Comparative assessment of commercially available wound gels in ex vivo human skin reveals major differences in immune response-modulatory effects.

Wound healing is a crucial process for maintaining the function of human skin as a protective barrier to pathogens and other external stress factors. Hydrogels-in combination with antimicrobials-are often used, as moist wound care has been widely accepted as standard therapy. Recently, we reported about immune response-modulatory effects of an octenidine-based hydrogel, however little is known about the mechanism of action of other hydrogels including antiseptic molecules or chlorine-based and chlorine-releasing agents, respectively. The aim of this study was the comparative assessment of commercially available wound gels (octenilin®, Prontosan®, Lavanid®, Betadona®, ActiMaris®, Microdacyn60®, VeriforteTMmed) with regard to their effects on the secretion of distinct cytokines (IL-6, IL-8, IL-10), matrix-metalloproteinases as well as their potential to cause alterations in skin structure and apoptosis. Hence, tape-stripped human ex vivo skin biopsies were treated topically with wound gels and cultured for 48 h. Enzyme-linked immunosorbent assays and an enzyme activity assay of culture supernatants revealed that octenilin® demonstrates significantly broader anti-inflammatory and protease-inhibitory capacities than other wound gels. Further, haematoxylin & eosin as well as caspase-3 staining of treated biopsies showed that octenilin® does not alter skin morphology and shows the least interfering effect on human epidermal cells compared to untreated controls. Overall, this study clearly demonstrates totally different effects for several commercially available hydrogels in our wound model, which gives also new insight into their tissue compatibility and mode of action.

Fetal and neonatal murine skin harbors Langerhans cell precursors.

Resident epidermal Langerhans cells (LC) in adult mice express ADPase, major histocompatibility complex (MHC) class II, and CD205 and CD207 molecules, while the first dendritic leukocytes that colonize the fetal and newborn epidermis are only ADPase(+). In this study, we tested whether dendritic epidermal leukocytes (DEL) are end-stage cells or represent LC precursors. In epidermal sheets of fetal and neonatal mice, we found no apoptotic leukocytes, suggesting that these cells do not die in situ. To address whether DEL can give rise to LC, sorted DEL from murine newborn skin were cultured with cytokines used to generate LC from human CD34(+) precursors. After 7-14 days, DEL proliferated and acquired the morphology and phenotype of cells reminiscent of LC. In concordance with this finding, we show that neonatal epidermis harbors 10-20 times the number of cycling MHC class II(+) leukocytes as adult tissue. To test whether LC can differentiate from skin precursors in vivo, we developed a transplantation model. As it was impossible to transplant fetal epidermis, whole fetal skin was grafted onto adult severe combined immunodeficient mice. As opposed to the uniform absence of donor LC at the time of transplantation, examination of the epidermis from the grafts after 2-4 weeks revealed MHC class II(+) donor cells, which had acquired CD205 and CD207, thus qualifying them as LC. Finally, we present evidence that endogenous LC persist in skin grafts for the observation period of 45 days. These studies show that hematopoietic precursors seed the skin during embryonic life and can give rise to LC.

MHC class I+/II- dendritic cells induce hapten-specific immune responses in vitro and in vivo.

