A Novel Model of Inflammatory Bowel Disease: Mice Deficient for the Multiple Drug Resistance Gene, mdr1a, Spontaneously Develop Colitis

Mice

FVB control mice and mdr1a^−/− mice were initially purchased from Taconic Farms (Germantown, NY) and then bred and maintained in the SPF facility at Immunex (Seattle, WA) in accordance with approved ethical guidelines. mdr1a^−/− mice had been backcrossed onto the FVB strain for at least seven generations at Taconic Farms before shipping. Control and knockout mice used for experiments were age matched and received treatment simultaneously. Mice were monitored on alternate days for the presence of diarrhea and mucous discharge from the anus. All mice were bred and maintained under SPF conditions in Thoren isolation racks under positive pressure and fed autoclaved food and water ad libitum. The quality of the SPF facility is monitored through the Charles River Health Monitoring Plus program (Charles River, Wilmington MA) using the Charles River tracking profile. Sentinel mice receiving bedding from donor cages are tested for viral, parasite, and bacterial infection. There were no viruses, parasites, or pathogenic bacteria detected in our colony throughout the course of the study. Of particular note, we did not detect any evidence of Helicobacter infection (Helicobacter bilis, Helicobacter hepaticus, or Helicobacter spp.) in any mice.

Treatment of mice with oral antibiotics

Streptomycin sulfate (1.25% w/v), neomycin (1% w/v), bacitracin (1% w/v), and amphotericin (0.25% w/v) were dissolved in autoclaved drinking water (pH 2.5–2.8) supplemented with saccharine (1% w/v) and then filtered through a 0.22 μm Nalgene filter (Nalgene, Milwaukee, WI). As a control, mice received saccharine alone (1% w/v) in the drinking water. All reagents were obtained from Sigma (St. Louis, MO). Regular drinking water was replaced with antibiotic or saccharine drinking water during treatment.

Antibodies

The following primary Abs were used for flow cytometric analysis and immunoperoxidase histochemistry: anti-CD4 (rat IgG; L3T4), anti-CD8α (rat IgG; 53-6.7), anti-TCRαβ (hamster IgG; H57-597), anti-TCRγδ (hamster IgG; GL3), anti-GR1 (rat IgG; RB6–8C5), anti-B220 (CD45R) (rat IgG; RA3–6B2), and anti-CD3ε (hamster IgG; 500A2; Immunex). The primary Abs were conjugated either with biotin or R-phycoerythrin (PE). All Abs were obtained from PharMingen (San Diego, CA) except where indicated. The following secondary Abs were used for immunoperoxidase histochemistry: biotinylated sheep anti-rat IgG (Amersham, Arlington Heights, IL) and biotinylated goat anti-hamster IgG (Caltag, South San Francisco, CA).

Flow cytometric analysis of isolated cells

Isolated cells were incubated with the appropriate PE- or biotin-conjugated primary Ab for 30 min on ice in PBS supplemented with 5% BSA and 0.02% sodium azide essentially as described previously (35). Stained cells were washed two times, incubated with streptavidin-allophycocyanin (Molecular Probes, Eugene, OR), and then fixed in 1% paraformaldahyde before analysis. Stained cells were analyzed on a Becton Dickinson FACScan flow cytometer (Immunocytochemistry Systems, San Jose, CA) using Becton Dickinson Cellquest software.

Rhodamine efflux assay

Lymphocytes were isolated from mice and incubated with 0.4 μg/ml rhodamine 123 (Sigma) for 15 min at 37°C in complete RPMI 1640 supplemented with 10% FCS. For two-color analysis, cells were simultaneously stained with PE-conjugated primary Abs. The stained, rhodamine-loaded cells were then washed two times and incubated in complete medium without rhodamine 123 for an additional 2 h at 37°C. Cells were then washed two times in complete medium and analyzed on a Becton Dickinson FACScan flow cytometer (Immunocytochemistry Systems) using Becton Dickinson Cellquest software.

Immunoperoxidase histochemistry analysis of frozen sections

Large intestines were removed, embedded in OCT compound (Tissue-Tek, Torrence, CA), frozen in a dry ice/ethanol bath, and stored at −80°C until use. Frozen sections (5 μm) of intestine were cut, fixed for 15 min in acetone, and stored at −80°C until staining. Peroxidase staining was performed using the Vectastain ABC system (Vector Laboratories, Burlingame, CA) according to specified directions as described previously (35). Briefly, tissue sections were preincubated in 5% goat serum, 5% bovine serum, and 1% mouse serum for 30 min at room temperature and then incubated with biotin-conjugated primary Ab for 1 h at room temperature. Slides were washed three times in Tris-buffered saline, further incubated with a biotin-conjugated secondary Ab for 30 min at room temperature, and washed again three times in Tris-buffered saline. Sections were then incubated with avidin peroxidase (Vectastain ABC system), washed, and finally incubated with diaminobenzidine substrate (Sigma) before counterstaining in hematoxylin. Positive cells were identified by the presence of a brown reaction product.

Immunofluorescence analysis of frozen sections

Tissues were frozen in liquid nitrogen as described above, and 5-μm cryosections were cut, air dried, and acetone fixed before staining. Tissue sections from both mdr1a^−/− and control FVB mice were stained with either 20 μg/ml FITC-conjugated anti-MDR mAb (C219; Signet Laboratories, Dedham, MA) or FITC-conjugated IgG2a isotype control mAb (PharMingen) in the presence of 2 mM TOTO-3 (Molecular Probes) and 30% mouse serum. Sections were visualized using a confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA).

Intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) cell isolations

IEL were isolated according to a modification of published procedures (35, 36). Briefly, the small and large intestines were removed and washed in Ca²⁺/Mg²⁺-free HBSS (Life Technologies, Bethesda, MD). Peyer’s patches were removed, and the intestines were opened longitudinally, cut into 0.5-cm pieces, and placed in 1 mM EDTA in Ca²⁺/Mg²⁺-free HBSS at 37°C for three sequential 15-min incubations with intermittent vortexing to remove epithelial cells and IEL. Supernatants were collected and pooled for IEL purification. The remaining tissues were then washed in PBS and incubated in trypsin-EDTA two times to remove any residual epithelial cells, and the denuded tissues were digested with 90 U/ml collagenase type VIII (Sigma) in RPMI 1640/10% FCS for three sequential 20-min incubations at 37°C with intermittent vortex mixing to release LPL. Supernatants were collected and pooled for LPL purification. IEL and LPL were purified by passing cells over a prewet glass wool column before spinning them through a 40%/80% discontinuous Percoll gradient (Pharmacia, Uppsala, Sweden) as previously described (35). The cells at the 40%/80% interface were collected, washed, and resuspended in complete medium for analysis.

Intestinal bacterial Ag preparation

Intestinal bacterial Ags were prepared from fecal contents harvested from the cecum according to a modification of published procedures (37). Briefly, individual preparations were processed from three different mouse sources: 1) an FVB control mouse, 2) an mdr1a^−/− mouse with no signs of inflammation, and 3) an mdr1a^−/− mouse with active colitis. The cecal contents from the individual mice were placed in a 2-ml bead shaker tube half-filled with 2-μm zirconium beads suspended in PBS. The mixture was vortexed in a bead beater (Biospec Products, Bartlesville, OK) for 30 s at 5000 rpm and placed on ice. The intestinal bacterial slurry was then microfuged at 14,000 × g, and the supernatant was harvested and filtered through a 0.22 μm filter. The relative protein content in each sample was estimated using a Bradford assay (Bio-Rad, Hercules, CA).

Proliferation assay

Cell suspensions were prepared by gently mashing mesenteric lymph nodes (MLN) between ground glass slides and passing them through nylon mesh. MLN cells were cultured at a density of 2 × 10⁵ cells/well in 96-well flat-bottom plates in complete RPMI supplemented with 10% FCS in a final volume of 200 μl in a humidified 6% CO₂ incubator at 37°C for 48 h. Cells were cultured either in the presence of titrating amounts of intestinal bacterial Ag preparations, in the presence of 1 μg/ml Con A, or in anti-CD3 mAb-coated wells (1 μg/ml). Proliferation was assessed by addition of 1 μCi/well [³H]TdR 18 h before harvesting using a Matrix-96 cell harvester (Inotech, Lansing, MI). The amount of radioactivity incorporated into DNA was measured using a direct β counter (Packard, Meriden, CT). All cultures were performed in triplicate. Data are reported as mean cpm ± 1 SD of triplicate wells.

