Lateral membrane LXA4 receptors mediate LXA4's anti-inflammatory actions on intestinal epithelium

Torsten Kucharzik, Andrew T. Gewirtz, Didier Merlin, James L. Madara, and Ifor R. Williams

Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lipoxin A4 (LXA4) and its stable analogs downregulate chemokine secretion in polarized epithelia. This anti-inflammatory effect has been suggested to be mediated by the LXA4 receptor (LXA4R), a G protein-coupled receptor. To determine whether LXA4R is expressed on the apical, basolateral, or both poles of intestinal epithelia, an NH2-terminal c-myc epitope tag was added to the human LXA4R cDNA and recombinant retroviruses were used to transduce polarized epithelial cells. In polarized T84 intestinal epithelial cells, c-myc-LXA4R was preferentially expressed on the basolateral surface as indicated by cell surface-selective biotinylation and confocal microscopy. Furthermore, expression of c-myc-LXA4R and a truncation mutant lacking the cytoplasmic terminus was primarily confined to the lateral subdomain. We also observed that the expression of myc-LXA4 conferred enhanced downregulation of IL-8 expression in response to LXA4 analog and that blockade of the CysLT1 receptor by montelukast did not prevent this response to LXA4 analog. Thus LXA4 generated in or near the paracellular space via neutrophil-epithelial interactions can rapidly act on epithelial LXA4R to downregulate epithelial promotion of intestinal inflammation.

G protein-coupled receptor; polarized epithelium; eicosanoid; epitope tag


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EPITHELIAL LINING of the gastrointestinal tract forms a vital protective barrier that separates luminal antigens and toxins from the underlying tissue compartments. Intestinal epithelial cells are increasingly recognized to play an important role in host defense against microorganisms in the intestinal lumen and in inflammatory responses. They contribute to regulation of immune responses by releasing a variety of proinflammatory cytokines and chemokines. For example, intestinal epithelial cells release IL-8 after stimulation with TNF-alpha or a variety of pathogens, thereby facilitating neutrophil migration into the intestinal epithelium (6, 7, 21). Homeostatic regulation of epithelial inflammation in the intestine involves the concerted action of both proinflammatory and counterregulatory pathways. One of the active counterregulatory pathways involves the action of eicosanoid mediators, including lipoxin A4 (LXA4) and 15-epimeric aspirin-triggered lipoxins (4, 16). These mediators are generated at sites of inflammation and exert anti-inflammatory effects on both neutrophils (9) and intestinal epithelial cells, including the downregulation of epithelial secretion of chemokines that direct neutrophil movement (10).

Intestinal epithelial cells express at least two G protein-coupled receptors for which LXA4 and its analogs have been demonstrated to have biological activity (14, 15). One of these receptors is the same LXA4 receptor (LXA4R) initially cloned from human and mouse myeloid cells on the basis of its high affinity for LXA4 (8, 31). This receptor also binds the 15-epi-lipoxins, which are synthesized by acetylated COX-2 in aspirin-treated cells (4). An alternate name used for this receptor is N-formyl peptide receptor-like 1 (FPRL1), because it also binds N-formylmethionyl-leucyl-phenylalanine (fMLP) as well as several other peptide ligands with lower affinities (3). The second known epithelial cell receptor for LXA4 is the CysLT1 receptor for leukotriene D4 (15, 22). LXA4 and its analogs act as partial agonists and/or antagonists at this receptor and block the function of cysteinyl leukotriene agonists. The CysLT1 receptor is also expressed by intestinal epithelial cells (15), suggesting the possibility that the anti-inflammatory effects of LXA4 could be mediated through this molecule rather than through LXA4R.

Intestinal epithelial cells are polarized cells expressing a distinct set of membrane proteins on their apical and basolateral plasma membranes. The polarized expression of a variety of surface receptors is necessary for epithelial cells to maintain normal structure and function (2, 24). It is not currently known whether receptors for LXA4 are polarized in their pattern of expression on intestinal epithelial cells. Further understanding of the mechanism of LXA4's anti-inflammatory action in polarized epithelia depends, in part, on identifying the subcellular location at which these receptors normally encounter their ligands. However, such studies have been stymied by the fact that the LXA4R, like other G protein-coupled receptors, are difficult to raise antibodies to and further that highly polarized intestinal epithelial cell lines are difficult to transfect. In this study, we overcame these technical problems by using retroviral transduction to express an epitope-tagged version of the LXA4R in several polarized epithelial cell lines. Our results indicate that LXA4R is preferentially expressed on the basolateral surface and further localized to the lateral membrane domain of such polarized epithelia. We also find that stable overexpression of the LXA4 receptor in intestinal epithelial cells is associated with enhanced LXA4-mediated inhibition of TNF-alpha -induced IL-8 secretion by epithelial cells, thus showing that the counterregulatory effects of LXA4 and its analogs on intestinal epithelial cells are at least in part mediated through the basolateral LXA4R.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. The stable LXA4 analog 15-R/S-methyl-LXA4-methyl ester was kindly provided by Nikos Petassis (University of Southern California, Los Angeles, CA). It was prepared by total organic synthesis, and its structure was confirmed by HPLC, NMR, and mass spectral analysis (29). Daily working stocks of 15-R/S-methyl-LXA4 (100 µm) were verified by UV spectroscopy using a molar extinction coefficient of 50,000 cm-1 · M-1 at 302 nm. These solutions were stored at -70°C in 99% ethanol. Flagellin was purified from Salmonella typhimurium-conditioned medium as described previously (12). Recombinant human TNF-alpha was purchased from R&D Systems (Minneapolis, MN). A stock solution of montelukast (Merck) was prepared by dissolving tablets containing 10.4 mg of the sodium salt in water.

