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Originally published online as doi:10.1189/jlb.0307152 on June 26, 2007

Published online before print June 26, 2007
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(Journal of Leukocyte Biology. 2007;82:519-531.)
© 2007 by Society for Leukocyte Biology

Endothelial selectins regulate skin wound healing in cooperation with L-selectin and ICAM-1

Toru Yukami*, Minoru Hasegawa*,1, Yukiyo Matsushita*, Tomoyuki Fujita*, Takashi Matsushita*, Mayuka Horikawa*, Kazuhiro Komura*, Koichi Yanaba*, Yasuhito Hamaguchi*, Tetsuya Nagaoka*, Fumihide Ogawa{dagger}, Manabu Fujimoto*, Douglas A. Steeber{ddagger}, Thomas F. Tedder§, Kazuhiko Takehara* and Shinichi Sato{dagger}

* Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan;
{dagger} Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan;
{ddagger} Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA; and
§ Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA

1 Correspondence: Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1, Takara-machi, Kanazawa 920-8641, Japan. E-mail: minoruha{at}derma.m.kanazawa-u.ac.jp

ABSTRACT

Skin wound healing is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. Mice lacking intercellular adhesion molecule-1 (ICAM-1) delayed skin wound healing and mice lacking both L-selectin and ICAM-1 (L-selectin/ICAM-1–/–) show more delayed wound healing. Deficiency of both endothelial selectins (E-selectin or P-selectin) also delays wound healing. However, the relative contribution and interaction of selectins and ICAM-1 to the wound healing remain unknown. To clarify them, repair of excisional wounds was examined in L-selectin/ICAM-1–/– mice, wild-type mice with both E- and P-selectin blockade, and L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade. Wild-type mice with both E- and P-selectin blockade showed delayed wound healing that was comparable with that in L-selectin/ICAM-1–/– mice. Combined E- and P-selectin blockade in L-selectin/ICAM-1–/– mice resulted in more significant delay. Mice lacking or blocked for adhesion molecules also showed suppressed keratinocyte migration, angiogenesis, granulation tissue formation, leukocyte infiltration, and cytokine expression, including transforming growth factor-ß and interleukin-6. Application of basic fibroblast growth factor (bFGF) but not platelet-derived growth factor to the wounds significantly improved wound healing in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade. bFGF significantly increased the leukocyte infiltration and subsequent fibrogenic cytokine production, as well as keratinocyte migration, angiogenesis, and collagen synthesis despite the loss of four kinds of adhesion molecules. These results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM-1 and may provide critical information for the therapy of skin wounds.

Key Words: adhesion molecule • cytokine • animal model

INTRODUCTION

Leukocyte recruitment from the circulation into inflammatory sites is a multistep process that is regulated by multiple cell-surface adhesion molecules [1 , 2 ]. Leukocytes first tether and roll on vascular endothelial cells, before they are activated to adhere firmly and subsequently immigrate into the extravascular space. The selectins primarily mediate tethering and rolling of leukocytes, whereas immunoglobulin (Ig) superfamily members, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and their integrin ligands, including lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) and very late antigen-4 (VLA-4, CD49d/CD29), are critical for the firm adhesion that follows. The selectin family consists of three cell-surface molecules expressed by leukocytes (L-selectin), vascular endothelium (E- and P-selectins), and platelets (P-selectin) [3 ]. While P-selectin is rapidly mobilized to the surface of activated endothelium or platelets, E-selectin expression is induced within several hours after activation with inflammatory cytokines [3 ]. By contrast, L-selectin is constitutively expressed on most leukocytes [3 ]. ICAM-1 is constitutively expressed at low levels by endothelial cells but is rapidly up-regulated during inflammation [4 ]. Interaction of adhesion molecules during the process of leukocyte migration into inflammatory sites is complex and highly regulated. Three selectins have partially overlapping functions for leukocyte rolling [5 6 7 ]. Furthermore, interactions between integrins and their Ig superfamily ligands can mediate rolling, as well as firm adhesion synergistically with selectins [8 9 10 ]. In addition, E-selectin does not only mediate rolling but also serves to promote firm adhesion [11 ]. This functional redundancy of adhesion molecules makes predicting relative contribution of each adhesion molecule to a certain inflammatory process difficult. In addition, the relative role of each adhesion molecule in inflammation varies according to the tissue site and the nature of the inflammatory stimuli [12 13 14 ]. We have previously demonstrated that various adhesion molecules play important roles with different relative contributions in the development of the Arthus reaction [14 ]. Therefore, to precisely determine the relative contribution of each adhesion molecule to a given disease model, the role of each adhesion molecule must be systematically examined.

