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The Journal of Immunology, 2004, 172: 1848-1855.
Copyright © 2004 by The American Association of Immunologists

The Essential Involvement of Cross-Talk between IFN-{gamma} and TGF-{beta} in the Skin Wound-Healing Process1

Yuko Ishida*,{dagger}, Toshikazu Kondo{dagger}, Tatsunori Takayasu{ddagger}, Yoichiro Iwakura§ and Naofumi Mukaida2,*

* Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; {dagger} Department of Legal Medicine, Wakayama Medical University, Wakayama, Japan; {ddagger} Department of Forensic and Social Environmental Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan; and § Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several lines of in vitro evidence suggest the potential role of IFN-{gamma} in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-{gamma} in the skin wound healing process utilizing WT and IFN-{gamma} KO mice. In WT mice, excisional wounding induced IFN-{gamma} mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-{gamma} KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-{gamma} KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-{gamma} KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-{beta}1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-{gamma} augmented the TGF-{beta}1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-{gamma}/Stat1 and TGF-{beta}1/Smad signaling pathways in the wound healing process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Skin wound healing starts immediately after an injury and consists of three phases; inflammation, proliferation, and maturation. These phases proceed with a complicated but well organized interaction among various types of tissues and cells (1, 2). During the inflammatory phase, platelet aggregation at the injury site is followed by infiltration of leukocytes, including neutrophils and macrophages, into the wound site. In the proliferative phase, re-epithelialization and newly formed granulation tissue begin to cover the wound area to repair tissue destruction. Moreover, collagen deposition is indispensable for granulation tissue formation and accumulating evidence implicates TGF-{beta}1 as one of the essential factors that can regulate collagen deposition (3, 4, 5, 6). However, the mechanisms regulating the production and activity of TGF-{beta}1 in vivo remain elusive.

IFN-{gamma} is mainly produced by NK cells and CD4+ Th1 cells and has multiple effects on macrophages, NK cells, and T lymphocytes (7). Moreover, IFN-{gamma} can inhibit collagen synthesis by fibroblasts in vitro (8, 9, 10, 11, 12). In line with these observations, the administration of exogenous IFN-{gamma} impaired collagen accumulation and disrupted wound strength, suggesting that IFN-{gamma} was deleterious to skin wound healing (13, 14, 15, 16). However, the role of endogenous IFN-{gamma} in the skin wound healing process remains to be investigated.

After binding its specific receptor on the cell surface, IFN-{gamma} activates receptor-associated Janus kinases, leading to the phosphorylation of specific tyrosine residues of Stat1 (17, 18). Stat1 mediates the biological activity of IFN-{gamma} by inducing the transcription of the target genes (19, 20). Accumulating evidence suggests that the IFN-{gamma}/Stat1 system can modulate TGF-{beta}1 activity in vitro by interfering with its signaling molecules, the Smad proteins (21, 22). However, it remains to be investigated whether or not there is crosstalk between the IFN-{gamma}/Stat1 and TGF-{beta}1/Smad signaling pathways in vivo, particularly in pathological conditions. In this report, we investigated the role of endogenous IFN-{gamma} in the skin wound healing process, particularly focusing on its interaction with the TGF-{beta}1/Smad system. In this study, we provided the first definitive evidence to indicate that endogenous IFN-{gamma} can negatively regulate the TGF-{beta}1 signaling pathway at wound sites in vivo, resulting in a retardation of the wound healing process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

The following mAb or polyclonal Abs (pAbs)3 and recombinant protein were used in this study; rat anti-mouse F4/80 mAb and rat anti-mouse CD3 mAb (Dainippon Pharmaceutical Company, Osaka, Japan), rat anti-mouse IFN-{gamma} mAb (clone XMG 1.2) and rat anti-mouse CD31 mAb (BD PharMingen, San Diego, CA), rabbit anti-myeloperoxidase (MPO) pAb (Neomarkers, Fremont, CA, USA), goat anti-mouse Smad2 pAb, rabbit anti-Smad3 pAb, goat anti-mouse Smad7 pAb (Santa Cruz, CA, USA), rabbit anti-phosphorylated Smad 2 (p-Smad 2) pAb (Upstate, USA), mouse anti-Stat-1 mAb, and mouse anti-p-Stat-1 mAb (Transduction Laboratories, Burlington, CA, USA), mouse anti-{alpha}-smooth muscle actin (SMA) mAb (clone asm-1, Boehringer Mannheim GmbH, Mannheim, Germany), rat anti-mouse IFN-{gamma} neutralizing mAb (a kind gift of Dr. H. Fujiwara, Osaka University), and recombinant murine IFN-{gamma} (PeproTech, London, U.K.).

