F.W. Bauer

Flow cytometry as a tool for the study of cell kinetics in epidermis

Flow cytometric measurements of the DNA content were performed on a large number of skin biopsies by an automated technique. Expressed as a percentage of all viable cells in the epidermis, the figures for cells in S‐phase averaged 1.8% and for G2M 0.9%. No significant differences due to sex were found. Concomitantly with age the ratio S/G2M (representing the duration of S to the duration of G2M) increased. Also seasonal effects were clear, showing higher values for S and G2M in June compared to November and December. Lastly we found small differences dependent on body‐site, the ratio S/G2M being greater in legs than in arms. The present status is discussed together with future lines of development.

Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4

The percentage of non‐diploid epidermal cells was determined by flow cytometry following application of leukotriene B4 (LTB4) to human skin. Doses in the range 35–500 ng were shown to cause a marked increase in proliferation, the non‐diploid cells reaching a maximum between 72 and 96 h after LTB4 application. No difference was observed between the response of healthy controls and the uninvolved skin of psoriatic patients. We suggest, therefore, that the hyperproliferation is a consequence of the physical disruption of the stratum corneum accompanying the rupture of microabscesses.

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Abstract. Tape stripping of human skin elicits a proliferative response of a synchronously‐dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid‐S phase and the other centred around G2+ M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram.

Methotrexate inhibits the leukotriene B4 induced intraepidermal accumulation of polymorphonuclear leukocytes.

The penetration of polymorphonuclear leukocytes (PMNs) into the epidermis following topical application of leukotriene B4 was assessed in the clinically uninvolved skin of psoriatic patients treated with methotrexate and of psoriatic patients without treatment, and in normal controls. Inhibition of PMN infiltration was observed in those patients treated with methotrexate while there was no significant difference between untreated psoriatic patients and normal controls.

Impulse cytophotometry in psoriasis.

There seems to be no general agreement in the literature regarding cell cycle parameters in psoriasis; the mitotic duration is reported to be lengthened (Fisher & Wells, 1968) or shortened (Weinstein & Frost, 1968), the DNA synthesis time doubled (Pullmann, Lennartz & Steigleder, 1974; Born & Kalkoff, 1969)5 or halved (Weinstein & Frost, 1968), the Gi time shortened by a factor of 10 (Weinstein, 1972) or by a factor of 2 (PuUmann et al., 1974). Some of these disagreements might be due to variations in the maturity of the psoriatic lesions and inter-individual variations, while others are certainly caused by pitfalls in the methods being used. Another problem is that until recently cell kinetic studies have been very time consuming. We describe here the apphcation of a rapid method for the analysis of cell kinetic parameters (up to 1000 cells/s) and some preliminary results obtained with it in the kinetics of psoriasis. The instrument used is a pulse cytophotometer (PCP) (Gohde & Dittrich, 1971; Lancet editorial, 1975). Cytophotometers measure light intensities as a function of cell characteristics. In pulse cytophotometry single cells are transported through the field of a microscope-photometer. In our study we made an epidermal cell suspension from skin biopsies (4inm diameter punch) by treating them with i % trypsin solution for 30 min at 37°C. The cells were fixed in absolute ethanol and stained with ethidixmi bromide, a fiuorescent dye which stains DNA quantitatively. Each cell produces a fiuorescent signal, the intensity of which is proportional to the amount of DNA per cell. The signals are electronically processed and classified into 128 intensity catagories to produce a DNA histogram. Fig. I (A) and (B) gives examples of such a DNA histogram from normal and psoriatic skin respectively. In Fig. i(A) 64,355 ^nd in Fig. (B) 83,699 cells were measured. The area under the major peak (channel number 30) represents cells which contain the 2n amount of DNA (cells in Gj); cells in G2 and mitosis contain 4n DNA and consequently one can find them around the double channel number. The region in between contains cells in various stages of DNA synthesis (S phase). With a simple arithmetical calculation one can find the percentages of the cells in each phase of the cell cycle. Table i gives the percentages of the cells in the various phases of the cycle and therefore the relative lengths of the phases, if we asstime that there is an asynchronous distribution of the germinative population over the cell cycle. These values have to be corrected since not all the epidermal population is proliferative. The cells of the stratum spinosum, for instance, also contain the 2n amoimt of DNA. One should therefore know the ratio proliferative/nonproliferative cells, or one could attempt to fractionate the cells into two populations. The former problem can be tackled by measuring two parameters with the PCP (e.g. DNA and RNA or DNA and protein) since one might expect different ratios of DNA/RNA or DNA/protein in the differentiated skin layers compared to the basal layer. The absolute time length of the different phases could be determined by synchronization procedures in vivo or in vitro. Even without these modifications the method already seems to be very promising

