Human epidermal acetylcholinesterase (AchE) is regulated by hydrogen peroxide (H2O2)

Extraneuronally, acetylcholine (Ach) is synthesized, stored and secreted in the tegumental cells covering the inner and outer surfaces of the body. It acts via nicotinic (nAchR) and muscarinic (mAchR) receptors in a paracrine and autocrine fashion influencing various keratinocyte functions. Using in vitro studies, we present evidence that effects of cholinergic agonists and antagonists on cutaneous biology are dependent upon specific Ach‐R, localised in the different compartments of the skin. Using immunohistochemistry on frozen section of normal skin we could previously demonstrate a differential distribution of alpha 3*, alpha 7, alpha 9 and beta 1 nAchR and m1, m3–5 mAchR in human epidermis. Here we show the functional significance of AchR blockage using organotypical keratinocytes cultures. Cholinergic agonists and antagonists were added on the day of the lift to the air‐liquid interface. After 5 days, blockage of all AchR using mecamylamine and atropine resulted in complete inhibition of terminal differentiation. Mecamylamine and atropine alone had only a retarding effect suggesting an additive mechanism of nAchR and mAchR blockage. Specific inhibition of α9 homooligomers with strychnine produced the strongest effects leaving behind only a keratinocytes monolayer. Stimulation of AchR with either nicotine or muscarine in short term organotypic cultures did not produce dramatic changes of epidermal morphology. We conclude that alpha 9 AchR that are located in the basal and lower suprabasal layers of the epidermis, are crucially involved in terminal differentiation of keratinocytes. The functional significance of other nAchR expressed in the epidermis remains unclear, due to the lack of specific agonists and antagonists.     

Immunohistochemical localization of the Ca2+ binding S100 proteins in normal human skin and melanocytic lesions

The purpose of this study was to evaluate the expression of the Ca²⁺-binding S100 proteins S100A1. S100A2, S100A3, S100A4, S100A6 and S100B in normal skin. These immunohistochemical staining patterns were compared with those in melanocytic lesions. Parafin-embedded tissue of normal skin adjacent to 26 naevi. 39 primary cutaneous melanomas and 14 cutaneous melanoma metastases was incubated with polyclonal antibodies against the recombinant human S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A6, S100B) using the alkaline phosphatase anti-alkaline phosphatase method. The S100A2 antibody stained the basal layer of the epidermis an hair follicles of normal skin. Four of 39 primary cutaneous melanomas were positive for S100A2, whereas none of the methastases or naevi showed any immunoreactivity. The S100A3 antibody only stained the inner root sheath cuticle of some hair follicles but no melanocytes or melanocytic lesions. Staining of S100A4 was weak and thus omitted to further analysis. S100A6 faintly labelled keratinocytes. Langerhans' cells, melanocytes and sweat glands. S100A6 immunoreaction was found in two of seven junctional naevi, five of seven compund naevi, and all dermal and blue naevi. There was an intense cytoplasmatic reaction for S100A6 in all primary cutaneous melanomas and in nine of 14 (64%) metastases. S100B was positive in melanocytes and Langerhans' cells, all primaries as well as in the metastases. S100A1 protein was not detected on any of the tissue specimens examined. Whereas S100B and S100A6 antibodies are useful markers for malignant melanoma, expression of S100A4 antibody is too low to be used for immunohistochemical staining. S100A1 and S100A3 antibodies are not expressed in melanocytic lesions and S100A2 is only found in selected tumours. The investigated S100 proteins, including S100B and S100A6, are also expressed in selected elements of normal skin. These findings are important for correct interpretation of staining patterns, when S100 antibodies are used as markers for melanoma or other tumours.     

PUVA therapy decreases HLA-DR+ CD1a+ Langerhans cells and epidermal cell antigen-presenting capacity in human skin, but flow cytometrically-sorted residual HLA-DR+ CD1a+Langerhans cells exhibit normal alloantigen-presenting function

We have investigated the effects of PUVA therapy on human Langerhans cell (LC) immunophenotype and function. Epidermal sheets were obtained from exposed, and control shielded, forearm skin at the end of a course of PUVA therapy, in patients receiving treatment routinely for a variety of dermatoses. PUVA therapy decreased the overall number of HLA-DR+CDIa+ LCs in epidermal sheets, and in epidermal cell (EC) suspensions examined using a fluorescence activated cell sorter (FACS). PUVA therapy also reduced the overall EC allostimulatory capacity in the allogeneic epidermal cell-lymphocyte reaction (ELR), and the capacity of ECs to present tetanus toxoid to, and augment concanavalin A-mediated stimulation of, lymphocytes in the autologous ELR. Depressed allostimulation by ECs from PUVA-treated skin could not be restored by indomethacin (added to block prostaglandin synthesis). The reductions in LC numbers and EC allostimulatory capacity varied according to dose, and time since cessation, of PUVA therapy, and in individual patients were of comparable degree. By contrast, the allostimulatory capacity of residual LCs from PUVA-treated skin (purified using the FACS) did not differ from that of purified control LCs. PUVA-induced suppression of cutaneous immune responses, therefore, results at least in part from an overall impairment of EC antigen-presenting capacity. Residual HLA-DR+CDIa+ LCs in PUVA-treated skin which retain their alloantigen-presenting function may represent a subgroup of PUVA-resistant LCs; alternatively, these cells may be as yet unaffected because they have only recently migrated into the epidermis.     

Antigen-presenting capacity in normal human dermis is mainly, subserved by CDla+ cells

A proposed role for antigen-presenting dermal dendrocytes in the pathogenesis of many dermal inflammatory skin diseases remains speculative. We therefore sought to determine the phenotype and functional characteristics of antigen-presenting cells isolated from normal human dermis. Normal adult human skin was incubated overnight with dispase at 4°C, the epidermis was removed, and the residual dermal preparation was then minced and digested with a mixture of hyaluronidase, collagenase, and DNAase at 37°C, prior to filtration through mesh. Dermal cell suspensions thus obtained were stained using specific monoclonal antibodies, and analysed by fluorescence micro- scopy or flow cytometry. Mean values were as follows: CD45⁺ leucocytes 39%, HLA-DR⁺ cells 39%, Ulex europaeus agglutinin I⁺ endothelial cells 26%, CD1a⁺ cells 3.9%, CD11b⁺ cells 16%, CDllc⁺ cells 6%. Mitomycin C-treated crude dermal cell suspensions induced allostimulation of peripheral blood mononuclear cells in a 7-day culture, as assessed by ³H-TdR incorporation. Depletion of CDla⁺ Langerhans-like cells from the dermal cell preparation, by 95, 74 and 90% in three separate experiments using immunomagnetic beads, reduced ³H-TdR incorporation at optimal responder-to- stimulator cell ratios by 90, 64, and 87%, respectively. Our findings suggest that, in normal human dermis, the great majority of the alloantigen-presenting capacity resides in the CDla⁺ Langerhans cell-like dendritic antigen-presenting cell population, and not to any great extent in either CDla^? macrophage-like cells, or HLA-DR⁺ endothelial cells. The relationship of the CDla⁺ dermal antigen presenting cells to the Langerhans cell lineage remains to be determined.     

The effect of H1 and H2 histamine antagonists on symptomatic dermographism

In ten patients suffering from symptomatic dermographism the combined administration of chlorpheniramine + cimetidine produced a greater reduction in the weal and flare response provoked by a standardized scratch than the administration of chlorpheniramine alone. There was a statistically significant improvement in the overall assessment of the patient's skin condition with the combined administration of chlorpheniramine + cimetidine. Chlorpheniramine given alone produced no significant benefit whilst cimetidine alone produced a marked exacerbation in itching in nearly half the patients who initially entered the study and was sufficient to require withdrawal.     

Suppression of alcohol-induced flushing by a combination of H1 and H2 histamine antagonists

Alcohol-induced flushing of the face and neck regions is a well recognized condition, probably genetically determined and due to individual differences in alcohol metabolism. We have shown that the flushing appears only after a threshold level of blood alcohol (20–35 mg per 100 ml) is exceeded. The rate of alcohol absorption and peak blood levels were reduced by a combination of H₁ and H₂ antagonists. This reduction was accompanied by abolition of flushing in sensitive individuals.     

