Epidermal Cell Populations Research Articles

Tacrolimus: A new topical immunomodulatory therapy for atopic dermatitis

Tacrolimus: A new topical immunomodulatory therapy for atopic dermatitis

Immunopathologic characterization of the tissue response in endemic pemphigus foliaceus (fogo selvagem)

Background: The research on endemic pemphigus foliaceus (fogo selvagem) has mainly focused on the humoral immune response, but little attention has been given to the function of cell-mediated immune response and the nature of the cellular elements of the tissue reaction in the lesions of fogo selvagem. Objective: The purpose of this study was the immunophenotype characterization of the inflammatory cells as well as the expression of adhesion molecules and HLA-DR in the perilesional and lesional skin of fogo selvagem. Methods: Twenty biopsy specimens of lesional and perilesional skin were analyzed by immunohistochemical techniques. The panel of monoclonal antibodies consisted of CD8, CD4, CD1a, HLA-DR, IL-2R, LFA-1, ICAM-1, and PAN-B. Results: The semiquantitative analysis of the cell population revealed a predominance of CD4 T lymphocytes in the tissue response of perilesional and lesional skin. The population of epidermal Langerhans cells was decreased in lesional skin when compared with the perilesional skin, whereas CD1a+ dermal dendritic cells predominated in lesional skin. Keratinocyte expression of ICAM-1 and HLA-DR was negative in both lesional and perilesional skin. Conclusion: The overall results suggest the participation of the cell-mediated immunity in endemic pemphigus foliaceus (fogo selvagem). The lack of keratinocyte ICAM-1 expression may be related to the pattern of cytokines secreted by the CD4+ T cells of the tissue reaction in fogo selvagem. (J Am Acad Dermatol 2001;44:446-50.)

Isolating a pure population of epidermal stem cells for use in tissue engineering.

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.

Atopic dermatitis: from the genes to skin lesions.

Atopic dermatitis: from the genes to skin lesions.

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

Characterization of the expression pattern of p63α and δnp63α in benign and malignant oral epithelial lesions

The p53 homologue p63 is essential for ectodermal differentiation, such that p63-/- mice lack all squamous epithelia and teeth. The p63 gene expresses at least 6 different transcripts, but information regarding the expression, regulation and function of the different isoforms has remained sparse, due to the lack of adequate reagents directed specifically against the individual proteins. Here we characterize the expression of p63 alpha/delta Np63 alpha in benign and malignant lesions of the oral epithelium, using a specific antibody raised against a peptide derived from the C-terminus of p63 alpha, which does not cross-react with p53 or the other p53 homologue, p73. By immunohistochemical analysis, we show that these p63 isoforms are expressed in the nucleus of many cells. In normal and benign lesions, p63 alpha/delta Np63 alpha-expressing cells are mainly found suprabasally, whereas p53-expressing cells are restricted to the basal-cell layer. By RT-PCR, we show that delta Np63 alpha is the predominant isoform in cell lines from squamous-cell carcinomas of the head and neck, confirming our immunochemical observations. Our data are consistent with studies suggesting a role for p63 in the transit-amplifying population of epidermal cells. Over-expression of p63 alpha, and in particular the delta N form, was frequently seen in carcinomas. Taken together with previous analyses of p63 expression, our data suggest distinct roles for different p63 isoforms in the regulation of growth and/or differentiation of epithelial cells. Moreover, our data are compatible with the notion that p63 can act to promote neoplastic growth in the oral epithelium.

