Laboratory of Mucosal Immunology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
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ABSTRACT |
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Human intestinal
epithelial cells secrete an array of chemokines known to signal the
trafficking of neutrophils and monocytes important in innate mucosal
immunity. We hypothesized that intestinal epithelium may also have the
capacity to play a role in signaling host adaptive immunity. The CC
chemokine macrophage inflammatory protein (MIP)-3/CCL20 is
chemotactic for immature dendritic cells and CD45RO+ T
cells that are important components of the host adaptive immune system.
In these studies, we demonstrate the widespread production and
regulated expression of MIP-3
by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3
mRNA. Moreover, MIP-3
mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-
or
interleukin-1
or in response to infection with the enteric bacterial
pathogens Salmonella or enteroinvasive Escherichia
coli. In addition, MIP-3
was shown to function as a nuclear
factor-
B target gene. In vitro findings were paralleled in vivo by
increased expression of MIP-3
in the epithelium of
cytokine-stimulated or bacteria-infected human intestinal xenografts
and in the epithelium of inflamed human colon. Mucosal T cells, other
mucosal mononuclear cells, and intestinal epithelial cells expressed
CCR6, the cognate receptor for MIP-3
. The constitutive and regulated
expression of MIP-3
by human intestinal epithelium is consistent
with a role for epithelial cell-produced MIP-3
in modulating mucosal adaptive immune responses.
chemokines; dendritic cells; infectious immunity; inflammation; T lymphocytes
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INTRODUCTION |
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THE EPITHELIAL CELL
LINING of the human intestine forms a physical barrier
that separates the internal milieu of the host from luminal contents.
During the course of intestinal inflammation or microbial infection,
intestinal epithelial cells can, in addition to their normal absorptive
and secretory functions, develop additional characteristics usually
attributed to classic inflammatory cell types. Thus, after stimulation
with proinflammatory mediators such as tumor necrosis factor (TNF)-
or interleukin (IL)-1
or in response to infection with enteric
pathogens, intestinal epithelial cells upregulate a program of genes
whose products can signal the onset of an acute mucosal inflammatory
response characterized by an influx of neutrophils and monocytes
(9, 10, 21, 23, 29, 32, 37, 42). Many of the genes,
including several of the chemokine genes that are activated in
intestinal epithelial cells in response to agonist stimulation or
bacterial infection, are target genes of the transcription factor
nuclear factor (NF)-
B (13, 19, 20, 32). In this regard,
NF-
B can be viewed as a central regulator of the intestinal
epithelial cell response to a set of signals, activated by
proinflammatory stimuli and bacterial infection, that are thought to be
important for the initiation of mucosal innate immune responses and
acute mucosal inflammatory responses (13). Whereas
intestinal epithelial cells do not produce the cytokines interferon
(IFN)-
, IL-2, IL-4, and IL-5, which are essential components of host
adaptive immune responses (9, 21), recent studies have
shown that they do produce three IFN-inducible T cell chemoattractants
that may play a role in physiological inflammation characteristic of
the normal intestinal mucosa and in the chemoattraction of T helper
(Th)1-type CD4+ T cells within the intestinal mucosa
(8).
Chemokines are low-molecular-weight chemotactic cytokines that have a diverse set of activities after binding and signaling through their cognate receptors on target cells (2, 30). Chemokines play a key role in the directional trafficking of leukocytes and dendritic cells (DCs) and may play a further role in angiogenesis, hematopoiesis, organogenesis, and viral pathogenesis (2, 25, 30). Chemokines signal target cells through G protein-coupled seven-transmembrane-spanning receptors that, in many cases, are promiscuous in that a single chemokine receptor frequently binds several different chemokines. In addition, several known chemokines can bind to more than one chemokine receptor (2, 26, 30). The chemokine superfamily can be divided into four groups based on the number and spacing of the amino-terminal cysteines. In CC chemokines, two amino-terminal cysteines are adjacent, whereas in CXC chemokines, the two amino-terminal cysteines are separated by an intervening amino acid (2, 30).
Macrophage inflammatory protein (MIP)-3/CCL20 (31),
also known as liver and activation-regulated chemokine (LARC)
(15) or Exodus (16), is a member of the CC
chemokine subfamily initially noted to be expressed in human liver,
lung, appendix, and tonsillar crypts (5, 6, 15, 16, 38).
