| Backgrounds and ObjectivesThe function of epidermal permeability barrier is carried out in the outer layer of the epidermis, such as the granular layer and stratum corneum. Terminally differentiated keratinocytes migrate to the granular layer from basal layer begin to synthesize lipid-enriched particles known as lamellar bodies. Lamellar bodies are released by keratinocytes into the intercellular space of the stratum corneum (SC), by which constitutes the basis of permeability barrier. The cells of stratum corneum are called "brick", and lamellar membranes in the intercellular space of the SC are called "mortar". It is also referred to as the "brick and mortar" hypothesis to explain the molecular structure of epidermal permeability barrier.The exact mechanisms underlying the secretion regulation of lamellar hodies and maintenance of lamellar membrane integrity remain unknown. Under the physiological condition, there is an extracellular calcium gradient existed in the epidermis with a change of the lowest of calcium concentration in the basal layer and of the highest in the stratum granulosum. Subsequently, the calcium slightly declines while entering the stratum corneum. It has been considered that the extracellular calcium gradient contributes to the maintenance of KC differentiation and the regulation of lamellar body secretion by granular layer keratinocytes.However, the mechanisms how to form calcium gradient in the epidermis is still unknown.We speculate that calcium was pre-loaded by melanin enclosed in melanosomes and then released into the extracellular milieu because of melanosome degradation occurring in the granular layer.In clinical, vitiliginous skin with the loss or absence of melanin provides a good model to investigate the relationship among melanin, calcium gradient, and skin barrier. In this study, we determined the transepidermal water loss (TEWL) and examined the epidermal calcium gradient and the cytokine expression profiles in the depigmentary skin of stable vitiligo. The purpose of this study is to investigate the effects of melanin on ultrastructure, function, and epidermal calcium gradient as well as the molecular mechanisms regarding epidermal permeability barrier.The function of the epidermal permeability barrier is to effectively prevent the excessive loss of water from the body or passing through skin into the body. Lamellar membranes in the intercellular space of the SC forms the structural basis of epidermal permeability barrier. Therefore, we first investigated the ultrastructural changes of lamellar membranes of the SC in the normal skin and vitiligo lesions using a transmission electron microscopy (TEM) technique, assessing the effect of melanin loss on the ultra-structures of epidermal permeability barrier, as detailed in Part One. After that, we determined the function of epidermal permeability barrier such as transepidermal water loss (TEWL) in the normal skin and vitiligo lesions as detailed in Part Two. The TEWL value and the recovery rate of permeability barrier function after damage represent the reliable procedures to assess epidermal permeability barrier. At present, there are a lot of controversies on the barrier fanction in dark skin and light skin. For this reason, the comparison of TEWL on both vitiligo-involved and contralateral uninvolved sites is important to understand the impact of melanin on permeability barrier function.Emerging data demonstrated that there is a concentration gradient of calcium ion in the normal epidermis.The calcium gradient possibly plays an important role in the proliferation, differentiation, cell adhesions, and barrier function of the epidermis.Meanwhile, it was also showed that low calcium promoted keratinocyte proliferation and high calcium promoted keratinocyte differentiation and cell adhesion.Full-functional epidermal barrier function is a prerequisite to maintain the concentration gradient of calcium ion in the epidermis.To further investigate whether the loss of melanin affects the calcium gradient, the calcium distribution in the normal skin and vitiligo lesions was visualized using an ion-capture cytochemistry with a combination of TEM technique, as detailed in Part Three.In addition, it remains to be investigated whether the disorder of calcium gradient affects terminal differentiation of KC and the homeostasis of epidermal cells. At last, the expressions of HMB-45, K15, Ki67, and FHL2 in the normal skin and vitiligo lesions were analyzed using an immunohistochemical staining, as detailed in Part Four. HMB-45/gp100, a structural protein contributing to melanin synthesis, is an important marker of melanocyte differentiation and maturation and specifically localized in the envelope membranes of melanin. Ki67 is a nuclear antigen that contributes to cell proliferation, the expression level of Ki67 protein in the skin is used for estimating cell mitosis activity. Under the physiological condition, the skin is a renewable stratified squamous epithelium, its renewal and homeostasis are largely depended on the balance of proliferation and differentiation of epidermal stem cells (ESCs). K15 is considered to be one of marker protein representing the stem cell in the basal layer of the skin. FHL2 (four and half LIM domains-2) is a family member which consists of four complete and one half LIM domain.FHL2 is involved