Differential effects of topical corticosteroid and calcineurin inhibitor on the epidermal tight junction

Tight junction (TJ) is one of the functional barriers present in the skin. Although topical corticosteroids and calcineurin inhibitors are used widely for atopic dermatitis, the effect of these agents on TJs has not been reported. We investigated the structural changes of TJs in mice skin after application of 0.05% clobetasol propionate or 0.1% tacrolimus ointment for 10 days. Clobetasol caused epidermal thinning and decreased collagen density. Basal transepidermal water loss was significantly increased in clobetasol‐treated versus vehicle‐ or tacrolimus‐treated skin. Confocal immunofluorescence showed that clobetasol altered the structure of claudin‐1,‐4 and occludin. Tacrolimus also caused morphological alteration of occludin. Western blot and real‐time PCR revealed that clobetasol significantly decreased claudin‐1,‐4 and occludin, whereas tacrolimus did not significantly affect claudin‐1 and ‐4 but downregulated occludin to a lesser extent compared to clobetasol. In conclusion, we suggest that downregulation of TJ proteins expression is another pathomechanism of corticosteroid‐induced permeability barrier disruption.     

Tight junction proteins: a novel class of integral membrane proteins. Expression in human epidermis and in HaCaT keratinocytes

Tight junction proteins comprise a novel group of integral membrane proteins necessary for cell-to-cell contacts and responsible for the barrier function in epithelial and endothelial cells in various tissues. The tight junction membrane domain contains at least three distinct proteins, named occludin, claudin and junctional adhesion molecule. Claudins are products of a gene family consisting of more than 20 members. We investigated mRNA expression of occludin and 13 different claudins in neonatal foreskin, adult skin and cultivated HaCaT keratinocytes by the Northern blot technique, and performed immunohistochemical staining of adult skin for occludin, claudin 1 and claudin 2. Occludin, claudin 1 and claudin 3 mRNAs were expressed in human neonatal and adult keratinocytes as well as in HaCaT keratinocytes. All other tested claudins were negative. Immunohistochemical staining of adult skin was positive for occludin in the intercellular space of the granular layer, and for claudin 1 in the inter-cellular space of the spinosum layer and basal layer, but negative for claudin 2 in all skin layers. Claudin 1 was also positive in the outer root sheath of hair follicles. Our results indicate that occludin, claudin 1 and claudin 3 are involved in cell-to-cell contacts between keratinocytes in human epidermis, although their functional importance remains unknown.     

Tight junction-associated proteins (occludin, ZO-1, claudin-1, claudin-4) in squamous cell carcinoma and Bowen's disease

BACKGROUND: The epidermis, which is a typical stratified epithelium, has tight junctions (TJs) in the granular layer, as do simple epithelia. So far, abnormalities of TJs and involvement of claudin-1 have been reported in tumours of simple epithelia. OBJECTIVES: To examine the expression of TJ-associated proteins (occludin, ZO-1, claudin-1 and claudin-4) in normal human epidermis and in malignant disorders of keratinization. METHODS: Expression of the proteins in normal human epidermis, five cases of squamous cell carcinoma (SCC) of the skin and five cases of Bowen's disease (BD) was examined by immunofluorescence staining. RESULTS: In normal human epidermis, occludin, ZO-1 and claudin-4 were expressed at the cell-cell borders in the granular layer specifically or dominantly, whereas claudin-1 was expressed in the whole epithelium. In SCC, occludin, ZO-1, claudin-1 and claudin-4 were strongly expressed in tumour cells with keratinization such as cancer pearls. Claudin-1 was heterogeneously expressed in unkeratinized tumour cells, whereas expression of occludin, ZO-1 and claudin-4 was decreased or absent. In BD, aberrant expression of occludin, ZO-1, claudin-1 and claudin-4 was observed at the cell-cell borders in addition to their expression patterns observed in normal epidermis. CONCLUSIONS: Expression of occludin, ZO-1 and claudin-4 is associated with keratinization in SCC and BD. However, the heterogeneous expression of claudin-1 in SCC is not determined only by keratinization.     

