Proliferation and interferon-γ receptor expression in psoriatic and healthy keratinocytes are influenced by interactions between keratinocytes and fibroblasts in a skin equivalent model

Epidermal-dermal interactions were studied in a skin equivalent model. Six combinations of keratinocytes and fibroblasts from healthy and psoriatic skin were used. TPA (12-O-tetradecanoylphorbol-13-acetate) was used to determine whether the expression of the IFN- receptors in keratinocytes was related to epidermal differentiation and proliferation. These phenomena were assessed by immunohistochemistry. In all epidermal outgrowths, the epidermal growth factor receptor was expressed throughout the epidermis, cytokeratin 16 suprabasally, and filaggrin and involucrin in its superficial part. The IFN- receptor was expressed throughout the epidermis, but was unevenly distributed. The expression of the IFN- receptor was quantified by confocal laser scanning microscopy both in the whole of epidermis and in areas with the strongest intensity. The total amount varied to a minor degree in the epidermal outgrowths of different origins and was unaffected by TPA. In high-intensity areas interactions between keratinocytes and fibroblasts did influence the amount of IFN- receptor expression and TPA decreased the expression by 13%. There was no correlation between the proliferation rate and the expression of the IFN- receptor. Psoriatic and healthy keratinocytes were equally well differentiated in the skin equivalents. The interferon- receptor was similarly expressed under these conditions. The growth rate, assessed by Ki-67-positive nuclei in the basal layer, was highest in healthy keratinocytes. Keratinocytes from psoriatic lesions increased their growth rate when cocultured with psoriatic fibroblasts compared with normal ones, indicating that fibroblasts may be of importance for epidermal hyperproliferation in psoriatic lesions.     

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

Effects of interferon-γ treatment on the cutaneous DTH reaction in rats

Summary The capacity of interferon- (IFN-) to induce class II histocompatibility antigens on different cell types including keratinocytes, is well known, but the impact of IFN- on the immune response is still unclear. Lewis rats sensitized with dinitrofluorobenzene (DNFB) were injected with recombinant rat IFN- (10⁵ U) or phosphate-buffered saline (PBS) once daily on 3 successive days at the bases of the ears either before or after they were challenged on the ears. As expected, the PBS-treated animals showed about a 30% increase in ear thickness and there was an induced expression of class II antigens on the keratinocytes as judged by immunohistochemistry 72 h after challenge. Exogenously added IFN- prior to DNFB challenge resulted in a significantly reduced ear swelling at 24 (p<0.01) and 48 h (p<0.05) after challenge. In this case the keratinocytes expressed class II antigens already at the time of challenge. When IFN- injections were given during the contact allergic reaction there was no significant reduction of ear swelling until 72 h (p<0.01). At that time point there was a more pronounced expression of class II antigens on the keratinocytes compared with PBS-injected animals, due to the IFN- treatment. These in vivo data support our previous observations that IFN- may play a self-limiting role in certain immune responses.     

Activation of phospholipase C-γ1 in human keratinocytes by hyperosmolar shock without enzyme phosphorylation

Human keratinocytes are exposed to strong physical changes, and have the potentiality to react to external stimuli by switching on adaptation mechanisms. In hyperosmotically shocked keratinocytes a rapid and strong increase in calcium has been observed. We showed that this increase could not be prevented by growing the cells in medium devoid of calcium and in the presence of EGTA, indicating that the intracellular calcium increase was due to delivery from internal stores. Further, we observed an increased synthesis of dyacylglycerol and inositol trisphosphates after shock, suggesting that phospholipase C mediates both events. Our experiments demonstrated that osmotic shock in human keratinocytes leads to activation of phospholipase C-1, as measured using an in vitro assay system. This activation is independent of protein tyrosine phosphorylation and corresponded to a relocation of the enzyme to perinuclear membranes as shown by immunofluorescence.Abbreviations 2-APB 2-aminoethoxydiphenylborate - IP inositol phosphate - PA phosphatidic acid - PBS phosphate-buffered saline - PI-PLC phosphatidyl inositide-specific PLC - PIP phosphatidylinositolphosphate - PIP₂ phosphatidylinositol(4,5)bisphosphate - PIP₃ phosphatidylinositol(3,4,5)trisphosphate - PLC phospholipase C - PLD phospholipase D     

A single intradermal injection of IFN-γ induces an inflammatory state in both non-lesional psoriatic and healthy skin