Mice

Pathogen-free eight- to 12-wk old male BALB/c mice were obtained from Sankyo Laboratories (Tokyo, Japan) and designated as WT mice in the following experiments. IFN-{gamma} KO mice, backcrossed to BALB/c mice for more than eight generations, were used in the experiments (23, 24). Age-matched male BALB/c-SCID mice were purchased from CLEA Japan, Inc (Tokyo, Japan). All of the mice were used for the experiments complied with the standards set out in the Guidelines for the Care and Laboratory Animals at the Takara-machi Campus of Kanazawa University and housed individually in cages under specific pathogen-free conditions during the whole course of the study.

Excisional wound preparation and macroscopic examination

Mice were anesthetized with i.p. administration of pentobarbital (50 µg/g weight), and full-thickness skin wounds were made in the dorsal skin under sterile conditions as described previously (25). Briefly, after shaving and cleaning with 70% ethanol, excisional full-thickness skin wounds were made in the dorsal skin by picking up a fold skin at the midline and punching through two layers of skin with a sterile disposable biopsy punch (diameter of 4 mm, Kai Industries, Tokyo, Japan). Two wounds with a diameter of 4 mm were made at the same time, one wound on each side of midline. The same procedure was repeated on the same animals three times, generating six wounds, with three wounds at each side. Each wound site was digitally photographed at the indicated time intervals, and wound areas were determined on photographs using PhotoShop (Version 7.0 Adobe Systems, Tokyo, Japan) without a prior knowledge of the experimental procedures. Changes in wound areas were expressed as the percentage of the initial wound areas. In another series of experiments, WT mice received i.p. injection of neutralizing anti-IFN-{gamma} mAb or control IgG (250 µg/mouse) once a day from Day 0 to 3, starting immediately after the wound preparation. In some experiments, wounds and their surrounding areas, including the scab and epithelial margins, were cut with a sterile disposable biopsy punch (diameter 8 mm, Kai Industries, Tokyo, Japan) at the indicated time intervals.

Histopathological analyses of wound sites

At the indicated intervals after the injury, wound specimens were removed and fixed in 4% formaldehyde buffered with PBS (pH 7.2) and then embedded with paraffin. Six-µm thick sections were stained with hematoxylin and eosin for histological analysis. Immunohistochemical analyses were performed for the evaluation of leukocyte infiltration, angiogenesis, and IFN-{gamma} expression as described previously (25). A double-color immunofluorescence analysis was also conducted to identify the types of IFN-{gamma}-expressing cells and p-Smad2-positive cells, as described previously (26). In some experiments, the anti-IFN-{gamma} mAb was incubated with the indicated concentration of recombinant mouse IFN-{gamma} at 4°C overnight before use.

MPO assay

Myeloperoxidase activity was measured to evaluate neutrophil recruitment (25). Briefly, the excised wound samples were washed in PBS and homogenized in 1 ml of 50 mM potassium phosphate buffer solution with 0.5% hexadecyl trimethyl ammonium bromide (Sigma-Aldrich, St. Louis, MO) and 5 mM EDTA. The samples were sonicated for 20 s, freeze-thawed three times, and centrifuged at 12,000 rpm at 4°C. MPO activities in the supernatants were assayed using the SUMILON peroxidase assay kit (Sumitomo Bekuraito, Tokyo, Japan), according to the manufacturer’s instructions. The data were expressed as absorbance divided by total dry weight (mg).

Measurement of hydroxyproline (HP) contents at wound sites

At the indicated time intervals after the injury, skin wound sites were removed using a sterile disposable biopsy punch (diameter 8 mm) and were dried for 16 h at 120 °C. As HP is a major constituent of and found almost exclusively in collagen, HP contents were measured as the index of collagen accumulation at the wound sites, as described previously (27). HP content was calculated by comparison to standards and expressed as the amount (µg) per wound.