MIP-3
is selectively chemotactic for CD34+ bone marrow
cell-derived immature DCs and CD45RO+ memory T cells that
express the cognate receptor CCR6 (1, 14, 24, 28).
MIP-3
produced at sites of inflammation may chemoattract
CCR6-expressing immature DCs to the subepithelial region of mucosal
surfaces (5, 18, 34). As immature DCs capture antigen at
mucosal surfaces, they undergo a functional and phenotypic change that
includes a decrease in CCR6 expression and a concomitant increase in
expression of CCR7, the receptor for secondary lymphoid tissue
chemokine (SLC) and MIP-3
. This change enables DCs to traffic out of
the tissues, where they encounter antigen and migrate to the blood and
secondary lymphoid organs (5, 34, 40). Almost all of the
memory T cells in the circulation that express the
4
7 integrin characteristic of mucosal
homing lymphocytes in humans (3) also coexpress CCR6
(24), suggesting that MIP-3
may also be important for
chemoattracting mucosal T cells.
The orthologue of human MIP-3 in mice, termed mLARC, was recently
cloned and shown to have a selective distribution in
follicle-associated epithelium overlying murine Peyer's patches and
other mucosal lymphoid follicles (38). Unlike human
MIP-3
, mLARC is not expressed in liver or lung (38),
and mLARC differs functionally from human MIP-3
in that mLARC, but
not human MIP-3
, is chemotactic for CCR6-expressing B cells
(24). mRNA for MIP-3
has been demonstrated in
epithelial cells in human appendix and in inflamed epithelial crypts of
tonsils (5, 38), but little is known regarding the overall
distribution and regulation of MIP-3
production in the normal or
inflamed human intestinal tract. We hypothesized that human intestinal
epithelial cells might have the capacity to link mucosal innate and
acquired immunity through the regulated production of MIP-3
, a
chemokine capable of signaling immature DCs and CD45RO+ T
cells. The studies herein describe the constitutive and regulated expression and production of MIP-3
mRNA and protein by human intestinal epithelial cells, using in vitro and in vivo models, and the
presence of cells in the intestinal mucosa that express CCR6, the
cognate receptor for MIP-3
.
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MATERIALS AND METHODS |
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Reagents.
Recombinant human (rh)IL-1, IFN-
, MIP-3
, and TNF-
were from
PeproTech (Rocky Hill, NJ). Biotin-conjugated affinity-purified goat
anti-human MIP-3
, murine monoclonal antibody (MAb) to human CCR6
(clone 53103.111), and murine MAb to human MIP-3
(clone 67310.111)
were from R&D Systems (Minneapolis, MN). Mouse IgG1 and
IgG2b as well as MG-132 were from Sigma Chemical (St.
Louis, MO). Rabbit anti-human CD3 was from Dako (Carpenteria, CA).
Alexa 488-conjugated goat anti-rabbit IgG was from Molecular Probes (Eugene, OR), and Cy3-conjugated goat anti-mouse IgG was from Jackson
ImmunoResearch Laboratories (West Grove, PA).
Cell culture and stimulation protocols. The human colon adenocarcinoma cell lines HT-29, HCA-7, LS174T, HCT-8, and I-407 were grown in DMEM supplemented with 10% heat-inactivated FCS and 2 mM L-glutamine as described previously (7), and the Caco-2 cell line was grown in DMEM supplemented with 15% heat-inactivated FCS. T84 human colon carcinoma cells were grown in 50% DMEM-50% Ham's F12 medium supplemented with 5% newborn calf serum and 2 mM L-glutamine (21). HT-29, LS174T, HCT-8, I-407, and Caco-2 were from the American Type Culture Collection, HCA-7 colony 29 was a gift from S. C. Kirkland (Royal Postgraduate Medical School, London, UK), and T84 was initially obtained from K. Dharmsathaphorn (UCSD). Cells were cultured at 37°C under 5% CO2-95% air.
For agonist stimulation, confluent monolayers of HT-29, Caco-2, or T84 cells grown in six-well plates (Costar, Cambridge, MA) or Caco-2 cells grown in Transwell cultures (24-mm diameter, 0.4-µm pore size; Costar) were stimulated with TNF-Human fetal intestinal xenografts.
