Development of a dermal matrix from glycerol preserved allogeneic skin.

Dermal substitutes can be used to improve the wound healing of deep burns when placed underneath expanded, thin autologous skin grafts. Such dermal matrix material can be derived from xenogeneic or human tissue. Antigenic structures, such as cells and hairs must be removed to avoid adverse inflammatory response after implantation. In this study, a cost-effective method using low concentrations of NaOH for the de-cellularization of human donor skin preserved in 85% glycerol is described. The donor skin was incubated into NaOH for different time periods; 2, 4, 6 or 8 weeks. These dermal matrix prototypes were analyzed using standard histology techniques. Functional tests were performed in a rat subcutaneous implant model and in a porcine transplantation model; the prototypes were placed in full thickness excision wounds covered with autologous skin grafts.An incubation period of 6 weeks was most optimal, longer periods caused damage to the collagen fibers. Elastin fibers were well preserved. All prototypes showed intact biocompatibility in the rat model by the presence of ingrowing blood vessels and fibroblasts at 4 weeks after implantation. An inflammatory response was observed in the prototypes that were treated for only 2 or 4 weeks with NaOH. The prototypes treated with 6 or 8 weeks NaOH were capable to reduce wound contraction in the porcine model. In neo-dermis of these wounds, elastin fibers derived from the prototype could be observed at 8 weeks after operation, surrounded by more random orientated collagen fibers. Thus, using this effective low cost method, a dermal matrix can be obtained from human donor skin. Further clinical studies will be performed to test this material for dermal substitution in deep (burn) wounds.

Immunology of skin transplantation.

Untreated viable allogeneic skin is highly immunogenic. Epidermal Langerhans migrate after transplantation out of the donor skin into the lymph node of the recipient where they can activate T cells capable to mediate rejection. Allogeneic skin is used as a temporary coverage of burn wounds, often in combination with autologous skin grafts. Several methods to pretreat the allogeneic skin have been used to delay the rejection process. Processing of allogeneic skin in 85% glycerol results in a non-viable skin with a well-preserved structure. Experiments in a full thickness porcine wound model showed that rejection of glycerol treated allogeneic skin grafts was up to six days delayed. Viable, untreated allogeneic skin grafts were rejected predominantly by CD8 positive T cells whereas in the glycerol treated grafts the influx of host cells was lower and the majority of the cells were macrophages. The outgrowth of the autologous skin grafts underneath glycerol treated allogeneic skin was three days earlier completed when compared to grafts in combination with untreated allogeneic skin. Thus, by processing the allogeneic skin into 85% glycerol, the direct route to induce graft rejection is blocked since the Langerhans cells are non-viable. The glycerol-preserved skin grafts are finally rejected via an indirect route mediated by macrophages; this process is less disturbing for the outgrowth of autologous cells.

Rat monocyte-derived dendritic cells function and migrate in the same way as isolated tissue dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells and are therefore useful to induce immune responses against tumor cells in patients. DC can be generated in vitro from monocytes using GM-CSF and IL-4, the so-called monocyte-derived DC (MoDC). To achieve antitumor responses, MoDC must be able to migrate to the draining lymph nodes after injection to induce cytotoxic T cells. Therefore, we studied migration of MoDC in a rat model. Functional rat MoDC were generated from PVG-RT7B rats and injected subcutaneously into PVG rats. These rat strains differ only at one epitope of the leukocyte-common antigen, which can be recognized by the antibody His 41. The advantage is that migrated cells can be detected in the draining lymph nodes by staining sections with His 41+; thus, migration is not influenced by labeling procedures. Rat MoDC migrated to the T-cell areas of the draining lymph nodes, just as isolated Langerhans cells or spleen DC do. In contrast, monocytes also migrated to the B-cell areas and the medulla.

Isolation of human skin dendritic cells by in vitro migration.

