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(The FASEB Journal. 2002;16:963-974.)
© 2002 FASEB

Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration

RYOICHI MORI, TOSHIKAZU KONDO, TOHRU OHSHIMA1, YUKO ISHIDA and NAOFUMI MUKAIDA*

Division of Environmental Science, Forensic and Social Environmental Medicine, Graduate School of Medical Science; and
* Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan

1Correspondence: Division of Environmental Science, Forensic and Social Environmental Medicine, Graduate School of Medical Science, Kanazawa University, 13–1 Takara-machi, 920-8640 Kanazawa, Japan. E-mail: ohshimat{at}med.kanazawa-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To clarify biological roles of tumor necrosis factor receptor p55 (TNF-Rp55) -mediated signals in wound healing, skin excisions were prepared in BALB/c (WT) and TNF-Rp55-deficient (KO) mice. In WT mice, the wound area was reduced to 50% of the original area 6 days after injury, with angiogenesis and collagen accumulation. Histopathologically, reepithelialization rate was ~80% 6 days. Myeloperoxidase activity and macrophage recruitment were the most evident 1 and 6 days after injury, respectively. Gene expression of adhesion molecules, interleukin 1{alpha} (IL-1{alpha}), IL-1ß, monocyte chemoattractant protein 1, macrophage inflammatory protein 1{alpha} (MIP-1{alpha}), MIP-2, transforming growth factor ß1 (TGF-ß1) connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), Flt-1, and Flk-1 was enhanced at the wound site. In KO mice, an enhancement in angiogenesis, collagen content, and reepithelialization was accelerated with the increased gene expression of TGF-ß1, CTGF, VEGF, Flt-1, and Flk-1 at the wound sites, resulting in accelerated wound healing compared with WT mice. In contrast, leukocyte infiltration, mRNA expression of adhesion molecules, and cytokines were significantly reduced in KO mice. These observations suggest that TNF-Rp55-mediated signals have some role in promoting leukocyte infiltration at the wound site and negatively affect wound healing, probably by reducing angiogenesis and collagen accumulation.—Mori, R., Kondo, T., Ohshima, T., Ishida, Y., Mukaida, N. Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration.


Key Words: TNF-Rp55 • angiogenesis • collagen production


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WOUND HEALING IN the skin starts immediately after an injury and consists of three phases: inflammation, proliferation, and maturation. These phases proceed with a complicated but well-organized interaction between various types of tissues and cells (1 , 2) . During the inflammatory phase, platelet aggregation at the injury site is followed by infiltration of leukocytes such as neutrophils and macrophages into the wound site. In the proliferative phase, reepithelialization and newly formed granulation tissue begin to cover the wound area to complete tissue repair. Angiogenesis is indispensable for sustaining granulation tissue.

Tumor necrosis factor {alpha} (TNF-{alpha}) is a pleiotropic cytokine produced by a variety of cell types including macrophages, T cells, mast cells, and keratinocytes (3) . Several lines of evidence have demonstrated the crucial involvement of TNF-{alpha} in life-threatening conditions such as endotoxin shock and cancer cachexia (3) . There are two types of receptors for TNF-{alpha} encoded by distinct genes: TNF receptor with a molecular mass of 55 kDa (TNF-Rp55) and one with a molecular mass of 75 kDa (TNF-Rp75) (4) . These two receptors show 30% homology at the amino acid level in their extracellular, cysteine-rich, and ligand binding regions. TNF-Rp55 is expressed ubiquitously on almost all cell types except for red blood cells whereas TNF-Rp75 expression is predominantly restricted to hematopoietic and endothelial cells (3) . TNF-Rp55 mediates various activities of TNF-{alpha}, including cytotoxicity, fibroblast proliferation, and induction of superoxide dismutase whereas TNF-Rp75 mediates thymocyte and cytotoxic T cell proliferation (5) . To elucidate the biological role of TNF-Rp55-mediated signals, TNF-Rp55-deficient (TNF-Rp55-/-) mice have been generated. There was no significant difference in phenotype between TNF-Rp55-/- and control wild-type (WT) mice under nonchallenging conditions. TNF-Rp55-/- mice were resistant to lipopolysaccharide- or staphylococcal enterotoxin-induced shock but were highly susceptible to infection with Listeria monocytogenes (6 , 7) .