Skin wound healing starts immediately after an injury and consists of three general stages: 1) an inflammatory stage, which consists of platelet aggregation and recruitment of inflammatory cells to the wound site; 2) a proliferative phase which involves the migration and proliferation of keratinocytes, fibroblasts, and endothelial cells, leading to re-epithelialization and granulation tissue formation; and 3) a long remodeling phase [15 16 17 ]. Migration of inflammatory cells to the wound site is important in wound repair. Initially, neutrophils begin accumulating at the wound sites within minutes of injury [16 ]. Infiltrating neutrophils form a first line of defense against local infections by clearing foreign particles and bacteria. Neutrophils are also a source of proinflammatory cytokines that likely serve as some of the earliest signals to activate fibroblasts and keratinocytes [17 ]. Neutrophils are then extruded with the eschar or phagocytosed by macrophages. Macrophages enhance the debridement by phagocytosis of microorganisms and fragments of extracellular matrix [18 ]. In addition, macrophages are important producers of a battery of growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor-ß (TGF-ß), heparin binding epidermal growth factor, and TGF-{alpha} [15 , 16 ]. These factors stimulate the synthesis of extracellular matrix by local fibroblasts, generate new blood vessels, promote granulation tissue formation, and enhance re-epithelialization that takes place by the migration of keratinocytes from the edges of the wound toward the center [15 , 16 ]. Mast cells are also considered an important source of mediators during wound healing and are detected at higher frequencies than in noninjured skin [19 , 20 ]. Otherwise, previous papers demonstrate that many factors affect the process of wound healing. Estrogens accelerate wound repair by inhibiting prolonged inflammatory cell infiltration and by enhancing collagen deposition [21 , 22 ]. By contrast, androgens negatively regulate the wound healing process via prolonged inflammatory response and reduced collagen deposition [23 ].

We previously showed that mice lacking ICAM-1 had delayed wound healing that was associated with decreased infiltration of neutrophils and macrophages [24 ]. Although the loss of L-selectin did not significantly affect wound healing, the absence of both L-selectin and ICAM-1 delayed wound healing beyond that caused by loss of ICAM-1 alone. Similarly, although the loss of either E-selectin or P-selectin does not affect cutaneous wound repair, deficiency of both E- and P-selectin does delay wound healing [25 ]. However, the relative contribution and interaction of selectin members and ICAM-1 to wound healing remain unknown. In this study, to clarify the roles of endothelial selectins in the absence of L-selectin and ICAM-1 expression during wound healing, we investigated a wound healing model in L-selectin/ICAM-1–/– mice treated with both E- and P-selectin blockade. The results of this study indicate that endothelial selectins contribute predominantly to wound healing by regulating the accumulation of inflammatory cells in the absence of L-selectin and ICAM-1, and also demonstrate that optimal wound healing is mediated cooperatively by all selectins and ICAM-1.

MATERIALS AND METHODS

Animals
L-selectin–/– mice were produced as described previously [26 ]. ICAM-1–/– mice [27 ] were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). These ICAM-1–/– mice express residual amounts of ICAM-1 splice variants in the thymus and spleen but not in other organs, including skin [28 ]. Mice lacking both L-selectin and ICAM-1 were generated by crossing F1 offspring from crosses of homozygous L-selectin–/– mice with homozygous ICAM-1–/– mice as described previously [10 ]. All mice were healthy, fertile, and did not display any evidence of infection or disease. All mice were backcrossed between 5 to 10 generations onto the C57BL/6 background. Seven to 12 week-old male mice were used for all experiments, and age- and gender-matched wild-type littermates or C57BL/6 mice (The Jackson Laboratory) were used as controls. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science.

Wounding and macroscopic examination
Mice were anesthetized with diethyl ether and their backs were shaved and cleaned with 70% alcohol. For a blocking study using mAbs to E- or P-selectin, mAbs were injected intravenously before wounding and on day 3. Abs used in this blocking study included mAbs to murine E-selectin (10E9.6, rat IgG2a, 30 µg per mouse; BD PharMingen, San Diego, CA, USA) [29 ] and mAbs to murine P-selectin (RB40.34, rat IgG1, 30 µg per mouse; BD PharMingen) [29 ]. These were the mAb concentrations required to inhibit E-selectin- and P-selectin-dependent leukocyte rolling in vivo as described previously [30 ]. Four full-thickness excisional wounds per mouse were made using a disposable sterile 6-mm biopsy punch (Kai, Tokyo, Japan), as described elsewhere [25 ]. After surgery, mice were caged individually. At 3 and 7 days after wounding, mice were anesthetized, and the areas of the open wounds were measured by tracing the wound openings onto a transparency. No signs suggestive of local infection were detected in the wounded skin. For macroscopic analysis of wound closure, 14 mice were used in each group.

Histologic examination and immunohistochemistry
After the mice were killed, wounds were harvested with a 2 mm rim of unwounded skin tissue. The wounds were cut into halves laterally, fixed in 3.5% paraformaldehyde, and were then paraffin embedded. Six-micrometer sections were stained with hematoxylin and eosin (H&E), toluidine blue staining, or immunostaining. For immunohistochemistry, deparafinized sections were treated with endogenous peroxidase blocking reagent (DAKO Cytomation A/S, Copenhagen, Denmark) and proteinase K (DAKO Cytomation A/S) for 6 min at room temperature. Sections were then incubated with rat mAbs specific for macrophages (clone F4/80, American Type Culture Collection, Rockville, MD, USA), mouse CD3 (Dainippon Pharamaceutical Company, Osaka, Japan), mouse CD31 (BD PharMingen), mouse E- and P-selectin (Coulter, Inc., Miami, FL, USA), and rabbit anti-myeloperoxidase polyclonal Ab (Neomarkers, Fremont, CA, USA). Rat IgG (Southern Biotechnology Associates Inc., Birmingham, AL, USA) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 min, 37°C) with a biotinylated rabbit anti-rat IgG secondary Ab (Vectastain ABC method, Vector Laboratories, Burlingame, CA, USA), then horseradish peroxidase-conjugated avidin-biotin complexes. Sections were washed 3 times with PBS between incubations. Sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide, and then counterstained with methyl green.

