Human leukocyte elastase induces keratinocyte proliferation in vitro and in vivo

Neutrophil infiltration and epidermal hyperproliferation are major histopathologic changes observed in psoriasis. Neutrophils contain human leukocyte elastase, which is thought to be released during neutrophil infiltration of the epidermis. As active human leukocyte elastase is known to be present in psoriatic lesions we were interested whether human leukocyte elastase induces hyperproliferation in keratinocytes in vitro and in vivo. In the cultured murine keratinocyte cell line PAM-212 concentrations of human leukocyte elastase from 1 to 30 nM induced significant proliferation as determined by 5-bromo-2'-deoxy-uridine-incorporation. Daily topical application of 0.043-434.8 pmol human leukocyte elastase per cm2 skin on hairless mice induced a concentration-dependent epidermal hyperproliferation and an increase in 5-bromo-2'-deoxy-uridine incorporation of up to 5-fold in basal keratinocytes within 3 d. Hyperproliferation resulted in a up to 2-fold increase of keratinocyte layers. Histologic analysis revealed marked vasodilatation but no inflammatory infiltrate. Application of porcine pancreatic elastase (3-300 pmol per cm2 skin) resulted in similar epidermal changes as observed for human leukocyte elastase. Hyperproliferative effects of human leukocyte elastase in vitro and in vivo were abolished by the addition of elastase inhibitors, such as elafin, anti-leukoprotease, and alpha1-protease inhibitor. In summary, human leukocyte elastase induces proliferation in murine keratinocytes in concentrations, which can be found on the skin surface of psoriatic lesions. These results may provide an explanation for the epidermal hyperproliferation observed in psoriasis.     

Comparisons of in vivo and in vitro photosensitivities and DNA repair in fibroblast and keratinocyte cells

Summary Minimal erythema dose of ultraviolet light correlated significantly with the UV-sensitivity of fibroblast cells from 5 patients with xeroderma pigmentosum, 13 patients with keratinocytic neoplasms of the skin, and 21 control subjects, but not with that of cells from 6 patients with photosensitive dermatitis. In unscheduled DNA synthesis after UV irradiation, the number of grains per nucleus was much less in keratinocytes than in fibroblasts, but the relative doseresponse relationship was similar. This indicates that keratinocytes can also be used in vitro UV-sensitivity studies.Supported in part by a Grant for Cancer Research from the Ministry of Education, Science and Culture of Japan     

Directing stem cells into the keratinocyte lineage in vitro

A major area of research in regenerative medicine is the potential application of stem cells in skin grafting and tissue engineering. This would require well defined and efficient protocols for directing the commitment and differentiation of stem cells into the keratinocyte lineage, together with their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying skin tissue biology, as well as facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for skin-related biomaterials and drugs could also utilize protocols developed for the commitment and differentiation of stem cells into the keratinocyte lineage. Hence, this review critically examines the various strategies that could be employed to direct the commitment and differentiation of stem cells into the keratinocyte lineage in vitro.     

The human hair follicle: glycoprotein-related antigenic profile of distinct keratinocyte populations in vivo and their alterations in vitro

