Abstract
Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed “3D keratinocytes”), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.
Highlights
Atopic dermatitis (AD) is the most common chronically relapsing allergic skin inflammatory disease
When a nitrocellulose filter containing 2 mU/ml Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) was placed onto the stratum corneum of 3D keratinocytes and incubated for 24 h, TNF-a mRNA expression by the keratinocytes was unaltered (Fig. 1A), the concentration of PaCDase in the assay system was sufficient to degrade ceramide [35]
These findings suggest that PaCDase degraded ceramide in the Triton X-100-damaged stratum corneum and that the resulting degradation products enhanced TNF-a mRNA expression by the 3D keratinocytes
Summary
Atopic dermatitis (AD) is the most common chronically relapsing allergic skin inflammatory disease. The stratum corneum is composed of terminally differentiated keratinocytes interspersed with intercellular lipids that are involved in both water retention and permeability barrier functions [5]. Ceramides are markedly reduced in the stratum corneum of AD skin, irrespective of the presence of lesions [8], and the skin of most patients with AD is colonized by Staphylococcus aureus (S. aureus) [9]. Decreases in ceramides may lead to increased transepidermal water loss, contributing to the dry and cracked skin that predisposes to S. aureus colonization [10]. S. aureus contains cardiolipin and phosphatidylglycerol, which enhance PaCDase hydrolysis in AD skin [13]. Several strains of P. aeruginosa, including AN17, secrete significant amounts of staphylolytic proteases that lyse S. aureus cells, resulting in their release of cardiolipin and phosphatidylglycerol. It is likely that PaCDase hydrolyzes ceramide in atopic skin
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