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• W2023398504 abstract "There is a continuous need for methods to evaluate the biologic effects of topically applied drugs in the skin. Irritation of the epidermis with sodium dodecyl sulfate leads to an upregulation of E-selectin on endothelial cells and E-selectin mRNA can be detected in vivo within a short time. This study was aimed to investigate whether this biologic response can be used as a read-out for the anti-inflammatory effect of topically administered corticosteroids. We investigated skin of healthy volunteers treated according to the two following experimental protocols: (i) topical application of different corticosteroids (versus basic ointments as controls) for 12 h and irritation with sodium dodecyl sulfate 1% for 4 h; (ii) irritation with sodium dodecyl sulfate 1% for 12 h and application of the corticosteroids for 5 h. The biopsy specimens were subjected to RNA extraction and reverse transcription and competitive reverse transcriptase–polymerase chain reaction was performed using defined concentrations of a preconstructed mimic DNA. As result, we found strong positive signals for wild-type E-selectin mRNA in all biopsies pretreated with basic ointments, whereas in biopsies from areas pretreated with corticosteroids the bands for wild-type E-selectin DNA could be detected at 10–1000 lower levels of mimic DNA concentrations. The reverse experiment, application of corticosteroids after the irritation, again yielded significantly reduced signals for E-selectin mRNA. In both experimental settings, the different strength of the topical corticosteroids used was reflected by significant differences in the amount of E-selectin mRNA found in the biopsies. This study demonstrates the pharmacologic effect of topical corticosteroids on the irritation-induced E-selectin mRNA expression on dermal endothelial cells in vivo using very small tissue samples and this approach may be of value for further pharmaceutical studies. There is a continuous need for methods to evaluate the biologic effects of topically applied drugs in the skin. Irritation of the epidermis with sodium dodecyl sulfate leads to an upregulation of E-selectin on endothelial cells and E-selectin mRNA can be detected in vivo within a short time. This study was aimed to investigate whether this biologic response can be used as a read-out for the anti-inflammatory effect of topically administered corticosteroids. We investigated skin of healthy volunteers treated according to the two following experimental protocols: (i) topical application of different corticosteroids (versus basic ointments as controls) for 12 h and irritation with sodium dodecyl sulfate 1% for 4 h; (ii) irritation with sodium dodecyl sulfate 1% for 12 h and application of the corticosteroids for 5 h. The biopsy specimens were subjected to RNA extraction and reverse transcription and competitive reverse transcriptase–polymerase chain reaction was performed using defined concentrations of a preconstructed mimic DNA. As result, we found strong positive signals for wild-type E-selectin mRNA in all biopsies pretreated with basic ointments, whereas in biopsies from areas pretreated with corticosteroids the bands for wild-type E-selectin DNA could be detected at 10–1000 lower levels of mimic DNA concentrations. The reverse experiment, application of corticosteroids after the irritation, again yielded significantly reduced signals for E-selectin mRNA. In both experimental settings, the different strength of the topical corticosteroids used was reflected by significant differences in the amount of E-selectin mRNA found in the biopsies. This study demonstrates the pharmacologic effect of topical corticosteroids on the irritation-induced E-selectin mRNA expression on dermal endothelial cells in vivo using very small tissue samples and this approach may be of value for further pharmaceutical studies. Irritation of the epidermis is a well-known model to study the mechanisms of inflammation (Nickoloff and Naidu, 1994Nickoloff B.J. Naidu Y. Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.J Am Acad Dermatol. 