The number of neutrophils determined with myeloperoxidase was counted in the entire section at 1 and 4 h after wounding. Mast cell infiltration identified with toluidine blue staining was evaluated by counting in the entire section at 3 and 7 days after wounding, respectively. At days 3 and 7, numbers of F4/80-positive macrophages and CD3-positive T cells were determined by counting in nine high-power fields (0.07 mm2, magnification, x400) in the wound bed per section. Among the 9 fields, 6 fields were selected from both edges of the wound bed, and the remaining 3 fields were chosen from the middle of the wound bed. The epithelial gap, which represents distance between the leading edge of migrating keratinocytes, was measured in H&E-stained sections of wounds. We identified the area that consisted of newly formed capillaries and the collection of fibroblasts and macrophages as granulation tissue. Wound sections were visualized by a microscope (BX50, OLYMPUS, Tokyo, Japan) with images collected using a digital camera (DP70, OLYMPUS). Then, the area of granulation tissue was gated and measured using the same system. Using free-hand tool of Photoshop (Version 7.0, Adobe Systems, Tokyo, Japan), vessel density defined as CD31-positive ones was measured in the whole wound bed areas and expressed as a percentage of the whole wound bed areas. The number of mice used for each examination was as follows: 10 in each group for analysis of neutrophils, macrophages, mast cells and CD3-positive T cells and 14 in each group for measurement of the epithelial gap, the area of granulation tissue, and vessel density.

Application of growth factors
Optimal concentration of growth factors was applied to each wound in 20-µl aqueous buffer, and wounds were covered with an occlusive dressing (Tegaderm, 3M, Minneapolis, MN, USA). Growth factors were applied to wounds immediately after wounding and 12 h after wounding. Growth factors and their amounts used in this study were as follows: PDGF B-B isoform (AUSTRAL Biologicals, San Ramon, CA, USA) 800 ng/20 µl and bFGF (Kaken Pharmaceutical, Tokyo, Japan) 1000 ng/20 µl. These optimal amounts of growth factors were determined elsewhere [31 ]. Skin samples from 14 mice for each genotype were harvested at 3 and 7 days after wounding.

Real-time reverse transcription-polymerase chain reaction
Total RNAs were extracted from injured skin samples using QIAGEN RNeasy spin columns (QIAGEN, Crawley, UK) and digested with DNase I (QIAGEN) to remove chromosomal DNA in accordance with the manufacturer’s protocols. Total RNA was reverse transcribed to cDNA using Reverse Transcription System with random hexamers (Promega, Madison, WI, USA). Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using the TaqMan® system (Applied Biosystems, Foster City, CA, USA) on an ABI Prism 7000 Sequence Detector (Applied Biosystems) according to the manufacturer’s instructions. TaqMan probes and primers for {alpha}2 chain of the type I collagen (COL1A2), tumor necrosis factor (TNF)-{alpha}, IL-6, IL-10, TGF-ß, vascular endothelial growth factor (VEGF), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems. Relative expression of real-time PCR products was determined using the {Delta}{Delta}CT technique. Briefly, each set of samples was normalized using the difference in threshold cycle (CT) between the target gene and housekeeping gene (GAPDH): {Delta}CT = (CT target gene - CT GAPDH). Relative mRNA levels were calculated by the expression 2-{Delta}{Delta}CT, where {Delta}{Delta}CT = {Delta}CT sample – {Delta}CT calibrator. Each reaction was performed in, at least, triplicate.

Statistical analysis
The Kruskal-Wallis test was used for determining the level of significance of differences between samples, and Bonferroni’s test was used for multiple comparisons. A P value <0.05 was considered statistically significant.

RESULTS

Area of open wound
Four full-thickness excisional wounds per mouse were made using a 6-mm biopsy punch in L-selectin/ICAM-1–/– mice, wild-type mice with both E- and P-selectin blockade, and L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade. Each open wound area was calculated as a percentage of original wound areas at 3 and 7 days after wounding to assess macroscopic healing defects (Fig. 1A and 1B ). The mean % value of 4 open wounds was considered to be the remaining wound area in each mouse. At 3 days after injury, the open wound area was significantly larger in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.001), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05) than in wild-type mice. Furthermore, the open wound area in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was larger than that in L-selectin/ICAM-1–/– mice (P<0.05), and wild-type mice with both E- and P-selectin blockade (P<0.05). Similarly, at day 7, % open wound area of original wound was larger in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.01), and L-selectin/ICAM-1–/– mice (P<0.05) than that in wild-type mice. Furthermore, combined blockade of both E- and P-selectin in L-selectin/ICAM-1–/– mice significantly suppressed wound repair compared with L-selectin/ICAM-1–/– mice (P<0.05), and wild-type mice with both E- and P-selectin blockade (P<0.01). In L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, % open areas of original wound at day 7 was similar to that at day 3, indicating a complete lack of wound repair in these mice. Thus, both E- and P-selectin blockade significantly delayed macroscopic wound repair in L-selectin/ICAM-1–/– mice.


