ToxSci Advance Access originally published online on September 1, 2005
Toxicological Sciences 2006 90(2):549-557; doi:10.1093/toxsci/kfi306
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Characterization of the Initial Response of Engineered Human Skin to Sulfur Mustard
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* Department of Oral Biology and Pathology, School of Dental Medicine, SUNY at Stony Brook, Stony Brook, New York 11794-8702; US Army Medical Research and Material Command, Fort Detrick, Maryland, LifeCell Inc., One Millenium Way, Branchburg, New Jersey 08876; and Department of Dermatology, School of Medicine, SUNY at Stony Brook, Stony Brook, New York 11794
1 To whom correspondence should be addressed at present address: Department of Oral and Maxillofacial Pathology, Division of Cancer Biology and Tissue Engineering, Tufts University, School of Dental Medicine, Boston, MA 02111. Fax: (617) 636-2915. E-mail: jonathan.garlick{at}tufts.edu.
Received July 21, 2005; accepted August 24, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have used a new approach to identify early events in sulfur mustard-induced, cutaneous injury by exposing human, bioengineered tissues that mimic human skin to this agent to determine the morphologic, apoptotic, inflammatory, ultrastructural, and basement membrane alterations that lead to dermal-epidermal separation. We found distinct prevesication and post-vesication phases of tissue damage that were identified 6 and 24 h after sulfur mustard (SM) exposure, respectively. Prevesication (6 h) injury was restricted to small groups of basal keratinocytes that underwent apoptotic cell death independent of SM dose. Immunoreactivity for basement membrane proteins was preserved and basement membrane ultrastructure was intact 6 h after exposure. Dermal-epidermal separation was seen by the presence of microvesicles 24 h after SM exposure. This change was accompanied by the dose-dependent induction of apoptosis, focal loss of basement membrane immunoreactivity, increase in acute inflammatory cell infiltration, and ultrastructural evidence of altered basement membrane integrity. These studies provide important proof of concept that bioengineered, human skin demonstrates many alterations previously found in animal models of cutaneous SM injury. These findings further our understanding of mechanisms of SM-induced damage and can help development of new countermeasures designed to limit the morbidity and mortality caused by this chemical agent.
Key Words: sulfur mustard; basement membrane; bioengineered human skin; human keratinocytes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sulfur mustard (SM: 2,2'dichlorodiethyl sulfide) is an alkylating agent (Auerbach, 1949
; Heston, 1953; Papirmeister et al., 1985) that has cytotoxic and vesicant properties (Gross et al., 1985; Papirmeister et al., 1984b) and has been used as a chemical warfare agent. As this agent continues to pose a threat as a chemical weapon, tissue models used in skin biology can play an important role in further understanding the pathogenesis of SM-induced, cutaneous lesions and may help lead to the development of new approaches for the prophylaxis and treatment of these lesions. While its vesicant properties have been well-documented in human skin (Papirmeister et al., 1984a,b), mechanisms through which this agent induces blisters remain unclear (Smith, 1999). Although a sub-epidermal split through the lamina lucida is seen in SM injury (Papirmeister et al., 1984b), it is not known if this is an initiating site of vesication or a nonspecific, secondary response that occurs subsequent to injury at a different site. Epidermal targets for initiation of SM-induced blister formation in human skin remain to be determined.
Detailed mechanistic studies have not been possible in humans for obvious ethical reasons. This has limited our understanding of the initiation of SM injury in human skin. As a result, a variety of animal models have been the mainstay of research on the pathophysiology of cutaneous SM injury (reviewed in Smith, 1999). These studies have shown that early events in SM-induced skin injury include alteration of epidermal basal cells, disruption of hemidesmosomal attachment, and progressive inflammatory changes at the basement membrane (BM) zone. However, while all animal species used, including hairless guinea pig, weanling pig, mouse ear and hairless mouse demonstrate microvesicle formation, no clinical blister formation has been seen after SM exposure (Smith et al., 1997). Similarly, SM exposure to human skin grafted to nude mice has demonstrated several responses that were similar to those seen in human skin, but in the absence of clinical blister formation (Vogt et al., 1984). This may be explained by differences between human and animal epidermis such as elevated levels of basal cells proliferation, increased stromal and vascular changes, and differences in adnexal organization and structure. It is therefore important to develop models that more fully simulate the response of human skin to SM exposure.
