The dimensions and characteristics of the subepidermal nerve plexus in human skin--terminal Schwann cells constitute a substantial cell population within the superficial dermis.

BACKGROUND: The skin constitutes the largest sensorial organ. Its nervous system consists of different types of afferent nerve fibers which spread out immediately beneath the skin surface to sense temperature, touch and pain. OBJECTIVE: Our aim was to investigate the dimension and topographic relationship of the different nerve fibers of the subepidermal nerve plexus in human hairy skin and to analyze numbers and marker expression of terminal Schwann cells. METHODS: Nerve fibers and Schwann cells were investigated on dermal sheet preparations and thick sections of skin from various body regions of 10 individuals. RESULTS: The dimension of subepidermal nerve fibers varied between different body sites with highest values in chest skin (100 ± 18 mm/mm(2)) and lowest in posterior forearm skin (53 ± 10 mm/mm(2)). The majority of fibers (85.79%) were unmyelinated, thus representing C-fibers, of which 7.84% were peptidergic. Neurofilament-positive fibers (A-fibers) accounted for 14.21% and fibers positive for both neurofilament and myelin (Aß-fibers) for only 0.18%. The number of Schwann cells varied in accordance with nerve fiber length from 453 ± 108 on chest skin to 184 ± 58/mm(2) in skin of the posterior forearm. Terminal Schwann cells showed a marker profile comparable to Schwann cells in peripheral nerves with the notable exception of expression of NGFr, NCAM, L1CAM and CD146 on myelinating Schwann cells in the dermis but not in peripheral nerves. CONCLUSION: Our data show that terminal Schwann cells constitute a substantial cell population within the papillary dermis and that both nerve fiber length and Schwann cell numbers vary considerably between different body sites.

Embryonic stem cell factors undifferentiated transcription factor-1 (UFT-1) and reduced expression protein-1 (REX-1) are widely expressed in human skin and may be involved in cutaneous differentiation but not in stem cell fate determination.

Undifferentiated transcription factor-1 (UTF-1) and reduced expression protein-1 (REX-1) are used as markers for the undifferentiated state of pluripotent stem cells. Because no highly specific cytochemical marker for epidermal stem cells has yet been identified, we investigated the expression pattern of these markers in human epidermis and skin tumours by immunohistochemistry and in keratinocyte cell cultures. Both presumed stem cell markers were widely expressed in the epidermis and skin appendages. Distinct expression was found in the matrix cells of the hair shaft. Differentiation of human primary keratinocytes (KC) in vitro strongly downregulated UTF-1 and REX-1 expression. In addition, REX-1 was upregulated in squamous cell carcinomas, indicating a possible role of this transcription factor in malignant tumour formation. Our data point to a role for these proteins not only in maintaining KC stem cell populations, but also in proliferation and differentiation of matrix cells of the shaft and also suprabasal KC.

Expression of VEGF-A/C, VEGF-R2, PDGF-alpha/beta, c-kit, EGFR, Her-2/Neu, Mcl-1 and Bmi-1 in Merkel cell carcinoma.

Merkel cell carcinoma is a rare but very aggressive tumor of the skin. With current treatment options, Merkel cell carcinoma is associated with a high incidence of recurrence and metastasis. Targeted anticancer therapies such as receptor tyrosine kinase inhibitors and antisense oligonucleotides have been found to be a promising new type of treatment for various types of cancer. To evaluate whether the use of targeted therapies is a possible treatment option in Merkel cell carcinoma, we determined the expression of the target molecules c-kit, Mcl-1, Bmi-1, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-receptor 2 (VEGF-R2), platelet-derived growth factor (PDGF)-alpha, PDGF-beta, epidermal growth factor receptor (EGFR) and Her-2/Neu in a tissue microarray of 32 samples of 29 patients with Merkel cell carcinoma. C-kit-positive samples were analyzed for mutations in exons 9 and 11. The tissue microarray was stained immunohistochemically with antibodies directed against the above-mentioned proteins, and an immunoreactivity score was calculated. DNA was extracted from c-kit-positive samples and was analyzed for exon 9 and 11 mutations using direct DNA sequencing. We found that c-kit (7%), Mcl-1 (88%), Bmi-1 (78%), VEGF-A (91%), VEGF-C (75%) VEGF-R2 (88%), PDGF-alpha (72%) and PDGF-beta (13%) were expressed in Merkel cell carcinomas. All samples showed a lack of EGFR and Her-2/Neu expression. Analysis of c-kit revealed no mutations. As VEGF-A, VEGF-C, VEGF-R2, PDGFs and c-kit are targets of new cytostatic agents used in the treatment of other cancers, inhibition by a multitargeted chemotherapy could be a very promising treatment option. High expression of Bmi-1 and Mcl-1 warrants further studies on the use of antisense oligonucleotides in Merkel cell carcinoma.

