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Departments of
*
Molecular Genetics and
Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; and
Division of Cardiology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PP inducer
cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature
lymphocytes by whole-mount immunostaining of 17.5 days postcoitus to 2
days postpartum mouse gut. Our results show that compartmentalization
of PP anlagen commences at day 18.5 of gestation by clustering and
subsequent follicle formation of IL-7R+,
VCAM-1+, and CD11c+ cells. This process adds
the primitive architecture of PP anlage with several follicles in which
IL-7R+ cells localize in the center, while
VCAM-1+ and CD11c+ cells localize at the
fringe. This follicle formation is accompanied by the establishment of
PP-specific vascular network expressing mucosal addressin cellular
adhesion molecule-1. Mature B and T lymphocytes entering in the PP
anlage are distributed promptly to their own target zones; B cells to
the follicle and T cells to nonfollicular zones. Our analysis of
scid/scid mouse indicate that the initial processes
including formation of PP-specific vascular network occur in the
absence of lymphocytes. These observations indicate that the basic
architecture of PP is formed by a set of cell lineages assembled during
the initial phase of induction of PP anlagen before entry of mature
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, CD11c, and
c-Fms, accumulate, though no mature lymphocytes are found at this time.
Entry of mature lymphocytes expressing CD3 or B220 occurs after 18.5
dpc. We have demonstrated that the initial step of PP organogenesis,
represented by the appearance of
VCAM-1+/ICAM-1+ spots, is
induced by IL-7R+ cells that express
lymphotoxin (LT) 1
2
upon stimulation of IL-7R/common 
/Jak3 signaling pathway
(2). Analysis of mice bearing null mutations of genes
involved in this induction phase concurs with the three-step model
proposed above (3, 4, 5). How primitive PP anlagen develops
into more complex structures as found in the fully developed PP is
unknown. The structure of the mature PP is similar to that of other peripheral lymphoid tissues in that it consists of a number of discrete lymphoid follicles in which T, B, and dendritic cells are located in an organized manner (6). By antigenic stimulation, primary follicles undergo structural changes, producing highly developed secondary architectures with germinal centers (7, 8). While this latter process generates extensive complexity in the peripheral lymphoid tissues, the basic tissue framework has to be prepared before Ag stimulation. Indeed, studies of germfree mice show that lymphoid follicles with segregated areas of T and B cells are formed in an Ag-independent manner (9). Furthermore, Noelle et al. clearly demonstrated that the cellular organization that directs T and B cells into segregated regions is established before the entry of lymphocytes (10). These results suggest that the primitive PP anlage identified initially as a simple structure in which VCAM-1/ICAM-1+ cells are scattered rather homogeneously becomes more complex via a series of compartmentalizing events before lymphocyte entry.
The first primary follicles, indicated by B cell aggregates, are seen 2 wk after birth in the rat (11, 12, 13) and 10 days after birth in the mouse (14, 15). In this study, we investigated the process by which the basic structure of PP with segregated compartments is established from the primitive PP anlagen. Our results show that the formation of basic architecture of PP with separate follicle-like structures commences from 18.5 dpc and is completed within a few days after birth. Moreover, this process is independent of mature lymphocytes, which have been shown to be the chief components inducing PP architecture in later life.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 strain and C.B17/Icr-SCID Jcl were purchased from Japan SLC (Shizuoka, Japan) and Japan CLEA (Tokyo, Japan), respectively. Female and male mice were allowed to mate overnight, and those with vaginal plugs were judged pregnant. Noon of the day when the vaginal plug was found was designated 0.5 dpc.
Monoclonal Abs
Monoclonal Abs against VCAM-1 (429 MVCAM.A; BD PharMingen, San
Diego, CA), CD11c (HL3; BD PharMingen), mucosal addressin cellular
adhesion molecule (MAdCAM)-1 (MECA367; BD PharMingen), platelet
endothelial cellular adhesion molecule (PECAM)-1 (MEC13.3; BD
PharMingen), CD3 (Y65.372; Seikagaku, Tokyo, Japan; and 2C11; BD
PharMingen) were purchased. Monoclonal Ab against IL-7R (A7R34)
(16), B220 (RA3-6B2) and Flk-1(AVAS12) (17)
were purified from hybridoma culture supernatant as described.
