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*
Department of Medicine, Division of Gastroenterology and Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine, Los Angeles, CA 90048;
Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA 90048; and
LeukoSite, Cambridge, MA 02142
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7. Here we show that TECK is expressed
in the human small bowel but not colon by endothelial cells and a
subset of cells in intestinal crypts and lamina propria. CCR9 is
expressed in the majority of freshly isolated small bowel lamina
propria mononuclear cells (LPMC) and at significantly higher levels
compared with colonic LPMC or PBL. TECK was selectively chemotactic for
small bowel but not colonic LPMC in vitro. The TECK-induced chemotaxis
was sensitive to pertussis toxin and partially inhibited by Abs to
CCR9. TECK attracts predominantly the T cell fraction of small bowel
LPMC, whereas sorted CD3+CCR9+ and
CD3+CCR9- lymphocytes produce similar Th1 or
Th2 cytokines at the single cell level. Collectively, our data suggest
that the selective expression of TECK in the small bowel underlie the
homing of CCR9+ intestinal memory T cells to the small
bowel rather than to the colon. This regional specialization implies a
segregation of small intestinal from colonic immune
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, CXCR5,
which binds B cell-attracting chemokine 1, and CCR9, which binds
thymus-expressed chemokine (TECK) only, among others
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Chemokines were identified by their ability to
direct extravasation of inflammatory cells during infection (1, 2). However, recent data identified several chemokines that
are expressed constitutively in lymphoid and extra-lymphoid
tissues, indicating that these chemokines might have homeostatic
function by regulating lymphocyte trafficking to or within lymphoid
organs and in peripheral tissues (3, 16, 17, 18, 19, 20, 21). Certain
chemokines, such as stromal cell-derived factor 1 (SDF-1), 6-C-kine,
and MIP-3ß can also stimulate leukocyte adhesion and arrest on
endothelium by triggering integrin activation (22, 23). It
is thought that the combined expression of adhesion molecules and
chemokine receptors on the cell surface provide an "address
code" for leukocyte migration to different sites
(24).
In contrast to naive T cells, memory/effector cells migrate mostly
through peripheral tissues, and this process is controlled by the
expression of different sets of integrins and chemokine receptors
(25, 26, 27, 28). Thus, naive T cells express CXCR4, the receptor
for SDF-1, and CCR7, the receptor for EBV-induced molecule 1 ligand
chemokine and secondary lymphoid tissue chemokine (also called
6-C-kine). Mice deficient in CCR7 or secondary lymphoid tissue
chemokine have defective homing of naive T cells to secondary lymphoid
organs (16, 29). Gene knockout studies have established
that CXCR5 is required for B cell migration to B cell follicles of
spleen and Peyer’s patches (PP; Ref. 30). In addition,
the defects in lymph node (LN) development observed in lymphotoxin-
and TNF knockout mice have been attributed, at least in part, to
decreased production of chemokines by stromal LN cells
(31). Collectively, these data suggest an important role
of certain chemokines in regulating the homing of specific T cell
subsets and other immune cells into microanatomic compartments of
secondary lymphoid organs. Certain chemokine receptors are also
preferentially expressed on naive T cells under Th1 or Th2 polarizing
conditions in vitro. Th1 cells predominantly express CXCR3 and CCR5
(32, 33, 34, 35, 36). In contrast, Th2 cells express CCR3, CCR4, and
CCR8 (3, 24, 32, 33, 35, 36). Recently, the orphan
chemokine receptor GPR-9-6, now designated CCR9, was found to be
expressed on a small percentage (2–4%) of circulating memory T cells,
all of which express the mucosal homing ligand
4ß7 (9).
The ligand for CCR9, TECK, also CCL25/Ckß-15 according to the recent
chemokine/chemokine receptor nomenclature (3), is
selectively expressed in the thymus and small intestine (9, 13, 37, 38). In addition, CCR9 mRNA is expressed in the thymus,
small intestine, and at lower levels in the spleen, suggesting that the
CC-chemokine TECK and its receptor, CCR9, may play an important role in
T cell maturation and in the intestinal immune response
(9).
