By
From the * Center for Experimental Therapeutics and Reperfusion Injury, Epithelial cells of the alimentary tract play a central role in mucosal immunophysiology. Pathogens and/or agonists that interact with mucosal surfaces often elicit epithelial responses that upregulate inflammation. Therefore, it was of interest to explore potential epithelial targeted antiinflammatory signals. Here we identified and sequenced a human enterocyte lipoxin (LX) A4
[5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid] receptor, and demonstrate that transcription of this receptor was controlled by cytokines, of which lymphocyte-derived interleukin (IL)-13 and interferon Epithelial cells that line the alimentary tract serve as the
barrier separating the lumen from underlying tissues. It
is now apparent that enterocytes play a critical role in mucosal immunophysiology that, in part, consists of a paracrine network between enterocytes and the underlying
immune and inflammatory cells (1). This view is supported by the observation that the release of lymphocyte-derived mediators alters enterocyte phenotype and function
as evidenced by the impact of IFN- Enterocytes can contribute to the regulation of mucosal
immune responses by releasing cytokines and chemokines
that can in turn activate and recruit inflammatory as well
as immune cells to the mucosa. For example, TNF- It is therefore of interest to elucidate regulatory signals
that could attenuate mucosal immune functions. In this
regard, endogenously generated lipoxin A4 (LXA4: 5(S),
6(R),15(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic
acid)1 is of interest because it inhibits the action of proinflammatory stimuli both in vitro and in vivo (for review see
reference 9). LXA4 is a potent inhibitor of both neutrophil
adhesion and transmigration across endothelia or epithelia.
It is then of special interest that acetylsalicylic acid (aspirin)
triggers the biosynthesis of a recently discovered novel
pathway that leads to transcellular production of 15-epi-LXA4, since enterocytes are probably among the first cell
types to encounter orally administered aspirin. This transcellular route involves acetylation of cyclooxygenase (COX) II by aspirin in both human endothelia and lung
epithelia (10, 11) and formation of 15-epi-LXA4 by neighboring neutrophils. The C-15 alcohol of 15-epi-LXA4 is in
the rectus (R) configuration, rather than sinister (S) as in native LXA4. This switch in chirality enhances its bioactivity as
well as resistance to metabolic inactivation (9). The inhibitory actions of LXA4 and the aspirin-triggered 15-epi-LXA4
provided an intriguing opportunity to develop stable LXA4
and 15-epi-LXA4 analogs. These analogs were recently
evaluated in a mouse in vivo inflammation model and proved
to be topically active, and also more potent inhibitors of
LTB4-initiated inflammation than was the well-known antiinflammatory steroid, namely dexamethasone (12).
The bioactions of LXA4, 15-epi LXA4, and LXA4 stable
analogs are transduced by a high affinity myeloid G protein-coupled receptor that has been sequenced and cloned
for both mouse (12) and human leukocytes (13, 14). In addition, LXA4 actions with vascular endothelial and mesangial cells are mediated via a distinct nonmyeloid receptor
that remains to be cloned (9). In the gastrointestinal tract,
COX II upregulation is associated with human colorectal adenocarcinomas (15, and see reference 16 for review),
mucosal lesions (17), colitis (18), and acute pathogen invasion (19). This localization of the enzyme and its regulation
can provide a strategic in vivo milieu "primed" for the biosynthesis of aspirin-triggered 15-epi-lipoxins.
Since enterocytes are in close proximity to cell types of
the immune system and are among the first cell types to encounter orally administered aspirin, the actions of 15-epi-LXA4 on these cells are of potential interest. Also, in view
of cytokine-mediated upregulation of epithelial immune
function and its associated increase in IL-8 release in human ulcerative colitis (20) and Crohn's colitis (21), as well
as increased levels of TNF- Materials.