Serum Ab titers

Mice were exsanguinated, and the serum was collected. Polyclonal serum Ab titers were determined by isotype-specific ELISAs for IgA, IgE, IgG1, IgG2a, IgG2b, IgG3, and IgM as previously described (38). Briefly, 96-well Nunc Maxisorp plates (Nunc, Naperville, IL) were coated overnight at 4°C with isotype-specific anti-mouse Ig Abs (0.2 mg/well), blocked with PBS/10% FCS for 2 h at room temperature, and washed with PBS/0.1% Tween-20. Serum samples and control samples were serially diluted in PBS/10% FCS starting at 1:100. Plates were incubated for 2 h at room temperature, washed, and incubated with the appropriate peroxidase-conjugated specific anti-isotype detecting Ab for an additional 2 h at room temperature. Plates were washed again, and enzyme activity was detected using TMB microwell peroxidase substrate reagents (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and 2 N H₂SO₄. The amount of reaction product was assessed on an ELISA plate reader at OD 450 nm using the Deltasoft program (DeltaPoint, Monterey, CA). The affinity-purified Abs used for determining isotype-specific serum Ab titers by ELISA were obtained from Southern Biotechnology Associates (Birmingham, AL), except for anti-IgE, which was obtained from Serotec (Oxford, U.K.), and antiIgG2a, which was obtained from PharMingen. Ig concentrations were determined by comparing test sample dilution curves with known concentrations of isotype controls. Results are indicated as mean concentration ± 1 SD of two separate mice for each group.

Bone marrow chimeric mice

mdr1a^−/− mice and FVB mice were irradiated with 10 Gy using a ¹³⁷Cs source, rested for 4–6 h, and then reconstituted by i.v. injection with 2 × 10⁶ bone marrow cells collected from the femurs of appropriate donors.

Bacterial identification

mdr1a is functionally expressed by mucosal lymphocytes and is on the apical surface of colonic epithelial cells

Efflux of rhodamine 123 from preloaded lymphocytes has been used to demonstrate the presence of mdr activity in peripheral T cells (19, 30). However, the assay does not distinguish whether rhodamine efflux is due to mdr1a activity, mdr3 activity, or perhaps even another unidentified transporter. We therefore analyzed rhodamine 123 efflux in lymphocytes from normal FVB mice and mdr1a^−/− mice to determine whether rhodamine 123 efflux was associated with a functional mdr1a gene and to identify which mucosal and peripheral lymphocyte subsets might exhibit the pump activity. mdr1a-associated functional activity was detected in MLN cells from normal FVB mice but not from mdr1a^−/− mice, as determined by ability to efflux rhodamine 123 (Fig. 1⇓A). These data indicate that a functional mdr1a gene product is necessary for rhodamine 123 efflux. To determine which subsets of mucosal lymphocytes have the mdr1a activity, we performed the rhodamine efflux assay on IEL and LPL isolated from the large intestines of FVB mice. We simultaneously stained these cells with mAbs against CD3, CD4, CD8, TCRαβ, and TCRγδ (Fig. 1⇓B). The individual CD3, CD4, CD8, TCRαβ, and TCRγδ subsets within the IEL and LPL compartments had similar efflux profiles. Virtually all of the CD8⁺ cells and TCRγδ⁺ cells appeared to exhibit mdr activity and efflux rhodamine 123, whereas only half of the CD4⁺ cells and half of the TCRαβ⁺ cells were able to efflux the dye. None of the mucosal lymphocyte populations from the mdr1a^−/− mice were able to efflux the rhodamine 123 (data not shown), further suggesting that mdr1a is necessary for rhodamine 123 efflux activity in mucosal lymphocytes.

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FIGURE 1.

Mucosal lymphocytes from FVB mice, but not mdr1a^−/− mice, exhibit mdr1a functional activity. MLN cells (A) and mucosal IEL and LPL (B) were loaded with rhodamine 123, simultaneously stained with PE-conjugated mAb (to CD3, CD4, CD8, TCRαβ, or TCRγδ), and then allowed to efflux rhodamine for 2 h. Cells were then analyzed by flow cytometry, and mdr1a activity was determined by decreased rhodamine intensity. Axes show log₁₀ fluorescence intensity measured at 525 nm for rhodamine 123 and 575 nm for PE after excitation with a 488-nm argon laser. The FACS profiles represent data from one of two separate experiments.