The potential importance of CCR9 and TECK in mucosal immunity prompted
us to study the expression of this chemokine/chemokine receptor pair in
the normal small and large bowel. Our data suggest that the TECK/CCR9
chemokine/chemokine receptor pair is important for the regional
specialization of intestinal immunity and the combined expression of
CCR9/4ß7 on the cell
surface may provide a small intestinal "address code" for
circulating intestinal memory T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intestinal specimens were obtained from patients undergoing surgical resection of the colon (with colon carcinoma) or small intestine usually during the second stage of a prior ileal anal-pouch anastomosis at Cedars-Sinai Medical Center (Los Angeles, CA). Approval for the use of human subjects was obtained from the Institutional Review Board at Cedars-Sinai Medical Center. In this study, all tissue specimens were taken from an uninvolved area of resected colon or small bowel. LPMC were isolated using a technique modified from that described previously (39). PBMC were isolated from normal healthy volunteers by separation on Ficoll-Hypaque gradients. The cells were subsequently washed three times with HBSS and resuspended in culture media (RPMI 1640 with 10% FCS) at a concentration of 2 x 106/ml. MLN lymphocytes were isolated following mechanical disruption of LNs. IECs from small bowel or colon were isolated as previously described (40).
Abs and reagents
Anti-CD3, -CD4, -CD8, -CD19, -CD56, -HLA-DR, -CD45RO dye-linked
mAbs for immunofluorescence studies were obtained from Caltag (South
San Francisco, CA). Anti-CD25, -CD69, and -CD95 were obtained from
PharMingen (San Diego, CA). TECK and SDF-1 were purchased from
PeproTech (Rocky Hill, NJ).
Generation of anti-TECK and anti-CCR9 mAbs
The mAb to human TECK (hTECK; clone LS202 5A9, IgG1) was generated by i.p immunization of BALB/c mice at 3-wk intervals with 10 µg of TECK (Peprotech) in CFA, IFA, and finally PBS. Fusions were performed after at least four immunizations 3 days after the last boost by fusion with SP2/0 myeloma cells (American Type Culture Collection, Manassas, VA). Fusions were screened by ELISA with plates coated with hTECK, and the positive hybridomas were subcloned. Of 20 anti-TECK mAbs tested only two, designated 4G1 and 5A9, were found to stain frozen sections of thymus. The mAb to human CCR9 has been previously described (9).
Flow cytometry
Freshly isolated or cultured LPMC or PBMC (2.5–5.0 x 105) were washed twice with 1 ml of PBS supplemented with 0.1% BSA and 0.1% azide. The cells were resuspended in 100 µl of 10% human Ab serum to block nonspecific Fc binding for 15 min. For the staining of surface Ags, cells were incubated with the mAb 3C3 for 30 min on ice, washed with PBS/BSA/azide, and incubated with a secondary goat anti-mouse anti-Fab (H+L) tricolor or goat anti-mouse IgG2b-PE for 30 min on ice. The cells were washed again with PBS/BSA/azide and incubated with mouse IgG for 15 min. FITC- and PE-conjugated mAb for surface Ag were used for 30 min. After washing twice, cells were resuspended in 400 µl of 1% paraformaldehyde in PBS and analyzed by FACS (Becton Dickinson, Mountain View, CA). Events (104) were routinely collected and analyzed using Lysis II software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Both the percentage of positive cells and the mean fluorescence intensity (MFI) of the cells were determined.
RT-PCR
Total RNA was extracted from small bowel or colonic intestinal mucosa and freshly isolated small bowel or colonic IECs and LPMC using the RNeasy Kit as recommended by the manufacturer (Qiagen, Valencia, CA). One microgram of RNA was reverse-transcribed into cDNA with oligo(dT) in a 20-µl volume using a Thermoscript RT-PCR System (Life Technologies, Grand Island, NY) according to a standard protocol. Primers were designed as described elsewhere (9). Primers for TECK were: sense 5'-TCGAAGAAGCTTATGAACCTGTGGCTCCTG-3' antisense 5'-AAGAAGTCTAGATCACAGTCCTGAATTAGC-3' (product 453 bp). Two microliters of the reaction cDNA was mixed with 10 mM dNTP, 10 µM primers, 50 mM MgCl2, and 5 U/µl of Platinum Taq DNA polymerase in a 50-µl volume as recommended by the manufacturer (Life Technologies). The cycle parameters were an initial melt at 95°C for 2 min, then 35 cycles: 95°C, 30 s; 55°C, 30 s; and 72°C, 1 min, followed by a final extension of 72°C, 7 min. Amplification with G3PDH primers (sense 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', antisense 5'-CATGTGGGCCATGAGGTCCACCAC-3') (product 983 bp) (Clontech, Palo Alto, CA) was examined in identical conditions as an internal control to demonstrate equivalence of template. The PCR products were visualized with ethidium bromide after 1.5% agarose gel electrophoresis.
