Published online before print August 24, 2004
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* Centre for Experimental Medicine, Nephrology & Critical Care, The William Harvey Research Institute, Queen Mary, University of London, United Kingdom;
Department of Experimental Pharmacology, Università ‘Federico II’, Naples, Italy; and
Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Italy
1 Correspondence: Centre for Experimental Medicine, Nephrology & Critical Care, William Harvey Research Institute, Queen Mary, University of London, Charterhouse Square, London, EC1M 6BQ, UK. E-mail: c.thiemermann{at}qmul.ac.uk
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-glutamyl transferase, alkaline phosphatase, bilirubin), renal dysfunction (creatinine), and pancreatic injury (lipase, amylase). Absence of functional 5-LOX (zileuton treatment or targeted disruption of the 5-LOX gene) reduced the multiple organ injury/dysfunction caused by endotoxemia. Polymorphonuclear leukocyte infiltration (myeloperoxidase activity) in the lung and ileum as well as pulmonary injury (histology) were markedly reduced in 5-LOX–/– mice. Zileuton also reduced the LPS-induced expression of CD11b/CD18 on rat leukocytes. We propose that endogenous 5-LOX metabolites enhance the degree of multiple organ injury/dysfunction caused by severe endotoxemia by promoting the expression of the adhesion molecule CD11b/CD18 and that inhibitors of 5-LOX may be useful in the therapy of the organ injury/dysfunction associated with endotoxic shock.
Key Words: shock • ß2-integrins • CD11a/CD18 • CD11b/CD18 • leukotrienes
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LTB4 is a proinflammatory mediator that activates polymorphonuclear leukocytes (PMN), thus changing their shape and promoting their binding to endothelium by inducing the expression of cell-adhesion molecules. The localization of leukocytes to the site of inflammation requires several families of adhesion molecules. Firm adhesion of leukocytes to the microvascular endothelium is dependent on the function of the class of adhesion molecules called ß2-integrins, which are expressed on neutrophil surface and interact with members of the immunoglobulin (Ig) supergene family expressed on endothelial cells such as intercellular adhesion molecule-1 (ICAM-1) [19
20
21
22
]. The ß2-integrins are heterodimeric molecules consisting of a common ß-subunit (CD18), which is associated with one of four different 
-subunits (CD11a–d) [23
, 24
]. CD11a/CD18 and CD11b/CD18 have been reported to mediate chemoattractant-induced leukocyte adhesion [25
26
27
28
29
30
31
32
33
34
]. It is known that LTB4 stimulates ß2-integrin-dependent adhesion of neutrophils [35
]. The 5-LOX inhibitor, zileuton, is highly effective at preventing the formation of LTs in vitro, ex vivo, and in vivo [13
] and is currently in clinical use for the treatment of patients with asthma [36
].
Here, we investigate the effects of zileuton on the organ injury and dysfunction caused by severe endotoxemia in the rat in vivo. To ensure that an enhanced formation of LTs from 5-LOX does indeed contribute to the organ injury and dysfunction, we have compared the degree of multiple organ injury and dysfunction caused by endotoxemia in wild-type mice with that in mice in which the gene for 5-LOX is absent (5-LOX–/–). To investigate the mechanism by which zileuton exerts its effects, we investigated the expression of adhesion molecules CD11a/CD18 and CD11b/CD18 on rat whole blood neutrophils by flow cytometry.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Forty mice (4–5 weeks old, 20–22 g) with a targeted disruption of the 5-LOX gene (5-LOX–/–) and littermate wild-type controls were purchased from Jackson Laboratories (Harlan Nossan, Italy). The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).