Epithelial cell lines and culture. Human colonic adenocarcinoma cell lines were grown and passaged with culture conditions previously described for T84 (5) and HT29 clone 19A (HT29cl.19A; Ref. 19) in an atmosphere of 5% CO2. In brief, T84 cells were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium supplemented with 14 mM NaHCO3, 40 mg/l penicillin, 9 mg/l streptomycin, 8 mg/l ampicillin, 5% newborn calf serum, and 15 nM Na+ HEPES buffer, pH 7.5. HT29cl.19A cells were cultured in DMEM containing a standard glucose concentration (4.5 g/l) and supplemented with 14 mM NaHCO3, 40 mg/l penicillin, 9 mg/l streptomycin, 8 mg/l ampicillin, 10% fetal bovine serum, and 15 mM HEPES buffer, pH 7.5. Polarized colonic epithelial cells were split near confluence by incubating cells with 0.1% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free PBS for 5-20 min. HT29cl.19A cells that were used for flow cytometry were harvested with 15 mM EDTA to avoid trypsin cleavage of cell surface receptors. Cells were grown on 0.33-mm2 collagen-coated semipermeable supports (0.4 µm) and maintained for 7-10 days until complete confluence and a steady-state transepithelial resistance were achieved.

Retroviral transduction of epithelial cells with myc-tagged LXA4R. A c-myc epitope tag was added to the NH2-terminal end of the human LXA4R with the pCMV-Myc mammalian expression vector (Clontech). A plasmid clone of the human LXA4R cDNA was amplified by PCR with the primers 5'-AGG CCA TGG AGG CCA TGG AAA CCA ACT TCT CC-3' (sense) and 5'-TCA CAT TGC CTG TAA CTC-3' (antisense). The purified PCR product was first cloned into PCR-Script cloning vector (Stratagene) and then excised with SfiI and NotI for cloning between the SfiI and NotI sites of pCMV-Myc to make pCMV-Myc-LXA4R. For retroviral expression of the same myc-tagged LXA4R construct, the open reading frame from pCMV-Myc-LXA4R was amplified with the primers 5'-AAA GCT TAG ATC TCC ACC ATG GCA TCA ATG C-3' (sense) and 5'-TCA CAT TGC CTG TAA CTC-3' (antisense) and initially cloned into PCR-Script. This plasmid was cut with BglII and NotI, and the insert was subcloned into the BglII and NotI sites of the pLPCX retroviral expression vector (Clontech). The resulting pLPCX-myc-LXA4R construct was transiently transfected into the packaging cell line 293-10A1 (Imgenex, San Diego) to make recombinant retrovirus. A control construct consisting of the coding region of enhanced green fluorescent protein (EGFP) cloned into pLPCX was used to assess the efficiency of cell transduction by the recombinant retroviruses. Transfections were done by using a CaPO4-mediated transfection procedure. Briefly, DNA was mixed with 0.25 M CaCl2 in a tube and 2× N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)-buffered saline was added a small amount at a time with mixing. After the CaCl2-DNA-BES-buffered saline solution was added dropwise, the cells were incubated for 16 h before the medium was changed. After 2 days, the supernatant of the packaging cell line was used to transduce intestinal epithelial cell lines. Briefly, target cells were split 24 h before they were used for transfection at a confluence of ~50%. For infection, fresh retroviral supernatant was added to the target cells on a six-well plate for 15 min at 32°C in the presence of 8 µg/ml Polybrene (Sigma). Cells were then spun at 2,500 rpm for 30 min at 32°C. After the spin the retroviral supernatant was replaced with fresh growth medium. Infection cycles were repeated two more times. Transduction efficiency was ~50% in HT29cl.19A cells and ~15% in T84 cells as detected by flow cytometry with cells transduced with EGFP-encoding retroviruses. Stably transfected HT29cl.19A cells were derived by puromycin selection.

