Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1{beta}- and COX-2-dependent mechanism

Donnie E. Shifflett,1 Frank G. Bottone, Jr.,2 Karen M. Young,1 Adam J. Moeser,1 Samuel L. Jones,1 and Anthony T. Blikslager1

1Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh 27606 and 2Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Submitted 14 February 2003 ; accepted in final form 9 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Polymorphonuclear neutrophils (PMNs) play a critical role in intestinal mucosal injury and repair. To study effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation or peritoneal fluid from animals with chemically induced peritonitis to ischemia-injured porcine ileal mucosa. In preliminary experiments, PMNs enhanced recovery of transepithelial electrical resistance (TER), and this action was inhibited by pretreatment with the nonselective cyclooxygenase (COX) inhibitor indomethacin. Because COX-2 is upregulated by inflammatory mediators such as IL-1{beta}, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1{beta} and COX-2. Application of 5 x 106 PMNs to the serosal surface of ischemia-injured mucosa significantly enhanced recovery of TER (P < 0.05), an effect that was inhibited by the selective COX-2 inhibitor NS-398 (5 µM) and by an IL-1{beta} receptor antagonist (0.1 mg/ml). Addition of 10 ng/ml IL-1{beta} to the serosal surface of injured tissues caused a significant increase in TER (P < 0.05) that was inhibited by pretreatment with NS-398. Western blot analysis of mucosal homogenates revealed dramatic upregulation of COX-2 in response to IL-1{beta} or peritoneal PMNs, and the latter was inhibited by an IL-1{beta} receptor antagonist. Real-time PCR revealed that increased mRNA COX-2 expression preceded increased COX-2 protein expression in response to IL-1{beta}. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1{beta}-dependent upregulation of COX-2.

intestine; prostaglandin; transepithelial electrical resistance; tight junction


POLYMORPHONUCLEAR NEUTROPHILS (PMNs) are a critical component of the inflammatory response associated with a variety of gastrointestinal pathophysiological conditions, including microbial invasion (18) and ischemia-reperfusion injury (25). Once PMNs have reached sites of tissue invasion or injury, they release a number of inflammatory mediators that may exacerbate injury but that are also vital to host defense. These neutrophilic mediators include reactive oxygen metabolites, proteases, and cytokines such as IL-1{beta}.

IL-1{beta} is a proinflammatory cytokine whose primary role is to modulate and amplify inflammatory responses (13). Previous studies (1, 7, 42) have shown that PMNs, on exposure to either LPS or granulocyte-macrophage colony-stimulating factor, release IL-1{beta}. Furthermore, elevated levels of IL-1{beta} have been consistently observed in tissues from humans and animals with a variety of inflammatory bowel diseases (1, 10, 30, 31, 44). IL-1{beta} appears to mediate its inflammatory effects via the cyclooxygenase (COX) (21, 22, 32, 39, 41) and MAPK pathways (14–15). However, the precise effects of the IL-1 family of cytokines on tissues are variable. For example, IL-1 has been shown to be harmful to cultured intestinal epithelial monolayers (29), but when IL-1 was added to T84 cells in the presence of subepithelial cocultured myofibroblasts, epithelial secretion was the most notable effect associated with upregulation of COX-2 (21, 32).

At least three isoforms of COX exist: COX-1, which is constitutively expressed in many tissues, an inducible COX-2 isoform (36), and COX-3, which is a COX-1 variant (11). COX-2 can be upregulated by a number of stimuli including growth factors and cytokines (20, 35) and appears to play an important role in recovery of injured intestinal mucosa (5, 33). For example, COX-2 was expressed in epithelium at the margins of experimentally induced gastric ulcers in rats, and selective inhibition of COX-2 retarded epithelial repair (33). Alternatively, whereas inhibition of COX-2 did not interrupt recovery of barrier function in ischemia-injured mucosa in previous studies from our laboratory (5), COX-2 prostanoids were able to stimulate recovery in the absence of COX-1 prostanoids.

We (17) have recently shown that PMNs migrate across restituting epithelium during the inflammatory phase of epithelial wound healing, thereby disrupting recovery of mucosal barrier function. However, in preliminary studies in which PMNs harvested from circulation were incubated with acutely injured mucosa, we noticed an enhancement rather than disruption of the recovery process that was sensitive to the nonselective COX inhibitor indomethacin. We hypothesized that this effect resulted from upregulation of COX-2, and we sought to determine the critical elements of the signaling pathway that led to PMN-enhanced recovery of transepithelial electrical resistance (TER) in ischemia-injured porcine ileum.


    METHODS AND MATERIALS
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental Surgery

All procedures were approved by the North Carolina State University Institutional Animal Care and Use Committee and have been previously described in detail (3, 4). Briefly, 6- to 8-wk-old Yorkshire-cross pigs of both sexes were anesthetized by using a combination of xylazine (1.5 mg/kg) and ketamine (11 mg/kg). Pigs were intubated via a tracheostomy, placed on a heating pad, and ventilated with 100% O2 via a tracheotomy using a time-cycled ventilator. Anesthesia was maintained by using periodic intravenous administration of a 5% sodium thiopental solution via a jugular catheter. Maintenance fluids were administered at a rate of 15 ml·kg–1·min–1 throughout the surgery. A carotid cutdown and catheterization was performed for collection of blood for harvesting of PMNs. The ileum was located via a midline incision, after which 10-cm ileal segments were ligated and subjected to ischemia by ligating the local mesenteric blood supply. Additional 10-cm ileal loops not subjected to ischemia were utilized as control tissues. After 45 min of ischemia, pigs were euthanized with an overdose of pentobarbital and 10-cm ileal loops were promptly removed and placed in oxygenated Ringer solution (95% O2-5% CO2).

