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of May 24, 2018. This information is current as Wound-Healing Process in the Skin β and TGF- γ between IFN- The Essential Involvement of Cross-Talk Yoichiro Iwakura and Naofumi Mukaida Yuko Ishida, Toshikazu Kondo, Tatsunori Takayasu, http://www.jimmunol.org/content/172/3/1848 doi: 10.4049/jimmunol.172.3.1848 2004; 172:1848-1855; ; J Immunol References http://www.jimmunol.org/content/172/3/1848.full#ref-list-1 , 18 of which you can access for free at: cites 57 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 2004 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on May 24, 2018 http://www.jimmunol.org/ Downloaded from by guest on May 24, 2018 http://www.jimmunol.org/ Downloaded from

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of May 24, 2018.This information is current as

Wound-Healing Process in the Skinβ and TGF-γbetween IFN-

The Essential Involvement of Cross-Talk

Yoichiro Iwakura and Naofumi MukaidaYuko Ishida, Toshikazu Kondo, Tatsunori Takayasu,

http://www.jimmunol.org/content/172/3/1848doi: 10.4049/jimmunol.172.3.1848

2004; 172:1848-1855; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/172/3/1848.full#ref-list-1

, 18 of which you can access for free at: cites 57 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Essential Involvement of Cross-Talk between IFN-� andTGF-� in the Skin Wound-Healing Process1

Yuko Ishida,* † Toshikazu Kondo,† Tatsunori Takayasu,‡ Yoichiro Iwakura, § andNaofumi Mukaida2*

Several lines of in vitro evidence suggest the potential role of IFN-� in angiogenesis and collagen deposition, two crucial steps inthe wound healing process. In this report, we examined the role of IFN-� in the skin wound healing process utilizing WT andIFN-� KO mice. In WT mice, excisional wounding induced IFN-� mRNA and protein expression by infiltrating macrophages andT cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-� KO mice exhibitedan accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-� KO miceexhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, comparedwith WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-� KO mice also exhibitedaccelerated collagen deposition with enhanced production of TGF-�1 protein in wound sites, compared with WT mice. Further-more, the absence of IFN-� augmented the TGF-�1-mediated signaling pathway, as evidenced by increases in the levels of totaland phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalkbetween the IFN-�/Stat1 and TGF-�1/Smad signaling pathways in the wound healing process.The Journal of Immunology, 2004,172: 1848–1855.

S kin wound healing starts immediately after an injury andconsists of three phases; inflammation, proliferation, andmaturation. 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 theinjury site is followed by infiltration of leukocytes, including neu-trophils and macrophages, into the wound site. In the proliferativephase, re-epithelialization and newly formed granulation tissue be-gin to cover the wound area to repair tissue destruction. Moreover,collagen deposition is indispensable for granulation tissue forma-tion and accumulating evidence implicates TGF-�1 as one of theessential factors that can regulate collagen deposition (3–6). How-ever, the mechanisms regulating the production and activity ofTGF-�1 in vivo remain elusive.

IFN-� is mainly produced by NK cells and CD4� Th1 cells andhas multiple effects on macrophages, NK cells, and T lymphocytes(7). Moreover, IFN-� can inhibit collagen synthesis by fibroblastsin vitro (8–12). In line with these observations, the administrationof exogenous IFN-� impaired collagen accumulation and disruptedwound strength, suggesting that IFN-� was deleterious to skinwound healing (13–16). However, the role of endogenous IFN-� inthe skin wound healing process remains to be investigated.

After binding its specific receptor on the cell surface, IFN-�activates receptor-associated Janus kinases, leading to the phos-phorylation of specific tyrosine residues of Stat1 (17, 18). Stat1mediates the biological activity of IFN-� by inducing the tran-scription of the target genes (19, 20). Accumulating evidence sug-gests that the IFN-�/Stat1 system can modulate TGF-�1 activity invitro by interfering with its signaling molecules, the Smad proteins(21, 22). However, it remains to be investigated whether or notthere is crosstalk between the IFN-�/Stat1 and TGF-�1/Smad sig-naling pathways in vivo, particularly in pathological conditions. Inthis report, we investigated the role of endogenous IFN-� in theskin wound healing process, particularly focusing on its interactionwith the TGF-�1/Smad system. In this study, we provided the firstdefinitive evidence to indicate that endogenous IFN-� can nega-tively regulate the TGF-�1 signaling pathway at wound sites invivo, resulting in a retardation of the wound healing process.

