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Am J Physiol Gastrointest Liver Physiol 289: G1075-G1083, 2005. First published August 11, 2005; doi:10.1152/ajpgi.00073.2005
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MUCOSAL BIOLOGY

Topical antisense oligonucleotide therapy against LIX, an enterocyte-expressed CXC chemokine, reduces murine colitis

John H. Kwon,1 Andrew C. Keates,2 Pauline M. Anton,2 Maria Botero,3 Jeffrey D. Goldsmith,3 and Ciarán P. Kelly2

1Division of Gastroenterology, Johns Hopkins University, Baltimore, Maryland; and 2Division of Gastroenterology and 3Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Submitted 16 February 2005 ; accepted in final form 9 August 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.

chemokine; colitis; neutrophil; murine

INFLAMMATORY BOWEL DISEASE (IBD) is thought to arise from an aberrant regulation of the mucosal immune system, resulting in an inappropriate recruitment of inflammatory cells to the gastrointestinal tract. Histopathologically, both Crohn’s disease and ulcerative colitis have acute and chronic phases, which can be distinguished by identifying the specific leukocytes populating the inflamed bowel (34). The acute phase of IBD is characterized by the recruitment of neutrophils to the mucosa and submucosa, whereas the chronic phase is characterized by the recruitment of a variety of cell types, including neutrophils, T lymphocytes, macrophages, mast cells, and eosinophils. In general, this leukocyte recruitment involves a multistep process of endothelial cell activation, cell adhesion molecule expression, and chemokine or chemoattractant cytokine production. The dysregulation of any or all of these steps may contribute to the pathogenesis of IBD.

The role of chemokines in the pathogenesis of IBD has received increasing attention. In general, chemokines comprise a superfamily of 8- to 12-kDa inducible peptides that specialize in the recruitment of leukocytes to areas of injury or inflammation (29). Chemokines are divided into four subfamilies based on their number and arrangement of conserved NH2-terminal cysteine residues (C, CC, CXC, and CX3C). Each chemokine subfamily directs the recruitment and activation of specific leukocyte populations. For example, CC chemokines exert their effects on activated T lymphocytes, basophils, eosinophils, monocytes, and macrophages. The CXC chemokines, which contain a specific glutamic acid-leucine-arginine (ELR) motif, predominantly direct neutrophil functions.

In both the acute and chronic phase of IBD, the recruitment and activation of neutrophils to the mucosal layer of the inflamed bowel are likely influenced by alterations in ELR-CXC chemokine production. Human ELR-CXC chemokines include interleukin (IL)-8 and epithelial neutrophil-activating peptide (ENA)-78 (29). Both IL-8 and ENA-78 have been implicated in the pathogenesis of IBD (1, 17, 21, 36, 37). Increased IL-8 production has been noted in IBD tissues; however, the cell types responsible for the IL-8 production in the inflamed bowel have not been fully elucidated (12, 22, 28). Although IL-8 is predominantly expressed in macrophages and neutrophils, colonic epithelial cell localization has been noted only on occasion (1, 12). In vitro data have supported a potential role of epithelial cells in the production of IL-8 in IBD. Isolated colonic epithelial cells and colonic cell lines have been shown to respond to inflammatory cytokine stimuli, bacterial lipopolysaccharide (LPS), and enteropathogenic bacteria by increasing IL-8 mRNA and protein production (9–11, 14, 32).

Although the role of colonic epithelial cell IL-8 production in neutrophil chemotaxis is unresolved, there exists evidence of in vivo colonic epithelial cell ENA-78 production. In Crohn’s colitis, increased ENA-78 immunohistochemical staining has been demonstrated, and modest increases in mRNA and protein have been reported (17). In ulcerative colitis, however, marked increases in ENA-78 mRNA and protein have been identified (17). Immunohistochemically, ENA-78 has been shown to be localized to colonic epithelial cells in IBD tissues (17, 36). Isolated colonic epithelial cells and cell lines exhibit significantly increased ENA-78 production in response to IL-1{beta}, tumor necrosis factor (TNF)-{alpha}, and enteropathogenic bacteria (17, 36). The stimulation of colonic cell lines with inflammatory cytokines and enteropathogenic bacteria results in a marked and prolonged upregulation in ENA-78 mRNA and protein compared with only a transitory increase in IL-8 production (17). This transitory IL-8 production and prolonged ENA-78 production has also been demonstrated in human monocytes (31).

