Homeostatic Regulation of Intestinal Villous Epithelia by B Lymphocytes

Mice

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-δ mutant (δ^−/−) mice (9) (backcrossed 10 times to B6 parent) and TCR-β mutant (β^−/−) 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, δ^−/−, β^−/− and δ × β^−/− 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-αβ mAb (H57-597; BD PharMingen, San Diego, CA), anti-γδ mAb (GL3; BD PharMingen), anti-Thy-1.2 mAb (30-H12; BD Biosciences, San Jose, CA), anti-CD4 mAb (GK1.5; BD PharMingen), anti-CD8α mAb (53-6.7; BD PharMingen), and anti-CD8β mAb (53-5.8) (BD PharMingen). We also used anti-I-A^b mAb (AF6–120.1; BD PharMingen), anti-I-A^d 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-FcRγII/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 × 10^{3 51}Cr-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-αβ TCR mAb (H57-597; BD PharMingen), or 1 μg/ml anti-γδ TCR mAb (GL3; BD PharMingen) in 75 μl complete medium (RPMI 1640 containing 10% FCS, 10 mM HEPES, 5 × 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) × 100.

Generation of IEC is increased and expression of MHC class II molecules on IEC is up-regulated in μm^−/− mice that lack B cells

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-A^b (MHC class II) molecules on IEC were substantially increased in μm^−/− mice compared with B6 and μm^+/− mice (Fig. 1⇓C).

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FIGURE 1.

Generation and differentiation of IEC are up-regulated in μm^−/− mice. Three independent experiments on the small intestine of B6 (n = 7), μm^+/− (n = 10), and μm^−/− (n = 10) mice were performed. A, Representative immunohistochemical visualization of BrdU-incorporated IEC (magnification, ×100). B, Number of BrdU-incorporated IEC per one villous section ± SD. The p value was determined using Student’s t test. C, Representative immunofluorescence profile of MHC class II molecules on IEC.

Development and cytolytic activity of IEL in μm^−/− mice that lack B cells

In the mouse small intestine numerous IEL expressing either TCR-αβ (αβ-IEL) or TCR-γδ (γδ-IEL) reside above the basement membrane together with the columnar IEC. IEL are unusual among mouse peripheral T cells in that most γδ-IEL and many αβ-IEL, unlike thymus-derived T cells, express a unique CD8αα homodimer (14) instead of a CD8αβ 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 αβ- and γδ-IEL were not impaired in the μm^−/− condition (Fig. 2⇓). On the contrary, while absolute numbers of γδ-IEL were not altered in μm^−/− mice (2.5 ± 0.4 × 10⁶; n = 8) compared with those in μm^+/− mice (2.7 ± 0.5 × 10⁶; n = 7), the population size of αβ-IEL in μm^−/− mice (4.4 ± 1.3 × 10⁶; n = 8) was about two times larger than that in μm^+/− mice (2.5 ± 0.4 × 10⁶; n = 7). Interestingly, this increase in number was caused by the selective expansion of CD8αβ⁺ αβ-IEL subset (Fig. 2⇓B), although further studies are required before we evaluate the mechanism and functional significance of the expansion we observed.

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FIGURE 2.

Composition and cytolytic activity of αβ- and γδ-IEL recovered from μm^+/− and μm^−/− mice. A, Composition (percentage) of Thy-1⁺ and Thy-1⁻ subsets in αβ- and γδ-IEL from μm^+/− (n = 7) and μm^−/− (n = 8) mice. □, 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-αβ TCR mAb (FITC-conjugated) or streptavidin-PE and anti-γδ TCR mAb (FITC-conjugated). B, Composition of various αβ- and γδ-IEL subsets from μm^+/− (n = 7) and μm^−/− (n = 8) mice. IEL were incubated first with anti-γδ mAb (biotinylated) and then with streptavidin-PE and anti-αβ TCR mAb (FITC-conjugated). IEL were incubated first with anti-CD4 mAbs (PE-conjugated), anti-CD8α mAb (biotinylated), or anti-CD8β mAbs (PE-conjugated) and then with either streptavidin-PE (only in the case of biotinylated anti-CD8α mAb) and anti-αβ TCR mAb (FITC-conjugated) or streptavidin-PE (only in the case of biotinylated anti-CD8α mAb) and anti-γδ TCR mAb (FITC-conjugated). IEL were incubated with a mixture of PE-conjugated anti-CD4 mAb and FITC-conjugated anti-CD8α mAb and also with a mixture of PE-conjugated anti-CD4 mAb and FITC-conjugated anti-CD8β mAb. For FACScan analysis, dead cells were excluded by PI gating. The quadrantal analysis using FACScan with CellQuest software revealed that the CD4⁺TCR-αβ⁻, CD4⁺TCR-γδ⁺, or CD4⁺CD8β⁺ IEL subset was negligible (data not shown). Thus, CD4⁺CD8⁺ IEL are αβ-IEL expressing CD4 and CD8αα molecules. The composition (percentage) of CD8αα⁺ αβ-IEL was calculated using the following equation: % CD8α⁺ αβ IEL − (% CD8β⁺ αβ-IEL plus % CD4⁺CD8⁺ αβ-IEL); and that of CD4⁺ αβ-IEL was calculated using the following equation: % CD4⁺ αβ-IEL − % CD4⁺CD8⁺ αβ-IEL. Likewise, the composition (percentage) of CD8αα⁺ γδ-IEL was calculated using the following equation: % CD8α⁺ γδ-IEL − % CD8β⁺ γδ-IEL; and that of CD4⁻CD8⁻ γδ-IEL was calculated using the following equation: % γδ-IEL − % CD8α⁺ γδ-IEL. C, Cytolytic activity of αβ- and γδ-IEL in μm^+/− (•) and μm^−/− (○) 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

Generation of IEC is increased and expression of MHC class II molecules on IEC is up-regulated in RAG-2^−/− and SCID mice that lack both B and T cells

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. 3⇓A), the number of BrdU-positive IEC (Fig. 3⇓B), and the expression of I-A^d (MHC class II) molecules on IEC (Fig. 3⇓C) 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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FIGURE 3.