Cytokine detection at the single cell level
Freshly isolated LPMC or sorted mucosal T cells were stimulated
with 10-7 M PMA and 1 µg/ml ionomycin for
4 h. Brefeldin A (10 µg/ml) was added to the culture after
2 h of stimulation to block cytokine secretion. Unseparated cells
(106 cells) were surface stained and washed twice
as mentioned above and subsequently fixed with 4% paraformaldehyde and
permeabilized with saponin. Fixed and permeabilized cells were stained
with FITC anti-IFN-
and PE anti-IL-4 mAbs (PharMingen) and
analyzed by FACS (Becton Dickinson).
Chemotaxis assay
Cell migration was evaluated using a 48-well chemotaxis chamber (Neuroprobe, Cabin John, MD). TECK, diluted in HEPES-buffered RPMI 1640 supplemented with 1% BSA, was added to the lower wells, and 105 cells in the same buffer to the upper wells. Polyvinylpyrrolidone-free polycarbonate membranes (Neuroprobe) with 3-µm pores were used. After incubation for 120 min at 37°C, the cells that had migrated through the pores to the lower wells were counted by FACS. A known number of 3.2-µm fluorescent microsphere beads (PharMingen) was added to each sample before analysis to determine the absolute number of migrating cells. The assay was performed in triplicate.
Immunohistochemistry
Sections were fixed and stained as previously described (41). Briefly, fixed sections (6 µm) from normal small bowel and colonic mucosa, or thymus, were deparaffinized and treated with citrate buffer (pH 6). The sections were then incubated with anti-hTECK Ab (clone LS202 5A9, IgG1) followed by a goat anti-mouse Ab (Dako, Carpinteria, CA) at a 1:20 dilution. The sections were subsequently incubated with a mouse peroxidase anti-peroxidase (Dako) at 1:100 dilution. Following wash, the sections were developed with DAB substrate-chromogen (Dako). Sections were counterstained with hematoxylin (Fisher Scientific, Pittsburgh, PA).
Statistical analysis
Where indicated Student’s t test was used to calculate statistical significance for difference in a particular measurement between different groups. Values of p < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We measured the percentage CCR9+ LPMC in the
small bowel and colon by flow cytometry using a CCR9-specific mAb,
designated 3C3. In cross-reactivity studies, 3C3 did not cross-react
with CCR1-7 or CXCR1-4 transfectants (9). Three color
immunofluorescence analysis of LPMC from small bowel or colon and PBMC
showed that CCR9 is expressed predominantly in small bowel compared
with colonic or peripheral blood CD3+ T
lymphocytes. CCR9 was expressed at high levels in both the CD8 and CD4
subsets of small bowel CD3+ cells. As shown in
Fig. 1
, CCR9 was expressed in 67%
(range, 57–76%) of
CD3+CD4+ small bowel
compared with 20% (range, 15–25%) of colonic
(p < 0.0001) and 4% (range, 3–5%) of
peripheral blood CD3+CD4+
lymphocytes. The differences between groups were statistically
significant (p < 0.0001). Similar differences
in CCR9 expression were observed between small bowel, colonic, or
peripheral blood CD3+CD8+
lymphocytes. CCR9 was expressed in 58% (range, 47–70%) compared with
10% (range, 6–14%) and 2% (range, 1.4–2.6%) of small bowel,
colonic, or peripheral blood
CD3+CD8+ cells,
respectively (Fig. 1). In addition, the density of CCR9 expression, as
assessed by the MFI in each lymphocyte subset showed that CCR9 is
expressed at higher levels in small bowel compared with colonic or
peripheral blood T cells in both the CD4 and CD8 compartment (Table I). We further analyzed the expression of
CCR9 by flow cytometry on freshly isolated MLN lymphocytes draining
small bowel and colon.
