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*
Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine, Osaka, Japan;
First Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan;
Departments of Biochemistry and Internal Medicine, School of Medicine, Keio University, Tokyo, Japan;
§
Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan; and
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Department of Pathology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7. After
birth, MAdCAM-1 expression in thymus blood vessels disappeared and
concomitantly, the soluble MAdCAM-1-reactive thymocytes were rapidly
down-regulated. Our results suggest that MAdCAM-1 functions as a
vascular addressin in not only mucosal, but also nonmucosal lymphoid
tissues during ontogeny. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7
(2) and vascular adhesion molecule MAdCAM-1 (3, 4), the interaction of which is essential in both rolling and
sticking of lymphocytes in PP HEV (5).
MAdCAM-1 is induced on endothelial cells by various proinflammatory
cytokines such as TNF-, IL-1 (6), and lymphotoxin
(7), or by inflammation-inducing agents such as LPS
(6), and enhanced expression of this molecule is
considered to play an important role in the recruitment of inflammatory
cells to the sites of inflammation. For instance, MAdCAM-1 expression
is up-regulated in endothelial cells of the intestinal lamina propria
in a mouse model of colitis (8, 9, 10), and induction of
colitis is inhibited by the administration of anti-MAdCAM-1 mAb or
anti-integrin ß7 mAb (10). In
the islet of nonobese diabetic mouse, MAdCAM-1 expression is induced on
endothelial cells during the course of insulitis (11),
which is inhibited by administration of anti-MAdCAM-1 or
anti-integrin ß7 mAb (12, 13).
In humans, MAdCAM-1 expression is enhanced in the intestine of patients
with inflammatory bowel diseases (14).
Although MAdCAM-1 is exclusively expressed on HEV in PP and mesenteric
lymph nodes (MLN) in adult mice, it is also expressed in peripheral
lymph nodes (PLN) in fetal and neonatal mice (15) and is
implicated in the entry of
CD4+CD3- cells and 
T cells into LN (15). MAdCAM-1 is also expressed in
follicular dendritic cells (FDC) in chronically stimulated LN and
spleen of mice (16). These observations suggest that the
expression of MAdCAM-1 is regulated by an intrinsic developmental
program as well as external immunological stimuli.
We have previously cloned cDNA encoding the rat MAdCAM-1,
and reported that the nucleotide sequence is highly homologous to
that of the mouse, and that its mRNA expression was largely restricted
in GALT in adult rats (17). In the present study, we
examined the functional role of MAdCAM-1 in rats by establishing a
panel of mAb. Our results demonstrated that MAdCAM-1 functions as a
principal rolling and adhesion receptor for circulating lymphocytes in
adult rats. Furthermore, we report herein that MAdCAM-1 is expressed in
not only mucosal, but also nonmucosal, lymphoid tissues in a
developmentally regulated manner. Of note was the presence of
MAdCAM-1-positive blood vessels mainly in the cortico-medullary
junction of the thymus in neonatal rats. In addition, a significant
fraction of thymocytes in situ expressed integrin
4ß7 and were
potentially reactive with MAdCAM-1 at this period of development, as
assessed by flow cytometry.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats were purchased from Sankyo Laboratory (Tokyo, Japan). Female BALB/c mice were purchased from SLC (Hamamatsu, Japan). They were reared under specific pathogen-free conditions. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University Medical School.
Cell lines
Mouse T cell lymphoma cell line, TK-1, was a kind gift from Dr. B. Holzmann (Technische Universität, Munich, Germany) (1). Cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM nonessential amino acids, and 50 µM 2-ME. Human embryonic kidney 293 cell (18) and its transfectants were maintained in DMEM supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Monoclonal Ab
Anti-human c-myc (CT14-G4, mouse IgG1) was purchased
from RIKEN cell bank (Tsukuba, Japan). Anti-rat ICAM-1 (1A29, mouse
IgG1) (19) was obtained in our laboratory. FITC-conjugated
anti-rat CD3 (1F4, mouse IgM) (20) and FITC-conjugated
anti-rat CD8 (OX8, mouse IgG1) (21) were purchased
from Seikagaku (Tokyo, Japan) and from Cedar Lane (Ontario, Canada),
respectively. Cy-Chrome-conjugated anti-rat CD4 (OX-35, mouse
IgG2a) (22) was purchased from PharMingen (San Diego, CA).