A novel approach to identifying pathways initiating SM-induced vesication would be to use bioengineered, human skin that mimics the clinical and histologic features of this tissue in vivo. Biologically meaningful signaling pathways can function optimally to normalize growth and differentiation when cells are spatially organized with in vivo-like architectural features, but not when grown in two-dimensional cultures (Hagios et al., 1998; O'Brien et al., 2002). Over the last decade, the development of tissue-engineered models that mimic human skin, such as cultured, organotypic skin equivalents, have provided important experimental systems to study epidermal biology. By growing keratinocytes at an air-liquid interface on a collagen matrix populated with dermal fibroblasts, these tissues undergo full biochemical and morphologic differentiation and rapidly assembled structured BM (Andriani et al., 2003). When these cultures were grafted to nude mice as surface transplants, they underwent long-term engraftment, rapid normalization of keratinocyte growth, and demonstrated the presence of keratinocyte stem cells (Kolodka et al., 1998).
In the current study, we have used bioengineered human skin grafted to nude mice to establish dose/time responses of this tissue to SM exposure by characterizing morphologic, apoptotic, ultrastructural, inflammatory, and BM alterations in response to this agent. We have identified two distinct stages of epithelial damage that include an initial, prevesication stage and a subsequent stage resulting in dermal-epidermal separation. These sequential events are associated with characteristic apoptotic and BM changes that are linked to the initiation of vesication. These findings demonstrate that bioengineered human skin responds to SM in a manner that mimics cutaneous alterations previously found in animal studies. These novel human tissue models should greatly advance our understanding of functional mechanisms of SM injury and can lead to the development of new strategies that are designed to limit SM-induced skin injury.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Organotypic cell culture and in vivo transplantation of engineered skin.
Normal human epidermal keratinocytes (NHK) were cultured from newborn foreskin by the method of Rheinwald and Green (Rheinwald, 1989
) in keratinocyte medium described by Wu et al. (1982). Cultures were established through trypsinization of foreskin fragments and grown on irradiated 3T3 fibroblasts. Human dermal fibroblasts used to generate organotypic cultures were derived from newborn foreskins and grown in media containing Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal calf serum. Three-dimensional (3-D), organotypic cultures were prepared as previously described (Greenberg et al., 2005). Briefly, early passage human dermal fibroblasts were added to neutralized Type I collagen (Organogenesis, Canton, MA) to a final concentration of 2.5 x 104 cells/ml. Three ml of this mixture was added to each 35 mm well insert of a 6-well plate and incubated for 4 to 6 days in media containing DMEM and 10% fetal calf serum, until the collagen matrix showed no further shrinkage. At this time, a total of 5 x 105 cells were seeded on the contracted collagen gel. Cultures were maintained submerged in low calcium epidermal growth media for two days, submerged for two days in normal calcium epidermal growth media, and raised to the air-liquid interface by feeding from below with normal calcium cornification medium for an additional three days before grafting. Animal grafting was performed with all institutional approvals from Stony Brook's IACUC review committee as previously described (Kolodka et al., 1998). Six week-old male Swiss nude mice (N:NIHS-nuf DF, Taconic Farms, Germantown, NY) were anesthetized using Xylazine:Ketamine (3:2) and a 1.3 cm diameter of full-thickness dorsal skin was removed. Organotypic cultures were trimmed using a surgical punch 1.4 cm in diameter and were placed onto this area, covered with petrolatum Gauze (Sherwood Pharmaceuticals, St. Louis, MO) and secured with bandages (Baxter Scientific). Dressings were changed after 7 days and removed completely after 14 days. Grafted animals were sent to the Medical Research and Materiel Command in Aberdeen, MD, six weeks after grafting. Animal care was provided according to protocols approved by the IACUC Review Board of SUNY Stony Brook and approved by the US Army Medical Research and Materiel Command.
Sulfur mustard exposure of bioengineered human skin grafts.
