胚胎组织提取液影响表皮干细胞表型变化的初步研究

目的： 模拟表皮干细胞周围生态微环境,研究表皮干细胞在此环境中的表型变化,表皮干细胞微环境在表皮干细胞"命运"决定过程中的作用。方法:分离、培养表皮干细胞,观察其形态,并进行免疫组织化学鉴定及免疫荧光检测。制备小鼠胚胎组织匀浆液,荧光活细胞示踪法和逆转录聚合酶链式反应检测胚胎组织提取液对表皮干细胞表型变化的影响。结果: 原代分离培养的表皮基底层干细胞表达α6、β1整合素、K19、K14、p63、Nestin、PCNA为阳性,而K10不表达。激光共聚焦检测显示α6整合素和CD71在表皮干细胞中的分布存在着区域性差异。此外,流式细胞检测显示胚胎组织提取液作用后α+6CD71-表达细胞由细胞总数的22.49%减少至10.92%,而α+6CD71+、α-6CD71+表达细胞则分别由诱导前的62.29%及0.19%增加至诱导后的68. 34%及4.51%。RT-PCR结果表明胚胎组织提取液作用后,表皮干细胞中β1整合素、K19、K10表达增强,而K14表达减弱。结论: Ⅳ型胶原黏附法分离的表皮干细胞具有干细胞的特点。α6整合素、CD71在表皮干细胞中分布的区域差异及CD71表达位点的空间变化可以作为区分表皮干细胞及其子代细胞标志之一。胚胎组织提取液对表皮干细胞的增殖分化具有促进作用。胚胎组织提取液中的某些因子可能抑制了K14表达的TA细胞的终末分化,甚至诱导其发生逆向分化,导致诱导后K14的总体表达水平下调。     

Identification of candidate murine esophageal stem cells using a combination of cell kinetic studies and cell surface markers.

The identification and characterization of esophageal stem cells are critical to our understanding of the biology of the esophageal epithelium in health and disease. However, the proliferative compartment within the mouse esophageal epithelium remains poorly characterized. Here, we report that the basal cells of the mouse esophagus can be separated into three phenotypically and functionally distinct subpopulations based on the expression of alpha(6) integrin and transferrin receptor (CD71). Cells that express high levels of alpha(6) integrin and low levels of CD71, termed alpha(6)(bri)CD71(dim), are a minor subpopulation of small and undifferentiated cells that are enriched for label-retaining cells and thus represent a putative esophageal stem cell population. Conversely, cells expressing high levels of both alpha(6) integrin and CD71 (alpha(6)(bri)CD71(bri)), the majority of basal esophageal cells, are enriched for actively cycling cells and therefore represent a transit-amplifying population. Kinetic analyses revealed that a third cell population, which is alpha(6) integrin-dim and CD71-bright (alpha(6)(dim)), is destined to leave the basal layer and differentiate.     

Enrichment of human beta 1 bri/alpha 6 bri/CD71 dim keratinocytes after culture in defined media

Stem cell-like keratinocytes are responsible for the high regenerative potential of the skin. For clinical applications using keratinocytes in artificial skin constructs, it is suitable to work with serum-free medium under defined conditions. This is also true for the preceding expansion of the stem cell-like keratinocyte population. Therefore, we analyzed the effect of a serum-free medium on the population distribution in comparison to an established serum-containing standard medium for keratinocyte culture. We quantified the freshly isolated as well as cultured primary human keratinocytes by their expression of the beta(1) integrin (CD29) in combination with the expression of the alpha(6) integrin (CD49f) and the transferrin receptor (CD71) by flow cytometric methods. We were able to show that cultivation with serum-free medium induces a switch of the cell population to higher expression of the beta(1) integrin. In addition, the proportion of the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population was enhanced about 35.4 +/- 6.56% after cultivation with serum-free medium. Culture in serum-containing medium increased this proportion of the keratinocyte cell population only about 17.3 +/- 8.06%, when compared to the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population measured directly after isolation. Our data show that the applied culture conditions already have an enormous impact on the development of a stem cell-like phenotype of keratinocytes. This work demonstrates that the serum-free medium significantly increases the proportion of beta(1)(bri)/alpha(6)(bri)/CD71(dim)-expressing keratinocytes. In conclusion, these findings implicate new applications in keratinocyte stem cell research and regenerative medicine.     

