Role of Ceramide from Glycosphingolipids and Its Metabolites in Immunological and Inflammatory Responses in Humans

Glycosphingolipids (GSLs) are composed of hydrophobic ceramide and hydrophilic sugar chains. GSLs cluster to form membrane microdomains (lipid rafts) on plasma membranes, along with several kinds of transducer molecules, including Src family kinases and small G proteins. However, GSL-mediated biological functions remain unclear. Lactosylceramide (LacCer, CDw17) is highly expressed on the plasma membranes of human phagocytes and mediates several immunological and inflammatory reactions, including phagocytosis, chemotaxis, and superoxide generation. LacCer forms membrane microdomains with the Src family tyrosine kinase Lyn and the Gαi subunit of heterotrimeric G proteins. The very long fatty acids C24:0 and C24:1 are the main ceramide components of LacCer in neutrophil plasma membranes and are directly connected with the fatty acids of Lyn and Gαi. These observations suggest that the very long fatty acid chains of ceramide are critical for GSL-mediated outside-in signaling. Sphingosine is another component of ceramide, with the hydrolysis of ceramide by ceramidase producing sphingosine and fatty acids. Sphingosine is phosphorylated by sphingosine kinase to sphingosine-1-phosphate, which is involved in a wide range of cellular functions, including growth, differentiation, survival, chemotaxis, angiogenesis, and embryogenesis, in various types of cells. This review describes the role of ceramide moiety of GSLs and its metabolites in immunological and inflammatory reactions in human

Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

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

Enhancement of NK Cell Cytotoxicity Induced by Long-Term Living in Negatively Charged-Particle Dominant Indoor Air-Conditions

<div><p>Investigation of house conditions that promote health revealed that negatively charged-particle dominant indoor air-conditions (NCPDIAC) induced immune stimulation. Negatively charged air-conditions were established using a fine charcoal powder on walls and ceilings and utilizing forced negatively charged particles (approximate diameter: 20 nm) dominant in indoor air-conditions created by applying an electric voltage (72 V) between the backside of the walls and the ground. We reported previously that these conditions induced a slight and significant increase of interleukin-2 during a 2.5-h stay and an increase of NK cell cytotoxicity when examining human subjects after a two-week night stay under these conditions. In the present study, seven healthy volunteers had a device installed to create NCPDIAC in the living or sleeping rooms of their own homes. Every three months the volunteers then turned the NCPDIAC device on or off. A total of 16 ON and 13 OFF trials were conducted and their biological effects were analyzed. NK activity increased during ON trials and decreased during OFF trials, although no other adverse effects were found. In addition, there were slight increases of epidermal growth factor (EGF) during ON trials. Furthermore, a comparison of the cytokine status between ON and OFF trials showed that basic immune status was stimulated slightly during ON trials under NCPIADC. Our overall findings indicate that the NCPDIAC device caused activation of NK activity and stimulated immune status, particularly only on NK activity, and therefore could be set in the home or office buildings.</p></div

The 20 genes most strongly upregulated by S1P in 3D cultured keratinocytes.

<p>The results shown are representative data of 3 experiments.</p

Effect of S1P on levels of expression of genes encoding sphingosine kinases and S1P receptors.

<p>Nitrocellulose filters with 5 µM S1P or 0.1% DMSO (-) in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and DNA microarray analysis was performed. The results shown are representative data of 3 experiments.</p

PaCDase-produced S1P induces endothelin-1 and IL-8 production by keratinocytes.

<p>(<i>A</i>) Involvement of SphK and S1P receptor in PaCDase-enhanced endothelin-1 and IL-8 gene expression. Nitrocellulose filters without (-) or with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum, and endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 3 independent experiments. **P<0.01; ***P<0.001. (<i>B</i>) Immunohistochemical analysis. Nitrocellulose filters with Tris-buffered saline alone (-/-), or without (+/−) or with 1 mU/ml PaCDase (+/PaCD) or 5 µM S1P (+/S1P) in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum. The cells were incubated for 24 h, embedded in paraffin, sectioned, and incubated with rabbit anti-endothelin-1 IgG (endothelin-s) or mouse anti-human IL-8 IgG. The data shown represent 3 independent experiments. Bar: 25 µm.</p

PaCDase-induced endothelin-1 and IL-8 production by keratinocytes.

<p>(<i>A</i>) Expression of endothelin-1 and IL-8 mRNA is dependent on PaCDase enzymatic activity. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml mutant H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated) in TBS containing 0.1% Triton X-100 were placed on the stratum corneum. The cells were incubated for 24 h, and endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. An S18 ribosomal protein gene was used for normalization. Each bar represents the mean±SD of 5 independent experiments. **P<0.01; ***P<0.001. (<i>B</i>) Sphingosine and S1P enhance expression of endothelin-1 and IL-8 mRNA. Nitrocellulose filters without (-) or with 5 µM sphingosine, S1P, or α-hydroxy myristic acid in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum. Endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 3 independent experiments. *P<0.05; ***P<0.001.</p

PaCDase enhances TNF-α gene expression via S1P and S1P receptors in 3D keratinocytes.

<p>(<i>A</i>) PaCDase induces TNF-α gene expression in 3D keratinocytes. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated), with or without 0.1% Triton X-100, were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 10 independent experiments. **P<0.01. (<i>B</i>) Sphingosine and S1P enhance TNF-α gene expression. Nitrocellulose filters with 1 mU/ml PaCDase, 5 µM sphingosine, phytosphingosine, C14 sphingoid base, S1P, α-hydroxy myristic acid, or 0.1% DMSO (solvent control) in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR and normalized relative to a RNA encoding an S18 ribosomal protein gene. Each bar represents the mean±SD of 5 independent experiments. **P<0.01; ***P<0.001. (<i>C</i>) Involvement of SphK and S1P receptor. Nitrocellulose filters with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 5 independent experiments. *P<0.05; **P<0.01; ***P<0.001.</p

The 20 genes most strongly upregulated by PaCDase in 3D cultured keratinocytes.

<p>The results shown are representative data of 3 experiments.</p