Grafted nude mice were anesthetized and grafts exposed to SM vapor by wetting a disc of Whatman filter paper (#2) with 10 µl undiluted SM that was placed into a polyethylene cap (Columbia Diagnostics Inc, Springfield, VA). These vapor caps were placed over grafted human skin and adjacent mouse skin and were secured with double-sided tape. The dose of SM administered was established by varying the duration of exposure to SM vapor for 5, 8, 10, and 12 min. After removal of the caps, mice were placed in individual holding cages under a ventilation hood to allow elimination of any residual SM. At either 6 h or 24 h post-exposure times, mice were euthanized and skin grafts and adjacent mouse skin were removed with a dermatologic punch. Grafted animals were exposed in triplicate for each SM exposure time.
Analysis of apoptosis, morphologic alterations, and inflammatory cell infiltrate in SM exposed skin.
Skin specimens were fixed in 10% neutral buffered formalin and in situ TUNEL assay was performed using ISOL (In Situ Oligo Ligation) end-labeling and immunoperoxidase detection of apoptotic cells in paraffin sections (Roche). Numbers of apoptotic cells were calculated by counting 1000 nuclei in the basal and suprabasal layers present in four serial sections found 100 µm apart in each specimen. Percentages of TUNEL-positive basal and suprabasal keratinocytes were calculated for each SM dose at both 6 h or 24 h after exposure. For analysis of tissue morphology, paraffin-embedded tissues were cut as 4 µm sections and stained with hematoxylin and eosin. Identification of inflammatory cell infiltrates in SM-exposed skin was studied in serial sections stained with Hematoxylin and Eosin and Giemsa stain. The distribution of the predominant inflammatory cell type, polymorphonuclear leukocytes (PMN), was determined in grafts 6 h or 24 h after SM exposure. Duplicate tissues were studied at each of these timepoints for 5, 8, 10, and 12 min exposures.
Immunohistochemical detection of BM proteins.
Specimens were frozen in embedding media (Triangle Biomedical, Durham, NC) in liquid nitrogen vapors after being placed in 2M sucrose for 2 h at 4°C. Tissues were serial sectioned at 6 µm and mounted onto gelatin-chrome alum-coated slides. Tissue sections were washed with PBS and blocked with 10 µg/ml goat IgG, 0.05% goat serum, and 0.2% BSA, vol/vol in PBS without fixation. Sections were incubated with monoclonal antibody to Type VII collagen (Sigma, St. Louis, MO) that was detected with Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR). Laminin 5 was detected using the GB-3 monoclonal antibody (Matsui et al., 1995) directed against the intact heterotrimeric molecule (gift of Dr. G. Meneguzzi). Slides were coverslipped with Vectashield containing 1 µg/ml DAPI (Vector Laboratories) and fluorescence was visualized using a Nikon Eclipse microscope and double exposure photomicroscopy was performed using FITC and Texas Red filters.
Transmission electron microscopy.
SM-exposed grafts and controls were cut into small pieces of approximately 2 x 2 mm and fixed in 2% glutaraldehyde in 0.1 M cacodylate and 0.1 M sucrose at pH 7.2. The samples were then post-fixed in 2% osmium tetroxide in 0.1 M cacodylate and 1% tannic acid in 0.1 M cacodylate. Samples were dehydrated in graded ethanol, cleared with propylene oxide, and infiltrated with Spurr's resin. Following polymerization of the resin, thick sections were produced using a Reichert Ultracut E microtome and sections were stained with toluidine blue to determine orientation. The blocks were then thin sectioned at approximately 90 nm and mounted on copper grids. Grids were stained with 5% Uranyl acetate in deionized water and Reynold's lead citrate. Stained grids were examined at various magnifications using a Hitachi H-600 transmission electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prevesication injury seen 6 h after SM exposure was limited to focal groups of basal keratinocytes while dermal-epidermal separation was seen only 24 h after SM exposure.
