Investigation of the Role of TNF-α Converting Enzyme (TACE) in the Inhibition of Cell Surface and Soluble TNF-α Production by Acute Ethanol Exposure

Toll-like receptors (TLRs) play a fundamental role in the immune system by detecting pathogen associated molecular patterns (PAMPs) to sense host infection. Ethanol at doses relevant for humans inhibits the pathogen induced cytokine response mediated through TLRs. The current study was designed to investigate the mechanisms of this effect by determining whether ethanol inhibits TLR3 and TLR4 mediated TNF-α secretion through inhibition of transcription factor activation or post-transcriptional effects. In NF-κB reporter mice, activation of NF-κB in vivo by LPS was inhibited by ethanol (LPS alone yielded 170,000±35,300 arbitrary units of light emission; LPS plus ethanol yielded 56,120±16880, p = 0.04). Inhibition of protein synthesis by cycloheximide revealed that poly I:C- or LPS-induced secreted TNF-α is synthesized de novo, not released from cellular stores. Using real time RT-PCR, we found inhibition of LPS and poly I:C induced TNF-α gene transcription by ethanol. Using an inhibitor of tumor necrosis factor alpha converting enzyme (TACE), we found that shedding caused by TACE is a prerequisite for TNF-α release after pathogen challenge. Flow cytometry was used to investigate if ethanol decreases TNF-α secretion by inhibition of TACE. In cells treated with LPS, ethanol decreased both TNF-α cell surface expression and secretion. For example, 4.69±0.60% of untreated cells were positive for cell surface TNF-α, LPS increased this to 25.18±0.85%, which was inhibited by ethanol (86.8 mM) to 14.29±0.39% and increased by a TACE inhibitor to 57.88±0.62%. In contrast, cells treated with poly I:C had decreased secretion of TNF-α but not cell surface expression. There was some evidence for inhibition of TACE by ethanol in the case of LPS, but decreased TNF-α gene expression seems to be the major mechanism of ethanol action in this system

Structural changes in the cells of some bacteria during population growth: a fourier transform infrared-attenuated total reflectance study

Structural changes occurring in the cells of several bacteria during their growth curves have been investigated by Fourier transform infrared (FT-IR) spectroscopy using the sampling technique of attenuated total reflectance (ATR). Spectra reflect all of the components of the cells including the cell walls, cell membranes, internal structures and the cytoplasm. The bacteria studied were Bacillus stearothermophilus, Halobacterium salinarium, Halococcus morrhuae and Acetobacter aceti. All species showed significant spectral changes during their growth curves, indicating structural changes in the cells during increases in cell numbers. The major change for B. stearothermophilus was in the lipid content which was at a maximum during the exponential phase of the growth curve. For the halophiles H. salinarium and H. morrhuae the major change was that the concentration of sulfate ion in the cells varied during the growth curve and was at a maximum during the mid-part of the exponential phase of the growth curve. A. aceti cells showed increasing polysaccharide content during the growth curve as well as maximum lipid content during the exponential phase of growth

Investigation of the role of TNF-a converting enzyme (TACE) in the inhibition of cell surface and soluble TNF-a production by acute ethanol exposure.

Toll-like receptors (TLRs) play a fundamental role in the immune system by detecting pathogen associated molecular patterns (PAMPs) to sense host infection. Ethanol at doses relevant for humans inhibits the pathogen induced cytokine response mediated through TLRs. The current study was designed to investigate the mechanisms of this effect by determining whether ethanol inhibits TLR3 and TLR4 mediated TNF-? secretion through inhibition of transcription factor activation or post-transcriptional effects. In NF-?B reporter mice, activation of NF-?B in vivo by LPS was inhibited by ethanol (LPS alone yielded 170,000?35,300 arbitrary units of light emission; LPS plus ethanol yielded 56,120?16880, p = 0.04). Inhibition of protein synthesis by cycloheximide revealed that poly I:C- or LPS-induced secreted TNF-? is synthesized de novo, not released from cellular stores. Using real time RT-PCR, we found inhibition of LPS and poly I:C induced TNF-? gene transcription by ethanol. Using an inhibitor of tumor necrosis factor alpha converting enzyme (TACE), we found that shedding caused by TACE is a prerequisite for TNF-? release after pathogen challenge. Flow cytometry was used to investigate if ethanol decreases TNF-? secretion by inhibition of TACE. In cells treated with LPS, ethanol decreased both TNF-? cell surface expression and secretion. For example, 4.69?0.60% of untreated cells were positive for cell surface TNF-?, LPS increased this to 25.18?0.85%, which was inhibited by ethanol (86.8 mM) to 14.29?0.39% and increased by a TACE inhibitor to 57.88?0.62%. In contrast, cells treated with poly I:C had decreased secretion of TNF-? but not cell surface expression. There was some evidence for inhibition of TACE by ethanol in the case of LPS, but decreased TNF-? gene expression seems to be the major mechanism of ethanol action in this system

Effect of TACE inhibition on the LPS and poly I:C induced TNF-α response in RAW264.7 cells.