Human fetal intestine (obtained from Advanced Biosciences Resources,
Alameda, CA), gestational age 12-18 wk, was transplanted subcutaneously onto the backs of C57BL/6 severe combined
immunodeficiency (SCID) mice as described previously (11, 17,
23). Human fetal intestinal xenografts were allowed to develop
for 10 wk after implantation, at which time the epithelium, which is
strictly of human origin, is fully differentiated (35).
Littermate SCID mice were injected subcutaneously with
~106 HT-29 cells, and tumors were allowed to develop for
5 wk. Mice carrying mature xenografts or HT-29 tumors were injected
intraperitoneally with 1 µg of human IL-1 in 200 µl of PBS or
with 200 µl of PBS alone. Intestinal xenografts and HT-29 tumors were
removed 5 h later, and adjacent segments of intestine and HT-29
tumors were frozen in liquid nitrogen for RNA isolation or were
embedded in optimum cutting temperature compound (TissueTek, Torrance,
CA) and frozen in isopentane-dry ice or fixed in 10% neutral buffered formalin for immunohistochemical analysis. In additional
experiments, intestinal xenografts were infected with ~5 × 107 of an attenuated aroA aroC
S. typhi, in DMEM-F12 medium at a 100-µl volume, injected
intraluminally by subcutaneous injection (17, 27). Those
xenografts were removed 6 h after infection, after which mucosal
scrapings were prepared and immediately frozen in liquid nitrogen.
These studies were approved by the University of California, San Diego
Human and Animal Subjects Committees.
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 previously (13,
20). 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
NF-
B activation (13, 20). The HA epitope tag enables
identification of the exogenous superrepressor with anti-hemagglutinin
antibodies. Viral titers were determined by plaque assay. Recombinant
virus was stored in PBS containing 10% (vol/vol) glycerol at
80°C.
RNA isolation.
Total cellular RNA was extracted using an acid
guanidinium-phenol-chloroform method (TRIzol Reagent, Gibco Life
Technologies, Grand Island, NY) and treated with RNase-free DNase
(Stratagene, La Jolla, CA) (7, 21). For RT-PCR, 1 µg of
total cellular RNA was reverse transcribed and cDNA was amplified as
described previously (21). The primers for human MIP-3
(sense 5'-ACC ATG TGC TGT ACC AAG AGT TTG-3' and antisense 5'-CTA AAC
CCT CCA TGA TGT GCA AGT GA-3') and MIP-3
(sense 5'-CAG CCT GCT GGT
TCT CTG GAC TTC-3' and antisense 5'-GCC CCT CAG TGT GGT GAA CAC TAC-3') were designed from available sequences from GenBank (NM004591 and
AJ223410) and yielded PCR products of 390 bp and 276 bp, respectively.
The amplification profile was 30 cycles of 45-s denaturation at 95°C
and 2.5-min annealing and extension at 60°C for cell lines and
tissues. Negative control reactions had no added RNA in the RT and
subsequent PCR amplification. Peripheral blood mononuclear cells
stimulated overnight with 10 µg/ml phytohemagglutinin-P and 2 µg/ml
lipopolysaccharide (E. coli O111:B4) were used as a source
of cDNA for positive controls. The MIP-3
primers did not
amplify sequences in murine or human genomic DNA or murine cDNA. After
amplification, aliquots of the PCR reaction were size separated on a
1.0% agarose gel containing ethidium bromide and photographed.
ELISA.
Polystyrene 96-well plates (Immulon-4, Dynex Technologies, Chantilly,
VA) were coated with murine MAb to human MIP-3 diluted in carbonate
buffer as the capture antibody. Affinity-purified biotinylated goat
anti-human MIP-3
diluted in PBS, 1.0% BSA, and 0.1% Tween-20 was
used as the detection antibody. The second step reagent was horseradish
peroxidase (HRP)-conjugated streptavidin. Bound HRP was visualized with
3,3',5,5'-tetramethylbenzidine dihydrochloride 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. MIP-3
concentration was calculated from a standard curve
using rhMIP-3
. The ELISA was sensitive to 50 pg/ml.
