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The exact pathogenesis of hypertrophic scar and keloid formation is still unknown and a good therapy to prevent or treat these scars is lacking. Because immunological processes seem to be important in excessive scar formation, immunological cells and parameters were studied in a standardized breast reduction wound-healing model in the present study. Standardized scar samples were taken from infra-mammary breast reduction scars, 3 and 12 months following surgery. The samples were investigated for their number of mast cells, Langerhans cells, T-lymphocytes, and macrophages, and the presence of interleukin-4 (IL-4) and counter-regulating interferon-gamma (gamma-IFN), in relation to the scar's clinical appearance--normal or hypertrophic. In this study, hypertrophic scar formation was significantly associated with an increased number of epidermal Langerhans cells (p=0.0001) and significantly (p<0.05) increased expression of epidermal IL-4. No relationship was found between mast cell, T-lymphocyte and macrophage numbers or gamma-IFN staining and the formation of normal or hypertrophic scars. These results, combined with previous observation of abnormal keratinocyte behaviour in this context, indicate that the epidermal immune barrier plays an important role in the development of hypertrophic scars., (Copyright 2003 John Wiley & Sons, Ltd.)

In predisposed individuals, wound healing can lead to hypertrophic scar or keloid formation, characterized by an overabundant extracellular matrix. It has recently been shown that hypertrophic scars are accompanied by abnormal keratinocyte differentiation and proliferation, and significantly increased acanthosis, compared with normal scars. This study addressed the question of whether the development of normal and hypertrophic scars is regulated by differences in the growth factor profiles of both the epidermis and the dermis. The presence of interleukin-1alpha (IL-1alpha), IL-1beta, tumour necrosis factor-alpha (TNF-alpha), platelet-derived growth factor (PDGF), transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) was investigated in biopsies taken from breast reduction scars at 3 and 12 months following surgery. The samples were analysed by immunohistological methods and categorized as scars that remained hypertrophic (HH), became normal (HN) or remained normal after 12 months (NN). The epidermal expression of IL-1alpha was significantly increased in NN scars compared with HN and HH scars 3 and 12 months following operation, whereas the dermal expression showed no difference. PDGF was significantly increased in the dermis of normal scars after 3 months and in both the epidermis and the dermis of hypertrophic scars after 12 months. IL-1beta, TNF-alpha, TGF-beta and bFGF showed no differences. It is hypothesized that impaired production of keratinocyte-derived growth factors, such as IL-1alpha, leads to a decrease in the catabolism of the dermal matrix, whereas augmented epidermal PDGF production leads to increased formation of the dermal matrix in hypertrophic scars. These observations support the possibility that the epidermis is involved in preventing the formation of hypertrophic scars., (Copyright 2001 John Wiley & Sons, Ltd.)

Ultraviolet B (UVB) irradiation is known to induce activation of cellular stress response pathways in cultured cells or intact human skin, as demonstrated by phosphorylation of MAP kinase family members and up- or down-stream targets, using biochemical assays. This study demonstrates by immunohistochemistry that low-dose UVB irradiation of normal human skin induces rapid and reversible phosphorylation of c-jun (a target of c-jun N-terminal kinase) and p38 mitogen activated protein kinase (p38 MAP kinase). Phosphorylation was maximal at 4-8 h and returned to normal levels at 48 h after irradiation. Nuclear localization of these phosphorylated substrates was found using antisera against the epitope containing the phosphorylated serine-73 of c-jun, and the dually phosphorylated epitope (threonine-180 and tyrosine-182) of p38 MAP kinase. Nearly all epidermal cells were positive for c-jun phosphorylation, whereas p38 phosphorylation was seen predominantly in the differentiated layers. In contrast to the massive activation of c-jun and p38, only a small population of the suprabasal cells showed nuclear translocation of nuclear factor kappa B (NFkappaB), and a few scattered cells became apoptotic, as determined by TUNEL (TdT mediated dUTP nick end labelling) staining. The expression of involucrin and skin-derived anti-leukoproteinase (SKALP)/elafin, two genes putatively under control of the c-jun and p38 pathways, was found to be increased. These findings establish the first cellular localization of UVB-induced protein phosphorylation of stress response proteins in human epidermis, thereby providing a link between cellular activation and gene expression in defined cell populations.

