Published online before print May 17, 2006
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Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham
1Correspondence: Center for Surgical Research, University of Alabama at Birmingham, Volker Hall G094, 1670 University Boulevard, Birmingham, AL 35294. E-mail: mashkoor.choudhry{at}ccc.uab.edu
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250 g) were gavaged with EtOH to achieve a blood EtOH level of 100 mg/dL prior to burn or sham injury (25% total body surface area). Immediately after injury, a group of rats was treated with CORT synthesis inhibitor metyrapone (25 mg/kg), with or without recombinant (r)IL-18 (50 µg/kg). Another group of rats was treated with caspase-1 inhibitor Ac-YVAD-CHO to block IL-18 production. On Day 1 after injury, there was a significant increase in blood CORT levels, intestinal levels of IL-18, neutrophil chemokines [cytokine-induced neutrophil chemoattractant 1 (CINC-1) and CINC-3], intercellular adhesion molecule-1, myeloperoxidase activity, and intestinal permeability in rats receiving a combined insult of EtOH and burn injury. Treatment of rats with CORT inhibitor or with caspase-1 inhibitor prevented the increase in all of the above parameters following a combined insult of EtOH and burn injury. Moreover, coadministration of rIL-18 in metyrapone-treated rats restored the above parameters, similar to those observed in rats receiving EtOH and burn injury. These findings suggest that a combined insult of EtOH and burn injury results in increased CORT levels, which in turn up-regulates intestinal IL-18 levels and thereby causes altered intestinal barrier function following a combined insult of EtOH intoxication and burn injury.
Key Words: ethanol • thermal injury • neutrophil • inflammatory mediators • adhesion molecules • chemokines
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There is evidence suggesting that EtOH intoxication and burn injury independently activate the hypothalamic-pituitary-adrenal (HPA) axis [4 , 15 16 17 18 ], and corticosterone (CORT), the end glucocorticoids product of the HPA axis in rodents, plays a role in shaping the immune response under those conditions [4 , 15 , 16 ]. In a recent study, we found that an elevation in CORT, following a combined insult of EtOH intoxication and burn injury, suppresses intestinal T cell function and potentiates bacterial translocation [12 ]. As we also found an increase in interleukin (IL)-18 and intestinal permeability under those conditions [11 ], we were interested in learning whether the effects of CORT are restricted to intestinal immune function or also include intestinal barrier function and whether this is a direct effect of CORT or is mediated via IL-18 production. IL-18 is synthesized as a nonfunctional precursor, pro-IL-18. It has been shown that protease activity mediated by caspase-1, also referred to as IL-1-converting enzyme, is needed for conversion of pro-IL-18 (23 kDa) to a mature, functional 18.3-kDa IL-18 protein [19 , 20 ].
Using approaches to modulate endogenous CORT and IL-18 levels via treating animals with CORT synthesis inhibitor metyrapone alone or in combination with recombinant (r)IL-18, the present study examined the mechanism of altered intestinal permeability following a combined insult of EtOH intoxication and burn injury. The findings reported in this manuscript suggest that CORT up-regulates IL-18 production, which in turn plays a major role in intestinal barrier dysfunction by potentiating neutrophil activation, neutrophil chemokines, and adhesion molecules.
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250 g) were obtained from Charles River Laboratories (Wilmington, MA). rIL-18 was purchased from Biosource International (Camarillo, CA). Metyrapone was obtained from Sigma Chemical Co. (St. Louis, MO). Caspase-1 inhibitor (Ac-YVAD-CHO) was obtained from Axxora LLC (San Diego, CA).