Figure 1
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  		Figure 1. (A) Representative photographs of open wounds in wild-type mice, L-selectin/ICAM-1–/– mice, wild type mice with both E- and P-selectin blockade, L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade at 3 and 7 days after wounding. Full-thickness cutaneous wounds were made using a 6-mm biopsy punch. Magnification, x4. (B) The area of the open wound was determined by tracing of the wound openings onto a transparency. The area was demonstrated as a percentage of the original wound. The mean percentage of four wounds was used for analysis in each mouse. (C) The distance between the migrating edges of keratinocytes under the eschar (epithelial gap) and (D) the area of granulation tissue were both measured in tissue sections. (E) Vessel density was determined as CD31-positive areas using PhotoShop. All values represent the mean ± SEM. These results were obtained from 14 mice in each group.

Epithelial gap
Migration of keratinocytes under the eschar was assessed by microscopically measuring the epithelial gap, that is the distance between the migrating edges of keratinocytes (Fig. 1C) . Keratinocyte migration was significantly inhibited in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade relative to wild-type mice at day 3 postwounding (P<0.001) and day 7 postwounding (P<0.0001). Wild-type mice with both E- and P-selectin blockade exhibited significant inhibition of keratinocyte migration at both day 3 (P<0.05) and day 7 postwounding (P<0.05) when compared with wild-type mice. Similarly, L-selectin/ICAM-1–/– mice exhibited significant inhibition of keratinocyte migration at both day 3 (P<0.001) and day 7 post-wounding (p<0.05) when compared with wild type mice. At 3 days after injury, migration of keratinocytes in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was reduced compared with wild-type mice with both E- and P-selectin blockade (P<0.01), and L-selectin/ICAM-1–/– mice (P<0.05). At 7 days after injury, migration of keratinocytes in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was inhibited compared with wild-type mice with blockade of both E- and P-selectins (P<0.01), and L-selectin/ICAM-1–/– mice (P<0.01). Thus, combined blockade of both E- and P-selectin further inhibited keratinocyte migration in L-selectin/ICAM-1–/– mice.

Granulation tissue formation
The area of granulation tissue was microscopically measured since granulation tissue formation is one of the most important components in wound repair (Fig. 1D) . At 3 days after wounding, granulation tissue formation was significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.0001), and L-selectin/ICAM-1–/– mice (P<0.0001) relative to wild-type controls. At 3 days after injury, granulation tissue formation in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was smaller than that in wild-type mice with both E- and P-selectin blockade (P<0.01), and than L-selectin/ICAM-1–/– mice (P<0.05). At 7 days after injury, granulation tissue formation was also inhibited in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.0001), and in L-selectin/ICAM-1–/– mice (P<0.0001) when compared with wild-type controls. Granulation tissue formation in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was smaller than that in wild-type mice with both E- and P-selectin blockade (P<0.05), and than L-selectin/ICAM-1–/– mice (P<0.01) by day 7 after wounding. Thus, the combined blockade of both E- and P-selectin significantly suppressed granulation tissue formation in L-selectin/ICAM-1–/– mice during wound healing.

Angiogenesis
Angiogenesis is an important event observed in the proliferative phase of wound healing. To assess the extent of angiogenesis, we performed immunohistochemical staining with anti-CD31 mAb (Fig. 1E) . At 3 days after wounding, the vascular density in the wound bed was significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.001), wild-type mice with both E- and P-selectin blockade (P<0.001), and L-selectin/ICAM-1–/– mice (P<0.001) relative to wild-type controls. At 3 days after injury, vessel density in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was smaller than that in wild-type mice with both E- and P-selectin blockade (P<0.01), and than L-selectin/ICAM-1–/– mice (P<0.01). At 7 days after injury, vessel density formation was also inhibited in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.0001), and in L-selectin/ICAM-1–/– mice (P<0.0001) when compared with wild-type controls. Vessel density in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade was smaller than that in wild-type mice with both E- and P-selectin blockade (P<0.01), and than L-selectin/ICAM-1–/– mice (P<0.01). Thus, the combined blockade of both E- and P-selectin significantly suppressed angiogenesis in L-selectin/ICAM-1–/– mice during wound healing.

Infiltration of neutrophils
Numbers of neutrophils that migrated outside the blood vessels were assessed in the wound tissues by immunohistochemical analysis using anti-myeloperoxidase mAb (Fig. 2A and 2B ). At 1 h after wounding, neutrophil numbers were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.01), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls. Neutrophil numbers in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were decreased compared with wild-type mice with both E- and P-selectin blockade (P<0.001), and L-selectin/ICAM-1–/– mice (P<0.001). Similarly, at 4 h after wounding, neutrophil numbers were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls. At 4 h after injury, neutrophil numbers in L-selectin/ICAM-1–/– mice with E- and P-selectin blockade were lower than wild-type mice with both E- and P-selectins blockade (P<0.05), and than that in L-selectin/ICAM-1–/– mice (P<0.01). Thus, both E- and P-selectin blockade significantly decreased neutrophil infiltration in L-selectin/ICAM-1–/– mice during the very early stage of wound repair.


Figure 2
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  		Figure 2. Representative histologic sections showing inflammatory cell infiltration into wounded skin of L-selectin/ICAM-1–/– mice, wild-type mice with both E- and P-selectin blockade, L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, and wild-type mice at 3 and 7 days after injury. (A) Neutrophils were detected in myeloperoxidase-stained sections at 1 and 4 h after injury. Magnification, x100. (B) Number of neutrophils per section was determined by counting in myeloperoxidase-stained sections. (C) Sections were stained with mAbs specific for macrophages (F4/80). Magnification, x200. (D) Numbers of F4/80-positive macrophages per field (0.07 mm2). (E) Numbers of mast cells per section determined by toluidine blue-staining. (F) Numbers of CD3-positive T cells per field (0.07 mm2). All values represent the mean ± SEM. These results were obtained from 10 mice in each group.