Immunohistochemistry
Whole-mount immunostaining was performed as previously described
(18) with slight modifications. In brief, excised guts
were incubated in fixing solution (4% paraformaldehyde in PBS) for 30
min at 4°C. After absorbing excessive paraformaldehyde with 4%
glycine in PBS for 30 min at 4°C, specimens were dehydrated by
incubating 30 min each with 50, 75, 100, 100% methanol in PBS at
4°C. To block endogenous peroxidase, the fixed specimens were
bleached (methanol: 30%
H2O2 20:1) for 30 min at
room temperature. For staining, the dehydrated specimens were first
blocked by incubating twice in PBSMT (1.5% skim milk and 0.1% Triton
X-100 in PBS) for 1 h at room temperature, incubated with PBSMT
containing biotinylated anti-CD11c mAb HL3 (1 µg/ml),
anti-IL7R mAb A7R34 (2 µg/ml), anti-VCAM-1 mAb 429 (2
µg/ml), anti-MAdCAM-1 mAb MECA-367 (2 µg/ml), anti-PECAM-1
mAb MEC13.3 (1 µg/ml), biotinylated anti-CD3 mAb Y65.372 (1
µg/ml), biotinylated anti-CD3 mAb 2C11 (0.5 µg/ml), or
anti-B220 mAb RA3-6B2 (2 µg/ml) overnight at 4°C. After washing
five times in PBSMT each for 1 h at 4°C, the primary Ab was
detected by incubating 1 mg/ml HRP-conjugated anti-rat Ig Ab
(Biosource, Camarillo, CA) overnight at 4°C. To biotinylated mAbs,
ABC kits (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame,
CA) were used at 1:20 dilution in PBS, incubated for 2 h, and
washed three times for 30 min at 4°C. After extensive washing with
more than five exchanges of PBSMT, including the final 20 min wash in
PBST (0.1% Triton X-100 in PBS) at room temperature, specimens were
soaked in PBST containing 0.05% NiCl2 and 250
mg/ml diaminobenzidine (DAB; Dojin Chem, Kumamoto, Japan) for 10–30
min, and H2O2 was added to
0.01%. The enzymatic reaction was allowed to proceed until the desired
color intensity was reached, and the specimens were rinsed three or
four times in PBST. Finally, the specimens were dehydrated and solution
was exchanged to glycerol before being photographed. Then 14.5-dpc
spleen was whole-mount immunostained by MAdCAM-1 and was embedded in
polyester wax and sectioned by microtome. The specimen was further
immunostained by a rat anti-mouse Flk-1 mAb (10 µg/ml), followed
by the peroxidase-labeled anti-rat IgG. To obtain a brown color,
the coloration reaction was performed in the absence of
NiCl2. It was observed by
differential-interference-contrast microscopy.
Whole-mount immunofluorescent staining was conducted in combination with the whole-mount immunostaining described above. FITC-conjugated HL3 (0.5 µg/ml) with Alexa594-conjugated A7R34 (1 µg/ml) in combination with biotinylated 429 (2 µg/ml) was used as a first step Ab in the above protocol. The location of each PP was identified by detecting 429 mAb binding by DAB/NiCl2 color-substrate reaction. Single PPs were then excised and mounted flat. Similarly, combinations of HL3-FITC/429-biotin followed by streptavidin (SA)-Alexa594 and anti-rat Ig HRP, 492-FITC/Alexa594-A7R/biotin-CD11c followed by SA-HRP, as well as A7R/2C11-biotin followed by SA-Texas Red (Molecular Probes, Eugene, OR) and anti-rat Ig HRP were also developed by DAB/NiCl2 and were excised and mounted flat. Fluorescent signals were detected by confocal microscopy (TCS-NT/Leica, Bensheim, Germany) or light microscopy (Axiophoto/Zeiss, München, Germany).
Preparation of single-cell suspensions for flow cytometry and cell sorting
The whole gut of embryonic and neonatal mice was chopped to small pieces with dissecting scissors and incubated in 2 U/ml(2.60 mg/ml) dispase (Life Technologies, Rockville, MD) in PBS for 30 min at 37°C. In some experiments, two to three PP in the upper intestine were dissected from intestines using a stereomicroscope and treated with the dispase solution. After pipetting 20 times, dissociated cells were washed with HBSS containing 2% BSA (HBSS/BSA; Sigma, St. Louis, MO). Cells were filtered through nylon mesh to remove large clumps, washed with HBSS/BSA, and centrifuged for 10 min at 1000 rpm at 4°C. Nonspecific binding of mAb was blocked by suspending cells (1 x 106) in 50 µl of normal mouse serum for 20 min at 4°C, stained with FITC-conjugated 429 (10 µg/ml) or Alexa488-conjugated A7R34 (5 µg/ml) with PE-conjugated HL3 (10 µg/ml) or biotin-conjugated A7R34 (5 µg/ml) added with PE-conjugated SA (5 µg/ml). Stained cells were washed twice in HBSS/BSA and analyzed using FACSVantage (Becton Dickinson, Mountain View, CA).
Cell preparations for RT-PCR analysis
For RT-PCR analysis, cells were labeled with A7R34-Alexa488 and
HL3-PE. After living cells were selected by staining with propidium
iodide (Sigma), IL-7R single-positive cells and CD11c
single-positive cells was sorted using FACSVantage (Becton Dickinson).
To obtain pure cell populations, each population was subjected to two
cycles of sorting. The purity of each cell population ranged from 89
to 96%.
RT-PCR detection of chemokine receptors in sorted mouse gut cell populations
Total RNA was extracted from 70,000 cells directly sorted into vials containing ISOGEN LS (Nippon Gene, Tokyo, Japan), and total RNA was isolated according to the manufacturer’s protocol. First-strand cDNA was prepared from total RNA by reverse transcriptase using oligo(dT) primers (Superscript2; Life Technologies). In each PCR for detection of CXCR5, CCR7, and GAPDH gene expression, cDNA corresponding to an equivalent of 3500 cells was incubated with 100 pg of the following primer sets; B lymphocyte chemoattractant (BLC), sense 5'-TCACCTTGTTGGGTACCCCAGCAA-3', antisense 5'-ATACACAGACTTCTGCGCAC-3'; CXCR5, sense 5'-TTGTTGGCAGTGCCTATCACTGTCC-3', antisense 5'-CTCGTGTACCATAACGACCCGTAC-3'; EBI1-ligand chemokine (ELC), sense 5'-CTGCCTCAGATTATCTGCCAT, antisense 5'-GCCAGAGTGATTCACATCTCT; secondary lymphoid-tissue chemokine (SLC), sense 5'-ATGGCTCAGATGATGACTCTG, antisense 5'-GTGTCTGTTCAGTTCTCTTGC; CCR7, sense 5'-ACAGCGGCCTCCAGAAGAACAGCGG, antisense 5'-TGACGTCATAGGCAATGTTGAGCTG; GAPDH, sense 5'-ATGGTGAAGGTCGGTGTGAACGGATTTGGC-3', antisense 5'-GC ATCGAAGGTGGAAGAGTGGGAGTTGCTG-3'.
Semiquantitative RT-PCR analysis was performed as follows. The amount of the template cDNA from each sample was equalized by comparing the concentration of RT-PCR products with GAPDH primer sets at various amplifying cycles. The minimum number of amplifying cycles that detected the PCR products were determined for each specimen by each primer set.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies defined the primitive PP anlage as a discrete
region expressing VCAM-1/ICAM-1 (1). Judging from the
homogeneous staining pattern of VCAM-1 (Fig. 1
A) and IL-7R (Fig. 1C) within the primitive anlagen, the structure appears
relatively simple with evenly distributed VCAM-1+
and IL-7R+ cells. However, in the adult PP,
VCAM-1+ (Fig. 1B) and
IL-7R+ (Fig. 1D) cells have
separated into distinct units in which T and B lymphocytes and
dendritic cells are segregated within different compartments. Thus, the
precisely regulated process of cell segregation in the PP must take
place between the formation of the primitive anlagen and mature PP.