The cell surface expression of three glycoprotein antigens, as defined by the monoclonal antibodies BT 15, T 43, and MH 99, was investigated in follicular keratinocyte populations in vivo. In addition, the regulation of glycoprotein synthesis was studied in follicular and interfollicular keratinocytes cultured in vitro. The BT 15 antigen was strongly expressed in the inner root sheath and the area above Auber's line of the hair bulb, whereas the T43 antigen was mainly seen in the outer root sheath. Selectively high expression of the MH 99 antigen was found only in outgrowing germ buds of early anagen follicles. Radioimmunoprecipitation revealed strong signals with BT 15 in freshly prepared follicular keratinocytes, two to three times stronger than those in interfollicular keratinocytes, but the signals clearly decreased by 80% under continuing culture conditions. The T 43 antigen was found by FACS analysis and radioimmunoprecipitation in initially low amounts in both populations, but the signals increased dramatically (up to 50 times) in long-term cultures and in subcultures. The MH 99 antigen was also initially present only in low amounts, in interfollicular rather than in follicular keratinocytes, but its expression increased up to 15-fold with continuing culture and any differences between the two populations disappeared. Our investigation revealed that at least three populations of hair follicle keratinocytes are characterized by different surface glycoprotein antigens, clearly related to their state of differentiation and proliferation. The BT 15 antigen seemed in vivo characteristic of well-differentiated keratinocytes of the inner root sheath and the keratogenous zone of the hair bulb, whereas the T 43 antigen appeared to mark a less differentiated keratinocyte population that formed the outer root sheath, clearly related to the basal epidermal layer. The MH 99 antigen clearly marked an early germ-cell phenotypic cell type to be seen only in the embryonal hair germ that showed high proliferative activity. Quantitative antigenic differences were initially found in vitro between follicular and interfollicular keratinocytes, but these differences gradually disappeared with continuing primary culture and in subcultures.     

Stimulation of PPARalpha promotes epidermal keratinocyte differentiation in vivo

Our recent studies have demonstrated that PPARalpha activators stimulate differentiation and inhibit proliferation in cultured human keratinocytes and accelerate epidermal development and permeability barrier formation in fetal rat skin explants. As the role of PPARalpha activation in adult epidermis is not known, the aim of this study was to determine if topically applied PPARalpha ligands regulate keratinocyte differentiation in murine epidermis. Topical treatment with PPARalpha activators resulted in decreased epidermal thickness. Expression of structural proteins of the upper spinous/granular layers (involucrin, profilaggrin-filaggrin, loricrin) increased following topical treatment with PPARalpha activators. Furthermore, topically applied PPARalpha activators also increased apoptosis, decreased cell proliferation, and accelerated recovery of barrier function following acute barrier abrogation. Experiments with PPARalpha-/- knockout mice showed that these effects are specifically mediated via PPARalpha. Compared with the epidermis of PPARalpha+/+ mice, involucrin, profilaggrin-filaggrin, and loricrin expression were slightly decreased in PPARalpha-/- mice. Moreover, topical clofibrate treatment did not increase epidermal differentiation in PPARalpha-/- mice. Furthermore, in cultured human keratinocytes we have demonstrated that PPARalpha activators induce an increase in involucrin mRNA levels. We have also shown that this increase in gene expression requires an intact AP-1 response element at -2117 to -2111 bp. Thus, stimulation of PPARalpha stimulates keratinocyte/epidermal differentiation and inhibits proliferation.     

Difference between the hair follicle cell and epidermal keratinocyte in vitro

We cultured human hair follicle cells (HFC) and epidermal keratinocytes (EK) using mixed collagen membranes as the substrate for the culture, and studied the degree of differentiation of these cells. Morphologically, polygonal epithelioid arrangements were seen in both cultures. However, in the culture of EK compared with HFC, large denucleated cells appeared within shorter periods of culture and stratification of cells were more prominent. In the culture of HFC, keratohyalin granules were not observed and cornified envelope formation was suppressed compared with that in EK. Then it is suggested that HFC and EK are different in the degree of keratinization and of other nature.     

隐球菌与体外培养角质形成细胞的相互作用

目的：研究隐球菌与体外培养的角质形成细胞的相互作用。方法：检测新生隐球菌对体外培养的角质形成细胞HaCaT株（简称HaCaT细胞）的时间-浓度黏附率、通透率;检测新生隐球菌对细胞的损伤;透射电镜观察二者相互作用的超微结构。结果：新生隐球菌标准野生株B3501（简称B3501）可以对HaCaT细胞产生黏附与侵袭,黏附率与侵袭率呈现时间依赖性;同时,B3501还可以使HacaT细胞凋亡率升高,对其造成损伤,这与菌体的活力相关。超微结构可见新生隐球菌与角质形成细胞的黏附与侵袭过程。结论：活的隐球菌黏附与侵袭角质形成细胞是其感染皮肤的重要条件。进一步明确二者的相互作用对隐球菌发病机制的研究具有重要意义。     

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.     