Infiltration of macrophages
Macrophage infiltration was assessed by immunohistochemistry using the F4/80 mAb (Fig. 2C and 2D) . Macrophage numbers were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.001) compared with wild-type mice at 3 days after injury. Furthermore, macrophage numbers in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were reduced compared with wild-type mice with both E- and P-selectin blockade (P<0.0001), and L-selectin/ICAM-1–/– mice (P<0.05). At 7 days after wounding, macrophage numbers were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), L-selectin/ICAM-1–/– mice (P<0.01) relative to wild-type controls. However, the blockade of both E- and P-selectins in wild-type mice did not significantly affect the number of infiltrating macrophages at this time point. Combined blockade of E- and P-selectin was not additive for the reduction of macrophage infiltration in L-selectin/ICAM-1–/– mice at 7 days after injury. Thus, E- and P-selectin function to support macrophage infiltration only during the early stages of wound healing.

Infiltration of mast cells
Mast cell infiltration was assessed by toluidine blue staining (Fig. 2E) . Mast cell numbers were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), wild-type mice with both E- and P-selectin blockade (P<0.05), L-selectin/ICAM-1–/– mice (P<0.05) when compared with wild-type mice at 3 days after injury. Furthermore, mast cell numbers in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were lower than these in wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05). At 7 days after wounding, mast cell numbers were also reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001), wild-type mice with both E- and P-selectin blockade (P<0.0001), and L-selectin/ICAM-1–/– mice (P<0.01) relative to wild-type controls. Furthermore, L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade showed lower mast cell numbers than L-selectin/ICAM-1–/– mice (P<0.01). Thus, both E- and P-selectin blockade significantly decreased mast cell infiltration in L-selectin/ICAM-1–/– mice.

Infiltration of CD3-positive T cells
T cell infiltration was assessed using immunohistochemical staining for CD3 (Fig. 2F) . The loss of both L-selectin and ICAM-1 or both E- and P-selectin blockade did not significantly affect CD3-positive T cell infiltration at 3 and 7 days after injury. However, CD3-positive T cell numbers were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.0001) when compared with wild-type mice at 3 days after injury. Furthermore, CD3-positive T cell numbers in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were lower than that in wild-type mice with both E- and P-selectin blockade (P<0.01) and L-selectin/ICAM-1–/– mice (P<0.001). At 7 days after wounding, CD3-positive T cell numbers were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade relative to wild-type mice (P<0.05) or L-selectin/ICAM-1–/– mice (P<0.01). Thus, both E- and P-selectin blockade decreased CD3-positive T cell infiltration in L-selectin/ICAM-1–/– mice of wound healing.

E- and P-selectin expression in wound healing
P-selectin is preformed and stored in the Weibel-Palade bodies of endothelial cells and is rapidly expressed on the surface after activation, while E-selectin requires synthesis before being expressed on the surface of activated endothelial cells. Therefore, E- and P-selectin expression was assessed immunohistochemically in normal and wounded skin. E- and P-selectin expression was not detected in normal skin (Fig. 3 ). By contrast, in wounded skin of wild-type mice, E- and P-selectin were expressed exclusively on endothelial cells of granulation tissue at both 3 and 7 days after wounding (Fig. 3) . The expression of E- and P-selectin in the granulation tissue was comparable between wild-type and L-selectin/ICAM-1–/– mice (data not shown). Thus, E- and P-selectin expression was up-regulated on wound endothelial cells.


Figure 3
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  		Figure 3. E- and P-selectin expression in normal and wounded skin from wild-type mice at 3 and 7 days after injury. Sections were stained with mAbs specific for E-selectin or P-selectin immunohistochemically. Magnification, x200.

Effect of growth factors on delayed wound healing
As neutrophils, macrophages, and mast cells in the initial inflammatory phase are a source of cytokines and growth factors that promote wound repair, the effect of growth factors on the delayed wound healing observed in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were examined. At 3 days after injury, the open wound area was not changed in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade by the addition of bFGF or PDGF (Fig. 4A ). At 7 days after injury, the addition of bFGF but not PDGF significantly reduced the wound area of these mice to a similar level of wild-type mice. Keratinocyte migration was almost normalized in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade by the addition of bFGF but not PDGF at day 3 and day 7 postwounding (Fig. 4B) . Granulation tissue formation was significantly increased in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade by the topical administration of bFGF at day 3 (P<0.0001) and day 7 postwounding (P<0.0001, Fig. 4C ). In fact, granulation tissue was comparable with that of wild-type mice at 7 days after injury. Although PDGF treatment also increased granulation tissue formation at days 3 and 7 (P<0.01 and P<0.001, respectively), it was still smaller than in wild-type mice even after 7 days (P<0.01). At 3 days after injury, vessel density was normalized in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade by the addition of bFGF but not PDGF at day 3 (Fig. 4D and 4E) . However, the normalized vascular injury by the treatment of topical bFGF had not been maintained until day 7 (P<0.0001 vs. wild type). Thus, topical administration of bFGF but not PDGF normalized macroscopic wound repair in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade by day 7 after wounding.