Anti-rat integrin 4ß7
(TA-6) (23) was a generous gift from Dr. T. B.
Issekutz (Dalhousie University, Halifax, Canada).
Construction of stable transfectants of rat MAdCAM-1
The cDNA encoding rat MAdCAM-1 was subcloned into the EcoRI site of the expression vector pME18S and cotransfected with the plasmid RSV-Neo into 293 cells using Lipofectin (Life Technologies, Rockville, MD), according to the instructions provided by the manufacturer. Stable transfectants were selected in a medium containing 600 µg/ml of G418 (Life Technologies) and screened for the transcripts by Northern blot analysis using the rat MAdCAM-1 cDNA as a probe.
Production of rat MAdCAM-1/human IgG-Fc chimeric proteins in 293 cells
The cDNA encoding the extracellular region of rat MAdCAM-1 was amplified by PCR using LA-TAQ (Takara, Tokyo, Japan) and primers including BamHI site. PCR products were digested with BamHI and subcloned into BamHI site of the plasmid vector pcDNAI-IgG (a generous gift from Dr. M. Fukuda, Burnham Institute, La Jolla, CA) (24) to generate pcDNAI-MAdCAM1-IgG. The primers used were as follows: RB3 (sense), 5'-CGGGATCCAGACAGAGAAAGGCATGGA-3'; RB2 (antisense), 5'-CGGGATCCGTAACATACAGACTGGTCAC-3'. After confirming the sequence of the pcDNAI-MAdCAM-1-IgG, it was cotransfected with the plasmid RSV-Neo into 293 cells using Lipofectin. Transfected cells were selected in a medium containing 600 µg/ml G418. Stable transfectants were cultured in large scale in a serum-free medium (ASF; Ajinomoto, Tokyo, Japan), and the supernatant of the culture was precipitated with 50% ammonium sulfate. After the precipitates were dissolved in PBS(-) and dialyzed against PBS(-), the chimeric protein was purified using HiTrap protein G affinity columns (Pharmacia, Uppsala, Sweden), as recommended by the manufacturer.
In vitro cell adhesion assay
The MAdCAM-1/IgG chimeric protein was absorbed onto 96-well
plastic plates (SUMILON H type; Sumitomo, Tokyo, Japan) by incubating
wells with 50 µl/well of the PBS solution containing 10 µg/ml of
the chimeric protein at 4°C overnight. After discarding the solution,
nonspecific protein-binding sites were blocked by incubating the wells
with 3% BSA (Sigma, St. Louis, MO) in PBS(-) at room temperature for
2 h. Mouse T cell lymphoma cell line TK-1, which expresses
integrin 4ß7, was
labeled with 1 mM 3'-O-acetyl-2', 7'-bis (carboxyethyl)-4 or
5-carboxyfluorescein diacetoxymethyl ester (BCECF-AM; Dojindo, Osaka,
Japan) by incubating at 37°C for 1 h. After washing three times
with RPMI 1640 medium, the cells were resuspended in RPMI 1640 with
10% FCS at a concentration of 1 x 106
cells/ml. After the wells were preincubated with anti-MAdCAM-1
or control mAb CT14-G4, the labeled cells (1 x
105 cells/well) were incubated in the wells for
30 min at 37°C. Then the wells were filled with RPMI 1640 containing
10% FCS, sealed with parafilm, and incubated at room temperature for
30 min in an inverted position. Then the supernatants were carefully
aspirated to remove unattached cells, and adherent cells were
lysed by adding 50 µl of 1% Nonidet P-40 in PBS per well. The plates
were measured by a fluorescence plate reader (Labosystems,
Fluoroskan, II).