Response of the clinically uninvolved skin of psoriatic patients to standardized injury.

Test sites on healthy controls and on the clinically uninvolved skin of psoriatic patients were stripped with tape, and eight variables were quantified at intervals during the subsequent healing process.

Metabolic changes at the margin of the spreading psoriatic lesion

Keratotome slices were cut across the margins of rapidly‐spreading psoriatic plaques. Each slice was divided into eight sections and in each section we measured the percentage cells in S phase and the levels of glucose‐6‐phosphate dehydrogenase (both related to epidermal proliferation), acid phosphatase (associated with keratinization) and alkaline phosphatase (a marker for dermal capillaries).

Flow cytometry as a tool for the study of cell kinetics in skin 2. Cell kinetic data in psoriasis.

Flow cytometry was used to measure the DNA content of epidermal keratinocytes from psoriatic patients. Gross deviations were found in the lesions and minor but significant changes in the uninvolved skin. Statistical analysis revealed that the rather large variation in the DNA distributions of the lesions was due to inter‐individual differences rather than to inter‐individual differences. The duration of the S‐phase seemed to be prolonged in the lesion, but the length of the overall cell cycle might be of the same order as that of normal skin.

Hyperdiploid cells in skin infiltrates.

We have developed a simple system based largely on parts of the Olympus OM camera system. The backbone of the apparatus is the bar with the bellows, equipped either with a 38 mm (/ 2 8) or a 20 mm {/2} macrolens. The camera body is a 35 mm SLR OM2 with an autoexposure electronic shutter. The bar carries a footplate tnade of 5 mm aluminium with a glass window with a diameter of about 35 mm along the optical axis. On one side the footplate carries an arm with the flash unit attached. The flash unit, equipped with a bright pilot light, is connected by a cable to the power control unit. The latter is fitted to the camera body by means of the flash shoe. The metering systetn of the camera automatically controls flash output, thus giving correct exposure under all conditions (Fig. i). Equipped with the 20 mm or 38 mm macrolens, the unit allows photographs to be taken with maximal magniflcations of up to x 16 or x 6 7 respectively. Photography of the skin with this apparatus is very simple. The footplate is put onto the skin with the pilot light on, either with or without immersion oil. Focusing and magniflcation are set by the knobs on the bellows. Once set, they can be kept fixed indeflnitely by tightening the knobs. Photos are taken, as with all cameras, by pressing the shutter release. Any of the vast number of different 35 mm films available may be used, including the new instant fllms. Our investigation of pigmented skin lesions (unpublished) has been done mainly with standard colour slide film, which allows easy secondary magnification by projecting the slides. Black and white films are also suitable. The simple apparatus described greatly facilitates macrophotography of the skin surface and skin lesions. Apart from possibly aiding in the differential diagnosis of pigmented skin lesions and their macroscopic changes with time, the system might also be of value for the documentation of vascular lesions before and after treatment. Other possible applications, e.g. in microlymphangiography, remain to be developed.

Long-distance cytotoxicity of parachlorophenol and Formalin in vitro

The “long-distance” cytotoxicity of 10% parachlorophenol in 80 alcohol solution and 25% Formalin in 80% alcohol solution has been tested in an in vitro experiment on Vero cells. No difference could be found between the tubes containing Formalin and those of the controls. However, parachlorophenol appeared far more toxic for the mammalian cell line used in this study.