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Mouse tail interscale epidermis contains numerous Langerhans cells, whereas the adjacent scale regions are devoid of these cells. No difference in a) proliferative activity and b) inhibitory effect of the epidermal G1 chalone can be demonstrated in both regions. A direct relationship between Langerhans cells and growth control may be excluded.
Dendritic antigen-presenting cells derived from epidermis (Langerhans cells), bone marrow, and peripheral blood can present a wide variety of antigens, including tumor-associated antigens, for various immune responses. The development and function of dendritic cells is dependent upon a number of cytokines including granulocyte-macrophage-colony-stimulating factor. For example, Langerhans cells can present tumor-associated antigens for the induction of substantial in vivo anti-tumor immunity but only after activation in vitro by granulocyte-macrophage-colony-stimulating factor. Thus, we reasoned that insertion of a cDNA for granulocyte-macrophage-colony-stimulating factor into dendritic antigen-presenting cells may allow for autocrine stimulation and increased antigen-presenting capability. To test this possibility, we utilized an adenovirus vector to insert a cDNA for murine granulocyte-macrophage-colony-stimulating factor into the dendritic cell lines XS52-4D and XS106 (derived from neonatal mouse epidermis), bone marrow-derived dendritic cells, and epidermal cells that contain Langerhans cells. Infection of each of these cell types resulted in release of abundant quantities of granulocyte-macrophage-colony-stimulating factor. XS52-4D and XS106 cells infected with adenovirus granulocyte-macrophage-colony-stimulating factor exhibited prolonged dendrites and greater expression of major histocompatibility complex class II molecules and CD86 compared with cells infected with a null vector. Granulocyte-macrophage-colony-stimulating factor cDNA-containing XS cells, bone marrow-derived dendritic cells, and epidermal cells had more potent alloantigen presenting capability than cells infected with a null vector. Most importantly, granulocyte-macrophage-colony-stimulating factor gene-transferred epidermal cells were able to present tumor-associated antigens for in vivo anti-tumor immunity against challenge with the S1509a spindle-cell tumor whereas null vector-infected cells were unable to prime for immunity. These results suggest that introduction of a cDNA for granulocyte-macrophage-colony-stimulating factor into dendritic cells may be an effective means to augment their antigen-presenting capability and that granulocyte-macrophage-colony-stimulating factor gene-transfer- red epidermal cells may be useful in tumor vaccination strategies.
Epidermal cells from cadaver skin grown in culture into confluent sheets of stratified cells were grafted on to partial thickness burn wounds in three patients. The burn areas covered with these allogeneic cultured epidermal allografts were tangentially excised deep second-degree burns which routinely would have been covered with split-thickness autografts. The burn wounds grafted with cultured allografts healed within three days and remained healthy for the 9 months of observation. Since epidermal cell cultures may be grown continuously, cultured allografts may serve as alternative biological dressings, or grafts, for deep second-degree burn wounds. They produce accelerated healing and an excellent cosmetic result, and they reduce the need for split-thickness autografts.
Wnt signaling orchestrates morphogenetic processes in which changes in gene expression are associated with dramatic changes in cell organization within developing tissue/organs. Upon signaling, excess beta-catenin not utilized at cell-cell junctions becomes stabilized, where it can provide the transcriptional activating domain for Lef/Tcf DNA binding proteins. In skin epithelium, forced stabilization of beta-catenin in epidermis promotes hair follicle morphogenesis, whereas conditional removal of beta-catenin in hair progenitor cells specifies an epidermal fate. We now report that a single protein, a stabilized version of beta-catenin lacking the COOH-terminal transactivation domain, acts in epidermis to promote hair fates and in hair cells to promote epidermal fate. This reveals fundamental differences in ways that epidermal and hair cells naturally respond to beta-catenin signaling. In exploring the phenotype, we uncovered mechanistic insights into the complexities of Lef1/Tcf/beta-catenin signaling. Importantly, how a cell will respond to the transgene product, where it will be localized, and whether it can lead to activation of endogenous beta-catenin/Tcf/Lef complexes is specifically tailored to skin stem cells, their particular lineage and their relative stage of differentiation. Finally, by varying the level of beta-catenin signaling during a cell fate program, the skin cell appears to be pliable, switching fates multiple times.
Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis response compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent. Flow cytometry showed that Ia-positive epidermal cells demonstrated the greatest UV fluorescence when treated with TCSA + UVA compared to both cells irradiated with UVA and subsequently treated with TCSA and untreated cells. This is consistent with the enhanced antigen presentation capability of TCSA + UVA treated LC-EC, which leads to the conclusion that LC photohapten-modified in vitro with TCSA + UVA demonstrate enhanced TCSA fluorescence and are capable of stimulating lymphocytes from TCSA photosensitized mice in an antigen-specific manner.
Epidermal cells from 8 patients with psoriasis (involved and uninvolved areas) and from 7 normal individuals, were propagated in vitro. DNA synthesis was determined autoradiographically by measuring the labeling index (1 hr) and a continuous labeling index (72 hr) on 8-10 day old cell cultures. No differences in these 2 kinetic parameters were obtained with respect to involved and uninvolved psoriatic cells. However, when psoriatic cells (involved and uninvolved) were compared to normal cells, the psoriatic cells exhibited a much higher labeling index and continuous labeling index, 200-300% and 60-70% respectively. These data clearly show that in vitro (1) epidermal cells from clinically uninvolved skin of psoriatics proliferate at a rate equal to that of involved psoriatic cells and (2) psoriatic epidermal cells proliferate at a rate greater than normal cells.
Epidermal cells produce an interleukin 1 (IL-1)-related molecule known as epidermal cell-derived thymocyte activating factor (ETAF). Given their similar physical and biologic properties, it was presumed that ETAF would share homology to known IL-1 sequences. A murine IL-1 alpha complimentary DNA (cDNA) probe hybridized with a 2.1 kb RNA from the murine keratinocyte cell line PAMM 212. Further, a 24-base synthetic IL-1 beta probe complementary to a region 700 bases from the putative 5'-end of human IL-1 beta hybridized with 2.7- and 1.6-kb RNAs in human keratinocytes, monocytes, and promyelocytic leukemia cells. Reverse transcripts primed with the IL-1 beta oligonucleotide revealed major first-strand cDNAs from human keratinocyte COLO-16 cell mRNA and human monocyte mRNA of about 700 bases, consistent with the expected cDNA from IL-1 beta. A partial library constructed from this cDNA contained clones that hybridized with the IL-1 beta oligonucleotide. The 750 bp-IL-1 beta-like cDNA clones were not identical to IL-1 beta as judged by restriction mapping and Northern analysis. These cDNA clones hybridized with the 1.6-kb keratinocyte mRNA but not to the 1.6-kb monocyte nor to the 2.7-kb mRNA. The 1.6-kb keratinocyte mRNA may encode a new IL-1-like species.
Suspensions of pig epidermal cells were prepared by trypsinisation of epidermis and incubated with I-14C-acetate in Eagle's minimum essential medium containing 10% serum. The incorporation of radioactivity into the total lipids of the cells increased with time despite the decrease in the proportion of viable cells which occurred during the period of incubation. The pattern of incorporation into the phospholipids and glycolipids showed similar changes with time to those observed previously with rat epidermal cell suspensions. Pig epidermal slices were incubated with I-14C-acetate in Eagle's minimum essential medium in the presence and absence of 10% serum. The proportion of radioactivity incorporated into the polar lipids was much greater in the epidermal slices than in the suspensions of epidermal cells. The pattern of incorporation in the phospholipids and glycolipids of epidermal slices differed from that found with epidermal cell suspensions and the pattern did not alter so markedly with the time of incubation. However, in each case reproducible patterns of incorporation were obtained under controlled conditions. The method of analysis can therefore be used to study lipid changes in small biopsies of normal and pathological skin.
Colloidal gold particles are well suited as markers in electron microscopy. Indirect immunogold staining was used to identify cell membrane antigens defined by monoclonal antibodies OKT6 and BL6 on human Langerhans cells (LC) in suspensions. Isolated epidermal cells were obtained by skin trypsinization and enriched or depleted in OKT6 positive on BL6 positive LC using the panning method: incubation of OKT6 or BL6 preincubated cells on immunoglobulin coated dishes. Indirect immunogold staining was then performed after prefixation in 2% paraformaldehyde. In LC enriched suspensions, only LC exhibited a specific membrane labelling with OKT6 or BL6 recognized by the presence of small evently distributed gold granules. Neither Birbeck granules, nor other cytoplasmic organelles, were labelled. No other epidermal cells were found positive. In LC depleted suspensions, no labelling was observed. Immunogold labelling on LC enriched suspensions after panning is now in progress for the qualitative evaluation and the quantitative analysis of cell surface constituents and antigens expressed by human dendritic epidermal cells.
