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Department of Microbiology, Keio University School of Medicine,
Department of Microbiology and Immunology and
Second Department of Internal Medicine, Nippon Medical School, and
Yakult Central Institute for Microbiological Research, Tokyo, Japan; and
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Department of Immunology, Graduate School of Pharmaceutical Science, Osaka University, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and -
double-mutant mice. As far as the goblet cells of villous
epithelium are concerned, absolute numbers of them remained the same
among these mutant mice that have no B and/or T cells. Alymphoplasia
(aly/aly) mutant mice that lacked Peyer’s patches and
Ig-producing cells in the lamina propria, but harbored a large number
of intestinal mucosal T cells, also displayed a significant
acceleration of epithelial cell turnover and, to some extent,
up-regulated expression of MHC class II molecules. Notably, the
accelerated epithelial cell turnover was not observed and returned to
normalcy in the Ig µ-chain mutant mice that had been given
antibiotic-containing water. These findings indicate that B cells
down-regulate the generation and differentiation of intestinal
epithelial cells in the normal wild-type condition and suggest that
enteric microorganisms are implicated in the accelerated generation of
epithelial cells in mice that have no B cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have indicated functional cross-talk between IEC and
intestinal intraepithelial T cells (IEL) (2, 3, 4, 5). For
instance, the absence of 
IEL was associated with a reduction of
IEC turnover and a down-regulation of the expression of MHC class II
molecules (4). Golovkina et al. (6) have also
demonstrated that the development of specialized Ag-handling epithelial
cells called microfold (M) cells, which overlay the intestinal Peyer’s
patches (PP), is impaired in mice that have no B cells. Thus, in mice
lacking B cells because of the targeted disruption of the membrane exon
of the Ig µ-chain gene (µm-/-), M
cells were almost entirely absent from the lymphoid follicle
(PP)-associated epithelium (FAE). In contrast, the lack of T cells in
TCR- and - double-mutant mice did not have a significant effect
on M cell development (6). B cell-mediated, but not T
cell-mediated, conversion of IEC into M cells was also verified in a
previous in vitro study (7).
In view of this distinctive function of B cells in M cell formation (6, 7), we realized that it is important to evaluate the role of B cells in the generation and differentiation of villous IEC. In the present study we investigated the same B and/or T cell-deficient mice previously analyzed by Golovkina et al. (6) and revealed that the absence of B, but not T, cells was associated with acceleration of IEC turnover and up-regulation of the expression of MHC class II molecules. Taken together, B cells are required for the development of M cells, whereas in the same gut microenvironment B cells are capable of exerting an inhibitory effect on the generation, migration, and differentiation of villous IEC. Remarkably, the accelerated generation of IEC in µm-/- mice was canceled when they were treated with antibiotics. The significance of these findings is discussed from the viewpoint that there is a functional dialog between enteric microorganisms and intestinal mucosal B cells by which the intestinal epithelial homeostasis is maintained.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J Jcl (B6), alymphoplasia (aly) mutant
aly/aly (8) and CB-17/Icr Jcl SCID and their
corresponding wild-type (WT) aly/+ and CB-17/Icr Jcl mice
were purchased from CLEA Japan (Tokyo, Japan).
RAG-2-/- mice carrying the genetic
background of BALB/c (B/c) mice were provided by Dr. S. Koyasu (Keio
University School of Medicine, Tokyo, Japan), and
RAG-2-/- mice carrying the genetic
background of B6 mice have been described previously (9).
µm-/- mice (10) that had been
backcrossed 12 times to B6 parent were a gift from Dr. H. Karasuyama
(Tokyo Medical and Dental University, School of Medicine, Tokyo,
Japan). TCR- mutant (-/-) mice
(9) (backcrossed 10 times to B6 parent) and TCR- mutant
(-/-) mice (9) (backcrossed
eight times to B6 parent) were also used. By intercrossing two
appropriate mouse strains we obtained µm-/-
and µm+/-,
RAG-2-/- and
RAG-2+/-, and WT,
-/-, -/- and
x -/- littermate mice. These mice
were maintained in our animal facility, and mice of both sexes, 11–18
wk of age, were used in the experiments.
