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.