Experimental designs
Rats were assigned to four experimental groups. Sham group: Rats were treated with 10% dimethyl sulfoxide (DMSO; 1 ml/kg i.v., n=13) without causing endotoxic shock [received saline rather than lipopolysaccharide (LPS)]. Sham + Zileuton group: Rats were treated with zileuton (3 mg/kg i.v., n=13) without causing endotoxic shock. LPS group: Rats were treated with 10% DMSO (1 ml/kg i.v., n=12) 15 min before they were subjected to endotoxic shock. Escherichia coli LPS (6 mg/kg i.v., serotype 0127:B8) was given slowly over 10 min. LPS + Zileuton group: Rats were treated with zileuton (3 mg/kg i.v., n=13) 15 min before they were subjected to endotoxic shock.
Following administration of endotoxin or saline, rats were given saline (1 ml/kg/h i.v.) throughout the experiment. The dose of zileuton used in this study (3 mg/kg i.v.) has been shown to significantly attenuate the LTB4 synthesis in vivo [17 ].
Mice were randomly divided into four groups. Sham-Wild-type group: Mice were treated with saline (i.p., n=10) without causing endotoxic shock. Sham-5-LOX–/– group: Mice were treated with saline (i.p., n=10) without causing endotoxic shock. LPS-Wild-type group: Mice were subjected to endotoxic shock by administration of E. coli LPS (20 mg/kg i.p., n=10). LPS-5-LOX–/– group: Mice were subjected to endotoxic shock by administration of E. coli LPS (20 mg/kg i.p., n=10).
Quantification of organ injury and dysfunction
Samples were collected 6 h after administration of endotoxin in rats and 16 h after administration of endotoxin in mice (endotoxic shock). Blood samples were collected into a serum gel S/1.3 tube (Sarstedt, Germany) and centrifuged to separate plasma. All plasma samples were analyzed within 24 h by a veterinary clinical laboratory using standard laboratory techniques. Plasma values were examined as described previously for indices of renal dysfunction [38
], liver injury [39
, 40
], and pancreatic injury [41
].
LTB4 measurement
Plasma LTB4 levels of four animals from each group of rats and mice were determined using a commercial enzymatic immunoassay kit (Cayman Chemicals, Inalco, Milan, Italy).
Myeloperoxidase (MPO) activity
MPO activity, an indicator of PMN accumulation, was determined as described previously [42
]. Sixteen hours after the injection of LPS, samples of lung and ileum tissue were obtained and weighed. Each sample was homogenized in a solution containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and hydrogen peroxide (0.1 mM). The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol peroxide/min at 37°C and was expressed in U/100 mg wet tissue.
Histological examination
Lung biopsies were taken 16 h after LPS administration. Lung biopsies were fixed for 1 week in 10% (w/v) phosphate-buffered saline (PBS)-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (8 µm) were then deparaffinized with xylene and stained with hematoxylin and eosin (H&E). All sections were studied using light microscopy (Dialux 22 Leitz). The histopathologic score was determined by a pathologist who was unaware of the infection status of the animals. This score assigned the samples values between 0 and 21 (the higher the score, the greater the inflammatory changes in the lung). Lungs were evaluated for inflammatory infiltrates and graded according to a scheme similar to that described previously [43
]. Each section was graded on the basis of a cumulative score from five categories: focal thickening of the alveolar membranes; pulmonary edema; perivascular infiltrates; intra-alveolar hemorrhage; and congestion.
Flow cytometry
Antibodies and reagents
Phycoerythrin- and fluorescein isothiocyanate-conjugated mouse monoclonal antibodies (mAb) against CD11a (IgG2a) and CD11b (IgA), respectively, were purchased from Becton Dickinson (Oxford, UK). Isotype-, fluorochrome-, and protein concentration-matched controls (Becton Dickinson) were run in parallel to the mAb.
