title: Chronic ethanol enhances ectodomain shedding in T cells and monocytes.
abstract: BACKGROUND
  Chronic ethanol (EtOH) has been shown to augment tumor necrosis factor (TNF)-alpha production, and this has been associated with EtOH-induced liver injury. We have recently described a chronic in vitro cell culture model where chronic ethanol exposure results in significantly augmented TNF production in Mono Mac 6 cells, a human monocytic cell line. This enhanced TNF production was redox regulated and associated with increased levels of TNF messenger RNA (mRNA) as well as increased processing of TNF by TNF converting enzyme (TACE), the enzymatic activity of which is regulated by the cellular redox state. We hypothesized that chronic ethanol through oxidative stress activates TACE-mediated ectodomain shedding of the preformed substrates p75 and p55 TNF receptors in Mono Mac 6 cells and L-selectin in Jurkat T cells.
  
  
  METHODS
  Mono Mac 6 or Jurkat T cells were treated with EtOH (0, 50, or 100 mM) for 4 to 6 days. Shedding of p75 and p55 TNF receptors (Mono Mac 6 cells) or L-selectin (Jurkat T cells) was induced by stimulation with lipopolysaccharide and phorbol myristate acetate for Mono Mac 6 cells and PMA alone for Jurkat T cells. Shedding was assessed by enzyme-linked immunosorbent assay for shed molecules in the cell supernatant as well as the cell-associated proteins recovered from cell pellets. Steady-state mRNA levels for p75 TNF receptor and L-selectin were determined by ribonuclease protection assay. Cell surface L-selectin and TACE were measured by flow cytometry, and cell associated p55 and p75 TNF receptors were measured by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  Chronic EtOH exposure for 6 days resulted in a significant dose-dependent increase in shedding of p75 and p55 TNF receptors from Mono Mac 6 cells and L-selectin from Jurkat T-cells. The enhanced shedding was correlated with an alcohol-induced increase in mRNA levels and cell surface protein levels for these TACE substrates. Although chronic EtOH exposure increased the total amount of p75 and p55 TNF receptor and L-selectin shed into the media, the efficiency of shedding was suppressed by EtOH. In the case of Mono Mac 6 cells, the EtOH exposure increased superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase and hydrogen peroxide partially prevented the increased production of p75 TNF receptor in these cells.
  
  
  CONCLUSIONS
  These results suggest that chronic EtOH up-regulates p75 and p55 TNF receptors on monocytes and L-selectin on T-cells. However, the TACE-mediated shedding efficiency of these substrates may be inhibited in the presence of EtOH. These results may have implications in monocyte signaling and T-cell trafficking, which may, in part, contribute to immune dysregulation associated with chronic ethanol.
authors.affiliation: Department of Pediatrics, University of Pittsburgh, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Xue-Jun
authors.initials: XJ
authors.lastname: Zhao
authors.forename: Peter
authors.initials: P
authors.lastname: Oliver
authors.forename: Kejing
authors.initials: K
authors.lastname: Song
authors.forename: Jill
authors.initials: J
authors.lastname: Schurr
authors.forename: Zili
authors.initials: Z
authors.lastname: Zhang
authors.forename: Jay K
authors.initials: JK
authors.lastname: Kolls
mesh_terms: D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000008:administration & dosage*; D006801:Humans; D019169:Jurkat Cells; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol