Laboratory of Mucosal Immunology, Department of Medicine, University of California at San Diego, La Jolla, California 92093
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ABSTRACT |
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The intestinal mucosa contains a subset of lymphocytes that
produce Th2 cytokines, yet the signals responsible for the recruitment of these cells are poorly understood. Macrophage-derived chemokine (MDC/CCL22) is a recently described CC chemokine known to
chemoattract the Th2 cytokine producing cells that express the receptor
CCR4. The studies herein demonstrate the constitutive production of MDC/CCL22 in vivo by human colon epithelium and by epithelium of human
intestinal xenografts. MDC/CCL22 mRNA expression and protein secretion
was upregulated in colon epithelial cell lines in response to
proinflammatory cytokines or infection with enteroinvasive bacteria.
Inhibition of nuclear factor (NF)-B activation abolished MDC/CCL22
expression in response to proinflammatory stimuli, demonstrating that
MDC/CCL22 is a NF-
B target gene. In addition, tumor necrosis factor-
-induced MDC/CCL22 secretion was differentially modulated by
Th1 and Th2 cytokines. Supernatants from the basal, but not apical,
side of polarized epithelial cells induced a MDC/CCL22-dependent chemotaxis of CCR4-positive T cells. These studies demonstrate the
constitutive and regulated production by intestinal epithelial cells of
a chemokine known to function in the trafficking of T cells that
produce anti-inflammatory cytokines.
mucosa; cytokines; Th1/Th2; chemotaxis
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INTRODUCTION |
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THE INTESTINAL
EPITHELIUM forms a barrier that restricts entry of intestinal
luminal contents and microbes into the host. Nonetheless, the lamina
propria underlying the surface epithelium is "physiologically
inflamed" in that it contains abundant numbers of B and T cells,
monocytes, and dendritic cells, as well as variable numbers of
eosinophils and mast cells. T cells that encounter luminally derived
antigens within the organized lymphoid tissue of the Peyer's patches
can exit from those structures, recirculate, and eventually home to the
intestinal lamina propria. T cells in the lamina propria are mostly
CD45RO+, consistent with a memory phenotype, and also
express the mucosal homing 4
7-integrin as
well as human leukocyte antigen-DR and CD25, reflecting an
activated state compared with T cells in other peripheral sites
(27). Lamina propria T cells play an important role in
host defense but can also contribute to the pathophysiology of
intestinal inflammation. The cytokine profile of lamina propria lymphocytes is predominantly Th1 in nature [i.e., interferon (IFN)-
producing] under physiological conditions (21) as well as
in Crohn's disease (18) and most murine models of
intestinal inflammation (5). Nonetheless, interleukin
(IL)-4-producing Th2 cells are also present in the normal human
intestinal mucosa and are increased in certain murine models of
colitis, such as the TCR
/
mutant mouse (25, 35) and
the oxazolone-induced colitis model (6). Despite the
association of those models of colitis with elevated Th2 cytokines,
most evidence supports the hypothesis that Th2 cytokines, primarily
IL-4 and IL-10, are anti-inflammatory in the intestinal mucosa through
their actions on macrophages and Th1 cells (3, 23).
Little is known about the signals responsible for the recruitment of T
cells within the intestinal mucosa. Although an initial interaction
between 4
7-integrin on T cells and
mucosal addressin cell adhesion molecule on venules is required
for homing of T cells to the gut, additional activation signals
provided by chemokines are also thought to be necessary (8,
31). T cells express a number of cell surface chemokine
receptors, and different subsets of T cells express different chemokine
receptors, allowing for differential recruitment of those subsets. For
example, Th2-type cells (IL-4, IL-5, IL-10, and IL-13 producing)
express the receptor CCR4 on their surface (11, 37),
whereas Th1-type cells (IFN-
and IL-2 producing) express the
receptor CXCR3.