Rat model of acute EtOH and burn injury
As described previously [11
, 13
], rats were divided randomly into four groups: saline + sham, EtOH + sham, saline + burn, and EtOH + burn. In EtOH groups, rats were gavaged with 5 ml 20% EtOH in saline. In saline groups, animals were gavaged with 5 ml saline. Four hours after gavaging, a time at which blood EtOH levels in EtOH-treated groups are in the range of 90–100 mg/dL (21.7 mM/L), rats were anesthetized and transferred into a template, which was fabricated to expose 25% of the total body surface area. Animals were then immersed in a boiling water bath (95–97°C) for 10 s. Sham-injured rats were subjected to identical anesthesia and immersed in lukewarm water. The animals were dried immediately and given fluid resuscitation [intraperitoneally (i.p.)] with 10 ml physiological saline.
Metyrapone (25 mg/kg body weight) or caspase-1 inhibitor (Ac-YVAD-CHO, 5 mg/kg) was administered i.p. at the time of injury [12
, 13
]. In some animals, metyrapone was coadministered with rIL-18 (50 µg/kg). Animals were allowed to recover from anesthesia, returned to their cages, and allowed food and water ad libitum. Animals were killed on Day 1 after injury. A diagrammatic presentation of various groups included in this study is shown Figure 1
. During the study, we stuck to a uniform time-frame for performing the procedure and for sacrificing the animals so that the rhythmic release of CORT levels would not interfere with our measurements. In general, rats were gavaged at 10:00 a.m., received sham or burn injury at 2:00 p.m., and were killed the next morning between 9:00 and 10:00 a.m. Thus, the animals were killed nearly 18–19 h after burn injury and 24 h after the animals were gavaged with EtOH.
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Figure 1. Diagrammatic presentation of various groups included in the study. The numbers in parentheses represent the number of animals in the group. Veh, Vehicle; Cas, caspase-1 inhibitor; Met, metyrapone.
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Measurements of blood CORT levels
Rats were anesthetized, blood was drawn via cardiac puncture, and plasma was separated [12
]. Plasma CORT levels were measured using a radioimmunoassay kit, Coat-A-Count (Diagnostic Products, Los Angeles, CA).
Preparation of intestinal homogenates
Immediately after drawing the blood, the intestine was exposed. Leaving approximately the first 5 cm-long proximal segment of intestine, 3 cm-long segments of jejunum and ileum were removed, cleaned, and snap-frozen in liquid nitrogen. The samples were stored at –70°C. Equal weights (100 mg wet weight) of intestine from various groups were suspended in 1 ml phosphate-buffered saline (PBS) and sonicated (30 cycles, twice, for 30 s) on ice [13
]. Homogenates were cleared by centrifuging at 12,000 revolutions per minute (rpm) at 4°C, and the supernatants were stored at –70°C. Protein levels in the homogenates were determined using the Bio-Rad (Hercules, CA) assay kit.
Measurement of myeloperoxidase (MPO) levels
MPO activity in the intestinal tissue homogenates was measured using the procedure described previously [13
]. Briefly, samples were incubated with a substrate o-dianisidine hydrochloride. The reaction was carried out in a 96-well plate by adding 290 µl 50 mM phosphate buffer, 3 µl substrate solution (containing 20 mg/ml o-dianisidine hydrochloride), and 3 µl H2O2 (20 mM). Sample (10 µl) was added to each well to start the reaction. Standard MPO (Sigma Chemical Co.) was used in parallel to determine MPO activity in the sample. The reaction was stopped by adding 3 µL sodium azide (30%). Plates were read at 460 nm. MPO activity was determined by using the curve obtained from the standard MPO.
Measurement of IL-18, cytokine-induced neutrophil chemoattractant 1 (CINC-1), CINC-3, and intercellular adhesion molecule-1 (ICAM-1)
IL-18, CINC-1, CINC-3, and ICAM-1 levels in intestinal tissue homogenates were measured using enzyme-linked immunosorbent assay kits (IL-18 from Biosource International, Camarillo, CA; CINC-1, CINC-3, and ICAM-1 from R&D Systems, Minneapolis, MN) following the manufacturers’ instructions.