Oral administration of antibiotics
Mice were given drinking water containing broad spectrum antibiotics, i.e., 200 µg/ml ampicillin sodium (Meijiseika, Tokyo, Japan) and 50 µg/ml imipenem/cilastatin (Banyu Pharmaceutical, Tokyo, Japan) for 10 days before the experiments, and this antibiotic-containing water was renewed every second day.
Antibodies
The following FITC-conjugated, biotinylated, and PE-conjugated
mAbs described previously (11) were used for flow
cytometric analysis: anti-
mAb (H57-597; BD PharMingen, San
Diego, CA), anti- mAb (GL3; BD PharMingen), anti-Thy-1.2
mAb (30-H12; BD Biosciences, San Jose, CA), anti-CD4 mAb (GK1.5; BD
PharMingen), anti-CD8 mAb (53-6.7; BD PharMingen), and
anti-CD8 mAb (53-5.8) (BD PharMingen). We also used
anti-I-Ab mAb (AF6–120.1; BD PharMingen),
anti-I-Ad mAb (AMS-32.1; BD PharMingen), and
the second biotinylated goat anti-mouse IgG (H and L chains)
(Zymed Laboratories, San Francisco, CA).
In vivo labeling and in situ immunohistochemical visualization of proliferating IEC
Mice were given drinking water containing 1 mg/ml
bromodeoxyuridine (BrdU) (12) for 38 h. The small
intestines were removed and opened along the mesenteric wall. Then
intestines, 
10 mm in length, that had been rolled up were embedded
in OCT compound (Tissue-Tek; Miles, Elkhart, IN) at -80°C.
Nine-micrometer-thick cryostat tissue sections 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) and
the second biotinylated goat anti-mouse Ig Ab (Cappel, Aurora, OH)
was performed according to methods described previously
(9). Because the difference in the number of
BrdU-incorporated IEC between various mutant and the corresponding WT
specimens was maintained throughout the length of the small intestine,
we mainly examined the jejunal tissues. For quantification of
proliferating villous IEC, 10 arbitrary villi/representative section
that exhibited exactly the vertical profile were chosen, and
BrdU-incorporated IEC located above the crypt-villus junction were
enumerated. Three rolls (see above) from each mouse and four mice from
each strain of mice were examined (120 villi in total).
Flow cytometry
We isolated IEL and IEC according to methods described
previously (4, 13). In brief, an inverted intestine was
cut into four segments, and the segments were transferred to a 50-ml
conical tube containing 45 ml RPMI 1640, 5% FCS, 25 mM HEPES, 100 U/ml
penicillin, and 100 µg/ml streptomycin. The tube was shaken at 37°C
for 45 min (horizontal position; orbital shaker at 150 rpm). Cell
suspensions were collected and passed through a glass-wool column to
deplete cell debris and sticky cells (crude cell preparation).
Subsequently, the cells were suspended in 30% Percoll solution
(Amersham Pharmacia Biotech, Uppsala, Sweden) and centrifuged for 20
min at 1800 rpm. After centrifugation, cells at the top of the 30%
Percoll solution were found to be enriched with IEC devoid of
CD3-positive cells. Cells at the bottom of the solution were then
subjected to Percoll discontinuous gradient centrifugation, and IEL
were recovered at the interphase of 44 and 70% Percoll solutions
(>95% were CD3 positive). IEL were incubated first with biotinylated
mAb or PE-conjugated mAb and then with FITC-conjugated second mAb with
or without (in case of PE-conjugated mAb) streptavidin-PE (BD
Biosciences). Stained cells were suspended in staining medium
(HBSS 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).
IEL were incubated with anti-FcRII/III mAb (2.4G2; BD
PharMingen) before staining to block nonspecific binding of labeled
mAbs to FcR. We also measured MHC class II molecules on IEC by flow
cytometry as described previously (4). Dead cells were
excluded by PI gating.
