Acute and chronic alcohol exposure impair the phagocytosis of apoptotic cells and enhance the pulmonary inflammatory response

Abstract

Background: Alcohol abuse increases the risk for acute respiratory distress syndrome (ARDS). Efferocytosis, the clearance of apoptotic cells, is important in the resolution of inflammation and is regulated by RhoA and rho kinase (ROCK) activation. The effects of alcohol on pulmonary Rho pathway activation and efferocytosis have not been determined. We hypothesize that acute and chronic alcohol exposure impair pulmonary efferocytosis, leading to heightened inflammation during ARDS.

Methods: For in vivo experiments, C57BL/6 mice received either a single intraperitoneal injection of alcohol or chronic ethanol-in-water for 8 weeks prior to intratracheal instillation of apoptotic cells or lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed for cells counts, calculation of the phagocytic index (PI), and Rho activity measurements. For in vitro studies, primary alveolar macrophages were cultured in alcohol (25-100 mM) and then co-cultured with apoptotic cells. RhoA activity was determined following alcohol exposure, and the PI was determined before and after treatment with the ROCK inhibitor, Y27632.

Results: Acute alcohol exposure was associated with impaired efferocytosis. Following LPS exposure, acute alcohol exposure was also associated with increased BAL neutrophils. Chronic alcohol exposure alone did not alter efferocytosis. However, following exposure to LPS, chronic alcohol exposure was associated with both impaired efferocytosis and increased BAL neutrophils. In vitro alcohol exposure caused a dose-dependent decrease in efferocytosis. Despite the fact that RhoA activity was decreased by alcohol exposure and RhoA inhibition did not alter the effects of alcohol on efferocytosis, treatment with the Rho kinase inhibitor, Y27632, reversed the effects of alcohol on efferocytosis.

Conclusions: Acute alcohol exposure impairs pulmonary efferocytosis, whereas exposure to chronic alcohol is only associated with impaired efferocytosis following LPS-induced lung injury. Both forms of alcohol exposure are associated with increased alveolar neutrophil numbers in response to LPS. The acute effects of alcohol on efferocytosis appear to be mediated, at least in part, by RhoA-independent activation of ROCK. Further studies are needed to dissect the differences between the effects of acute and chronic alcohol exposure on efferocytosis and to determine the effects of alcohol on alternative activators of ROCK.

Figure 1

Representative photomicrograph (60×) demonstrating phagocytosis of apoptotic thymocytes by alveolar macrophages. Ingestions are indicated by arrows, which are manually counted to determine the phagocytic index. The arrowheads demonstrate remnants of apoptotic thymocytes that have been destroyed during the cytospin process.

Figure 2

In vitro exposure of alveolar macrophages to alcohol for 24h impairs phagocytosis of apoptotic cells in a dose dependent fashion. Data shown represent combined data from three separate experiments, each performed in duplicate. *p<0.05 when compared to control.

Figure 3

In vivo acute alcohol exposure is associated with impaired phagocytosis of apoptotic cells by alveolar macrophages. Data shown represent combined data from three separate experiments. *p<0.05 when compared to control.

Figure 4

In vivo acute alcohol exposure is associated with increased numbers of alveolar neutrophils (A) but does not alter alveolar macrophage (B) numbers following LPS-induced lung injury. n=6-8 for each experimental group. *p<0.05 when compared to time-matched control.

Figure 5

In vivo chronic alcohol exposure did not alter phagocytosis of apoptotic cells by alveolar macrophages at baseline (A) or 24h following i.t. LPS instillation (B). However, at 7 days following LPS, chronic alcohol exposure was associated with impaired efferocytosis (C). Data shown represent combined data from three separate experiments. *p<0.05 when compared to time-matched control.

Figure 6

In vivo chronic alcohol exposure is associated with increased numbers of alveolar neutrophils (A) at 48h and 72h following i.t. instillation of LPS. Chronic alcohol exposure was also associated with decreased numbers of alveolar macrophages (B) at baseline, although alveolar macrophage numbers were similar at all times measured following LPS instillation. n=6-8 for each experimental group. *p<0.05 when compared to time-matched control. † p=0.07.

Figure 7

In vitro exposure of alveolar macrophages to alcohol for 24h impairs phagocytosis of apoptotic cells. This effect is reversed by pre-treatment with the Rho kinase inhibitor, Y27632 (1-10μM). Data shown represent combined data from three separate experiments, each performed in duplicate. *p<0.05 when compared to control.

Figure 8

In vitro exposure of alveolar macrophages to alcohol for 24h impairs phagocytosis of apoptotic cells. This effect is not reversed by pre-treatment with the cell permeable Rho inhibitor, C3 transferase (1-10μg/ml) (A). RhoA activity is not increased by either in vitro (B) or in vivo (C) exposure to alcohol, suggesting that the ROCK-dependent effects of alcohol on efferocytosis are independent of RhoA. In fact, in vivo alcohol exposure is associated with significantly decreased RhoA activity. *p<0.05 when compared to control.