1994; 30: 535-546Abstract Full Text PDF PubMed Scopus (408) Google Scholar). Immigration of leukocytes in tissues such as the skin is dependent on their attachment to activated endothelial cells (Springer, 1990Springer T.A. Adhesion receptors of the immune system.Nature. 1990; 346: 425-434Crossref PubMed Scopus (5701) Google Scholar). This has been shown to be a multistep procedure involving the upregulation of cellular adhesion molecules (Friedmann et al., 1993Friedmann P.S. Strickland I. Memon A.A. et al.Early time course of recruitment of immune surveillance in human skin after chemical provocation.Clin Exp Immunol. 1993; 91: 351-356Crossref PubMed Scopus (32) Google Scholar;Saito et al., 1993Saito I. Terauchi K. Shimuta M. et al.Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjögren's syndrome.J Clin Lab Anal. 1993; 7: 180-187Crossref PubMed Scopus (97) Google Scholar;Meagher et al., 1994Meagher L. Mahiouz D. Sugars K. et al.Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.J Immunol Methods. 1994; 175: 237-246Crossref PubMed Scopus (46) Google Scholar). Recent data suggest a crucial part for E-selectin to recruit P-selectin glycoprotein ligand-1 expressing T cells into the inflamed tissue (Austrup et al., 1997Austrup F. Vestweber D. Borges E. et al.P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues.Nature. 1997; 385: 81-83Crossref PubMed Scopus (643) Google Scholar;Fuhlbrigge et al., 1997Fuhlbrigge R.C. Kieffer J.D. Armerding D. et al.Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells.Nature. 1997; 389: 978-981Crossref PubMed Scopus (452) Google Scholar). Upregulation of E-selectin on upper dermal vessels has been described to be an early event after irritation of the epidermis (Friedmann et al., 1993Friedmann P.S. Strickland I. Memon A.A. et al.Early time course of recruitment of immune surveillance in human skin after chemical provocation.Clin Exp Immunol. 1993; 91: 351-356Crossref PubMed Scopus (32) Google Scholar) and, in a pilot study, we found the presence of the corresponding E-selectin mRNA as soon as 30 min after application of the irritant and a maximum after 4 h (unpublished data). Because the amount of E-selectin expression correlates with the extent of dermal inflammation (von den Driesch et al., 1993von den Driesch P. Gruschwitz M. Hornstein O.P. et al.Adhesion molecule modulation in Sweet's syndrome compared to erythema multiforme.Eur J Dermatol. 1993; 3: 393-397Google Scholar;von den Driesch and Simon, 1994von den Driesch P. Simon Jr, M. Cellular adhesion antigen modulation in pupura pigmentosa chronica.J Am Acad Dermatol. 1994; 30: 193-200Abstract Full Text PDF PubMed Scopus (26) Google Scholar;Henseleit et al., 1996Henseleit U. Steinbrink K. Goebeler M. et al.E-selectin expression in experimental models of inflammation in mice.J Pathol. 1996; 180: 317-325Crossref PubMed Scopus (36) Google Scholar), we questioned whether the mRNA response for E-selectin could be useful to monitor the effect of an anti-inflammatory drug in vivo. In other studies on the effects of corticosteroid treatment in inflammatory disorders such as allergic rhinitis, downregulation of VCAM-1 and E-selectin proteins were shown by means of immunohistochemistry (Karlsson and Hellquist, 1996Karlsson M.G. Hellquist H.B. Endothelial adhesion molecules for nasal-homing T cells in allergy.Virchows Arch. 1996; 429: 49-54Crossref PubMed Scopus (9) Google Scholar). The regulation of E-selectin on mRNA level, however, has not been used in this regard so far. Thus, this study investigated the influence of topically applied corticosteroids on irritation-induced E-selectin mRNA signals in vivo. Thirty healthy volunteers, 17 male and 13 female, mean age 33, participated in this study. After written informed consent up to five test areas on the skin of the back were chosen. The presence of any inflammatory systemic or skin disease as well as any need for systemic or topical anti-inflammatory therapy, and a history of sunburn 3 mo prior to the experiment, were strictly used as exclusion criteria. The first set of experiments was carried out treating four