In order to determine the SM dose at which epithelial damage would occur, preliminary experiments were conducted to establish the length of vapor exposure and times after exposure that could induce structural alterations in grafted human skin. It was found that grafts exposed to SM for 2 min yielded little epithelial damage, while 5 min exposures produced alterations limited to the basal cell layer that were characterized by the presence of pyknotic nuclei (data not shown). Based on these findings, grafts were exposed to SM for either 5, 8, 10, or 12 min and animals were sacrificed 6 or 24 h after exposure so that early events leading to SM-injury could be monitored. When the morphology of SM-exposed grafts was analyzed by Hematoxylin and Eosin staining 6 h after SM exposure, tissues demonstrated alterations confined to small numbers of cells in the basal layer while suprabasal layers showed normal tissue architecture (Fig. 1). In addition, no morphologic alterations or vesication was seen at the dermal-epidermal junction at this time. SM-induced cellular alterations were characterized by pyknotic nuclei limited to small cell clusters and individual cells in the basal layer, thus demonstrating a selectivity of SM damage to focal subsets of cells in the basal layer. These nuclear changes seen 6 h after exposure were similar for 8 min (Fig. 1A), 10 min (Fig. 1B), and 12 min (Fig. 1C) exposure times, showing that these early changes were independent of SM dose. In contrast, control grafts demonstrated normal tissue architecture and morphology (Fig. 1G). These findings showed that prevesication damage was limited to the basal layer of the epithelium and involved only small numbers of basal cells. This suggested that only subpopulations of basal cells were susceptible to the intitial injury induced by SM, while other cells in this layer were resistant to these effects.
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Grafts examined 24 h after SM exposures showed significantly greater tissue damage. These changes were seen after dose/exposure times as short as 5 min and were characterized by large numbers of pyknotic nuclei in basal and suprabasal layers (Fig. 1D). In addition, some basal cells showed ballooning degeneration and initiation of separation from the BM interface 24 h after a relatively short, 5-min SM exposure (Fig. 1D). Upon longer SM exposures of 8 min (Fig. 1E) and 10 min (Fig. 1F), areas of damage at the dermal-epidermal junction were characterized by the presence of acantholytic cells and complete separation along the BM zone to form well-defined microvesicles. In addition, these longer exposure times were correlated with a dose-dependent increase in damage to suprabasal cells and thinning of the epithelium. These findings demonstrated two distinct stages of SM-induced injury. An initiating, prevesication stage that was seen 6 h after SM exposure and resulted in very limited and focal damage to small numbers of basal cells without altering the integrity of the BM zone. Longer post-exposure latencies were required to induce microvesication at the dermal-epidermal interface as seen 24 h after SM exposure.
Apoptotic cell death was not dependent on SM dose and was restricted to basal cells during the prevesication stage, but was dose-dependent at the post-vesication stage.
SM-exposed grafts were analyzed by in situ TUNEL assay (ISOL) to determine indices of apoptotic cell death. This was measured by calculating both the percentage of TUNEL-positive cells seen in all layers of the tissue as well as the percentage of basal cells that were TUNEL-positive (Fig. 2). Roughly 5% of basal cells were TUNEL-positive 6 h after an 8 min SM exposure and percentages of apoptotic cells did not increase with elevated length of SM exposures (10 and 12 min) when measured 6 h after SM exposure. The finding that only a consistent number of basal cells had undergone apoptosis 6 h after exposure indicated that a limited number of cells were susceptible to initiating events during the prevesication stage of SM damage. In contrast, a dose-dependent increase in the percentage of TUNEL-positive basal cells was seen when determined 24 h after SM exposure. In addition, the percentage of TUNEL-positive cells in the entire tissue seen 24 h after SM exposure increased from 5% for SM exposures of 5 min, 15% for exposures of 8 min and 35% for 10 min exposures. Apoptotic cells seen after 5 min exposures were found only in the basal cell layer, as seen by the superimposition of the percentage of apoptotic cells at this exposure time (Fig. 2), and were not accompanied by vesication. In contrast, most apoptotic cells seen after 8 and 10 min exposures were found in the suprabasal layers and were associated with a split at the dermal-epidermal interface. The fact that a small number of apoptotic basal cells were seen 6 h after SM exposure and a significantly greater number of apoptotic cells was present 24 h after SM exposure suggests that only a limited number of basal cells were targeted by SM and underwent an apoptotic response upon the initiation of SM injury.
