Repeated injections of antibodies against murine BP180/CXVII induce extensive skin blistering.

<p>The extent of disease was scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031066#s4" target="_blank">Methods</a>. 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.</p

Neutrophil depletion partly inhibits skin blistering induced by BP180/CXVII-specific IgG in mice.

<p>Mice pre-immunized with rabbit IgG and injected i.p. with BP180/CXVII-specific rabbit IgG were treated with (<b>A</b>) a Ly-6G-specific monoclonal or (<b>B</b>) a mock antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031066#s4" target="_blank">Materials and Methods</a> (arrows). (<b>C</b>) 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.</p

Intraperitoneal injection of BP180/CXVII-specific IgG induces blister formation in adult mice.

<p>Skin lesions, including blisters, erosions, and crusts developed on the (<b>A</b>) ear, (<b>B</b>) front leg and (<b>C</b>) 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. (<b>D, E, F</b>) A control mouse pre-sensitized with rabbit IgG and challenged with the same dose of normal rabbit IgG showed no skin alterations.</p

<i>In vivo</i> luteolin treatment does not significantly inhibit blistering in adult mice.

<p>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 (<b>A</b>) luteolin at concentrations of 5.24 µM/ear did not result in different outcome compared with (<b>B</b>) animals treated topically with vehicle alone. (<b>C</b>) 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.</p

Complement-binding capacity of BP180/CXVII-specific rabbit antibodies.

<p>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. (<b>A</b>) Deposits of C3 in sections incubated with BP180/CXVII-specific rabbit antibody. (<b>B</b>) No C3 deposition in sections incubated with normal rabbit IgG (magnification, ×400).</p

IgG and complement C3 deposition at the basement membrane in experimental bullous pemphigoid.

<p>IF microscopy, performed on frozen sections of a perilesional mouse skin biopsy reveals linear deposition of (<b>A</b>) rabbit IgG, (<b>B</b>) murine C3, and (<b>C</b>) murine IgG at the epidermal basement membrane in a diseased mouse. No deposits of (<b>D</b>) rabbit IgG, (<b>E</b>) murine C3, and (<b>F</b>) murine IgG of a control mouse showing no skin lesions (magnification, ×400).</p

Subcutaneous injection of BP180/CXVII-specific IgG induces blister formation in adult mice.

<p>Skin lesions, including blisters, erosions crusts, and alopecia developed in (<b>A</b>) BALB/c, (<b>B</b>) C57BL6, and (<b>C</b>) 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. (<b>D</b>) A control mouse pre-sensitized with rabbit IgG and challenged with the same dose of normal rabbit IgG showed no skin alterations.</p

Dermal-epidermal separation in mice injected with BP180/CXVII-specific antibodies.

<p>Skin biopsies from mice pre-sensitized with rabbit IgG and subsequently injected with rabbit IgG were stained with hematoxylin and eosin. (<b>A</b>) Dense inflammatory infiltrates dominated by granulocytes in a BALB/c mouse receiving BP180/CXVII-specific IgG; (<b>B</b>) Subepidermal split and dense inflammatory infiltrates dominated by granulocytes; (<b>C</b>) Extensive dermal-epidermal separation; (<b>D</b>) Normal histological appearance in a control mouse receiving normal rabbit IgG (magnification, ×400).</p

Luteolin inhibits ROS production, but not the release of MMP-9 from IC-stimulated leukocytes.

<p>(<b>A</b>) 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>B</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). (<b>C</b>) Luteolin inhibits dermal-epidermal separation in cryosection assays <i>ex vivo</i>. 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).</p

<i>Ex vivo</i> granulocyte-activation capacity of BP180/CXVII-specific rabbit IgG antibodies.

<p>(<b>A</b>) 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>B, C</b>) Frozen skin sections were incubated with IgG and with 3×10⁷ leukocytes/ml. Dermal-epidermal separation was observed in sections treated with (<b>B</b>) BP180/CXVII-specific IgG, but not with (<b>C</b>) normal rabbit IgG (magnification, ×400).</p