| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Department of Internal Medicine,* Washington University School of Medicine, St. Louis, Missouri; the Department of Medicine B, University of Muenster, Muenster, Germany; and the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Deficiency of CC chemokine receptor 6 (CCR6) has been demonstrated to adversely affect the development of mucosal immune responses,10 but the mechanisms resulting in this defect are still being elucidated.11-14 In contrast to most chemokine receptors, CCR6 pairs monogamously with its ligand, CCL20.15 ß-Defensins have also been identified as potential ligands for CCR6; however, these ligands have a significantly reduced affinity for binding CCR6 and a reduced ability to induce CCR6-dependent chemotaxis when compared with CCL20.16 CCL20 is preferentially produced at mucosal surfaces by a variety of cell types including monocytes, endothelial cells, dendritic cells, fibroblasts, and epithelial cells in the follicle-associated epithelium (FAE).17 Multiple cell types express CCR6, including immature dendritic cells, memory T lymphocytes, and B lymphocytes.15,18,19 Most investigations into the role of CCR6 in mucosal immune responses have revolved around alterations in the function of PP dendritic cells in CCR6-deficient mice.11-14 The contributions of other cell types and the effects of CCR6 deficiency on the development of ILFs have not been previously addressed.
In this study, we evaluated the role for CCR6 in the development of ILFs. We observed elevated expression of CCR6 by ILF B lymphocytes when compared with B lymphocytes from other tissues and elevated expression of CCL20 within ILFs. We found that CCR6–/– mice have significantly reduced numbers of ILFs, but the formation of CPs, as well as CPs containing a large population of dendritic cells, was unaffected. Consistent with this block in ILF formation at a stage before the influx of B lymphocytes, we observed that CCR6–/– mice had a significantly reduced population of non-PP intestinal B lymphocytes, and this defect was most pronounced in the distal small intestine where we have observed the majority of ILFs in wild-type mice. Bone marrow reconstitution demonstrated that CCR6-sufficient B lymphocytes are required for the formation of ILFs, and adoptive transfer studies confirmed that CCR6–/– B lymphocytes have a deficiency in their ability to localize to wild-type PPs and CP/ILFs. Expanding previous reports, and paralleling our observations with ILFs, we observed that CCR6–/– mice have a reduced proportion of B lymphocytes within the PP with an associated decrease in the number of follicles within each PP and a decreased surface area of each PP follicular dome. These findings demonstrate a role for CCR6 expression by B lymphocytes in localizing these cells to organized intestinal lymphoid structures and suggest this requirement is particularly important for ILF development. The profound defect we observed in ILF development in CCR6–/– mice, coupled with the inflammatory nature of CCL20 expression, further highlights the dynamic nature of ILF development and suggests that CCL20 expression facilitates the transition of CPs into ILFs.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Mice used for this study were housed in a specific pathogen-free facility and fed routine chow diet. Animal procedures and protocols were performed in accordance with the institutional review board at Washington University School of Medicine. C57BL/6 mice, C57BL/6 congenic mice (B6.Cg-IgHa Thy1a Gpi1a/J), and B-cell-deficient JH–/– mice20
on the C57BL/6 background were purchased from The Jackson Laboratory, Bar Harbor, ME. Lymphotoxin (LT)
-deficient mice21
(a gift from Dr. Dave Chaplin, University of Alabama, Birmingham, AL) were bred onto the C57BL/6 background for more than 10 generations before use in experiments. CCR6-deficient mice22
were bred on to the C57BL/6 background for four generations before use in experiments. Timed pregnant C57BL/6 female mice and CCR6–/– mice for use in experiments involving the injection of lymphotoxin ß receptor (LTßR)-Ig fusion protein, were generated by matings with C57BL/6 male mice or CCR6–/– male mice, respectively. Six- to 10-week-old LT –/– mice were used as recipients for bone marrow transfers.
