ARTICLE |
Correspondence to: Lucie Germain, LOEX, Hôpital du Saint-Sacrement, 1050 Chemin Ste-Foy, Québec, PQ G1S 4L8, Canada..
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Summary |
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Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound. (J Histochem Cytochem 46:12911301, 1998)
Key Words: wound healing, epidermis, keratinocytes, skin, mice, keratin, heat shock proteins, molecular chaperones, immunohistochemistry
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Introduction |
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The wound healing process can be divided into three consecutive and overlapping phases: inflammation; organization of the inflammatory exudate, leading to reepithelialization and granulation tissue formation; and remodeling of the extracellular matrix (
During wound healing, cells are exposed to a variety of stresses originating from the wound environment. Cells within and surrounding the wound are subjected to dehydration and exposure to oxygen because the epithelial barrier is broken down (
Cells defend themselves from stresses by expressing heat shock proteins (Hsps). These proteins, first reported to be induced by heat shock (
Considering that several stressful cellular conditions are present in the wound environment, we examined the expression of Hsps during wound healing of mouse skin. Hsp60, Hsp90, Hsp27, and Hsp70 were detected by immunofluorescence during inflammation and organization of the inflammatory exudate that leads to granulation tissue formation and reepithelialization. This report focuses on the epidermal compartment.
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Materials and Methods |
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Surgery
Six-week-old CD-1 mice (Charles River Laboratories of Canada; St-Constant, PQ, Canada) were used in these studies. Anesthesia was produced with 0.9 ml of ketamine (20 mg/ml) and xylazine (2.5 mg/ml) injected into the leg. One full-thickness skin wound (4 cm2 surface area) was made on the back. Wounds were left to heal by second intention without any device. Mice were sacrificed by overdose of anesthetics and biopsied at different times from 24 hr to 21 days after wounding (two mice per time of biopsy). Wound healing tissue and normal skin were embedded in OCT and frozen sections (4 µm thick) were prepared. This study was repeated twice. The Ethics Committee of Laval University approved animal experiments in accordance with the guidelines of the Canadian Council on Animal Care.
Antibodies
The primary antibodies used for the immunodetection of the different Hsps and keratin 6 were all polyclonal rabbit antibodies. The anti-human keratin 6 antibody was a gift of Dr. Pierre A. Coulombe (
Gel Electrophoresis and Immunoblot Analysis
The specificity of the primary anti-Hsp antibodies was demonstrated using total proteins from normal mouse skin and Western blot analysis as described previously (
Immunofluorescence Microscopy
Indirect immunofluorescence labeling of frozen sections (fixed for 10 min in -20C methanol) was realized using polyclonal anti-keratin 6 (1:500), Hsp60 (1:100), Hsp90 (1:100), Hsp27 (1:100), and Hsp70 (1:100) antibodies and rhodamine-conjugated secondary antibody (1:100) as described previously (
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Results |
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Antibody Specificities
The specificity of the primary antibodies against Hsp27, Hsp60, Hsp70, and Hsp90 was demonstrated by Western blot analysis of total proteins from normal mouse skin and is shown in Figure 1.
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Keratin 6
Keratinocytes of normal skin did not express keratin 6, except in the hair follicles, but this keratin was present in the regenerating epidermis. Figure 2 shows a low magnification of normal skin adjacent to the regenerating epidermis (48 hr after wounding) labeled with an anti-keratin 6 antibody. The different locations with regard to the wound margin that will be stated in this report are identified. Because this photograph was taken 48 hr after wounding, the thickened epidermis (TE) above the granulation tissue was short compared to that present above the dermis (at the wound margin). The length of the TE over the granulation tissue increased as a function of time after wounding. Keratinocytes of normal epidermis were not labeled, whereas keratin 6 was expressed by low suprabasal cells in the TE (see Figure 2 inset). Behind the migrating epithelial tongue (MET), a suprabasal pattern of keratin 6 was preserved. In the MET, uniform labeling of all cell layers was observed. Therefore, the pattern of expression of keratin 6 varied within the epithelium regenerating over the granulation tissue, which constitutes the neoepidermis.
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Variations in Expression of Hsps as a Function of the Location of Keratinocytes in the Regenerating Epidermis
Each Hsp showed a specific pattern of expression in normal mouse epidermis, and different modifications of these patterns were observed in wound healing tissue. In the regenerating epidermis, the variations were dependent on the location of keratinocytes with regard to the wound margins rather than on the time after wounding. Therefore, our observations are presented in terms of location within the wound (TE, behind MET, MET, regenerating epidermis, and neoepidermis; see Figure 2).
