Epidermal Sheets Research Articles

Thioredoxin inhibits epidermal Langerhans’ cell (LC) migration in man and mouse (44.6)

Abstract Epidermal LC play a critical role in the initiation of skin immune responses. LC migration is dependent upon cytokine signals, provided by tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β. We have now examined the ability of human recombinant thioredoxin (hTrx), a redox-active protein, to influence LC migration. Wild type hTrx and various cysteine to alanine mutants were produced in E coli. LC migration in response to chemical contact allergen (oxazolone; Ox) or to intradermal (id) injection of recombinant murine TNF-α or IL-1β was assessed in BALB/c mice. LC migration was provoked in healthy human volunteers (n=10) by id injection of recombinant human TNF-α. Various forms of hTrx were administered by topical application 2h prior to the migratory stimuli. LC densities were assessed in epidermal sheets by immunofluorescent staining for MHC class II (mouse) or CD1a (human). Topical application of hTrx inhibited significantly Ox- and TNF-α-induced LC migration in BALB/c strain mice, but failed to impact on IL-1β-induced migration. Use of mutant forms of hTrx demonstrated that this ability was redox-independent. Application to the skin of wild type hTrx to human volunteers inhibited TNF-α-induced LC migration also. Inhibition of LC migration was selective for IL-1β-dependent stimuli (contact allergen and TNF-α). These data suggest that thioredoxin possesses novel immunomodulatory properties as a result of its ability to modulate IL-1β production and/or signalling.

Sec61α is required for dorsal closure duringDrosophilaembryogenesis through its regulation of Dpp signaling

During dorsal closure in Drosophila, signaling events in the dorsalmost row of epidermal cells (DME cells) direct the migration of lateral epidermal sheets towards the dorsal midline where they fuse to enclose the embryo. A Jun amino-terminal kinase (JNK) cascade in the DME cells induces the expression of Decapentaplegic (Dpp). Dpp signaling then regulates the cytoskeleton in the DME cells and amnioserosa to affect the cell shape changes necessary to complete dorsal closure. We identified a mutation in Sec61alpha that specifically perturbs dorsal closure. Sec61alpha encodes the main subunit of the translocon complex for co-translational import of proteins into the ER. JNK signaling is normal in Sec61alpha mutant embryos, but Dpp signaling is attenuated and the DME cells fail to maintain an actinomyosin cable as epithelial migration fails. Consistent with this model, dorsal closure is rescued in Sec61alpha mutant embryos by an activated form of the Dpp receptor Thick veins.

Quantitative Analysis of Cell Edge Dynamics and Cell Shape in Non-Polarized Fish Epidermal Keratocytes

Cell migration requires the coordination of several processes such as leading edge protrusion, adhesion formation and disassembly, and trailing edge retraction. The quantitative analysis concerning the correlation between these processes will be useful to understand the coordination mechanism. A major difficulty in the quantitative analysis stems from the complex morphology of migrating cells. To cope with this difficulty, we selected the non-polarized fish epidermal keratocytes as a simplified experimental system that includes basal migratory mechanisms. We acquired a mixture of non-polarized stationary keratocytes and polarized highly motile keratocytes by disaggregating the large epidermal sheets. The time-lapse micrographs of non-polarized keratocytes were used to analyze cell edge dynamics and cell peripheral shape. We adopted the protrusion and retraction rate and cell peripheral curvature as quantitative parameters. Non-polarized keratocytes did not exhibit net translocation, however, active protrusion and retraction were observed around the cell periphery. Protrusion rate was negatively correlated with the cell peripheral curvature. In contrast, retraction rate was positively correlated with the cell peripheral curvature. The plot of protrusion and retraction rates over the entire cell periphery showed that protrusion and retraction waves were traveling laterally in both directions along the cell periphery. The lateral traveling velocity of each wave was constant. The wave persistence varied from 10 s to 100 s. These results indicate that the cell has the positive feedback mechanism maintaining stable protrusion and retraction and that the rate of protrusion and retraction is related to the cell peripheral shape. Quantitative analysis together with theoretical and molecular biological studies will shed light on the mechanism of cell migration.