Bone Marrow Transfers
Bone marrow transfers were performed after lethal irradiation as previously described.23
A total of 1 x 107 T-lymphocyte-depleted bone marrow cells from gender-matched donors were injected intravenously into recipients on the second day of irradiation. Mice receiving bone marrow from multiple donors (C57BL/6 and JH–/– or CCR6–/– and JH–/–; see Figure 4
) received 5 x 106 cells from each donor. Mice were allowed 12 weeks for reconstitution with donor bone marrow before use in experiments. Flow cytometric analysis was performed on splenocytes from recipients at the time of sacrifice to document appropriate B- and T-lymphocyte reconstitution.
|
LTRß-Ig was purified from supernatants generated by a Chinese hamster ovary cell line producing the LTRß-Ig fusion protein (a gift from Dr. W. Yokoyama, Washington University School of Medicine, St. Louis, MO) as previously described.23
Timed pregnant female mice were injected with 100 µg of LTßR-Ig or 100 µg of human Ig (Bayer Corporation, Elkhart, IN) via tail vein on day 16 postcoitus. Mice receiving LTßR-Ig or human Ig in utero were analyzed for the presence of ILFs at 7 weeks of age (see Figure 2
).
|
Small intestines were removed intact, flushed with ice-cold phosphate-buffered saline (PBS), and opened along the mesenteric border. Intestines were mounted, lumen facing up, and fixed with ice-cold 10% phosphate-buffered formyl saline (Fisher Scientific, Pittsburgh, PA) for 1 hour at 4°C. Intestines were washed three times in ice-cold PBS, incubated in a solution of 20 mmol/L dithiothreitol, 150 mmol/L Tris, and 20% ethanol at room temperature for 45 minutes, washed three times in ice-cold PBS, and incubated in a solution of 1% H2O2 for 15 minutes at room temperature to block endogenous peroxidases. Intestines were washed three times in PBS, followed by incubation in PBS containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 for 30 minutes. Intestines were incubated with horseradish peroxidase-conjugated lectin from Ulex europaeus (UEA-I; Sigma-Aldrich, St. Louis, MO) in PBS, BSA, and Triton X-100 solution overnight at 4°C to facilitate the identification of PPs and mature ILFs (mILFs). The following day intestines were washed three times in PBS, incubated in DAB metal peroxide substrate (Pierce Chemical Co., Rockford, IL) for 15 minutes, rinsed twice in distilled water, and returned to PBS for further analysis. Investigators unaware of the treatment groups determined the presence of mILF. Under low-power microscopy (25 to 65x) previously established criteria were used to determine the presence of mILF2 : 1) presence of a nodular structure with size equal to or greater than the width of one villus; 2i) nodular structure possessing an overlying dome resembling the FAE of PP; and 3) nodular structures occurring singly or in groups of two.
For the analysis of PPs, whole mounts of intestines were stained with UEA-I as above. The following criteria were used for the purpose of enumerating PPs. PPs were defined as nodular structures more than five villi wide, located along the anti-mesenteric border of the small intestine. These criteria overlap with the criteria used to enumerate mILFs but were necessary because some of the PPs of the CCR6–/– mice contained only one follicle. PPs were identified using a dissecting microscope at 32x magnification and photographs obtained of each PP. Photographs of PPs and a scale were enlarged to 10 x 12 inches, and the diameter of each dome was measured. The surface area of each dome was calculated using the following formula: surface area = 
(diameter/2).2
For anti-B220 and anti-CD11c staining of whole mounts to determine the numbers of iILFs and CD11c+ clusters, intestines were removed intact, flushed with PBS, opened along the mesenteric border, and mounted as above. Intestines were then incubated three times in Hanks’ balanced salt solution (BioWhittaker, Walkersville, MD) containing 5 mmol/L EDTA at 37°C with shaking for 10 minutes to remove epithelial cells. Intestines were then fixed in 10% phosphate-buffered formyl saline and treated with 1% H2O2 for 15 minutes at room temperature as above. Intestines were incubated in a solution of 50 mmol/L Tris, pH 7.2, 150 mmol/L NaCl, 0.6% Triton X-100, and 0.1% BSA for 1 hour at 4°C to block nonspecific antibody binding and then incubated with rat anti-mouse B220 antibody (PharMingen, San Diego, CA) or biotin-conjugated hamster anti-mouse CD11c (eBiosciences, San Diego, CA) diluted in the above solution overnight at 4°C. Intestines were washed three times in the above solution and incubated with a horseradish peroxidase-conjugated goat anti-rat IgG antibody or streptavidin-conjugated horseradish peroxidase (Jackson Immuno-Research Laboratories, West Grove, PA) diluted in the above solution at room temperature for 1 hour. Intestines were washed three times and incubated in DAB metal peroxide substrate as above. Intestine whole mounts were examined under a dissecting microscope at 25 to 65x. Immature ILFs (iILFs) were counted as clusters of B220+ cells occurring at the base of villi and not containing an overlying dome. Dendritic cell clusters were counted as clusters of CD11c+ cells occurring at the base of villi.
