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₄β₇/MAdCAM-1 Interactions Play an Essential Role in Transitioning Cryptopatches into Isolated Lymphoid Follicles and a Nonessential Role in Cryptopatch Formation¹

Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The ₄ integrins ₄β₇ and ₄β₁, and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of ₄ integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express ₄β₇ and ₄β₁, including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal β₇^–/– mice and adult and neonatal mice given blocking Abs to ₄β₇, MAdCAM-1, or VCAM-1 did not identify a role for ₄ integrins in cryptopatch (CP) development; however, these studies demonstrated that ₄β₇ and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that β₇^–/– LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that β₇ expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for ₄β₇/MAdCAM-1 in ILF development corresponding to the influx of β₇-expressing lymphocytes and a nonessential role for β₇-localizing LTi-like cells to the small intestine.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Integrins are heterodimeric proteins composed of and β subunits that promote cell-cell interactions and consequently perform diverse roles in the immune system. Only two integrins are known to contain an ₄ subunit, ₄β₇ and ₄β₁. These integrins and their ligands, mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1)³ and VCAM-1, are expressed in restricted manners on hematopoietic cells, high endothelial venules (HEVs), and stromal cells (3, 12, 13, 18, 24, 28), respectively, and have critical roles in secondary lymphoid structure formation and inflammatory responses (1, 2, 3, 4, 5, 6, 7).

One of the earliest events in secondary lymphoid structure organogenesis is the clustering of fetal CD3^–CD4⁺CD45⁺ lymphoid tissue inducer (LTi) cells, which express lymphotoxin (LT), and stromal organizer cells, which express the lymphotoxin β receptor (LTβR) (8, 9, 10). Interactions of the ₄ integrins expressed on the fetal LTi and VCAM-1 expressed by the LTβR⁺ stromal cells are felt to be instrumental in maintaining the early cellular clusters, thus sustaining LT-LTβR interactions and leading to a cascade of events resulting in the formation of secondary lymphoid tissues (2, 10, 11, 12). Likewise, the expression of MAdCAM-1 on lymph node HEVs during embryogenesis contributes to the colonization of ₄β₇-expressing fetal LTi cells (12). However, this interaction is not solely responsible for fetal LTi cell retention, since Abs that block this interaction only partially inhibit fetal LTi cell colonization (12). Fetal LTi cells also express ₄β₁ and therefore interactions with VCAM-1-expressing stromal cells may also contribute to stabilizing this cellular interaction. Collectively, these observations suggest that both ₄β₇ and ₄β₁ expression by fetal LTi cells and VCAM-1 expression by organizer cells are important in the early steps of secondary lymphoid tissue formation.

Following the formation of a self-sustaining cluster of fetal LTi cells and organizer cells, mature hematopoietic cells are recruited to the forming lymph node or Peyer’s patch (PP); ₄ integrins are crucial to this process. Studies using knockout mice and Ab blockade demonstrate a critical role for ₄β₇ and its ligand, MAdCAM-1, in mature lymphocyte homing to the gut (4, 13, 14, 15). This role also extends to lymphocyte trafficking in intestinal inflammation where MAdCAM-1 expression is aberrantly up-regulated in chronically inflamed intestines of patients with inflammatory bowel disease (16, 17, 18, 19, 20), and accordingly ₄β₇/MAdCAM-1 blockade has been considered as a novel organ-specific therapeutic target for the treatment of inflammatory bowel disease (21, 22, 23, 24, 25, 26). Collectively, these observations suggest that ₄β₇ interactions with MAdCAM-1 play critical roles in later stages of secondary lymphoid tissue development and in lymphocyte trafficking in the intestine during inflammation.

Isolated lymphoid follicles (ILFs) are intestinal lymphoid aggregates that can resemble a single-domed PP. Recently, these aggregates have become appreciated as distinct members of the gastrointestinal-associated lymphoid tissues (27, 28, 29, 30, 31, 32). In contrast to PP, ILFs are part of a spectrum of lymphoid aggregates in various stages of development. Cryptopatches (CP) are collections of unique bone marrow-derived cells clustered at the base of the villi and are believed to be the precursor cellular aggregate giving rise to ILFs. These unique cells lack the expression of mature lineage markers (lin^–), but express c-kit and share many phenotypic and developmental features with the fetal LTi cells. Accordingly, the lin^–c-kit⁺ CP cells are believed to carry out an analogous function as organizing cells delivering the early LT signals resulting in the formation of CP, which subsequently progress to become ILFs (33). ILF and PP development share many characteristics; however, a primary distinction is that PP formation is developmentally driven, with critical events occurring during embryogenesis, conversely ILF development initiates after birth and its progression is augmented by exogenous stimuli including normal intestinal microbiota (27, 28, 34, 35). Although the function of ₄ integrins in ILF development was previously uninvestigated, a role for ₄ integrins in this process is suggested by observations of VCAM-1 expression by stromal cells in CP and the role of inflammatory stimuli augmenting ILF development (36, 37, 38, 39). Paralleling the events in PP formation, ₄ integrins could be important at multiple points in CP and ILF formation, including early events required for the clustering of lin^–c-kit⁺ cells to form CP and later events related to the recruitment of lymphocytes to form the mature ILFs.

