Our current study indicates that acute alcohol intoxication suppresses the pulmonary MIG, IP-10, and I-TAC response to airway endotoxin challenge. This inhibition occurs, at least, at the level of transcriptional regulation as evidenced by decreased chemokine mRNA expression in vivo. In agreement with prior studies showing inhibition of accessory cell function as a result of acute ethanol exposure (Szabo et al., 2001
; Szabo et al., 2004
), the in vitro data demonstrate that ethanol directly and dose-dependently inhibits the MHS cell response to LPS or LPS + IFN-γ. Furthermore, to our knowledge, these data are the first to show that the early pulmonary IFN-γ response to LPS is attenuated by a single dose of ethanol. Hence, these data support both direct (decreased MH-S cell chemokine mRNA) and indirect (decreased lung IFN-γ transcripts) mechanisms underlying inhibition of ELR-
CXC chemokine expression in the lung.
The association of alcohol abuse and poor outcome from pneumonia has been well documented and is associated with substantial cost (Saitz et al., 1997
). Recruitment of immune effector cells is critical to optimal response to respiratory infection and host survival. Alcohol impairs the neutrophil response to pulmonary infection (Astry et al., 1983
; Zhang et al., 1999
), and this effect is at least partially due to the attenuated expression of chemokines (Boe et al., 2001
). While it is clear that alcohol intoxication alters the early neutrophil response, it is plausible that no such effect would be seen on later cellular recruitment events since ethanol is rapidly metabolized in the mouse, with little to no detectable blood alcohol content 4 to 6 hours following intoxication. It is therefore interesting that we found a measurable defect in lymphocyte recruitment 4 days after challenge as a result of a single ethanol injection. While products of ethanol metabolism may contribute to the in vivo finding, we speculate that the in vitro finding of impaired chemokine release in LPS-stimulated intoxicated MHS cells supports a direct effect of ethanol, as other studies have shown that defects in inflammatory responses by alveolar macrophages are observed despite blockade of ethanol metabolism (Greenberg et al., 1999
Two weeks of ethanol feeding in CD-1 mice can impair the IFN-γ response to pulmonary K. pneumoniae
challenge, a response which occurs 3 days after challenge (Zisman et al., 1998
). Perhaps as a result of our using a relatively high dose of LPS, we were able to readily demonstrate pulmonary IFN-γ transcripts as early as 6 hours after challenge. We speculate that gamma delta-T cells and NK cells are important early sources of IFN-γ in our model, based on prior reports identifying these cells as producing (NK cells) or critical for the expression of (gamma delta-T cells) IFN-γ during bacterial pneumonia (Deng et al., 2001
; Moore et al., 2000
Toll-like receptors are well-characterized pattern recognition receptors which recognize a diverse group of conserved microbial products (Medzhitov, 2001
). Significant progress has been made in our understanding of ethanol’s modulation of Toll-signaling, with varying effects of alcohol exposure depending on the Toll ligand of interest (Oak et al., 2006; Pruett et al., 2004b
; Pruett et al., 2004a
). CD14/TLR4 activation by LPS is accompanied by rapid nuclear translocation of NF-kB, and prior studies have shown inhibition of LPS-induced NF-kB induction by acute ethanol exposure both in vivo (human monocytes) and in vitro (murine Raw 264.7 cells). It is interesting that our current studies did not show a decrease in the nuclear presence of NF-kB RelA (p65) in stimulated MH-S cells acutely cultured in ethanol. Although it is possible that other NF-kB family members responsible for induction of MIG, IP-10, and I-TAC were downregulated by ethanol, we find this an unlikely explanation of our results since RelA is the most abundant trans-activating member of this transcription factor family involved in monocye/macrophage responses to LPS (Guha and Mackman, 2001
). Furthermore, others have shown that increases in nuclear p65 as a result of TLR3 (Pruett et al., 2004a
) or TLR4 (Mandrekar et al., 1999
) activation are abrogated by ethanol. Discrepancies between these and our observations may be related to differences in the cell type studied, dosing of alcohol, or amount of LPS exposure. Nonetheless, we conclude that acute alcohol inhibits MH-S cell ELR-
chemokine elaboration in a manner not reliant on NF-kB suppression. This finding suggests alternative intracellular events downstream of TLR4 activation are affected by acute alcohol.
TLR4 activation by LPS has been shown to result in activation of several MAP kinases, including p38 (Lu et al., 1999
), Erk 1/2 (Weinstein et al., 1992
), and JNK (Hambleton et al., 1996
). Because no effects on NF-kB were observed in MH-S cells, we then sought to determine if MAPK activity was affected by acute intoxication of this cell line. We observed a dose-dependent inhibition of Erk 1/2 phosphorylation as a result of acute intoxication. These results are similar to prior studies which indicate defects in Erk activation after acute in vivo ethanol treatment followed by ex vivo LPS stimulation of macrophages (Goral et al., 2004
; Kato et al., 2005).
In contrast, we found no ethanol effect on LPS-induced phosphorylation of p38 or JNK (JNK data not shown). Others have shown acute ethanol (25mM) actually increases human monocyte p38 activation following in vitro LPS stimulation (Drechsler et al., 2006
). Our finding of no effect on LPS-induced JNK activation by acute intoxication is consistent with previous work (Oak et al., 2006). To support our hypothesis that ethanol’s inhibition of Erk may underlie the defect in chemokine secretion during intoxication, we observed that two specific MEK inhibitors also strongly down-regulate ELR- chemokine expression during LPS challenge. These results demonstrate a strong dependence on Erk signaling for expression of these chemokines, and they support our conclusion that Erk inhibition is a critical mechanism underlying the effect of acute intoxication on these cytokines.
Ethanol-induced suppression of lung neutrophil recruitment has been extensively studied (as reviewed by Zhang et al., 2002
), but the effect on pulmonary lymphocyte influx has not been described. Because CXCR3 is expressed on activated T cells, we hypothesized that suppression of MIG, IP-10, and I-TAC by acute ethanol administration would be accompanied by an attenuation of LPS-induced pulmonary T cell recruitment. Following acute intoxication, we observed a modest yet statistically significant decrease in T cells recruited into the alveolar space 4 days after LPS instillation. We find these results interesting since this T cell recruitment defect occurs 4 days after the acute intoxication event. Although we suggest that this defect is at least partially a result of the defective ELR-
chemokine expression currently observed, several caveats must be addressed. First, T cells express other chemokine receptors such as CCR2 and CCR5, and inhibition of the ligands for these receptors, such as the monocyte chemotactic protein (MCP) family for CCR2 and the macrophage inflammatory protein (MIP) family for CCR5, may contribute to decreased T cell presence in BAL following LPS challenge. Furthermore, direct effects of alcohol on the T cells themselves may impair their ability to traffic into inflamed tissues, as has been described in neutrophils (MacGregor et al., 1974
; Zhang et al., 1998
). It is also possible that acute alcohol’s induction of IL-10, either in the lung (Happel et al., 2006
) or the systemic compartment (Szabo et al., 2001
) may contribute to the blunted recruitment of T cells into inflamed lung tissue.
In summary, our work shows that acute alcohol intoxication impairs the lung’s expression of the ELR- CXC chemokines MIG, IP-10, and I-TAC, and this suppression may be the result of ethanol’s inhibition of Erk MAPK activation. Acute ethanol decrease the alveolar presence of T cells 4 days after intoxication, indicating that even a single dose of ethanol can affect cellular recruitment days after the disappearance of ethanol from the circulation. These data further our understanding of the specific mechanisms by which acute alcohol intoxication interferes with the pulmonary inflammatory response to bacterial challenge.