Cell Isolation from Spleen, PPs, and mILFs
Spleens and PPs were removed from unmanipulated C57BL/6 mice and disrupted by mechanical dissociation. Intestines were removed from C57BL/6 mice receiving LTßR-Ig in utero, flushed with ice-cold PBS, opened along the mesenteric border, and mounted with the lumen facing up in cold PBS, as described above. Using the dissecting microscope and a blunt-end 26-gauge needle and syringe, multiple mILFs were aspirated and placed in cold PBS. Red blood cells 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/intestine.
Flow Cytometric Analysis
Single-cell suspensions were obtained as above and flow cytometric analysis performed as previously described.23
Reagents used for analysis were fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated rat anti-mouse CD19, fluorescein isothiocyanate-conjugated anti-mouse CD45, streptavidin-conjugated phycoerythrin, appropriate isotype control antibodies (all from BD Biosciences, San Diego, CA), and rat anti-mouse CCR6 (R&D Systems, Minneapolis, MN). Dead cells were excluded based on forward and side light scatter. Gates for positive staining were defined such that 1% of the analyzed population stained positive with the appropriate isotype control antibody. Flow cytometric analysis for the expression of CCR6 was performed using directly conjugated anti-CCR6 antibodies in some replicates as well as anti-CCR6 antibodies with an anti-rat IgG secondary antibody (eBiosciences) to augment the fluorescence intensity in some replicates (Figure 1, c and d)
; both methods gave equivalent results.
|
Paraffin-embedded sections containing PPs from whole-mount intestines (performed as described above) were deparaffinized, treated with antigen-unmasking solution (Vector Laboratories, Burlingame, CA), treated with avidin/biotin blocking kit (Vector Laboratories), washed three times in PBS, and blocked for 15 minutes at room temperature in PBS containing 1% BSA and 0.1% Triton X-100. Sections were then incubated with biotin-conjugated lectin from Arachis hypogaea (PNA) (Sigma-Aldrich) or biotin-conjugated anti-B220 antibody (BD Biosciences) diluted in PBS containing 1% BSA and 0.1% Triton X-100 overnight at 4°C. Biotinyl-tyramide signal amplification (DuPont/NEN, Boston, MA) followed by incubation with streptavidin-conjugated cyanine 2 dye (Jackson ImmunoResearch) was used for detection of PNA staining. Anti-B220 staining was detected using streptavidin-conjugated cyanine 3 dye (Jackson ImmunoResearch). Sections were counterstained with Hoescht dye (Sigma-Aldrich) to visualize nuclei.
Immunohistochemistry on frozen sections of intestine was used to enumerate CD90+ cellular clusters and to evaluate the localization of adoptively transferred B lymphocytes. Intestines were embedded in OCT compound and serial 8-µm sections were obtained from each block. Slides were fixed with a 1:1 solution of acetone and methanol for 15 minutes, dried, rehydrated, and blocked with PBS containing 1% BSA. Sections were incubated with primary antibodies overnight at 4°C, washed with PBS, and incubated with fluorescently labeled secondary reagents for 1 hour at room temperature. Sections were stained with Hoescht dye (Sigma-Aldrich) to visualize nuclei.
