Accelerated Wound Closure in Mice Deficient for Interleukin-10
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060370
ISSN1525-2191
AutoresSabine A. Eming, Sabine Werner, Philippe Bugnon, Claudia Wickenhauser, Lisa Siewe, Olaf Utermöhlen, Jeffrey M. Davidson, Thomas Krieg, Axel Roers,
Tópico(s)Pressure Ulcer Prevention and Management
ResumoThe impact of the local inflammatory response on the process of wound healing has been debated for decades. In particular, the question whether infiltrating macrophages and granulocytes promote or impede tissue repair has received much attention. In the present study, we show that wound healing is accelerated in mice deficient for the anti-inflammatory cytokine interleukin (IL)-10. IL-10−/− mice closed excisional wounds significantly earlier compared with IL-10-competent control littermates. This effect was attributable to accelerated epithelialization as well as enhanced contraction of the wound tissue in the mutant animals. Increased α-smooth muscle actin expression in IL-10-deficient mice suggests that augmented myofibroblast differentiation is responsible for the enhanced contraction of wounds in mutant mice. The number of macrophages infiltrating the wound tissue was significantly increased in IL-10−/− mice compared with control littermates suggesting that this cell type mediates the accelerated tissue repair. These results show for the first time that IL-10 can impede wound repair. The impact of the local inflammatory response on the process of wound healing has been debated for decades. In particular, the question whether infiltrating macrophages and granulocytes promote or impede tissue repair has received much attention. In the present study, we show that wound healing is accelerated in mice deficient for the anti-inflammatory cytokine interleukin (IL)-10. IL-10−/− mice closed excisional wounds significantly earlier compared with IL-10-competent control littermates. This effect was attributable to accelerated epithelialization as well as enhanced contraction of the wound tissue in the mutant animals. Increased α-smooth muscle actin expression in IL-10-deficient mice suggests that augmented myofibroblast differentiation is responsible for the enhanced contraction of wounds in mutant mice. The number of macrophages infiltrating the wound tissue was significantly increased in IL-10−/− mice compared with control littermates suggesting that this cell type mediates the accelerated tissue repair. These results show for the first time that IL-10 can impede wound repair. The process of wound repair requires a complex interplay of resident epithelial and mesenchymal cells with resident and recruited hematopoietic cells to accomplish the three stages of wound healing: inflammation, formation of new tissue, and tissue remodeling.1Martin P Wound healing—aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3762) Google Scholar The initial inflammatory phase is characterized by a local activation of innate immune mechanisms resulting in an initial influx of neutrophilic granulocytes into the damaged tissue followed by an accumulation of macrophages.1Martin P Wound healing—aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3762) Google Scholar The innate response of resident and recruited cells combats invading microbes but supposedly also critically influences the repair process by liberation of a wide spectrum of mediators. However, it remains unclear whether the inflammatory response hampers or accelerates wound repair and whether it affects the quality of the repaired tissues.2Eming SA, Krieg T, Davidson JM: Inflammation in wound repair: molecular and cellular mechanisms. J Invest Dermatol (in press)Google Scholar Interestingly, analyses of wound healing in a number of murine knockout models deficient for individual inflammatory mediators or their receptor including tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, and interferon (IFN)-γ have yielded heterogeneous results.3Werner S Grose R Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2625) Google Scholar Wound healing is accelerated in TNF-receptor-554Mori R Kondo T Ohshima T Ishida Y Mukaida N Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration.FASEB J. 2002; 16: 963-974Crossref PubMed Scopus (222) Google Scholar or IFN-γ-deficient mice5Ishida Y Kondo T Takayasu T Iwakura Y Mukaida N The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process.J Immunol. 2004; 172: 1848-1855PubMed Google Scholar but is impaired in mice deficient for IL-66Gallucci RM Simeonova PP Matheson JM Kommineni C Guriel JL Sugawara T Luster MI Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.FASEB J. 2000; 14: 2525-2531Crossref PubMed Scopus (330) Google Scholar or MCP-1.7Low QE Drugea IA Duffner LA Quinn DG Cook DN Rollins BJ Kovacs EJ DiPietro LA Wound healing in MIP-1alpha(−/−) and MCP-1(−/−) mice.Am J Pathol. 