Intestinal epithelial cells are an important source of chemokines in
the intestinal mucosa. Epithelial cells are the first line of defense
against luminal pathogens and, in response to bacterial invasion, can
secrete an array of neutrophil and macrophage chemoattractants (e.g.,
IL-8/CXCL8, ENA-78/CXCL5, GRO/CXCL1, MCP-1/CCL2, and MIP-1
/CCL3)
(14, 42). Human intestinal epithelium can also produce
IP-10/CXCL10, Mig/CXCL9, and I-TAC/CXCL11 (13, 40), chemokines that are known to chemoattract
CXCR3-expressing T cells that have a Th1 phenotype (i.e.,
IFN-
-producing CD4+ T cells) and MIP-3
/CCL20, a
chemoattractant for CD45RO+ T cells and immature dendritic
cells (26). These findings suggest that epithelial cells
may have a role not only in the acute host response to infection but
also in T cell trafficking in the intestine.
The CC chemokine receptor CCR4 is preferentially expressed by T lymphocytes that produce Th2 cytokines (11, 37). The production of a chemokine specific for CCR4 by the intestinal epithelium could provide a signal for the specific recruitment of Th2 cells. Macrophage-derived chemokine (MDC/CCL22) is a recently identified chemokine of the CC chemokine family that uses CCR4 as its receptor and is known to selectively chemoattract Th2 cytokine-producing cells (2, 20). MDC/CCL22 is constitutively expressed by macrophages, mature dendritic cells, and B cells, and upregulated expression of MDC/CCL22 has been noted in T lymphocytes that concomitantly produce the Th2 cytokines IL-4, IL-5, and IL-6 and in monocytes stimulated with the Th2 cytokines IL-4 and IL-13 (2, 19).
We hypothesized that human intestinal epithelial cells may have the capacity to signal Th2 cells through the production of the CCR4 ligand MDC/CCL22. In the studies herein, we report on the constitutive expression of MDC/CCL22 by epithelium in normal human colon and in human intestinal xenografts in vivo and the regulated expression of MDC/CCL22 by cultured human intestinal epithelial cells. These data suggest the notion that intestinal epithelial cells have the capacity to regulate mucosal T cell trafficking through the release of T cell chemoattractants and the potential to modulate the local mucosal cytokine milieu through the recruitment of specific cytokine-producing T cell subsets.
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MATERIALS AND METHODS |
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Reagents.
Recombinant human (rh) tumor necrosis factor (TNF)-, IL-1
,
MDC/CCL22, and transforming growth factor-
1 were from R&D Systems (Minneapolis, MN). rhIFN-
was from Biosource International
(Camarillo, CA), and rhIL-4 and IL-13 were from PeproTech (Rocky Hill,
NJ). Mouse anti-human MDC/CCL22 monoclonal antibody (MAb) (IgG2b) and affinity-purified goat anti-human TNF-
were from R&D Systems, and
rabbit anti-human MDC/CCL22 IgG was from PeproTech. MG-132 was from
Sigma Chemical (St. Louis, MO).
Cell culture.
The human colon adenocarcinoma cell lines HT-29 (ATCC HTB 38, Rockville, MD) and HCA-7 colony 29 (a gift from S. C. Kirkland) were grown in DMEM supplemented with 10% heat-inactivated FCS and 2 mM
L-glutamine. Epithelial cells were grown to confluence in
six-well plates before stimulation with cytokines or bacterial infection. To obtain polarized monolayers, HCA-7 cells were seeded onto
tissue culture-treated transwell filters (0.4 µm pore size, 1.2 cm2 surface area; Costar, Cambridge, MA) and allowed to
grow for ~7 days, at which time a mean resistance of ~500
· cm2 was established. Peripheral blood
monocytes were prepared by separation of whole blood on a Ficoll
gradient, followed by adherence of the harvested buffy coat to tissue
culture plastic wells overnight. Monocyte-derived macrophages were
prepared as previously described (32).
Bacterial infection.