Extraction of total RNAs and RT-PCR

Total RNAs were extracted from uninjured and injured skin samples using ISOGENE (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. Five µg of total RNA was reverse-transcribed at 42°C for 1 h in 20 µl reaction mixture containing mouse Moloney leukemia virus reverse transcriptase (Toyobo, Osaka, Japan) with oligo(dT) primers (Amersham-Pharmacia Biotech Japan, Tokyo, Japan). The resultant cDNAs were amplified together with Taq polymerase (Nippon Gene) using specific sets of primers for IFN-{gamma}, IL-12p35, IL-12p40, IL-18, vascular endothelial growth factor (VEGF), COL1A1, and {beta}-actin (Table I). PCR amplification of each gene was conducted with the optimal cycles consisting of 94°C for 1 min, optimal annealing temperature shown in Table I for 1 min, and 72°C for 1 min, followed by incubation at 72°C for 3 min. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities were measured using Image Analysis software (version 1.61; National Institutes of Health, Bethesda, MD) and the ratios to {beta}-actin were calculated (23, 25).


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  		Table I. Sequences of the primers used for RT-PCRa

 
Western blotting

At the indicating time intervals after the injury, wound samples were homogenized with a lysis buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton, 1 mM EDTA) containing Complete Protease Inhibitor Cocktail (Roche, Tokyo, Japan), and Phosphatase Inhibitor Cocktails for serine/threonine protein phosphatases and tyrosine protein phosphatases (P2850 and P5726; Sigma-Aldrich) and centrifuged to obtain lysates. The lysates (30 µg) were electrophoresed in a 10% SDS-polyacrylamide gel and transferred onto a nylon membrane. The membrane was then incubated with Abs to TGF-{beta}1, Stat1, p-Stat1, Smad2, p-Smad2, Smad3, or Smad7 diluted at 1: 1,000. After the incubation of HRP-conjugated secondary Abs, the immune complexes were visualized using ECL® System (Amersham, Japan) according to the manufacturer’s instructions.

Statistical analysis

The means and SEMs were calculated for all parameters determined in this study. Statistical significance was evaluated by using ANOVA or Mann-Whitney’s U test. p < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma}, IL-12, and IL-18 expression during wound healing

In our initial experiments, we examined IFN-{gamma} expression at the skin excisional wound sites. In uninjured skin of WT mice, IFN-{gamma} mRNA was only weakly expressed (Fig. 1a). IFN-{gamma} mRNA expression increased significantly 3 days after the injury and remained elevated until 6 days after the injury (Fig. 1b). Moreover, there were no significant differences in IFN-{gamma} gene expression between WT and SCID mice, suggesting that non-lymphoid cells were a major cellular source of IFN-{gamma} at the skin wound sites (Fig. 1, a and b). Immunohistochemical analysis revealed that IFN-{gamma} protein was very low in wound sites of WT mice 1 day after the injury (Fig. 2a). In contrast, a large number of cells were positive for IFN-{gamma} at wound sites 3 and 6 days after injury (Fig. 2, b and c). Preadsorption of the Ab with an excess amount of IFN-{gamma} abolished the positive signals (Fig. 2d), indicating the specificity of the reaction. A double-color immunofluorescence analysis demonstrated that IFN-{gamma}-positive cells were also positive for F4/80 at 3 days and 6 days after injury (Fig. 3a). IFN-{gamma}-positive and F4/80-negative cells were judged as resident fibroblasts based on their morphology. At 6 days after the injury, a few CD3-positive cells were also positive for IFN-{gamma} (Fig. 3b). Considering that IFN-{gamma} gene was expressed to a similar extent at the wound sites of WT and SCID mice, these observations suggest that non-lymphoid cells were a major cellular source of IFN-{gamma} in skin wound healing. We also analyzed IL-12 and IL-18 gene expression, which when combined can induce IFN-{gamma} production in macrophages (28, 29, 30). The expression of both genes was enhanced to similar levels at skin wound sites in WT and SCID mice (Fig. 1, a and c–e). These results indicate that F4/80-positive macrophages and to a lesser degree, T cells, might produce IFN-{gamma} 3 days after injury, under the combined effects of IL-12 and IL-18.



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  		FIGURE 1. The analysis of the gene expression of IFN-{gamma}, IL-12p35, IL-12p40, and IL-18 at excisional skin wound sites of WT and SCID mice. a, RT-PCR analysis for gene expression of these cytokines. RT-PCR was performed as described in Materials and Methods and representative results from six independent experiments are shown in a. Under the conditions used, mRNA of all cytokines was weakly detected in the uninjured skin. The ratios of IFN-{gamma} (b), IL-12p35 (c), IL-12p40 (d), and IL-18 (e) to {beta}-actin at the wound sites of WT ({square}) and SCID mice ({blacksquare}) were determined by RT-PCR at 1, 3, and 6 days after the injury. Each value represents mean ± SEM (n = 6). *, p < 0.05' vs uninjured skin of BALB/c; #, p < 0.05' vs uninjured skin of SCID.