Potential role of Wnt signaling pathway in epidermal cell dedifferentiation under traumatic microenvironment

Objective To investigate the inductive role of traumatic microenvironment in dedifferentiation of epidermal cells and explore the potential role of Wnt signaling pathway in this biological process. Methods The sheets of human foreskin were digested overnight after removal of adipose tissue, and then the epidermis was separated from the dermis. The separated epidermis sheets were repeatedly adhered to type Ⅳ collagen and flushed to remove the epidermal stem cells. The obtained epidermis sheets were transplanted onto the full-thickness skin wounds on the back of BALB/c nude mice, five days after which the cell lineage was evaluated by immunohistochemistry and the expressions of Wnts and downstream components in the grafted epidermal sheets examined by RT-PCR and Western blot. Results The cells in the basal layer of full-thickness epidermal sheets were positive for CK19 and β1 integrin and negative for CK10. While the cells in uhrathin epidermal sheets treated with type Ⅳ collagen were fully positive for CK10. Five days after transplantation of the ultrathin epidermal sheets, cells negative for CK10 but positive for CK19 and β1 integrin emerged at the wound-neighboring side of the skin grafts. At the same time, the expressions of Wnt-10b, Wnt-4 and Wnt-7a mRNA were increased by about 3.1-fold, 2.2-fold and 1.4-fold independently after transplantation. Furthermore, the expressions of β-catenin and β-catenin target genes (cyclin D1 and c-myc) were elevated by about 3-fold, 1.5-fold and 2-fold respectively in the grafted epidermal sheets (P < 0.01). Conclusion Traumatic microenvironment can induce epidermal cell dedifferentiation, when the Wnt/β -catenin signaling pathway may play an important role. Key words: Wounds and injuries; Epidermis; Dedifferentiation; Signal transduction; Wnt/β-catenin signaling pathway

Conservation of CD1 protein expression patterns in the chicken

The CD1 molecules are cell-surface proteins that bind and present foreign lipids and glycolipids to T cells in a manner similar to the MHC system. While the mammalian CD1 antigen presentation pathway is well characterized, little is known about CD1 in non-mammalian vertebrates. Previous studies have identified two CD1 homologues in the chicken. We developed a monoclonal antibody designated NL1-1.A1 specific for the chCD1-1 isoform and have used this to characterize CD1 expression in tissues and cells of normal adult and embryonic chickens. The chCD1-1 isoform is expressed on a high proportion of cells in the spleen and bursa. Cells in the spleen that stain for CD1 are also positive for IgM and consistent with identification of these as B cells. In the skin, chCD1-1 is expressed on cells with dendritic morphology along the dermal–epidermal boundary and in epidermal sheets consistent with chicken Langerhans cells. Staining of cells in the medullary region of the chicken thymus was also observed. The CD1 proteins in mammals traffic to intracellular compartments to acquire lipid antigens for subsequent presentation to T cells on the surface. Consistent with data from mammal CD1 proteins, chCD1-1 partially co-localized with a lysosomal marker in the myeloid cell line BM2. Taken together, these data support broad distribution of chCD1-1 in both lymphoid and non-lymphoid tissues of the chicken that is remarkably similar to the distribution of CD1 isoforms in mammals.

Research Highlights: Immunotherapy

Research Highlights: Immunotherapy

An organotypic culture system of Merkel cells using isolated epidermal sheets

Merkel cells (MCs) exist in the epidermal basal layer, in contact with keratinocytes. This direct contact seems critical for maintaining MCs in vitro. To estimate the effects of nerve cells on the maintenance of MCs within epidermal sheets in a new organotypic culture system of MCs. We developed a new organotypic culture system of MCs, using MC-containing epidermal sheets embedded in collagen gel. To estimate the effects of nerve cells on the maintenance of MCs within the epidermal sheets, we cocultured nerve cells and MC-containing epidermal sheets. In these culture assemblies, cellular behaviour was analysed by histochemistry, immunohistochemistry, electron microscopy and enzyme-linked immunosorbent assay. This culture, even in the absence of neurotrophin (NT)-3 and nerve growth factor (NGF) (which are crucial for MC biology), retained cytokeratin (CK)-20-positive and neuroendocrine granule-containing MCs within the sheets for over 2 weeks. Coculture of MCs with PC-12 nerve cells significantly increased the number of MCs within the epidermal sheets, and the keratinocytes had almost identical expression levels of CK1, CK10, CK14 and the progenitor marker p63 to those produced by keratinocytes in vivo. Uptake of the growth marker bromodeoxyuridine by MCs and levels of NT-3 and NGF in the culture supernatants were undetectable in this system, regardless of the presence or absence of PC-12. The data suggest, first, that direct contact between MCs and keratinocytes may be critical for retaining MCs in vitro; second, that nerve cell-affected maintenance of keratinocyte differentiation, but not NT-3 and NGF, may contribute to MC maintenance; and third, that MCs are not able to grow, at least in our system. Our method would be useful for studying MC biology.

Transplantation of chronic wounds with epidermal sheets derived from autologous hair follicles—the Leipzig experience

An increasing number of chronic wounds in our society require strategies to improve wound healing and wound closure. One of several options is skin transplantation. In this article, we focus on the transplantation of tissue engineered autologous epidermal sheets derived from outer root sheath (ORS) cells of the patients' hair. Out of the stem cells of the ORS of anagen hair, autologous keratinocytes are cultured ex vivo in organotypic cultures to form a multilayered epidermal equivalent (EpiDex, EuroDerm Biotech & Aesthetics, Stuttgart, Germany). These sheets are placed on the wound bed. Patients were observed twice a week in the first 2 weeks, then once weekly for 4 weeks, then every 4 weeks for up to 12 weeks after transplantation. A total of 23 patients with (n = 18) and without (n = 5) therapeutic improvement were analyzed retrospectively. We evaluated only the effect of a single transplantation in a selected ulcer per patient. Furthermore, a subgroup-analysis for responder patients with an ulcer area < 25 cm2 (n = 12) was performed. In the responder patients (n = 18), a total wound reduction of 23% was observed. Patients (n = 12) with ulcer area < 25 cm2 had an improvement of 64%. Complete wound closure in this subgroup after a single transplantation was achieved in 33 % (n = 4) cases. Autologous keratinocyte transplantation with EpiDex can be performed easily and safely in patients with chronic wounds with satisfying results. Our data suggest that patients with small ulcer area < 25 cm2 might profit the most from this method.

Dedifferentiation-derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell-like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation-derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c-nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β1-integrin emerged at the wound-neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β1-integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation-derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β1-integrin, Oct4 and Nanog in dedifferentiation-derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation-derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9-fold and 6.7-fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 105 cells versus 1 × 1012 cells) were all significantly increased and (4) dedifferentiation-derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation-derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Treatment of giant congenital melanocytic nevi with enzymatically separated epidermal sheet grafting

Treatment of giant congenital melanocytic nevi (GCMNs) must be considered from both a cosmetic and a medical aspect of reducing the risk of a growing malignancy. We treated five patients who had a GCMN on the trunk with an enzymatically separated epidermal sheet taken from their excised GCMN skin. All patients underwent enzymatically separated epidermal sheet grafting at the age of 2 months. With the patient under general anaesthesia, we removed the affected area with an electric dermatome until the brown colour had almost completely disappeared. The removed skin was immersed in dispase I solution (1000Uml(-1)) dissolved in Ringer's solution at 37 degrees C for 75-105min until the epidermal sheet separated from the dermis without tension. Three back lesions and two abdominal lesions were treated. In all cases, the enzymatically separated epidermis grafted well, without infection. The amount of discharge that accumulated from immediately after the operation until the first dressing change (performed at 3 days after the operation) was reduced compared with that from curettage. The skin colour was light brown at first and gradually became lighter within a few months. The grafted areas were soft and did not show significant re-pigmentation. In most cases, hard hair did not grow on the treated area. Histological samples taken 1 year after the operation showed that the dermal layer was reconstituted without pigmented nevus cells. Enzymatically separated epidermal grafting is a useful method for the treatment of GCMN, is easy to perform and leaves cosmetically satisfactory results.

Cutaneous immune responses to 2, 4-dinitrochlorobenzene (DNCB): role of interleukin (IL)-2 (97.15)

Abstract Prolonged topical exposure of BALB/c strain mice to the contact allergen DNCB, or to the respiratory sensitiser trimellitic anhydride, results in T helper (h) 1 and Th2 cell polarisation, respectively. In addition, a single application of allergen results in discrete cutaneous cytokine profiles and the migration of Langerhans' cells (LC) with different tempos. Of note, DNCB only induced a rapid and transient increase in cutaneous IL-2. We have now explored the role of IL-2 in DNCB-induced LC migration. BALB/c mice received an intradermal injection of recombinant IL-2 or IL-1β (50ng/ear); the latter is known to provoke LC migration. LC densities were determined in epidermal sheets by immunofluorescence staining. Cytokine production by skin explants was measured by protein array. Intradermal injection of IL-1β decreased epidermal LC numbers significantly after 4 and 16h whereas IL-2 was without effect. Prior systemic administration of neutralising anti-IL-2 antibody failed to influence LC migration monitored 6h after DNCB treatment. Migration was blocked by treatment with an anti-tumor necrosis factor-α antibody, a cytokine regulator of LC migration. Administration of IL-2 did result in increased expression of cutaneous IL-17. These data show that IL-2 is not involved in LC migration, however, the selective up-regulation of IL-2 by DNCB may play a role in the subsequent polarisation of the immune response.