Fibronectin-mediated keratinocyte migration and initiation of fibronectin receptor function in vitro

Cell suspensions of human keratinocytes, freshly isolated from skin specimens, did not express plasma fibronectin (pFN) receptor function in short-term assays for cell attachment and spreading on pFN-coated culture dishes and binding and phagocytosis of pFN-coated latex beads. These activities were expressed, however, by the cells harvested from primary keratinocyte cultures after 2-4 days of culture. Analysis of the cell types arising during primary culture, based on staining with antikeratin antibodies and bullous pemphigoid (BP) serum, revealed that about 90% of the originally isolated cell population consisted of keratinocytes (keratin-positive) and 30% were basal cells (BP antigen-positive). After 2 days of culture, 95% of the cells were keratinocytes and 70% were basal cells. In vitro initiation of pFN receptor function also was observed in cells harvested from epidermal explants. After 9 days in culture, the cells that migrated out of the explants also were active in short-term cell adhesion assays, while cells remaining in the central region of the explant had much less activity. In related studies, the role of pFN in epidermal cell migration was analyzed, and it was found that anti-pFN antibodies inhibited migration of keratinocytes out of epidermal explants. Addition of preimmune IgG, however, had no effect. It appears, therefore, that pFN is important in all aspects of keratinocyte adhesion, and the expression of pFN receptor function may be a critical activation step necessary for basal cell phagocytosis and migration during wound healing.     

A rapid fluorometric assay for the determination of keratinocyte proliferation in vitro

In the present study we describe a simple technique for the determination of keratinocyte proliferation in vitro, based on the hydrolysis of a fluorogenic substrate by cell esterases. Normal and transformed human keratinocytes were grown in microtiter plates and were incubated with 4-methylumbelliferyl heptanoate after 3, 5, and 7 days. The fluorescence was quantified using an automatic fluorescence detection unit. The fluorescence showed a strong correlation with the cell number at various growth phases. In addition, the method reliably detected the growth inhibitory effect of recombinant interferon gamma on human keratinocytes. The fluorometric assay is a simple, fast and reliable method to assess cell number in keratinocyte cultures.     

Substance P and keratinocyte activation markers: An in vitro approach

Substance P (SP) released by cutaneous C fibres is involved in the physiopathology of cutaneous lesions. As normal human keratinocytes have been reported to express SP receptors, we studied the effects of SP on keratinocyte activation markers such as ICAM-1 induction and cytokine production. Human keratinocytes derived from skin obtained during plastic surgery were cultured in defined medium (MCDB 153) and were stimulated by SP. Flow cytometry analysis showed that SP (10 ^–7 and 10 ^–5 M ) as well as the specific NK1 agonist Sar ⁹ Met(O ₂ ) ¹¹ SP (Sar Met) induced a slight but significant expression of ICAM-1 at the cell surface during treatment periods of 24 h and 48 h. SP (10 ^–5 M ) also induced a significant but transient increase in the production of IL-1α, IL-1β, IL-1 receptor antagonist and IL-8 which was detectable by ELISA techniques 6 h after stimulation. This elevation returned to constitutive levels 24 or 48 h postinduction. TNFα secretion was detected in stimulated cells only after 48 h. These results suggest that SP can activate keratinocytes and support its role in the local inflammatory reaction. Received: 18 November 1994     

Anthralin decreases keratinocyte TGF-alpha expression and EGF-receptor binding in vitro.