The role of epidermal keratinocytes in the early phases of normal unimpaired wound healing has been studied extensively. However, little is known about the cell biological processes in the epidermis and the basal membrane zone during the later phases of dermal matrix formation and remodelling of the scar tissue. This study investigated epidermal growth and differentiation and maturation of the basal membrane zone. Biopsies were taken from (clinically) hypertrophic and non-hypertrophic scars at 3 and 12 months after a breast-reduction operation. Tissues were analysed using immunohistochemical techniques. The data showed that epidermal abnormalities with respect to differentiation persist up to 3 months, as witnessed by the expression of cytokeratin 16. Remarkably, hypertrophic scars that remained hypertrophic throughout the period of analysis (up to 12 months) showed significantly more cytokeratin 16 expression at 3 months, when compared either with normal scars or with hypertrophic scars that became normal after 12 months. Staining for Ki-67 antigen, a marker for cell proliferation, revealed an increase in basal keratinocyte proliferation rate in 3-month-old hypertrophic scars compared with non-hypertrophic scars. After 12 months, this difference had disappeared completely and the number of cycling basal cells had returned to normal values. Three-month-old hypertrophic scars showed more acanthosis than non-hypertrophic scars of the same age, irrespective of whether they remained hypertrophic or became normal scars. After 12 months, this difference was no longer present. Staining for various heparan sulphate proteoglycan epitopes revealed that restoration of the basal membrane was incomplete at 3 months, but was complete at 12 months with respect to this component. No differences in the expression of several components of the basal membrane zone (heparan sulphate proteoglycan, laminin, tenascin) were noted between hypertrophic and non-hypertrophic scars. These data show that in the early phase of hypertrophic scarring, epidermal abnormalities are found compared with normal wound healing. In addition, early (3 months) epidermal abnormalities are associated with the clinical outcome at 12 months. These findings raise the possibility that the epidermal compartment is involved in the pathogenic process.

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing.

In adult human skin, the expression of the extracellular matrix glycoprotein tenascin is limited. Under hyperproliferative conditions such as psoriasis and epidermal tumours, dermal tenascin expression is strongly upregulated. The aim of this study was to investigate the pattern and kinetics of tenascin expression in human skin during wound healing and to address the question of whether keratinocytes can directly interact with tenascin during re-epithelialization. Tenascin expression was investigated in excisional wounds in normal human skin, in explants of normal human skin, and in chronic venous ulcers, using immunohistochemistry. No tenascin staining was found directly underneath the leading edge of the sheet of migrating keratinocytes in the excisional wounds and explants. In the excisional wounds and the ulcers, dermal tenascin was strongly upregulated in areas adjacent to hyperproliferative epidermis. These hyperproliferative areas are located approximately 10-50 cells behind the leading edge, as assessed by staining for the Ki-67 antigen and the proliferating cell nuclear antigen (PCNA). At the later stages of normal wound healing and in the chronic ulcers, tenascin was also detected in the wound bed. In these areas, the dermal-epidermal junction stained positive for laminin but was negative for heparan sulphate. The absence of the latter basement membrane component suggests that the formation of a new basement membrane is not completed in these wounds. These findings suggest that tenascin is not a substrate for migrating keratinocytes; that the rapid induction of tenascin expression in the papillary dermis during wound healing results from interaction with the hyperproliferative epidermis; and that in the later stages of wound healing, keratinocytes can potentially interact with tenascin in the wound bed, because the basement membrane of the neo-epidermis is incomplete.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a strong and specific inhibitor of elastase and proteinase 3. SKALP is not present in normal epidermis, but is expressed by epidermal keratinocytes under hyperproliferative conditions such as psoriasis, wound healing, and in cell culture. In human epidermal tumours, SKALP is differentially expressed and restricted to tumours with distinct squamous differentiation. We have studied the presence of both SKALP and one of its known target enzymes, leukocyte elastase, in 41 squamous cell carcinomas of the skin. SKALP expression correlated with the degree of differentiation: strong expression was seen in well-differentiated cells and expression was absent in poorly differentiated tumour cells. Most of the squamous cell carcinomas showed elastase-positive cells in the tumour stroma and also within the tumour cell nests. SKALP may interfere with the proteolytic activity of infiltrating inflammatory cells or with hitherto unknown proteinases from the tumour cells. We hypothesize that in squamous cell carcinoma progressive loss of SKALP expression could facilitate tumour spread.