While Ag-induced reorganization of the architecture of peripheral
lymphoid tissues has been defined to some extent, earlier processes
remains completely obscure. Therefore, we first attempted to define the
process in which a simple PP anlage develops higher order
structures.
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+,
VCAM-1+, and CD11c+ cells
was analyzed after the formation of primitive PP anlage. We have
proposed that IL-7R+ and
VCAM-1+ cells form a mutually interacting unit
for making an organizing center of the PP anlage (19).
Whole-mount preparations of 17.5-dpc to 2-days postpartum (dpp)
embryonic intestines were stained by anti-IL-7R,
anti-VCAM-1, and anti-CD11c mAbs. In Fig. 2, two types of staining pattern were
observed. IL-7R+ or
VCAM-1+ cells, which are distributed diffusely in
the PP anlagen (Fig. 2, A and F), first formed
irregularly segregated subregions (Fig. 2, B, C,
G, and H), which subsequently split into several
apparent compartments of similar size (Fig. 2, D,
E, I, and J).
CD11c+ cells first appear in the periphery of PP
anlagen (Fig. 2K) but eventually fused to the
IL-7R+ area after birth (Fig. 2, L–O). These observations indicated that the three markers
that we have used to define PP inducers, PP organizers (which we
defined as a population activated by the inducer), and dendritic cells
are sufficient to detect the initial process toward
compartmentalization of PP from 18.5 dpc. Within 2 days of birth,
positioning of each component is complete, thereby forming several
follicle like structures (Fig. 2, E, J, and
O).
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+ cells are concentrated in the center
(Fig. 3, B and H),
while VCAM-1+(Fig. 3, E and
G) and CD11c+ (Fig. 3, A
and D) components accumulate at the fringe. However, these
regions are not demarcated sharply.
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+
cells are distinct populations, we prepared single-cell suspensions
from two to three PPs from upper intestines of 2-day-old neonates. As
the size of PP at this stage is too small to dissect specifically, the
cell preparation may contain surrounding tissues. Thus, the percentage
of each population in Fig. 4 may not
represent the actual proportion in the PP. Nevertheless, Fig. 4
demonstrated clearly that expression of VCAM-1, IL-7R, and CD11c are
completely segregated into distinct cell populations. These results
suggest that initial compartmentalization of the PP anlagen is
established through coordinated migration of distinct cell types that
assembled in the primitive PP anlagen.
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Chemokines and their receptors play an essential role in
regulating the compartmentalization of hemopoietic cells in the
secondary lymphoid organs (20, 21, 22, 23, 24). Among the three
cellular components, CD11c+ and
IL-7R+ cells are CD45+
hemopoietic cells (25). To gain insight into the
mechanisms underlying the distinct movement of the two hemopoietic cell
populations during the initial phase of compartmentalization,
CD11c+ and IL-7R+ cells
were sorted to a purity of 89–96%, and the expression of chemokine
receptors was analyzed by RT-PCR. As shown in Fig. 4
B,
IL-7R+ cells expressed both CXCR5 and CCR7,
whereas CD11c+ cells expressed only CCR7. While
expression level may decrease during the process of
compartmentalization, expression of CXCR5 and CCR7 in each population
is maintained throughout the process from 17.5 dpc to 2 dpp. As BLC,
ELC, and SLC are expressed in the VCAM-1+ stromal
cell component in the PP anlagen (Ref. 38 and K. Honda, unpublished
observation), IL-7R+ and
CD11c+ cells are likely to have different
affinities for regions expressing these chemokines.
Development of vascular structures
High endothelial venules (HEVs) are a hallmark of peripheral lymphoid organs. In adult PP, MAdCAM-1 is specifically expressed on HEV and functions as an adhesion molecule during lymphocyte homing (26, 27). Mebius et al. demonstrated that MAdCAM-1 is expressed by HEV in lymph nodes from the neonatal stage (28), suggesting that this specialized vascular structure may also be established in parallel with the compartmentalization process of PP described above. Thus, we investigated the temporal relationship between compartmentalization of PP and development of vascular architecture.