Psoriasis is a chronic, debilitating, immune-mediated inflammatory skin disease. As IFN-γ is involved in many cellular processes, including activation of dendritic cells (DCs), antigen processing and presentation, cell adhesion and trafficking, and cytokine and chemokine production, IFN-γ-producing Th1 cells were proposed to be integral to the pathogenesis of psoriasis. Recently, IFN-γ was shown to enhance IL-23 and IL-1 production by DCs and subsequently induce Th17 cells, which are important contributors to the inflammatory cascade in psoriatic lesions. To determine whether IFN-γ indeed induces the pathways expressed in psoriatic lesions, a single intradermal injection of IFN-γ was administered to an area of clinically normal, non-lesional (NL) skin of psoriasis patients and biopsies were collected 24 hours later. Although there were no visible changes in the skin, IFN-γ induced many molecular and histological features characteristic of psoriatic lesions. IFN-γ increased a number of differentially expressed genes in the skin, including many chemokines concomitant with an influx of T cells and inflammatory DCs. Furthermore, inflammatory DC products tumor necrosis factor (TNF), inducible nitric oxide synthase, IL-23, and TNF-related apoptosis-inducing ligand were present in IFN-γ-treated skin. Thus, IFN-γ, which is significantly elevated in NL skin compared with healthy skin, appears to be a key pathogenic cytokine that can induce many features of the inflammatory cascade of psoriasis.     

Inhibition of benzalkonium chloride-induced skin inflammation in mice by an indol-1-ylpropan-2-one inhibitor of cytosolic phospholipase A2 α

Background Irritant contact dermatitis (ICD) is a frequent and often underrated problem for which the major efficacious therapy is still local glucocorticoids, although they have known adverse effects due to their wide spectrum of action. A more focused therapeutic strategy would be the inhibition of a key enzyme for biosynthesis of the lipid mediators, cytosolic phospholipase A₂α (cPLA₂α), in ICD. We are analysing the pharmacological and biological effects of a selective cPLA₂α inhibitor. Objectives To examine the usefulness of the potent and selective cPLA₂α inhibitor 3‐(5‐carboxypentanoyl)‐1‐[3‐(4‐octylphenoxy)‐2‐oxopropyl]indole‐5‐carboxylic acid (compound 1) for therapy of inflammatory skin disorders. Methods We examined clinical and cellular effects of a selective cPLA₂α inhibitor (compound 1) on ICD in mice. Results Topical application of the compound significantly reduced ear swelling after induction by the irritant benzalkonium chloride. Concomitantly, compound 1 inhibited the accumulation of granulocytes as well as the expression of inflammatory proteins such as tumour necrosis factor‐α, interleukin‐1β and macrophage inflammatory proteins 1α and 1β in the ear tissue. In primary murine keratinocytes, the benzalkonium chloride‐induced expression of these proteins was also downregulated after treatment with compound 1 in vitro. Conclusions Compound 1 is a well‐aimed agent for the treatment of nonspecific skin inflammation as it selectively inhibits cPLA₂α and as it acts on an early stage of skin inflammation after its elicitation.     

What is really in control of skin immunity: lymphocytes,dendritic cells,or keratinocytes? facts and controversies

The pathophysiology of atopic dermatitis is still under discussion. Although it is widely accepted that environmental factors and a genetic predisposition are essential, the role of the innate and adaptive immune system and the functional cascade of the cells involved is still unclear. A concept that integrates all immune cells as equally essential has allure. In addition, barrier abnormalities due to mutations of the gene coding for filaggrin and down-regulation of antimicrobial peptides, such as LL-37 and β-defensins 2 and 3, were very recently found to be relevant for the pathogenesis of atopic dermatitis.     

Bacterial skin infections in children hospitalized with varicella: a possible negative impact of non-steroidal anti-inflammatory drugs?

This 1-year multicentre prospective study in northern France sought to evaluate the incidence of secondary bacterial skin complications related to varicella, describe these superinfections, and analyse risk factors for their onset. The study included every child admitted to a district paediatric unit with a varicella infection. Patients with varicella infection, with and without secondary bacterial skin complication, were compared. The study included 159 children, 43 of whom had a secondary bacterial skin complication on admission, 21 of them had a severe secondary bacterial skin complication (respective incidence: 7.5 and 3.7/100,000 children younger than 16 years old). Persistence or recurrence of fever > or =38.5 degrees C for > or =3 days after the beginning of varicella infection (adjusted odds ratio (aOR)=8.1; 95% confidence interval (CI): 2.3-28.4) and the use of non-steroidal anti-inflammatory drugs (aOR=4.8; 95% CI: 1.6-14.4) were independent factors associated with severe secondary bacterial skin complication.     