Activation requirements and biologic properties of hapten-specific, major histocompatibility complex class I-restricted CD8+ T lymphocytes are not fully understood. To address this issue, a novel CD45+/major histocompatibility complex class I+ (H-2k)/II-/CD80+ dendritic cell line, termed 80/1, which is capable of stimulating naïve, allogeneic CD8+ but not CD4+ T cells in vitro, was derivatized with trinitrobenzenesulfonic acid and co-cultured for 4 d with syngeneic, naïve CD8+ T cells. Results obtained showed that trinitrophenyl-derivatized, but not underivatized 80/1 dendritic cells, can induce vigorous proliferation ofCD8+ T cells. T-cell blasts generated in this fashion were able to lyse syngeneic, trinitrophenyl-derivatized targets but failed to lyse underivatized or trinitrophenyl-derivatized syngeneic, major histocompatibility complex class I- mutant cells or allogeneic targets. The ability of 80/1 dendritic cells to prime naïve, syngeneic T cells in vivo was tested in a contact hypersensitivity model. C3H/HeN mice were injected subcutaneously with identical numbers of (i) trinitrophenyl-derivatized 80/1 dendritic cells; (ii) trinitrophenyl-derivatized 80/1 dendritic cells fragmented by freeze-thawing cycles; (iii) trinitrophenyl-derivatized fibrosarcoma L929; and (iv) trinitrophenyl-derivatized lymphoma R1.1 cells. Whereas live trinitrophenyl-derivatized 80/1 dendritic cells were able to sensitize for contact hypersensitivity, killed hapten-derivatized 80/1 dendritic cells or control cells failed to do so. Thus, we conclude that 80/1 dendritic cells, when compared with major histocompatibility complex class I+ non-dendritic cells, can effectively prime naïve, syngeneic CD8+ T cells for hapten-specific responses, probably due to their better costimulatory and migratory properties.

Erythema multiforme associated human autoantibodies against desmoplakin I and II: biochemical characterization and passive transfer studies into newborn mice.

The demonstration of circulating autoantibodies directed against the constitutive desmosomal plaque proteins desmoplakin (dp) I and II in mucocutaneous lesions in a subset of patients with erythema multiforme major, suggests that humoral immune mechanisms may play a role in the pathogenesis of this severe skin disease. In this study we identified a specific peptide sequence--YSYSYS--representing an antigenic binding site for the human autoantibodies. This epitope is localized at the extreme carboxy terminal domain of dp thought to be responsible for the assembly of keratin filaments with desmosomes. To test the possibility whether these antibodies may exert any pathologic effects in vivo, human autoantibodies were affinity purified on a corresponding synthetic peptide matrix and peptide-specific antibodies were raised in rabbits. After repeated subcutaneous injections into newborn mice, affinity-purified human autoantibodies and anti-peptide rabbit IgG were detected on desmosomal plaques of keratinocytes overlying the injection site. Histologic and electron microscopic examinations showed hydropic degeneration of basal and suprabasal keratinocytes, dyskeratosis, signs of suprabasal acantholysis, and keratin filaments detached from the desmosomal plaques clumping around the nucleus. We demonstrate that autoantibodies are directed to an epitope within a dp domain crucial for the interaction of keratin filaments with desmosomes, and, when injected subcutaneously into newborn mice, produce pathologic changes. These findings imply that autoantibodies to dp could impair the function of desmosome-keratin filament complexes suggesting a pathogenic role in vivo.

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice.

It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle.

Vitamin D receptor ablation alters skin architecture and homeostasis of dendritic epidermal T cells.

BACKGROUND: 1alpha,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)], the active metabolite of vitamin D, exerts its activities by binding to the vitamin D receptor (VDR) with subsequent function as a transcription factor. Targeted ablation of the VDR in mice results in rickets and alopecia. OBJECTIVES: To study the consequences of VDR deficiency for skin physiology, and to investigate the mechanisms of the immunosuppressive effect of 1,25(OH)(2)D(3) on LC. METHODS: We studied the structural, phenotypic and functional properties of skin and individual skin leucocyte populations in VDR(-/-) mice. RESULTS: The lack of VDR induced a wide spectrum of pathologies including dermal deposition of collagen, enlargement of sebaceous glands, dilation of the hair follicles, development of epidermal cysts, increased numbers of dendritic epidermal T cells (DETC) and hyperkeratosis. Ageing aggravated these changes. Intriguingly, Langerhans cells (LC) were indistinguishable in distribution, morphology and number compared with controls. In vitro, LC underwent a maturation/migration process similar to LC from control mice. Pretreatment of epidermal cells or LC-enriched epidermal cell suspensions with 1,25(OH)(2)D(3) impaired LC maturation and T-cell stimulatory capacity from VDR(+/+) but not VDR(-/-) mice, demonstrating that LC are targets of vitamin D(3) and that interaction between vitamin D(3) and LC results in a suppression of LC activity. CONCLUSIONS: Our data imply that VDR expression controls dermal collagen production, hair development and growth, proliferation of sebaceous glands and the homeostasis of DETC. Surprisingly, VDR deficiency does not influence LC phenotype and function.