2001; 159: 457-463Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar IL-10 is an immunoregulatory cytokine that limits innate as well as adaptive immune responses protecting the host from immune-mediated tissue damage.8Moore KW de Waal Malefytqq R Coffman RL O'Garra A Interleukin-10 and the interleukin-10 receptor.Annu Rev Immunol. 2001; 19: 683-765Crossref PubMed Scopus (5322) Google Scholar In a variety of different cell types, IL-10 mediates down-regulation of a broad spectrum of proinflammatory mediators such as IL-1, IL-6, IL-12, IFN-γ, TNF-α, regulated on activation normal T cell expressed and secreted, MCP-1, macrophage inflammatory protein (MIP)-1α, IL-8, interferon-γ-inducible protein 10 (IP-10), and prostaglandin E2 (PGE2), whereas anti-inflammatory molecules such as IL-1 receptor antagonist (IL-1 ra) or soluble TNF-receptor are up-regulated.8Moore KW de Waal Malefytqq R Coffman RL O'Garra A Interleukin-10 and the interleukin-10 receptor.Annu Rev Immunol. 2001; 19: 683-765Crossref PubMed Scopus (5322) Google Scholar, 9Fiorentino DF Zlotnik A Mosmann TR Howard M O'Garra A IL-10 inhibits cytokine production by activated macrophages.J Immunol. 1991; 147: 3815-3822PubMed Google Scholar In IL-10−/− mice, uncontrolled Th1 responses to intestinal bacterial antigens result in the spontaneous development of inflammatory bowel disease.10Kühn R Löhler J Rennick D Rajewsky K Müller W Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3668) Google Scholar On infection with a number of different pathogens these animals develop severe immunopathology.11Kullberg MC Ward JM Gorelick PL Caspar P Hieny S Cheever A Jankovic D Sher A Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism.Infect Immun. 1998; 66: 5157-5166PubMed Google Scholar, 12Gazzinelli RT Wysocka M Hieny S Scharton-Kersten T Cheever A Kühn R Müller W Trinchieri G Sher A In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha.J Immunol. 1996; 157: 798-805PubMed Google Scholar, 13Deckert M Soltek S Geginat G Lutjen S Montesinos-Rongen M Hof H Schlüter D Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis.Infect Immun. 2001; 69: 4561-4571Crossref PubMed Scopus (68) Google Scholar, 14Namangala B Noel W De Baetselier P Brys L Beschin A Relative contribution of interferon-gamma and interleukin-10 to resistance to murine African trypanosomosis.J Infect Dis. 2001; 183: 1794-1800Crossref PubMed Scopus (102) Google Scholar The importance of IL-10 for the control of innate responses was demonstrated by an increased sensitivity of IL-10-deficient animals to lipopolysaccharide. IL-10−/− mice develop lethal septic shock because of overproduction of TNF-α after the intraperitoneal administration of 20-fold lower doses of lipopolysaccharide compared with wild-type animals.15Berg DJ Kühn R Rajewsky K Muller W Menon S Davidson N Grunig G Rennick D Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance.J Clin Invest. 1995; 96: 2339-2347Crossref PubMed Scopus (481) Google Scholar In addition, the local inflammatory response to lipopolysaccharide and the local response to bacterial DNA or CpG oligodeoxynucleotides follow a more vigorous course in IL-10−/− mice.16Siewe L, Bollati-Fogolin M, Wickenhauser C, Krieg T, Müller W, Roers A: Interleukin-10 derived from macrophages and/or neutrophils regulates the inflammatory response to LPS but not to CpG oligonucleotides. Eur J Immunol (in press)Google Scholar Likewise, the irritant response of the skin to tetradecanoylphorbol-acetate-containing irritants, which is a function of cutaneous innate immunity, is clearly enhanced in IL-10-deficient mice in comparison with wild-type animals.17Berg DJ Leach MW Kühn R Rajewsky K Müller W Davidson NJ Rennick D Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar A role for IL-10 in wound repair has been suggested by the results of several earlier studies. An analysis of IL-10 expression during wound healing revealed an early peak of IL-10 production 3 hours after wounding and a second peak 3 days after injury.18Sato Y Ohshima T Kondo T Regulatory role of endogenous interleukin-10 in cutaneous inflammatory response of murine wound healing.Biochem Biophys Res Commun. 1999; 265: 194-199Crossref PubMed Scopus (134) Google Scholar Keratinocytes at the wound margins and infiltrating mononuclear cells were identified as the major sources of IL-10 mRNA and protein. A function of IL-10 in repair was suggested by a study in which IL-10-neutralizing antibodies were locally applied to incisional wounds.18Sato Y Ohshima T Kondo T Regulatory role of endogenous interleukin-10 in cutaneous inflammatory response of murine wound healing.Biochem Biophys Res Commun. 