Figure 4
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  		Figure 4. Effect of growth factors (bFGF and PDGF) on delayed wound repair in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade at 3 and 7 days after injury. Growth factors were applied to each wound in 20 µl aqueous buffer immediately after wounding and 12 h after wounding. (A) The area of the open wound was determined by tracing of the wound openings onto a transparency. (B) The distance between the migrating edges of keratinocytes under the eschar (epithelial gap) and (C) the area of granulation tissue were both measured in tissue sections. (D) Vessel density was determined by the endothelium within the wound bed. (E) Vessel density was determined as CD31-positive areas using PhotoShop. Magnification, x100. All values represent the mean ± SEM. These results were obtained from 14 mice in each group.

Effect of growth factors on infiltration of inflammatory cells
Topical application of bFGF or PDGF significantly increased macrophage infiltration in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade at day 3 (P<0.0001 and P<0.001, respectively) and day 7 (P<0.01 and P<0.05, respectively), with the macrophage numbers in mice treated with bFGF at day 7 being comparable to those of wild-type mice (Fig. 5A ). Topical treatment of bFGF significantly increased mast cell infiltration in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade at day 3 (P<0.01) and day 7 (P<0.001), although these numbers were still lower than wild-type mice (Fig. 5B) . Mast cell numbers were also increased by PDGF treatment at day 3 (P<0.05). At day 3, CD3+ T cell number was significantly increased by the treatment with bFGF but not with PDGF in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.05, Fig. 5C ). At day 7, both treatments normalized the CD3+ T cell infiltration. Thus, the addition of bFGF or PDGF increased the number of macrophages, mast cells, and T cells in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade.


Figure 5
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  		Figure 5. Effect of growth factors on macrophage and mast cell recruitment in wounded skin from L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade at 3 and 7 days after injury. Mice were treated with growth factors as in Fig. 4 . Numbers of macrophages (A), mast cells (B), and CD3+ T cells (C) were determined as in Figure 2 . All values represent the mean ± SEM. These results were obtained from 10 mice in each group.

Expression of collagen mRNA
We next assessed mRNA expression levels of collagen (COL1A2) during granulation tissue formation. In uninjured skin, there was no significant difference in COL1A2 mRNA expression between wild-type and L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (data not shown). At 3 days after injury, COL1A2 mRNA levels were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (46%, P<0.05) relative to wild-type controls (Fig. 6A ). At 7 days after injury, COL1A2 mRNA levels were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls. At day 3 and 7, bFGF treatment significantly increased COL1A2 expression in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), and the levels were comparable with that in wild-type mice. By contrast, PDGF treatment did not significantly affect to the collagen expression. Thus, bFGF but not PDGF remarkably increased collagen mRNA expression in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade.


Figure 6
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  		Figure 6. Relative mRNA expression of (A) COL1A2, (B) TNF-{alpha}, IL-6, IL-10, TGF-ß, and VEGF at open wounds in wild-type mice, L-selectin/ICAM-1–/– mice, wild-type mice with both E- and P-selectin blockade, L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, and L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade treated with bFGF or PDGF. Relative mRNA expression was quantified by real-time RT-PCR. All values represent the mean ± SEM. These results were obtained from 10 mice in each group.

Expression of cytokines
mRNA levels of TNF-{alpha}, IL-6, IL-10, TGF-ß, and VEGF were also examined by real-time RT-PCR (Fig. 6B) . At 3 days after injury, TNF-{alpha} mRNA levels were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls. TNF-{alpha} mRNA levels in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were decreased compared with L-selectin/ICAM-1–/– mice (P<0.05). Similarly, at 7 days after wounding, TNF-{alpha} mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls. TNF-{alpha} mRNA levels in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade were lower than that in L-selectin/ICAM-1–/– mice (P<0.05) and wild-type mice with both E- and P-selectin blockade (P<0.01). Three days after injury, IL-6 mRNA levels were significantly reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01), wild-type mice with both E- and P-selectin blockade (P<0.01), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls. IL-6 mRNA levels were also reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade relative to wild-type controls (P<0.01) or L-selectin/ICAM-1–/– mice at day 7 (P<0.05). At 3 days after injury, IL-10 mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.05), and wild-type mice with both E- and P-selectin blockade (P<0.05) relative to wild-type controls. At 7 days after wounding, IL-10 mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.05) and wild-type mice with both E- and P-selectin blockade (P<0.05) relative to wild-type controls. Three days after injury, TGF-ß mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.001), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.01) relative to wild-type controls. At 7 days after wounding, TGF-ß mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.05) and wild-type mice with both E- and P-selectin blockade (P<0.05) relative to wild-type controls. Three days after injury, VEGF mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.01) relative to wild-type controls. At 7 days after wounding, VEGF mRNA levels were reduced in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (P<0.05), wild-type mice with both E- and P-selectin blockade (P<0.05), and L-selectin/ICAM-1–/– mice (P<0.05) relative to wild-type controls.

In L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, topical application of bFGF but not PDGF, significantly increased mRNA expression levels of TNF-{alpha} (P<0.01 and P<0.05), IL-6 (P<0.05 and P<0.01), IL-10 (P<0.05 and P<0.05), TGF-ß (P<0.001 and P<0.01), at day 3 and 7, respectively. bFGF did not significantly affect VEGF expression levels in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade. By contrast, PDGF treatment significantly increased only TNF-{alpha} levels at day 3 (P<0.05). Thus, the addition of bFGF significantly increased the mRNA levels of TNF-{alpha}, IL-6, IL-10, and TGF-ß in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade.

DISCUSSION

In the present study, macroscopic wound healing, keratinocyte migration, angiogenesis, and granulation tissue formation were significantly inhibited by the loss of both L-selectin and ICAM-1 (Fig. 1) . Blockade of both E- and P-selectins significantly suppressed wound healing to similar levels found in L-selectin/ICAM–/– mice (Fig. 1) . Combined blockade of both E- and P-selectins remarkably inhibited keratinocyte migration, angiogenesis, and granulation tissue formation, which resulted in a further delay of wound healing compared with L-selectin/ICAM-1–/– mice, indicating the cooperative function of these adhesion molecules (Fig. 1) . The impaired wound repair was accompanied by significantly reduced infiltration of neutrophils, macrophages, mast cells, and T cells and subsequent cytokine production (Fig. 2) . Therefore, the results of this study indicate distinct and cooperative roles of ICAM-1 and selectins in the process of wound healing. Interestingly, topical application of bFGF but not PDGF normalized wound repair by day 7 in association with improved keratinocyte migration, angiogenesis, granulation tissue formation, inflammatory cell infiltration, and fibrogenic cytokine or growth factor expression (Figs. 4 5 6) . Taken together, these results indicate that endothelial selectins and ICAM-1 cooperatively regulate optimal wound healing.

Wound healing is normal in single selectin-deficient mice, such as E-selectin–/–, L-selectin–/–, and P-selectin–/– mice, although neutrophil infiltration is reduced in P-selectin–/– mice at 1 and 4 h after injury [24 , 25 ]. Therefore, the loss of a single selectin member is not sufficient to cause impaired wound healing. Although L-, P-, and E-selectins have distinct roles, these selectins support optimal leukocyte rolling through overlapping functions [32 ]. In the absence of E-selectin or L-selectin, P-selectin primarily mediates rolling, whereas L-selectin primarily mediates rolling in P-selectin–/– mice [5 6 7 , 33 ]. Interestingly, the loss of both P- and E-selectin expression leads to delayed wound repair and inhibited keratinocyte migration after wounding [25 ], suggesting that L-selectin function alone cannot compensate for loss of the endothelial selectins. By contrast, the loss of ICAM-1 expression alone is able to inhibit wound healing [24 ]. Therefore, ICAM-1 may not be compensable by expression of other adhesion molecules when compared with each selectin in the process of wound healing. Alternatively, ICAM-1-mediated firm adhesion and transmigration of leukocytes may contribute to wound repair more than selectin-mediated rolling. ICAM-1 expression on fibroblasts may also mediate migration of neutrophils and macrophages through fibroblast layers [34 , 35 ]. A role for L-selectin in wound healing was revealed in the absence of ICAM-1 expression [24 ]. Therefore, it is likely that ICAM-1 and each selectin member have overlapping roles in wound healing. In the present study, P- and E-selectin expression were increased on endothelial cells during the wound healing process in wild-type and ICAM-1/L-selectin–/– mice (Fig. 3 and data not shown). This suggests that the delayed wound healing observed with mAb blockade of both E- and P-selectin is attributed mainly to impaired interaction between leukocytes and endothelial cells by functional inhibition of E- and P-selectin on endothelial cells.

In this study, the loss of all selectins and ICAM-1 remarkably reduced mast cell infiltration into the skin during wound healing as well as neutrophils, macrophages, and T cells (Fig. 2E) . Mast cell recruitment into tissues is considered to occur by release of immature mast cell precursors from the bone marrow into the peripheral blood, followed by migration of these precursors into tissue and their subsequent differentiation into mature mast cells [36 ]. In addition, increased numbers of mast cells are noted at sites of inflammation [37 ]. It is important to clarify whether adhesion molecules are critical for mast cell proliferation or recruitment of mast cells/mast cell precursors during wound healing. It has been demonstrated that the increase of mast cells within the first 10 days in the wounded skin is the result of increased recruitment/survival of mast cells or mast cell precursors [20 ]. Several studies have shown that rolling of immature bone marrow-derived mast cell precursors is mediated by the interaction of P-selectin and P-selectin glycoprotein ligand-1 [38 , 39 ]. In addition, in the passive Arthus reaction, cutaneous and peritoneal mast cell recruitment is reduced in mice lacking P-selectin or E-selectin [14 ]. Therefore, our findings suggest that all selectins and ICAM-1 regulate mast cell recruitment, presumably through the peripheral blood during skin wound healing.

Despite the loss or blockade of all four adhesion receptors, a complete inhibition of neutrophils, macrophages, mast cells, or CD3+ T cells was not found in this study (Fig. 2) . This raised the possibility that an adhesion pathway independent of all selectin members and ICAM-1 exists. Intravital microscopy studies have shown that a selectin-independent pathway is dependent on VLA-4, the ligand for VCAM-1, in the cremasteric microvasculature from mice challenged with TNF-{alpha} [40 ]. VLA-4 is the predominant selectin-independent mechanism for leukocyte migration to skin in response to various inflammatory stimuli [41 ]. Thus, the remaining leukocyte accumulation during wound healing may be mediated by VLA-4. In our previous study, the loss or blockade of all four adhesion molecules completely abrogated the development of both cutaneous and peritoneal Arthus reaction [14 ]. By contrast, wound healing was delayed but was not completely injured by the loss or blockade of these adhesion molecules (Fig. 1) . Therefore, remaining infiltrating leukocytes may partly be able to compensate for the role of optimal cell infiltration in wound healing model (Figs. 2 and 6) . Another possibility is that regional cells including keratinocytes, endothelial cells, and fibroblasts may have a significant role for wound healing without amounts of leukocyte infiltration. Leukocyte infiltration, including macrophage has been considered to be critical for the wound healing [42 , 43 ]. However, several recent papers have shown that angiogenesis and collagen deposition can compensate the skin wound healing independent of leukocyte infiltration under the specific pathogen-free conditions [44 45 46 ].

