Ectopic Expression of Epidermal Antigens
Renders the Lung a Target Organ in
Paraneoplastic Pemphigus
This information is current as
of June 17, 2017.
Tsuyoshi Hata, Shuhei Nishimoto, Keisuke Nagao, Hayato
Takahashi, Kazue Yoshida, Manabu Ohyama, Taketo
Yamada, Koichiro Asano and Masayuki Amagai
J Immunol published online 31 May 2013
http://www.jimmunol.org/content/early/2013/05/31/jimmun
ol.1203536
http://www.jimmunol.org/content/suppl/2013/05/31/jimmunol.120353
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Supplementary
Material
Published May 31, 2013, doi:10.4049/jimmunol.1203536
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*
A
mong tissue-specific autoimmune diseases, pemphigus
comprises a group of IgG autoantibody-mediated blistering diseases of the skin and mucous membranes,
characterized histologically by intraepidermal blisters due to the loss
of keratinocyte cell–cell adhesion, and immunopathologically by
the in vivo finding of bound and circulating IgG autoantibodies
directed against the surface of keratinocytes. Pemphigus has three
major forms: pemphigus foliaceus (PF), pemphigus vulgaris (PV),
and paraneoplastic pemphigus (PNP) (1). The classic forms of
pemphigus are PF and PV, which are mediated solely by humoral
autoimmunity involving IgG autoantibodies against desmoglein
(Dsg)1 and Dsg3, respectively, both of which are cadherin-type cell–
cell adhesion molecules in desmosomes (2, 3).
PNP shares the basic feature of pemphigus in that IgG autoantibodies against Dsg3 or Dsg1 play a pathogenic role by mediating blister formation in the mucous membranes and skin (4),
but its phenotype is more complex than those of PV and PF. PNP
*Department of Dermatology, Keio University School of Medicine, Tokyo 160-8582,
Japan; †Cutaneous Research Section, Fundamental Research Laboratories, KOSÉ
Corporation, Tokyo 174-0051, Japan; ‡Department of Pathology, Keio University
School of Medicine, Tokyo 160-8582, Japan; xDepartment of Respiratory Medicine,
Keio University School of Medicine, Tokyo 160-8582, Japan; and {Division of
Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Kanagawa 259-1193, Japan
Received for publication December 27, 2012. Accepted for publication April 30,
2013.
This work was supported by Grants-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology of Japan, Health and Labor
Sciences Research Grants for Research on Measures for Intractable Diseases from the
Ministry of Health, Labor, and Welfare of Japan, and Keio Gijuku Academic Development Funds.
Address correspondence and reprint requests to Prof. Masayuki Amagai, Department
of Dermatology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: B6, C57BL/6J; Dsg, desmoglein; PF, pemphigus
foliaceus; PNP, paraneoplastic pemphigus; PV, pemphigus vulgaris.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203536
occurs in association with underlying neoplasms, mainly of
lymphoid origin, and has characteristic IgG autoantibodies
against the plakin family (plectin, desmoplakins I and II, BP230,
envoplakin, and periplakin) (5, 6). Additionally, PNP patients
exhibit a cellular immune response in the skin and mucous membranes. Histological examination shows not only blister formation
caused by IgG autoantibodies against Dsg3 and/or Dsg1, but also
interface dermatitis showing keratinocyte apoptosis and T cell infiltration in the epidermis and mucosal epithelia. Furthermore, some
patients with PNP develop pulmonary involvement, such as bronchiolitis obliterans, which can cause fatal respiratory failure (7–11).
Further investigation is required to determine the target Ag(s) of
T cells that infiltrate the skin and mucous membranes as well as the
reason that such T cells also attack the lungs. The technical hurdles
to identification of these T cell Ags and the rarity of the disease
hamper progress in solving these questions.
