requires rapid proliferation and sarily show increased mortality rates (H. Takahashi and M. Amagai, differentiation into the appropriate epithelial cell subsets that unpublished observations). Additionally, it was previously reported constitute the normal lungs (19). To achieve this, pulmonary ep- that CD4+ T cells were required for efficient CD8+ T cell function in ithelial cells undergo squamous metaplasia with dynamic changes general (41, 42). These findings together suggest that the combina- in gene expression and ectopic expression of various epidermal tion of CD4+ and CD8+ T cells is more efficient than each population Ags. In the presence of Dsg3- or other epidermal Ag-specific IgG alone to induce the lung injury. Further investigations will elucidate and/or T cells, pulmonary epithelia with squamous metaplasia the exact roles of CD4+ and CD8+ T cells in PNP mice model. might represent an unexpected target. Thus, the ectopic expression Squamous metaplasia is often found in the lungs of patients with of Dsg3 and other epidermal Ags provides a missing link in PNP (8, 10, 31, 43). Linear IgG deposition on epithelial cell pulmonary involvement in PNP in an Ag-specific autoimmune 90 CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS setting, although further studies are needed to clarify the mecha- 19. Park, K. S., J. M. Wells, A. M. Zorn, S. E. Wert, V. E. Laubach, L. G. Fernandez, and J. A. Whitsett. 2006. 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