Immunohistochemistry.
Segments of human colon from the histologically normal-appearing
resection margin of a surgical specimen from an individual undergoing
partial colectomy for diverticular disease, mucosal biopsies from four
healthy individuals obtained during screening colonoscopy, and mucosal
biopsies from three individuals with inflammatory bowel disease were
embedded in optimum cutting temperature compound and snap frozen in
isopentane-dry ice as described previously (7, 27). Serial
cryostat sections (5 µm) were prepared, fixed in acetone for 10 min,
and blocked in PBS-1% BSA for 1 h at room temperature. For
MIP-3 and CCR6 immunostaining, sections were incubated overnight at
4°C with 5 µg/ml mouse MAb to human MIP-3
or control mouse
IgG1 or 5 µg/ml mouse MAb to human CCR6 or control mouse
IgG2b, and sections were subsequently stained with
Cy3-conjugated goat anti-mouse IgG antibody. The isotype-matched
control antibodies were used at the same concentrations as the MIP-3
and CCR6 antibodies, respectively. For CD3 immunostaining, sections
were incubated as above with rabbit anti-human CD3 or normal rabbit
serum and subsequently stained with Alexa 488-conjugated goat
anti-rabbit IgG.
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RESULTS |
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MIP-3 expression by epithelium of normal and inflamed adult
human colon.
MIP-3
is a chemokine known to chemoattract populations of immature
DCs and CD45RO+ T cells. To test whether epithelial cells
are the major source of MIP-3
production in human colon, sections of
normal and inflamed human colon were immunostained for MIP-3
. As
shown in Fig. 1B, MIP-3
is
minimally expressed by normal colon epithelium. In contrast, epithelial
MIP-3
immunostaining was markedly increased in sections of inflamed
human colon and the epithelium was the major site of MIP-3
production (Fig. 1A). No immunostaining was seen in sections
of inflamed human colon incubated with an isotype-matched control
antibody.
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Constitutive and regulated MIP-3 mRNA expression in human
intestinal epithelial cell lines.
To better characterize the expression of MIP-3
seen in vivo, we used
several human colon epithelial cell lines. As shown in Fig.
2A, several human colon
epithelial cell lines, namely, HT-29, Caco-2, LS174T, and, to a lesser
extent, I-407, constitutively expressed MIP-3
mRNA. In contrast,
none of the cell lines expressed mRNA for MIP-3
, a chemoattractant
for CCR7-expressing mature DCs. As shown in Fig. 2B,
MIP-3
mRNA levels in HT-29 and Caco-2 cells were upregulated after
stimulation with the proinflammatory mediators TNF-
or IL-1
,
which are cytokines produced by mononuclear cells in the intestinal
mucosa during the course of mucosal inflammation. MIP-3
mRNA levels
were not upregulated by stimulation of the human colon epithelial cell
lines with IFN-
, granulocyte-macrophage colony-stimulating factor
(GM-CSF), or IL-4 (data not shown). None of the cytokines tested
(i.e., TNF-
, IL-1
, IFN-
, GM-CSF, or IL-4)
upregulated expression of the mature DC chemoattractant MIP-3
(data
not shown).
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Regulated production of MIP-3 by human intestinal epithelial
cell lines.
We next assessed whether the constitutive and regulated expression of
MIP-3
mRNA by the epithelial cell lines was paralleled by protein
production, using an ELISA to measure secreted levels of MIP-3
. As
shown in Fig. 3, unstimulated HT-29,
Caco-2, and T84 cell lines secreted 2.1, 18.6, and 1.4 ng/ml MIP-3
,
respectively, as determined after 18-h incubation. Stimulation of those
cells with IL-1
or TNF-
markedly increased MIP-3
secretion
within 3 h, with MIP-3
levels ranging as high as 200-500
ng/ml at 18 h after stimulation. Consistent with the lack of
effect on mRNA expression, stimulation of the same cell lines with
IFN-
, GM-CSF, or IL-4 did not increase MIP-3
secretion (data not
shown). Consistent with the data for T84, the cell lines HCA-7 and
HCT-8, which did not constitutively express MIP-3
mRNA, could also
be induced to upregulate the production of MIP-3
protein in response
to IL-1
or TNF-
stimulation (data not shown).
