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*
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan;
Department of Immunology, Graduate School of Pharmaceutical Science, and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
Department of Microbiology, Nippon Medical School, Tokyo, Japan;
¶ Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan;
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Department of Anatomy, School of Medicine, and
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Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-, and Ig
µ-chain mutant (µm-/-) mice, although
c-kit+ cells outnumber B220+ cells
in germfree and athymic nude mice, and most lymphoid residents are
c-kit+B220- in
RAG-2-/-, TCR--/-, and
µm-/- mice. ILF develop normally in the progeny
of transplacentally manipulated Peyer’s patch (PP)-deficient mice, and
decreased numbers of conspicuously atrophied ILF are present in
IL-7R
-/- PPnull mice. Neither ILF nor PP
are detectable in lymphotoxin -/- and
aly/aly mice that retain well-developed cryptopatches
(CP) and thymus-independent subsets of intraepithelial T cells, whereas
ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells
disappear from common cytokine receptor 
-chain mutant mice. These
findings indicate that ILF, PP, and CP constitute three distinct
organized gut-associated lymphoid tissues that reside in the lamina
propria of the mouse small intestine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now clear that IgA is one of the key hallmarks of the intestinal
humoral immune system (1, 2, 3) and that PP are the major
inductive sites for initiation of Ag-specific IgA responses to a
variety of intestinal luminal Ags (4, 5). However, it has
been reported that PP are not of paramount importance for establishing
strong local IgA Ab responses to foreign protein Ag in rats
(6) and to live locally invasive bacteria in rabbit
(7). Consistent with these earlier observations, we
(8) have recently demonstrated that PP also are not a
strict requirement in mice for induction of Ag-specific intestinal IgA
Ab responses. Oral immunization of the progeny of mice treated with
lymphotoxin receptor (LTR) and Ig chimeric protein (LTR-Ig)
that lacked PP but retained MLN (9) with OVA plus cholera
toxin as mucosal adjuvant resulted in OVA-specific intestinal IgA Ab
responses (8). In contrast, TNF and lymphotoxin (LT)
double-knockout mice that lacked both PP and MLN (10)
failed to elicit the responses (8). On the basis of these
findings, we underscored the compensatory role of MLN for the induction
of mucosal immune responses in LTR-Ig-treated
PPnull mice.
Other forms of lymphoid aggregations that have been identified in the wall of human (11), rabbit (7), and guinea pig (12) small intestines are isolated lymphoid follicles (ILF) which are invisible from the serosal or mucosal surface of the small intestine. ILF are structurally and functionally similar to the follicular units that compose PP and are believed to be an equivalent or complementary system to PP for the induction of intestinal IgA Ab responses. In contrast to ILF identified in the above mammals, however, ILF in the mouse small intestine have not been described in the immunological literature to date. Recently, we revealed multiple tiny clusters filled with c-kit+IL-7R+Thy-1+ lymphohemopoietic progenitors in crypt lamina propria (LP) of the mouse small intestine (cryptopatches; CP) (13, 14, 15, 16) and verified that CP were extrathymic anatomical sites indispensable for intestinal T lymphopoiesis (14, 15, 16). The modified nonclassical villi occupied mostly by immature lymphocytes (lymphocyte-filled villi; LFV) have also been identified in the rat small intestine and are regarded as candidates for specialized sites of primary extrathymic T lymphopoiesis (17). In this context, such a continued description of novel GALT is viewed as a good indication of the complexity of the intestinal immune system that has been driven by millennia of evolutionary pressures (18).