Division of Gastrointestinal Pathology,
Abstract
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
were the most potent. When lipoxins and LXA4
stable analogs were evaluated for enterocyte functional as well as immune responses, lipoxins
sharply inhibited TNF-
-induced IL-8 release but did not alter either barrier function or agonist-stimulated chloride secretion. 15R/S-methyl-LXA4 and 16-phenoxy-LXA4 each attenuated (IC50 ~10 nM) IL-8 release. Cyclooxygenase (COX) II is emerging as an important component in wound healing and proliferation in intestinal epithelia and when acetylated by
acetylsalicylic acid (aspirin) initiates the biosynthesis of a LXA4 receptor ligand. We therefore
determined whether colonic cell lines (HT-29 Cl.19A, Caco-2, or T84) express the COX II
isozyme. Results for RT-PCR and Western blot analysis showed that COX I as well as an IL-1
- and TNF-
-inducible COX II are expressed in HT-29 Cl.19A. In addition, aspirin-treated enterocytes generated 15R-HETE, a precursor of 15-epi-LXA4 biosynthesis, whose
potent bioactions were mimicked by the stable analog 15R/S-methyl-LXA4. Taken together,
these results identify an endogenous pathway for downregulating mucosal inflammatory events
and suggest a potential therapeutic benefit for LXA4 stable analogs.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
on human enterocytes in vitro. IFN-
attenuates barrier function and agonist-stimulated chloride secretion as well as induces expression of MHC class II molecules for enterocyte immune
accessory functions (1, 6). In addition, lymphocytes can
convert enterocytes into M cells that transport antigen to underlying immune cells, an action constituting an important component of oral vaccination and immunization (4).
or
pathogens can induce release of a potent leukocyte chemoattractant, namely IL-8, by intestinal epithelial cell lines as
well as freshly isolated human colon enterocytes (7, 8).
Only pathways that upregulate the inflammatory/immune response are recognized to date in agonist- or pathogen-stimulated enterocytes (for review see reference 1).
(22), it was of interest to elucidate endogenous signals that might attenuate mucosal inflammatory responses. To this end, we identified the first
nonmyeloid LXA4R in human enterocytes, and we demonstrate here that expression of this receptor is regulated by
cytokines and that stable analogs of LXA4 and aspirin-triggered 15-epi-LXA4 are potent inhibitors of agonist-induced
IL-8 secretion by enterocytes. These results provide the
first evidence that LXA4R gene expression is associated
with immune functions of human enterocytes.
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, rIL-4, rIL-6,
rIL-13, and rIFN-
were purchased from R&D Systems (Minneapolis, MN). Dulbecco's PBS (DPBS), DMEM, and Ham's F-12
were obtained from Bio Whittaker (Walkersville, MD) and
HBSS was from GIBCO BRL (Gaithersburg, MD). First strand
cDNA synthesis kit was purchased from Promega (Madison, WI),
and all other molecular biology reagents were obtained from
Boehringer Mannheim Corp. (Indianapolis, IN). Human colonic epithelial cell lines (Caco-2 and T84) and alveolar epithelial cells
(A549) were purchased from the American Type Culture Collection (Rockville, MD), and HT-29 Cl.19A were generously provided by Dr. Laboisse (Universite de Nantes, Nantes, France).
Oligonucleotide primers were purchased from Integrated DNA
Technologies (Coralville, IA). Sep-Pak C18 cartridges were obtained from Alltech (Deerfield, IL). Methyl formate was purchased from Eastman Kodak Co. (Rochester, NY). Hexane and
all HPLC solvents were purchased from J.T. Baker (Phillipsburg,
NJ). Calcium ionophore (A23187) and all other reagents were
obtained from Sigma Chemical Co. (St. Louis, MO).
Epithelial Cell Lines and Culture.
Human colonic adenocarcinoma cell lines were grown and passaged in culture conditions as previously described for T84 (24), Caco-2 (25), and HT-29 Cl.19A (26) at 37°C in an atmosphere of 5% CO2. In brief, T84 cells were propagated in 1:1 mixture of DMEM and Ham's F-12 medium supplemented with 14 mM NaHCO3, 40 mg/liter penicillin, 9 mg/liter streptomycin, 8 mg/liter ampicillin, 5% newborn calf serum, and 15 mM Na+ Hepes buffer, pH 7.5. HT-29 Cl.19A and Caco-2 were propagated in DMEM containing a standard (4.5 gram/liter) glucose concentration and supplemented with 14 mM NaHCO3, 40 mg/liter penicillin, 9 mg/liter streptomycin, 8 mg/liter ampicillin, 10% fetal bovine serum, and 15 mM Hepes buffer, pH 7.5. The human lung adenocarcinoma cell line A549 was grown and passaged as in reference 11. Polarized colonic epithelial cells were split near confluency by incubating cells with 0.1% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free DPBS for 5-20 min. Cells were diluted in media alone or in media containing IL-1Reverse Transcription and PCR.