In this study, we evaluated the role of ₄ integrins in the development of ILFs. We observed that a significant population of the lin^–c-kit⁺ cells, ILF B lymphocytes, and ILF T lymphocytes express ₄β₇ and ₄β₁. In a related manner, we found that stromal cells within the ILFs express VCAM-1, while MAdCAM-1 expression was restricted to nonlymphatic vascular structures within ILFs. A functional role for ₄ integrins in CP and ILF development was defined by knockout mice, Ab blockade, and bone marrow reconstitution. Surprisingly, we observed that β₇ is dispensable for the formation of CP and the recruitment of dendritic cells to CP; however, β₇ is essential for the development of ILFs. Parallel studies using Ab blockade in adult and neonatal mice demonstrated that Abs specific for murine ₄β₇ or MAdCAM-1, but not VCAM-1, significantly decreased the numbers of ILFs in adult mice, while the number of CP remained unaffected in all treatment groups of adult and neonatal mice. Consistent with this block corresponding to the influx of B lymphocytes into the developing ILFs, bone marrow reconstitution and adoptive transfer of lymphocytes demonstrated an absolute requirement for β₇ expression by lymphocytes for their localization to developing ILFs and a redundant role for β₇ in localizing the LTi-like cells to the small intestine. Collectively, these findings demonstrate an absolute role for ₄β₇-MAdCAM-1 interactions at late stages in ILF development corresponding to the influx of mature β₇- expressing lymphocytes into the developing ILFs and a redundant role for β₇ in the localization of the LTi-like cells to the small intestine and CP development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

BALB/c mice, C57BL/6 mice, β₇^–/– mice on the C57BL/6 background (stock no. 002965), C57BL/6-congenic mice expressing the CD90.1 allele (stock no. 001317), C57BL/6-congenic mice expressing the CD45.1 allele (stock no. 002014), and RAG^–/– mice on the C57BL/6 background (stock no. 002216) were purchased from The Jackson Laboratory. Animals were housed in a specific pathogen-free facility and fed routine chow diet. Animals were 8–16 wk of age at the time of analysis except where noted otherwise. Animal procedures and protocols were conducted in accordance with the institutional review board at Washington University School of Medicine.

Isolation of cellular populations from spleen, PP, and ILFs

Spleens and PP were removed from mice and disrupted by mechanical dissociation. Small intestines were removed from mice, flushed with cold PBS, opened along the mesenteric border, and mounted with the lumen facing up in cold PBS. Using the dissecting microscope and a 26-gauge needle and syringe, the contents of multiple mature ILFs were aspirated, placed in cold PBS, and mechanically disrupted. RBC were lysed from cellular suspensions and then used for flow cytometric analysis as described below. Average yield of viable mononuclear ILF cells ranged from 3 to 7 x 10⁵ cells per small intestine.

Flow cytometric analysis

Single-cell suspensions from spleen, PP, and ILFs obtained as above were used for flow cytometric analysis. Abs used for analysis were anti-mouse β₁(BD Biosciences), anti-mouse β₇, anti-mouse ₄, anti-mouse CD3, anti-mouse CD19, anti-mouse c-kit, anti-mouse lineage marker mixture (anti-mouse CD3, CD11b, B220, Gr-1, TER119, CD11c), and appropriate isotype control Abs (all from eBioscience). Data acquisition was performed on a FACScan cytometer (BD Biosciences) retrofitted with a second laser using CellQuest (BD Biosciences) and Rainbow (Cytek) software. Data analysis was performed on a Macintosh G4 computer running FlowJo software (Tree Star) or CellQuest software (BD Biosciences). Dead cells were excluded based on forward and side light scatter. Gates for positive staining was defined such that 1% of the analyzed population stained positive with the appropriate isotype control Abs.

Immunohistochemistry

Small intestines were opened and 1.5-cm sections were snap frozen in OCT medium (Sakura Finetek). For the purpose of evaluating MAdCAM-1 and VCAM-1 expression, 7-µm sections were cut parallel to the axis of the villi (longitudinal sections). For the purpose of evaluating CP, 7-µm sections were cut perpendicular to the axis of the villi (horizontal sections). Endogenous peroxidase activity was quenched with 3% H₂O₂ in PBS for 10 min at room temperature and endogenous biotin was blocked with an avidin/biotin blocking kit (Vector Laboratories). Sections were washed in PBS three times, blocked with PBS plus 1% BSA for 30 min at room temperature, and incubated with the primary Ab for 1 h at room temperature. Sections were washed in PBS three times. Sections incubated with unconjugated primary Abs were subsequently incubated with biotinylated secondary Abs for 1 h at room temperature and washed three times in PBS. Tyramide signal amplification (PerkinElmer LAS) was used for the detection of VCAM-1 per the manufacturer’s recommendations. For detection of other Abs, we used streptavidin-conjugated Cy2 or streptavidin-conjugated Cy3 (Jackson ImmunoResearch Laboratories). In experiments using multiple fluorophores, sections were treated with an avidin/biotin blocking kit (Vector Laboratories), and the above protocol was repeated using a second fluorophore for detection. Sections were counterstained with Hoechst dye (Sigma-Aldrich) to visualize nuclei (see blue staining in photomicrographs in Figs. 2, 3, 5, and 6).

Determination of cluster density and size

To enumerate and determine the density of clusters, sections corresponding to identical regions of the small intestine were obtained from experimental and control mice. The entire small intestine was mounted in four equivalent pieces and a 1- cm-long segment from each end of the piece, totaling eight small intestine sections from each animal, was embedded in OCT compound and frozen. Immunohistochemistry on sections cut perpendicular to the villi was performed as described above. Under x100 magnification, clusters located in the crypt area were counted using an immunofluorescence microscope. The same section was then stained with H&E and the total crypt surface area was determined using MetaVue software (Molecular Device). The density of clusters was calculated by dividing the total number of clusters by the total crypt area for each animal. The surface area of clusters was determined using Image J software (http://rsb.info.nih.gov/ij/; National Institutes of Health).