<p>TNF-α levels were measured by ELISA from cell culture medium immediately after collection. Each group contained 6 samples. Naive groups received no treatment. <b>A</b> Cells were treated with 25 µg/ml TAPI-0, 5 µl/ml DMSO, and/or 100 ng/ml LPS. Treatments were given at the same time point and cells were incubated 2 h. <b>B</b> Appropriate groups were treated with 86.8 mM EtOH and incubated 30 min. Cells were treated with 25 µg/ml TAPI-0, 5 µl DMSO, and/or 50 µg/ml poly I:C, and incubated further 2 h. Bars with no shared letters are significantly different (p<0.05; nd = not detectable).</p

Comparison of NF-κB expression in reporter mice treated with LPS alone or ethanol and LPS.

<p><b>A</b> For the naive mouse, luminescence was 1.627×10⁴ CCD camera counts. <b>B</b> In the group treated with LPS only, 2.406×10⁵ counts were measured for mouse one, 1.347×10⁵ counts for mouse two, and 8.485×10⁴ counts for mouse three. <b>C</b> In the group treated with LPS+86.8 mM EtOH, 4.754×10⁴ counts were measured for mouse one, 3.213×10⁴ for mouse two, and 8.869×10⁴ for mouse three. <b>D</b> Mice treated with 6 g/kg ethanol before LPS showed significant reduction of luminescence (* = LPS plus EtOH vs. LPS only: P<0.05).</p

Mouse TNF-α ELISA assay from cell culture supernatants.

<p>Each group contained 6 samples. Cells were treated with LPS/poly I:C alone, with cycloheximide and LPS/poly I:C at the same time point, or with LPS/poly I:C first and cycloheximide 30 min later. Naive, untreated cells served as control. Bars with no shared letters are significantly different (p<0.05).</p

Surface expression of TNF-α on RAW264.7 cells measured by flow cytometry, and TNF-α ELISA from cell culture supernatants of the same experiments.

<p>Results shown in <b>A</b> and <b>B</b> were obtained in one experiment and results shown in <b>C</b> and <b>D</b> were obtained in an independent experiment. In some groups, no TNF-α was detected (nd). Each group contained 5 samples. Two repeat experiments are shown. Cells were treated with ethanol (either 43.4 mM or 86.8 mM), 100 ng/ml LPS or 20 µg/ml TAPI-0, or a combination of these treatments. ELISA was performed from cell culture supernatants of each group. The percentage of gated cells (positive for TNF-α surface expression by flow cytometry) for each group is shown in <b>A</b> & <b>C</b>. The ELISA results from cell culture supernatants of the cells used for flow cytometry are depicted in <b>B</b> and <b>D</b>. Bars designated by the same letter are not significantly different (p>0.05); bars with no shared letters are significantly different (p<0.05). Histograms for representative samples from key groups for the experiment shown in A are shown below panels <b>C</b> and <b>D</b>.</p

Real time RT-PCR with mRNA isolated from RAW264.7 cells treated with ethanol and LPS or poly I:C.

<p>Naive groups received no treatment. <b>A</b> Appropriate groups were treated with ethanol for 30 min, then with LPS for further two hours. The average of two experiments, each with three replicates, were pooled. Each sample replicate was doubled in the PCR plate. 86.8 mM ethanol significantly reduced the LPS induced TNF-α mRNA expression (P<0.001). Treatment with ethanol only was not significantly different from naive. <b>B</b> Appropriate groups were treated with different concentrations of ethanol for 30 min, then with poly I:C for further two hours. The average of two experiments, one with three replicates, and one with two replicates, is shown. Each sample replicate was doubled in the PCR plate. A concentration of 43.4 mM EtOH and higher significantly reduced the poly I:C induced TNF-α mRNA expression (P<0.01). Ethanol without poly I:C was not significantly different from naive. Results were normalized to 18S and analyzed using the ΔΔCt method. Bars with no shared letters are significantly different (p<0.05).</p