Immunofluorescent staining was used to localize mdr1a expression in the colon. Fig. 2⇓ shows mdr1a staining (green) on transverse sections of colons from FVB control (+/+) and mdr1a-deficient (−/−) mice. The sections were counterstained with TOTO-3 (red) to visualize gut architecture. mdr1a staining appears to be restricted to the apical surface of epithelial cells in FVB mice. No mdr1a staining was apparent on sections from mdr1a^−/− mice or on sections from FVB mice stained with an isotype control (data not shown). We were also unable to detect any mdr1a staining on LPL or on IEL in FVB mice, despite functional evidence that these cells express mdr1a (see above). It is unclear whether this was due to low expression levels of mdr1a on mucosal lymphocytes or to the relatively low frequency of lymphocytes within each section.

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FIGURE 2.

mdr1a is expressed on the apical surface of the intestinal epithelial cells in the colon. Frozen sections of large intestine from FVB control mice (+/+) and mdr1a-deficient mice (−/−) were stained for mdr1a expression with anti-mdr FITC mAb C219 (green) and counterstained with TOTO-3 (red). The scale bars represent 20 μm. The staining shown is representative of two mice analyzed. Similar staining was seen in two separate experiments.

Clinical characteristics and incidence of colitis in mdr1a^−/− mice

Approximately 20–25% of the 169 mdr1a knockout mice included in this part of the study appeared to develop loose stools and anal mucous discharge by 1 year of age (Table I⇓). Histologic analysis of mice with clinical signs of colitis confirmed the presence of active intestinal inflammation that was primarily restricted to the large intestine. The difference in incidence between males (20%) and females (26%) was not significant. The average age at onset was approximately 20 wk, although the first signs of colitis development could be seen in mice ranging between 8 and 36 wk of age. Although some mice with colitis developed a wasting-type disease, the majority of colitic animals were maintained for up to 3 mo without signs of cachexia. Disease was nontransmissible, since mdr1a^−/− mice both with and without colitis were housed in the same cage. We observed no clinical or histologic signs of colitis in any FVB control mice, heterozygous mdr1a^+/− mice, or other immunocompetent mouse strains of any age housed within the facility.

Histopathologic changes associated with colitis in mdr1a^−/− mice

The severe inflammation of the large intestine that develops in mdr1a^−/− mice bears resemblance to the human IBD, ulcerative colitis. Histopathologic analysis of multiple colitic mdr1a^−/− animals revealed inflammation spread along the entire length of the colon. The intestinal inflammation was characterized by a massive thickening of the mucosa and evidence of inflammatory cell infiltrates into the lamina propria in mdr1a^−/− mice with active colitis (Fig. 3⇓a) compared with mdr1a^−/− mice without colitis (Fig. 3⇓b). Occasional crypt abscesses and ulcerations extending through the mucosa to the muscular layer were also often noted in the colitic animals (Fig. 3⇓c). Crypt length was greatly increased in colitic animals, with approximately three- to fivefold more epithelial cells per crypt in colitic mice (Fig. 3⇓a) compared with noncolitic mice (Fig. 3⇓b). There also appeared to be some evidence of dysregulated epithelial cell growth in colitic mdr1a^−/− mice in areas of inflammation (Fig. 3⇓d).

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FIGURE 3.

mdr1a^−/− mice develop severe spontaneous inflammation of the large intestine. Frozen sections stained with hematoxylin and eosin revealed a massive thickening of the colonic tissues when mdr1a^−/− mice with established colitis (a) were compared with mdr1a^−/− mice without colitis (b); original magnification, ×63.5. c, Crypt abscesses and ulcers extending through the mucosa to the muscularis were prevalent in many sections of intestine from mdr1a^−/− mice with active colitis (original magnification, ×250). d, Evidence of dysregulated epithelial cell growth in colitic mice (original magnification, ×500). a–d are from individual mice and are representative of multiple tissue sections analyzed from >15 mice per group.

Immunohistochemical analysis of the gastrointestinal tissues of animals with prevalent colitis or animals with early stages of colitis was used to further characterize the lymphocytic infiltrate into the mucosa of the large intestine compared with control FVB mice (Fig. 4⇓). There was an increased number of infiltrating CD3⁺ T cells scattered diffusely throughout the lamina propria of inflamed colonic tissue from mice with active colitis (Fig. 4⇓a) compared with colonic tissue from control FVB mice (Fig. 4⇓c). A less dramatic CD3⁺ T cell infiltrate was observed in mice with early colitis (Fig. 4⇓b).