While we initially expected MAdCAM-1 staining to reveal the process of
HEV development in PP, we found that MAdCAM-1 is detectable in the
intestine before the formation of PP anlagen. As shown in Fig. 5A, MAdCAM-1 expression was
detectable in the embryo as early as 9.5 dpc. As compared with PECAM-1,
which is expressed by all vascular endothelial cells (ECs)
(29) (Fig. 5D), MAdCAM-1 is expressed
specifically by venous ECs. The vein-specific expression of MAdCAM-1 is
most clearly displayed in the mesenteric veins that run parallel to
arteries and lymphatics (Fig. 5B) that express PECAM-1 but
not MAdCAM-1 (Fig. 5, E and F). Moreover,
analyses of other regions demonstrated that venous specific expression
of MAdCAM-1 is a general phenomenon during development of vascular
system (data not shown). While most venous EC express MAdCAM-1 during
embryogenesis, the expression disappears by birth except in PP (Fig. 5C), spleen, and mesenteric lymph nodes (MLN). As detected
by expression of nkx-2.5, the primordium of the spleen is first formed
at 10.5 dpc (30). Expression of MAdCAM-1 is detected in
the vascular structures of the developing spleen from 12.5 dpc. Double
staining of 14.5-dpc spleen by anti-Flk-1 and anti-MAdCAM-1
(Fig. 5G) shows that all vascular structures are
Flk-1+, whereas only a portion are stained with
MAdCAM-1. The primordium of MLN can also be detected as aggregates of
IL-7R+ cells at 12.5 dpc (data not shown).
MAdCAM-1 expression on vascular structures is seen from 15.5 dpc in
MLNs (data not shown). These results suggests that venous-specific
expression of MAdCAM-1 is a general phenomenon during organogenesis of
peripheral lymphoid tissues.
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P). While MAdCAM-1 expression in the PP anlagen
starts from a homogeneous pattern, a strong net-like expression emerges
on 17.5 dpc (Fig. 2P) and eventually becomes restricted to
HEV (Fig. 2T). Expression in surrounding tissues disappears
gradually. From 18.5 dpc (Fig. 2Q) to 0 dpp (Fig. 2R), when entry of mature lymphocytes is observed, MAdCAM-1
is strongly expressed on vessels in the PP anlagen, though
compartmentalization is not yet observed. From 1 dpp, the distribution
pattern of MAdCAM-1+ vessels changes in parallel
with the compartmentalization of VCAM-1+,
IL-7R+, and CD11c+
cells. MAdCAM-1+ vessels become associated with
the developing follicle-like structure by 1 dpp (Fig. 2S)
and start to surround them from 2 dpp (Fig. 2T), while the
compartmentalization appeared to occur earlier. This time course
indicates that the process of cell compartmentalization is followed by
reorganization of vascular architecture, thereby each newly formed
compartment is supplied with discrete vascular units. Entry of mature lymphocytes
Our previous study demonstrated that entry of mature lymphocytes
to PP anlagen is detected primarily at around 18.5 dpc
(1). B220+ cells (Fig. 6, A and B) and
CD3+ (Fig. 6, E and F)
cells were distributed evenly over the PP anlagen from 18.5 dpc to 0
dpp. This diffuse pattern is consistent with the architecture of
vascular network before compartmentalization. Along with formation of
follicle-like structure with MAdCAM-1+ HEV,
B220+ cells accumulate in the central region of
each follicle-like structure (Fig. 6C) and eventually show a
similar distribution pattern as that of IL-7R+
cells (Fig. 6D). In contrast, T cells remain over the
outside space of follicle-like structures (Fig. 6, G and
H). Note that this pattern is different from those of
IL-7R+, CD11c+, and
VCAM-1+ cells. However, in the fully mature PP, T
cell distribution became similar to that of CD11c, indicating that the
segregation pattern of these two cell types found in early neonates is
transitional.