Materials and MethodsAbs and reagents

The following mAb or polyclonal Abs (pAbs)3 and recombinant proteinwere used in this study; rat anti-mouse F4/80 mAb and rat anti-mouse CD3mAb (Dainippon Pharmaceutical Company, Osaka, Japan), rat anti-mouseIFN-� mAb (clone XMG 1.2) and rat anti-mouse CD31 mAb (BD Phar-Mingen, San Diego, CA), rabbit anti-myeloperoxidase (MPO) pAb (Neo-markers, Fremont, CA, USA), goat anti-mouse Smad2 pAb, rabbit anti-Smad3 pAb, goat anti-mouse Smad7 pAb (Santa Cruz, CA, USA), rabbitanti-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-�-smooth muscle actin (SMA) mAb(clone asm-1, Boehringer Mannheim GmbH, Mannheim, Germany), ratanti-mouse IFN-� neutralizing mAb (a kind gift of Dr. H. Fujiwara, OsakaUniversity), and recombinant murine IFN-� (PeproTech, London, U.K.).

*Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa Univer-sity, Kanazawa, Japan; †Department of Legal Medicine, Wakayama Medical Univer-sity, Wakayama, Japan; ‡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 ofTokyo, Tokyo, Japan

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

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.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.2 Address correspondence and reprint requests to Dr. Naofumi Mukaida, Division ofMolecular Bioregulation, Cancer Research Institute, Kanazawa University, Ka-nazawa, Japan, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail address:[email protected]

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; �-SMA, �-smooth muscle actin; VEGF, vascular endothelialgrowth factor.

The Journal of Immunology

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Mice

Pathogen-free eight- to 12-wk old male BALB/c mice were obtained fromSankyo Laboratories (Tokyo, Japan) and designated as WT mice in thefollowing experiments. IFN-� KO mice, backcrossed to BALB/c mice formore 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 compliedwith the standards set out in the Guidelines for the Care and LaboratoryAnimals at the Takara-machi Campus of Kanazawa University and housedindividually in cages under specific pathogen-free conditions during thewhole course of the study.

Excisional wound preparation and macroscopic examination

Mice were anesthetized with i.p. administration of pentobarbital (50 �g/gweight), and full-thickness skin wounds were made in the dorsal skin understerile conditions as described previously (25). Briefly, after shaving andcleaning with 70% ethanol, excisional full-thickness skin wounds weremade in the dorsal skin by picking up a fold skin at the midline and punch-ing through two layers of skin with a sterile disposable biopsy punch (di-ameter of 4 mm, Kai Industries, Tokyo, Japan). Two wounds with a di-ameter of 4 mm were made at the same time, one wound on each side ofmidline. The same procedure was repeated on the same animals threetimes, generating six wounds, with three wounds at each side. Each woundsite was digitally photographed at the indicated time intervals, and woundareas were determined on photographs using PhotoShop (Version 7.0Adobe Systems, Tokyo, Japan) without a prior knowledge of the experi-mental procedures. Changes in wound areas were expressed as the per-centage of the initial wound areas. In another series of experiments, WTmice received i.p. injection of neutralizing anti-IFN-� mAb or control IgG(250 �g/mouse) once a day from Day 0 to 3, starting immediately after thewound preparation. In some experiments, wounds and their surroundingareas, including the scab and epithelial margins, were cut with a steriledisposable biopsy punch (diameter 8 mm, Kai Industries, Tokyo, Japan) atthe indicated time intervals.

Histopathological analyses of wound sites

At the indicated intervals after the injury, wound specimens were removedand fixed in 4% formaldehyde buffered with PBS (pH 7.2) and then em-bedded with paraffin. Six-�m thick sections were stained with hematoxylinand eosin for histological analysis. Immunohistochemical analyses wereperformed for the evaluation of leukocyte infiltration, angiogenesis, andIFN-� expression as described previously (25). A double-color immuno-fluorescence analysis was also conducted to identify the types of IFN-�-expressing cells and p-Smad2-positive cells, as described previously (26).In some experiments, the anti-IFN-� mAb was incubated with the indicatedconcentration of recombinant mouse IFN-� 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 homog-enized 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-thawedthree times, and centrifuged at 12,000 rpm at 4°C. MPO activities in thesupernatants were assayed using the SUMILON peroxidase assay kit(Sumitomo Bekuraito, Tokyo, Japan), according to the manufacturer’s in-structions. The data were expressed as absorbance divided by total dryweight (mg).