Divalent cations (Mg2+, Ca2+) differentially influence the β1 integrin-mediated migration of human fibroblasts and keratinocytes to different extracellular matrix proteins

Abstract Directed migration of keratinocytes and fibroblasts is a fundamental prerequisite in wound healing. Cation-dependent affinity changes of integrins are responsible for cell adhesion to and deadhesion from extracellular matrix proteins and have been implicated in driving cell migration. The specific requirements for divalent cations in the integrin-dependent migration of human dermal fibroblasts and human epidermal keratinocytes to various extracellular matrix proteins have been studied in vitro using blindwell Boyden chambers. The migration of the tested cells to collagen type I was mediated by the α₂β₂ integrins, to fibronectin by the combined action of the α₃β₂ and the α₅β₁ integrin, and the migration of fibroblasts to laminin dependent both on the α₂β₁ and the α₆β₁ integrins. No migration of keratinocytes to laminin was detected. Mg²⁺ alone induced cell migration with an optimum at 2 mM for fibroblasts and at 10 mM for keratinocytes. Ca²⁺ alone at 2 mM only marginally enhanced fibroblast and keratinocyte migration. At higher concentrations Ca²⁺ suppressed the stimulatory Mg²⁺ effect. 2 mM Ca²⁺ combined with 2 mM Mg²⁺ showed an additive stimulatory effect on the migration of fibroblasts to fibronectin. These data suggest that extracellular divalent cations differentially influence the integrin-mediated cell migration. A concentration gradient of Mg²⁺/Ca²⁺, as reported in tissue injury, thus may play a regulatory role in cell migration required for tissue remodelling.     

H2 Blockers in Chronic Urticaria

Two hundred unselected patients with chronic idiopathic urticaria were treated with adequate doses of conventional antihistamines given at frequent intervals; 98.5% were completely freed of disease while on therapy. In three cases, the lesions subsided but did not clear completely. The addition of 800 mg of cimetidine in divided doses cleared the lesions in two cases, and in the third case the dose of H1 blocker was increased, which resulted in complete clearance of wheals and symptoms. Addition of H2 blockers play a role, but only in a very small percentage of patients with chronic idiopathic urticaria who do not completely respond to adequate doses of H1 blockers.     

Lack of antagonism to Ni2+ and Co2+ contact allergy from other essential divalent metal ions

The potential antagonistic effects of Ca²⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ on contact allergy to Co²⁺ and Ni²⁺ were studied. The immune response was characterized by the Co²⁺ or Ni²⁺ mediated cellular [methyl-³H]thymidine uptake in peripheral blood mononuclear cell (PBMC) cultures from 6 subjects contact-allergic to Co²⁺ and Ni²⁺ and 6 non-allergic control individuals. Results from the in vitro experiments were further evaluated with Co²⁺-sensitized guinea pigs according to the modified Freund's complete adjuvant test. Ni²⁺ and Co²⁺ (10–50 μM) significantly increased the lymphocyte proliferation in PBMC cultures from contact-allergic subjects in comparison with those from control individuals. Pretreatment of the PBMCs with Ca²⁺, Fe²⁺, Mg²⁺ (10–100 μM) or Mn²⁺ (1–10 μM) did not influence, while Zn²⁺ (100 μM) enhanced, and Cu²⁺ (5 and 10 μM) markedly reduced the Ni²⁺ and Co²⁺ mediated cellular [methyl-³H]thymidine uptake. The inhibition of the Ni²⁺- and Co²⁺-induced cell proliferation by Cu²⁺ in vitro was probably related to toxicity, since the viability of the cells was significantly reduced by applied combinations of Ni²⁺ or Co²⁺ with Cu²⁺. Topical pretreatment of Co²⁺-sensitized guinea pigs with maximum non-irritating doses of CuCl₂· 2H₂O (0.8%) did not affect the challenge testing to CoCl₂· 6H₂O (0.1 and 0.3%). In conclusion, our combined in vitro and in vivo results indicate that Ca²⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ are not able to antagonise the formation of Ni²⁺ and Co²⁺ antigens.     