1999; 265: 194-199Crossref PubMed Scopus (134) Google Scholar In this model, the infiltration of myeloid cells toward the site of injury, as well as the expression of chemokines and proinflammatory mediators was increased, indicating a suppressive role for IL-10 in wound inflammation. Furthermore, a study of fetal wound healing in a skin transplant model suggested that IL-10 may be responsible for the scarless tissue repair observed in fetal skin.19Liechty KW Kim HB Adzick NS Crombleholme TM Fetal wound repair results in scar formation in interleukin-10-deficient mice in a syngeneic murine model of scarless fetal wound repair.J Pediatr Surg. 2000; 35: 866-872Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar Herein we report that IL-10 deficiency results in accelerated wound closure. These results show for the first time that IL-10 can impede wound repair and support the view that the local inflammatory response can promote tissue repair. IL-10−/− mice10Kühn R Löhler J Rennick D Rajewsky K Müller W Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3668) Google Scholar on the C57BL/6 background were maintained and bred under standard pathogen-free conditions and genotyped by Southern blotting. Ten- to 12-week-old male IL-10−/− mice and IL-10wt/− or IL-10w/w control littermates (offspring from heterozygote breedings) were used for the experiments. Only healthy mice without any sign of inflammatory bowel disease were included in the study. Mice were anesthetized by intraperitoneal injection of Ketanest/Rompun (Ketanest S: Park Davis GmbH, Karlsruhe, Germany; Rompun 2%: Bayer, Leverkusen, Germany). The back was shaved and four circular excisional wounds of 6-mm diameter were generated that extended beyond the panniculus carnosus (full thickness wounds) using a standard biopsy punch (Stiefel, Offenbach, Germany). The four wounds on the back of one animal were at least 5 mm apart from each other. For histological analysis, mice were sacrificed, and an area of 8 μm in diameter, which included the complete epithelial margins, was excised. Wounds were bisected in caudocranial direction, and the tissue was either fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS) or embedded in OCT compound (Tissue Tek; Miles, Elkhart, IN), immediately frozen in liquid nitrogen, and stored at −80°C. Histological analysis was performed on serial sections from the central portion of the wound. For immunohistochemical staining of macrophages, paraffin sections (5 μm) were incubated with rat anti-mouse F4/80 antibody (MCA497GA; Serotec, Duesseldorf, Germany) at 4°C overnight. The section was then incubated with biotin-labeled polyclonal rabbit anti-rat Ig (DakoCytomation, Hamburg, Germany) for 30 minutes followed by incubation with streptavidin-conjugated horseradish peroxidase (30 minutes) and aminoethyl carbazole as chromogen (10 minutes). Nuclei were counterstained with hematoxylin. The naphthol-AS-d-chloroacetate esterase reaction was performed according to standard procedures (IHC World, Online Information Center For Histochemistry) for detection of neutrophilic granulocytes. To process tissue sections for the detection of CD31 (PECAM-1), 5-μm cryosections were fixed in acetone, endogenous peroxidase was inactivated (0.03% H2O2, 0.15 mol/L NaN3), and unspecific binding sites were blocked with 3% bovine serum in PBS. Sections were incubated with polyclonal rat antisera against murine CD31 (1 hour, room temperature, 1:500) (Pharmingen, Heidelberg, Germany); bound primary antibodies were detected using a peroxidase-conjugated goat anti-rat antibody (Southern Biotechnology, Birmingham, AL). Aminoethyl carbazole was used as a substrate and sections were counterstained with hematoxylin. For immunofluorescent staining, bound primary CD31 antibody was detected using an Alexa Fluor 488-conjugated polyclonal goat anti-rat antibody (1 hour, 1:500; Molecular Probes, Leiden, The Netherlands). For staining of α-smooth muscle actin (α-SMA) the cryosections were fixed in acetone, blocked with 3% bovine serum albumin in PBS, and incubated with Cy3-conjugated monoclonal anti-α-SMA antibody (1 hour, room temperature, 1:200; Sigma, St. Louis, MO). For vascular endothelial growth factor (VEGF)-A detection, 5-μm cryosections were fixed in 4% paraformaldehyde, blocked with 3% bovine serum albumin in PBS, and incubated overnight at room temperature with polyclonal rabbit anti-VEGF-A antibody (1:100, sc-507; Santa Cruz, Heidelberg, Germany), followed by detection using the DakoCytomation Envision system (labeled polymer horseradish peroxidase anti-rabbit; DakoCytomation) following the provider's instructions. For immunofluorescent staining of VEGF-A, sections were incubated with an Alexa Fluor 594-conjugated polyclonal goat anti-rabbit antisera (Molecular Probes). For immunofluorescent staining of macrophages, cryosections (5 μm) were incubated