Download presentation
Presentation is loading. Please wait.
1
Volume 102, Issue 5, Pages 647-655 (September 2000)
The Serpin α1-Proteinase Inhibitor Is a Critical Substrate for Gelatinase B/MMP-9 In Vivo Zhi Liu, Xiaoye Zhou, Steven D Shapiro, J.Michael Shipley, Sally S Twining, Luis A Diaz, Robert M Senior, Zena Werb Cell Volume 102, Issue 5, Pages (September 2000) DOI: /S (00)
2
Figure 1 Histological Evaluation of Neonatal GB−/− and NE−/− Mice Injected with Pathogenic anti-mBP180 IgG and Reconstituted with Mouse PMN (A) Pathogenic rabbit anti-murine BP180 IgG (i.d. injection, 2.5 mg/g body weight) produced subepidermal blistering in neonatal GB−/− mice reconstituted with 5 × 105 PMN from (a) GB +/+, (c) NE−/−, but not (b) GB−/− mice. (B) Pathogenic rabbit anti-murine BP180 IgG (i.d. injection, 2.5 mg/g body weight) produced clinical (data not shown) and histological subepidermal blistering in neonatal NE−/− mice reconstituted with 5 × 105 PMN from (a) NE +/+, (c) GB−/−, but not (b) NE−/− mice (×184). Insets, higher magnifications of H&E staining sections, demonstrate infiltrating PMN in the dermis (×920). Basal keratinocyte (arrow), dermis (d), epidermis (e), blister vesicle (v), PMN (arrowhead). Cell , DOI: ( /S (00) )
3
Figure 2 BP180 Cleavage and Epidermal–Dermal Separation
(A) To identify BP180 degradation in the lesional skin of experimental BP neonatal NE−/− (lanes 1–4), NE +/+ (lane 5), and GB−/− (lanes 6–7) mice were injected i.d. with anti-mBP180 IgG. 2 hr later, the IgG-injected mice were reconstituted with 5 × 105 mouse PMN. Skin samples were obtained at 12 hr after IgG injection and protein extracts (15 μg/lane) were analyzed by immunoblotting using the anti-mBP180 IgG. Both the intact and degraded BP180 were seen in lesional skin samples of IgG-injected NE +/+ mice (lane 5), NE−/− mice injected with IgG and reconstituted with PMN from NE +/+ (lane 2) and GB−/− (lane 4) mice, and GB−/− mice injected with IgG and reconstituted with PMN from NE−/− (lane 6) mice. In contrast, no degraded BP180 was found in skin samples of IgG-injected NE−/− mice (lane 1), IgG-injected NE−/− mice reconstituted with PMN from NE−/− mice (lane 3), or GB−/− mice reconstituted with PMN from GB−/− mice (lane 7). (B) To generate BP-like dermal–epidermal separation in mouse skin by NE neonatal mouse skin sections were incubated in (a) MEM alone with (b) 25 μg/ml NE or (c) 80 μg/ml GB at 37°C for 24 hr and were examined by H&E staining. Dermal–epidermal separation was observed in sections treated with NE, but not GB. Control sections incubated in MEM without proteinases showed no dermal–epidermal separation (panel A). Site of basal keratinocytes (arrow), epidermis (e), dermis (d), blister vesicle (V) (×368). Cell , DOI: ( /S (00) )
4
Figure 3 Comparison of Levels of PMN Infiltration in the Lesional Skin in Experimental BP GB +/+, GB−/−, NE +/+, and NE−/− mice were injected with pathogenic anti-mBP180 IgG. Recruitment of PMN (as assayed by MPO, GB and NE in the IgG injection sites) was analyzed at 12 hr after IgG injection. (A) MPO activity assay showed significantly higher levels of PMN recruitment in the lesional skin of GB +/+ (bar 1) and NE +/+ (bar 3) mice as compared to GB−/− (bar 2) and NE−/− (bar 4) mice. (B) Gelatin zymography revealed GB bands in lesional skin samples of GB +/+ (lane 1), NE +/+ (lane 3), and nonlesional skin samples of NE−/− (lane 4), but not in nonlesional skin samples of GB−/− (lane 2). 