In the human skin, various types of antigen-presenting cells (APC) are present. In the epidermis, they are identified ultrastructurally as Langerhans cells (LC) by the presence of Birbeck granules. LC are considered to belong to the family of dendritic cells (DC) that are important for the initiation of immune responses (1). In the dermis, macrophages and DC are present (2,3). The expression of CD1a molecules can be used to identify DC in the skin (4,5), because macrophages do not express this marker. In vivo, these skin DC are supposed to take up antigens penetrating in the skin. Thereafter, they migrate via the afferent lymphatics into the draining lymph nodes, where a T-cell response can be initiated (6,7). During migration, the DC mature into potent APC. Besides an increase in MHC class II expression, adhesion (8,9) and B7 co-stimulatory molecules (10) are up-regulated. Most research on skin DC has been carried out with cells isolated from enzyme digested skin (8-10). In this chapter, we describe a method to obtain DC from human skin without enzymes, by making use of their migratory capacities. The cells migrate "spontaneously" out of the skin during culture. Characterization of the cells shows that mature DC are obtained with a marker expression not influenced by enzymes.

Migration of dendritic cells to the draining lymph node after allogeneic or congeneic rat skin transplantation.

BACKGROUND: After transplantation, donor dendritic cells (DC) migrating to the draining lymph node of the recipient are thought to play an important role in the initiation of graft rejection. In this study, we compared the in vivo migration of DC after allogeneic skin transplantation with that after congeneic skin transplantation. METHODS: A rat model was used with the PVG-RT7b rats as donor animals. These rats have leukocytes bearing an epitope of the leukocyte common antigen that can be recognized by the monoclonal antibody His 41. The cells of the allogeneic (ACI) and congeneic (PVG) recipient animals do not express this marker. RESULTS: In both recipient rat strains, graft-derived His 41+ DC could be detected in the T cell areas of the draining lymph nodes after skin transplantation. However, the number of migrated His 41+ cells present was lower in the allogeneic recipients. Similar results were obtained when skin DC isolated from the PVG-RT7b rats were injected subcutaneously into the hind footpads of allogeneic and congeneic recipients. Although the numbers of migrated His 41+ DC present were lower, the lymph nodes of the allogeneic recipients were much more enlarged and the grafts were rejected which did not occur in the congeneic recipients. CONCLUSIONS: The presence of donor-derived DC in the graft draining lymph nodes underlines the importance of the direct route of allo-activation. The lower numbers of migrated His 41+ DC in lymph nodes of allogeneic recipients may be the result of killing of the cells after presentation of the allo-antigens to the recipient T cells.

MHC class II and invariant chain biosynthesis and transport during maturation of human precursor dendritic cells.

Dendritic cells (DC) are highly potent activators of the immune response. The precise mechanisms that give rise to the DC phenotype are not known. To investigate the mechanisms that contribute to the generation of the DC phenotype, precursor DC were freshly isolated from human blood and allowed to mature in vitro. These matured DC showed the phenotypical and functional characteristics of DC. Analysis of the MHC class II and invariant chain (li) biosynthesis revealed that upon maturation, class II synthesis was induced whereas li synthesis was significantly up-regulated. In mature DC, despite the presence of large amounts of li, export of MHC class II molecules from the endoplasmic reticulum was incomplete, up to 4 h after biosynthesis. Thus, MHC class II-li synthesis and transport in DC is highly regulated during maturation of DC. Analysis of the regulatory mechanisms may contribute to a better understanding of antigen-presenting capacities during the differentiation of DC.

Immunogenicity of glycerol-preserved human cadaver skin in vitro.

Donor allograft skin preserved in 85% glycerol is used as a temporary coverage for large burn wounds. Glycerol treatment does not affect the structural integrity of the skin; cells are well preserved but dead. However, cells expressing major histocompatibility class II molecules can still be observed. In this study we investigated the mechanism underlying the clinical observation that glycerol-treated alloskin will be destroyed but after a prolonged period. We compared the in vitro immunogenicity of untreated and 85% glycerol-treated human skin cells. Human purified blood T cells did not proliferate when cultured with allogeneic treated skin cells, whereas untreated cells induced a distinct response. A moderate response was measured after adding T cells and viable antigen presenting cells, such as monocytes, to the allogeneic treated skin cells. However, the response on untreated skin cells was much higher. These results favor the suggestion that after transplantation of glycerol preserved skin is performed, an inflammatory process mediated by infiltrating host monocytes occurs rather than a rejection process mediated by T cells.