Enteroinvasive Escherichia coli strain O29:NM was obtained
from the ATCC (43892), and Salmonella dublin was provided by
Dr. J. Fierer (University of California, San Diego). For infection, HT-29 cells were grown to confluence in six-well plates and incubated with 108 S. dublin or 5 × 108
enteroinvasive E. coli for 1 h to allow invasion to
occur, after which the extracellular bacteria were removed by washing
and cells were incubated for an additional 24 h in the presence of
50 µg/ml of the non-membrane-permeant antibiotic gentamicin to kill
remaining extracellular bacteria (15). Culture
supernatants were removed 24 h after infection and stored at
20°C before measurement of MDC/CCL22 by ELISA.
Adenovirus constructs and adenovirus infection.
Recombinant adenovirus 5 (Ad5) containing an IB
-AA superrepressor
(Ad5I
B-A32/36) or the E. coli
-galactosidase gene
(Ad5LacZ) was constructed as described before (17).
Ad5I
B-A32/36 expresses a hemagglutinin (HA) epitope-tagged
mutant form of I
B
in which serine residues 32 and 36 are replaced
by alanine residues. The mutant I
B
cannot be phosphorylated at
positions 32 and 36 and acts as a superrepressor of nuclear factor
(NF)-
B activation (17). The HA epitope tag enables
identification of the exogenous superrepressor with anti-HA antibodies.
Viral titers were determined by plaque assay. Recombinant virus was
stored in PBS containing 10% (vol/vol) glycerol at
80°C.
RT-PCR.
Total cellular RNA was extracted with TRIzol reagent (GIBCO BRL,
Gaithersburg, MD), treated with RNase-free DNase (Stratagene, La Jolla,
CA) to remove contaminating genomic DNA, and reverse transcribed using
1 µg of total RNA and 2.5 units Superscript II RT (GIBCO BRL).
Sequences were amplified from cDNA with primers specific for human
MDC/CCL22 and -actin. Primer sequences for MDC/CCL22 were
5'-GAGACATACAGGACAGAGCATGGC T-3' (sense) and
5'-ATGGAGATCAGGGAATGCAGAGAG-3' (antisense), and those for
-actin
were 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (sense) and
5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3' (antisense). Five microliters of RT
reaction were used for PCR amplification in a volume of 50 µl of PCR
buffer (GIBCO BRL), including 25 pM of each primer, 1.5 mM
MgCl2, and 200 µM of each dNTP. After an initial hot
start at 95°C for 5 min, 4.0 units Taq polymerase (GIBCO
BRL) was added. The amplification profile consisted of 45 s
denaturation at 95°C, 2.5 min annealing, and extension at 64°C
(MDC/CCL22) or 72°C (
-actin) for 38 (MDC/CCL22) or 28 (
-actin)
cycles. Each experiment included negative controls in which RNA was
omitted from the RT mixture and cDNA was omitted from the PCR
reactions. RNA from human monocyte-derived macrophages was used as a
positive control for MDC/CCL22.
Real-time PCR.
Real-time PCR was performed using an ABI Prism 7700 sequence detection
system (PE Applied Biosystems, Foster City, CA). Each reaction
contained 25 µl of 2× SYBR Green Master Mix (containing 200 nM dATP,
dGTP, and dCTP, 400 nM dUTP, 2 mM MgCl2, 0. 25 units uracil
N-glycosylase, and 0. 625 units Amplitaq Gold DNA
polymerase), 25 pmol of the sense and antisense primers, and 2 µl of
cDNA in a final volume of 50 µl. MDC/CCL22 primers used were those
described above, and -actin primers were as follows: sense,
5'-CAAAGACCTGTACGCCAACAC-3'; antisense,
5'-CATACTCCTGCTTGCTGATCC-3'. Reactions were incubated at 50°C
for 2 min followed by 95°C for 10 min. The amplification profile was
95°C for 15 s and 62°C for 1 min for 40 cycles. Amplification of the expected single PCR product was confirmed by electrophoresis of
the product on a 1% agarose gel that was stained with ethidium bromide. Data analysis was performed using PE Biosystems 7700 sequence
detection system software. Real-time PCR data were plotted as the
Rn fluorescence signal vs. the cycle number.
Rn was calculated using the equation
Rn = R
R
MDC/CCL22 ELISA. Polystyrene 96-well plates (Immulon-4; Dynex Technologies, Chantilly, VA) were coated with mouse anti-human MDC/CCL22 MAb (R&D Systems), diluted in carbonate buffer, as the capture antibody. Affinity-purified rabbit polyclonal anti-human MDC/CCL22 (PeproTech) was diluted in PBS, 1.0% BSA, and 0.1% Tween 20 and used as the detection antibody. The second step reagent was horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science, Arlington Heights, IL). Bound horseradish peroxidase was visualized with tetramethylbenzidine and H2O2 diluted in sodium acetate buffer, pH 6.0. The color reaction was stopped by addition of 1.2 M H2SO4, and absorbance was measured at 450 nm. MDC/CCL22 concentrations were calculated from standard curves using rhMDC/CCL22 (R&D Systems). The MDC/CCL22 ELISA was sensitive to 50 pg/ml.
Human intestinal xenografts. Human fetal intestine, gestational age 14-20 wk (Advanced Biosciences Resources, Alameda, CA), was transplanted subcutaneously onto the backs of C57BL/6 SCID mice as described before (16, 24). Human fetal intestinal xenografts were allowed to develop for at least 10 wk following transplantation, at which time the epithelium, which is strictly of human origin, is fully differentiated (38). Intestinal xenografts were removed, and segments were embedded in optimum cutting temperature (OCT) compound and frozen in isopentane/dry ice for immunohistochemical analysis.
Immunohistochemical detection of MDC/CCL22 in human colon and human intestinal xenografts. Sections of histologically normal human colon from surgical specimens from individuals undergoing partial colectomy were imbedded in OCT and snap frozen in isopentane/dry ice. Cryostat sections (5 µm) of human colon or intestinal xenografts were fixed in acetone for 10 min and blocked in PBS-1% BSA with 10% goat serum for 1 h at room temperature. Sections were incubated overnight at 4°C with 2.5 µg/ml mouse anti-human MDC/CCL22 MAb, an isotype-matched control MAb (mouse IgG2b, 2.5 µg/ml, Sigma), or anti-MDC/CCL22 antibody that was preabsorbed with rhMDC/CCL22 (2.5 µg/ml), and sections were subsequently stained with Cy3-labeled goat anti-mouse IgG. Sections were counterstained with the nuclear dye Hoechst 33258 (Calbiochem, San Diego, CA).
Chemotaxis assay.
To determine whether epithelial cell-derived MDC/CCL22 chemoattracts T
lymphocytes, chemotaxis assays were performed using the CEM T cell
line, which expresses CCR4 as we verified by RT-PCR using previously
described primers (12). Supernatants were obtained from
the apical or basolateral chambers of polarized HCA-7 cells cultured in
Transwells in RPMI-0.1% BSA 24 h after stimulation with TNF-
or IL-1
plus IFN-
. Recombinant human MDC/CCL22 or HCA-7
supernatants were added to the bottom well of ChemoTx chemotaxis microplates (Neuroprobe, Gaithersburg, MD), and a 5-µm-pore filter plate was placed over the bottom wells. To assess chemotaxis, 1.25 × 105 CEM cells labeled with the fluorescent dye
calcein-AM (Molecular Probes, Eugene, OR) in a total volume of 25 µl
were added to the top of the filter. After 2 h incubation,
fluorescence in the bottom wells was measured with a fluorescence
microplate reader and number of migrated cells were calculated from a
standard curve of labeled cells. To determine the role of MDC/CCL22 in
supernatant-induced chemotaxis, 5 µg/ml of a neutralizing chicken IgY
anti-MDC/CCL22 antibody (R&D Systems) or an equivalent concentration of
control IgY (Jackson ImmunoChemical Laboratories, West Grove, PA) were added to the supernatants before addition to the chemotaxis plate.
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RESULTS |
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Constitutive MDC/CCL22 expression by epithelial cells in human
colon and human intestinal xenografts.