Enumeration of CD11c and B220 clusters

Enumeration of B220⁺ and CD11c⁺ cellular clusters was performed using anti-B220- or anti-CD11c-stained whole mounts as previously described (30). The numbers of B220⁺ and CD11c⁺ clusters were determined using a dissecting microscope at a magnification of x25 or greater.

In vivo Ab blockade

The hybridoma cell line producing rat anti-mouse ₄β₇ (clone DATK 32; American Type Culture Collection) was cultured in CD hybridoma serum-free medium (Life Technologies) and Ab was purified from the culture supernatant by protein G chromatography (Pierce) under endotoxin-free conditions. The endotoxin level was determined with a QCL-1000 kit (BioWhittaker) using the Limulus amebocyte lysate method. The concentration of purified Ab was determined using ELISA specific for rat IgG2a and the activity of the purified Ab was assessed by flow cytometry.

To evaluate CP and ILF development in adults, 7- to 8-wk- old mice were injected i.p. with 200 µg of rat anti-mouse MAdCAM-1 (BioExpress), rat anti-mouse VCAM-1 (BioExpress), rat anti-mouse ₄β₇, purified as above, or rat IgG (Southern Biotechnology Associates) every other day for 2 wk, at which time they were sacrificed for analysis. To examine CP development in the neonatal period, 200 µg of anti-₄β₇, MAdCAM-1, VCAM-1, or rat IgG was injected i.p. at 17 days of gestation and every other day after birth until analysis on day 19 of neonatal life.

Bone marrow transfers

Bone marrow chimeric mice were generated as previously described (28). Seven-week-old bone marrow recipients received 1000 Gy of gamma irradiation in divided doses over 2 sequential days and were injected i.v. with 1 x 10⁷ T lymphocyte-depleted bone marrow cells from gender-matched donors. In experiments using mixed chimeras, recipients received 5 x 10⁶ cells from each donor. Mice were allowed 12 wk for reconstitution with donor bone marrow before use for experiments. Appropriate reconstitution of lymphocyte compartments were examined by flow cytometry at the time of sacrifice.

Adoptive transfer of lymphocytes

To assess a role for β₇ expression by B lymphocytes in localizing to ILFs, wild-type and β₇^–/– deficient mature B lymphocytes were cotransferred into RAG^–/– recipients. Splenocytes were isolated from wild-type (CD45.1) and β₇^–/– (CD45.2) mice and the number of B lymphocytes in each population was determined by flow cytometry. Splenic B lymphocytes (1.9 x 10⁷) from each donor genotype were coinjected i.v. into RAG^–/– recipients. Recipients were sacrificed 1 wk later and evaluated for the presence of transferred cellular populations by flow cytometry and immunohistochemistry. The number of total B lymphocytes (B220⁺ cells) and the number of wild-type B lymphocytes (B220⁺CD45.1⁺) in the ILFs were determined by examining intestinal sections stained for B220 and CD45.1 at x200 or greater magnification. Flow cytometric analysis was performed by gating on live B lymphocytes (B220⁺CD19⁺) and evaluating the ratio of CD45.1⁺ (wild-type) vs CD45.2⁺ (β₇^–/–) cells.

Statistical analysis

Data analysis using Student’s t test and one-way ANOVA followed by Tukey’s multiple comparison posttest was performed using GraphPad Prism. A value of p < 0.05 was used as a cutoff for statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
₄ integrins and their ligands are expressed within ILFs

To assess a role for ₄ integrins in ILF formation, we examined the expression of 4β₇ and ₄β₁ by ILF cell types by flow cytometry. Similar to PP, the majority of ILF B lymphocytes (CD19⁺) were ₄β₇⁺, while in comparison fewer ILF T lymphocytes (CD3⁺) were ₄β₇⁺ (Table I). Approximately one-half of the B and T lymphocytes from ILFs and PP were ₄β₁⁺ (Table I). Approximately one-half of the ₄β₇⁺ B lymphocytes and T lymphocytes also expressed ₄β₁ (Fig. 1A). To assess a potential role for ₄ integrins in the early steps of ILF development, we evaluated ₄β₇ and ₄β₁ expression by lin^–c-kit⁺ cells in ILFs and found that 32% of these cells expressed ₄β₇ and 44% of these cells expressed ₄β₁ (Table I), and a significant proportion expressed both ₄β₇ and ₄β₁ (Fig. 1B).

View this table: [in this window] [in a new window]   Table I. 4 integrin expression by PP and ILF cellular populations

View larger version (34K): [in this window] [in a new window]   FIGURE 1. 4 integrins are expressed by ILF lymphocytes and LTi-like cells. The expression of 4β7 and 4β1 on ILFs and PP cellular populations from BALB/c mice was performed using multicolor flow cytometry. Gates were set such that 1% of the cellular population stained positive with isotype control Abs. Isotype control staining for A is shown for PP CD3+4+ cells and CD19+4+ cells; no difference was observed in isotype control staining between the PP and ILFs in these cellular populations. Similar to PP, the majority of ILF B lymphocytes express 4β7 and in comparison fewer ILF T lymphocytes express 4β7 (Table I). Approximately one-half of PP and ILF T and B lymphocytes express 4β1 (Table I), and the majority of these 4β1-expressing cells also express 4β7 (A). The lin–c-kit+ cells make up 10% of the ILF cellular population, and a significant proportion of these cells express both 4β7 and 4β1 (Table I and B). These findings are consistent with a role for the 4 integrins and their ligands in ILF development and/or function. Dot plots in A and B are representative of one of two independent experiments using pooled cellular populations from three mice.

View larger version (55K): [in this window] [in a new window]   FIGURE 2. Vessels within ILFs express MAdCAM-1 and stromal cells within ILFs express VCAM-1. To evaluate the expression of the ligands for 4β1 and 4β7, frozen sections of ILFs from BALB/c were stained with anti-mouse MAdCAM-1 and VCAM-1 as described in Materials and Methods. The expression of MAdCAM-1 localized to structures that had the appearance of vessels (A), which was confirmed by positive staining for CD31 (B and C: serial sections of a vessel at higher magnification stained with anti-MAdCAM-1 and CD31, respectively). These vascular structures displayed low or no PNAd expression (data not shown). VCAM-1 expression was diffusely distributed in the ILF and primarily restricted to CD45– (nonhematopoietic) stromal cells (D). Bar in A,100 µm.

Recent observations indicate that ILFs are a spectrum of lymphoid structures ranging from CP, small clusters of intestinal lineage marker-negative c-kit-positive cells located at the base of the villi, to larger isolated lymphoid follicles rich in B cells (33, 41). To determine whether β₇ integrins play an important role in the formation of CP and ILFs and to determine the stage in which this role exists, we examined β₇^–/– mice for the presence of CP and ILFs. An early event in CP development is the localization of the lin^–c-kit⁺ cells to the small intestine. We examined the percentage of lin^–c-kit⁺ cells among lamina propria cellular population by flow cytometry and found no difference between β₇^–/– and wild-type mice (Fig. 3A). Once localized to the small intestine, the lin^–c-kit⁺ cells cluster to form CP. The lin^–c-kit⁺ CP cells also express CD90 and, due to its intensity in immunohistochemistry, anti-CD90 staining is useful to identify and to enumerate every structure in the CP/ILF continuum. We observed that β₇^–/– mice had an equivalent number of CD90⁺ clusters when compared with wild-type mice (Fig. 3B), indicating that β₇ is not required for the early steps of CP development and that in the absence of β₇ the total number of structures in the CP/ILF continuum is unchanged.

View larger version (88K): [in this window] [in a new window]   FIGURE 3. ILF formation in β7–/– mice is arrested at a stage corresponding to the influx of B lymphocytes. To assess a role for β7 in CP and ILF development, we evaluated β7–/– mice for the presence of intestinal lin–c-kit+ cells and the presence of cellular aggregates within the CP/ILF continuum. Photomicrographs in H, I, and J are from intestinal sections from wild-type mice and photomicrographs from K, L, and M are from intestinal sections from β7–/– mice. There was no difference in the percentage of lamina propria cells that were lin–c-kit+ between wild-type and β7-deficient mice (A). The lin–c-kit+ CP cells also express CD90 and, due to its intensity, anti-CD90 staining is useful to identify cellular clusters that encompass all of the cellular aggregates in the CP/ILF spectrum. There was no difference in the density of CD90+ clusters when comparing wild-type and β7–/– mice (B). β7–/– mice had an increased number of intestinal CD11c+ clusters, corresponding to an increase in the number of CD90+ clusters that are infiltrated with a large population of CD11c+ cells (C, H, and K). The size of the CD11c+ clusters in β7–/– mice was not significantly different from those in wild-type mice (E). In contrast, β7–/– mice had a dramatically reduced number of intestinal B220+ clusters (D, I, and L) and a reduced population of T lymphocytes infiltrating the CD90+ clusters (J and M). CD90 clusters in β7–/– mice were significantly smaller (F) and few clusters were larger than 10,000 µm2 (G), corresponding to the absence of structures with a higher cellular complexity. The data in A–D are generated from four mice in each group and presented as the mean ± 1 SD. The data in E–G are generated from three mice in each group. The photomicrographs H–M are representative of one of four or more mice from each group. Bar in H, 100 µm. *, p < 0.05; ns, not significant.

The progression of CP to ILFs occurs when a subset of CP are infiltrated by B lymphocytes and accordingly enumerating the total number of B lymphocyte clusters (B220⁺) is an effective way to evaluate the presence of ILFs. To evaluate the role of β₇ integrins in the progression to ILFs, we compared the numbers of B220⁺ clusters between β₇^–/– mice and wild-type mice. We observed a striking decrease in B220⁺ clusters in the small intestine of the β₇^–/– mice (Fig. 3, D, I, and L), indicating a profound defect in the ability of CP to progress to ILFs in the absence of β₇.