Ussing Chamber Studies

The mucosa was stripped from the seromuscular layer and mounted in 3.14 cm2-aperture Ussing chambers. The tissues were bathed on both serosal and mucosal sides with 10 ml of oxygenated (95% O2-5% CO2) Ringer solution. In addition, the serosal bathing solution contained 10 mM of glucose, which was osmotically balanced by 10 mM mannitol on the mucosal side. Bathing solutions were circulated in water-jacketed reservoirs and maintained at 37°C. The spontaneous potential difference (PD) was measured by using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. Resistance ({Omega}·cm2) was calculated from the spontaneous PD and short-circuit current (Isc). If the spontaneous PD was between –1.0 and 1.0 mV, tissues were current clamped at ±100 µA for 5 s and the PD was recorded. Isc and PD were recorded every 15 min for 180 min.

Blood PMN Collection

Arterial blood (20 ml) was collected from the carotid catheter, heparinized, and thoroughly mixed with 6% dextran (4 ml). Red blood cells were allowed to settle for 60 min. The leukocyte-rich plasma was drawn off and overlaid onto 5 ml of Ficoll-Paque and centrifuged at 1,800 rpm for 20 min. The plasma and Ficoll were removed, and the remaining pellet was suspended in 40 ml HBSS and counted by using a hemacytometer. Cell viability was determined by using Trypan blue. Viability was consistently >98%. The suspended cells were then centrifuged at 1,000 rpm for 10 min. The HBSS was drawn off, and the cells were resuspended in Ringer solution for use. Neutrophil purity using this method was >97% (data not shown). PMNs (1 or 5 x 106 total cells) were applied to the serosal side of the tissue 45 min after equilibration of the tissues on the Ussing chamber.

Extravasated PMN Collection

Peritonitis was induced in pigs for collection of extravasated PMNs. Briefly, 6- to 8-wk-old Yorkshire-cross pigs of both sexes were anesthetized by using a combination of xylazine (1.5 mg/kg im) and ketamine (11 mg/kg im). Buprenorphine was administered (0.05 mg/kg im) for analgesia. Subsequently, 15–20 ml of thioglycollate was injected into the peritoneal cavity. Each pig was then allowed to recover for 4 h, after which they were again anesthetized by using a combination of xylazine (1.5 mg/kg im) and ketamine (11 mg/kg im). Pigs were then euthanized with an overdose of pentobarbital, and a midline incision was performed to allow entry to the peritoneal cavity. Fluid was collected from the peritoneum. Collected cells were spun down, and the pellet was resuspended in 20 ml HBSS and counted by using a hemacytometer. Cell viability was determined by using Trypan blue. Viability was consistently >98%. The suspended cells were then centrifuged at 1,000 rpm for 10 min. The HBSS was drawn off, and the cells were resuspended in Ringer solution for use.

Experimental Treatments

Recombinant porcine IL-1{beta} and recombinant porcine IL-1{beta} receptor antagonist were purchased from R&D Systems (Minneapolis, MN). NS-398 was purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Gel Electrophoresis and Western Blotting

Tissues mounted in the Ussing chambers were removed at 75 min after the initiation of the experiment. Mucosal scraping was performed to remove additional connective tissue, after which tissue was snap-frozen in liquid nitrogen and stored at –70°C until analysis for COX 1 and 2 proteins. In preparation for SDS-PAGE, tissues were thawed to 4°C. One-gram tissue portions were added to 3 ml of chilled radioimmunoprecipitation assay buffer [0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS], including protease inhibitors (PMSF and aprotinin). The mixture was homogenized on ice and then centrifuged twice at 10,000 g for 10 min at 4°C, and the supernatant was saved. Protein analysis of extract aliquots was performed (DC protein assay; Bio-Rad, Hercules, CA). Tissue extracts (amounts equalized by protein concentration) were mixed with an equal volume of 2x SDS-PAGE sample buffer and boiled for 4 min at 100°C. Lysates were loaded on a 10% SDS-polyacrylamide gel, and electrophoresis was carried out according to standard protocols. Proteins were transferred to a nitrocellulose membrane (Hybond ECL; Amersham Life Science, Birmingham, UK) using an electroblotting minitransfer apparatus according to the manufacturer's protocol. Membranes were blocked overnight at 4°C in TBS and 5% dry powered milk. Membranes were washed two times with TBS containing 0.05% Tween (TBS-T) and incubated for 2 h at room temperature in primary antibody (COX-1 or COX-2 affinity-purified goat polyclonal antibodies; Santa Cruz Biotechnology, Santa Cruz, CA). After being washed three times for 5 min each with TBS-T, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. After washing two times for 5 min each with TBS-T and one time with TBS for 15 min, the membranes were developed for visualization of protein by the addition of enhanced chemiluminescence reagent (Amersham).

mRNA Analysis

RT. RNA was treated with 1 unit of amplification-grade deoxyribonuclease I (Life Technologies, Gaithersburg, MD) per microgram of RNA at room temperature for 15 min to remove genomic DNA followed by inactivation of the deoxyribonuclease I with 2.5 mM EDTA (pH 8.0). The RNA was then incubated at 65°C for 5 min followed by quantitation. Two micrograms of the RNA was reverse transcribed by using 100 units of Superscript-II reverse transcriptase according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). After RT, cDNA was treated with 1 unit RNase H (Life Technologies) per microgram RNA at 37°C for 20 min. RT was performed by using Qiagen's Omniscript RT kit according to the manufacturer's instructions. A negative control containing all of the RT reagents in the absence of RT enzyme (no RT control) was also routinely performed. The cDNA was diluted five times with RNase-free water (Ambion, Austin, TX) and stored at –80°C until used.

