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Am J Physiol Gastrointest Liver Physiol 294: G477-G488, 2008. First published November 29, 2007; doi:10.1152/ajpgi.00229.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

5-Lipoxygenase-derived lipid mediators are not required for the development of NSAID-induced inflammatory bowel disease in IL-10–/– mice

Seiko Narushima,1 Daniel DiMeo,2 Jun Tian,1 Juan Zhang,1 Dahzi Liu,1 and Daniel J. Berg1

1Department of Internal Medicine, 2Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa

Submitted 21 May 2007 ; accepted in final form 29 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
Leukotrienes are potent lipid mediators derived from the metabolism of arachidonic acid by the enzyme 5-lipoxygenase (5-LO). Elevated levels of the proinflammatory leukotriene LTB4 have been found in preclinical models of inflammatory bowel disease (IBD) as well as in colon tissue from individuals with IBD. We therefore determined the extent to which absence of 5-LO-derived lipid mediators would alter the colitis in IL-10–/– mice, a model of human IBD. IL-10–/–/5-LO–/– mice were generated and were healthy. Absence of 5-LO did not alter the development of spontaneous colitis in IL-10-deficient mice. We then evaluated the extent to which absence of 5-LO would alter the development of NSAID-induced colitis in IL-10–/– mice. Absence of 5-LO did not delay the onset or alter the severity of inflammation in NSAID-treated IL-10–/– mice. At an early time point, 3 days after NSAID treatment was initiated, a qualitative increase in the number of dendritic cells and CD4+ T cells was noted in the colons of IL-10–/–/5-LO–/–; however, this difference was no longer present after 14 days of NSAID treatment. Absence of 5-LO did not alter the degree of neutrophil infiltration into the in this model. Absence of 5-LO does not alter the development of IFN-{gamma} producing Th1-type CD4+ T cells or IL-17 producing CD4+ T cells. Absence of 5-LO-derived mediators did not alter the expression of the adhesion molecules ICAM-1 and P-selectin. Development of colitis in IL-10–/– mice was associated with increased levels of the 5-LO-derived anti-inflammatory lipoxin LXA4. These studies demonstrate that 5-LO-derived leukotrienes are not required for the development or maintenance of spontaneous or NSAID-induced colonic inflammation in IL-10–/– mice.

cytokines; leukotriene; lipid mediators; inflammation

LEUKOTRIENES ARE A CLASS OF potent biological mediators called eicosanoids that are derived from arachidonic acid. The eicosanoids include both cyclooxygenase products (prostaglandins and thromboxanes) and the 5-lipoxygenase (5-LO) products, known as leukotrienes. Leukotrienes have been implicated in the pathophysiology of multiple diseases including asthma, atopic dermatitis, psoriasis, arthritis, and inflammatory bowel disease (IBD) (8, 13, 17, 18, 21, 22).

The synthesis of leukotrienes is tightly regulated. Arachidonic acid released from endogenous phospholipids by phospholipase A2 is subsequently sequestered at the nuclear envelope and brought into contact with the 5-LO enzyme by an accessory protein termed 5-LO activating protein (FLAP). The enzyme 5-LO catalyzes the formation of 5-hydroperoxy-6,8,11,14-eicosotetraenoic acid (5-HPETE) and its subsequent conversion to leukotriene A4 (LTA4). The conversion of arachidonic acid to LTA4 by the 5-LO is the critical initiation enzyme in the formation of all leukotrienes (20). 5-LO is expressed primarily in immune cells including granulocytes, macrophages, dendritic cells, and mast cells (10, 26, 41, 36). Low levels of 5-LO expression have also been detected in nonimmune cells such as keratinocytes (28) and colon epithelial cells (46, 50).

LTA4, the initial product of 5-LO, is subsequently metabolized to the proinflammatory leukotriene LTB4, or LTC4 and its metabolites LTD4 and LTE4, also known as the slow-reacting substances of anaphylaxis. LTB4 has multiple proinflammatory biological activities. LTB4 is a potent chemoattractant for neutrophils, eosinophils, monocytes, and dendritic cells (31, 39). LTB4 can also increased adhesion of leukocytes to vascular endothelium and induce neutrophil degranulation (15). LTB4 is also a chemotactic factor for CD4+ and CD8+ T cells (47, 48). LTA4 may also be conjugated to glutathione by the enzyme leukotriene C4 synthase to produce cysteinyl-leukotrienes that originally were described as the slow-reacting substances of anaphylaxis. These leukotrienes are important mediators of pulmonary allergic responses (16). Unlike LTB4, the cysteinyl-leukotrienes do not induce leukocyte migration into inflamed tissue. However, cysteinyl-leukotrienes may contribute to inflammation via increased vascular permeability and subsequent edema formation (15).

The role of leukotrienes in the pathogenesis and pathophysiology of chronic IBD is unclear. Leukotrienes are key mediators in the development of acute inflammation. Moreover, leukotrienes may regulate the adaptive immune response via their effects on dendritic cells (29) and chemotactic effects on T cells. Thus it is reasonable to hypothesize that leukotriene production may be an important mediator in the development of intestinal inflammation. In normal colonic mucosa there is little to no leukotriene synthesized (35). In contrast, leukotriene production has been clearly shown in multiple studies to the increased in individuals with chronic IBD (46). This increased concentration of LTB4 clearly correlates with disease activity, being increased with active inflammation and decreased when inflammation subsides (33). In preclinical models of intestinal inflammation, decreased levels of inflammation have been reported when leukotriene production is inhibited pharmacologically (11) or in the absence of 5-LO (14). Clinical studies in individuals with chronic IBD have not shown a significant impact of inhibition of leukotriene production on disease activity (40); however, the role of 5-LO-derived mediators in NSAID-exacerbated or induced colitis is unknown.

Nonsteroidal anti-inflammatory drugs, or NSAIDs, are inhibitors of prostaglandin production via inhibition of the cyclooxygenase enzymes (COX1 and COX2). NSAIDs are potent inhibitors of prostaglandin production but do not inhibit the leukotriene production pathway. Although inhibition of prostaglandin production is useful in a number of inflammatory diseases (rheumatoid arthritis, for example), there is no role for NSAIDS in the treatment of IBD. In fact, there is evidence that cyclooxygenase inhibitors actually aggravate or reactivate IBD (1, 7, 30). The mechanisms by which NSAIDs induce intestinal inflammation have not been clearly determined. Recently we reported on a novel model of NSAID-induced colitis, NSAID-treated IL-10–/– mice (6). A short course of nonsteroidal treatment rapidly activates intestinal inflammation in this model of IBD. In this study we assessed the extent to which 5-LO-derived leukotrienes contribute to spontaneous and NSAID-induced IBD in IL-10-deficient mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
Animals. Wild-type (wt) 129/SvEv mice were obtained from Taconic Farms (Germantown, NY). 5-LO-deficient mice (5-LO–/–) on a mixed 129/B6 background were obtained from Jackson Laboratories (Bar Harbor, ME) and backcrossed five generations to the 129/SvEv strain. IL-10–/– mice on the 129/SvEv strain were from the investigators breeding colony. Mice were maintained in microisolator cages under specific pathogen-free, Helicobacter-free conditions at the animal care facility at the University of Iowa.

IL-10–/– and 5-LO–/– mice were backcrossed to generate double-deficient mice. These mice were backcrossed greater than five generations onto the 129/SvEv background. Although both 5-LO-deficient mice and IL-10-deficient mice can be bred as homozygous knockouts, double-deficient (5-LO–/–/IL-10–/–) mice were poor breeders and generated limited numbers of mice. Mice for these studies were offspring of 5-LO–/–/IL-10+/– breeders. The breeders were healthy and had no evidence of colitis.