different areas on the back of healthy subjects. Area I was irritated for 4 h by applying 50 μl of sodium dodecyl sulfate (SDS) (1% in water) using an occlusive Finn chamber patch test on Scanpore tape (Hermal, Reinbeck, Germany), area II was pretreated with basic ointment (Dermatop basic cream, Hoechst, Bad Soden, Germany) for 12 h before application of the irritant for 4 h, area III was pretreated with 0.25% prednicarbate (Dermatop) for 12 h, and area IV was left untreated. After removing the Finn chamber, 4 mm biopsies were obtained under local anesthesia. Epidermis and dermis were separated after 45 s of microwave heating (490 W) and frozen at 70°C until use. In the second set of experiments one area was treated with Unguentum leniens (according to the Deutsche Rezept Formulierungen DRF) as basic ointment and three other areas were treated with hydrocortisone acetate 0.4%, prednisolone 1%, and triamcinolone acetonide 0.1%, respectively, each dissolved in Unguentum leniens, for 12 h. After that the areas were treated with SDS 1% for 4 h before the biopsy specimens were done. In the third series of experiments the test areas were treated with SDS 1% for 12 h, followed by an application of hydrocortisone acetate 0.4%, prednisolone 1%, and triamcinolone acetonide 0.1%, each dissolved in Unguentum leniens, and Unguentum leniens as control. After another 5 h the biopsy specimens were taken. Total RNA was extracted from upper dermal tissue using an RNeasy Total RNA kit (Quiagen, Hilden, Germany). The dermal tissue was thoroughly homogenized with a rotar-stator homogenizer (Ultra Turrax T8, IKA Analyzentechnik GmbH, Staufen im Breisgau, Germany) in 2 × 300 μl lysis buffer containing β-mercaptoethanol. The extraction was then performed according to the user protocol. Total RNA was eluted in 40 μl of diethylpyrocarbonate-treated water. The concentration and purity of the RNA was determined by measuring the absorbance at 230, 260, 280, 320 nm using a 10 μl ultramicrocuvette (Gene Quant II, Pharmacia Biotech, Freiburg, Germany). The ratio between the absorbance values at 260 and 280 nm gave an estimate of RNA purity. One hundred nanograms of RNA was subjected to 100 U of a Moloney leukemia virus reverse transcriptase (GIBCO-BRL, Eggenstein, Germany) in a reaction mixture containing 5 × buffer, 0.1 M ditriothritol, 0.3 mM deoxynucleotide triphosphates (Boehringer, Mannheim, Germany) and 360 pmol of random primer (GIBCO-BRL) for 60 min at 39°C. Afterwards, competitive PCR was performed using a PCR Mimic Construction Kit (Clontech, Palo Alto, CA). Composite E-selectin primers were designed (Table I) and used in the PCR amplification with the mimic DNA fragment provided with the kit. The templates were amplified and purified according to the manufacturer's instructions. Pure PCR mimics were then quantitated and diluted from 103 amol per μl to 10−4 amol per μl and used as competitive internal standards for quantitation of the E-selectin wild-type DNA levels after reverse transcription. Primers for E-selectin were used based on a previously published sequence (Saito et al., 1993Saito I. Terauchi K. Shimuta M. et al.Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjögren's syndrome.J Clin Lab Anal. 1993; 7: 180-187Crossref PubMed Scopus (97) Google Scholar) (Table I). Afterwards, 2 μl of cDNA and 2 μl of mimic DNA were added to 45 μl of a PCR mixture containing 10 × buffer, MgCl2, dNTP, 11 pmol (each) of E-selectin sense and anti-sense primers and 0.2 U DNA polymerase (Eurogentec, Seraing, Belgium). The mixtures were covered with 50 μl mineral oil (Roth, Karlsruhe, Germany) and amplification was performed with 35 cycles in an Omni Gene temperature cycler (Hybaid, Teddington, U.K.) with denaturation for 1 min at 94°C, annealing for 1 min at 55°C and primer extension at 72°C. After amplification, 8 μl aliquots of each reaction mixed with 1.5 μl loading buffer were subjected to electrophoresis through a 2% agarose gel (Roth) and stained with ethidium bromide. Restriction analysis of the E-selectin wild-type amplificate using PvuII revealed the expected bands (205 bp and 83 bp, respectively) in