Treatment with bFGF normalized the delayed wound repair by day 7 in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade (Fig. 6) . bFGF is released at the wound site by macrophages and damaged endothelial cells [15 , 16 ]. Therefore, the decreased release of bFGF by reduced numbers of wound macrophage might account for the impaired wound repair in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade. Since bFGF treatment compensated for the suppressed keratinocyte migration, granulation tissue formation, and collagen expression in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, the effect of bFGF may be due to its potential effect on keratinocytes and fibroblasts. Additionally, down-regulated angiogenesis was normalized at day 3 in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade. Unexpectedly, application of bFGF was accompanied to varying extents by increased numbers of infiltrating cells despite the lack of adhesion molecules (Fig. 5) . Especially, the number of macrophages and T cells was normalized by day 7. It has been reported that bFGF treatment induces the infiltration of a large number of leukocytes in the wound exudates of diabetic mice, although the mechanism remains unclear [47 ]. bFGF acts mainly as a potent angiogenic factor during wound healing, since wound angiogenesis is remarkably inhibited when this growth factor is depleted with monospecific Abs raised against bFGF or genetically deleted [48 , 49 ]. In addition, bFGF promotes angiogenesis during the early stage of wound healing [50 ]. Therefore, the increased number of infiltrating cells despite the lack of adhesion molecules after topical application of bFGF is probably due to the augmented angiogenesis. Another possibility is that bFGF may enhance the role of chemokines or remaining adhesion molecules such as VCAM-1, although the mechanism remains unclear. Furthermore, some of proinflammatory or fibrogenic cytokines suppressed by the blockade of adhesion molecules were also improved by bFGF treatment (Fig. 6) . Thus, the effect of bFGF treatment on delayed wound healing observed in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade may not only be due to its potential effect on keratinocytes and fibroblasts but also to the augmented infiltration of inflammatory cells following angiogenesis and their subsequent production of cytokines or growth factors. By contrast, PDGF application did not normalize the delayed wound healing observed in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, although inflammatory cell infiltration and granulation tissue formation were increased to some extent (Fig. 4 and 5) . PDGF accelerates deposition of provisional wound matrix and collagen synthesis by fibroblasts [51 ]. Early in the repair process, PDGF augments the acute inflammatory response, specifically recruiting and activating wound macrophages [52 ]. PDGF is also a chemoattractant for wound fibroblasts [52 ]. These various functions of PDGF were found to cooperatively normalize the delayed wound healing in ICAM-1–/– mice [24 ]. However, PDGF activities were not sufficient for normalization of impaired wound repair in L-selectin/ICAM-1–/– mice [24 ] or L-selectin/ICAM-1–/– mice with both E-selectin and P-selectin blockade (Fig. 5) . PDGF did not increase keratinocyte migration, angiogenesis, collagen expression, and fibrogenic cytokine production, such as TGF-ß and IL-6, during wound repair process in L-selectin/ICAM-1–/– mice with both E- and P-selectin blockade, although PDGF treatment increased infiltrating cells and granulation tissue formation in some extent (Fig. 4 5 6) . Therefore, PDGF likely does not have enough roles in this adhesion molecule-disrupted wound model.

The loss of adhesion molecule decreased TNF-{alpha}, IL-6, IL-10, TGF-ß, and VEGF (Fig. 6) . Among them, TGF-ß was most remarkably inhibited by the loss of these adhesion molecules and completely recovered by bFGF treatment by day 3. Previous studies have demonstrated that TGF-ß improved skin wound healing, especially via augmented collagen production [53 , 54 ]. Furthermore, TGF-ß promotes reepithelialization of skin wounds of rats [53 ] and stimulates epidermal outgrowth in vitro [55 ]. IL-6 also may have critical roles in wound healing since IL-6-deficient mice show impaired skin wound healing probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation [56 , 57 ]. By contrast, TNF-{alpha} has been considered to inhibit TGF-ß expression, as well as collagen synthesis leading impaired wound healing [58 ]. IL-10 likely impedes wound repair via decreasing macrophage infiltration [59 ]. VEGF is a representative angiogenic factor in skin wound healing [60 ]. TGF-ß and IL-6 can augment VEGF transcription in various cell types [61 , 62 ]. Therefore, the change of TGF-ß and IL-6 expression levels by the loss or blockade of adhesion molecules and bFGF treatment may affect epidermal growth, collagen synthesis, and angiogenesis during wound healing (Fig. 1E 4D 4E ; and 6 ).

In summary, these results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM-1, and may provide critical information for the therapy of skin wounds.

ACKNOWLEDGEMENTS

We thank Ms. M. Matsubara and Y. Yamada for technical assistance. This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (to M. H. and S. S.) and U.S. National Institutes of Health grants CA54464 and CA81776 (to T. F. T.).

Received March 13, 2007; revised May 14, 2007; accepted May 23, 2007.

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