To determine if human intestinal epithelium in vivo expresses
MDC/CCL22, we first immunostained sections of normal human colon for
that chemokine. As shown in Fig.
1A, MDC/CCL22 was
constitutively expressed by epithelial cells in normal human colon.
Sections stained with an isotype-matched control antibody at the same
concentration (data not shown) or anti-MDC/CCL22 antibody preabsorbed
with rhMDC/CCL22 (Fig. 1B) did not demonstrate any specific
immunoreactivity. Surface epithelium as well as crypt epithelium
stained with the anti-MDC/CCL22 antibody.
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Constitutive and regulated MDC/CCL22 mRNA expression in human
intestinal epithelial cells.
To determine if the expression of MDC/CCL22 by intestinal epithelial
cells is regulated, we took advantage of cultured human colon
epithelial cell lines. As shown in Fig.
3A, MDC/CCL22 mRNA expression
was upregulated in the human colon epithelial cell line (HT-29) by
3 h and continued to increase up to 12 h after stimulation
with TNF- or IL-1
. MDC/CCL22 mRNA expression also increased after
IFN-
stimulation, but increased expression was delayed for up to
12 h after IFN-
stimulation. Furthermore, costimulation of
HT-29 cells with a combination of IFN-
plus TNF-
or IFN-
plus
IL-1
resulted in a synergistic increase MDC/CCL22 mRNA expression (Fig. 3). Similar data were obtained using HCA-7 cells (data not shown). Real-time PCR was performed to quantify the increase in MDC/CCL22 mRNA expression after cytokine stimulation. These data confirmed that costimulation of HT-29 cells with TNF-
or IL-1
plus IFN-
induced a synergistic increase in MDC/CCL22 mRNA
expression (Fig. 3, B and C). Thus IFN-
and
TNF-
costimulation induced a 123-fold increase in MDC/CCL22
expression over unstimulated controls, compared with a 35-fold increase
after TNF-
stimulation and an 8-fold increase after IFN-
stimulation.
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Cytokine-stimulated MDC/CCL22 secretion.
To determine if agonist-stimulated MDC/CCL22 mRNA expression was
accompanied by increased MDC/CCL22 protein secretion, HT-29 cells were
left unstimulated or were stimulated for up to 24 h with TNF-,
IL-1
, or IFN-
, after which MDC/CCL22 levels in culture supernatants were assayed by ELISA. As shown in Fig.
4A, TNF-
stimulation
markedly increased MDC/CCL22 secretion (130-fold increase over
unstimulated cells at 24 h), and this was also the case, although
to a lesser extent, for IL-1
stimulation (36-fold increase). In
contrast, MDC/CCL22 secretion in response to IFN-
stimulation was
significantly less than that induced by TNF-
or IL-1
(10-fold increase over control at 24 h).
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Polarized secretion of MDC/CCL22.
Intestinal epithelial cells in vivo are polarized with functionally
distinct apical and basolateral domains. For epithelial MDC/CCL22 to
act as a physiological chemoattractant for target cells in the lamina
propria, it predictably would be secreted in a polarized manner from
the basolateral epithelial cell membrane rather than apically into the
intestinal lumen. To determine if this was the case, HCA-7 cells were
grown as polarized monolayers and stimulated with TNF- or IL-1
either alone or in combination with IFN-
. As shown in Table
1, for each stimulus, ~90% of
MDC/CCL22 was secreted into the basal compartment.
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Epithelial cell-derived MDC/CCL22 induces T cell chemotaxis.