Quantification of CD90+ Cellular Clusters
Segments of small intestine of 1.5 cm were embedded in OCT compound, frozen, and cut at an axis perpendicular to the villi into 8-µm sections. The segments comprised more than one-half of the entire intestine, and identical areas of the intestine were obtained from each animal evaluated. Two serial sections of small intestine were stained with anti-CD90 (eBioscience) and anti-CD3 (eBioscience) as above using two-color immunofluorescence. The serial sections were examined at a magnification of 100x or higher, and the number of CD90+CD3– cellular clusters falling within the crypt area on each section was counted and averaged between the serial sections. Additional sections were stained with anti-CD90 and anti-c-kit to confirm that the CD90 clusters are also c-kit+, stained with anti-B220 to assess the numbers of B lymphocytes associating with the CD90 clusters (Figure 2, d and e)
, and stained with anti-CD11c (BD Biosciences) to assess the clustering of dendritic cells associated with the CD90 clusters. Photomicrographs of the sections taken with a 25x objective were assembled, and the crypt area of each section was determined using AxioVision software (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). The average number of CD90+ clusters/mm2 of crypt surface area was then determined.
RNA Isolation and Real-Time Polymerase Chain Reaction
PPs were removed from unmanipulated C57BL/6 mice. Mature ILFs were isolated from C57BL/6 mice receiving LTßR-Ig in utero using a dissection microscope and 26-gauge needle as described above. Non-PP, non-mILF-bearing intestine from the distal small intestine of C57BL/6 mice was identified using a dissecting microscope and removed. The PP and mILF tissue contained the overlying FAE, stromal elements, and mononuclear cells.
RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and treated with DNase I (Ambion, Austin, TX) to remove contaminating DNA, and cDNA was synthesized from 2 µg of total RNA using Superscript II RNase H– reverse transcriptase (Invitrogen). Expression of targets was detected by real-time polymerase chain reaction using ABI prism 7700 sequence detection system and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The following primers were used for detection of the targets, forward primers are listed first, followed by reverse primers: 18S 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3', ß-actin 5'-GCTTCTTTGCAGCTCCTTCGT-3' and 5'-ATATCGTCATCCATGGCGAAC-3', CCL20 5'-TGATGCTTTTTTGGGATGGAA-3' and 5'-AGCCTTCAACCCCAGCTGT-3', and CCR6 5'-TGTTCTGCTATCTGTTCATTATCAAGA-3' and 5'-CACGACTCGGATGGCTCTGT-3'. Samples were measured in triplicate. Relative quantitation of target expression using 18S or ß-actin as a housekeeping gene was determined using the comparative crossing threshold method as described in the ABI Prism 7700 sequence detection system user bulletin.
Adoptive Transfers
To assess the ability of wild-type and CCR6–/– B lymphocytes to localize to the organized intestinal lymphoid tissues, splenocytes isolated from CCR6–/–, C57BL/6 congenic mice (expressing the IgHa allotype) were injected intravenously into JH–/– (B lymphocyte-deficient) recipients. Intestinal B lymphocytes could not be used for these studies because the CCR6–/– mice had few intestinal B lymphocytes (Figure 2f)
. Recipients received a total 3 x 107 cells from either CCR6–/– mice, C57BL6 congenic mice, or combined CCR6–/– and C57BL/6 congenic mice. Flow cytometric analysis was performed on the cellular populations at the time of transfer to confirm that equivalent numbers of B lymphocytes from each donor were injected into recipients. Mice were sacrificed 1 week later and frozen sections from PPs and small intestines were evaluated for the presence of transferred cellular populations using immunohistochemistry with anti-IgMb (BioLegend, San Diego, CA), anti-IgMa (BD Biosciences), and anti-B220 antibodies. In co-transfer experiments, serial sections of intestine were stained with either IgMa or IgMb. In experiments involving separately transferred populations, intestinal sections were stained with anti-CD11c and anti-B220. Sections were examined with fluorescence microscopy at a magnification of 200x or higher. The number of IgMa- or IgMb-positive cells in each PP serial section or the number of B220+ cells in each CD11c+ cluster was determined by an individual unaware of the treatment groups.