Hybridoma production
Five-week-old female BALB/c mice were immunized every week by footpad injection of MAdCAM-1/IgG chimeric protein (5 µg/animal). CFA was used in the first immunization and IFA was used thereafter. Three days after the fifth injection, the popliteal LN cells were fused with the mouse myeloma cell line, PAI (25), using PEG 4000 (Merck, Darmstadt, Germany), and the supernatants of hybridomas were screened for their reactivity with the rat MAdCAM-1 cDNA transfectants, but not with mock transfectants.
Flow-cytometric analysis
To detect the immunoreactivity of mAb, rat MAdCAM-1
transfectants or mock transfectants were first incubated with the mAb
on ice for 30 min. The cells were washed twice and stained with
FITC-labeled anti-mouse Ig (G+M) polyclonal Ab (American Qualex,
San Clemente, CA). To detect the reactivity of MAdCAM-1/IgG chimeric
protein with rat thymocytes, freshly prepared thymocytes were first
incubated in TBS/Mn2+ (25 mM Tris-HCl, pH 7.4,
140 mM NaCl, 2.7 mM KCl, 2 mM MnCl2) containing
MAdCAM-1/IgG (3 µg/ml) at room temperature for 30 min. After washing
in TBS/Mn2+, cells were stained with biotinylated
anti-human IgG polyclonal Ab (Immunotech, Marseille, France),
followed by PE-streptavidin (Immunotech) and FITC anti-CD3 in
TBS/Mn2+. For mAb-blocking studies, cells were
preincubated with OST2 (30 µg/ml), OST20 (30 µg/ml), or TA-6
(10 µg/ml) in TBS/Mn2+ before MAdCAM-1/IgG was
added to the cells. In three-color analysis, thymocytes were first
incubated with MAdCAM-1/IgG or anti-integrin
4ß7, followed by
appropriate biotinylated secondary Ab. After washing, the cells were
stained with a mixture of PE-streptavidin, Cy-Chrome anti-CD4,
and FITC anti-CD8. Background staining was determined using an
equal amount of human IgG1 for MAdCAM-1/IgG and CT14-G4 for
anti-integrin 4ß7.
Immunofluorescence was analyzed on an EPICS-XL or EPICS-ELITE flow
cytometer (Coulter, Miami, FL).
Immunoprecipitation
MAdCAM-1 transfectants or mock transfectants (3 x 106 cells) were incubated in PBS(-) containing 25 µg/ml ImmunoPure NHS-LC-biotin (Pierce, Rockford, IL) on ice for 1 h. After washing in Tris-buffered saline (25 mM Tris-HCl, pH 7.4, 140 mM NaCl), the cells were solubilized by lysis buffer (PBS(-), 0.5% Nonidet P-40, 1 mM PMSF). After being precleared with protein G-Sepharose (Pharmacia), the lysates (5 x 105 cells per lane) were incubated with mAb (1 µg) and protein G-Sepharose at 4°C overnight. After washing in the lysis buffer, the immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions using 4–20% gradient gel. The proteins were then transferred to polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, MA), and the membranes were blocked using 3% BSA in PBS(-). The bands of biotin-labeled proteins were visualized by avidin-biotin-HRP complex (Vector Laboratory, Burlingame, CA) and ECL Western blotting detection reagents (Amersham, Buckinghamshire, U.K.), according to the manufacturer’s instructions.