Generation and differentiation of IEC are up-regulated in RAG-2^−/− and SCID mice. Three independent experiments on the small intestine of RAG-2^+/− (n = 9), RAG-2^−/− (n = 10), CB-17 (n = 7), and SCID (n = 9) mice were performed. A, Representative immunohistochemical visualization of BrdU-incorporated IEC (magnification, ×100). B, Number of BrdU-incorporated IEC per one villous section ± SD. The p values were determined using Student’s t test. C, Representative immunofluorescence profile of MHC class II molecules on IEC.

Generation of IEC and expression of MHC class II molecules on IEC are not altered in δ × β^−/− mice that lack T cells

We (4) have reported that the absence of γδ, but not αβ, 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 δ^+/− (WT) and δ^−/− littermates from an intercross between δ^+/− and δ^−/− mice and β^+/− (WT) and β^−/− littermates from an intercross between β^+/− and β^−/− mice. We also analyzed several WT, δ^−/−, and β^−/− mice as well as two δ × β^−/− double-mutant littermates obtained from the F₂ generation of an intercross between δ^−/− and β^−/− 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 δ × β^−/− mice. With these previous observations in mind, we reinvestigated the intestinal epithelia of δ × β^−/− double-mutant mice that lacked γδ and αβ T cells. Because we now have δ^−/− and β^−/− mice that have been backcrossed more than eight times to the B6 parent, we intercrossed these two mutant strains of mice to obtain WT, δ^−/−, β^−/−, and δ × β^−/− littermates and extensively examined the effects of T cell deprivation on the generation and differentiation of IEC.

The absence of γδ T cells in δ^−/− 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. 4⇓C). No such effects were observed in β^−/− mice that lacked αβ T cells (Fig. 4⇓). Obviously, these results were in line with our earlier observations (4). However, in T cell-deficient δ × β^−/− 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 β^−/− 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 δ^−/− mice is cancelled by the simultaneous disappearance of αβ T cells in δ × β^−/− mice.

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FIGURE 4.

Generation and differentiation of IEC are down-regulated only in δ^−/− mice among WT, δ^−/−, β^−/−, and δ × β^−/− littermates. Four independent experiments on the small intestine of WT (n = 10), δ^−/− (n = 12), β^−/− (n = 9), and δ × β^−/− (n = 12) mice were performed. A, Representative immunohistochemical visualization of BrdU-incorporated IEC (magnification, ×100). B, Number of BrdU-incorporated IEC per one villous section ± SD. The p value was determined using Student’s t test. C, Representative immunofluorescence profile of MHC class II molecules on IEC.

Generation of IEC is increased and MHC class II expression on IEC is up-regulated to some extent in aly/aly mutant mice

The aly/aly mice (8), which carry a homozygous recessive point mutation in the nuclear factor κB-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. 5⇓C) in aly/aly mice compared with WT aly/+ mice.

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FIGURE 5.

Generation and differentiation of IEC are up-regulated in aly/aly mice. Three independent experiments on the small intestine of aly/+ (n = 9) and aly/aly (n = 9) mice were performed. A, Representative immunohistochemical visualization of BrdU-incorporated IEC (magnification, ×100). B, Number of BrdU-incorporated IEC per one villous section ± SD. The p value was determined using Student’s t test. C, Representative immunofluorescence profile of MHC class II molecules on IEC.

Goblet cells in villous epithelia develop normally in the absence of B and/or T cells

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. 6⇓A). This impression was confirmed by counting the number of goblet cells in the small intestinal villi of these various strains of mice (Fig. 6⇓B).

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FIGURE 6.

Goblet cells develop normally in the absence of B and/or T cells. A, Detection of goblet cells in H&E-stained distal jejunal tissues of the small intestine from various mice (magnification, ×200). Arrows indicate the representative goblet cells. B, Number of goblet cells per 10 villous sections ± SD. Three arbitrary jejunal and two arbitrary ileal fields of the small intestine from each mouse were photographed. Ten representative villi in each field were chosen from three mice (150 villi in total), and goblet cells were enumerated.

Effect of antibiotics on the generation of IEC in μm^−/− and their WT μm^+/− littermate mice

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-A^b 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. 7⇓C). 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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FIGURE 7.

The acceleration of IEC turnover is not observed in μm^−/− mice treated with antibiotics. Two independent experiments on the small intestine of μm^−/− mice given antibiotics (AB +; n = 6) and not given antibiotics (AB −; n = 6), and μm^+/− mice given antibiotics (AB +; n = 5) and not given antibiotics (AB −; n = 5) were performed. A, Representative immunohistochemical visualization of BrdU-incorporated IEC (magnification, ×100). B, Number of BrdU-incorporated IEC per one villous section ± SD. The p value was determined using Student’s t test. C, Representative immunofluorescence profile of MHC class II molecules on IEC.