Thus, the continued identification of new organized GALT and a
consideration of the reasoning behind this, in conjunction with the
significance of our novel findings from LTR-Ig-treated
PPnull mice (8), led us to
investigate whether lymphoid aggregations equivalent in all respects to
ILF are present in the small intestine of laboratory mice. We found
that 100–200 lymphoid aggregations that fulfilled the criteria of ILF
were aligned along the antimesenteric wall of the mucosa. In the
present paper, anatomical structure, organogenesis, lymphocyte
composition, and strain-to-strain variation of these newly identified
ILF are described in relation to those of the other organized GALT of
the mouse small intestine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cA/Jcl (B/c), C57BL/6J/Jcl (B6), DBA/2J/Jcl, C3H/HeN/Jcl,
athymic nude (nu/nu) BALB/cA/Jcl, and alymphoplasia
(aly) mutant aly/aly Jcl (19) mice
were purchased from CLEA Japan (Tokyo, Japan). LT mutant C57BL/6J
(LT-/-) mice (20) were
purchased from The Jackson Laboratory (Bar Harbor, ME). IL-7R
-chain-deficient (IL-7R-/-) mice
(21), TCR-C mutant (TCR--/-)
mice (22), male mice carrying a truncated mutation of
common cytokine receptor gene
(CR-/Y) (23), and
germfree (GF) BALB/cA mice (24) have been described
previously (13, 15). Recombination activating gene-2
mutant (RAG-2-/-) (25) B/c
mice were provided by Dr. S. Koyasu (Keio University School of
Medicine, Tokyo, Japan). Ig µ-chain membrane exon-deficient
(µm-/-) mice (26) were a
generous gift from Dr. H. Karasuyama (Tokyo Medical and Dental
University, School of Medicine, Tokyo, Japan). We obtained
transplacentally manipulated PP-deficient mice according to the two
methods described elsewhere (8, 9, 27). In brief, timed
pregnant B/c mice were injected i.v. with 2 mg of an antagonistic mAb
to IL-7R (A7R34) on gestational day 14.5 (27) or 200 µg
LTR-Ig chimeric protein on gestational days 14 and 17
(8). All animal procedures described in this study were
performed in according with the guidelines for animal experiments of
Keio University School of Medicine.
Abs and lectin
The following mAbs described elsewhere (13, 14, 15, 16) were used. For immunohistochemical staining: anti-B220 (RA3-6B2; 2 µg/ml), anti-c-kit (ACK-2; 5 µg/ml), anti-CD3 (145-2C11; 5 µg/ml), anti-IL-7R (A7R34; 5 µg/ml) and anti-CD11c (N418; supernatant of the cultured hybridoma) mAbs. Biotinylated peanut agglutinin (PNA; 7.5 µg/ml) (Vector Laboratories) was also used in this study. For flow cytometric analysis: 3–15 x 105 cells were stained in 50 µl staining medium containing the following mAbs described elsewhere (13, 14, 15, 16). FITC-conjugated anti-B220 (RA3-6B2; 5 µg/ml), anti-c-kit (ACK-4; 25 µg/ml), and anti-Mac-1 (M1/70; 10 µg/ml) mAbs; and biotinylated anti-IL-7R (A7R34; 30 µg/ml) mAb. FITC-conjugated anti-IgD (11–26c.2a; 20 µg/ml; BD PharMingen, San Diego, CA), anti-IgA (C10-3; 20 µg/ml; BD PharMingen), and anti-T and B cell activation Ag (GL-7; 10 µg/ml; BD PharMingen) mAbs; and biotinylated anti-IgM (R6-60.2; 20 µg/ml, BD PharMingen), anti-B220 (RA3-6B2; 10 µg/ml, BD PharMingen), anti-CD11c (HL3; 10 µg/ml, BD PharMingen), anti-CD5 (53-7.3; 10 µg/ml, BD PharMingen), anti-CD19 (1D3; 10 µg/ml; BD PharMingen), and anti-CD23 (B3B4; 20 µg/ml; BD PharMingen) mAbs as well as biotinylated PNA (0.5 µg/ml; Vector Laboratories) were also used in this study.
Immunohistochemical procedure
Small intestine was longitudinally opened along the mesenteric
wall, and then intestine 
10 mm long that had been either kept flat
for horizontal section or rolled up for vertical section was embedded
in OCT compound (Tissue-Tek; Miles, Elkhalt, IN) at -80°C.
The tissue segments were sectioned with a cryostat at 6 µm, and
sections were preincubated with Block-ace (Dainippon Pharmaceutical,
Osaka, Japan) to block nonspecific binding of mAbs. The sections were
then incubated with rat or hamster mAb for 30 min at 37°C and rinsed
three times with PBS, followed by incubation with biotin-conjugated
goat anti-rat IgG Ab (5 µg/ml; Cedarlane Laboratories, Hornby,
Ontario, Canada) or with biotin-conjugated goat anti-hamster IgG (5
µg/ml; Cedarlane Laboratories). In staining with biotinylated PNA,
the second biotin-conjugated anti-IgG Ab was not used.
Subsequently, the sections were washed three times with PBS and then
incubated with avidin-biotin peroxidase complexes (Vectastain ABC kit;
Vector Laboratories). The histochemical color development was achieved
by the Vectastain DAB (3,3'-diaminobenzidine) substrate kit (Vector
Laboratories) according to the manufacturer’s instructions. Finally,
the sections were counterstained with hematoxylin for microscopy.
Endogenous peroxidase activity was blocked with 0.3%
H2O2 and 0.1%
NaN3 in distilled water for 10 min at room
temperature. Tissue sections incubated either with isotype-matched
normal rat IgG or with nonimmune hamster serum showed only minimal
background staining.
In vivo labeling and in situ immunohistochemical visualization of proliferating lymphocytes
Mice were given drinking water containing 1 mg/ml
bromodeoxyuridine (BrdU) for 38 h. The small intestines were
removed and opened along the mesenteric wall. Then intestines 10 mm
long that had been rolled up were embedded in OCT compound at -80°C.
Cryostat tissue sections 9 µm thick that included PP and/or ILF were
fixed in 4% paraformaldehyde for 15 min at 4°C, washed three times
with PBS, and treated with 2 M HCl for 20 min at 37°C, followed by
neutralization with 0.1 M sodium tetraborate. Subsequent
immunohistochemical color development using the first anti-BrdU mAb
(B44; BD Biosciences, San Jose, CA) and the second biotinylated goat
anti-mouse Ig Ab (20 µg/ml; Cappel, Aurora, OH) was performed
according to the method described in the preceding section.
Flow cytometry
A single lymphoid cell suspension was prepared, and nucleated
cells were counted using a hemocytometer. Resident lymphoid cells in
the peritoneal cavity (PEC) were obtained by rinsing PEC with 10 ml
ice-cold PBS without Ca2+ and
Mg2+. ILF cells were isolated by essentially the
same technique used for the isolation of CP cells
(14, 15, 16). In brief, the small intestine was opened
longitudinally along the mesenteric wall, and mucus and feces were
removed with filter paper. Subsequently, intestine 10 mm long was
pasted on a plastic culture dish. We amputated a needle (18-gauge;
inner diameter, 940 µm) at the proximal end of the tapering tip. We
then bent the needle in the middle, sharpened its cross-section with
the aid of a small electric grinder, i.e., a dental instrument (UA12A;
Urawa Kogyo, Saitama, Japan), and finally fitted it on a 1-ml syringe.
We located ILF under a transillumination stereomicroscope and isolated
a tiny fragment of the small intestine containing one ILF using the
needle described above. Lymphoid cells were incubated first with
biotinylated mAb and then with streptavidin PE (BD Biosciences) and
FITC-conjugated second mAb. Stained cells were suspended in staining
medium (Hanks’ solution without phenol red, 0.02%
NaN3, and 2% heat-inactivated FBS) containing
0.5 µg/ml propidium iodide (PI) and analyzed using FACScan with
CellQuest software (BD Biosciences). Dead cells were excluded by PI
gating. Lymphoid cells were incubated with anti-FcRII/III mAb
(2.4G2; 10 µg/ml; BD PharMingen) before staining to block nonspecific
binding of labeled mAbs to FcR.
Electron microscopy
Under anesthesia with sodium pentobarbital, B/c mice were perfused transcardially with Ringer’s solution and subsequently with a mixture of 2.5% glutaraldehyde and 0.5% paraformaldehyde buffered at pH 7.3 with 0.1 M cacodylate. The small intestine was excised and immersed overnight in the same fixative. After fixation, ILF on the antimesenteric wall were cut out with iridectomy scissors under a dissecting microscope, rinsed in 0.1 M cacodylate buffer (pH 7.3), and postfixed with 1% OsO4 buffered with cacodylate (0.1 M, pH 7.2) for 2 h. For scanning electron microscopy, the specimens were then dehydrated through a graded series of ethanol, transferred to isoamyl acetate, and critical-point dried with liquid CO2. The dried specimens were coated with osmium in a plasma osmium coater (Nippon Laser and Electronics Lab, Nagoya, Japan), and examined in a Hitachi H-4500 scanning electron microscope (Hitachi, Tokyo, Japan) at an acceleration voltage of 10 kV. For transmission electron microscopy, the osmicated tissue pieces were dehydrated through a series of ethanol and embedded in Epon 812. Ultrathin sections were examined in a Hitachi H-7100 transmission electron microscope (Hitachi) after double staining with uranyl acetate and lead citrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the course of our study on CP (13, 14, 15, 16), we noticed the presence of tiny and macroscopically invisible lymphoid clusters filled with B220+ cells in the mouse small intestine, although they are far less frequent but much larger than CP. Recently, we (8) verified that PP are not necessarily obligate GALT for the induction of Ag-specific intestinal IgA and serum IgG Ab responses to an orally administered protein Ag. With these observations in mind, we aimed at investigating the cluster filled with B220+ cells (we use hereafter the term ILF for these B cell aggregations; see below) in detail.