Phenotyping of epidermal dendritic cells: Clinical applications of a flow cytometric micromethod

The differential diagnosis of inflammatory skin diseases is largely based on the patient's history and the morphological analysis of the skin lesion. Laboratory data, such as serum IgE-level and prick and patch tests, may be helpful but do not assess individual lesions. The assumption of our approach is that each individual lesion is associated with a specific microenvironment and that the immunophenotype of the two epidermal dendritic cell populations, Langerhans cells (LC) and inflammatory dendritic epidermal cells (IDEC), reflects this environment in a disease-specific manner. A flow cytometric micromethod was developed to directly analyze individual inflammatory human skin lesions. Crude epidermal single cell suspensions were prepared by trypsinization, stained for three-color analysis with different monoclonal antibodies and the vital stain 7-amino-actinomycin-D, and finally analyzed on a single laser equipped FACScan flow cytometer. With a limited set of cell surface markers, such as FcepsilonRI, FcgammaRII/CD32, CD1b and CD36, highly specific diagnostic criteria for atopic dermatitis and inflamed human skin could be established. Phenotyping of epidermal dendritic cells is a useful procedure helpful in differential diagnosis of inflammatory skin diseases.

The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin.

Gene knockout technology has provided a powerful tool for functional analyses of genes expressed preferentially in a particular tissue. Given marked similarities between human and mouse skin, such studies with epidermally expressed genes have often provided valuable insights into human genetic skin disorders. Efficient silencing of a specified gene in a temporally regulated and epidermal-specific fashion could extend functional analyses to broadly expressed genes and increase the categories of human skin disorders to which parallels could be drawn. We have generated transgenic mice expressing Cre and a fusion protein between Cre recombinase and the tamoxifen responsive hormone-binding domain of the estrogen receptor (CreER(tam)) under the control of the human keratin 14 (K14) promoter. This promoter is strongly active in dividing cells of epidermis and some other stratified squamous epithelia. With K14-Cre, transgenic embryos recombine genetically introduced loxP sequences efficiently and selectively in the genomes of keratinocytes that reside in embryonic day 14.5 skin, tongue, and esophagus. With K14-CreER(tam), postnatal transgenic mice show no Cre activity until tamoxifen is administered. If orally administered, tamoxifen activates keratinocyte-specific CreER(tam), allowing recombination of loxP sequences in epidermis, tongue, and esophagus. If topically administered, tamoxifen allows recombination in the area of skin where tamoxifen was applied. Finally, we show that epidermal cells harboring a Cre-dependent rearranged genome persist for many months after tamoxifen application, indicating that the epidermal stem cell population has been targeted efficiently. These tools now pave the way for testing the functional role of different somatic mutations that may exist in mosaic disorders of the skin, including squamous and basal cell carcinomas.

Generation of a spacing pattern: the role of triptychon in trichome patterning in Arabidopsis.

Trichomes in Arabidopsis are single-celled hairs that exhibit a regular spacing pattern. Here, the role of TRIPTYCHON (TRY) in the generation of this spacing pattern is studied. By using genetic mosaics, we demonstrate that the formation of trichome clusters in try mutants is not correlated with cell lineage, indicating that TRY is required to single out trichome cells in a process involving cellular interactions. The genetic interactions of TRY, GLABRA1 (GL1), and TRANSPARENT TESTA GLABRA (T TG) in trichome patterning are assessed by determining the cluster frequency in various genetic combinations. It is shown that TRY acts as a negative regulator of GL1- and TTG-dependent pathways. Furthermore, it is demonstrated that trichome initiation in ttg-1, a strong ttg allele, is rescued almost to wild-type levels in a try background in which GL1 is expressed under the control of the cauliflower mosaic virus 35S promoter, indicating that T TG acts upstream of GL1 and TRY. These findings are incorporated into a model to explain the generation of a trichome spacing pattern from a homogeneous population of epidermal cells.

Transduction of a preselected population of human epidermal stem cells: consequences for gene therapy.

Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.