overnight at room temperature with rat anti-mouse F4/80 antibody (1:100, MCA497GA; Serotec), followed by detection using an Alexa Fluor 488-conjugated polyclonal goat anti-rat antibody (Molecular Probes). Double fluorescence was analyzed in a laser-scanning confocal microscope (True Confocal Scanner Leica TCS SL; Leica Microsystems, Heidelberg, Germany) at ×200 magnification (CD31 and α-SMA) or ×1000 magnification (VEGF-A and F4/80). Isotype-matched rat antibodies were used as negative controls. For Fizz1 and Ym1 detection, cryosections were incubated for 1 hour at room temperature with rabbit anti-Fizz1 (Peprotech, London, UK) or goat anti-Ym1 (R&R Systems, Wiesbaden, Germany) antibody (1:50); bound primary antibodies were detected using a peroxidase-conjugated anti-rabbit or anti-goat antibody (DakoCytomation), respectively. Organization and maturation of collagen bundles was assessed on paraffin sections of day 14 wounds stained by Masson trichrome or by polarized light microscopy after Sirius Red staining. Immunofluorescence/immunohistochemical microscopy was conducted at indicated magnifications (Microscope Eclipse 800E; Nikon, Düsseldorf, Germany). Morphometric analysis was performed on digital images using the Imaging Software Lucia G 4.80 (Laboratory Imaging Ltd., Prague, Czechoslovakia). The wound area was quantified by processing of photographs taken at various time points and was calculated as the percentage of the wound area immediately after surgery. The extent of epithelialization and granulation tissue formation was determined on hematoxylin and eosin (H&E)-stained paraffin tissue sections. The length of the epithelial tongue was determined as the distance that the neo-epithelium had migrated from the margin of the wound as defined by the presence of hair follicles in nonwounded skin. In addition, the width of the gap between the epithelial tips was measured. The distance between the edges of the panniculus carnosus was determined as a measure of wound contraction. Numbers of macrophages and neutrophilic granulocytes in the wound tissues were determined by counting cells in representative squares of 250 × 250 μm2. Three squares were selected at each wound margin and the center of the wound, on each of three serial sections per wound. The data are presented as the mean number of macrophages counted in the nine squares; three serial wound sections per wound were analyzed. For quantitative analysis of CD31 or α-SMA expression, the percentage of the area of granulation tissue, which stained positive for CD31 or α-SMA, was calculated. All histomorphometric analyses were performed in a blinded manner by two independent investigators. Mice were anesthetized and the dorsal region was shaved and treated with a depilatory agent (Pilca Perfect; Stafford-Miller Continental, Oevel, Belgium). Two full-thickness incisions (1 cm) were made at one anterior and one posterior dorsal site, and the skin margins were closed with strips of a wound plaster (Fixomull stretch; Beiersdorf, Hamburg, Germany). Mice were sacrificed on day 5 after wounding, and bursting strength of the wounds was determined in situ using the BTC-2000 system (SRLI Technologies, Nashville, TN) according to the manufacturer's protocol for the nonhuman disruptive linear incision analysis. The experiments were performed with permission from the local veterinary authorities of Zurich, Switzerland. Circular excisional wounds of 6-mm diameter were generated as described above. The mice were sacrificed and wound tissue as well as nonwounded back skin was excised, frozen, and immediately lyophilized. Amino acid composition of 6 N HCl-hydrolyzed tissue specimens was determined by phenylisothiocyanate derivatization and reverse phase high performance liquid chromatography, as previously described.20Buckley A Hill KE Davidson JM Collagen metabolism.Methods Enzymol. 1988; 163: 674-694Crossref PubMed Scopus (21) Google Scholar The percent collagen content was calculated based on the relative hydroxyproline and proline contents of collagenous and noncollagenous proteins. Statistical analyses were performed using SPSS version 12.0.2 (SPSS GmbH, München, Germany). Significance of difference was analyzed using the Mann-Whitney U-test for non-Gaussian distribution and the unpaired t-test for Gaussian distribution. All data are presented as mean ± SD. A P value less than 0.05 was considered significant. If several statistical tests will be performed with an unadjusted type I error rate, the P values may only be interpreted in an explorative way. Full-thickness wounds (extending beyond the panniculus carnosus, ie, the subcutaneous muscle layer of murine skin) were generated by circular excisions of 6-mm diameter on the shaved back of IL-10−/− (20 wounds on five animals) or control