Total RNA from colonic (T84, Caco-2, HT-29 Cl.19A) or alveolar epithelial cells (A549), which were exposed to either IL-1Identification and cDNA Cloning of the Intestinal Epithelial LXA4R.
Total RNA was isolated from T84, HT-29 Cl.19A, Caco-2, or A549 as described above, and 0.5 µg of RNA was reverse transcribed. Oligonucleotide primers, sense primer 5'-CACCAGGTGCTGCTGGCAAG-3' and antisense primer 5'-AATATCCCTGACCCCATCCTCA-3', were designed to amplify the published human myeloid LXA4R coding region (~1.1 kb; reference 12). Amplification protocols, using the high fidelity polymerase PCR system Expand (Boehringer Mannheim Corp.), consisted of 35 repetitive cycles of denaturing at 94°C (30 s), annealing at 64°C (45 s), and extension at 72°C (80 s). In parallel analysis, oligonucleotide primers designed for the recently sequenced myeloid LTB4 receptor (32), sense primer 5'-GGCAAGCTTATGAACACT ACATCTTCT-3' and antisense primer 5'-GGCAGGACCTCTAGTTCAGTTCGTTTA-3', were used to amplify the LTB4 receptor coding region (~1.1 kb). The amplification protocol, using vent (New England Biolabs, Beverly, MA) as the polymerase, consisted of 35 repetitive cycles of denaturing at 98°C (60 s), annealing at 60°C (60 s), and extension at 72°C (70 s). PCR analysis of LXA4R demonstrated a single PCR product from T84 cDNA with an apparent size of ~1.1 kb, which was subcloned into pBluescript KS(+), and three independent clones were isolated and selected for analysis at the Children's Hospital sequencing facility (Boston, MA). The following oligonucleotide primers were used to sequence the 1,053-bp coding region of the human enterocyte LXA4R: nucleotides 0-264 [pBluescript KS (+) reverse primer] and 884-1053 (M13-20 sense primer); nucleotides 210-631 (5'-ATCTGTTACCTGAACCTGGC-3') and 514-914 (5'-GTCTCTCTCGGAAGTCTTGG-3'); and nucleotides 477- 907 (5'-TTGCTCTAGTCCTTACCTTGC-3') and 461-818 (5'-GATGTCAATGATTTTGTACTTGC-3').Northern Blot Analysis of Enterocyte LXA4R Messenger RNA.
Confluent T84 cells (resistance >600Barrier Function and Chloride Secretion in Human Enterocytes.
Polarized monolayers of T84 (resistance >600TNF--induced IL-8 Release by Human Enterocytes.
Western Blot Analysis of COX I and II Proteins in Human Enterocytes.
Subconfluent HT-29 Cl.19A were lifted with trypsin/ EDTA and resuspended in media containing IL-1Analysis of Mono-HETE Generation by Human Enterocytes.
Confluent HT-29 Cl.19A cells that were exposed for 24 h to either media alone or to both IL-1Statistical Analysis.
Unless otherwise indicated, all values are represented as mean values ± SEM. Results were analyzed using Student's t test. Differences were considered significant at the P <0.05 level. ![]() |
Results |
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To elucidate potential bioactions of LXA4 and 15-epi-LXA4 with human enterocytes, we first sought to determine whether LXA4Rs are present in human colonic epithelia using established model cell lines. Reverse transcription (RT)-PCR analysis of enterocyte-derived cDNA demonstrated a single band with the expected size of ~0.7 kb present in T84, HT-29 Cl.19A, and Caco-2. It is of interest to note that another epithelial cell line from the airway, A549, did not show appreciable levels of the LXA4R cDNA (data not shown). To determine the sequence homology of the human enterocyte LXA4R and those of the human and mouse myeloid receptors (9, 12), specific LXA4 primers were designed to amplify the complete coding region (~1.1 kb; see Materials and Methods). T84 cells gave an ~1.1 kb PCR product that was isolated, cloned, and sequenced. Three independent clones contained a 1,053-bp cDNA that was identical to the human myeloid LXA4R. Of interest, RT-PCR analysis did not provide evidence for expression of the recently identified human myeloid LTB4 receptor in these particular epithelial cell lines. These findings clearly demonstrated that the LXA4R is not only expressed in enterocytes but that it is conserved in both human leukocytes and enterocytes. The cDNA sequence of the human enterocyte LXA4R is available from EMBL/ GenBank/DDBJ under accession number AF054013.
Selected cytokines were recently found to be modulators
of phenotype and enterocyte function (6, 27). To evaluate
whether LXA4R expression was altered by these cytokines,
Northern blot analysis of poly+ RNA was used to monitor
LXA4R transcription in T84 cells. Cytokines for which enterocytes bear functional receptors were chosen for evaluation (i.e., see Fig. 1) (6, 27). After 24 h, exposure to these
cytokines increased LXA4R mRNA levels compared with
those of cells that were exposed to media alone (Fig. 1).
Among the panel of cytokines evaluated, IL-1 and LPS
were the least potent cytokines, inducing a 1.8-fold increase in LXA4R mRNA, whereas exposure to either IL-4
or IL-6 increased receptor message levels by ~3-fold. The
most potent cytokines proved to be IFN-
and IL-13, which increased LXA4R mRNA by ~6.8- and 8.6-fold,
respectively. This pattern of cytokine selective upregulation
of LXA4R transcription was also observed for the 48-h interval. LXA4R mRNA levels increased in each experimental group from 24 to 48 h compared with those of the control group in which message levels remained unchanged. After 48 h of exposure, IL-1
and IL-4 increased LXA4R
message levels by a total of ~4.0- to 4.5-fold, whereas IL-6
gave an ~6.6-fold and LPS an ~7.3-fold increase. The
most potent of the cytokines examined proved to be IL-13
and IFN-
, as they increased LXA4R mRNA by 8.4- and
17.3-fold in 48 h when compared with the parallel controls. Similar patterns of selective regulation of the receptor message levels by these cytokines were also observed using
RT-PCR analysis of total RNA from T84 (data not
shown). Together these results indicate that IL-13 and
IFN-
each selectively increase LXA4R mRNA levels.
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Epithelial cells that line
the intestine perform a primary function as a selective barrier and secretory cell (1, 26). We next examined whether
LXA4 or LXB4 could have an impact on epithelial barrier
function and/or agonist-induced chloride secretion when
compared with the established effect of IFN- on enterocyte function (6). Neither LXA4 nor LXB4 altered T84
barrier function after 24 or 48 h of exposure when compared with vehicle alone (data not shown; n = 3). In addition, exposure to either LXA4 or LXB4 for 48 h did not
stimulate chloride secretion or alter agonist-activated (forskolin, 10 µM) chloride secretion in enterocytes when
compared with the vehicle control (data not shown; n = 3). Essentially similar findings were observed after 24 h of
exposure to either LXA4 or LXB4. Furthermore, results obtained with 15R/S-methyl-LXA4 and 16-phenoxy-LXA4
confirm these findings, since neither analog in a concentration range of 1 nM to 1 µM and an exposure of 30 min to
24 h altered these enterocyte functional responses (data not
shown; n = 3). Together these results indicate that LXA4R-mediated actions include neither modulation of epithelial
barrier function nor chloride secretion.
Intestinal epithelia constitutively express low levels of IL-8 that can be
markedly upregulated by pathogens and proinflammatory cytokines such as TNF-. This increase in basolateral IL-8
secretion is held to be important in PMN recruitment to
the epithelium (7, 8). To evaluate whether LXA4 and
LXA4 stable analogs modulate IL-8 release, T84 monolayers were stimulated with TNF-
, and IL-8 release was determined after epithelial exposure to LXA4 and/or stable
analogs. IL-8 secretion in untreated T84 cells ranged from less than the ELISA detection limit to ~20 pg/epithelial
cell monolayer. TNF-
, in the concentration range expected to use the high affinity receptor (Kd ~0.1 nM) (34),
induced epithelial IL-8 secretion that was significantly inhibited (P <0.006) by both 15R/S-methyl-LXA4 and 16-phenoxy-LXA4 (Fig. 2 A).
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15R/S-methyl-LXA4 inhibition of TNF--induced IL-8
secretion was concentration dependent (Fig. 2 B) and maximal inhibition (65 ± 14%) was observed at the highest
concentration tested, namely, 100 nM. 16-phenoxy-LXA4
at 100 nM proved to be even more potent, inhibiting agonist-induced IL-8 secretion by 82 ± 11% (Fig. 2 A). Furthermore, IL-8 secretion induced by 0.02 and 0.2 nM
TNF-
was also significantly inhibited (~50%) by 15R/S-methyl-LXA4 (data not shown; n = 3). Native LXA4 did
not exhibit a statistically significant effect on TNF-
-induced
IL-8 (Fig. 2 A), although a trend toward suppression of
IL-8 secretion was consistently noted. It is likely that a decrease in LXA4 concentration during the incubation (5 h)
brought about by metabolic inactivation may explain its diminished action compared with its stable LXA4 analogs. Stimulation of IL-8 secretion with higher concentrations of
TNF-
that were in the range of the low affinity TNF-
receptor (~0.5 nM) (34) led to greater IL-8 secretion that
was not subject to inhibition by 100 nM 15R/S-methyl-LXA4 (data not shown). These results indicate that LXA4
stable analogs are potent inhibitors of TNF-
-induced IL-8
release by intestinal epithelial cells and appear to selectively
inhibit signals transduced via the CD120b TNF receptor
(35, 36).
Upregulation of COX II in enterocytes, an enzymatic target for
nonsteroidal antiinflammatory drug action, is associated with intestinal inflammation and cell proliferation (16). To determine whether human enterocytes have the enzymatic
capacity to serve as a site or donor for aspirin-triggered 15-epi-lipoxin production, cDNA from untreated as well as
IL-1-primed enterocyte cell lines was examined for the
presence of COX II. In addition, we evaluated the presence
of the lipoxygenases 5-LO, 12-LO, and myeloid 15-LO, which are other components of the transcellular pathways
for LXA4 and 15-epi-LXA4 biosynthesis (for review see
reference 9). In the absence of cytokine stimulation, HT-29
Cl.19A and Caco-2, but not T84, expressed low levels of
COX II. Exposure to IL-1
enhanced COX II expression
in HT-29 Cl.19A cells, but no significant change in COX
II RNA levels was observed for Caco-2 cells. T84 cells did
not express appreciable amounts of COX II (Fig. 3). Indeed, the level of enterocyte basal and cytokine-induced
COX II mRNA was less than that observed with the airway epithelial cell line A549 (Fig. 3). It is of interest to
note that each of these colonic epithelial cell lines (HT-29
Cl.19A, Caco-2, and T84) expressed 5-LO mRNA to
some extent. In contrast, these enterocyte cell lines expressed neither 12-LO nor 15-LO RNA (data not shown). These findings indicate that human enterocytes (HT-29
Cl.19A and Caco-2) exposed to cytokines have the enzymatic capacity to generate COX II-derived eicosanoids.
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Do changes in COX II mRNA translate to
protein levels and active enzyme in cytokine-primed enterocytes? To achieve high levels of COX II expression,
HT-29 Cl.19A were treated with TNF-, a cytokine that
also induces COX II in a variety of cell types (1), or were
treated in combination with IL-1
. COX II protein levels were quantitated and compared with COX I by Western
blot. COX II protein was not observed with the untreated
cells but was induced by both IL-1
and TNF-
after 24 h
(Fig. 4 A). TNF-
-induced COX II levels were approximately twofold higher than IL-1
-induced protein levels.
Treating cells with both cytokines gave an approximately fourfold greater increase in COX II protein as compared
with IL-1
treatment alone. Extending exposure to these
cytokines from 24 to 48 h decreased COX II protein levels.
These findings indicate that upregulated COX II expression was transient. In addition, increasing TNF-
concentration from 50 to 100 ng/ml (TNF-
+ IL-1
exposed
for 24 h) induced COX II protein at levels that were
higher than those observed with IL-1
alone but less than
levels observed with 50 ng/ml of TNF-
alone or in combination with IL-1
(24 h of exposure). These findings are
consistent with the well-characterized cytotoxic effect of
TNF-
with tumor cells such as HT-29 (38, 39).
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For the purpose of direct comparison, COX I protein
levels were analyzed by Western blot. Results in Fig. 4 indicate that COX I was a highly abundant protein in HT-29
Cl.19A (note that protein concentrations were reduced by
10-fold in comparison to the COX II immunoblot to
achieve visualization of immunoreactive bands). COX I
was detected in untreated cells as well as in cytokine-primed enterocytes (Fig. 4 B). However, protein levels for
COX I were not significantly altered when these cells were
exposed to either IL-1 and/or TNF-
. Thus, in enterocytes, COX II levels were most effectively increased by
combined exposure to TNF-
and IL-1
. This "upregulation" of COX II was transient and did not extend to COX
I, whose protein levels were not significantly altered by exposure to these cytokines.