Intestinal permeability
The assay of intestinal permeability was modified from the method described by Choudhry et al. [11
]. In brief, the rat’s right femoral artery was cannulated under anesthesia, using PE-50 tubing filled with heparin saline (10 U/ml), and midline laparotomy was performed. Renal artery and vein in both kidneys were ligated. A 20-cm segment of the small intestine (ileum) was isolated without damaging intestinal and mesenteric structures, and PE-50 tubing was inserted into the isolated intestine from the proximal end. Solution [1 ml; 25 mg/ml 4 kDa fluorescein isothiocyanate (FITC)-conjugated dextran, Sigma Chemical Co.] was injected into the isolated intestine. Blood samples were collected from femoral artery and portal vein at 0, 15, 30, 60, and 90 min after infusion of FITC-dextran. Plasma was separated by centrifuging at 4°C, 8000 rpm for 7 min, and was analyzed for FITC-dextran concentration using a fluorometer (FL500, Bio-Tek Instruments, Inc., Winooski, VT) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Standard curves to calculate FITC-dextran concentration in the plasma samples were prepared from dilutions of FITC-dextran in PBS [21
].
Statistical analysis
Results are presented as mean ± SE and were analyzed using ANOVA. The significance between the groups was determined using Tukey’s test (Statistical Package for Social Sciences Software program, Version 2.0, Sigma Stat). A P < 0.05 between two groups was considered statistically significant.
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Figure 2. Blood CORT levels, intestinal IL-18 levels, and intestinal MPO activity following EtOH intoxication and burn injury. On Day 1 after EtOH and burn injury, blood was drawn via cardiac puncture, and plasma CORT levels (A) were measured using a radioimmunoassay kit. For IL-18 and MPO activity, equal weights of jejunum and ileum from various groups were homogenized. IL-18 level (B) and MPO activity (C) were normalized to the jejunum and ileum homogenates’ protein contents. Values are mean ± SE from at least six animals in each group; *, P < 0.05, versus other groups; #, P < 0.05, versus sham + saline.
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Similarly, intestinal tissue MPO activity (Fig. 2C) following burn injury alone was not found to be significantly different compared with sham animals. However, a significant increase in jejunum and ileum MPO activity was observed in rats receiving a combined insult of EtOH intoxication and burn injury (P<0.05) compared with burn-injured rats gavaged with saline or sham rats, regardless of their EtOH intoxication.
Effect of metyrapone treatment on CORT and IL-18 levels
In these experiments, it is to be noted that in evaluating the effects of CORT inhibitor (metyrapone), we have not included sham plus EtOH group, as data in Figure 1A
1B
1C
, did not show any significant differences in CORT, IL-18, and MPO activity in sham-injured rats gavaged with saline or EtOH. Results as presented in Figure 3A
suggest that treatment of rats with metyrapone significantly lowered CORT levels in burn-injured rats gavaged with saline or EtOH intoxication. Similar to CORT, metyrapone administration also significantly prevented the increase in IL-18 levels in jejunum (Fig. 3B)
and ileum (Fig. 3C)
of rats receiving a combined insult of EtOH intoxication and burn injury. No significant effect of metyrapone treatment was observed on IL-18 levels in burn-injured rats gavaged with saline. Moreover, the administration of metyrapone did not influence CORT and IL-18 levels in sham-injured rats gavaged with saline compared with untreated rats.
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Figure 3. Effect of CORT inhibitor metyrapone on blood CORT levels and intestinal IL-18 levels following EtOH intoxication and burn injury. Rats were treated i.p. with metyrapone (25 mg/kg) at the time of injury. On Day 1 after EtOH and burn injury, plasma CORT levels (A) and IL-18 levels in jejunum (B) and ileum (C) were measured. Values are mean ± SE from at least six animals in each group; *, P < 0.05, versus other groups; #, P < 0.05, versus sham + saline.