the gel electrophoresis (not shown). Bands were visualized under UV light and photographed.Table IPrimers used in the studyPrimerDirectionChosen sequenceLength of amplification productE-SelectinUpstreamAGAAGAAGCTTGCCCTATGC315 bpDownstreamAGGCTGGAATAGGAGCACTCCAUpstreamAGAAGAAGCTTGCCCTATGCCAGTGAAATCTCCTCCG442 bpDownstreamAGGCTGGAATAGGAGCACTCCATCTGTCAATGCAGTTTGTAG Open table in a new tab Photographs were scanned and densitometry was performed using ScanPack 2.0 software (Biometra, Göttingen, Germany). Equal bands for mimic and wild-type DNA indicated the corresponding wild-type DNA concentration. If no equal bands could be detected, the value of wild-type DNA was considered to be the 5-fold of the weaker mimic DNA concentration. Statistical evaluation of the results was performed using Fisher's exact test. In the first series of experiments, E-selectin wild-type mRNA could not be found in all biopsy specimens from the control area without any treatment (Figure 1a, summary of results in Table II). In test sites treated with SDS 1% for 4 h mRNA for E-selectin was found in eight of 10 biopsy specimens at a dilution level of the mimic DNA of 10−2 amol per μl and in two of 10 at 0.5 × 10−2 amol per μl (Figure 1b).Table IIResults of competitive reverse transcriptase–PCR for E-selectin with different treatment conditionsaSkin biopsy specimens were obtained from the back of healthy individuals. Area I was irritated with SDS 1% for 4 h, area II was pretreated for 12 h with a basic ointment and irritated with SDS 1% for 4 h, area III with 0.25% prednicarbate for 12 h and with SDS for 4 h; area IV was not pretreated or irritated. After RNA extraction and reverse transcription, competitive reverse transcriptase–PCR was performed using defined concentrations of a preconstructed mimic DNA. The results show a significant (p > 0.001, Fisher's exact test) increase in amounts of E-selectin mRNA in SDS-treated (areas I and II) versus SDS-untreated area IV and significant (p > 0.001, Fisher's exact test) decreases in E-selectin mRNA for SDS-pretreated and prednicarbate-pretreated area III compared with area I pretreated with SDS only and with area II pretreated with SDS and basic ointment.amol per μl mimic DNAArea I No pretreatment SDS 4 hArea II Pretreatment with basic ointment SDS 4 hArea III Pretreatment with prednicarbate SDS 4 hArea IV No pretreatment No irritation010/1010−44/1010−36/1010−28/106/100.5 × 10−22/102/1010−12/10a Skin biopsy specimens were obtained from the back of healthy individuals. Area I was irritated with SDS 1% for 4 h, area II was pretreated for 12 h with a basic ointment and irritated with SDS 1% for 4 h, area III with 0.25% prednicarbate for 12 h and with SDS for 4 h; area IV was not pretreated or irritated. After RNA extraction and reverse transcription, competitive reverse transcriptase–PCR was performed using defined concentrations of a preconstructed mimic DNA. The results show a significant (p > 0.001, Fisher's exact test) increase in amounts of E-selectin mRNA in SDS-treated (areas I and II) versus SDS-untreated area IV and significant (p > 0.001, Fisher's exact test) decreases in E-selectin mRNA for SDS-pretreated and prednicarbate-pretreated area III compared with area I pretreated with SDS only and with area II pretreated with SDS and basic ointment. Open table in a new tab In six of 10 biopsy specimens from the areas which were pretreated with an identical basic ointment for 12 h and with SDS 1% for 4 h a similar amount of wild-type E-selectin amplificate was found (Figure 1c). In two of 10 cases, the signal was equal to 0.5 × 10−2 amol per μl and the other two biopsy specimens revealed a concentration equal to 10−1 amol per μl mimic DNA. Thus both test sites treated with SDS showed a significant (p < 0.001, Fisher's exact test) upregulation of E-selectin mRNA compared with the untreated test sites. In six of 10 of the biopsy specimens derived from the areas which were pretreated for 12 h with topically applied prednicarbate 0.25% and for 4 h with SDS 1% an E-selectin mRNA concentration equal to 10−3 amol mimic DNA per μl was found, whereas four of 