Epidermal cells were isolated from adult inbred SENCAR (SSIN) mice and separated by density-gradient centrifugation. The cells were pooled into three fractions shown by previous work to differ in their state of differentiation and proliferative potential. The three fractions were examined for their capacity to metabolize exogenous 14C-arachidonic acid (AA) into prostaglandins (PG) and hydroxyeicosatetraenoic acids (HETE). Cells found in the upper two fractions, which are less dense, have less proliferative potential in vitro, and are more differentiated than cells in the lower more dense fraction, are much more active in producing PG from exogenous AA than are the more dense cells. This was observed in intact cells as well as cells disrupted by freeze-thawing following density separation. The same relationship was found for HETE production in that cytoplasmic preparations from the two fractions containing the less dense cells were much more active in the production of HETE than cytoplasmic preparations from the more dense fraction. The two upper fractions differed little from each other in the production of PG or HETE. These results indicate the presence of higher levels of active cyclooxygenase and lipoxygenases in fractions containing the less dense, more differentiated cells than in the fraction containing the more dense, less differentiated cells which are highly enriched for basal keratinocytes.
Epidermal spongiosis, invasion of mononuclear cells into the epidermis, and epidermal cell destruction are regular findings in allergic contact dermatitis. The mechanism(s) by which these changes occur is not known. We have examined the possibility that some of the pathological changes observed in allergic contact dermatitis could be accounted for by invasion of the epidermis by cytotoxic effector cells which recognize hapten-modified self-antigens and therein cause epidermal cell destruction. C3H and BALB/c mice were sensitized by epicutaneously applied 7% trinitrochlorobenzene (TNCB). 14 days later spleen cells from these mice were stimulated in vitro to trinitrophenylated- (TNP-conjugated) syngeneic spleen cells and their responses were compared to the in vitro responses of spleen cells from unsensitized mice. After 5 days of culture, effector cell activity was assayed on 51Cr-labeled TNP-conjugated syngeneic epidermal cells and on unconjugated epidermal cells. Cytotoxic activity was detected in the spleens of both mouse strains, but was greater in the C3H than the BALB/c strain. The cytotoxic effector cell activity was hapten specific in that spleen cells from TNCB sensitized mice did not cause lysis of fluorescein isothiocyanate (FITC) conjugated epidermal cells and spleen cells from FITC sensitized mice did not cause lysis of TNP-conjugated epidermal cells. No significant cytotoxic activity was detected on unconjugated epidermal cells. These findings suggest that destruction of the epidermis in allergic contact dermatitis may be contributed to by sensitized cytotoxic effector cells.
Epidermal basal cells have been prepared using trypsin or dispase to remove the epidermal sheet from the skin. In addition, for trypsin, the "immersion method" and the "flotation method" have been described. However, there has been no study that quantitatively compares these methods using the same animal, making their usefulness difficult to evaluate. In this report, comparative studies on these methods were undertaken concurrently with newborn and adult rats. The flotation method using trypsin was found to be preferable to the immersion method, because separation of the epidermal sheet by the former method was easier than by the latter. Then, the flotation method using trypsin was compared with the isolation method using dispase, and quantitative estimation was made of the yield, viability, and the number of attached epidermal cells. The dispase method was found to be superior to the trypsin method, especially for cell yield, which was about four times as high as with the latter method. Furthermore, cells dissociated with dispase showed a higher rate of viability and attachment to culture dishes than those done with trypsin. These results clearly demonstrated that the dispase method is more useful for preparing epidermal basal cells than the trypsin method.