Preparation of blood leukocytes for flow cytometry
Heparinized, arterial blood (
600 µl) was collected from rats at times 0, 100, and 400 min of the experiment. For flow cytometric analysis of leukocyte adhesion molecules, 100 µl whole blood in 12 x 75 mm polystyrene test tubes (Becton Dickinson) was mixed with mAb against rat CD11a (5 µl) or CD11b (3 µl). The test tubes were then incubated on ice for 30 min with continuous shaking, protected from light. Blood with no antibody served as a control of autofluorescence. After finishing incubation with anti-CD11a/CD11b, erythrocytes were lysed by adding 2 ml Becton Dickinson FACSTM lysing solution to the test tubes, after which they were incubated for 10 min on ice in the dark and centrifuged at 1000 g for 3 min at 4°C. The supernatant was discarded, and the leukocyte pellet was resuspended and washed twice in 2 ml ice-cold optimized PBS (Cell Wash, Becton Dickinson). Finally, leukocytes were fixed in 0.3 ml 1% w/v paraformaldehyde in PBS, pH 7.4, and the tubes were stored in the dark at 4°C for up to 24 h until flow cytometric analysis could be performed.
Flow cytometric analysis
The samples were analyzed within 24 h using a Becton Dickinson FACScan flow cytometer equipped with the Cell-Quest software (Becton Dickinson). CaliBRITE-3® beads (Becton Dickinson) and FACS COMP software (Becton Dickinson) were used on a weekly basis to calibrate the fluorescence intensity in accordance with the manufacturer’s instructions. Ten thousand neutrophils were collected from each sample with light-scatter gain set in the linear mode and fluorescence gain set in the logarithmic mode. The neutrophil population was identified by means of their light-scatter characteristics (forward- vs. side-scatter), enclosed in an electronic gate (Fig. 1
), and fluorescence histogram analysis was then performed. Antibody binding was expressed as median fluorescence intensity (MFI), whose values were corrected for nonspecific binding by subtracting the MFI measured for the matched isotype-control sample.
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Figure 1. Forward-scatter (FSH-H) versus side-scatter (SSH-H) dot-plot showing location of neutrophils (n) and mononuclear cells (mnc).
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Statistical evaluation
All data are presented as means ± SEM of n observations, where n represents the number of animals or blood samples studied. For repeated measurements (hemodynamics), a two-way ANOVA was performed. Data without repeated measurements (multiple organ injury/failure) were analyzed by one-way ANOVA, followed by a Dunnett’s test for multiple comparisons. A Pvalue less than 0.05 was considered statistically significant.
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Figure 2. Changes in (A) MAP and (B) HR in rats subjected to the surgical procedure without administration of LPS and pretreated with 10% DMSO (Sham Control, 1 ml/kg i.v., n=13) or zileuton (Sham+Zil, 3 mg/kg i.v., n=13) or endotoxemia for 6 h. Rats were pretreated with the vehicle (LPS Control, 6 mg/kg i.v., n=12) or zileuton (LPS+Zil, 3 mg/kg i.v., n=13). *, P< 0.05, when compared with the LPS group; #, P< 0.05, when compared with the Sham group.
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Organ injury and dysfunction caused by endotoxemia in rats treated with zileuton
When compared with the rats treated with vehicle rather than endotoxin (Sham group), endotoxemia (LPS group) resulted in significant rises in the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and -glutamyltransferase (-GT; liver injury, Fig. 3
), and creatinine (renal dysfunction, Fig. 4
). Pretreatment with zileuton prior to administration of endotoxin (LPS+Zileuton group) attenuated the rises in the serum levels of AST, ALT, and -GT (P<0.05, Fig. 3
) and hence, the liver injury caused by LPS. Zileuton also attenuated the rise in serum level of creatinine in rats subjected to endotoxemia, thus ameliorating the renal dysfunction caused by endotoxin (P<0.05, Fig. 4
). In sham-operated animals, administration of zileuton (Sham+Zileuton group) did not result in any significant alterations in the serum levels of any of the parameters measured when compared with sham-operated animals treated with 10% DMSO (Sham group).