10 showed a concentration equal to 10−4 amol mimic DNA per μl (Figure 1d). Thus significantly less mRNA for E-selectin was found in the prednicarbate-pretreated test sites (significance p < 0.001, Fisher's exact test). In the second series of experiments, hydrocortisone acetate 0.4%, prednisolone 1.0%, and triamcinolone acetonide 0.1% were dissolved in Unguentum leniens and their inhibiting effect on SDS-induced E-selectin expression was compared with the basic ointment and to each other. As a result (Table III) we found that the effect of topical hydrocortisone compared with the basic ointment did not reach significance, whereas prednisolone and triamcinolone caused a significant (p < 0.001, Fisher's exact test) decrease in E-selectin mRNA levels compared with basic ointment. Furthermore, comparison of triamcinolone versus hydrocortisone revealed significant lower mRNA levels in the triamcinolone-treated skin biopsy specimens (p < 0.01, Fisher's exact test), whereas comparison of prednisolone versus triamcinolone and prednisolone versus hydrocortisone did not reach clear significance (p = 0.33 and p = 0.06, respectively, Fishers exact test).Table IIIResults of competitive reverse transcriptase–PCR for E-selectin with different treatment conditionsaSkin biopsy specimens were obtained from the back of healthy individuals. Area I was pretreated for 12 h with a basic ointment and irritated with SDS 1% for 4 h, area II with hydrocortisone 0.4%, area III with prednisolone 1.0%, and area IV with triamcinolone 0.1%. SDS 1% was then applied for 4 h. After RNA extraction and reverse transcription, competitive reverse transcriptase–PCR was performed using defined concentrations of a preconstructed mimic DNA. The results show a significant (p > 0.001, Fisher's exact test) decrease in amounts of E-selectin mRNA in the prednisolone 1% and triamcinolone 0.1% treated areas (III and IV) versus area I treated with basic ointment only. The differences between triamcinolone 0.1% treated area IV and hydrocortisone-treated area II additionally reach significance (p > 0.01, Fisher's exact test).amol per μl mimic DNAArea I Pretreatment with basic ointment SDS 4 hArea II Pretreatment with hydrocortisone 0.4% SDS 4 hArea III Pretreatment with prednisolone 1% SDS 4 hArea IV Pretreatment with triamcinolone 0.1% SDS 4 h03/106/106/1010−43/105/104/1010−32/104/102/1010−23/106/100.5 × 10−22/1010−1a Skin biopsy specimens were obtained from the back of healthy individuals. Area I was pretreated for 12 h with a basic ointment and irritated with SDS 1% for 4 h, area II with hydrocortisone 0.4%, area III with prednisolone 1.0%, and area IV with triamcinolone 0.1%. SDS 1% was then applied for 4 h. After RNA extraction and reverse transcription, competitive reverse transcriptase–PCR was performed using defined concentrations of a preconstructed mimic DNA. The results show a significant (p > 0.001, Fisher's exact test) decrease in amounts of E-selectin mRNA in the prednisolone 1% and triamcinolone 0.1% treated areas (III and IV) versus area I treated with basic ointment only. The differences between triamcinolone 0.1% treated area IV and hydrocortisone-treated area II additionally reach significance (p > 0.01, Fisher's exact test). Open table in a new tab The reverse experiments, treating a skin site which had been irritated for 12 h with SDS 1% with the three topical corticosteroids, revealed similar results (Table IV). Prednisolone 1% and triamcinolone acetonide 0.1% significantly reduced the E-selectin mRNA levels compared with the basic ointment Unguentum leniens (p < 0.001, Fisher's exact test) and hydrocortisone acetate 0.4% (p < 0.01, Fisher's exact test). Comparison of hydrocortisone versus basic ointment and triamcinolone versus prednisolone were statistically borderline (p = 0.071 and p = 0.044, respectively, Fisher's exact test).Table IVResults of competitive reverse transcriptase–PCR for E-selectin with different treatment conditionsaSkin biopsy specimens were obtained from the back of healthy individuals. All areas were irritated for 12 h with SDS 1% for 12 h and area II treated with hydrocortisone 