Bacteriology, inflammation, and healing: a study of nanocrystalline silver dressings in chronic venous leg ulcers

BACKGROUND: Healing of venous leg ulcers (VLUs) is often stalled despite compression therapy. Increased bacterial burden and chronic inflammation are 2 factors that may prevent these chronic VLUs (CVLUs) from healing. There is evidence that nanocrystalline silver dressings may reduce bacterial levels, decrease the chronic inflammatory response, and thus promote wound healing. OBJECTIVE: To determine the effects of a nanocrystalline silver barrier dressing on wound microflora, wound inflammation, and healing in CVLUs. METHOD: Stalled VLUs in 15 patients were managed using nanocrystalline silver dressings under 4-layer compression bandages. Paired skin biopsies at baseline and at an average of study week 6.5 were analyzed for bacteria and inflammatory infiltrates. Serum silver levels were monitored, and wound healing was assessed using planimetry. RESULTS: VLUs in 4 patients healed, and 8 other patients completed the 12-week study. There was a significant reduction in the log10 total bacterial count between baseline and final biopsies (P = .011). Greater numbers of lymphocytes were associated with an increased reduction of ulcer size at week 6.5 and final assessment at week 12 (P < .05). Heavy neutrophilic infiltration in skin biopsies at week 6.5 was associated with high bacterial counts and delayed healing (P = .037). The median reduction in ulcer surface area for all patients was 83.5%. Serum silver levels increased slightly, but values were within the normal range. CONCLUSION: A nanocrystalline silver dressing combined with 4-layer bandaging was safe and successful in promoting healing in stalled CVLUs. Healing was associated with a reduction in wound bacteria and neutrophilic inflammation with an associated persistent or high lymphocyte count, as determined by wound biopsy.     

Tight junction properties change during epidermis development

In terrestrial animals, the epidermal barrier transitions from covering an organism suspended in a liquid environment in utero, to protecting a terrestrial animal postnatally from air and environmental exposure. Tight junctions (TJ) are essential for establishing the epidermal permeability barrier during embryonic development and modulate normal epidermal development and barrier functions postnatally. We now report that TJ function, as well as claudin‐1 and occludin expression, change in parallel during late epidermal development. Specifically, TJ block the paracellular movement of Lanthanum (La³⁺) early in rat in vivo prenatal epidermal development, at gestational days 18–19, with concurrent upregulation of claudin‐1 and occludin. TJ then become more permeable to ions and water as the fetus approaches parturition, concomitant with development of the lipid epidermal permeability barrier, at days 20–21. This sequence is recapitulated in cultured human epidermal equivalents (HEE), as assessed both by ultrastructural studies comparing permeation of large and small molecules and by the standard electrophysiologic parameter of resistance (R), suggesting further that this pattern of development is intrinsic to mammalian epidermal development. These findings demonstrate that the role of TJ changes during epidermal development, and further suggest that the TJ‐based and lipid‐based epidermal permeability barriers are interdependent.     

Expression of claudin‐11 by tumor cells in cutaneous squamous cell carcinoma is dependent on the activity of p38δ

The incidence of cutaneous squamous cell carcinoma (cSCC) is rapidly increasing, and the prognosis of patients with metastatic disease is poor. There is an emerging need to identify molecular markers for predicting aggressive behaviour of cSCC. Here, we have examined the role of tight junction (TJ) components in the progression of cSCC. The expression pattern of mRNAs for TJ components was determined with RNA sequencing and oligonucleotide array‐based expression analysis from cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK, n=5). The expression of CLDN11 was specifically elevated in primary cSCC cell lines (n=5), but low or absent in metastatic cSCC cell lines (n=3) and NHEKs. Claudin‐11 was detected in cell‐cell contacts of primary cSCC cells in culture by indirect immunofluorescence analysis. Analysis of a large panel of tissue samples from sporadic UV‐induced cSCC (n=65), cSCC in situ (n=56), actinic keratoses (n=31), seborrhoeic keratoses (n=7) and normal skin (n=16) by immunohistochemistry showed specific staining for claudin‐11 in intercellular junctions of keratinizing tumor cells in well and moderately differentiated cSCCs, whereas no staining for claudin‐11 was detected in poorly differentiated tumors. The expression of claudin‐11 in cSCC cells was dependent on the activity of p38δ MAPK and knock‐down of claudin‐11 enhanced cSCC cell invasion. These findings provide evidence for the role of claudin‐11 in regulation of cSCC invasion and suggest loss of claudin‐11 expression in tumor cells as a biomarker for advanced stage of cSCC.     

Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 in cultured keratinocytes

Mutations in the ATP2C1 gene encoding Ca²⁺/Mn²⁺ ATPase SPCA1 cause Hailey–Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes, in which the SPCA1 mRNA was down‐regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down‐regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by Western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1‐deficient and control keratinocytes cultured in low (0.06 mm ) or high (1.2 mm ) calcium concentration. The mRNA and protein levels of most TJ components were up‐regulated in non‐treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1‐deficient keratinocytes displayed high levels of TJ proteins claudins 1 and 4 even in low calcium. ZO‐1 did not, however, follow similar expression patterns. Protein levels of occludin, beta‐catenin, E‐cadherin, desmoplakin, desmogleins 1–3, desmocollin 2/desmocollin 3 and plakoglobin did not show marked changes in SPCA1‐deficient keratinocytes. Indirect immunofluorescence labelling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes and increased intracellular pools of TJ and desmosomal components in SPCA1‐inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD.     

Reversed cellular polarity in primary cutaneous mucinous carcinoma: A study on tight junction protein expression in sweat gland tumors

Primary cutaneous mucinous carcinoma (PCMC) is a rare sweat gland tumor characterized by the presence of abundant mucin around the tumor islands, but the molecular mechanisms for this structure are not well elucidated. Because mucin is epithelial in nature, it is likely to be produced by epithelial tumor cells, not by surrounding stromal cells. We hypothesized that the abundant mucin is a result of reversed cellular polarity of the tumor. To test this hypothesis, we conducted an immunohistological study to investigate expression of tight junction (TJ) proteins occludin and ZO‐1 in PCMC, as well as in normal sweat glands and other sweat gland tumors. Dot‐like or linear expression of TJ proteins was observed at ductal structures of sweat glands, and ductal or cystic structures of related tumors. In PCMC, however, TJ protein expression was clearly visible at the edges of tumor cell islands. This study provides evidence to show that the characteristic histological structure of PCMC is caused by inverse polarization of the tumor cells, and that TJ proteins are useful markers of ductal differentiation in sweat gland tumors.     

Transcutaneous measurement of oxygen and carbon dioxide pressure in necrobiosis lipoidica

Transcutaneous measurement of oxygen pressure (PcO2) and carbon dioxide pressure (PcCO2) was performed in nine patients with histologically confirmed necrobiosis lipoidica. None of the patients had diabetes mellitus. All measurements were taken at the lower leg. In each case, the atrophic center, the inflamed border, and the surrounding clinically normal skin of necrobiosis lipoidica were examined at 44 degrees C sensor temperature (maximal vasodilatation). Statistically significant hypoxia was found in the area of necrobiosis lipoidica, which was even more pronounced in the inflamed border. Inhalation of 100% oxygen provoked a marked increase in the PcO2 in the lesion, but the values were still significantly lower than in the normal skin. At the edge of the lesions the PcCO2 was significantly elevated. These findings support a vascular origin of necrobiosis lipoidica, involving reduced vascular perfusion combined with diffusion block.     

Visualizing CD4 T‐cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model

Background Chemokines are critical mediators of T‐cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. Objectives To visualize directly T‐cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. Materials and methods A mouse model of allergic contact dermatitis was used. T cells from oxazolone‐sensitized and ‐challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c‐SCID mice. The recipient mice were challenged with oxazolone and CD4 T‐cell homing into inflamed skin was visualized. Results T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T‐cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T‐cell migration and skin inflammation. Conclusions Our data indicate that these tissue‐selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T‐cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.     