Redirected cytotoxicity assay
Redirected cytolytic activity of freshly isolated IEL was
measured in a standard 51Cr release assay. The
fresh effector IEL were incubated with 3 x
103 51Cr-labeled
FcR-positive P815 mastocytoma target cells for 6 h at 37°C
without addition of any mAbs or in the presence of 0.2 µg/ml
anti-CD3 mAb (145-2C11; BD PharMingen), 0.2 µg/ml anti-
TCR mAb (H57-597; BD PharMingen), or 1 µg/ml anti- TCR mAb
(GL3; BD PharMingen) in 75 µl complete medium (RPMI 1640 containing
10% FCS, 10 mM HEPES, 5 x 10-5 M 2-ME, 4
mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) in
each well of flat-bottom 96-well microtiter plates (Nunc, Copenhagen,
Denmark). Two hundred microliters of complete medium was added, and 100
µl supernatant was collected after centrifugation for assay of the
51Cr released. The percentage of specific
51Cr release was calculated using the following
equation: (experimental release - spontaneous
release)/(detergent-induced release - spontaneous release)
x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epithelial stem cells proliferate at just above the crypt base in
the small intestine, and newly formed cells differentiate into mature
IEC during their upward migration toward the top of the villi. In an
attempt to evaluate the effect of B cell deprivation on the generation
of IEC, we administered BrdU to µm-/- mice to
visualize the IEC that had passed through the S phase of the cell cycle
(4). Maturation of IEC corresponds to the expression of
various brush border enzymes and MHC class II Ags (4). To
determine whether B cells have any effect on upward-moving IEC, we
examined the expression of MHC class II molecules by flow cytometry of
IEC isolated from µm-/- and their WT
µm+/- littermate mice. As shown in Fig. 1
, A and B, the
migration of proliferated IEC toward the top of the villi and the
number of IEC that had incorporated BrdU were augmented markedly in
µm-/- mice. In addition,
I-Ab (MHC class II) molecules on IEC were
substantially increased in µm-/- mice
compared with B6 and µm+/- mice (Fig. 1C).
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In the mouse small intestine numerous IEL expressing either
TCR- (-IEL) or TCR- (-IEL) reside above the
basement membrane together with the columnar IEC. IEL are unusual among
mouse peripheral T cells in that most -IEL and many -IEL,
unlike thymus-derived T cells, express a unique CD8 homodimer
(14) instead of a CD8 heterodimer and develop
extrathymically in the intestinal mucosa (15) and in that
freshly isolated IEL are capable of killing FcR-bearing target cells
after bridging them with anti-CD3 or anti-TCR mAbs
(16). Moreover, recent studies have indicated functional
cross-talk between IEL and IEC (2, 3, 4, 5, 17, 18).
With these noteworthy findings in mind we addressed the question of
whether the absence of B cells in µm-/- mice
undermines the development and in situ function, such as the cytolytic
activity of IEL, that lead, in turn, to the up-regulation of epithelial
stem cell proliferation and differentiation. Flow cytometric and
redirected cytolysis analyses revealed that the development and
cytolytic activity of - and -IEL were not impaired in the
µm-/- condition (Fig. 2). On the contrary, while absolute
numbers of -IEL were not altered in
µm-/- mice (2.5 ± 0.4 x
106; n = 8) compared with those
in µm+/- mice (2.7 ± 0.5 x
106; n = 7), the population size
of -IEL in µm-/- mice (4.4 ±
1.3 x 106; n = 8) was about
two times larger than that in µm+/- mice
(2.5 ± 0.4 x 106; n =
7). Interestingly, this increase in number was caused by the selective
expansion of CD8+ -IEL subset (Fig. 2B), although further studies are required before we
evaluate the mechanism and functional significance of the expansion we
observed.