Size has also been used to classify structures within the CP/ILF spectrum (35). Although this approach does not formally assess cellular composition, increasing cellular complexity generally correlates with the increasing size of the structures as they progress to become mature ILFs (35, 41). We observed that the CD90⁺ clusters were significantly smaller in the β₇^–/– mice (Fig. 3F). The discrepancy between the normal size of the CD11c⁺ clusters and the smaller size of the CD90⁺ clusters in the β₇^–/– mice can be accounted for by the location of these cell types within the CP, which make up the majority of the intestinal cellular aggregates. We have observed that CD90⁺ cells are preferentially located in the center of the CP, thus fewer CD90⁺ cells will result in the appearance of a smaller CD90⁺ cluster, while CD11c⁺ cells are preferentially located in a ring around the CD90⁺ cells. Therefore, an influx of a large population of CD11c⁺ cells into a small CD90⁺ cluster can result in a CD11c⁺ cluster with a normal appearance. We also observed that the β₇^–/– mice lacked structures >10,000 µm², corresponding to structures containing B lymphocytes (Fig. 3G), further supporting the observation of a decrease in B220⁺ clusters in the β₇^–/– mice. T lymphocytes are not required for CP and ILF development and do not make up a large component of the cellular population of these structures (27, 30, 44). However, related to our above findings regarding B lymphocytes, we also observed a relative absence of CD3⁺ cells associated with the CD90⁺ clusters in the β₇^–/– mice (Fig. 3, J and M). Overall, these findings suggest that β₇ is not required for the clustering of the LTi-like cells to form CP or for the initial step progressing to ILFs, the infiltration by a substantial population of CD11c⁺ cells; however, β₇ is essential for later steps in ILF development related to the influx of lymphocytes into the maturing ILFs.

Blockade of ₄β₇/MAdCAM-1 pathway inhibits ILF formation at a stage corresponding to the influx of B lymphocytes

β₇ associates with both the _E subunit and the ₄ subunits. _Eβ₇ is expressed selectively on intestinal intraepithelial lymphocytes and plays an important role in mediating the selective localization or retention of intraepithelial lymphocytes by interactions with E-cadherin expressed on intestinal epithelial cells (45). We observed that <10% of the ILF cellular population expresses _Eβ₇⁺ (data not shown). To determine whether the deficiencies in ILF development in the β₇^–/– mice are mediated by the loss of ₄β₇ or _Eβ₇, we treated C57BL/6 mice with blocking Abs specific for ₄β₇ and examined them for the presence of CP and ILFs. In a correlative manner, we treated mice with blocking Abs against MAdCAM-1 and VCAM-1 to evaluate the role of these ligands in ILF development. We observed no significant differences in the percentage of lin^–c-kit⁺ cells in the lamina propria (Fig. 4A), the number of CD90⁺ clusters (Fig. 4B), and the numbers of CD11c⁺ clusters (Fig. 4C) between the four treatment groups. However, mice receiving Abs to ₄β₇, MAdCAM-1 contained significantly fewer B220 clusters, while mice receiving anti-mouse VCAM-1 Ab showed no significant difference in the numbers of B220⁺ clusters when compared with control Ig-treated mice (Fig. 4D). The defect in ILF formation in the mice receiving Abs to MAdCAM-1 correlated with a decrease in the lamina propria B lymphocyte population (Fig. 4E). There was no significant difference in the lamina propria B lymphocyte population in animals in the other treatment groups. This suggests that the blockade of ILF formation seen in β₇^–/– mice is mediated by the ₄β₇/MAdCAM-1 pathway, and this blockade occurs at the later stage of ILF formation corresponding to the influx of B lymphocytes.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Blockade of the 4β7/MAdCAM-1 pathway in adult mice inhibits ILF formation at a stage corresponding to the influx of B lymphocytes. C57BL/6 mice receiving blocking Abs to mouse 4β7, mouse MAdCAM-1, mouse VCAM-1, or rat IgG (control) were evaluated for the density of CD90+ clusters, the percentage of lin–c-kit+ cells and CD19+ B lymphocytes in the lamina propria and the presence of CD11c clusters and B220 clusters. There were no significant differences in the percentage of lamina propria lin– cells expressing c-kit (A), the number of CD90+ clusters (B), and the numbers of CD11c clusters (C) between the four groups. Mice receiving Abs to 4β7 or MAdCAM-1 contained significantly fewer B220+ clusters when compared with controls (D). Mice receiving anti-mouse VCAM-1 Ab showed no significant difference in the numbers of B220+ clusters (D). The defect in ILF formation in the mice receiving Abs to MAdCAM-1 correlated with a decrease in the lamina propria B lymphocyte population (E). The data are presented as the mean ± the SEM of data generated from three or more mice in each group. *, p < 0.05; ns, not significant.

In addition to being substantial constituents of ILFs, B lymphocytes play an important role in ILF development by facilitating the transition of ILFs from a loose cellular cluster to an organized lymphoid tissue with an overlying follicule-associated epithelium (30). The above findings indicate that the ₄β₇/MAdCAM-1 pathway is important for ILF formation and imply that ₄β₇ expression by B lymphocytes may be required for their localization and subsequent contribution to the developing ILFs. To evaluate this, we injected bone marrow from gender-matched β₇^–/– (CD90.2, IgM^b) mice or C57BL/6 (CD90.2, IgM^b) mice into irradiated B6.Cg-IgH^a Thy1a Gpi1a/J (CD90.1, IgM^a) mice. This approach allowed us to distinguish donor (β₇^–/– or wild-type) CD90.2⁺ CP cells from recipient (wild-type) CD90.1⁺ CP cells and to evaluate the ability of wild-type or β₇^–/– lymphocytes to localize to the wild-type CP. Analysis of splenocytes from the recipients demonstrated effective reconstitution with IgM^b+ cells and CD90.2⁺ cells from donors, as well as a deficiency in β₇ in the recipients of β₇^–/– bone marrow (Fig. 5, A and B). We did not observe any difference in the density of total CD90⁺ clusters or CD11c⁺ clusters between mice receiving bone marrow from β₇^–/– and C57BL/6 donors; however, the density of B220 clusters in mice receiving β₇^–/– bone marrow was significantly lower than that of mice receiving wild-type bone marrow (Fig. 5, C, D, F, G, and I). Irradiation did not eliminate recipient CD90.1⁺ CP cells (Fig. 5, D and G). We found that all CD90⁺ clusters in both groups of animals contained a large population of CD90.1⁺ (recipient-derived) CP cells. Although the density of total CD90⁺ clusters was not different, we observed fewer CD90.2⁺ (donor-derived) cells in the CD90⁺ clusters in recipients of β₇^–/– bone marrow (Fig. 5, C and F). This implied a relative defect in the ability of the CD90⁺ CP cells from β₇^–/– mice to localize to the sites of CP when competing with the endogenous wild-type CP cells. The presence of wild-type (CD90.1⁺) CP cells in all of the clusters and ability of the CD90⁺ clusters to recruit in CD11c⁺ cells (Fig. 5, E and H) indicate that a functional deficiency in CD90⁺ cells is unlikely to account for the diminished number of B220⁺ clusters in the β₇^–/– bone marrow recipients.

