Alcohol reversibly disrupts TNF-alpha/TACE interactions in the cell membrane

Abstract

Background: Alcohol abuse has long been known to adversely affect innate and adaptive immune responses and pre-dispose to infections. One cellular mechanism responsible for this effect is alcohol-induced suppression of TNF-alpha (TNF) by mononuclear phagocytes. We have previously shown that alcohol in part inhibits TNF-alpha processing by TNF converting enzyme (TACE) in human monocytes. We hypothesized that the chain length of the alcohol is critical for post-transcriptional suppression of TNF secretion.

Methods: Due to the complex transcriptional and post-transcriptional regulation of TNF in macrophages, to specifically study TNF processing at the cell membrane we performed transient transfections of A549 cells with the TNF cDNA driven by the heterologous CMV promoter. TNF/TACE interactions at the cell surface were assessed using fluorescent resonance energy transfer (FRET) microscopy.

Results: The single carbon alcohol, methanol suppressed neither TNF secretion nor FRET efficiency between TNF and TACE. However, 2, 3, and 4 carbon alcohols were potent suppressors of TNF processing and FRET efficiency. The effect of ethanol, a 2-carbon alcohol was reversible.

Conclusion: These data show that inhibition of TNF-alpha processing by acute ethanol is a direct affect of ethanol on the cell membrane and is reversible upon cessation or metabolism.

Figure 1

Panel A: Acute EtOH reversibly suppresses TNF-α secretion in transfected A549 cells. A549 cells transfected with the hTNF-α cDNA were re-seeded 24 hours after transfection and subjected to the addition of 0, 50 or 100 mM EtOH for 1 hour (n = 6, *. Denoted p < 0.05). For the washout experiments, a subgroup of transfected cells had media changed after one hour to fresh media without alcohol for an additional hour (n = 6, * denotes p < 0.05). Panel B: Effect of one to four carbon alcohols on TNF-α secretion (n = 4–6, * denotes p < 0.05 compared to EtOH). Panel C: Effect of one to four carbon alcohols on cell-associated TNF-α levels (n = 4–6, * denotes p < 0.05 compared to EtOH). Transfected A549 cells were treated as in Materials and Methods and TNF-α was measured in cell lysates as the cell-associated level (all data are per mg of protein). Panel D: Effect of one to four carbon alcohols on TNF processing as assessed by the ratio of shed vs. cell-associated TNF-α (n = 4–6, * denotes p < 0.05 compared to EtOH). Transfected A549 cells were treated as in Materials and Methods and TNF-α was measured in cell lysates and media and graphed as the ratio of the shed over the cell-associated level.

Figure 2

Immunofluorescent detection, co-localization, and FRET intensity in representative transfected A549 cells. Cells were stained with anti-TACE (Cy3) and anti-TNF-α (FITC) as FRET acceptor and donor, respectively (panels a-t). The staining pattern is suggestive of both cell membrane and membrane vesicle localization. In photo-bleached samples (Panels c, h, m, r) there was less than 2% initial anti-TACE (Cy3) intensity post-photobleaching. Panels u-x: FRET intensity, calculated from the difference between donor pre- and post-photobleaching intensities shown in pseudocolor.

Figure 3

Mean FRET intensity, calculated from the difference between donor (TNF-α) pre- and post-photobleaching intensities in 20 high power fields per group (n = 4, * denotes p < 0.05 compared to 0 mM control).