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The findings presented in the preceding section indicate that the
generation and differentiation of villous IEC are up-regulated in the
absence of B cells. It appears possible that T cells in the absence of
B cells up-regulate the generation and differentiation of IEC. To
determine whether T cells were required for the up-regulation observed
in µm-/- mice, we examined the intestinal
epithelia of mice that lacked both B and T cells. In B and T
cell-deficient RAG-2-/- and SCID mutant
animals, which have the genetic background of the B/c strain of mice,
the migration of proliferated IEC toward the top of the villi (Fig. 3A), the number of
BrdU-positive IEC (Fig. 3B), and the expression of
I-Ad (MHC class II) molecules on IEC (Fig. 3C) were all up-regulated significantly compared with those
of IEC from their corresponding WT
RAG-2+/- and CB-17 mice, respectively.
The same up-regulations were observed in
RAG-2-/- mice carrying the B6 genetic
background (data not shown). Overall, these results are inconsistent
with the proposition described above.
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x -/- mice that lack T
cells
We (4) have reported that the absence of , but
not , T cells is associated with a significant reduction of IEC
turnover and a down-regulation of the expression of MHC class II
molecules. During the course of the study, however, we examined mostly
+/- (WT) and -/-
littermates from an intercross between +/-
and -/- mice and
+/- (WT) and -/-
littermates from an intercross between +/-
and -/- mice. We also analyzed several WT,
-/-, and -/- mice
as well as two x -/- double-mutant
littermates obtained from the F2 generation of an
intercross between -/- and
-/- mice. Although the data were not shown
(4), it appeared that the expression of MHC class II
molecules on IEC was reduced to a certain extent in x
-/- mice. With these previous observations
in mind, we reinvestigated the intestinal epithelia of x
-/- double-mutant mice that lacked
and T cells. Because we now have -/-
and -/- mice that have been backcrossed more
than eight times to the B6 parent, we intercrossed these two mutant
strains of mice to obtain WT, -/-,
-/-, and x
-/- littermates and extensively examined the
effects of T cell deprivation on the generation and differentiation
of IEC.
The absence of T cells in -/- mice
was associated with a reduction in IEC turnover (Fig. 4, A and B) and a
marked down-regulation of the expression of MHC class II molecules
(Fig. 4C). No such effects were observed in
-/- mice that lacked T cells (Fig. 4). Obviously, these results were in line with our earlier observations
(4). However, in T cell-deficient x
-/- mice, not only the number of
BrdU-positive upward-moving IEC but also the expression of MHC class II
molecules on villous IEC remained unaltered and were almost comparable
to those displayed by WT and -/- IEC (Fig. 4). Thus, taking all present and previous findings together, it is now
evident that complete T cell deficiency does not affect the generation
and differentiation of IEC, indicating that the down-regulation of
intestinal epithelia inherent in -/- mice is
cancelled by the simultaneous disappearance of T cells in
x -/- mice.
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The aly/aly mice (8), which carry a
homozygous recessive point mutation in the nuclear factor 
B-inducing
kinase (19), are characterized by a systemic defect of
lymph nodes and PP, drastically reduced levels of serum Ig and severe
immunodeficiency. While the number of T cells colonized in the
epithelial and lamina propria (LP) compartments of the small intestine
was only slightly diminished, IgA+ B cells were
virtually absent in the LP of aly/aly mice
(13). We determined the effect of aly mutation
on the generation and differentiation of IEC, and the results are
presented in Fig. 5. The IEC turnover was
conspicuously accelerated (Fig. 5, A and B), and
the expression of MHC class II molecules on IEC appeared to be
up-regulated to some extent (Fig. 5C) in aly/aly
mice compared with WT aly/+ mice.
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The development of M cells is impaired in mice that have no B
cells (6). In contrast, the results obtained to date
indicate that the generation and differentiation of villous IEC are
up-regulated in B cell-null mice. Mucus-producing goblet cells are
another major descendant of the stem cells and develop during their
upward migration to the top of the villi. We examined H&E-stained
tissue sections of the small intestines prepared from the
aforementioned mice and noticed that the intraepithelial development of
goblet cells was not significantly altered in mice that lacked B and/or
T cells (Fig. 6A). This
impression was confirmed by counting the number of goblet cells in the
small intestinal villi of these various strains of mice (Fig. 6B).
