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-Stimulated Neutrophil-Enterocyte Interactions In Vitro and Attenuate TNF--Induced Chemokine Release and Colonocyte Apoptosis in Human Intestinal Mucosa Ex Vivo1
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Departments of
*
Gastroenterology, and
Medicine and Therapeutics, Mater Misericordiae Hospital, and
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, and the Dublin Molecular Medicine Centre, Dublin, Ireland; and
Department of Chemistry, University of Southern California, Los Angeles, CA 90089
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated neutrophil-enterocyte
interaction in vitro and TNF--stimulated chemokine release, changes
in mucosal architecture, and enterocyte apoptosis in cytokine-activated
intact human colonic mucosa ex vivo. LXA4,
15-(R/S)-epi-LXA4, and
16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA4 methyl
ester (16-phenoxy-LXA4) inhibited TNF--stimulated
neutrophil adherence to epithelial monolayers at nanomolar
concentrations. In parallel experiments involving human colonic mucosa
ex vivo, LXA4potently attenuated TNF--stimulated release
of the C-X-C chemokine IL-8, and the C-C chemokines
monocyte-chemoattractant protein-1 (MCP-1) and RANTES. Exposure of
strips of normal human colonic mucosa to TNF- induced disruption of
mucosa architecture and enhanced colonocyte apoptosis via a
caspase-3-independent mechanism. Prior exposure of the mucosa strips to
15-(R/S)-methyl-LXA4 attenuated
TNF--stimulated colonocyte apoptosis and protected the mucosa
against TNF--induced mucosal damage. In aggregate, our data
demonstrate that lipoxins and aspirin-triggered 15-epi-LXA4
are potent antagonists of TNF--mediated neutrophil-enterocyte
interactions in vitro, attenuate TNF--triggered chemokine release
and colonocyte apoptosis, and are protective against TNF--induced
morphological disruption in human colonic strips ex vivo. Our
observations further expand the anti-inflammatory profile of these
lipoxygenase-derived eicosanoids and suggest new therapeutic approaches
for the treatment of inflammatory bowel disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recruitment of neutrophils to the gut is a complex process coordinated by cytokines, chemokines, and adhesion molecules. Chemokines are 8- to 10-kDa peptides whose principal actions are to serve as chemotactic cytokines for leukocytes (9). In vitro studies indicate that transformed human colonic adenocarcinoma cell lines secrete most chemokines upon cytokine activation or bacterial infection (10). Freshly isolated intestinal epithelial cells are also a source of chemokines (11). Potential cellular sources of chemokines in this environment include colonocytes, and resident and recruited leukocytes (9, 12, 13).
The relapsing-remitting course and frequent spontaneous remission observed in IBD imply the existence of endogenous anti-inflammatory mechanisms. Adding to the known endogenousanti-inflammatory armamentarium (which includes Th1/Th2 imbalance, IL-1 receptor antagonist) are a class of eicosanoidsknown as lipoxins (lipoxygenase interaction products; LXs). The major bioactive LXs in mammalian systems, 5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (LXA4) and 5(S),14(R),15(S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid (LXB)4, are formed via pathways initiated by the dual lipoxygenation of arachidonic acid during cell-cell interactions (14, 15). They are rapidly metabolized in vitro, and stable analogs have been designed that retain the bioactivity of the native compounds (16).
LXs are endogenous eicosanoids, generated during cell-cell interactions, that appear to serve as braking signals for neutrophil-mediated tissue damage (14, 15, 17). LXs inhibit recruitment of neutrophils by attenuating their chemotaxis, adhesion, and transmigration across vascular endothelial (18, 19) and epithelial cells (7, 8) and by down-regulating chemokine production (20, 21). LXs may further contribute to the resolution of inflammation by stimulating nonphlogistic phagocytosis of apoptotic neutrophils by macrophages (22).
Recently, a seven-transmembrane G protein-coupled
LXA4 receptor has been cloned from T84 cells, a
human colonic adenocarcinoma cell line (21). This is
identical with the previously described LXA4
receptor (termed ALXR) cloned from cells of myeloid lineage. LXs
attenuate TNF--induced IL-8 secretion by these cells in vitro
(21). Furthermore, LXs also inhibit Salmonella
typhimurium-induced secretion of IL-8 and pathogen-elicited
epithelial chemoattractant by model intestinal epithelial cells
(23). Intriguingly, aspirin-acetylated cyclooxygenase-2
generates 15(R)-hydroxyeicosatetraenoic acid which,
in the context of cell-cell interactions, can be converted by
neutrophils to a series of 15-epimers (aspirin-triggered 15-epi-LXs;
ATLs) that may share many anti-inflammatory activities with the
native LXs (14, 15, 17). ATLs may contribute to the
bioactivity profile of the prototype nonsteroidal anti-inflammatory
drugs in vivo. Taken together, these data suggest that LXs may play
important anti-inflammatory roles in intestinal inflammation that
include neutrophil-independent modulation of cell function.
Here we assessed the influence of LXs, stable LX analogs, and
aspirin-triggered 15-epi-LXA4 on TNF--stimulated
neutrophil-intestinal epithelial cell interaction in vitro and
TNF--stimulated chemokine release by intestinal mucosa ex vivo. In
addition, we monitored the effects of LXs on TNF--induced changes in
colonocyte apoptosis and tissue architecture in normal human intestine
ex vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T84 cell culture.
T84, a human colonic adenocarcinoma cell line with a well characterized
epithelial phenotype and polarity (24), was obtained from
American Type Culture Collection (Manassas, VA) and grown to confluence
in T75 tissue culture flasks in a 1:1 mixture of DMEM and Ham’s F-12
medium supplemented with 15 mM HEPES buffer (pH 7.5), 10% FCS, 1%
penicillin/streptomycin, and 1% L-glutamine. To facilitate
study of neutrophil trafficking in a physiological
(basolateral-to-apical) direction, T84 epithelial monolayers were grown
on the undersurface of polycarbonate filters according to methods
described by Colgan et al. (8). Briefly, the inverted
monolayers were grown on the undersurface of ring-supported
0.33-cm2 polycarbonate filters (Corning Costar,
Cambridge, MA), and confluence was assessed electrophysiologically by
measurement of transepithelial resistance (TER) with Endohm-6 and the
EVOM resistance meter (World Precision Instruments, Sarasota,
FL). Consistent with the known ability of T84 to form tight
intercellular junctions, mean TER of the monolayers on filters at time
of confluence was 1367.7 ± 50.4 
.cm2
(n = 45). The monolayer on the filter effectively
divided the well into an upper (basolateral) compartment and a lower
(apical) compartment.
Neutrophil isolation and transmigration/adherence assays. Human neutrophils from healthy volunteers were isolated by density gradient centrifugation and dextran sedimentation and labeled with a fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein ester (Calbiochem, San Diego, CA) as described previously (19). Viability was determined by trypan blue exclusion test. One million fluorescence-labeled neutrophils suspended in 100 µl Dulbecco’s PBS and 1% bovine calf serum were added to the upper (basolateral) compartment of the filter inserts. In all experiments, a positive control for neutrophil transmigration comprising the chemotactic peptide fMLP (6 µM; Sigma, St. Louis, MO) in the lower apical compartment was included. At the end of the 120-min experimental period at 37°C, the nonadherent neutrophils were removed by washing twice with Dulbecco’s PBS. The number of neutrophils that adhered to the epithelial monolayers or transmigrated into the lower (apical) compartment were determined fluorometrically on the Cytoflour II (Perspective Biosystems, Framingham, MA).
For studies of the influence of cytokine activation of epithelium on
neutrophil trafficking in vitro, both sides of the confluent epithelial
monolayers were preincubated with TNF-, IL-1
(100 ng/ml; R&D
Systems, Minneapolis, MN), or vehicle control for 24 h at 37°C
before initiation of transmigration experiments, and washed before
addition of neutrophils. TER of the cytokine-treated monolayers was
determined before and after 24 h of cytokine preincubation.
LXA4 was purchased from Cascade Biologicals (Berkshire, U.K.). The synthetic LXA4 analog 15-(R/S)-methyl-11,12-dehydro-LXA4 methyl ester (15-(R/S)-methyl-LXA4) covers the biological activity of 15-epi-LXA4. This and the synthetic stable LXA4 analog 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were gifts from Dr. Nicos Petasis (University of Southern California, Los Angeles, CA) and prepared by total organic synthesis as previously described (16). Structures were confirmed by reversed phase-HPLC, nuclear magnetic resonance, and mass spectral analysis. The solutions were stored at -70°C in 99% ethanol. The modulatory effects of LXs on neutrophil-epithelial cell interaction were assessed by preincubation of human neutrophils and epithelial monolayers, respectively, with LX (0.1–100 nM, 37°C). Neutrophils were preincubated with LX for 15 min before addition to the epithelial monolayers. T84 monolayers were treated with LX for 20 min before incubation with cytokine. Vehicle controls comprised dilutions of the solvent (absolute ethanol) to the highest concentration of LXs used in the experiments (maximal ethanol concentration 0.01%). All individual experiments were performed in duplicate.