To determine if MDC/CCL22 secreted by agonist-stimulated epithelial
cells could contribute to the recruitment of T cells, chemotaxis assays
were performed using supernatants obtained from polarized HCA-7 cells
(Fig. 5). rhMDC/CCL22 induced a
dose-dependent increase in chemotaxis of the CCR4-expressing T cell
line CEM. Supernatants taken from the basal chamber of Transwell-grown
HCA-7 cells stimulated with IL-1 and IFN-
induced an increase in
T cell chemotaxis compared with media from unstimulated monolayers (18.6 vs. 8.3% of input cells migrated). This was comparable to the level of chemotaxis induced by similar concentrations of
rhMDC/CCL22. In contrast, supernatants from the apical chamber of
filter-grown, stimulated HCA-7 cells did not induce any increase in
chemotaxis (7.7% of input cells migrated). IL-1 and IFN-
at a
concentration of 20 ng/ml in RPMI did not affect chemotaxis of CEM
cells (7.3% of input cells migrated). Incubation of the basal chamber
supernatants from TNF-
- and IFN-
-stimulated HCA-7 cells with a
neutralizing anti-MDC/CCL22 antibody, but not an isotype-matched
control antibody, completely abrogated chemotaxis induced by the
supernatants (8.8% of input cells migrated in the presence of anti-MDC
compared with 15.1% of input cells in the presence of an
isotype-matched control antibody).
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Epithelial MDC/CCL22 secretion in response to infection with
enteroinvasive bacteria.
Epithelial cells can function as sensors for microbial infection and,
in response to bacterial infection, secrete chemokines that can
chemoattract neutrophils and monocyte/macrophages within the intestinal
mucosa (22, 29, 34). To determine if epithelial cell
infection with enteroinvasive bacteria upregulates MDC/CCL22 secretion,
HT-29 cells were infected with S. dublin or enteroinvasive E. coli O29:NM. As shown in Fig.
6, infection with those bacteria increased MDC/CCL22 secretion. To determine if IFN- synergized with
bacterial infection to increase MDC/CCL22 production, HT-29 cells were
stimulated with IFN-
immediately after Salmonella infection. As shown in Fig. 6, MDC/CCL22 secretion synergistically increased when bacteria-infected cultures were stimulated with IFN-
.
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MDC/CCL22 expression is regulated by NF-B.
The transcription factor NF-
B plays a central role in the activation
of several genes that are upregulated in intestinal epithelial cells in
response to stimulation with proinflammatory cytokines or bacterial
infection (17). However, little is known about the
transcriptional regulation of MDC/CCL22 in different cell types. To
determine if MDC/CCL22 functions as an NF-
B target gene in
intestinal epithelial cells, HT-29 cells were treated for 1 h with
the proteasome inhibitor MG-132 (10 µM). MG-132 completely abrogated
TNF-
-stimulated MDC/CCL22 mRNA expression (Fig.
7A) and protein secretion
(data not shown). Since pharmacological agents are not always
completely specific, we used an additional approach in which HT-29
cells were infected with a recombinant adenovirus expressing a mutant
I
B
protein that has serine-to-alanine substitutions at positions
32 and 36 (Ad5I
B-A32/36) and acts as a superrepressor of NF-
B
activation (17). As shown in Fig. 7B, MDC/CCL22
secretion was markedly inhibited in Ad5I
B-A32/36-infected cells, but
not in cells infected with control adenovirus, in response to TNF-
or IL-1
stimulation or S. dublin or E. coli
O29:NM infection. Real-time RT-PCR was performed on mRNA isolated
12 h after cytokine stimulation to examine the effect of
inhibiting NF-
B activation at a time when maximal changes were
observed in MDC/CCL22 mRNA expression. TNF-
induced a 15-fold
increase in MDC/CCL22 mRNA expression in HT-29 cells infected with
control virus compared with a 4-fold increase in cells infected with
virus containing the Ad5I
B-A32/36 superrepressor. At 12 h after
stimulation, MDC/CCL22 protein secretion induced by TNF-
was reduced
by 91% in Ad5I
B-A32/36-infected HT-29 cells compared with cells
infected with the control virus. Similar results were obtained in HT-29
cells stimulated with TNF-
plus IFN-
at 12 h. Inhibition of
NF-
B had no effect on the induction of MDC/CCL22 expression in
response to stimulation of cells with IFN-
alone (data not shown),
which is consistent with the lack of NF-
B activation in these cells
after IFN-
stimulation.