 


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  		FIGURE 2. Immunohistochemical analysis of IFN-{gamma} protein expression in skin wound sites. Skin wound samples were obtained from WT at days 1 (a), 3 (b and d), and 6 (c) after the wound preparation. Samples were immunostained with either untreated anti-IFN-{gamma} mAb (a–c) or that preadsorbed with an excess amount of rIFN-{gamma} (d) as described in Materials and Methods. Representative results from three independent experiments are shown here. Original magnification, x100.

 


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  		FIGURE 3. A double-color immunofluorescence analysis of wound sites. Wound sites were obtained from WT mice at 3 (a) or 6 (b) days after the injury. The samples were immunostained with anti-F4/80 (a-i, Cy3), anti-CD3 (b-i, Cy3), or anti-IFN-{gamma} mAb (a-ii and b-ii, FITC) as described in Materials and Methods and observed under a fluorescence microscopy (original magnification, x100). Signals in i and ii were digitally merged in panels iii. Representative results from three independent experiments are shown.

 
Macroscopic wound closure in IFN-{gamma} KO and WT mice

To evaluate the pathophysiological role of locally produced IFN-{gamma} in the wound healing process, we made excisional skin wounds in IFN-{gamma} KO and WT mice. In IFN-{gamma} KO mice, the wound areas were reduced to 40% at 3 days after injury. In contrast, wound areas in WT mice still remained at 50% even 6 days after injury (Fig. 4). Furthermore, the administration of a neutralizing anti-IFN-{gamma} mAb also increased the wound closure rates in WT mice (Fig. 5). These observations indicate that wound closure and subsequent wound healing were accelerated in the absence of IFN-{gamma}.



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  		FIGURE 4. Macroscopic changes in skin excisional wound sites. a, The wound sites were photographed at the time indicated. Day 0 picture was taken immediately after the injury. Representative results from 12 individual animals in each group are shown here. b, Changes in percentage of wound area at each time point in comparison to the original wound area. Values represent mean ± SEM. {square}, WT; {blacksquare}, IFN-{gamma} KO (n = 12 animals). *, p < 0.05; **, p < 0.01, IFN-{gamma} KO compared with WT.

 


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  		FIGURE 5. Macroscopic appearance of wound healing process in WT mice administered with a control or an anti-IFN-{gamma} mAb. a, The wound sites were photographed at the time indicated. Day 0 picture was taken immediately after the injury. Representative results from 12 individual animals in each group are shown. b, Changes in percentage of wound area at each time point in comparison to the original wound area in WT mice administered with a control or an anti-IFN-{gamma} mAb. Values represent mean ± SEM. {square}, WT treated with control Ig G (IgG); {blacksquare}, WT treated with anti-IFN-{gamma} mAb (n = 12 animals). *, p < 0.05; **, p < 0.01, anti-IFN-{gamma} mAb compared with control.

 
Leukocyte infiltration at the wound sites in IFN-{gamma} KO and WT mice

We next examined the effects of IFN-{gamma} deficiency on leukocyte infiltration at the excisional wound sites. Consistent with our previous observations, neutrophil infiltration in WT mice was maximal at 1 day after injury. In contrast, macrophages and CD3-positive cells started to accumulate at 1 day after injury and reached maximal levels at 6 days after injury. The infiltration of these cells was remarkably attenuated in IFN-{gamma} KO mice, compared with WT mice, at every time interval examined. Only macrophage infiltration 1 day after injury was similar in WT and IFN-{gamma} KO mice (Fig. 6).



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  		FIGURE 6. Immunohistochemical analyses on leukocyte recruitment in skin excisional wound sites. a–f, Immunohistochemical analysis was performed using anti-MPO at day 1 (a and d), anti-F4/80 at day 6 (b and e), or anti-CD3 Abs at day 6 (c and f) in skin wound samples from WT (a–c) and IFN-{gamma} KO (d–f) mice (x200). Representative results from three independent experiments are shown here. g, MPO activity at the wound site of IFN-{gamma} KO ({blacksquare}) and WT ({square}) was determined to evaluate neutrophil accumulation. The numbers of macrophages (h) or those of T cells (i) per a high-power microscopic field (original magnification, x200) were counted. All values represent the mean ± SEM (n = 6 animals). *, p < 0.05, IFN-{gamma} KO compared with WT.