Immunoperoxidase staining of frozen sections
Tissues from Wistar rats were frozen in OCT compound (Sakura, Tokyo, Japan) and sectioned at a thickness of 6–8 µm. The cryostat sections were fixed in ice-cold acetone for 3 min and blocked by incubating with RPMI 1640 medium containing 10% FCS at room temperature for 30 min. For detection of MAdCAM-1, tissue sections were incubated with 10 µg/ml OST2 or OST12 diluted with RPMI 1640 containing 10% FCS at room temperature for 1 h. mAb CT14-G4 was used for the isotype-matched negative control. After washing with PBS(-) for three times, tissue sections were incubated with biotinylated anti-mouse IgG polyclonal Ab (Chemicon, Temecula, CA). After washing with PBS(-) twice, the endogenous peroxidase was blocked by incubation in 1.5% H2O2 in methanol for 10 min at room temperature. After washing with PBS(-) twice, specimens were further incubated with HRP-conjugated streptavidin (Vector Laboratory) for 15 min, followed by washing three times with PBS(-). Immunoreactivity was detected with 0.1% 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0.02% H2O2 in PBS(-), and counterstained with Giemsa solution.
Visualization of MAdCAM-1 expression in rat PP in vivo
Expression of MAdCAM-1 in rat PP was examined in vivo, as described previously (26, 27). Briefly, male Wistar rats (250–300 g) were anesthetized with pentobarbital sodium (50 mg/kg i.m.). The femoral vein was cannulated with a polyethylene catheter for infusion of FITC-labeled mAb. The abdomen was opened via a midline incision, and ileal PP were mounted on a plastic stage. The expression of MAdCAM-1 in PP in vivo was examined from the serosal side with a fluorescence microscope (Meridian Far East, Tokyo, Japan) assisted by an intensified color CCD camera (C5810; Hamamatsu Photonics, Shizuoka, Japan), 30 min after the infusion of mAb. The infusion dose of FITC-labeled mAb was 1 mg/kg. FITC-labeled CT14-G4 was used for isotype-matched control.
Intravital examination of effects of anti-rat MAdCAM-1 mAb on lymphocyte rolling and adhesion in PP
The interaction of lymphocytes with the rat PP HEV was visualized as described previously (28). Briefly, lymphocytes were collected from the mesenteric lymphatic ducts of male Wistar rats (250–300 g) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR). Anti-rat MAdCAM-1 mAb (OST2 or OST20, 2 mg/kg) was infused via a cannula in the jugular vein of littermate rats. Twenty minutes after mAb infusion, 3 x 107 CFSE-labeled lymphocytes in 1 ml of PBS were infused i.v. The interaction of infused lymphocytes with venules in PP was visualized on the monitor screen by using a fluorescence microscope equipped with a silicon-intensified target image tube camera and a contrast enhancement unit (C-2400-08; Hamamatsu Photonics) and recorded on videotapes. CFSE-positive cells per 1 mm2 in the examined areas that remained at the same position of venules throughout the observation periods (30 s each) were counted as adherent cells. Four animals were used for each mAb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Supernatants of hybridoma were screened for their ability to bind
to rat MAdCAM-1-transfected 293 cells. Of 
400 samples, 13 were
specifically reactive with rat MAdCAM-1 transfectants, and three of
these (OST2, OST12, and OST20, all IgG1, 
) were used for further
analysis. Flow-cytometric analysis (Fig. 1
A) showed immunoreactivity of
these mAb with MAdCAM-1 transfectants, but not with mock-transfected
293 cells. Immunoprecipitation studies (Fig. 1B)
demonstrated that OST2 specifically recognized a molecule with an
expected size (55 kDa) in MAdCAM-1 transfectants. Similar results were
obtained with OST12 and OST20 (data not shown). We next examined the
effects of anti-rat MAdCAM-1 mAb on the binding of TK-1 cells
expressing 4ß7
integrin to immobilized MAdCAM-1/IgG chimeric protein. As shown in Fig. 1C, OST2 and OST12 abolished the binding of TK-1 cells to
MAdCAM-1, whereas OST20 had minimal effect, suggesting that OST2 and
OST12 recognize the binding epitope(s) of MAdCAM-1 to
4ß7 integrin.