Pimecrolimus does not affect Langerhans cells in murine epidermis.

BACKGROUND: Langerhans cells (LCs) function as specialized antigen-presenting cells in the epidermis, and therefore play a critical role in cutaneous immunological reactions. Topical treatment with corticosteroids is associated with a decrease in epidermal LC number and antigen-presenting capacity in laboratory animals and humans. OBJECTIVES: To examine whether pimecrolimus, a nonsteroidal inflammatory cytokine inhibitor recently introduced for the topical treatment of atopic dermatitis, differs from corticosteroids in effects on LCs. METHODS: Groups of BALB/c mice were treated twice daily on one to five consecutive days on the inner surface of the right ear with 10 micro L of ethanolic solutions of the test compounds at their clinically used concentrations (1% pimecrolimus, 0.1% betamethasone-17-valerate, 1% hydrocortisone and 0.05% clobetasol propionate) or with the vehicle (controls) alone. At selected time points after the treatment epidermal sheets were prepared and examined histomorphometrically for LCs immunolabelled with antibodies to major histocompatibility complex (MHC) class II and DEC 205, and adenosine diphosphatase staining. RESULTS: No changes in number or morphology of LCs were observed in epidermal sheets of mice treated for 5 days with pimecrolimus. In contrast, an almost complete depletion of LCs was observed in skin samples treated with hydrocortisone, betamethasone or clobetasol. Even a single-day treatment schedule with hydrocortisone, betamethasone or clobetasol caused a significant reduction in MHC class II+ LCs, by 31%, 62% and 87%, respectively. CONCLUSIONS: It is therefore unlikely that topically applied pimecrolimus affects epidermal LCs, in contrast to corticosteroids.

Dendritic cells presenting equine herpesvirus-1 antigens induce protective anti-viral immunity.

Equine herpesvirus-1 (EHV-1) causes rhinopneumonitis, abortion and CNS disorders in horses. Using intranasal inoculation, the mouse model of this disease mimics the major pathogenic and clinical features of the equine disease. The aim of this study was to investigate whether murine dendritic cells (DC) can be infected with EHV-1 and whether they can be used as cellular vaccines for the induction of prophylactic anti-EHV-1 immunity. It was found that the DC lines FSDC, D2SC1, 18 (all H-2d) and 80/1 (H-2k), when incubated with the Ab4 strain of EHV-1, do not change their morphology and phenotype but sustain virus replication and induce proliferation of naive, syngeneic T cells. An even stronger proliferation of T cells was seen when DC were used that had been pre-exposed to heat-inactivated virus. DC lines were therefore pulsed with inactivated virus and were then administered intranasally to either BALB/c or C3H mice on days -25, -15 and -5. Control groups received either medium, unpulsed DC or inactivated virus only. Animals were challenged with EHV-1. Whereas mice of control panels showed clinical signs of EHV-1 disease and 27% died, animals immunized with the pulsed DC lines showed only subtle clinical symptoms, lost significantly less weight, exhibited a reduced virus load in their lungs and CNS and did not succumb to the disease during the observation period. These results show that murine DC can present EHV-1 and initiate a protective anti-viral immunity in vivo.

Murine Langerhans cells cultured under serum-free conditions mature into potent stimulators of primary immune responses in vitro and in vivo.