Quantification of epidermal cell populations in the centre and margin of stable psoriatic plaques

The histological picture of psoriasis has been studied extensively. Several authors have investigated the differences between the centre and the margin of spreading plaques, because the margin is of great pathogenic interest as lesions enlarge by centrifugal expansion. However, little is known about the differences between the centre and the margin of stable plaques. In the present study we investigated quantitatively the differences between the centre and margin of stable psoriatic plaques with respect to differentiation, inflammation and proliferation. To quantify these parameters, we used flow cytometry. From nine patients with nonspreading, stable psoriasis, we obtained punch biopsies from the centre and from the lesional margin of a plaque, and performed a flow cytometric assessment, using the markers keratin 10 for differentiation, vimentin for inflammation, and TO-PRO-3 iodide for proliferation. We found that the quantitative parameters showed a large interindividual variability, and that there was no significant difference in the quantitative parameters for inflammation and proliferation between the centre and margin of stable plaques. However, the percentage of differentiated cells was significantly higher in the margin than in the centre. We conclude that there is a great heterogeneity within stable psoriatic plaques with respect to differentiation, inflammation and proliferation, but further quantitative studies are needed to substantiate the pathogenic relevance of the significant difference in keratinization between the centre and the margin of stable psoriatic plaques.

Telomerase is Not an Epidermal Stem Cell Marker and is Downregulated by Calcium

The ribonucleoprotein complex telomerase, which was found to be active in germ line, immortal, and tumor cells, and in cells from continuously renewing normal tissues such as epidermis or bone marrow, is thought to be correlated with an indefinite life span. Therefore, it has been postulated that in the normal tissues, telomerase activity may be restricted to stem cells, the possible precursors of tumor cells. Here, we demonstrate that a 56% enriched population of epidermal stem cells exhibited less telomerase activity than the more actively proliferating transit amplifying cells, which are destined to differentiate after a finite number of cell divisions. Thus telomerase is not a stem cell marker. In human epidermis we found a heterogeneous expression of the telomerase RNA component (hTR) within the basal layer, with clusters of hTR-positive cells showing variable activities. Histone-3 expressing S-phase basal cells were distributed evenly, illustrating that hTR upregulation may not strictly be correlated with proliferation. We further show for human epidermal cells that differentiation-dependent downregulation of telomerase correlates with Ca++-induced cell differentiation and that increasing the amount of Ca++ but not Mg++ or Zn++ reduced telomerase activity in a dose-dependent manner in a cell-free system (differentiation-independent). Furthermore, addition of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid completely reversed this Ca++-induced inhibition. These data indicate that Ca++ is not only an important regulator of epidermal differentiation but also a key regulator of telomerase.

Human Melanocytes and Keratinocytes Exposed to UVB or UVA In Vivo Show Comparable Levels of Thymine Dimers

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation.

The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m2. The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of beta1 integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing beta1 integrin were divided into two subpopulations, denoted beta1high or beta1low. The proportion of beta1high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of beta1low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that beta1high and beta1low populations of basal epidermal cells in HSEs respond differently to UV irradiation.

Therapeutic photoimmunology: photoimmunological mechanisms in photo (chemo) therapy

Ultraviolet radiation, either alone or in combination with photosensitizing agents, is widely used for the treatment of skin diseases. The efficacy of photo- and photochemotherapeutic modalities is thought to result, at least in part, from the induction of immunomodulatory effects. In particular, UV radiation has been shown to affect (i) the production of soluble mediators, (ii) the expression of cell-surface receptors and (iii) to induce apoptosis in pathogenetically relevant cells. UVB radiation-induced immunomodulatory effects are limited to the epidermis, whereas UVA radiation affects both epidermal and dermal cell populations. UVB and UVA radiation can exert essentially identical immunomodulatory effects, which result, however, from different photobiological mechanisms.

Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.

Despite the central role of human epidermal stem cells in tissue homeostasis, wound repair, and neoplasia, remarkably little is known about these cells, largely due to the absence of molecular markers that distinguish them from other proliferative cells within the germinative/basal layer. Epidermal stem cells can be distinguished from other cells in the basal layer by their quiescent nature in vivo and their greater overall proliferative capacity. In this study, we demonstrate enrichment and isolation of a subpopulation of basal epidermal cells from neonatal human foreskin based on cell surface phenotype, which satisfy these criteria. These putative stem cells are distinguished from other basal cells by their characteristic expression of high levels of the adhesion molecule alpha6, a member of the integrin family (alpha6bri), and low levels of a proliferation-associated cell surface marker recognized by recently described mAb 10G7 (10G7(dim)). We conclude that cells with the phenotype alpha6bri10G7(dim) represent the epidermal stem cell population based on the demonstration that these cells (i) exhibit the greatest regenerative capacity of any basal cells, (ii) represent a minor subpopulation (approximately 10%) of immature epidermal cells, which (iii) are quiescent at the time of isolation from the epidermis, as determined by cell cycle analysis.