ILF were located in tandem on the antimesenteric wall down from
duodenum to ileum in B/c (Fig. 1
A,
left) and B6 (data not shown) mice, whereas CP filled with
c-kit+ cells were interspersed throughout
the mucosa (Fig. 1A, middle). We also confirmed
antimesenteric localization of multiple ILF in the small intestines of
DBA/2J/Jcl and C3H/HeN/Jcl mice (data not shown). ILF were smaller than
B cell-enriched follicular units of PP and lacked interfollicular PP
regions that contained mainly T cells (Fig. 1B). Absolute
numbers of ILF in the small bowels of 8- to 20-wk-old adult B/c and B6
mice were 150–200 and 100–150, respectively, and neonatal intestines
from these mice lacked ILF. In B/c mice, ILF were not detected at 4
days after birth but became detectable without exception
(preferentially in the duodenal and proximal jejunal mucosa) at the 7th
postnatal day, the day when CP were first detected in these mice.
Postnatal development of ILF in B/c mice is shown in Fig. 2A. We also evaluated the
frequency DNA replicating cells in ILF and verified that the net
accumulation of BrdU-incorporated DNA-replicating cells in ILF was
comparable with that in PP (Fig. 2B). We (13)
have previously reported that CP can be first detected at 14–17 days
after birth in the small intestine of B6 mice. However, ILF in B6
intestine remained unidentifiable until day 25 of postnatal life. Thus,
although these observations confirm the difference in the time course
of postnatal formation of ILF and CP between B/c and B6 animals,
histogenetic events behind this difference remain to be explored.
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CP are located mostly in the crypt LP and are not subdivided into
specific areas based on their cellular composition (13).
We found in the present study that ILF were larger than CP (Fig. 1
A) and appeared to occupy both the crypt and villous LP
(Fig. 1B). To compare more precisely the anatomical and
cellular features of ILF with those of CP, immunohistochemical analysis
was conducted using five representative mAbs that were reactive with B
cells (B220), mature T cells (CD3), lymphohemopoietic precursor cells
(c-kit and IL-7R) or dendritic stromal cells (CD11c), and the results
are presented in Fig. 3A. On
the basis of vertical profile, villi containing ILF were thicker
(barrel-shaped) and shorter than surrounding villi not containing ILF
(Figs. 1B and 3A, top). The collection
of B cells that resided the central region of ILF was surrounded by the
layer of cells expressing c-kit and IL-7R molecules, and a
considerable number of dendritic CD11c+ cells
were distributed in the peripheral region of ILF (Fig. 3A,
top and middle). Flow cytometric analysis of lymphoid
cells isolated from ILF confirmed that the B220+,
c-kit+IL-7R+, and
CD11c+ cells constituted three discrete
nonoverlapping populations (Fig. 3B). A small number of
CD3+ T cells was also interspersed within the ILF
(Figs. 1B and 3A). Finally, transverse profiles
clearly showed that the average diameter of ILF was longer by a factor
of 2–5 compared with that of CP, and, as verified previously
(13), neither B cells nor mature T cells were localized in
CP (Fig. 3A, bottom). These findings suggest that ILF and CP
constitute two distinct organized GALT in the mouse small
intestine.