The ability of Ag-pulsed dendritic cells (DC) to induce primary immune responses has led them to be used for vaccination purposes. However, irrelevant Ags (e.g., FCS) can also be taken up by DC during their isolation and culture and then presented in vivo. To circumvent this, we have established a serum-free (SF) culture system. Murine epidermal cell (EC) suspensions were prepared with and without FCS and cultured for 3 days either in SF or FCS-containing medium. In spite of the lower Langerhans cell (LC) yields under SF conditions, both SF- and FCS-cultured LC (SF-cLC, FCS-cLC) underwent a similar maturation process, as evidenced by a similar increase in the cell surface expression of MHC class II and of costimulatory molecules. The further observation that SF-EC cultures elaborated comparable amounts of granulocyte-macrophage (GM)-CSF as FCS-cultured EC, but were relatively impaired in their IL-1alpha and TNF-alpha production, supports the role of GM-CSF in LC maturation and, less so, in LC survival. Functionally, freshly isolated SF-LC compared with FCS-LC in their Ag-processing capacity. Three-day-cultured SF-LC were as potent stimulators of polyclonal T cell responses and of the primary allogeneic MLR as FCS-cLC, but were relatively poor activators of naive, syngeneic CD4+ T cells. In vivo, hapten-modified SF-cLC induced a contact hypersensitivity response similar in magnitude and kinetics to that evoked by FCS-cLC. Our data show that, in the absence of serum and exogenous cytokines, LC mature into potent activators of T cell responses and could thus be a valuable cellular source for DC-based immunotherapy.

Major histocompatibility complex class II- fetal skin dendritic cells are potent accessory cells of polyclonal T-cell responses.

Whereas dendritic cells (DC) and Langerhans cells (LC) isolated from organs of adult individuals express surface major histocompatibility complex (MHC) class II antigens, DC lines generated from fetal murine skin, while capable of activating naive, allogeneic CD8+ T cells in a MHC class I-restricted fashion, do not exhibit anti-MHC class II surface reactivity and fail to stimulate the proliferation of naive, allogeneic CD4+ T cells. To test whether the CD45+ MHC class I+ CD80+ DC line 80/1 expresses incompetent, or fails to transcribe, MHC class II molecules, we performed biochemical and molecular studies using Western blot and polymerase chain reaction analysis. We found that 80/1 DC express MHC class II molecules neither at the protein nor at the transcriptional level. Ultrastructural examination of these cells revealed the presence of a LC-like morphology with indented nuclei, active cytoplasm, intermediate filaments and dendritic processes. In contrast to adult LC, no LC-specific cytoplasmic organelles (Birbeck granules) were present. Functionally, 80/1 DC in the presence, but not in the absence, of concanavalin A and anti-T-cell receptor monoclonal antibodies stimulated a vigorous proliferative response of naive CD4+ and CD8+ T cells. Furthermore, we found that the anti-CD3-induced stimulation of naive CD4+ and CD8+ T cells was critically dependent on the expression of FcgammaR on 80/1 DC and that the requirement for co-stimulation depends on the intensity of T-cell receptor signalling.

A novel splice variant of the transcription factor Nrf1 interacts with the TNFalpha promoter and stimulates transcription.

Common signaling chains of various receptor families, despite some similarities, are able to provoke quite different cellular responses. This suggests that they are linked to different cascades and transcription factors, dependent on the context of the ligand binding moiety and the cell type. The ITAM (immunoreceptor tyrosine-based activation motif) containing gamma chain of the FcepsilonRI, FcgammaRI, FcgammaRIII and the T-cell receptor is one of these shared signaling molecules. Here, we show that in the context of the FcgammaRIII, the gamma chain activates the transcription factor Nrf1 or a closely related protein that specifically interacts with the extended kappa3 site in the TNFalpha promoter. A novel splice variant of Nrf1 with a 411 bp deletion of the serine-rich region, resulting in an overall structure reminiscent of the BTB and CNC homology (Bach) proteins, was isolated from the corresponding DC18 cells. In a gel shift analysis, this bacterially expressed splice variant binds to the TNFalpha promoter site after in vitro phosphorylation by casein kinase II (CKII). In addition, cotransfection studies demonstrate that this splice variant mediates induced transcription at the TNFalpha promoter after stimulation/activation in a heterologous system.