Published online before print February 23, 2005
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Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham
1 Correspondence: Center for Surgical Research, University of Alabama at Birmingham, Volker Hall G 094, 1670 University Boulevard, Birmingham, AL 35294. E-mail: mashkoor.choudhry{at}ccc.uab.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: thermal injury • alcohol intoxication • cytokine • intestinal permeability • caspase-1 inhibitor • tissue injury
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, interleukin (IL)-1, IL-10, prostaglandin E2, and transforming growth factor-ß [4
, 5
, 14
, 16
17
18
]. Although burn or trauma-mediated, initial release of cytokines or inflammatory mediators is the normal host response to injury, if it remains unchecked, it can lead to multiple organ dysfunction (MODS) and multiple organ failure (MOF). Thus, MODS/MOF becomes the primary cause of death in patients who survive initial injury.
Previous studies have shown that a combined insult of EtOH and burn injury exacerbates the suppression of immune response, resulting in decreased host resistance and enhanced susceptibility to infection [5
6
7
8
9
10
11
12
13
14
15
, 19
, 20
]. Recent studies from our laboratory and reported by others suggest that acute EtOH ingestion prior to burn injury results in a several-fold increase in bacterial accumulation in the mesenteric lymph nodes (MLN) compared with the animals receiving burn or EtOH alone [8
, 20
]. Furthermore, we found that increased bacterial accumulation in MLN was accompanied by an increase in intestinal permeability and a decrease in intestinal lymphoid T cell proliferation, IL-2, and interferon-
(IFN-) production [8
]. Previous studies have implicated intestinal barrier failure and the subsequent translocation of bacteria and/or endotoxin in the development of systemic infection and MOF [16
, 21
22
23
24
]. In addition to bacteria, the intestine is also being increasingly recognized as the organ that initiates many of the inflammatory responses [5
, 16
, 21
22
23
24
25
26
27
28
29
30
31
]. These studies have shown that mesenteric lymph from rats subjected to hemorrhagic shock can cause neutrophil activation, endothelial cell permeability, and expression of adhesion molecules on endothelial cells [21
, 26
].
A multitude of factors, such as neutrophil infiltration and the production of cytokines (e.g., IL-6 and IL-10) and chemokines [e.g., IL-8, cytokine-induced neutrophil chemoattractant (CINC)], have been proposed to contribute to loss of mucosal integrity in burn, hemorrhagic shock, and sepsis [5
, 16
, 21
, 26
, 31
]. In addition, recent studies have implicated IL-18 in tissue damage in patients and in experimental models of colitis and arthritis as well as in experimental models of sepsis [32
33
34
35
36
37
38
39
40
41
42
43
44
]. IL-18 was discovered initially to be a factor that enhances IFN- production [33
34
35
36
]; however, recent studies have suggested that IL-18 alone does not induce IFN- but rather potentiates an IL-12-mediated increase in IFN- (reviewed in ref. [36
]). Consistent with these findings, our preliminary studies suggested that the decrease in MLN T cell IFN- following a combined insult of EtOH intoxication and burn injury is likely a result of a decrease in IL-12 and is independent of IL-18 up-regulation [37
]. In addition, recent studies suggested that IL-18 plays a role in neutrophil activation and superoxide production [39
40
41
42
43
44
], although a definitive mechanism by which IL-18 activates neutrophils remains to be established. Previous studies have shown that IL-18 induces production of chemokines including CINC, IL-8, and macrophage-inflammatory protein-1 [39
, 42
]. These chemokines in turn can influence neutrophil activation as well as their recruitment. Furthermore, neutrophils are shown to express IL-18 receptors (IL-18Rs) [36
, 39
, 42
43
44
], and it is likely that an up-regulation of IL-18 following EtOH and burn injury may cause increased neutrophil infiltration in the intestinal tissue. As neutrophil infiltration has been considered a cause for tissue injury [21
, 26
, 29
, 30
, 39
, 43
44
45
46
], in this study, we determined if an up-regulation of IL-18 is responsible for neutrophil infiltration in the intestine following EtOH and burn injury.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rat model of acute EtOH and burn injury