Enhancement of delayed hypersensitivity in guineapigs by H2-receptor antagonist

Guinea-pigs have been sensitized with dinitrochlorobenzene (DNCB) to produce type IV-reactions. The reactions spontaneously faded in the untreated animals. In contrast, in the group treated with cimetidine, the reaction remained essentially unchanged. Cimetidine thus caused an augmentation by preventing the spontaneous fading. The enhancement of the type IV-reaction by cimetidine is possibly caused by supression of regulatory T-lymphocytes through their H₂-receptors. The histamine content of the sensitized and challenged sites increased both in the untreated and cimetidine-treated groups.     

p120 catenin is associated with desmogleins when desmosomes are assembled in high-Ca2+ medium but not when disassembled in low-Ca2+ medium in DJM-1 cells

We recently showed that p120 catenin (p120ctn), which is an armadillo family protein member that binds to E-cadherin (E-cad), is also localized to desmosomes by directly or indirectly binding to desmogleins (Dsg). We examined whether p120ctn is associated with Dsg1 and Dsg3, as compared with E-cad and plakoglobin (PG), in keratinocytes grown in high or low Ca²⁺, using a human squamous cell carcinoma cell line, DJM-1 cells. The cell lysate of DJM-1 cells grown in high- or low-Ca²⁺ media was immunoprecipitated with anti-Dsg1/2 and Dsg3 antibodies, and we examined whether p120ctn is associated with Dsg1 and Dsg3. Then, we observed the co-localization between Dsg3 and p120ctn in cells grown in high- or low-Ca²⁺ medium on double-staining immunofluorescence microscopy using anti-p120ctn and anti-Dsg3 antibodies. Immunoprecipitates with anti-Dsg1/2 and Dsg3 antibodies in cells grown in high-Ca²⁺ medium contained p120ctn. In contrast, in low-Ca²⁺ medium, p120ctn was co-immunoprecipitated with neither Dsg1 nor Dsg3, but was co-immunoprecipitated with E-cad in cells grown in both high- and low-Ca²⁺ media. Dsg3 was associated with PG in cells grown in both low- and high-Ca²⁺ media. On immunofluorescence microscopy, p120ctn and Dsg3 were independently observed in cells grown in low-Ca²⁺ medium; p120ctn, but not Dsg3, was observed in a linear pattern at the cell–cell boundary. However, they were co-localized at cell–cell contacts in cells grown in high-Ca²⁺ medium. Thus, these proteins are not co-localized in low Ca²⁺ medium. These results suggest that p120ctn plays an important role in Ca²⁺-induced desmosome formation.     

T6+ and HLA-DR+ cell numbers in epidermis of immunosuppressed renal transplant recipients

The increased susceptibility of the skin of chronically immunosuppressed individuals to viral infections and sunlight-induced malignancies suggests specific drug-induced, dysfunction of local immune mechanisms within the sun-exposed skin of these individuals. To help understand the effect of immunosuppressive therapy alone in the absence of ultraviolet light on the immune system of skin, biopsies were collected from non-sun-exposed buttock skin of control, healthy volunteers and kidney transplant recipients immunosuppressed with either azathioprine/prednisone or cyclosporin A/prednisone and examined for incidences of T6+, and HLA-DR+ cells. No significant differences in the incidences of these 2 cell types were found (a) between control individuals and transplants recipients, (b) between transplant recipients receiving either of the immunosuppressive drug regimes, or (c) between transplant recipients who either had or had not developed skin cancer.     

In-vivo administration of recombinant IL-2 increases the number of Thy-1+ dendritic epidermal cells

The number, morphology and response of Thy-1+ dendritic epidermal cells to recombinant interleukin-2 (rIL-2) were investigated in young and aged mice. The Thy-1+ dendritic cells in the aged mice continued to express T-cell receptor (TCR) gamma/delta but differed morphologically from those of the young mice. The aged mice had 50% fewer cells than the young ones. In the rIL-2 treated mice all the Thy-1+ cells were expressed as TCR gamma/delta and exposure to rIL-2 increased the number of these cells in a time- and dose-dependent manner when given systemically and locally. In the aged mice daily injection of rIL-2 increased the number of Thy-1+ dendritic cells within 2 weeks to almost that of young mice, however they had a lower response in the earlier stage. Nude mice showed no response to rIL-2.     

