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Lymphocyte recruitment to intestinal tissues depends on β7 integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn’s disease. To assess the role of β7 integrins in chronic ileitis, we generated SAMP1/YitFc lacking β7 integrins (SAMP1/YitFc Itgb7−/−) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7−/− mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20–50 wk) SAMP1/YitFc Itgb7−/− mice, ileitis was reduced by 30–50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7−/− mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7−/− mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7−/− MLN B cells along with CD4+ T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that β7 integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.
Inflammatory bowel diseases (IBDs), represented mainly by Crohn’s disease (CD) and ulcerative colitis, are chronic inflammatory disorders of the intestine. The etiology of these diseases is thought to involve increased host genetic susceptibility, immune system dysregulation, and altered interactions of host cells with pathogens and normal flora within the intestinal mucosa (1, 2). Dysregulated recruitment of lymphocytes into inflamed intestinal tissues has been implicated in the pathogenesis of IBD (2–5).
The trafficking of lymphocytes to inductive sites of the GALTs, such as Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs), and diffuse effector sites, such as the epithelium and lamina propria (LP), require their orchestrated adhesion to vascular endothelium and migration through the blood vessel walls (6–8). These processes follow a sequential engagement of cell adhesion molecules and chemokines with their receptors (9–11). The efficient homing and retention of lymphocytes to the gut is dependent on the β7 integrins, the chemokine CCL25, and its receptor CCR9 (8, 12–15). The β7 integrin subunit can pair with α4 (CD49d) or αE (CD103) subunits. α4β7 integrin binds mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is constitutively expressed on high endothelial venules of PPs and MLNs and on postcapillary venules of gut LP (12). Under inflammatory conditions, MAdCAM-1 expression is increased and more widespread (16). Both naive and effector T lymphocytes use α4β7 integrin to extravasate from the blood to gut mucosal tissues via interactions with MAdCAM-1 (6, 12, 14, 17). B cells also use α4β7/MAdCAM-1 pathways to migrate to PPs and MLNs, where they proliferate and differentiate into IgA-secreting plasma cells (PCs), which then home to intestinal LP (8, 14, 18). Thus, β7 integrin-deficient mice exhibit a severely reduced PP cellularity, decreased lymphocyte adhesion to PP high endothelial venules, and significantly reduced LP lymphocytes (14), as well as a defective specific IgA Ab response to rotavirus (19). Similarly, MAdCAM-1–deficient mice display a compromised migration of IgA-secreting PCs, resulting in a paucity of PCs in the LP and a defective intestinal IgA response following oral immunization with cholera toxin (18).
Despite a large body of knowledge on their role in homeostatic intestinal lymphocyte recruitment, the contribution of β7 integrins in the setting of intestinal inflammation remains controversial. In colitis of IL-2–deficient mice and in an adoptive transfer model of CD4+CD45Rbhigh cells into SCID mice, Sydora et al. (20) showed that β7 integrins are not required for T cell localization to the intestine and pathogenesis of colitis. However, using a similar adoptive transfer model of chronic colitis, reduced colitis was shown in mice reconstituted with CD4+CD45RBhigh T cells from β7 integrin-deficient mice and aberrantly activated α4β7 integrin knockin mice (D146A) compared with wild-type (WT) mice (21). mAbs to β7 integrins or MAdCAM-1 reduced colitis in the same adoptive transfer model (22). Discrepant roles of β7 integrins in the pathogenesis of ileitis have also been reported in two mouse models of chronic ileitis. Thus, genetic deletion of β7 integrin in the TNFΔARE model resulted in amelioration of the ileitis (23), whereas administration of anti-α4β7 Ab alone in SAMP1/YitFc model showed no effect on ileitis (24).
We recently reported expansion of MLN B cells in SAMP1/YitFc mice compared with WT mice (25). Adoptive transfer experiments showed that B cells contribute to the development of SAMP1/YitFc ileitis (25). The severity of ileitis in these mice correlates with the number of MLN B cells, and cotransfer of SAMP1/YitFc MLN B cells with CD4+ T cells increases ileitis severity in SCID mice compared with transfer of CD4+ T cells alone (25).
In the current study, we used the SAMP1/YitFc mouse, a model that develops spontaneous ileitis similar to human CD. We assessed the role of β7 integrins in the pathogenesis of ileitis by generating SAMP1/YitFc mice with a null mutation in the Itgb7 gene encoding the β7 integrin subunit. Our data demonstrate that β7 integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of B cells. β7 integrin-deficient B cells were unable to exacerbate disease in a combined T/B cell adoptive transfer model.
The SAMP1/YitFc substrain was developed at the University of Virginia (Charlottesville, VA) after >20 generations of continuous brother-sister mating of a parental colony of SAMP1/Yit mice kindly provided by S. Matsumoto (Yakult Central Institute for Microbiological Research, Tokyo, Japan) (26). SAMP1/Yit mice were originally derived from the AKR/J mouse strain purchased from The Jackson Laboratory (Bar Harbor, ME) after an unintentional outcross to an unknown strain (27). The SAMP1/YitFc Itgb7−/− mice were generated by crossing a null allele for β7 integrin from C57BL/6-Itgb7tm1Cgn/J donors into SAMP1/YitFc using microsatellite-based marker-assisted selection. C57BL/6J, C57BL/6J-CD45.1, AKR/J mice, and SCID mice on the C3H/HeJ background were obtained from The Jackson Laboratory. C57BL/6-Itgb7tm1Cgn/J breeders were provided by N. Wagner (Rheinisch-Westfaelische Technische Hochschule, Aachen University, Aachen, Germany) (14). All mice were kept under specific pathogen-free conditions. All animal procedures were approved by the Institutional Animal Care and Use Committees at the University of Virginia and La Jolla Institute for Allergy and Immunology (La Jolla, CA).
To generate the SAMP.B6-Itgb7tmCgn congenic strain, two potential C57BL/6-Itgb7tmCgn male founders were screened at 95 highly polymorphic microsatellite loci distributed throughout the genome, as previously described (28). We identified non-C57BL/6 background genetic markers at 17 loci on 11 chromosomes (data not shown), with heterozygosity at 15 of 17, indicating a recent outcross to another unidentified strain. These loci were used to select a polymorphic map for a cross to SAMP1/YitFc. Using 52 widely distributed microsatellite loci (Table I; genomic locations annotated using the National Center for Biotechnology Information m37), we generated a SAMP.B6-Itgb7tmCgn congenic strain by backcrossing donor males to SAMP1/YitFc females for four generations with genotypic selection. Five Itgb7−/− female and three male offspring were produced from three closely related pens from the fourth backcross to initiate breeding for the congenic strain. All potential breeders were con-firmed to carry homozygous SAMP1/YitFc alleles at the test loci, with the exception of the interval linked to Itgb7 on chromosome 15. Using a panel of six informative microsatellites on chromosome 15 (Table I), we determined that all were homozygous for SAMP1 alleles, with the exception of the tightly linked D15Mit16 locus 800 kb telomeric to Itgb7. Thus, the non-SAMP1 alleles flanking Itgb7 are limited to an interval of <16.2 Mb (iD15Mit159->telomere).
SAMP1/YitFc and SAMP1/YitFc Itgb7−/− mice were euthanized at the ages required by the experimental design, and the distal ilea (10 cm) and MLNs were harvested. The distal ileum was opened, rinsed of debris, oriented from distal to proximal, and pinned longitudinally on corkboard. Alternatively, the distal ileum was rinsed of debris and rolled into a Swiss roll configuration. Samples were then fixed in Bouin’s fixative (Fisher, Newark, DE), embedded in paraffin, and cut into 3–5-μm sections. The tissues were stained with H&E for histological assessment by a single experienced pathologist who was blinded to the experimental design using a standardized semiquantitative scoring system, as previously described (29).
Single-cell suspensions were prepared from spleen, MLNs, PPs, and LP as previously described (24, 29). Spleen RBCs were lysed by RBC lysis buffer (eBioscience, San Diego, CA) following the manufacturer’s instruction. mAbs used include allophycocyanin-Cy7 or FITC-CD3, PE-Texas Red or PerCP-CD45, PE- or allophycocyanin-CD19, allophycocyanin- or PE-CD8, FITC-, PE- or Pacific Blue-CD4, FITC-CD44, and PE-Cy7-CD62L. All mAbs were obtained from BD Pharmingen (San Diego, CA). Data were analyzed after eliminating doublet cells by pulse width and dead cells by using aqua dead cell stain (Invitrogen, Molecular Probes, Eugene, OR).