As described previously [8
], rats were divided randomly into four experimental groups: saline + sham; EtOH + sham; saline + burn; and EtOH + burn. In EtOH-treated groups, rats were gavaged with 5 ml 20% EtOH in saline. This resulted in a blood EtOH equivalent to 90–100 mg/dl nearly 4 h after gavage. Earlier, we showed that gavaging rats with 5 ml 20% EtOH results in blood EtOH levels in the range of 170–180 mg/dl within 30 min of EtOH administration. Furthermore, we found that gavaging with 5 ml 40% EtOH resulted in higher circulatory EtOH levels in a range of 380–400 mg/dl within 30 min of EtOH administration [8
]. These differences in blood EtOH levels, however, were maintained only in the first 4 h, and at the end of 4 h, these levels were 90–100 mg/dl in rats gavaged with 5 ml 20% EtOH and 240–250 mg/dl in those receiving 5 ml 40% EtOH. Regardless of the initial EtOH dose, nearly 70–80% of the circulating EtOH was metabolized within the first 8 h and 100%, 24 h after administration [8
]. Thus, we used 5 ml 20% EtOH in subsequent studies, as it gave a blood EtOH in the range of 90–100 mg/dl, which is more or less equivalent to legal limits and thus is comparable with those found in a clinical setting [5
6
7
8
9
10
11
12
13
14
15
]. In saline groups, animals were gavaged with 5 ml saline. Four hours after gavage, all animals were anesthetized with pentobarbital sodium (65 mg/kg). Hairs were shaved from their dorsal body surface. For burn procedure, rats were transferred into a template, which was fabricated to expose 25% of the total body surface area (TBSA). Animals were then immersed in boiling water (95–97°C) for 10 s. Sham-injured animals were subjected to identical anesthesia and other treatments, except that they were immersed in luke-warm water. The animals were dried immediately and given intraperitoneal (i.p.) fluid resuscitation with 
10 ml physiological saline. Animals were allowed to recover from anesthesia and were returned to their cages. Animals were allowed food and water ad libitum. This procedure resulted in a third-degree, full scald burn injury, which results in total loss of nerve endings, and thus, animal do not feel pain. After injury, we did not administer any pain killer, as such treatment of animals will interfere with the outcome of our experiments. The experiments described here were carried out in adherence with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and are approved by the Institutional Animal Care and Use Committee, University of Alabama at Birmingham.
Measurement of IL-1ß and mature IL-18 levels in culture supernatants prepared from cells of spleen, MLN, and Peyer’s patches (PP)
One day (24 h) after EtOH and burn injury, animals were anesthetized. The abdominal cavity was exposed, blood was drawn via cardiac puncture, and plasma was separated and stored at –70°C. Spleen, MLN, and PP were collected and were gently crushed to prepare single-cell suspensions in Hanks’ balanced salt solution supplemented with 10 mM HEPES and 50 µg gentamicin/ml. Mixed splenocytes and cells from MLN and PP were suspended in RPMI 1640, supplemented with L-glutamine (2 mM), 2-mercaptoethanol (50 µM), HEPES (10 mM), gentamicin (50 µg/ml), and 10% fetal calf serum, at a density of 5 x 106 cells/ml. The cell suspensions (100 µL) were added to the wells of a 96-well plate [8
]. Cells were cultured in the absence or presence of 1 or 10 µg/ml LPS. Supernatants were harvested 24 and 48 h later. IL-1ß and mature IL-18 levels were measured in the supernatants using an enzyme-linked immunosorbent assay (ELISA) kit (Biosource International).
Preparation of intestinal homogenates
Immediately after anesthetizing the rats, intestine was exposed. Leaving approximately the first 15-cm-long proximal segment of intestine, a 3-cm-long piece of intestine was removed, cleaned, and snap-frozen. Equal weights (100 mg wet weight) of intestine from various groups were suspended in 1 ml buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer, pH 6.0) and sonicated at 30 cycles, twice, for 30 s on ice [47
]. Homogenates were cleared by centrifuging at 12,000 rpm at 4°C, and the supernatants were stored at –70°C. Protein levels in the homogenates were determined using the BioRad (Hercules, CA) assay kit.