Genotyping of 5-LO–/– and IL-10–/– mice. Genotyping of mice was based upon detection of the wt allele and the allele disrupted by insertion of the neo gene. Tail DNA was isolated by using DirectPCR (Viagen Biotech, Los Angeles, CA) exactly as per the manufacturer's directions. The PCR primers for used for detection of wt and disrupted 5-LO gene were as follows: IMR 0510, ATC GCC TTC TTG ACG AGT TC; IMR 0511, GCA GGA AGT TAC TGT GGA; IMR 0512, TGC AAC CCA GTA CTC ATC AAG. The wt allele was detected by using primers IMR 0510 and IMR 0512 that yield a 164-bp DNA fragment. The combination of primers IMR 0510 and IMR 0511 amplify a DNA fragment of ~600 bp. The PCR primers used for the detection of wt and disrupted IL-10 gene were as follows: T2.2, GTG GGT GCA GTT ATT GTC TTC CCG; T1.4, GCC TTC AGT ATA AAA GGG GGA CC; Neo5, CCT GCG TGC AAT CCA TCT TG. The combination of T1.4 and Neo5 for the disrupted allele yields a 400-bp fragment. The combination of T2.2 and T1.4 for the wt allele yields a fragment of ~200 bp.

Reagents. Piroxicam was obtained from Sigma (St. Louis, MO). NIH-31M powdered diet was obtained from Harlan-Teklad (Indianapolis, IN). DirectPCR for tail DNA extraction was obtained from Viagen Biotech (Los Angeles, CA) and used exactly per manufacturer's instructions. PCR primers were obtained from IDT (Coralville, IA). Nuclease resistant phosphorothioate-modified oligodeoxynucleotides were obtained from the Coley Pharmaceutical Group (Wellesley, MA). LPS from Escherichia coli(serotype 0111:B4) was obtained from Sigma.

Experimental protocol for NSAID treatment. Piroxicam was fed to weanling mice (4 wk of age) for varying time periods (3 days or 14 days) at a dose of 200 ppm piroxicam in the diet. Piroxicam was mixed with rodent chow (NIH-31M) by the technique of geometric dilution to ensure uniform distribution of the NSAID in the diet. Briefly, an equivalent amount of mouse diet was added to the NSAID and then thoroughly mixed. Successive equivalent amounts of the mouse diet were added and mixed well after each dilution, until the entire quantity of the mouse diet was incorporated. After the NSAID-feeding period mice were euthanized and colons were removed for further studies (as described below).

Histological analysis. Tissues from wt, 5-LO–/–, IL-10–/–, and 5-LO–/–/IL-10–/– mice were fixed in 10% neutral buffered formalin, routinely processed, sectioned at 6 µm, and stained with hematoxylin and eosin for light microscopic examination. Samples from the entire gastrointestinal tract were examined without knowledge of which treatment group the samples were from. Because intestinal lesions were multifocal and of variable severity, the grades given to any section of intestine took into account the number of lesions as well as their severity. A score from 0 to 4 was based on the following criteria: Grade 0, no change from normal tissue. Grade 1, one or a few multifocal mononuclear cell infiltrates in the lamina propria accompanied by minimal epithelial hyperplasia and slight to no depletion of mucus from goblet cells. Grade 2, lesions were more frequent and typical changes included several multifocal, mild inflammatory cell infiltrates in the lamina propria composed primarily of mononuclear cells with a few neutrophils. Mild epithelial hyperplasia and mucin depletion were also seen. Small epithelial erosions were occasionally present and inflammation rarely involved the submucosa. Grade 3, lesions involved a large area of the mucosa or were more frequent than grade 2 lesions. Inflammation was moderate and often involved the submucosa but was rarely transmural. Inflammatory cells were a mixture of mononuclear cells as well as neutrophils, and crypt abscesses were sometimes observed. Moderate epithelial hyperplasia and mucin depletion were seen. Ulcers were occasionally observed. Grade 4, lesions usually involved most of the intestinal section and were more severe than grade 3 lesions. Inflammation was intense, including mononuclear cells and neutrophils, and was sometimes transmural. Epithelial hyperplasia was marked with crowding of epithelial cells in elongated glands. Few mucin-containing cells were seen. Crypt abscesses and ulcers were present and foci of fibrinoid necrosis were present in the submucosa contiguous to ulcerations and crypt abscesses.

Induction of colitis with anti-IL-10R antibody. Five-week-old mice (wt or 5-LO–/–) were treated with anti-IL-10R Ab [1B13A (32), 1 mg ip twice weekly] and fed control diet or diet containing piroxicam, 200 ppm 3 wk. Mice were subsequently euthanized and colons evaluated by standard histological techniques as described above.

Immunohistochemical analysis. Colons from control and NSAID-treated IL-10–/– and 5-LO–/–/IL-10–/– mice were carefully dissected from PBS-irrigated colons and snap frozen in optimum cutting temperature compound. The cryostat sections (6 µm) were fixed in methanol-acetone (1:1) for 5 min at –20°C. After being washed with PBS, the sections were incubated in PBS containing 0.3% H2O2 with 0.2 M sodium azide to inhibit endogenous peroxidase. For CD4, CD8{alpha}, ICAM-1, and F4/80 staining, sections were incubated in PBS containing 5% rabbit serum. Hamster serum and rat serum were used for CD11c and B220, respectively. Avidin and biotin blocking was performed by using the Avidin/Biotin blocking kit from Vector Laboratories (Burlingame, CA) following the manufacturer's protocol (Vector Laboratories). Standard immunohistochemical procedures were used. After incubation with the primary antibody sections were incubated with biotinylated rabbit anti-rat IgG (1:250, Vector). Diaminobenzidine with Vectastain Elite ABC Kit (Vector) was used to visualize stained cells and sections were lightly counterstained with hematoxylin. Slides were dehydrated with graded alcohol and xylene and subsequently mounted with Permount under glass coverslips. Abs used for section staining included the following: purified rat anti-mouse CD4 (clone L3/T4; 10 µg/ml, Caltag, South San Francisco, CA); purified rat anti-mouse F4/80 tissue culture supernatant (clone A3-1; 10 µg/ml, Caltag); rat anti-mouse neutrophil (clone 8C5, 10 µg/ml, Caltag); purified rat anti-mouse CD45R (B220) (clone RA3-6B2, 10 µg/ml, Caltag), rat anti-mouse CD8{alpha} (clone CT-CD8{alpha}, 10 µg/ml, Caltag), rat anti-mouse ICAM (clone 3E2, 10 µg/ml), rat anti-mouse P-selectin (clone RB40.34, 10 µg/ml), and biotinylated hamster anti-CD11c (clone HL3, BD PharMingen, San Diego, CA). Rat and hamster immunoglobulin isotype control antibodies were obtained from BD PharMingen.

MPO assay. The myeloperoxidase (MPO) activity in colonic tissues was determined by a method reported previously with a slight modification (24). Briefly, colonic tissues were homogenized in 50 mM phosphate buffer containing 0.5% (wt/vol) hexadecyltrimethyl ammonium bromide (Sigma) and then sonicated for 10 s. The samples were freeze-thawed three times and centrifuged at 15,000 g for 20 min at 4°C. The supernatants were diluted in 50 mM phosphate buffer, and sample was added to phosphate buffer containing 0.5 mM 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate (Sigma), 0.00001% H2O2. The change in absorbance at 630 nm was measured with a spectrophotometer. Data are presented as MPO activity units per gram protein.