CD3+CCR9+ lymphocytes were
significantly enriched in small bowel vs colonic MLN (mean, 69 vs 11%,
n = 3, p < 0.05). A representative
FACS analysis of CCR9 expression in MLN lymphocytes is shown in Fig. 2. Collectively, our data show that the
chemokine receptor CCR9 is expressed in a much larger percentage and at
a higher density in small bowel compared with colonic lamina propria
and draining MLN T lymphocytes.
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, CCR9 was expressed
predominantly on CD3+ small bowel LPMC, although
a significant percentage of CD3- cells also
express CCR9. Also CCR9 was expressed on a subset of B and NK cells.
Almost all CCR9+ LPMC, whether from small bowel
or colon, coexpressed CD45RO, CD69, CD95, and a significant percentage
were also HLA-DR+ (Fig. 3, and data not shown).
This is consistent with the known phenotype of mucosal lymphocytes as
highly activated memory cells (39).
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It has been shown that hTECK message was detected by RT-PCR and
Northern blot in the thymus and small bowel, but not colon (9, 37). In mice, murine TECK (mTECK) was initially reported to be
expressed by thymic dendritic cells (37) but two recent
reports showed it to be expressed by thymic and small IECs (13, 38). Therefore, we studied the expression of TECK in human
intestinal tissues. First we analyzed TECK expression by RT-PCR in
whole small bowel and colonic mucosa, as well as in IECs and LPMC
isolated from small bowel and colon. Fig. 4 shows that TECK was detected by RT-PCR
in whole small bowel mucosa, as well as in small bowel IECs and LPMC.
TECK message was absent from whole colonic mucosa and colonic IECs or
LPMC. To investigate more precisely the distribution of TECK expression
in small bowel, we performed immunohistochemistry using a mAb,
designated 5A9, against hTECK. Thymus tissue was used as positive
control for hTECK staining. As shown in Fig. 5, TECK staining was detected in the
small bowel and thymus but not colon (compare a–c, and
f with d). Strong TECK immunoreactivity was
detected in a subset of cells with elongated processes in the small
bowel intestinal crypts and the lamina propria predominantly in areas
of lymphocyte aggregates (Fig. 5b). TECK staining was not
detected in mucosal lymphocytes or surface epithelial cells.
TECK-immunoreactive cells were also detected in small bowel but not
colonic endothelial cells (compare Fig. 5, a inset and
c with d). In thymic sections, TECK
immunoreactivity was observed in the stromal component of the thymic
medulla and cortex, as has been shown previously for TECK expression in
murine thymus (Fig. 5f).
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TECK has been shown to induce Ca2+
mobilization and chemotaxis of CCR9 transfectants (9, 10, 14), suggesting that CCR9 is a specific chemotactic receptor for
TECK. In addition, the small percentage of PBMC that respond by
chemotaxis to TECK express high levels of the mucosal homing ligand
4ß7 (9).
In addition, the selective expression of the chemokine TECK in the
small bowel and its receptor CCR9 in small bowel lymphocytes suggest
that TECK may be involved in the selective trafficking of
CCR9-expressing lymphocytes to the small bowel instead of colon.
Therefore, we examined whether small bowel and colonic LPMC respond
differentially to TECK in a chemotaxis assay. In bulk migration
experiments, TECK chemoattracts freshly isolated small bowel but not
colonic LPMC or PBMC (Fig. 6a). The migrating small bowel
LPMC when plotted vs increasing concentration of TECK revealed a
bell-shaped curve typically observed with other chemokines. The optimal
chemotactic concentration of TECK was 500 nM for small bowel
lymphocytes, which is within the range (1–1000 nM) of chemotactic
activity seen with other chemokines (9) (Fig. 6a). Checkerboard analysis established that TECK induced
chemotaxis and not chemokinesis of small bowel LPMC (data not shown).