Primer design. Primers were designed by using PrimerExpress Software (Applied Biosystems, Foster City, CA) from known pig (Sus scrofa) sequences found in GenBank. Primers were from Life Technologies and dissolved in 10 mM Tris, pH 7.0. Primers are listed as follows: accession no. (common name) forward primer; reverse primer [product size]. U07786 [GenBank] (Actin) 5'-CTCCTTCCTGGGCATGGA-3', 5'-CGCACTTCATGATCGAGTTA-3' [65]; AY028583 [GenBank] (prostaglandin G/H synthase-2, PGHS-2) 5'-TGTATCCTCCGACAGCCAAAG-3', 5'-GCGGAGGTGTTCAGGAGTGT-3' [71].

Real-time RT-PCR with SYBRgreen detection. Traditional and real-time RT-PCR were performed by using an ABI Prism 7700 (Applied Biosystems) as previously described (8). Real-time RT-PCR fluorescence detection was performed in 96-well plates using Quantitect SYBRgreen buffer (Qiagen). Each 50-µl PCR reaction contained cDNA, 0.5 units of Amp Erase uracil-N-glycosylase (UNG) (PerkinElmer Life Sciences, Boston, MA), forward and reverse primers, and the passive reference dye (carboxy-X-rhodamine; ROX) to normalize the SYBRgreen/double-stranded DNA complex signal during analysis to correct for well-to-well variations, whereas primer concentrations were optimized to yield the lowest concentration of primers that yielded the same threshold cycle (Ct) values as recommended by Applied Biosystems. A no-RT control RNA sample was used with each real-time RT-PCR experiment containing human actin primers to verify no genomic DNA contamination. Amplification parameters were UNG incubation for one cycle at 50°C for 2 min to prevent amplification of carryover DNA; denaturation/UNG inactivation at 94°C for 10 min; amplification, 40 cycles of 95°C/15 s and 60°C/60 s. Amplification products using SYBRgreen detection were checked by using dissociation curve software (PerkinElmer) and by gel electrophoresis on a 1% agarose gel and were then visualized under UV light after staining with 0.05% ethidium bromide to confirm the size of the DNA fragment and that only one product was formed. Samples were compared by using the relative (comparative) Ct method. The Ct value, which is inversely proportional to the initial template copy number, is the calculated cycle number where the fluorescence signal emitted is significantly above background levels. Fold induction by real-time RT-PCR was measured in triplicate wells to account for RT-PCR and repeated with two animals and calculated after adjusting for actin using 2{Delta}{Delta}Ct, where {Delta}Ct = target gene Ct-actin Ct and {Delta}{Delta}Ct = {Delta}Ct control – {Delta}Ct treatment.

Data Analysis

All data were analyzed by using a statistical software package (SigmaStat; Jandel Scientific, San Rafael, CA). Data were reported as means ± SE for a given number of animals for each experiment. All treatments for each experiment were applied to tissues from each of the animals, but there were no duplicate treatments on any of the animals. Electrical data were analyzed by repeated-measures, two-way ANOVA for the effects of time and treatment on TER using the animal number as the subject. For a significant result on the initial two-way ANOVA for each set of data, a Tukey's test was utilized for pairwise multiple comparisons to discern differences between treatments. P values reported for each experiment on electrical data represent the result of the repeated-measures ANOVA for the time period over which treatments were applied, rather than the differences between treatments at a single time point. For data on IL-1{beta} levels, a two-way ANOVA for the effects of time and treatment was utilized, followed by a post hoc Tukey's test for pairwise comparisons. The {alpha}-level for statistical significance was set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neutrophils Increase TER

Because of our interest in the effects of PMNs on recovery of acutely injured mucosa, we initially applied either 1 or 5 x 106 PMNs harvested from arterial blood to the mucosal or serosal surface of ischemia-injured ileum from the same animal after an initial 45-min equilibration period. The number of PMNs for application was based on the results of work by Gayle et al. (17) showing peak PMN infiltration of 700 PMNs/mm2 in porcine postischemic ileal mucosa. Mathematical calculations utilizing the surface area of the tissue exposed to PMNs within Ussing chambers (3.14 cm2) and the effect of villus surface area amplification (3), indicated that 1–10 million PMNs per chamber would approximate this peak number of PMNs. These PMNs were allowed to interact with the tissue within Ussing chambers for 15 min before circulating 10 ml of Ringer through the chambers from the mucosal and serosal fluid reservoirs. Although there was no significant effect of low numbers of PMNs (1 x 106 cells), there was a sustained increase in TER after the addition of 5 x 106 PMNs to the serosal surface of tissues (Fig. 1). Conversely, there was no significant effect of PMNs applied to the mucosal surface of tissues, and there was no effect of PMNs on normal tissues (data not shown). Orienting the chambers vertically in an attempt to facilitate PMN contact with tissues did not alter the magnitude of change in TER after serosal application of PMNs. Microscopic evaluation of tissues did not reveal a significant increase in the number of adherent PMNs compared with tissues in the absence of added PMNs (data not shown). Additionally, there was no effect of pretreatment of PMNs with the anti-{beta}2-integrin monoclonal antibodies IB4 or R15.7 (40 µg/ml each, data not shown). Taken together, these data suggested that PMNs enhanced recovery of TER when applied to the serosal surface of ischemia-injured tissues by a mechanism independent of PMN infiltration of the tissue.



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Fig. 1. Electrical response of ischemia (Isch)-injured porcine ileal mucosa to serosal addition of neutrophils (PMNs). Tissue treated with PMNs (5 x 106) exhibited a significant (P < 0.05) increase in transepithelial electrical resistance (TER) over time compared with untreated ischemia-injured tissue as determined using 2-way ANOVA on repeated measures. Pretreatment of tissue with the cyclooxygenase (COX) inhibitor indomethacin (Indo; 5 µM) significantly (P < 0.05) prevented the enhanced TER observed with 5 x 106 PMNs. Values represent means ± SE; n = 6 animals.