Statistical Analysis
Data analysis using Student’s t-test and a one-way analysis of variance with a Dunnett’s multiple comparison post-test was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
To assess the role of CCR6 in ILF formation, we examined the mRNA expression of CCR6 in ILFs using real-time polymerase chain reaction. We noted that CCR6 expression was significantly higher in ILFs when compared with non-PP, non-ILF-bearing intestine (Figure 1a)
. To evaluate CCR6 protein expression and to determine the cell types expressing CCR6, we examined ILF cellular populations for CCR6 expression by flow cytometry. We found that a large proportion of the ILF cellular population expresses CCR6; gates for positive staining were set such that less than 1% of cells stain positive for the isotype control (Figure 1c)
. The majority of cells falling within the CCR6+ gate are B lymphocytes (CD19+) (Figure 1c)
. Directly comparing B-lymphocyte populations from the spleen, PPs, and ILFs, we observed that ILF B lymphocytes displayed greater cell surface expression of CCR6 when compared with splenic B lymphocytes and Peyer’s patch B lymphocytes (Figure 1d)
. We observed that T lymphocytes (CD3+) and dendritic cells (CD11c+) displayed staining consistent with CCR6+ and CCR6– populations, which comprised approximately 3% and 2% of the CCR6+ ILF cellular population, respectively; the remaining CCR6+ population of the ILFs was predominantly comprised of lineage c-kit+ cells (data not shown). In attempts to understand further the significance of CCR6 in ILF and PP formation, we examined the expression of CCL20 in the spleen, PPs, and ILFs. We found that both ILFs and PPs displayed CCL20 mRNA expression that was significantly greater than that seen in the non-PP, non-mILF-bearing intestine (Figure 1b)
.
ILF Development Is Arrested at a Stage Corresponding to the Influx of B Lymphocytes in the Absence of CCR6
To determine whether CCR6 played an important role in the formation of CPs and ILFs, we examined the intestines of CCR6–/– and C57BL/6 mice for the presence of CPs and ILFs. CPs are clusters of lineage marker-negative c-kit-positive cells randomly distributed throughout the small intestine.24
These CP cells also express other markers such as CD90. CPs can only be identified using immunohistochemistry on frozen sections of intestine cut on an axis perpendicular to the villi. We observed that anti-CD90 is a useful marker for enumerating CPs, as the anti-CD90 staining is robust and CPs can easily be identified using 100x magnification. In contrast, anti-c-kit staining is relatively weak and therefore less useful to enumerate CPs. We observed that CCR6–/– mice had an equivalent number of CD90+ clusters when compared with wild-type mice (Figure 2a)
. CPs and ILFs also contain a surrounding population of CD11c+ dendritic cells. In some CPs, this CD11c+ population becomes prominent. We can enumerate these CPs containing a large population of CD11c+ cells as well as CD11c+ clusters within ILFs using whole mounts with anti-CD11c staining. We observed no differences in the numbers of large CD11c+ clusters when comparing CCR6–/– mice to wild-type mice (Figure 2b)
.