The inability of colonic LPMC to migrate in response to TECK was not
due to a general migratory defect of those cells compared with small
bowel LPMC because both migrated to SDF-1, which signals through
CXCR4 (23) (data not shown). Migration in response to TECK
could be partially blocked by preincubation of lymphocytes with
anti-CCR9, but not by anti-CCR3 mAb (Fig. 6b).
Preincubation of the cells with pertussis toxin completely inhibited
the migration in response to TECK, consistent with
Gi protein-coupled signaling through CCR9
(9), as has been shown for other chemokine receptors
(1, 2, 3).
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To further characterize the mononuclear cell subsets that respond
to TECK, we analyzed the phenotype of small bowel LPMC that migrated to
optimal concentrations (500 nM) of TECK. We used flow cytometry to
identify T cells (CD3+), B cells
(CD19+), or non-T/non-B cells
(CD3- CD19-) cells and
CD4+ or CD8+ subsets of T
cells. LPMC that migrated to the lower wells of the chemotaxis chamber
were pooled and stained with mAb for CD3, CD4, CD8, CD45, and CD19. The
percentage of cells of each phenotype that migrated to TECK was
analyzed by FACS. As shown in Fig. 7, TECK was primarily chemotactic for T cells, both the
CD4+ and CD8+ subsets. The
percentage of T cells was 85% (60% CD4+, 20%
CD8+) in the migrating population compared with
56% (39% CD4+, 13% CD8+)
in the starting population. TECK was not chemotactic for B lymphocytes
and non-T/non-B mononuclear cells (Fig. 7).
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Distinct profiles of chemokine receptors are acquired during in
vitro differentiation of naive T lymphocytes into Th1 or Th2 subsets.
For example, Th1 cells preferentially express CXCR3 and CCR5
(32, 33, 34, 35, 36), and Th2 cells express CCR3, CCR4, and CCR8
(24, 32, 33, 35, 36). Therefore, we examined whether CCR9
expression in mucosal T cells defines a Th1 or Th2 cytokine-producing
phenotype. We examined cytokine production at the single cell level by
intracellular cytokine staining. In initial experiments, we found no
differences in the percentage of IFN--producing (Th1) or
IL-4-producing (Th2) cells between small bowel and colonic
CD3+ lymphocytes despite a significant difference
in the expression of CCR9 between these lymphocyte subsets (data not
shown). To further confirm the cytokine profile of
CCR9+ mucosal T cells, CD3+
cells from the small bowel were sorted into
CD3+CCR9+ and
CD3+CCR9- subsets using
flow cytometry and analyzed for cytokine production. The percentage of
Th1 or Th2 cells among
CD3+CCR9+ and
CD3+CCR9- small bowel
lymphocytes was similar, although IFN--producing cells were slightly
higher in the CD3+CCR9+
subset and the percentage of cells producing neither IFN- nor IL-4
were higher in the
CD3+CCR9- subset (Fig. 8). These data show that small bowel
mucosal T cells expressing CCR9 exhibit a diverse cytokine profile and
that CCR9 expression is rather linked to a phenotype with selective
homing potential to the small bowel mucosa.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7
(43).
MAdCAM-1/4ß7
interaction seems to play an important role in the preferential homing
of
4ß7high
memory lymphocytes, which carry memory for intestinal Ags
(44) to the gastrointestinal tract and mucosal immune
system (28, 42). Indeed,
ß7-deficient mice have hypoplastic
gut-associated lymphoid tissue (GALT) and a significant reduction in
the number of CD4+, CD8+,
and plasma cells in the lamina propria (45). Therefore,
the 4ß7 expression in
peripheral memory T cells provides a mechanism for the segregation of
intestinal from systemic immune responses (42).