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Figure 4. Intestinal MPO activity in rats treated with metyrapone, caspase-1 inhibitor, or metyrapone plus rIL-18 following EtOH intoxication and burn injury. Rats were treated i.p. with metyrapone (25 mg/kg) or caspase-1 inhibitor (Ac-YVAD-CHO, 5 mg/kg) at the time of injury. In a group of EtOH-intoxicated and burn-injured rats, metyrapone was coadministered with rIL-18 (50 µg/kg). On Day 1 after EtOH and burn injury, MPO activity was measured in jejunum (A) and ileum (B) homogenates. Values are mean ± SE from six animals in each group, except the metyrapone + rIL-18 group, which has three animals. *, P < 0.05, versus other groups, except EtOH + burn + rIL-18. #, P < 0.05, versus other groups except EtOH + burn.
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Effect of metyrapone and caspase-1 inhibitor on intestinal CINC-1 and CINC-3 levels
There was no significant difference in intestinal CINC-1 and CINC-3 levels between sham animals gavaged with EtOH or saline (data not shown). Furthermore, no significant difference in the CINC-1 (Fig. 5A
and 5B
) and CINC-3 (Fig. 5C
and 5D)
levels was observed in jejunum and ileum in rats receiving burn injury alone in the absence of EtOH intoxication compared with sham-injured rats gavaged with saline. A significant increase in CINC-1 and CINC-3 levels was observed in jejunum and ileum following a combined insult of EtOH intoxication and burn injury compared with rats receiving burn injury alone in the absence of EtOH intoxication or sham-injured rats gavaged with saline. Treatment of rats with CORT or caspase-1 inhibitor significantly prevented the increase in intestinal CINC-1 and CINC-3 levels following EtOH and burn injury. Similar treatment did not influence CINC-1 and CINC-3 levels in rats receiving sham or burn injury in the absence of EtOH intoxication. However, coadministration of rIL-18 along with metyrapone in EtOH-intoxicated, burn-injured rats restored CINC-1 and CINC-3 levels, similar to those observed in vehicle-treated rats following a combined insult of EtOH intoxication and burn injury.
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Figure 5. Intestinal CINC-1 and CINC-3 levels in rats treated with metyrapone, caspase-1 inhibitor, or metyrapone plus rIL-18 following EtOH intoxication and burn injury. Rats were treated i.p. with metyrapone (25 mg/kg) or caspase-1 inhibitor (Ac-YVAD-CHO, 5 mg/kg) at the time of injury. In a group of EtOH-intoxicated and burn-injured rats, metyrapone was coadministered with rIL-18 (50 µg/kg). On Day 1 after EtOH and burn injury, CINC-1 and CINC-3 levels were measured in jejunum (A and C) and ileum (B and D) homogenates. Values are mean ± SE from six animals in each group, except the metyrapone + rIL-18 group, which has three animals. *, P < 0.05, versus other groups except EtOH + burn + rIL-18. #, P < 0.05, versus other groups except EtOH + burn.
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Figure 6. Intestinal ICAM-1 expression in rats treated with metyrapone, caspase-1 inhibitor, or metyrapone plus rIL-18 following EtOH intoxication and burn injury. Rats were treated i.p. with metyrapone (25 mg/kg) or caspase-1 inhibitor (Ac-YVAD-CHO, 5 mg/kg) at the time of injury. In a group of EtOH-intoxicated and burn-injured rats, metyrapone was coadministered with rIL-18 (50 µg/kg). On Day 1 after EtOH and burn injury, ICAM-1 expression was measured in jejunum (A) and ileum (B) homogenates. Values are mean ± SE from six animals in each group, except the metyrapone + rIL-18 group, which has three animals. *, P < 0.05, versus other groups except EtOH + burn + rIL-18. #, P < 0.05, versus other groups except EtOH + burn.
