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.

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.

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.

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.

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.

Transport of immune complexes from the subcapsular sinus into the lymph node follicles of the rat.

To study the mode of transport of immune complexes from the subcapsular sinus into the follicles of draining popliteal lymph nodes, horse radish peroxidase (HRP)-anti HRP was injected in rat footpads. Within six minutes, complexes were already present in the subcapsular sinus freely or attached to the plasma membrane of different types of cell including cells forming the stroma. A few minutes later, complexes were also seen in the deeper part of the outer cortex, and after two hours they had reached the periphery of the follicles. They were always seen scattered between lymphoid and non-lymphoid cells. After one day, complexes were present on well-developed follicular dendritic cells. After injection of HRP, no localization of this antigen was observed in the deeper part of the outer cortex including the follicles. These results strongly suggest that HRP-anti HRP complexes are passively transported through the outer cortex into the follicles where they are trapped and retained by follicular dendritic cells.

Relation between langerhans cells, veiled cells, and interdigitating cells.

Tissue macrophages are bone marrow derived mononuclear cells which play an important role in the immune response, especially as antigen presenting cells. They comprise a heterogeneous population of cells with phagocytic activity. On morphological functional and cytochemical criteria it is likely that the Langerhans cell (LC) in the epidermis, the veiled cell (VC) in the afferent lymph and the interdigitating cell (IDC) in the thymus dependent area of peripheral lymphoid organs and the thymus medulla belong to a subpopulation of the macrophages. They are low phagocytic, Ia positive and are highly immunogenic. VC and IDC may contain Birbeck granules, the characteristic organelles of the LC, suggesting a relationship between these cell types. An epithelial micro-environment as present in the skin epidermis and the thymus is necessary for the induction of these granules, which appear to have no immunological significance. In a scheme the development from monocyte into LC or into VC and subsequently IDC is postulated. Probably VC transport antigen from the skin area via the afferent lymphatics into the draining lymph node. In the thymus dependent area of this organ they present this antigen to T cells and mature into IDC. IDC in the medullary area of the thymus may also be involved in antigen presentation to immunocompetent T cells. However, in this central lymphoid organ a function in instruction of helper T cells may not be excluded.

Morphological and functional characteristics of rat steady state peritoneal dendritic cells.

Dendritic cells (DC) are present in lymphoid organs and also in many non-lymphoid tissues. In this study, DC in the steady state peritoneal cavity of rats were identified morphologically and functionally. Approximately 1% of the peritoneal cells are DC. On cytocentrifuge preparations these cells had the same characteristics as lymph node and spleen DC: they had an irregular outline, all were strongly MHC class II positive and had acid phosphatase activity in a spot in a juxtanuclear position. Also ultrastructurally, peritoneal DC were similar to DC isolated from lymph node and spleen. Enrichment of peritoneal DC, using overnight culture and a Nycodenz gradient, resulted in a highly purified DC fraction. Functionally, peritoneal DC appeared to be very potent antigen-presenting cells, far more potent than peritoneal macrophages, which had an inhibitory rather than an accessory function.

Induction of an increased number of dendritic cells in the peritoneal cavity of rats by intraperitoneal administration of Bacillus Calmette-Guérin.

Recently we described the presence of a small number of DC among the peritoneal cells of steady state rats. These DC had the same morphological characteristics and a similar antigen-presenting capacity as DC isolated from the spleen. This study shows that in the peritoneal cavity, which is a non-lymphoid microenvironment, the number of DC increases after i.p. administration of BCG. Next to this relatively small influx of DC, the approximately three-fold increase of the total number of cells is predominantly caused by an enormous influx of neutrophilic granulocytes, and to a lesser extent by an influx of macrophages. The phenotype and the antigen-presenting capacity of peritoneal DC has not changed, while the number of Ia-positive M phi has increased. Nevertheless, due to a suppressive effect of the peritoneal M phi, the total peritoneal cell suspension is no longer capable of presenting antigen.

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.

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.

Differential effect of cyclosporin application on epithelial cells of the rat thymus. Immunohistochemical study.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. A panel of monoclonal antibodies was used to study the phenotype of thymic epithelial cells. After treatment with CS, subcapsular epithelial cells, although phenotypically similar to medullary epithelial cells, were changed in a similar manner to phenotypically distinct epithelial cells of the deep cortex. These cells became enlarged, stockier and their cytoplasmic prolongations were thicker and coarser compared with control cells and their number was not decreased. In contrast, the number of medullary epithelial cells was markedly reduced, whereby the cells with the most mature phenotype (CK8+10-19- and CK8+10+19-) were the most prominently depleted. No proliferation of thymic epithelial cells was detected as monitored by incorporation of 5-bromodeoxyuridine.

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.