LPS-inducible CXC-chemokine (LIX) has been identified as the murine homolog of ENA-78 (33). Rovai et al. (30) identified LIX mRNA expression in the lungs of untreated mice and marked increases in LIX mRNA expression in the heart, lung, spleen, bowel, kidney, and muscle after an intravenous injection of LPS. The potential role for LIX in moderating acute inflammation has recently been noted by Chandrasekar et al. (5). In that study, administration of LIX neutralizing antibodies before the induction of myocardial ischemia and subsequent reperfusion resulted in a marked reduction in neutrophil infiltration. Neutralization of other murine CXC chemokines, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP2), resulted in only modest reductions in neutrophil infiltration. Although LIX appears to be functionally important for inflammatory responses during myocardial reperfusion after ischemia, the role of LIX in murine models of colitis is not known. The purpose of this study was to evaluate LIX expression in murine colonic epithelial cell lines and to examine the significance of LIX expression in the pathogenesis of murine colitis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Cell culture. MODE-K cells were maintained in DMEM supplemented with 10% FBS (Sigma, St. Louis, MO). Cells were incubated at 37°C in an atmosphere of 5% CO2 and 95% air. All cell culture experiments were carried out using confluent monolayers of MODE-K cells incubated with serum-free medium. Cytokine stimulation was carried out with 10 ng/ml IL-1{beta} (R&D Systems, Minneapolis, MN). Transcription inhibition was carried out with 10 µg/ml actinomycin D (Fisher Scientific, Fair Lawn, NJ). To assess the mRNA and protein expression of LIX in MODE-K cells, cells were seeded on 24-well tissue culture plates in DMEM with 10% FBS at a density of 2 x 105 cells. Before IL-1{beta} stimulation (1 day), cells were washed two times with PBS, and fresh serum-free medium was added. On day 0, cell monolayers were washed and refed with serum-free medium for 2 h in the presence or absence of 10 ng/ml IL-1{beta}. Two hours before each specified time point, the medium was replaced with new serum-free medium with or without IL-1{beta}. At each specified time point, the cells and media were harvested and assayed for LIX mRNA and protein expression.

LIX protein measurements by ELISA. A sandwich ELISA was developed to assess LIX protein expression. A Maxisorp 96-well microtiter plate (NUNC, Rochester, NY) was coated with 0.5 µg/ml goat antimouse LIX IgG antibody (R&D Systems) overnight at 4°C. Specificity screening by the manufacturer demonstrated <5% cross-reactivity with human granulocyte chemoattractant protein-2, rat cytokine-induced neutrophil chemoattractant-2{alpha}, and cytokine-induced neutrophil chemoattractant-2{beta}, as well as murine KC and MIP2 (27). After being washed with PBS containing 0.05% Tween 20 (PBS-T), wells were blocked with 5% BSA in PBS-T for 1 h at room temperature. After further washing, duplicate 50-µl aliquots of sample or recombinant LIX (R&D Systems) were added for 1 h at room temperature. To detect bound LIX, the goat anti-mouse LIX antibody was biotinylated using NHS-LC biotin (Pierce Biotechnology, Rockford, IL) and incubated at 1 µg/ml in blocking buffer for 1 h at room temperature. After additional washing, streptavidin-biotin-horseradish complex (Amersham Biosciences, Piscataway, NJ), diluted 1:5,000 in PBS-T, was added, and the plate was incubated at room temperature for 30 min. After being washed, 50 µl tetramethylbenzidine substrate solution (Kirkegaard and Perry Labs, Gaithersburg, MD) were added to each well, and, after color development, the reaction was stopped with 50 µl 1 M o-phosphoric acid. The optical density at 450 nm was read using an automated microtiter plate photometer (Dynatech, Chantilly, VA). The sample concentration of LIX was determined by comparison with a standard curve using recombinant LIX. Results are expressed as the amount of LIX protein levels (ng/ml) produced per hour in MODE-K at each specific time point.