Measurement of IL-1ß and mature IL-18 in intestinal tissue
Using ELISA kits (Biosource International), IL-1ß and mature IL-18 levels in intestinal homogenates were determined.
Measurement of myeloperoxidase (MPO) levels
MPO activity in the intestinal homogenates was measured using the procedure described previously [47
]. Briefly, samples were incubated with a substrate o-dianisidine hydrochloride. This 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., St. Louis, MO) 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 water content
In a separate cohort, intestinal segments were removed, weighed, and dried for 24 h at 80°C [45
]. Water content (%) of intestinal tissue was calculated as (wet wt–dry wt)/wet wt x 100.
Histological analysis of intestine
As described previously [8
], three 1-cm-long pieces of small intestine were fixed in 10% formalin in phosphate-buffered saline for 24 h and were sent to the Histology Laboratory at the University of Alabama at Birmingham for further processing. Briefly, the sections were embedded in paraffin. These were then cut (4–5 µm) and mounted on glass slides. Intestine sections were stained with hematoxylin-eosin, observed under the microscope (Nikon Eclipse TS100) at a magnification x200 for changes in intestinal morphology, and photographed using a camera (SPOT, RTcolor, Diagnostic Instrument, Inc, Iowa City, Iowa) attached with the microscope.
Statistical analysis
Results are presented as means ± SE and were analyzed using an ANOVA statistical program (Statistical Package for Social Sciences Software program, Version 2.0, Sigma Stat). A P < 0.05 between two groups was considered as statistically significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 pg/ml. Compared with the sham group, there was no difference in IL-18 levels in rats receiving EtOH alone. Similarly, no change in serum IL-18 levels was observed in rats subjected to burn injury in the absence of prior EtOH intoxication. The EtOH-plus-burn group also did not exhibit any noticeable increase as compared with sham, as we found the values to be 25 pg/ml after 24 h of EtOH plus burn injury. Hence, no significant differences in IL-18 levels were noted in any groups.
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Figure 1. Circulatory levels (A) and the production of IL-18 by splenocytes (B), MLN (C), and PP (D) following EtOH and burn injury. Animals were killed 24 h after EtOH and burn injury. Blood was drawn via cardiac puncture, and plasma was separated. Mixed cells prepared from spleen, MLN, and PP (5x105 cells/well) were cultured in the presence of LPS (10 µg/ml) for 24 h at 37°C. IL-18 in plasma and in the culture supernatants was determined using ELISA. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham-injured rats regardless of EtOH ingestion and burn-injured rats gavaged with saline; @, P < 0.05, compared with sham-injured rats gavaged with saline.
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IL-18 levels in small intestinal tissue
As shown in Figure 2
, a tendency of an increase in IL-18 levels was observed in homogenates prepared from the small intestine of sham-injured rats gavaged with EtOH compared with sham-injured rats gavaged with saline; however, this increase was not significantly different (P>0.05). A significant (P<0.05) increase in intestinal IL-18 levels was noted in animals in burn-injured rats gavaged with saline compared with sham-injured rats gavaged with saline. The increase in IL-18 levels in intestinal homogenates in burn-injured rats gavaged with saline was not found to be significantly different from IL-18 levels in the intestinal homogenates prepared from sham rats gavaged with EtOH. A further increase (P<0.05) in IL-18 levels was observed in the intestine of EtOH and burn-injured rats compared with sham-injured rats, regardless of their EtOH intoxication. Although IL-18 levels in intestinal tissue following a combined insult of EtOH intoxication and burn injury were higher than those observed in rats receiving burn injury alone in the absence of EtOH, this increase was not found to be significantly different.
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Figure 2. Intestinal tissue levels of IL-18 following EtOH and burn injury. Animals were killed 24 h after EtOH and burn injury. Equal weights (100 mg) of intestine from various groups were sonicated. Homogenates were cleared by centrifugation, and IL-18 levels in the clear supernatants were determined using ELISA. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham-injured rats regardless of EtOH ingestion; #, P > 0.05, compared with burn-injured rats gavaged with saline; @, P < 0.05, compared with sham-injured rats gavaged with saline.