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FIGURE 4.

mdr1a^−/− mice with established colitis have extensive T cell, B cell, and granulocyte infiltrates into the colonic lamina propria in areas of intestinal inflammation. Frozen sections of colon from mdr1a^−/− mice with active colitis (a, d, and g), mdr1a^−/− mice with early/intermediate colitis (b, e, and h) and control FVB mice (c, f, and i) were stained by the immunoperoxidase technique for expression of CD3 (a–c), B220 (d–f), and Gr1 (g–i). Positive staining is indicated by the presence of a brown reaction product. Note a dramatic increase in the number of CD3⁺ T cells in the lamina propria of mdr1a^−/− mice with active colitis (a) compared with early/intermediate colitic mdr1a^−/− (b) and FVB (c) mice. B220 staining revealed the presence of B cells in follicular clusters in mdr1a^−/− mice with active colitis (d). mdr1a^−/− mice with early/intermediate colitis (e) did not have defined B cell clusters but had more lamina propria B cells than FVB mice (f). Increased frequencies of Gr1⁺ cells could be seen aggregating in areas of inflammation in mdr1a^−/− mice (g), particularly where breaches in the epithelium were apparent. Few Gr1⁺ cells were present in the lamina propria of mdr1a^−/− mice with early/intermediate colitis (h) or FVB mice (i). Sections are representative of data from two colitic mice, one early/intermediate colitic animal, and five control animals. At least three regions of intestine from the proximal to the distal end of each individual colon was analyzed.

FACS analysis of the large-intestine IEL and LPL isolated from colitic animals revealed that the majority of the infiltrating T cells were CD4⁺TCRαβ⁺ (Table II⇓). Although there was an apparent decrease in the relative proportion of CD8⁺ T cells in the lamina propria of colitic animals, the absolute number of CD8⁺TCRαβ⁺ T cells was not diminished, probably owing to the fourfold increase in the total number of LPL isolated from colitic animals (data not shown). There was no apparent increase in TCRγδ⁺ T cells in the inflamed tissue (data not shown).

In addition to T cell infiltrates, immunohistochemical analysis also revealed the presence of numerous B220⁺ B cell clusters/follicles within the lamina propria of animals with active colitis (Fig. 4⇑d) compared with control FVB animals (Fig. 4⇑f). Whereas the T cells were diffusely scattered throughout the lamina propria, the B cell infiltrates appeared to be localized to follicular clusters within the mucosa. The follicular clusters varied in size and were only observed in the colon. In contrast to animals with fully developed colitis, animals with intermediate colitis mostly had individual B220⁺ cells scattered throughout the lamina propria and the few clusters that were present were reduced in size (Fig. 4⇑e).

Gr1⁺ cellular infiltrates were also prominent throughout the lamina propria of the large intestine in mice with active colitis (Fig. 4⇑g) compared with control FVB mice, which appeared devoid of Gr1⁺ cells (Fig. 4⇑i). The Gr1⁺ infiltrates were uniformly present throughout the lamina propria, although an increased number of cells did appear to be migrating across the epithelium into the gut lumen in regions where the epithelialbarrier may have been compromised (Fig. 4⇑g). Mice with early colitis had relatively few Gr1⁺ infiltrating cells, and these were restricted to small focal regions in the lamina propria (Fig. 4⇑h).

Prophylactic treatment with oral antibiotics can prevent spontaneous colitis in mdr1a^−/− mice

It has been well established in a majority of mouse models that spontaneous colitis can be prevented if mice are housed under germfree conditions, suggesting that the presence of the gut flora is necessary for the development of colitis (4, 12, 14). It has also been previously demonstrated in another model of spontaneous colitis that treating mice with antibiotics can prevent inflammation (13). In the first part of the study, we treated mdr1a^−/− mice with broad-spectrum oral antibiotics to eliminate the commensal gut flora and then monitored for the development of colitis. Control mice were given water treated with saccharine alone. None of the mice had detectable colitis at the beginning of the study.

The incidence of colitis in mdr1a^−/− mice treated with antibiotics was greatly reduced compared with mdr1a^−/− mice treated with saccharine alone (Fig. 5⇓). In the 16-wk treatment, 14 of 31 saccharine-treated mdr1a^−/− mice developed colitis (loose stools and mucous discharge), whereas only 1 of 39 antibiotic-treated mdr1a^−/− mice developed any signs of inflammation (no clinical signs, histopathologic evidence only). The one antibiotic-treated mdr1a^−/− mouse that showed signs of colitis had only one minor focal region of inflammatory cell accumulation (data not shown) and did not show any other overt signs of intestinal disease.