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Mature lymphocytes play an important role in the formation of
lymphoid follicles (10). Thus, it is likely that
lymphocyte entry into the PP anlagen provides momentum to induce cell
segregation and subsequent outward movement of the follicle-like
structures within the PP. To test this possibility, we stained the gut
of scid/scid mice in which mature lymphocytes are rarely
observed (31). As shown in Fig. 7, all processes of compartmentalization
described in the preceding section occur normally in the gut of
scid/scid mouse, although the size of scid/scid
PP were much smaller. This conclusion was confirmed by staining >10
PPs from each scid/scid mouse. As Croitoru et al and Falk et
al. have shown that adult scid/scid (32) and
rag1 or rag2 null mutant mice have
CD3+ intraepithelial lymphocytes
(33), it is possible that these mature lymphocytes are
involved in PP organogenesis in scid/scid mice. However, we
could not detect any CD3- nor B220-positive cells by whole-mount
immunostaining of guts of 17.5-dpc to 2-dpp scid/scid mice
(Fig. 8; data not shown). Defective
lymphogenesis in each scid/scid mouse was also confirmed by
staining the spleen by anti-CD3 and anti-B220. This result
indicates that the early compartmentalization process with concomitant
formation of the vascular system is independent of entry of mature
lymphocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using whole-mount immunostaining, we succeeded to detect the first
processes of compartmentalization as early as 18.5 dpc. The first event
detected in this study is outward spreading of
IL-7R+ cells that rapidly develop into several
segregated compartments. As this population is also present in
scid/scid mice, IL-7R+ cells may
represent
LT12-producing PP
inducers that we have characterized in previous studies
(2). Almost concomitant with this event, diffusely stained
VCAM-1+ regions segregate to form highly complex
structures by 2 dpp. It should be noted that
IL-7R+ and VCAM-1+ cells
behave similarly during the initial step of the compartmentalization.
This is consistent with our hypothesis that two populations interact
reciprocally to form an organizing center for PP organogenesis.
CD11c+ dendritic cells are also one of the major
cell populations within the secondary lymphoid organs and are found
from an early phase of PP organogenesis. As compared with
IL-7R+ cells, which are distributed
homogeneously in the PP anlage, CD11c+ cells
distributed more densely in the outer rim of PP anlagen at 17.5 dpc. At
18.5 dpc, when segregation of IL-7R+ cells
starts, they tend to fuse to the developing follicle-like structures
where IL-7R+ cells are concentrated. In the
follicle-like structures of 2-dpp mouse,
IL-7R+ cells localize more preferentially in
the central region of the follicle-like structure, whereas
CD11c+ cells display a tendency to localize at
the fringe. This distribution pattern of
IL-7R+ and CD11c+ cells
suggests that each follicle-like structure can be divided to the core
and fringe region in the next step, although the two regions are not
sharply demarcated. Nevertheless, the behaviors of
IL-7R+ and CD11c+ cells
imply that the complexity of PP architecture is induced in a stepwise
manner by creating distinct, novel compartments within previously
formed compartment. In this study, we analyzed expression of CXCR5 and
CCR7 in these two hemopoietic populations, as they were shown to be
essential to establishing an organized PP architecture (19, 23). Interestingly, IL-7R+ cells
express both CXCR5 and CCR7, while CD11c+ cells
express only CCR7, with these expression patterns remaining unchanged
during the initial phase of compartmentalization from 18.5 dpc to 2
dpp. This difference in the chemokine receptor expression may correlate
with the distinct distribution patterns of the two populations. In
fact, about a half of cxcr5 -/- mice were
reported to lack PP (20), implicating its role in the
induction phase of PP organogenesis. However, induction of PP anlage
and subsequent compartmentalization of IL-7R+
and CD11c+ cells are normal in plt
mutant mice which have a defect in SLC and ELC expression (H. Hashi et
al., unpublished observation). Consistent with this,
ccr7-/- mice have been shown to develop
normal numbers of PP, though the ultimate PP architecture is disturbed
(24). Thus, the significance of the differential
expression pattern of these chemokine receptors remains to be
elucidated in future studies. Nevertheless, comparison of mice with
mutations of various chemokine and chemokine receptor genes in terms of
the processes defined here will be important to fully understand the
molecular mechanisms regulating the compartmentalization process.