Intracellular cytokine staining for flow cytometric analysis. Cells were stimulated for 4–6 h with 10 ng/ml PMA (Sigma) and 1 µg/ml ionomycin (Sigma). Control cells received no stimulation. After 1–2 h, GolgiStop or GolgiPlug (BD PharMingen) was added to the culture to block cytokine secretion. Cells were surfaced stained for 15 min at room temperature with FITC-conjugated anti-CD4 mAb (L3T4, PharMingen) in PBS supplemented with 2% fetal calf serum and 0.04% sodium azide. T cells were fixed and permeabilized with Cytofix/Cytoperm (PharMingen) and stained intracellularly with PE-conjugated anti-cytokine antibodies (anti-IL-17, TC11-18H10, BD PharMingen; anti-IFN-{gamma}, XMG1.2; anti-IL-4, BVD4-1D11 from BD PharMingen). Samples were acquired on a Becton-Dickinson FACS LSRII. Data analysis was conducted by use of CellQuest Pro software (BD Biosciences, San Diego, CA).

RNA isolation, cDNA synthesis, and real-time PCR. Total RNA was isolated by TRIzol extraction (Invitrogen). Total RNA was further purified by use of the RNease Mini Kit (Qiagen, Valencia, CA), using DNase I (Qiagen) to remove contaminating genomic DNA. RNA quality was then assessed with agarose gel electrophoresis. First-strand cDNA synthesis was performed with 1 µg total RNA for each 20-µl reaction by using SuperScript III (Invitrogen) according to the manufacturer's directions. Real-time PCR was performed using an ABI Prism 7000 instrument (Applied Biosystems, Foster City, CA) with FAM-labeled primer pairs and probes for murine iIL-17 (Mm00439619_m1), obtained from Applied Biosystems. Sample normalizations were performed using VIC-labeled mouse actin (4352341E) measurements and relative gene expression was calculated by the {Delta}{Delta}CT method.

Quantification of LXA4. Colon tissue lipoxin A4 (LXA4) levels were determined by use of an LXA4 ELISA kit from Neogen (Lexington, KY), as per manufacturer's instructions. Colons were snap frozen in liquid N2 and then homogenized in PBS containing 1 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µg/ml PMSF, and 100 µM piroxicam (to inhibit prostaglandin production during sample preparation). Debris was removed by centrifugation at 15,000 rpm for 20 min at 4°C. The supernatant was removed for determination of LXA4 concentration. Protein concentration in the supernatant was measured by using a commercial reagent based on BCA staining (Pierce, Rockford, IL), using BSA as an internal standard.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
Generation of mice deficient in leukotriene production and IL-10 production (5-LO–/–/IL-10–/– mice). To assess the extent to which leukotriene production contributes to IBD in IL-10–/– mice, we generated mice that were deficient both in the production of leukotrienes (5-LO–/–) and IL-10 (IL-10–/–). Mice deficient in 5-LO were backcrossed onto a 129/SvEv background. Double-deficient mice (IL-10–/–/5-LO–/–) were generated by crossbreeding the two mutant strains. No differences were seen in the growth and development, survival, and overall health of mice deficient in 5-LO and IL-10 compared with IL-10–/– mice. The IL-10–/–/5-LO–/– mice at 4 wk of age appeared healthy. Examination at necropsy revealed no evidence of any disease. The spleen and lymph nodes, including the mesenteric lymph nodes, were normal in size and appearance. The colon was normal in appearance. Histologically, the absence of leukotriene production did not alter the microscopic appearance of the colon. At 4 wk of age there was no evidence of IBD in any of the mice evaluated (wt, 5-LO–/–, IL-10–/–, and 5-LO–/–/IL-10–/–) (data not shown).

Absence of 5-LO does not prevent the development or alter the severity of spontaneous IBD in IL-10–/– mice. To evaluate the effect of absence of 5-LO-derived leukotrienes on the development of spontaneous colitis in IL-10-deficient mice, we evaluated IL-10–/–/5-LO–/– and IL-10–/– mice at 6 mo of age, a time point at which IL-10-deficient mice have developed spontaneous IBD (4). The absence of 5-LO did not prevent the development of spontaneous IBD in IL-10-deficient mice (pathological score: 5-LO–/–/IL-10–/–: 3.0 + 0.4 vs. IL-10–/– 3.2 + 0.2, P > 0.05, not significant; n = 12 in both groups). The cellular infiltrate in both groups consisted of mainly mononuclear cells in the lamina propria and submucosa with occasional transmural inflammation. Neutrophils were noted in areas of ulceration in both strains (IL-10–/– and IL-10–/–/5-LO–/–) but were not a significant component of the infiltrate in nonulcerated regions. Thus the cellular infiltrates in the colonic lamina propria in double-deficient mice with spontaneous colitis were not histologically different from those in single deficient (IL-10–/–) mice with spontaneous colitis.

Effect of absence of 5-LO on the development of NSAID-induced colitis in IL-10–/– mice. We next tested whether absence of 5-LO-derived leukotrienes would alter the development of NSAID-induced IBD. The nonselective NSAID piroxicam (200 ppm) was fed for 2 wk to young (5- to 6-wk-old) wt, 5-LO–/–, IL-10–/–, and IL-10–/–/5-LO–/– mice. NSAID treatment of wt and 5-LO-deficient mice (wt for IL-10) did not induce IBD (Fig. 1). Rare aggregates of mononuclear cells could be seen in the lamina propria of wt and 5-LO–/– mice treated with the nonsteroidal drug piroxicam. There was no alteration of the overlying epithelium.


Figure 1
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  		Fig. 1. Pathology of NSAID-fed 5-lipoxygenase-deficient (5-LO–/–) and wild-type (wt) mice. A: colon from a 5-LO–/– mouse treated with piroxicam for 2 wk (x20). Occasional mononuclear cells are noted in the lamina propria. B: colon from a wt mouse treated with piroxicam for 2 wk (x20). There is no active colitis.

 
In contrast, NSAID-treatment of IL-10–/– mice and 5-LO–/–/IL-10–/– mice resulted in the development of moderate to severe colitis in both strains. There was no difference in the severity of colitis scores between NSAID-treated IL-10–/– and 5-LO–/–/IL-10–/– mice after 3 or 14 days of piroxicam (Table 1 and Fig. 2). At necropsy, thickening of the proximal colon and enlargement of mesenteric lymph nodes were noted in both in NSAID-treated IL-10–/– mice and 5-LO–/–/IL-10–/– mice. Thus absence of 5-LO-derived leukotrienes did not alter the severity of NSAID-induced colitis in IL-10–/– mice.


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  		Table 1. Gastrointestinal inflammation in NSAID-treated IL-10–/– and 5-LO–/–/IL-10–/– mice

 

Figure 2
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  		Fig. 2. Pathology of NSAID-induced colitis. Six-week-old IL-10–/– and 5-LO–/–/IL-10–/– mice were treated with piroxicam for 14 days and colons were subsequently evaluated for pathology. A: colon from a 6-wk-old IL-10–/– mouse after 14 days of piroxicam (x10). The lamina propria contains a predominantly mononuclear cellular infiltration. B: colon from a 6-wk-old 5-LO–/–/IL-10–/– mouse with NSAID-induced colitis, day 14 (x10). Similar to the spontaneous colitis in IL-10–/– mice there is a mononuclear infiltrate in the lamina propria. C: higher power view of lamina propria infiltrate from an IL-10–/– mouse with NSAID-induced colitis, day 14 (x20). Note the mononuclear cell infiltration in the lamina propria. D: colon from a 5-LO–/–/IL-10–/– mouse with NSAID-induced colitis (x20). Similar to the colitis in IL-10–/– mice there is a dense mononuclear infiltrate in the lamina propria. E: ulcer in colon from an NSAID-treated IL-10–/– mouse (x10). Transmural inflammation is present. Neutrophils are a component of the inflammatory infiltrate. F: ulcer in colon from an NSAID-treated 5-LO–/–/IL-10–/– mouse with spontaneous colitis (x10). Similar to the ulcer in IL-10–/– mice, there is severe inflammation and neutrophils are present in the infiltrate. G: higher power view of infiltrate at the base of an ulcer from a NSAID-treated IL-10–/– mouse (x40). Neutrophils are present in the infiltrate. H: higher power view of infiltrate at the base of an ulcer from a NSAID-treated 5-LO–/–/IL-10–/– mouse (x40). Large numbers of neutrophils are present at the base of the ulcer.