Migration of rat skin dendritic cells.

This study examines the in vivo migration of rat skin dendritic cells (including Langerhans cells) after skin transplantation. As donor animals, PVG-RT7b rats were used. The leukocytes of these rats bear an epitope of the leukocyte common antigen that can be recognized by use of the antibody His 41. The cells of allogeneic (ACI) recipient strains do not label with this antibody. Four days after transplantation of PVG-RT7b skin on allogeneic recipients, His 41+ cells showing a dendritic morphology were present in the T cell area of the draining lymph nodes. During culture of rat skin explants, dendritic cells migrated spontaneously into the medium. These in vitro migrated cells showed a high capacity to stimulate allogeneic T cells. When these cells, obtained from PVG-RT7b skin, were injected into the hind footpads of allogeneic recipients, they migrated to the same compartments of the draining lymph node. These data indicate that the cells that migrate from a transplanted allogeneic skin grafts are the same cells that migrate in vitro from explants. Most probably, they initiate graft rejection in the draining lymph nodes of the recipient.

Effect of low dose UVB irradiation on the migratory properties and functional capacities of human skin dendritic cells.

We recently described the 'spontaneous' migration of skin dendritic cells out of human split skin during culture. Since newly infiltrating cells from the circulation are excluded, this in vitro model is very suitable for studying the effect of UVB irradiation on the migratory properties, phenotype and functional capacities of skin cells. In the present study, we show that UVB irradiation of the skin before the culture period results in a significantly lower number of migrated cells that could be obtained compared with untreated skin. Relatively more dendritic cells of the population that migrated from UVB-irradiated skin were of dermal origin, as indicated by a higher percentage of CD1b+ cells. These data imply that UVB irradiation inhibits migration, especially of the epidermal Langerhans cells. Ultrastructural analysis of the irradiated skin revealed that the UVB dose used did not cause any directly visible damage to the cells. However, the cell population that had migrated from UVB-irradiated skin showed a significantly lower capacity to stimulate allogeneic T cells. This was not due to a lower expression of MHC class II on these cells. The percentage of cells expressing B7.1, B7.2 and LFA-3 was decreased in the population migrated from irradiated skin. The possible mechanism underlying the UVB-induced suppression is discussed.

Morphology of glycerol-preserved human cadaver skin.

Donor allograft skin preserved in 85 per cent glycerol has been used successfully as a temporary coverage for large burn wounds. The glycerol preservation is a method with low costs and has practical advantages such as antibacterial and virucidal effects. This report shows that the glycerol treatment did not affect the fundamental structural integrity of the skin. Intact keratinocytes and Langerhans cells with their characteristic Birbeck granules were still present in the glycerol-treated skin. After treatment with glycerol, the cells in the prepared epidermal cell suspensions were non-viable. MHC class II positive and CD1a positive cells could still be identified in situ and in the suspension.

Omental milky spots in the local immune response in the peritoneal cavity of rats.

BACKGROUND: Milky spots have been described as reactive structures, their classification varying from inflamed or haematopoietic tissue to lymphoid organs. In this study we investigated the reactivity of the milky spots in the omentum of rats upon induction of a chronic immune response in the peritoneal cavity. METHODS: At different time points after intraperitoneal administration of Bacillus Calmette-Guérin (BCG), a peritoneal lavage was made, and the omentum and the draining parathymic lymph nodes were taken out. The cellular composition of these tissues was examined on the light microscopic level, using a panel of monoclonal antibodies, and also by electron microscopy. RESULTS: During the first 4 months after administering BCG, the number and size of the milky spots increased enormously. Separate macrophage, T, and B cell areas were formed, but interdigitating cells and follicular dendritic cells were not observed. The number of cells in the peritoneal cavity also increased, and the cellular composition showed a strong similarity with that of the milky spots. Especially during the onset of the experiment, most bacteria were observed in the macrophages in the milky spots rather than in the draining lymph nodes. A cellular immune response was observed in the parathymic lymph nodes but not in the milky spots. CONCLUSIONS: Milky spots, either unstimulated or stimulated, should be classified as perivascular infiltrates. They play a role in the initial clearance of bacteria from the peritoneal cavity. Although the large increase in cell number is predominantly caused by immigration of cells, the results do support the role of milky spots as a site for local proliferation and maturation of especially macrophages and also B cells. The obtained data, however, do not support the earlier made assumption that milky spots function as a secondary lymphoid organ in the peritoneal cavity.