In order to find a model system which best preserves human Langerhans cells (LC) outside of the human body, three possibilities were examined: epidermal cell culture, skin explants, and skin grafts onto "nude" mice. Using OKT-6 and anti-HLA-DR monoclonal antibodies, we quantified LC in epidermal sheets or epidermal cell cultures. All observations were carried out over a period of 4 weeks. We found that under epidermal cell culture conditions, LC rapidly disappeared, to the extent that after 10 days only rare HLA-DR-positive cells could be observed. In contrast, in the presence of intact dermis (explants and grafts), 60%-80% of the original number of LC, morphologically unchanged, dendritic and OKT-6 and HLA-DR-positive, were seen. These findings suggest that human LC are either a long-lived cell population or else can proliferate locally. The systems studied may be a useful tool for future investigation of LC function.
Epidermal cells from ligated day-3 last instar larvae of Galleria mellonella secrete a larval cuticle if exposed to ecdysone immediately following ligation; whereas, they deposit a pupal cuticle if exposed to ecdysone three to four days after ligation. During the period of reprogramming the genome in the diploid cells of the wax moth larvae no DNA synthesis occurs. This is direct proof that DNA synthesis is not required for reprogramming in the epidermis of Galleria during larval-pupal transformation.
It has been postulated that ultraviolet radiation (UVR) alters antigen presentation by macrophages. This is thought to be due, in part, to inhibition of macrophage-derived interleukin 1 (IL-1), which is a hormone-like factor with immunoregulatory functions. Conventional stimulator cells for antigen presentation are macrophages; however, other cell types such as epidermal Langerhans cells are capable of antigen presentation. Keratinocytes also play a role in the immune system by providing a factor with IL-1-like activity, termed Epidermal cell-derived Thymocyte-Activating Factor (ETAF). The purpose of this study was to determine whether UVR affects alloantigen presentation by epidermal cells and if so, whether the UV-induced change is due to UVR alteration in ETAF activity. Epidermal cells from UV-treated BALB/c mice (UV-EC) or from non-UV-treated mice (EC) were x-irradiated and then cocultured for 5 days with allogeneic T cells from C57Bl/6 mice. UV-EC caused less T-cell stimulation than did EC from non-UV-treated animals. When chromatography purified fractions of ETAF were added to cultured UV-EC, partial restoration of T-cell stimulation was seen. These results suggest that this UV-induced defect in alloantigen presentation is due, in part, to decreased ETAF activity.
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In this report we introduce an alternative procedure for enrichment of human epidermal Langerhans cells (LC) from epidermal cell suspensions of normal skin. By means of discontinuous Ficoll-Metrizoate density gradient centrifugation, a fraction containing high numbers of viable, more than 80% pure LC was recovered, as judged by CD1a expression. The purity of the LC-enriched fraction appeared to be dependent on the percentage LC in the crude epidermal cell suspension. LC enriched by this method retained their accessory and antigen-presenting capacities, as determined in the Concanavalin-A induced T-cell response, in the allogeneic mixed leukocyte reaction and in the antigen-specific T-cell proliferation assay in vitro. The great advantage of this method is that it is simple and rapid and that the isolated LC are unlabeled.
We have investigated the effects of PUVA therapy on human Langerhans cell (LC) immunophenotype and function. Epidermal sheets were obtained from exposed, and control shielded, forearm skin at the end of a course of PUVA therapy, in patients receiving treatment routinely for a variety of dermatoses. PUVA therapy decreased the overall number of HLA-DR+CDIa+ LCs in epidermal sheets, and in epidermal cell (EC) suspensions examined using a fluorescence activated cell sorter (FACS). PUVA therapy also reduced the overall EC allostimulatory capacity in the allogeneic epidermal cell-lymphocyte reaction (ELR), and the capacity of ECs to present tetanus toxoid to, and augment concanavalin A-mediated stimulation of, lymphocytes in the autologous ELR. Depressed allostimulation by ECs from PUVA-treated skin could not be restored by indomethacin (added to block prostaglandin synthesis). The reductions in LC numbers and EC allostimulatory capacity varied according to dose, and time since cessation, of PUVA therapy, and in individual patients were of comparable degree. By contrast, the allostimulatory capacity of residual LCs from PUVA-treated skin (purified using the FACS) did not differ from that of purified control LCs. PUVA-induced suppression of cutaneous immune responses, therefore, results at least in part from an overall impairment of EC antigen-presenting capacity. Residual HLA-DR+CDIa+ LCs in PUVA-treated skin which retain their alloantigen-presenting function may represent a subgroup of PUVA-resistant LCs; alternatively, these cells may be as yet unaffected because they have only recently migrated into the epidermis.