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) and 24 h (
) after SM exposures while the percentage of total numbers of apoptotic cells in the entire tissue was calculated after 6 h (
) and 24 h (•). Only one point is seen at 5 min exposure due to the presence of apoptotic cells only in the basal layer.Localization of BM proteins was preserved only during the prevesication stage of tissue injury.
Immunohistochemical stains for BM proteins laminin 5 and Type VII collagen were performed on SM-exposed grafts in order to determine if the distribution of these proteins was altered during the prevesication and post-vesication stages. Six h after SM injury, grafts exposed for 8 min or 12 min did not demonstrate any disruption of laminin 5 (Figs. 3A and 3B) or Type VII collagen staining (Figs. 3E and 3F) as seen by the linear and continuous pattern of immunoreactivity that was identical to vehicle-exposed, control grafts for laminin 5 (Fig. 3D) and Type VII collagen (Fig. 3H). These findings showed that immunohistochemical localization of BM proteins was preserved during the prevesication stage in spite of the apoptotic cell death seen in basal cells 6 h after exposure. In contrast, SM exposure of 8 min were sufficient to lead to a discontinuous pattern of laminin 5 (Fig. 3C, arrow) and Type VII Collagen (Fig. 3G, arrow) when determined 24 h after SM exposure. These results showed that prevesication changes, including the initial apoptotic response in selected basal cells, occurred before the onset of significant BM disruption. Interestingly, the distribution of these proteins was only focally-disrupted even after vesication (Figs. 3C and 3G), demonstrating that immunoreactivity for laminin 5 and Type VII collagen was generally preserved even after loss of epithelial attachment had occurred. These results suggested that preserved BM proteins could still play a role in mediating the initial events in SM-injury seen 6 h after SM exposure.
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A dose-and time-dependent increase in acute inflammation is linked to SM exposure.
Infiltration of inflammatory cells into the dermis demonstrated a dose- and time-dependency following SM exposure. The predominant cell type seen was polymorphonuclear leukocytes (PMN), and this was the only inflammatory cell seen 6 h after SM exposure. After a 6 min SM exposure at this time, PMNs were confined to the vasculature in the lower dermis as no evidence of extravasation was seen. This was in contrast to the infiltration of PMNs seen 6 h after an 8 min SM exposure, where clusters of PMNs demonstrated margination, diapedesis, and extravasation. Most of this infiltrate was seen in post-capillary venules in the periphery of the grafted epithelium in the middle to lower part of the dermis. No PMNs were seen in the upper dermis or along the BM interface at this time. Infiltration of PMNs was seen throughout the dermis 6 h after a 10 min SM exposure. While PMNs were seen near the BM zone, there was no evidence of their infiltration into the epithelium at this timepoint. In contrast, 12 min SM exposure was linked to the infiltration of PMNs into the epithelium and a dense PMN infiltration throughout the dermis. Thus, the prevesication stage of SM exposure showed a dose-dependent increase in PMN infiltration, as increased SM exposure time was associated with an increase in the density of PMNs after 6 h. When studied 24 h after 6, 8, and 10 min SM exposures, all tissues demonstrated focal PMN infiltration of the epithelium. These focal infiltrates were associated with loss of cell-cell adhesion in basal and suprabasal cells but were not linked to vesication after 6 and 8 min exposures. In contrast, focal aggregates of PMNs found after 10 min exposures to SM were associated with areas of vesication. Small numbers of perivascular lymphocytes were first seen at this timepoint. These findings demonstrated that temporal and spatial changes in the infiltration of PMNs in response to SM exposure were linked to the specific stage of SM injury.
Ultrastructural analysis demonstrates distinct pre-vesication and post-vesication alterations.