Competitive homing assays were conducted as previously described (21). Equal numbers of splenocytes (2 × 107) from C57BL/6J and Itgb7−/− were labeled with 5 μM 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR) and CFSE (Invitrogen, Molecular Probes), respectively, in 1% FCS/DMEM for 20 min at 37°C. After washing with PBS containing 1% FCS, the labeled cells were mixed and injected i.v. into C57BL/6J-CD45.1. An aliquot was saved to determine the input ratio, and in some experiments, the dyes were switched. Recipient mice were sacrificed 18 h postinjection. Spleen and MLNs were made into single-cell suspensions and stained with anti-CD3, anti-CD19, and anti-CD45 mAb. CFSE- and CMTMR-labeled cell populations as a fraction of the CD45+ cell population in spleen and MLNs were determined by flow cytometry. The homing index was calculated as the ratio of CFSE-labeled to CMTMR-labeled cells in the tissue divided by the input ratio. Similarly, an equal number of splenocytes (2 × 107) from SAMP1/YitFc and SAMP1/YitFc Itgb7−/− were labeled with 5 μM CMTMR and CFSE, respectively, processed as above, and injected i.v. into SAMP1/YitFc mice.
SAMP1/YitFc CD4+ T cells were isolated from MLNs by negative selection with the mouse CD4+ negative isolation kit (Miltenyi Biotec, Auburn, CA). B cells were isolated by positive selection with anti-mouse CD19 microbeads (Miltenyi Biotec). All selections were performed according to the manufacturer’s instructions. For adoptive transfer, cells were counted, washed, and resuspended in PBS for injection into SCID recipients. A total of 5 × 105 T cells and 2 × 106 B cells were injected in 500 μl PBS i.p. into SCID recipient mice. The ilea of SCID recipients were harvested 6 wk posttransfer.
MLN Ig concentrations were determined by the mouse Ig isotyping cytometric bead array (BD Pharmingen). MLNs were crushed, resuspended in 5 ml master buffer, centrifuged for 5 min (350 × g), and supernatants assayed according to the manufacturer’s instructions, using 1:100 and 1:1 dilutions of supernatant for detection of κ and λ L chain Igs, respectively.
Data were expressed as mean ± SD. The level of significance between groups was set at *p < 0.05, **p < 0.01, and ***p < 0.001 by two- or one-tailed t test.
To assess the role of β7 integrin in the pathogenesis of ileitis, we compared the severity of inflammation in the terminal ileum between SAMP1/YitFc and SAMP1/YitFc Itgb7−/− mice (microsatellite markers shown in Table I) in groups of mice at 8–19 and 20–50 wk of age (Fig. 1). Histopathological assessment of the severity of the ileal inflammation revealed significantly lower total inflammatory scores in both age groups of SAMP1/YitFc Itgb7−/− mice (Fig. 1D). To assess the impact of loss of β7 integrins on the individual components of the inflammatory index used to score diseases severity in this model, we compared the individual scores for villus distortion and active and chronic inflammation in 8–19-and 20–50-wk-old mice. All three components of inflammation were reduced in SAMP1/YitFc Itgb7−/− mice (Fig. 1A–C). This is illustrated by a pair of representative photomicrographs (Fig. 1E).
A marked increase in the size and cellularity of the MLN is one of the prominent features of established ileitis in SAMP1/YitFc mice (26). In the current study, β7 integrin deficiency resulted in reduction of MLN size by 67% (Fig. 2A, 2B) and of the total number of leukocytes in MLN by 85% (Fig. 2C, 2D) compared with SAMP1/YitFc mice. Using flow cytometry, we found a considerable decrease in the percentage of B cells in 20–40-wk-old MLN of SAMP1/YitFc Itgb7−/− mice compared with SAMP1/YitFc mice (Fig. 3A, 3E). In young mice (12–19 wk), this difference was not seen (Fig. 3B, 3C). T and B cell content of the spleen was not different in SAMP1/YitFc and SAMP1/YitFc Itgb7−/− at either age (Fig. 3B, 3D). The composition and size of MLN in SAMP1/YitFc and SAMP1/YitFc Itgb7−/− mice is summarized in Fig. 3G. The reduced number of B cells in the MLN did not result in reduced secretion of IgG1, IgA, or IgM (Supplemental Fig. 1).
The reduction in the number of CD19+ B cells in MLNs of SAMP1/YitFc Itgb7−/− may be caused by a reduced capacity of B cells to migrate to these tissue sites. To test this hypothesis, we performed competitive homing assays in which 2 × 107 isolated splenocytes from C57BL/6J and Itgb7−/− mice were fluorescently labeled with CMTMR and CFSE, respectively, and injected i.v. into C57BL/6J-CD45.1 mice. After 18 h, the mice were euthanized, and the homing indices were determined in spleen and MLNs. Whereas CD3+ T and CD19+ B lymphocytes from C57BL/6J and Itgb7−/− mice homed equally well to the spleen, CD19+ cells from Itgb7−/− mice showed slightly diminished homing to MLNs compared with CD19+ cells from C57BL/6J mice, which is not statistically significant (data not shown).