 
Microscopic examination revealed no significant changes in the inflammatory infiltrate in the colons of NSAID-treated 5-LO–/–/IL-10–/– mice compared with IL-10–/– mice (Fig. 2). NSAID-treated IL-10–/– mice demonstrated severe inflammatory infiltrates, which were most commonly found of the proximal colon (including the cecum). In some instances, the mice had a pancolitis. The cellular infiltrate predominantly involved the lamina propria and was composed predominately of mononuclear cells. Neutrophils and eosinophils were present in the lamina propria of both strains of mice although the cells were not a prominent feature of the infiltration and neutrophils were mainly seen in association with ulcerations (Fig. 2).

Immunohistochemical studies were performed to characterize the effect of absence of 5-LO on the constituents of the cellular infiltrate in NSAID-treated IL-10–/– mice (Fig. 3). Similar to previous studies (5), NSAID-treated IL-10–/– mice had a large increase in CD4+ T cells within the lamina propria. CD4+ T cells were present in the lamina propria as well as association with ulcerations. NSAID-treated IL-10–/– mice also had a marked increase in lamina propria macrophages. Dendritic cells were present scattered throughout the lamina propria. A small increase in B220+ lymphocytes, mainly located at the base of lamina propria, was noted (not shown). The numbers of CD8+ leukocytes were minimally increased and their location was similar to that of control (no colitis) IL-10–/– mice. Similarly, NSAID-treated 5-LO–/–/IL-10–/– mice had increased numbers of infiltrating CD4+ T cells and F4/80+ macrophages in the lamina propria. Similar to IL-10–/– mice, CD11c+ dendritic cells were noted in 5-LO–/–/IL-10–/– mice. There was no qualitative difference in the cellular constituents of the infiltrate in 5-LO–/–/IL-10–/– mice compared with wt mice.


Figure 3
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  		Fig. 3. Immunohistochemical staining of colon tissue from IL-10–/– or 5-LO–/–/IL-10–/– mice after 14 days of piroxicam treatment. A: F4/80+ macrophages in 5-LO–/–/IL-10–/– colon. B: CD11c+ dendritic cells in 5-LO–/–/IL-10–/– colon. C: CD4+ T cells in 5-LO–/–/IL-10–/– colon. D: F4/80+ macrophages in IL-10–/– colon (x10). E: CD11c+ dendritic cells in IL-10–/– colon (x10). F: CD4+ T cells in IL-10–/– colon (x10).

 
LTB4 is one of the most potent chemoattractant for neutrophils. We therefore further assessed the effect of absence of 5-LO on neutrophil infiltration in NSAID-induced colitis. In previous studies we found that there are few neutrophils in the colon of healthy IL-10 knockout mice (4). With NSAID-induced colitis mild infiltration of neutrophils was noted; however, this was always in association with ulcerations (6). MPO assays were performed to determine whether absence of 5-LO altered neutrophil infiltration. Consistent with our observations from histology, there was no difference in the MPO activity in NSAID-treated 5-LO–/–/IL-10–/– mice compared with IL-10–/– mice with NSAID-induced colitis (5-LO–/–/IL-10–/– mice with NSAID-induced colitis: 1.6 ± 0.41 U/g protein vs. control IL-10–/– mice with NSAID-induced colitis: 1.64 ± 0.83 U/mg protein; P not significant).

We further evaluated the role of IL-10 and 5-LO on the development of colitis using a blocking antibody to the IL-10 receptor. The nonselective NSAID piroxicam (200 ppm) was fed for 3 wk to control wt and 5-LO–/– mice or anti-IL-10R-treated wt and 5-LO–/– mice (32). Similar to our previous studies (6), NSAID treatment of wt did not induce IBD nor did NSAID treatment induce colitis in 5-LO-deficient mice (pathological score of colon from mice treated with piroxicam: wt 0.2 ± 0.4 vs. 5-LO 0.6 ± 0.5, n = 5/group; Fig. 4). In contrast, NSAID feeding to wt or 5-LO–/– mice treated with anti-IL-10 receptor resulted in colitis in both groups (Fig. 4). The severity of colitis did not differ between wt and 5-LO–/– mice (pathological score of colon from mice treated with piroxicam and anti-IL-10 receptor antibody: wt 2.6 ± 0.5 vs. 5-LO 2.4 ± 0.5, n = 5/group).


Figure 4
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  		Fig. 4. Pathological evaluation of NSAID-fed 5-LO–/– mice treated with anti-IL-10 receptor antibody. A: colon from a 5-LO–/– mouse on control diet. B: colon from a 5-LO–/– mouse on diet containing piroxicam (200 ppm for 3 wk). C: colon from a 5-LO–/– mouse treated with anti-IL-10 receptor antibody (1B13A, 1 mg ip, biw for 3 wk) on a diet containing piroxicam (200 ppm for 3 wk).

 
Effect of NSAID treatment at early time points in 5-LO–/–/IL-10–/– mice. It has been reported that LTB4 is a key mediator of neutrophil (15) and T cell infiltration (38, 47) early in the development of inflammatory and immune responses. We therefore evaluated an early time point (3 days) in the development of the NSAID-induced colitis to determine whether absence of leukotrienes would alter the cellular infiltrate. IL-10–/– and 5-LO–/–/IL-10–/– mice were treated with NSAIDs for 3 days, and the colons were subsequently assessed for severity of inflammation by histopathology. There was no difference in the severity of colitis scores between NSAID-treated IL-10–/– and 5-LO–/–/IL-10–/– mice after 3 days of piroxicam (Table 2 and Fig. 5). At this time point a mononuclear infiltrate was noted in the lamina propria. No ulcerations were noted. We next evaluated the constituents of the cellular infiltrate to determine whether absence of 5-LO-derived leukotrienes altered the types of cells that infiltrate of the colon at early time points in NSAID-induced colitis. Immunohistochemistry demonstrated similar levels of infiltrating F4/80+ macrophages. A qualitative increase in CD11c+ dendritic cells and CD4+ T cells was noted in the 5-LO–/–/IL-10–/– mice compared with the IL-10–/– mice (Fig. 6). Similar levels of CD8+ T cells and B220+ B cells were noted in the NSAID-treated 5-LO–/–/IL-10–/– mice compared with IL-10–/– mice (not shown).


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  		Table 2. Intracellular cytokine expression of CD4+ T cells from mesenteric lymph node

 

Figure 5
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  		Fig. 5. Pathological evaluation of the colon after 3 days of NSAID-treatment. Six-wk-old IL-10–/– and 5-LO–/–/IL-10–/– mice were treated with piroxicam for 3 days and colons were subsequently evaluated for pathology. A: colon from an IL-10–/– mouse after 3 days of piroxicam (x10). The lamina propria contains a predominantly mononuclear cellular infiltration. B: colon from a 5-LO–/–/IL-10–/– mouse with NSAID-induced colitis, day 3 (x10). A mononuclear infiltrate is noted in the lamina propria. C: higher power view of lamina propria infiltrate from an IL-10–/– mouse with NSAID-induced colitis (x20). Note the mononuclear cell infiltration in the lamina propria. Neutrophils are rarely seen. D: colon from a 5-LO–/–/IL-10–/– mouse after 3 days of NSAID treatment (x20). The lamina propria infiltrate is composed of mononuclear cells.