An evaluation of Na+,Cl− and pH ion-specific electrodes in the study of the electrolyte contents of epidermal transudate and sweat

Ion-specific electrode detectors have been used to measure electrolytes in various biological fluids, including sweat. As part of a study of skin barrier function, we have used such electrodes to detect the presence of sodium, chloride and hydrogen (pH) in the transepidermal water loss. This paper describes the effects of cellophane tape stripping (of human forearm epidermis in vivo) on surface electrolyte concentrations, in parallel with observations on transepidermal water loss and galvanic skin resistance.     

The ICAM-3/LFA-1 interaction is critical for epidermal Langerhans cell alloantigen presentation to CD4+ T cells

Intercellular adhesion molecule (ICAM)-3 is a recently described member of the immunoglobulin superfamily and, as such, is closely related to ICAM-1 and ICAM-2. All three ICAMS are cognate for the counter-receptor lymphocyte function associated antigen-1 (LFA-L CD11a/CD18). Unlike ICAM-1 and ICAM-2. ICAM-3 is constitutively expressed at high levels on resting leucocytes. We investigated the expression and function of ICAM-3 in normal skin (n= 5), as well as its expression in psoriasis (n= 4). atopic eczema (n= 4), allergic (rhus) contact dermatitis (n=3). and cutaneous T-cell lymphoma (CTCL. n=2). Five-micrometre cryostat sections of skin were stained using monoclonal antibodies to ICAM-3 and A well characterized immunoperoxidase technique. In normal skin. ICAM-3 was expressed by all cutaneous leucocytes hut most striking was the strong expression of ICAM-3 by Langerhans cells within both epidermis and dermis. This observation was confirmed by double-labelling with CD1a and negative staining with an IgG1 isotype control. In psoriasis, atopic eczema, allergic contact dermatitis, and CTCL. ICAM-3 was co-expressed on all CD1a⁺ cells, although, in psoriasis, the intensity of ICAM-3 expression was reduced. Functional blocking experiments were performed to determine whether the observed ICAM-3 expression on Langerhans cells was functionally important in antigen presentation. CD4⁺ T cells were prepared from peripheral blood and 10⁵ CD4⁺ T cells combined with 10⁵ epidermal cells harvested from keratome biopsies of normal skin of an individual allogeneic to the T-cell donor. Addition of 50 μg anti-ICAM-3 to the co-culture resulted in a consistent (50%) reduction in degree of alloantigen presentation by Langerhans cells to T cells. Inhibition was 77% of that produced by the addition of anti-LFA-1. These data indicate that ICAM-3 is constitutively expressed by Langerhans cells and is a major ligand for LFA-1 on CD4⁺ T cells during their response to Langerhans cells. Because fresh Langerhans ceils constitutively express little ICAM-1. whereas ICAM-3 is constitutively expressed at high levels, it would appear that 1CAM-3 is the dominant functional ICAM on in situ Langerhans cells in the normal epidermis.     

Roles of CD4+ and CD8+ cells in UVB-induced suppression of splenic alloantigen presenting function

Abstract Whole body ultraviolet light (UV) radiation causes systemic immunosuppression. Splenic antigen-presenting cell (APC) activity is decreased by UV radiation. To determine whether splenic CD4⁺ or CD8⁺ cells are involved in the UV-induced depression of splenic alloantigen presenting function, we investigated the effect of in vivo UV radiation on the splenic stimulatory function in allogeneic mixed lymphocyte reaction in mice after the elimination of CD4⁺ or CD8⁺ cells by administering anti-CD8 or anti-CD4 Ab. Ab-treated and non-treated mice were exposed to UVB radiation (2.5 k.J/m²) twice or eight times. Two exposures to UVB radiation significantly suppressed the splenic alloantigen-presenting function of mice previously treated with anti-CD4 Ab, but hardly affected that of anti-CD8 Ab-treated or non-treated mice 2 days after the last radiation. On the other hand, eight exposures to UVB radiation suppressed this function in all mice. FACS analysis revealed that the UV induced suppression is not associated with a significant decrease in the number of IA⁺ cell, as stimulator cells. It is suggested that CD4⁺ cells are somewhat preventive of the UV-induced depression of splenic alloantigen-presenting function.     

