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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
(IL-1), IL-1ß, monocyte chemoattractant protein 1, macrophage inflammatory protein 1 (MIP-1), 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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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 (TNF-) 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- in life-threatening conditions such as endotoxin shock and cancer cachexia (3)
. There are two types of receptors for TNF- 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-, 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- 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- increased wound disruption strength in incisional wounds and eventually accelerated wound healing in rats (10)
; the other groups reported that local application of TNF- (11
, 12)
or inoculation of TNF--producing cells (13)
impaired skin wound healing. Regan and colleagues (14)
observed the beneficial effects of rabbit anti-TNF- 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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- 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-, interleukin 1 (IL-1), IL-1ß, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), 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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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-, MIP-1, 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, MCP-1 and MIP-2 levels were measured by ELISA using purified rabbit pAb against IL-1, MCP-1 or MIP-2 and rabbit anti-IL-1, 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 > 10pg/mL, IL-1ß > 7pg/mL, TNF- > 5 pg/mL, MIP-1 > 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, TNF-Rp55, and -Rp75 during wound healing 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- 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--TNF-Rp55 signals may be involved mainly in skin wound healing.
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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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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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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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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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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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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, IL-1ß, MCP-1, MIP-1, 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, IL-1ß, MCP-1, MIP-1, 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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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, IL-1ß, MCP-1, MIP-1, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on granulation tissue formation in skin wound healing (10
11
12)
, indicating there is no consensus on the effects of local TNF- application. The administration of rabbit anti-mouse TNF- pAb demonstrated that endogenous TNF- 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- can reduce transcription of collagen 1(I) gene in vitro (26
, 27)
. In vivo, nude mice with the implant of TNF--producing cells exhibited a decrease in collagen synthesis and collagen 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- 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- 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--TNF-Rp55 signal pathways. In contrast, in silica- or bleomycin-induced lung inflammation and fibrosis, endogenously produced TNF- was presumed to be a profibrotic factor (29
, 30)
. Several lines of evidence demonstrated that an organ-specific expression of TNF- 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- expression was just transiently up-regulated in our wound model in contrast to other models, where overexpression of TNF- 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--producing cells, implying that TNF- can inhibit in vivo TGF-ß1 as well as collagen gene expression. A recent study revealed that TNF- 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- in angiogenesis. Leibovich and colleagues (41)
reported that TNF- induced the formation of capillary tube-like structure in vitro. In contrast, Frater-Schröder and colleagues (42)
demonstrated that TNF- was a potent inhibitor of endothelial cell growth in vitro. These discrepancies may be explained by several independent observations that low doses of TNF- 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- 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- can induce expression of IL-1 (53)
and several chemokines with chemotactic activities against neutrophils and macrophages such as MCP-1, MIP-1, and MIP-2 (54
, 55)
. The expression of these chemokines can be up-regulated by other proinflammatory cytokines, IL-1, and IL-1ß (54)
. We observed that TNF-Rp55-/- mice exhibited reduced gene expression of IL-1 (IL-1, IL-1ß) and these chemokines with concomitant decreases in IL-1, IL-1ß, MCP-1, MIP-1, 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 -smooth muscle actin (-SMA) and desmin between WT and TNF-Rp55 -/- mice (data not shown). This finding was supported by an earlier observation that -SMA was induced by TGF-ß1 but not TNF- (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.
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ACKNOWLEDGMENTS |
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Received for publication September 25, 2001.
Revision received March 7, 2002.