 
Angiogenesis and VEGF gene expression at the wound sites in IFN-{gamma} KO and WT mice

We next examined the effects of IFN-{gamma} on the angiogenic process; one of the important events in the proliferative phase of wound healing (Fig. 7). No significant difference was observed in the vessel density of the uninjured skin when WT and IFN-{gamma} KO mice were compared (2.2 ± 0.4% vs 2.7 ± 0.4%) as measured by CD31-positive areas. Six days after the injury, the vessel density within the wound bed was increased in both the WT and IFN-{gamma} KO mice, and the vessel density of IFN-{gamma} KO mice was significantly higher than WT mice (Fig. 7, a–e). VEGF mRNA was weak but similar in uninjured skin of both WT and IFN-{gamma} KO mice. VEGF mRNA expression was enhanced at the wound sites in both mice 3 days after injury but the enhancement was significantly greater in IFN-{gamma} KO than WT mice (Fig. 7, f and g). These observations imply that the lack of IFN-{gamma} may augment angiogenesis in skin wound sites, partly by enhancing VEGF expression.



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  		FIGURE 7. a–d, Immunohistochemical analyses on excisional skin wound sites of WT (a and c) and IFN-{gamma} KO mice (b and d) at 6 days after injury. The sections were stained with a mAb for the endothelium (CD31) (a and b, x10; c and d, x100). Representative results from six independent animals in each group are shown. e, Vascular areas were determined as CD31-positive areas in IFN-{gamma} KO ({blacksquare}) and WT mice ({square}) mice with the help of PhotoShop. All values represent the mean ± SEM (n = 6 animals). *, p < 0.05, IFN-{gamma} KO compared with WT. f and g, RT-PCR analysis of VEGF gene expression at wound sites in WT and IFN-{gamma} KO mice. Representative results from 10 independent animals are shown in f. Under the conditions used, VEGF mRNA was faintly detected in uninjured skin samples of WT and IFN-{gamma} KO mice. The ratios of VEGF to {beta}-actin of WT ({square}) and IFN-{gamma} KO mice ({blacksquare}) were determined by RT-PCR and are shown in g. Each value represents mean ± SEM (n = 10 animals). *, p < 0.05; **, p < 0.01, IFN-{gamma} KO compared with WT.

 
Granulation tissue formation at the wound sites in IFN-{gamma} KO and WT mice

We next explored the effects of IFN-{gamma} on collagen content in the extracellular matrix, another crucial molecule for the wound healing process. Histopathologically, at 3 days and after, granulation tissue was evident at wound sites in WT mice (Fig. 8a) and the granulation tissue formation was more prominent at the wound sites in IFN-{gamma} KO mice (Fig. 8b). In uninjured skin, there was no significant difference in terms of HP content and COL1A1 mRNA expression between WT and IFN-{gamma} KO mice (Fig. 8, c–e). In WT mice, HP content and COL1A1 mRNA expression at the wound sites started to increase progressively 3 days after injury. However, the increases in HP content and COL1A1 mRNA expression at the wound sites were consistently and significantly higher in IFN-{gamma} KO mice than WT mice (Fig. 8, c–e). These observations indicate that the absence of IFN-{gamma} augmented collagen gene expression and eventually collagen production at the wound sites.



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  		FIGURE 8. a and b, Histopathological analyses on skin wound sites of WT (a) and IFN-{gamma} KO (b) mice at 6 days after injury. Granulation tissue formation was more evident in IFN-{gamma} KO mice than in WT mice. c, HP contents in the excisional wound sites in WT ({square}) and IFN-{gamma} KO ({blacksquare}) mice. HP contents were determined as an indicator of collagen contents. All values represent the mean ± SEM (n = 6 animals). *, p < 0.05, IFN-{gamma} KO compared with WT. d and e, RT-PCR analysis of collagen gene expression in the wound sites in WT and IFN-{gamma} KO mice. Under the conditions used, RT-PCR analysis did not detect the mRNA of COL1A1 in uninjured skin samples of WT and IFN-{gamma} KO mice. Representative results from six animals in each group are shown in d. The ratios of COL1A1 to {beta}-actin of WT ({square}) and IFN-{gamma} KO ({blacksquare}) were determined by RT-PCR at 1, 3 and 6 days after injury (e). Each value represents mean ± SEM (n = 6 animals). *, p < 0.05; **, p < 0.01, IFN-{gamma} KO compared with WT.