5-Lipoxygenase expression in Langerhans cells of normal human epidermis.

We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.

Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha-ras genotype

Mirex is a potent tumor promoter in 7,1 2-dimethylbenz[a]anthracene (DMBA)-initiated female CD-1 mouse skin. Like 12-O-tetradecanoylphorbol-13-acetate (TPA), mirex promotes papillomas that have a Ha-ras mutation; however, unlike TPA promotion, mirex promotion does not involve a general hyperplastic response. We used proliferating cell nuclear antigen (PCNA) and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining to further examine the proliferative capacity of mirex. The numbers of PCNA- and BrdU-positive epidermal S-phase cells were highly concordant in all treatment groups. Unlike a single application of TPA, a single application of mirex had little or no effect on the number of S-phase epidermal cells, and chronic application of mirex to mouse skin produced only minimal increases in S-phase cells. Moreover, mirex did not significantly alter the growth of BALB/MK-2 keratinocytes in media containing either 0.05 or 1.2 mM Ca++. These results suggest that mirex may have highly specific effects on the proliferation of initiated cells and support the existence of a unique mirex mechanism and/or distinct population of mirex-promotable mutant Ha-ras epidermal cells. To begin to address this issue of a distinct population of mirex-promotable mutant Ha-ras cells, we conducted a tandem experiment in which DMBA-initiated mice were treated twice weekly with a maximal promoting dose of mirex. Then, when the number of papillomas reached a plateau, these same mice were treated twice weekly with a maximal promoting dose of TPA. Mice treated with mirex developed a maximum of 6.4 papillomas/mouse. These mice were then promoted with TPA, which produced 8.9 additional papillomas/mouse for a total of 15.3 papillomas/mouse. The maximum tumor yields from other groups of mice treated with only TPA or mirex were 9.8 and 7.3 papillomas/mouse, respectively. Therefore, under these tandem conditions, tumor yields were additive, indicating that there are at least two distinct populations of mutant Ha-ras cells: one promoted by mirex and the other by TPA.

Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha‐ras genotype

Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha‐ras genotype

Calcitonin gene-related peptide and Langerhans cell function.

Morphologic studies have indicated that Langerhans cells (LC) are frequently in anatomic apposition with epidermal nerves containing the neuropeptide calcitonin gene-related peptide (CGRP). Experiments were undertaken to examine whether CGRP modulates LC function. The effect of pre-exposure of LC to CGRP in vitro on alloantigen presentation and specific protein presentation to a responsive T-cell line were studied using freshly prepared murine epidermal cell populations enriched for LC content (EC) by treatment with antibody to Thy-1 and complement. The ability of EC to present tumor-associated antigens for induction and elicitation of delayed-type hypersensitivity (DTH) in tumor-immune mice was also examined. Inhibitory effects of CGRP on antigen presentation were observed in each of these assays. Experiments were also performed examining the ability of intradermally administered CGRP to modulate induction of contact hypersensitivity (CHS) to a hapten applied at the injected site. Administration of CGRP led to a decrease in the CHS response after immunization at the site of injection. Intracellular cAMP was significantly increased in freshly prepared LC after exposure to CGRP, and this process could be inhibited by a specific inhibitor of the CGRP receptor, demonstrating the existence of CGRP receptors on LC. B7-2 expression induced by LPS and GM-CSF in the LC-like line XS52, and by LPS in peritoneal macrophages, was suppressed by CGRP. This suppression may account, in part, for the inhibitory effect of CGRP. As a whole, these observation suggest that regulation of antigen presentation by nerves may occur in the epidermis.