12 μg protein/lane for lanes 1 and 2; 30 μg protein/lane for lanes 3 and 4. (C) NE activity assay was significantly higher in the lesional skin of GB +/+ (bar 1) and NE +/+ (bar 3) mice, as compared to the skin of GB−/− (bar 2) and NE−/− (bar 4) mice. (n = 8 for each group, *p < 0.001, for paired samples: bar 1 versus 2 and 3 versus 4). Cell , DOI: ( /S (00) )
5
Figure 4 Dependence of α1-PI Degradation on GB
GB +/+, GB−/−, NE +/+, and NE−/− mice were injected i.d. with pathogenic IgG. 2 hr later, GB−/− and NE−/− mice were reconstituted with 2.5 × 106 mouse PMN. These animals were examined at 12 hr after IgG injection. (A) Clinical and histological examination showed that IgG-injected GB−/− mice reconstituted with 2.5 × 106 PMN from GB−/− mice developed skin lesions (a and b), while the NE−/− reconstituted with 2.5 × 106 PMN from NE−/− mice remained resistant to experimental BP (c and d). Site of basal keratinocytes (arrow), epidermis (e), dermis (d), blister vesicle (V), PMN (arrowhead). (B) Immunoblotting using the anti-mBP180 IgG demonstrates that both the intact and degraded BP180 were present in lesional skin extracts of GB−/− mice reconstituted with 2.5 × 106 GB−/− PMN (lane 1), but not in skin extracts of NE−/− mice reconstituted with 2.5 × 106 NE−/− PMN (lane 2). 15 μg protein/lane. (C) Reverse zymography (38 μg protein/lane) protein showed significantly higher levels of α1-PI in the nonlesional skin of GB−/− (1.90 ± 0.24 units/mg protein) and NE−/− (1.17 ± 0.13 units/mg) mice, as compared to the lesional skin of GB +/+ (0.53 ± 0.06 units/mg; p < 0.001) and NE +/+ (0.41 ± 0.05 units/mg; p < 0.001) mice. (D) Immunoblotting using the anti-α1-PI IgG shows both the intact and degraded α1-PI in lesional skin samples of GB +/+ mice injected with IgG (lane 6) and GB−/− mice injected with IgG and reconstituted with 5 × 105 PMN from GB +/+ (lane 2) or NE−/− (lane 5) mice. In contrast, only the intact α1-PI was found in skin samples of GB−/− mice injected with pathogenic IgG only (lane 1) or IgG-injected GB−/− mice reconstituted with 5 × 105 PMN from GB−/− mice (lane 3). GB−/− mice reconstituted with 2.5 × 106 PMN from GB−/− mice developed blisters, but showed no detectable degraded α1-PI in the skin (lane 4). Cell , DOI: ( /S (00) )
6
Figure 5 In Vivo Inhibition of Dermal–Epidermal Separation by α1-PI
(A) Histology of mouse skin sections: (a) Blister in mice injected with pathogenic IgG alone. (b) No blister in mice pretreated with α1-PI. (c) Blister in mice pretreated with the neutrophil cathepsin G inhibitor, α1-antichymotrypsin (α1-AC). Site of basal keratinocytes (arrow), epidermis (e), dermis (d), blister vesicle (V). (B) MPO assay at 4 hr (hatched bars) after injection of wild-type mice with pathogenic IgG, shows that the same number of PMN were present in mice pretreated with α1-PI (bar 3), as those injected with PBS alone (bar 1) and α1-AC (bar 5). However, by 12 hr (black bars) the mice pretreated with α1-PI (bar 4) had significantly fewer PMN than controls (bars 2 and 6) (*, p < 0.001). n = 8 for each group. Cell , DOI: ( /S (00) )
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.