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Figure 3. MPO activity in intestinal tissue 24 h after EtOH and burn injury. Animals were killed 24 h after EtOH and burn injury. Equal weights (100 mg) of intestine from various groups were suspended in 1 ml buffer containing 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0) and sonicated. Homogenates were cleared by centrifugation, and MPO activity in the supernatants was determined as described in Materials and Methods. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham-injured rats regardless of EtOH ingestion and burn-injured rats gavaged with saline.
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Figure 4. Representative photomicrographs of small intestine of sham gavaged with saline (A), sham gavaged with EtOH (B), burn gavaged with saline (C), and burn gavaged with EtOH (D). Animals were killed 24 h after EtOH and burn injury. Small intestine was removed and processed as described in Materials and Methods. Intestine sections were stained with hematoxylin-eosin, examined at an original magnification x200, and photographed.
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Effect of caspase-1 inhibitor treatment on IL-18 production by splenic and intestinal lymphoid cells
There was no significant difference in circulatory levels of IL-18 in caspase-1 inhibitor-treated or -untreated rats following EtOH and burn injury (data not shown). As shown in the Figure 5
, administration of casapse-1 inhibitor did not affect splenic (Fig. 5A)
MLN (Fig. 5B)
, and PP (Fig. 5C)
IL-18 production in sham animals; however, similar treatment of rats significantly attenuated the increase in IL-18 in splenic and intestinal lymphoid organs (MLN and PP) after EtOH and burn injury.
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Figure 5. Effect of caspase-1 inhibitor on IL-18 production by splenocytes (A), MLN (B), and PP (C) following EtOH and burn injury. Immediately after sham or burn injury, rats were divided randomly into two groups. One group of sham, burn, and EtOH-plus-burn rats was injected i.p. with caspase-1 inhibitor (Ac-YVAD-CHO) at a dose of 5 mg/kg. The other group received a similar amount of vehicle [dimethyl sulfoxide (DMSO)]. Twenty-four hours after injury, rats were killed. Mixed cells prepared from spleen, MLN, and PP (5x105 cells/well) were cultured in the presence of LPS (10 µg/ml) for 24 h at 37°C. IL-18 in the culture supernatants was determined using ELISA. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham- and burn-injured rats gavaged with saline; @, P < 0.05, compared with sham-injured rats gavaged with saline; #, P > 0.05, compared with sham-injured rats.
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Figure 6. In vitro treatment of spleen, MLN, and PP cells with caspase-1 inhibitor prevented IL-18 production following EtOH and burn injury. Twenty-four hours after injury, rats were killed. Mixed cells prepared from spleen, MLN, and PP (5x105 cells/well) were cultured first, in the presence of vehicle (DMSO) or caspase-1 inhibitor (Ac-YVAD-CHO) at doses 50 µM and 25 µM for 30 min at 37°C. At the end of 30 min, LPS was added to each well, and plates were incubated for 24 h at 37°C. Supernatants were harvested for IL-18 measurement. Data are means ± SE from four animals in each group. *, P < 0.05, compared with sham- and burn-injured rats gavaged with the EtOH group treated with caspase-1 inhibitor.
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Figure 7. Effect of caspase-1 inhibitor on intestinal tissue levels of IL-18 following EtOH and burn injury. Immediately after sham or burn injury, rats were divided randomly into two groups. One group of sham, burn, and EtOH-plus-burn rats was injected i.p. with caspase-1 inhibitor (Ac-YVAD-CHO) at a dose of 5 mg/kg. The other group received a similar amount of vehicle (DMSO). Animals were killed 24 h after EtOH and burn injury. Equal weights (100 mg) of intestine from various groups were sonicated, and IL-18 levels in the homogenates were determined using ELISA. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham-injured rats; @, P < 0.05, compared with sham-injured rats; #, P > 0.05, compared with sham-injured rats.