To determine the role of the C-X-C chemokine, IL-8, and the adhesion
molecule, ICAM-1, in TNF--stimulated neutrophil-epithelial cell
interactions, the blocking mAbs to the respective chemokine and
adhesion molecule were added to both the basolateral and apical
compartments at the beginning of transmigration assays (both mAbs, 50
µg/ml; R&D Systems).
Assessment of T84 viability and apoptosis in vitro.
In experiments assessing TNF--induced changes in viability and
apoptosis, T84 monolayers were grown to confluence on 12- and 24-well
tissue culture plates. Monolayers were incubated with TNF- (20–100
ng/ml) or vehicle control for 24 h. Gross morphological assessment
of the monolayers was performed by standard light microscopy. The
monolayers were subsequently trypsinized. Viability was assessed by
flow cytometry following incorporation with propidium iodide. Apoptosis
was quantified by flow cytometry as the percentage of cells with
hypodiploid DNA. Cell suspensions were centrifuged at 200 x
g for 10 min. The cell pellets were resuspended in 500 µl
of hypotonic fluorochrome solution (50 µg/ml propidium iodide, 3.4 nM
sodium citrate, 1 mM EDTA, and 0.1% Triton X-100) and kept in the dark
at 4°C before they were analyzed using a Coulter Elite
cytofluorometer (Coulter, Bedfordshire, U.K.). A human prostate
adenocarcinoma cell line, LNCaP androgen-dependent prostate cancer
cells, treated with etoposide was used as positive control.
Studies of human intestinal mucosa ex vivo
Harvesting of human intestinal mucosa.
Normal human colonic mucosa was harvested from colonic resection
specimens for colorectal cancer following informed consent from
patients preoperatively. Patients with known IBD, or ischemic bowel or
familial colon cancer syndromes were excluded. The resected bowel
specimen was transported to the pathology laboratory for processing
within 30 min of resection. Twelve 
1-cm2
strips of macroscopically normal colonic mucosa at least 10 cm away
from the tumor margin were harvested and transported to the laboratory
in prewarmed, preoxygenated Kreb’s physiological solution (Sigma) at
37°C. The resection specimen was immersed in 10% formalin and
processed routinely.
A purpose-built oxygenation manifold comprising six oxygenation ports was arranged in a longitudinal 2 x 3 fashion. Oxygenation of the colonic mucosa strips was delivered via the oxygenation ports fitted through perforations on the lid of a 12-well culture plate in the water bath at 37°C. A duplicate of the oxygenation manifold and culture well enabled six conditions in duplicate at one experiment. Two milliliters of prewarmed Kreb’s physiological solution was added to each of the 12 wells and preoxygenated for 10 min. The 12 colonic mucosa strips harvested as described above were removed from the transport medium, placed in each well individually containing the physiological solution, and allowed to equilibrate for 10 min and maintained ex vivo at 37°C for up to 8 h.
Treatment of colonic mucosa with cytokine or LXs.
Following equilibration, the mucosal strips were preincubated with LX
at doses ranging from 0.1, 1, 10, and 100 nM in 2 ml physiological
solution for 20 min. The vehicle control was 0.1% ethanol. At the end
of the preincubation period, the experiment was started with activation
by TNF- (20 µg/ml; R&D Systems) or vehicle control (Dulbecco’s
PBS-BSA). Each well was wrapped in cling film and allowed to
incubate for 8 h. To determine the time course of chemokine
production and modulation by LX, two 100-µl aliquots were
removed from each well at times 0, 3, and 8 h, snap-frozen, and
stored at -70°C. The volume sampled was replaced with the
appropriate physiological solution containing TNF-.
At the end of the experimental period, the colonic mucosa strips were snap-frozen and stored at -70°C for protein assay. In separate experiments, the strips were stored in 10% neutral formalin, paraffin-mounted, and processed for H&E staining, or fixed in glutaraldehyde and processed for electron microscopy.
Chemokine assay in colonic mucosa.
TNF--stimulated chemokines IL-8, monocyte-chemoattractant
protein-1 (MCP-1), and RANTES release by the colonic mucosa ex vivo in
the supernatant were assayed separately by ELISA according to
manufacturer’s protocol (Quantikine; R&D Systems). To account for
individual variation in the sampling size of the colonic strips,
chemokine release was expressed as picograms of chemokine per milligram
of protein, the latter being determined by the standard Bradford
protein assay (Bio-Rad, Hertfordshire, U.K.).
Histological assessment and apoptosis in colonic mucosa. At the end of the cytokine stimulation experiments, the colonic strips were processed for 1) H&E staining of paraffin-embedded sections or 2) electron microscopy on glutaraldehyde-fixed sections. The architectural integrity of the epithelial cells and crypt cells was assessed on H&E-stained slides.
Apoptosis of the surface colonocytes was defined as cells displaying the classical stigmata of cytoplasmic condensation and nuclear fragmentation on H&E-stained sections. The apoptotic count was taken as the average number of apoptotic epithelial cells per five high-power fields under light microscopy. The final figure was the average of two apoptotic counts assessed by an experienced pathologist blinded to the experimental conditions.
For electron microscopy, colonic strips were fixed in glutaraldehyde followed by osmium tetroxide and embedded in epoxy resin using routine methods. Sections (50-nm thick) were stained with uranyl acetate and lead citrate and examined in a JEOL 2000 transmission electron microscope.
Caspase 3 immunohistochemistry.
To study the potential role of caspase 3, a cell death protease
involved in apoptosis, on the observed TNF--induced colonocyte
apoptosis, immunohistochemistry was performed on paraffin-embedded
sections of colonic mucosa as previously described (25).
Briefly, 4- to 5-µm thin sections of paraffin-embedded sections of
colonic mucosa were cut and placed on polylysine coated slides.
Following deparaffinization, endogenous peroxidase activity was
quenched by 3% hydrogen peroxide in methanol. Nonspecific staining was
reduced by preincubation with normal blocking serum. The sections were
then incubated with the primary Ab CPP32 p20 (N19), a goat polyclonal
IgG (Santa Cruz Biotechnology, Santa Cruz, CA), for 60 min at room
temperature. After washing, sections were incubated with a biotinylated
secondary Ab for 30 min, followed by Avidin Biotin enzyme reagent and
DAB chromogen, a peroxidase substrate (ABC staining system; Santa Cruz
Biotechnology). The sections were then counterstained with methyl green
and mounted with DPX mountant (neutral solution of polystyrene
and plasticizers in xylene) for microscopic assessment. Sections of
human prostate gland with benign prostatic hypertrophy served as
positive control for caspase 3 staining.
Ethics approval
This study was approved by the Ethics Committee of the Mater Misericordiae Hospital, Dublin, Ireland. Written informed consent for the harvesting of colonic mucosa was obtained from all patients preoperatively.
Statistical analysis
Data are expressed as mean ± SEM and were compared using Student’s t test. Statistical significance was considered when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on
neutrophil adherence and transmigration
Incubation of the T84 monolayers with TNF- (100 ng/ml; 24
h at 37°C) enhanced neutrophil adherence (69.5 ± 4.6 vs
9.6 ± 2.8%, control; p < 0.001;
n = 6) via an IL-8- and ICAM-1-independent mechanism
(Fig. 1
). Conditioning of T84 with
TNF- was not associated with alteration in TER (1467 ± 77.9 vs
1535 ± 132.3 .cm2; TNF vs control;
p = 0.67; n = 6), epithelial cell
viability, or apoptosis. Similarly, neutrophil adherence to
TNF--activated T84 monolayers did not affect TER (1465 ± 72.1
vs 1247.4 ± 120.8 .cm2; pre- and
postneutrophil adherence; p = 0.63; n
= 4).
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) in keeping with previous reports
(6, 7, 8). Incubation of T84 with TNF- did not induce
neutrophil transmigration, but did prime monolayers such that the fMLP
response was enhanced (40.2 ± 2.9 vs 59.5 ± 3.7%,
respectively, n = 4; p < 0.05).
Transmigration was not further observed with TNF- in studies
examining extended experimental periods up to 4 and 6 h.
Incubation of epithelial monolayers with IL-1 (100 ng/ml; 24 h)
failed to induce neutrophil adherence or transmigration and was not
studied further (data not shown).
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-induced neutrophil adherence: in vitro
evidence for direct LX-epithelial cell interaction
LXA4 did not affect basal neutrophil
adherence or transmigration (Fig. 1
). Prior exposure of both the
basolateral and apical aspects of T84 epithelial monolayers to
LXA4 (10 nM for 20 min) significantly attenuated
TNF--induced neutrophil adherence during subsequent coincubations
(percent inhibition = 53.5 ± 4.5%; p =
0.003; n = 3; Fig. 1) and blunted fMLP-induced
transmigration across TNF--activated monolayers (59.5 ± 3.7 vs
15.8 ± 8.7% + LXA4; p <
0.05; n = 4). These actions were shared by
15-(R/S)-methyl-LXA4 and
16-phenoxy-LXA4 at nanomolar concentrations (Fig. 1). In parallel experiments, pretreatment of human neutrophils with
15-(R/S)-methyl-LXA4 (10 nM; 15 min)
significantly attenuated TNF--induced neutrophil adherence
(76.6 ± 6.2% control vs 45.2 ± 5.4% + LX;
p < 0.005; n = 3).
The dose-response relationship among the three LX compounds (0.1–100
nM dose range) and TNF--induced neutrophil adherence was determined
(Fig. 3). Maximal inhibition of
neutrophil adherence by native LXA4 was observed
at 10 nM (53.5 ± 4.5%; p < 0.005;
n = 3). At 10 nM, inhibition of neutrophil adherence by
15-(R/S)-methyl-LXA4 and
16-phenoxy-LXA4 was 58.2 ± 7.4 and
52.5 ± 7.5%, respectively; p < 0.005 and
p < 0.05, respectively; n = 3). For
the dose range studied, the most potent analog was
15-(R/S)-methyl-LXA4 with maximal
inhibition of neutrophil adherence at 100 nM (65.3 ± 3.6%;
n = 3; p < 0.001) and statistically
significant inhibition from 1 to 100 nM. At 0.1 nM, no statistically
significant effect on TNF--induced neutrophil adherence was observed
for all three compounds.
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on chemokine release by intestinal mucosa ex
vivo: modulation by LXs
TNF- (20 ng/ml) significantly enhanced release of MCP-1, IL-8,
and RANTES by colonic strips ex vivo (Fig. 4). RANTES was detected in much smaller
quantities relative to the other two chemokines but was also
induced by TNF-. The influence of LXs on TNF--stimulated
chemokine release was examined by pretreatment of the colonic mucosa
strips with LX for 20 min before the addition of cytokine. At a 10-nM
concentration, 15-(R/S)-methyl-LXA4
and 16-phenoxy-LXA4 significantly attenuated
TNF--stimulated IL-8 production from 5102.8 ± 528.3 to
2617.9 ± 124.4 and 1783.8 ± 241.4 pg/mg, respectively
(p < 0.005 and p < 0.05,
respectively, n = 4) (Fig. 5). Native LXA4 at
10 nM attenuated IL-8 production, although this did not reach
statistical significance (5102.8 ± 528.3 vs 1728.9 ± 711.1
pg/mg; p = 0.07; n = 3) (Fig. 5).
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Although lower levels of RANTES were produced relative to the other chemokines, a trend toward attenuation by LX was observed. Statistically significant attenuation was only seen with the stable analogs at maximal dose, i.e., 100 nM. The effect of LXs on RANTES production was as follows: 1) LXA4: 91.4 ± 24.4 vs 292.5 ± 95.8 pg/mg (control) (n = 3; p = 0.16), 2) 15-(R/S)-methyl-LXA4: 128.4 ± 32.1 vs 389 ± 63.3 pg/mg (control) (n = 3; p < 0.05), and 3) 16-phenoxy-LXA4: 175.7 ± 37.8 vs 389 ± 63.3 pg/mg (control) (n = 3, p < 0.05).
A representative dose-response relationship between LX concentration
and inhibition of TNF--stimulated chemokine release (RANTES) is
shown in Fig. 6. A similar relationship
was observed for the other chemokines.
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on colonic mucosal architecture and
modulation by LX
In separate experiments, the colonic mucosal strips were fixed,
following exposure to TNF- ± LXs, in 10% formalin,
paraffin-embedded, and stained with H&E. At the end of the 8-h
experimental period, colonic mucosa under basal unstimulated condition
(vehicle control) exhibited relative preservation of the surface
epithelial, crypt, and lamina propria architecture (Fig. 7A).
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(20 ng/ml; 8 h) induced significant
distortion in mucosa architecture of the surface colonocytes, crypt
cells, and the lamina propria (Fig. 7B). Some of the surface
enterocytes have begun to slough off the basement membrane and appeared
necrotic. Apoptosis was also increased (see below). Treatment with the
LXA4 analog
15-(R/S)-methyl-LXA4 (10 nM for 20
min) before the addition of TNF- conferred cytoprotection against
TNF--induced structural disruption (Fig. 7C). This
protective effect was similarly observed with
16-phenoxy-LXA4 and native
LXA4 (10 nM) (data not shown).
Effect of TNF- on apoptosis of surface enterocytes and
modulation by LX
Incubation of the colonic strips with TNF- (20 ng/ml; 8 h)
was associated with increased colonocyte apoptosis (apoptotic count of
surface colonocytes: 38 ± 4 TNF- vs 5 ± 1 control;
n = 3; p < 0.005). To investigate
whether the observed TNF--stimulated apoptosis of surface
enterocytes was caspase 3-dependent, paraffin-embedded sections of
TNF--stimulated colonic mucosa was processed for caspase 3
immunohistochemistry (described in Materialsand
Methods). In contrast to the strong (grade 4) staining seen in
human prostate with benign prostatic hypertrophy, which served as
positive control, cytoplasmic or nuclear caspase 3 immunostaining was
not observed in the TNF--stimulated colonic mucosa.
Prior exposure of colonic strips to
15-(R/S)-methyl-LXA4 (10 nM; 20 min)
significantly attenuated this TNF--stimulated apoptotic response
(apoptotic count 18 ± 3 LX vs 38 ± 4; TNF-;
p < 0.005; n = 3; Fig. 8A).
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-stimulated intestinal mucosa.
The classical appearance of TNF--stimulated apoptotic colonocytes is
shown in Fig. 8B. Pretreatment with
15-(R/S)-methyl-LXA4 at 10 nM for 20
min conferred cytoprotection and relative ultrastructural preservation
that approximated that of control tissue. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated
neutrophil adherence and transmigration in an in vitro model of
neutrophil trafficking across intestinal epithelia and 2)
TNF--induced chemokine production, architectural disruption, and
colonocyte apoptosis in an ex vivo model of intestinal inflammation
involving human colonic mucosa.
Using the well-characterized T84 cell line in an in vitro model of
intestinal inflammation, we have demonstrated that TNF- activation
of these cells promotes adherence of neutrophils via an IL-8 and
ICAM-1-independent mechanism and further enhanced fMLP-directed
neutrophil transmigration. The neutrophil 2
integrin CD11b/CD18 is required for neutrophil transepithelial
migration (26). However, its ligand mediating
neutrophil-endothelial adhesion, ICAM-1, is apically expressed in the
intestinal epithelium (26) and therefore does not interact
with CD11b/CD18 during the transmigration process (2, 5, 26). Indeed, neutralizing Ab to ICAM-1 failed to inhibit
fMLP-directed neutrophil transepithelial migration (2) and
TNF--stimulated neutrophil adherence in vitro. The basolaterally
expressed IL-8 may act as chemotactic signal for neutrophil trafficking
across the vascular endothelium and subepithelial matrix (2, 27), and is probably not crucial for neutrophil-epithelial
interaction. The precise molecular mechanisms underlying
TNF--promoted neutrophil adherence remain to be elucidated, and
candidate molecules include the neutrophilic CD11b/CD18 and epithelial
CD47 (2, 5, 26). In contrast to alteration of epithelial
barrier function following neutrophil transmigration (6, 28), TER was not affected by TNF--promoted neutrophil
adherence. This may infer that the initial neutrophil adherence process
elicited by TNF- in the absence of a chemotactic gradient may
involve interaction between neutrophils with the basement membrane or
basolateral membrane of the enterocytes. Further transit of the
neutrophils across the paracellular space cannot take place unless
other cofactors are present, such as chemoattractants in the lumen.
Indeed, fMLP-directed neutrophil transmigration was more efficient
following TNF- conditioning of the epithelia. TNF-, at the dose
and duration studied, was not associated with alteration in T84
epithelial barrier function, basal viability, and apoptosis rates.
Dynamic cross-talk between intestinal epithelial cells and
immune cells of the lamina propria results in the production of
chemokines that serve to recruit inflammatory cells, a feature central
to the pathogenesis of IBD (3, 4, 9). Increased levels of
chemokines have been detected in IBD tissues and in vitro models of
intestinal inflammation (9, 29, 30, 31). It has previously
been shown that IL-1, TNF-, and IFN-
stimulated
IL-8, MCP-1, andRANTES mRNA expression and secretion from HT29
and Caco-2 cells, both human colonic epithelial cell lines (11, 29, 30, 31). Furthermore, T84 cells also secrete a wide array of
chemokines in response to cytokine stimulation and bacterial infection
(10). In vivo studies showed that cellular sources of
MCP-1 include lamina propria macrophages, endothelial cells, spindle
cells, and intestinal epithelial cells (11). Colonic
mucosa constitutively express MCP-1 and the C-X-C chemokine, ENA-78,
which are up-regulated during inflammation (11, 32).
However, this model system did not distinguish the cells of origin of
the chemokines studied. We demonstrated that TNF- stimulated the
release of IL-8, MCP-1, and RANTES over an 8-h period from normal
colonic mucosa. The low level of RANTES detected in the time course
studied relative to IL-8 and MCP-1 suggested a differential effect of
cytokine stimulation on chemokine release.
Absence of autolysis and preservation of normal colonic architecture
and cellular ultrastructure (electron microscopy data not shown)
were demonstrable in the tissue maintained under vehicle control
conditions. The observed architectural and structural damage of
intestinal mucosa induced by TNF- did not resemble that seen
classically in infective colitis or IBD. Importantly, apoptosis of
surface colonocytes appeared to be a cardinal feature. The influence of
cytokines on colonocyte apoptosis has been examined in in vitro studies
involving intestinal cell lines. For instance, TNF- induces
apoptosis in certain intestinal epithelial cell lines such as HT-29
(33, 34), but not in others such as T84 cells. In
addition, antagonism of TNF- and NO inhibit apoptosis of HT-29 cells
following infection with Salmonella (34). In
the presence of TNF- alone or in synergy with IFN-, HIEC, a
nontransformed human intestinal epithelial cell line, become highly
susceptible to Fas-induced apoptosis (35). This enhanced
sensitivity is mediated via TNF-- and IFN--induced up-regulation
of Fas expression, and the effect of TNF- on Fas is mediated via the
p55 TNFR. Similarly, in colonic organ cultures, IFN- and TNF-
also enhance colonocyte Fas expression, resulting in markedly enhanced
apoptotic response to stimulation of this receptor
(35).
Because TNF--induced chemokine production is NF-
B-mediated
(36, 37) and NF-B is generally considered
anti-apoptotic (38, 39), the concurrent finding of
TNF--mediated apoptosis and chemokine production is surprising. In
this regard, we postulate that TNF- induced enterocyte apoptosis via
activation of the classical TNFR death domain mechanism, but the
influence of NF-B remains unclear. Although it is intriguing that
this form of apoptosis is not associated with activation of caspase-3,
one of the key enzymes involved in the lethal proteolytic cascade of
apoptosis execution, our finding complemented findings by Ruemmele et
al. (40) that caspase 3 is not activated in
TNF--induced apoptosis in another intestinal cell line, IEC-6.
The anti-inflammatory properties of LXs have been most extensively
described with reference to neutrophil function. These include
attenuation of neutrophil chemotaxis, adhesion to endothelial cells,
transendothelial and transepithelial migration, degranulation,
superoxide anion generation, and neutrophilic expression of L-selectin
and CD11/CD18 expression (8, 15, 16, 17, 18, 41). In this context,
Hachicha et al. (41) provided the first in vivo example of
LX-induced cytokine regulation by demonstrating that
LXA4, 15-epi-LXA4, and its
analogs are potent regulators for gene expression of individual
cytokines relevant in inflammation. Godson et al. (22)
recently showed that LXs also stimulate human macrophages to
phagocytose apoptotic neutrophils. The identification and cloning of
human nonmyeloid LXA4 receptors on intestinal
epithelial cells (21), synovial fibroblasts
(20), and mesangial cells (42) infer a
potential local immunoregulatory role of LXs in these organ systems. In
the gastrointestinal tract, direct incubation of intestinal epithelial
cells with LXs results in attenuation of TNF--induced IL-8 secretion
by T84 cells (21) and inhibition of S.
typhimurium-induced secretion of IL-8 and pathogen-elicited
epithelial chemoattractant by model intestinal epithelial cells
(23). In the current study, we show that LX-epithelial
interactions are potent inhibitors of TNF--stimulated neutrophil
adherence and blunt fMLP-directed neutrophil transmigration across
TNF--primed epithelium. There are few agents that display potency in
neutrophil-epithelial cell interactions within the 1- to 10-nM range.
In addition, this potency range confirms and is consistent with
observations in the literature (8, 16, 19).
