Mashkoor A. Choudhry

Heme oxygenase-1 (HO-1) has been demonstrated to protect against tissue injury. Furthermore, HO-1 is also shown to be antioxidant. Our recent findings indicate that acute alcohol (EtOH) intoxication exacerbates postburn intestinal and lung tissue damage, and this was found to be neutrophil dependent. Because neutrophil-mediated tissue injury involves the release of superoxide anions (O₂⁻), the present study examined the role of HO-1 in neutrophil O₂⁻ production following EtOH and burn injury. Furthermore, we investigated whether HO-1 antioxidant properties are mediated via modulation of p47^phox and/or p67^phox proteins. Male rats (∼250 g) were gavaged with EtOH to achieve a blood EtOH level of ∼100 mg/dL before burn or sham injury (∼12.5% total body surface area). Some rats were treated with HO-1 activator cobalt protoporphyrin IX chloride (Copp; 25 mg/kg body weight) at the time of injury. On day 1 after injury, we found that EtOH combined with burn injury significantly increased neutrophil O₂⁻ production and p47^phox and p67^phox activation and decreased caspase-3 activity and apoptosis. This was accompanied with a decrease in neutrophil HO-1 levels. The treatment of animals with HO-1 activator Copp normalized neutrophil HO-1, O₂⁻, p47^phox, and p67^phox following EtOH and burn injury. The expression of caspase-3, however, was further decreased in Copp-treated sham and EtOH plus burn groups. Moreover, Copp treatment also prevented the increase in intestinal edema and permeability following EtOH and burn injury. Altogether, these findings provide a new insight into the mechanism by which HO-1 regulates neutrophil O₂⁻ production and protect the intestine from damage following EtOH and burn injury.

This study examined whether acute alcohol (EtOH) intoxication before burn injury potentiates postburn intestinal tissue damage and whether neutrophils have any role in the damage under those conditions. Male rats ( approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dL or with saline and received either approximately 12.5% or approximately 25% total body surface area (TBSA) burn or sham injury. Rats were killed at 4 or 24 h after injury, and various parameters were measured. As compared with sham animals, burn injury alone (regardless of size) resulted in a significant increase in intestinal tissue myeloperoxidase (MPO; an index of neutrophil infiltration) activity and IL-18 levels 4 h after injury. Furthermore, rats receiving 25% TBSA, but not 12.5%, burn exhibited intestine edema. The IL-18 and MPO activity were normalized at 24 h after injury in rats receiving 12.5% TBSA burn, whereas these parameters remained elevated at 24 h in rats with 25% burn. The presence of EtOH in rats at the time of burn injury exacerbated the levels of IL-18, MPO activity, and edema at 4 and 24 h after burn injury. Treatment of rats with anti-IL-18 antibodies or with antineutrophil antiserum prevented the increase in the above parameters after EtOH and burn injury, except that the depletion of neutrophils did not prevent the IL-18 increase. In summary, these findings suggest that acute EtOH intoxication exacerbates postburn intestinal tissue damage after burn injury, and that it is, in part, neutrophil mediated.

Results from the studies discussed in this article suggest that alcohol (EtOH) intoxication is a major public health problem. While the effects of injury and EtOH intoxication independent of each other have been studied in detail, only few studies have evaluated the effect of a combined insult of EtOH intoxication and burn injury on host defense. An analysis of the studies conducted in the clinical setting suggests that intoxicated patients require frequent intubations, experience delayed wound healing and longer hospital stay. Furthermore, there is a greater risk of mortality in these patients compared to those who sustained injuries in the absence of EtOH intoxication. On the other hand, there are a few studies that do not support this notion. The results obtained in experimental models clearly suggest that acute EtOH intoxication before burn injury impairs host defense and increases susceptibility to infection. Additionally, experimental data from our laboratory also indicate that EtOH intoxication before burn injury suppresses intestinal immune defense, impairs gut barrier functions and increases bacterial growth. This results in increased bacterial translocation in EtOH and burn injury. In addition, a decrease in cardiac function is also reported following a combined insult of EtOH intoxication and burn injury. Altogether, these findings suggest that EtOH intoxication before burn injury diminishes host resistance resulting in increased susceptibility to infection. Moreover, the findings of a higher incidence of infectious complications in burn and trauma patients who sustained injury in the presence of EtOH compared to those in its absence suggest that EtOH intoxication at the time of injury is a risk factor. Therefore blood EtOH should be monitored in burn/trauma patients at the time of admission in the emergency room.

Previous studies showed that alcohol (EtOH) intoxication before burn injury suppresses mesenteric lymph node (MLN) T cell functions and increases gut bacterial translocation. In this study, we examined whether corticosterone (Cort) plays any role in suppressing MLN T cell function and bacterial accumulation after EtOH intoxication and burn injury. Rats were gavaged with EtOH to achieve a blood EtOH level of ∼100 mg/dl before receiving 25% total body surface area burn or sham injury. A group of rats was treated with the Cort synthesis inhibitor metyrapone (25 mg/kg) at the time of injury and on day 1 after injury. Two days after injury, a significant increase in blood Cort levels and suppression of MLN T cell proliferation and IL-2 production was observed in rats receiving combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. There was no change in T cell apoptosis after combined insult of EtOH and burn injury. Furthermore, T cell suppression was accompanied by a significant decrease in p38 and ERK1/2 activation (phosphorylation). There was no difference in JNK activation after EtOH and burn injury. Treatment of rats with metyrapone prevented the suppression of MLN T cell proliferation, IL-2 production, and p38 and ERK1/2 phosphorylation. Restoration of T cell function in metyrapone-treated animals was also associated with the decrease in bacterial accumulation in MLN. These findings suggest that EtOH intoxication before burn injury augments Cort release, which suppresses MLN T cell function by inhibiting p38 and ERK1/2 activation and promotes bacterial accumulation in MLN after EtOH and burn injury.

Recent studies from our laboratory have shown that alcohol and burn injury impair intestinal barrier and immune functions. Although multiple factors can contribute to impaired intestinal barrier function, such an alteration could result from a decrease in intestinal blood flow (BF) and oxygen delivery (Do₂). Therefore, in this study, we tested the hypothesis that alcohol ingestion before burn injury reduces splanchnic blood flow and oxygen delivery. Rats (250 g) were gavaged with alcohol to achieve a blood ethanol level in the range of 100 mg/dl before burn or sham injury (25% total body surface area). Day 1 after injury, animals were anesthetized with methoxyflurane. Blood pressure, cardiac output (CO), ±dP/d t, organ BF (in ml·min⁻¹·100 g⁻¹), and Do₂ (in mg·ml⁻¹·100 g⁻¹) were determined. CO and organ BF were determined using a radioactive microsphere technique. Our results indicate that blood pressure, CO, and +dP/d t were decreased in rats receiving a combined insult of alcohol and burn injury compared with rats receiving either burn injury or alcohol alone. This is accompanied by a decrease in BF and Do₂ to the liver and intestine. No significant change in BF to the coronary arteries (heart), brain, lung, skin, and muscles was observed after alcohol and burn injury. In conclusion, the results presented here suggest that alcohol ingestion before burn injury reduces splanchnic BF and Do₂. Such decreases in BF and Do₂ may cause hypoxic insult to the intestine and liver. Although a hypoxic insult to the liver would result in a release of proinflammatory mediators, a similar insult to the intestine will likely perturb both intestinal immune cell and barrier functions, as observed in our previous study.