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as a critical macrophage chemoattractant in murine wound repair. J. Clin. Invest. 101,1693-1698[Medline]
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 R. Mori, T. J. Shaw, and P. Martin Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring J. Exp. Med., January 21, 2008; 205(1): 43 - 51. [Abstract] [Full Text] [PDF]
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 Y. Ishida, J.-L. Gao, and P. M. Murphy Chemokine Receptor CX3CR1 Mediates Skin Wound Healing by Promoting Macrophage and Fibroblast Accumulation and Function J. Immunol., January 1, 2008; 180(1): 569 - 579. [Abstract] [Full Text] [PDF]
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 T. Yukami, M. Hasegawa, Y. Matsushita, T. Fujita, T. Matsushita, M. Horikawa, K. Komura, K. Yanaba, Y. Hamaguchi, T. Nagaoka, et al. Endothelial selectins regulate skin wound healing in cooperation with L-selectin and ICAM-1 J. Leukoc. Biol., September 1, 2007; 82(3): 519 - 531. [Abstract] [Full Text] [PDF]
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 F. J. S. Rocha, U. Schleicher, J. Mattner, G. Alber, and C. Bogdan Cytokines, Signaling Pathways, and Effector Molecules Required for the Control of Leishmania (Viannia) braziliensis in Mice Infect. Immun., August 1, 2007; 75(8): 3823 - 3832. [Abstract] [Full Text] [PDF]
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T. Hayashi, Y. Ishida, A. Kimura, Y. Iwakura, N. Mukaida, and T. Kondo IFN-{gamma} Protects Cerulein-Induced Acute Pancreatitis by Repressing NF-{kappa}B Activation J. Immunol., June 1, 2007; 178(11): 7385 - 7394. [Abstract] [Full Text] [PDF]
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S. A. Eming, S. Werner, P. Bugnon, C. Wickenhauser, L. Siewe, O. Utermohlen, J. M. Davidson, T. Krieg, and A. Roers Accelerated Wound Closure in Mice Deficient for Interleukin-10 Am. J. Pathol., January 1, 2007; 170(1): 188 - 202. [Abstract] [Full Text] [PDF]
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 J. M. Peterson, K. D. Feeback, J. H. Baas, and F. X. Pizza Tumor necrosis factor-{alpha} promotes the accumulation of neutrophils and macrophages in skeletal muscle J Appl Physiol, November 1, 2006; 101(5): 1394 - 1399. [Abstract] [Full Text] [PDF]
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S. Saika, K. Ikeda, O. Yamanaka, K. C. Flanders, Y. Okada, T. Miyamoto, A. Kitano, A. Ooshima, Y. Nakajima, Y. Ohnishi, et al. Loss of Tumor Necrosis Factor {alpha} Potentiates Transforming Growth Factor {beta}-mediated Pathogenic Tissue Response during Wound Healing Am. J. Pathol., June 1, 2006; 168(6): 1848 - 1860. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Kondo, A. Kimura, K. Matsushima, and N. Mukaida Absence of IL-1 Receptor Antagonist Impaired Wound Healing along with Aberrant NF-{kappa}B Activation and a Reciprocal Suppression of TGF-beta Signal Pathway J. Immunol., May 1, 2006; 176(9): 5598 - 5606. [Abstract] [Full Text] [PDF]
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R. Liu, H. S. Bal, T. Desta, Y. Behl, and D. T. Graves Tumor Necrosis Factor-{alpha} Mediates Diabetes-Enhanced Apoptosis of Matrix-Producing Cells and Impairs Diabetic Healing Am. J. Pathol., March 1, 2006; 168(3): 757 - 764. [Abstract] [Full Text] [PDF]
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L. S. Barcelos, A. Talvani, A. S. Teixeira, L. Q. Vieira, G. D. Cassali, S. P. Andrade, and M. M. Teixeira Impaired inflammatory angiogenesis, but not leukocyte influx, in mice lacking TNFR1 J. Leukoc. Biol., August 1, 2005; 78(2): 352 - 358. [Abstract] [Full Text] [PDF]
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C. Cataisson, A. J. Pearson, S. Torgerson, S. A. Nedospasov, and S. H. Yuspa Protein Kinase C{alpha}-Mediated Chemotaxis of Neutrophils Requires NF-{kappa}B Activity but Is Independent of TNF{alpha} Signaling in Mouse Skin In Vivo J. Immunol., February 1, 2005; 174(3): 1686 - 1692. [Abstract] [Full Text] [PDF]
