Large and Sustained Induction of Chemokines during Impaired Wound Healing in the Genetically Diabetic Mouse: Prolonged Persistence of Neutrophils and Macrophages during the Late Phase of Repair  Christian Wetzler, Heiko Kämpfer, Birgit Stallmeyer, Josef Pfeilschifter, Stefan Frank  Journal of Investigative Dermatology  Volume 115, Issue 2, Pages 245-253 (August 2000) DOI: 10.1046/j.1523-1747.2000.00029.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Regulation of IL-1β and TNF-α expression during wound healing in control and db/db mice. Expression of inflammatory cytokines during wound healing in control mice (BALB/C, C57BLKS) and genetically diabetic mice (db/db) is shown for IL-1β in (A) and for TNF-α in (B) as indicated. Total cellular RNA (20 μg) from nonwounded and wounded back skin of control mice or diabetic mice was analyzed by RNase protection assay at the indicated time points after injury for the presence of IL-1β (A) and TNF-α (B) mRNA. Sixteen wounds (n = 16) from the backs of four animals were excised for each experimental time point and used for RNA isolation. Control skin refers to nonwounded skin of control and db/db mice. One representative experiment is shown. 1000 cpm of the hybridization probe were used as a size marker (probe). Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 MIP-2 and MCP-1 mRNA expression is dysregulated during repair in db/db mice. Total cellular RNA (20 μg) from normal and wounded back skin isolated from control mice (BALB/C, C57BLKS) and diabetic mice (db/db) was analyzed by RNase protection assay for expression of MIP-2 (A) and MCP-1 (B) as indicated. Sixteen wounds (n = 16) from the backs of four animals were excised for each experimental time point and used for RNA isolation. The time after injury is indicated at the top of each lane. Control skin refers to nonwounded skin of control and db/db mice. 1000 cpm of the hybridization probe were added to the lane labeled probe. Expression of GAPDH mRNA is shown as a loading control. The degree of MIP-2 and MCP-1 induction as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gels is shown schematically in the right panels. For the right panels, note that every experimental time point represents a total of 32 wounds (n = 32) from two independent animal experiments. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Elevated levels of MIP-2 and MCP-1 protein during impaired repair. Total protein (50 μg) from lysates of nonwounded and wounded back skin (day 1, 3, 5, 7, and 13 after injury, as indicated) isolated from control mice (BALB/C, C57BLKS) and db/db mice as indicated were analyzed by ELISA for the presence of MIP-2 (A) or MCP-1 (B) specific proteins. Eight wounds (n = 8) from the backs of four animals were excised for each experimental time point and used for protein isolation. Control skin refers to nonwounded back skin of control (BALB/C, C57BLKS) and db/db mice. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cellular sources of MIP-2, MCP-1, or CXCR2 expression at the wound site and colocalization of PMN and macrophages within the granulation tissue. A-H, frozen serial sections from 5 d mouse wounds isolated from control animals (BALB/C). Sections were incubated with polyclonal antibodies against murine MIP-2 (A, B), CXCR2 (C, D), MCP-1 (E, F), or with monoclonal antibodies against murine PMN-specific Gr-1 (G) or macrophage-specific F4/80 antigen (H) as indicated. All sections (A-H) were stained with the avidin-biotin-peroxidase complex system using 3-amino-9-ethylcarbazole as a chromogenic substrate. Nuclei were counterstained with hematoxylin. Scale bars: (A, C) 200 μm; (E, F) 100 μm; (B, D, G, H) 50 μm. Strongly immunopositive signals within the sections are indicated with arrows. g, granulation tissue; h, hair follicle; he, hyperproliferative epithelium; mac, macrophages; pmn, polymorphonuclear neutrophils. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Sustained presence of PMN and macrophages during the late phase of repair in db/db mice. Total protein (50 μg) from lysates of nonwounded and wounded back skin (day 1, 3, 5, 7, and 13 after injury, indicated at the top of each lane) isolated from control mice (BALB/C, C57BLKS) and db/db mice as indicated were analyzed by immunoblotting for the presence of the PMN-specific marker protein Gr-1 (A) and the macrophage-specific F4/80 antigen (B). Eight wounds (n = 8) from the backs of four animals were excised for each experimental time point and used for protein isolation. Control skin refers to nonwounded skin of control and db/db mice. Gr-1 or F4/80 protein is indicated by an arrow. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Localization of PMN and macrophages during late repair in normal wounds and wounds from diabetic mice. (A) and (C, BALB/C), frozen serial sections from 13 d mouse wounds isolated from BALB/C control animals. (B), (D), (E), (F, db/db), frozen serial sections from 13 d mouse wounds isolated from db/db animals. For (A), (B), (E), sections were incubated with a monospecific monoclonal antibody directed against murine PMN-specific Gr-1 as indicated. For (C), (D), (F), sections were incubated with a monospecific monoclonal antibody directed against murine macrophage-specific F4/80 antigen as indicated. Note that panels E and F represent directly neighbored serial 6 μm sections of the same wound. All sections (A-F) were stained with the avidin-biotin-peroxidase complex system using 3-amino-9-ethylcarbazole as a chromogenic substrate. Nuclei were counterstained with hematoxylin. Scale bars: (A-D) 50 μm; (E, F) 200 μm. Strongly immunopositive signals within the sections are indicated with arrows. e, epidermis; g, granulation tissue; mac, macrophages; pmn, polymorphonuclear neutrophils. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Topical treatment of 13 d wounds in db/db mice using neutralizing MIP-2 and MCP-1 antibodies results in reduced PMN and macrophage infiltration. Expression of PMN- (lipocalin, left panels) and macrophage- (lysozyme M, right panels) specific markers in 13 d wounds of vehicle- (+ PBS), or antibody- (+ anti-MIP-2/MCP-1) treated db/db mice. Total cellular RNA (20 μg) from PBS- or antibody-treated db/db animals was analyzed by RNase protection assay. Presence of the constitutively expressed lipocalin and lysozyme M mRNA expression was used as a readout for PMN (lipocalin) or macrophage (lysozyme M) cell numbers. Twelve wounds (n = 12) from the backs of three animals were excised for each experimental time point and used for RNA isolation. One representative experiment is shown. GAPDH mRNA is shown as a loading control. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Expressional regulation of the murine equivalent to human CXCR2 and murine CCR2 at the mRNA level during healing in normal and diabetic mice. Total cellular RNA (20 μg) from nonwounded and wounded back skin of control mice (BALB/C, C57BLKS) and diabetic mice (db/db) was analyzed by RNase protection assay for the presence of lipocalin and CXCR2 (A), or lysozyme M and CCR2 (B) mRNA expression as indicated by arrows. For every experimental time point, four wounds each from four animals (total of n = 16 wounds) were pooled for analysis. The time after injury is indicated at the top of each lane. Control skin refers to nonwounded skin. 1000 cpm of the hybridization probe were added to the lane labeled probe. Expression of GAPDH mRNA is shown as a loading control. Representative experiments are shown. (C) The degree of lipocalin and CXCR2 mRNA induction (left panel) and lysozyme M and CCR2 mRNA induction (right panel) as assessed by PhosphoImager analysis of the radiolabeled gels is shown schematically. Every experimental time point represents a total of 32 wounds (n = 32) from two independent animal experiments. Journal of Investigative Dermatology 2000 115, 245-253DOI: (10.1046/j.1523-1747.2000.00029.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions