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Lipoxin A₄ Analogs Attenuate Induction of Intestinal Epithelial Proinflammatory Gene Expression and Reduce the Severity of Dextran Sodium Sulfate-Induced Colitis¹

Andrew T. Gewirtz^2,*, Lauren S. Collier-Hyams^*, Andrew N. Young^*, Torsten Kucharzik^*, William J. Guilford, John F. Parkinson, Ifor R. Williams^*, Andrew S. Neish^* and James L. Madara^*

^* Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322; and Departments of Medicinal Chemistry and Immunology, Berlex Biosciences, Richmond, CA 94804

	Abstract

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The anti-inflammatory eicosanoid lipoxin A₄ (LXA₄), aspirin-triggered 15-epi-LXA₄, and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA₄ analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA₄ analog pretreatment attenuated induction of 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA₄ analog pretreatment is regulated by NF-B, suggesting that LXA₄ analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA₄ analog reduced NF-B-mediated transcriptional activation in a LXA₄ receptor-dependent manner and inhibited induced degradation of IB. LXA₄ analog did not affect earlier stimulus-induced signaling events that lead to IB degradation, such as S. typhimurium-induced epithelial Ca²⁺ mobilization or TNF--induced phosphorylation of IB. To establish the in vivo relevance of these findings, we examined whether LXA₄ analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA₄ analog (15-epi-16-para-fluoro-phenoxy-LXA₄, 10 µg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA₄ analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-B pathway can be therapeutic for diseases characterized by mucosal inflammation.

	Introduction

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As an interface with the outside world, the intestinal epithelium dynamically alters its gene expression in response to the changing environments in both the intestinal lumen and the subepithelial domain. For example, in response to either lumenal enteric pathogens or subepithelial proinflammatory cytokines, the intestinal epithelium activates the expression of a panel of genes that promote an acute inflammatory response (1). As uncontrolled inflammation can result in tissue damage, the intestinal epithelium is also influenced by exogenous and endogenous anti-inflammatory mediators that attenuate proinflammatory responses. One example of this type of mediator and the focus of this study is that of the anti-inflammatory eicosanoid lipoxin (lipoxin A₄ (LXA₄)³).

Lipoxins such as LXA₄ are derived from arachidonate as a result of its exposure to the unique combinations of lipoxygenases that occur during specific heterotypic cell-cell interactions such as those occurring in inflammation (e.g., epithelial-neutrophil interactions). LXA₄-induced responses down-regulate events associated with inflammation in a variety of in vitro and in vivo models (2). Acetylation of cyclooxygenase by aspirin results in the biosynthesis of the 15-epimer of LXA₄ (3). Such 15-epi-LXA₄ as well synthetic analogs of LXA₄ resist enzymatic degradation and thus have longer-lasting anti-inflammatory bioactivity than the native eicosanoid (4). LXA₄ and its synthetic stable analogs attenuate the IL-8 expression that is induced in model epithelia in response to the gastroenteritis-causing pathogen Salmonella typhimurium and the proinflammatory cytokine TNF- (5, 6). Lipoxin analogs also attenuate chemokine secretion by human colon, resulting in reduced neutrophil adherence and tissue damage (7).

The mechanism by which LXA₄ analogs down-regulate IL-8 expression is largely unknown, although we have shown that IL-8 mRNA levels are reduced (5), implying action at the level of transcription. While this bioaction is at least somewhat specific, in that the mRNA levels of actin are not affected by LXA₄ analogs, technology to broadly evaluate gene expression has not, until recently, been available. Microarray technology now permits simultaneous parallel measurement of the expression of thousands of genes, making it possible to evaluate the effect of a given mediator on global gene expression. We sought to use this technology to characterize LXA₄ bioaction and perhaps better predict its in vivo behavior. In this study, we use this approach to test the hypothesis that LXA₄ exerts its effects primarily on the transcriptional activation of genes involved in the proinflammatory epithelial response.

We observed that LXA₄ analogs did not directly affect gene expression but broadly induced proinflammatory gene expression, particularly that regulated by NF-B. This result is not surprising when one considers that LXA₄ analogs are known to antagonize the effects of a number of proinflammatory agonists that signal through this transcription factor (as discussed above). Having identified a role for this factor, we next explored which elements of the signaling pathway were affected. Last, we tested the in vivo relevance of these findings in a mouse model of colitis.