0.4%, area III with prednisolone 1.0%, and area IV with triamcinolone 0.1% for 5 h. After RNA extraction and reverse transcription, competitive reverse transcriptase–PCR was performed using defined concentrations of a preconstructed mimic DNA. The results show a significant (p > 0.001, Fisher's exact test) decrease in the amount of E-selectin mRNA in the prednisolone 1% and triamcinolone 0.1% (areas III and IV) versus area I treated with the basic ointment only. The differences between prednisolone 1.0% and triamcinolone 0.1% treated areas versus hydrocortisone-treated areas II additionally reach significance (p > 0.01, Fisher's exact test). Comparison of triamcinolone 0.1% versus prednisolone 1.0% revealed lower statistical significance (p = 0.044, Fisher's exact test).amol per μl mimic DNAArea I SDS 12 h Pretreatment with basic ointment 5 hArea II SDS 12 h Pretreatment with hydrocortisone 0.4% 5 hArea III SDS 12 h Pretreatment with prednisolone 1% 5 hArea IV SDS 12 h Pretreatment with triamcinolone 0.1% 5 h02/107/1010−45/103/1010−31/105/103/1010−24/105/100.5 × 10−23/1010−12/10a Skin biopsy specimens were obtained from the back of healthy individuals. All areas were irritated for 12 h with SDS 1% for 12 h and area II treated with hydrocortisone 0.4%, area III with prednisolone 1.0%, and area IV with triamcinolone 0.1% for 5 h. After RNA extraction and reverse transcription, competitive reverse transcriptase–PCR was performed using defined concentrations of a preconstructed mimic DNA. The results show a significant (p > 0.001, Fisher's exact test) decrease in the amount of E-selectin mRNA in the prednisolone 1% and triamcinolone 0.1% (areas III and IV) versus area I treated with the basic ointment only. The differences between prednisolone 1.0% and triamcinolone 0.1% treated areas versus hydrocortisone-treated areas II additionally reach significance (p > 0.01, Fisher's exact test). Comparison of triamcinolone 0.1% versus prednisolone 1.0% revealed lower statistical significance (p = 0.044, Fisher's exact test). Open table in a new tab Several read-out systems have been established that try to evaluate and quantitate the amount of cutaneous irritation and its corresponding inflammatory response. Most studies are based on noninvasive clinical and physiologic methods such as laser Doppler blood flow, color reflectance (Wilhelm et al., 1989Wilhelm K.P. Surber C. Maibach H.I. Quantification of sodium lauryl sulfate irritant dermatitis in man: comparison of four techniques: skin color reflectance, transepidermal water loss, laser doppler flow measurements and visual scores.Arch Dermatol Res. 1989; 281: 293-295Crossref PubMed Scopus (150) Google Scholar), clinical scoring (York et al., 1996York M. Griffiths H.A. Whittle E. et al.Evaluation of a human patch test for the identification and classification of skin irritation potential.Contact Dermatitis. 1996; 34: 204-212Crossref PubMed Scopus (96) Google Scholar), measurements of the transepidermal water loss (Iliev et al., 1997Iliev D. Hinnen U. Elsner P. Reproducibility of a non-invasive skin irritancy test in a cohort of metalworker trainees.Contact Dermatitis. 1997; 36: 101-103Crossref PubMed Scopus (11) Google Scholar), or the skin redness (colorimetry). Sorption-desorption and moisture accumulation tests were additionally considered to be suitable to detect changes of the stratum corneum properties even if there are no visible modifications of the skin (Treffel and Gabard, 1996Treffel P. Gabard B. Measurement of sodium lauryl sulfate-induced skin irritation.Acta Derm Venereol (Stockh). 