Flow cytometric analysis of transduced intestinal epithelial cells. The level of myc-tagged LXA4R expression by individual HT29cl.19A clones was assessed by flow cytometry with the 9E10 anti-myc MAb (Clontech) or a mouse IgG1 isotype control (Caltag, Burlingame, CA) as the primary antibody. Primary antibody binding was detected with a biotinylated polyclonal goat-anti-mouse IgG secondary antibody (PharMingen, San Diego, CA) followed by phycoerythrin-labeled streptavidin (Immunotech, Portland, ME).

Expression of a truncated LXA4R mutant in intestinal epithelial cells. A mutant myc-LXA4R construct was engineered in which the CAA codon encoding the glutamine present immediately following the seventh transmembrane segment (31) was converted into a premature termination codon (TAA). This point mutation (abbreviated as Q307X; amino acid designation based on the wild-type LXA4R sequence) results in a truncated LXA4R protein lacking the final 45 amino acids of the protein that make up the COOH-terminal cytoplasmic tail. The myc-LXA4R-Q307X construct was subcloned into pLPCX for preparation of recombinant retrovirus with the same approach as for the wild-type myc-LXA4R construct.

Intracellular Ca2+ measurement in fMLP-stimulated cells. Intracellular Ca2+ recordings in monolayers of HT29cl.19A cells were performed as described previously (11) with minor modifications. Briefly, HT29cl.19A cells were plated on 0.4-µm polyester filters coated with collagen that were mounted over the window of a polycarbonate holder that fit diagonally into a standard fluorescence cuvette. After 7 days of cell growth, the filters were washed, loaded with fura 2-acetoxymethyl ester (Molecular Probes), and placed into a Hitachi (Sunnyvale, CA) F-4500 spectrofluorometer thermostated to 37°C. The bottom and side edges of the polycarbonate holder were not coated with vacuum grease for these experiments, so diffusion of added compounds between the apical and basolateral sides was possible. Fluorescence emission was read at 505 nm, whereas the excitation wavelength was changed between 340 and 380 nm four times per second with Intracellular Cation software (Hitachi). After baseline fluorescence was read for 3-5 min, fMLP (10-4 M) was added to the basolateral side of the cuvette. Carbachol (100 µm) was subsequently added to the basolateral side of the cuvette to verify that the cells could respond to a strong agonist with an increase in Ca2+.

Immunofluorescence microscopy. Matrix-coated 0.4-µm filters on which retrovirally transduced T84 cells were cultured were cut out and washed three times with HBSS. Fixation was done with 3% paraformaldehyde [10 min at room temperature (RT)] followed by permeabilization with 1% Triton X-100 for 20 min at RT. After being washed in HBSS, filters were then blocked for 1 h with 5% goat serum in a moist chamber. After being washed three times with HBSS, filters were incubated for 1 h with c-myc MAb (2 µg/ml). Sections were washed three times with HBSS again and incubated for 1 h with FITC-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch). After another wash, the sections were incubated with 5 U/ml rhodamine-phalloidin (Molecular Probes) and washed again. Filters were then mounted on glass slides with p-phenylenediamine. Mounted sections were viewed with a scanning laser confocal microscope (Zeiss, Jena, Germany).

Immunoblotting of c-myc-tagged LXA4R in intestinal epithelial cells. Expression of the c-myc-tagged LXA4R by transfected and transduced cells was detected by immunoblotting. Tissue samples were lysed in 500 µl of HBSS containing 1% Triton X-100 (Sigma) and a proteinase inhibitor cocktail. Protein concentration in the lysate was determined by using the Pierce bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). After sample buffer was added, 10 µg of total protein of each sample was separated under reducing conditions on a 12% SDS-polyacrylamide gel. Protein transfer to Hybond-C nitrocellulose membranes (Amersham Pharmacia) was performed at 200 mA for 90 min or overnight with a Trans-Blot SemiDry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Richmond, CA). Membranes were blocked with 5% fat-free milk powder in TTBS buffer (0.01% Tween 20, 0.05 M Tris · HCl, 0.15 M NaCl, pH 7.5) for 60 min at RT and then incubated for 1 h with c-myc MAb (1:100 dilution; Clontech). Membranes were washed three times with TTBS and incubated with a peroxidase-conjugated goat anti-mouse IgG (1:5,000 dilution; Jackson ImmunoResearch) for 1 h. After extensive washing with TTBS and Tris-buffered saline (TBS), the reaction was developed by enhanced chemiluminescent staining (Amersham).

Apical and basolateral cell surface biotinylation. Biotinylation of the apical or basolateral surfaces of filter-grown T84 cells (4 filters for each condition) was done as described previously (23). Briefly, the apical or basolateral sides of the monolayers were incubated for 30 min at RT with a 1 mg/ml solution of freshly prepared sulfo-NHS-biotin (Pierce Chemical) diluted in PBS with 1 mM CaCl2 and 1 mM MgCl2. The reaction was quenched with 50 mM NH4Cl, and cells were lysed with a solution of 1% (wt/vol) Triton X-100 in 20 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA, and 0.2% (wt/vol) bovine serum albumin supplemented with protease inhibitors. The protein solution was diluted with 1 ml of lysis buffer and then incubated with streptavidin-agarose (Pierce) for 24 h at 4°C to bind biotinylated proteins. The bound proteins were separated by SDS-PAGE and blotted to nitrocellulose membranes. The blots were sequentially incubated with anti-c-myc MAb (1:100 dilution) or 1 µg/ml of anti-beta 1-integrin MAb (clone P4C10; Life Technologies) followed by peroxidase-conjugated goat anti-mouse IgG (1:10,000 dilution; Jackson ImmunoResearch) and developed with the ECL chemiluminescence system (Amersham).

LXA4 effects on spontaneous, TNF-alpha induced, and flagellin-induced IL-8 secretion. Confluent monolayers of HT29cl.19A cells were grown on 0.33-cm2 collagen-coated permeable supports, washed three times with HBSS, and placed into 500 µl of serum-free medium that contained 100 nM 15-R/S-methyl-LXA4 or vehicle (0.1% ethanol). Sixty minutes later, various concentrations of TNF-alpha (0.01-1 µg/ml) or Salmonella flagellin (5-20 ng/ml) were added to the basolateral compartment. After 5 h HT29cl.19A supernatants were removed and assayed for IL-8. IL-8 was measured by ELISA as previously described (21) with minor changes. The 96-well plates (Linbro/Titertek; ICN Biomedicals, Costa Mesa, CA) were coated overnight with goat anti-human IL-8 (R&D Systems), and the detecting antibody used was rabbit anti-human IL-8 (Endogen, Woburn, MA).

Statistical analysis. Unless otherwise indicated, results are represented as means ± SD. Results were analyzed with Student's t-test. Differences were considered significant if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epithelial surface expression of functional epitope-tagged LXA4R by retroviral transduction. An epitope-tagged version of the human LXA4R was constructed by subcloning the wild-type human LXA4R cDNA sequence into a mammalian expression vector (pCMV-Myc) that added the 13-amino acid c-myc epitope tag to the NH2-terminal end of the LXA4R protein. To achieve increased efficiency of transduction in human intestinal epithelial cell lines (HT29cl.19A and T84), the c-myc-tagged construct was subcloned into the pLPCX retroviral expression and transfectants were selected in the presence of puromycin. We verified by immunoblotting that stable transfectants of HT29cl.19A indeed expressed c-myc-LXA4R (Fig. 1A) and that this protein was the expected size. Furthermore, flow cytometric analysis of these transfectants indicated that this epitope-tagged LXA4R was expressed on the cell surface (Fig. 1B). We next sought to examine whether this epitope-tagged receptor expressed in this manner retained previously demonstrated signaling ability. One known signal transduced by LXA4R is elevation of intracellular Ca2+ in response to high concentrations of fMLP (30). Thus we compared cytosolic Ca2+ concentration ([Ca2+]) of wild-type HT29cl.19A cells and myc-LXA4R-transfected cells after stimulation with 10-4 M fMLP. HT29cl.19A transfectants expressing the myc-LXA4R demonstrated a gradual increase in intracellular [Ca2+] after fMLP stimulation that was not detected in nontransfected HT29cl.19A cells (Fig. 2). This result confirms that the myc-tagged LXA4R retains the capacity to signal through G proteins and indicates that transfected cells likely express more total (i.e., endogenous plus transfected) functional LXA4R than their parental HT29cl.19A cells.


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Fig. 1.   HT29cl.19A cells express a myc-tagged lipoxin A4 receptor (LXA4R) after retroviral transduction. HT29cl.19A cells were transduced with myc-LXA4R/pLPCX, and stable clones were derived by puromycin selection. The size of the myc-expressing LXA4R in these cells was determined by immunoblotting (A). myc-LXA4R-transfected cells demonstrated a single band at ~40 kDa, whereas there was no signal in nontransfected cells. Cell surface expression of c-myc was detected by flow cytometry with anti-c-myc followed by biotin-conjugated goat anti-mouse IgG and phycoerythrin-conjugated streptavidin (B). The results for nontransfected cells and a representative clone of the transfectants are shown as overlays of histograms obtained with the anti-myc primary antibody (solid line) and with a mouse IgG1 isotype control antibody (dotted line).



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Fig. 2.   Calcium flux in myc-LXA4R-transfected HT29cl.19A cells after stimulation with N-formylmethionyl-leucyl-phenylalanine (fMLP). Control (A) and transfected (B) HT29cl.19A cells were grown on matrix-coated semipermeable filters, loaded for 1 h with fura-2, placed in a spectrofluorometer, and stimulated with 10-4 M fMLP. An increase of intracellular Ca2+ was observed only after fMLP treatment of filters with myc-LXA4R-transfected cells. The functional integrity of each filter was demonstrated by subsequent stimulation with 100 µM carbachol. The data are representative of 2 independent experiments with a total of 8 different filters that showed similar results.

LXA4R is localized to basolateral surface and most prominent on lateral membrane of polarized epithelia. The polarity of expression cell surface receptors can be a very important aspect of their biology. Attempts to raise antibodies to LXA4R that could determine the polarity of the endogenous receptor have failed, probably as a consequence of the combination of low levels of the endogenous LXA4R in these cells and the lack of sufficient specificity of the affinity-purified anti-peptide antibodies that were used (data not shown). Thus we transiently transfected T84 cells with recombinant retroviruses encoding myc-LXA4R or EGFP (as a control) without antibiotic (i.e., puromycin) selection as an alternative approach to expression of the myc-tagged LXA4R in these cells. Using three infection cycles with EGFP-encoding retroviruses, we were able to detect expression by ~15% of T84 cells (data not shown). T84 cells were transduced with supernatant containing myc-LXA4R retroviruses with the same conditions as for the EGFP retroviruses and plated on collagen-coated semipermeable supports for 7-10 days, allowing them to form confluent polarized epithelial monolayers. We then used domain-selective biotinylation of just the apical or basolateral membrane of these polarized model epithelia to determine the polarity of specific cell surface proteins as described previously (23, 26). Briefly, the apical or basolateral surface was biotinylated and quenched and biotinylated proteins were collected with streptavidin-coated agarose beads and immunoblotted with anti-myc. Basolaterally biotinylated surfaces contained much more c-myc-LXA4R (migrating as a 40-kDa band) than did similarly treated apical surfaces (Fig. 3A). As a control, we verified with the same lysates that the beta 1-integrin molecule previously shown to be basolaterally polarized (23) was predominantly detected in the basolaterally biotinylated cells. Thus LXA4R appears to be preferentially expressed on the basolateral surface.


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Fig. 3.   Polarity of the LXA4R in T84 cells. A: myc-LXA4R-transduced T84 cells were biotinylated on the apical or basolateral surface, followed by isolation of biotinylated proteins with streptavidin-agarose. The biotinylated proteins were separated by SDS-PAGE and immunoblotted with anti-myc-MAb, showing that a much larger amount of biotinylated LXA4R was recovered after biotinylation of the basolateral surface of T84 cells. Antibody to beta 1-integrin was used as a positive control for basolateral expression. B: T84 cells were transduced with retroviruses coding for a myc-tagged LXA4R. The transduced cells were grown for 7-10 days on a matrix-coated filter and then stained with anti-myc MAb (green fluorescence) and rhodamine-phalloidin (red fluorescence). An X-Z image (apical surface at top; basal surface at bottom) acquired by laser confocal microscopy showed that the myc-tagged LXA4R was located exclusively along the lateral membrane of T84 cells. C: T84 cells were transduced with retroviruses encoding a mutant c-myc-LXA4R receptor (Q307X mutant) lacking the COOH-terminal 45 amino acids from the COOH-terminal intracellular domain. An X-Z image demonstrated the same lateral membrane localization observed for the full-length c-myc-LXA4R construct.

We next sought to further define the localization of LXA4R expression via confocal microscopy. Polarized c-myc-LXA4R-expressing T84 cells were fixed, permeabilized, and stained with antibody to the c-myc epitope along with rhodamine-phalloidin to visualize cytoplasmic actin. Twenty X-Y plane images (1-µm thickness) were used to create an X-Z (i.e., orthogonal) view of our retrovirally transfected epithelial monolayer (Fig. 3B). Consistent with the results of our cell surface-selective biotinylation studies, this view showed that c-myc-LXA4R was clearly located below the perijunctional actin ring. However, c-myc-LXA4R was not uniformly distributed along the basolateral membrane but rather showed specific areas along the lateral membrane of high expression, whereas, in contrast, staining was not observed along the basal membrane. We next sought to confirm this pattern of expression with another epithelial cell line. Madin-Darby canine kidney (MDCK) is a well-characterized polarized renal epithelial cell line that has been used extensively to determine the subcellular distribution of other G protein-coupled receptors (1, 25). We transduced MDCK cells with the myc-LXA4R by the same technique used for T84 cells and again examined the distribution of anti-myc staining by confocal microscopy. A similar pattern of preferentially lateral staining was observed, indicating that this result was not specific to a single polarized cell line (data not shown).