 

Figure 6
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  		Fig. 6. Immunohistochemical staining of colon tissue from IL-10–/– or 5-LO–/–/IL-10–/– mice after 3 days of piroxicam treatment. A: F4/80+ macrophages in 5-LO–/–/IL-10–/– colon (x10). B: CD11c+ dendritic cells in 5-LO–/–/IL-10–/– colon (x10). C: CD4+ T cells in 5-LO–/–/IL-10–/– colon (x10). D: F4/80+ macrophages in IL-10–/– colon after 3 days of NSAID treatment. (x10). E: CD11c+ dendritic cells in IL-10–/– colon (x10). F: CD4+ T cells in IL-10–/– colon. (x10).

 
The effect of deficiency in 5-LO on cytokine production in double-deficient (5-LO–/–/IL-10–/–) mice. We previously demonstrated that colitis in NSAID-treated IL-10–/– mice is correlated with the development of Th1-type CD4+ T cells (6). Therefore we assessed whether absence of 5-LO-derived leukotrienes would alter the development of T helper subsets in NSAID-induced colitis. Flow cytometry with intracellular cytokine staining was performed to determine the percentage of mesenteric lymph node CD4+ T cells producing IFN-{gamma}, IL-17, and IL-4 from mesenteric lymph nodes of NSAID-treated IL-10–/– mice and 5-LO–/–/IL-10–/– mice. The percent of CD4+ T cells producing IFN-{gamma}, IL-17, or IL-4 from 5-LO–/–/IL-10–/– mice was not statistically different from that of IL-10–/– CD4 T cells (Fig. 7 and Table 2). No differences in IL-17 mRNA levels in colon or mesenteric lymph nodes from piroxicam-treated 5-LO–/–/IL-10–/– and IL-10–/– mice were detected by real-time PCR analysis (data not shown).


Figure 7
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  		Fig. 7. Effect of absence of 5-LO on cytokine production from mesenteric lymph nodes CD4+ T cells from mice with NSAID-induced colitis. Representative flow cytometry dot plots from IL-10–/– or 5-LO–/–/IL-10–/– mice are presented. Percentage of total cells is indicated in the dot plot quadrants.

 
The effect of deficiency in 5-LO on ICAM-1 and p-selectin expression in NSAID-treated IL-10–/– mice. Previous reports suggested that absence of 5-LO altered adhesion molecule expression in a 2,4-dinitrobenzene sulfonic acid (DNBS) model of colitis (14). We therefore assessed expression of ICAM-1 and P-selectin in NSAID-induced colitis in IL-10–/– mice. Constitutive staining for ICAM-1 was seen in colons from both 5-LO–/–/IL-10–/– and IL-10–/– mice. There was no difference in the intensity or localization of staining in the 5-LO–/–/IL-10–/– mice compared with the IL-10–/– mice with NSAID-induced colitis (Fig. 8, A and B). Low levels of P-selectin expression were noted on endothelial cells in colons from both 5-LO–/–/IL-10–/– and IL-10–/– mice with NSAID-induced colitis. There was no difference in the intensity or localization of staining in the 5-LO–/–/IL-10–/– mice compared with the IL-10–/– mice (Fig. 8, C–F).


Figure 8
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  		Fig. 8. Immunohistochemical staining for ICAM-1 and P-selectin in colon tissue from IL-10–/– or 5-LO–/–/IL-10–/– mice after 14 days of piroxicam treatment. A: ICAM-1 expression in IL-10–/– colon. B: ICAM-1 expression in 5-LO–/–/IL-10–/– colon (x10). C: low-power view of P-selectin expression in IL-10–/– colon; arrow indicates staining of endothelial cells. D: low-power view of P-selectin expression in 5LO–/–/IL10–/– colon; arrow indicates staining of endothelial cells. E: P-selectin expression in IL-10–/– colon (x40). F: P-selectin expression in 5LO–/–/IL10–/– colon (x40).

 
Lipoxins are produced in IL-10–/– mice with NSAID-induced colitis. Lipoxins are potent anti-inflammatory mediators in vivo by 5-LO-dependent metabolism of arachidonic acid. We therefore assessed the levels of LXA4 in colons from healthy control IL-10–/– (no colitis) and IL-10–/– mice with colitis. Low levels of lipoxin were detected in IL-10–/– control colons. With the development of NSAID-induced inflammation, there was a statistically significant elevation in the level of LXA4 in the IL-10–/– colon with colitis (Fig. 9). No increase in LXA4 was seen in NSAID-treated wt mice (data not shown).


Figure 9
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  		Fig. 9. Lipoxin A4 concentration in colons from 5-wk-old IL-10–/– mice maintained on control diet or IL-10–/– mice maintained on piroxicam for 7 days. Tissue LXA4 concentrations were measured by enzyme immunoassay in snap-frozen tissues homogenized in buffer containing protease inhibitors. Values are means + SD, n = 4 per group, *P < 0.05 control vs. piroxicam-treated mice.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
In this study we have characterized of the role of 5-LO-derived lipid mediators in the development of spontaneous and NSAID-induced IBD in IL-10–/– mice. The development of spontaneous IBD in IL-10–/– mice was not altered by the absence of 5-LO-derived leukotrienes nor was there any alteration in the severity of spontaneous IBD. We further characterized the role of 5-LO-derived leukotrienes in the development of NSAID-induced colitis in IL-10–/– mice. Our studies demonstrate that absence of 5-LO-derived leukotrienes did not alter the development, severity, or cellular infiltrate in NSAID-treated 5-LO–/–/IL-10–/– mice. Absence of 5-LO did not alter the development of IFN-{gamma}- or IL-17-producing cells in NSAID-induced colitis. Interestingly, we found that production of the potent anti-inflammatory mediator LXA4 is increased in IL-10–/– mice with NSAID-induced IBD, suggesting a potential role for this 5-LO-derived anti-inflammatory mediator. Prostaglandins are potent lipid mediators derived from arachidonic acid and have an important role in regulating the development of colitis in IL-10-deficient mice. Absence of prostaglandins leads to the development of IBD in IL-10–/– mice, and treatment or IL-10–/– mice with PGE2 inhibits the development of NSAID-induced IBD (6). In contrast, our studies presented herein indicate that proinflammatory 5-LO-derived leukotrienes are not essential for the development or maintenance of spontaneous or NSAID-induced IBD in IL-10–/– mice.

Leukotrienes are potent lipid mediators produced at sites of inflammation. Because of their potent proinflammatory activities, the synthesis of leukotrienes is tightly controlled. LTB4 is a very potent chemoattractant for neutrophils, eosinophils, monocytes, and dendritic cells (31). Given the proinflammatory properties of LTB4 we hypothesized that absence of 5-LO, and consequently LTB4, would result in decreased inflammation in IL-10–/– mice. However, both IL-10-deficient and 5-LO–/–/IL-10–/– mice developed spontaneous IBD. The severity of disease was similar in both groups of mice and there was no significant difference in the constituents of the cellular infiltrate. These data indicate that 5-LO-derived leukotrienes are not necessary for the development or maintenance of spontaneous IBD.

