The term dermal cell trafficking has been used to describe the dynamic nature of cell movement in the dermis reflecting the skin's role as an immunological organ. A light microscopic experimental model for qualitative and quantitative counting of the dermal inflammatory cell infiltrate in allergic, acute irritant and cumulative irritant contact reactions has been developed In human studies use of microdialysis technique has enabled observation of biochemical events in the skin, in vivo over a period of time. This method might allow measurement of cytokines and other inflammatory mediators in the intercellular space of the dermis without the need of multiple biopsies. For confirmation that cytokines detected/not detected in the dialysate are present /absent in the dermis, and to determine the possible cellular source of the cytokines, a return to biopsy technique at time points of special interest would be required.
The general aims of this thesis have been to extend the experimental studies on skin reactions to immediate reaction types and to develop the use of microdialysis technique for the measurement of cytokines.
Plastic embedding, thin sectioning and optimal staining were the basis for inflammatory cell counting. The immediate hypersensitivity reaction to ovalbumin and the non immunological immediate contact reaction to dimethyl sulfoxide were studied. The effect of topical glucocorticosteroid on delayed contact reaction types was also studied. A polyethersulfone membrane, with a cut-off value of 100,000 Daltons was used. Reliable sample volumes and high analyte recovery was achieved either by push pull pumping or standard pumping using a perfusate consisting of Ringer Dextran 60. ELISA and flow cytometry based analysis of polystyrene bead conjugated antibodies (suspension array) was used to estimate the levels of interleukins 1 beta, 2, 4, 5, 6, 8, 10, granulocyte monocyte colony stimulating factor, interferon gamma and tumor necrosis factor alpha in normal skin for up to 24 -28 hours. Tissue sections from the area of the microdialysis membrane were examined with double immunofluorescence for cytokines and resident dermal cells.
The immediate Type 1 hypersensitivity reaction to ovalbumin showed an early phase with basophil granulocytes and a late lymphocytic phase. 100% DMSO gave a basophil rich non immunological immediate reaction while repeated applications of 12% DMSO gave a reaction most like the cumulative irritant reaction. Topical glucocorticosteroid and its acetone vehicle showed anti inflammatory effects most pronounced on the acute irritant reaction. Microdialysis showed IL6, IL8 and IL1b in response to insertion with a slow equilibration period. Other cytokines were detected less frequently and in smaller amounts. The biopsies revealed intracellular cytokines in general concordance with the microdialysis fmdings. Confocal microscopy using double immunofluorescence allowed demonstration of cytokines and cellular markers in the same preparation.
The experimental model illustrates differences in dermal inflammatory histological patterns in various common reaction types. Findings are relevant for discussion of pathogenetic mechanisms and as background information for continued clinical studies. Microdialysis is well suited to chronological studies of cytokine patterns in vivo. Suspension array technique allows measurement of multiple cytokines and other analytes, the results of which need interpretation against background knowledge of the particular analyte. End point biopsy for immunofluorescence studies enable intracellular localization of cytokines and even speculation about cellular origin.
Linköping: Linköpings universitet , 2005. , 73 p.
2005-03-18, Elsa Brändströmsalen, Hälsouniversitetet, inköping, 09:00 (Swedish)