Several peptide motifs have been identified that are associated with basolateral targeting of membrane proteins (18). Putative basolateral targeting determinants for other G protein-coupled receptors including the FSH receptor (1) and the mGluR7 glutamate receptor have been mapped to the COOH-terminal intracellular domain. Because some basolateral sorting signals are localized to the COOH-terminal cytoplasmic tail, we asked whether truncation of the final 45 amino acids of the myc-tagged LXA4R would interfere with targeting of this receptor to the basolateral membrane. A premature stop codon was inserted in place of a glutamine codon just beyond the seventh and last transmembrane segment of the protein to generate a Q307X mutant of myc-LXA4R. Recombinant retroviruses coding for the truncated form of the LXA4R were prepared and used to transiently transduce T84 and MDCK cells. Immunostaining of confluent monolayers of T84 cells grown on a semipermeable membrane showed the same pattern of basolateral membrane staining as obtained with the nonmutated myc-LXA4R (Fig. 3C). Thus the cytoplasmic tail is not necessary to bring about the basolateral targeting of the LXA4R in polarized epithelial cells.

Overexpression of LXA4R in intestinal epithelial cells enhances inhibitory effect of LXA4 on IL-8 secretion. Polarized intestinal epithelial cell lines constitutively secrete a low basal level of IL-8 (6, 10) that is markedly upregulated in response to stimulation with pathogens or proinflammatory cytokines such as TNF-alpha . We previously showed (10, 14) that stable analogs of LXA4 partially attenuate the IL-8 secretion induced in T84 cells by S. typhimurium or subsaturating concentrations of TNF-alpha . Such attenuation of epithelial IL-8 secretion was suggested to be mediated via the LXA4R but was not directly investigated. Because, as shown above, the c-myc-tagged LXA4R we expressed in epithelial cells was capable of signaling in response to ligand, we investigated whether the increased expression of LXA4R in our retrovirally transfected cells might lead to a greater inhibitory effect of an LXA4 analog on IL-8 secretion. Specifically, we measured the effect of 100 nM 15-R/S-methyl-LXA4 on IL-8 secretion on both LXA4R-transfected and nontransfected HT29cl.19A cells. We observed that addition of 15-R/S-methyl LXA4 suppressed TNF-alpha -induced IL-8 release to a greater extent in the LXA4R-transfected cells (Fig. 4A). Inhibition of induced IL-8 release was dependent on the concentration of TNF-alpha that was used. IL-8 secretion induced by TNF-alpha concentrations between 100 and 1,000 pg/ml could be inhibited by 15-R/S-methyl-LXA4, with progressive loss of the inhibitory effect at higher concentrations of TNF-alpha (data not shown), consistent with previous studies (14). Salmonella-induced IL-8 secretion by model intestinal epithelia is largely the result of the interaction of Salmonella flagellin with Toll-like receptor 5 (TLR5) (12). Thus we also tested whether the myc-LXA4R-transfected cells were more sensitive to the effects of an LXA4 analog on IL-8 secretion elicited after TLR5 interaction with Salmonella flagellin (Fig. 4B). 15-R/S-methyl-LXA4 inhibited flagellin-stimulated IL-8 secretion to a greater extent in LXA4R-transfected cells than control EGFP transfectants, with the most inhibition again observed at a subsaturating dose of flagellin.


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Fig. 4.   Intestinal epithelial cells overexpressing the LXA4R show increased sensitivity to the inhibitory effects of LXA4 on TNF-alpha - and flagellin-induced IL-8 secretion. Wild-type, myc-LXA4R overexpressing, or EGFP-transfected HT29cl.19A cells were grown to confluence on semipermeable collagen-coated filters. The cells were pretreated with 100 nM 15-R/S-methyl-LXA4 for 1 h, and some of the cultures were stimulated basolaterally with human TNF-alpha (1 ng/ml) or Salmonella flagellin (5 or 20 ng/ml). The supernatant was collected after 5 h of stimulation and assayed for IL-8 by ELISA. A: LXA4 analog inhibited TNF-alpha -induced IL-8 secretion to a greater extent in myc-LXA4R-transfected cells than in nontransfected cells. In this experiment, addition of the LXA4 analog reduced TNF-alpha -induced IL-8 secretion by 60% (*P < 0.05). B: addition of LXA4 analog inhibited flagellin-induced IL-8 secretion in myc-LXA4R-transfected cells stimulated with 5 ng/ml flagellin (57% inhibition; P < 0.05) or 20 ng/ml flagellin (43% inhibition; P < 0.05). LXA4 analog inhibited IL-8 production by EGFP-transfected cells stimulated with 5 ng/ml flagellin (35% inhibition; P < 0.05) but not by cells stimulated with 20 ng/ml flagellin.