Ultrastructural studies demonstrated distinct prevesication (Figs. 4A and 4B) and post-vesication (Figs. 4C, 4D, 4E, and 4F) changes that correlated with morphologic, apoptotic, and immunohistochemical findings described above. Minimal alterations were seen 6 h after an 8 min SM exposure in the basal layer (Fig. 4A). These changes included nuclear alterations characterized by condensation and margination of heterochromatin at the nuclear membrane in basal cells (Fig. 4A, arrows). Cell-cell and cell-stroma adhesion were not disturbed as large numbers of intact desmosomes (Fig. 4A) and hemidesmosomes (Fig. 4B) were seen at this time. No vesication at the dermal-epidermal interface was identified and intact lamina densa, lamina lucida, hemidesmosomal plaques (Fig. 4B) and anchoring fibrils (Fig. 4B, inset) were present along the dermal-epidermal interface, demonstrating that BM structure was not disturbed 6 h after exposure. In contrast, significant cellular and BM alterations were seen 24 h after SM exposure and were characterized by disruption of tissue architecture that included breakage of desmosomes (Fig. 4C). Apoptotic cells showed progressive stages of nuclear alterations including margination of heterochromatin (Fig. 4C, longest arrow) and varying degrees of nuclear pyknosis characterized by partial nuclear fragmentation (Fig. 4C, medium arrow) and complete nuclear pyknosis (Fig. 4C, short arrow). These pyknotic cells demonstrated no perinuclear intermediate filaments and were devoid of cytoplasmic structures. Cytoplasmic changes were even more clearly seen in cells undergoing separation from the BM zone. Individual basal cells showed a focal loss of attachment that led to the initiation of microvesicles as cells began to detach from the BM zone (Fig. 4D). These cells showed a gradation from a dense, structureless cytoplasm (Fig. 4D, star) to one that was pale and rarified (Fig. 4D, asterisks). This cytoplasmic rarification was found adjacent to early microvesicle formation as the cytoplasm underwent degradation adjacent to the BM (Fig. 4E, right side). BM beneath these areas of rarification was destroyed, while BM structure was better preserved immediately adjacent to these areas in spite of significant cytoplasmic alterations (Fig. 4E, inset). These findings suggested that initial damage induced by SM seen 6 h after SM exposure was intracellular and that BM damage occurred subsequent to these events. The presence of patches of residual BM structure seen 24 h after SM exposure are in agreement with the immunohistochemical pattern of BM proteins described above. Finally, microvesication was identified by clear spaces that completely separated the epithelium from the underlying BM zone (Fig. 4F, asterisks). Remnants of electron-dense, BM structure were seen just below these clear spaces while cells just above this space demonstrated nuclear pyknosis and cytoplasmic vacuolization. Taken together, the ultrastructural appearance 24 h after SM exposure demonstrated the advanced stages of cellular apoptosis characterized by nuclear and cytoplasmic degradation, disruption of BM integrity, and the initial stages of microvesicle formation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For ethical reasons, it is not possible to perform in vivo experiments in humans to further understand mechanisms of action of the cutaneous response to SM exposure. An important approach to identifying molecular and biochemical pathways of SM-induced injury would therefore be to use engineered human skin that mimics the in vivo features of this tissue. We have studied the early response of bioengineered human skin to SM and have characterized two distinct phases of tissue damage upon SM exposure, including an initiating, prevesication phase and a subsequent vesication phase that demonstrates early dermal-epidermal separation. These sequential events were very similar to findings reported in a variety of animal models (Mershon et al., 1990
; Smith, 1999) and show the suitability of engineered human skin to further our understanding of mechanisms of SM injury.
It is of particular importance to understand the initial events in SM injury that occur shortly after cutaneous exposure to this vesicating agent. We observed that the prevesication phase of tissue damage was characterized by subsets of basal cells targeted for cell death, as seen by the presence of clusters of apoptotic basal cells 6 h after SM. The finding that the earliest, apoptotic cell death was restricted both to the basal layer and to selected foci of cells within this layer suggests that a limited number of basal cells were susceptible to SM damage while other basal cells could survive initial SM exposure. This raises the question of which basal cell population was being targeted by SM. It has been shown that SM can selectively target and inactivate cells previously "initiated" during two-stage mouse skin carcinogenesis (De Young et al., 1977). Since SM could specifically target and eliminate these early progenitor cells, it is possible that this basal cell population was specifically susceptible to initial SM damage in our studies, as well. We have also found that most basal cells were resistant to initiating damage induced by SM exposure. It has previously been shown that extracellular matrix engagement by basal epithelia results in a reduced susceptibility to apoptosis (Dowling et al., 1996; Weaver et al., 2002). Furthermore, it is known that disruption of cell polarity and the concomitant loss of ß4-integrin signaling can sensitize epithelial cells to apoptotic cell death (Dowling et al., 1996). It is possible that the loss of hemidesmosomal attachment known to occur during early SM injury (Werrlein et al., 2003) may act in a similar manner to render cells susceptible to apoptosis. In this light, BM proteins may be involved directly in the induction of SM-induced lesion initiation.