We had observed that immunoreactive TNF-{alpha} proteins and its mRNA were transiently detected in neutrophils and macrophages with maximal protein levels 6 h after an incisional wound of the skin, which returned to basal levels within 24 h (8 , 9) . One group reported that local application of TNF-{alpha} increased wound disruption strength in incisional wounds and eventually accelerated wound healing in rats (10) ; the other groups reported that local application of TNF-{alpha} (11 , 12) or inoculation of TNF-{alpha}-producing cells (13) impaired skin wound healing. Regan and colleagues (14) observed the beneficial effects of rabbit anti-TNF-{alpha} serum on murine skin wounds without examining the histological changes in more detail. Because enhanced expression of TNF-Rp55 was observed at the wound site in our preliminary experiments, we examined the biological roles of TNF-Rp55 in the wound healing by making skin excisional wounds in TNF-Rp55-/- mice. TNF-Rp55-/- mice showed enhanced angiogenesis and collagen accumulation along with reduced infiltration of neutrophils and macrophages compared with WT mice, implying that wound healing was accelerated in TNF-Rp55-/- mice compared with WT mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
The following monoclonal or polyclonal antibodies (mAbs or pAbs) were used in this study: rat anti-mouse F4/80 and rat anti-mouse CD3 mAbs (Dainippon Pharmaceutical Company, Osaka, Japan), rat anti-mouse CD31 (platelet endothelial cell adhesion molecule 1, PECAM-1) mAb (PharMingen, Burlington, CA), rabbit anti-TNF-{alpha} pAb, and rabbit anti-myeloperoxidase (MPO) pAb (Neomarkers, Fremont, CA).

Mice
After obtaining BALB/c mice from Sankyo Laboratories (Tokyo, Japan), we kept their colonies in specific pathogen-free conditions in our animal facility and designated them as WT mice thereafter. TNF-Rp55-/- mice, backcrossed to BALB/c mice for 8 to 10 generations, were kept under the same conditions as WT mice and were used in the experiment (15) . Eight- to 12-wk-old male TNF-Rp55-/- and age-matched male WT mice 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.

Wound preparation and macroscopic examination
Mice were deeply anesthetized with intraperitoneal (i.p.) administration of Nembutal® (5 µg/g weight). After shaving and cleaning with 70% ethanol, excisional full-thickness skin wounds were aseptically made on 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 with a 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 with a Nikon FX-35A (Nikon, Tokyo, Japan) at the indicated time intervals, and wound areas were determined on photographs using PhotoShop (Version 5.5, Adobe Systems, Tokyo, Japan) without prior knowledge of the experimental procedures. Changes in wound areas were expressed as percentage of the initial wound areas. In another series of experiments, wounds and their surrounding areas, including the scab and epithelial margins, were cut with a sterile disposable biopsy punch with a diameter of 8 mm (Kai Industries, Tokyo, Japan) after the mice were killed with an overdose of Nembutal®. Only the central areas, which were previously excised, were used for the following experiments.

Histopathological analyses of wound sites
Excised wound specimens was fixed in 4% formaldehyde buffered with PBS (pH 7.2) and embedded with paraffin. Sections 6 µm thick were stained with hematoxylin and eosin for histological analysis. Deparaffinized sections were immersed in 0.3% H2O2 in methanol for 30 min to eliminate endogenous peroxidase activities. The sections were further incubated with PBS containing 1% normal serum corresponding to the secondary Ab and 1% bovine serum albumin to reduce nonspecific reactions. The sections were incubated with anti-MPO, anti-F4/80, anti-CD3, or anti-CD31 Ab at a concentration of 1 µg/mL at 4°C overnight. After the incubation of biotinylated secondary Ab, immune complexes were visualized using Catalyzed Signal Amplification System (Dako, Kyoto, Japan) according to the manufacturer’s instructions. A double-color immunofluorescent analysis was performed to determine TNF-{alpha} expressing cell types as described previously (16) .

Analysis of reepithelialization
We analyzed the degree of reepithelialization (17) . The central portion of the wound was viewed at x100 or x400; the width of the wound and the distance, which epithelium had traversed were measured as reepithelialization. The percent of reepithelialization in total wound was calculated.

Measurements of leukocyte recruitment and angiogenesis at wound sites
The wound bed was defined as the area surrounded by unwounded skin and fascia, regenerated epidermis, and eschar (18) . Two visual fields (x200) were chosen from each edge of the wound bed; the other three were chosen from the middle of the wound bed. The numbers of F4/80-positive macrophages and CD3-positive T cells within a wound bed were enumerated on these five visual fields (18) . Using free-hand tool of PhotoShop, vascular areas (defined as CD31-positive ones) were measured in the whole wound bed areas 6 and 14 days after the injury and expressed as percentage of the whole wound bed areas (17) . All measurements were performed without prior knowledge of the experimental procedures.

MPO assay
Myeloperoxidase activity was measured for quantitation of neutrophil recruitment (19 , 20) . 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 Chemical, 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 SUMILON peroxidase assay kit (Sumitomo Bekuraito, Tokyo, Japan) according to the manufacturer’s instructions. Data were expressed as absorbance/total dry weight (mg).

Measurement of hydroxyproline (HP) content in wound sites
Skin wound sites were excised using a sterile disposable biopsy punch with a diameter of 8 mm and dried for 16 h at 120°C. HP contents were measured according to a previous study (17) . HP amount was calculated by comparison to standards and the data were expressed as the amount (µg) per skin wound.