	Materials and Methods

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Materials

15-(R/S)-methyl-LXA₄ was synthesized by Dr. N. Petasis (University of Southern California, Los Angeles, CA) as previously described (4). 15-epi-16-parafluoro-phenoxy-LXA₄ was supplied by Berlex Biosciences (Richmond, CA). Dextran sodium sulfate (DSS; m.w. 40,000) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). MG-132 was obtained from Calbiochem (La Jolla, CA).

Cell culture

Polarized model intestinal epithelia were prepared via culturing T84 cells on permeable supports as previously described (8). Model epithelia were used 6–14 days after plating after verification (for T84) that they had achieved a transepithelial electrical resistance of at least 1000 cm². S. typhimurium was cultured and used to colonize model epithelia as previously described (5).

Microarray analysis

Total RNA was isolated with TRIzol (GIBCO, Gaithersburg, MD) following instructions by the manufacturer. mRNA was isolated, hybridization was performed by Incyte Genomics (Palo Alto, CA), and mRNA were analyzed as we have recently described (9). Each array condition was performed on RNA pooled from six individual 5-cm² model epithelia so as to minimize the effect of experimental variability that might occasionally arise in individual samples. Changes in gene expression of 2-fold or more are highly likely to be significant (9).

Transient transfections and CAT assays

HeLa cells (40–60% confluent) were transiently transfected using Superfect Reagent (Qiagen, Valencia, CA) with 2 µg of the reporter plasmid pIL-8-CAT (10) and variable quantities of the pCMV-myc-LXA₄R expression plasmid (encoding LXA₄ receptor) according to the manufacturer’s instructions. All cotransfection reactions were balanced for total amount of expression plasmid DNA with pCMV-myc vector. Approximately 16–24 h after transfection, cells were washed with HBSS and incubated with 0–100 nM 15-(R/S)-methyl-LXA₄ for 1 h followed by TNF- for 8 h. Cell lysates were prepared and assayed for chloramphenicol acetyl transferase (CAT) using the CAT ELISA kit from Roche (Basel, Switzerland).

IB assays

Levels of IB and phospho-IB were assayed from whole cell lysates of model intestinal epithelia via immunoblotting using an IB Ab (Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (11).

Ca²⁺ mobilization

Intracellular Ca²⁺ was measured in fura 2-loaded polarized model epithelia via spectrofluorometry as previously described (11). Briefly, polarized model epithelia were prepared on customized supports permitting their insertion into a standard fluorometry cuvette. Model epithelia were incubated with 5 mM fura 2-AM (Molecular Probes, Eugene, OR) added for 60 min, and unincorporated probe was removed with a 10-min washing. Fluorescence was read with emission at 505 nm while the excitation wavelength is changed from 340 to 380 nm. Values of intracellular Ca²⁺ were calculated via the Grynciewitz equation (R - R_min)/(R_max - R) x K_d. R_max and R_min are measured by adding digitonin (10 µM) and then EGTA (20 mM), respectively.

DSS colitis

Six- to 8-wk-old BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). One week following arrival, mice were given drinking water containing 4% DSS and 0.05% ethanol (vehicle) or 10 µg/ml 15-epi-16-parafluoro-LXA₄ (following internal review board approved protocol). Water consumption was measured per cage and corrected for leakage/evaporation by comparison to identical water bottles placed in empty cages. Water was changed every 5 days, a time at which HPLC analysis indicated that degradation of the LXA₄ analog had not occurred. Body mass was measured daily by a technician blinded to the drug protocol, who also checked mice daily for gross rectal bleeding and occult blood in stools via Hemoccult Sensa (Beckman Coulter, Fullerton, CA). Bleeding score was 0 (hemoccult negative), 1 (mild positive), 2 (strong positive), and 4 (gross blood) following the well-established procedure developed by Cooper et al. (12). We did not consistently observe diarrhea in these mice and therefore did not include this parameter in our data presentation. Mice that were judged (by the blinded technician) to be moribund were sacrificed (and classified as nonsurvivors), although only one mouse in this study fell into this category (others were found dead upon daily check). Seven days following administration of DSS, mice were switched to DSS-free water containing vehicle or LXA₄ analog. Statistical significance was determined for body mass and survival data by Student’s t test with p < 0.05 termed significant.