Measurement of hydroxyproline (HP) contents at wound sites

At the indicated time intervals after the injury, skin wound sites wereremoved using a sterile disposable biopsy punch (diameter 8 mm) and weredried for 16 h at 120 °C. As HP is a major constituent of and found almostexclusively in collagen, HP contents were measured as the index of col-lagen accumulation at the wound sites, as described previously (27). HPcontent was calculated by comparison to standards and expressed as theamount (�g) per wound.

Extraction of total RNAs and RT-PCR

Total RNAs were extracted from uninjured and injured skin samples usingISOGENE (Nippon Gene, Toyama, Japan) according to the manufacturer’sinstructions. Five �g of total RNA was reverse-transcribed at 42°C for 1 hin 20 �l reaction mixture containing mouse Moloney leukemia virus re-verse transcriptase (Toyobo, Osaka, Japan) with oligo(dT) primers (Am-ersham-Pharmacia Biotech Japan, Tokyo, Japan). The resultant cDNAswere amplified together with Taq polymerase (Nippon Gene) using specificsets of primers for IFN-�, IL-12p35, IL-12p40, IL-18, vascular endothelialgrowth factor (VEGF), COL1A1, and �-actin (Table I). PCR amplificationof each gene was conducted with the optimal cycles consisting of 94°C for1 min, optimal annealing temperature shown in Table I for 1 min, and 72°Cfor 1 min, followed by incubation at 72°C for 3 min. The amplified PCRproducts were fractionated on a 2% agarose gel and visualized by ethidiumbromide staining. The band intensities were measured using Image Anal-ysis software (version 1.61; National Institutes of Health, Bethesda, MD)and the ratios to �-actin were calculated (23, 25).

Western blotting

At the indicating time intervals after the injury, wound samples were ho-mogenized 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 (P2850and P5726; Sigma-Aldrich) and centrifuged to obtain lysates. The lysates(30 �g) were electrophoresed in a 10% SDS-polyacrylamide gel and trans-ferred onto a nylon membrane. The membrane was then incubated withAbs to TGF-�1, Stat1, p-Stat1, Smad2, p-Smad2, Smad3, or Smad7 dilutedat 1: 1,000. After the incubation of HRP-conjugated secondary Abs, theimmune complexes were visualized using ECL® System (Amersham, Ja-pan) according to the manufacturer’s instructions.

Statistical analysis

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

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

Transcript SequenceAnnealing

Temperature (°C) CycleProduct

Size (bp)

IFN-� (F) 5�-AGCGGCTGACTGAACTCAGATTGTAG-3� 60 30 247(R) 5�-GTCACAGTTTTCAGCTGTATAGGG-3�

IL-12p35 (F) 5�-AACAAGAGGGAGCTGCCTGCC-3� 60 36 300(R) 5�-CGGGTGCTGAAGGCGTGAAGC-3�

IL-12p40 (F) 5�-CGTGCTCATGGCTGGTGCAAAG-3� 55 36 576(R) 5�-GAACACATGCCCACTTGCTG-3�

IL-18 (F) 5�-CGTGCTCATGGCTGGTGCAAAG-3� 55 32 434(R) 5�-GAACACATGCCCACTTGCTG-3�

VEGF (F) 5�-TGAACTTTCTGCTCTCTTGG-3� 60 32 457(R) 5�-AACAAATGCTTTCTCCGCTC-3�

COLIAI (F) 5�-GCCAAGAAGACATCCCTGAAG-3� 60 34 138(R) 5�-TCATTGCATTGCACGTCATC-3�

�-Actin (F) 5�-TTCTACAATGAGCTGCGTGTGGC-3� 62 26 456(R) 5�-CTCATAGCTCTTCTCCAGGGAGGA-3�

a (F), Forward primer; (R), reverse primer.