 
Neutrophil Effects on Ischemia-Injured Ileum are COX-2 and IL-1{beta} Dependent

Because we (4) have previously shown an important role of prostaglandins in orchestrating recovery of TER in ischemia-injured porcine ileum, tissues were pretreated with the nonselective COX inhibitor indomethacin (5 µM) or the selective COX-2 inhibitor NS-398 (5 µM) followed by serosal application of 5 x 106 PMNs harvested from whole blood after an initial 45-min equilibration period. Indomethacin inhibited both baseline recovery of TER and PMN-stimulated recovery of TER (Fig. 1), whereas NS-398 inhibited the PMN-induced increases in TER observed after the addition of PMNs alone but had no effect on baseline recovery of TER (Fig. 2). We then considered potential PMN mediators that might be responsible for stimulating COX-2. Although there was no effect of pretreatment of tissues with the antioxidant catalase (1,000 U/ml), pretreatment of tissues with 0.1 mg/ml of an IL-1{beta} receptor antagonist prevented the PMN-induced rise in TER (Fig. 2). Analysis of fluid for IL-1{beta} showed that PMNs suspended in Ringer (5 x 106 PMNs/ml), before addition to tissues in the Ussing chamber, contained ~100 ng/ml IL-1{beta}, whereas fluid bathing ischemia-injured tissues before PMN addition contained ~20 ng/ml. After 180 min of in vitro recovery, tissues exposed to PMNs had significantly greater levels of IL-1{beta} than untreated tissues (Fig. 3). Collectively, these data indicated that the pathway utilized by PMNs to stimulate increases in TER involved both IL-1{beta} and COX-2.



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Fig. 2. Electrical response of ischemia-injured porcine ileal mucosa to serosal addition of PMNs and pretreatment with NS-398 or IL-1{beta} receptor antagonist. Tissue pretreated with the selective COX-2 inhibitor, NS-398 (5 µM), or an IL-1{beta} receptor antagonist (0.1 mg/ml) each significantly (P < 0.05) prevented the effect of application of 5 x 106 PMNs on TER over the experimental time course. PMNs were added at 45 min. Values represent means ± SE; n = 6 animals.

 


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Fig. 3. Analysis of tissue bathing solutions and stock PMN solution for IL-1{beta}. The amount of IL-1{beta} was significantly higher in tissue treated with PMN at 180 min compared with untreated tissue at 180 min (#P < 0.05). Additionally, all tissue at 180 min had significantly higher levels of IL-1{beta} compared with baseline (*P < 0.05). Values represent means ± SE; n = 4 animals.

 
IL-1{beta} Dose Response

To determine whether the addition of IL-1{beta} could improve TER in the absence of PMNs and to determine whether this effect was dose dependent, IL-1{beta} was added to the serosal side of ischemia-injured porcine ileal mucosa after an initial 45-min equilibration period. As shown in Fig. 4, IL-1{beta} at a dose of 10 ng/ml had a significant effect (P < 0.05) on improving TER compared with untreated tissue, whereas lower or higher doses (1 ng/ml and 100 ng/ml, respectively) were without effect. The specific dose response improvement in TER observed was only noted after the addition of IL-1{beta} to the serosal side of tissue, because there was no effect when IL-1{beta} was applied to the mucosal side of tissue (data not shown). IL-1{beta} was also applied at the same doses to uninjured tissue but had no effect on TER when applied to the serosal side of control tissue (data not shown). On the basis of this information, it appears that IL-1{beta} interacts with subepithelial tissues primed by ischemia to induce early recovery of TER.



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Fig. 4. Dose response of ischemia-injured porcine ileal mucosa to IL-1{beta}. Application of 10 ng/ml of IL-1{beta} significantly (P < 0.05) enhanced TER compared with untreated ischemia-injured mucosa over the experimental time course. IL-1{beta} at 1 ng/ml or 100 ng/ml had no effect on TER compared with untreated ischemia-injured mucosa. Values represent means ± SE; n = 7 animals.

 
COX-2 Role in IL-1{beta}-Induced Increases in TER

We then determined whether COX-2 played a role in the increased TER seen with 10 ng/ml IL-1{beta} by pretreating tissues with the selective COX-2 inhibitor NS-398 (5 µM). Similar to the effect of NS-398 on tissues exposed to PMNs, pretreatment of ischemia-injured tissues with NS-398 blocked the increase in TER that was observed after serosal addition of 10 ng/ml of IL-1{beta} after an initial 45-min equilibration period (Fig. 5). To further investigate the relationship between IL-1{beta} and COX isoforms, Western blot analysis of IL-1{beta}-treated ischemia-injured tissues was performed. Tissues from six animals were removed from the Ussing chambers after the addition of 10 ng/ml IL-1{beta} at the earliest time point at which increases in TER were noted (15 min after the addition of IL-1{beta} and 60 min after initiation of the experiment). The same tissue was also subjected to Western blot analysis for COX-1 protein. As seen in Fig. 6, IL-1{beta} at 10 ng/ml appreciably upregulated COX-2 compared with untreated ischemia-injured tissue, whereas there was no notable effect on COX-1 expression (not shown). On additional blots not shown, COX-2 could be appreciated in untreated ischemia-injured tissue lanes, but IL-1{beta}-treated tissue resulted in marked upregulation of COX-2. Due to the rapid upregulation of COX-2 in IL-1{beta}-treated mucosa, mRNA analysis was performed at 5, 10, and 15 min after IL-1{beta} treatment. Figure 7 shows that COX-2 mRNA is elevated in IL-1{beta}-treated ischemia-injured tissue compared with untreated ischemia-injured tissue at 5 min. However, expression levels were similar in both sets of tissues at 10 min, and by 15 min, COX-2 mRNA expression was decreased compared with untreated ischemia-injured mucosa, suggesting that mRNA had largely been transcribed and degraded at this point, giving rise to increased protein expression. Overall, RT-PCR and Western blot analyses supported the hypothesis that IL-1{beta} upregulates COX-2. Furthermore, the blockade of IL-1{beta}-induced increases in TER with the COX-2 inhibitor NS-398 indicates that increased COX-2 expression is responsible for enhanced recovery of ischemia-injured mucosa.



