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*
Department of Internal Medicine,
Division of Gastroenterology, and
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
, and
IL-1
. These findings, in conjunction with the known immunomodulatory
capacities of PGs, suggest that COX-2 expression by the small intestine
lamina propria is a basal state contributing to the hyporesponsiveness
of the intestinal immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COXs perform the committed step in the conversion of AA to PGH2, an intermediate metabolite in the pathway to the production of PGs and thromboxanes (TXs) by cell-specific reductases and isomerases (12). Mammalian COXs are known to exist in two isoforms encoded by distinct genes, COX-1, whose expression is constitutive, and COX-2, whose expression is predominantly inducible and transient (13, 14). COX-1 and -2 enzymes perform identical steps in the production of AA metabolites; however, their physiological roles are viewed differently, largely due to differences in the patterns of expression of these isoenzymes. COX-2-dependent AA metabolites are rapidly and transiently produced at sites of inflammation (15). This observation, along with the anti-inflammatory properties of selective COX-2 inhibitors, led to the concept that COX-2 and COX-2-dependent AA metabolites act predominantly as inflammatory mediators. Conversely, COX-1 and COX-1-dependent AA metabolites are largely viewed as having housekeeping functions based on their constitutive expression and production (16). These concepts are challenged by descriptions of constitutive COX-2 expression in the kidney (17), the brain (18), and in colorectal carcinomas (19, 20) and by descriptions of the immunomodulatory/anti-inflammatory roles of PGs (21, 22).
We have previously shown that the small intestine LP produces abundant
PGE2 in a COX-2-dependent manner
(11). To better understand the role of the COX-2-dependent
AA metabolites produced by the small intestine LP, we have identified
the source of COX-2-dependent AA metabolites and examined the pattern
of COX-2 expression in the small intestine LP. Here we report that
non-bone marrow-derived stromal cells in the murine small intestine
spontaneously and continuously express COX-2 and abundantly produce a
COX-2-dependent AA metabolite, PGE2. Development
and maintenance of this phenotype is not dependent on "typical"
proinflammatory mediators, including bacterial flora, LPS, TNF-,
IL-1, IFN-, or IL-12. We also demonstrate that that these cells
do not produce a soluble mediator with the ability to induce
PGE2 production in resident peritoneal
macrophages. These observations, combined with previous descriptions of
the immunomodulatory role of PGs, suggest that COX-2 expression and
COX-2-dependent PGE2 production by the murine
small intestine LP is a basal state establishing the tone of the
intestinal immune response to luminal Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice used for this study were 6–12 wk of age and, with the
exception of germfree mice, were housed in a specific pathogen-free
facility and fed routine chow diet. Germfree mice were housed in a
gnotobiotic facility and fed an elemental diet. At the time of
sacrifice, cecal cultures from germfree animals were obtained to
confirm the absence of bacterial flora. B10.BR/SgSnJ, C3H/HeJ,
C3H/HeSnJ, C57BL/6, TNFR II-deficient, and IL-12 p40-deficient mice on
the C57BL/6 background were purchased from The Jackson Laboratory (Bar
Harbor, ME). IFN-R-deficient mice (23) on the 129/SvImJ
background were a gift from R. Schrieber (Washington University School
of Medicine, St. Louis, MO). IL-1-deficient mice (24)
and TNFR I-deficient mice (25) on the C57BL/6 background
were a gift from D. Chaplin (Washington University School of Medicine).
Germfree and conventionally housed FVB/NJ mice were a gift from J.
Gordon (Washington University School of Medicine). COX-2-deficient mice
on a mixed C57BL/6 x 129/SvImJ background were a gift from R.
Langenbach (National Institute of Environmental Health Sciences,
Research Triangle Park, NC) (15).
Cell isolation and culture
LP cells, Peyer’s patch (PP) cells, and splenocytes (SPLs) were isolated as previously described (11). Briefly, single-cell suspensions were prepared from spleens, small intestines, and PPs by dispase and collagenase digestion. Resident peritoneal cells (PC) were harvested by flushing the peritoneal cavity with 15 ml ice-cold PBS. Cells were counted for viability by trypan blue exclusion. Viability was >75% for all cell populations. The typical yield for LP cell isolation was 2 x 107 viable cells/intestine, and the typical yield for resident PC was 5 x 106 viable cells/mouse.
