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The loss of ICAM-1 inhibited wound healing, keratinocyte migration from the edges of the wound toward the center, and granulation tissue formation. By contrast, L-selectin deficiency alone did not affect any of these parameters. However, the loss of both L-selectin and ICAM-1 resulted in inhibition of keratinocyte migration and granulation tissue formation beyond those caused by loss of ICAM-1 alone.
These results demonstrate a distinct role of ICAM-1 and L-selectin in wound healing and that the delayed wound healing in the absence of these molecules is likely because of decreased leukocyte accumulation into the wound site. Leukocyte recruitment into inflammatory sites is achieved using distinct constitutive or inducible adhesion molecules. The generation of adhesion molecule-deficient mice has provided considerable insight into the molecular interactions that occur during inflammation in vivo.
Healing of cutaneous wounds is a complex process that progresses through 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. Initially, neutrophils begin accumulating at the wound sites within minutes of injury.
Recent studies have shown that neutrophils are also a source of pro-inflammatory cytokines that probably serve as some of the earliest signals to activate fibroblasts and keratinocytes. Macrophages enhance the debridement by phagocytosis of microorganisms and fragments of extracellular matrix. The later stages of wound repair have been suggested to be strongly dependent on the initial inflammatory phase of the healing process. Depletion of macrophages by corticosteroids and anti-macrophage serum delays wound healing.
For this purpose, we analyzed cutaneous wound repair in mice lacking either L-selectin or ICAM-1, or both. The results demonstrate that ICAM-1 and L-selectin contribute to wound healing by mediating accumulation of leukocytes through synergistic functions. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens.
Four full-thickness excisional wounds per mouse were made using a disposable sterile 6-mm punch biopsy Maruho, Osaka, Japan , as described elsewhere. At 3 days and 7 days after wounding, mice were anesthetized, and areas of open wounds were measured by tracing the wound openings onto a transparency.
Any signs suggestive for local infection were not detected in the wounded skin. For macroscopic analysis of wound closure, 14 mice were used in each group. After distorted wounds were excluded, one wound was randomly selected in each mouse for the analysis.
After the mice were sacrificed, wounds were harvested with a 2-mm rim of unwounded skin tissue. The wounds were cut into halves laterally, fixed in 3. All sections were derived from the center of the wounds. Neutrophils were counted in the entire section outside the blood vessels at 1 hour and 4 hours after wounding. Numbers of macrophages per field 0.
We identified the area that consists of newly formed capillaries and the collection of fibroblasts and macrophages as granulation tissue. The number of mice used for each examination was as follows: 10 in each group for count of neutrophils and macrophages and 14 in each group for measurement of the epithelial gap and the granulation tissue area.
After distorted wounds were excluded, one wound was randomly selected in each mouse for every microscopic analysis. Sections were washed 3 times with PBS between incubations. We identified the area surrounded by both sides of unwounded skin, fascia, regenerated epidermis, and eschar as the wound bed.
Among the nine fields, six fields were selected from both edges of the wound bed, and the remaining three fields were chosen from the middle of the wound bed. Growth factors were applied to wounds immediately after wounding and 12 hours after wounding. These optimal amounts of growth factors were determined elsewhere.
For the analysis, 14 mice were used in each group, and one wound was randomly selected after distorted wounds were excluded in each mouse. Analysis of variance was used to analyze the data and Mann-Whitney U test was used to determine the level of significance of differences in sample means.
The areas of open wounds were measured at 3 days and 7 days after wounding to assess macroscopic healing defects Figure 1A. Therefore, macroscopic wound healing was delayed in the absence of ICAM-1 whereas L-selectin loss did not affect macroscopic wound healing. Wound closure and granulation tissue formation in mutant and wild-type mice at 3 days and 7 days after wounding.
Full-thickness cutaneous wounds were made using a 6-mm punch biopsy. The area of open wound was determined by tracing of the wound openings onto a transparency A. The distance between the migrating edges of keratinocytes under the eschar epithelial gap; B and the area of granulation tissue C were measured in the tissue sections.
These results were obtained from 14 mice in each group. Migration of keratinocytes under the eschar was assessed by microscopically measuring the epithelial gap that is the distance between the migrating edges of keratinocytes Figure 1B. The L-selectin deficiency alone did not significantly impair migration of keratinocytes at either day 3 or day 7.
The area of granulation tissue was microscopically measured because granulation tissue formation is one of the most important components in wound repair Figure 1C. L-selectin deficiency alone did not affect granulation tissue formation at either day 3 or day 7. Numbers of neutrophils that migrated outside the blood vessels were assessed in the wound tissues Figures 2 and 3.