View larger version (55K): [in this window] [in a new window]   FIGURE 5. β7-sufficient bone marrow-derived cells are essential for the formation of ILFs. Lethally irradiated C57BL/6-congenic (IgMa, CD90.1) mice were given bone marrow from gender-matched β7–/– (IgMb, CD90.2) or C57BL/6 (IgMb, CD90.2) donors and examined for the presence of donor lymphocytes in the spleen, intestinal CD90 clusters, and intestinal B220 clusters. Flow cytometric analysis of splenocytes demonstrated effective engraftment of donor B lymphocytes (IgMb+) and T lymphocytes (CD90.2+) and the absence of β7 expression in splenocytes from recipients of β7–/– bone marrow (A and B). There were no differences in the infiltration of the CD90+ clusters with CD11c+ cells (E and H). Few CD90+ clusters from recipients receiving β7–/– mice bone marrow contained B lymphocytes (C, D, F, and G), and quantitatively this was associated with a significant decrease in the number of B220+ clusters in these recipients (I). All CD90 clusters in both groups contained a population of CD90.1+ (recipient-derived, wild-type) CP cells (D and G). However, the CD90+ clusters in the recipients of β7–/– bone marrow contained few CD90.2+ (donor-derived) CP cells (cf C and F), suggesting a relative defect in the ability of these cells to localize to the CP in the absence of β7. Data in I are presented as the mean ± the SEM of data generated from three mice in each group. Bar in C, 100 µm. *, p < 0.05.

₄β₇ plays a redundant role in localizing LTi-like cells to the small intestine and a nonessential role in CP formation

In contrast to the above findings, we observed a normal number of CD90⁺ clusters in the β₇^–/– mice (Fig. 3B). CP development occurs over several days in the postnatal period. This relatively protracted period may allow time for other pathways to compensate in the absence of ₄β₇ function and consequently evaluation of adult animals may not reveal subtle defects such as a delay in CP formation. To investigate this possibility, we evaluated the ability of β₇^–/– and wild-type CP cells to compete for the CP niche following bone marrow transfer, and we evaluated the development of CP in the neonatal period in mice lacking ₄β₇ function.

In competitive bone marrow transfer experiments, equal numbers of wild-type (CD45.2, CD90.1) and β₇^–/– (CD45.2, CD90.2) bone marrow cells were injected into wild-type (CD45.1, CD90.2) recipients. Recipients were analyzed by flow cytometry for the presence of donor wild-type and β₇^–/–lin⁺ splenocytes and donor wild-type and β₇^–/–lin⁺ and lin^– lamina propria cells. CD45.2 marks all donor-derived cells and CD90.1 and CD90.2 distinguishes between wild-type and β₇^–/– LTi-like cells and lymphocytes. Flow cytometry revealed that recipient lin⁺ splenocytes were enriched in β₇^–/– (CD90.2⁺CD45.2⁺) donor cells when compared with wild-type (CD90.1⁺CD45.2⁺) donor cells and conversely the lin⁺ lamina propria cells contained significantly fewer β₇^–/– donor cells when compared with wild-type cells (Fig. 6A). The spleen contained too few lin^–CD90⁺ cells for analysis; however, analysis of the lamina propria revealed a trend toward fewer β₇^–/–lin^– donor cells localizing to the lamina propria when compared with wild-type lin^– donor cells; this trend did not reach statistical significance when compared with the ratio of donor and wild-type lin⁺ splenocytes (Fig. 6A). Analysis of the composition of CP by immunohistochemistry revealed that approximately equal populations of CD90⁺ cells were of wild-type (CD90.1⁺) donor origin and β₇^–/– (CD90.2⁺) donor origin or recipient origin (CD90.2⁺) (Fig. 6B). However, the majority of the donor (CD45.2⁺) CP cells were of wild-type (CD90.1⁺) donor origin (Fig. 6C) and the vast majority of these CD90⁺ cells were CD3^– and c-kit⁺ (data not shown). These findings suggest a relative inefficiency of the β₇^–/– CD90⁺ cells to localize to the CP.