Our findings indicate that targeted apoptotic cell death is an early event that is induced shortly after SM exposure and precedes dermal-epidermal separation. It has previously been shown that basal keratinocytes in grafted neonatal foreskins and hairless guinea pig skin demonstrate nuclear pyknosis that precedes the appearance of blister formation after SM exposure (Petrali and Oglesby-Megee, 1997). Ultrastructural alterations and focal clusters of TUNEL-positive, basal cells that are identical to those shown in our study have been observed 6 h after SM exposure using a hairless guinea pig model (J. Petrali, personal communication). Our finding of numerous apoptotic cells throughout the tissue 24 h after SM exposure showed that additional apoptotic events occurred during the period of latency leading to epidermal-dermal separation. SM is now known to induce caspase-mediated apoptotic cell death through Fas/TNF receptor-mediated pathways that are activated when cultured keratinocytes and human skin grafted to nude mice were exposed to SM (Rosenthal et al., 1998, 2003). In addition, these studies have shown that human keratinocytes can be protected from SM-induced apoptosis by expressing a dominant-negative form of Fas-associated death domain. While the in situ TUNEL assay was used to measure numbers of apoptotic cells, it is possible that SM also induced necrosis as an alternative form of cell death. This is likely to be the case since SM exposure induced morphologic nuclear features, such as nuclear condensation and margination, that are not unique to apoptosis and can be seen in cells undergoing necrotic cell death as well (Leist and Jäättelä, 2001). Although TUNEL stain is specific for apoptotic cells, our morphologic evidence of leterodromatin magination is similar to that seen in cells that are caspare-deficient and apoptosis-incompetent (Leist and Jäättelä, 2001). Thus, although a large majority of cells in our model system have undergone apoptosis, an alternative pathway of necrotic cell death cannot be ruled out. However, more definite determination of the nature of cell death pathways, such as through caspare activation, will need to be established in future studies. Together with previous animal studies, our results demonstrate that apoptotic cell death was an important early event necessary to initiate tissue alterations but was not sufficient to induce immediate microvesication 6 h after SM exposure.
Changes in BM integrity have previously been identified in SM-induced lesions, but it remains unclear if these alterations are directly involved in the initiation of blister formation or are the consequence of it (Papirmeister et al., 1984b). Vesicating lesions have shown a split through the lamina lucida so that intact hemidesmosomal components and attached anchoring filaments form the roof of the blister and the lamina densa form its base (Papirmeister et al., 1984b). More recent studies have demonstrated that low levels of SM vapor exposure induced microblisters in animal models without marked epidermal necrosis or inflammatory infiltrate (Smith, 1999), thus implicating changes in BM integrity as being associated with SM-induced blistering. However, when human skin explants (Smith, 1999; Smith et al., 1998) and Yorkshire pigs (Smith et al., 1997) were exposed to SM, a decrease in laminin 5 reactivity was seen shortly after exposure, while Type IV and VII collagens were preserved. We have determined that the immunoreactivity of these BM components was intact during the prevesication stage (6 h) but was altered upon vesication (24 h). Our findings of preserved immunoreactivity of laminin 5 and Type VII collagen during the prevesication stage of blister pathogenesis support previous findings in hairless guinea pigs (Petrali and Oglesby-Megee, 1997) and suggest that intact BM proteins may play a role in mediating early changes in SM injury. Recent studies using multiphoton microscopy of SM-treated human epidermal keratinocyte cultures have shown that SM-induced alkylation disrupts 
6ß4 integrin-laminin 5-mediated adhesion and leads to destabilization of the K14 cytoskeleton (Werrlein and Madren-Whalley, 2003). Taken together with our findings, these studies suggest that loss of BM-mediated adhesion is likely to be associated with the initiating mechanism of SM injury.