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ResultsIFN-�, IL-12, and IL-18 expression during wound healing

In our initial experiments, we examined IFN-� expression at theskin excisional wound sites. In uninjured skin of WT mice, IFN-�mRNA was only weakly expressed (Fig. 1a). IFN-� mRNA ex-pression increased significantly 3 days after the injury and re-mained elevated until 6 days after the injury (Fig. 1b). Moreover,there were no significant differences in IFN-� gene expression be-tween WT and SCID mice, suggesting that non-lymphoid cellswere a major cellular source of IFN-� at the skin wound sites (Fig.1, a and b). Immunohistochemical analysis revealed that IFN-�protein was very low in wound sites of WT mice 1 day after theinjury (Fig. 2a). In contrast, a large number of cells were positivefor IFN-� at wound sites 3 and 6 days after injury (Fig. 2, b and c).Preadsorption of the Ab with an excess amount of IFN-� abolishedthe positive signals (Fig. 2d), indicating the specificity of the re-action. A double-color immunofluorescence analysis demonstratedthat IFN-�-positive cells were also positive for F4/80 at 3 days and6 days after injury (Fig. 3a). IFN-�-positive and F4/80-negativecells were judged as resident fibroblasts based on their morphol-ogy. At 6 days after the injury, a few CD3-positive cells were also

positive for IFN-� (Fig. 3b). Considering that IFN-� gene wasexpressed to a similar extent at the wound sites of WT and SCIDmice, these observations suggest that non-lymphoid cells were amajor cellular source of IFN-� in skin wound healing. We alsoanalyzed IL-12 and IL-18 gene expression, which when combinedcan induce IFN-� production in macrophages (28–30). The ex-pression of both genes was enhanced to similar levels at skinwound sites in WT and SCID mice (Fig. 1, a and c–e). Theseresults indicate that F4/80-positive macrophages and to a lesserdegree, T cells, might produce IFN-� 3 days after injury, under thecombined effects of IL-12 and IL-18.

FIGURE 3. A double-color immunofluorescence analysis of woundsites. Wound sites were obtained from WT mice at 3 (a) or 6 (b) days afterthe injury. The samples were immunostained with anti-F4/80 (a-i, Cy3),anti-CD3 (b-i, Cy3), or anti-IFN-� mAb (a-ii and b-ii, FITC) as describedin Materials and Methods and observed under a fluorescence microscopy(original magnification, �100). Signals in i and ii were digitally merged inpanels iii. Representative results from three independent experiments areshown.

FIGURE 1. The analysis of the gene expression of IFN-�, 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 wasperformed as described in Materials and Methods and representative re-sults from six independent experiments are shown in a. Under the condi-tions used, mRNA of all cytokines was weakly detected in the uninjuredskin. The ratios of IFN-� (b), IL-12p35 (c), IL-12p40 (d), and IL-18 (e) to�-actin at the wound sites of WT (�) and SCID mice (f) were determinedby RT-PCR at 1, 3, and 6 days after the injury. Each value representsmean � SEM (n � 6). �, p � 0.05� vs uninjured skin of BALB/c; #, p �0.05� vs uninjured skin of SCID.

FIGURE 2. Immunohistochemical analysis of IFN-� protein expressionin skin wound sites. Skin wound samples were obtained from WT at days1 (a), 3 (b and d), and 6 (c) after the wound preparation. Samples wereimmunostained with either untreated anti-IFN-� mAb (a–c) or that pread-sorbed with an excess amount of rIFN-� (d) as described in Materials andMethods. Representative results from three independent experiments areshown here. Original magnification, �100.

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Macroscopic wound closure in IFN-� KO and WT mice

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

Leukocyte infiltration at the wound sites in IFN-� KO andWT mice

We next examined the effects of IFN-� deficiency on leukocyteinfiltration at the excisional wound sites. Consistent with our pre-vious observations, neutrophil infiltration in WT mice was maxi-mal at 1 day after injury. In contrast, macrophages and CD3-pos-itive cells started to accumulate at 1 day after injury and reachedmaximal levels at 6 days after injury. The infiltration of these cellswas remarkably attenuated in IFN-� KO mice, compared with WTmice, at every time interval examined. Only macrophage infiltration 1day after injury was similar in WT and IFN-� KO mice (Fig. 6).