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FIGURE 5.

Treating mdr1a^−/− mice with oral antibiotics can prevent the onset of colitis. Seventy mdr1a^−/− mice with no visible signs of colitis were selected for the study. Regular drinking water was replaced with antibiotics and saccharine dissolved in drinking water (n = 31) or saccharine alone dissolved in drinking water (n = 39). Mice were monitored on alternate days for clinical signs of colitis for up to 16 wk. Mice were then killed and analyzed for histopathologic signs of colitis. Data represent the percentage of mice with clinical or histopathologic evidence of colitis.

Therapeutic antibiotic treatment can reverse ongoing active inflammation in mdr1a^−/− mice

To ascertain whether the intestinal inflammation in mice with active colitis could be reversed by antibiotic treatment, mice with active colitis (as determined by evidence of loose stools and mucous discharge from the anus) were therapeutically treated with antibiotics in the drinking water. Within 3 wk of continuous antibiotic therapy, the previously colitic mice had no residual visible signs of anal discharge or loose stools. To determine whether the pathology associated with colitis could also be reversed along with the gross symptoms, mice treated with antibiotics for 10 wk were killed and the colons analyzed histopathologically. As expected, the colons of untreated colitic animals were grossly thickened (Fig. 6⇓a), compared with control animals (Fig. 6⇓b). Interestingly, the gross thickening of the colon associated with active IBD was significantly reduced in mice that had received therapeutic antibiotic treatment (Fig. 6⇓c). On further histopathologic examination of these intestines, we observed little evidence of inflammation, and the general gut architecture appeared similar to that of control mice (data not shown). Immunohistochemical analysis revealed that the mice treated with antibiotics had no evidence of persisting Gr1⁺ infiltrates or B cell follicles (data not shown). However, despite the apparent reduction in inflammation arising from antibiotic treatment, there still appeared to be an increased number of CD3⁺ T cells in the lamina propria of these animals (Fig. 6⇓d) compared with control mice (Fig. 4⇑c).

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FIGURE 6.

Therapeutic antibiotic treatment can reverse the gut inflammation associated with established colitis in mdr1a^−/− mice. Mice with active signs of colitis (loose stools and anal discharge) were placed on antibiotic treatment for 10 wk (n = 3). At the end of the treatment period, colons were removed from untreated mdr1a^−/− mice with active colitis (a), control FVB mice (b), and mdr1a^−/− mice therapeutically treated with antibiotics (c). Note that the colon from the antibiotic-treated mouse does not appear inflamed and shows little residual evidence of thickening. d, Immunohistochemical analysis of colonic tissues from mice treated with antibiotics showed persistence of CD3⁺ T cells despite significant reduction in inflammation.

Analysis of bacterial flora in colitic and control mice

Although the commensal bacterial flora resident in the intestinal lumen of the gut appears to play a major role in the induction of colitis, little is known about the role of individual bacteria in the initiation and perpetuation of intestinal inflammation. In this study we analyzed the bacterial flora present in the cecum and colon of mdr1a^−/− mice with colitis, mdr1a^−/− mice with no evidence of active colitis, and control FVB mice. Table III⇓ lists the major bacterial species recovered from the animals. All of the mice appeared to have a gut bacterial profile devoid of any pathogenic organisms.

Recently, it has been shown that Helicobacter infection in immunocompromised mice can give rise to a pathology resembling colitis (39, 40, 41). To ensure that colitis in the mdr1a^−/− mice was not due to Helicobacter infection, samples from colitic, noncolitic, and control mice were analyzed by PCR for the presence of H. bilis, H. hepaticus, and Helicobacter spp. All mice were negative (data not shown).

In vitro proliferative reactivity of cells from the gut-associated lymphoid tissue following stimulation with intestinal bacterial Ags

Since bacterial colonization of the intestine appears to be crucial for the initiation of spontaneous colitis in mdr1a^−/− mice, we analyzed the in vitro proliferative response of cells isolated from the MLNs following stimulation with intestinal Ags (Fig. 7⇓). Intestinal Ags were prepared from cecal contents obtained from FVB control mice, mdr1a^−/− mice with no overt signs of colitis, and mdr1a^−/− mice with active colitis. MLN were removed from the same animals, and cell suspensions were tested for proliferative reactivity against each of the intestinal bacterial Ag preparations.

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FIGURE 7.