Next, we studied the role of β7 integrin-mediated lymphocyte migration during ileitis by injecting CMTMR- and CFSE-labeled splenocytes from SAMP1/YitFc and SAMP1/YitFc Itgb7−/− i.v. into SAMP1/YitFc mice. The number of CD3+ and CD19+ lymphocytes from SAMP1/YitFc Itgb7−/− mice homing to MLNs was much lower compared with the same cells from SAMP1/YitFc mice (Fig. 4A, 4B; p < 0.01). Although a homing defect was also evident in T cells, the homing defect was much stronger in the CD19+ B cell population (Fig. 4B; p < 0.01). Expression of α4β7 integrin was similar on B cells and T cells, but a higher percentage of B cells was positive for α4β7 (data not shown). Within the 18-h experiment, almost no SAMP1/YitFc Itgb7−/− B cells (<5%) homed to the MLNs. No defect was seen in the spleen.
To test whether the reduced capacity of B cells to migrate to intestinal tissue sites contribute to attenuation of ileitis, we co-transfered 5 × 105 SAMP1/YitFc MLN CD4+ T cells with 2 × 106 SAMP1/YitFc or SAMP1/YitFc Itgb7−/− B cells into SCID recipients (Fig. 5). Out of 20 mice transferred with combinations of T and B cells, 17 developed inflammation (total score >3) and were included in the analysis. As was shown previously (25), SCID recipient mice receiving CD4+ T cells and CD19+ B cells from SAMP1/YitFc mice had greater inflammatory scores (Fig. 5A) than those receiving SAMP1/YitFc CD4+ T cells only. By contrast, mice receiving SAMP1/YitFc Itgb7−/− B cells with SAMP1/YitFc CD4+ T cells showed significantly lower scores that were similar to those seen in mice receiving T cells only. This is illustrated by representative H&E histopathological micrographs from terminal ileum of SCID recipient mice (Fig. 5B). In both SAMP1/YitFc and SAMP1/YitFc Itgb7−/− mice, the percentage of L-selectin–positive naive T cells was ~60%, and the percentage of CD44+-activated T cells was ~25% (Supplemental Fig. 2).
Dysregulated recruitment of lymphocytes into inflamed intestinal tissues has been implicated in the pathogenesis of IBD (2–5, 8). In this paper, we studied the in vivo role of β7 integrin for lymphocyte homing and disease progression during ileitis by crossing the null allele for the β7 integrin subunit into SAMP1/YitFc mice, a spontaneous model of chronic ileitis. Disease severity was attenuated by 30–50% at all ages tested. The percentage of B cells was much lower in MLNs of SAMP1/YitFc Itgb7−/− than SAMP1/YitFc mice. In short-term homing experiments, B lymphocytes from SAMP1/YitFc Itgb7−/− mice failed to home to MLNs in vivo.
The phenotype of the inflammation in SAMP1/YitFc mice is well characterized (26). In this mouse model of human CD, Th1 and Th2 adaptive immune responses are detectable at distinct stages of the disease. Disease initiation in this model is Th1 mediated and requires expression of TNF-α and INF-γ, but in the chronic phase, two classical Th2 cytokines, IL-5 and IL-13, are expressed (30, 31). The distinct, stage-specific role of these major subsets of CD4+ effector T cells may in part be explained by the differential role of adhesion molecules and chemokines expressed. In this regard, it has been reported that Abs to CCR9 and CCL25 attenuated early disease but not late stages of the chronic ileitis (32). Our present study suggests that β7 integrins play a role during both progression and initiation of ileitis.
In a previous study, Sydora et al. (20) found no role for β7 integrins for T lymphocyte localization to intestinal tissues and colitis development using two colitis models, a spontaneous colitis model of IL-2–deficient mice and adoptive transfer of CD4+ CD45Rbhigh cells into SCID mice. They noted no difference in the onset and severity of colitis in IL-2–deficient mice crossed to Itgb7−/− mice. In contrast, in the adoptive transfer model, delayed onset of colitis was noted over 9 wk in recipients of T cells from β7-deficient mice, though these mice progressed to severe inflammation at 25 wk (20). Using a similar adoptive transfer model of chronic colitis, Park et al. (21) reported reduced colitis over 11 wk in mice reconstituted with CD4+CD45RBhigh T cells from β7 integrin-deficient mice and aberrantly activated α4β7 integrin knockin mice compared with WT mice. A critical role of β7 integrin in chronic ileitis has also been reported in the TNFΔARE model of ileitis. In these mice, TNF-α is chronically overexpressed. TNFΔARE mice genetically lacking β7 integrin showed very mild intestinal pathology (23).