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
to Dsg3-expressing Rag22/2 mice (12, 13). PV model mice persistently produce anti-Dsg3 IgG and develop blisters on the skin
and mucous membranes, as found in PV patients. Subsequently,
Dsg3-specific CD4+ T cell clones were isolated, and Dsg3-specific
TCR transgenic (Dsg3H1) mice were generated (14, 15). Interestingly, Dsg3-specific transgenic CD4+ T cells, which developed
in the absence of Dsg3 or in Dsg32/2 mice, induced not only the
blister formation caused by anti-Dsg3 IgG, but also interface
dermatitis with CD4+ T cell infiltration in the epidermis and
keratinocyte necrosis upon adoptive transfer with Dsg32/2 B cells
(15). This demonstrates that the cellular autoimmune response
against Dsg3 can lead to the formation of interface dermatitis,
suggesting that Dsg3 may be a target autoantigen of the cellular
immune reaction seen in PNP.
In this study, by adoptive transfer of lymphocytes from Dsg32/2
mice immunized with wild-type (WT) skin grafts, we generated
a PNP model mouse that showed both humoral and cellular immune reactions against Dsg3. PNP model mice showed supra-
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Paraneoplastic pemphigus (PNP) is an autoimmune disease of the skin and mucous membranes that can involve fatal lung complications. IgG autoantibodies target the cell adhesion molecules desmoglein (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 represents 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 acantholysis and interface dermatitis, 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
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 pulmonary 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: 000–000.
2
CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS
basilar acantholysis caused by anti-Dsg3 IgG Abs as well as interface dermatitis with CD4+ as well as CD8+ T cell infiltration
and keratinocyte apoptosis. Furthermore, these mice demonstrated
bronchial inflammation with a high mortality rate. Surprisingly,
Dsg3 was ectopically expressed in the lungs of the PNP model
mice, but not in the lungs of WT or PV model mice. Ectopic
expression of Dsg3 and other epidermal Ags in the lungs was
detected during epithelial remodeling after naphthalene-induced
pulmonary injury. CD4+ T cells containing retrovirally transduced
Dsg3-specific TCR (15) preferentially infiltrated into bronchial
epithelia in naphthalene-injected mice, but not in those that received
the control corn oil injection. Squamous metaplasia in the lungs,
which showed ectopic expression of various epidermal Ags, was
indeed frequently identified in patients with PNP. These findings
using PNP model mice provide valuable clues to solving a longstanding mystery in PNP: why the lungs, which were thought to exhibit no epidermal Ag expression, are involved in this skin-specific
autoimmune condition.
Immunization of Dsg32/2 mice via Dsg3+/+ skin grafts
Dorsal skin from Dsg3+/+ WT mice (B6) was grafted onto the backs of sexmatched Dsg32/2 or Dsg3+/2 mice, as described previously (17), and the
grafted skin was biopsied at various times after transplantation. A second
skin graft was performed 28 d after the first graft for adoptive transfer
studies. Graft survival was determined in accordance with a method described previously (18). For adoptive transfer studies, splenocytes from
mice that received a second skin graft for 14 d or more were transferred
adoptively into Rag22/2 mice via the tail vein. In control PV model mice,
Dsg32/2 mice were immunized with recombinant mouse Dsg3 in CFA, as
described previously (12).
Induction of acute lung injury by naphthalene
To examine whether a skin-specific protein could be expressed ectopically
in bronchial epithelium under certain conditions, we injected naphthalene
(Sigma-Aldrich, St. Louis, MO), which selectively injures Clara cells, and
observed bronchial epithelium during the repair process. Naphthalene was
dissolved in corn oil at 10 or 20 mg/ml and administered to mice (100 or 200
mg/kg) by i.p. injection weekly for 4 wk (19). Control mice received an
identical volume of corn oil (Sigma-Aldrich).
Retroviral transduction
Mice
Dsg1tg/tgDsg32/2 mice with the C57BL/6J (B6) background, in which
Dsg3-deficient phenotypes are rescued by ectopic Dsg1 expression (16),
were used as Dsg32/2 mice. In this study, “Dsg32/2 mice” refers to
“Dsg1tg/tgDsg32/2 mice” unless noted otherwise. These mice and Rag22/2
mice in a B6 background (Central Institute for Experimental Animals,
Tokyo, Japan) were maintained under specific pathogen-free conditions at
Keio University. All mouse studies were approved by the Animal Ethics
Review Board of Keio University.
FIGURE 1. WT skin grafted onto Dsg32/2 mice
induced both humoral and cellular immune
responses with resultant graft rejection. (A) Experimental 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 symbols) 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 littermates (bottom) on day 21. Scale bars, 20 mm.