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Figure 3. Serum levels of (A) AST, (B) ALT, and (C) -GT in rats subjected to the surgical procedure and pretreated with 10% DMSO (Sham group, n=13) or zileuton (Sham+Zil, n=13). Rats subjected to endotoxic shock (LPS 6 mg/kg i.v.) were pretreated with 10% DMSO (LPS group, n=12) or zileuton (LPS+Zil, n=13). *, P< 0.05, when compared with the LPS group; #, P< 0.05, when compared with the Sham group.
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Figure 4. Serum levels of creatinine in rats subjected to the surgical procedure and pretreated with 10% DMSO (Sham group, n=13) or zileuton (Sham+Zil, n=13). Rats subjected to endotoxic shock (LPS, 6 mg/kg i.v.) were pretreated with 10% DMSO (LPS group, n=12) or zileuton (LPS+Zil n=13). *, P< 0.05, when compared with the LPS group; #, P< 0.05, when compared with the Sham group.
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Figure 5. Serum levels of (A) AST, (B) ALT, (C) bilirubin, and (D) alkaline phosphatase in wild-type (WT) and 5-LOX–/– mice treated with saline (Sham) or LPS (20 mg/kg i.p.). n = 10 in each group. *, P< 0.05, when compared with LPS-WT; #, P< 0.05, when compared with Sham-WT.
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Figure 6. Serum levels of (A) amylase, (B) lipase, and (C) creatinine in wild-type (WT) and 5-LOX–/– mice treated with saline (Sham) or LPS (20 mg/kg i.p.). n = 10 in each group. *, P< 0.05, when compared with LPS-WT; #, P< 0.05, when compared with Sham-WT.
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Plasma LTB4 levels in rats treated with zileuton and in wild-type and 5-LOX–/– mice
In rats, endotoxemia caused a significant increase in the plasma level of LTB4 when compared with sham-operated rats, suggesting a significant increase in 5-LOX activity (Fig. 7A
). Administration of zileuton significantly attenuated LTB4 synthesis caused by endotoxemia (Fig. 7A)
.
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Figure 7. Plasma levels of LTB4 in (A) rats subjected to the surgical procedure and pretreated with 10% DMSO (Sham group, n=13) or zileuton (Sham+Zil, n=13). Rats subjected to endotoxic shock (LPS, 6 mg/kg i.v.) were pretreated with 10% DMSO (LPS group, n=12) or zileuton (LPS+Zil, n=13). (B) Plasma levels of LTB4 in wild-type (WT) and 5-LOX–/– mice treated with saline (Sham) or LPS (20 mg/kg i.p.). n = 10 in each group. *, P< 0.05, when compared with (A) LPS group or (B) LPS-WT; #, P< 0.05, when compared with (A) Sham group or (B) Sham-WT .
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Expression of CD11a/CD18 and CD11b/CD18 on rat leukocytes measured by flow cytometry
Endotoxemia for 6 h in rats resulted in a significant increase in the expression of CD11b/CD18 on neutrophils in rat whole blood when compared with sham-operated rats (P<0.05, Fig. 8B
). In contrast, the expression of CD11a/CD18 was decreased during endotoxemia when compared with sham-operated rats (P<0.05, Fig. 8A
). Treatment with zileuton of the rats subjected to endotoxemia significantly reduced the LPS-stimulated expression of CD11b/CD18 on neutrophils (P<0.05, Fig. 8B
) and attenuated the decline in the expression of CD11a/CD18 (P<0.05, Fig. 8A ).
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Figure 8. The effect of zileuton on the expression of neutrophil ß2-integrins (A) CD11a/CD18 and (B) CD11b/CD18 at 1 and 6 h post-LPS. Rats were administered LPS 15 min after the administration of zileuton (3 mg/kg i.v.). Values are mean ± SEM. *, P< 0.05, versus LPS; #, P< 0.05, versus baseline.
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Figure 9. MPO activity in the lung (A) and (B) ileum from wild-type (WT) and 5-LOX–/– mice treated with saline (Sham) or LPS (20 mg/kg i.p.). n = 10 in each group. *, P< 0.05, when compared with LPS-WT; #, P< 0.05, when compared with Sham-WT.
