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Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertion
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 Cytokines play important roles in steering homeostatic and inflammtory activities in human tissues not the least skin. In vivo, human, large pore membrane microdialysis technique can be used to measure cytokines in human tissues such as skin, muscle and brain. The new analytical technique of flow cytometry based analysis of polystyrene bead conjugated antibodies (suspension array) allows the analysis of multiple cytokines or other proteins on the typically small sample volumes (10 - 15 ul) provided by microdialysis. Another challenge for microdialysis is the differentiation of observations due to the pathological process being studied from those caused by the insertion and presence in the tissue of the catheter itself.

Objectives The objective of the present study was to use suspension array analysis of 10 cytokines to illustrate the chronology of the response of normal living human skin to the introduction of a microdialysis probe in-vivo.

Methods CE-marked, commercially manufactured microdialysis catheters equipped with a 100 kiloDalton cut-off polyethersulphone membrane were introduced into the normal skin of the ventral forearm in 10 volunteers. Probes were perfused with Ringer Dextran Braun at a speed of 0.3 ul min-I. Samples were collected at 1 hour intervals for the first 7-8 hours, for a period of 9- 14 hours during the evening and during a 15-21 hours night period. Hourly sampling was again done the following morning until at least 24 hours after catheter insertion. Total protein was analysed by a protein assay kit. A cytokine suspension array was used to estimate the levels ofinterlenkin (IL) 1beta (b), 2, 4, 5, 6, 8, 10, granulocyte monocyte colony stimulating factor (GM-CSF), interferon gamma (IFNg) and turnor necrosis factor alpha (TNFa).

Results All probes were in place as planned for 24-28 hours with a small and acceptable degree of discomfort to the subjects. Total protein levels confirmed that probes were functioning with high initial levels followed by a fall. The cytokines IL6, IL8 and IL-1 beta were detected in all patients at all time points after the first hour. IL-6 levels reached a peak at 5-8 hours, falling to levels between 25 - 820 pg ul-1 at 24 hours. IL-8 and IL-1b showed similar time courses. IL-2, GM-CSF and TNFalpha were detected at levels above the lowest standard for the technique at some time points in some subjects. IL-10, IL-5, and IL-4 were only detected in isolated samples in a few individuals. IFNg was not detected at any time point, in any of the ten subjects.

Conclusions The previously observed elevation of IL-6 immediately after microdialysis catheter insertion into normal skin was confirmed with levels peaking at 5-8 hours and then falling to lower levels by 24 hours. IL-8 and IL-1b showed a similar time course though absolute levels at peak were lower. Other cytokines showed variable outcomes from similarly high levels, to variably low levels as well as absent levels. The cytokine spectrum was pro-inflammatory with a profile of non-Th2 character according to the Th1/Th2 paradigm. Suspension array represents a significant analytical advance in the applicability of in-vivo microdialysis whether it be experimental or clinical since most analytical capture molecules can be conjugated to the beads. The levels of cytokines seen after probe insertion provide a basis for normal biology and chronology of the cytokines as well as the interpretation of levels in the study of pathological reactions.

National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-84290OAI: diva2:558504
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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