 
The effects of IFN-{gamma} deficiency on the TGF-{beta}1-mediated signaling pathway

As TGF-{beta}1 has been considered to be a major regulator of collagen biosynthesis (3, 4, 5, 6), we examined the changes in the TGF-{beta}1 signaling pathway at the wound sites (Fig. 9). The amounts of both total and phosphorylated Stat1 were increased at the wound sites of WT mice 1 day after injury. In contrast, the amount of total and phosphorylated Stat1 was not significantly changed at the wound sites of IFN-{gamma} KO mice, due to the absence of IFN-{gamma}-mediated signals. The amount of TGF-{beta}1 protein was increased at the wound sites although the increase was more marked in IFN-{gamma} KO mice than WT mice. Moreover, although total Smad7 levels were increased in the wound sites of WT mice, a corresponding increase in Smad7 levels was not observed in IFN-{gamma} KO mice. Although wound injury increased the amount of Smad3 to similar extents in the wound sites of both WT and IFN-{gamma} KO mice, the amounts of total and phosphorylated Smad2 remained at similar levels at the wound sites in WT mice. In contrast, the amount of total and phosphorylated Smad2 was strongly increased in the wound sites of IFN-{gamma} KO mice. A double color immunofluorescence analysis demonstrated that phosphorylated Smad2 was detected mainly in {alpha}-SMA-positive fibroblasts, which are presumed to express IFN-{gamma} receptors (Fig. 10). Thus, IFN-{gamma} may negatively regulate TGF-{beta}1 signaling pathway by down-regulating the expression of TGF-{beta}1 and its downstream intracellular molecules at the wound sites.



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  		FIGURE 9. Western blotting analysis on the expression of TGF-{beta}, Stat1, phosphorylated Stat1, Smad2, phosphorylated Smad2, Smad3, and Smad7, at the wound sites. Under the conditions used, these molecules were faintly detected in uninjured skin sites of WT and IFN-{gamma} KO mice. Western blotting analysis using anti-{alpha}-tubulin Ab confirmed that an equal amount of protein was loaded onto each lane. Representative results from six individual animals in each group are shown.

 


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  		FIGURE 10. A double-color immunofluorescence analysis of phosphorylated Smad2-positive cells in skin excisional wound site at 6 days after the injury in WT mice. A double-color immunofluorescence analysis was performed using anti-{alpha}-SMA (i, Cy3) and anti-pSmad2 Abs (ii, FITC) and observed under a fluorescence microscope (x400). Signals were digitally merged in panel iii (derived from i and ii). Representative results from six individual animals are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFN-{gamma} has pleiotropic actions on various types of immune cells and has been implicated as one of main regulatory factors for CD4+ Th1 polarization (7). In addition to its immunoregulatory actions, IFN-{gamma} exerts multiple effects on non-immune cells, particularly fibroblasts. Several lines of evidence have demonstrated that IFN-{gamma} can inhibit collagen synthesis by fibroblasts (8, 9, 10, 11, 12) and accumulating evidence suggests that administration of exogenous IFN-{gamma} impairs skin wound healing (13, 14, 15, 16). In this report, we examined the roles of endogenous IFN-{gamma} in wound healing process, by using IFN-{gamma} KO mice. We found that the lack of endogenous IFN-{gamma} significantly accelerated wound healing process as evidenced by rapid wound closure and enhanced granulation tissue formation.

In WT mice, we observed enhanced IFN-{gamma} mRNA and protein expression at the skin excisional wound sites. A double-color immunofluorescence analysis detected IFN-{gamma} protein in F4/80-positive macrophages recruited to the wound sites. Several lines of evidence have demonstrated that the combined stimulation of IL-12 and IL-18 induces bioactive IFN-{gamma} protein in macrophages (28, 29, 30). Consistent with this observation, IL-12 and IL-18 gene expression was cooperatively enhanced at the wound sites of WT mice after injury. Moreover, in SCID mice, IFN-{gamma} protein could also be detected in F4/80-positive macrophages at the wound sites with a concomitant enhancement of IL-12 and IL-18 gene expression. These observations indicate that, in addition to T cells, macrophages are a cellular source of IFN-{gamma} during skin wound healing.