Moreover, further investigations of the expression of different
chemokines in each newly emerging compartment is needed.
The mature PP architecture should be equipped with a specific vascular structure including HEV and identified by expression of MAdCAM-1. In the intestine, expression of MAdCAM-1 becomes detectable in venous ECs over the intestine at 13.5 dpc. Thus, MAdCAM-1 expression is not specific to HEV and is induced at restricted stages of development of venous ECs in a spatio-temporally regulated manner. This notion is consistent with the observation by Iizuka et al. that in rat embryo MAdCAM-1 is also expressed on nonmucosal tissues such as in blood vascular ECs in the fetal skin and neonatal thymus (35).
While PP organogenesis commences from 15.5 dpc, clear MAdCAM-1
expression in this region is first detected at 16.5 dpc. Judging from
the staining pattern, possibly all newly developing venous ECs in the
PP anlagen are MAdCAM-1+. However, unlike other
regions, high expression is maintained in PP. This is likely to be due
to expression of LT12
in the IL-7R+ PP inducers, as
LT12 was shown to be
a potent inducer of MAdCAM-1 (36) as are IL-1 and TNF-
(37). Along with compartmentalization of
IL-7R+ cells, MAdCAM-1 expression became
restricted to the ECs surrounding each follicle-like structure. It
appears that MAdCAM-1+ vessels are pushed
away to the rim of the follicle-like structure by increasing entry of
hemopoietic cell lineages. Correspondingly, in the scid/scid
mouse in which entry of mature lymphocytes is defective, the average
diameter of each follicle-like structures surrounded by
MAdCAM-1+ vessels appears to be smaller than that
of the wild type.
Interestingly, even before the formation of discrete follicle-like
structures with HEV, mature lymphocytes enter the PP anlage from 18.5
dpc. The first mature lymphocytes detected in PP are distributed rather
evenly with a tendency to concentrate in the central part of PP, but B
and T cells are not segregated yet. As soon as the follicle-like
structures are formed, B220+ cells migrate to the
these area, while most CD3+ cells are distributed
within the space between these follicle-like structures. Noelle et al.
has shown that the segregation of T and B cells is regulated by LT
produced by nonlymphoid cells (10). Thus, it is likely
that LT12 produced in
IL-7R+ PP inducer cells also play a role in
this process by forming a B cell target zone before lymphocyte
entry.
It is still unclear how segregation is initiated in the apparently
simple and homogeneous primitive PP anlagen. We speculate that
CD11c+ cells play a role in this initial
segregation. Unlike VCAM-1+ or
IL-7R+ cells, which are distributed evenly
over the PP anlagen, CD11c+ cells first
accumulated in the periphery of the PP anlagen at 17.5 dpc. Thus entry
of CD11c+ cells from the periphery of the PP
anlage potentially adds a different influence by disturbing the
interactions between IL-7R+ and
VCAM-1+ cells.
In conclusion, we defined the initial process in formation of follicle-like structures in the PP. To our knowledge, this is the first description of how the rudiments of primary follicles are established. Thus, our present study, though remaining largely phenomenological, succeeded to specify the new process that should be examined in future investigation of PP organogenesis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiroyuki Hashi, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, Japan 606-8507.
3 Abbreviations used in this paper: PP, Peyer’s patch; dpc, days postcoitus; dpp, days postpartum; MAdCAM-1, mucosal addressin cell adhesion molecule; LT, lymphotoxin; PECAM-1, platelet endothelial cell adhesion molecule-1; HEV, high endothelial venule; EC, endothelial cell; BLC, B lymphocyte chemoattractant; SLC,secondary lymphoid-tissue chemokine; ELC, EBI1-ligand chemokine; DAB, diaminobenzidine; SA, streptavidin; MLN, mesenteric lymph node.
