Induction of HSP27 nuclear immunoreactivity during stress is modulated by vitamin C
Boxman, Ingeborg L.A. ; Kempenaar, Johanna ; Haas, Ellis De; Ponec, Maria H. - \ 2002
Experimental Dermatology 11 (2002)6. - ISSN 0906-6705 - p. 509 - 517.
Ascorbic acid - Human skin equivalent - Irritation - SLS - UV
For the investigation of the skin irritancy potential of chemicals in an in vitro model it is necessary to have sensitive endpoints that predict the effects of those compounds on native human skin. Recently, we have identified that 27-kDa heat shock protein (HSP27) can serve as a sensitive marker of skin irritation, as exposure of human skin to sodium lauryl sulfate (SLS) both in vitro and in vivo induced relocalization of HSP27 from the cytoplasm to the cell nucleus. The aim of the present study was to determine whether nuclear localization of HSP27 could be used as a parameter for evaluation of potential skin irritants in screening assays in vitro. For this purpose, human skin equivalent consisting of epidermis reconstructed on de-epidermized dermis was exposed to SLS or UV light. Stress-induced nuclear relocalization of HSP27 was observed in excised skin exposed to SLS or UV light and in reconstructed epidermis only when the latter was generated in the absence of vitamin C. The omission of vitamin C results in an impaired barrier function. In the presence of vitamin C, however, the barrier function was comparable with excised skin, suggesting that vitamin C may control the response to stress in the reconstructed epidermis. Besides the presence of vitamin C, the response of skin equivalents may strongly depend on other conditions under which they are generated, because the stress-induced HSP27 relocalization was not detected in the commercially available epidermal kit EpiDerm. The results of the present study show that HSP27 nuclear staining can serve as a sensitive marker for skin irritation or cellular stress in excised skin as well as in certain well-characterized human skin equivalents in vitro.
Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin
Boxman, I.L.A. ; Hensbergen, P.J. ; Schors, R.C. Van Der; Bruynzeel, D.P. ; Tensen, C.P. ; Ponec, M. - \ 2002
British journal of dermatology 146 (2002)5. - ISSN 0007-0963 - p. 777 - 785.
Proteomics - Screening - Sodium lauryl sulphate
Background: There is an increasing need for screening of mild irritants in vitro to reduce animal testing. Objectives: Proteomics were used to search for new markers of which the expression changes after mild irritation. Methods: Sodium lauryl sulphate (SLS) was applied topically on excised human skin. Epidermal proteins were isolated from SLS-treated skin specimens that showed hardly any morphological changes. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and proteins that significantly increased or decreased after SLS treatment in a dose-dependent way were characterized by mass spectrometry. Subsequently, immunohistochemistry was performed on skin samples treated with SLS in vivo and nonanoic acid (NAA) or benzalkonium chloride (BC) in vitro to evaluate one of the identified proteins for its predictive value. Results: We identified seven proteins as potentially new epidermal markers for skin irritation. Among these seven proteins, the 27 kDa heat shock protein (HSP27) was identified as the most prominently upregulated protein. A strong nuclear HSP27 staining was seen in the SLS-treated skin, whereas in the vehicle controls only cytoplasmic staining was observed. Moreover, nuclear staining was also observed after topical application of SLS in vivo and after exposure to NAA and BC in vitro. Conclusions: Our findings suggest that HSP27 may serve as a sensitive marker of skin irritation and eventually as a novel tool in clinics for testing the sensitivity of the patient for a panel of irritants.
HPV-DNA is not detectable in outgrowing cells from explant cultures of skin lesions established at the air-liquid-interface
Boxman, Ingeborg L.A. ; Mulder, Linda H.C. ; Vermeer, Bert Jan ; Bouwes Bavinck, Jan Nico ; Schegget, Jan Ter; Ponec, Maria - \ 2000
Journal of medical virology 61 (2000)3. - ISSN 0146-6615 - p. 281 - 288.
Epidermodysplasia verruciformis - Organotypic culture - Papillomavirus - PCR - Skin cancer
Keratinocyte cultures established from HPV containing skin cancers were described earlier to lose their HPV DNA after passaging in vitro. A different approach was therefore used in this study. Explant cultures were generated by depositing small pieces of various benign and (pre)malignant skin specimens of renal transplant recipients and non-immunosuppressed patients on fibroblast-populated collagen lattices or on de-epidermized dermis. Subsequently, the cultures were maintained at the air-liquid interface. At various time points, samples were collected for both HPV analysis, using a nested PCR approach, and morphology. The outgrowing keratinocytes developed into multilayered epithelial structures showing terminal differentiation. No histological differences were observed between cultures established from HPV positive and negative lesions. Eighteen biopsy specimens were tested for their HPV content before and after culture. Before culture 11 out of these skin specimens contained DNA of the Epidermodysplasia Verruciformis-related HPV types (EV-HPV). Comparison of the HPV types detected in two different parts of the same skin specimen before culture was strongly suggestive for a non-homogeneous distribution of EV-HPV in the lesions. From the explant cultures derived from the 11 HPV-positive biopsies, 31 samples from the originally explanted pieces of tissue and 38 samples from the outgrowing multilayered epithelial sections were collected. HPV DNA was detected in 10 of the 31 and in 3 of the 38 samples (Chi-square test, P = 0.01), respectively. These results indicate that EV-HPV positive keratinocytes do not efficiently proliferate or lose their HPV DNA in this culture system or EV-HPV DNA is present in only a few basal cells, making it improbable that these cells are located at the outgrowing margins. (C) 2000 Wiley-Liss, Inc.
Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1
Boxman, Ingeborg L.A. ; Ruwhof, Cindy ; Boerman, Otto C. ; Löwik, Clemens W.G.M. ; Ponec, Maria - \ 1996
Archives of Dermatological Research 288 (1996)7. - ISSN 0340-3696 - p. 391 - 398.
Fibroblast interaction - IL-1 receptor - Interleukin-1 - Interleukin-8 - Keratinocyte
In the epidermis, the keratinocytes are the first cells to be encountered by external stimuli and they are able to promote the inflammatory response by increased production and release of various cytokines. In their turn, these cytokines may directly affect the production of proinflammatory cytokines in human dermal fibroblasts. In addition, in both epithelial and mesenchymal cells cytokine production may be modulated by their mutual interaction, and thereby regulate the inflammatory response. The present study aimed to examine the role of fibroblasts in the regulation of proinflammatory IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. The data show that in fibroblasts exposed to conditioned media derived from cultures of normal human keratinocytes or squamous carcinoma cells (SCC-4), both the IL-8 and IL-6 mRNA expression as well as protein production were elevated. In addition, it was shown that these effects were induced by IL-1α. The IL-1α-induced increase in IL-8 and IL-6 production, both on the protein level as well as on the mRNA level, were concentration dependent and occurred almost simultaneously. While the induction of IL-6 and IL-8 occurred simultaneously, the IL-6 mRNA remained elevated for longer. In contrast to increased IL-6 and IL-8 production the IL-1α levels markedly decreased upon culturing of fibroblasts in keratinocyte-derived conditioned medium. From internalization experiments it could be concluded that binding of IL-1 to IL-1 receptors, and its subsequent internalization and intracellular degradation is the most likely mechanism involved in the reduction of IL-1 levels by fibroblasts. Comparing the rate of IL-1 reduction in the presence of various cell types indicated that the rate of IL-1 reduction is directly related to the number of IL-1 receptors found on these cell types. In conclusion, these results indicate that the release of IL-1α by activated keratinocytes may act as an inducer of IL-8 and IL-6 production in neighbouring fibroblasts. This may be an important pathway for the amplification of the inflammatory response. The amounts of both cytokines produced by fibroblasts were at least two to three orders of magnitude higher than those produced by keratinocytes, suggesting an important role of fibroblasts in the general inflammatory response. Furthermore, fibroblasts might be involved in turning off this inflammatory response by reducing IL-1 levels, most likely via IL-1 receptor-mediated uptake.
Differential regulation of plasminogen activation in normal keratinocytes and SCC-4 cells by fibroblasts
Boxman, I.L.A. ; Quax, P.H.A. ; Lowik, C.W.G.M. ; Papapoulos, S.E. ; Verheijen, J. ; Ponec, M. - \ 1995
Journal of Investigative Dermatology 104 (1995)3. - ISSN 0022-202X - p. 374 - 378.
cocultures - PAI-1 - PAI-2 - squamous carcinoma cells - u-PA - u-PA receptor
The plasminogen activator (PA)/plasmin system is thought to be involved in processes such as tumor invasion and wound healing, during which epithelial and mesenchymal cells come close together. However, information on regulation of the PA/plasmin system during epithelial-mesenchymal interactions is scarce. Therefore, we examined the in vitro modulation of the production and activity of the components of the PA/plasmin system in squamous carcinoma cells (SCC-4) and normal human keratinocytes in relation to cell density and the presence or absence of fibroblasts (3T3 cells). There was an inverse relation between cell density and mRNA expression for urokinase-type plasminogen activator (u-PA) and u-PA receptor in both SCC-4 cells and keratinocytes. In addition, such a relation was found for plasminogen-activator inhibitor types 1 (PAI-1) and 2 (PAI-2) in SCC-4 monocultures, but not in keratinocyte monocultures. In contrast to monocultures, variation of cell density did not affect the mRNA expression of the components of the PA/plasmin system in cocultures of SCC-4 cells or keratinocytes with 3T3 cells. However, the relative expression of mRNAs in co-cultures was clearly different from that in monocultures, especially at low cell density. For most of the components of the PA/plasmin system, a decrease in mRNA expression and u-PA receptor protein was observed at most cell densities, whereas for PAI-1 only in keratinocytes a marked increase was documented. Zymography of supernatants revealed that the levels of both free u-PA and PA-PAI were increased in SCC-4/3T3 co-cultures, whereas in keratinocyte/3T3 co-cultures, only levels of the PA-PAI complex were increased, while the amount of free u-PA activity decreased. This occurred despite the increased u-PA immunoreactivity and was probably caused by the markedly elevated levels of immunoreactive PAI-1. The results of the present study reveal that the production and synthesis of various components of the PA/plasmin system in keratinocytes and SCC-4 cells depend on the density of epithelial cells and are modulated by fibroblasts, probably through a direct cell-cell or cell-matrix contact. Fibroblast-induced modulations are similar in keratinocytes and SCC-4 cells except for the regulation of PAI-1, which is markedly enhanced only in keratinocytes. This suggests that the modulation of PA activity in the direct microenvironment may be different under physiologic and pathologic conditions.