Angiogenesis and VEGF gene expression at the wound sites inIFN-� KO and WT mice

We next examined the effects of IFN-� on the angiogenic process;one of the important events in the proliferative phase of woundhealing (Fig. 7). No significant difference was observed in the ves-sel density of the uninjured skin when WT and IFN-� KO micewere compared (2.2 � 0.4% vs 2.7 � 0.4%) as measured byCD31-positive areas. Six days after the injury, the vessel densitywithin the wound bed was increased in both the WT and IFN-� KOmice, and the vessel density of IFN-� KO mice was significantlyhigher than WT mice (Fig. 7, a–e). VEGF mRNA was weak butsimilar in uninjured skin of both WT and IFN-� KO mice. VEGFmRNA expression was enhanced at the wound sites in both mice3 days after injury but the enhancement was significantly greater inIFN-� KO than WT mice (Fig. 7, f and g). These observationsimply that the lack of IFN-� may augment angiogenesis in skinwound sites, partly by enhancing VEGF expression.

Granulation tissue formation at the wound sites in IFN-� KOand WT mice

We next explored the effects of IFN-� on collagen content in theextracellular matrix, another crucial molecule for the wound heal-ing process. Histopathologically, at 3 days and after, granulationtissue was evident at wound sites in WT mice (Fig. 8a) and thegranulation tissue formation was more prominent at the woundsites in IFN-� KO mice (Fig. 8b). In uninjured skin, there was nosignificant difference in terms of HP content and COL1A1 mRNAexpression between WT and IFN-� KO mice (Fig. 8, c–e). In WTmice, HP content and COL1A1 mRNA expression at the woundsites started to increase progressively 3 days after injury. However,the increases in HP content and COL1A1 mRNA expression at thewound sites were consistently and significantly higher in IFN-�KO mice than WT mice (Fig. 8, c–e). These observations indicatethat the absence of IFN-� augmented collagen gene expression andeventually collagen production at the wound sites.

The effects of IFN-� deficiency on the TGF-�1-mediatedsignaling pathway

As TGF-�1 has been considered to be a major regulator of colla-gen biosynthesis (3–6), we examined the changes in the TGF-�1signaling pathway at the wound sites (Fig. 9). The amounts of bothtotal and phosphorylated Stat1 were increased at the wound sites ofWT mice 1 day after injury. In contrast, the amount of total andphosphorylated Stat1 was not significantly changed at the woundsites of IFN-� KO mice, due to the absence of IFN-�-mediatedsignals. The amount of TGF-�1 protein was increased at thewound sites although the increase was more marked in IFN-� KOmice than WT mice. Moreover, although total Smad7 levels wereincreased in the wound sites of WT mice, a corresponding increasein Smad7 levels was not observed in IFN-� KO mice. Althoughwound injury increased the amount of Smad3 to similar extents inthe wound sites of both WT and IFN-� KO mice, the amounts oftotal and phosphorylated Smad2 remained at similar levels at thewound sites in WT mice. In contrast, the amount of total andphosphorylated Smad2 was strongly increased in the wound sitesof IFN-� KO mice. A double color immunofluorescence analysisdemonstrated that phosphorylated Smad2 was detected mainly in

FIGURE 5. Macroscopic appearance of wound healing process in WTmice administered with a control or an anti-IFN-� mAb. a, The wound siteswere photographed at the time indicated. Day 0 picture was taken imme-diately after the injury. Representative results from 12 individual animalsin each group are shown. b, Changes in percentage of wound area at eachtime point in comparison to the original wound area in WT mice admin-istered with a control or an anti-IFN-� mAb. Values represent mean �SEM. �, WT treated with control Ig G (IgG); f, WT treated with anti-IFN-� mAb (n � 12 animals). �, p � 0.05; ��, p � 0.01, anti-IFN-� mAbcompared with control.

FIGURE 4. Macroscopic changes in skin excisional wound sites. a, Thewound sites were photographed at the time indicated. Day 0 picture wastaken immediately after the injury. Representative results from 12 individ-ual animals in each group are shown here. b, Changes in percentage ofwound area at each time point in comparison to the original wound area.Values represent mean � SEM. �, WT; f, IFN-� KO (n � 12 animals).�, p � 0.05; ��, p � 0.01, IFN-� KO compared with WT.