Survivin identifies keratinocyte stem cells and is downregulated by anti-beta1 integrin during anoikis

Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-DeltaEx3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2alpha tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking beta1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of beta1 integrin upregulates mRNA expression of survivin-2alpha. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the "niche" protection by preventing anoikis in KSCs.     

Rapid adhesion to collagen isolates murine keratinocytes with limited long-term repopulating ability in vivo despite high clonogenicity in vitro

A prevalent belief in epidermal biology is that stem cells are highly clonogenic; that is, they have the ability to produce many large colonies in vitro. However, it has been well-established in hematology, and recently suggested in epithelial biology, that short-term in vitro clonogenic assays may not be reliable predictors of long-term in vivo repopulating ability. Numerous groups have shown that rapid adhesion to collagen selects for highly clonogenic keratinocytes, but it has not been demonstrated whether this subpopulation is enriched in stem cells as defined by long-term repopulating ability in vivo. We found that although rapid adhesion to collagen (within 5 minutes) selected for cells with increased short-term colony forming ability in vitro, these cells were not enriched in long-term proliferative ability in vitro or in repopulating ability in vivo after 9 weeks. Conversely, keratinocytes that did not adhere to collagen (after 20 minutes) were less clonogenic in short-term assays but possessed equivalent long-term proliferative ability in vitro and superior long-term repopulating ability in vivo. Both the rapidly adherent cell and not rapidly adherent cell populations contained small, noncomplex basaloid cells, expressed integrin alpha2 (a collagen IV receptor), and expressed the putative epidermal stem cell phenotype integrin alpha6(hi)CD71(lo). Our results indicate that the superior short-term colony forming ability of collagen-adherent murine keratinocytes does not correlate with long-term repopulating ability in vitro or in vivo and that proliferation in vitro is not a reliable surrogate for stem cell behavior in vivo.     

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.     

A subpopulation of human basal keratinocytes has a low/negative MHC class I expression

By means of flow cytometry, we demonstrate that a subpopulation of cells in the basal layer of the human epidermis has undetectable levels of major histocompatibility complex (MHC) class I molecule. The percentage of MHC class I-negative cells in the basal layer ranged 0.5%-2%. MHC class I-negative cells were characterized by small size (low forward scatter) and low granularity (low side scatter). Upon culturing of MHC class I-negative cells, increase of MHC class I expression was observed. This expression was lower than the expression of MHC class I molecule both in cultured MHC class I-positive cells and in ex vivo keratinocytes. Furthermore, stimulation of MHC class I-negative keratinocytes with interferon gamma (IFN-gamma) did not bring about further increase in MHC class I expression. MHC class I-negative cells were identified as keratinocytes as they expressed keratin 14 and formed keratinocyte colonies in vitro.     

Normalization of keratinocyte-type integrins during the establishment of the oral mucosa phenotype in vitro.

In stratified epithelia, integrins play a fundamental role in mediating basal cell attachment to a variety of extracellular matrix molecules. To assess whether keratinocyte-specific integrins are expressed in a similar way as in the normal situation also under in vivo related conditions, we processed oral mucosa equivalents consisting of keratinocytes and fibroblasts from non-cornified gingiva. In this model histomorphology, the expression of differentiation-specific keratins and keratinocyte-type integrins exhibited similarity to the tissue of origin. The stages of tissue normalization were assessed on frozen sections by indirect immunofluorescence. The initial activated stage (1 week) was characterized by (i) incomplete epithelial organization and a weak presence of the suprabasal mucosa type keratin K4, (ii) diffuse expression of the integrin chains beta 1 and alpha 6 and (iii) abundance of the wound healing-associated integrin alpha v throughout the whole epithelium. After 2 weeks, the increase in epithelial organization was characterized by (i) the presence of a basal and suprabasal cell compartment, (ii) extension of K4 in the suprabasal compartment, (iii) extended expression of the keratinocyte integrins beta 1 and alpha 6 and (iv) concentration of alpha v integrin underneath basal cells. Further normalization of tissue architecture was indicated by (i) a slight increase in K4 extension, (ii) appearance of keratinocyte integrins beta 1 and alpha 6 in basal and parabasal cells and (iii) interruption of the band-like alpha v integrin immunolocalization at the subepithelial site. The findings in the in vitro model system indicate that these oral mucosa equivalents exhibit similarities to the in vivo situation of non-cornified gingiva, thus rendering them a suitable model for the assessment of stages during epithelial reconstruction or in vivo relevant studies on material effects.     

Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis

Although homeostatic renewal of human skin epidermis is achieved by the combined activity of quiescent stem cells (SCs) and their actively cycling progeny, whether these two populations are equipotent in their capacity to regenerate tissue has not been determined in biological assays that mimic lifelong renewal. Using fluorescence activated cell separation strategy validated previously by us, human epidermis was fractionated into three distinct subsets: that is, α?6briCD71(dim) , α?6briCD71(bri) , and α?6dim with characteristics of keratinocyte stem, transient amplifying, and early differentiating cells, respectively. The global gene expression profile of these fractions was determined by microarray, confirming that the α?6briCD71(dim) subset was quiescent, the α?6briCD71(bri) was actively cycling, and the α?6dim subset expressed markers of differentiation. More importantly, functional evaluation of these populations in an in vivo model for tissue reconstitution at limiting cell dilutions revealed that the quiescent α?6briCD71(dim) fraction was the most potent proliferative and tissue regenerative population of the epidermis, capable of long-term (LT) epidermal renewal from as little as 100 cells for up to 10 weeks. In contrast, the cycling α?6briCD71(bri) fraction was the first to initiate tissue reconstitution, although this was not sustained in the LT, while differentiating α?6dim cells possessed the lowest demonstrable tissue regenerative capacity. Our data suggest that in human skin, the epidermal proliferative compartment is not composed of equipotent cells, but rather is organized in a functionally hierarchical manner with the most potent quiescent SCs at its apex (i.e., α?6briCD71(dim) ) followed by cycling progenitors (i.e., α?6briCD71(bri) ) and finally early differentiating keratinocytes (i.e., α?6dim).     

Adenoviral-encoded antigens are presented efficiently by a subset of dendritic cells expressing high levels of alpha(v)beta3 integrins

Dendritic cells (DC) play a central role in antigen presentation and are often targeted by adenoviral (Ad)-based gene therapy. However, DC lack the coxsackie-Ad receptor, and little is known about the process by which they acquire and present Ad-encoded antigens. We examined the expression of alpha(v)beta3 integrins (CD51/CD61) on mouse bone marrow-derived DC (BM-DC) and their susceptibility to transduction by Ad vectors. Less than 10% of BM-DC precursors expressed CD51, but expression increased over time in culture with granulocyte macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. After 7 days, 28 +/- 1.7% of CD11c+ DC expressed high levels of CD51 (CD51(hi)), and the remaining DC expressed low levels of CD51 (CD51(lo)). CD51(hi) CD express higher major histocompatibility complex type 1 (MHC I); however, both of the DC subsets expressed similar levels of MHC II and costimulatory molecules. When exposed to a first-generation Ad vector, transgene expression was restricted to the CD51(hi) DC subset and blocked by soluble peptides expressing an arginine, glycine, aspartic acid (RGD) sequence, confirming the role of integrins in viral entry. Consistent with this, a modified Ad expressing an RGD-binding sequence in its fiber knob (Ad-RGD) transduced the CD51(hi) DC subset with significantly higher efficiency. When BM-DC were transduced with an Ad-expressing ovalbumin (Ad-OVA), the CD51(hi) subset proved superior in activating OT-I (T cell receptor-OVA) T cells. Similar to in vitro effects, systemic administration of GM-CSF/IL-4 increased the expression of CD51 on splenic DC and rendered these cells susceptible to Ad transduction. These results suggest that a limited subset of DC expressing high levels of alpha(v)beta3 integrins is preferentially transduced by Ad vectors and activates CD8+ T cell responses against Ad-encoded antigens.     

MHC antigen expression in human oral squamous carcinoma cell lines.