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Modulation of MDC/CCL22 secretion by Th2 cytokines.
IL-4 and IL-13 increase MDC/CCL22 expression by monocytes and
macrophages (2, 7). To determine if this is the case also for MDC/CCL22 production by intestinal epithelial cells, HT-29 cells
were stimulated with IL-4 or IL-13, alone or in the presence of
TNF-. IL-4 or IL-13 alone had no significant effect on constitutive MDC/CCL22 secretion. However, IL-4 and IL-13 inhibited
TNF-
-stimulated MDC/CCL22 secretion by ~60% (Table
2).
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DISCUSSION |
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The normal adult human intestine is considered to be "physiologically inflamed." Thus the normal intestinal mucosa contains B and T lymphocytes, mononuclear phagocytes, and dendritic cells, as well as smaller numbers of eosinophils and mast cells that, if present in other tissues, would be regarded as an abnormal chronic inflammatory cell infiltrate. A requirement for the presence of cells producing downregulatory immune mediators in the intestinal mucosa is evident in the IL-10 knockout mouse, which develops spontaneous inflammation of the colonic mucosa (30). We hypothesized that the intestinal epithelium may play a role in regulating physiological mucosal inflammation by producing signals with the capacity to chemoattract T cells that can, in turn, produce anti-inflammatory cytokines (e.g., IL-4, IL-10) capable of downregulating mucosal inflammation (3, 23). As we demonstrate herein, normal human intestinal epithelial cells produce MDC/CCL22, a CC chemokine that could fulfill that role.
Using cultured human intestinal epithelial cells, we demonstrated that
the regulation of MDC/CCL22 production differs between intestinal
epithelial cells and macrophages and monocytes. Whereas TNF- and
IL-1 were potent agonists for MDC/CCL22 protein production by cultured
human intestinal epithelial cells, this was not the case for other
MDC/CCL22-expressing cells (including monocytes, macrophages, and B
cells from human peripheral blood) in vitro (2), although
one report described small increases of MDC/CCL22 mRNA expression in
TNF-
- or IL-1-stimulated macrophages (36). In contrast,
IL-4 and IL-13 increased MDC/CCL22 production by macrophages (2,
7) but had no effect by themselves on MDC/CCL22 secretion by
cultured intestinal epithelial cells and modestly inhibited
TNF-
-stimulated MDC/CCL22 secretion. Whereas IFN-
alone
stimulated low levels of MDC/CCL22 production in human intestinal epithelial cells and synergistically increased TNF-
- and
IL-1
-stimulated MDC/CCL22 production in those cells, IFN-
inhibited MDC/CCL22 secretion by macrophages and monocytes
(7). Together, these findings suggest that MDC/CCL22
production by different cell types is dependent on the cytokine milieu
in their microenvironment, with inflammatory stimuli appearing to favor
epithelial but not macrophage-derived MDC/CCL22 production. The
predicted outcome of this epithelial cell response would be an influx
of T cells known to produce cytokines that can potentially downregulate
mucosal inflammation. The observation of constitutive MDC/CCL22 protein expression in the human intestinal xenograft tissue demonstrates that
epithelial-derived MDC/CCL22 is not completely dependent on the
presence of an inflammatory state and may be stimulated by low basal
levels of cytokines released by resident immune cells or by other
factors that remain to be identified.
Expression of the chemoattractants MDC/CCL22 and TARC/CCL17, which are both ligands for CCR4, appears to differ between different mucosal sites. We show herein that MDC/CCL22, but not TARC/CCL17, is produced by intestinal epithelial cells in response to proinflammatory stimuli. In addition, chemotactic activity for the CCR4-expressing T cell line CEM in supernatants of intestinal epithelial cells was completely abolished by neutralizing anti-MDC/CCL22 antibodies. This finding indicates that MDC/CCL22 may be the sole intestinal epithelial-derived factor responsible for recruiting CCR4-positive T cells. In contrast to intestinal epithelial cells, bronchial epithelial cells produce TARC/CCL17, but not MDC/CCL22, after stimulation with the same proinflammatory cytokines (4, 39). The significance of this regional compartmentalization of chemokine expression is unclear since both chemokines are known to signal through CCR4 as their receptor and both can act as Th2-type T cell chemoattractants. Nonetheless, these findings suggest that there may be divergent functions between MDC/CCL22 and TARC/CCL17.