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�-SMA-positive fibroblasts, which are presumed to express IFN-�receptors (Fig. 10). Thus, IFN-� may negatively regulate TGF-�1signaling pathway by down-regulating the expression of TGF-�1and its downstream intracellular molecules at the wound sites.

DiscussionIFN-� has pleiotropic actions on various types of immune cells andhas been implicated as one of main regulatory factors for CD4�

Th1 polarization (7). In addition to its immunoregulatory actions,IFN-� exerts multiple effects on non-immune cells, particularlyfibroblasts. Several lines of evidence have demonstrated thatIFN-� can inhibit collagen synthesis by fibroblasts (8–12) andaccumulating evidence suggests that administration of exogenousIFN-� impairs skin wound healing (13–16). In this report, we ex-amined the roles of endogenous IFN-� in wound healing process,by using IFN-� KO mice. We found that the lack of endogenous

FIGURE 7. a–d, Immunohistochemical analyses on excisional skin wound sites of WT (a and c) and IFN-� KO mice (b and d) at 6 days after injury. Thesections were stained with a mAb for the endothelium (CD31) (a and b, �10; c and d, �100). Representative results from six independent animals in each groupare shown. e, Vascular areas were determined as CD31-positive areas in IFN-� KO (f) and WT mice (�) mice with the help of PhotoShop. All values representthe mean � SEM (n � 6 animals). �, p � 0.05, IFN-� KO compared with WT. f and g, RT-PCR analysis of VEGF gene expression at wound sites in WT andIFN-� KO mice. Representative results from 10 independent animals are shown in f. Under the conditions used, VEGF mRNA was faintly detected in uninjuredskin samples of WT and IFN-� KO mice. The ratios of VEGF to �-actin of WT (�) and IFN-� KO mice (f) were determined by RT-PCR and are shown ing. Each value represents mean � SEM (n � 10 animals). �, p � 0.05; ��, p � 0.01, IFN-� KO compared with WT.

FIGURE 6. Immunohistochemical analyses on leukocyte recruitment in skin excisional wound sites. a–f, Immunohistochemical analysis was performedusing 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-�KO (d–f) mice (�200). Representative results from three independent experiments are shown here. g, MPO activity at the wound site of IFN-� KO (f)and WT (�) was determined to evaluate neutrophil accumulation. The numbers of macrophages (h) or those of T cells (i) per a high-power microscopicfield (original magnification, �200) were counted. All values represent the mean � SEM (n � 6 animals). �, p � 0.05, IFN-� KO compared with WT.

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IFN-� significantly accelerated wound healing process as evi-denced by rapid wound closure and enhanced granulation tissueformation.

In WT mice, we observed enhanced IFN-� mRNA and proteinexpression at the skin excisional wound sites. A double-color im-munofluorescence analysis detected IFN-� protein in F4/80-posi-tive macrophages recruited to the wound sites. Several lines ofevidence have demonstrated that the combined stimulation ofIL-12 and IL-18 induces bioactive IFN-� protein in macrophages(28–30). Consistent with this observation, IL-12 and IL-18 geneexpression was cooperatively enhanced at the wound sites of WTmice after injury. Moreover, in SCID mice, IFN-� protein couldalso be detected in F4/80-positive macrophages at the wound siteswith a concomitant enhancement of IL-12 and IL-18 gene expres-sion. These observations indicate that, in addition to T cells, mac-rophages are a cellular source of IFN-� during skin wound healing.

Immediately after skin wounding, neutrophils infiltrate to thewound site, followed by macrophages. The infiltration of theseinflammatory cells is regulated by coordinate expression of che-mokines and ICAM-1. IFN-� can up-regulate ICAM-1 expression,which is important for cell adhesion (7). In line with this obser-vation, we previously reported that leukocyte infiltration wasmarkedly attenuated in the liver of IFN-� KO mice treated withacetaminophen, with a concomitant reduction in chemokine andICAM-1 gene expression, compared with WT mice (23). Also inthis skin wound model, leukocyte infiltration was remarkably at-tenuated in IFN-�-deficient mice, with a concomitant reduction inchemokine and ICAM-1 gene expression (data not shown), result-ing in reduced neutrophil infiltration during skin wound healing(31). In contrast, cardiac allografts in IFN-� KO recipient miceexhibited a massive neutrophil infiltration with accelerated tissue

necrosis, compared with allografts in WT recipients (32). How-ever, CD8� lymphocytes control the cardiac allograft process,whereas T lymphocytes have little, if any, role in the skin woundhealing process, as evidenced by no apparent morphological dif-ferences between WT and SCID mice (data not shown). Moreover,reduced CD8� lymphocyte infiltration induced aberrant chemo-kine gene expression and eventually augmented neutrophil infil-tration in cardiac allograft in IFN-� KO mice. Thus, in a specificcontext, IFN-� may have seemingly contradictory effects on neu-trophil infiltration.