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To examine the cellular distribution of rat MAdCAM-1 in various
tissues of adult rats, we conducted immunohistochemistry with OST2 and
OST12. Both mAb showed essentially the same immunoreactivity in all
tissues examined in this study. MAdCAM-1 was specifically expressed on
HEV of PP and MLN, and on the venules of the intestinal lamina propria
and submucosa of the small intestine (Fig. 2A–C) and colon (data not
shown). The expression in the lamina propria was largely restricted to
venules around crypts and was absent on those in intestinal villi (Fig. 2C), while ICAM-1 was widely expressed on all venules in the
lamina propria (data not shown). In PLN, low levels of MAdCAM-1
expression were occasionally observed on HEV (data not shown). Previous
studies in mice demonstrated that MAdCAM-1 is expressed also in
sinus-lining cells in the marginal zone of the spleen
(29). However, no immunoreactivity with any of the
anti-MAdCAM-1 mAb was observed in the marginal sinus of the rat
spleen (Fig. 2D). In addition, the expression of MAdCAM-1
was absent in all other tissues examined, including the thymus,
esophagus, trachea, bladder, skin, submandibular gland, and thyroid
gland in adult rats. These results indicate that the expression of
MAdCAM-1 in adult rats is GALT specific.
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We next examined the expression and functional roles of
MAdCAM-1 in PP HEV in vivo by using intravital microscopy. When
FITC-labeled anti-MAdCAM-1 mAb were injected i.v., both OST2 and
OST20 selectively reacted with the luminal surface of PP HEV in vivo
(Fig. 3A–D). However, as
shown in Fig. 3E, only a function-blocking mAb OST2
completely abolished lymphocyte rolling and subsequent lymphocyte
adhesion. These results demonstrate that MAdCAM-1 is selectively
expressed in PP HEV and functions as a principal rolling and adhesion
receptor for circulating lymphocytes.
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Although MAdCAM-1 expression has been documented on HEV of PLN in
fetal and neonatal mice (15), the expression in other
tissues during embryogenesis has not been reported. Immunohistochemical
examination using OST2 revealed that the intestinal lamina propria
contained numerous small blood vessels expressing MAdCAM-1 in fetal and
neonatal rats (Fig. 4, A,
C, and E). The expression of MAdCAM-1 in the
lamina propria was detected as early as 16 days of gestation (data not
shown), and was more widely distributed than in adult rats, in that it
was expressed on not only venules in the lamina propria around the
crypts, but also those in the villi (Fig. 4, A,
C, and E). In contrast, ICAM-1 was absent in the
gastrointestinal tract of fetal rats (Fig. 4, B and
D), but was rapidly induced after birth (Fig. 4F). MAdCAM-1 was also expressed at low levels in FDC with
dendritic morphology in the follicle of neonatal PP (Fig. 5), while it was absent in FDC of adult
PP (Fig. 2
A).
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Further immunohistochemical studies revealed that MAdCAM-1 was
constitutively expressed in endothelial cells in the skin and thymus in
fetal and neonatal rats (Fig. 6). In the
fetal skin, MAdCAM-1 was found in endothelial cells in the dermis
around gestation day 18, which was gradually down-regulated after birth
(Fig. 6, A, C, and E). In the thymus,
MAdCAM-1 expression was transiently but strongly detected in
endothelial cells of small blood vessels at the cortico-medullary
junctions and those in interlobular connective tissues around birth
(Fig. 6, B, D, and F).