1996; 76: 341-343PubMed Google Scholar). These methods, however, all have a relatively high interobserver variation or show a great variation from site to site or from day to day (e.g., transepidermal water loss). Furthermore, their exact correlation to the extent of the dermal inflammation has not been established. Other possible methods to evaluate directly the amount of inflammation, e.g., counting of the number of inflammatory cells or vessels positive for an inflammatory adhesion molecule such as ICAM-1, VCAM-1, or E-selectin are time-consuming and may greatly depend on which sections were chosen for staining and evaluation. Additionally, they may have a high interobserver variation. The aim of this study was therefore to develop a read-out system that: (i) is observer independent; (ii) can be performed in different laboratories using the same standardized protocol; (iii) evaluates the whole biopsy specimen and not only single sections; and that (iv) needs only small tissue samples. The evaluation of the expression of the adhesion molecule E-selectin was chosen for the following reasons: It is not expressed in healthy skin, but can be found on the surface of dermal endothelial cells in cutaneous inflammatory reactions (Groves et al., 1991Groves R.W. Allen M.H. Barker J.N. et al.Endothelial leucocyte adhesion molecule-1 (ELAM-1) expression in cutaneous inflammation.Br J Dermatol. 1991; 124: 117-123Crossref PubMed Scopus (195) Google Scholar;Brasch et al., 1992Brasch J. Burgard J. Sterry W. Common pathogenetic pathways in allergic and irritant contact dermatitis.J Invest Dermatol. 1992; 98: 166-170Crossref PubMed Scopus (118) Google Scholar;Henseleit et al., 1996Henseleit U. Steinbrink K. Goebeler M. et al.E-selectin expression in experimental models of inflammation in mice.J Pathol. 1996; 180: 317-325Crossref PubMed Scopus (36) Google Scholar). Some immunohistochemical studies suggested a good correlation between the expression of E-selectin on vessels and the amount of cutaneous inflammation (von den Driesch et al., 1993von den Driesch P. Gruschwitz M. Hornstein O.P. et al.Adhesion molecule modulation in Sweet's syndrome compared to erythema multiforme.Eur J Dermatol. 1993; 3: 393-397Google Scholar;von den Driesch and Simon, 1994von den Driesch P. Simon Jr, M. Cellular adhesion antigen modulation in pupura pigmentosa chronica.J Am Acad Dermatol. 1994; 30: 193-200Abstract Full Text PDF PubMed Scopus (26) Google Scholar). It is known that E-selectin is induced after challenge of the epidermis with agents causing an irritant or allergic contact dermatitis (Brasch et al., 1992Brasch J. Burgard J. Sterry W. Common pathogenetic pathways in allergic and irritant contact dermatitis.J Invest Dermatol. 1992; 98: 166-170Crossref PubMed Scopus (118) Google Scholar;Friedmann et al., 1993Friedmann P.S. Strickland I. Memon A.A. et al.Early time course of recruitment of immune surveillance in human skin after chemical provocation.Clin Exp Immunol. 1993; 91: 351-356Crossref PubMed Scopus (32) Google Scholar). Another group demonstrated upregulation of E-selectin after induction of irritant or allergic contact dermatitis in a mouse model and found a good correlation between E-selectin expression and the amount of cutaneous inflammatory infiltrates (Henseleit et al., 1996Henseleit U. Steinbrink K. Goebeler M. et al.E-selectin expression in experimental models of inflammation in mice.J Pathol. 1996; 180: 317-325Crossref PubMed Scopus (36) Google Scholar). To avoid an immunohistochemical evaluation of single sections and to get representative data for the whole biopsy specimen, we decided to monitor E-selectin mRNA regulation. Little is known so far about the in vivo mRNA expression of E-selectin in cutaneous irritation. We therefore established in a pilot study that by means of a sensitive reverse transcriptase–PCR the occurence of E-selectin mRNA can be demonstrated as soon as 30 min after the application of SDS 1% on healthy epidermis and reaches a maximum at 4 h. We found the corresponding protein expression 2–3 h after application (unpublished data). To allow a comparison between biopsy specimens from test areas treated with different modalities, the establishment of a competitive reverse transcriptase–PCR was essential. A similar method was recently used to quantitate mRNA of several adhesion molecules (VCAM-1, ICAM-1, and E-selectin) in skin biopsies taken from delayed-type hypersensitivity reactions (Meagher et al., 1994Meagher L. Mahiouz D. Sugars K. et al.Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.J Immunol Methods. 