We also observed a consistent trend toward increased suppression of basal IL-8 production by 15- R/S-methyl-LXA4 in the myc-LXA4R-overexpressing HT29cl.19A cells. Figure 5 summarizes data from a series of five experiments that showed an average of 41% inhibition (±SD of 21%) of basal IL-8 production in transfected cells by 15-R/S-methyl-LXA4 compared with an average of 15% inhibition (±SD of 22%) in nontransfected cells. The enhanced lipoxin responsiveness of the LXA4R-transfected cells for both agonist-induced and basal IL-8 production supports the hypothesis that at least part of the inhibitory effect of lipoxin analogs on the IL-8 response of intestinal epithelial cells is mediated through the LXA4R and is more efficient when a larger number of receptors are available.


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Fig. 5.   Intestinal epithelial cells overexpressing the LXA4R show increased sensitivity to the inhibitory effects of LXA4 on basal IL-8 secretion. Wild-type and myc-LXA4R-overexpressing HT29cl.19A cells were grown to confluence on semipermeable collagen-coated filters. The medium was replaced, and some wells received 100 nM 15-R/S-methyl-LXA4. The supernatant was collected after 6 h and assayed for IL-8 by ELISA. The graph shows the % decrease in basal IL-8 production by both cell types in the presence of 15-R/S-methyl-LXA4 in 5 independent experiments. The horizontal bars indicate the mean % decrease in IL-8 production. The difference between the 2 cell types was statistically significant (P = 0.04).

Pharmacological inhibition of CysLT1 receptor by montelukast does not interfere with inhibitory effect of LXA4 on TNF-alpha -stimulated IL-8 secretion. To determine whether the CysLT1 receptor was involved in the observed inhibitory effects of LXA4 analog on IL-8 secretion, we tested whether the CysLT1 receptor antagonist montelukast influences TNF-alpha -stimulated IL-8 production in myc-LXA4R-overexpressing HT29cl.19A cells or the inhibition of this response by 15-R/S-methyl-LXA4. At a concentration of 100 nM (significantly higher than the reported half-maximal inhibitory concentration for this antagonist at the CysLT1 receptor; Ref. 17), montelukast had no significant effect on TNF-alpha -elicited IL-8 production or the inhibition of this response by 15-R/S-methyl-LXA4 (Fig. 6). We conclude that the effects of LXA4 analog in this system are not inhibited when CysLT1 receptors on HT29cl.19A cells are subjected to pharmacological blockade.


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Fig. 6.   The CysLT1 receptor inhibitor montelukast does not interfere with the inhibitory effect of LXA4 on TNF-alpha -stimulated IL-8 production. myc-LXA4R-overexpressing HT29cl.19A cells on filters were left unstimulated or were stimulated basolaterally with human TNF-alpha (5 ng/ml). Some wells were pretreated for 1 h with 100 nM 15-R/S-methyl-LXA4, 100 nM montelukast, or both reagents. The supernatant was collected after 5 h of stimulation and assayed for IL-8 by ELISA. Addition of montelukast did not result in a statistically significant change in the IL-8 responses examined.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LXA4 and its stable analogs downregulate chemokine secretion by polarized intestinal epithelial cell lines and colonic epithelium (13). Epithelial cells express two different high-affinity binding sites for LXA4, namely LXA4R and CysLT1, that have been proposed to mediate the anti-inflammatory effects of this eicosanoid. We show here that overexpression of LXA4R led to a greater reduction in chemokine secretion in the presence of LXA4 analog, indicating that LXA4 attenuation of epithelial chemokine secretion is mediated, at least in part, by the LXA4R. Furthermore, the selective CysLT1 receptor antagonist montelukast (17) did not interfere with the inhibitory effect of LXA4 analog. These results indicate that the effects of LXA4 analog on intestinal epithelial cells are primarily mediated through the LXA4R and do not require availability of the CysLT1 receptor. Thus pharmacological analogs that seek to mimic LXA4's anti-inflammatory bioaction on epithelial cells should retain the ability to ligate LXA4R.

Polarized intestinal epithelial cells selectively sort a variety of cell surface proteins to their apical and basolateral membranes, and proper sorting can be essential for specific absorptive, secretory, endocrine, and signal transduction functions (2). For this reason, we sought to determine the polarity of the LXA4R. Methods of determining the polarity of a specific protein generally depend on either being able to generate antibodies to the protein of interest or transfection with epitope-tagged or EGFP fusion constructs. Insertion of short epitope tags into proteins generally does not affect their intracellular trafficking or function, although interruption of important protein motifs with resultant effects on protein trafficking and/or function is possible with this approach (32). Lack of antigenicity of the protein of interest and transfection inefficiency of polarized intestinal epithelial cells can provide significant technical obstacles. We circumvented these problems by high-efficiency retroviral transduction of polarized epithelial cells with epitope-tagged LXA4R. This retroviral expression method should be a versatile approach to defining the polarity of other cell surface molecules in polarized cell lines that are inefficiently transfected with standard plasmid DNA constructs. Here, we utilized this approach for three distinct polarized epithelial cell lines and determined that LXA4R is expressed preferentially on the basolateral surface. LXA4R expression was not uniform on this membrane but rather further localized to lateral areas of this membrane domain.

