Different responses to oxidized low-density lipoproteins in human polarized macrophages

<p>Abstract</p> <p>Background</p> <p>Oxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages.</p> <p>Results</p> <p>Human monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-β1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. <it>HMOX1 </it>gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment.</p> <p>Conclusions</p> <p>The present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-β1- and NF-κB-mediated functions of M1 macrophages, but not those of M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL.</p

Epidemiology of Infectious Hepatitis 1st. Chapter Epidemiologic Observations for Infectious Hepatitis in Akaiwa District (No. 1)

Since August 1951 to Feb. 1953, there was a great epidemic of serious infectious hepatitis over the area including Toyota, Onoda, Kama villages in Akaiwa County, as well as Kumayama village in wake County; observing its state, the following results were obtained. 1. Death toll has amounted to 13, out of 93 patients: its mortality rate, 13.98%; in number of the patients, Kama at the top, next, Toyoda, Onoda and Kumayama village, in literal order. 2. The epidemic took the direction, from the south extending to the north in a slow march, taking a hamlet as unit, showed a lack of specific seasonal occurence. 3. As to age, it has ranged roughly from 10 to 60, amid which 21-30 figured out as top years; below 10, only 4 cases; above 70, 5 cases. As to sex, almost similar in number. 4. As to profession, principally, farming people; as one can suppose from its locality. In making investigations into the cause of disease. certain consideration must be taken on the great responsibility imposed on farmers' wives. 5. The infection for this disease was classified as village infection or familiary infection; among 22 cases (23.4% of total patients) of familary infection, there were 4 families, each yielding even 3 sickened members; 5 families whose two members taken infection; in total, amnunted to 9 families. Among these tribal infection, report has been submitted about 1 family whose infection tract was clearly traced, as well as a family in which, if man did not expose certain inapparent infections, all cases have proved utterly whimsical. 6. The incubation period for this disease may probably be estimated to be about 24-27 days, if it may be supposed from these infectious state, and would be considered perorale infection

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

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-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 production of TNF-α and SphK1 by keratinocytes.

<p>PaCDase-induced TNF-α production. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase without or with 10 µM SphK inhibitor in TBS containing 0.1% Triton X-100 were placed on the stratum corneum. After incubation for 24 h, the cells were washed and solubilized and lysates cleared by centrifugation. The equivalent amounts of protein of the lysates were loaded onto polyacrylamide gels. Membranes were incubated with anti-TNF-α (A) or anti-SphK1 (B) antibody. To determine the amount of membrane-bound form TNF-α or SphK1 in each band, the membranes were re-probed with anti-β-actin. The blots shown are representative of 3 independent experiments. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. *P<0.05.</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

Infliximab inhibits PaCDase-induced production of IL-8, but not TNF-α, by keratinocytes.

<p>(<i>A</i>) PaCDase-induced TNF-α gene expression is not inhibited by infliximab. Nitrocellulose filters with Tris-buffered saline with 0.1% Triton X-100 (-/-) or with 1 mU/ml PaCDase (-), PaCDase plus 100 µg/ml normal human IgG or 10 or 100 µg/ml infliximab were placed on the stratum corneum. The cells were incubated for 4 h or 24 h, and the level of TNF-α mRNA was determined. An S18 ribosomal protein gene was used for normalization. Each bar represents the mean±SD of 3 independent experiments. **P<0.01. (<i>B</i>) Infliximab inhibition of PaCDase-induced IL-8 gene expression. IL-8 mRNA levels were assayed in the samples described in A. Each bar represents the mean±SD of 3 independent experiments. **P<0.01; ***P<0.001.</p