This study quantified the constitutive and interferon-gamma (IFN-gamma) stimulated expression of MHC class I (HLA-ABC and beta 2 microglobulin) and class II antigens (HLA-DR, -DP, -DQ) on normal and malignant oral keratinocytes using radioimmunoassay and immunocytochemical techniques. Normal keratinocytes and three of four malignant cell lines (H103, H157, H314) expressed MHC class I antigens constitutively; IFN-gamma increased MHC class I expression with significant changes in normals, H157 and H314. Normal keratinocytes expressed significantly more constitutive MHC class I antigens than H103 and H157 and significantly more IFN-gamma stimulated MHC class I antigens than H103, H157 and H314. MHC class II antigens predominantly were not expressed constitutively on normals, H103 and H157 but, in H314, HLA-DR, -DP and -DQ antigens were demonstrated on 35, 11 and 5 per cent of cells, respectively, and resulted in a non-coordinated pattern of expression (HLA-DR greater than -DP = -DQ). IFN-gamma induced HLA-DR on normals, H103 and H157, whilst HLA-DP and -DQ remained undetectable. In H314, IFN-gamma enhanced HLA-DR, -DP and -DQ (significant increase of HLA-DQ) but the interrelationship between these antigens was maintained (HLA-DR greater than -DP = -DQ). Normal keratinocytes expressed significantly more IFN-gamma stimulated HLA-DR than H103 and H157 but significantly less HLA-DR than H314 under similar experimental conditions. One oral malignant cell line (H191) did not express MHC class I and MHC class II antigens either constitutively or in response to IFN-gamma. The results demonstrate aberrant patterns of MHC expression (absence, enhanced, diminished) in the different malignant oral keratinocyte cell lines.     

Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.

Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry. Wounding was associated with alterations in extracellular matrix proteins, namely, loss of laminin and type IV collagen in the region of the wound and expression of tenascin and fibronectin. Changes were also noted in the integrins on the migrating keratinocytes. There was marked up-regulation of the alpha v subunit and de novo expression of the fibronectin receptor (alpha 5 beta 1) during the stage of active migration (days 1 to 3 after wounding). In the later stages of wound healing, after epithelial integrity had been established, redistribution of the alpha 2, alpha 3, alpha 6, and beta 4 collagen/laminin-binding integrin subunits to suprabasal epidermal layers was noted. Thus, during cutaneous wound healing, keratinocytes up-regulate fibronectin/fibrinogen-binding integrins and redistribute collagen/laminin-binding integrins. This study demonstrates that the human skin/severe combined immunodeficient chimera provides a useful model to study events during human wound repair.     

Resting porcine T lymphocytes expressing class II major histocompatibility antigen.

The immune system of swine is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting, extrathymic (CD1-) T lymphocytes in the circulation as well as in lymphoid tissue. Here it is documented that the specialty of the porcine T lymphocyte compartment extends to the expression of class II MHC (SLA) antigens. While the TCR gamma/delta CD4-CD8- as well as the TCR alpha/beta CD4+CD8- subpopulation both lack MHC II, the TCR alpha/beta CD4-CD8+ and the CD4+CD8+ subpopulation, the latter of which is private to swine, both do express MHC II. As opposed to human T lymphocytes, expression of porcine MHC II is not transient and restricted to lymphoblasts but is imminent in small, resting T lymphocytes of the two CD8-expressing subsets, even though also in swine activation can induce MHC II. Activation-induced extrathymic acquisition of MHC II without reversal can be discussed as one possible way of how MHC II+ T lymphocytes are generated. Alternatively, MHC II antigens should already be expressed by thymic progenitors. Remarkably, all CD1+CD4-CD8+ and most CD1+CD4+CD8+ thymocytes lack MHC II, yet, a minor subset with the phenotype CD4hiCD8hi expresses MHC II. One may speculate that these cells do not undergo thymic selection and represent the progenitors of the unusual, swine-typic MHC II+CD4+CD8+ extrathymic T lymphocytes.     

Expression of CD1d in human scalp skin and hair follicles: hair cycle related alterations