Migratory properties and functional capacities of human skin dendritic cells.

The different cell types which migrated 'spontaneously' out of human skin explants during different periods of culture were characterized. Before culture, CD1a+ dendritic cells were observed not only in the epidermis but also in the dermis, whereas CD1b+ dendritic cells were present exclusively in the dermis. The populations of migrating cells were harvested and phenotyped on 3 successive days of culture. They always contained high percentages of CD1a+ cells. The other cells that migrated were T cells and macrophages. A relatively high proportion of the CD1a+ cells that migrated during the first 24 h culture period was also CD1b+. The number of cells which were positive for both CD1a and CD1b decreased in the following 2 days of culture. However, the purified CD1a+ cell populations isolated on the 3 consecutive days did not show any difference in their capacity to stimulate allogeneic T cells. The CD1a+ cells possess potent allo-activating capacities that are independent of whether or not they are positive for CD1b+. Three days after culture about half of the CD1a+ cells were still present in the epidermis and dermis, but no CD1b+ cells could be detected in the dermis. This suggests that the CD1b+ cells represent a population of active migrating cells.

Localization and morphology of antigen-presenting cells in the adenoid of children with otitis media with effusion.

This study describes the localization of antigen-presenting cells (APC) in the different compartments in adenoids of children with otitis media with effusion (OME) and "healthy" children and adults. It is shown that the adenoid of children with OME contains a relatively high number of OKT6 and RFD1 positive cells. Moreover, accumulations of these cells are present in the extrafollicular areas of these adenoids. Very occasionally dendritic cells in the epithelial microenvironment contain Birbeck granules, indicating characteristic Langerhans cells. These OKT6 positive cells are not seen in the adenoids of the control group. Our results clearly show a relation between the presence of dendritic cells and the occurrence of OME. No differences are seen in localization and morphology of the APC in the studied adenoids.

Isolation and characterization of migratory human skin dendritic cells.

A method is described to isolate and characterize human skin dendritic cells (DC). This method is based on the migratory capacities of these cells. The cells migrated 'spontaneously' out of split-skin explants into the medium during a 24-h culture period and contained up to 75% CD1a+ cells. After removal of co-migrated T cells and macrophages, the highly enriched (> 95% CD1a+) DC showed potent allo-antigen-presenting capacities. About 25% of the CD1a+ cells were also positive for the dermal DC marker CD1b, whereas only 15-20% of the cells contained Birbeck granules, the characteristic cell organelle of the epidermal Langerhans cell. Before culture, CD1a+ DC were observed on cryostat sections not only in the epidermis but also in the dermis. After culture, the number of CD1a+ cells in both epidermis and dermis had decreased. Not all the cells had migrated during the culture period; some CD1a+ cells could still be detected in the epidermis and dermis after culture. Thus, using this method, potent allo-stimulating CD1a+ cells, migrating from both epidermis and dermis, can be obtained without the use of enzymes.

Ultrastructural study of macrophages of the rat thymus after cyclosporin treatment.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. After CS treatment thymic medulla virtually disappears and the thymus is almost entirely composed of cortical tissue. Macrophages are scattered throughout the thymic cortex. These cells are very large, rounded, with inconspicuous prolongations and euchromatic nucleus with prominent nucleoli. These cells are loaded with lipid bodies and vacuolar cytoplasmic inclusions of different size and diverse content, but very rarely contain phagocytosed lymphocyte remnants. The cytoplasm between inclusions has very active aspect with abundance of polyribosomes, granular endoplasmic reticulum and vesicles. Mitoses of lymphocytes in the vicinity of macrophages are frequently seen. We discuss the morphological similarity between cortical macrophages of CS-treated thymus and macrophages of cortico-medullary zone (CMZ) of the normal rat thymus, as well as functional significance of described morphological characteristics of this type of thymic macrophages, which probably reflect the metabolism of arachidonic acid.