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Figure 10. Histological examination of the lung from (a) sham, (b) wild-type mice subjected to endotoxemia for 16 h (E. coli LPS 20 mg/kg i.p.), and (c) 5-LOX–/– mice subjected to endotoxemia for 16 h (E .coli LPS 20 mg/kg i.p.). The examination of the lung biopsies revealed extravasation of red cells and neutrophils and macrophage accumulation in wild-type mice subjected to endotoxemia (b). The absence of a functional 5-LOX gene in 5-LOX–/– mice resulted in a significant reduction of pulmonary injury caused by LPS (20 mg/kg i.p.; c). H&E, Original magnification, x150; figures are representative of at least three experiments performed on different experimental days.
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-GT, alkaline phosphatase, and bilirubin, indicating liver injury and dysfunction, and creatinine, indicating the development of acute renal dysfunction. In endotoxemic mice, we also observed significant increases in serum levels of amylase and lipase, indicating pancreatic injury. Histological analysis also revealed marked lung injury in endotoxemic, wild-type mice. In rats, endotoxemia resulted in circulatory failure (hypotension and tachycardia). Zileuton is a selective and potent inhibitor of 5-LOX, which is currently being used in the therapy of patients suffering from asthma [36 ]. Here, we demonstrate that pretreatment of rats subjected to endotoxemia with zileuton markedly attenuated the liver injury. The degree of inhibition of renal dysfunction, however, is less pronounced, suggesting that activation of 5-LOX may not fully account for the renal dysfunction seen during endotoxemia. In contrast, 5-LOX–/– mice show a much better profile of protection. The dose of zileuton used in this study (3 mg/kg i.v.) has been shown to significantly attenuate the LTB4 synthesis in vivo [17 ]. However, it is possible that a higher dose of zileuton might have afforded better protection against the renal dysfunction caused by endotoxemia.
As the circulatory failure induced by endotoxemia was not prevented by zileuton, favorable hemodynamic effects do not contribute to the protection of the organs afforded by the 5-LOX inhibitor. What then is the mechanism(s) by which 5-LOX or its products amplify the inflammatory response and ultimately the organ injury and dysfunction caused by endotoxemia? Endotoxemia resulted in significant increases in the plasma levels of LTB4 in rats. This increase in 5-LOX activity caused by endotoxemia was reduced by treatment with zileuton. To confirm that the beneficial effects of zileuton are indeed a result of inhibition of 5-LOX (rather than a nonspecific effect), we have compared the effects of severe endotoxemia in 5-LOX–/– mice (no detectable levels of LTB4 in sham-operated mice or 5-LOX–/– mice subjected to endotoxemia) with those obtained using their wild-type littermates. We report here for the first time that the degree of multiple organ injury and dysfunction caused by severe endotoxemia is reduced in 5-LOX–/– mice. Thus, we propose that metabolites of 5-LOX contribute to the pathophysiology of endotoxemic organ injury and dysfunction and that inhibition of 5-LOX activity with zileuton reduces the multiple organ injury and dysfunction associated with severe endotoxemia.
