Eccrine sweat glands are major contributors to reepithelialization of human wounds

Abstract

Eccrine sweat glands are skin-associated epithelial structures (appendages) that are unique to some primates including humans and are absent in the skin of most laboratory animals including rodents, rabbits, and pigs. On the basis of the known importance of other skin appendages (hair follicles, apocrine glands, and sebaceous glands) for wound repair in model animals, the present study was designed to assess the role of eccrine glands in the repair of wounded human skin. Partial-thickness wounds were generated on healthy human forearms, and epidermal repair was studied in skin biopsy samples obtained at precise times during the first week after wounding. Wound reepithelialization was assessed using immunohistochemistry and computer-assisted 3-dimensional reconstruction of in vivo wounded skin samples. Our data demonstrate a key role for eccrine sweat glands in reconstituting the epidermis after wounding in humans. More specifically, (i) eccrine sweat glands generate keratinocyte outgrowths that ultimately form new epidermis; (ii) eccrine sweat glands are the most abundant appendages in human skin, outnumbering hair follicles by a factor close to 3; and (iii) the rate of expansion of keratinocyte outgrowths from eccrine sweat glands parallels the rate of reepithelialization. This novel appreciation of the unique importance of eccrine sweat glands for epidermal repair may be exploited to improve our approaches to understanding and treating human wounds.

Figure 1

Partial-thickness wounds trigger a proliferative response in eccrine sweat glands and pilosebaceous units underlying the wound. A: CO₂ laser treatment efficiently ablates the entire epidermis and the superficial dermis as noted at 24 hours after wounding. Collagen VII staining (red) locates the basement membrane for reference. B: Paucity of basal Ki-67 staining in appendages of unwounded forearm skin. Hair follicle (left) and eccrine gland duct (right) with overlying epidermis, respectively. C: Ki-67 staining of an eccrine sweat gland duct in wounded forearm skin at 4 days after CO₂ laser treatment. Note that almost all cells of the outermost layer of the eccrine duct are positive for Ki-67 (arrow). D: Fourteen consecutive sections of wounded human skin were taken 3 days after CO₂ laser treatment, stained for Ki-67, and imaged. Eight of the 14 sections with corresponding stack number are shown. Digital images were used to generate a 3D reconstruction of the eccrine gland duct and the Ki-67–positive cells within the duct. Inset: Positive Ki-67 staining in outermost layer of the eccrine duct (arrow) as shown in C. Arbitrary colors are magenta for eccrine gland and maroon for Ki-67–positive staining. E: Ki-67 staining in a pilosebaceous unit at 3 days after CO₂ laser treatment. Black arrow denotes keratinocyte outgrowth, and white arrow indicates overall direction of hair follicle. A–E: Staining patterns are representative of at least 4 subjects. Scale bars = 100 μm. Positive staining is shown in red, and hematoxylin counterstaining in blue.

Figure 2

Eccrine glands and pilosebaceous units give rise to individual keratinocyte outgrowths during wound repair in human skin. 3D reconstruction from immunohistochemistry of whole skin biopsy samples obtained 3 days after wounding. Consecutive sections were cut parallel to the skin surface, and the topmost 135 sections (7 μm thick) were used for reconstruction (representing the topmost 945 μm of skin). Arbitrary colors are cyan for pilosebaceous units, magenta for eccrine sweat glands, yellow for new epidermis, and gray mesh for sample contours.

Figure 3

Keratinocyte outgrowths expand above appendages until they merge with each other, thereby reconstituting the new interfollicular epidermis. 3D reconstruction seen from the underside of epidermis, generated from immunohistochemistry of whole skin biopsy samples obtained at 3, 4, 5, 6, and 7 days after wounding (A–E, respectively). Sample A is identical, albeit rotated, to sample in Figure 2. Arbitrary colors are cyan for pilosebaceous units, magenta for eccrine sweat glands, yellow for new epidermis, and gray mesh for biopsy contours. Revolving animations are presented in Supplemental Videos S1–S5. F: Quantification of epidermal coverage from 3D reconstructed images. Epidermal areas (in micrometers squared) were normalized to the total biopsy area for each sample. Results represent percentage of biopsy coverage (mean ± SEM, n = 2 to 3 per time point).

Figure 4

Relative contribution of eccrine glands and pilosebaceous units to the new interfollicular epidermis. A: Quantification of eccrine sweat glands and pilosebaceous units in human forearm skin. Appendages were counted in 4-mm diameter (∼0.126 cm² area) reconstructed skin samples. Results are given as mean ± SEM, n = 12. B: Comparison of total epidermal growth rate (straight line, lefty axis) with rate of expansion of eccrine gland–derived outgrowths (dashed line, righty axis). Total epidermis growth rate was quantified as described in Figure 3F; eccrine gland growth rate represents the area of new epidermis overlying eccrine glands per eccrine gland. Total eccrine glands from 2 to 3 individuals were combined for each time point; results are given as mean ± SEM. C: 3D reconstruction of skin sample obtained at 5 days after wounding showing a close-up of an outgrowth merging area. Pan-keratin immunostaining of selected sections (top panels), according to the cutting planes represented on 3D reconstruction in the bottom panel. Arbitrary colors are cyan for pilosebaceous units, magenta for eccrine sweat glands, and yellow for new epidermis. Revolving animation is presented in Supplemental Video S6.

Figure 5

Eccrine sweat glands are sole appendages involved in the reepithelialization of palm human skin. Top panel: 3D reconstruction from immunohistochemistry of whole skin palm biopsy sample obtained at 4 days after wounding. Biopsy encompasses the wounded area (foreground) and adjacent nonwounded skin (far back). Arbitrary colors are cyan for pilosebaceous units, magenta for eccrine sweat glands, darker yellow for new epidermis, lighter yellow for nonwounded epidermis, and gray mesh for biopsy contours. Note lack of pilosebaceous unit in palmar skin. Bottom panel represents a close-up of 3 isolated eccrine glands located from within the wounded area, and shows that each gland gives rise to individual epidermal outgrowths. Revolving animation is presented in Supplemental Video S7.