Transcriptomic and lipidomic profiling of eicosanoid/docosanoid signalling in affected and non‐affected skin of human atopic dermatitis patients

Lipoxygenases (LOX) and cyclooxygenase (COX) are the main enzymes for PUFA metabolism to highly bio‐active prostaglandins, leukotrienes, thromboxanes, lipoxins, resolvins and protectins. LOX and COX pathways are important for the regulation of pro‐inflammatory or pro‐resolving metabolite synthesis and metabolism for various inflammatory diseases such as atopic dermatitis (AD). In this study, we determined PUFAs and PUFA metabolites in serum as well as affected and non‐affected skin samples from AD patients and the dermal expression of various enzymes, binding proteins and receptors involved in these LOX and COX pathways. Decreased EPA and DHA levels in serum and reduced EPA level in affected and non‐affected skin were found; in addition, n3/n6‐PUFA ratios were lower in affected and non‐affected skin and serum. Mono‐hydroxylated PUFA metabolites of AA, EPA, DHA and the sum of AA, EPA and DHA metabolites were increased in affected and non‐affected skin. COX1 and ALOX12B expression, COX and 12/15‐LOX metabolites as well as various lipids, which are known to induce itch (12‐HETE, LTB4, TXB2, PGE2 and PGF2) and the ratio of pro‐inflammatory vs pro‐resolving lipid mediators in non‐affected and affected skin as well as in the serum of AD patients were increased, while n3/n6‐PUFAs and metabolite ratios were lower in non‐affected and affected AD skin. Expression of COX1 and COX‐metabolites was even higher in non‐affected AD skin. To conclude, 12/15‐LOX and COX pathways were mainly upregulated, while n3/n6‐PUFA and metabolite ratios were lower in AD patients skin. All these parameters are a hallmark of a pro‐inflammatory and non‐resolving environment in affected and partly in non‐affected skin of AD patients.     

E‐cadherin and p120ctn protein expression are lost in hidradenitis suppurativa lesions

Hidradenitis suppurativa (HS) is a chronic, inflammatory skin disease affecting the pilosebaceous units in the axilla, groin and buttocks. While the pathogenesis of HS is not clear, mechanical stress exacerbates HS. In this study, we aimed to determine whether intracellular adhesive junctions may be aberrant in HS patient skin. Strikingly, we observed loss of E‐cadherin and p120ctn protein expression, two key adherens junction proteins, in ~85% of HS severe skin lesions. Moreover, loss of protein expression was apparent in non‐lesional skin from HS patients and the degree of loss positively correlated with HS Hurley Stage of disease. E‐cadherin expression was unaltered in other inflammatory skin conditions including chronic wound epithelium, atopic dermatitis, and acne vulgaris compared with healthy skin suggesting that its loss may be uniquely relevant to HS pathogenesis. A complete loss of α‐catenin, β‐catenin and ZO‐1 was not observed; however, some cytoplasmic staining of the catenins was noted in HS epithelium. We also demonstrated diminished desmosome size in HS lesional skin. Overall, our data suggested that loss of adherens junction proteins and diminished desmosome size in HS skin contributes to the skin's inability to withstand mechanical stress and provides rationale as to why mechanical stress exacerbates HS symptoms.     

Expression of Fc receptors for IgG during acute and chronic cutaneous inflammation in atopic dermatitis

Atopic dermatitis is an allergic skin disease characterized by elevated total and antigen-specific serum IgE and IgG4 levels. In acute and chronic cutaneous inflammation, large cellular infiltrates including T cells, dendritic cells and macrophages are found, especially in the dermis. These cells play an important part in the regulation of local inflammatory reactions. Receptors binding IgG (FcgammaR) are involved in dendritic cell and macrophage function. In this study, we examined the in vivo distribution and cellular expression of the three classes of leucocyte FcgammaR in human skin during acute and chronic cutaneous inflammation in atopic dermatitis. Atopy patch test skin was used as a model for acute inflammation in atopic dermatitis, while chronic lesional skin was used to investigate FcgammaR expression in chronically inflamed skin. In atopy patch test sites no increase in the number of CD1a+ dendritic cells and a slight increase in macrophages compared with non-lesional skin was observed. Our results showed increased expression of FcgammaRI (CD64) and FcgammaRIII (CD16) in acutely inflamed skin as well as in chronically inflamed lesional skin, compared with healthy and non-lesional atopic dermatitis skin. FcgammaRI was expressed by RFD1+, RFD7+ and CD68+, but not by CD1a+ dermal dendritic cells. RFD1+ dendritic cells and CD68+ macrophages were the main FcgammaRIII-expressing cells during the acute inflammatory reaction. The significant increase in expression of FcgammaRIII (CD16) and FcgammaRI (CD64) probably results from upregulation of the receptors on resident cells. Insight into the presence of FcgammaR+ cells in human skin during inflammation is important both for our understanding of skin immune reactions and the development of new therapeutic concepts.     