Retroviral transduction of a Dsg3-specific TCR (Dsg3H1) to CD4+ T cells
from B6 mice was performed using a set of TCRs (Va8 and Vb6) of
a CD4+ T cell clone that recognizes the peptide Dsg3301–315, as described
previously (14, 15, 20).
Histology and immunostaining in mice and humans
To quantify the induction of cellular immune responses, infiltrating CD4+
and CD8+ cells were counted in frozen sections. The numbers of lymphocytes
and TUNEL+ cells in the oral epithelia (hard palate) and the peribronchial
area (cells in the epithelia and within 100-mm depth from the basement
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Materials and Methods
The Journal of Immunology
3
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 immunoprecipitation
and immunoblotting were performed to detect the production of antiplakin family Abs (22). Statistical analyses were performed using an unpaired 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
B cell lymphoma and positive for anti-Dsg3, anti-envoplakin, and antiperiplakin 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
Results
Grafting Dsg3-expressing skin onto Dsg32/2 mice elicits
Dsg3-specific cellular and humoral immune responses
To generate both humoral and cellular immune responses against
Dsg3, we sought to use the skin graft technique as an immunization
procedure instead of immunizing Dsg32/2 mice with recombinant
Dsg3 protein, because skin allografting is known to induce immune
responses mediated by CD4+ and CD8+ T cells, as well as Abs
(24–26). When Dsg3+/+ WT skin was grafted onto Dsg32/2 mice
or Dsg3+/2 littermates, the skin grafts on Dsg32/2 mice (n = 8), but
not on Dsg3+/2 littermates (n = 4), were rejected on days 14–28
(Fig. 1A, 1C). Second, Dsg3+/+ skin grafts onto the same mice
resulted in even more rapid rejection (days 7–10, n = 7). Histologically, the first skin grafts on Dsg32/2 mice showed acanthosis
with mild lymphocytic infiltration on day 14, interface dermatitis
with significant lymphocytic infiltration and occasional keratinocyte apoptosis on day 21, and complete necrosis of the grafted skin
on day 28, in contrast to the grafts on Dsg3+/2 littermates, which
showed no apparent change (Fig. 1B). The infiltrating lymphocytes
included both CD4+ and CD8+ T cells, and the presence of apoptotic basal keratinocytes was confirmed via in situ TUNEL assay
(Fig. 1E). In addition to the cellular immune response, Dsg32/2
mice with WT skin grafts demonstrated circulating anti-Dsg3 IgG
on day 14 with continuous increases in their titers thereafter (Fig.
1D), as well as in vivo IgG deposition on keratinocyte cell surfaces
of grafted skin (Fig. 1E, day 21).
To determine whether the T cell infiltration in the grafted skin
was Dsg3-specific, WT skin and Dsg32/2 skin were grafted simultaneously onto Dsg32/2 mice that had been primed with WT
skin 28 d prior (Fig. 2A). The WT-grafted skin showed interface
dermatitis with marked CD4+ and CD8+ T cell infiltration and
liquefaction degeneration, whereas the Dsg32/2 grafted skin
showed no apparent histological changes and no T cell infiltration
(Fig. 2B, n = 3). These findings indicate that grafting of Dsg3expressing WT skin onto Dsg32/2 mice induces both cellular and
humoral Dsg3-specific immune responses.
FIGURE 2. T cells infiltrating WT skin grafts are Dsg3-specific. (A)
Experimental protocol for determination of Dsg3-specific immune reactions caused by skin graft immunization. Dsg32/2 mice (n = 3) were first
primed with WT (Dsg3+/+) skin grafts and challenged with second skin
grafts side-by-side with WT skin and Dsg32/2 skin. Biopsies were taken
from the grafted skin for histology and CD4/CD8 staining. (B) Histology,
CD4+ and CD8+ T cells (red) of WT skin grafts (left panel) and Dsg32/2
skin grafts (right panel) on Dsg32/2 mice after priming with WT skin
grafts. Scale bar, 20 mm.