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Fig. 5. Electrical response of ischemia-injured porcine ileal mucosa to treatment with IL-1{beta} (10 ng/ml) and NS-398 (5 µM). Pretreatment of tissue with the selective COX-2 inhibitor, NS-398 (5 µM), significantly (P < 0.05) inhibited the rise in TER after application of 10 ng/ml IL-1{beta} over the experimental time course. Values represent means ± SE; n = 7 animals.

 


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Fig. 6. The effect of IL-1{beta} on COX-2 protein expression in ischemia-injured porcine ileal mucosa. COX-2 protein was assessed by Western blot analysis. Ischemia-injured ileal mucosa was treated with IL-1{beta} (10 ng/ml) or untreated. COX-2 protein expression was markedly upregulated in IL-1{beta}-treated tissue compared with untreated ischemia-injured tissue. Shown are blots of tissue homogenates from 3 separate pigs on the same gel, representative of 6 pigs.

 


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Fig. 7. Fold change in COX-2 mRNA gene expression in ischemia-injured tissues subjected to IL-1{beta} treatment. mRNA gene expression of actin and COX-2 were measured by real-time RT-PCR in ischemia-injured tissues from 2 animals (Pigs 1 and 2). Tissues were either untreated or treated with IL-1{beta} (10 ng/ml) for 5, 10, or 15 min. Values represent means ± SE for triplicate analyses of COX-2 mRNA expression relative to untreated ischemia-injured tissues, adjusted for actin.

 
Extravasated PMNs Enhance TER Recovery

In further experiments, we wished to study the effects of extravasated PMNs to determine whether postcirculatory PMNs would have a similar effect on recovery of TER in ischemia-injured mucosa. Extravasated PMNs were harvested from peritoneal fluid from a separate pig with peritonitis. A separate pig was utilized for the induction of peritonitis to ensure no effect of peritoneal PMN infiltration on experimental intestine. These extravasated PMNs were considered to be a physiologically relevant population of cells, because PMNs migrating in repairing mucosa in vivo would have already exited the microcirculation. As shown in Fig. 8, extravasated PMNs had a similar effect on TER when applied at a dose of 5 x 106 cells on the serosal side after an initial 45-min equilibration period.



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Fig. 8. Transepithelial electrical resistance (TER) response of ischemia-injured porcine ileal mucosa with the addition of extravasated PMNs. Tissue treated with extravasated neutrophils (5 x 106) exhibited a significant (P < 0.05) increase in TER compared with untreated ischemia-injured tissue over the experimental time course. Values represent means ± SE; n = 8 animals.

 
COX-2 Role in PMN-Induced Increases in TER

To further investigate the hypothesis that the improvement in TER after the addition of 5 x 106 PMN was due to an increase in COX-2 protein expression that was IL-1{beta} dependent, tissues equilibrated for 45 min were exposed to extravasated PMNs for 30 min in the presence or absence of an IL-1{beta} receptor antagonist added at the beginning of the equilibration period and Western blotted for COX-2. Mucosal homogenates from untreated, ischemia-injured ileum revealed notable expression of COX-2, whereas mucosal homogenates from tissues exposed to extravasated PMNs had dramatically increased expression of COX-2 (Fig. 9). Furthermore, the increase in COX-2 protein in PMN-treated tissue was prevented by pretreatment of tissues with an IL-1{beta} receptor antagonist. Thus these data support the hypothesis that PMNs are capable of improving TER in ischemia-injured tissue via an IL-1{beta}- and COX-2-dependent pathway.



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Fig. 9. Effect of PMNs on COX-2 protein expression in ischemia-injured porcine ileal mucosa. COX-2 protein was assessed by Western blot analysis. Ischemia-injured ileal mucosa was left untreated, treated with PMN (5 x 106), treated with IL-1{beta} receptor antagonist (IL-1{beta}RA), or treated with a combination of PMN and IL-1{beta}RA. COX-2 protein expression was markedly upregulated in PMN-treated tissue compared with untreated ischemia-injured tissue. Additionally, application of an IL-1{beta}RA prevented the PMN-induced upregulation in COX-2 protein expression.

 

    DISCUSSION
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 
It has been shown previously that PMNs are attracted to sites of injury, during which time they are activated and may exacerbate injury as a result of release of proteases, oxidants, or proinflammatory cytokines (16, 25, 37, 38). However, PMNs are a critical component of innate immunity that also appear to have a beneficial role when applied to acutely injured porcine ileal mucosa. Specifically, we have shown that the addition of 5 x 106 PMNs hastens recovery of TER via an IL-1{beta}- and COX-2-dependent mechanism. We do not know the physiological relevance of these findings, because PMN infiltration into acutely injured mucosa is not immediate after ischemic injury. For example, peak numbers of infiltrating PMNs recorded in our previous studies (17) occurred at 6 h after the initial ischemic event. However, there are resident PMNs in mucosa that would become activated in the early phases of the injury-repair cycle (26) that could presumably release IL-1{beta} in the immediate vicinity of repairing epithelium. Furthermore, the beneficial effects of neutrophilic IL-1{beta} release would be counterbalanced by PMN transmigration across repairing epithelium, which we have previously shown is capable of retarding the mucosal recovery response (17), although this effect is dependent on the number of transmigrating PMNs (34). Thus the net effect of PMNs on repairing epithelium would relate to factors such as the number of PMNs activated within the vicinity of repairing epithelium and the concentration of IL-1{beta} released by these PMNs.