Isolated cells were cultured in 96-well tissue culture plates at a
density of 2.5 x 106 cells/ml in RPMI 1640
medium (BioWhittaker, Walkersville, MD) containing 2 mM GLUTAMAX I
(L-Alanyl-L-Glutamine; Life Technologies,
Gaithersburg, MD), 10 mM HEPES, 1 mM sodium pyruvate, 50 U/ml
penicillin-50 µg/ml streptomycin, 5 x
10-5 M 2-ME, and 10% FCS (HyClone, Logan, UT)
at 37°C and 5% CO2. NS-398 (Biomol, Plymouth
Meeting, PA) was used to selectively inhibit COX-2, and indomethacin
(Sigma, St. Louis, MO) was used as a nonselective COX inhibitor. NS-398
selectively inhibits COX-2 (IC50 = 1 µM) with
no inhibition of COX-1 at concentrations up to 100 µM
(26). LPS from Salmonella typhimurium (Sigma)
was used to stimulate resident PC (Table II
).
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LP cells were isolated as described above and cultured on polystyrene tissue culture plates at a density of 2 x 106 cells/ml in RPMI 1640 medium containing 2 mM GLUTAMAX I, 10 mM HEPES, 1 mM sodium pyruvate, 50 U/ml penicillin-50 µg/ml streptomycin, 5 x 10-5 M 2-ME, and 0.5% normal mouse serum. After 1 h, nonadherent cells were removed by washing with cold PBS, and the remaining adherent cells were cultured overnight in the medium described above. After overnight culture, the plates were washed again with cold PBS to remove nonadherent cells (adherent 1 h), and the remaining adherent cells were incubated in HBSS (BioWhittaker) containing 2 mM EDTA at 4°C for 15 min to release the remaining adherent cell population (adherent overnight).
For experiments examining the profile of PG and TX production (Table I), LP cells were isolated and cultured as described above. After
24 h, nonadherent cells were removed and the remaining adherent
cell population was cultured for an additional 24 h, and
supernatants were then removed for analysis.
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For the measurement of PGs and TXB2,
cellular populations were isolated and cultured as described above, and
supernatants were removed and stored at -80°C until analysis.
PGE2, PGF2, 6-keto
PGF1, TXB2, and
11-PGF2 measurements were performed using
specific ELISAs (Cayman Chemicals, Ann Arbor, MI). For the measurement
of PGD2, samples were treated with methoxime-HCl
and analyzed for the presence of PGD2-methoxime
using a PGD2-methoxime-specific ELISA (Cayman
Chemicals). Each supernatant was measured in duplicate in at least
three dilutions using a Bio-Tek microplate reader (Bio-Tek,
Winooski, VT).
Western blot assays
Western blot analysis for COX-2 production was performed as previously described (15). Briefly, cells were washed twice in PBS, resuspended in a buffer consisting of 100 mM Tris-HCl, 3% SDS, 6% 2-ME, 30% glycerol, and 0.05% bromophenol blue and heated to 90°C for 5 min. Suspensions underwent electrophoresis on a 5–10% gradient Tris-glycine gel (Bio-Rad, Hercules, CA) and were transferred to polyvinylidene difluoride membranes (Tropix, Bedford, MA). Detection of COX-2 protein was performed using Western light detection system (Tropix), rabbit anti-COX-2 Ab (Cayman Chemicals), and goat anti-rabbit IgG alkaline phosphatase-conjugated Ab (Jackson ImmunoResearch, West Grove, PA). Polyvinylidene difluoride membranes were stripped and reprobed with rabbit anti-actin Ab followed by goat anti-rabbit IgG alkaline phosphatase-conjugated Ab to detect actin as a control for protein loading and transfer.