Anthralin is an effective topical treatment for active psoriasis; however, its mechanism of action is unknown. Both TGF-alpha and its receptor, the EGF receptor, are overexpressed in active psoriatic plaques and might, therefore, play a role in psoriatic epidermal hyperplasia. In order to assess whether anthralin might act via alteration of this growth factor pathway, we examined the in vitro effects of pharmacologic concentrations of anthralin on cultured normal human keratinocytes. Keratinocyte proliferation was inhibited by 98% at an anthralin concentration of 10 ng/ml. In contrast, lymphocyte proliferation was inhibited by only 50% at an anthralin concentration of 10 micrograms/ml. Anthralin treatment did not induce cell-cycle-specific growth arrest as assessed by flow-cytometric analysis of acridine-orange-stained keratinocytes. Northern analysis of anthralin-treated keratinocytes demonstrated a marked decrease in TGF-alpha mRNA expression. Anthralin-treated keratinocytes showed decreased binding of 125I-EGF and 125I-IGF-I to their respective receptors, but EGF receptor binding was inhibited to a greater extent. Anthralin decreased ligand-binding affinity and cell-surface numbers of EGF receptors as assessed by Scatchard analysis of 125I-EGF binding to anthralin-treated keratinocytes. These results indicate that anthralin alters components of the EGF receptor pathway in cultured keratinocytes and that these effects might contribute to the clinical efficacy of anthralin in the treatment of active psoriasis.     

姜黄油对角质形成细胞体外增殖分化影响的研究

目的：探讨姜黄油对角质形成细胞体外增殖分化的影响。方法：以人角质形成细胞株COLO-16细胞为模型，噻唑盐比色法(MTT)检测细胞活性和生长情况；免疫组化SABC法检测姜黄油对增殖细胞核抗原(PCNA)表达的影响；电镜观察姜黄油对细胞超微结构的影响。结果：姜黄油抑制角质形成细胞增殖，随药物浓度增加，其抑制增殖能力增加，PCNA表达逐渐减弱，且对角质形成细胞超微结构有直接损伤作用。结论：姜黄油具有抑制体外角质形成细胞增殖分化的作用，有望成为一种治疗增殖性皮肤病的外用药物。     

Methotrexate reduces keratinocyte proliferation,migration and induces apoptosis in HaCaT keratinocytes in vitro and reduces wound closure in Skh1 mice in vivo

The aim of the present work was to evaluate MTX treatment (0.1, 1 and 10 μg mL^?1) in vitro in order to characterize its effects on cell proliferation alterations in cell cycle of HaCaT keratinocytes and wound healing in a Skh1 mice treated with MTX (low doses 30 mg kg^?1, high doses 200 mg kg^?1 and repeated doses at 1.5 mg kg^?1). We analyzed the cytotoxic effect of methotrexate by a resazurin assay. The effects in the proliferation, cell cycle and apoptosis of HaCaT cells were analyzed by flow cytometry. The effects of MTX on wound healing in vivo were also analyzed. A trend toward reduction in the resazurin assay was found (p > 0.05). Reduced proliferation was also identified in a clonogenic assay and a CFSE assay (p < 0.05) due to the MTX treatment. A reduction in the G₂/M and S phases was observed accompanied by apoptosis induction with increased sub G₀ phase and annexin V FITC staining. Effect of MTX was evidenced in vivo on the wound closure process after day 10 (p < 0.05) with alterations in tissue architecture and remodeling. There is a marked effect of MTX on wound healing in vivo in Skh1 mice with implications for long-term therapy and surgical interventions.     

Cyclosporin A inhibits directly in vivo keratinocyte proliferation of living human skin

A direct in vivo antiproliferative effect of cyclosporin A (CsA) on human epidermal keratinocytes (EK) grafted onto nude mice was evaluated. Using pulse-labeling of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue incorporated into the nuclei of DNA-synthesizing (S-phase) cells, the antiproliferative effect of CsA was revealed as a decrease in the number of BrdU-positive human EK grafted onto nude mice receiving a daily subcutaneous injection of 50 mg/kg of CsA. The blood level of CsA in the treated mice, evaluated by a radioimmunologic assay, was 679 +/- 501 ng/ml (n = 3). Using an antibody to leukocyte common antigen, it was shown that no human lymphocytes were present in the grafted skin. Therefore, this antiproliferative effect of CsA on human EK seems to be due to a direct effect on EK rather than to lymphocyte regulation.