Immediately after skin wounding, neutrophils infiltrate to the wound site, followed by macrophages. The infiltration of these inflammatory cells is regulated by coordinate expression of chemokines and ICAM-1. IFN-{gamma} can up-regulate ICAM-1 expression, which is important for cell adhesion (7). In line with this observation, we previously reported that leukocyte infiltration was markedly attenuated in the liver of IFN-{gamma} KO mice treated with acetaminophen, with a concomitant reduction in chemokine and ICAM-1 gene expression, compared with WT mice (23). Also in this skin wound model, leukocyte infiltration was remarkably attenuated in IFN-{gamma}-deficient mice, with a concomitant reduction in chemokine and ICAM-1 gene expression (data not shown), resulting in reduced neutrophil infiltration during skin wound healing (31). In contrast, cardiac allografts in IFN-{gamma} KO recipient mice exhibited a massive neutrophil infiltration with accelerated tissue necrosis, compared with allografts in WT recipients (32). However, CD8+ lymphocytes control the cardiac allograft process, whereas T lymphocytes have little, if any, role in the skin wound healing process, as evidenced by no apparent morphological differences between WT and SCID mice (data not shown). Moreover, reduced CD8+ lymphocyte infiltration induced aberrant chemokine gene expression and eventually augmented neutrophil infiltration in cardiac allograft in IFN-{gamma} KO mice. Thus, in a specific context, IFN-{gamma} may have seemingly contradictory effects on neutrophil infiltration.

Macrophages, which infiltrate into the wound sites, have been presumed to promote wound healing by producing various types of bioactive substances (33, 34). However, several recent reports raised questions regarding the validity of this hypothesis. Secretory leukocyte protease inhibitor-deficient mice exhibited impaired wound healing despite or because of exaggerated leukocyte infiltration (35). Moreover, skin wound healing was accelerated despite reduced leukocyte infiltration in mice deficient in the TNF receptor p55 (25). In line with the latter observations, IFN-{gamma} KO mice exhibited accelerated wound healing with a concomitant reduction in leukocyte infiltration. Moreover, two indispensable steps for wound healing, angiogenesis and collagen deposition, were enhanced at the wound sites of IFN-{gamma} KO mice, as similarly observed in TNF receptor p55 KO mice (25). Thus, under the specific pathogen free conditions, angiogenesis and collagen deposition can proceed in wound sites, independent of leukocyte infiltration in this skin excisional wound model.

Accumulating evidence indicates that IFN-{gamma} has a negative effect on collagen deposition, one of the most crucial events for wound healing (13, 14, 15, 16), although the precise molecular mechanisms involved in this inhibition remain elusive. We also observed that the absence of IFN-{gamma} resulted in enhanced collagen deposition in wound sites as evidenced by increased COL1A1 mRNA expression and HP contents. As TGF-{beta}1 has been implicated as a key mediator of collagen synthesis, we examined the TGF-{beta}1 signaling pathway in the wound sites of IFN-{gamma} KO mice. We observed that mature TGF-{beta}1 protein was significantly increased in the wound sites of IFN-{gamma} KO mice, compared with WT mice, consistent with the previous in vitro observations that IFN-{gamma} inhibited TGF-{beta}1 protein synthesis at a posttranslational level (36, 37).

TGF-{beta}1 mediates its signals mainly by phosphorylating stimulatory Smads, Smad2 and 3, whereas another Smad, Smad7, antagonizes its signaling pathways (38, 39, 40, 41, 42, 43, 44). This intracellular signaling machinery also plays a role in fibrotic changes in bleomycin-induced pulmonary fibrosis as in vivo gene transfer of Smad7 reduced collagen expression at the mRNA and protein levels by reducing the phosphorylation of Smad2 and eventually attenuated pulmonary fibrosis (45). By using different types of cell lines, independent groups reported that in vitro, IFN-{gamma}/Stat1 signals can increase the amount of an inhibitory Smad, Smad7 and prevent the phosphorylation of Smad2 and 3, thereby inhibiting the actions of TGF-{beta}1 (21, 22). We observed increases in the amount of total and phosphorylated Smad2 and a reciprocal decrease in the amount of an inhibitory Smad, Smad7, at the wound sites in IFN-{gamma} KO mice, compared with WT mice, with a concomitant reduction in the amount of total and phosphorylated Stat1 (Fig. 9). Moreover, immunohistochemical analyses detected IFN-{gamma} protein in various types of cells including macrophages and fibroblasts, whereas phosphorylated Smad2 was detected predominantly in fibroblasts. Thus, crosstalk between IFN-{gamma}/Stat1 and TGF-{beta}1/Smad systems appears to operate in the skin wound healing processes in an autocrine and/or paracrine manner.