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A, flow-cytometric analysis
showed that about 14% of neonatal thymocytes were reactive with
MAdCAM-1/IgG chimeric protein in the presence of
Mn2+, whereas only a small proportion (3.6%) of
adult thymocytes bound MAdCAM-1/IgG under the same condition. Two-color
flow-cytometric analysis showed that MAdCAM-1/IgG-reactive cells were
found in both CD3+ and
CD3- thymocyte subsets and, as shown in Fig. 7B, a subpopulation of CD3 high positive cells was strongly
reactive with soluble MAdCAM-1. MAdCAM-1/IgG binding was abrogated by
blocking mAb OST2 and also by blocking mAb to integrin
4ß7, but not by a
nonblocking anti-MAdCAM-1 mAb OST20 (Fig. 7B). To
further characterize the thymocyte subset reactive with soluble
MAdCAM-1/IgG, we conducted three-color analysis. Significant
proportions of CD4+ and
CD8+ subsets were reactive (about 64% of these
cells were double positive), while double-negative
CD4-CD8- subset was
nonreactive (Fig. 8). Similar results
were obtained for cells positive for integrin
4ß7 expression. Taken
together, these results suggest that a relatively mature
subpopulation(s) of neonatal rat thymocytes is potentially reactive
with MAdCAM-1 via integrin
4ß7.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 interaction
(15). Previous studies suggested that
CD3-CD4+ cells are
lineage-restricted progenitor cells that can differentiate into
Ag-presenting dendritic cells and NK cells and may play a role in the
initiation of lymphoid organogenesis during ontogeny (30).
Accumulation of similar
CD3-CD4+ cells during PP
formation has also been reported (31). Therefore, we
speculate that widely distributed MAdCAM-1 in the developing
gastrointestinal tract, whose expression is probably under the
developmental control and not dependent on external Ag, may play a
critical role in the colonization of such progenitor-type cells
required for the formation of GALT.
It is of interest that FDC in PP of neonatal rats expressed readily
detectable levels of MAdCAM-1, whereas those in PP of adult rats did
not. This finding is in sharp contrast to the previous observation in
adult mice in that FDC in adult mouse PP constitutively express
MAdCAM-1 (16). MAdCAM-1 expression in mouse FDC is thought
to play a role in the retention of
4ß7 integrin-bearing T
and B cells in the follicles. However, the absence of MAdCAM-1
expression in FDC in adult rats may indicate the presence of an
alternative adhesive pathway for the Ag-driven recruitment and
subsequent retention of lymphocytes in the follicle.
Expression of MAdCAM-1 was also transiently detected in the thymic
medulla in neonatal rats. Although the level of resolution in the
present study does not allow definitive conclusions to be made,
MAdCAM-1 staining appeared to be localized in endothelial cells of
these blood vessels. Flow-cytometric analysis showed that about
10–20% of thymocytes were potentially reactive with MAdCAM-1 at
birth through integrin
4ß7-dependent pathway,
suggesting that a subpopulation of neonatal thymocytes is capable of
binding to MAdCAM-1+ endothelium in the medulla.
Of note is that MAdCAM-1+ thymic blood vessels
were detectable only during the neonatal period, but not at other time
points during development, and that MAdCAM-1-reactive thymocytes were
also relatively abundant only at neonatal period. In this regard, Surh
et al. (32) recently reported interesting findings; they
showed that, contrary to adult thymus, newborn thymus can accommodate
circulating T cells with a CD44low,
CD45RBhigh, L-selectinlow
phenotype and allow them to lodge selectively in the medulla and to
remain for prolonged periods, which may possibly be important for
self-tolerance induction (32). It should be mentioned that
thymocytes with a CD3high phenotype reacted
strongly with soluble MAdCAM-1 in our study, and it would be
interesting to examine whether this subset is the same as that
identified by Surh et al. (32). Expression of MAdCAM-1 in
the thymic medullary blood vessels has been documented under disease
conditions and implicated in enhanced lymphocyte traffic into the
thymus, leading to the marked thymic hypertrophy in AKR/J mice
(33). However, it is also possible that MAdCAM-1 on the
thymic medullary vasculature contributes to emigration rather than
immigration of lymphocytes. The thymic medulla has been considered the
site in which single positive cells are predominantly localized
awaiting exportation to the periphery (34). Consistent
with this notion, Gabor et al. (35) previously
demonstrated that a small subpopulation of medullary single positive
thymocytes and CD4-CD8+
recent thymic emigrants strongly expressed integrin
ß7 in mice. Collectively, the precise role of
MAdCAM-1 on the neonatal medullary blood vessels is currently a matter
of speculation, and should be examined by the Stamper-Woodruff in vitro
lymphocyte-binding assay as well as in vivo lymphocyte-homing assay
employing a function-blocking anti-MAdCAM-1 mAb.