Although PMNs release numerous cytokines, we selected IL-1{beta} as a possible candidate for the PMN-induced recovery response, because our preliminary experiments indicated that the response to PMNs was COX dependent and because there are studies showing that IL-1{beta} is capable of upregulating COX-2 in gastrointestinal tissue. For example, IL-1{beta} upregulated COX-2 in a human colonic subepithelial myofibroblast cell line (32) and in gastric cancer cells (14). IL-1{beta} has also been shown to increase PGE2 levels in Mode-K intestinal epithelial cells (22), and IL-1{beta} is present in gastric ulcers (39). Such IL-1{beta} accumulation at gastric ulcers could be related to increased expression of COX-2 at the margins of ulcers, which appears to facilitate ulcer healing (33). The link between IL-1{beta} and COX-2 is not restricted to gastrointestinal tissues. For example, IL-1{beta} has also been shown to elevate COX-2 protein levels and PGE2 production in endometrial stromal cells (40). Interestingly, IL-1 also has the capability of activating chloride secretion in T84 cells (21) and increasing CFTR expression (9) that may be relevant to intestinal epithelial repair, because we (3, 28) have previously shown that chloride secretion is a critical signaling component of the recovery process in ischemia-injured porcine ileum.

Remarkably rapid detection of COX-2 protein in IL-1{beta}-stimulated mucosa could be explained by mRNA stabilization and subsequent translation (12, 19). In the present study, mucosa was subjected to 45 min of ischemia in vivo with an additional 45 min of recovery time within the Ussing chambers before the addition of IL-1{beta}. Thus the tissue was likely producing the COX-2 transcript before the addition of IL-1{beta} or PMNs. For stimuli such as IL-1{beta}, message stabilization appears to be the primary mechanism of COX-2 induction (2, 23). Our data support the role of IL-1{beta} on COX-2 message stabilization, because increased mRNA was detected at 5 min after 10 ng/ml IL-1{beta} treatment. The decline in COX-2 mRNA at 15 min after IL-1{beta} application corresponded to the elevated COX-2 protein we detected through Western blot analysis, suggesting utilization and degradation of COX-2 transcripts by this time. As for the signaling mechanism involved in mRNA stabilization, studies (32) in basic FGF-stimulated human intestinal epithelial cells and IL-1-stimulated intestinal myofibroblasts have suggested that p38 MAPK regulates COX-2 expression at the posttranscriptional level by increasing mRNA stability. Thus it is possible that IL-1{beta} is capable of increasing COX-2 protein expression via a p38 MAPK-dependent mechanism resulting in mRNA stability.

Beneficial effects of IL-1{beta} are dose specific with low doses (1 ng/ml) or high doses (100 ng/ml) having no effect on TER, whereas 10 ng/ml consistently augmented recovery of TER. Not all studies show beneficial effects of IL-1{beta} on intestinal tissues. For example, blockade of IL-1 and TNF-{alpha} during ischemia-reperfusion of the rat small intestine alleviated intestinal injury (43). Furthermore, addition of IL-1{beta} directly to intestinal epithelium resulted in declining measurements of TER (6) and increases in horseradish peroxidase fluxes, which indicates altered epithelial permeability (29). Dose specificity in the present study may relate to dose-dependent effects on COX-2 expression counterbalanced by damaging effects on epithelium at higher doses. However, we did not note any change in the histological appearance of tissues exposed to higher doses of IL-1{beta}. A similar dose response to IL-1{beta} has also been observed on CFTR expression in T84 cells (9). In studies by Hinterleitner et al. (21), 10–1,000 pg/ml of IL-1{alpha} applied to 18Co cells dose-dependently increased PGE2 levels with a maximum effect achieved at 100 pg/ml of IL-1{alpha}, suggesting that peak PGE2 release may be achieved with submaximal dosages, which may explain the results in the present study. The beneficial effect of 10 ng/ml IL-1{beta} was blocked by application of a COX-2-specific inhibitor NS-398. Additionally, at the 10 ng/ml dose, there was upregulation of COX-2 as determined by Western blot analysis. Furthermore, the beneficial effects of IL-1{beta} only occurred when it was added to the serosal side of tissues, suggesting upregulation of COX-2 within cells localized to the subepithelial layer. One potential population of subepithelial cells are myofibroblasts, which have previously been shown to be capable of expressing COX-2 when exposed to IL-1{beta} in cell culture conditions (20, 32). Our previous studies (5) on porcine ischemia-injured mucosa have shown expression of COX-2 in repairing epithelium and within mononuclear cells adjacent to the epithelium. Based on the requirement for addition of IL-1{beta} to the serosal surface of tissues, subepithelial mononuclear cells would appear to be one potential target of PMNs and IL-1{beta} in the present studies.

The implications of this study are that PMNs have a beneficial role in recovery of ischemia-injured ileum, which must be balanced with studies indicating a deleterious role of PMNs in mucosal recovery (17). Thus although inhibiting PMN infiltration can augment PMN recovery, other important PMN functions, such as innate defense and upregulation of proreparative COX-2, suggest that this nonspecific approach to blocking PMN function may have detrimental effects. Ultimately, by understanding the signaling pathways utilized by PMNs and recovering intestinal mucosa, targeted therapy that retains the beneficial elements of the inflammatory cascade may result in hastening of mucosal repair in patients suffering from a broad range of inflammatory bowel diseases.


    GRANTS
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grants DK-53284 and DK-34987 and United States Department of Agriculture National Research Initiative Grant 0102490.


    ACKNOWLEDGMENTS
 
We thank the Immunotechnology Core of the Center for Gastrointestinal Biology and Disease (Chapel Hill, NC) for assistance with IL-1{beta} assays.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. T. Blikslager, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606 (E-mail: Anthony_Blikslager{at}ncsu.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.