Splenocytes from Dsg3+/+ skin-grafted Dsg32/2 mice induce
characteristic features of PNP in recipient mice
Patients with PV and PNP present with mucocutaneous blisters and
erosions that histologically show suprabasilar acantholysis or the
loss of keratinocyte cell–cell adhesion just above the basal cell
layer, which results from the binding of anti-Dsg3 IgG autoantibodies to keratinocyte surfaces (Fig. 3A, 3B, Table I) (2, 4, 27).
However, the oral lesions of PNP clinically appear more inflamed
with necrotic and lichenoid changes than do those of PV, and
histological examination shows suprabasilar acantholysis with CD4+
and CD8+ T cell infiltration and keratinocyte apoptosis (6, 28).
When lymphocytes from naive Dsg32/2 mice or recombinant
Dsg3-immunized Dsg32/2 mice were adoptively transferred to
Rag22/2 mice, the recipient mice began to produce anti-Dsg3
IgG, which mediates suprabasilar acantholysis. However, no obvious T cell infiltration was identified in the skin or mucous
membranes (Fig. 3C, Supplemental Fig. 1) (12, 29). In contrast,
when splenocytes from Dsg32/2 mice immunized with Dsg3+/+
skin grafts were transferred adoptively, the recipient mice developed oral lesions with combined suprabasilar acantholysis caused
by anti-Dsg3 IgG and interface dermatitis with CD4+ and CD8+
T cell infiltration and keratinocyte apoptosis (Fig. 3D). The numbers
of infiltrating CD4+ and CD8+ T cells and TUNEL+ apoptotic cells
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The following Abs were used: anti–E-tag mAb (GE Healthcare BioSciences) conjugated with Alexa Fluor 488; anti-fluorescein/Oregon
Green goat IgG fraction conjugated with Alexa Fluor 488; goat anti-rat
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 antienvoplakin Ab (Santa Cruz Biotechnology). TUNEL+ cells were detected
using the In Situ Cell Death Detection kit and TMR red (Roche, Mannheim, Germany).
4
CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS
in the oral epithelia were significantly higher in these mice than in
PV model mice (Fig. 3E). Furthermore, by day 30, 4 of 12 mice
produced anti-envoplakin IgG, a characteristic autoantibody in PNP,
whereas none of the 40 PV model mice did so (Fig. 3F). Thus, mice
receiving splenocytes from Dsg3+/+ skin graft–immunized Dsg32/2
mice developed characteristic histological and immunological features seen in PNP. Consequently, these mice constitute a model for
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
mice
High mortality rate and lung involvement in PNP mice
Human
Clinical phenotype
Blisters and erosions
Histology
Acantholysis
Interface dermatitis
Humoral autoimmunity
Anti-Dsg3 IgG
Anti-plakin IgG
Cellular autoimmunity
T cell–mediated cytotoxicity
Dsg3-specific T cell attack
Lung involvement
Mouse
PV
PNP
+
+a
+
+
+
2
+
+
+
2
+
+
+
2
+
+
+
2
+
+
2
2
2
+
+
+
2
+
2 N.D.
2 +b
PV Model PNP Model
a
Oral PNP lesions showed more necrosis and lichenoid changes than did PV
lesions.
b
Human PNP shows bronchiolitis obliterans.
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 =
0.02 [Wilcoxon test]). To investigate the cause of this difference,
we performed whole-body histological analyses and found intense
mononuclear cell infiltrates in the peribronchial and perivascular
spaces in the lungs of PNP mice, but not in those of PV mice (Fig.
4B). Most of the infiltrating cells were CD4+or CD8+ T cells,
which were significantly more plentiful in PNP mice than in PV
mice (Fig. 4C, 4D). The degree of inflammation was profound in
some animals, and robust infiltration of neutrophils and mononuclear cells was observed in the alveolar spaces; other organs, including the liver, kidney, heart, and small intestine, showed no such
findings (Supplemental Fig. 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.
Surprisingly, the PNP mice expressed various levels of Dsg3, as
shown by RT-PCR, whereas no Dsg3 was detected in PV mice (Fig.
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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 immunized 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 immunoprecipitation and immunoblotting.
