Analysis of human epidermal Langerhans' cells and allergens with confocal laser scanning microscopy

Abstract: Analysis of human epidermal Langerhans' cells and allergenswith confocal laser scanning microscopy By Axel Emilson From the Department of Laboratory Medicine, Division of ClinicalImmunology, Karolinska Institute and Hospital, and the Department of Medical Biophysics,MBB, Karolinska Institute, Stockholm, Sweden The aim of the thesis was to explore confocal laser scanning microscopy (CLSM)as a tool for 1) quantitative and 3-dimensional (3-D) analysis of the human antigen-presentingLangerhans' cell (LC) in epidermis and 2) for localization of allergens in yeastcells and in birch pollen. Epidermal LCs were investigated after occlusion with patchtests and in basal cell carcinoma (BCC) skin. Occlusion with patch test produceda slight transient inflammatory reaction in the skin. The density of LCs was notsignificantly affected, but an altered distribution of the LCs dendrites towardsthe skin surface was observed, which suggests a LC reaction to the change in theenvironment. The number and density of LCs in BCC was decreased and the morphologyof LCs was altered compared to normal skin. There was an increased expression ofICAM-I on keratinocytes overlying BCC compared to normal skin. These findings mightbe due to secondary changes in the local envlronment. Two different skin techniques - epidermal sheets and vertical skin sections -were used to study and compare the density and 3-D morphology of epidermal LCs withCLSM. The results indicate that both skin forms are suitable for quantitative studies.Due to lower background intensity and larger tissue volume, detailed 3-D analysisof LCs is preferably performed on epidermal sheets rather than on vertical sections. The epidermal volume of HLA-DR and invariant chain (Ii) reactivity and the totalepidermal volume of biopsy specimens from nickel allergic contact dermatitis (ACD)and detergent induced irritant contact dermatitis (ICD) at 6-72 hours after patchtesting were analyzed with CLSM. An increased epidermal volume was recorded overtime, at 24 h in ICD and at 72 h in both groups, suggesting a direct effect of thedetergent in ICD on the epidermal environment. There was a reduced epidermal volumeof Ii reactivity at 72 h compared to 24 h in the ICD group and compared to 72 h inthe ACD group. Furthermore, the Ii expression was significantly lower than the HLA-DRreactivity in the ICD group at 72 h. This could point to differences in the biosynthesisrate of the Ii betwcen ACD and ICD due to variance in the local cytokine production. CLSM and flow cytometry were used to determine the subcellular localization oftwo major yeast allergens in the Pityrosporum genus and various other yeast genera.All members of the Pityrosporum genus expressed the two major allergens on the cellsurface, whereas these proteins were virtually undetectable in the Candida genusand Saccharomyces cerevisiae. The expression of the two major allergens was significantlydecreased after more than 4 days of culture, indicating that extracts from the exponentialphase of the yeast culture should be used in studies of IgE responses to P. orbiculare.The localization of the major allergen, Bet v I in birch pollen, was investigatedwith CLSM. When the pollen grains had been prefixed, Bet v I was found in the cytoplasm.When the prefixation was omitted, a minor portion of Bet v I also appeared in theexine in the aperture regions. This suggests that the normal route for excretionof Bet v I is via the apertures on contact between pollen and the stigmatic surfaceof the pistil. It is concluded that CLSM is an advantageous tool in the study of LCs and allergens. Key words: 3-D analysis, allergen, allergic contact dermatitis, basal cellcarcinoma, Bet v I, birch, confocal laser scanning microscopy, epidermis, HLA-DR,image analysis, invariant chain, irritant contact dermatitis, Langerhans' cell, occlusion,P. orbiculare, pollen. ISBN 91-628-2734-0 Stockholm 1997