The absence of 5-LO-derived leukotrienes may have altered early events in the development of spontaneous IBD. It has been previously demonstrated that leukotriene B4 is an important mediator of early infiltration of both neutrophils and CD4+ and CD8+ T cells into inflamed tissue (47). Because we did not evaluate the spontaneous IBD at early time points we may have missed early changes in the 5-LO-deficient mice. Although early events may be modified by leukotrienes, the cellular infiltration at later time points (chronic inflammation) is likely regulated by the multiple chemokine mediators that are expressed in chronic inflammation (9). Multiple chemokines and their receptors are known to be induced in IL-10–/– mice with spontaneous colitis (42). The redundancy of the system may be such that leukotrienes are not necessary for the development or maintenance of this chronic inflammatory infiltrate.

Although leukotrienes were not necessary for the development of spontaneous colitis in IL-10–/– mice, we hypothesized that leukotrienes may be important mediators in the development of nonsteroidal induced IBD in IL-10-deficient mice. We previously demonstrated that a short course of NSAID, with consequent inhibition of prostaglandin production, results in the rapid development of IBD in IL-10–/– mice (6). A potential explanation for the rapid development of IBD in IL-10-deficient mice with NSAID treatment would be the shunting of arachidonic acid substrate away from the cyclooxygenase pathway into the leukotriene pathway with excess production of proinflammatory leukotrienes. To test this hypothesis, we treated young IL-10–/– or 5-LO–/–/IL-10–/– mice with piroxicam and assessed the development of inflammation at early time points (3 days) and later time points (7 and 14 days). Our studies clearly show that the severity of NSAID-induced IBD in the double-deficient mice was the same as that for the single-deficient mice. These studies indicate that absence of 5-LO-derived leukotrienes was not necessary for the development of inflammation in NSAID-treated IL-10–/– mice.

Although the histological inflammation scores were similar in 5-LO–/–/IL-10–/– mice and IL-10–/– mice, absence of leukotrienes could potentially have altered the constituents of the inflammatory infiltrate. Leukotrienes are generally thought of as mediators of acute inflammatory responses; however, recent studies have suggested an important role for leukotrienes in the regulation of T lymphocyte trafficking (47). T lymphocytes are known to express receptors for both LTB4 and cysteinyl leukotrienes (34). LTB4 is a chemotactic factor for CD4+- and CD8+-positive T lymphocytes. This effect is mediated through the BLT1 receptor, which in mouse is expressed on effector CD4+ and CD8+ T lymphocytes (23), demonstrating that leukotrienes can interact directly with T cells. To assess whether absence of 5-LO-derived leukotrienes altered the early inflammatory infiltrate in NSAID-induced IBD, we assessed by immunohistochemistry the cellular infiltrate in 3-day treated IL-10–/– and 5-LO–/–/IL-10–/– mice. In models of TH2-mediated pulmonary inflammation, leukotrienes, specifically LTB4, were clearly important in attracting CD4+ T and CD8+ T cells into the lung (34). In contrast, in this Th1/Th17-driven model of colitis we saw a qualitative increase in the infiltration of CD4+ T cells in 5-LO–/–/IL-10–/– at an early (3-day) time point. After 14 days, there was no difference in the cellular infiltrate in 5-LO–/–/IL-10–/– compared with IL-10–/– mice. The absence of a significant effect of 5-LO deficiency on the cellular infiltrate may be due to redundancy in the system of chemoattractants in intestinal inflammation in IL-10–/– mice (42).

Multiple studies have demonstrated that T cells are necessary for the development of IBD in IL-10-deficient mice (4). The colitis in IL-10–/– mice is characterized by an increase in Th1-type CD4+ T cells (4) and IL-17-producing T cells (51). Given their pleiotropic effects, 5-LO-derived leukotrienes potentially could alter the T cell immune response. Dendritic cells, the central antigen-presenting cell, express 5-LO (25), and leukotrienes can modulate cytokine release from dendritic cells (29). T cells also express the high-affinity receptor for LTB4 (BLT1) and can respond to LTB4 (49). This study, however, demonstrated that the absence of leukotrienes was not necessary for the development of the pathogenic Th1 and Th17 subset of CD4+ T cells.

The effect of 5-LO deficiency on T cell phenotype is variable depending on the system tested and strain background of the mice. In some studies a skew toward Th1 T cells has been documented (44), although in our studies we found that absence of 5-LO resulted in increased IL-10 production and a skewing toward Th2 type CD4+ T cells (unpublished observations, D. Berg). In contrast, flow cytometric evaluation of T cells from double-deficient mice with NSAID-induced colitis demonstrated that absence of leukotrienes did not alter the percentage of Th1-type T cells or IL-17-producing T cells from the draining mesenteric lymph nodes of mice with IBD. This study indicates that absence of 5-LO-derived leukotrienes did not skew the development of these subsets.

Leukotriene antagonists are important parts of clinical armament for the treatment of asthma (37, 43). Multiple studies have demonstrated improvement in a several different types of asthma due to blockade of cysteinyl leukotriene production. In contrast to these positive studies in pulmonary inflammation, the role of leukotriene antagonist in IBD has not been clear. Multiple studies have demonstrated increased leukotriene production in colitis tissue with inflammation (12, 27). Animal studies of colitis have suggested benefits for leukotriene production inhibition (14). For example, recently it has been reported that 5-LO-deficient mice have decreased inflammation in the DNBS colitis model (14). The decrease in inflammation in the 5-LO-deficient mice was attributed to altered expression of adhesion molecules with subsequent alteration in neutrophil migration. In contrast, neutrophils are not a major component of the inflammatory infiltrate in spontaneous colitis or NSAID-induced colitis (4). We did not find a significant difference in neutrophil infiltration in NSAID-treated 5-LO–/–/IL-10–/– mice compared with IL-10–/– mice. In contrast to the DNBS model of colitis (14), absence of 5-LO did not alter expression of ICAM-1 or P-selectin in 5-LO–/–/IL-10–/– compared with IL-10–/– mice with NSAID-induced colitis. The difference between these two studies is likely due to the difference in the underlying mechanism of the colitis (hapten induced vs. NSAID induced) with resulting differences in cellular infiltrates. Neutrophils are a major component of the inflammatory infiltrate in DNBS colitis (14) whereas the inflammatory infiltrate in NSAID-treated IL-10–/– mice is composed mainly of T cells and macrophages.

Why was the severity of inflammation not decreased in IL-10–/– mice deficient for 5-LO? One explanation may be due to the fact that, in addition to generating a proinflammatory mediator LTB4, 5-LO is also important for the biosynthesis of lipoxins. Lipoxins are lipid mediators derived locally from 5-LO biosynthetic pathways and are key anti-inflammatory regulators of immune and inflammatory responses (45). For example, lipoxins have been shown to act in vitro as a negative regulator of dendritic cell IL-12 production triggered by the intracellular parasite Toxoplasma gondii (2). In a study of the immune response to Mycobacterium tuberculosis, it was clearly demonstrated that 5-LO dependent lipoxins were important regulators of the immune response to this intracellular organism (3). In the absence of 5-LO, M. tuberculosis-infected mice had significantly increased mRNA levels for IL-12 and IFN-{gamma} compared with wt mice (3), demonstrating that these mediators are important both in vitro and in vivo. Lipoxins may be important anti-inflammatory mediators regulating intestinal inflammation. In a model of hapten-induced colitis, treatment with a LXA4 analog resulted in attenuation of inflammation (19). In our studies we demonstrated increased production of LXA4 in the colons of IL-10–/– mice with NSAID-induced colitis. Because lipoxins are potent anti-inflammatory compounds, the increased production of LXA4 in IL-10–/– mice may regulate inflammation in NSAID-induced colitis. The fact that absence of 5-LO results in loss of both a proinflammatory mediator, LTB4, and the anti-inflammatory lipoxins may explain why absence of 5-LO did not in the final analysis result in altered severity of pathology in NSAID-treated IL-10–/– mice. Indeed, inhibition of anti-inflammatory lipoxin production could be one reason why previous clinical trials using 5-LO inhibitors did not show clinical benefit in patients with ulcerative colitis (40).