The transition of CPs to ILFs is thought to occur when a subset of CPs are infiltrated by B lymphocytes. Initially, B lymphocytes cluster in the CP at the base of the villi and form a loosely organized structure that we have termed the iILFs. A subset of these immature structures continue to accumulate B lymphocytes and develop a follicle-associated epithelium and a loosely organized germinal center; we have termed these structures mILFs. Using whole mounts of the intestine to enumerate ILFs, we observed that CCR6–/– have a near absence of both iILFs and mILFs, and the reduced number of iILFs was significantly different from wild-type mice (Figure 2c)
. Mature ILFs are rare in C57BL/6 mice in our colony, making it difficult to assess a defect in mILF development by comparing wild-type and CCR6–/– mice. In addition, underdeveloped PPs in the CCR6–/– mice would be counted as mILFs in the current scoring system, further complicating this assessment. However, the numbers of mILFs can be augmented by LTßR-Ig treatment in utero. In utero LTßR-Ig treatment on day 16 of gestation ablates PP development and results in a transient IgA deficiency in the progeny, which is subsequently associated with augmented development of ILFs.25
Therefore, to identify a defect in mILF development, mice were given LTßR-Ig in utero and examined for the presence of ILFs. Treatment of C57BL/6 mice with LTßR-Ig in utero augmented the development of both iILFs and mILFs (Figure 2c)
. CCR6–/– mice showed a modest increase in the numbers of iILFs following LTßR-Ig treatment; however, the numbers of iILFs formed were still significantly less than unmanipulated C57BL/6 mice (Figure 2c)
. This indicates that in the absence of CCR6 other pathways can facilitate iILF formation, but these pathways are less effective. Treatment of CCR6–/– mice with LTßR-Ig in utero failed to induce the formation of mILFs (Figure 2c)
. Consistent with a block in the ability of B lymphocytes to infiltrate the CP in CCR6–/– mice, we observed few B220+ cells within CD90+ clusters in CCR6–/– mice (Figure 2, d and e)
. In C57BL/6 mice in our colony, ILFs are located preferentially in the distal small intestine.2
Consistent with the loss of ILFs and the inability of CCR6–/– CP to become infiltrated with B lymphocytes, we observed that CCR6–/– mice have a diminished population of B lymphocytes in their diffuse lamina propria, which contains the CPs and ILFs (Figure 2f)
. This defect was most pronounced in the distal small intestine where the majority of ILFs are located in the wild-type mice (Figure 2f)
. These findings indicate that in the absence of CCR6 the development of ILFs is blocked at a stage of B-lymphocyte infiltration into CPs, or the formation of iILFs.
PPs Have a Reduced B-Lymphocyte Population and a Reduced FAE in the Absence of CCR6
To determine whether CCR6 deficiency affected the B-lymphocyte population in other intestinal organized lymphoid structures, we examined the PPs of the CCR6–/– mice. The initial observations regarding the PP phenotype in the CCR6–/– mice was unclear, with one group reporting normal PP development and a second group reporting that the CCR6–/– PPs contained fewer domes and had reduced total cellularity.10,13
We analyzed the numbers of PPs, the numbers of domes, and the surface area of each dome in CCR6–/– and wild-type mice. We observed that CCR6–/– mice had an equal number of PPs when compared with wild-type mice (data not shown); however, each PP in the CCR6–/– mice contained significantly fewer domes when compared with wild-type mice (Figure 3, a, e, and f)
. Moreover, the average surface area of each PP in the CCR6–/– mice was significantly less than that seen in the wild-type mice (Figure 3, b, e, f, g, and h)
. Consistent with these observations, we observed significantly decreased total cellularity of PPs from CCR6–/– mice when compared with wild-type mice (Figure 3c)
, and we observed a decrease in the B-lymphocyte population in the PPs of CCR6–/– mice when compared with wild-type mice (Figure 3d)
. We did not observe any differences in the proportion of CD4+ or CD8+ T lymphocytes or in the proportion of dendritic cells (MHCII+, CD11c+) within the PP when comparing CCR6–/– and wild-type mice (data not shown). The architecture and organization of the PP from CCR6–/– mice and wild-type mice were similar. Each contained a large population of B220+ cells with a central lucent area with PNA+ staining consistent with the presence of germinal centers (Figure 3, g–l)
. These findings confirm and extend earlier findings regarding the PPs of CCR6–/– mice and demonstrate that although CCR6 deficiency affects B-lymphocyte populations in both types of organized intestinal lymphoid structures, this deficiency more dramatically effects ILF development.