HPLC

HPLC analyses were conducted on LXA₄ analog-containing drinking water via a LUNA 5 µm C18(2) column (250 x 4.60 mm) using a ProStar HPLC (Varian, Palo Alto, CA) equipped with a diode array detector.

	Results

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LXA₄ analog attenuates changes in gene expression induced by S. typhimurium

To explore the mechanism of the anti-inflammatory bioactivity of LXA₄ and its stable analogs, the effect of this eicosanoid on global gene expression in control and inflamed (S. typhimurium-infected) model intestinal epithelia was examined by high-density cDNA microarray. First, we asked whether LXA₄ analogs alone globally influenced gene regulation, potentially up-regulating anti-inflammatory effector molecules. Model epithelia (6 x 5 cm² epithelia per condition) were treated with vehicle (0.05% ethanol) or 100 nM of 15-(R/S)-methyl-LXA₄ (a concentration known to attenuate agonist-induced IL-8 mRNA expression (5)) for 4 h. mRNA was isolated from each set of samples and pooled, and microarray analysis was performed by hybridization to 7075 independent cDNA targets according to the protocol of Incyte Genomics as previously described (9). The expression levels of each gene from untreated (labeled with Cy3 before hybridization) epithelia vs that of LXA₄ analog-treated epithelia (Cy5 labeled) are plotted in Fig. 1. Accordingly, genes whose expression are unchanged lie on the central diagonal while, for example, genes whose expression is induced 2- to 5-fold are plotted between the upper diagonals labeled 2 and 5 (Fig. 1). Interestingly, LXA₄ analog by itself did not induce any significant changes (2-fold or more (9)) in any of the genes included in the array used. These data suggest that LXA₄ does not directly affect gene expression, and hence more likely modulates the signaling pathways by which proinflammatory agonists regulate gene expression.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. LXA4 analog attenuates changes in epithelial gene expression induced by S. typhimurium: scatter plot of expression levels from control and LXA4 analog-treated cells. Paired hextuplicate sets of T84 model epithelia were treated as follows for 4 h, at which time mRNA was isolated and analyzed via cDNA microarray hybridization. A, Epithelia were treated with 100 nM 15-(R/S)-methyl-LXA4 and compared with untreated (except vehicle) control. B, Epithelia were treated with vehicle for 1 h followed by apical colonization with 109 CFU S. typhimurium and compared with untreated control. C, Epithelia were treated with 100 nM 15-(R/S)-methyl-LXA4 vehicle for 1 h followed by apical colonization with 109 CFU S. typhimurium and compared with untreated control. Expression of each gene plotted as treated vs untreated so the diagonal represents no change from the control state. The diagonals represent indicated fold changes (above for positive) from control. The total number of genes between each set of diagonals is indicated.