Immunohistochemical analysis
Cell culture. Small intestine LP cells were isolated as described above and cultured in chamber slides (Nunc, Naperville, IL) for 72 h until they became densely adherent, and nonadherent cells were removed by washing with PBS. CD45 and CD11b were detected using biotin-conjugated anti-CD45 (BD PharMingen, San Diego, CA) and biotin-conjugated anti-CD11b (BD PharMingen), respectively, followed by FITC-conjugated streptavidin (BD PharMingen). Cells were then fixed and permeabilized using a Cytoperm/Cytofix kit (BD PharMingen), and COX-2 was detected with goat anti-COX-2 Ab (SC-1747; Santa Cruz Biotechnology, Santa Cruz, CA) followed by Cy3-conjugated donkey anti-goat Ab. Cells were counterstained with Hoescht dye (bis-benzimide, B2883; Sigma) to detect nuclei.
To determine the percentage of cells expressing COX-2, CD45, and CD11b,
stained cell populations were examined by fluorescent microscopy at
x200 magnification. Random fields were selected from each group, and
the number of nuclei, COX-2+ cells, and double
positive (COX-2+ and CD45+
or CD11b+) cells were counted. Random fields were
counted until 
500 cells had been examined. Percentage of single
positive and double positive cells were recorded for each field and
used to calculate the mean ± SD of single and double positive
cells.
Small intestine sections.
Two-centimeter sections of small intestines from 6- to 8-wk-old C57BL/6
mice were flushed with ice-cold PBS, embeded in Tissue Tek OCT compound
(VWR Scientific Products, West Chester, PA), frozen, and cut into
7-µm thick acetone-fixed sections. Endogenous peroxidases were
inactivated by treating sections with 0.3% hydrogen peroxide followed
by treatment with avidin/biotin blocking kit (Vector Laboratories,
Burlingame, CA) and blocking of nonspecific staining with PBS
containing 1% BSA and 0.2% powdered skim milk. COX-2 protein was
identified using goat anti-COX-2 Ab (SC-1747; Santa Cruz
Biotechnology) followed by biotin-conjugated donkey anti-goat Ab
and streptavidin-conjugated HRP. For detection, we used a Cy3-tyramide
signal amplification kit (DuPont/NEN, Boston, MA). Following this
signal amplification, peroxidases were again inactivated, and avidin
and biotin was blocked as described above. -smooth muscle actin,
CD45, and CD11b were detected using FITC-conjugated -smooth muscle
actin (clone 1A4; Sigma), FITC-conjugated anti-CD45 (BD PharMingen)
or biotin-conjugated CD11b (BD PharMingen) followed by FITC-conjugated
streptavidin (BD PharMingen). Adjacent sections were stained with
appropriate isotype Abs to serve as negative controls. Sections were
counterstained with Hoescht dye to detect nuclei.
Flow cytometric analysis.
Single-cell suspensions of adherent small intestine LP cells were
obtained as described above and resuspended in PBS with 1% BSA (Fisher
Scientific, Pittsburgh, PA) and 1 mg/ml human IgG (Novartis
Pharmaceuticals, East Hanover, NJ) at 2 x
107 cells/ml. Cells were stained with
FITC-conjugated rat anti-mouse CD45 Ab, PE-conjugated rat
anti-mouse CD19, biotin-conjugated rat anti-mouse CD11b,
biotin-conjugated hamster anti-mouse TCR-, (all obtained from BD
PharMingen), rat anti-mouse c-kit (Caltag,
Burlingame, CA), or appropriate isotype control Abs (BD PharMingen) for
30 min on ice, washed twice, and resuspended in the above-described
buffer. To detect CD11b, TCR-, and c-kit, cellular
suspensions were then stained with streptavidin-PE or FITC-conjugated
goat anti-rat Ig (both obtained from BD PharMingen) for 30 min on
ice. Cellular suspensions were washed twice and fixed with 1%
paraformaldehyde in PBS. Flow cytometric analysis was done on a
triple-laser flow cytometer (FACScan; Becton Dickinson, Mountain View,
CA), and analysis was performed on a Macintosh G3 using the CellQuest
program (Becton Dickinson). Dead cells were excluded based on forward
and side light scatter, and the 10,000 cells from remaining population
were analyzed for CD45, CD19, CD11b, TCR-, and c-kit
expression. Gates for positive staining were defined such that <1% of
the analyzed population stained positive with the appropriate isotype
control Ab.