Extraction of total RNAs and reverse transcriptase-polymerase chain reaction (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 micrograms 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), followed by PCR amplification to quantitate gene expression in a semiquantitative manner according to published methods (21 22 23) . cDNA was amplified together with Taq polymerase (Nippon Gene) using specific primers for intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, TNF-{alpha}, interleukin 1{alpha} (IL-1{alpha}), IL-1ß, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1{alpha} (MIP-1{alpha}), MIP-2, transforming growth factor ß1 (TGF-ß1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), Flt-1, Flk-1, and ß-actin as described in Table 1 . To determine the optimal cycle number and annealing temperature, we performed PCR on each molecule by increasing PCR cycle numbers from 20 to 40 by two at different annealing temperatures. The PCR products were run on a 2% agarose gel stained with ethidium bromide. The intensities of specific bands were quantified with a charge-coupled device imaging system (GelDoc 2000, Bio-Rad, Hercules, CA) and NIH Image Analysis Software Ver 1.61 (National Institutes of Health, Bethesda, MD). The band intensities were plotted against cycle numbers on semilogarithmic graphs, and the most optimal cycle number and annealing temperature were determined on each molecule as indicated in Table 1 . RT-PCR was performed at least three times for each sample and the band intensities were determined as described above. A constant amount of a standard DNA was electrophoresed each time to standardize the band intensities; the ratios to ß-actin were determined.


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  		Table 1. Sequences of the primers used for RT-PCRa

Enzyme-linked immunosorbent assay (ELISA)
Wound tissue were excised with a punch biopsy (8 mm) and homogenized with 0.3 mL lysis buffer (10 mM PBS, 0.1% SDS, 1% Nonidet P-40, and 5 mM EDTA) containing Complete Protease Inhibitor Mixture (Roche Diagnostics, Tokyo, Japan). The homogenates were centrifuged at 12000 rpm for 15 min. The supernatant was applied to ELISA. IL-1ß, TNF-{alpha}, MIP-1{alpha}, TGF-ß1 and VEGF protein levels were measured with commercial ELISA kits (IL-1ß; BioSource, Inc., Camarillo, CA; others; Quantikine M, R&D, USA) according to the manufacturers’ recommendation. IL-1{alpha}, MCP-1 and MIP-2 levels were measured by ELISA using purified rabbit pAb against IL-1{alpha}, MCP-1 or MIP-2 and rabbit anti-IL-1{alpha}, MCP-1, or -MIP-2 pAb conjugated with horseradish peroxidase. Four-amino-antipyrine in potassium phosphate buffer was used as a substrate, and the color intensity was measured at 492 nm. The detection limits in each method was as follows: IL-1{alpha} > 10pg/mL, IL-1ß > 7pg/mL, TNF-{alpha} > 5 pg/mL, MIP-1{alpha} > 1.5pg/mL, VEGF > 3pg/mL, TGF-ß1 > 7 pg/mL, MCP-1 > 10 pg/mL, and MIP-2 > 10pg/mL. Total protein in the supernatant was measured with a commercial kit (BCA Protein Assay Kit, Pierce, Rockford, IL). The data were expressed as cytokine or growth factor (pg/mL)/total protein (mg/mL) for each sample.

Statistical analysis
The means and SE were calculated for all parameters determined. 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
 
Expression of TNF-{alpha}, TNF-Rp55, and -Rp75 during wound healing
In WT mice, TNF-{alpha} was up-regulated at levels of protein and mRNA injury whereas enhanced expression was significantly attenuated in TNF-Rp55-/- mice (Fig. 1 A–C). A double-color immunofluorescent analysis revealed that neutrophils and macrophages were the main cellular source of TNF-{alpha} in wound healing (Fig. 1D, E ). The mRNA expression of TNF-Rp55 was conspicuously up-regulated at the wound site continuously after day 1 whereas TNF-Rp75 gene expression was enhanced just transiently at day 3 (Fig. 2 ). These observations suggest that TNF-{alpha}-TNF-Rp55 signals may be involved mainly in skin wound healing.



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  		Figure 1. Analysis of TNF-{alpha} expression during wound healing. A) RT-PCR analysis for gene expression of TNF-{alpha}. Under the conditions used, RT-PCR analysis did not detect TNF-{alpha} mRNA in uninjured skin. B) The ratio of TNF-{alpha} to ß-actin of WT (open bars) and TNF-Rp55-/- (filled bars) was determined by RT-PCR 1, 3, and 6 days after injury. Each value represents mean ± SE (n=10 animals). C) Protein level of TNF-{alpha} in wound healing by ELISA. Each value represents mean ± SE (n=10 animals). *P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT. D, E) A double-color immunofluorescent analysis of cells expressing TNF-{alpha} during wound healing. Immunostaining with anti-MPO, which is a marker for neutrophils (D-I, cy3), anti-F4/80 (E-I, cy3), or anti-TNF-{alpha} (D-II and E-II, FITC) by fluorescent microscopy (x400). Signals were digitally merged in panel D-III (derived from D-I and D-II) or E-III (E-I and E-II).