In SAMP1/YitFc mice, Ab blockade of β7 integrin or MAdCAM-1 alone failed to attenuate the ileitis, but these same Abs were effective when combined with L-selectin blockade (24). In a separate study, anti–MAdCAM-1 Ab inhibited adhesion of T cell to microvessels of the terminal ileum and attenuated established ileitis in the SAMP1/YitFc model of ileitis (33). The discrepancies between these studies may arise due to the start of therapy relative to onset of inflammation, age of mice used, the dose, or the treatment regimen of anti-MAdCAM-1 mAb used. Our genetic approach in a spontaneous mouse model of chronic ileitis circumvents these confounding parameters.
The constitutive trafficking of lymphocytes to intestinal lymphoid tissues and extralymphoid effector sites has been well studied in mice deficient for β7 integrin (14, 34–36). Although lymphocyte migration into MLN is slightly diminished, lymphocyte migration into LP and PPs is severely reduced in β7 integrin-deficient mice (14). The remaining lymphocyte homing is largely dependent on L-selectin (14, 34–36). In this study, we confirm a partial role for β7 integrins in MLN homing and extend our studies to examining lymphocyte recruitment to MLNs during intestinal inflammation. In SAMP1/YitFc mice, unlike lymphocyte migration into MLNs in WT mice, migration of Itgb7−/− lymphocytes into MLN was found to be severely reduced during an 18-h in vivo homing assay. The loss of β7 integrin expression almost completely prevented B cell entry into MLN of SAMP1/YitFc mice. We also noticed that the effect of β7 loss was more prominent on B cell than T cell migration to MLN in this ileitis model. These findings suggest that L-selectin–dependent homing found in uninflamed WT mice (14, 34–36) is less important under conditions of chronic ileitis and that β7 integrin-dependent homing dominates.
Previous studies indicated that the differential migration of B cells and T lymphocyte subsets into lymphoid tissues is dictated by intrinsic differences in expression levels of cell surface L-selectin and β7 integrin (37). Thus, whereas T cells express L-selectin at 50–100% higher levels than B cells, B cells express twice as much α4β7 integrin as T cells (37). Despite these surface expression differences, more T cells migrate to PPs and all other lymphoid tissues at a faster rate than B cells (36). Our finding that B cell homing to MLNs becomes more β7 integrin dependent in inflammation suggests that therapeutic approaches directed at β7 integrins might be effective.
In a previous study, we observed that SAMP1/YitFc MLN B cells contribute to the development of SAMP1/YitFc ileitis (25). The severity of ileitis in the SAMP1/YitFc mice correlates with the number of MLN B cells, and cotransfer of SAMP1/YitFc MLN B cells with CD4+ T cells in SCID mice increased ileitis severity compared with transfer of CD4+ T cells alone (25). In the current study, we confirmed these findings: SCID recipient mice receiving MLN B cells from SAMP1/YitFc Itgb7−/− mice with MLN CD4+ T cells from SAMP1/YitFc mice exhibited lower ileitis inflammatory indices than mice receiving SAMP1/YitFc B cells. These data show that the suppressed migration of B cells to MLN in SAMP1/YitFc Itgb7−/− mice attenuates inflammation.
In conclusion, our study suggests that β7 integrins are essential for lymphocyte recruitment during chronic ileitis in the SAMP1/YitFc model. This effect is explained in part by the significant decrease in the percentage of pathogenic B cells in MLNs of SAMP1/YitFc Itgb7−/− mice. The reduced in vivo homing of B cells to MLNs in SAMP1/YitFc Itgb7−/− mice suggests that β7 integrins are indispensable for B cell homing in chronic ileitis. Moreover, cotransfer of SAMP1/YitFc Itgb7−/− MLN B cells along with CD4+ T cells resulted in attenuated ileitis severity in SCID mice. Taken together with the known proinflammatory role of B cells in ileitis (25), our findings have implications for revisiting the possible use of anti-α4β7 humanized mAbs in CD (38).
This work was supported by National Institutes of Health Grant DK 57880.
We thank Hui Ouyang and Anthony Bruce for expert animal husbandry.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.