Neutrophil recruitment in wounded skin from mutant and wild-type mice at 1 hour and 4 hours after injury. These results were obtained from 10 mice in each group. Macrophage recruitment in wounded skin from mutant and wild-type mice at 3 days and 7 days after injury. ICAM-1 has been found on the surface of keratinocytes and endothelial cells of inflamed skin and several pro-inflammatory cytokines up-regulate ICAM-1 expression on various types of cells.
ICAM-1 expression was up-regulated on endothelial cells in granulation tissues at both 3 days and 7 days after wounding Figure 6, B and C. By day 7, ICAM-1 expression was detected in keratinocytes, particularly basal keratinocytes, above granulation tissue, although its staining intensity was much weaker than that of endothelial cells Figure 6C.
In addition, the loss of L-selectin expression did not affect ICAM-1 expression in the intact or wounded skin data not shown. Thus, ICAM-1 was predominantly expressed on wound endothelial cells.
ICAM-1 expression in wild-type mice during wound healing. Wound healing was assessed by macroscopic area of the open wound at 3 days and 7 days after wounding. The area of open wound at 3 days and 7 days after wounding was determined by tracing of the wound openings onto a transparency.
In the present study, macroscopic wound healing, keratinocyte migration, and granulation tissue formation were significantly inhibited in the absence of ICAM-1 expression Figure 1. By contrast, the L-selectin deficiency alone did not affect any of these parameters in the wound healing process. However, the loss of both L-selectin and ICAM-1 led to inhibited keratinocyte migration and retarded granulation tissue formation beyond those caused by loss of ICAM-1 alone at an early time point after wounding.
However, L-selectin may also contribute to wound repair through overlapping and synergistic functions with ICAM This is consistent with the finding that L-selectin and ICAM-1 function synergistically to mediate optimal rolling as well as to recruit leukocytes into sites of inflammation. Therefore, the results of this study demonstrate a distinct role of ICAM-1 and L-selectin in a process of wound healing and that the delayed wound healing in the absence of these molecules is likely because of decreased leukocyte accumulation into the wound site.
Therefore, the loss of a single selectin member is not sufficient to cause impaired wound healing. Although L-, P-, and E-selectin have distinct roles, the selectins support optimal leukocyte rolling through overlapping functions. Therefore, reduced leukocyte rolling by combined loss of the selectins may cause the defect in the wound healing process. The loss of ICAM-1 expression resulted in dramatic inhibition of wound healing at both 3 days and 7 days after wounding Figure 1. This suggests that ICAMmediated firm adhesion and transmigration of leukocytes contribute to wound repair more than selectin-mediated rolling.
However, ICAM-1 expression on various types of cells, including keratinocytes and fibroblasts, is induced by stimulation with several pro-inflammatory cytokines in vitro. ICAM-1 expression on endothelial cells was up-regulated in granulation tissues of the wounded skin Figure 6. However, fibroblasts in granulation tissues did not express ICAM-1 and basal keratinocytes of the epidermis above granulation tissues were weakly positive for ICAM This suggests that the delayed wound healing in the absence of ICAM-1 is attributed mainly to impaired interaction between leukocytes and endothelial cells by loss of ICAM-1 expression on endothelial cells.
Taken together, these findings demonstrate a critical role of ICAM-1 in wound healing. PDGF accelerates deposition of provisional wound matrix and collagen synthesis by fibroblasts. This reinforces an important role of L-selectin in wound healing.
Understanding regulation of cutaneous wound repair at the molecular level is important because there are many disorders based on abnormal wound repair, including stasis ulcer, diabetic ulcer, keloids, and hypertrophic scars.
Address reprint requests to Shinichi Sato, M. Read article at publisher's site DOI : Physiol Rev , 99 1 , 01 Jan Review Free to read. Front Pharmacol , , 23 Aug Front Immunol , , 09 Apr Sci Rep , 7 1 , 12 Sep This data has been provided by curated databases and other sources that have cited the article. To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. J Leukoc Biol , 82 3 , 26 Jun Cited by 21 articles PMID: Am J Pathol , 5 , 01 Nov Am J Pathol , 5 , 01 May J Immunol , 6 , 01 Mar Cited by 25 articles PMID: J Immunol , 4 , 01 Aug Cited by 75 articles PMID: Coronavirus: Find the latest articles and preprints.
Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression.
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