Do cytokine-primed enterocytes generate 15R-HETE when treated with aspirin? It was of interest to determine if enterocytes (HT-29 Cl.19A) that demonstrated cytokine-regulated COX II at both RNA and protein level (Figs. 3 and 4) also had the capacity to generate 15R-HETE when exposed to aspirin. To this end cytokine-primed enterocytes were incubated with aspirin, and lipid extracts were subjected to analysis using RP-HPLC. Aspirin-treated enterocytes gave a product that coeluted with authentic 15-HETE standard and displayed a characteristic conjugated diene UV chromophore with a maximum absorbance at 234 nm (Fig. 5, chromophore inset). 15-HETE production by cytokine-primed enterocytes exposed to aspirin increased by approximately threefold (Fig. 5), a finding that is consistent with the notion of aspirin-dependent 15R-HETE generation demonstrated in both endothelial cells (10) and airway epithelial cells (11). These findings are also consistent with results obtained with isolated recombinant COX II and 15R-HETE biosynthesis (40). Of interest, RP-HPLC profiles from permeabilized cytokine-treated enterocytes did not show 5-HETE generation, although we found message for this enzyme to be present. These results are consistent with previous studies that detected no 5-LO activity in intact HT-29, and only after sonication were small amounts of 5-LO products observed (41). Together, these findings indicate that cytokine-primed enterocytes in the presence of aspirin can generate 15R-HETE.
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Discussion |
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Our results demonstrate that human intestinal epithelial
cells express LXA4Rs and that the stable analogs of LXA4
and aspirin-triggered 15-epi-LXA4 attenuated TNF--
induced IL-8 release in human enterocytes. Also, lymphocyte-derived cytokines exhibiting potent actions on enterocyte function and phenotype (6, 27) were shown here for the first time to upregulate gene expression of this receptor. RT-PCR analysis demonstrated that the LXA4R is
expressed in established model cell lines of colonic epithelial cells. These cells form functional columnar epithelia
that resemble natural in vivo crypt and brush border epithelia (6, 25, 26, 42). Sequence analysis revealed that the
enterocyte LXA4R is identical to the human myeloid receptor (13, 14) and 76% homologous to the mouse leukocyte receptor (12). It is therefore of interest to note that
RT-PCR analysis of RNA from other epithelial tissues including human cornea also proved positive for the LXA4R
(data not shown), yet epithelial cells from the airway were
negative. The finding of LXA4R expression in human epithelial cells suggests conserved receptor function and provides support for the immunoregulatory role of enterocytes.
This conclusion is further supported by results from experiments with human neutrophils indicating that the LXA4R transduces signals that counterregulate proinflammatory
mediators such as LTB4 and FMLP both in vitro (9) and in
vivo (12).
The intestinal mucosa forms the most extensive barrier
separating the external environment from the internal milieu (for review see reference 1). Enterocytes are in communication with the interdispersed intraepithelial lymphocytes and submucosal inflammatory cells to fulfill their
function as an absorptive/secretory and immune accessory
cell. In this respect, enterocytes are considered a primary
lymphoid organ and a component of the mucosal immune system (for review see references 1 and 3). It is thus of particular interest that lymphocyte-derived cytokines upregulated gene expression of the epithelial LXA4R (Fig. 1).
These cytokines initiate enterocyte immune functions that
include antigen presentation, expression of secretory components, and transport of immunoglobulin A into the intestinal lumen. Cytokines associated with these phenotypic changes, namely IL-13 (27) and IFN- (6), were the most
potent in inducing LXA4R transcription. Of interest, IL-13
also induces 15-LO, an important enzyme in native lipoxin
biosynthesis (43).
Our results also suggest that signals transduced by the LXA4R are associated with the immune function of human enterocytes. Evidence to support this hypothesis is twofold. First, LXA4, LXB4, and stable LXA4 analogs did not alter enterocyte barrier function or cAMP-mediated chloride secretion, a finding consistent with previous results showing that exposure to LXA4 for 2 h did not impact monolayer integrity (44). This further distinguishes lipoxin A4 from the bioaction of prostaglandin E2, whose receptors and action have been characterized in human enterocytes and include stimulation of cAMP-mediated chloride secretion (45). Also, our results indicate that lipoxins do not activate secondary mediators such as prostaglandin or arachidonic acid release in enterocytes, since these compounds are known to stimulate chloride secretion (45, 46). Second, unlike the lack of impact on human enterocyte secretory and barrier function, LXA4 and stable LXA4 analogs attenuated epithelial immune function, namely the inhibition of IL-8 release.