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Figure 7. Intestinal permeability in rats treated with metyrapone, caspase-1 inhibitor, or metyrapone plus rIL-18 following EtOH intoxication and burn injury. Rats were treated i.p. with metyrapone (25 mg/kg) or caspase-1 inhibitor (Ac-YVAD-CHO, 5 mg/kg) at the time of injury. In a group of EtOH-intoxicated and burn-injured rats, metyrapone was coadministered with rIL-18 (50 µg/kg). On Day 1 after EtOH and burn injury, intestinal permeability was determined by monitoring the transfer of FITC-dextran from the isolated intestinal segment to blood drawn from femoral artery (A) and portal vein (B) at 0, 15, 30, 60, and 90 min. (C) Data (90 min) obtained following the treatment of rats with metyrapone, caspase-1 inhibitor, or rIL-18 plus metyrapone. Values are mean ± SE from six animals in each group, except the metyrapone + rIL-18 group, which has three animals. *, P < 0.05, versus other groups; #, P < 0.05, versus sham + saline.
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Glucocorticoids are pleiotropic hormones with a wide spectrum of immunomodulatory and anti-inflammatory properties [4 , 6 , 22 23 24 25 ]. In addition, there is evidence of adrenal insufficiency in patients with septic complications, and thus, in these situations, glucocorticoids were used as therapeutic regimens [26 ]. Faunce et al [27 ] suggested a somewhat similar, protective role of glucocorticoids, following a combined insult of EtOH intoxication and burn injury. Faunce et al. [27 ] have shown that although the individual insult of EtOH intoxication and burn injury resulted in increased CORT levels, a combined insult of EtOH intoxication and burn injury caused a decrease in CORT levels. Their findings suggested that treatment of animals with CORT prevented splenic T cell suppression following EtOH and burn injury [27 ]. In contrast, we found an increase in CORT levels after EtOH intoxication and burn injury. Although a definitive cause for the differences observed between our study and the study of Faunce et al. [27 ] is not known, it is possible that those differences could be a result of different animal models used in the two studies. In many previous studies, elevated levels of CORT are shown to cause apoptosis as well as a suppression in macrophage and T cell functions [4 , 6 , 12 ]. In addition, a role of CORT is shown in increased intestinal permeability in a recent study [23 ]. Our findings suggest that although there is a close relationship between the elevation in CORT levels and increased intestinal permeability following a combined insult of EtOH intoxication and burn injury, CORT does not directly alter intestinal permeability, rather it up-regulates IL-18, which in turn contributes to increased intestinal permeability.
IL-18, like IL-12, was discovered initially to be a factor that enhances T cell interferon-
(IFN-) production [19
, 28
]. However, recent findings indicated that IL-18 can cause tissue damage in arthritis and has been found to be the central mediator in intestinal damage in an animal model of colitis [29
30
31
32
33
]. Although under those conditions, IL-18-mediated T cell activation is likely the cause for tissue damage [19
, 20
, 31
, 33
, 34
], the findings included in this manuscript suggest a role for neutrophil. It is likely that IL-18 up-regulates neutrophil recruitment to the intestine by potentiating the production of neutrophil chemokines and ICAM-1 expression in the intestinal tissue. Previous studies suggest that EtOH intoxication or burn injury results in neutrophil activation and release of free oxygen radicals (O2–) [35
36
37
]. Although an intracellular O2– release in neutrophils leads to oxidant-mediated pathogen killing and thus to an efficient host defense, excessive O2– release in the environment in close proximity of outside neutrophils can cause tissue damage. In the present study, we have not determined neutrophil O2– release capacity; however, our unpublished findings suggest a significant increase in neutrophil p-38 phosphorylation in rats receiving a combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. Such increase in p-38 activation has been correlated with an increase in neutrophil tissue-damaging actions [36
, 38
].