The chemokine receptor CCR9 has been shown to be expressed on
thymocytes and on a subset of memory
4ß7+
intestinal trafficking CD4 and CD8 PBL. In addition, all small
intestinal LPL and intraepithelial lymphocytes (IELs) express CCR9
(9). In this study, we directly compared the expression of
CCR9 between small bowel and colonic LPL and MLN lymphocytes. Although
transcript of CCR9 is absent in colon, we found that a small percentage
of colonic lymphocytes express CCR9 by flow cytometry. We show the
selective expression of CCR9 on the majority of small bowel lymphocytes
as was also shown previously (9) and, in addition, CCR9
expression on a much lower percentage of colonic lymphocytes. Moreover,
we demonstrate that CCR9+ lymphocytes are
significantly enriched in MLN draining small bowel vs colon. In
addition, phenotypic analysis of CCR9-expressing small bowel LPL
reveals that CCR9 is expressed predominantly on
CD3+ lymphocytes, although a subset of B and NK
cells are also CCR9+. It was shown previously
that CCR9 is expressed on a significant percentage of B cells but not
NK cells on PBL (9). Based on three-color flow cytometry,
we show in this study that CCR9 is expressed on CD4 and CD8 small bowel
T lymphocytes and virtually all
CD3+CCR9+ lymphocytes
coexpress CD45RO, CD69, and CD95. A subset of
CD3+CCR9+ small bowel
lymphocytes also coexpress CD25 and HLA-DR.
The ligand for CCR9, TECK, has been shown to be expressed in the thymus
and small intestine (9, 13, 37). The mTECK was reported to
be expressed in the thymus by dendritic cells as well as endothelial
cells (37). Another report showed mTECK expression in the
thymus and small intestine by epithelial cells (13). In
this study, we define for the first time the cells expressing TECK in
human intestinal tissues. Human TECK is selectively expressed in
the small bowel but not colonic mucosa by a subset of cells in
intestinal crypts and lamina propria as well as endothelial cells.
In a recent report mTECK mRNA expression was reported to be restricted
to villus epithelial cells, the expression beginning at or just below
the crypt-villus junction, increasing to maximum level approximately
one-third of the way up the villus, and subsequently decreasing toward
the villus tip (13). Interestingly, mTECK mRNA was
detected in the follicle-associated epithelium of the murine PP by in
situ hybridization. Although we have not directly examined hTECK
protein expression in human PP, immunohistochemistry of small bowel
mucosa shows distinct hTECK protein expression compared with mTECK mRNA
expression profile. In human tissue, scattered crypt but not villus
epithelial cells stain for TECK protein. The reason for the discrepancy
in TECK mRNA and protein expression in the small bowel mucosa between
mouse and human is unknown. Importantly, we show that hTECK is
expressed on small bowel but not colonic endothelial cells. In addition
to the expression profile of hTECK protein in intestinal tissues, we
demonstrate that TECK chemoattracts small bowel but not colonic
lymphocytes in vitro. Therefore, the selective expression of TECK in
small bowel compared with colon may account for the selective
recruitment and retention of
CCR9+4ß7+
T cells in small bowel lamina propria. The small number of colonic
lymphocytes expressing CCR9, which are predominantly
CD3+CD4+, may be recruited
to the colonic lamina propria in response to other chemokine(s) and may
only coincidentally express CCR9. It has been hypothesized that CCR9
expression is induced on naive T lymphocytes in the mucosal environment
(9). We further propose that CCR9 induction must be unique
to the small bowel mucosal environment. This idea is supported by the
finding that draining MLN from small bowel contain a higher percentage
of CCR9+ lymphocytes than do MLN draining colon.
However, in naive T lymphocytes CCR9 expression could not be induced
during culture with several cytokines, including TGF-ß or IL-10,
which are highly expressed in the mucosal environment
(46). Therefore, the mechanism by which CCR9 is induced or
up-regulated by local microenvironmental factors in the small bowel
inductive sites of the mucosal immune system is currently unknown.
Nevertheless, activated CCR9+ cells in draining
LNs may subsequently recirculate and home selectively to intestinal
sites that express TECK, such as the small bowel mucosa.
The precise mechanism by which TECK is involved in the homing of
CCR9+ lymphocytes to the small bowel mucosa is
currently unknown. A likely explanation is that TECK presented on the
small bowel endothelial cell surface may trigger firm adhesion of
circulating CCR9+ lymphocytes to the vessel wall
via 4ß7/MAdCAM-1
interactions, and subsequent transmigration into the lamina propria as
has been shown for other chemokines (20, 21, 22, 23). TECK
expressed by resident small bowel mucosal stromal cells may help retain
these CCR9+ T lymphocytes into the small bowel
mucosa. It is also tempting to speculate that TECK, which is
constitutively expressed in the small bowel intestinal crypts, may be
involved in the homing and retention of small bowel IELs. Indeed, most
of small bowel IELs express CCR9 (9), whereas the majority
of colonic IELs are CCR9- (K. A. Papadakis,
unpublished data). Our data show for the first time a phenotypic
difference between small bowel and colonic LPMC in humans, based on the
selective expression of the chemokine receptor CCR9, which strongly
suggests a mechanism for regulated trafficking of intestinal memory T
cells to the small bowel vs colon.