Hsp60
In normal mouse epidermis, Hsp60 was present at a low level with a uniform distribution (Figure 3A). In the regenerating epidermis, the pattern of expression differed from that observed in normal skin. In the TE (Figure 3D and Figure 3E), many granules were observed in the basal and low suprabasal keratinocytes, whereas the high suprabasal cells were only lightly labeled. The location of this deep granular labeling varied according to the length of the regenerating epidermis; the longer it was, the further towards the center of the wound the labeling was. The neoepidermis behind the MET had this deep granular labeling at its maximum of intensity. In the MET, fewer granules were observed (Figure 3C) and these were uniformly spread, with an intermediate intensity between the labeling of normal epidermis (Figure 3A) and that of adjacent neoepidermis (Figure 3D and Figure 3E). Controls (for the four Hsps studied) of normal and regenerating mouse epidermis (with normal rabbit serum) showed minor background (Figure 3B and Figure 3F).
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Hsp90
Normal mouse epidermis had a high suprabasal pattern of expression, basal and low suprabasal cells having very light labeling and nuclei being negative (Figure 4A). During healing, Hsp90 preserved a suprabasal pattern in the TE (Figure 4B and Figure 4C) and the intensity was comparable to that observed in normal epidermis (Figure 4A). The labeling was located in the very high suprabasal cells and the stratum corneum progressively lost this labeling as the cells were reaching the surface. Basal and low suprabasal keratinocytes were slightly more labeled in the thickened neoepidermis (Figure 4B and Figure 4C) than in the normal epidermis (Figure 4A) or in the TE above dermis (Figure 4B). Behind the MET (Figure 4D), the neoepidermis preserved the same suprabasal pattern of expression, with a reduction in the intensity of both basal and suprabasal labeling. The MET showed faint and uniform labeling (Figure 4E).
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Hsp27
Normal mouse epidermis had a suprabasal pattern of expression, but the stratum corneum was not labeled (Figure 5A). The labeling was mainly cytoplasmic, with nuclei showing only faint staining. Basal and low suprabasal keratinocytes showed a slight expression of Hsp27. In the wound, the TE above the dermis (at the wound margin) had a pattern and level of expression (Figure 5C and Figure 5D) similar to those of normal epidermis (Figure 5A). In the TE over the granulation tissue (in the wound), the labeling intensity of superficial cells was reduced whereas that of deep cells was lightly increased (Figure 5C and Figure 5D). Instead of being concentrated in a thin suprabasal band, as in the normal epidermis (Figure 5A), the labeling was spread over a wider band (Figure 5BD). In some areas, basal keratinocytes showed a higher labeling (Figure 5D), which was more abundant in the TE over dermis (at the wound margin) and decreased towards the MET. Behind the MET, the neoepidermis was characterized by a granular distribution of Hsp27 in the suprabasal keratinocytes (Figure 5B). From the edge of the wound, these granules increased in number and intensity, producing a higher level of labeling. The MET was characterized by a uniform pattern and the intensity of the labeling was comparable to that of adjacent basal or low suprabasal cells (Figure 5E). The expression of Hsp27 by suprabasal keratinocytes in normal and TE (Figure 5AD) was seen in lower epidermal layers than that observed for Hsp90 (Figure 4AD).
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Inducible Hsp70
In normal mouse epidermis, the Hsp70 labeling was diffuse rather than granular and was slightly more visible in suprabasal keratinocytes than in basal ones (Figure 6A). The stratum corneum was not labeled. Some nuclei were lightly stained with a granular pattern. In the wound, the neoepidermis (Figure 6BE) had a different pattern of expression and a reduced labeling intensity compared to normal epidermis (Figure 6A) or to adjacent TE above dermis (Figure 6D). The thickened neoepidermis showed reduced and uniform Hsp70 labeling (Figure 6D). Many positive nuclei could be observed (Figure 6C and Figure 6D). Distal to the wound margin, basal and low suprabasal keratinocytes were more labeled than high suprabasal cells, producing a gradient of labeling (Figure 6B and Figure 6C). The pattern of expression was therefore reversed in comparison with normal epidermis (Figure 6B and Figure 6C vs Figure 6A). Fewer positive nuclei were found behind the MET (Figure 6B), and almost none were observed in the MET (Figure 6E). Keratinocytes in the MET had a lower expression of Hsp70 and the gradient of labeling from basal to suprabasal cells was lost (Figure 6E vs Figure 6B and Figure 6C).