Rarefaction of the peripheral nerve network in diabetic patients is associated with a pronounced reduction of terminal Schwann cells.

OBJECTIVE: Peripheral neuropathy is the most frequent neurological complication in diabetic patients. The diagnosis is established by both clinical neurological examination and demonstration of reduced epidermal nerve fibers in skin biopsies (1). Whereas the decrease of free nerve endings has been extensively studied in diabetic patients (2,3), no data are available on possible changes of terminal Schwann cells. Besides their role as scaffold for peripheral nerves, they also play an important role in supporting survival and function of peripheral nerves (4). RESEARCH DESIGN AND METHODS: We analyzed the subepidermal nerve plexus in dermal sheet preparations of deceased diabetic and nondiabetic patients by immunostaining for detection of the neural cell adhesion molecule and quantification of the subepidermal nerve plexus. RESULTS AND CONCLUSIONS: The subepidermal nerve plexus, comprising nerve fibers and ensheathing Schwann cells, was significantly reduced in diabetic patients. Whether the reduction in terminal Schwann cells is cause or consequence of the loss of peripheral nerve fibers remains to be investigated.

Expression of BMI-1 in normal skin and inflammatory and neoplastic skin lesions.

BACKGROUND: BMI-1 is involved in the maintenance of stem cells and functions as an oncogene in both lymphomas and solid carcinomas, acting by downregulation of p16ink4a. We have investigated the expression profile of BMI-1 in normal and inflamed skin as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). METHODS: BMI-1 expression was determined by immunohistochemistry and immunofluorescence, and evaluated semiquantitatively. RESULTS: BMI-1 was weakly expressed in nuclei of basal and sometimes suprabasal keratinocytes, in basal cells of sebaceous glands, weakly to moderately in the bulge area and the external root sheath of hair follicles, and strongly in sweat glands. Whereas BCCs showed strong and diffuse BMI-1 expression, SCCs expressed BMI-1 heterogeneously. Strong cytoplasmic expression of BMI-1 was found in dividing cells. CONCLUSIONS: BMI-1 expression marks stem cells within the hair follicle. As BMI-1 was also found in suprabasal keratinocytes and a variety of specialized cells, the distribution of BMI-1 only partly reflects the known distribution of stem cell compartments. BMI-1 is strongly overexpressed in BCCs, tumors linked to dysregulation of the sonic hedgehog pathway, which has been shown to upregulate BMI-1, suggesting a contribution of the BMI-1 oncogene in their pathogenesis.

The touch dome in human skin is supplied by different types of nerve fibers.

Receptor end organs and free-nerve endings in the skin are the peripheral sentinels of the sensorial nervous system encoding for touch, temperature, and pain. Using a novel approach to analyze the outermost nerves of the skin, we visualized for the first time the distinct microanatomical structure of the touch dome of human hairy skin. The dermal nerve fibers of this slowly adapting type 1 mechanoreceptor were embedded in dermal protrusions that could be readily discerned by Laminin-5 staining. Concerning the nerves supplying the touch domes, we found, unexpectedly, that besides Abeta-fibers, Adelta- and C-fibers also were regularly present. The epidermis overlying the nerve convolutes showed a distinctive architecture of the rete ridges clearly demarcated from the surroundings and extending over 0.193 +/- 0.138 mm(2) (mean +/- standard deviation). Within this area, 756 +/- 386 Merkel cells/mm(2) (mean +/- standard deviation) were present compared with less than 50/mm(2) outside the touch dome, demonstrating for the first time a highly discontinuous distribution of these cells in nonglabrous skin. Our findings strongly suggest that the receptive qualities of human touch domes exceed mechanosensation, and that they may serve as multifunctional nerve end organs in human skin.

Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.

Since vertical tissue sections used for the study of the human cutaneous nervous system inherently allow visualization of only a small part of the mainly horizontally oriented cutaneous nerves, we searched for possibilities to extend this view. We now propose a method based on the immuno-staining of dermal sheet preparations for subsequent analysis by electron-, light- or laser scanning microscopy. Dermal sheet preparations for the first time allowed the imaging of the complex structure of the nerve end organ over several cm2, and facilitated viewing of its topological relationship to other tissue components. We could visualize that the bulk of free ending nerve fibers ramified within 25 microm of the dermo-epidermal junction, whereas below that only larger nerve bundles were present. This method further allowed the detection and quantification of NCAM/CD56+ non-myelinating Schwann cells which envelope terminal axons within the dermis. Depending on the body region, we detected between 140 to over 300 individual terminal Schwann cells per mm2 skin surface. Our method should allow the acquisition of new insights into the highly organized architecture of the skin nerve end organ. Its further application will give new impetus in the investigation of alterations of this skin compartment under pathological conditions.

