Use of Human Skin Recombinants as an in vitro Model for Testing the Irritation Potential of Cutaneous Irritants

Abstract

Two human skin recombinants, the epidermis reconstructed on the deepidermized dermis (RE-DED) or on fibroblast-populated collagen matrix (Living Skin Equivalent, LSE^TM), were used to study the irritating effect of sodium lauryl sulfate (SLS). The extent of cytotoxicity induced after a 24-hour exposure period to increasing concentrations of SLS (0-5%) was evaluated on the basis of (1) morphological perturbations, (2) changes in the expression of differentiation-specific protein markers (keratin 1, 10, 6, 16, involucrin and transglutaminase), (3) cell membrane integrity (LDH leakage) and (4) release of proinflammatory mediators (PGE₂, IL-1, IL-6 and IL-8). SLS induced significant changes in epidermal morphology and changes in the expression and localization of differentiation-specific protein markers when applied topically in concentrations higher than 1 % on RE-DED and higher than 0.1% on LSE. The exposure of both human skin recombinants to SLS resulted in a dose-dependent release of LDH, PGE₂and IL-1Α and in an increase in keratinocyte intracellular IL-1 levels. Upon application of 5% SLS on RE-DED the total (intra- and extracellular) IL-1 levels remained high but due to cell damage the intracellular IL-1 level was markedly decreased and the extracellular IL-1 level increased. Similar observations have been made with LSE after application of 0.5% SLS. However, with LSE the extracellular IL-1Α levels were found to be about 100 times lower than those measured with RE-DED. Exposure of LSE to SLS induced a marked increase of IL-6 production in fibroblasts incorporated in the collagen matrix. Contrary to LSE, both intra- and extracellular levels of IL-6 were low in unexposed controls and were only marginally modulated by the exposure of the RE-DED to SLS. In addition, a dose-dependent increase in IL-8 release was observed upon application of SLS on RE-DED. The results of the present study indicate that concentrations of SLS required to induce epidermal irritancy in vitro approximate those inducing irritation in human skin in vivo. All parameters used in the present study for evaluation of toxicity can serve as useful endpoints for screening of contact skin irritancy in vitro. Compared to RE-DED, the LSE seems to be more susceptible to SLS. The differences in sensitivity between LSE and RE-DED can be ascribed to reported differences in their stratum corneum barrier function.

Human Skin in Organ Culture and Human Skin Cells (Keratinocytes and Fibroblasts) in Monolayer Culture for Assessment of Chemically Induced Skin Damage

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contact-sensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995; Boxman et al, 1996; Corsini and Galli, 1998; Bernhofer et al, 1999a, 1999b; Coquette et al, 2003). While in our small sample of test reagents we found no definitive differences between the contact irritants and contact sensitizers (as groups), the finding that the contact sensitizers did, in fact, raise the levels of the 2 cytokines is significant in that it shows that these agents are not inert in the ex vivo assays utilized here.…”

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contactsensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995;Boxman et al, 1996; 700 VARANI ET AL.…”

Human Skin in Organ Culture and Human Skin Cells (Keratinocytes and Fibroblasts) in Monolayer Culture for Assessment of Chemically Induced Skin Damage

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contact-sensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995; Boxman et al, 1996; Corsini and Galli, 1998; Bernhofer et al, 1999a, 1999b; Coquette et al, 2003). While in our small sample of test reagents we found no definitive differences between the contact irritants and contact sensitizers (as groups), the finding that the contact sensitizers did, in fact, raise the levels of the 2 cytokines is significant in that it shows that these agents are not inert in the ex vivo assays utilized here.…”

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contactsensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995;Boxman et al, 1996; 700 VARANI ET AL.…”

Skin Barrier Disruption by Sodium Lauryl Sulfate-Exposure Alters the Expressions of Involucrin, Transglutaminase 1, Profilaggrin, and Kallikreins during the Repair Phase in Human Skin In Vivo