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Figure 8. Effect of caspase-1 inhibitor on intestinal tissue levels of IL-1 following EtOH and burn injury. Immediately after sham or burn injury, rats were divided randomly into two groups. One group of sham, burn, and EtOH-plus-burn rats was injected i.p. with caspase-1 inhibitor (Ac-YVAD-CHO) at a dose of 5 mg/kg. The other group received a similar amount of vehicle (DMSO). Animals were killed 24 h after EtOH and burn injury. Equal weights (100 mg) of intestine from various groups were sonicated, and IL-1 levels in the homogenates were determined using ELISA. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham-injured rats.
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Figure 9. Effect of caspase-1 inhibitor on intestinal tissue MPO activity following EtOH and burn injury. Immediately after sham or burn injury, rats were divided randomly into two groups. One group of sham, burn, and EtOH-plus-burn rats was injected i.p. with caspase-1 inhibitor (Ac-YVAD-CHO) at a dose of 5 mg/kg. The other group received a similar amount of vehicle (DMSO). Animals were killed 24 h after EtOH and burn injury. Equal weights (100 mg) of intestine from various groups were sonicated, and MPO activity in the supernatants was determined as described in Materials and Methods. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham- and burn-injured rats gavaged with saline; #, P > 0.05, compared with sham-injured rats.
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Figure 10. Effect of caspase-1 inhibitor on intestinal tissue edema following EtOH and burn injury. Immediately after injury, EtOH and burn-injured rats were divided randomly into two groups. One group of EtOH-plus-burn rats was injected i.p. with caspase-1 inhibitor (Ac-YVAD-CHO) at a dose of 5 mg/kg. The other group received a similar amount of vehicle (DMSO). Animals were killed 24 h after EtOH and burn injury. Intestinal segments were removed, weighed, and dried for 24 h at 80°C. Water content (%) of intestinal tissue was calculated as (wet wt–dry wt)/wet wt x 100. Data are means ± SE from at least six animals in each group. *, P < 0.05, compared with sham-injured rats, regardless of EtOH ingestion, burn-injured rats gavaged with saline and EtOH-plus-burn-injured rats treated with caspase-1 inhibitor. #, P > 0.05, compared with sham-injured rats.
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IL-18 is mainly produced by activated macrophages, Kupffer cells, dendritic cells, Langerhans cells, and B cells [32 , 36 ]. In addition IL-18 is shown to be produced in the lamina propria as well as by intestinal epithelial cells [33 34 35 36 ]. IL-18 is synthesized as a nonfunctional precursor, pro-IL-18. Studies have shown that protease activity mediated by IL-1-converting enzyme, also referred to as caspase-1, is needed for conversion of pro-IL-18 to the mature, functional protein [33 34 35 36 ]. The activity of IL-18 can also be regulated after release by IL-18-binding protein (IL-18bp), which is normally present in serum. IL-18bp can bind IL-18 and block bioavailability and subsequent function [38 ]. In our study, we treated rats with caspase-1 inhibitor Ac-YVAD-CHO and then examined the effects of IL-18 on post-EtOH and burn injury-mediated neutrophil accumulation and edema in small intestine. Ac-YVAD-CHO has been used in previous studies and is found to be specific for its action on caspase-1 [32 , 40 ]. As caspase-1 also plays a role in IL-1 production, we determined IL-1 levels in our experiments to rule out the role of IL-1 [33 34 35 36 ]. As discussed in Results, we did not observe detectable/significant IL-1 levels in plasma or in the immune cell culture supernatants in any experimental group. However, in intestinal tissue homogenates, there was a significant decrease in IL-1 following EtOH and burn injury compared with sham-injured rats. This is in contrast to IL-18, which is up-regulated following EtOH and burn injury. The treatment of rats with caspase-1 inhibitor (Ac-YVAD-CHO) at 5 mg/kg did not influence further intestinal IL-1 levels in EtOH and burn-injured rats, and a similar treatment clearly blocked IL-18 synthesis. The mechanism of such differential roles of caspase-1 in regulation of IL-1 and IL-18 remains to be established. Previous studies have shown that IL-18 is constitutively expressed, and IL-1 is not [32 ]. It is likely that caspase-1 does not regulate immature forms of IL-1 and IL-18 but rather participates only in the maturation process of available biologically immature IL-1 or IL-18. Furthermore, it is possible that IL-1 and IL-18 production follows different kinetics following EtOH and burn injury. Although we plan to explore all these possibilities in future investigations, the results presented in this manuscript collectively suggest a relationship between IL-18 up-regulation and neutrophil recruitment as well as in the development of intestinal edema following EtOH and burn injury.