Real-time quantitative RT-PCR. MODE-K total RNA was harvested using phenol-chloroform-isoamyl extraction as previously described (7). cDNA was generated using random hexamer primers and Moloney murine leukemia virus RT, as previously described (17). Real-time TaqMan quantitative RT-PCR using a fluorogenic 5'-nuclease PCR assay with a GeneAmp 5700 sequence detection system (ABI/Perkin-Elmer, Boston, MA) was used for assessment of LIX, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and KC mRNA (13). For each standard or sample, duplicate reactions containing 2.5 µl cDNA were incubated for 2 min at 50°C, denatured for 10 min at 95°C, and subjected to 40 cycles of annealing at 55°C for 20 s, extension at 60°C for 1 min, followed by denaturation at 95°C for 15 s. The gene-specific primers used were as follows: LIX sense primer, 5'-TTC-CAG-CTC-GCC-ATT-CAT-G-3'; LIX antisense primer, 5'-TGC-GGC-AGC-GTG-AAC-A-3'; GAPDH sense primer, 5'-TGT-GTC-CGT-CGT-GGA-TCT-GA-3'; GAPDH antisense primer, 5'-ACC-ACC-TTC-TTG-TGT-CAT-CAT-ACT-T-3'; KC sense primer, 5'-CAA-GAA-CAT-CCA-GAG-CTT-GAA-GTT-3'; and KC antisense primer 5'-GTG-GCT-ATG-ACT-TCG-GTT-TGG-3'. To detect amplicons generated using the gene-specific primers, dual-labeled fluorogenic (TaqMan) probes containing FAM (at the 5'-end) and TAMRA (at the 3'-end) were synthesized (Sigma-Genosys Fisher, The Woodlands, TX). The TaqMan probes used were as follows: LIX, 5'-CAG-AAATGC-CAG-CGG-CGC-CAT-3'; GAPDH, 5' TGC-CGC-CTG-GAG-AAA-CCT-GC-3'; and KC, 5'-TTG-CCC-TCA-GGG-CCC-CAC-TG-3'. A comparative threshold cycle method was used to compare each condition with the unstimulated control samples (35).

Dextran sodium sulfate model of colitis. Male BALB/c mice (4–6 wk old; Charles River Laboratories, Wilmington, MA) were used in this study. Experimental colitis was induced via the addition of 5% dextran sodium sulfate (DSS), molecular mass 36–50 kDa (ICN Biomedicals, Aurora, OH), to the drinking water for 7 days. Weight change was assessed though 7 days. On day 7, mice were killed, and colectomy was performed. Colon length was measured, and tissues were harvested for the assessment of colitis, LIX mRNA and protein levels, and myeloperoxidase (MPO) activity. To assess the protein expression of LIX in the colons of BALB/c mice, tissues were transected and snap-frozen in liquid nitrogen until further assessment. Tissues were thawed and homogenized in the presence of protease inhibitors (Roche Boehringer Mannheim, Indianapolis, IN) using a tissue homogenizer. LIX protein levels were assessed using an ELISA, as described above.

Assessment of colitis. The severity of colitis was assessed as previously described (20). Briefly, 6-µm sections of formalin-fixed tissues were embedded in paraffin and stained with hematoxylin and eosin. Sections from each mouse were coded with an accession number and assessed independently by two pathologists (Botero and Goldsmith) without access to the codes. Each section was assessed based on a point scale (0–11) according to severity of colitis (0–3: 0, normal; 1, mild; 2, moderate; and 3, severe), ulceration (0–1: 0, not present; 1, present), hyperplasia (0–3: 0, normal; 1, mild; 2, moderate; and 3, severe), and area involved (0–4: 0, normal; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%).

Administration of phosphorothioate oligonucleotides. The administration of antisense oligonucleotides was performed as previously described (24). Fluorescein-labeled 19-mer phosphorothioate sense and antisense oligonucleotides against LIX were synthesized (Sigma-Genosys Fisher). The LIX sense and antisense oligonucleotides were as follows: sense, 5' ACA-ATG-AGC-CTC-CAG-CTC-C 3'; and antisense, 5' GGA-GCT-GGA-GGC-TCA-TTG-T 3'. Briefly, 4- to 6-wk-old male BALB/c mice were fasted overnight and anesthetized with ketamine and xylazine. Mice were randomized into four groups. The mice in the first group received a 0.1 ml distilled water enema followed by tap water for 7 days. The mice in the second group received an intracolonic enema consisting of 20 nmol LIX antisense oligonucleotide diluted in 0.1 ml distilled water followed by 7 days of 5% DSS. The mice in the third group received an intracolonic enema consisting of 20 nmol LIX sense oligonucleotide diluted in 0.1 ml distilled water followed by 7 days of 5% DSS. The mice in the fourth group received a 0.1-ml distilled water enema followed by 7 days of 5% DSS. For each mouse, the intracolonic administration was performed by slow infusion through a polyethylene catheter inserted 3 cm in the anus. DSS treatment was begun immediately after recovery from anesthesia. Weight change was noted on days 1, 3, 5, and 7. Mice were killed under anesthesia on day 7. Colon length was measured, and tissues were processed for confocal microscopy, immunohistochemistry, MPO activity, and LIX protein levels by ELISA.

Confocal microscopy. Colon tissue from mice pretreated with fluorescein-labeled sense and antisense oligonucleotides followed by DSS were excised, mounted in optimum cutting temperature (OCT) medium, and snap-frozen in liquid nitrogen. Tissues were stored at –80°C until ready for processing. Colon tissue was sectioned into 6-µm sections and mounted on glass slides. A Bio-Rad MRC 1024 confocal microscope was used to localize fluorescein-labeled sense and antisense oligonucleotides within each section.

Immunohistochemistry. Colon tissue from mice was excised and mounted in OCT medium and snap-frozen in liquid nitrogen. Tissues were stored at –80°C until ready for processing. Colon tissue was sectioned into 6-µm sections. Sections were mounted on glass slides. Immunohistochemistry was performed using a 0.5-mg/ml goat antimouse LIX IgG polyclonal antibody (R&D Systems) diluted to 1:50 followed by a standard horseradish peroxidase reaction. A normal goat polyclonal IgG antibody (R&D Systems) was used as a negative control. Sections were counterstained using methyl green. Images were obtained using a Nikon microscope.

MPO assay. MPO activity was determined by a modified method of Bradley et al. (4). After homogenization, loop samples were frozen and thawed three times and then sonicated (Heat Systems; Ultrasonics, Plain View, NY) in 1.5 ml of 50 mM phosphate buffer containing 0.5% hexadecyl-trimethyl ammonium bromide. Samples were then centrifuged (10,000 g for 15 min at 4°C), and supernatants were further diluted in the same phosphate buffer containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. MPO activity was measured spectrophotometrically (Lambda 20, UV/VIS spectophotometer; Perkin-Elmer, Norwalk, CT) at 450 nm using human MPO (0.1 U/100 µl; Sigma) as a standard. Sample protein concentrations were determined by the Bio-Rad Detergent Compatible Protein Assay (Bio-Rad Inc, Hercules, CA), and MPO activity was expressed as MPO units per gram tissue.

Statistical methods. Experimental results are expressed as means ± SE. Statistical analysis was performed with unpaired, two-tailed Student’s t-tests and one-way ANOVA for comparing all pairs of groups (SPSS software, version 12.0). P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
LIX expression is inducible in a colonic enterocyte cell line. The human CXC chemokine, ENA-78, is expressed predominantly in epithelial cells, including colonic enterocytes and colonic cell lines (17, 36, 37). Its expression in colonic cell lines can be induced by IL-1{beta}, TNF-{alpha}, and enteropathogenic bacteria (17, 36). To determine whether murine colonic cell lines respond similarly to cytokine stimulation, LIX expression was examined in MODE-K cells.

In untreated MODE-K cells, LIX mRNA and protein were expressed at low levels (Fig. 1). When stimulated by IL-1{beta}, LIX mRNA and protein expression increased within the 1st h. Maximal concentrations of LIX mRNA and protein were observed after 4 h and remained elevated for 24 h. At 4 h, LIX mRNA was 73.8 times that of untreated cells, whereas LIX protein was 17.6 times that produced by untreated cells. Although there were no obvious differences noted in the number of cells per well at each time point or treatment condition, specific cell counts as well as proliferation and apoptosis assays were not conducted. Similar results were seen in other colon cell lines, including MCA-26 and MCA-38 cell lines (data not shown).



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  		Fig. 1. Lipopolysaccharide-induced CXC-chemokine (LIX) mRNA and protein expression is increased in MODE-K cells in response to interleukin (IL)-1{beta}. MODE-K cells were treated with 10 ng/ml IL-1{beta}, and LIX mRNA (A) and protein (B) were measured by real-time TaqMan RT-PCR and ELISA, respectively. Increased LIX mRNA and protein levels were detected at 1 h, maximal at 4 h, and remained elevated through 24 h. mRNA data are presented as a comparative threshold cycle method ({Delta}{Delta}CT) relative to the 0-h time point (n = 2 mice/condition). LIX protein levels are presented as ng/ml supernatant (n = 3/condition). *P < 0.05 vs. 0-h control. ^P < 0.005 vs. 0-h control.

 
Chemokine expression is predominantly regulated at the level of transcription (3, 23). To determine whether the IL-1{beta}-induced LIX expression required de novo transcription, MODE-K cells were pretreated with actinomycin D followed by a 4-h treatment with IL-1{beta} (Fig. 2). IL-1{beta}-induced LIX mRNA and protein expression was completely inhibited by pretreatment with actinomycin D, indicating the IL-1{beta}-stimulated LIX expression required new gene transcription.



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  		Fig. 2. Cytokine-stimulated LIX expression in MODE-K cells requires new transcription. MODE-K cells were pretreated with 10 µg/ml actinomycin D (ActD) followed by IL-1{beta} for 4 h. LIX mRNA (A) and protein (B) production was measured by real-time TaqMan RT-PCR and ELISA, respectively. ActD pretreatment completely inhibited the IL-1{beta}-induced stimulation of LIX mRNA and protein production. mRNA data are presented as {Delta}{Delta}CT relative to untreated cells (n = 3/condition). LIX protein levels are presented as ng/ml supernatant (n = 3/condition). *P < 0.001 vs. control (Con).

 
Although IL-1{beta}-stimulated LIX expression required de novo transcription, the time course of LIX mRNA expression in Fig. 1 indicated that mRNA stability may be contributing to the prolonged LIX expression. The relative quantities of LIX, GAPDH, and KC mRNA were assessed in MODE-K cells pretreated with IL-1{beta} followed by actinomycin D (Fig. 3). After 6 and 8 h of actinomycin D, 88.9 and 66.5% GAPDH mRNA was still present, respectively. Similarly, after 6 and 8 h of actinomycin D, 74.8 and 98.8% LIX mRNA was still present, respectively. In contrast, KC levels decreased to 11.9 and 6.5% of their original levels after 6 and 8 h of actinomycin D exposure, respectively. Therefore, LIX mRNA was highly stable in colonic cell lines compared with KC.



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  		Fig. 3. LIX mRNA is stable compared with keratinocyte-derived chemokine (KC). MODE-K cells were pretreated with IL-1{beta} for 4 h and then treated with ActD for 2, 4, 6, and 8 h. Total RNA was isolated, and real-time TaqMan RT-PCR was used to assess mRNA levels for LIX (A), KC (B), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; C). LIX and GAPDH mRNA levels remain stable through 8 h, whereas KC mRNA levels rapidly decrease after IL-1{beta} treatment. mRNA data are presented as {Delta}{Delta}CT relative to untreated cells (n = 3/condition). *P < 0.05 vs. control. ^P < 0.001 vs. control.

 
LIX expression in murine colon and in experimental colitis. Although LIX mRNA and protein were clearly expressed in MODE-K cells and previous studies identified LIX mRNA expression in colonic tissue of mice injected with LPS (30), LIX localization in colonic tissues has not been reported previously. To determine the sites of LIX protein expression, immunohistochemistry for LIX was performed on colonic tissues from untreated mice and mice treated with 5% DSS for 7 days (Fig. 4).



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  		Fig. 4. LIX protein is predominantly expressed in the epithelial cell layer in control and dextran sodium sulfate (DSS)-treated mice. Colon sections from control (A–C) and DSS-treated (D–F) mice were assessed after 7 days. Immunohistochemical staining for LIX is predominantly localized to colonic epithelial cells and some individual cells in the lamina propria both in control and in DSS-treated mice (A and D). Minimal staining of enterocytes was observed in the colons of control and DSS-treated mice using a normal goat polyclonal IgG antibody (B and E) or with the omission of the primary antibody (C and F). Magnification = x100; scale bars = 50 µM.

 
LIX protein was identified in both untreated mice and after a 7-day treatment with DSS (Fig. 4, A and D). LIX immunostaining was predominantly noted in the colonic enterocytes with some additional staining in individual cells in the muscle layer and lamina propria. Surface enterocytes and crypt epithelial cells appeared to express LIX. Sections stained after exposure to a normal goat polyclonal IgG (Fig. 4, B and E) or when the primary antibody had been excluded (Fig. 4, C and F) showed minimal immunohistochemical staining.

Although both the untreated and treated mice showed LIX protein expression by immunohistochemistry, it was difficult to assess whether there was a significant difference between the two groups. To determine whether LIX expression was altered in the presence of colonic inflammation, mice were assessed for severity of colitis and for LIX protein expression (Fig. 5). Colitis was assessed using a previously published scoring system based on area involved, severity of colitis, hyperplasia, and ulcerations (20). Consistent with previous studies, DSS induced a 2.9-fold increase in mean colitis score (Fig. 5A). A 2.0-fold increase in colonic LIX protein expression was seen in DSS-treated mice (Fig. 5B).



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  		Fig. 5. DSS treatment significantly increases the colitis score and colonic LIX protein expression in BALB/c mice. A: colon sections were harvested from untreated mice and mice treated with DSS for 7 days. The colitis score ± SE was assessed in colon sections (n = 8 mice per condition). B: LIX protein expression was assessed via ELISA in colon sections (n = 6 mice/condition). *P < 0.05 vs. control. ^P < 0.001 vs. control.

 
LIX antisense oligonucleotide administration inhibits experimental colitis. Although LIX expression in DSS-treated mice paralleled the level of colitis, the role of LIX expression in the pathogenesis of DSS-induced colitis was still uncertain. To determine whether LIX expression was necessary for DSS-induced colitis, fluorescein-labeled antisense and sense oligonucleotides to LIX were delivered via rectal enemas. A single treatment of sense or antisense oligonucleotides was given to fasted mice just before initiating DSS treatment. Mice were then assessed for oligonucleotide localization, LIX expression, neutrophil activity, and severity of colitis.

With the use of confocal microscopy, antisense oligonucleotides to LIX were visualized in the surface epithelium and surface mucus layer after 3 h (Fig. 6B). A similar distribution of LIX sense oligonucleotide was seen after 3 h (data not shown). After 7 days, both the sense and antisense oligonucleotides were visualized in the surface enterocytes (Fig. 6, C–H). At high power, LIX sense and antisense oligonucleotides could be clearly visualized in individual surface enterocytes. Interestingly, crypt epithelial cells and lamina propria cells exhibited very little oligonucleotide localization. Also, even after 7 days, both the sense and the antisense oligonucleotides were still visible in the surface epithelium (Fig. 6, C–H).



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  		Fig. 6. LIX antisense (AS) and sense (S) oligonucleotides are localized to surface colonic enterocytes after intracolonic administration. Fluorescein-labeled AS and S oligonucleotides to LIX were delivered via enemas on the day of DSS treatment initiation. Colons were excised after 3 h and 7 days. Confocal fluorescence microscopy was performed for oligonucleotide localization. A: control (tap-water enema) colonic tissue demonstrated minimal autofluorescence. B: 3 h after AS oligonucleotide delivery, marked fluorescein labeling was noted within the lumen and epithelial cell layer. After 7 days, fluorescein-labeled S oligonucleotide was localized to the epithelial cell layer of control (C) and DSS-treated (E) mice. Less fluorescein-labeled AS oligonucleotide was noted in the epithelial cell layer after 7 days in the control (D) and DSS-treated (F) mice. At high magnification, S (G) and AS (H) oligonucleotide labeling is clearly noted in the colonic enterocytes of DSS-treated mice. A–F: magnification = x200. G and H: magnification = x500.

 
LIX expression was assessed in mice treated with LIX sense and antisense oligonucleotides followed by DSS for 7 days (Fig. 7A). A 58.5% reduction in LIX expression was seen in mice given the LIX antisense oligonucleotide enema followed by DSS compared with mice given a tap water enema followed by DSS. LIX antisense oligonucleotide delivery before DSS treatment resulted in a LIX expression level similar to the control mice that did not receive DSS. There was no significant difference in LIX expression between mice given tap water enemas followed by DSS and mice given the LIX sense oligonucleotide followed by DSS. Of note, pretreatment with either sense or antisense LIX oligonucleotides did not significantly reduce KC chemokine expression in DSS-treated mice (data not shown).



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  		Fig. 7. LIX AS oligonucleotide delivery before DSS treatment results in reduced colonic LIX protein expression and colonic MPO activity in BALB/c mice. Mice were pretreated with LIX S or AS oligonucleotide followed by DSS for 7 days. A: colon sections were assessed for LIX protein expression by ELISA and expressed as pg/mg total protein (n = 6 mice/condition). B: colon sections were assessed for MPO activity and expressed as U/mg tissue (n = 5 mice/condition). NS, not significant.

 
To determine whether the reduction in LIX expression in mice given the LIX antisense oligonucleotide enema resulted in reduced neutrophil activation and recruitment, MPO assays were performed (Fig. 7B). Mice given the LIX antisense oligonucleotide followed by DSS displayed a 63.5% reduction in colonic MPO activity compared with the mice given tap water enemas followed by DSS. In contrast, there was no difference in MPO activity in colonic tissues of mice given the LIX sense oligonucleotide followed by DSS compared with mice given tap water enemas followed by DSS. These findings indicate that LIX antisense oligonucleotide treatment, but not LIX sense oligonucleotide treatment, reduced both LIX protein expression and subsequent neutrophil infiltration in DSS-treated mice.

To determine whether this reduction in LIX protein expression and MPO activity resulted in a corresponding reduction in colitis, several parameters were assessed at 7 days after DSS treatment. Mice were assessed for weight loss, colon length, and colitis score. There was no significant difference in weight loss in mice given LIX sense oligonucleotides, antisense oligonucleotides, or tap water enemas followed by DSS for 7 days. The mice given tap water enemas exhibited a 14.8% weight loss over the 7 days of DSS exposure, whereas the mice given the LIX sense and antisense oligonucleotides exhibited an 11.0 and 10.6% weight loss over the same time period, respectively (data not shown).

Although weight loss was not affected by LIX antisense oligonucleotide treatment, there was a significant reduction in colon shortening in mice given LIX antisense oligonucleotides (Fig. 8A). Mice pretreated with tap water enemas demonstrated a 42% reduction in colon length after 7 days of DSS exposure. Mice given LIX antisense oligonucleotides followed by DSS demonstrated only a 28% reduction in colon length. Mice given the LIX sense oligonucleotide followed by DSS demonstrated a 38% reduction in colon length, which was not significantly different from the tap water enema group.



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  		Fig. 8. LIX AS oligonucleotide treatment before DSS results in decreased colon shortening and improved colitis scores in BALB/c mice. Mice were treated with LIX S or AS oligonucleotide enemas followed by DSS for 7 days. Colons were excised, and tissues were assessed for colon length (A) and colitis score (B). AS but not S oligonucleotide resulted in a significantly decreased DSS-induced colon shortening (n = 10 mice/condition). AS but not S oligonucleotide delivery resulted in a significantly decreased DSS-induced colitis score (n = 10 mice/condition).

 
The mean colitis score was also significantly reduced by the LIX antisense oligonucleotide treatment (Fig. 8B). Pretreatment with LIX antisense oligonucleotides followed by DSS resulted in a 53.2% reduction in the mean colitis score, whereas pretreatment with the LIX sense oligonucleotide resulted in a 26.3% reduction in the mean colitis score. The mean colitis score of mice given the LIX sense oligonucleotide enema was not significantly different from the mice given tap water enemas. Thus the LIX antisense oligonucleotide, but not the LIX sense oligonucleotide, resulted in reduced LIX expression and neutrophil infiltration that was also associated with an overall reduction in the severity of DSS colitis.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrate that the neutrophil-directed CXC chemokine LIX is expressed in colonic enterocytes and in colonic epithelial cell lines. LIX expression in colonic epithelial cells is upregulated by the proinflammatory cytokine IL-1{beta}. Furthermore, pretreatment with an antisense oligonucleotide to LIX reduces DSS-induced murine colitis. These findings support and advance previous studies demonstrating the efficacy of topical delivery of antisense oligonucleotides to inhibit murine colitis. Specifically, previous studies demonstrated that the rectal delivery of antisense oligonucleotides to the p65 subunit of nuclear factor (NF)-{kappa}B was effective in inhibiting colitis induced by DSS and trinitrobenzenesulfonic acid (19, 24, 26).

The NF-kB family comprises several transcription factors known to mediate the activation of numerous cell adhesion molecules and inflammatory cytokines (2, 15, 16, 18, 23–25). For example, Murano et al. (24) demonstrated that IL-1{beta}, TNF-{alpha}, and IL-6 levels were all reduced in mice pretreated with the NF-{kappa}B antisense oligonucleotide before the induction of colitis. Neurath et al. (26) demonstrated a similar reduction in IL-1{beta}, TNF-{alpha}, and IL-6 levels in lamina propria macrophages isolated from mice pretreated with the NF-{kappa}B antisense oligonucleotide before the induction of colitis. Our laboratory and others have previously demonstrated that NF-{kappa}B is a key transcriptional regulator of ENA-78 expression (6, 8, 16). An analysis of the LIX 5'-untranslated region indicates an NF-{kappa}B binding site 173 nucleotides upstream from the start codon (data not shown). Regardless of the mechanism by which the LIX antisense oligonucleotide inhibits murine colitis, this study demonstrates that the inhibition of neutrophil chemokine activity with the LIX antisense oligonucleotide parallels the results observed using the general inflammatory cytokine transcriptional regulator.

In our study, the LIX sense oligonucleotide did not result in significant reductions in colon shortening, colitis score, MPO activity, and LIX expression levels. In contrast, the LIX antisense oligonucleotide therapy did have significant effects in each of these parameters. Although the LIX antisense oligonucleotide therapy was quite effective, it did not completely prevent DSS-induced colonic injury and inflammation. Further studies would be necessary to determine whether increasing the frequency or concentration of antisense oligonucleotide delivery could lead to greater reductions in LIX expression and a greater inhibition of DSS-induced colitis. The ability of LIX antisense oligonucleotides to reduce colitis should also be validated in other animal models of colitis.

The findings of this study clearly demonstrate that targeting a specific enterocyte-expressed CXC chemokine can significantly reduce neutrophil infiltration and colonic injury in an in vivo model of acute colitis. In humans, both infectious diarrheal illnesses and acute, active IBD have been shown to involve neutrophil recruitment to areas of inflammation. The reduction of murine colitis by downregulating LIX chemokine expression via topical antisense oligonucleotide delivery provides insight into the potential therapeutic efficacy of an ENA-78 antisense oligonucleotide in human intestinal inflammatory processes characterized by neutrophil recruitment. Specific targeting of colonic enterocyte neutrophil chemokine expression with topical antisense oligonucleotide therapy may prove to be a feasible and efficacious treatment option.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-58858 (to C. P. Kelly) and T32 DK-007760 (to J. H. Kwon).


   ACKNOWLEDGMENTS
 
We thank Dan Brown for technical assistance and Dr. Peter Ernst (University of Texas, Galveston) for the kind donation of MODE-K cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. P Kelly, Div. of Gastroenterology, Dana 501, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (e-mail: ckelly2{at}bidmc.harvard.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
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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