Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-
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--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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 cm1 · 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-
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 (104 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-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- 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-
(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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
104 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.
|
|
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 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.
|
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-. 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-
. 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-
-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-
that was used. IL-8 secretion induced by TNF-
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-
(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.
|
|
Pharmacological inhibition of CysLT1 receptor by montelukast does
not interfere with inhibitory effect of LXA4 on
TNF--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-
-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-
-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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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
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
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Beau, I,
Groyer-Picard MT,
Le Bivic A,
Vannier B,
Loosfelt H,
Milgrom E,
and
Misrahi M.
The basolateral localization signal of the follicle-stimulating hormone receptor.
J Biol Chem
273:
18610-18616,
1998
2.
Brown, D,
and
Breton S.
Sorting proteins to their target membranes.
Kidney Int
57:
816-824,
2000[ISI][Medline].
3.
Christophe, T,
Karlsson A,
Dugave C,
Rabiet MJ,
Boulay F,
and
Dahlgren C.
The synthetic peptide Trp-Lys-Tyr-Met-Val-Met-NH2 specifically activates neutrophils through FPRL1/lipoxin A4 receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2.
J Biol Chem
276:
21585-21593,
2001
4.
Claria, J,
and
Serhan CN.
Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions.
Proc Natl Acad Sci USA
92:
9475-9479,
1995[Abstract].
5.
Dharmsathaphorn, K,
and
Madara JL.
Established intestinal cell lines as model systems for electrolyte transport studies.
Methods Enzymol
192:
354-389,
1990[Medline].
6.
Eckmann, L,
Jung HC,
Schurer-Maly C,
Panja A,
Morzycka-Wroblewska E,
and
Kagnoff MF.
Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin 8.
Gastroenterology
105:
1689-1697,
1993[ISI][Medline].
7.
Eckmann, L,
Kagnoff MF,
and
Fierer J.
Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry.
Infect Immun
61:
4569-4574,
1993[Abstract].
8.
Fiore, S,
Maddox JF,
Perez HD,
and
Serhan CN.
Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor.
J Exp Med
180:
253-260,
1994[Abstract].
9.
Gewirtz, AT,
Fokin VV,
Petasis NA,
Serhan CN,
and
Madara JL.
LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively downregulate PMN azurophilic degranulation.
Am J Physiol Cell Physiol
276:
C988-C994,
1999
10.
Gewirtz, AT,
McCormick B,
Neish AS,
Petasis NA,
Gronert K,
Serhan CN,
and
Madara JL.
Pathogen-induced chemokine secretion from model intestinal epithelium is inhibited by lipoxin A4 analogs.
J Clin Invest
101:
1860-1869,
1998
11.
Gewirtz, AT,
Rao AS,
Simon PO, Jr,
Merlin D,
Carnes D,
Madara JL,
and
Neish AS.
Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-B pathway.
J Clin Invest
105:
79-92,
2000
12.
Gewirtz, AT,
Simon PO, Jr,
Schmitt CK,
Taylor LJ,
Hagedorn CH,
O'Brien AD,
Neish AS,
and
Madara JL.
Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response.
J Clin Invest
107:
99-109,
2001
13.
Goh, J,
Baird AW,
O'Keane C,
Watson RW,
Cottell D,
Bernasconi G,
Petasis NA,
Godson C,
Brady HR,
and
MacMathuna P.
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:
2772-2780,
2001
14.
Gronert, K,
Gewirtz A,
Madara JL,
and
Serhan CN.
Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon and inhibits tumor necrosis factor
-induced IL-8 release.
J Exp Med
187:
1285-1294,
1998
15.
Gronert, K,
Martinsson-Niskanen T,
Ravasi S,
Chiang N,
and
Serhan CN.
Selectivity of recombinant human leukotriene D4, leukotriene B4, and lipoxin A4 receptors with aspirin-triggered 15-epi-LXA4 and regulation of vascular and inflammatory responses.
Am J Pathol
158:
3-9,
2001
16.
Levy, BD,
Clish CB,
Schmidt B,
Gronert K,
and
Serhan CN.
Lipid mediator class switching during acute inflammation: signals in resolution.
Nat Immunol
2:
612-619,
2001[ISI][Medline].
17.
Lynch, KR,
O'Neill GP,
Liu Q,
Im DS,
Sawyer N,
Metters KM,
Coulombe N,
Abramovitz M,
Figueroa DJ,
Zeng Z,
Connolly BM,
Bai C,
Austin CP,
Chateauneuf A,
Stocco R,
Greig GM,
Kargman S,
Hooks SB,
Hosfield E,
Williams DL, Jr,
Ford-Hutchinson AW,
Caskey CT,
and
Evans JF.