Distinct subsets of human skin dendritic cells differ on their ability to initiate Th17 responses (90.24)

Abstract Activated human cutaneous dendritic cells (DCs) have the ability to initiate Th1 and Th2 immunity. However, whether human cutaneous DCs are capable of biasing pro-inflammatory Th17 responses remains little understood. Utilizing human skin explants composed by both epidermis and dermis or epidermal or dermal sheets to collect activated skin-migratory DCs (smiDCs), we demonstrated that smiDCs stimulate allogeneic naïve CD4+ T cells to differentiate simultaneously into two distinct effector Th17 and Th1 populations with ability of skin homing and inducing severe tissue damage. Of the two main myeloid DC populations resident in the skin, the subset of epidermal skin-migratory Langerhans cells (smiLCs) were capable of inducing Th17 responses. This effect depended on the combined effects of IL-15 and stabilized IL-6 trans-signaling of naïve CD4+ T cells. Purified skin-migratory DDCs did not synthesize IL-15 and were unable to bias Th17 responses however, they acquired the ability to bias Th17 cells in co-cultures with CD4+ T cells supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human cutaneous LCs induce Th17 responses by mechanisms different from those previously described for mouse DCs and human monocytes and highlight the need to target clinical treatments based on these variations. Supported by NIH grants: R01 CA100893 (ATL)

Epidermal Langerhans cells in actinic prurigo: a comparison between lesional and non-lesional skin

Actinic Prurigo (AP) is a chronic pruritic dermatosis of unknown cause affecting sun exposed skin in defined ethnic groups with characteristic MHC alleles. However, the cutaneous dendritic cells have not been assessed. To assess in situ the epidermal Langerhans Cell (LC) status in Actinic Prurigo. Fresh skin samples from three AP patients were used to evaluate in situ the epidermal LC, comparing lesional and non-lesional sites in each subject. AP patients attending the Dermatology Department at the Hospital M. Gea-Gonzalez in Mexico city. Lesional and non-lesional skin samples were taken from each subject to prepare both epidermal sheets and conventional tissue sections. Three markers restricted to LC in epidermis (CD1a, ATPase, MHC-II) were used to quantify the LC per area in epidermal sheets. Compared to non-lesional skin from the same subject, a significant reduction in the number of LC per area of epidermis was found in lesional skin; with any of the three markers evaluated. The frequency of epidermal LC decreases importantly in lesional skin from AP patients.

A TR(I)P to Pruritus Research: Role of TRPV3 in Inflammation and Itch

Pruritus in eczema and other inflammatory skin diseases is an unsolved problem. Recent findings strongly indicate that histamine is not the only mediator of pruritus. Subtypes of the transient receptor potential (TRP) ion channel superfamily are expressed by sensory nerves, keratinocytes, and certain leukocytes. Temperature, pH changes, and certain toxins activate TRPs. New evidence indicates that the vanilloid type 3 (TRPV3) channel is crucially involved in pruritic dermatitis, making it a good candidate for future therapy in skin inflammation and pruritus.

Endogenous Galectin-3 Is Localized in Membrane Lipid Rafts and Regulates Migration of Dendritic Cells

This study reveals a function of endogenous galectin-3, an animal lectin recognizing beta-galactosides, in regulating dendritic cell motility both in vitro and in vivo, which to our knowledge is unreported. First, galectin-3-deficient (gal3(-/-)) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3(+/+) cells. Second, cutaneous dendritic cells in gal3(-/-) mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3(+/+) mice. Moreover, gal3(-/-) mice were impaired in the development of contact hypersensitivity relative to gal3(+/+) mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3(-/-) cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3(-/-) cells contained structures with lower complexity compared to gal3(+/+) cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.

The Essential Role of p53 in Hyperpigmentation of the Skin via Regulation of Paracrine Melanogenic Cytokine Receptor Signaling