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Much attention is now focused on the immunobacterial homeostasis
in the gut, namely, mutual functional cross-talks between indigenous
commensal microorganisms, IEC, and intestinal mucosal lymphocytes
(2, 3, 4, 5, 6, 7, 17, 20, 21, 22). For instance, evidence has been
presented that primitive T cell-independent B cells play a role in
defending intestinal mucosal surface from environmental organisms by
producing IgA Abs to commensal bacteria (20). To evaluate
the bacterial involvement in the accelerated IEC turnover observed in
mice that have no B cells, we administered antibiotics to
µm-/- and µm+/- mice
and determined the generation and differentiation of IEC (Fig. 7). The treatment of
µm-/- mice with antibiotics markedly
decelerated the migration of IEC toward the top of the villi and
decreased the net accumulation of BrdU-incorporated IEC (Fig. 7, A and B). Expression of
I-Ab molecules on IEC from
µm-/- mice also appeared to be reduced to
some extent by treatment with antibiotics compared with that on IEC
from untreated µm-/- mice (Fig. 7C). Because the generation and differentiation of IEC
remained almost the same between antibiotic-treated and -untreated WT
µm+/- mice (Fig. 7), the down-regulatory
effect of antibiotics on IEC turnover in
µm-/- mice was brought about through the
quantitative and/or qualitative modulations of intestinal flora, not by
the noxious effect on IEC themselves.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this context the results obtained by oral administration of broad
spectrum antibiotics to µm-/- mice provide an
important clue to elucidate the B cell-mediated regulation of IEC
growth. As shown in Fig. 7
, treatment with antibiotics abrogated the
accelerated generation of IEC observed in
µm-/- mice, indicating that intestinal
microorganisms play a critical role in accelerating IEC turnover in
mice that have no B cells. These findings support the idea that B cells
and/or secreted Abs, in turn, are capable of shifting the generation
and differentiation of IEC into low gear in the normal WT gut
microenvironment. Because many aspects of complex functional links
between intestinal flora, IEC, and gut-associated lymphoid tissues
(2, 3, 4, 5, 6, 7, 17, 20, 21, 22, 30, 31) are beginning to come together,
the precise mechanism underlying this B cell-mediated regulation of
intestinal epithelia and the question of how many immunologically
relevant players other than B cells are involved in this phenomenon
remain the subject of considerable importance.
In -/- mice, the development of IEC is
down-regulated, as reported in our previous (4) and
current (Fig. 4) studies. Taking this effect of T cells on IEC
growth at face value, IEL are capable of accelerating the
generation and differentiation of adjacent IEC (
). In the
complete absence of T cells in x
-/- mice, however, the generation and
differentiation of IEC were unaltered (Fig. 4). In view of these
findings, we consider that T cells residing in the
intraepithelial ( IEL) and/or LP compartments may have the
ability to decelerate the generation and differentiation of IEC
(
). Based on this concept, and/or T
cell-mediated controls of intestinal epithelia were formulated and are
presented in Table I. IEC growth remained
almost the same in WT mice with / activities and
x -/- mice without
/ activities and was down-regulated in
-/- mice that lack activity but
possess activity. Thus, these findings are compatible with
the concept presented. Then why was it not up-regulated in
-/- mice that possess activity but
lack activity (Fig. 4)? It has been demonstrated that the
cytolytic (32, 33) and IFN--producing (33)
activities of IEL were attenuated sharply in
-/- mice, whereas IEL were irrelevant
to such activities of IEL (33). In this context, it
is conceivable that the activity of IEL is also
dependent on T cells and, therefore, drastically reduced in
-/- mice (Table I). Whatever the functional
link between and activities, it is corroborated
that the total disappearance of T cells does not exert any significant
effect on the generation and differentiation of villous IEC.