LXA₄ analog attenuates activation of NF-B in model epithelia

In light of both the above microarray data and our previous finding that LXA₄ analogs down-regulate IL-8 secretion (5, 6) (also a NF-B regulated gene), we next investigated whether LXA₄ analogs directly attenuated epithelial cell activation of NF-B. As polarized model epithelia do not permit direct quantitation of NF-B promoter activity (because they are not transfectable), we used HeLa epithelial cells, which were transiently cotransfected with plasmids encoding an NF-B-responsive reporter gene (derived from the IL-8 promoter) in the presence and absence of a plasmid encoding the LXA₄ receptor. While such nonpolarized cells respond very differently to bacteria than polarized ones, both cell types respond to classic cytokine agonists such as TNF- in a very similar manner. Thus, the effects of LXA₄ analog on NF-B-mediated transcriptional activity was determined by comparing TNF--induced CAT reporter activity in LXA₄ analog-pretreated and untreated cells. LXA₄ analog attenuated NF-B-mediated gene expression by 50% (Fig. 2), consistent with the notion that attenuation of proinflammatory gene expression by LXA₄ analog is mediated via signaling through this transcription factor. Such LXA₄ analog attenuation of NF-B activity was not seen in the absence of cotransfected LXA₄ receptor, indicating that this receptor is specifically required for the observed anti-inflammatory bioactivity.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. LXA4 analog down-regulates epithelial activation of NF-B. HeLa cells were transiently transfected with plasmids encoding NF-B-CAT and empty vector or LXA4R as described in Materials and Methods, treated with 100 nM 15-(R/S)-methyl-LXA4 for 1 h, and then stimulated with TNF- for 8 h. Cells were then lysed and CAT activity was measured via ELISA. Data are means of two separate experiments, each of which showed a similar pattern of results. *, Statistically significant difference (p < 0.05).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. LXA4 analog reduces degradation, but not phosphorylation, of IB. Model epithelia were treated with vehicle (0.1% ethanol or 15-(R/S)-methyl-LXA4) for 1 h and stimulated, and whole cell lysates were assayed for IB levels via immunoblotting. A, Epithelia were colonized by 109 CFU S. typhimurium for 1 h in the presence or absence of 100 nM 15-(R/S)-methyl-LXA4. B, Epithelia were treated with vehicle or indicated concentration of 15-(R/S)-methyl-LXA4 in the presence or absence of the proteasome inihibitor MG-132 (50 µM), and then stimulated with TNF- (10 ng/ml) for 45 min. Whole cell lysates were analyzed via SDS-PAGE immunoblotting using anti-IB. Note that phospho-IB is visible only upon proteasomal inhibition with MG-132. Data are individual experiments and are representative of three separate experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. LXA4 analog did not affect Ca2+ mobilization in response to S. typhimurium. Model epithelia were loaded with the Ca2+ indicator fura 2 (5 µM for 1 h) and intracellular Ca2+ measured by spectrofluorometry. A, Monitor of baseline Ca2+ in untreated epithelia. B and C, Fura 2-loaded epithelia were treated for 1 h with vehicle (B) (0.1% ethanol) or 100 nM 15-(R/S)-methyl-LXA4 (C) and then apically treated with 109 CFU S. typhimurium during fluorometric measurement (addition of bacteria is indicated by arrow). Data are individual experiments and are representative of three separate experiments.

Chronic inflammatory diseases of the intestine such as Crohn’s disease and ulcerative colitis (i.e., inflammatory bowel disease (IBD)) are associated with, and possibly mediated by, increased levels of proinflammatory cytokines, many of which are NF-B regulated, in the intestinal mucosa (13). Activated NF-B is detectable in biopsies of IBD patients (14), and therapeutic agents that are effective in IBD are known to act, at least in part, through inhibition of NF-B activation. Because LXA₄ analogs attenuated NF-B-mediated gene expression in vitro, we next asked whether LXA₄ analogs might be therapeutic for intestinal inflammation in vivo. Due to the lack of established murine models of infectious gastroenteritis (mice get systemic illness rather than intestinal inflammation from S. typhimurium), we used a well-established chemically induced murine colitis model. Specifically, we examined whether an orally administered LXA₄ analog affected the colitis induced by DSS by measuring the clinical parameters that are the defined disease indicators in this widely used colitis model (Fig. 5). Colitis was induced in groups of five 8-wk-old mice via the addition of 4% DSS to their drinking water for 7 days. Simultaneous to the DSS administration, mice were also administered, via their drinking water, vehicle (0.05% ethanol) or 10 µg/ml 15-epi-16-para-fuoro-phenoxy-LXA₄, an analog of LXA₄ that has been shown to have local and systemic in vivo anti-inflammatory bioactivity (15). HPLC analysis of the LXA₄ analog recovered from the drinking water solutions demonstrated the structural integrity of the tetraene chromophore with only ester hydrolysis byproducts seen. Daily measurements of weight, occult blood, gross bleeding, and average (per group) water consumption were made. Because each mouse drank 1 ml per day, the approximate daily dose of ingested compound was 10 µg per mouse.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Oral administration of LXA4 analog attenuates DSS-induced colitis. BALB/c mice were given 4% DSS in their drinking water along with vehicle or 10 µg/ml 15-epi-16-parafluoro-phenoxy-LXA4. Body mass and bleeding (gross and occult, when stools could be obtained) were checked daily. A and B, Results (±SEM for body mass) of an experiment using six mice for each condition. The pattern is similar to two additional experiments using five mice per condition. C, Pooled results from the three replicated experiments. *, Statistically significant differences (p < 0.05).