Hyperpigmentation of the skin is characterized by increases in melanin synthesis and deposition. Although considered a significant psychosocial distress, little is known about the detailed mechanisms of hyperpigmentation. Recently, the tumor suppressor protein p53 has been demonstrated to promote ultraviolet B-induced skin pigmentation by stimulating the transcription of a melanogenic cytokine, POMC (pro-opiomelanocortin), in keratinocytes. Given that p53 can be activated by various kinds of diverse stresses, including sun exposure, inflammation, and aging, this finding led us to examine the involvement of p53 in cytokine receptor signaling, which might result in skin hyperpigmentation. Immunohistochemical and reverse transcription-PCR analyses revealed the increased expression and phosphorylation of p53 in the epidermis of hyperpigmented spots, accompanied by the higher expression of melanogenic cytokines, including stem cell factor, endothelin-1, and POMC. The involvement of p53 in hyperpigmentation was also indicated by the significantly higher expression of p53 transcriptional targets in the epidermis of hyperpigmented spots. Treatment of human keratinocytes and melanocytes with known p53 activators or inhibitors, including pifithrin-alpha (PFT), demonstrated significant increases or decreases, respectively, in the expression of melanogenic factors, including cytokines and their receptors. Additionally, PFT administration abolished stem cell factor-induced phosphorylation of mitogen-activated protein kinase in human melanocytes. Furthermore, when organ-cultured hyperpigmented spots, in vitro human skin substitutes, and mouse skin were treated with PFT or p53 small interfering RNA, the expression of melanogenic cytokines and their receptors was significantly decreased, as were levels of tyrosinase and melanogenesis. Taken together, these data reveal the essential role of p53 in hyperpigmentation of the skin via the regulation of paracrine-cytokine signaling, both in keratinocytes and in melanocytes.

The effects of multiple passes on the epidermal thermal damage pattern in nonablative fractional resurfacing

Nonablative fractional resurfacing is a concept of cutaneous re-modeling whereby laser-induced microscopic treatment zones (MTZs) are surrounded by normal viable tissue. Such thermal damage pattern with a small diameter of individual lesions allows fast re-epithelialization with minimal side effects. The purpose of this in vitro study was to determine the fraction of thermal injury per unit surface area (fill factor) and lesion size in relation to pulse energy and number of passes. Full thickness abdominal skin samples were exposed ex vivo to the Fraxel SR 750 laser (Reliant Technologies, Mountain View, CA). One set of exposures was performed for pulse energies in the range of 8 to 40 mJ for a single pass at 250 MTZ/cm(2). A second set of exposures was performed at 10 mJ with number of passes from 1 to 30. The thermal damage pattern was assessed by incubation of epidermal sheets with NitroBlueTetrazoliumChloride (NBTC) stain. Size of individual MTZ and fill factor were determined by image analysis (ImageJ, NIH, Bethesda, MD) of digital micrographs. Width of the thermal injury zone was directly related to the pulse energy used. The fill factor did not have a uniform relationship with the number of passes. Due to the stochastic placement of individual MTZs, even for greater number of passes, some residual undamaged tissue was found. Due to formation of thermal damage clusters, defined as overlapping individual MTZs, the size of the resulting clustering lesions which we defined as microscopic treatment cluster (MTC) increased linearly as a function of the number of passes. We have described the fill factor as it relates to the number of passes and have demonstrated that the average size of individual lesions depends on the number of passes. Clustering of MTZs lead to the development of MTC, the average size of which increased with the number of passes. The clinical implications of these findings are contingent on further studies.

Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status

Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes.

The involvement of Ca2+ and integrins in directional responses of zebrafish keratocytes to electric fields

Many cells respond directionally to small DC electrical fields (EFs) by an unknown mechanism, but changes in intracellular Ca(2+) are widely assumed to be involved. We have used zebrafish (Danio rerio) keratocytes in an effort to understand the nature of the EF-cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm(-1), and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca(2+) spikes and basal Ca(2+) levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two-photon imaging failed to detect a pattern of gradients of Ca(2+) across the lamellipodia during normal or EF-induced turning but did detect a sharp, stable Ca(2+) gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca(2+) within the lamellipodium are not required for the EF response. Immunostaining revealed an anode to cathode gradient of integrin beta1 during EF-induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro-osmotic water flow mediate EF-cell interactions via effects on focal adhesions.

CXCL12 is essential for migration of activated Langerhans cells from epidermis to dermis

The initial step in Langerhans cell (LC) migration from the epidermis to the lymph node involves migration of maturing LC into the dermis. Here, we investigated the migration of LC out of the epidermis after exposure of the skin to contact allergens. Ex vivo intact human skin, epidermal sheets, and LC derived from the MUTZ-3 cell line (MUTZ-LC) were used to determine whether dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of epidermal sheets or MUTZ-LC to allergens (nickel sulphate, 2,4-dinitrochlorobenzene, and cinnamaldehyde) or a cytokine maturation cocktail resulted in LC migration towards dermal fibroblasts. This was due to upregulation of CXCR4 on maturing LC and secretion of CXCL12/stromal derived factor-1 chemokine by fibroblasts. Neutralizing antibodies to either CXCL12 or CXCR4 completely blocked migration. Injection of CXCL12 neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. In contrast, neutralizing antibodies to CCL19/CCL21 did not inhibit migration into the dermis. We describe a novel and essential role of dermis-derived CXCL12 in initiating migration of maturing human LC to the dermis thus permitting their further journey to the draining lymph nodes.