We have recently reported a reduction of renal I/R injury in 5-LOX–/– mice and by the 5-LOX inhibitor zileuton [17 ]. In that study, we demonstrated that zileuton reduces the expression of ICAM-1 caused by I/R of the kidney in wild-type mice, with a similar reduction in ICAM-1 expression in 5-LOX–/– mice. We proposed that endogenous 5-LOX metabolites enhance the degree of renal injury, dysfunction, and inflammation caused by I/R of the kidney by promoting the expression of adhesion molecules [17 ]. In models of I/R of the skin, zileuton reduces the expression of CD18 and ß2-integrins [14 , 46 ], but it is not known whether this effect is indeed a result of inhibition of 5-LOX. We demonstrate here that in endotoxemic rats, expression of ß2-integrin CD11b/CD18 on neutrophils was increased and that zileuton reduced the endotoxin-stimulated expression of CD11b/CD18 on neutrophils. In contrast, we found that the expression of CD11a/CD18 was decreased by endotoxin, and zileuton treatment of the rats attenuated this decline. We also report here that the accumulation of neutrophils was reduced in the lung and ileum from 5-LOX–/– mice subjected to endotoxemia when compared with wild-type littermates. This was confirmed in the histological analysis, which revealed that the extravasation of red cells and neutrophils and macrophage accumulation in the lung were also significantly reduced in 5-LOX–/– mice. Hence, inhibition of 5-LOX activity reduces expression of adhesion molecule(s) and neutrophil accumulation in tissues in response to endotoxemia and thus, the organ injury and dysfunction. This is not entirely surprising, as it is known that LTB4 stimulates ß2-integrin-dependent adhesion of neutrophils [35 ]. There is also recent evidence that inhibition of LT synthesis reduces firm adhesion of neutrophils to cerebral vessels in the guinea pig brain [47 ]. As leukocyte recruitment plays a pivotal role in the organ injury caused by endotoxin [20 , 22 , 27 , 48 , 49 ], we propose that zileuton protects the organs against endotoxin-induced injury and dysfunction by inhibiting the synthesis of 5-LOX-derived LTs, consequently reducing the stimulation of ß2-integrin-dependent adhesion and finally, the recruitment of neutrophils. Our results suggest that CD11b/CD18 plays a more important role in the neutrophil recruitment than CD11a/CD18. These results are in accordance with previous studies, in which CD11b/CD18 expression on leukocytes from endotoxin-injected mice was greatly increased when compared with that of controls [25 ]. The decrease in CD11a/CD18 expression has also been observed previously on human whole blood leukocytes in response to lipotechoic acid [50 ]. The authors speculated that the observed down-regulation of the CD11a/CD18 expression was a result of shedding of the receptor. The role of this phenomenon warrants further investigation. However, the literature about the role of individual ß2-integrins is, to date, complex and contradictory, and the relative importance of the individual ß2-integrins appears to be stimulus- and organ-dependent [25 26 27 28 29 30 31 32 33 34 ].
It should be noted that inhibition of 5-LOX activity also reduces the formation of lipoxins (LXs). LXA4 (or its analogs) reduces the degree of inflammation [51 , 52 ]. Thus, it has been suggested that LXA4 may play an important role in the resolution of inflammation. Our findings indicate that the proinflammatory effects of LTB4 (and other LTs) are more important in the regulation of the acute inflammatory response associated with acute severe endotoxemia than LXA4 (and other endogenous lipoxins). However, the contribution of 5-LOX metabolites to the regulation of the inflammatory response in more chronic settings of inflammation cannot be discounted.
In conclusion, our results support the view that metabolites of 5-LOX contribute to the multiple organ injury and dysfunction caused by severe endotoxemia. We propose that zileuton protects the organs against endotoxin-induced injury and dysfunction by inhibiting the LT synthesis, thereby reducing the LT-induced stimulation of ß2-integrin-dependent adhesion and the subsequent recruitment of neutrophils. We propose that zileuton or other potent inhibitors of 5-LOX may be useful in the therapy of the organ injury associated with endotoxic shock.
Received June 14, 2004; accepted August 3, 2004.
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-induced leucocyte adhesion in venules Br. J. Haematol. 110,424-429[CrossRef][Medline]-induced inflammation by LFA-1 and Mac-1 Blood 99,336-341 and neutrophils in endotoxin-induced liver injury Am. J. Physiol. 265,G1011-G1015 agonists modulate macrophage activation by gram-negative and gram-positive bacterial stimuli Shock 20,56-62[CrossRef][Medline]-stimulated neutrophil-enterocyte interactions in vitro and attenuate TNF--induced chemokine release and colonocyte apoptosis in human intestinal mucosa ex vivo J. Immunol. 167,2772-2780This article has been cited by other articles:
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