Analysis of human epidermal Langerhans' cells and allergens with confocal laser scanning microscopy
Author: Emilson, Axel
Date: 1997-11-21
Location: CMMs hörsal L8:00, Karolinska sjukhuset
Time: 9.00
Department: Inst för laboratoriemedicin / Dept of Laboratory Medicine
Abstract
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-presenting Langerhans' cell (LC) in epidermis and 2) for localization of allergens in yeast cells and in birch pollen. Epidermal LCs were investigated after occlusion with patch tests and in basal cell carcinoma (BCC) skin. Occlusion with patch test produced a slight transient inflammatory reaction in the skin. The density of LCs was not significantly affected, but an altered distribution of the LCs dendrites towards the skin surface was observed, which suggests a LC reaction to the change in the environment. The number and density of LCs in BCC was decreased and the morphology of LCs was altered compared to normal skin. There was an increased expression of ICAM-I on keratinocytes overlying BCC compared to normal skin. These findings might be due to secondary changes in the local environment.
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 with CLSM. The results indicate that both skin forms are suitable for quantitative studies. Due to lower background intensity and larger tissue volume, detailed 3-D analysis of 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 total epidermal volume of biopsy specimens from nickel allergic contact dermatitis (ACD)and detergent induced irritant contact dermatitis (ICD) at 6-72 hours after patch testing 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 the detergent in ICD on the epidermal environment. There was a reduced epidermal volume of Ii reactivity at 72 h compared to 24 h in the ICD group and compared to 72 h in the ACD group. Furthermore, the Ii expression was significantly lower than the HLA-DR reactivity in the ICD group at 72 h. This could point to differences in the biosynthesis rate of the Ii between ACD and ICD due to variance in the local cytokine production.
CLSM and flow cytometry were used to determine the subcellular localization of two 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 cell surface, whereas these proteins were virtually undetectable in the Candida genus and Saccharomyces cerevisiae. The expression of the two major allergens was significantly decreased after more than 4 days of culture, indicating that extracts from the exponential phase 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 investigated with 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 the exine in the aperture regions. This suggests that the normal route for excretion of Bet v I is via the apertures on contact between pollen and the stigmatic surface of the pistil. It is concluded that CLSM is an advantageous tool in the study of LCs and allergens.
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 with CLSM. The results indicate that both skin forms are suitable for quantitative studies. Due to lower background intensity and larger tissue volume, detailed 3-D analysis of 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 total epidermal volume of biopsy specimens from nickel allergic contact dermatitis (ACD)and detergent induced irritant contact dermatitis (ICD) at 6-72 hours after patch testing 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 the detergent in ICD on the epidermal environment. There was a reduced epidermal volume of Ii reactivity at 72 h compared to 24 h in the ICD group and compared to 72 h in the ACD group. Furthermore, the Ii expression was significantly lower than the HLA-DR reactivity in the ICD group at 72 h. This could point to differences in the biosynthesis rate of the Ii between ACD and ICD due to variance in the local cytokine production.
CLSM and flow cytometry were used to determine the subcellular localization of two 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 cell surface, whereas these proteins were virtually undetectable in the Candida genus and Saccharomyces cerevisiae. The expression of the two major allergens was significantly decreased after more than 4 days of culture, indicating that extracts from the exponential phase 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 investigated with 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 the exine in the aperture regions. This suggests that the normal route for excretion of Bet v I is via the apertures on contact between pollen and the stigmatic surface of the pistil. It is concluded that CLSM is an advantageous tool in the study of LCs and allergens.
Issue date: 1997-10-31
Publication year: 1997
ISBN: 91-628-2734-0
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