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Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skin
Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background Classically, participation of cytokines in a tissue process is demonstrated by biopsy, immunostaining and fluorescence microscopy or by PCR technique. If the chronology of a reaction is to be studied, multiple biopsies are required. This can pose practical and ethical problems. Cutaneous microdialysis is a technique which allows the qualitative and quantitative, chronological study of endogenous molecules including cytokines in living, human skin. For confumation 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 is required.

Objectives The first objective of the study was to use a single immunofluorescence staining technique to demonstrate the presence or absence of ten pro-inflammatory and lymphocyte regulatory cytokines in biopsies from the site of a microdialysis catheter. Findings were to be compared with analysis of the same cytokines in microdialysates from the same subjects immediately prior to the biopsy. A further objective was to investigate use of double immunostaining, confocal microscopy to compare the localisation of individual cytokines with cellular markers for four major candidate cell types -fibroblasts, endothelial cells, mast cells and dendritic/Langerhans cells.

Methods In 10 volunteers studied by dermal microdialysis in normal skin for 24 -28 hours, a biopsy was taken from the area of the microdialysis membrane at the tip of the catheter. Biopsies were frozen in liquid nitrogen. Single immunostaining for interleukin (IL) I beta (b), 2, 4, 5, 6, 8, 10, granulocyte monocyte colony stimulating factor (GM-CSF), interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa), was performed and assessed semiquantitatively. Results were then compared with the microdialysis cytokine levels in the same subject innnediately prior to biopsy. Double immunostaining confocal microscopy for the cytokine and markers of the four main cell types in the dermis was performed on biopsies positive for the cytokines.

Results Negative controls contained no fluorescence and there was reproducibility of results in multiple sections. Cellular localisation of cytokines was noted to varying degrees: IL8 was seen in all subjects; IL6 was seen in 70% of the subjects; TNFa in 50%; IL1b, IL2, GM-CSF and IFNg were seen in 30-40% of subjects; IL4, IL5 and IL10 were seen least often, 10 - 20% of subjects. At an individual subject level, concordance between positive or negative results for fluorescence microscopy and microdialysis was highest for IL8 (100%), lowest for TNFa (50%) with the remaining cytokines distributed between these two values. Double immunofluorescence and confocal microscopy enabled study of cytokine and cellular markers in the same section. Apparent co-localisation of the two markers was seen to varying degrees.

Conclusions Fluorescence microscopy and microdialysis illustrate different aspects of cytokine presence in tissue reactions. Though individual variability can be seen in both methods and concordance of results was not seen in all subjects, the methodology was useful for better interpretation of microdialysis data. The individual concordance for the three main cytokines, IL8, IL6 and IL1b, seen after probe insertion were 100%, 80% and 60% respectively. Microdialysis fmdings in the 24 hours up to the biopsy bad suggested a non-Th2 cytokine pattern according to the Th1/Th2 paradigm. The immunofluorescence findings in the present paper indicate an even higher degree of positivity for TNFa and IFNg. Two important Th2 cytokines, IL4 and IL5 found in 1 of 10 subjects in microdialysis were not detected more frequently with immunofluorescence. The microdialysis and fluorescence findings together support a conclusion that dermal stimulation as reflected by catheter insertion creates a Th1 cytokine environment. The use of end point biopsy in the experimental design of microdialysis studies seems capable of producing worthwhlle data in regard to expression of cytokines and possible even cellular origin of cytokines.

Keyword [en]
Immunostaining, cytokines, cutaneous microdialysis, confocal microscopy normal skin
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-84291OAI: diva2:558506
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-03Bibliographically approved
In thesis
1. Dermal cell trafficking: from microscopy to microdialysis
Open this publication in new window or tab >>Dermal cell trafficking: from microscopy to microdialysis
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2005. 73 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 883
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-31154 (URN)16891 (Local ID)91-7373-865-4 (ISBN)16891 (Archive number)16891 (OAI)
Public defence
2005-03-18, Elsa Brändströmsalen, Hälsouniversitetet, inköping, 09:00 (Swedish)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-03Bibliographically approved

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