	Discussion

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Chronic inflammatory diseases of the intestine (e.g., Crohn’s disease and ulcerative colitis, collectively referred to as IBD) are a serious public health problem, particularly in the developed world. While chronic inflammatory events play a definitive role in these diseases, clinically significant flares are often associated with augmentation of the innate immune response, specifically neutrophil flux across the epithelial surface. Although the underlying causes of this response are generally unknown, proinflammatory cytokines, including those of epithelial origin, are thought to be essential mediators of this process (16). As shown in this work, stable analogs of the endogenous anti-inflammatory eicosanoid LXA₄ can attenuate epithelial proinflammatory gene expression. LXA₄ analog attenuation of proinflammatory gene expression was mediated through a central proinflammatory signaling pathway and lessened the disease activity in a well-established mouse model of colitis, thus suggesting that these compounds could be therapeutic for the active phase of IBD.

Acute flares of IBD resemble infectious colitis (e.g., salmonellosis) both histologically (characterized by massive neutrophil influx and transepithelial migration) and clinically (i.e., diarrhea, cramping). The clinical manifestations of salmonellosis are thought to be largely attributable to the mucosal innate (neutrophil-mediated) immune response to this organism, leading to the suggestion that IBD flares may result from an aberrant mucosal innate immune response to normally nonpathogenic gut flora (perhaps the end result of signals originating from cells of specific immunity) (1). Thus, it is encouraging that LXA₄ analogs broadly attenuated the changes in epithelial gene expression induced by S. typhimurium. Interestingly, LXA₄ analogs did not uniformly attenuate all such changes in gene expression but rather diminished some nearly completely while other induced changes in gene expression were unaffected. Categorization of many of these genes as well as their possible roles in inflammation are not yet well defined. One specific class of genes uniformly down-regulated by LXA₄ analogs were the heat shock proteins (HSP); the up-regulation of the HSP genes induced by S. typhimurium was completely reversed in the presence of LXA₄ analogs. Such regulation of HSP genes may play a role in LXA₄ analog bioactivity or may simply reflect the general reduced stress level of LXA₄ analog-treated epithelial cells. Consistent with the latter possibility, another family of stress-induced genes, the NF-B-dependent genes, was also clearly down-regulated. As these genes are known to encode mediators of mucosal inflammation, this LXA₄ analog bioactivity likely plays a role in the observed therapeutic effects of this eicosanoid on DSS colitis in vivo.

Several nonsteroidal anti-inflammatory drugs (NSAID) including aspirin have also been shown to attenuate activation of the proinflammatory transcription factor NF-B (17). However, LXA₄ analog attenuation of NF-B differs from that of such agents in several important ways. LXA₄ analogs act via a specific receptor, whereas NSAID primarily directly inhibit proinflammatory enzymes. While both LXA₄ analogs and NSAID reduce IB degradation, LXA₄ analogs did so at nanomolar concentrations, while NSAID require much higher concentrations for this activity. In vitro studies indicate that the mechanism by which LXA₄ analog attenuated IB degradation also differs significantly from that of NSAID, in that LXA₄ analogs did not reduce the phosphorylation of IB, while NSAID, like other pharmacological attenuators of NF-B, reduce IB degradation by preventing this phosphorylation event (18). However, because LXA₄ analogs are structurally similar to 15-epi-LXA₄, which is biosynthesized by cyclooxygenase that has been acetylated by aspirin (3), some of aspirin’s attenuation of proinflammatory gene expression may yet result via this route. Mechanistically, LXA₄ analog attenuation of NF-B more closely resembles that of non-proinflammatory bacteria that also block IB degradation but not phosphorylation (19). Such phosphorylation-independent regulation of IB may thus be common to agonists that activate endogenous anti-inflammatory pathways and may hold more promise to have fewer unwanted effects than global inhibitors of IB kinase.

LXA₄ analogs have in vivo anti-inflammatory activity when applied both locally (topically in the mouse ear) and systemically via tail vein (15, 20). Oral administration is somewhat equivalent to topical application in the gut, as it provides direct delivery of LXA₄ analog to the intestinal epithelium. However, this LXA₄ analog is rapidly absorbed following oral gavage in rodents with 17% oral availability (B. Subramanyam, W. Guilford, J. Bauman, and J. Parkinson, unpublished observations) and thus may act systemically. While LXA₄ analog is short-lived in plasma (t_1/2 <30 min for i.v.; B. Subramanyam, W. Guilford, J. Bauman, and J. Parkinson, unpublished observations) placement in the drinking water provides semicontinuous systemic delivery. A particularly likely target are neutrophils, which are known to be targets of LXA₄ and major immune mediators of this colitis model. LXA₄ analogs attenuate neutrophil chemotaxis (21), oxidative burst (22), and the release of granule proteases (23). Delivery of LXA₄ analogs via tail vein showed a similar trend as oral administration on DSS colitis but did not differ statistically significantly from control (DSS-treated) mice (data not shown). This could have resulted from a less-targeted delivery of compound to the inflammatory site or may have occurred because delivery in the drinking water maintained a continued presence of the LXA₄ analog while the injected compound is rapidly cleared from the circulation (24).