The Journal of Immunology
5
4E). Dsg3 in the lungs of PNP mice could not be detected at
protein level using either various mAbs (23) or sera from patients
with PV, probably due to its low expression or poor stability on
cell membranes that lacked proper formation of desmosomes at
the time points examined (up to day 49).
These findings suggest that ectopic pulmonary Dsg3 expression
might redirect Dsg3-specific CD4+ and CD8+ T cells to the lungs
and there induce inflammation.
Squamous metaplasia is frequently found in the lungs of
patients with PNP
It is well established that Dsg3 is not expressed in the normal lungs of
mice and humans (8, 30). However, the lungs of patients with PNP
frequently show squamous metaplasia at autopsy or in bronchial
biopsy specimens, which is often accompanied by CD4+ and CD8+
T cell infiltration (Fig. 5A) (8, 10, 31). Squamous metaplasia
develops in response to acute or chronic inflammation caused by
smoking (32) or viral infection (33–35), and it shows striking
similarities to the skin and mucosa not only in terms of morphology,
but also gene expression (36). Immunohistochemical analysis of
human lungs with squamous metaplasia confirmed ectopic Dsg3
expression in 18 of 19 samples (representative data in Fig. 5B).
Ectopic Dsg3 expression during the tissue repair process in the
lungs is sufficient to recruit Dsg3-specific CD4+ T cells
The above findings indicate that the stratified squamous epithelia–
specific adhesion molecule Dsg3 may be ectopically expressed
under certain conditions. To further explore a setting in which ectopic expression of Dsg3 is induced in bronchial epithelium, a single
dose of naphthalene (200 mg/kg), which selectively injures nonciliated Clara respiratory epithelial cells, was administered i.p.
Naphthalene is concentrated in Clara cells that are enriched in p450
enzymes, and its metabolism generates toxic metabolites that result
in selective injury to Clara cells (37). On day 1, the bronchiolar
surface appeared to be denuded, and exfoliated cells were evident
in the bronchiolar lumen. On day 3, squamous cells were lining the
injured bronchioles, which exhibited a relatively homogeneous cuboidal cell appearance (Fig. 6A), as described previously (19, 37).
Ectopic expression of Dsg3 was transiently found on days 1–3,
which declined after day 4, as determined by RT-PCR (Fig. 6B). To
examine the effects of naphthalene during the chronic phase, a lower
dose (100 mg/kg) was administered once per week for 4 wk and the
expression of epidermal Ags was examined. Interestingly, not only
Dsg3, but also other epidermal Ags such as keratin 5 and keratin 14
were detected by RT-PCR throughout the course (Fig. 6C); however,
non-epidermal peripheral Ags, such as a-fibrinogen (liver-specific
Ag), were not detected. Thus, these results demonstrate that Dsg3
and other epidermal Ags may be ectopically expressed during epithelial remodeling or squamous metaplasia as part of the wound
healing process after tissue injury.
Next, we determined whether Dsg3-reactive CD4+ T cells are
preferentially recruited to lung in which Dsg3 was ectopically
expressed. CD4+ T cells from CD45.1+ congenic B6 mice and B6
mice (CD45.2+) were retrovirally transduced with Dsg3-specific
TCR (Dsg3H1) and mock T cells, respectively. Equal numbers
(1 3 106) of Dsg3H1 (CD45.1+) and mock T cells (CD45.2+) were
adoptively transferred into Rag22/2 mice (n = 5), followed by
weekly naphthalene administration (100 mg/kg) (Fig. 6D). Dsg3H1
T cells infiltrated to the skin and induced interface dermatitis, with
the gross phenotype becoming apparent ∼4 wk after adoptive
transfer (15). We therefore examined the lungs of naphthalenetreated mice at the time points after adoptive transfer of T cells
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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) Staining of CD4+ and CD8+ T cells in the intraepithelial 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)
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).
6
CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS
at which the skin phenotype became apparent. Compared with
corn oil–treated control mice, naphthalene-treated mice showed
greater T cell infiltration in the lungs. The lungs of naphthalenetreated mice showed significantly higher numbers of infiltrating
CD45.1+CD4+ T cells in bronchial epithelia with squamous metaplasia than did mock CD45.2+CD4+ T cells (Fig. 6E, 6F). These
findings indicate that ectopic Dsg3 expression by bronchial epithelial
cells is sufficient to recruit Dsg3-specific CD4+ T cells.