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  		Figure 2. Analysis of TNF-Rp55 and -Rp75 expression during wound healing. A) RT-PCR analysis for gene expression of TNF-Rp55 during wound healing of WT mice. RT-PCR analysis did not detect TNF-Rp55 mRNA in the uninjured skin. B) Ratios of TNF-Rp55 to ß-actin were determined by RT-PCR 1, 3, and 6 days after injury. After excisional wound, the gene expression of TNF-Rp55 was up-regulated significantly in WT mice. C) RT-PCR analysis of gene expression for TNF-Rp75 during wound healing of WT and TNF-Rp55-/- (KO) mice. RT-PCR analysis did not detect the mRNA of TNF-Rp75 in uninjured skin. D) The ratios of TNF-Rp75 to ß-actin were determined by RT-PCR 1, 3, and 6 days after injury. After excisional wound, there was no difference in gene expression of TNF-Rp75 between WT and TNF-Rp55-/- mice and no difference in the expression of TNF-Rp75 mRNA between them. Each value represents mean ± SE (n=10 animals).

Macroscopic wound closure
We evaluated the changes in wound areas in TNF-Rp55-/- and WT mice as indices of wound closure. One day after the injury, wound sites in TNF-Rp55-/- mice exhibited similar morphology as WT mice (Fig. 3 A). Three days after injury, wound areas in TNF-Rp55-/- mice were reduced to 50% of the original area (Fig. 3B ). In contrast, wound areas in WT mice were reduced to 50% only 6 days after injury. Three days after the injury and thereafter, the wound areas in TNF-Rp55-/- mice were consistently smaller than in WT mice. These observations demonstrated that wound closure and eventually healing were accelerated in the absence of TNF-Rp55.



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  		Figure 3. Skin wound healing in TNF-Rp55-/- and WT mice. A) Macroscopic changes in skin wound sites in a TNF-Rp55-/- mouse and a WT one. Day 0 picture was taken immediately after injury. Representative results from 12 individual animals in each group are shown. B) Changes in percentage of wound area at each time point to the original wound area. Values represent mean ± SE Open bars, WT; filled bars, TNF-Rp55-/- (n=12 animals)., P < 0.05, **P < 0.01, TNF-Rp55-/- compared with WT.

Histological reepithelialization
We evaluated the degree of histological reepithelialization in wounds from TNF-Rp55-/- mice and WT mice (Fig. 4 ). One day after injury, reepithelialization could not be observed in TNF-Rp55-/- and WT mice (data not shown). In WT mice, the rate of reepithelialization was still ~60 and 80% 3 and 6 days after injury, respectively; the reepithelialization was complete 10 days in all of the WT mice examined (Fig. 4A, B, E ). TNF-Rp55-/- mice showed a significantly accelerated reepithelialization compared with WT mice (Fig. 4C-E ). These observations were consistent with the macroscopic results mentioned above and suggested that the absence of TNF-Rp55 promoted reepithelialization in wound healing.



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  		Figure 4. Histological reepithelialization of skin wounds in WT (A, B) and TNF-Rp55-/- (C, D) mice 3 (A, C) and 6 (B, D) days after injury (HE staining, low magnification: x10; high magnification: x200). Arrowheads and arrows indicate original wound edges and reepithelialized edges, respectively. E) The ratio of reepithelialization was evaluated in WT (open bars) and TNF-Rp55-/- (filled bars). All values represent the mean ± SE (n=6 animals). *P < 0.05, **P < 0.01, TNF-Rp55-/- compared with WT.

MPO activity and leukocyte infiltration in wound sites
In the inflammatory phase of the wound healing process, different types of leukocytes infiltrate into the wound site depending on the types of wound and the time intervals after injury. In WT mice, a large number of neutrophils infiltrated to the wound site 1 day after the injury (Fig. 5 A, B) but almost disappeared 6 days after the injury. It is difficult to accurately determine neutrophil number because many of neutrophils were trapped within the clot. Thus, we measured MPO activity to evaluate neutrophil recruitment. MPO activity increased 6 h after wounding, and reached a peak 1 day after injury (Fig. 5E ). F4/80-positive macrophages infiltrated the wound site beginning 1 day after the injury and massive infiltration was observed 6 days in WT mice (Fig. 6 A–D). In TNF-Rp55-/- mice, infiltration of neutrophils was reduced impressively at day 1 (Fig. 5C, D ) and MPO activity was significantly attenuated 6 h, 1, and 3 days in TNF-Rp55-/- mice compared with WT mice (Fig. 5E ). Recruitment of macrophages was significantly reduced at days 3 and 6 compared with WT mice (Fig. 6E-I ). CD3-positive T cell infiltration was not diminished in TNF-Rp55-/- mice compared with WT mice (Fig. 7 ). These results demonstrated that neutrophil and macrophage recruitment occurring during the course of the inflammatory phase after skin excision was markedly reduced in the absence of TNF-Rp55.