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 Y. Maekawa, T. Anzai, T. Yoshikawa, Y. Sugano, K. Mahara, T. Kohno, T. Takahashi, and S. Ogawa Effect of granulocyte-macrophage colony-stimulating factor inducer on left ventricular remodeling after acute myocardial infarction J. Am. Coll. Cardiol., October 6, 2004; 44(7): 1510 - 1520. [Abstract] [Full Text] [PDF]
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X. Zhang, M. Kohli, Q. Zhou, D. T. Graves, and S. Amar Short- and Long-Term Effects of IL-1 and TNF Antagonists on Periodontal Wound Healing J. Immunol., September 1, 2004; 173(5): 3514 - 3523. [Abstract] [Full Text] [PDF]
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 A. LEASK and D. J. ABRAHAM TGF-{beta} signaling and the fibrotic response FASEB J, May 1, 2004; 18(7): 816 - 827. [Abstract] [Full Text] [PDF]
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 T. Wada, K. Furuichi, N. Sakai, Y. Iwata, K. Kitagawa, Y. Ishida, T. Kondo, H. Hashimoto, Y. Ishiwata, N. Mukaida, et al. Gene Therapy via Blockade of Monocyte Chemoattractant Protein-1 for Renal Fibrosis J. Am. Soc. Nephrol., April 1, 2004; 15(4): 940 - 948. [Abstract] [Full Text] [PDF]
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R. Mori, T. Kondo, T. Nishie, T. Ohshima, and M. Asano Impairment of Skin Wound Healing in {beta}-1,4-Galactosyltransferase-Deficient Mice with Reduced Leukocyte Recruitment Am. J. Pathol., April 1, 2004; 164(4): 1303 - 1314. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Maegawa, T. Kondo, A. Kimura, Y. Iwakura, S. Nakamura, and N. Mukaida Essential Involvement of IFN-{gamma} in Clostridium difficile Toxin A-Induced Enteritis J. Immunol., March 1, 2004; 172(5): 3018 - 3025. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Kondo, T. Takayasu, Y. Iwakura, and N. Mukaida The Essential Involvement of Cross-Talk between IFN-{gamma} and TGF-{beta} in the Skin Wound-Healing Process J. Immunol., February 1, 2004; 172(3): 1848 - 1855. [Abstract] [Full Text] [PDF]
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N. Zhang, Z. Fang, P. R. Contag, A. F. Purchio, and D. B. West Tracking angiogenesis induced by skin wounding and contact hypersensitivity using a Vegfr2-luciferase transgenic mouse Blood, January 15, 2004; 103(2): 617 - 626. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Kondo, K. Tsuneyama, P. Lu, T. Takayasu, and N. Mukaida The pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen-induced liver injury in mice J. Leukoc. Biol., January 1, 2004; 75(1): 59 - 67. [Abstract] [Full Text] [PDF]
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 G. H. Zhu and E. L. Schwartz Expression of the Angiogenic Factor Thymidine Phosphorylase in THP-1 Monocytes: Induction by Autocrine Tumor Necrosis Factor-{alpha} and Inhibition by Aspirin Mol. Pharmacol., November 1, 2003; 64(5): 1251 - 1258. [Abstract] [Full Text] [PDF]
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 S. WERNER and R. GROSE Regulation of Wound Healing by Growth Factors and Cytokines Physiol Rev, July 1, 2003; 83(3): 835 - 870. [Abstract] [Full Text] [PDF]
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Z.-Q. Lin, T. Kondo, Y. Ishida, T. Takayasu, and N. Mukaida Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice J. Leukoc. Biol., June 1, 2003; 73(6): 713 - 721. [Abstract] [Full Text] [PDF]
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 K. Chida, T. Hara, T. Hirai, C. Konishi, K. Nakamura, K. Nakao, A. Aiba, M. Katsuki, and T. Kuroki Disruption of Protein Kinase C{eta} Results in Impairment of Wound Healing and Enhancement of Tumor Formation in Mouse Skin Carcinogenesis Cancer Res., May 15, 2003; 63(10): 2404 - 2408. [Abstract] [Full Text] [PDF]
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