Although the mechanism by which CORT up-regulates IL-18 remains to be established, recent findings suggest protective and deleterious roles of IL-18 in burn and sepsis. Ami et al. [39
] have shown that IL-18 administration following burn injury restored IFN- production and prevented mortality from subsequent sepsis induced by cecal ligation and puncture. In contrast, Emmanuilidis et al. [40
] have suggested that although IL-12 may contribute to protective immune reactions against a septic challenge, IL-18 may preferentially promote organ injury and lethal shock. Findings included in this manuscript further suggest that an IL-18-mediated increase in chemokines and adhesion molecules is likely to cause increased neutrophil accumulation in the intestinal tissue, which in turn, may lead to altered intestinal barrier function following EtOH and burn injury. Napolitano et al. [41
] have shown that chronic EtOH exposure before burn injury resulted in substantial damage to the intestine. In contrast, in our model, as reported earlier [11
, 13
], we did not observe any demonstrable physical damage to the intestine following EtOH intoxication and burn injury. Furthermore, there was no difference in the tight junctions in the intestine following EtOH intoxication and/or burn injury compared with sham-injured rats. The differences in these findings could be a result of the fact that rats in our studies received a single dose of EtOH 4 h before burn injury, whereas rats in the studies reported by Napolitano et al. [41
] received chronic exposure (rats were gavaged daily for 14 days) before burn injury. Nevertheless, the mechanism by which acute EtOH intoxication and burn injury increases intestinal permeability in the absence of demonstrable physical damage to intestine (observed in our study) remains to be established. It is likely that EtOH intoxication, with or without burn injury, may alter the physiological regulation of tight junctions and paracellular spaces without causing visible damage. Such regulatory alterations in tight junctions/paracellular spaces may contribute to the enhanced permeability to FITC-dextran in rats following EtOH intoxication and burn injury.
We recognize that the present study was performed at a single time-point (i.e., Day 1 after injury) and that burn injury alone in the absence of EtOH intoxication did not influence the intestinal permeability. Many previous studies have shown that the suppression in immune cell function after burn injury in the absence of EtOH intoxication is observed up to 7–10 days after injury [42 , 43 ]. Consistent with those studies, we had shown in a previous study that on Day 2 after injury, there was an increase in intestinal permeability following burn injury alone in the absence of EtOH intoxication; however, the intensity of the tissue damage was significantly higher in the group of rats receiving the combined insult of EtOH intoxication and burn injury compared with rats receiving burn or sham injury alone [11 , 44 ]. These findings support the suggestion that EtOH consumption before injury may have a synergistic effect on intestinal permeability.
It is also to be noted that in this study, we found an increase in intestinal permeability following EtOH intoxication alone in the absence of IL-18, suggesting that an additional, potential mechanism may also exist by which EtOH intoxication can cause an increase in intestinal permeability in the absence of IL-18. Alternatively, it is possible that after EtOH intoxication, IL-18 releases early and follows a kinetic, which is different from that observed following a combined insult of EtOH intoxication and burn injury, so that by 24 h, IL-18 levels in EtOH-intoxicated animals returned to basal levels. However, as a result of combined insult of EtOH intoxication and burn injury, they remain elevated at 24 h after injury. Therefore, more studies should be performed at multiple time-points, which will establish the kinetics of CORT and IL-18 release following EtOH intoxication and burn injury. Furthermore, these studies will also help in delineating if there are differences in the mechanism of altered intestinal permeability following EtOH intoxication alone or in combination with burn injury.
In summary, results presented in this manuscript suggest that although there is an association between the CORT levels and increased intestinal permeability following a combined insult of EtOH and burn injury, CORT does not directly influence the intestinal permeability; rather, it up-regulates IL-18, which in turn contributes to increased intestinal permeability.
Received December 20, 2005; revised March 15, 2006; accepted April 11, 2006.
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production late postinjury J. Clin. Immunol. 14,340-352[CrossRef][Medline] production by T cells Nature 378,88-91[CrossRef][Medline] production from liver mononuclear cells of mice in burn injury as well as in postburn bacterial infection models and the therapeutic effect of IL-18 J. Immunol. 169,4437-4442This article has been cited by other articles:
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