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  		Figure 5. Immunohistochemical analyses using anti-MPO antibody in skin wound samples from WT (A, B) and TNF-Rp55-/- (C, D) mice 1 day after injury (A, C: x10; B, D: x200). F) MPO activity at the wound site of TNF-Rp55-/- (filled bars) and WT (open bars) was evaluated 6 h, 1, and 3 days after injury for the examination of neutrophil recruitment. All values represent the mean ± SE (n=6 animals). **P < 0.01, TNF-Rp55-/- compared with WT.



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  		Figure 6. Immunohistochemical analyses of skin wound samples of WT (A–D) and TNF-Rp55-/- (E–H) 3 (A, B, E, F) and 6 days (C, D, G, H) after injury. The sections were stained with a monoclonal antibody for murine macrophages (F4/80) (A, C, E, G: x10; B, D, F, H: x200). I) Macrophage recruitment in the wounded skin of TNF-Rp55-/- (filled bar) and WT (open bar) 3, 6, and 14 days after injury. The number of macrophages per a high-power microscopic field (x200) was counted. All values represent the mean ± SE (n=6 animals). *P < 0.05, TNF-Rp55-/- compared with WT.



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  		Figure 7. Immunohistochemical analyses of skin wound samples of WT (A, B) and TNF-Rp55-/- mice (C, D) 6 days after injury. The sections were stained with a monoclonal antibody for CD-3 (A, C: x10; B, D: x100). E) T cell recruitment in wounded skin from TNF-Rp55-/- (filled bar) and WT (open bar) 3 and 6 days after injury was evaluated. The number of T cell was counted per section. All values represent the mean ± SE (n=6). * P < 0.05, TNF-Rp55-/- compared with WT.

Angiogenesis at wound sites
Angiogenesis is an important morphological change observed in the proliferative phase of wound healing. Using immunohistochemical staining for CD31, angiogenesis was assessed in normal and injured skin of WT and TNF-Rp55-/- mice. No significant difference was found in vessel density of the uninjured skin between WT and TNF-Rp55-/- mice (2.3±0.4% vs. 2.6±0.5%) (Fig. 8 C). Six days after injury, the vessel density within the wound bed increased in WT and TNF-Rp55-/- mice and the vessel density of TNF-Rp55-/- mice was nearly twice that of WT mice (5.2±0.4% vs. 10.2±1.8%, P<0.05) (Fig. 8A-E ). However, by 14 days after injury, there were no significant differences between WT and TNF-Rp55-/- mice in terms of vessel density (4.3±0.8% vs. 4.6±0.5%) (Fig. 8E ).



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  		Figure 8. Immunohistochemical sections of skin wound samples of WT (A, B) and TNF-Rp55-/- mice (C, D) 6 days after injury. Sections were stained with a monoclonal antibody for the endothelium (CD-31) (A, C: x10; B, D: x100). Representative results from 6 animals in each group are shown. E) Vascular density within the wound bed 6 and 14 days after injury) and that in uninjured skin samples of TNF-Rp55-/- (filled bar) and WT (open bar) mice were determined using PhotoShop. All values represent the mean ± SE (n=6 animals). *P < 0.05, TNF-Rp55-/- compared with WT. F) Protein levels of VEGF at the wound samples from WT (open bars) and TNF-Rp55-/- mice (filled bars). All values represent the mean ± SE (n=6 animals). *P < 0.05, TNF-Rp55-/- compared with WT. (G) RT-PCR analysis of gene expression for VEGF, Flk-1 and Flt-1 at wound sites in WT and TNF-Rp55-/- (KO) mice. RT-PCR analysis did not detect the mRNA of these factors in uninjured skin samples of WT and TNF-Rp55-/- mice. The ratios of VEGF (H), Flk-1 (I), and Flt-1 (J) to ß-actin of WT (open bars) and TNF-Rp55-/- (filled bars) were determined by RT-PCR 3 and 6 days after injury. Each value represents mean ± SE (n=10 animals). *P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT.

HP content in wound site
An increased collagen content in extracellular matrix is another characteristic change observed in the proliferative phase of wound healing process. Because HP is a major constituent and found almost exclusively in collagen (24) , we determined HP content in the wound sites. In uninjured skin, there was no significant difference in HP content between WT and TNF-Rp55-/- mice (data not shown). HP content in wound sites increased progressively in WT mice, starting immediately after the injury (Fig. 9 A). HP contents of TNF-Rp55-/- mice increased more rapidly after injury, with significantly higher levels 3 and 6 days after injury than WT mice. These results suggest that collagen production was enhanced in the absence of TNF-Rp55.