In the mucosal milieu, enterocytes play an important
role in leukocyte recruitment by releasing basolaterally the
potent chemoattractant IL-8 (8), which is associated with
the pathogenesis of diseases such as Crohn's (21) and ulcerative colitis (20). The stable LXA4 analogs 15R/S-methyl-LXA4 and 16-phenoxy-LXA4 proved to be potent inhibitors of IL-8 release induced by the high affinity (CD120b)
TNF- receptor (Fig. 2), attenuating IL-8 release by as
much as 62 ± 14% and 82 ± 11%, respectively. Therefore,
these are the first results demonstrating that a lipid mediator
inhibits IL-8 release. These findings suggest a potentially
important new role for LXA4 as an endogenous "stop-signal" in the progression of an enterocyte-initiated inflammatory response.
COX II is expressed in human colonic tumors and adenocarcinoma epithelial cell lines (for review see references 1 and 16) as well as in normal epithelial tissue in acute bacterial infections (19), mucosal lesions (17), and colitis (18). Activation of this early response gene (e.g., COX II) is associated with both epithelial cell proliferation (for review see reference 16) and wound healing, and selective inhibition of COX II exacerbates colon injury in several in vivo models (1, 17, 18). It is of interest to point out that the analog 15R/S-methyl-LXA4, which inhibited enterocyte immune function (Fig. 2), is a structurally related mimetic of aspirin-triggered 15-epi-LXA4 (9). The observations that cytokines upregulated enterocyte COX II and that these cells (HT-29 Cl.19A) generate 15-HETE in the presence of aspirin are in agreement with results reported with both human vascular endothelial and lung epithelial cells (10, 11). Together, these in vitro findings suggest that, in scenarios where COX II is induced, enterocytes have the potential in vivo to augment the biosynthesis of 15-epi-LXA4, which may serve to inhibit further PMN accumulation during mucosal inflammation (Fig. 6).
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In summary, this is the first evidence demonstrating that a cytokine regulates LXA4R expression in intestinal epithelial cells, suggesting that the LXA4R is associated with enterocyte immune functions. In addition, our findings indicate that LXA4 directly modulates the initiation of inflammatory events by inhibiting the release of the potent chemokine IL-8 at the initial site of pathogen exposure, namely at the epithelial barrier. These results indicate that the potential for antiinflammatory actions of LXA4 in the gastrointestinal tract are multifaceted by acting on both myeloid cells to counterregulate proinflammatory mediators (Fig. 6 and for review see reference 9) and directly attenuating the initiation of inflammation by modulating IL-8 release from epithelia (Fig. 2). Thus, these findings with enterocytes and lipoxins expand the bioactivity of these endogenous lipid mediators and provide potential new uses for LXA4 stable analogs in mucosal immunobiology.
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Footnotes |
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Address correspondence to Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital and Harvard Medical School, 75 Francis St., Boston, MA 02115. Phone: 617-732-8822; Fax: 617-278-6957; E-mail: cnserhan{at}zeus.bwh.harvard.edu
Received for publication 10 November 1997 and in revised form 17 February 1998.
1Abbreviations used in this paper: 15-epi-LXA4, 5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid; 15 (R/S)-methyl LXA4, 5(S),6(R), 15(R/S)-trihydroxy-15-methyl-7,9,13-trans-11-cis-eicosatetraenoic acid; 16-phenoxy-LXA4, 16-phenoxy-17,18,19,20-tetranor-LXA4; aspirin, acetylsalicylic acid; COX, cyclooxygenase (PGHS); 15-HETE, 15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid; LO, lipoxygenase; LXA4, 5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid; LXA4R, lipoxin A4 receptor; RP-HPLC, reverse phase-HPLC.We thank Mary Halm Small for assistance in manuscript preparation.
These studies were supported in part by National Institutes of Health (NIH) grant GM-38765, a research grant from Schering AG (to C.N. Serhan), and NIH grants DK-47662 and DK-35392 (to J.L. Madara). K. Gronert is the recipient of a postdoctoral fellowship from the National Arthritis Foundation and A. Gewirtz is a recipient of an individual National Research Service Award.
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