Although lipoxins may modulate the severity of inflammation, their absence is not central for the development of colitis, as demonstrated by the fact that 5-LO–/– mice do not develop colitis or any other spontaneous inflammation. Our studies using blockade of IL-10 function with an anti-IL-10 receptor antibody demonstrated that absence of IL-10 was the central determinant for the development of NSAID-induced colitis; the severity of colitis was similar in both anti-IL-10 receptor-treated wt and 5-LO–/– mice on NSAIDs. 5-LO-derived lipoxins may have their anti-inflammatory effects after infiltration of activated myeloid cells expressing 5-LO.

In summary, in this study we have demonstrated that absence of 5-LO does not alter the severity of IBD in NSAID-treated IL-10–/– mice, a model of NSAID-induced colitis. Despite the absence of LTB4, neutrophil and T cell infiltration into inflamed colons in 5-LO–/–/IL-10–/– mice was similar to that seen in IL-10–/– mice. Absence of 5-LO-derived lipid mediators did not alter the development of pathogenic Th1 or Th17 T cells in mesenteric lymph nodes of IL-10–/– mice with NSAID-induced colitis. Interestingly, our studies demonstrated increased production of the anti-inflammatory mediator LXA4 in IL-10–/– mice with NSAID-induced colitis, suggesting a potential role for this lipid mediator in the regulation of NSAID-induced colitis. This study demonstrates that 5-LO-derived lipid mediators are not required for the development or maintenance of colitis in this model of a T cell-mediated IBD.


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
GRANTS
 REFERENCES
 
When this manuscript was first posted to the Articles in PresS website, the photomicrograph in Fig. 4A contained a digital artifact that obscured a portion of the image obtained. This problem has been corrected in the final published version. The photomicrographs in Fig. 8, D and F, were incorrectly labeled in the figure legend of this manuscript as posted to Articles in PresS, and photomicrographs 8, E and F were reversed. The figure legends for Figs. 3, 5, and 6 contained redundant text. These problems have been corrected in the final-published version. The authors apologize for the previous errors. The problems identified did not alter the conclusions reached in this study.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
REFERENCES
 