|
Given the above findings, we wished to determine whether CCR6-sufficient B lymphocytes were required for the development of ILFs and to examine the stage of ILF development for which a requirement for CCR6-sufficient B lymphocytes might exist. LT–/– mice lack CPs, iILFs, and mILFs, but the formation of ILFs can be reliably induced in LT–/– mice following the transfer of LT-sufficient bone marrow reconstituting the LT/LTßR, as well as the LT/TNFR and LT/HVEM axes.2,25
This provides a useful model to study the requirements of bone marrow-derived cell populations on ILF formation. LT–/– mice reconstituted with CCR6–/– bone marrow, a combination of CCR6–/– and JH–/– bone marrow, such that all B lymphocytes are CCR6-deficient, or a combination of C57BL/6 and JH–/– bone marrow were examined for the presence of iILFs and mILFs. LT–/– recipients of CCR6–/– bone marrow and recipients of a combination of CCR6–/– and JH–/– bone marrow were deficient in the formation of iILFs and mILFs when compared with recipients of a combination of C57BL/6 and JH–/– bone marrow (Figure 4)
, thus demonstrating a requirement for CCR6-sufficient B lymphocytes in the development of ILFs.
CCR6–/– B Lymphocytes Have a Diminished Ability to Localize to Organized Gastrointestinal-Associated Lymphoid Tissues
The above findings suggest that CCR6–/– B lymphocytes have a diminished capacity to localize to intestinal lymphoid structures. To confirm this defect we injected equal numbers of CCR6–/– (IgHb allotype) and wild-type (IgHa allotype) splenic B lymphocytes into B-cell-deficient JH–/– mice and looked for their presence in organized intestinal lymphoid structures. When equal numbers of CCR6–/– and wild-type B lymphocytes were injected into the same JH–/– recipient, we observed that wild-type B lymphocytes have a threefold increased ability to localize to PPs as determined by the number of IgMbright cells (Figure 5, a–c)
. We also observed a larger number of wild-type IgMdull cells when compared with CCR6–/– IgMdull cells in the recipients’ PPs; however, definitive quantification of these IgMdull cells was not feasible due to the intensity of staining. We were unable to identify many donor B lymphocytes within CD90+ or CD11c+ clusters when using IgM allotype staining, despite being able to identify B220+ CD11c– cells within these clusters. We feel this is due to the relatively lower expression of IgM by the majority of ILF B lymphocytes, making their allotype-specific identification more difficult. To examine the ability of CCR6–/– B lymphocytes to localize to CD11c+ clusters, we transferred equivalent numbers of wild-type or CCR6–/– splenic B lymphocytes to individual recipients and examined the recipients for the presence of B220+ CD11c– cells within the clusters. We observed that wild-type B lymphocytes had approximately a twofold better ability to localize to CD11c+ clusters when compared with CCR6–/– B lymphocytes (Figure 5, d–f)
. These findings demonstrate that CCR6–/– B lymphocytes have an impaired ability to localize to organized intestinal lymphoid structures.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
CCL20 has several properties suggesting it is a candidate for participating in ILF development. CCL20 is produced at mucosal surfaces, and specifically produced by the FAE under normal circumstances.17 Similar to ILF formation, CCL20 expression is induced by inflammatory stimuli, and intestinal CCL20 expression is elevated in inflammatory conditions associated with the formation of lymphoid structures.17,27-29 In addition, CCL20 functions to recruit dendritic cells and B lymphocytes; these cell types make up a large proportion of the ILF cellular population.4 CCR6, the only identified receptor for CCL20, plays an important role in mucosal immune responses as evidenced by diminished production of antigen-specific mucosal IgA following oral immunization of CCR6-deficient mice.10 The specific defects resulting in this phenotype are still being elucidated. Initial studies attributed this defect to the absence of CD11b+ dendritic cells in the PP subepithelial dome in CCR6-deficient mice.13 However, subsequent studies demonstrated that this dendritic cell population was present but reduced in CCR6-deficient mice.14 More recent investigations revealed that CCR6 expression by dendritic cells in the subepithelial dome is required to initiate T-cell-dependent responses to an oral pathogen.12 Despite these observations, the role of CCL20 and CCR6 in the development of ILFs is largely uninvestigated with the exception of one report of normal CP development in CCR6–/– mice.30