Flow cytometric analysis and sorting of HLA-DR+CD1+ Langerhans cells

There is a considerable need for reliable methods for enumeration and enrichment of Langerhans cells (LCs), since they continue to be the subject of intensive investigation in normal and diseased skin. It has been claimed that standard labelling with either anti-HLA-DR or OKT6 antibodies alone may fail to identify potentially important subsets of LCs with the phenotypes HLA-DR+CD1- and HLA-DR-CD1+. We report here on flow cytometric analysis of suction blister-derived normal epidermal cell (EC) suspensions, double stained with phycoerythrin-conjugated anti-HLA-DR and fluoresceinated OKT6. In seven separate experiments, no evidence for the existence of either HLA-DR+CDI- or HLA-DR-CDI+ ECs was obtained. We found that HLA-DR+CDI+LCs, which constituted a mean of 2.5% (+/- 0.3 SEM) of all ECs, could be readily identified on the basis of fluorescence, and that their light scatter characteristics were those of moderately sized cells of low granularity. We further describe our method for flow cytometric enrichment of such HLA-DR+CDi+ LCs for functional studies, based on selection on both fluorescence and light scatter criteria. Enrichment is to greater than 90% purity, and the method is applicable to the small number of ECs (approximately 1 x 10(6] obtained from a suction blister.     

Histamine H2-receptor antagonists inhibit enzymic histamine degradation in human skin

The histamine H₂-receptor antagonists burimamide and cimetidine cause inhibition of human skin histamine-N-methyl transferase (HMT) in the dose range 10^-3–10^-4 M. Amodiaquin, a recognized inhibitor of animal HMT, also produced dose-related inhibition of human HMT. These findings may explain the observation that H₂-receptor antagonists enhance certain types of inflammatory response in skin.     

Oral submucosal dendrocytes: factor XIIIa+ and CD34+ dendritic cell populations in normal tissue and fibrovascular lesions

Factor XIIIa+ and CD34+ dendritic cells, believed to be subsets of monocyte/macrophages, have been identified in dermis and in dermal tumors. The purpose of this study was to determine the presence and distribution of analogous cell types in oral submucosa and oral fibro-vascular lesions. Antibodies to XIIIa, CD34, S-100 protein, and macrophage antigen (MAC 387) were tested on formalin-fixed, paraffin-embedded tissue sections from normal mucosa, peripheral fibroma (PF), peripheral ossifying fibroma (POF), peripheral giant cell granuloma (PGCG), pyogenic granuloma (PG), lymphangioma (La), benign fibrous histiocytoma (BFH), idiopathic histiocytosis (IH), angiofibroma (Af) using an ABC immunoperoxidase technique. Numbers of positively stained cells were compared to unstained cells in the tumors. XIIIa positive submucosal dendrocytes (CD34-, S-100-, MAC 387-) were found in abundance in normal tissue in characteristic distributions: collagen-associated, vessel-associated, and lymphoid-associated. The percentage of XIIIa+ cells in the oral tumors was as follows: PF: 10-30%, POF: 5-10%, PGCG: 0-5%, PG: 5-20%, La: 0%, BFH: 5-25%, IH: 0%, and Af: 10-20%. CD34+ dendrocytes (XIIIa-, S-100-, MAC 387-) were few in number and were found in deeper submucosa, especially around skeletal muscle. Other than blood vascular endothelium, CD34+ cells were not generally seen in the oral tumors studied. It is concluded that two previously unrecognized dendrocyte populations reside in normal submucosa. XIIIa+ cells participate in the formation of some oral reactive and neoplastic lesions.