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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.
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.
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.
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.
Epidermal spinous and granular cells from the newborn rat neither replicate their nuclear DNA nor proliferate in vitro under conditions which support both processes in basal cells. However, as shown by autoradiography, (3H)thymidine and (14C)bromodeoxyuridine do label nuclei removed from spinous cells but not from granular cells. CsCl density gradient centrifugation of DNA obtained from early differentiated nuclei which had been exposed in vitro to (14C)bromodeoxyuridine, indicated that a considerable level of the tracer was present in the nucleic acid and suggested that replication of the genome had occurred. Therefore, spinous cells appear to retain the capability of reproducing nuclear DNA. Since differentiated cells appear to have the "diploid" level of DNA, these observations point to the replication of DNA as a possible locus of the mitotic inhibition which is coincident with epidermal differentiation.
Human epidermal cells dissociated by repeated trypsinization of skin explants were stimulatory to allogeneic lymphocytes in mixed lymphocyte skin-cell cultures. After elimination of the lymphocytes proliferating in response to a particular allogeneic lymphocyte donor by a "hot" pulse of 3H-thymidine of high specific activity, the viable lymphocytes remaining in culture were still capable of responding to stimulation by epidermal cells from the same donor. The response towards allogeneic epidermal cells could only partially be eliminated by 3H-thymidine treatment. This treatment, however, also partially eliminated the secondary stimulation response towards lymphocytes from the epidermal cell donor used initially, but did not alter the response to lymphoid or epidermal cells from an unrelated third party donor. In addition, HL-A antisera, which specifically inhibited the stimulatory capacity of mitomycin-treated lymphocytes that carried the relevant HL-A antigen also inhibited the stimulatory properties of epidermal cells from the same donor.
Immunoglobulin from pemphigus patients binds to the surface of mouse epidermal cells in culture. Cells incubated with the pemphigus antibody are easily detached from culture plates whereas cells incubated with serum from normal patients remain on the plate. Pemphigus antibody-mediated cell detachment is blocked by the addition of the proteinase inhibitors soybean trypsin inhibitor and alpha2-macroglobulin to the culture media. Detachable cells are viable, and activation of the complement cascade is not necessary for cell detachment. The anti-cell-surface antibody of pemphigus appears to disrupt adhesion between viable epidermal cells by activation of proteinase.
Swiss albino mouse 3T3 cells in various states were inoculated onto one side of Millipore filters. The other side of the filter was then coated with type I collagen and inoculated with newborn rat skin epidermal cells. On coculture of these cells, the attachment, growth and keratin synthesis of epidermal cells were found to depend on the state of the 3T3 cells: 3T3 cells in the stationary phase of growth were the most effective, followed by those in the logarithmic growth phase, those in the lag phase and plasmolyzed fibroblasts being only slightly effective. The effects of 3T3 cells in different states correlated well with their abilities to synthesize type IV collagen, but not type I collagen: with an increase in type IV collagen synthesis by the 3T3 cells, attachment of epidermal cells to the cell support, and their growth and synthesis of keratins increased. This culture system is concluded to mimic conditions in skin in vivo, and therefore to be suitable for studies on the effects of fibroblasts on the growth of epidermal cells.
Epidermal cells (EC) are a rich source of cytokines that can regulate the function of cells in skin and in other tissues. To organize the array of data pertaining to cytokine expression by EC subpopulations, we have tabulated such data according to cell source, state of cell activation, and type of assay employed. This information forms a background for our own studies, in which reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show that Langerhans cells (LC) are the principal source of mRNA for interleukin 1 beta and macrophage inflammatory protein-1 alpha (MIP-1 alpha) among unstimulated mouse EC.
Cultured human epidermal cells and human stratum corneum (callus) contain a number of keratins of different molecular size, but the size distribution is not the same in the two cases. To characterize these keratins in more detail, we compared them by amino acid analysis, immunological reactivity and one-dimensional peptide mapping (Cleveland et al., 1977). No differences in amino acid compositon could be detected among keratins of stratum corneum differing in molecular size by as much as 50%, suggesting that some repeating structure may be present in these molecules. Examination of polypeptide fragments produced by partial enzymatic hydrolysis showed strong similarities among all the keratins of stratum corneum and of cultured epidermal cells, even extending to the keratins of rodents; but the keratins of similar size, whether of stratum corneum or cultured cells, were more closely related than keratins of different size. This conclusion was supported by studies of the immunological reactivity of the keratins. How the epidermal cell generates a family of keratins is a problem of considerable interest. The differences in size and structure between the keratins of stratum corneum and cultured epidermal cells suggest that the epidermal cell can modify the expression its keratin genes.