View larger version (66K): [in this window] [in a new window]   FIGURE 6. β7 plays a redundant role in localizing LTi-like cells to the small intestine and a nonessential role in CP development in neonatal mice. To more definitively address the role for β7 in localizing the CD90+ cells to the small intestine and CP, we performed competitive transfers of wild-type (CD90.1, CD45.2) and β7–/– (CD90.2, CD45.2) bone marrow into wild-type (CD90.2, CD45.1) recipients. Recipients were analyzed to identify the origin of CD45.2 (donor) lin+ CD90+, and lin–CD90+ cells with flow cytometry and with immunohistochemistry to evaluate the localization of wild-type and β7–/– CD90+ cells to the CP. Flow cytometric analysis of gated on the CD45.2+ (donor) population revealed an enrichment of lin+CD90+ cells of β7–/– origin (CD90.2) in the spleen while conversely the lamina propria contained significantly fewer CD45.2+ (donor) lin+CD90+ cells of β7–/– origin (A). There were too few lin–CD90+ splenocytes for analysis; however, the donor (CD45.2+) lin–CD90+ cells in the lamina propria were relatively lacking in cells from β7–/– donors (CD90.2) in comparison to cells from wild-type donors (CD90.1; A). This did not reach statistical significance when compared with the origins of lin+ donor splenocytes. Immunohistochemistry revealed equal populations of CD90.1+ (wild-type donor origin) and CD90.2+ (β7–/– donor origin or recipient origin) cells in the CP (B). The donor (CD45.2+) CP cells were largely from wild-type donors (CD90.1+) (yellow costaining, C), thus demonstrating a relative deficiency of β7–/– cells to compete for this niche. To evaluate a role for β7 in the development of CP in the neonatal period, we examined small intestines from neonatal wild-type and β7–/– mice and wild-type mice given blocking Abs to 4β7, MAdCAM-1, VCAM-1, or control IgG from day 17 of gestation until analysis at day 19 of neonatal life. CP are detectable in both wild-type and β7–/– mice on day 17 of neonatal life (D), and there was no difference in the density of CP on day 19 of neonatal life when CP formation is rapidly increasing in β7–/– mice (E) or mice given blocking Abs to 4β7, MAdCAM-1, or VCAM-1 (F). Consistent with the observation of Ab blockade in adult mice, we observed that anti-4β7 and anti-MadCAM-1 inhibited the influx of B lymphocytes into the cellular clusters and anti-VCAM-1 had no effect on B lymphocyte influx into the cellular clusters at day 19 of neonatal life (G). Data in A and E are generated from four mice in each group. Data in F are generated from three mice in each group. *, p < 0.05; ns, not significant. Bar in B, 100 µm; D, 50 µm; and G, 50 µm.

₄β₇ expression by B lymphocytes is required for their localization to the transitioning CP

Our above observations demonstrate that ₄β₇ and MAdCAM-1 play important roles in localizing lymphocytes to the transitioning CP. This role could involve the expression of ₄β₇ directly on lymphocytes or ₄β₇ expression by other cell types needed to subsequently recruit lymphocytes to the transitioning CP. To evaluate the role of ₄β₇ expression on B lymphocytes in their localization to ILFs, we performed competitive adoptive transfers of wild-type (CD45.1) and β₇^–/– (CD45.2) splenic B lymphocytes into RAG^–/– mice and analyzed the ability of the B lymphocytes to localize to the small intestine and CP by flow cytometry and immunohistochemistry. We observed that β₇^–/– B lymphocytes were less efficient at localizing to the small intestine when compared with wild-type B lymphocytes (Fig. 7A). Moreover, analysis of the B220⁺ cells within the CP/ILFs revealed that in contrast to the spleen, the majority of cells were of the wild-type (CD45.1⁺) donor origin; the costaining for CD45.1 (red) and B220 (green) appears yellow within the ILFs (Fig. 7, B–D). Thus confirming that ₄β₇ expression on B lymphocytes plays a critical role in recruiting these cells into the transitioning CP and its subsequent progression into ILFs.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. β7-deficient B lymphocytes have impaired localization to ILFs. To evaluate the role of β7 in localizing B lymphocytes to ILFs, equivalent numbers of splenic B lymphocytes from β7–/– (CD45.2) and B6SJL (CD45.1) donors were transferred into RAG–/– recipients and analyzed for the presence of donor B lymphocyte populations in the spleen and lamina propria by flow cytometry and in the ILFs by immunohistochemistry. Flow cytometry analysis demonstrated enrichment in β7–/– (CD45.2) B lymphocytes in the spleen and a relative paucity of β7–/– B lymphocytes in the lamina propria (A). Immunohistochemistry demonstrated that the majority of the B lymphocytes (B220+) within the ILFs were of the wild-type (CD45.1+) origin (B–D; costaining for CD45.1, red, and B220, green appears yellow). Data in A and B are displayed as mean ± SD of data generated from four mice in each group. *, p < 0.01; bar in C, 20 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

₄ integrins play diverse roles in the immune systems, including essential functions in the development and maintenance of lymphoid tissues. ₄β₇ and its ligand MAdCAM-1 have well-described roles in lymphocyte homing to the gut (4, 13, 14, 15) and function in a related manner localizing fetal LTi cells to the intestine to deliver the early signals for PP organogenesis (47). ₄β₁ and its ligand VCAM-1 also play a critical role in PP organogenesis. ₄β₁ is expressed by the fetal LTi cells and interactions with VCAM-1 expressed by the stromal organizer cells are believed to be important for maintaining a self-sustaining cellular cluster and formation of the PP anlagen (2, 10, 11, 12). The similarities between PP and ILF suggest that ₄ integrins might also have important and diverse roles in ILF development; however, a potential role for ₄ integrins in ILF development has not been previously addressed.