Inhibition of human neutrophil chemotaxis toward interleukin 8 with six clinical antithrombin concentrates in vitro.

OBJECTIVE: Antithrombin exerts direct effects on neutrophils by inhibiting chemokine-induced migration. This study examined the potency of different pharmaceutical antithrombin preparations in inhibiting neutrophil chemotaxis toward interleukin 8. METHODS: Cell migration was tested by the leading front assay in modified Boyden microchemotaxis chambers bearing nitrocellulose filters. Human neutrophils were incubated with six different antithrombin concentrates or an immunopurified antithrombin preparation at concentrations of 1 micro IUeth-5 IU/ml. RESULTS: All antithrombin concentrates irrespective of the pharmaceutical source deactivated neutrophil chemotaxis. At concentrations below 100 mIU/ml neutrophil chemotaxis toward interleukin 8 was decreased by the antithrombin preparations with varying potency, but at 1 mIU/ml no significant differences were observed. CONCLUSIONS: As the ability of antithrombin to deactivate neutrophil chemotaxis toward interleukin 8 shows differences depending on the source of commercial antithrombin, these results suggest that at equivalent WHO standard concentrations clinical antithrombin concentrates may differ in anti-inflammatory potential.

Inhibition of plasma-dependent monocyte chemokinesis and cytokine-triggered endothelial activation for neutrophil transmigration by administration of clopidogrel in man.

Mediators released by spontaneously activated platelets may contribute to alterations in endothelial and leukocyte dysfunctions. We investigated the roles of clopidogrel and aspirin in ex vivo endothelial activation for interactions with leukocytes. Eight healthy volunteers received clopidogrel or aspirin for 8 days. Blood samples were taken before, during, and after treatment. Levels of adhesion molecules and platelet-derived mediators in these samples were measured using commercially available test kits, and effects of plasma on endothelial cells and leukocytes were investigated in neutrophil transendothelial migration, monocyte-endothelial adhesion and leukocyte migration assays. Plasma samples from clopidogrel-treated persons induced diminished chemokinesis of monocytes. Tumour necrosis factor-induced priming of endothelial cells for enhanced neutrophil transmigration was also diminished by pretreatment of endothelial cells, but not of neutrophils, with plasma derived from subjects during clopidogrel treatment. Plasma from the aspirin group had no such effects. Administration of clopidogrel but not aspirin significantly decreased serum levels of soluble intercellular adhesion molecule-1, whereas no changes in levels of soluble vascular cell adhesion molecule-1, P-selectin, L-selectin, von Willebrand factor, platelet-derived growth factor, vascular-endothelial growth factor, and transforming growth factor-beta were observed. Inhibition of plasma-promoted endothelial activation by clopidogrel may indicate a novel role in the prevention of atherosclerosis.

Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes.

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Whether antithrombin affects migration of other types of leukocytes is not known. We investigated the effects of antithrombin on spontaneous and chemokine-triggered migration of lymphocytes and monocytes from human peripheral blood in modified Boyden chamber micropore filter assays. Lymphocyte and monocyte populations from human peripheral blood were purified using magnetic antibody cell sorting. The signaling mechanisms required for antithrombin-dependent migration were studied using signaling enzyme blockers. Expression of heparan sulfate proteoglycan core protein was studied by RT-PCR and flow cytometry. The antithrombins used were Kybernin P from human plasma and a monoclonal-antibody-purified preparation from this plasma. Pretreatment of lymphocytes and monocytes with antithrombin inhibited chemotaxis toward optimal concentrations of interleukin-8 or Rantes (regulated upon activation normal T-cell expressed and activated) at concentrations of antithrombin as low as 10 nU/ml. In the absence of the chemokines, direct exposure of cells to gradients of antithrombin stimulated migration. Effects of antithrombin were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in monocytes and lymphocytes was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assays, respectively. In the presence of pentasaccharide, antithrombin lost its effect on cells. Data indicate that antithrombin directly inhibits chemokine-stimulated migration of monocytes and lymphocytes via the effects of its heparin-binding site on cell surface syndecan-4 by activation of protein kinase C and Rho signaling.