Mast-cell-derived mediators showed mitogenic activities on mouse-transformed epidermal cell line Pam 212 cells. These activities were eluted into the low-molecular-weight fractions below a molecular weight of 10 kD on a high performance liquid chromatography TSK 2000G column, and were partially abrogated by antihistamines or anticytokine antibodies, including anti-IL1 alpha, -IL1 beta or IL6 antibodies. Pretreatment of mast cell lines with sodium butyrate enhanced the production of these factors. Calcium ionophore or Concanavalin A (ConA) stimulated mast cells to generate factor production. These results suggest that mast-cell-derived mediators might play some role in epidermal hyperplasia seen in lichenified lesions in atopic dermatitis.
In the present study we demonstrate that supernatants of highly enriched cultured Langerhans cells (cLC) display IL-1, IL-6, granulocyte/macrophage (GM)-CSF, and TNF-alpha, but no IL-2, IL-3, IL-4, and IFN-gamma activities. We further show that IL-6, GM-CSF, and TNF-alpha bioactivities can be specifically blocked in the presence of the respective neutralizing mAb. Concerning the IL-1 bioactivity, the combined use of anti-IL-1 alpha and anti-IL-1 beta mAb was needed to completely inhibit the proliferative response of the indicator cell line D10. One of the difficulties in studying the secretory potential of LC is that even highly enriched cLC are contaminated with keratinocytes (KC), which are known to be a rich source of cytokines. To overcome this problem we compared cytokine bioactivities in supernatants of cell cultures consisting of selected cLC:cKC ratios. These cell mixing experiments revealed that cLC are the major source of the IL-6 bioactivity, whereas IL-1, GM-CSF, and TNF-alpha are predominantly generated by cKC. In order to determine whether the cytokine bioactivities measured in supernatants of epidermal cell cultures are simply caused by an increased release or by de novo synthesis, we performed molecular biologic studies. Polymerase chain reaction analysis of cLC and cKC revealed that IL-1 beta and IL-6 transcripts are virtually limited to cLC, whereas IL-1 alpha, GM-CSF, and TNF-alpha messages are preferentially exhibited by cKC. mRNA coding for IL-2, IL-3, IL-4, and IFN-gamma could neither be amplified from cLC nor from cKC. Furthermore, the quantitative comparison of cytokine transcripts in cLC vs cKC using Northern blot analysis and mRNA detection on the single cell level using in situ hybridization confirmed that cLC generate IL-6, whereas cKC synthesize IL-1 alpha and GM-CSF. Taken together our results demonstrate that cultured murine LC synthesize and secrete IL-1 beta and IL-6, cytokines known to be important accessory molecules in T cell activation.
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Mouse epidermal cells in primary culture differentiate rapidly over a 2-week period leading to keratinization and sloughing of most of the plated cells. Cell replication was partially synchronized in these cultures with peaks of DNA synthesis at the 2nd and 8th day. The ability of epidermal cells to metabolize 7,12-dimethylbenz[a]anthracene and the subsequent binding of activated products to epidermal DNA was a function of the culture time. Constitutive and induced levels of aryl hydrocarbon hydroxylase in cells cultured for 10 days were half of those in cells grown for 3 days. Likewise, 7,12-dimethylbenz[a]anthracene binding to epidermal DNA was two- to fourfold lower in 10-day than 3-day cultures. This decrease in metabolism and binding between 3-day and 10-day cultures could be eliminated by the inclusion of 1.25% dimethyl sulfoxide in the culture medium during the entire culture period.
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Cyclopentenone prostaglandins (PG) such as delta 12-PGJ2 and PGA are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of PG cytotoxicity in human epidermal cells, we examined the effects of delta 12-PGJ2 on the induction of a heat shock protein (HSP), and on the organization of cytoskeletons in the HSC-I-transformed human epidermal cell line. Immunoblot analysis using a monoclonal antibody specific for the 72-kD heat shock protein (HSP72) revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced HSP72 formation in HSC-I cells. HSP72 was also induced by heat shock treatment at 43 degrees C for 90 min. The quantity of HSP72 produced was markedly decreased by co-treatment with 1 microgram/ml of cycloheximide in delta 12-PGJ2-treated cells, and similarly reduced in HSC-I cells following heat treatment. Immunofluorescence using a monoclonal antibody to HSP72 demonstrated that HSP72 was localized mainly in the cytoplasm of HSC-I cells. Following treatment with 5 micrograms/ml of delta 12-PGJ2, however, HSP72 was found in the nucleolus as well as in the cytoplasm. The accumulation of HSP in the nucleolus was similarly prominent in HSC-I cells after treatment at 43 degrees C for 90 min. Addition of delta 12-PGJ2 to confluent HSC-1 cells resulted in the disappearance of actin filaments and the disarrangement of keratin filaments, as visualized with fluorescent-labeled phallacidine or immunofluorescence. These results suggest that the cytotoxicity of cyclopentenone PG is related to the induction of HSP72, and to cytoskeleton damage in transformed human epidermal cells in culture.