Macrophages, which infiltrate into the wound sites, have beenpresumed to promote wound healing by producing various types ofbioactive substances (33, 34). However, several recent reportsraised questions regarding the validity of this hypothesis. Secretoryleukocyte protease inhibitor-deficient mice exhibited impairedwound healing despite or because of exaggerated leukocyte infil-tration (35). Moreover, skin wound healing was accelerated de-spite reduced leukocyte infiltration in mice deficient in the TNFreceptor p55 (25). In line with the latter observations, IFN-� KOmice exhibited accelerated wound healing with a concomitant re-duction in leukocyte infiltration. Moreover, two indispensablesteps for wound healing, angiogenesis and collagen deposition,were enhanced at the wound sites of IFN-� KO mice, as similarlyobserved in TNF receptor p55 KO mice (25). Thus, under thespecific pathogen free conditions, angiogenesis and collagen dep-osition can proceed in wound sites, independent of leukocyte in-filtration in this skin excisional wound model.

Accumulating evidence indicates that IFN-� has a negative ef-fect on collagen deposition, one of the most crucial events forwound healing (13–16), although the precise molecular mecha-nisms involved in this inhibition remain elusive. We also observedthat the absence of IFN-� resulted in enhanced collagen depositionin wound sites as evidenced by increased COL1A1 mRNA expres-sion and HP contents. As TGF-�1 has been implicated as a keymediator of collagen synthesis, we examined the TGF-�1 signal-ing pathway in the wound sites of IFN-� KO mice. We observedthat mature TGF-�1 protein was significantly increased in the

FIGURE 8. a and b, Histopathological analyses on skin wound sites ofWT (a) and IFN-� KO (b) mice at 6 days after injury. Granulation tissueformation was more evident in IFN-� KO mice than in WT mice. c, HPcontents in the excisional wound sites in WT (�) and IFN-� KO (f) mice.HP contents were determined as an indicator of collagen contents. Allvalues represent the mean � SEM (n � 6 animals). �, p � 0.05, IFN-� KOcompared with WT. d and e, RT-PCR analysis of collagen gene expressionin the wound sites in WT and IFN-� KO mice. Under the conditions used,RT-PCR analysis did not detect the mRNA of COL1A1 in uninjured skinsamples of WT and IFN-� KO mice. Representative results from six ani-mals in each group are shown in d. The ratios of COL1A1 to �-actin of WT(�) and IFN-� KO (f) were determined by RT-PCR at 1, 3 and 6 daysafter injury (e). Each value represents mean � SEM (n � 6 animals). �,p � 0.05; ��, p � 0.01, IFN-� KO compared with WT.

FIGURE 9. Western blotting analysis on the expression of TGF-�,Stat1, phosphorylated Stat1, Smad2, phosphorylated Smad2, Smad3, andSmad7, at the wound sites. Under the conditions used, these moleculeswere faintly detected in uninjured skin sites of WT and IFN-� KO mice.Western blotting analysis using anti-�-tubulin Ab confirmed that an equalamount of protein was loaded onto each lane. Representative results fromsix individual animals in each group are shown.

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wound sites of IFN-� KO mice, compared with WT mice, consis-tent with the previous in vitro observations that IFN-� inhibitedTGF-�1 protein synthesis at a posttranslational level (36, 37).