Matrix metalloproteinases as mediators of tissue injury in different forms of cutaneous lupus erythematosus

Background Matrix metalloproteinases (MMPs) contribute to tissue destruction, regeneration, inflammation and apoptosis and several of them are upregulated by ultraviolet (UV) radiation in skin. Although some MMPs associate with organ manifestations of systemic lupus erythematosus (SLE), their role in cutaneous lupus erythematosus (LE) is elusive. Objectives Our aim was to evaluate the expression of MMPs in SLE, subacute cutaneous LE (SCLE) and discoid LE (DLE) skin lesions and their relation to apoptosis and epidermal changes. Methods Lesional skin biopsies from 20 patients with SLE, 20 with DLE and 17 with SCLE, and from UVA/UVB‐photoprovoked skin of healthy volunteers were immunostained using antibodies to multiple MMPs and tissue inhibitors of metalloproteinases (TIMPs). The TUNEL (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labelling) method was used for detection of apoptosis. Results MMP‐3, ‐10, ‐19 and ‐26 were abundantly expressed by keratinocytes in SLE, DLE and SCLE skin samples. MMP‐7 was detected in keratinocytes in regions of oedema and vacuolization especially in SLE and SCLE, while MMP‐14 was only occasionally observed in keratinocytes. Photoprovocation did not induce MMP‐10 or ‐26 expression in skin of healthy volunteers. Epithelial TIMP‐1 expression was low while occasional positive fibroblasts were seen in the dermis. TIMP‐3 was abundantly expressed in the epidermis, endothelial cells and macrophages. Conclusions Different subtypes of cutaneous LE are fairly similar in their MMP expression profile. MMP‐3 and ‐10 mediate both epidermal changes and dermal tissue remodelling but are not present in lymphocytes. Low expression of TIMP‐1 suggests that lupus skin is characterized by proteolytic events, and targeted action using selective MMP inhibitors may reduce lupus‐induced damage in inflamed tissues.     

Somatostatin receptors are strongly expressed in palmoplantar sweat glands and ducts: studies of normal and palmoplantar pustulosis skin

Background. The acrosyringium is the target for inflammation in the chronic and intensely inflammatory skin disease palmoplantar pustulosis (PPP). The sweat‐gland apparatus seems to be an immunocompetent structure that probably contributes to skin defence. Furthermore, the sweat gland and duct may be a hitherto unrecognized neuroendocrine organ. Aim. To obtain further information about the neuroendocrine properties of the sweat‐gland apparatus by examining expression of the somatostatin receptors (SSTRs) 1–5 in healthy palmar skin and in PPP skin. Methods. Biopsy specimens were taken from 25 patients with PPP and 25 healthy controls. Immunohistochemical analysis was used to investigate expression of SSTRs 1–5. Results. SSTRs 1–5 were expressed in both epidermal and endothelial structures. The staining intensity of the sweat‐gland apparatus was more pronounced than that of the epidermis. Expression differed significantly between lesional PPP and normal plantar skin, with increased expression of SSTRs 3 and 4 in ducts in epidermis, and decreased expression of SSTR 1 in ducts in both papillary and reticular dermis. In specimens with pronounced inflammation, numerous dendritic cells with strong expression of SSTRs 1, 2 and 4 were seen, especially in the papillary dermis. Conclusions. The presence of SSTRs in palmoplantar skin, and specifically at high density in the sweat glands and ducts, might be of particular importance in skin neuroimmunoendocrinology. Although the relevance of the changes in SSTR expression in PPP skin compared with normal skin is unclear, our hypothesis is that these differences might influence the function of both the neuroendocrine and neuroimmunological properties of palmoplantar skin, especially in the sweat‐gland apparatus.