“…Others have reported increased expression of involucrin after acetone exposure of mice skin (Ekanayake- Mudiyanselage et al, 1998) and after SLS exposure of human skin in vivo (Le et al, 1997;van Ruissen et al, 1998) and reconstructed skin in vitro (Ponec and Kempenaar, 1995;Fletcher and Basketter, 2006). The increased expression and localization of the protein resembles that observed in lesional skin of atopic dermatitis (Jensen et al, 2004).…”

Cytokine and chemokine release upon prolonged mechanical loading of the epidermis

“…Cell swelling or cytoplasmic swelling is further known to be one of the first visible signs of cell damage (28). This cell damage is supported by the observed release in LDH (29)(30)(31). Severe tissue damage was observed after loading the EpiDerm cultures with 150 and 200 mmHg.…”

Human Skin in Organ Culture and Human Skin Cells (Keratinocytes and Fibroblasts) in Monolayer Culture for Assessment of Chemically Induced Skin Damage

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contact-sensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995; Boxman et al, 1996; Corsini and Galli, 1998; Bernhofer et al, 1999a, 1999b; Coquette et al, 2003). While in our small sample of test reagents we found no definitive differences between the contact irritants and contact sensitizers (as groups), the finding that the contact sensitizers did, in fact, raise the levels of the 2 cytokines is significant in that it shows that these agents are not inert in the ex vivo assays utilized here.…”

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contactsensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995;Boxman et al, 1996; 700 VARANI ET AL.…”

Skin Barrier Disruption by Sodium Lauryl Sulfate-Exposure Alters the Expressions of Involucrin, Transglutaminase 1, Profilaggrin, and Kallikreins during the Repair Phase in Human Skin In Vivo

“…Others have reported increased expression of involucrin after acetone exposure of mice skin (Ekanayake- Mudiyanselage et al, 1998) and after SLS exposure of human skin in vivo (Le et al, 1997;van Ruissen et al, 1998) and reconstructed skin in vitro (Ponec and Kempenaar, 1995;Fletcher and Basketter, 2006). The increased expression and localization of the protein resembles that observed in lesional skin of atopic dermatitis (Jensen et al, 2004).…”

Cytokine and chemokine release upon prolonged mechanical loading of the epidermis

“…Cell swelling or cytoplasmic swelling is further known to be one of the first visible signs of cell damage (28). This cell damage is supported by the observed release in LDH (29)(30)(31). Severe tissue damage was observed after loading the EpiDerm cultures with 150 and 200 mmHg.…”

Human Skin in Organ Culture and Human Skin Cells (Keratinocytes and Fibroblasts) in Monolayer Culture for Assessment of Chemically Induced Skin Damage

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contact-sensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995; Boxman et al, 1996; Corsini and Galli, 1998; Bernhofer et al, 1999a, 1999b; Coquette et al, 2003). While in our small sample of test reagents we found no definitive differences between the contact irritants and contact sensitizers (as groups), the finding that the contact sensitizers did, in fact, raise the levels of the 2 cytokines is significant in that it shows that these agents are not inert in the ex vivo assays utilized here.…”

“…The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contactsensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995;Boxman et al, 1996; 700 VARANI ET AL.…”

Skin Barrier Disruption by Sodium Lauryl Sulfate-Exposure Alters the Expressions of Involucrin, Transglutaminase 1, Profilaggrin, and Kallikreins during the Repair Phase in Human Skin In Vivo

“…Others have reported increased expression of involucrin after acetone exposure of mice skin (Ekanayake- Mudiyanselage et al, 1998) and after SLS exposure of human skin in vivo (Le et al, 1997;van Ruissen et al, 1998) and reconstructed skin in vitro (Ponec and Kempenaar, 1995;Fletcher and Basketter, 2006). The increased expression and localization of the protein resembles that observed in lesional skin of atopic dermatitis (Jensen et al, 2004).…”

Cytokine and chemokine release upon prolonged mechanical loading of the epidermis

“…Cell swelling or cytoplasmic swelling is further known to be one of the first visible signs of cell damage (28). This cell damage is supported by the observed release in LDH (29)(30)(31). Severe tissue damage was observed after loading the EpiDerm cultures with 150 and 200 mmHg.…”