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  		Figure 9. A) Collagen content in the excisional wounds in WT (open bar) and TNF-Rp55-/- (filled bar) mice. A biochemical assay for hydroxyproline (HP) as an indicator of collagen content was performed. All values represent the mean ± SE (n=5 animals). *P < 0.05, TNF-Rp55-/- compared with WT. B) Protein levels of TGF-ß1 at the wound samples from WT (open bars) and TNF-Rp55-/- mice (filled bars). Each value represents mean ± SE (n=6 animals). *P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT. C) RT-PCR analysis of gene expression for growth factors at wound sites in WT and TNF-Rp55-/- (KO) mice. RT-PCR analysis did not detect the mRNA of these growth factors in uninjured skin samples of WT and TNF-Rp55-/- mice. Representative results from 5 animals in each group are shown. Ratios of TGF-ß1 (D) and CTGF (E) to ß-actin of WT (open bar) and TNF-Rp55-/- (filled bar) were determined by RT-PCR 3 and 6 days after injury. Each value represents mean ± SE (n=10 animals). *P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT.

Gene expression of adhesion molecules, cytokines, growth factors, and VEGF receptors
RT-PCR analysis failed to detect the mRNA of E-selectin, ICAM-1, VCAM-1, IL-1{alpha}, IL-1ß, MCP-1, MIP-1{alpha}, MIP-2, TGF-ß1, CTGF, Flt-1, and Flk-1 in uninjured skin specimens from WT and TNF-Rp55-/- mice. Skin excision induced gene expression of all these molecules in wound sites at the times examined (Figs. 8 G, 9C, 10A, E, and 11A). TNF-Rp55-/- mice showed significantly reduced gene expression of adhesion molecules at the times examined (E-selectin: 3 and 6 h, VCAM-1: 6 h and 1 day, and ICAM-1: 6 h, 1 and 3 days) (Fig. 10 ). In TNF-Rp55-/- mice, enhanced gene expression of IL-1{alpha}, IL-1ß, MCP-1, MIP-1{alpha}, and MIP-2 was significantly attenuated 3 and 6 days after injury compared with WT mice (Fig. 11 ). These results showed that in the absence of TNF-Rp55, neutrophil and macrophage infiltration was reduced, with diminished expression of these adhesion molecules and cytokines. On the other hand, gene expression of TGF-ß1, CTGF, VEGF, and VEGF receptors was significantly enhanced in TNF-Rp55-/- mice compared with WT mice; TGF-ß1 at 1, 3, and 6 days, CTGF at 1 and 6 days, VEGF, Flt-1, and Flk-1 at 3 and 6 days (Figs. 8G-J and 9C- E ). These observations suggest that enhanced expression of these factors is responsible for augmented collagen accumulation and angiogenesis in TNF-Rp55-/- mice.



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  		Figure 10. A, E) RT-PCR analysis of gene expression for adhesion molecules (E-selectin, VCAM-1 and ICAM-1) at wound sites in WT and TNF-Rp55-/- (KO) mice. RT-PCR analysis did not detect the mRNA of these adhesion molecules in uninjured skin samples of WT and TNF-Rp55-/- mice. Representative results from 10 animals in each group are shown. The ratios of E-selectin (B, F), VCAM-1 (C, G), and ICAM-1(D, H) to ß-actin of WT (open bar) and TNF-Rp55-/- (filled bar) were determined by RT-PCR. Each value represents mean ± SE (n=10 animals). *P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT.



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  		Figure 11. A) RT-PCR analysis of gene expression for IL-1{alpha} and IL-1ß, MCP-1, MIP-1{alpha} and MIP-2 at wound sites in WT and TNF-Rp55-/- (KO) mice. RT-PCR analysis did not detect the mRNA of these cytokines and chemokines in uninjured skin samples of WT and TNF-Rp55-/- mice. Representative results from 10 animals in each group are shown. The ratios of IL-1{alpha} (B), IL-1ß (C), MCP-1 (D), MIP-1{alpha} (E), and MIP-2 (F) to ß-actin of WT (open bar) and TNF-Rp55-/- (filled bar) were determined by RT-PCR 3 and 6 days after injury. Each value represents mean ± SE (n=10 animals). *P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT.

Protein levels of cytokines, TGF-ß1, and VEGF
The protein levels of cytokines, chemokines, TGF-ß1, and VEGF at the wound site were measured with ELISA. In WT and TNF-Rp55-/- mice, there was no significant difference in these protein levels at the uninjured skin (data not shown); these protein levels were increased at the wound site after injury. Protein levels of IL-1{alpha}, IL-1ß, MCP-1, MIP-1{alpha}, and MIP-2 were significantly reduced in TNF-Rp55 -/- mice 3 and 6 days after injury, when compared with WT mice (Fig. 12 ). TGF-ß1 and VEGF levels were significantly increased in TNF-Rp55 -/- mice when compared with WT mice; VEGF: 3 and 6 days (Fig. 8F ) and TGF-ß1: 1, 3, and 6 days (Fig. 9B ). These results were consistent with the results of RT-PCR analysis.