Taken together, these findings indicate that Dsg3 and other
epidermal Ags can be expressed ectopically in the lungs during
tissue repair after acute inflammation, as well as in tissues showing
squamous metaplasia, rendering the bronchial epithelium prone to
Dsg3-specific or epidermal Ag-specific cellular autoimmune attack
in mice and humans.
Discussion
The autoimmune reaction in PNP is unique among the pemphigus
group in at least two immunological aspects (Table I): 1) patients
with PNP show cellular autoimmune attack of stratified squamous
epithelia in the skin and oral mucosa in addition to humoral autoimmune attack by anti-Dsg IgG autoantibodies; and 2) a subset
of patients with PNP develops fatal pulmonary involvement as
a form of bronchiolitis obliterans, although no epidermal Ags are
known to be expressed in the lungs. In this study, we attempted to
clarify the mechanisms underlying these unique features of PNP
using an experimental pemphigus mouse model.
PV model mice were originally generated by adoptive transfer
of lymphocytes from Dsg32/2 mice immunized with recombinant
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FIGURE 5. Squamous metaplasia in the lungs of PNP patients. (A)
Lung histology in a PNP patient. Transition of squamous metaplasia with
multilayered epithelial cells (left square in the top panel) from columnar
ciliated respiratory epithelial cells (right square; squares were enlarged in
the columns below) are shown. CD4+ and CD8+ cells were stained in the
area of squamous metaplasia. Scale bars, 100 mm. (B) Squamous metaplasia (top panel) in patients with chronic bronchitis (left) and lung cancer
(right) was stained for Dsg3 (bottom panel). Scale bars, 100 mm.
Dsg3 in Freund’s adjuvant or naive Dsg32/2 mice into Rag22/2
mice (12, 29). These PV model mice showed only a humoral immune reaction as a form of anti-Dsg3 IgG production, but no apparent cellular immune reaction to Dsg3 (Table I). When Dsg3specfic 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 transgenic 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 dermatitis (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 dermatitis.
To induce polyclonal Dsg3-specific CD4+ and CD8+ T cells, we
modified the immunization step and immunized Dsg32/2 mice
with WT skin grafts and transferred splenocytes to Rag22/2 mice.
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 understanding the pathophysiology of autoimmune diseases. However, it is more difficult to identify a T cell–targeted Ag than the
target of IgG, because TCRs recognize antigenic peptides presented in the context of MHC molecules. Additionally, direct
evidence for autoimmune diseases requires transmissibility of the
characteristic lesions from humans to animals, which is difficult to
do with T cells, primarily because of MHC mismatches. Currently,
it is not feasible to identify the target Ag of infiltrating individual
T cells in the skin or mucosal lesions of PNP.
When PV and PNP model mice were compared, the latter showed
significantly higher mortality, and intense CD4+ and CD8+ T cell
infiltrations were found in the peribronchial area in the lungs of
PNP model mice, but not in PV model mice (Fig. 4). It is not certain
whether the lung involvement is the direct cause of mortality in
PNP model mice, but it seems to be at least associated. It has been
long considered that the lung expresses Dsg2, but not Dsg3 (8, 30).
However, the expression profile had been examined only under
normal conditions. Our unexpected finding of Dsg3 expression in
the lungs of PNP mice suggests that Dsg3 and other epidermal Ags
are expressed in the lungs of mice during squamous metaplasia after
naphthalene-induced pulmonary epithelial injury (Fig. 5A, 5C).
Dsg3H1 CD4+ T cells transduced using a retrovirus did not infiltrate
in the lungs in the absence of ectopic Dsg3 expression; however,
they preferentially infiltrated into the pulmonary epithelia with
squamous metaplasia, in which Dsg3 was expressed ectopically, in
naphthalene-treated mice (Fig. 5D, 5E). These findings indicate that
the ectopic expression of Dsg3 in the pulmonary epithelial cells was
sufficient to recruit Dsg3-specific T cells.