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) that possessed IgA and
RAG-2-/- (Fig. 3) and in the
aly/aly (Fig. 5) mice that lacked IgA, it appeared that
IgA-producing B cells in LP were irrelevant to the up-regulation
observed in these three mutant conditions.
Finally, the fact that goblet cells were intact in the B cell-deficient
conditions (Fig. 6) is very intriguing. The result indicates that
whatever influence B cell deficiency has, B cell deficiency works past
the stage of goblet cell differentiation. In this context, the
up-regulation of intestinal epithelia in the absence of B cells appears
to take place at the level of mature IEC that keep migrating toward the
top of the villi, but not at the level of immature precursor IEC. In
conclusion, a better understanding of B cell-mediated regulation of
intestinal epithelia will not only offer additional clues for
elucidating the functional associations of IEC and immunologically
relevant cells and/or molecules, but may also shed light on the new
aspect of cellular events underlying intestinal mucosal
surveillance.
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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, Tokyo 160-8582, Japan. E-mail address: ishikawa{at}microb.med.keio.ac.jp
3 Abbreviations used in this paper: IEC, intestinal epithelial cell; aly, alymphoplasia; BrdU, bromodeoxyuridine; FAE, follicle-associated epithelium; IEL, intestinal intraepithelial T cell; LP, lamina propria; M, microfold; PI, propidium iodide; PP, Peyer’s patch; RAG, recombination-activating gene; WT, wild type.
Received for publication October 11, 2001. Accepted for publication January 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells. Science 266:1253. T cells. Proc. Natl. Acad. Sci. USA 92:6147. T cells and Peyer’s patches. J. Exp. Med. 191:1569.+ intraepithelial lymphocytes. Science 243:1716. intraepithelial lymphocytes as suggested by their transcriptional profile. Proc. Natl. Acad. Sci. USA 98:10261.B-inducing kinase. Nat. Genet. 22:74.[Medline]
2 integrins and ICAM-1 are involved in establishment of the intestinal mucosal T cell compartment. Immunity 5:263.[Medline]
-producing activities of T cells in the mouse intestinal epithelium are T cell receptor--chain dependent. Proc. Natl. Acad. Sci. USA 96:7451. chain expression in developing B cells. Nat. Immunol. 2:625.[Medline]This article has been cited by other articles:
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  R. Shaykhiev and R. Bals Interactions between epithelial cells and leukocytes in immunity and tissue homeostasis J. Leukoc. Biol., July 1, 2007; 82(1): 1 - 15. [Abstract] [Full Text] [PDF]
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S. Ueha, M. Murai, H. Yoneyama, M. Kitabatake, T. Imai, T. Shimaoka, S. Yonehara, S. Ishikawa, and K. Matsushima Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects J. Leukoc. Biol., January 1, 2007; 81(1): 176 - 185. [Abstract] [Full Text] [PDF]
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 S. Inouye, H. Izu, E. Takaki, H. Suzuki, M. Shirai, Y. Yokota, H. Ichikawa, M. Fujimoto, and A. Nakai Impaired IgG Production in Mice Deficient for Heat Shock Transcription Factor 1 J. Biol. Chem., September 10, 2004; 279(37): 38701 - 38709. [Abstract] [Full Text] [PDF]
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 T. Tsuchiya, S. Fukuda, H. Hamada, A. Nakamura, Y. Kohama, H. Ishikawa, K. Tsujikawa, and H. Yamamoto Role of {gamma}{delta}T Cells in the Inflammatory Response of Experimental Colitis Mice J. Immunol., November 15, 2003; 171(10): 5507 - 5513. [Abstract] [Full Text] [PDF]
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,
Thy-1+ IEL; 
, Thy-1- IEL. IEL were
incubated first with anti-Thy-1.2 mAb (biotinylated) and then with
either streptavidin-PE and anti-
) mice was determined according to the technique described in
Materials and Methods. Data are the mean values from two
µm+/- mice and the mean ± SD from four
µm-/- mice