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  		Figure 12. Protein levels of IL-1{alpha} (A), IL-1ß (B), MCP-1 (C), MIP-1{alpha} (D), and MIP-2 (E) by ELISA (open bars: WT, filled bars: TNF-Rp55-/-). Each value represents mean ± SE (n=10 animals). * P < 0.05, **P < 0.01 TNF-Rp55-/- compared with WT.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Granulation tissue formation is prerequisite for wound healing. There are contradictory reports on the effects of locally applied TNF-{alpha} on granulation tissue formation in skin wound healing (10 11 12) , indicating there is no consensus on the effects of local TNF-{alpha} application. The administration of rabbit anti-mouse TNF-{alpha} pAb demonstrated that endogenous TNF-{alpha} could reduce collagen deposition at skin wound sites, although the detailed mechanism was not explained (14) . Cutaneous barrier repair was delayed in TNF-Rp55 -/- mice (25) , where the cutaneous permeability barrier in the stratum corneum was disrupted by tape stripping. However, the roles of TNF-Rp55 in excisional skin wounds remain to be investigated. Hence, we evaluated the precise pathophysiological roles of TNF-Rp55 in healing process of skin wounds, focusing on granulation tissue formation and angiogenesis.

A characteristic feature of granulation formation is collagen accumulation at the wound site. TNF-{alpha} can reduce transcription of collagen {alpha}1(I) gene in vitro (26 , 27) . In vivo, nude mice with the implant of TNF-{alpha}-producing cells exhibited a decrease in collagen synthesis and collagen {alpha}1(I) gene mRNA in the skin and displayed impaired healing of skin wounds (13) . CH3He/J mice, with an impaired capacity to produce TNF-{alpha} in response to LPS, showed enhanced collagen gene expression at skin wound sites compared with control mice (28) . Our present results on TNF-Rp55-/- mice revealed that the lack of TNF-Rp55 resulted in enhanced collagen accumulation with reduced leukocyte infiltration at wound sites. We also observed that i.p. administration of anti-TNF-{alpha} Ab accelerated wound healing with a concomitant reduction in neutrophil and macrophage infiltration (data not shown). Thus, the phenotypes observed in skin wound sites of TNF-Rp55 -/- mice may be ascribed directly to the absence of TNF-{alpha}-TNF-Rp55 signal pathways. In contrast, in silica- or bleomycin-induced lung inflammation and fibrosis, endogenously produced TNF-{alpha} was presumed to be a profibrotic factor (29 , 30) . Several lines of evidence demonstrated that an organ-specific expression of TNF-{alpha} transgenes eventually resulted in fibrosis in the corresponding organs such as lung, heart, pancreas, and skin (31 32 33 34) . The discrepancies may be explained by the fact that TNF-{alpha} expression was just transiently up-regulated in our wound model in contrast to other models, where overexpression of TNF-{alpha} was sustained for a longer period.

Potent fibrogenic growth factors such as TGF-ß1 cause an increase in collagen production during skin wound healing (35 36 37 38) . After wound preparation, we observed enhanced gene expression of fibrogenic growth factors such as TGF-ß1 and CTGF with a concomitant increase in TGF-ß1 protein content in TNF-Rp55-/- mice compared with WT mice. Buck et al. (13) demonstrated that the expression of collagen and TGF-ß1 genes was suppressed in cachexic mice with inoculation of TNF-{alpha}-producing cells, implying that TNF-{alpha} can inhibit in vivo TGF-ß1 as well as collagen gene expression. A recent study revealed that TNF-{alpha} suppressed TGF-ß-induced expression of CTGF (39) . TGF-ß1 is involved in epithelial migration. Hebda (40) demonstrated TGF-ß1 promoted the expansion of the epidermal outgrowth in vitro. Mustoe and colleagues (37) demonstrated that TGF-ß1 promoted reepithelialization of skin wounds in rats. Collectively, TNF-Rp55-mediated signals may dampen the expression of fibrogenic factors in vivo; the absence of TNF-Rp55 resulted in enhanced expression of these factors and subsequently promoted collagen accumulation and reepithelialization.

Angiogenesis is another indispensable event for granulation tissue formation. Contradictory results have been reported on the roles of TNF-{alpha} in angiogenesis. Leibovich and colleagues (41) reported that TNF-{alpha} induced the formation of capillary tube-like structure in vitro. In contrast, Frater-Schröder and colleagues (42) demonstrated that TNF-{alpha} was a potent inhibitor of endothelial cell growth in vitro. These discrepancies may be explained by several independent observations that low doses of TNF-{alpha} promote angiogenesis whereas higher doses actually inhibit it (43 44 45) . Alternatively, TNF-Rp55-mediated signals can reduce angiogenesis indirectly by decreasing the expression of Flt-1 and Flk-1 in vitro (46) . We demonstrated that TNF-Rp55-/- mice exhibited the increment in vascular densities at wound sites, accompanied by an increase of gene expression in VEGF and its receptors such as Flt-1 and Flk-1, with a concomitant increase of VEGF protein content at the wound sites compared with WT mice. These observations suggest that the absence of TNF-Rp55-mediated signals promotes angiogenesis in wound healing through enhanced expression of VEGF, Flt-1, and Flk-1.