We finally demonstrate that CCR9+ small bowel T
lymphocytes have a diverse cytokine profile, as similar percentages of
sorted CD3+CCR9+ and
CD3+CCR9- small bowel
lymphocytes produce IFN- or IL-4. Distinct patterns of chemokine
receptors are acquired by T cells under Th1 (CXCR3, CCR5)- or Th2
(CCR3, CCR8)-polarizing conditions in vitro, which translates into new
migratory behavior toward their respective chemokines (24, 32, 33, 34, 35, 36). Therefore, the expression of certain chemokines will
influence the type of T cell immune response in a tissue, for instance,
Th1 or Th2. However, other chemokines may have a role in
tissue-selective recruitment of lymphocytes from the blood
(47). Thymus and activation-regulated chemokine and
macrophage-derived chemokine, for example, the ligands for CCR4,
strongly attract skin-homing but not intestinal memory T cells. The
receptor CCR4 is highly expressed on cutaneous lymphocyte Ag memory
CD4+ cells; therefore, CCR4 expression may define
a phenotype of predominantly skin homing subset of peripheral memory T
cells (47). Consistent with these observations, CCR4
expression was virtually absent in mucosal T cells (K. A.
Papadakis, unpublished results). Another example, cutaneous T
cell-attracting chemokine (CTACK or CCL27), a recently described
chemokine, is expressed in skin and selectively chemoattracts cutaneous
lymphocyte Ag memory T cells (19).
Our data suggest that the TECK/CCR9 ligand/receptor pair is important for the selective homing and retention of CCR9+ T lymphocytes to the small intestine instead of colon, and provides a mechanism for regional specialization of the mucosal immune system and the segregation of small bowel from colonic immune responses. The CCR9 phenotype is not linked to a specific cytokine profile because both small bowel and colonic lymphocytes as well as sorted CCR9+ and CCR9- small bowel T lymphocytes produce similar Th1 or Th2 cytokines at the single cell level. Consistent with these observations is the finding that CCR9 expression cannot be induced in naive T cells under Th1-, Th2, or T-regulatory 1 polarizing conditions (9).
In summary, the preferential expression of the chemokine receptor CCR9
in small bowel compared with colonic lymphocytes, the expression of its
ligand, TECK, by small bowel but not colonic endothelial and stromal
cells, and the in vitro migration of small bowel but not colonic
lymphocytes to TECK, suggest that the TECK/CCR9 ligand/receptor pair is
important for the regional specialization of the mucosal immune system.
The selective expression of a chemokine, such as TECK, in the small
intestine could provide the fine-tuning of mucosal T cell trafficking
in combination with the expression of the intestinal mucosal homing
ligand 4ß7. Therefore,
the combination of
CCR9/4ß7 expression
may provide a novel mechanism to segregate small intestinal from
colonic immune responses. The recent identification of the mouse
homologs of TECK and CCR9 will permit further examination of their role
in T cell development and intestinal immunity by selectively inhibiting
their expression. Further understanding of the selective
trafficking of T cell to the small bowel based on CCR9 expression will
also help us study immune mechanisms such as oral tolerance and
immune-mediated diseases of the small intestine.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Konstantinos A. Papadakis, Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, D-4062, Los Angeles, CA 90048.
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; LN, lymph node; LPMC, lamina propria mononuclear cells; PP, Peyer’s patches; TECK, thymus-expressed chemokine; hTECK, human TECK; IECs, intestinal epithelial cells; MAdCAM-1, mucosal addressin cell-adhesion molecule; mTECK, murine TECK; IELs, intraepithelial lymphocytes; SDF-1, stromal cell-derived factor 1; MLN, mesenteric lymph node; MFI, mean fluorescence intensity.
Received for publication May 22, 2000. Accepted for publication July 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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