Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec;31(4):331-8.
doi: 10.1016/j.jaut.2008.08.009. Epub 2008 Oct 14.

Subepidermal Blistering Induced by Human Autoantibodies to BP180 Requires Innate Immune Players in a Humanized Bullous Pemphigoid Mouse Model

Affiliations
Free PMC article

Subepidermal Blistering Induced by Human Autoantibodies to BP180 Requires Innate Immune Players in a Humanized Bullous Pemphigoid Mouse Model

Zhi Liu et al. J Autoimmun. .
Free PMC article

Abstract

Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease associated with subepidermal blistering and autoantibodies against BP180, a transmembrane collagen and major component of the hemidesmosome. Numerous inflammatory cells infiltrate the upper dermis in BP. IgG autoantibodies in BP fix complement and target multiple BP180 epitopes that are highly clustered within a non-collagen linker domain, termed NC16A. Anti-BP180 antibodies induce BP in mice. In this study, we generated a humanized mouse strain, in which the murine BP180NC14A is replaced with the homologous human BP180NC16A epitope cluster region. We show that the humanized NC16A (NC16A+/+) mice injected with anti-BP180NC16A autoantibodies develop BP-like subepidermal blisters. The F(ab')(2) fragments of pathogenic IgG fail to activate the complement cascade and are no longer pathogenic. The NC16A+/+ mice pretreated with mast cell activation blocker or depleted of complement or neutrophils become resistant to BP. These findings suggest that the humoral response in BP critically depends on innate immune system players.

Conflict of interest statement

Disclosures: The authors have no financial conflict of interest.