Although, classically, membrane proteins on polarized epithelia have been described as apical or basolateral, it is becoming clear that in fact more precise targeting exists. Localization to the lateral subdomain has been described for several other G protein-coupled receptors including the vasopressin V2 receptor (28), the alpha 2a-adrenergic receptor (27), and the human CTR-2 calcitonin receptor (26). One molecular mechanism contributing to the polarized expression of plasma membrane proteins is the presence of specific "targeting" motifs within the primary amino acid sequence. For example, basolateral targeting motifs have been identified for several G protein-coupled receptors including the alpha 2a-adrenergic receptor (27), the FSH receptor (1), the mGluR7 glutamate receptor (20), and the M3 acetylcholine receptor (25). In some cases, these basolateral targeting determinants are located within the COOH-terminal intracellular domain (1, 20). However, this does not appear to be the case for LXA4R because we observed that a mutant form of the human LXA4R that lacked the final 45 amino acids of the cytoplasmic domain beginning just distal to the seventh transmembrane segment was targeted similarly to parental myc-tagged LXA4R. Similarly, the basolateral targeting signals for the M3 acetylcholine receptor and the alpha 2a-adrenergic receptor do not map to the COOH-terminal intracellular domain (25, 27). Thus the basolateral sorting signal for LXA4R, like these other G protein-coupled receptors, is likely contained in another region of the molecule.

The major route of LXA4 biosynthesis in vivo is via the "transcellular pathway" whereby lipoxygenases from two distinct cell types (e.g., neutrophil 5-LO and epithelial 15-LO) act on the same molecule of arachidonate, resulting in generation of LXA4. As a result of such a mechanism, LXA4 concentrations are likely to be greatest in areas in which the cell types that cooperate in their synthesis are in close proximity. During active mucosal inflammation, the paracellular space between adjacent epithelial cells would be one such place because this condition is characterized by neutrophil transepithelial migration and such migration proceeds between epithelial cells. Thus it is interesting that we observed LXA4R to be expressed on the lateral surface of intestinal epithelial cells, because such localization should afford epithelial cells immediate exposure to the highest concentration of lipoxins synthesized during such an inflammatory process. Because epithelial exposure to LXA4 analogs downregulates the chemokine secretion that promotes neutrophil transmigration, lateral expression of LXA4R would seem to be an efficient component of a negative feedback loop that should prevent uncontrolled inflammation.

Another implication of LXA4R being expressed primarily laterally is that therapeutic strategies that seek to pharmacologically activate the LXA4R to reduce inappropriate intestinal inflammation will need to utilize compounds that have good epithelial permeability. Although LXA4 and LXA4 analogs are acids (i.e., negatively charged) at physiological pH and thus would not be expected to cross epithelia efficiently, addition of appropriate ester groups should improve permeability. Binding studies with LXA4 and its analogs indicate that methyl esters of these compounds retain the ability to ligate the LXA4R, indicating that structural alterations in this region of the compound may be permissible as long as the trihydroxytetraene moiety remains intact (29). Thus design of LXA4 analogs with adequate epithelial permeability that retain anti-inflammatory activity should be possible and may be therapeutic for diseases characterized by inappropriate mucosal inflammation such as inflammatory bowel disease.


    ACKNOWLEDGEMENTS

We thank James Hudson for technical assistance and Andrew Kowalczyk for suggesting use of the 293-10A1 packaging cells for production of retroviruses.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-47662 (to J. L. Madara), DK-09800 (to A. T. Gewirtz), DK-02831 (to D. Merlin), and AR-44268 (to I. R .Williams) and the Crohn's and Colitis Foundation of America (D. Merlin). T. Kucharzik was supported by a fellowship award (Ku 1328-1) from the Deutsche Forschungsgemeinschaft (DFG).

Present address of T. Kucharzik: Dept. of Medicine B, University of Münster, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany.

Address for reprint requests and other correspondence: I. R. Williams, Dept. of Pathology and Laboratory Medicine, Whitehead Biomedical Research Bldg. 105D, Emory Univ., 615 Michael St., Atlanta, GA 30322 (E-mail: irwilli{at}emory.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.

First published November 27, 2002;10.1152/ajpcell.00507.2001

Received 22 October 2001; accepted in final form 21 November 2002.


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