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in mouse skin: does S. aureus generally produce a biofilm on damaged skin?

BACKGROUND: Bacteria that adhere to damaged tissues encase themselves in a hydrated matrix of polysaccharides, forming a slimy layer known as a biofilm. This is the first report of detection of glycocalyx production by Staphylococcus aureus using confocal laser scanning microscopy (CLSM) on damaged skin tissues. OBJECTIVES: To analyse glycocalyx production by S. aureus cells on damaged skin tissues and the influence of polymorphonuclear leucocytes (PMNs) and various antimicrobial agents on its production using CLSM in cyclophosphamide (Cy)-treated (neutropenic) or non-Cy-treated (normal) mice. METHODS: S. aureus cells were inoculated on damaged skin tissues in neutropenic or normal mice with or without topical application of antimicrobial agents. S. aureus cells were stained with safranine, and positive staining with fluorescein isothiocyanate-conjugated concanavalin A was considered to indicate the presence of glycocalyx. RESULTS: All S. aureus cells tested on damaged skin tissues formed microcolonies encircled by glycocalyx. The colony counts of S. aureus cells on croton oil dermatitis in normal mice treated with 2% fusidic acid ointment were about 100 times lower than those in neutropenic mice (control). CONCLUSIONS: As S. aureus cells can generally produce a biofilm on damaged skin tissues, antimicrobial agents may not eradicate S. aureus cells without the help of PMNs. S. aureus glycocalyx may play a crucial role in colonization and adherence to damaged skin tissues.     

Organ culture of psoriatic skin: effect of TGF-α and TGF-Β on epidermal structure in vitro

Summary Normal skin and uninvolved and involved psoriatic skin specimens were maintained in vitro in organ culture. The 3–4 mm punch-biopsied skin specimens were put freely into the culture medium with or without fetal calf serum, under an atmosphere of 95% O₂ plus 5% CO₂, and rotated at 60 rpm at 37C. In the serum-free culture medium (vitamin A-free) granular layers appeared in the involved psoriatic epidermis in culture. Addition of TGF- caused normal skin and uninvolved and involved psoriatic skin specimens to become acanthotic and to degenerate easily almost to the full thickness of the epidermal layer in proportion to increasing concentrations of TGF- as well as with the duration of the culture, but without disappearance of their granular layers. TGF- caused the normal skin and uninvolved psoriatic skin specimens to become thinned without disappearance of granular layers, but caused the involved psoriatic skin specimens to be thinned without appearance of granular layers in serumcontaining medium or with their disappearance in the serum-free medium. TGF- also antagonized the acanthotic and degenerative effect of TGF-. The results suggest that TGF- and TGF- may partially be related to the induction of psoriatic epidermal lesions.     

Is there any barrier impairment in sensitive skin?: a quantitative analysis of sensitive skin by mathematical modeling of transepidermal water loss desorption curves

Background/purpose: Sensitive skin is a vague, subjective and difficult to characterize affliction. It affects a large part of the population and is accompanied with great interest by the cosmetic industry. Some studies have suggested that sensitive skin is the result of impaired barrier function, which leads to the exposure of immune system cells and sensitive nerves, resulting in marked cutaneous responses to otherwise harmless stimuli. This study aimed to investigate the cutaneous barrier integrity of individuals with sensitive skin by a novel approach: a plastic occlusion stress test followed by measurement of transepidermal water loss (TEWL) desorption curves. Methods: The study was conducted in volunteers with sensitive skin in the hands and a control group with no sensitivity complaints. A previously developed mathematical model was adjusted to the TEWL data points and two parameters were calculated: dynamic water mass and the evaporation half‐life period. Results: Statistically significant differences have been detected in the parameters obtained in the sensitive skin group, which supports the thesis that individuals with an increased skin susceptibility have impaired barrier function. Conclusion: Whereas in the studies based in basal TEWL measurements only discrete differences were reported, the dynamic approach followed in this study provided unequivocal evidence of barrier impairment. The methodology enabled a more objective characterization of sensitive skin and can potentially be applied to the diagnosis/prediction of sensitivity; as well as the efficacy assessment of cosmetic products that are specifically designed to fulfill the needs of consumers with this skin condition.