Stastical analysis.
Delta Soft 3 software (BioMetallics, Princeton, NJ) was used to
determine the weighted mean ± the SEM of each sample. Weighted
mean = 
wiconci/wi;
conci is the interpolated mean
concentration for dilution i;
wi =
1/(SEMi)2, and
SEMi is the SE of mean for
conci. Data analysis using an unpaired
Student’s t test was performed using GraphPad Prism
(GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently described an anti-inflammatory/immunomodulatory
role for COX-2-dependent AA metabolites produced by the small intestine
LP in the response to a dietary Ag (11). In most systems
studied to date, COX-2 expression and the subsequent production of AA
metabolites are an inducible and transient event. To better understand
the role of COX-2-dependent AA metabolites produced by the small
intestine LP, we examined the pattern of COX-2-dependent
PGE2 production by small intestine LP cells.
Small intestine LP cells display spontaneous and abundant
COX-2-dependent PGE2 production. This phenotype
is stable, persisting for days in culture (Fig. 1).
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, small
intestine LP cells produce abundant PGE2, which
can be inhibited by the selective COX-2 inhibitor NS-398. The level of
PGE2 production in the presence of NS-398 is
equivalent to the level of PGE2 production by
small intestine LP cells from COX-2-/- mice
(compare Fig. 1 and Table II). In
contrast, SPLs, resident PC, and PP cells produce little spontaneous
PGE2 (Fig. 2A). Evaluation of COX-2
protein expression further demonstrates the differences between
these cellular populations (Fig. 2B). Small intestine LP
cells, in contrast to the other cell populations, demonstrate
spontaneous expression of COX-2 by Western blot analysis. These
findings suggest that basal COX-2 expression represents a unique
property of the small intestine LP when compared with other mucosal and
nonmucosal lymphoid compartments.
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TCR-bearing T cells (11). Additionally, we have
noted that recombination activation gene-deficient mice (lacking T
cells and B cells) have small intestine LP PGE2
production that is equal to or greater than wild-type mice (data not
shown). To better identify the cellular population responsible for the
basal COX-2 expression and PGE2 production, we
separated cells based on the property of adherence. As shown in Fig. 3A, strongly adherent
populations are significantly enriched for
PGE2-producing cells. Others have described the
activation of adherent cells in response to physical stimuli, including
adherence to plastic or phagocytosis of beads (27). To
investigate the possibility that adherence to plastic was inducing
COX-2 expression in small intestine LP cells, we cultured small
intestine LP cells under conditions that would not allow adherence. As
shown in Fig. 3B, adherence was not required for production
of COX-2-dependent PGE2, and transferring cells
from nonadherent to adherent culture conditions did not augment
PGE2 production further.
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, the predominant
metabolite measured in these supernatants is
PGE2, and its production is significantly
inhibited by the selective COX-2 inhibitor NS-398 as well as the
nonselective COX inhibitor indomethacin, indicating that the vast
majority of PGE2 production by these cells is
COX-2-dependent. This is consistent with our previous findings
demonstrating that LP MNC from COX-1-/- mice
produce PGE2 levels equivalent to wild-type
littermates, whereas LP MNC from COX-2-/- mice
produce significantly reduced PGE2 levels
(11), equivalent to those seen when wild-type LP MNC are
treated with NS-398. PGF2 and 6-keto
PGF1, a breakdown product of prostacyclin,
were present in moderate amounts in these supernatants, and their
production was predominantly COX-2-dependent.
