Fig 1: MMP9 and ADAM10 are responsible for shedding of mCD46 in BP pathogenesis. HaCaT cells were incubated with activated MMP9 (50, 100 and 200 pM) and ADAM10 (100, 250 and 500 ng/mL) proteinases at 37 °C for 2 h. (A) The HaCaT cells were collected and CD46 were detected by western blot after incubation. (B) Elisa assay were used to value the sCD46 in the supernatant that shedded by MMP9 and ADAM10. Results represent the mean ± SEM from three independent experiments. P < 0.05 was considered significant. (C) IF staining was performed on normal skin sections and lesions from BP patient measure MMP9 and ADAM10 expression in the epidermal keratinocytes. DAPI staining of nuclei is shown in blue. Scale bar, 100 nm.
Fig 2: MMP1 and MMP9 generated collagen I fragments bind to and activate LAIR-1. (A) Analysis of MMP1 and MMP9 generated collagen I fragments by SDS-PAGE and silver staining. Representative image of n = 3. (B, C) LAIR-1-Fc protein binding to mock treated, MMP1 treated and MMP9 treated collagen I. (B) LAIR-1-Fc ELISA based immune binding assay (n = 3 with technical duplicates). (C) LAIR-1-Fc fluorescence based immune binding assay. Representative images show immunofluorescence by the AF647-conjugated secondary antibody alone or by secondary antibody binding to LAIR-1-Fc, representative of n = 3. (D) Flow cytometry analysis of NFAT-GFP reporter cells, representing LAIR-1 ligation (n = 3 with technical duplicates). Anti-human-LAIR-1 and anti-mouse-CD3 are positive controls for reporter cell activation. The isotype antibody and PBS are negative controls. (E) Representative images by live-cell IncuCyte imaging upon stimulation with indicated concentrations of collagen and collagen fragments (n = 3). (F) Quantification of the total green integrated intensity in the images from the live-cell IncuCyte imaging over time (n = 3 with technical duplicates). Significant differences at t = 24 hours compared to WT reporter cells are indicated, tested using a two-way ANOVA with Tukey’s multiple comparison correction. In all panels symbols represent the mean and whiskers indicate the standard deviation.
Fig 3: LAIR-2 fusion proteins block LAIR-1 activation by collagen I fragments. (A) LAIR-2-Fc and LAIR-2-Fc dead binding to 5 µg/ml coated mock treated, MMP1 treated and MMP9 treated collagen I by ELISA based immune binding assay (n = 3 with technical duplicates). Symbols represent the mean and whiskers indicate the standard deviation. (B) LAIR-2-Fc and LAIR-2-Fc dead binding by fluorescence based immune binding assay (representative of n = 3). (C) LAIR-1-Fc ELISA based immune binding assay after pre-treatment with LAIR-2-Fc and LAIR-2-Fc dead of 5 µg/ml mock treated, MMP1 treated and MMP9 treated collagen I (n = 3 with technical duplicates). (D) LAIR-1-Fc fluorescence based immune binding assay after pre-treatment with LAIR-2-Fc and LAIR-2-Fc dead (representative of n = 3). (E) Flow cytometry analysis of GFP+ reporter cells, representing LAIR-1 activation, after pre-treatment with LAIR-2 fusion proteins (n = 3 with technical duplicates). (F) Quantification of the total green integrated intensity in the images from the live-cell IncuCyte imaging at 24 hours upon stimulation with pre-treated 5 µg/ml coated, differently treated collagen I (n = 3 with technical duplicates). In (C, E, F) * indicates statistically significant differences compared to the respective isotype control (isotype for LAIR-2-Fc and isotype dead for LAIR-2-Fc dead). # indicates statistically significant differences compared to the respective 10 µg/ml pre-treatment. In all panels, significance is tested using a two-way ANOVA with Tukey’s multiple comparison correction.
Fig 4: MMP1 and MMP9 generated collagen I fragments inhibit T cell function through LAIR-1. (A) Flow cytometry analysis of parental (light grey) or full-length human LAIR-1 expressing (dark grey) NFAT-GFP CD3 reporter cells unstimulated, and stimulated with anti-mouse-CD3 in the presence of 5 µg/ml BSA, untreated collagen I, mock treated collagen I, MMP1 treated collagen I and MMP9 treated collagen I. (B) Inhibition of CD3-signaling-induced GFP expression by increasing concentrations of collagen or collagen fragments compared to BSA. (C) Inhibition of anti-CD3 induced IFN-? secretion by increasing concentrations of collagen or collagen fragments compared to BSA. Significant differences compared to the parental cell line are indicated, tested using a two-way ANOVA with Sidak’s multiple comparison correction.
Supplier Page from Abcam for Native human MMP9 protein (Proenzyme, monomer)