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During the course of immunohistochemical study on ILF, we realized
that the lymphocytes compartmentalized in a core area of the
B220+ cell aggregations are proliferating
vigorously (Fig. 2B) and appear to include B blasts. In
lymphoid follicles, B cells at active immune responses can be
identified by their distinctive ability to bind PNA (28)
and the mAb GL-7 (29, 30) and undergo clonal expansion in
forming GC. In this context, we examined whether or not these putative
B blasts were capable of binding PNA and found that, in addition to the
well-known GC formation in PP (Fig. 4A, bottom),
PNA+ cell clusters, namely GC, were present in
the central area of the B220+ cell aggregations
(Fig. 4A, upper left and middle). In contrast, CP
were void of such PNA-reactive cells (Fig. 4A, upper right).
Among cells isolated from ILF, PP, MLN, and spleen of nonimmunized
normal B/c mice examined, a substantial fraction of
B220+ ILF cells was PNA+
and/or GL-7+, and a large fraction of
B220+ PP cells was PNA+
and/or GL-7+, whereas only a minimal fraction of
B220+ MLN cells and B220+
splenic cells displayed PNA+ and/or
GL-7+ phenotypes (Fig. 4B), indicating
the persistent GC formation in ILF and PP due to the constant antigenic
challenge from the gastrointestinal tract.
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, A and B), and these M cells were found to enfold
lymphoid cells in their central hollow (Fig. 5C). Thus,
taking all of the above findings collectively, these
B220+ cell aggregations are the first
identification of ILF in the mouse small intestine.
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IgA is a predominant Ig which is secreted mainly across mucous
membranes, and the LP of intestines contains the largest collection of
IgA-producing plasma cells. These plasma cells have been shown to
derive either from the conventional Ag-specific IgA-committed B-2 cells
in PP (4, 32) or from the B-1 cells (33, 34)
that are enriched among B cells compartmentalized in the PEC
(35). To determine whether B cells that reside in ILF are
similar or dissimilar to those of PP, flow cytometric analysis was
conducted (Fig. 6). Both ILF and PP
harbored a large population of IgM+ B cells and
also a significant population of IgA+ B cells,
whereas MLN contained few IgA+ B cells (Fig. 6, top). B cells isolated from ILF, PP, and MLN appeared to be
IgMlowIgDhigh as compared
with those recovered from PEC, the majority of which displayed
IgMhighIgDlow phenotype
(Fig. 6, middle). In contrast to the fact that two-thirds of
B220+ PEC cells expressed CD5 (Fig. 6, bottom) and Mac-1 (data not shown) molecules, such
B220+CD5+ cells (Fig. 6, bottom) and
B220+Mac-1+ cells (data not
shown) were minimal in ILF, PP, and MLN compartments. Moreover, almost
all B220+ cells from ILF and PP were
CD19+, and a large fraction of them were
CD23+, whereas B220+ cells
from PEC remained CD23- (data not shown).
Because mucosal B cells have been classified into B-1 and B-2 cells
based on the differential expression of B220, IgM, IgD, CD5, Mac-1, and
CD23 molecules (34, 36), the present findings suggest that
ILF are inductive sites for initiation of IgA-committed B-2 cell
responses in the gastrointestinal tract.
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Immunohistochemical analysis was conducted to characterize ILF of
GF and various mutant mice (Fig. 7). We
evaluated the effects of microbial and thymic deprivations in GF (Fig. 7B) and nu/nu (Fig. 7C) mice,
respectively, on the development of ILF and found that the cellularity
of ILF remained the same in both animals. However,
c-kit+ cells outnumbered
B220+ cells (Fig. 7, B and
C) and the formation of GC was hardly detectable (data not
shown) under these conditions. The cellular mass of ILF was also not
significantly altered in the RAG-2-/- (Fig. 7D), TCR--/- (Fig. 7F), and µm-/- (Fig. 7G) conditions as compared with those of wild-type ILF from
B/c (Fig. 7A) and B6 (Fig. 7E) mice, whereas the
difference in cellular composition was striking. Thus, the population
size of B220+ cells was reduced drastically in
TCR--/- ILF (Fig. 7F), and few,
if any, B220+ cells were detected in
RAG-2-/- (Fig. 7D) and
µm-/- (Fig. 7G) ILF. In these three mutant
ILF, especially in RAG-2-/- and
µm-/- ILF, c-kit+
cells replaced B220+ cells as the predominant
population. As far as the lymphoid residents of PP are concerned,
c-kit+ cells occupied interfollicular T
cell regions in TCR--/- PP, follicular B
cell regions in µm-/- PP and entire T and B
cell regions in RAG-2-/- PP (data not shown).
As in the histogenesis of CP (13, 15), sharply emaciated
ILF were detected in IL-7R-/- mice (Fig. 7H).
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. Absolute numbers of ILF were not
significantly altered in GF, nu/nu,
RAG-2-/-, and
TCR--/- mice as compared with those of the
corresponding wild-type ILF, whereas absolute numbers of ILF in
µm-/- mice were about one-half of those in B6
mice. Finally, a drastically reduced number of ILF was detected in
IL-7R-/- PPnull mice.
It has been reported that PP are absent from the small intestines of
LT-/- (20) and
aly/aly (19) mutant mice. In this context, we
addressed whether these mutations affect the development of ILF and
found that histogenesis of ILF was also completely blocked. In these
mutant conditions, however, histogenesis of CP and intestinal
development of intraepithelial T cells (IEL) that derive from
c-kit+ CP cells (14, 16)
remained intact. It has recently been verified that not only PP but
also CP are absent from CR-/Y mice. We found
that ILF were hardly detectable in the intestinal LP of these
CR-/Y mice. We also determined the
development of ILF in the transplacentally manipulated
PPnull mice because previous studies have shown
that exposure to anti-IL-7R mAb (27) as well as
exposure to LTR-Ig chimeric protein (8, 9) during
gestation disrupted PP but not MLN formation. As a result, it was
confirmed that the offspring from these mothers lacked PP but possessed
well-developed ILF (Table I) and CP (data not shown). In conclusion,
ILF are not identical with PP or with CP with respect to their
histogenesis and lymphocyte composition, and it is quite intriguing
that they are aligned along the antimesenteric wall of the mouse small
intestine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100–200
lymphoid aggregations that satisfied the structural and cellular
requirements of ILF in mouse small intestine. Remarkably, they were
aligned at roughly regular intervals along the antimesenteric wall of
the small bowel (Fig. 1A, left). In addition to 10
aggregated lymphoid follicles (37), we also verified that
50 ILF were interspersed throughout the large intestinal mucosa of
B/c mice (data not shown). We do not know why they have not been
described to date, but we believe the existence of CP and ILF in the
murine intestinal mucosa has not been previously evaluated and reported
simply because they are very small and constitute only a thinly
scattered population. Immunohistochemical examination revealed that
villi housing ILF are thicker and shorter in a longitudinal section
than surrounding classical villi, and they are distinguished by
containing a LP that is replaced by closely packed lymphocytes (Figs. 1B and 3A). In this regard, ILF are structures
that resemble most closely LFV of the rat small intestine
(17), although their lymphoid residents are markedly
different from those of rat LFV, a large fraction of which displays an
immature phenotype (17). Thus, in agreement with the
properties of ILF described in other mammals (7, 11, 12),
it is evident that mouse ILF identified in the present study are
similar to the follicular units that compose PP.
As far as organogenesis is concerned, however, the difference between
ILF and PP is a matter of considerable interest. First, ILF are not
detectable until day 4 (B/c mice) or day 25 (B6 mice) of postnatal
life, whereas PP are already microscopically well developed by just
before birth in both strains of mouse (Ref. 27 and data
not shown). Second, in utero treatment with anti-IL-7R mAb or
LTR-Ig chimeric protein abrogates the development of PP, leaving
that of ILF unaffected. Third, IL-7R-/- mice
lack PP, whereas they possess ILF-like lymphoid aggregations although
they are atrophied markedly in both average size (Fig. 7H)
and numbers. Because the present findings (Figs. 4 and 6) support the
notion that ILF are also the inductive sites for intestinal IgA Ab
responses to a variety of luminal Ags, the biological significance of
the difference in ILF and PP formation is most likely to be failsafe
and/or mutually compensatory systems for the maintenance of intestinal
immune surveillance. In mice, organogenesis of 6 to 12 PP involves at
least 3 distinct steps in the late embryonic stage (27).
Exposure of day 14.5 to 15.5 fetuses, namely, the first step, to
anti-IL-7R mAb results in the generation of
PPnull but otherwise normal mice (Ref.