While it is important to sort out the precise cellular mechanism of LXA₄ analog in vivo bioactivity, regardless, oral administration seems to be an effective way for this compound to down-regulate intestinal inflammation. In contrast, NSAID, including cyclooxygenase-2-specific inhibitors, tend to damage the intestinal mucosa, resulting in causation or exacerbation of intestinal inflammation, while mAb-based drugs (e.g., imflixamab), although therapeutic, must be given i.v. Thus, this novel strategy of activating endogenous anti-inflammatory pathways with stable analogs of LXA₄ could be developed into an effective means of treating human colitis.

	Acknowledgments

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants DK-02792 (to A.T.G.), AR-44268 (to I.R.W.), AI-49741 (to A.S.N.), DK-35932 (to J.L.M.), and DK-47662 (to J.L.M.).

² Address correspondence and reprint requests to Dr. Andrew T. Gewirtz, Department of Pathology and Laboratory Medicine, Emory University, WRB 10SH, 615 Michael Street, Atlanta, GA 30322. E-mail address: agewirt{at}emory.edu

³ Abbreviations used in this paper: LXA₄, lipoxin A₄, CAT, chloramphenicol acetyl transferase; DSS, dextran sodium sulfate; IBD, inflammatory bowel disease; HSP, heat shock protein; NSAID, nonsteroidal anti-inflammatory drug.

