Figure 1.
Complement-binding capacity of BP180/CXVII-specific rabbit antibodies.
Frozen murine skin sections were incubated with rabbit antibodies and subsequently with fresh human serum as a source of complement. Bound C3 was visualized at the dermal-epidermal junction by fluorochrome-labeled antibody. (A) Deposits of C3 in sections incubated with BP180/CXVII-specific rabbit antibody. (B) No C3 deposition in sections incubated with normal rabbit IgG (magnification, ×400).
Figure 2.
Ex vivo granulocyte-activation capacity of BP180/CXVII-specific rabbit IgG antibodies.
(A) Leukocytes (3×107/ml) were stimulated with rabbit ICs consisting of 5 µg recombinant BP180/CXVII/well and 100 µl of 50-fold diluted BP180/CXVII-specific rabbit serum. ROS production was measured over a period of 60 min. Data are represented as mean ± SD; p<0.001. (B, C) Frozen skin sections were incubated with IgG and with 3×107 leukocytes/ml. Dermal-epidermal separation was observed in sections treated with (B) BP180/CXVII-specific IgG, but not with (C) normal rabbit IgG (magnification, ×400).
Figure 3.
Intraperitoneal injection of BP180/CXVII-specific IgG induces blister formation in adult mice.
Skin lesions, including blisters, erosions, and crusts developed on the (A) ear, (B) front leg and (C) periocular area in a BALB/c mouse pre-sensitized with rabbit IgG and subsequently receiving, over a period of 10 days, 6 i.p. injections of IgG, each containing 15 mg of IgG, from a rabbit immunized against murine BP180/CXVII. (D, E, F) A control mouse pre-sensitized with rabbit IgG and challenged with the same dose of normal rabbit IgG showed no skin alterations.
Figure 4.
Subcutaneous injection of BP180/CXVII-specific IgG induces blister formation in adult mice.
Skin lesions, including blisters, erosions crusts, and alopecia developed in (A) BALB/c, (B) C57BL6, and (C) SJL-1 mice pre-sensitized with rabbit IgG and subsequently receiving, over a period of 10 days, 6 s.c. injections of IgG, each containing 15 mg of IgG, from a rabbit immunized against murine BP180/CXVII. (D) A control mouse pre-sensitized with rabbit IgG and challenged with the same dose of normal rabbit IgG showed no skin alterations.
Figure 5.
Dermal-epidermal separation in mice injected with BP180/CXVII-specific antibodies.
Skin biopsies from mice pre-sensitized with rabbit IgG and subsequently injected with rabbit IgG were stained with hematoxylin and eosin. (A) Dense inflammatory infiltrates dominated by granulocytes in a BALB/c mouse receiving BP180/CXVII-specific IgG; (B) Subepidermal split and dense inflammatory infiltrates dominated by granulocytes; (C) Extensive dermal-epidermal separation; (D) Normal histological appearance in a control mouse receiving normal rabbit IgG (magnification, ×400).
Figure 6.
IgG and complement C3 deposition at the basement membrane in experimental bullous pemphigoid.
IF microscopy, performed on frozen sections of a perilesional mouse skin biopsy reveals linear deposition of (A) rabbit IgG, (B) murine C3, and (C) murine IgG at the epidermal basement membrane in a diseased mouse. No deposits of (D) rabbit IgG, (E) murine C3, and (F) murine IgG of a control mouse showing no skin lesions (magnification, ×400).
Figure 7.
Repeated injections of antibodies against murine BP180/CXVII induce extensive skin blistering.
The extent of disease was scored as described in Methods. Means of individual clinical scores of mice injected with BP180/CXVII-specific antibodies (n = 9) and mice injected with normal rabbit IgG (n = 5) are shown before the first injection as well as every subsequent second day for 19 days. The lower panel shows BALB/c mice at day 12 after the first i.p. injection.
Figure 8.
Neutrophil depletion partly inhibits skin blistering induced by BP180/CXVII-specific IgG in mice.
Mice pre-immunized with rabbit IgG and injected i.p. with BP180/CXVII-specific rabbit IgG were treated with (A) a Ly-6G-specific monoclonal or (B) a mock antibody as described in Materials and Methods (arrows). (C) Disease activity during the depletion of Ly-6G-positive cells is significantly reduced in the group of mice treated with Ly-6G-specific antibody (n = 5) compared with the group treated with mock antibody (n = 5; p<0.05). Data are shown as mean ± SD.
Figure 9.
Luteolin inhibits ROS production, but not the release of MMP-9 from IC-stimulated leukocytes.
(A) Leukocytes (3×107/ml) were pre-incubated with luteolin in different concentrations or vehicle alone and stimulated with rabbit ICs consisting of 5 µg recombinant BP180/CXVII/well and 100 µl of 50-fold diluted rabbit serum. ROS production was measured over a period of 60 min. Data are represented as mean ± SD; p<0.05. (B) 3×107/ml leukocytes were stimulated with rabbit ICs consisting of 5 µg recombinant BP180/CXVII/well and 100 µl of 50-fold diluted rabbit serum for 3 h at 37°C. MMP-9 activation was evaluated by zymography in 50-fold diluted supernatants of these cultures of cells stimulated with BP180/CXVII (lane 1), BP180/CXVII-specific rabbit IgG (lane 2), ICs (lane 3), ICs with 1000, 500 and 200 µg/ml Luteolin (lanes 4, 5, and 6 respectively). (C) Luteolin inhibits dermal-epidermal separation in cryosection assays ex vivo. Frozen murine skin sections were incubated with pre-immune rabbit serum or BP180/CXVII-specific rabbit serum. Significantly less dermal-epidermal separation was observed in sections treated with 500 µg/ml luteolin compared with those treated with vehicle alone (magnification, ×400).
Figure 10.
In vivo luteolin treatment does not significantly inhibit blistering in adult mice.
BALB/c mice were pre-immunized with rabbit IgG and injected s.c. with BP180/CXVII-specific IgG. Control mice were pre-sensitized with rabbit IgG and challenged with the same dose of normal rabbit IgG showed no skin alterations. Topical treatment of mice with (A) luteolin at concentrations of 5.24 µM/ear did not result in different outcome compared with (B) animals treated topically with vehicle alone. (C) Treatment of mice with 1 mg luteolin i.p. daily (n = 8) did not significantly influence disease activity when compared to mice treated with vehicle alone (n = 8, p>0.5). Data are shown as mean ± SEM.