TXB2 and PGD2 were produced
in predominantly COX-2-dependent manners but were present in more
modest concentrations. Notably, the majority of
TXB2 measured in these supernatants was present
in medium in the absence of cell culture and was not produced by the
adherent LP cell population. Supernatants from all groups contained
<10 pg/ml 11-PGF2, a breakdown product of
PGD2, consistent with the finding of low
PGD2 production by these cellular populations. By
FACS analysis, this adherent LP cell population was comprised of cells
that were 21% CD45+, 11%
CD11b+, 1% TCR-+, 1%
CD19+, and <1%
c-kit+. Non-bone marrow-derived stromal cells are the predominant COX-2-expressing cell types in the murine LP
To further characterize the cellular population responsible for
the basal expression of COX-2, immunohistochemical analysis of COX-2
expression on LP-adherent cell populations was performed. As shown in
Fig. 4, A and B,
the predominant population of cells expressing COX-2 were both
CD45-, a cell surface marker of bone
marrow-derived cells, and CD11b-, a cell surface
marker expressed on macrophages and dendritic cells that could be
enriched by adherence. Occasional CD11b+
COX-2-expressing cells were seen (inset, Fig. 4B); however, these cells were not the predominant
population of COX-2-expressing cells. By immunohistochemical analysis
of the adherent population, we found that 51.4 ± 2.8% of
nucleated cells expressed COX-2, 3.7 ± 1.3% were both
CD45+ and COX-2+, and
4.7 ± 1.9% were both CD11b+ and
COX-2+. Immunohistochemical analysis of
-smooth muscle actin expression was variable in the cultured
LP-adherent cell population, with 36.6 ± 23.9% of
COX-2+ cells expressing -smooth muscle actin.
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, D
and E, COX-2-expressing cells were present in the LP of
small intestine villi. These COX-2-expressing cells were preferentially
located near the tips of the villi and were predominantly
CD45- and CD11b-,
consistent with the immunohistochemical findings seen on cultured
cells described above. By immunohistochemical analysis, these
COX-2-expressing cells did not express -smooth muscle actin. This
finding, along with the location of these cells in the villus, suggests
that these cells are not subepithelial myofibroblasts (28, 29). These findings demonstrate that the population of
COX-2-expressing cells in the small intestine LP is a non-bone
marrow-derived stromal cell residing adjacent to lymphocytes and
monocytes. COX-2 expression and PGE2 production by LP cells does not require exogenous stimuli
LP MNC reside in an environment exposing them to multiple
inflammatory stimuli, such as LPS and other bacterial components, and
mediators of inflammation, such as TNF-, IFN-, and IL-1. These
stimuli have been noted to induce the expression of COX-2 in other
systems (30, 31, 32, 33). Therefore, we evaluated the requirement
for these inflammatory stimuli in the production of
PGE2 by small intestine LP cells through the
examination of various spontaneous and induced mutant strains of mice,
as well as germfree and conventionally housed mice. As shown in Fig. 5A, small intestine LP cells
from germfree FVB/NJ mice and conventionally housed FVB/NJ mice produce
equivalent levels of PGE2, indicating that the
presence of resident bacterial flora is not required to develop or
express this phenotype. This finding was expanded and confirmed by
examining PGE2 production by small intestine LP
cells from C3H/HeJ mice, which contain a spontaneous mutation in the
Toll-like receptor 4 gene, resulting in the inability to respond to LPS
via the CD14 receptor (34). Small intestine LP cells from
C3H/HeJ mice produce levels of PGE2 equivalent to
those of wild-type mice (Fig. 5). Notably, PC from C3H/HeJ mice produce
<5% of the PGE2 when compared with PC from the
congenic C3H/HeSnJ (LPS-responsive) mice in response to 10 ng/ml LPS
(Table II). This result documents the
requirement for LPS signaling through the Toll-like receptor 4 complex
for the optimal induction of COX-2-dependent PGs in response to LPS in
peritoneal macrophages, but not for the continuous production of
PGE2 by LP stromal cells.
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-deficient
(IL-1-/-), IFN-R-deficient
(IFN-R-/-), TNFR I-deficient (TNFR
I-/-), TNFR II-deficient (TNFR
II-/-), IL-12-deficient (IL-12
p40-/-), and C57BL/6 mice. As shown is Fig. 5B, all induced mutant strains of mice tested had
spontaneous PGE2 production that was equivalent
to wild-type mice. These findings convincingly document that
bacterial flora, LPS, IL-1, IFN-, TNF, and IL-12 are not
essential for the development or expression of this phenotype. LP cells do not produce a soluble factor with the ability to induce PGE2 production by peritoneal macrophages
To address the possibility that a soluble stimulus produced by LP
cells induces COX-2 expression and PGE2
production, we evaluated the ability of small intestine LP cell- and
SPL-conditioned medium to induce PGE2 production
by resident PC, a well studied cell for the induction of COX-2.