The endothelium of small blood vessels in the fetal skin also
transiently expressed MAdCAM-1. In rats and mice, T cells are
abundant in the skin (36, 37). In mice, previous studies
showed that dendritic epidermal T cells (dEC) are derived from a single
wave of precursor cell colonization from the fetal thymus
(38). Although the mechanism underlying the skin homing by
the dEC precursors remains to be identified, it is possible that the
developmentally regulated expression of endothelial addressin in the
skin is involved in the specific trafficking of dEC precursors. Whether
or not MAdCAM-1 can function as the skin addressin needs to be
experimentally verified.
In mice, MAdCAM-1 expression has been documented also in the sinus-lining cells in the marginal zone of the spleen (29). However, our previous observations by Northern blot analysis suggested that MAdCAM-1 mRNA was not expressed in the rat spleen (17). In the present study, we confirmed that sinus-lining cells in the rat splenic marginal zone were devoid of MAdCAM-1 expression at the protein level, in agreement with the notion that MAdCAM-1 does not play a major role in the physiological lymphocyte trafficking into the spleen (29).
In summary, we have demonstrated in the present study that MAdCAM-1 is specifically expressed in GALT, and functions as a principal PP HEV ligand for lymphocyte rolling and adhesion in adult rats. Furthermore, we also demonstrated, for the first time, a developmentally regulated expression of MAdCAM-1 in the endothelium of the gastrointestinal tract, skin, and thymus during ontogeny in rats. The stage-specific expression of MAdCAM-1 in these tissues may indicate a much broader function of MAdCAM-1 than documented to date: as an endothelial addressin that regulates trafficking of lymphoid cells and/or their progenitors to developing lymphoid and nonlymphoid tissues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masayuki Miyasaka, Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita 565-0871, Japan. E-mail address:
3 Abbreviations used in this paper: LN, lymph node; CFSE, carboxyfluorescein diacetate succinimidyl ester; dEC, dendritic epidermal T cell; FDC, follicular dendritic cell; GALT, gut-associated lymphoid tissue; HEV, high endothelial venule; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MLN, mesenteric lymph node; PLN, peripheral lymph node; PP, Peyer’s patch.
Received for publication July 23, 1999. Accepted for publication December 10, 1999.
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J. D. Mintern, M. M. Maurice, H. L. Ploegh, and E. Schott Thymic Selection and Peripheral Activation of CD8 T Cells by the Same Class I MHC/Peptide Complex J. Immunol., January 1, 2004; 172(1): 699 - 708. [Abstract] [Full Text] [PDF]
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E. Donskoy, D. Foss, and I. Goldschneider Gated Importation of Prothymocytes by Adult Mouse Thymus Is Coordinated with Their Periodic Mobilization from Bone Marrow J. Immunol., October 1, 2003; 171(7): 3568 - 3575. [Abstract] [Full Text] [PDF]
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I. Louis, G. Dulude, S. Corneau, S. Brochu, C. Boileau, C. Meunier, C. Cote, N. Labrecque, and C. Perreault Changes in the lymph node microenvironment induced by oncostatin M Blood, August 15, 2003; 102(4): 1397 - 1404. [Abstract] [Full Text] [PDF]
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C. G. Lo, T. T. Lu, and J. G. Cyster Integrin-dependence of Lymphocyte Entry into the Splenic White Pulp J. Exp. Med., February 3, 2003; 197(3): 353 - 361. [Abstract] [Full Text] [PDF]
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H. Hashi, H. Yoshida, K. Honda, S. Fraser, H. Kubo, M. Awane, A. Takabayashi, H. Nakano, Y. Yamaoka, and S.-I. Nishikawa Compartmentalization of Peyer's Patch Anlagen Before Lymphocyte Entry J. Immunol., March 15, 2001; 166(6): 3702 - 3709. [Abstract] [Full Text] [PDF]
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