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 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Arai Y, Takanashi H, Kitagawa H, and Okayasu I. Involvement of interleukin 1 in the development of ulcerative colitis induced by dextran sulfate sodium in mice. Cytokine 10: 890–896, 1998.[CrossRef][ISI][Medline]
  2. Barrios-Rodiles M, Tiraloche G, and Chadee K. Lipopolysaccharide modulates cyclooxygenase-2 transcriptionally and posttranscriptionally in human macrophages independently from endogenous IL-1{beta} and TNF-{alpha}. J Immunol 163: 963–969, 1999.[Abstract/Free Full Text]
  3. Blikslager AT, Roberts MC, and Argenzio RA. Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion. Am J Physiol Gastrointest Liver Physiol 276: G28–G36, 1999.[Abstract/Free Full Text]
  4. Blikslager AT, Roberts MC, Rhoads JM, and Argenzio RA. Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum. J Clin Invest 100: 1928–1933, 1997.[Abstract/Free Full Text]
  5. Blikslager AT, Zimmel DN, Young KM, Campbell NB, Little D, and Argenzio RA. Recovery of ischaemic injured porcine ileum: evidence for a contributory role of COX-1 and COX-2. Gut 50: 615–623, 2002.[Abstract/Free Full Text]
  6. Bode H, Schmitz H, Fromm M, Scholz P, Riecken E, and Schulzke J. IL-1{beta} and TNF-{alpha}, but not IFN-{alpha}, IFN-{gamma}, IL-6 or IL-8, are secretory mediators in human distal colon. Cytokine 10: 457–465, 1998.[CrossRef][ISI][Medline]
  7. Bossu P, Del Grosso E, Cesaroni MP, Maurizi G, Balestro N, Stoppacciaro A, Giudice E, Ruggiero P, and Boraschi D. Balance between autocrine interleukin-1{beta} and caspases defines life versus death of polymorphonuclear leukocytes. Eur Cytokine Netw 12: 177–186, 2001.[ISI][Medline]
  8. Bottone FG Jr, Martinez JM, Alston-Mills B, and Eling TE. Gene modulation by Cox-1 and Cox-2 specific inhibitors in human colorectal carcinoma cancer cells. Carcinogenesis 25: 349–357, 2004.[Abstract/Free Full Text]
  9. Cafferata EG, Gonzalez-Guerrico AM, Giordano L, Pivetta AH, and Santa-Coloma TA. Interleukin-1{beta} regulates CFTR expression in human intestinal T84 cells. Biochim Biophys Acta 1500: 241–248, 2000.[ISI][Medline]
  10. Casini-Raggi V, Kam L, Chong YJ, Fiocchi C, Pizzaro TT, and Cominelli F. Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease. A novel mechanism of chronic intestinal inflammation. J Immunol 154: 2434–2440, 1995.[Abstract/Free Full Text]
  11. Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, and Simmons DL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci USA 15: 13926–13931, 2002.[CrossRef]
  12. Cok SJ and Morrison AR. The 3'-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency. J Biol Chem 276: 23179–23185, 2001.[Abstract/Free Full Text]
  13. Dinarello CA. Biological basis for interleukin-1 in disease. Blood 87: 2095–2147, 1996.[Abstract/Free Full Text]
  14. Fan XM, Yu Wong BC, Lin MC, Cho CN, Wang WP, Kung HF, and Lam SK. Interleukin-1{beta} induces cyclo-oxygenase-2 expression in gastric cancer cells by the p38 and p44/42 mitogen-activated protein kinase signaling pathways. J Gastroenterol Hepatol 16: 1098–1104, 2001.[CrossRef][ISI][Medline]
  15. Faour WH, He Y, He QW, Ladurantaye M, Qunitero M, Mancini A, and Battista JA. Prostaglandin E2 regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1{beta} treated human synovial fibroblasts. J Biol Chem 276: 31720–31731, 2001.[Abstract/Free Full Text]
  16. Fitzal F, DeLano FA, Young C, Rosario HS, and Schmid-Schonbein GW. Pancreatic protease inhibition during shock attenuates cell activation and peripheral inflammation. J Vasc Res 39: 320–329, 2002.[CrossRef][ISI][Medline]
  17. Gayle J, Jones SL, Argenzio RA, and Blikslager AT. Neutrophils increase paracellular permeability of restituted ischemic-injured porcine ileum. Surgery 132: 461–470, 2002.[ISI][Medline]
  18. Gerwirtz AT, Siber AM, Madara JL, and McCormick BA. Orchestration of neutrophil movement by intestinal epithelial cells in response to salmonella typhimurium can be uncoupled from bacterial internalization. Infect Immun 67: 608–617, 1999.[Abstract/Free Full Text]
  19. Guan Z, Buckman SY, Miller BW, Springer LD, and Morrison AR. Interleukin-1{beta} induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells. J Biol Chem 273: 28670–28676, 1998.[Abstract/Free Full Text]
  20. Habbib A, Creminon C, and Frobert Y. Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2. J Biol Chem 268: 448–454, 1993.
  21. Hinterleitner TA, Saada JI, Berschneider HM, Powell DW, and Valentich JD. IL-1 stimulates intestinal myofibroblast COX gene expression and augments activation of Cl secretion in T84 cells. Am J Physiol Cell Physiol 271: C1262–C1268, 1996.[Abstract/Free Full Text]
  22. Homaiden FR, Chakroun I, Dbaibo GS, El-Assaad W, and El-Sabban ME. IL-1 activates two phospholipid signaling pathways in intestinal epithelial cells. Inflamm Res 50: 375–381, 2001.[ISI][Medline]