Lymphocytes from colitic mdr1a^−/− mice have enhanced proliferative reactivity to bacterial Ags. Cells were prepared from the MLN of mdr1a^−/− mice with active colitis (mdr1a^−/−, colitis), mdr1a^−/− mice with no signs of colitis (mdr1a^−/−), and age-matched FVB mice (FVB) and assayed for proliferative reactivity to intestinal bacterial Ag preparations (a) or Con A and anti-CD3 mAb (b) as described in Materials and Methods. a, Cells from mdr1a^−/− mice with active colitis demonstrated enhanced proliferative reactivity to the bacterial Ags (p < 0.05, Student’s t test) compared with other mice. Cells from mdr1a^−/− mice without colitis did not differ significantly from cells from FVB mice. b, Cells from all mice responded similarly following stimulation with Con A or anti-CD3. Values are expressed as mean cpm ± 1 SD of triplicate wells. The data shown are from one experiment and are representative of three similar experiments.

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FIGURE 8.

Serum Ab titers are elevated in mdr1a^−/− mice with active colitis. Isotype-specific ELISAs were performed on serum from mdr1a^−/− mice with active colitis (mdr1a^−/−, colitis), mdr1a^−/− mice without active colitis (mdr1a^−/−), and control FVB animals (FVB). Colitic mice had an increase in serum Ab titers of all isotypes compared with noncolitic and control mice. Serum Ab titers were similar in noncolitic and control mice. Values are expressed as μg/ml for each isotype. Data represent the mean plus the SD of two separate animals analyzed for each experimental group.

We observed a significant increase in proliferation in response to bacterial Ags when MLN from mdr1a^−/− mice with active colitis were compared with those of other mice (p < 0.05). In contrast, there was no statistically significant difference in the response to bacterial Ag stimulation when MLN from FVB mice were compared with MLN from noncolitic mdr1a^−/− mice (p > 0.1). Reactivity profiles of each source of MLN were similar regardless of the source of bacterial Ags (data not shown). No significant differences were noted in the proliferative responses to Con A or anti-CD3 between any of the three groups of mice analyzed in these experiments (p > 0.1). The data shown are from one experiment and are representative of three similar experiments.

mdr1a^−/− mice with active colitis have increased serum Ab titers

The increased frequency of B cell clusters in the lamina propria of colitic animals suggests that there may be a dysregulated humoral response in these animals. To test this, we measured the serum Ab titers of colitic, noncolitic, and control animals. Each of the Ig isotype levels was assessed by ELISA. All of the serum Ig isotype levels were elevated in colitic animals compared with control FVB mice or mdr1a^−/− mice without colitis, suggesting polyclonal activation of B cells associated with colitis. Serum IgA and IgG1 levels appeared most elevated in the mice with active colitis, although IgG2a, IgG2b, IgM, and IgE levels were also increased over control mice (Fig. 8).

Spontaneous colitis correlates with a lack of mdr1a expression on intestinal epithelial cells

Given that mdr1a is expressed on both lymphocytes and epithelial cells in the large intestine, we sought to determine whether the spontaneous development of colitis was the result of an mdr1a defect within the lymphoid or within the epithelial cell population. We generated irradiation bone marrow chimeras in which irradiated FVB animals were reconstituted with bone marrow from mdr1a^−/− or FVB donors, or irradiated mdr1a^−/− mice were reconstituted with bone marrow from FVB or mdr1a^−/− donors. As controls, additional groups of mice were generated in which FVB mice were reconstituted with FVB bone marrow and mdr1a^−/− mice were reconstituted with mdr1a^−/− bone marrow. The incidences of colitis in the groups are listed in Table IV⇓. mdr1a^−/− mice that were reconstituted with wild-type bone marrow developed signs of colitis within 12 wk after reconstitution. After being maintained for 52 wk, 25% of the mice had developed active colitis. In contrast, two groups of FVB mice, which were reconstituted with mdr1a^−/− bone marrow, were maintained for up to 38 wk without any overt signs of colitis. As expected, the control FVB mice reconstituted with FVB bone marrow did not develop colitis. Surprisingly, 100% of the mdr^−/− mice reconstituted with mdr1a^−/− bone marrow developed colitis. The reason for the increased incidence in the latter group compared with unmanipulated mdr1a^−/− mice is unclear, although it is possible that radiation damage to the mucosa sufficiently enhanced susceptibility to developing colitis.