27 and Table I), indicating that there exists a short and
critical time window during the initial step of PP formation. In
contrast, organogenesis of ILF commences in early postnatal life and
thereafter increases gradually in numbers and average size (Fig. 2A). In the context of these findings and given that PP have
evolved earlier than ILF, it is conceivable that the development of ILF
in the mouse small intestine is a failsafe system. Conversely, if the
ILF evolved earlier than PP, the development of PP is regarded as a
complementary system to the lack of ILF during early infantile life. In
any case, exploration of these possibilities is certainly an important
goal for future experiments.
Not only PP but also ILF are absent from
LT-/- and aly/aly mice, whereas
CP and their IEL descendants are present in these mutant animals
(14, 16, 38) and, in contrast to antimesenteric
distribution of PP and ILF, CP are situated randomly around the
circumference of the intestinal wall (Fig. 1A).
Intriguingly, however, organogeneses of ILF and CP commence at the same
postnatal age in B/c mice, although that of PP is completed just before
birth. Because it has recently been demonstrated that LT12
receptor (LTR) signaling is crucial for the first step of PP
formation (27) and that LTR-mediated activation of
NF-
B-inducing kinase is selectively cancelled in the aly
mutation (39), it is evident that the signal passing
through LTR at antimesenteric organizing centers is indispensable
for the formation of both PP and ILF anlagen. In sharp contrast,
neither LT12 nor the positional signals that emanate solely from
antimesenteric mucosa are essential for the formation of CP anlagen.
Also, the lymphoid follicles in the cecum (cecal patch), which are
surprisingly quite intact in IL-7R-/-
mice (27), are absent in
LT-/- and aly/aly mice (H.
Yoshida, unpublished observations). All in all,
LT-/- and aly/aly mice lack PP,
ILF, and cecal patch but possess well-developed CP,
IL-7R-/- mice lack PP but possess normal
cecal patch and conspicuously atrophied ILF and CP, and
CR-/Y mice are void of PP, ILF, and CP.
Taking these observations at face value, there exists remarkable
complexity in the organogenetic mechanism of different GALT, and much
remains to be learned about molecular level of cellular events
underlying the formation of these organized GALT before we elucidate
the biological significance of this complexity.
In addition to PP and CP, lymphoid aggregations analogous to ILF of
wild-type mice are present in nu/nu,
RAG-2-/-, and µm-/-
mice, indicating that organogenesis of ILF anlagen is dependent neither
on thymus-derived lymphocytes nor on the expression of Ag receptor
genes on T and B cells. However, ILF detected in
RAG-2-/- and µm-/-
mice are phenotypically abnormal because most lymphoid cells express
c-kit but not B220 molecules (Figs. 7, D and
G). These results not only indicate that the organogenesis
of ILF proceeds through at least several histologically distinct steps
but also suggest that B lineage-committed cells and/or mature B cells
immigrate from outside into ILF during the latter stage of ILF
formation. In conclusion, the current studies identified and
characterized ILF in mouse small intestine and illuminated the various
facets of their postnatal development. We consider that our findings
are of considerable importance because researchers exploring the
distinctive features of intestinal immune responses to luminal Ags such
as the regulation of mucosal IgA Ab responses and induction of oral
tolerance in the gastrointestinal tract, and the role of PP in these
processes have been performing their experiments using various
manipulated laboratory mice, including the mice that lack PP but
possess ILF.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiromichi Ishikawa, Department of Microbiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: ishikawa{at}microb.med.keio.ac.jp
3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissues; aly, alymphoplasia; BrdU, bromodeoxyuridine; CP, cryptopatches; CR, common cytokine receptor -chain; GC, germinal center; GF, germfree; ILF, isolated lymphoid follicles; IEL, intestinal intraepithelial T cells; LFV, lymphocyte-filled villi; LP, lamina propria; LT, lymphotoxin; LTR, lymphotoxin receptor; M cells, microfold cells; MLN, mesenteric lymph nodes; µm, Ig µ-chain membrane exon; nu/nu, athymic nude; PEC, peritoneal cavity; PI, propidium iodide; PNA, peanut agglutinin; PP, Peyer’s patches; RAG, recombination-activating gene.
Received for publication August 27, 2001. Accepted for publication October 30, 2001.
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