Conditioned medium was generated using SPLs and small intestine LP
cells from wild-type C57BL/6 mice, as well as
COX-2-/- mice, to reduce background levels of
PGE2 in the conditioned medium. To minimize any
effect that LPS contaminating the cellular preparations might have on
the induction of COX-2 in our responding resident PC, we used resident
PC from C3H/HeJ (LPS-nonresponsive) as well as congenic C3H/HeSnJ
(LPS-responsive) mice as responding cell populations. Conditioned
medium generated from culture of SPLs or LP MNC from C57BL/6 and
COX-2-/- mice was diluted 1:1 with fresh medium
and cultured in the absence of cells (no responder cells) to assess the
basal level of PGE2 in the conditioned medium or
used to culture resident PC from C3H/HeJ (LPS-nonresponsive) or
C3H/HeSnJ (LPS-responsive) mice at a density of 2.5 x
106/ml for 48 h. After 48 h,
supernatants were removed, and the PGE2
concentration was measured by ELISA. As shown in Table II, SPL- and
small intestine LP cell-conditioned medium does not contain factors
with the ability to induce PGE2 production by
resident PC. These findings further suggest that the phenotype of
spontaneous COX-2 expression and abundant PGE2
production is not an acute response to a stimulus, but represents a
stable phenotype unique to the murine small intestine LP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(36), whereas pathogenic
states such as inflammatory bowel disease are associated with the
production of inflammatory cytokines IL-12, TNF-, and IFN-
(37, 38, 39). In addition to these observations, we have
recently described a role for a COX-2-dependent AA metabolite,
PGE2, produced by the small intestine LP in the
immune response to a dietary Ag (11).
PGE2 is known to have immunomodulatory effects
including down-regulation of MHC class II (40) and IL-12R
(41) expression, increased production of IL-10
(42), and decreased production of TNF-
(43) and IL-12 (44). In this context,
PGE2 production by the LP could represent a
proximal step in the development of the immunomodulatory tone of the
intestine.
Here we demonstrate the spontaneous and sustained expression of COX-2
and the resultant production of abundant PGE2 by
LP cells in the absence of a known exogenous stimulus. The spontaneous
production of COX-2-dependent PGE2 is a unique
property of the LP when compared with other lymphoid compartments,
including intestinal lymphoid compartments exposed to identical or
nearly identical luminal stimuli. We have observed a modest number of
COX-2-expressing cells in the subepithelial dome of the PP by
immunohistochemistry (our unpublished observations). This
observation could explain the low levels of COX-2 expression and
PGE2 production seen in PP (Fig. 2). In addition,
the pattern of COX-2 expression in the LP contrasts with the majority
of published descriptions of COX-2 expression in other organs, which
demonstrate inducible and transient expression in response to an
exogenous stimulus.
To better understand a potential role for the spontaneous and
continuous expression of COX-2 by LP stromal cells, we examined the
profile of COX-dependent AA metabolites produced by these cells. Here
we demonstrate that the predominant COX-dependent AA metabolite is
PGE2, and its production is overwhelmingly
COX-2-dependent. Other COX-dependent AA metabolites
(PGF2, 6-keto PGF1,
TXB2, and PGD2) were also
produced in a COX-2-dependent manner but in more modest amounts. This
finding, taken in the context of the previously described
immunomodulatory roles of PGE2, suggests that a
function of these stromal cells may be to contribute to the immunologic
hyporesponsiveness of the intestinal immune response.