IL-18, like IL-12, was discovered initially to be a factor that enhances IFN- production [33
34
35
36
]. It is not surprising, therefore, that it appears essential to host defenses against a variety of infections. IL-18 is particularly effective during the clearance of intracellular bacteria, fungi, and protozoa, requiring the induction of host-derived IFN-. In a recent study, Ami et al. [50
] have shown that IL-18 administration following burn injury restored IFN- production and prevented mortality from subsequent sepsis induced by cecal ligation and puncture (CLP). In contrast, our preliminary findings suggest that the decrease in IFN- following EtOH and burn injury is likely a result of a decrease in IL-12 and is independent of IL-18. Although the difference between the two studies remains to be established, such variations could result from a different experimental model used in the two studies. Ami et al. [50
] induced 20% TBSA burn injury in mice by a heated brass blade before CLP, and in our studies, rats were gavaged with EtOH 4 h before receiving 25% burn injury by immersing them into the 95°C hot water. Recent studies suggested that like many other proinflammatory cytokines, IL-18 possesses broad, immunomodulatory properties. Emmanuilidis et al. [51
] recently have shown that IL-12 was reduced significantly in sepsis patients compared with control, surgical patients without sepsis. In contrast to IL-12, IL-18 serum levels were significantly higher in sepsis patients than in controls. Furthermore, they observed that IL-18 levels were increased significantly in patients with lethal sepsis compared with sepsis survivors at all time-points studied. From these findings, Emmanuilidis et al. [51
] have concluded that IL-12 may contribute to protective immune reactions against a septic challenge, whereas IL-18 may preferentially promote organ injury and lethal shock. Additional findings suggest that IL-18 induces a wide array of inflammatory responses, such as the expression of adhesion molecules, intercellular adhesion molecule-1, in different cell types, activation of nuclear factor-
B, Fas ligand expression, and induction of chemokines [33
34
35
36
37
38
39
40
41
42
43
44
]. IL-18 is often expressed in inflammatory lesions and is found to be elevated in the blood of patients with a variety of diseases including Crohn’s disease, Sjogren syndrome, graft-versus-host disease, and rheumatoid arthritis [41
]. These studies suggested that inappropriate IL-18 production contributes to the pathogenesis of these diseases and may influence the clinical outcome of these patients. Although many of these studies suggested IL-18-induced IFN- as the cause for tissue damage [33
34
35
36
, 41
], others have supported the role of neutrophil recruited by IL-18 [39
, 40
, 42
43
44
]. As in our studies we observed a decrease in IFN- [7
], it is unlikely that IFN- is involved in intestinal tissue damage following EtOH and burn injury; instead, IL-18-mediated, increased neutrophil recruitment to intestine is likely to play a role in increased intestinal edema and permeability. The mechanism by which IL-18 up-regulation following EtOH and burn injury mediates neutrophil recruitment to intestine remains to be established.