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IB
superrepressor blocks inducible MIP-3
production in
HT-29 cells.
Many of the genes upregulated in response to bacterial infection of
intestinal epithelial cells, or after stimulation of those cells with
TNF-
or IL-1
, are NF-
B target genes (13, 20), although the role, if any, of NF-
B in the transcriptional regulation of the CC chemokine MIP-3
has not previously been reported. To determine whether MIP-3
in intestinal epithelial cells functions as
an NF-
B target gene, we first assessed whether blocking NF-
B activation with a proteasome inhibitor, MG-132, decreased MIP-3
secretion in response to TNF-
or IL-1
stimulation. Treatment of
HT-29 cells with 50 µM of MG-132 before stimulation with TNF-
decreased MIP-3
secretion by 90% compared with cells not pretreated with MG-132 (data not shown). Because pharmacological agents are not
always completely specific, we also used an additional approach to
block NF-
B activation. In this approach, 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). Ad5I
B-A32/36 acts as a superrepressor of NF-
B activation by preventing signal-induced I
B
phosphorylation
(13, 20). After infection with this recombinant
adenovirus, HT-29 cells were stimulated with TNF-
or IL-1
or
infected with S. dublin or enteroinvasive E. coli. As shown in Table 2, MIP-3
secretion was markedly inhibited in agonist-stimulated or
bacteria-infected HT-29 cells infected with recombinant adenovirus
expressing the superrepressor but not in cells infected with the
same recombinant adenovirus expressing the LacZ gene (Ad5LacZ).
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MIP-3 expression is upregulated in human intestinal xenografts
in response to IL-1
stimulation or Salmonella infection.
To determine whether regulated MIP-3
expression by the human
intestinal epithelial cell lines was paralleled in vivo, expression of
MIP-3
was assessed in human intestinal xenografts. As shown in Fig.
4, we first showed that HT-29 cells,
which respond to cytokine stimulation in vitro, also do so in vivo when
implanted subcutaneously in SCID mice. We next used human intestinal
xenografts implanted subcutaneously in SCID mice and cytokine treatment
to assess regulated MIP-3
production by normal human intestinal epithelium. For these studies, we first assessed MIP-3
mRNA
expression in xenografts after intraperitoneal injection of SCID mice
with human IL-1
. As shown in Fig. 4, MIP-3
mRNA expression
increased in the xenografts of IL-1
-injected mice, although
constitutive background levels of MIP-3
varied from xenograft to
xenograft. MIP-3
mRNA expression was also increased in xenografts
infected with an aroA aroC mutant of S. typhi. We note that IL-1
is not species specific and may have
stimulated additional murine mediators that potentially can act on
human cells in the xenografts. Nonetheless, the approach used herein
allows the assessment of early changes in epithelial cell chemokine
gene expression and regulation in response to a limited set of stimuli
in human intestine in vivo, which is not possible when studying
naturally infected human subjects or individuals with ongoing acute or
chronic intestinal mucosal inflammation.
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CCR6, the cognate receptor for MIP-3, is expressed by
mononuclear cells and epithelial cells in human colon mucosa.
The normal human intestinal mucosa contains an abundant population of T
cells and DCs. Because CCR6 is the only currently known cellular
receptor for MIP-3
, we sought to determine whether cells in human
colon mucosa express CCR6. As shown in Fig.
6A, cells within the lamina
propria and a lymphoid follicle, most markedly in the marginal zone
rather than the germinal center, expressed CCR6. In addition, human
colon epithelial cells expressed CCR6 (Fig. 6, A and
B), consistent with our prior report (7) of the
constitutive expression of CCR6 mRNA by several human intestinal epithelial cell lines. Whereas many CCR6-positive mononuclear cells
also stained for the T cell marker CD3, a number of CCR6-expressing cells in the lamina propria, some of which were in close proximity to
the epithelium, did not stain for CD3 (compare Fig. 6C with Fig. 6A). This finding is consistent with the reported
expression of CCR6 by both T cells and immature DCs (5,
24). In contrast to MIP-3
mRNA expression and protein
production, CCR6 mRNA expression was not significantly increased in
intestinal epithelial cell lines infected with Salmonella,
as we reported previously (12), and epithelial CCR6
immunostaining was not increased in sections of inflamed human colon
(data not shown).
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DISCUSSION |
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We report here that the CC chemokine MIP-3 is widely expressed
and regulated in human intestinal epithelium. Moreover, epithelial expression of MIP-3
is upregulated by stimulation with the
proinflammatory cytokines IL-1
and TNF-
, cytokines that can be
produced by mononuclear cells in the intestinal mucosa during acute
inflammation, or in response to bacterial infection. Intestinal
epithelial cells activated by proinflammatory stimuli, infected with
enteric pathogens, or stimulated with bacterial products have been
shown to produce signals for the chemoattraction of neutrophils and
monocytes, cells important in host innate mucosal immunity (22,
29, 32, 37, 39, 42). Because MIP-3
is known to chemoattract
CCR6-expressing memory T cells and immature DCs (6, 24),
our data extend that current view and suggest that intestinal
epithelial cells also have the capacity to play a role in orchestrating
antigen-specific acquired mucosal immune responses. The pattern of
expression of MIP-3
throughout the epithelium of human intestine and
its expression in other sites such as human lung and liver contrasts
with its more localized expression in follicle-associated epithelium in mice and suggests that the functional activity of this chemokine goes
beyond a proposed role in the genesis and function of mucosal lymphoid
follicles (5, 6, 15, 16, 18, 38). In addition, the
upregulated expression of human MIP-3
in response to TNF-
or IL-1
stimulation of human intestinal epithelial cells contrasts with the
lack of response of MIP-3
production in J774 mouse monocytes to
those stimuli (38), indicating that there may be
differences in the signal transduction pathways important for
activating MIP-3
in different cell types and/or species.
Our finding of upregulated epithelial cell MIP-3 in
cytokine-stimulated or bacteria-infected human intestinal xenografts, and in inflamed human colon, indicates that human intestinal epithelial cells regulate the expression of a chemokine whose major known function
is to chemoattract cells important for antigen presentation and the
development of the host adaptive immune response. Thus, in response to
inflammatory stimuli, intestinal epithelial cells may develop the
capacity to chemoattract immature DCs and memory T cells in close
proximity to the single layer of intestinal epithelium that separates
the intestinal lumen from the host's internal milieu. After capturing
antigen, immature DCs at sites of inflammation undergo a phenotypic
change and migrate to regional lymph nodes. As part of this process,
they downregulate surface expression of several chemokine receptors
(i.e., CCR1, CCR5, and CCR6), concurrently lose their responsiveness to
the chemokine ligands for those receptors (i.e., MIP-1
, MIP-1
,
RANTES, and MIP-3
) (33, 34, 36), upregulate the
expression of CXCR4, CCR4, and CCR7, and become responsive to
chemokines expressed in blood vessels and secondary lymphoid organs
[e.g., SLC, MIP-3
, stromal cell-derived factor (SDF-1),
macrophage-derived chemokine, thymus- and activation-regulated chemokine] (5, 33, 34, 36). Consistent with this
phenotypic and functional change in DCs, intestinal epithelial cells do
not express the mature DC chemoattractant MIP-3
. In addition to
high-level MIP-3
production after stimulation, it is possible that
constitutive low levels of epithelial expression of MIP-3
may serve
to maintain immature DCs and memory T cells in close proximity to the
epithelial surface, the first site of host contact with antigen in the
intestinal lumen.
MIP-3 is shown to function as a NF-
B target gene in human
intestinal epithelial cells, and its expression is upregulated by
TNF-
or IL-1
stimulation or infection with enteric bacteria. The
same is true of IL-8 and growth-related protein-
(GRO-
) that,
like MIP-3
, are upregulated in the intestinal epithelium by IL-1 or
TNF-
stimulation or infection with enteric bacterial pathogens
(13, 19, 22, 42). However, MIP-3
mainly signals immature DC and CD45RO+ T cells important in host adaptive
immune responses, whereas IL-8 and GRO-
signal neutrophils that can
play a role in mucosal innate immune defense. We recently described
(8) the expression of three additional T cell
chemoattractants expressed by human intestinal epithelium, the CXC
chemokines IP-10, Mig, and I-TAC, which, in contrast to MIP-3
, are
known to signal activated/memory CD4+ CD45RO+ T
cells that express the receptor CXCR3. Unlike MIP-3
, those chemokines are preferentially upregulated by the Th1 cytokine IFN-
and are only minimally, if at all, regulated by TNF-
, IL-1, or
bacterial infection in the absence of IFN-
. Together, these data
suggest that the intestinal epithelium can play a role in regulating
both innate and acquired mucosal immune responses and demonstrate
differential regulation of epithelial cell-produced chemokines that act
on different target populations of T cells.