in regulation of gene expression, signal transduction, cytoskeletal formation and cell adhesion and metastasis, and it plays a very important role in normal tissue growth and development.The expression levels of the above-mentioned proteins in vitiligo lesions and normal skin were determined using an immunohistochemical method. We will make clear whether the loss of melanin affects epidermal cell homcostasis.Methods and results1. The ultrastructural changes of lipid lamellar membranes in the skin were assessed by using a transmission electron microscopy (TEM) technique in combination with ruthenium tetroxide (RuO4) staining. The result clearly revealed that the four to eight layers lipid lamellae existed within the intercellular space of the normal SC with a characteristic alternating electron-dense and electron-lucent pattern.The fragmentation and lamellar separation were also observed in the depigmented skin from vitiligo patients, the bulbous regions of lipid lamellae were filled with more electron-dense amorphous materials;2. The basal TEWL were measured on both vitiligo-involved and contralateral uninvolved sites by respective probes connected to a multifunctional skin physiological instrument, the results showed that TEWL in vitiligo-involved sites did not differ significantly compare to in uninvolved sites (11.13±1.04g/m2/h vs.10.38 ± 1.26g/m2/h; not significant); At the same time, the barrier recovery rate was assessed at Oh and 5h after barrier perturbation by repeated D-Squame applications. However, barrier recovery was significantly delayed in vitiligo-involved sites in comparison with contralateral uninvolved sites (41.29±4.03% vs.59.47±4.23%; P<0.05)3. The calcium ion distributions in the epidermis were detected using an ion-capture cytochemistry and a TEM technique. The large clumps of calcium precipitants were visualized in the stratum granulosum (SG) of normal skin, less calcium precipitates were noted within the normal stratum basale (SB).At the same time, base layer exist more oval IV melanosome; The calcium distribution in SG and SB of vitiligo lesion was dramatically decreased comparing to normal pigmented skin, and melanosome was absent.4. Expression of HMB45?Ki67% K15 and FHL2 in vitiligo-involved and normal skin were detected by immunohistochemistry. IPP6.0 software was used to analyze the semi-quantitive data of each cytokine and then the statistical analysis was carried out. The result showed that HMB45 protein expression were observed in normal skin,and a part of melanocyte cytoplasm was dyed brown-yellow, cells were large, round, vacuolar and small cell nuclei, surrounding the cytoplasm was stained in different shades of yellow colored. No positive signal was found in the vitiligo-involved sites;The K15 protein was expressed in the basal layer of cell cytoplasm, it displayed in a brown-yellow colored, and there were not nuclear staining, the cells above the basal layer were not stained. The results showed that K15 in vitiligo-involved sites did not differ significantly from that in normal skin(0.1267±0.0514 vs.0.1263±0.0172; not significant); The Ki-67 protein were located in the nucleus, and the nucleus were colored with clear boundary of brown and dark brown particles. Normal skin tissue is colored mainly confined in the proliferative basal layer, few of the spine cell were colored. The Ki-67 protein in Vitiligo was expressed not only in the basal layer but also in the layer of the spine, and there are more dense cells colored than normal skin. The expression of ki67 in vitiligo-involved site was higher than that in normal skin (0.1075±0.0802 vs.0.0608±0.0483; P<0.05); The FHL2 protein was mainly expressed in the cytoplasm and nucleus, and it displayed in brown-yellow particles. Expression of FHL2 in vitiligo-involved sites was staining strongly, and brown-yellow particles were thickly.The FHL2 protein was also expressed in normal skin, but it was weak. The expression of FHL2 in vitiligo- involved sites was higher than that in normal skin (0.2905±0.0332 vs.0.142810.0341; P<0.05)ConclusionThe TEM data showed that the disrupted ultrastructure of SC lamellar membranes was existed in the vitiligo-affected skin, indicating the loss of melanin in the skin might cause the dysfunction of epidermal lipid lamellar membranes.There is no statistic difference in basal TEWL values between vitiligo-involved sites and normal skin, the recovery time of barrier is significantly delayed in vitiligo-involved sites. The results indicate that the melanin distribution in the epidermis plays a essential role in the maintenance of the skin barrier function.The calcium gradient was disappeared in the vitiligo-affected skin, clearly indicating that the proper deposition of melanin in epidermis contributes to the maintenance and formation of calcium gradient. Comparing with normal skin, the positive cell numbers of HMB-45 or/ Ki67 or FHL2 immunostaining were increased in vitligo lesions, but no difference for K15 immunostaining. These results exhibit that proper melanin deposition slightly inhibits keratinocyte proliferation. We quite surely observe the mild upregulation of FHL2 protein in the lesional tissues from vitiligo, it remains to be verified whether FHL2 inhibits tyrosinase activity of residual melamnocytes in vitiligo lesions. |
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