Plasminogen activators are involved in keratinocyte and fibroblast migration in wounded cultures in vitro
Quax, P.H.A. ; Boxman, I.L.A. ; Kesteren, C.A.M. van; Verheijen, J.H. ; Ponec, M. - \ 1994
Fibrinolysis and Proteolysis 8 (1994)4. - ISSN 0268-9499 - p. 221 - 228.
During skin repair both keratinocytes and fibroblasts migrate into the wounded area. In the process of cell migration, controlled proteolytic degradation of the extracellular matrix occurs. It has been suggested that the plasminogen activator system is involved in such proteolytic processes occurring during wound healing. The role of plasmin, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) in keratinocyte and fibroblast migration in vitro was examined in the present study. Confluent cultures of normal human keratinocytes, human squamous cell carcinoma (SCC-4), SV40 transformed keratinocytes (SVK-14) and human fibroblasts were mechanically wounded and the repopulation of the denuded area was examined. Migration of cells into the denuded area could be inhibited by neutralizing antibodies against t-PA or u-PA, or the serine protease inhibitor Trasylol in all cell types studied. For detailed study on involvement of u-PA and t-PA in migration processes wounded cultures of SVK14 cells were used. Both t-PA and u-PA activity could be detected at the migrating edge of SVK14 cultures as revealed by zymography and by immunocytochemistry using polyclonal antibodies. Our results demonstrate the direct involvement of not only u-PA but also t-PA in migration of keratinocytes and fibroblasts in wounded cultures in vitro.
Modulation of IL-6 production and IL-1 activity by keratinocyte-fibroblast interaction
Boxman, Ingeborg ; Löwik, Clemens ; Aarden, Lucien ; Ponec, Maria - \ 1993
Journal of Investigative Dermatology 101 (1993)3. - ISSN 0022-202X - p. 316 - 324.
interleukin-6/interleukin-1/normal human keratinocytes/squamous carcinoma cells/fibroblast co-cultures
The present study was undertaken to investigate whether modulation of interleukin-6 and interleukin-1 production occurs owing to keratinocyte-fibroblast interaction. Normal human keratinocytes or squamous carcinoma cells were cultured either alone or in the presence of human foreskin fibroblasts or murine 3T3 cells. All cells tested produced interleukin-6, and interleukin-6 levels were markedly increased when normal or malignant keratinocytes were co-cultured with fibroblasts. The bioassay (species independent) and enzyme-linked immunosorbent assay (specific for human interleukin-6) together with use of complementary DNA probes specific for human or murine interleukin-6 revealed that fibroblasts are responsible for increased interleukin-6 levels. Moreover, interleukin-6 levels were increased when fibroblasts were cultured in conditioned media derived from normal human keratinocytes and squamous carcinoma cells-4 cultures. Interleukin-1α secreted by normal human keratinocytes and squamous carcinoma cells-4 cells was mainly responsible for the increased interleukin-6 production in fibroblasts. Although interleukin-1 activity increased linearly with the incubation time in squamous carcinoma cells-4 mono-cultures, interleukin-1 activity was low and remained unchanged in squamous carcinoma cells-4/3T3 co-cultures. Low interleukin-1 activity was most probably not due to inhibition of interleukin-1α production in squamous carcinoma cells-4/3T3 co-cultures because interleukin-lα messenger RNA expression in squamous carcinoma cells-4 cells remained unchanged in the presence of 3T3 cells. Furthermore, when 3T3 cells were incubated in conditioned medium derived from squamous carcinoma cells-4 cells, high interleukin-1 activity decreased to an undetectable level, suggesting that fibroblasts are involved in the suppression of interleukin-1 activity. The remaining interleukin-1 activity, however, was sufficient for maximal induction of interleukin-6 production in fibroblasts. These results suggest that the interaction between epithelial and mesenchymal cells is at least partly initiated by interleukin-lα secreted by the activated epithelial cell during skin injury or tumor invasion. Interleukin-1 in turn can induce modulation of the synthesis of various pro-inflammatory mediators and proteases in surrounding fibroblasts. An enhanced proteolytic activity and/or a possible induced production of an interleukin-1 inhibitor in fibroblasts and/or a receptor-mediated interleukin-1 consumption by fibroblasts will cause a decrease in interleukin-1 activity and thus exert a negative feedback.