Characterization of the human cysteinyl leukotriene CysLT1 receptor.
Nature
399:
789-793,
1999[ISI][Medline].
18.
Matter, K,
Yamamoto EM,
and
Mellman I.
Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells.
J Cell Biol
126:
991-1004,
1994[Abstract].
19.
Matthews, JB,
Smith JA,
Tally KJ,
Awtrey CS,
Nguyen H,
Rich J,
and
Madara JL.
Na-K-2Cl cotransport in intestinal epithelial cells. Influence of chloride efflux and F-actin on regulation of cotransporter activity and bumetanide binding.
J Biol Chem
269:
15703-15709,
1994
20.
McCarthy, JB,
Lim ST,
Elkind NB,
Trimmer JS,
Duvoisin RM,
Rodriguez-Boulan E,
and
Caplan MJ.
The C-terminal tail of the metabotropic glutamate receptor subtype 7 is necessary but not sufficient for cell surface delivery and polarized targeting in neurons and epithelia.
J Biol Chem
276:
9133-9140,
2001
21.
McCormick, BA,
Colgan SP,
Delp-Archer C,
Miller SI,
and
Madara JL.
Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.
J Cell Biol
123:
895-907,
1993[Abstract].
22.
McMahon, B,
Stenson C,
McPhillips F,
Fanning A,
Brady HR,
and
Godson C.
Lipoxin A4 antagonizes the mitogenic effects of leukotriene D4 in human renal mesangial cells. Differential activation of MAP kinases through distinct receptors.
J Biol Chem
275:
27566-27575,
2000
23.
Merlin, D,
Si-Tahar M,
Sitaraman SV,
Eastburn K,
Williams I,
Liu X,
Hediger MA,
and
Madara JL.
Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: transport of bacterial peptides influences expression of MHC class I molecules.
Gastroenterology
120:
1666-1679,
2001[ISI][Medline].
24.
Mostov, KE,
Verges M,
and
Altschuler Y.
Membrane traffic in polarized epithelial cells.
Curr Opin Cell Biol
12:
483-490,
2000[ISI][Medline].
25.
Nadler, LS,
Kumar G,
and
Nathanson NM.
Identification of a basolateral sorting signal for the M3 muscarinic acetylcholine receptor in Madin-Darby canine kidney cells.
J Biol Chem
276:
10539-10547,
2001
26.
Nussenzveig, DR,
Matos MD,
and
Thaw CN.
Human calcitonin receptor is directly targeted to and retained in the basolateral surface of MDCK cells.
Am J Physiol Cell Physiol
275:
C1264-C1276,
1998
27.
Saunders, C,
Keefer JR,
Bonner CA,
and
Limbird LE.
Targeting of G protein-coupled receptors to the basolateral surface of polarized renal epithelial cells involves multiple, non-contiguous structural signals.
J Biol Chem
273:
24196-24206,
1998
28.
Schulein, R,
Lorenz D,
Oksche A,
Wiesner B,
Hermosilla R,
Ebert J,
and
Rosenthal W.
Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells.
FEBS Lett
441:
170-176,
1998[ISI][Medline].
29.
Serhan, CN,
Maddox JF,
Petasis NA,
Akritopoulou-Zanze I,
Papayianni A,
Brady HR,
Colgan SP,
and
Madara JL.
Design of lipoxin A4 stable analogs that block transmigration and adhesion of human neutrophils.
Biochemistry
34:
14609-14615,
1995[ISI][Medline].
30.
Su, SB,
Gong W,
Gao JL,
Shen W,
Murphy PM,
Oppenheim JJ,
and
Wang JM.
A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates the chemotactic activity of serum amyloid A for human phagocytic cells.
J Exp Med
189:
395-402,
1999
31.
Takano, T,
Fiore S,
Maddox JF,
Brady HR,
Petasis NA,
and
Serhan CN.
Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors.
J Exp Med
185:
1693-1704,
1997
32.
Turner, JR,
Lencer WI,
Carlson S,
and
Madara JL.
Carboxy-terminal vesicular stomatitis virus G protein-tagged intestinal Na+-dependent glucose cotransporter (SGLT1): maintenance of surface expression and global transport function with selective perturbation of transport kinetics and polarized expression.
J Biol Chem
271:
7738-7744,
1996