BACKGROUND: CD1d belongs to a family of antigen presenting molecules that are structurally and distantly related to the classic major histocompatibility complex class I (MHC I) proteins. However, unlike MHC I molecules, which bind protein antigens, CD1d binds to lipid and glycolipid antigens. CD1d is expressed by cells of lymphoid and myeloid origin, and by cells outside of the lymphoid and myeloid lineages, such as human keratinocytes of psoriatic skin.AIMS: To investigate whether CD1d is also expressed in sun exposed skin and in the immuno-privileged anagen hair follicle.Materials/METHODS: CD1d immunoreactivity was studied in human scalp skin and hair follicles of healthy women in situ by immunofluorescent and light microscopic immunohistology. Skin biopsies were obtained from normal human scalp containing mainly anagen VI hair follicles from women (age, 53-57 years) undergoing elective plastic surgery.RESULTS: CD1d showed strong immunostaining in human scalp skin epidermis, pilosebaceous units, and eccrine glands. In the epidermis, CD1d was strongly expressed by basal and granular keratinocytes. In hair follicles, CD1d was expressed in the epithelial compartment and showed hair cycle related alterations, with an increase in the anagen and a reduction in the catagen and telogen phases.CONCLUSIONS: These results suggest that CD1d plays a role in human scalp skin immunology and protection against lipid antigen rich infectious microbes. They also raise the question of whether keratinocytes of the immuno-privileged anagen hair follicle can present lipid antigens to natural killer T cells. These data could help provide new strategies for the manipulation of hair related disorders.     

Two- and Three-Dimensional Culture of Keratinocyte Stem and Precursor Cells Derived from Primary Murine Epidermal Cultures

In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.     

不同氧分压时人脂肪干细胞细胞因子的分泌

背景：不同氧分压对人脂肪来源干细胞分泌细胞因子的影响目前尚未定论,这些差异可能由于研究者对于氧分压的选取不同而造成影响。 目的：检测不同氧分压对人脂肪来源干细胞分泌细胞因子的影响。 方法：体外分离培养人脂肪来源干细胞进行免疫表型进行鉴定。将人脂肪来源干细胞分别在1%,3%,5%,10%,21%氧分压的环境中培养24 h后,使用实时定量PCR和酶联免疫吸附法对人脂肪来源干细胞分泌的血管内皮生长因子、肝细胞生长因子、神经细胞生长因子、角质细胞生长因子,在基因水平以及蛋白水平上进行检测分析。 结果与结论：人脂肪来源的人脂肪来源干细胞阳性表达CD71,CD73,CD90,CD105,阴性表达CD34,CD45,CD54及HLA-DR。经单因素方差分析统计,在基因水平上,低氧环境(体积分数1%,3%氧)均可促进人脂肪来源干细胞显著性高表达血管内皮生长因子、神经生长因子(P均< 0.01);对人脂肪来源干细胞表达肝细胞生长因子有显著影响(P < 0.05);而对角质细胞生长因子的表达则无显著影响(P > 0.05)。在蛋白水平上,低氧促进人脂肪来源干细胞分泌肝细胞生长因子、血管内皮生长因子蛋白(P均<0.01),对神经生长因子、角质细胞生长因子的分泌无显著影响。结果提示,人脂肪来源干细胞在低氧环境中培养后,其生物学特性受到一定的改变,可促进血管内皮生长因子、肝细胞生长因子、神经生长因子基因的表达,提高肝细胞生长因子、血管内皮生长因子蛋白的分泌。     

Identification of human oral keratinocyte stem/progenitor cells by neurotrophin receptor p75 and the role of neurotrophin/p75 signaling

This study was undertaken to determine whether human oral keratinocyte stem cells characteristically express higher levels of the low-affinity neurotrophin receptor p75 and to elucidate the function of p75 in oral keratinocytes. Examination of their expression patterns and cell-cycling status in vivo showed that p75 was exclusively expressed in the basal cell layer of both the tips of the papillae and the deep rete ridges. These immunostaining patterns suggest a cluster organization; most p75(+) cells did not actively cycle in vivo. Cell sorting showed that cells in the p75(+) subset were smaller and possessed higher in vitro proliferative capacity and clonal growth potential than the p75(-) subset. Clonal analysis revealed that holoclone-type (stem cell compartment), meroclone-type (intermediate compartment), and paraclone-type (transient amplifying cell compartment) cells, previously identified in skin and the ocular surface, were present in human oral mucosal epithelium. Holoclone-type cells showed stronger p75 expression at both the mRNA and protein level than did meroclone- and paraclone-type cells. Among the several neurotrophins, nerve growth factor (NGF) and neurotrophin-3 stimulated p75(+) oral keratinocyte cell proliferation, and only NGF protected them from apoptosis. Our in vivo and in vitro findings indicate that p75 is a potential marker of oral keratinocyte stem/progenitor cells and that some neurotrophin/p75 signaling affects cell growth and survival.     

Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis.

The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes.