Monocyte/Macrophage MMP-14 Modulates Cell Infiltration and T-Cell Attraction in Contact Dermatitis But Not in Murine Wound Healing  Anke Klose, Paola Zigrino, Cornelia Mauch  The American Journal of Pathology  Volume 182, Issue 3, Pages 755-764 (March 2013) DOI: 10.1016/j.ajpath.2012.11.028 Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

Figure 1 Characterization of KO and control animals and wounds. A: H&E staining of mouse back skin. B: Genotyping and efficiency of MMP-14 ablation of bone marrow–derived macrophages. gDNA was isolated and subjected to genotyping by PCR (left gels). MMP-14 transcripts were visualized after amplification of cDNA (top right gels); mouse S26 served as internal control. MMP-14 protein was analyzed by immunoblotting of cell lysates (bottom right gels); equal loading was demonstrated by detection of β-actin. C: The size of the wound surface was measured at 0, 2, 3, 6, 8 and 10 days after wounding. *P < 0.05. n = 23 (KO) or 17 (control). Scale bar = 200 μm. Original magnification, ×100. ctrl, control; del, deletion. The American Journal of Pathology 2013 182, 755-764DOI: (10.1016/j.ajpath.2012.11.028) Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

Figure 2 Analysis of granulation tissue and wound infiltration on day 14 after wounding. A: The granulation tissue area (encircled by a dotted line) was measured on micrographs of H&E-stained wound sections. B and C: Infiltration of monocytes (B) and macrophages (C) was monitored after immunofluorescence analysis of CD11b and F4/80, respectively, and total signal intensities were determined. D: Infiltrated T cells were visualized by detection of CD3ε (KT3), and positive cells were counted per wound section. Data are expressed as means ± SD. n = 23 (KO) or n = 17 (control). Scale bars: 500 μm (B and C); 100 μm (D). Original magnification: ×40 (A–C); ×100 (D). The American Journal of Pathology 2013 182, 755-764DOI: (10.1016/j.ajpath.2012.11.028) Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

Figure 3 Ear swelling and analysis of infiltrate. A: Ears were challenged with croton oil, and swelling was documented over 72 hours. B: Infiltration of monocytes as visualized by immunofluorescence analysis of CD11b was reduced in KO mice at both 24 and 72 hours after challenge. C: Macrophage numbers as revealed by analysis of F4/80 were reduced in KO mice at 72 hours after challenge. Micrographs (C and D) show the corresponding ear tissue at 72 hours. D: To calculate the ratio of monocytes to macrophages, total signal intensities of CD11b and F4/80 were determined and normalized to tissue size. Data are expressed as means ± SD. n = 5 per group at 24 hours; n = 10 (KO) or n = 9 (control) at 72 hours. *P < 0.05, **P < 0.01. Scale bar = 100 μm. Original magnification, ×100. The American Journal of Pathology 2013 182, 755-764DOI: (10.1016/j.ajpath.2012.11.028) Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

Figure 4 Analysis of infiltrating T cells at 72 hours after croton oil challenge. A: T cells were visualized by immunofluorescence analysis of CD3ε. Micrographs of ear tissue are presented in color (upper panel) and in grayscale (lower panel) displaying more clearly bright positive cells. B: Immunofluorescence analysis of CD8 and CD4 markers of infiltrated cells. C: Double staining of CD3ε (red) and CD8 (green). CD3+CD8+ double-positive cells are indicated by arrows. Total signal intensities were determined (CD4) or positive cells (CD3ε, CD8) were counted and numbers were normalized to tissue size. Data are expressed as means ± SD. n = 10 (KO), n = 7 (heterozygous), and n = 8 (control). *P < 0.05. Scale bars: 100 μm (A); 50 μm (C). Original magnification: ×100 (A); ×200 (C). The American Journal of Pathology 2013 182, 755-764DOI: (10.1016/j.ajpath.2012.11.028) Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

Figure 5 Cytokine arrays and P-selectin expression of mouse ear lysates and serum. A, B, D, and E: Ear lysates (A and B) and serum samples (D and E) from control and KO animals with and without croton oil challenge were subjected to cytokine array analysis (A and D) or were analyzed by immunoblotting (B and E). A and D: Differentially regulated cytokines in ear lysates (A) and serum samples (D). Cytokine levels of samples obtained from challenged control and KO animals were compared to samples of unchallenged control and KO animals. First, the ratio of challenged versus unchallenged was determined. Thereafter, the ratio of KO versus control was calculated to clearly distinguish between effects caused by MMP-14 deletion and challenge. B: Immunoblot analysis of P-selectin expression in ear lysates. Ear lysates obtained from control and KO animals, both croton oil challenged (+) and unchallenged (−), were used for immunoblot analysis of P-selectin expression. Total amount of loaded protein was demonstrated by detection of β-actin. C: Quantification of P-selectin expression in ear lysates. Immunoblots were analyzed using ImageJ software version 1.32j (NIH) and P-selectin expression was plotted after calculation of the ratio of P-selectin and the corresponding β-actin amount (arbitrary units). E: Both dimeric and monomeric P-selectin were detected, along with a soluble 100-kDa form (arrow). Data are expressed as means ± SD of three independent experiments (C) or as fold change (A and D). *P < 0.05. NC, no change; ND, not detectable. The American Journal of Pathology 2013 182, 755-764DOI: (10.1016/j.ajpath.2012.11.028) Copyright © 2013 American Society for Investigative Pathology Terms and Conditions

Figure 6 Migratory capacity and transendothelial migration of bone marrow–derived macrophages. Bone marrow–derived macrophages were isolated and differentiated for 8 days in the presence of M-CSF. A: To analyze migratory capacity, cells were seeded on fibronectin and cell migration was monitored for 24 hours. Images were captured every 30 minutes, and the trajectory length and the total distance covered by the cells were calculated. Both distance and trajectory length were significantly reduced in KO cells, compared with control. B: Transendothelial migration of cells was monitored for 4 hours. The percentage of transmigrated cells was calculated by defining the number of originally seeded macrophages (1 × 106) as 100%. Data are expressed as means ± SD of three independent experiments. n = 200 cells per group (A). *P < 0.05, **P < 0.01. The American Journal of Pathology 2013 182, 755-764DOI: (10.1016/j.ajpath.2012.11.028) Copyright © 2013 American Society for Investigative Pathology Terms and Conditions