mice (20 wounds on five animals). Macroscopic wound closure was accelerated in IL-10−/− mice compared with control animals (Figure 1A). On day 7 after wounding, the wounds of the mutant mice had already lost their eschar and appeared completely epithelialized, whereas the control wounds showed only partial epithelialization and were still carrying a scab (Figure 1A). Measurement of the wound area on digital images showed that the differences between mutant and control mice were statistically significant on days 3, 5, and 7 after wounding (P < 0.01) (Figure 1B). These macroscopic findings were confirmed by histological assessment of epithelialization (Figure 2A). Mutant and control animals were sacrificed on each of days 1, 3, 5, 7, and 14 after injury and the wound tissue (eight wounds on three mice per time point for each group) was excised. On H&E-stained paraffin sections, representing the longitudinal diameter of the wound, significantly shorter distances between the tips of the epithelial tongues were measured for the wounds of IL-10−/− mice compared with control animals on days 3, 5, and 7 after wounding (P < 0.04) (Figure 2B). Likewise, the length of the epithelial tongues (see Materials and Methods) was significantly increased in IL-10−/− mice in comparison with control mice on day 5 after injury (P < 0.018) (Figure 2C). These findings show that enhanced epithelialization contributed to the accelerated wound closure of IL-10-deficient mice. To analyze dermal repair, we determined granulation tissue formation and angiogenesis in wound tissue of mutant and control mice. Differences in the amount of granulation tissue were analyzed in H&E-stained sections and were shown to be not statistically different between wounds of mutant and control mice (not shown). Morphometric quantification of the expression of the endothelial cell marker CD31 within the area of granulation tissue was used as read-out for neovascular processes at the wound site. In knockout and control mice, vascular density within the granulation tissue increased during the healing response, peaking at day 5 after wounding (Figure 3A). At day 3 after wounding in IL-10-deficient mice (four wounds on two animals), the density of vascular structures was significantly increased when compared with control mice (three wounds in two animals), reflecting an accelerated angiogenic response during the early phase of repair (P < 0.04) (Figure 3, A–C). After day 3, the density of vascular structures at the wound site was not significantly different in knockout and control mice. To identify factors that might mediate the accelerated vascular response in mutant mice, we stained wound tissue for VEGF-A, one of the most potent angiogenic mediators. Numerous mononuclear leukocytes were detected that stained positive for VEGF-A within the early granulation tissue of knockout mice (Figure 3, D and E). Double staining for F4/80 and VEGF-A indicated that macrophages present the major fraction of VEGF-A expressing mononuclear cells in early granulation tissue of mutant mice (Figure 3F). These results suggest that macrophage-derived VEGF-A contributes to the accelerated angiogenic response in IL-10 knockout mice.Figure 2IL-10 deficiency accelerates epithelialization. A: H&E staining of wounds of IL-10−/− and control mice at indicated time points after injury. In IL-10−/− mice, the length of the epithelial tongue was increased when compared with control mice. Correspondingly, the distance between the epithelial tips is reduced in knockout versus control mice. Whereas in control mice, the day 5 wound is filled with a fibrin-rich clot, the clot has been replaced by a cellular and highly vascularized granulation tissue in IL-10−/− mice; arrows point to the tips of epithelial tongues, arrowheads indicate wound edges defined by the presence of hair follicles in nonwounded skin. B: Distance between the epithelial tips (*P < 0.04); C: length of the epithelial tongue (*P < 0.018); D: distance between the edges of the panniculus carnosus (*P < 0.001) in wounds of IL-10−/− and control mice. Data are expressed as mean ± SD, n = 8 wounds for each time point and group. e, epidermis; d, dermis; f, subcutaneous fat layer; pc, panniculus carnosus; g, granulation tissue. Scale bars: 1 mm (left); 250 μm (right). Original magnifications: ×12.5 (A, left); ×50 (A, right).