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. 1998 Aug 3;188(3):475-82.
doi: 10.1084/jem.188.3.475.

Gelatinase B-deficient Mice Are Resistant to Experimental Bullous Pemphigoid

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Free PMC article

Gelatinase B-deficient Mice Are Resistant to Experimental Bullous Pemphigoid

Z Liu et al. J Exp Med. .
Free PMC article

Abstract

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by deposition of autoantibodies at the basement membrane zone. In an experimental BP model in mice, the subepidermal blistering is mediated by antibodies directed against the hemidesmosomal protein BP180 (collagen XVII, BPAG2), and depends on complement activation and neutrophil infiltration. Gelatinase B is present in BP blister fluid and can cleave BP180. In this study we investigated the role of gelatinase B in the immunopathogenesis of experimental BP using mice containing targeted disruption of the gelatinase B (MMP-9, 92 kD gelatinase) gene. Gelatinase B-deficient mice were resistant to the blistering effect of intracutaneous anti-mBP180 antibodies, although these mice showed deposition of autoantibodies at the basement membrane zone and neutrophil recruitment to the skin comparable to that observed in the control mice. Interleukin 8 given intradermally concomitantly with pathogenic anti-mBP180 elicited a significant neutrophil recruitment into the skin in gelatinase B-deficient mice, but blistering did not occur. However, gelatinase B-deficient mice reconstituted with neutrophils from normal mice developed blistering in response to anti-mBP180 antibodies. These results implicate neutrophil-derived gelatinase B in the pathogenesis of experimental BP and might lead to novel therapeutic strategies for BP.

Figures

Figure 1
Figure 1
Gelatin zymography of lesional skin in experimental BP. Skin samples (12 μg/lane) from BALB/c mice injected with PBS (lane 1), control rabbit IgG R50 (lanes 2, 5, and 8), or pathogenic anti-mBP180 IgG R621 (lanes 3, 6, 9, and 11) were analyzed by gelatin zymography. Purified murine neutrophil extract was used as a standard (1 μg/lane; lanes 4, 7, and 10). A 97-kD gelatinolytic band was seen in lesional skin samples of mice injected with R621 (lane 3) but not in samples from mice injected with PBS or R50 controls (lanes 1 and 2). This band comigrates with the proenzyme form of gelatinase B (pro) seen in purified PMN (lane 4). Gelatinase activities in lanes 2–4 were completely inhibited by the metalloproteinase inhibitor EDTA (lanes 5–7) but not by the serine proteinase inhibitor PMSF (lanes 8–10). The gelatinase B activity was converted to lower molecular weight species after treatment with APMA, an activator of matrix metalloproteinases (lane 11).
Figure 2
Figure 2
Clinical and histological evaluation of neonatal gelatinase B−/− and gelatinase B+/+ mice injected with pathogenic anti-mBP180 IgG. Pathogenic rabbit anti–murine BP180 IgG R621 (intradermal injection, 2.5 mg/g body weight) produced extensive epidermal blistering in neonatal gelatinase B+/+ mice (A). The skin of these animals showed linear deposition of R621 (B) and mouse C3 (C) at the BMZ by direct IF. Hematoxylin and eosin (H&E) staining of skin from these mice showed subepidermal vesicle formation with neutrophilic infiltration (D). The inset, a higher magnification of D, demonstrates a neutrophil at the lesional site in the dermis. In contrast, neonatal gelatinase B−/− mice injected intradermally with R621 IgG showed no clinical evidence of skin disease (E). Direct IF studies showed rabbit R621 IgG deposition (F) and mouse C3 (G) at the BMZ. These animals exhibited neutrophilic infiltration, but no evidence of subepidermal vesiculation at the light microscopic level (H) by H&E staining. The inset, a higher magnification of H, exhibits a neutrophil underneath the basal keratinocyte. Site of antibody labeling (white arrows), basal keratinocyte (black arrows). d, dermis; e, epidermis; v, vesicle. Original magnification: ×100. Insets (original magnification ×400): neutrophil (black arrowheads).
Figure 3
Figure 3
MPO activity of skin extracts from mice injected intradermally with pathogenic rabbit anti-mBP180 IgG. Neonatal gelatinase B−/− (bars 1 and 2), gelatinase B+/+ (bars 3 and 4), and BALB/c (bars 5 and 6) mice received 2.5 mg/g body weight anti-mBP180 IgG R621. Tissue MPO activities (mean ± SEM) in the injection sites were determined 4 (bars 1, 3, and 5) and 12 (bars 2, 4, and 6) h after the IgG injection. n = 8 for each group. *P <0.001, Student's t test for paired samples: (bar 2 versus 4). 0.58 ± 0.06 versus 1.27 ± 0.11 (P <0.001) and versus 1.19 ± 0.16, respectively (P <0.001). The MPO values shown were corrected for PBS controls. Each group of mice injected with PBS yielded an average of MPO activity of ∼0.1 OD460nm/mg protein.
Figure 4
Figure 4
Experimental BP in gelatinase B−/− mice reconstituted with normal neutrophils. Pathogenic rabbit anti-mBP180 IgG R621 (intradermal injection, 2.5 mg/g body weight) did not induce blisters in neonatal gelatinase B−/− mice reconstituted with neutrophils from gelatinase B−/− mice [A, mPMN(−/−)]. The skin of these animals showed no dermal–epidermal separation by H&E (B). In contrast, the pathogenic R621 IgG triggered separation in neonatal gelatinase B−/− mice reconstituted with neutrophils from gelatinase B+/+ mice [C, mPMN(+/+)]. H&E-stained section from these mice showing a subepidermal vesicle (D). d, dermis; e, epidermis; v, vesicle. Original magnification: ×100.
Figure 5
Figure 5
Gelatin zymography of lesional skin samples of gelatinase B−/− mice reconstituted with normal neutrophils. Neonatal gelatinase B−/− mice received intradermally 2.5 mg/g body weight of control IgG R50 (lanes 1 and 3), or pathogenic anti-mBP180 IgG R621 (lanes 2 and 4). 2 h later, these mice were injected at the same site with 5 × 105 neutrophils purified from gelatinase B−/− mice [mPMN(/); lanes 1 and 2], or gelatinase B+/+ mice [mPMN(+/+); lanes 3 and 4]. 12 h after IgG injections, these animals were examined for blistering, and skin biopsies at the injection site were analyzed by gelatin zymography (12 μg/lane). Purified control murine neutrophil extract (1 μg; lane 5) was used as a standard for gelatinase B. Gelatinase B−/− mice reconstituted with mPMN (−/−) showed no disease and no gelatinase B in the skin samples when injected with control R50 IgG (lane 1) or R621 IgG (lane 2). In contrast, gelatinase B−/− mice reconstituted with mPMN(+/+) developed disease when injected with R621 IgG (lane 4) but not control IgG R50 (lane 3). Gelatinase B activity is seen when gelatinase B−/− mice are reconstituted with normal neutrophils (lanes 3 and 4).

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