Neutrophil and macrophage infiltration into wound sites is a hallmark of the inflammatory phase of wound healing. TNF-Rp55-/- mice exhibited reduced neutrophil and macrophage infiltration at wound sites compared with WT mice. There is a report that LPS treatment induced significantly less mononuclear cell infiltration in Propionibacterium acnes-primed TNF-Rp55-/- mice than WT mice (15) . Accumulating evidence indicates that adhesion molecules and chemokines have prominent roles in leukocyte infiltration into tissues (47 48 49 50 51) . TNF-{alpha} induced the expression of several adhesion molecules, particularly VCAM-1 and E-selectin, through interaction with TNF-Rp55 (52) . Actually, gene expression of adhesion molecules such as E-selectin, ICAM-1, and VCAM-1 was significantly reduced in wound sites of TNF-Rp55-/- mice compared with WT mice. TNF-{alpha} can induce expression of IL-1 (53) and several chemokines with chemotactic activities against neutrophils and macrophages such as MCP-1, MIP-1{alpha}, and MIP-2 (54 , 55) . The expression of these chemokines can be up-regulated by other proinflammatory cytokines, IL-1{alpha}, and IL-1ß (54) . We observed that TNF-Rp55-/- mice exhibited reduced gene expression of IL-1 (IL-1{alpha}, IL-1ß) and these chemokines with concomitant decreases in IL-1{alpha}, IL-1ß, MCP-1, MIP-1{alpha}, and MIP-2 protein content at the wound sites. Thus, it can be considered that the lack of TNF-Rp55-mediated signals resulted in reduced expression of adhesion molecules and chemokines, thereby leading to attenuated leukocyte infiltration.

Wound contraction is an important factor for wound closure and myofibroblasts seem contribute to the wound contraction, although we failed to detect a significant difference in myofibroblast number and mRNA expression of myofibroblast markers, including {alpha}-smooth muscle actin ({alpha}-SMA) and desmin between WT and TNF-Rp55 -/- mice (data not shown). This finding was supported by an earlier observation that {alpha}-SMA was induced by TGF-ß1 but not TNF-{alpha} (56 , 57) . However, we cannot rule out the possibility that a marginal increase of myofibroblasts in TNF-Rp55 -/- mice may be responsible for the secondary up-regulation of TGF-ß1 and eventual accelerated wound healing in TNF-Rp55 -/- mice.

An absence or a decrease in macrophage number at wound sites impaired tissue repair (58) , and transfer of macrophage into aged mice accelerated wound healing (59) . Skin wound healing was impaired with decreased macrophage infiltration in mice deficient in ICAM-1 and those deficient in ICAM-1 and L-selectin (18) . These observations suggest that macrophage infiltration has an important role in wound healing, although several recent reports have raised questions on the validity of this notion. Secretory leukocyte protease inhibitor-deficient mice exhibited impaired wound healing despite or because of exaggerated leukocyte infiltration into wound sites (60) . Skin wound healing was accelerated despite reduced leukocyte infiltration in mice deficient in Smad3, an essential signal transducer of TGF-ß1 signal pathway, similarly as TNF-Rp55-/- mice (61) . These findings suggest that the reduction of leukocyte infiltration does not always result in impaired healing of skin wounds. Tissue remodeling of skin wounds is composed of extracellular matrix (ECM) production and matrix destruction by proteases. Reduced leukocyte infiltration may lead to the decrease of tissue destructive mechanisms and eventually promote wound healing, since leukocytes such as neutrophils and macrophages produce MMPs (62) . In TNF-Rp55 -/- mice, gene expression of MMPs was significantly reduced (our unpublished data), suggesting that ECM degradation was less evident in TNF-Rp55 -/- mice compared with WT mice.

Angiogenesis and collagen accumulation may proceed in wound healing processes independent of leukocyte infiltration. The lack of TNF-Rp55 may enhance gene expression of fibrogenic factors and VEGF receptors in skin wounds, thereby promoting angiogenesis and collagen accumulation; eventually, wound healing is accelerated. Hence, the manipulation of TNF-Rp55-mediated signal system might be a good therapeutic maneuver for skin wounds.


   ACKNOWLEDGMENTS
 
We would like to express our sincere gratitude to Dr. Joost J. Oppenheim and Dr. Dan Mellon (NCI-FCRDC) for their critical review of the paper. The work is supported by Grants-in-Aids from the Ministry of Education, Culture, Science, and Technology of the Japanese Government. This study was partly presented at III Roche Milano Ricerche Symposium (Milan, March 29–31, 2001).

Received for publication September 25, 2001. Revision received March 7, 2002.
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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