  23. Huang ZF, Massey JB, and Via DP. Differential regulation of cyclooxygenase-2 (COX-2) mRNA stability by interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}) in human in vitro differentiated macrophages. Biochem Pharmacol 59: 187–194, 2000.[CrossRef][ISI][Medline]
  24. Jenkins JK, Malyak M, and Arend WP. The effects of interleukin-10 on interleukin-1 receptor antagonist and interleukin-1{beta} production in human monocytes and neutrophils. Lymphokine Cytokine Res 13: 47–54, 1994.[ISI][Medline]
  25. Konaka A, Kato S, Tanaka A, Kunikata T, Korolkiewicz R, and Takeuchi K. Roles of enterobacteria, nitric oxide and neutrophil in pathogenesis of indomethacin-induced small intestinal lesions in rats. Pharm Res 40: 517–524, 1999.[CrossRef]
  26. Kubes P, Hunter J, and Granger DN. Ischemia/reperfusion induced feline intestinal dysfunction: importance of granulocyte recruitment. Gastroenterology 103: 807–812, 1992.[ISI][Medline]
  27. Lee JC, Kumar S, Griswold DE, Underwood DC, Votta VJ, and Adams JL. Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology 47: 185–201, 2000.[CrossRef][ISI][Medline]
  28. Little D, Dean RA, Young KM, McKane SA, Martin LA, Jones SL, and Blikslager AT. PI3K signaling is required for prostaglandin-induced recovery in ischemia-injured porcine ileum. Am J Physiol Gastrointest Liver Physiol 284: G46–G56, 2003.[Abstract/Free Full Text]
  29. Matysiak-Budnik T, Thomas-Collignon A, Megraud F, and Heyman M. Alterations in epithelial permeability by Helicobacter and IL-1{beta} in vitro: protective effect of rebamipide. Dig Dis Sci 46: 1558–1566, 2001.[CrossRef][ISI][Medline]
  30. McAlindon ME, Hawkey CJ, and Mahida YR. Expression of interleukin 1{beta} and interleukin 1{beta} converting enzyme by intestinal macrophages in health and inflammatory bowel disease. Gut 42: 214–219, 1998.[Abstract/Free Full Text]
  31. McCall RD, Haskill S, Zimmerman EM, Lund PK, Thompson RC, and Sartor RB. Tissue interleukin 1 and interleukin 1 receptor antagonist expression in enterocolitis in resistant and susceptible rats. Gastroenterology 106: 960–972, 1994.[ISI][Medline]
  32. Mifflin RC, Saada JI, DiMari JF, Adegboyega PA, Valentich JD, and Powell DW. Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1 mediated induction. Am J Physiol Cell Physiol 282: C824–C834, 2002.[Abstract/Free Full Text]
  33. Mizuno H, Sakamoto C, Matsuda K, Wada K, Uchida T, Noguchi H, Akamatsu T, and Kasuga M. Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice. Gastroenterology 112: 87–97, 1997.
  34. Nusrat A, Parkos CA, Liang TW, Carbnes DK, and Madara JL. Neutrophil migration across model intestinal epithelia: monolayer disruption and subsequent events in epithelial repair. Gastroenterology 113: 1489–500, 1997.[ISI][Medline]
  35. O'Banion KM, Winn VD, and Young DA. cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase. Proc Natl Acad Sci USA 89: 4888–4892, 1992.[Abstract]
  36. O'Neill GP and Ford-Hutchison AW. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett 330: 156–160, 1993.[ISI][Medline]
  37. Souza DG, Cassali GD, Poole S, and Teixeira MM. Effects of inhibition of PDE4 and TNF-{alpha} on local and remote injuries following ischaemia and reperfusion injury. Br J Phamacol 134: 985–994, 2001.[Abstract/Free Full Text]
  38. Sun Z, Wang X, Lasson A, Bojesson A, Annborn M, and Andersson R. Effects of inhibition of PAF, ICAM-1 and PECAM-1 on gut barrier failure caused by intestinal ischemia and reperfusion. Scand J Gastroenterol 36: 55–65, 2001.[CrossRef][ISI][Medline]
  39. Takahashi S, Kobayashi N, and Okabe S. Regulation of endogenous interleukin 1 of mRNA expression of healing related factors in gastric ulcers in rats. J Pharmacol Exp Ther 291: 634–641, 1999.[Abstract/Free Full Text]
  40. Tamura M, Sebastian S, Yang S, Gurates B, Fang Z, and Bulun SE. Interleukin-1{beta} elevates cyclooxygenase-2 protein level and enzyme activity via increasing its mRNA stability in human endometrial stromal cells: an effect mediated by extracellular regulated kinases 1 and 2. J Clin Endocrinol Metab 87: 3263–3273, 2002.[Abstract/Free Full Text]
  41. Thomas B, Thirion S, Humbert L, Tan L, Goldring MB, Bereziat G, and Berenbaum F. Differentiation regulates interleukin-1{beta}-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase. Biochem J 362: 367–373, 2002.[CrossRef][ISI][Medline]
  42. Watson RW, Rotstein OD, Parodo J, Bitar R, and Marshall JC. The IL-1{beta} converting enzyme (Caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1{beta}. J Immunol 161: 957–962, 1998.[Abstract/Free Full Text]
  43. Yamamoto S, Tanabe M, Wakabayashi G, Shimazu M, Matsumoto K, and Kitajima M. The role of tumor necrosis factor-{alpha} and interleukin-1{beta} in ischemia reperfusion injury of the rat small intestine. J Surg Res 99: 134–141, 2001.[CrossRef][ISI][Medline]
  44. Youngman KR, Simon PL, West GA, Cominelli F, Rachmilewitz D, Klein JS, and Fiocchi C. Localization of intestinal interleukin 1 activity and protein and gene expression to lamina propria cells. Gastroenterology 104: 749–758, 1993.[ISI][Medline]




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