It is unclear how the ectopic Dsg3 expression is induced in the
first place in the lung of PNP mice. One speculation is that
bronchial epithelia may be constantly remodeled after minor
physiological insults, followed by transient ectopic expression of
epidermal Ags, including Dsg3 (19). Another speculation is that
the inflammatory processes in the skin may produce certain
cytokines, such as IL-22, which are able to affect epithelial proliferation (38, 39). These humoral factors may get into circulation
and remotely affect bronchial epithelia to induce ectopic Dsg3
expression. Epidermal Ags, including Dsg3, keratin 14, and
involucrin, are able to be upregulated by TGF-b in human bron-
The Journal of Immunology
7
chial epithelial cells in vitro (40). Further intensive investigations
are necessary to clarify these issues.
T cell infiltration observed in the lungs of PNP model mice was
a mixture of CD4+ and CD8+ cells, and it remains to be elucidated
whether both populations are necessary or whether one population
is more important than the other for the lung injury. Based on the
observation that Dsg3H1 CD4+ T cells alone caused interface dermatitis (15), it is speculated that CD4+ T cells are sufficient to induce
tissue injury. However, mice receiving Dsg3H1 CD4+ T cells from
Dsg3H1 TCR transgenic mice by adoptive transfer did not necessarily show increased mortality rates (H. Takahashi and M. Amagai,
unpublished observations). Additionally, it was previously reported
that CD4+ T cells were required for efficient CD8+ T cell function in
general (41, 42). These findings together suggest that the combination of CD4+ and CD8+ T cells is more efficient than each population
alone to induce the lung injury. Further investigations will elucidate
the exact roles of CD4+ and CD8+ T cells in PNP mice model.
Squamous metaplasia is often found in the lungs of patients with
PNP (8, 10, 31, 43). Linear IgG deposition on epithelial cell
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FIGURE 6. Naphthalene-induced ectopic
Dsg3 expression in the lungs is sufficient to
recruit Dsg3-specific CD4+ T cells. (A) Histological 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
for a-Fib. (D) Experimental protocol for determination 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
(1 3 106) of CD45.1+ and CD45.2+ T cells were
adoptively transferred into Rag22/2 mice, followed 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 (arrowheads) were found in the intraepithelial area in
naphthalene-treated mice, but not in corn oiltreated mice. Scale bar, 50 mm. (F) The numbers (6SD) of CD45.1+CD4+ T cells and
CD45.2+CD4+ T cells infiltrating the intraepithelial area in the lungs of mice treated with
naphthalene or corn oil were counted per 200
mm basement membrane (BM).
surfaces and acantholysis of epithelial cells were found in the
lungs of PNP patients (8). However, at the time of the previous
study, no target Ag was known. It is tempting to speculate that the
autoantibodies deposited in pulmonary lesions with squamous
metaplasia are anti-Dsg3 IgG that recognize ectopic Dsg3, thereby
inducing acantholysis in bronchial epithelia.
Respiratory epithelia are constantly exposed to hazardous
conditions, including viral or bacterial infection, chemical inhalation, and smoking. Repair and maintenance of lung functions
under such hazardous conditions requires rapid proliferation and
differentiation into the appropriate epithelial cell subsets that
constitute the normal lungs (19). To achieve this, pulmonary epithelial cells undergo squamous metaplasia with dynamic changes
in gene expression and ectopic expression of various epidermal
Ags. In the presence of Dsg3- or other epidermal Ag-specific IgG
and/or T cells, pulmonary epithelia with squamous metaplasia
might represent an unexpected target. Thus, the ectopic expression
of Dsg3 and other epidermal Ags provides a missing link in
pulmonary involvement in PNP in an Ag-specific autoimmune
8
CELLULAR AUTOIMMUNITY IN PARANEOPLASTIC PEMPHIGUS
setting, although further studies are needed to clarify the mechanism(s) underlying the occurrence of bronchiolitis obliterans,
a form of pulmonary inflammation, in a subset of PNP patients.
Ectopic expression of tissue-specific Ags at different anatomical
sites under inflammatory conditions also provides important clues
as to the connections among the organs involved in tissue-specific
autoimmune diseases.
Acknowledgments
We thank H. Ito, S. Kagawa, and M. Suzuki for excellent technical support.
Disclosures
The authors have no financial conflicts of interest.
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