It has previously been shown that cutaneous injury in response to SM is associated with an increased inflammatory infiltrate in the dermis (Petrali and Oglesby-Megee, 1997; Vogt et al., 1984). Similarly, we have shown that an acute inflammatory infiltrate, consisting almost entirely of PMNs, demonstrated a dose-dependent elevation both 6 and 24 h after SM exposure. While early, prevesication changes were limited to a perivascular and dermal distribution, infiltration of the epithelium was seen during the vesication stage. Previous studies have demonstrated that inflammatory infiltrate varied greatly between animal species. In rabbits, PMN infiltration was seen shortly (1 h) after SM exposure at the BM interface (Vogt et al., 1984). In contrast, leukocyte infiltrate seen shortly after SM exposure was predominantly composed of basophils in guinea pig skin. While PMN infiltration was most common at later timepoints, very early changes in inflammatory infiltrate have been linked to an immediate increase in vascular permeability in capillaries in the uppermost dermis upon SM exposure (Vogt et al., 1984). More recently, it has been shown that increased expression of inflammatory cytokines, including IL-1B, GM-CSF, IL-6, and IL-1 was demonstrated shortly after SM exposure (Sabourin et al., 2000). This demonstrated that the release of these inflammatory mediators in response to SM occurred before SM-induced blister formation. The effect of these inflammatory mediators and the PMN infiltrate that we describe on the initiation of vesication at the BM will require further study with our models.
The development of tissue-engineered models that mimic human skin has provided novel experimental systems to study the behavior of normal and altered human stratified squamous epithelium. When these three-dimensional cultures were grafted to nude mice, we have previously shown that the resulting epithelium include features of human skin such as normalized tissue architecture, ultrastructural evidence of intact BM (Andriani et al., 2003), and the presence of stem cells in epidermal proliferation units (Kolodka et al., 1998). In this light, these in vivo tissues provide an advantage over previous efforts to use reconstructed, epidermis generated in vitro (Petrali et al., 1993). These in vitro models do not assemble intact BM and their application has been limited as a result. In the current study, we have demonstrated that the response of these in vivo tissues to SM bears a remarkable resemblance to previous animal models used in SM experiments. We describe ultrastructurally distinct prevesication alterations that were very similar to those seen 6 h after SM exposure in the hairless guinea pig model (Smith, 1999). In addition, our findings of microvesicles demarcated by degenerating basal cells at their roof and remnants of the lamina densa at their floor are similar to those described in human skin grafted to nude mice exposed to SM (Papirmeister et al., 1984b). The subepidermal microblister seen in the human tissue model presented in our report appear similar to those reported in the most commonly used animal models (Vogt et al., 1984). However, as is the case in these animal models, we do not see the presence of fluid-filled blisters that occur upon exposure of human skin to SM. This may be explained by differences between human and animal epidermis such as elevated levels of basal cells proliferation, increased stromal and vascular changes, and differences in adnexal organization and structure. However, due to the variable sensitivity of different animals to cellular necrosis and apoptosis it is necessary to search for models that more closely mimic the response of human skin to SM (Smith, 1999). The human tissue models described above may provide a new opportunity to study SM injury in tissues that more closely mimic the response of human skin to this agent.
A more complete understanding of the molecular and pathogenic mechanisms of vesication by SM is critical to the development of new countermeasures to control skin blistering induced by this agent. Since there is no current effective therapy for vesicant-induced injury of human skin, approaches to limit this debilitating injury need to be designed to protect skin to prevent this injury or to limit the extent of tissue damage. Since our findings and previous studies have shown that a short period of latency precedes dermal-epidermal separation, this interval would be an important window to protect skin from ensuing damage by developing new strategies to attenuate and modify the occurrence of these lesions. By using bioengineered human skin, it is now possible to further study mechanisms through which SM-induced, cutaneous lesions develop, and to test strategies designed to minimize or prevent their development.
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ACKNOWLEDGMENTS |
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We would like to thank Lina Nguyen, Taly Argov, Marc Mendelsohn, Jennifer Landmann, Louis Bertalloti, and Larry Pfeiffer for their technical assistance and Drs. Rebecca Morris and Lorne Taichman for critical comments. This work was supported by the U.S. Army Medical Research and Materiel Command under Grant DAMD17-01-1-0688.
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REFERENCES |
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