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Angiogenesis in wounds of IL-10−/− and control mice. A: Computer-assisted morphometric quantification of CD31-positive cells within the granulation tissue of IL-10−/− and control mice during healing (*P < 0.04); data are expressed as mean ± SD, n = 3 wounds for each time point and group. B and C: CD31-stained blood vessels (red) 3 days after wounding; microvascular density is significantly increased in early granulation tissue of IL-10−/− mice when compared with control mice; arrows indicate the epithelial wound edge. D and E: VEGF-A staining (red) 3 days after wounding; arrows indicate VEGF-A-expressing mononuclear cells within the granulation tissue of control (D) and mutant mice (E). F: Double labeling for F4/80 (green) and VEGF-A (red) in granulation tissue of IL-10−/− mice indicates VEGF-A expression in macrophages. e, epidermis; d, dermis. Scale bars: 100 μm (B, C); 50 μm (D, E). Original magnifications, ×1000 (F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) H&E staining (Figure 2A) as well as staining for the neutrophil-specific enzyme chloroacetate esterase (not shown) demonstrated a substantial influx of neutrophilic granulocytes into day 1 and day 3 wound tissue of mutant and control mice. Quantitative evaluation did not reveal significant differences in neutrophil numbers between mutant and control mice (not shown). In contrast, immunostaining of wound tissue for the macrophage marker F4/80 demonstrated that the influx of these cells occurred earlier, that they persisted longer at the wound site, and that the number of infiltrating macrophages was increased at the wound site of IL-10−/− mice in comparison with control wounds (Figure 4A). Thus, significantly more macrophages were counted in IL-10 mutant mice compared with controls on day 3 through day 14 after wounding (Figure 4B) (eight wounds from three animals for each experimental group, P < 0.024). To phenotypically characterize the macrophages infiltrating the wound tissue, we analyzed wound sections for the expression of Fizz1 (found in inflammatory zone)/RELM-α and Ym1/ECF (eosinophil chemotactic factor). Expression of both factors was recently described as a reliable marker of alternatively activated macrophages.21Raes G De Baetselier P Noel W Beschin A Brombacher F Hassanzadeh G Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages.J Leukoc Biol. 2002; 71: 597-602PubMed Google Scholar, 22Nair MG Cochrane DW Allen JE Macrophages in chronic type 2 inflammation have a novel phenotype characterized by the abundant expression of Ym1 and Fizz1 that can be partly replicated in vitro.Immunol Lett. 2003; 85: 173-180Crossref PubMed Scopus (187) Google Scholar In wound tissue 5 days after injury, both control (three wounds on three animals) and IL-10-deficient wound tissue (three wounds on three animals) contained numerous Fizz1- and Ym1-positive cells (Figure 5, A–D). Double staining for F4/80 and Fizz1 or Ym1 revealed that most cells expressing these markers were macrophages (Figure 5G). The fraction of Fizz1- or Ym1-positive macrophages was similar in knockout and control animals. To investigate whether, in addition to more rapid epithelialization, enhanced contraction of the wound tissue also contributed to the accelerated wound closure of IL-10-deficient mice, we measured the distance between the edges of the panniculus carnosus at the wound margins (Figure 2D). This distance was significantly shorter in wounds from IL-10 mutant mice than in the control wounds on day 3 and 5 after wounding (P < 0.001). In 5-day old wounds of mutant and control mice, immunostaining for α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, revealed that α-SMA was abundantly expressed at the wound margins and to a lesser extent in the wound bed (Figure 6A). In day 7 wounds, α-SMA was present throughout the entire layer of the late granulation tissue that had developed beneath the neoepidermis and which extended into deep dermal layers (Figure 6A). Although the pattern of α-SMA expression during repair was similar in both experimental groups, in day 5 and day 7 wounds from IL-10 mutant mice, the number of α-SMA-positive cells as well as the staining intensity of positive cells was significantly increased (Figure 6B), suggesting that augmented myofibroblast differentiation is responsible for the enhanced contraction of wounds from IL-10-deficient animals (five wounds from three animals for each experimental group per day, P < 0.03). To evaluate whether the increased staining for α-SMA in IL-10−/− wounds might be attributable to an increased number of vascular structures coated with α-SMA-positive perivascular cells, we performed immunofluorescent double labeling for CD31 and α-SMA. At days 5 and 7 after wounding, both IL-10−/− and control mice showed a highly vascularized granulation tissue (Figure 6C). In wounds of both mouse strains, the tissue between capillary structures stained positive for α-SMA, indicating a nonendothelial cell origin of this staining and suggesting the presence of myofibroblasts. To analyze whether the accelerated wound healing observed in IL-10-deficient mice results in altered biomech
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