Research support was provided by the Crohn's and Colitis Foundation of America, Research Award (D. J. Berg) and NIH RO1 DK-060718-01A2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Berg, Univ. of Iowa Hospitals, Dept. of Internal Medicine, C32-GH, 200 Hawkins Dr., Iowa City, IA 52242 (e-mail: daniel-j-berg{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
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 RESULTS
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  1. Aabakken L. Review article: non-steroidal, anti-inflammatory drugs—the extending scope of gastrointestinal side effects. Aliment Pharmacol Ther 6: 143–162, 1992.[Medline]
  2. Aliberti J, Hieny S, Reis e Sousa C, Serhan CN, Sher A. Lipoxin-mediated inhibition of IL-12 production by DCs: a mechanism for regulation of microbial immunity. Nat Immunol 3: 76–82, 2002.[CrossRef][Web of Science][Medline]
  3. Bafica A, Scanga CA, Serhan C, Machado F, White S, Sher A, Aliberti J. Host control of Mycobacterium tuberculosis is regulated by 5-lipoxygenase-dependent lipoxin production. J Clin Invest 115: 1601–1606, 2005.[CrossRef][Web of Science][Medline]
  4. Berg DJ, Davidson N, Kuhn R, Muller W, Menon S, Holland G, Thompson-Snipes L, Leach MW, Rennick D. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4+ TH1-like responses. J Clin Invest 98: 1010–1020, 1996.[Web of Science][Medline]
  5. Berg DJ, Lynch NA, Lynch RG, Lauricella DM. Rapid development of severe hyperplastic gastritis with gastric epithelial dedifferentiation in Helicobacter felis-infected IL-10(–/–) mice. Am J Pathol 152: 1377–1386, 1998.[Abstract]
  6. Berg DJ, Zhang J, Weinstock JV, Ismail HF, Earle KA, Alila H, Pamukcu R, Moore S, Lynch RG. Rapid development of colitis in NSAID-treated IL-10-deficient mice. Gastroenterology 123: 1527–1542, 2002.[CrossRef][Web of Science][Medline]
  7. Bjarnason I, Hayllar J, MacPherson AJ, Russell AS. Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans [see comments]. Gastroenterology 104: 1832–1847, 1993.[Web of Science][Medline]
  8. Capra V, Thompson MD, Sala A, Cole DE, Folco G, Rovati GE. Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends. Med Res Rev 27: 469–527, 2006.[CrossRef][Web of Science]
  9. Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 354: 610–621, 2006.[Free Full Text]
  10. Chen XS, Naumann TA, Kurre U, Jenkins NA, Copeland NG, Funk CD. cDNA cloning, expression, mutagenesis, intracellular localization, and gene chromosomal assignment of mouse 5-lipoxygenase. J Biol Chem 270: 17993–17999, 1995.[Abstract/Free Full Text]
  11. Clapp N, Henke M, Hansard R, Carson R, Fretland D. Anti-colitic efficacy of SC-41930 in colitic cotton-top tamarins. Agents Actions 39 Spec No: C36–C38, 1993.
  12. Cole AT, Pilkington BJ, McLaughlan J, Smith C, Balsitis M, Hawkey CJ. Mucosal factors inducing neutrophil movement in ulcerative colitis: the role of interleukin 8 and leukotriene B4. Gut 39: 248–254, 1996.[Abstract/Free Full Text]
  13. Crowther SD, Rees PJ. Current treatment of asthma—focus on leukotrienes. Expert Opin Pharmacother 1: 1021–1040, 2000.[CrossRef][Medline]
  14. Cuzzocrea S, Rossi A, Mazzon E, Di Paola R, Genovese T, Muia C, Caputi AP, Sautebin L. 5-Lipoxygenase modulates colitis through the regulation of adhesion molecule expression and neutrophil migration. Lab Invest 85: 808–822, 2005.[CrossRef][Medline]
  15. Dahlen SE, Bjork J, Hedqvist P, Arfors KE, Hammarstrom S, Lindgren JA, Samuelsson B. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response. Proc Natl Acad Sci USA 78: 3887–3891, 1981.[Abstract/Free Full Text]
  16. Dahlen SE, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 288: 484–486, 1980.[CrossRef][Medline]
  17. Fauler J, Neumann C, Tsikas D, Frolich J. Enhanced synthesis of cysteinyl leukotrienes in atopic dermatitis. Br J Dermatol 128: 627–630, 1993.[CrossRef][Medline]
  18. Fauler J, Neumann C, Tsikas D, Frolich J. Enhanced synthesis of cysteinyl leukotrienes in psoriasis. J Invest Dermatol 99: 8–11, 1992.[CrossRef][Medline]
  19. Fiorucci S, Wallace JL, Mencarelli A, Distrutti E, Rizzo G, Farneti S, Morelli A, Tseng JL, Suramanyam B, Guilford WJ, Parkinson JF. A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction. Proc Natl Acad Sci USA 101: 15736–15741, 2004.[Abstract/Free Full Text]
  20. Ford-Hutchinson AW, Gresser M, Young RN. 5-Lipoxygenase. Annu Rev Biochem 63: 383–417, 1994.[CrossRef][Web of Science][Medline]
  21. Fretland DJ, Djuric SW, Gaginella TS. Eicosanoids and inflammatory bowel disease: regulation and prospects for therapy. Prostaglandins Leukot Essent Fatty Acids 41: 215–233, 1990.[CrossRef][Medline]
  22. Fretland DJ, Widomski D, Tsai BS, Zemaitis JM, Levin S, Djuric SW, Shone RL, Gaginella TS. Effect of the leukotriene B4 receptor antagonist SC-41930 on colonic inflammation in rat, guinea pig and rabbit. J Pharmacol Exp Ther 255: 572–576, 1990.[Abstract/Free Full Text]
  23. Goodarzi K, Goodarzi M, Tager AM, Luster AD, von Andrian U. Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues. Nat Immunol 4: 965–973, 2003.[CrossRef][Web of Science][Medline]
  24. Grisham MB, Benoit JN, Granger DN. Assessment of leukocyte involvement during ischemia and reperfusion of intestine. Methods Enzymol 186: 729–742, 1990.[Medline]
  25. Harizi H, Juzan M, Moreau JF, Gualde N. Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism. J Immunol 170: 139–146, 2003.[Abstract/Free Full Text]
  26. Hedi H, Norbert G. 5-Lipoxygenase pathway, dendritic cells, and adaptive immunity. J Biomed Biotechnol 2004: 99–105, 2004.[CrossRef][Medline]
  27. Ikehata A, Hiwatashi N, Kinouchi Y, Yamazaki H, Ito K, Toyota T. Altered leukotriene B4 metabolism in colonic mucosa with inflammatory bowel disease. Scand J Gastroenterol 30: 44–49, 1995.[CrossRef][Medline]
  28. Janssen-Timmen U, Vickers PJ, Wittig U, Lehmann WD, Stark HJ, Fusenig NE, Rosenbach T, Radmark O, Samuelsson B, Habenicht AJ. Expression of 5-lipoxygenase in differentiating human skin keratinocytes. Proc Natl Acad Sci USA 92: 6966–6970, 1995.[Abstract/Free Full Text]
  29. Jozefowski S, Biedron R, Bobek M, Marcinkiewicz J. Leukotrienes modulate cytokine release from dendritic cells. Immunology 116: 418–428, 2005.[Medline]
  30. Kaufmann HJ, Taubin HL. Nonsteroidal anti-inflammatory drugs activate quiescent inflammatory bowel disease. Ann Intern Med 107: 513–516, 1987.[CrossRef][Web of Science][Medline]
  31. Klickstein LB, Shapleigh C, Goetzl EJ. Lipoxygenation of arachidonic acid as a source of polymorphonuclear leukocyte chemotactic factors in synovial fluid and tissue in rheumatoid arthritis and spondyloarthritis. J Clin Invest 66: 1166–1170, 1980.[Medline]
  32. Kullberg MC, Jankovic D, Feng CG, Hue S, Gorelick PL, McKenzie BS, Cua DJ, Powrie F, Cheever AW, Maloy KJ, Sher A. IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis. J Exp Med 203: 2485–2494, 2006.[Abstract/Free Full Text]
  33. Lauritsen K, Laursen LS, Bukhave K, Rask-Madsen J. Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene B4 levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. Gastroenterology 91: 837–844, 1986.[Web of Science][Medline]
  34. Luster AD, Tager AM. T-cell trafficking in asthma: lipid mediators grease the way. Nat Rev Immunol 4: 711–724, 2004.[CrossRef][Web of Science][Medline]
  35. MacDermott RP. Alterations in the mucosal immune system in ulcerative colitis and Crohn's disease. Med Clin North Am 78: 1207–1231, 1994.[Web of Science][Medline]
  36. Murakami M, Penrose JF, Urade Y, Austen KF, Arm JP. Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells. Proc Natl Acad Sci USA 92: 6107–6111, 1995.[Abstract/Free Full Text]
  37. O'Byrne PM, Parameswaran K. Pharmacological management of mild or moderate persistent asthma. Lancet 368: 794–803, 2006.[CrossRef][Medline]
  38. Ott VL, Cambier JC, Kappler J, Marrack P, Swanson BJ. Mast cell-dependent migration of effector CD8+ T cells through production of leukotriene B4. Nat Immunol 4: 974–981, 2003.[CrossRef][Web of Science][Medline]
  39. Robbiani DF, Finch RA, Jager D, Muller WA, Sartorelli AC, Randolph GJ. The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell 103: 757–768, 2000.[CrossRef][Web of Science][Medline]
  40. Roberts WG, Simon TJ, Berlin RG, Haggitt RC, Snyder ES, Stenson WF, Hanauer SB, Reagan JE, Cagliola A, Tanaka WK, Simon S, Berger ML. Leukotrienes in ulcerative colitis: results of a multicenter trial of a leukotriene biosynthesis inhibitor, MK-591. Gastroenterology 112: 725–732, 1997.[CrossRef][Web of Science][Medline]
  41. Rouzer CA, Shimizu T, Samuelsson B. On the nature of the 5-lipoxygenase reaction in human leukocytes: characterization of a membrane-associated stimulatory factor. Proc Natl Acad Sci USA 82: 7505–7509, 1985.[Abstract/Free Full Text]
  42. Scheerens H, Hessel E, de Waal-Malefyt R, Leach MW, Rennick D. Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10–/– mice and Rag-2–/– mice reconstituted with CD4+CD45RBhigh T cells. Eur J Immunol 31: 1465–1474, 2001.[CrossRef][Web of Science][Medline]
  43. Scow DT, Luttermoser GK, Dickerson KS. Leukotriene inhibitors in the treatment of allergy and asthma. Am Fam Physician 75: 65–70, 2007.[Medline]
  44. Secor WE, Powell MR, Morgan J, Wynn TA, Funk CD. Mice deficient for 5-lipoxygenase, but not leukocyte-type 12-lipoxygenase, display altered immune responses during infection with Schistosoma mansoni. Prostaglandins Other Lipid Mediat 56: 291–304, 1998.[CrossRef][Medline]
  45. Serhan CN. Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution. Prostaglandins Leukot Essent Fatty Acids 73: 141–162, 2005.[CrossRef][Web of Science][Medline]
  46. Sharon P, Stenson WF. Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease. Gastroenterology 86: 453–460, 1984.[Web of Science][Medline]
  47. Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD, Friedrich EB, Carafone AD, Gerszten RE, Luster AD. Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat Immunol 4: 982–990, 2003.[CrossRef][Web of Science][Medline]
  48. Tager AM, Dufour JH, Goodarzi K, Bercury SD, von Andrian UH, Luster AD. BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis. J Exp Med 192: 439–446, 2000.[Abstract/Free Full Text]
  49. Tager AM, Luster AD. BLT1 and BLT2: the leukotriene B(4) receptors. Prostaglandins Leukot Essent Fatty Acids 69: 123–134, 2003.[CrossRef][Web of Science][Medline]
  50. Ye YN, Liu ES, Shin VY, Wu WK, Cho CH. Contributory role of 5-lipoxygenase and its association with angiogenesis in the promotion of inflammation-associated colonic tumorigenesis by cigarette smoking. Toxicology 203: 179–188, 2004.[CrossRef][Medline]
  51. Yen D, Cheung J, Scheerens H, Poulet F, McClanahan T, McKenzie B, Kleinschek MA, Owyang A, Mattson J, Blumenschein W, Murphy E, Sathe M, Cua DJ, Kastelein RA, Rennick D. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 116: 1310–1316, 2006.[CrossRef][Web of Science][Medline]




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