Previous studies have shown that neutrophils constitutively express IL-18R ( and ß), and thus, IL-18 can activate multiple neutrophil functions including superoxide production [36
, 44
]. Jordan et al. [39
] have shown that intratracheal administration of IL-18 caused significant increases in lung vascular permeability and resulted in increased infiltration of neutrophils. Conversely, intratracheal instillation of anti-IL-18 in inflamed lung greatly reduced the recruitment of these cells and prevented an increase in vascular permeability. Similarly, intratracheal administration of IL-18bp also resulted in suppressed lung vascular permeability and decreased bronchoalveolar lavage content of neutrophils, cytokines, and chemokines. Consistent with these studies, Sir et al. [45
] have shown that neutrophil antiserum prevents the increase in intestinal permeability and edema following burn injury. As IL-18Rs are present on neutrophil, IL-18 can activate and recruit neutrophil directly. Alternatively, IL-18 can induce the production of chemokines such as CINC [39
, 42
, 43
], which in turn, can influence neutrophil activation and their recruitment to intestinal tissue. Although in the present study, we have not determined the mechanism by which neutrophil causes tissue injury following EtOH and burn injury, previous studies have shown that insult such as EtOH intoxication or burn results in neutrophil activation and production of free oxygen radical (O2–) [25
, 26
, 29
, 30
, 46
]. Although such intracellular O2– production in neutrophils is essential for pathogen killing, an excessive production of O2– and its release to the extracellular environment can cause tissue damage. Such neutrophil-mediated tissue injury is demonstrated in pathological conditions of acute and chronic EtOH intoxication, rheumatoid arthritis, acute respiratory distress syndrome, and tissue ischemia [25
, 26
, 29
, 30
, 39
40
41
42
43
44
45
46
]. It is interesting to note that in many of the injury conditions, the chemotactic factors for neutrophil recruitment to tissue/organ originate from the cells at sites of inflammation, and thus, inflamed tissue/organs themselves become the source for such chemotactic response. Consistent with this hypothesis, we observed a significant increase in IL-18 levels in intestinal homogenates prepared from EtOH and burn-injured rats compared with those from sham-injured rats. Thus, an increase in IL-18 level, locally in intestinal tissue, directly, or via inducing neutrophil chemotactic factor (e.g., CINC), may play a role in neutrophil recruitment to intestine following EtOH and burn injury.
In summary, results presented here suggest a role of IL-18 in increased neutrophil (determined by MPO activity) recruitment to the intestine following EtOH and burn injury. Whether neutrophil recruitment is a direct effect of IL-18 or is a result of an up-regulation of neutrophil chemotactic factors such as CINC remains to be established. Regardless of the mechanism, once neutrophils are recruited, they can potentiate a microvascular blockade, leading to tissue hypoperfusion, or penetrate through extracellular spaces into a submucosal region to cause an increase in intestinal permeability and edema (reviewed in ref. [30 ]). In the present study, we have not determined intestinal permeability; rather, we examined the development of intestinal edema, which is another important index of tissue injury. Consistent with increased intestinal permeability as reported previously [8 ], we observed a significant increase in intestinal tissue water content in rats receiving a combined insult of EtOH intoxication and burn injury compared with the rats receiving EtOH intoxication or injury alone. The finding of a decreased intestinal MPO activity and edema along with IL-18 in EtOH and burn-injured rats treated with caspase-1 (IL-18) inhibitor suggests that IL-18 is likely to play a role in neutrophil accumulation and increased edema in intestine following EtOH and burn injury.
Received July 12, 2004; revised January 14, 2005; accepted January 25, 2005.
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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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H.-P. Yu, S. Yang, Y.-C. Hsieh, M. A. Choudhry, K. I. Bland, and I. H. Chaudry Maintenance of lung myeloperoxidase activity in proestrus females after trauma-hemorrhage: upregulation of heme oxygenase-1 Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L400 - L406. [Abstract] [Full Text] [PDF]
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X. Li, S. N. Rana, M. G. Schwacha, I. H. Chaudry, and M. A. Choudhry A novel role for IL-18 in corticosterone-mediated intestinal damage in a two-hit rodent model of alcohol intoxication and injury J. Leukoc. Biol., August 1, 2006; 80(2): 367 - 375. [Abstract] [Full Text] [PDF]
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H.-P. Yu, T. Shimizu, Y.-C. Hsieh, T. Suzuki, M. A. Choudhry, M. G. Schwacha, and I. H. Chaudry Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage J. Leukoc. Biol., May 1, 2006; 79(5): 963 - 970. [Abstract] [Full Text] [PDF]
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H.-P. Yu, M. A. Choudhry, T. Shimizu, Y.-C. Hsieh, M. G. Schwacha, S. Yang, and I. H. Chaudry Mechanism of the salutary effects of flutamide on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of estrogen receptor-{beta}-dependent HO-1 J. Leukoc. Biol., February 1, 2006; 79(2): 277 - 284. [Abstract] [Full Text] [PDF]
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