The regulated production of MDC/CCL22 by intestinal epithelial cells
shares similarities with several other chemokines that are expressed by
the intestinal epithelium. Like MDC/CCL22, the CXC chemokines
IL-8/CXCL8 and GRO/CXCL1 and the CC chemokines MCP-1/CCL2 and
MIP-3
/CCL20 are upregulated in intestinal epithelial cells in
response to TNF-
and IL-1
stimulation or bacterial infection
(26, 42) and, like the genes that encode those other chemokines (17), we show that MDC/CCL22 is a NF-
B
target gene. Furthermore, synergy between IFN-
and TNF-
or
IL-1
was observed for MDC/CCL22 regulation, similar to that noted
for IL-8 (14), RANTES, MCP-1 (41), IP-10, and
I-TAC (13). Therefore, when the cytokine milieu includes
the proinflammatory stimuli TNF-
or IL-1 together with IFN-
,
chemoattractants for both CCR4-expressing (i.e., MDC/CCL22) and
CXCR3-expressing (i.e., IP-10, Mig, and I-TAC) cells could be secreted
by the intestinal epithelium.
The constitutive production of MDC/CCL22 by intestinal epithelium in
vivo suggests that this chemokine may contribute to the trafficking of
CCR4-expressing CD4+ T lymphocytes within the
microenvironment of the normal intestinal mucosa. Nonetheless, the
cytokine milieu in the normal human intestinal mucosa is thought to be
predominantly Th1 in nature, with 10-fold more IFN--producing than
IL-4-producing T cells (21). This may reflect the low
expression of CCR4, relative to CXCR3, on
4
7-integrin mucosal homing T cells
(1, 9). A recent study demonstrated that ~7-10% of
4
7-integrin-positive peripheral blood T
cells express CCR4 (9), which approximately corresponds with the fraction of Th2 cells found within the intestinal mucosa (21). It is clear that Th2 cytokine-producing cells are
present in the intestinal mucosa, under both basal conditions
(21) and in sufficient numbers to induce pathology, as
seen in the TCR
/
knockout mouse (25) and in the
oxazolone model of murine colitis (6). However, MDC/CCL22
may have broader functions beyond the chemoattraction of Th2 cells.
Recently, a CCR4 knockout mouse was described that exhibited impaired
immune responses and increased mortality after lipopolysaccharide
challenge, associated with a decrease in macrophage recruitment
(10). Furthermore, in vivo administration of MDC/CCL22 has
been shown to increase bacterial clearance in a murine model of sepsis
through the increased recruitment of macrophages (33).
These findings suggest that MDC/CCL22 has a role in macrophage
trafficking and that epithelial cell-derived MDC/CCL22 may also
function to chemoattract mononuclear cells other than CCR4-expressing T
cells to the subepithelial regions in the intestinal mucosa.
Alternatively, the relative paucity of CCR4-expressing T cells in the
intestinal mucosa could suggest that epithelial cell-derived MDC/CCL22
can also act on a chemokine receptor other than CCR4.
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ACKNOWLEDGEMENTS |
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We thank John Leopard for expert technical assistance with immunohistochemistry and thank Dr. D. Guiney for providing monocyte-derived macrophages.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-35108, DK-58960, and DK-02808, a Research Grant from the Crohn's and Colitis Foundation of America, and a Canadian Institute of Health Research Fellowship (M. C. Berin).
Address for reprint requests and other correspondence: M. F. Kagnoff, Univ. of California, San Diego, Dept. of Medicine (0623D), 9500 Gilman Drive, La Jolla, California 92093-0623.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 September 2000; accepted in final form 26 January 2001.
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