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Table 1 summarizes the results observed on the expression of Hsp60, Hsp90, Hsp27, and Hsp70 in the normal vs the regenerating epidermis.
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Discussion |
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Indirect immunofluorescence on frozen sections was used to follow the expression of keratin 6 and four Hsps during reepithelialization of mouse skin after wounding. Each Hsp showed a specific pattern of expression in normal epidermis and these patterns were modified in regenerating epidermis, as summarized in Table 1.
In normal skin, the basic keratin 6 is associated with the acidic keratin 16 and both are expressed only by cells in the outer root sheet of hair follicles (
Each Hsp was expressed constitutively with a specific pattern in normal epidermis. However, the patterns and levels of expression were modified during wound healing. Unexpectedly, the changes observed in the regenerating epidermis were not linked to the time after wounding but rather were dependent on the location of keratinocytes in the regenerating epidermis. Each Hsp was expressed by keratinocytes of specific layers in normal and regenerating epidermis. Hsp60 was located in the basal and low suprabasal cells. Hsp90 and Hsp27 were mostly found in the high suprabasal cells, Hsp90 being expressed higher in the differentiation process than Hsp27. Hsp70 could be found throughout the epidermal compartment. Although the pattern of expression of each Hsp was affected differently after wounding, the MET always had a uniform expression of Hsps, the labeling being similar for the basal and suprabasal cells.
Keratinocytes located in the regenerating epidermis expressed more Hsp60 than those in normal epidermis. The pattern of expression varied from a light and uniform labeling in normal epidermis to a granular pattern located mainly in the basal and low suprabasal cells of the TE and finally to a uniformly distributed granular pattern in the MET. This granular pattern can be explained by the mitochondrial origin of Hsp60 (
Hsp90 had a very high suprabasal pattern of expression in normal and regenerating epidermis (except in the MET). Hsp27 also had a suprabasal pattern of expression in normal epidermis, as already shown in human skin (
In the TE, the labeling pattern of Hsp90 was similar to that of normal epidermis, with maximal expression in the very high suprabasal cells. Although cultured human keratinocytes have been shown to respond to physical and chemical stresses by the induction of a 90-kD protein (
Hsp27 was mainly expressed by suprabasal keratinocytes in normal and regenerating epidermis (except in the MET). This expression of Hsp27 mainly by keratinocytes in a low proliferative and highly differentiated state is consistent with the data published by others on different cell types and tumors (
In normal epidermis, keratinocytes constitutively express the inducible form of Hsp70, as shown by others (
Several studies have been conducted to understand the migration of epithelial cells over the wound surface (
In summary, the present study shows that the wound environment, which is believed to be stressful for cells, affects the expression of Hsps in a more complex manner than expected. Instead of being in direct relation to the time after wounding, the expression of Hsps is dependent on the location of keratinocytes in the regenerating epidermis. However, the variations observed along the neoepidermis might be due to the wound environment as the healing process progresses in the granulation tissue from the wound margin to its center. Each Hsp shows a specific pattern of expression in the regenerating epidermis instead of showing a unique pattern of response to the wound environment. This might be explained by their different functions: Hsp60 and Hsp70 were associated with proliferation in the TE, whereas the expression of Hsp27 and Hsp90 was correlated with the differentiation of keratinocytes. As discussed, the patterns of expression of these Hsps are probably not unique to wound healing because similar observations have been reported in other pathologies. The present data should help in our understanding of the physiological significance of the yet unknown roles of Hsps in living organisms.
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Acknowledgments |
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Supported by grants from the Medical Research Council of Canada (MRC) to FAA, JL, RMT, and LG. AL is a recipient of a MRC studentship and FAA and LG are recipients of scholarships from the Fonds de Recherche en Santé du Québec (FRSQ).
We thank Julie Bergeron and Julie Tremblay for technical support and Dr Pierre A. Coulombe for the anti-keratin 6 antibody. We are grateful to Claude Marin for the photographs.
Received for publication December 22, 1997; accepted June 30, 1998.
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