The human intestinal epithelial cell lines tested in our studies
secreted up to 200-500 ng/ml of MIP-3 in response to IL-1
stimulation. It is not currently possible to determine the effective concentrations of MIP-3
in the microenvironment of the intestinal mucosa because this depends on several factors, including the biological half-life of MIP-3
in the mucosa, the extent to which it
binds proteoglycans and is available for binding to CCR6 on target
cells, and its effective diffusion distance from the epithelium. Nevertheless, the quantity of MIP-3
secreted by human intestinal epithelial cell lines is within the range shown in vitro to be chemotactic for lymphocytes. Thus 1 µg/ml recombinant MIP-3
was maximally chemotactic for freshly isolated human peripheral blood lymphocytes, and significant chemotaxis was observed at concentrations of 100 ng/ml (1).
The present studies report on the regulated expression of MIP-3 by
human intestinal epithelium. It will be important for future studies to
assess the functional activity of this chemokine on target cells in the
complex microenvironment of the human intestinal mucosa. Relevant to
MIP-3
function in the intestinal tract, a recent study in mice
indicated that a specific subset of mucosal DC within the subepithelial
region of Peyer's patches express CCR6 and migrate in response to
MIP-3
(18). However, we also note that CCR6, the
cognate receptor for MIP-3
, was abundantly expressed by epithelial
cells in normal human colon. This is consistent with our prior report
(7) that several human intestinal epithelial cell lines
express CCR6 mRNA as well as functional receptors for several other CC
and CXC chemokines, most notably CXCR4, which binds the CXC chemokine
SDF-1, and CCR5, which binds the CC chemokines MIP-1
, MIP-1
, and
RANTES. Like CXCR4 and CCR5, CCR6 on intestinal epithelial
cells acts as a functional signaling receptor after interaction with
its ligand MIP-3
(unpublished data). Thus, in addition to paracrine
effects on DCs and T cells, MIP-3
, like several other mediators
produced by intestinal epithelial cells, may mediate autocrine effects
on the intestinal epithelium. In contrast to MIP-3
, CCR6 was not
upregulated by intestinal epithelial cells in inflamed colon.
Finally, we note that CCR6 has been reported to act as a functional
receptor for a second epithelial cell-produced ligand, the
antimicrobial peptide human defensin-2 (hBD-2) (41).
In addition, we have reported (27) the upregulated
expression of hBD-2 by human intestinal epithelial cells in vitro and
in vivo in response to proinflammatory cytokine stimulation or
bacterial infection. However, we found (present study and Ref.
27) that MIP-3
secretion by agonist-stimulated or
bacteria-infected human intestinal epithelial cell lines was
~100-fold greater than that of hBD-2 in response to identical
proinflammatory stimuli. Coupled with a lower affinity of hBD-2 binding
to CCR6 compared with MIP-3
(41), hBD-2 produced by
intestinal epithelium may not compete effectively with MIP-3
for
binding to CCR6 on immature DC or mucosal T cells or to CCR6 expressed
by intestinal epithelium.
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ACKNOWLEDGEMENTS |
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We thank J. Leopard and D. McCole for helpful assistance and R. Lara for final preparation of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-35108. A. Izadpanah is a Howard Hughes Medical Institute Medical Student Research Training Fellow; M. B. Dwinell was supported by a Research Career Development Award from the Crohn's and Colitis Foundation of America (CCFA) and NIDDK Grant K01-DK-02808; and L. Eckmann was supported, in part, by a research grant from the CCFA.
Address for reprint requests and other correspondence: M. F. Kagnoff, Laboratory of Mucosal Immunology, Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 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 July 2000; accepted in final form 19 October 2000.
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