1994; 175: 237-246Crossref PubMed Scopus (46) Google Scholar). In the first series of experiments, a commercially available corticosteroid was chosen for which an identical basic ointment could be obtained. As a result, we found no E-selectin mRNA in untreated skin. It has to be kept in mind, however, that our subjects did not suffer from any skin or systemic inflammatory disease. In the biopsy specimens from skin irritated with SDS for 4 h, constant amounts of wild-type E-selectin mRNA comparable with a level of 10−2 amol per μl mimic DNA could be detected. These data suggest that the amount of the endothelial activation was similar between the 10 subjects tested. Topical preapplication of prednicarbate 0.25% 12 h before application of the irritant yielded 10–100-fold lower wild-type DNA concentrations. Again, the variation between the 10 subjects tested was surprisingly low resulting in clearly significant results. Application of the basic ointment showed in eight cases no difference to the untreated biopsies, but in two subjects even higher mRNA levels comparable with 10−1 amol mimic DNA per μl. The frequency of this result was too low, however, to reach significance but it has to be concluded at least that no preventing potency of the basic ointment could be demonstrated. An explanation for this phenomenon could be that the basic ointment increased the penetration of the SDS. The second and third series of experiments represent a comparison of topical corticosteroids of different strength which were dissolved in a standard basic ointment. Two models were used: (i) prevention of irritation-induced E-selectin mRNA induction and (ii) suppression after irritation-induced induction. According toGuzzo et al., 1997Guzzo C.A. Lazarus G.S. Werth V.P. Dermatological pharmacology.in: Hardmann J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman and Gilmans the Parmacological Basis of Therapeutics. McGraw-Hill, New York1997: 1593-1616Google Scholar, hydrocortisone acetate 0.4% belongs to group 6, prednisolone 1% to group 5, and triamcinolone acetonide 0.1% to group 3. Our results of suppression of E-selectin mRNA induction correlates well with these suggested strengths. The differences between the group 3 and 5 corticosteroids and the basic ointment as well as the difference between the group 3 and group 6 corticosteroids were highly significant. There was additionally a trend for differences between prednisolone and hydrocortisone and between triamcinolone and prednisolone, but the number of measurements was presently too limited to reach clear significance. We believe from our data, however, that optimizing the system question should make it possible to work out even relatively small strength differences between topical anti-inflammatory drugs. It is mandatory, however, to use the same basic ointment. Thus, a comparison of the data of the first experiments with prednicarbate and the second series with the three other corticosteroids was not reasonable. This study shows that it is possible to evaluate a possible anti-inflammatory effect of topically applied corticosteroids by monitoring irritation-induced mRNA signals for E-selectin in vivo. The method is invasive but due to the sensitivity of the reverse transcriptase–PCR only very small tissue samples are needed. Done routinely, this assay is not too time-consuming. We suggest this approach to be a tool for the evaluation of the anti-inflammatory potency of drugs applied topically to the skin. The authors thank Mrs. C. Wagner for her excellent technical assistence and Prof. Dr T. L. Diepgen for his help regarding statistics. The study was supported by the Johannes and Frieda-Marohn-Foundation (DRI-95)." @default.
• W2023398504 created "2016-06-24" @default.
• W2023398504 creator A5020977664 @default.
• W2023398504 creator A5032408571 @default.
• W2023398504 creator A5042344855 @default.
• W2023398504 creator A5059690035 @default.
• W2023398504 date "1999-08-01" @default.
• W2023398504 modified "2023-10-14" @default.
• W2023398504 title "Modulation of Irritation-Induced Increase of E-selectin mRNA In Vivo by Topically Applied Corticosteroids1" @default.
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