Received for publication September 24, 1999. Accepted for publication January 5, 2001.
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References |
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  T. Katakai, H. Suto, M. Sugai, H. Gonda, A. Togawa, S. Suematsu, Y. Ebisuno, K. Katagiri, T. Kinashi, and A. Shimizu Organizer-Like Reticular Stromal Cell Layer Common to Adult Secondary Lymphoid Organs J. Immunol., November 1, 2008; 181(9): 6189 - 6200. [Abstract] [Full Text] [PDF]
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M. Okuda, A. Togawa, H. Wada, and S.-i. Nishikawa Distinct Activities of Stromal Cells Involved in the Organogenesis of Lymph Nodes and Peyer's Patches J. Immunol., July 15, 2007; 179(2): 804 - 811. [Abstract] [Full Text] [PDF]
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R. T. Taylor, S. R. Patel, E. Lin, B. R. Butler, J. G. Lake, R. D. Newberry, and I. R. Williams Lymphotoxin-Independent Expression of TNF-Related Activation-Induced Cytokine by Stromal Cells in Cryptopatches, Isolated Lymphoid Follicles, and Peyer's Patches J. Immunol., May 1, 2007; 178(9): 5659 - 5667. [Abstract] [Full Text] [PDF]
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 T. Ara, K. Tokoyoda, R. Okamoto, P. A. Koni, and T. Nagasawa The role of CXCL12 in the organ-specific process of artery formation Blood, April 15, 2005; 105(8): 3155 - 3161. [Abstract] [Full Text] [PDF]
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R. T. Taylor, A. Lugering, K. A. Newell, and I. R. Williams Intestinal Cryptopatch Formation in Mice Requires Lymphotoxin {alpha} and the Lymphotoxin {beta} Receptor J. Immunol., December 15, 2004; 173(12): 7183 - 7189. [Abstract] [Full Text] [PDF]
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T. Cupedo, M. F. R. Vondenhoff, E. J. Heeregrave, A. E. de Weerd, W. Jansen, D. G. Jackson, G. Kraal, and R. E. Mebius Presumptive Lymph Node Organizers are Differentially Represented in Developing Mesenteric and Peripheral Nodes J. Immunol., September 1, 2004; 173(5): 2968 - 2975. [Abstract] [Full Text] [PDF]
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A. V. Tumanov, D. V. Kuprash, J. A. Mach, S. A. Nedospasov, and A. V. Chervonsky Lymphotoxin and TNF Produced by B Cells Are Dispensable for Maintenance of the Follicle-Associated Epithelium but Are Required for Development of Lymphoid Follicles in the Peyer's Patches J. Immunol., July 1, 2004; 173(1): 86 - 91. [Abstract] [Full Text] [PDF]
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T. Yamanaka, L. Helgeland, I. N. Farstad, H. Fukushima, T. Midtvedt, and P. Brandtzaeg Microbial Colonization Drives Lymphocyte Accumulation and Differentiation in the Follicle-Associated Epithelium of Peyer's Patches J. Immunol., January 15, 2003; 170(2): 816 - 822. [Abstract] [Full Text] [PDF]
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 A. Naito, H. Yoshida, E. Nishioka, M. Satoh, S. Azuma, T. Yamamoto, S.-i. Nishikawa, and J.-i. Inoue TRAF6-deficient mice display hypohidrotic ectodermal dysplasia PNAS, June 25, 2002; 99(13): 8766 - 8771. [Abstract] [Full Text] [PDF]
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H. Yoshida, H. Kawamoto, S. M. Santee, H. Hashi, K. Honda, S. Nishikawa, C. F. Ware, Y. Katsura, and S.-I. Nishikawa Expression of {alpha}4{beta}7 Integrin Defines a Distinct Pathway of Lymphoid Progenitors Committed to T Cells, Fetal Intestinal Lymphotoxin Producer, NK, and Dendritic Cells J. Immunol., September 1, 2001; 167(5): 2511 - 2521. [Abstract] [Full Text] [PDF]
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