In addition to the available in vitro data, we have shown that
incubation of human colonic mucosa with LXs results in dose-dependent
attenuation of TNF--induced release of IL-8, MCP-1, and RANTES by
human colonic mucosa ex vivo. This bioactivity was shared by
LXA4, 15-epi-LXA4, and LX analog and
observed at nanomolar doses. Although this model of intestinal
inflammation excludes recruitable circulating leukocytes, participation
from the resident inflammatory cells in the lamina propria cannot be
ruled out. Our results from the present study with human colonic
mucosal tissue ex vivo demonstrate that these compounds, namely
LXA4 and the 15-epi-LXA4 stable
analogs, are potent inhibitors of key responses of interest in mucosal
inflammation in an explant tissue milieu that is biologically relevant.
Our data enhance earlier findings with isolated cell types in vitro
(21, 23) and confirm and extend these earlier results.
Furthermore, the observation that LXA4 and the
15-epi-LXA4 are potent inhibitors of RANTES release is an
important addition to the bioactivity profile of these compounds and
suggests a role for LXs as potential therapeutic modifiers of other
diseases in which RANTES dysregulation plays an important role, such as
allograft rejection (43), atopy (44), and
viral infections (45). The net effect of LX-mediated
attenuation of neutrophil adherence and chemokine production may
translate into a reduction in the recruitment of inflammatory cells and
down-regulation of intestinal inflammation.
Aspirin and nonsteroidal anti-inflammatory drugs cause upper
gastrointestinal pathology including inflammation and ulceration on one
hand while may be protective against lower gastrointestinal
carcinogenesis on the other (46, 47). Aspirin acetylation
of cyclooxygenase-2 stimulates the production of
15(R)-hydroxyeicosatetraenoic acid from arachidonic acid in
endothelial cells, which may then be converted to ATLs (including
15-epi-LXA4 and 15-epi
LXB4) by neutrophil 5-lipoxygenase in the context
of cell-cell interaction (14). ATLs share many
anti-inflammatory properties of LXs and may account for much of the
favorable bioactivity profile of aspirin. In this context, we have
demonstrated that LXA4 and
15-epi-LXA4 ameliorated TNF--induced intestinal mucosal
structural disruption and colonocyte apoptosis. This observed
cytoprotective effect of 15-epi-LXA4 may seem at variance
with reports of apparent exacerbation of IBD in patients ingesting
nonsteroidal anti-inflammatory drugs (48). A potential
cytoprotective effect of LXA4 and
15-epi-LXA4 may be due to combination of inhibition of 1)
neutrophil-mediated tissue destruction through attenuation of
neutrophil recruitment and 2) cytokine-stimulated architectural
destruction and enterocyte apoptosis through mechanisms as yet unknown.
The ability of LXs to antagonize a wide array of TNF--mediated
inflammation as diverse as neutrophil activation (41),
chemokine release, and noninflammatory events such as apoptosis may
infer interaction with TNF- at an upstream level. Further studies
are needed to examine whether this interaction occurs at the receptor
binding, early signal transduction pathway, or nuclear transcription
level.
In summary, we have shown that LXA4 and
15-epi-LXA4 inhibited TNF--induced neutrophil-intestinal
epithelial cell interaction in vitro, chemokine release and enterocyte
apoptosis, and conferred cytoprotection in human intestinal mucosa ex
vivo. We also provided further evidence in support of
anti-inflammatory mechanisms of LXs that are mediated through their
direct interaction with epithelial cells independent of their
antineutrophil property. Furthermore, the counterregulatory role of LXs
in intestinal inflammation may in part be mediated through antagonism
of the proinflammatory actions of TNF-. Collectively, our data
support the important roles of the intestinal epithelial cells and LXs
in the regulation of immune function within the intestinal mucosa. The
accumulating evidence in favor of LXs as anti-inflammatory
eicosanoids in intestinal inflammation should provide the rationale for
the evaluation of their efficacy and safety as potential therapeutic
agent in IBD.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Padraic MacMathuna, Department of Gastroenterology, Mater Misericordiae Hospital, Eccles Street, Dublin 7, Ireland. E-mail address: gicancer{at}mater.ie
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; LX, lipoxin; LXA4, 5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; ATL, aspirin-triggered 15-epi-LX; 15-(R/S)-methyl-LXA4, 15-(R/S)-methyl-11,12-dehydro-LXA4 methyl ester; 16-phenoxy-LXA4, 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA4 methyl ester; MCP-1, monocyte-chemoattractant protein-1; TER, transepithelial resistance.
Received for publication January 26, 2001. Accepted for publication June 7, 2001.
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