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Dermal cell trafficking: from microscopy to microdialysis
Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 883
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-31154Local ID: 16891ISBN: 91-7373-865-4 (print)OAI: oai:DiVA.org:liu-31154DiVA: diva2:251977
Public defence
2005-03-18, Elsa Brändströmsalen, Hälsouniversitetet, inköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-03Bibliographically approved
List of papers
1. The cellular dermal infiltrate in experimental immediate type cutaneous hypersensitivity
Open this publication in new window or tab >>The cellular dermal infiltrate in experimental immediate type cutaneous hypersensitivity
1995 (English)In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 75, no 6, 417-421 p.Article in journal (Refereed) Published
Abstract [en]

A previously developed guinea pig model for the study of the dermal inflammatory cell infiltrate of allergic, toxic, and irritant reactions was adapted to the study of the immediate intradermal reaction to ovalbumin, Comparison of qualitative and quantitative counts of infiltrating cells at three levels in the dermis showed that counting 20 subepidermal fields starting from the injection point of the allergen gave reliable figures, The reaction showed microscopically two phases. The first was of rapid onset and characterized by a high proportion of neutrophils, unlike the picture seen in the previously studied (allergic and toxic) reaction types. In the second phase, which can be termed 'late phase' reaction, mononuclear cells and basophil granulocytes predominated. The late phase of the reaction bears similarities to the delayed allergic contact reaction at the same timepoint in that the response was rich in basophils. There were, however, other differences; e.g, eosinophils and neutrophils were more common.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84289 (URN)8651014 (PubMedID)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved
2. The spectrum of inflammatory cell response to dimethyl sulfoxide
Open this publication in new window or tab >>The spectrum of inflammatory cell response to dimethyl sulfoxide
2000 (English)In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 42, no 4, 216-221 p.Article in journal (Refereed) Published
Abstract [en]

Dimethyl sulfoxide (DMSO), depending upon the concentration and mode of application to the skin, can induce either a non-immunological immediate contact urticaria or an irritant reaction. The dermal cellular infiltrate after open application of varying concentrations of DMSO has been studied in an experimental guinea pig model. The composition of the dermal cellular infiltrate showed a spectrum dependent on the concentration and number of applications of DMSO. The immediate reaction infiltrate 3 h after application of 100% DMSO consisted of 50% granulocytes, basophils being predominant. On the other hand, 12% DMSO applied 3 × daily for 3 days (cumulative insult) caused histologically a cellular reaction in which 80% of the infiltrate consisted of mononuclear cells. The present findings are compared to the microscopic findings in 3 other cutaneous reactions previously studied in this animal model, namely, the Type I immediate hypersensitivity reaction, the Type IV delayed hypersensitivity reaction, and the irritant reaction. Differing cellular infiltrate patterns are discernible at the same time points. The study illustrates the spectrum of inflammatory reactions seen in the skin and provides background information for future clinical studies, for instance, on the role of the basophil granulocyte in immediate contact reactions.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24852 (URN)10.1034/j.1600-0536.2000.042004216.x (DOI)9251 (Local ID)9251 (Archive number)9251 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-03Bibliographically approved
3. Acetone has anti-inflammatory effects on experimental contact reactions
Open this publication in new window or tab >>Acetone has anti-inflammatory effects on experimental contact reactions
1999 (English)In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 41, no 1, 22-29 p.Article in journal (Refereed) Published
Abstract [en]

The effects of a topically applied corticosteroid and its acetone vehicle on experimental allergic, toxic and irritant reactions are presented. The corticosteroid budesonide in acetone or acetone alone was applied to reactions immediately after and at different time intervals within the 1st h after provocation. Classical naked eye observation was performed and the dermal cellular infiltrate was differentiated and counted using a previously well-characterized method. “Treatment”, whether with the steroid in acetone or acetone alone, had anti-inflammatory effects. For all reaction types, erythema and oedema diminished and a significant decrease in mononuclear cells was seen, when application occurred within the first 5 min after provocation. The effects were most marked for the toxic reaction to croton oil, the steroid and the vehicle being anti-inflammatory to the same extent. Application up to 60 min after provocation had anti-inflammatory effects for this reaction type. The mechanisms of acetone's anti-inflammatory effects are at present unclear. One possible explanation is that intercellular lipid organisation and, by extension, cellular membrane lipid organisation, are altered, influencing membrane receptor function. Possible anti-inflammatory effects of acetone should be considered in experimental and perhaps even clinical situations. Further investigation of the therapeutic possibilities of the finding seems warranted.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24846 (URN)10.1111/j.1600-0536.1999.tb06203.x (DOI)9244 (Local ID)9244 (Archive number)9244 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-03Bibliographically approved
4. Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis
Open this publication in new window or tab >>Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis
2002 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 146, no 3, 375-382 p.Article in journal (Refereed) Published
Abstract [en]

Background  Cutaneous microdialysis in vivoin human skin is demonstrably of use in the study of skin metabolism, percutaneous absorption and skin inflammation. A promising area for cutaneous microdialysis is the measurement of cytokines. This requires catheters equipped with membranes permeable to molecules of high molecular weight.

Objectives  To address technical problems of poor sample volume retrieval and analysis sensitivity in the simplest model of provocation, namely the insertion of the catheter itself in vivo into human dermis.

Methods  Use of a polyethylenesulphone membrane, with a cut-off value of 100 000 Da, allowed measurement of target molecules of large molecular weight. Using an adaptation of a commercially available high sensitivity enzyme-linked immunosorbent assay, the ubiquitous proinflammatory cytokine interleukin (IL)-6 was measured in the normal skin of six healthy volunteers after insertion of the microdialysis catheter.

Results  Reliable sample volumes and high analyte recovery were achieved either by push–pull pumping or by standard pumping using a perfusate consisting of Ringers Dextran 60 Braun. No IL-6 was detected in 25 of 26 samples taken during the first 100 min after catheter insertion. The IL-6 concentration then increased and remained elevated for the duration of the experiments.

Conclusions  Technical and analytical modifications in the microdialysis technique have allowed the measurement of IL-6 in vivo in human dermis. It is suggested that the cytokine production is the result of the dermal trauma caused by catheter insertion, but the cellular source of the IL-6 is at present unknown.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24850 (URN)10.1046/j.1365-2133.2001.144003650_146_3.x (DOI)9249 (Local ID)9249 (Archive number)9249 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-03Bibliographically approved
5. Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertion
Open this publication in new window or tab >>Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertion
(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
Identifiers
urn:nbn:se:liu:diva-84290 (URN)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-03Bibliographically approved
6. Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skin
Open this publication in new window or tab >>Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skin
(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
Immunostaining, cytokines, cutaneous microdialysis, confocal microscopy normal skin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84291 (URN)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-03Bibliographically approved

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