TGF-�1 mediates its signals mainly by phosphorylating stimu-latory Smads, Smad2 and 3, whereas another Smad, Smad7, an-tagonizes its signaling pathways (38–44). This intracellular sig-naling machinery also plays a role in fibrotic changes inbleomycin-induced pulmonary fibrosis as in vivo gene transfer ofSmad7 reduced collagen expression at the mRNA and protein lev-els by reducing the phosphorylation of Smad2 and eventually at-tenuated pulmonary fibrosis (45). By using different types of celllines, independent groups reported that in vitro, IFN-�/Stat1 sig-nals can increase the amount of an inhibitory Smad, Smad7 andprevent the phosphorylation of Smad2 and 3, thereby inhibiting theactions of TGF-�1 (21, 22). We observed increases in the amountof total and phosphorylated Smad2 and a reciprocal decrease in theamount of an inhibitory Smad, Smad7, at the wound sites in IFN-�KO mice, compared with WT mice, with a concomitant reductionin the amount of total and phosphorylated Stat1 (Fig. 9). Moreover,immunohistochemical analyses detected IFN-� protein in varioustypes of cells including macrophages and fibroblasts, whereasphosphorylated Smad2 was detected predominantly in fibroblasts.Thus, crosstalk between IFN-�/Stat1 and TGF-�1/Smad systemsappears to operate in the skin wound healing processes in an au-tocrine and/or paracrine manner.

Enhanced TGF-�1 production may account for augmentedTGF-�1 signaling in the skin wound site of IFN-� KO mice. Al-though it has been reported that TGF-�1 can rapidly and massivelyinduce Smad7 in several types of cells (42), in the wound sites ofIFN-� KO mice, Smad7 protein levels were not significantly in-creased despite increased levels of TGF-�1. Thus, it is more likelythat TGF-�1-mediated signaling pathways were mainly aug-mented by the absence of IFN-�/Stat1 signaling but not increasedTGF-�1 production.

Angiogenesis is another indispensable event for granulation tis-sue formation and subsequent wound healing. IFN-� can inhibitcapillary growth and development in vitro (46, 47) and can inducethe expression of a chemokine, IP-10, which exhibits potent anti-angiogenic activity (48). Thus, angiogenesis may be augmented bythe absence of a negative regulator, i.e., IFN-�. Although the ef-fects of IFN-� on the expression of a master regulator of angio-genesis, VEGF, are still controversial (49, 50), several lines ofevidence suggest that IFN-� inhibits VEGF expression (51), con-sistent with our present observations. Furthermore, TGF-�1 canaugment VEGF transcription in various cell types (52–56). Thus,the enhancement in TGF-�1 expression and its signaling path-ways, may be responsible for enhanced VEGF expression and sub-sequent enhanced angiogenesis in IFN-� KO mice.

Our present observations suggest that the absence of IFN-� mayaugment the expression and phosphorylation of a stimulatorySmad, Smad2, and thus accelerate excisional skin wound healing.However, mice deficient in another stimulatory Smad, Smad3, ex-hibited enhanced re-epithelialization, and eventually acceleratedincisional skin wound healing (57). In the healing process of in-cisional wounds, re-epithelialization is presumed to be the mostcrucial phenomenon. As TGF-�-mediated signals inhibit re-epi-thelialization, the lack of Smad3 might accelerate incisional skinwound healing (57). In contrast, collagen deposition might have amore important role in the healing process of an excisional skinwound. As collagen deposition was markedly attenuated through areduction in Smad2 phosphorylation induced by Smad7 in bleo-mycin-induced pulmonary fibrosis in mice, Smad2 may be moreimportant with respect to collagen deposition (45). This hypothesisis supported by our present observations that the amount of phos-phorylated Smad2 and total Smad2 was significantly increased inIFN-� KO mice compared with WT mice, despite a marginal dif-ference in total Smad3 amount.

Our observations suggest that IFN-� can negatively modulatethe wound healing process by suppressing the production andfunctional activity of TGF-�1. As TGF-�1 can inhibit IFN-� pro-duction and its receptor expression, both cytokines can antagonizeone another. Thus, the blockade of the IFN-� signal transductionpathway may enhance TGF-�1 production and TGF-�1 signalingin a positive feedback manner and may be an important strategy toaccelerate the healing process of skin wounds.

AcknowledgmentsWe express our sincere gratitude to Dr. Howard A. Young (National Can-cer Institute, Frederick, MD) for his invaluable comments on the manu-script. We thank Ryoichi Mori for his technical assistance with the deter-mination of HP content, and we are grateful to Dr. Yasuhiko Yamamoto(Department of Biochemistry and Molecular Vascular Biology, KanazawaUniversity) for his instructive advice about Western blotting.

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