Enhanced TGF-{beta}1 production may account for augmented TGF-{beta}1 signaling in the skin wound site of IFN-{gamma} KO mice. Although it has been reported that TGF-{beta}1 can rapidly and massively induce Smad7 in several types of cells (42), in the wound sites of IFN-{gamma} KO mice, Smad7 protein levels were not significantly increased despite increased levels of TGF-{beta}1. Thus, it is more likely that TGF-{beta}1-mediated signaling pathways were mainly augmented by the absence of IFN-{gamma}/Stat1 signaling but not increased TGF-{beta}1 production.

Angiogenesis is another indispensable event for granulation tissue formation and subsequent wound healing. IFN-{gamma} can inhibit capillary growth and development in vitro (46, 47) and can induce the expression of a chemokine, IP-10, which exhibits potent anti-angiogenic activity (48). Thus, angiogenesis may be augmented by the absence of a negative regulator, i.e., IFN-{gamma}. Although the effects of IFN-{gamma} on the expression of a master regulator of angiogenesis, VEGF, are still controversial (49, 50), several lines of evidence suggest that IFN-{gamma} inhibits VEGF expression (51), consistent with our present observations. Furthermore, TGF-{beta}1 can augment VEGF transcription in various cell types (52, 53, 54, 55, 56). Thus, the enhancement in TGF-{beta}1 expression and its signaling pathways, may be responsible for enhanced VEGF expression and subsequent enhanced angiogenesis in IFN-{gamma} KO mice.

Our present observations suggest that the absence of IFN-{gamma} may augment the expression and phosphorylation of a stimulatory Smad, Smad2, and thus accelerate excisional skin wound healing. However, mice deficient in another stimulatory Smad, Smad3, exhibited enhanced re-epithelialization, and eventually accelerated incisional skin wound healing (57). In the healing process of incisional wounds, re-epithelialization is presumed to be the most crucial phenomenon. As TGF-{beta}-mediated signals inhibit re-epithelialization, the lack of Smad3 might accelerate incisional skin wound healing (57). In contrast, collagen deposition might have a more important role in the healing process of an excisional skin wound. As collagen deposition was markedly attenuated through a reduction in Smad2 phosphorylation induced by Smad7 in bleomycin-induced pulmonary fibrosis in mice, Smad2 may be more important with respect to collagen deposition (45). This hypothesis is supported by our present observations that the amount of phosphorylated Smad2 and total Smad2 was significantly increased in IFN-{gamma} KO mice compared with WT mice, despite a marginal difference in total Smad3 amount.

Our observations suggest that IFN-{gamma} can negatively modulate the wound healing process by suppressing the production and functional activity of TGF-{beta}1. As TGF-{beta}1 can inhibit IFN-{gamma} production and its receptor expression, both cytokines can antagonize one another. Thus, the blockade of the IFN-{gamma} signal transduction pathway may enhance TGF-{beta}1 production and TGF-{beta}1 signaling in a positive feedback manner and may be an important strategy to accelerate the healing process of skin wounds.


   Acknowledgments
 
We express our sincere gratitude to Dr. Howard A. Young (National Cancer Institute, Frederick, MD) for his invaluable comments on the manuscript. We thank Ryoichi Mori for his technical assistance with the determination of HP content, and we are grateful to Dr. Yasuhiko Yamamoto (Department of Biochemistry and Molecular Vascular Biology, Kanazawa University) for his instructive advice about Western blotting.


   Footnotes
 
1 This work was supported in part by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government. Back

2 Address correspondence and reprint requests to Dr. Naofumi Mukaida, Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail address: naofumim{at}kenroku.kanazawa-u.ac.jp Back

3 Abbreviations used in this paper: pAb, polyclonal Ab; COL1A1, collagen 1A1; HP, hydroxyproline; MPO, myeloperoxidase; p-Smad2, phosphorylated Smad2; p-Stat-1, phosphorylated Stat-1; {alpha}-SMA, {alpha}-smooth muscle actin; VEGF, vascular endothelial growth factor. Back

Received for publication July 17, 2003. Accepted for publication November 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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