Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts.

The damaging effect of long-wave ultraviolet radiation (UVA) on the plasma membranes of cultured human skin fibroblasts was evaluated by cytofluorometry performed after vital staining with fluorescein diacetate. The damage was associated with lipid peroxidation, as shown by accumulation of malondialdehyde and 4-hydroxyalkenals; such accumulation was induced by a UVA dose of only 8 J/cm2. Pretreatment with the effective membrane peroxidation inhibitor alpha-tocopherol (added in the form of alpha-tocopherol succinate) or the singlet oxygen quencher beta-carotene protected the cells from membrane damage. Further, depletion of intracellular glutathione by exposure of the cells to buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase aggravated the membrane-damaging effects. The results confirm the photodynamic effects of UVA, which presupposes the excitation of endogenous photosensitizer(s) and the production of reactive oxygen species. The present results indicate that this method of detection of alterations in plasma membrane stability may be highly suitable for studying various photobiological phenomena and for use as a model for testing substances that could protect skin from UVA damage. The trypan blue exclusion test proved to be too insensitive to detect these changes.

The cellular infiltrate in experimental allergic and toxic contact dermatitis. Effect of systemically administered corticosteroids.

The effect of systemically administered corticosteroids on allergic and toxic contact dermatitis in the guinea pig was studied. Dermatitis was provoked with dinitrochlorobenzene (DNCB). The influence of corticosteroids on the sensitization phase was also investigated. The epicutaneous test reactions were assessed with the naked eye, low-power microscopy, and a method based on the counting of infiltrating cells in the upper corium. Prednisolone injected in a dose of 5 mg/kg body weight daily for 4 days starting on the 1st day of sensitization or the 1st day of testing tended to shorten the duration of the contract allergic skin reaction as seen with the naked eye. Microscopically, a decrease in the number of infiltrating basophil granulocytes was found. Prednisolone had no convincing influence on the toxic contract eczematous reaction macroscopically or microscopically. Responses to weakly toxic doses of DNCB tended to fade earlier and to show small displacements of granulocytes in the differential count of infiltrating cells compared to controls. The results suggest that prednisolone given systemically influences only slightly the inflammatory cell infiltration in contact dermatitis in the guinea pig.

Measurement and differentiation of the cellular infiltrate in experimental toxic contact dermatitis.

Toxic (irritant) contact dermatitis was elicited by epicutaneous application of dinitrochlorobenzene (DNCB) and croton oil in unsensitized guinea pigs. The course of the skin reaction was studied with the naked eye, low-power microscopy, and a method based on counting of infiltrating cells in the upper corium. A weakly toxic dose of DNCB (100 microgram/cm2) gave marked erythema and moderate swelling, more pronounced 6 h after application of the DNCB than at 24 h or 48 h. A significant increase in mononuclear cells in particular but also in neutrophil and eosinophil granulocytes was noted in the upper corium. A more strongly toxic dose (500 microgram/cm2) gave a similar visible reaction, this too most marked at 6 h, but here there was an increase in neutrophil granulocytes in particular. Over and above predominant mononuclear-cell infiltration, croton oil (10 microgram/cm2) caused a slight increase in basophil cells. The reaction thus resembled a contact allergic reaction. In the light of earlier findings in allergic contact reactions the results suggest that weak stimuli, toxic or allergenic, elicit a non-specific skin response. With stronger stimulation the histological picture is modified and the cellular response acquires a pattern characteristic of toxic or allergic contact dermatitis.

Measurement and differentiation of the cellular infiltrate in expermiental allergic contact dermatitis.

We describe a quantitative method for the grading of contact allergic reactions in guinea pigs. These reactions are characterized by marked cellular infiltration, and the method is based on total and differential counting of cells in the upper corium. A varying and objectively gradable increase in mononuclear and basophil polymorphonuclear cells was found. In naked-eye-positive reactions this increase was highly significant 24, 48, and 72 hours after epicutaneous application of dinitrochlorobenzene (DNCB). The degree of cellular infiltration reflects aspects of a cell-mediated immune response other than the visible reaction ordinarily made use of. The method can be used to study how systemically or topically administered drugs affect cellular features in contact allergy.

Effect of cyclophosphamide on the cellular infiltrate in experimental allergic contact dermatitis.

The influence of cyclophosphamide on contact allergy was studied by a method based on the count of cells infiltrating epicutaneous test sites in sensitized guinea pigs. Cyclophosphamide was injected 72 h before sensitization with dinitrochlorobenzene (DNCB). On testing 2 weeks later, marked mononuclear cell infiltration and dramatic increase in the count of basophil polymorphonuclear cells in the upper corium were found, compared to the control group. Cell infiltration in tests performed 1 and 3 weeks after sensitization differed from the reaction after 2 weeks. When cyclophosphamide was given 72 h before testing done 2 weeks after sensitization, both mononuclear and polymorphonuclear counts were found to be greatly reduced. This in vivo method of assessing the degree and composition of cellular infiltration in a contact allergic reaction after different periods of time throws new light on the influence of drugs on a cell-mediated immune response and also the variation in response with time.