Flow cytometric quantification of T6-positive cells in psoriatic epidermis after PUVA and methotrexate therapy.

We describe a flow cytometric technique for measuring the percentage of T6-positive cells in suspensions prepared by trypsinization of human epidermis. The value obtained for healthy controls (1.55 +/- 0.52%) corresponds to about 600 T6-positive cells per mm2 of skin surface, a figure in line with histological estimates. No significant change was found in the percentage of T6-positive cells in either the uninvolved or lesional epidermis of untreated psoriatic patients. PUVA treatment resulted in a significant reduction in T6-positive cells. A cyclic fluctuation in the numbers of T6-positive cells was shown to accompany methotrexate administration on a weekly divided dose schedule.

Isolation and preliminary biochemical characterization of the human epidermal Langerhans cell.

A method is described for the isolation of human epidermal Langerhans cells (LC). Following the disaggregation of the epidermis by gentle trypsinization, the cell suspension is incubated with the fluorescein-conjugated monoclonal antibody OKT6. Using a fluorescence-activated cell sorter, a subpopulation is selected on the basis of positive fluorescence and low forward-scatter, the latter parameter reducing contamination by LC-keratinocyte clusters. The LC averaged 1.7% of the original epidermal preparation. The purity averages 83% as judged either by OKT6 binding or by ATPase activity. Biochemical measurements indicated that the activity of lysosomal enzymes was low with the exception of alpha-mannosidase, which was about 12-fold higher than that of the keratinocyte. The activities of 3 enzymes of intermediary metabolism suggest that the utilization of glucose by the LC is considerably greater than that of the keratinocyte.

Monocyte function is normal in quiescent psoriasis.

We report an investigation of peripheral blood monocytes from untreated patients with mild, quiescent psoriasis. Possible metabolic changes were monitored by the determination of 3 enzymes representing different pathways of glucose metabolism and 2 lysosomal enzymes. Signal processing was evaluated by the measurement of cyclic AMP levels before and after hormonal stimulation. Luminol-amplified chemiluminescence provided an objective approach to assessing phagocytic capacity. Finally, the pattern of maturation of normal and psoriatic monocytes has been compared during culture in vitro. Our results were uniformly and wholly negative; we conclude that the concept of an "intrinsic" abnormality of the psoriatic monocyte may be excluded. Possible reasons for discrepancies in the literature are discussed.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 5. Lectin binding.

The glycocalyx of epidermal keratinocytes from psoriatic patients has been investigated by means of lectins. Striking changes were found in the levels of glucose and/or mannose (concanavalin A) and of N-acetylglucosamine and/or sialic acid (wheat germ agglutinin) on the surface of cells from the psoriatic lesion. Smaller but significant changes were seen in the clinically uninvolved epidermis of the patient. A marked increase in the affinity of the cell surface for Ulex europus agglutinin (fucose-specific) confirms our previous reports of structural alterations in fucose-containing oligosaccharides in psoriasis.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 4. Characterization of glycoconjugates.

A substantial proportion (20-50%) of radioactive sugar incorporated into glycoconjugates by normal human keratinocytes was soluble in chloroform-methanol; using (14C)-galactose as precursor about half of this fraction was neutral lipid. The incorporation of labelled sugars into the lipid fraction was consistently increased in keratinocytes derived from psoriatic lesions. The abnormality in fucose metabolism which we reported previously has been confirmed. In particular we have shown that the molecular weight of fucosylated glycopeptides appears to be abnormally high in the untreated psoriatic lesion, possibly reflecting an increased degree of branching. In psoriatic uninvolved epidermis and in treated psoriatic lesions the situation is reversed, the molecular weight being significantly lower than normal.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 3. Uptake of labelled sugars and their incorporation into glycoconjugates.

We report the uptake of four labelled sugars by keratinocytes isolated from normal epidermis, psoriatic 'uninvolved' skin and psoriatic lesions. Our findings include the following: (1) The rate of uptake of all sugars by the psoriatic lesion is increased. (2) This abnormally high uptake diminishes dramatically during 22 h incubation in vitro. (3) There is a striking abnormality in the metabolism of fucose by psoriatic keratinocytes; our data suggest an increased rate of incorporation of fucose into glycoconjugates.

Lectin binding studies on suspensions of isolated epidermal keratinocytes.

The sugar composition of the surface of isolated human keratinocytes has been investigated using lectins. A method is described in which the FITC-labelled lectin is displaced with competing sugar prior to measurement. This avoids errors due to aspecific binding and utilizes the minimum biological sample. Evidence is presented which indicates that little cell surface glycoconjugate is lost during the preparation of the keratinocyte suspension from human epidermal slices using the technique described here. Values for keratinocytes are similar to those reported for fibroblast lines, being much higher than those found using erythrocytes or lymphocytes.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 2. Cyclic AMP and its response to hormonal stimulation.

Cyclic AMP levels have been determined for the first time in isolated keratinocytes. Values were more reproducible than those reported using epidermal slices. Evidence is presented to show that damage to hormone receptors is minimal. Other observations include the following: (1) Keratinocytes from psoriatic lesions showed reduced 'resting' levels of cyclic AMP as well as a diminished response to adrenaline. (2) Cyclic AMP levels were maximal in the basal cells, falling dramatically in fully differentiated keratinocytes. (3) The topical application of a corticosteroid (fluocinolone acetonide) did not modulate the response of adenyl cyclase to hormonal stimulation.

Studies on the plasma membrane of normal and psoriatic keratinocytes. I. Preparation of material and morphological characterization.

A method is described for the preparation of isolated keratinocytes suitable for subsequent biochemical studies. Scanning electron microscopy showed that the maturation process is accompanied by an increase in cell size and a shortening and eventual loss of microvilli. Psoriatic keratinocytes are distinguishable by exhibiting longer microvilli at all levels of maturation.