Given the specialized microenvironment in which LP cells reside, it is
intriguing to hypothesize that agents such as LPS or other bacterial
products may play a role in the development or expression of this
unique phenotype. In this study, we demonstrate that neither bacterial
flora nor conventional LPS signaling through the Toll-like receptor 4
are required for the development of small intestine LP cells with this
phenotype. In contrast, signaling via Toll-like receptor 4 is required
to induce COX-2 expression in peritoneal macrophages in response to LPS
(Table II). We also document that other proinflammatory stimuli known
to induce COX-2 expression are not essential for the development or
expression of this phenotype. It is possible that the lack of
individual proinflammatory stimuli in the knockout mice may not lead to
loss of PGE2 production due to a redundancy in
the inflammatory stimuli inducing COX-2 in this system. However, we
further demonstrate that small intestine LP cells do not produce a
soluble factor with the ability to induce COX-2-dependent
PGE2 production in resident peritoneal
macrophages, suggesting that if redundant stimuli are responsible for
the COX-2 expression in this system, they are not the typical
inflammatory mediators known to induce COX-2 in peritoneal macrophages.
Together, these findings suggest that the abundant
PGE2 production by these LP cells does not
represent an acute and transient response to an inflammatory or
secreted stimulus.
We have shown that the basal production of COX-2-dependent
PGE2 in the small intestine LP is a phenotype of
non-bone marrow-derived stromal cells. These COX-2-expressing stromal
cells are preferentially located near the tips of the villi and in the
proximal small intestine (our unpublished observations). Based
on the location of these stromal cells in the villi, their lack of
-smooth muscle actin expression by immunohistochemistry of the small
intestine, and their spontaneous expression of COX-2, these cells do
not resemble previously described intestinal myofibroblasts (28, 29, 45, 46).
Multiple studies have documented a tolerant phenotype of LP lymphocytes in response to antigenic stimuli. This phenotype is manifest by decreased production of proinflammatory cytokines, increased production of immunomodulatory cytokines, and predominance of immunoregulatory Th cell subsets. Given previous descriptions of the ability of PGE2 to blunt the production of proinflammatory cytokines, enhance the production of immunomodulatory cytokines, and favor the development of immunomodulatory Th cell subsets, it is intriguing to postulate that a factor in the development of the immunological tolerance in the intestine is the production of PGE2 by resident stromal cells in the LP. In this context, it is logical that the production of PGE2 would be continuous and the source would be a cell that is intrinsic to the intestine and located adjacent to responding lymphocytes. These resident cells, through their production of AA metabolites, could help dictate the tone of the intestinal immune response produced by circulating lymphocytes and monocytes.
The physical convergence of bacteria, bacterial products, dietary Ags, and activated components of the cellular immune system in the gastrointestinal tract presents an unusual challenge to the mucosal immune system. The description of the immunomodulatory role of COX-2-dependent AA metabolites in the intestinal immune response adds another agent to the list of mechanisms used by the mucosal immune system. Understanding how these mechanisms allow the mucosal immune system to negotiate this continual challenge will broaden our understanding of basic immunology as well as yield insight into pathophysiologic conditions such as inflammatory bowel disease and celiac disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robin G. Lorenz, Department of Pathology and Immunology, Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
3 Abbreviations used in this paper: COX, cyclooxygenase; AA, arachidonic acid; TX, thromboxane; LP, lamina propria; PP, Peyer’s patch; SPL, splenocyte; PC, peritoneal cells; MNC, mononuclear cells.
Received for publication July 31, 2000. Accepted for publication January 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. D. Newberry, J. S. McDonough, K. G. McDonald, and R. G. Lorenz Postgestational Lymphotoxin/Lymphotoxin {beta} Receptor Interactions Are Essential for the Presence of Intestinal B Lymphocytes J. Immunol., May 15, 2002; 168(10): 4988 - 4997. [Abstract] [Full Text] [PDF]
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, supernatant PGE2
concentrations from C57BL/6 small intestine LP cells cultured in the
absence of a COX inhibitor. Every 3rd day (
), C57BL/6 small
intestine LP cells were cultured with the selective COX-2 inhibitor
NS-398 at 1 µg/ml to document the dependency of PGE2
production on COX-2 activity. Results are displayed as the weighted
mean of the concentration of PGE2 ± the SEM in
picograms per milliliter. Results are representative of one of two
experiments. *, p < 0.05 when compared with the
PGE2 concentration of the supernatant from the previous
day.