We observed that a significant proportion of ILF lymphocytes and LTi like cells express ₄β₁ and ₄β₇. In accord with previous observations, we found that VCAM-1 expression was primarily restricted to nonhematopoietic stromal cells within the CP and ILFs (39). MAdCAM-1 expression was primarily restricted to nonlymphatic vessels within the ILFs. In comparison to PP HEVs, these vessels universally had low or no PNAd expression and could represent immature HEVs (40).

Our findings provide convincing evidence of a role for ₄β₇ and MAdCAM-1 in recruiting B lymphocytes into the developing ILFs. Animals deficient in β₇ and animals receiving blocking Abs to ₄β₇ or MAdCAM-1 had similar phenotypes demonstrating a blockage of ILF development at a stage corresponding to the influx of B lymphocytes. Likewise, the bone marrow chimeric mice demonstrated a deficiency in the localization of β₇^–/– B lymphocytes to the developing ILFs, despite the presence of wild-type CD90⁺ cells and the ability to recruit dendritic cells to the growing cluster. Furthermore, adoptive cotransfer of wild-type and β₇^–/– mature B lymphocytes demonstrated that β₇^–/– B lymphocytes were less efficient at localizing to the small intestine and the transitioning CP. The recruitment of B lymphocytes into the developing ILFs is of particular importance, as LT-dependent signals delivered by this cell type are required for the maturation of ILFs into a functional immune inductive site containing a follicle-associated epithelium (29, 30). These observations are consistent with the role of ₄β₇ for localizing B lymphocytes to PP and add ILFs as another mucosal site highly dependent upon ₄β₇ and MAdCAM-1 for lymphocyte entry.

The role of ₄β₇ expression by the lin^–c-kit⁺ CP cells in ILF development is less critical. Mice deficient in β₇ have a normal number of PP that are hypoplastic, suggesting that the early events determining PP number are intact (4, 13, 14, 15). These early events are dependent upon signals delivered by the fetal LTi cells; therefore, implicating that the function of the fetal LTi cells inducing PP anlagen development is intact in β₇ deficiency. The lin^–c-kit⁺ CP cells share phenotypic and developmental features with the fetal LTi cells and are felt to function in a similar manner delivering the early signals that subsequently result in ILF development (33). We observed no deficiency in the localization of the lin^–c-kit⁺ cells to the small intestine or the clustering of these cells to form CP in the absence of β₇, suggesting that in accord with PP development in the β₇^–/– mice, these early events are preserved. In contrast to this, previous investigations revealed that MAdCAM-1 blockade during embryogenesis reduced fetal LTi cell recruitment to developing lymphoid tissues, thus implying a role for ₄β₇-MAdCAM-1 interactions in fetal LTi cell localization (47). Consistent with this, our bone marrow chimeric studies demonstrated that β₇^–/– CP cells were less efficient at localizing to the small intestine and CP when they were required to compete with wild-type CP cells for this niche. This contrasts with our observations that CP development in the neonatal period is intact in animals lacking ₄β₇/MAdCAM-1 function. In total, these observations are consistent with a role for ₄β₇ in efficiently localizing the lin^–c-kit⁺ cells to the small intestine and a nonessential role for ₄β₇ in CP development.

Despite identifying ₄β₁ and VCAM-1 expression by ILF cell types, our observations did not identify a role for VCAM-1 in CP and ILF development in adult and neonatal mice. This is in contrast to studies demonstrating a role for VCAM-1 and β₁ in embryonic events required for PP development (2, 47). Our observations could result from a functional difference between PP and CP development or, like ₄β₇, ₄β₁ may play a less essential role in CP development that was not revealed by these studies.

Blockade of the ₄β₇/MAdCAM-1pathway is a potential therapy for inflammatory bowel disease, with the appeal that the effects of blocking this pathway will largely be limited to mucosal sites and therefore in comparison to more global immunomodulators, systemic toxicity will be reduced. The findings presented here indicate that blocking this pathway will adversely effect the development and hence the function of ILFs. Although the function of these structures is still an ongoing area of investigation, ILFs have been shown to play an important role in generating immune responses to luminal Ags, and by extension protection from potential pathogens (29). These observations highlight the duality of the ₄β₇/MAdCAM-1 pathway contributing to intestinal inflammation and simultaneously playing an essential role in the development of lymphoid tissues that promote immune homeostasis.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by National Institutes of Health Grant DK-64798 (to R.D.N.), the Crohn’s and Colitis Foundation of America (to C.W.), the Washington University School of Medicine Digestive Diseases Research Core Center Grant (P30-DK52574), and The Siteman Cancer Center High Speed Cell Sorting Core supported in part by a National Cancer Institute Cancer Center Support Grant (P30 CA91842).

² Address correspondence and reprint requests to Dr. Rodney D. Newberry, 660 South Euclid Avenue, Box 8124, St. Louis, MO 63110. E-mail address: rnewberry{at}im.wustl.edu

³ Abbreviations used in this paper: MAdCAM-1, mucosal addressin cell adhesion molecule 1; LTi, lymphoid tissue inducer; CP, cryptopatch; ILF, isolated lymphoid follicle; LT, lymphotoxin; LTβR, LT β receptor; PP, Peyer’s patch; HEV, high endothelial venule; PNAd, peripheral lymph node addressin.

Received for publication August 27, 2007. Accepted for publication July 20, 2008.

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