Figures

Figure 1
Figure 1. Generation of humanized BP180NC16A mice
A The murine genomic segment encoding BP180 NC14A domain (exons 17 through 18) was replaced by the human NC16A genomic region (exons 18 through 19; black bars). TM, transmembrane. B. Tail DNA from wild-type (WT) and NC16A+/+ mice were PCR amplified with primer pairs specific for the mouse BP180 (m) or hBP180NC16A (h) sequence. A 2198-bp band corresponding to the mBP180 segment was obtained from WT (lane 1) but not in NC16A+/+ mice (Lane 3). A 571-bp stretch that contains only the human BP180 NC16A sequence was obtained from NC16A+/+ (lane 4) but not WT mice (Lane 2). C. Total RNA from the tails of WT and NC16A+/+ was assayed by RT-PCR with primer pairs specific for mouse BP180 or hBP180NC16A. A 331-bp band corresponding to mBP180 cDNA sequence was obtained from WT (lane 1) but not NC16A+/+ mice (lane 3), whereas a 191-bp band corresponding to hBP180NC16A cDNA was obtained from NC16A+/+ (lane 4) but not WT mice (Lane 2). D. Indirect IF showed that rabbit anti-mBP180NC14A stained the BMZ of WT (a) but not NC16A+/+ mouse skin (c), while anti-hBP180NC16A autoantibodies (BP1) stained the basement membrane zone of NC16A+/+ (d) but not WT mice (b). Arrow, basal keratinocytes. 100x magnification. E. Electron microscopic examination of WT (a) and NC16A+/+ (b) mouse skin showed no differences in the structure and distribution of hemidesmosomes. HD, hemidesmosome; LD, lamina densa; LL, lamina lucida. 30,000x magnification.
Figure 2
Figure 2. Anti-BP180NC16A autoantibodies induce subepidermal blisters in the humanized NC16A mice
Neonatal NC16A+/+ mice injected i.d. with BP180NC16A-specific autoantibodies (BP1, 0.29 mg/g body weight) developed clinical blistering (a). Direct IF showed deposition of human IgG (b) and murine C3 (d) at the basement membrane zone. H/E staining showed dermal-epidermal separation (d). Electron microscopic analysis revealed a split at the lamina lucida (e). Examination of toluidine blue stained skin sections revealed degranulating mast cells (f). H/E staining showed infiltrating neutrophils in the upper dermis (g). 400x magnification. Eosinophil-specific histochemical staining of the skin failed to identify eosinophils in the dermis (dark-brown staining for eosinophils) (h). NC16A+/+ mice injected with normal human IgG or rabbit anti-mBP180 IgG showed no skin lesions (Table 1). E, epidermis. D, dermis. V, vesicle. arrow, basal keratinocytes. 100x magnification for b–c and i. 400x magnification for g and h. 30,000x magnification for f.
Figure 3
Figure 3. Pathogenicity of anti-BP180NC16A autoantibodies in the NC16A+/+ mice requires complement activation
Neonatal NC16A+/+ mice injected with anti-BP180NC16A intact antibodies (BP1, 0.29 mg/g body weight) developed subepidermal blistering with C3 deposition at the basement membrane zone (c and d). In contrast, F(ab′)2 fragments (0.29 mg/g body weight) of the pathogenic antibodies failed to induce skin lesions or C3 deposition (e and d). NC16A+/+ mice injected with normal human IgG (NH IgG, 0.29 mg/g body weight) showed no skin lesions and complement activation (a and b). Arrow, basal keratinocytes. 100x magnification.
Figure 4
Figure 4. Anti-BP180NC16A autoantibody-induced BP depends on mast cell degranulation
Normal human IgG (0.29 mg/g body weight), when injected i.d. into NC16A+/+ mice, induced minimal mast cell degranulation (a) and no skin lesions (b). NC16A+/+ mice injected i.d. with pathogenic anti-BP180NC16A autoantibodies (BP1, 0.29 mg/g body weight) showed extensive mast cell degranulation (c) and subepidermal blistering (d). In contrast, NC16A+/+ mice pretreated with the mast cell degranulation inhibitor, cromolyn sodium (10 μg/g body weight), and then injected i.d. with pathogenic antibodies (BP1, 0.29 mg/g body weight) exhibited minimal mast cell degranulation (e) and no skin lesions (f).
Figure 5
Figure 5. Neutrophils are required for subepidermal blistering triggered by anti-BP180NC16A autoantibodies
Neonatal NC16A+/+ mice pretreated with normal control rabbit IgG (NR IgG, 100 μl/g body weight, i.p.) followed by an i.d. injection with pathogenic antibodies (BP1, 0.29 mg/g body weight) had normal numbers of neutrophils in circulation (a) and developed subepidermal blistering (b). In contrast, the mice depleted of neutrophils by i.p. injection of AI-A31140 (100 μl/g body weight) and then injected i.d. with pathogenic antibodies (BP1, 0.29 mg/g body weight) showed >90% reduction in circulating neutrophils (c) and developed no skin lesions (d). Arrow, neutrophil.
Figure 6
Figure 6. Blocking complement activation and mast cell degranulation impairs skin neutrophil infiltration in the pathogenic antibody-induced NC16A+/+ mice
NC16A+/+ mice injected i.d. with pathogenic anti-BP180NC16A antibodies (BP1, 0.29 mg/g body weight; HG1, 0.17 mg/g body weight) showed extensive neutrophil infiltration (bar 2). In contrast, NC16A+/+ mice injected i.d with F(ab′)2 fragments of anti-BP180NC16A (BP1, 0.29 mg/g body weight) (bar 3) showed a dramatically lower levels of neutrophil infiltration. Also showing a significant reduction in neutrophil infiltration were mice injected with pathogenic anti-BP180NC16A IgG following pretreatment with either the mast cell degranulation inhibitor cromolyn sodium (10 μg/g body weight) (bar 4) or the neutrophil-depleting antibody (AI-A31140, 100 μl/g body weight) (bar 5) n=6. *p< 0.01.

Similar articles

See all similar articles

Cited by 35 articles

See all "Cited by" articles

Publication types

Feedback