Received for publication December 6, 2001. Accepted for publication March 5, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. McCormick, B., A. Gewirtz, J. L. Madara. 1998. Epithelial crosstalk with bacteria and immune cells. Curr. Opin. Gastroenterol. 14:492.
2. Serhan, C. N., T. Takano, J. F. Maddox. 1999. Aspirin-triggered 15-epi-lipoxin A₄ and stable analogs on lipoxin A₄ are potent inhibitors of acute inflammation: receptors and pathways. Adv. Exp. Med. Biol. 447:133.[Medline]
3. Claria, J., C. N. Serhan. 1995. Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc. Natl. Acad. Sci. USA 92:9475.[Abstract/Free Full Text]
4. Serhan, C. N., J. F. Maddox, N. A. Petasis, I. Akritopoulou-Zanze, A. Papayianni, H. R. Brady, S. P. Colgan, J. L. Madara. 1995. Design of lipoxin A₄ stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 34:14609.[Medline]
5. Gewirtz, A. T., B. McCormick, A. S. Neish, N. A. Petasis, K. Gronert, C. N. Serhan, J. L. Madara. 1998. Pathogen-induced chemokine secretion from model intestinal epithelium is inhibited by lipoxin A₄ analogs. J. Clin. Invest. 101:1860.[Medline]
6. Gronert, K. G., A. T. Gewirtz, J. L. Madara, C. N. Serhan. 1998. Identification of a human enterocyte lipoxin A₄ receptor that is regulated by IL-13 and IFN- that inhibits TNF--induced IL-8 release. J. Exp. Med. 187:1285.[Abstract/Free Full Text]
7. Goh, J., A. W. Baird, C. O’Keane, R. W. Watson, D. Cottell, G. Bernasconi, N. A. Petasis, C. Godson, H. R. Brady, P. MacMathuna. 2001. Lipoxin A₄ and aspirin-triggered 15-epi-lipoxin A₄ antagonize TNF--stimulated neutrophil-enterocyte interactions in vitro and attenuate TNF--induced chemokine release and colonocyte apoptosis in human intestinal mucosa ex vivo. J. Immunol. 167:2772.[Abstract/Free Full Text]
8. Madara, J. L., S. P. Colgan, A. Nusrat, C. Delp, C. A. Parkos. 1992. A simple approach to measurement of electrical parameters of cultured epithelial monolayers: use in assessing neutrophil epithelial interactions. J. Tissue Cult. Methods 15:209.
9. Young, A. N., M. B. Amin, C. S. Moreno, S. D. Lim, C. Cohen, J. A. Petros, F. F. Marshall, A. S. Neish. 2001. Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers. Am. J. Pathol. 158:1639.[Abstract/Free Full Text]
10. Kunsch, C., R. K. Lang, C. A. Rosen, M. F. Shannon. 1994. Synergistic transcriptional activation of the IL-8 gene by NF-B p65 (RelA) and NF-IL-6. J. Immunol. 153:153.[Abstract]
11. Gewirtz, A. T., A. S. Rao, Jr P. O. Simon, D. Merlin, D. Carnes, J. L. Madara, A. S. Neish. 2000. Salmonella typhimurium induces epithelial IL-8 expression via Ca²⁺- mediated activation of the NF-B pathway. J. Clin. Invest. 105:79.[Medline]
12. Cooper, H. S., S. N. Murthy, R. S. Shah, D. J. Sedergran. 1993. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab. Invest. 69:238.[Medline]
13. Neurath, M. F., I. Fuss, G. Schurmann, S. Pettersson, K. Arnold, H. Muller-Lobeck, W. Strober, C. Herfarth, K. H. Buschenfelde. 1998. Cytokine gene transcription by NF-B family members in patients with inflammatory bowel disease. Ann. NY Acad. Sci. 859:149.[Medline]
14. Schreiber, S., S. Nikolaus, J. Hampe. 1998. Activation of nuclear factor B inflammatory bowel disease. Gut 42:477.[Abstract/Free Full Text]
15. Clish, C. B., K. Gronert, C. N. Serhan. 2000. Local and systemic delivery of an aspirin-triggered lipoxin stable analog inhibits neutrophil trafficking. Ann. NY Acad. Sci. 905:274.[Medline]
16. Papadakis, K. A., S. R. Targan. 2000. Role of cytokines in the pathogenesis of inflammatory bowel disease. Annu. Rev. Med. 51:289.[Medline]
17. Kopp, E., S. Ghosh. 1994. Inhibition of NF-B by sodium salicylate and aspirin. Science 265:956.[Abstract/Free Full Text]
18. Yin, M. J., Y. Yamamoto, R. B. Gaynor. 1998. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IB kinase-. Nature 396:77.[Medline]
19. Neish, A. S., A. T. Gewirtz, H. Zeng, A. N. Young, M. E. Hobert, V. Karmali, A. S. Rao, J. L. Madara. 2000. Prokaryotic regulation of epithelial responses by inhibition of IB- ubiquitination. Science 289:1560.[Abstract/Free Full Text]
20. Takano, T., S. Fiore, J. F. Maddox, H. R. Brady, N. A. Petasis, C. N. Serhan. 1997. Aspirin-triggered 15-epi-lipoxin A₄ and LXA₄ stable analogs are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors. J. Exp. Med. 185:1693.[Abstract/Free Full Text]
21. Lee, T. H., C. E. Horton, U. Kyan-Aung, D. Haskard, A. E. Crea, B. W. Spur. 1989. Lipoxin A₄ and lipoxin B₄ inhibit chemotactic responses of human neutrophils stimulated by leukotriene B₄ and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Clin. Sci. 77:195.[Medline]
22. Levy, B. D., V. V. Fokin, J. M. Clark, M. J. Wakelam, N. A. Petasis, C. N. Serhan. 1999. Polyisoprenyl phosphate (PIPP) signaling regulates phospholipase D activity: a "stop" signaling switch for aspirin-triggered lipoxin A₄. FASEB J. 13:903.[Abstract/Free Full Text]
23. Gewirtz, A. T., V. V. Fokin, N. A. Petasis, C. N. Serhan, J. L. Madara. 1999. LXA₄, aspirin-triggered 15-epi LXA₄, and their stable analogs selectively down-regulate PMN azurophilic degranulation. Am J. Physiol. 276:C988.[Abstract/Free Full Text]
24. Clish, C. B., J. A. O’Brien, K. Gronert, G. L. Stahl, N. A. Petasis, C. N. Serhan. 1999. Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitment in vivo. Proc. Natl. Acad. Sci. USA 96:8247.[Abstract/Free Full Text]

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