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. 2012 Jan;31(1):38-44.
doi: 10.1016/j.matbio.2011.09.003. Epub 2011 Sep 25.

Neutrophil Elastase Cleaves the Murine Hemidesmosomal Protein BP180/type XVII Collagen and Generates Degradation Products That Modulate Experimental Bullous Pemphigoid

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Neutrophil Elastase Cleaves the Murine Hemidesmosomal Protein BP180/type XVII Collagen and Generates Degradation Products That Modulate Experimental Bullous Pemphigoid

Lan Lin et al. Matrix Biol. .
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Abstract

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor α1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.

Figures

Figure 1
Figure 1. Neutrophil elastase cleaves BP180
mBP180ABC containing NC14A and N-terminal half of C13 domains (see panel C) (4 μg) was incubated with neutrophil elastase (NE, 0.02 μg) at 37°C for 0–2 h. Degradation products of mBP180ABC were then resolved by 21% SDS-PAGE, visualized by Coomassie blue (CB) staining and immunoblotting, and analyzed by mass spectrometry. (A) CB staining. Fragments of approximately 15 kD, 12 kD, and 8 kD were generated from the cleavage of mBP180ABC by NE (lanes 2–6). No degradation products were seen after incubation for 2 h without NE (lane 7). (B) Immunoblotting. Anti-mBP180ABC IgG identified three fragments with molecular weights of approximately 15 kD, 12 kD, and 8 kD (lanes 2–6). (C) NE cleavage sites. The location of mBP180ABC and the NE cleavage sites within the mBP180ABC are depicted. The schematic diagram at the left is a structural representation of mouse BP180. The horizontal black bar designates the transmembrane domain (TM). The COOH-terminal extracellular region is made up of 13 collagen triple-helical (open bar) and 14 noncollagenous (closed bar) domains. NC14A represents the 14th noncollagenous domain and C13 the 13th collagenous domain. The purified recombinant mBP180ABC protein is a 145 amino acid fragment of the mBP180 extracellular domain, made up of an 8-kD NC14A (closed bar) and an 8-kD half of C13 (open bar). The degradation products were excised from Coomassie-stained PVDF membranes and subjected to sequencing analysis. NE cleaved mBP180ABC at 3 sites (amino acid positions 506, 561, and 592).
Figure 2
Figure 2. Neutrophil elastase is the major neutrophil proteinase for BP180 degradation
mBP180ABC (4 μg) was incubated with buffer control (lane 1), 0.02 μg of NE (lane 2), degranulated neutrophil supernatant (5 μl) from wild-type mice (NE+/+, lane 3), NE+/+ in the presence of α1-PI (lane 4), or NE-deficient mice (NE−/−, lane 5) at 37°C for 30 min. Degradation products of mBP180ABC were separated on 21% SDS-PAGE and analyzed by immunoblotting using antibodies directed against the mBP180ABC. Degranulated neutrophil supernatants from NE+/+ and not NE−/− mice cleaved mBP180ABC and its proteolytic activity was completely abolished by addition of the NE inhibitor α1-PI.
Figure 3
Figure 3. Neutrophil elastase-cleaved BP180 fragments are chemotactic for neutrophils in vitro and in vivo
(A) Chemotaxis assays were performed using mouse neutrophils placed in the upper compartment of a modified Boyden chamber. Buffer control, peptides p506, p561, and p592 (10−5 – 10−7 M), or fMLP (10−5 M) were placed in the lower compartment as the chemoattractant. The average number of migrated neutrophils per high powered field in triplicate wells from three separate experiments was determined. The peptide p561, but not p506 and p592, exhibits chemotactic activity for neutrophils in a dose-dependent fashion (bars 6–8). *p<0.05 (p561 vs. buffer control), Student t-test. (B) Neonatal C57BL/6J mice (1–2 days old) were administrated intradermally with PBS, peptides p506, p561, and p592 (10−5 – 10−6 M in 50 μl PBS), or IL-8 (10−7 M). Four h later, skin sections at the injection sites were obtained, and infiltrating neutrophils were quantified by measuring MPO enzyme activity in the skin protein extracts. Significantly increased levels of infiltrating neutrophils are present in the p561-injected mouse skin (bars 5,6) as compared to the p506- (bars 3,4) and p592-injected (bars 7,8) mouse skin. Levels of neutrophil infiltration were expressed as relative MPO activity (mean OD460nm reading + SEM/mg protein). n=3 for each group, *p<0.05 (p561 vs. buffer control), Student t-test.
Figure 4
Figure 4. Local injection of NE induces neutrophil infiltration in mice
Neonatal mice were injected intradermally with 100 μl of NE (100 μg/ml in PBS) in the absence and presence of the NE inhibitor α1-proteinase inhibitor (α1-PI) and the skin sections were examined at different time points. (A) H/E staining. The skin sections were examined by routine H/E staining. The skin sections treated with NE showed neutrophil infiltration at 8 and 24 h and dermal-epidermal separation at 24 h post injection. X100 magnification. E, epidermis. D, dermis. V, vesicle. Arrow, basal keratinocyte. (B) MPO assay. Neutrophil infiltration in the NE injection sites were quantified by MPO assay at different time points post NE injection and expressed as relative MPO activity (mean OD460nm reading + SEM/mg protein). Significantly increased levels of infiltrating neutrophils in NE-injected mouse skin were seen at 4, 8, 12 and 24 h as compared to those injected with NE plus α1-PI. *p<0.05. n=3. (C) Mouse epidermis was incubated at 37°C in PBS alone for 0 (lane 1) or 30 minutes (lane 2), in the presence of NE (lane 3) or NE plus α1-PI (lane 4), for 30 minutes. Protein extracts were separated by SDS-PAGE and analyzed by immunoblotting with a rabbit anti-mouse BP180 antibody. Significant degradation of BP180 was observed only in the reaction containing NE. Addition of α1-PI completely abolished degradation of BP180.
Figure 5
Figure 5. NE cleaves native human BP180
(A). Human foreskin epidermis was incubated at 37°C in PBS without (lane 1) or with NE (lane 2) for 30 minutes. Protein extracts were separated by SDS-PAGE and analyzed by immunoblotting with a rabbit anti-BP180NC16A antibody. (B) Protein extracts of cultured human primary keratinocytes were incubated in the presence (lanes 3, 4) or absence (lanes 1, 2) of NE at 37°C for 30 min. The digestion mixtures were separated in 6% and 18% SDS under native (lanes 1, 2) or denaturing/reducing conditions (lanes 3, 4), and then probed with rabbit anti-BP180NC16A antibody. Under native conditions, the full-length BP180 trimer (top panel, lane 1) was degraded into a lower molecular weight trimer band (top panel, lane 3). A band corresponding to a small BP180 fragment was identified on the 18% SDS-PAGE gel.

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