Here, we demonstrate that CCL20 expression is increased in ILFs and that CCR6 is highly expressed by B lymphocytes within ILFs. We observed that CCR6-deficient mice have a defect in ILF development; however, the formation of CPs and the recruitment of a substantial population of dendritic cells into these CPs are not altered in these animals. This defect in ILF development correlated with a decreased population of B lymphocytes in the diffuse lamina propria, which contains the CPs and ILFs. Accordingly, this defect was most pronounced in the distal small intestine, the region in which we see the majority of ILFs in wild-type mice. These observations indicate that the requirement for CCR6 in ILF development coincides with the influx of B lymphocytes into the CPs and/or ILFs. Using bone marrow chimeric mice, we confirmed the requirement for CCR6 expression by B lymphocytes in ILF development. These findings were further supported by adoptive transfer studies demonstrating that CCR6–/– B lymphocytes had a reduced ability to localize to PPs and CPs. This demonstrates a specific requirement for CCR6 expression by B lymphocytes in localizing to the intestine and is consistent with a block in ILF development at a stage in which B lymphocytes localize to these structures in CCR6-deficient mice. Although not specifically addressed in the studies presented here, our findings do not preclude, but could suggest, that other CCR6-sufficient cell types play a role in ILF development. We observed a small number of iILFs in recipients of a combination of JH–/– and CCR6–/– bone marrow, suggesting that other pathways can partially compensate for the loss of CCR6 expression by B lymphocytes. In combination with this observation, the absence of ILFs in the recipients of CCR6–/– bone marrow suggests that a CCR6-sufficient non-B lymphocyte plays a role in ILF development. In addition to these observations, we noted a trend toward decreased numbers of large CD11c+ clusters in the CCR6–/– mice, suggesting other potential defects in ILF development.
PPs from CCR6–/– mice revealed parallel, but less dramatic, findings. CCR6–/– mice have a normal number of PPs but with diminished size and decreased number of follicles.13 We observed that as the CCR6–/– mice are crossed onto the C57BL/6 background, this phenotype has become more pronounced, such that on average PPs in the CCR6–/– mice have less than two follicles. In our current scoring system, these abnormal PPs are counted as mILFs, thus artificially increasing the number of mILFs in the CCR6–/– mice. Therefore, it is likely that the CCR6–/– mice on the C57BL/6 background are more deficient in mILFs than suggested by the data presented here. We also observed that the PPs in the CCR6–/– mice had small follicular domes, reduced total cellularity, and a reduced proportion of B lymphocytes. The reduced PP total cellular population is consistent with previous reports on the phenotype of the CCR6–/– mice11,13 ; however, the decreased percentage of PP B lymphocytes and the smaller PP follicular dome in the CCR6–/– mice has not been previously noted. The differences we observed in the PP B-lymphocyte populations were small but statistically significant. Evaluation of older animals revealed an increase in the PP B-lymphocyte populations in both wild-type and CCR6–/– mice; however, CCR6–/– mice maintained a statistically significant decrease in the proportion of PP cells that were B lymphocytes when compared with age-matched wild-type mice (data not shown). This indicates that the small, but statistically significant, difference we observed is maintained with aging. The decreased size of the follicle dome associated with a decreased population of PP B lymphocytes in the PPs of the CCR6–/– mice is supported by a prior study documenting a role for B lymphocytes in facilitating the development of the FAE31 as well as a recent study demonstrating a reduced number of M cells in the FAE of CCR6–/– mice.11 The reduced surface area of the FAE and decreased numbers of M cells could provide another explanation for the diminished capacity of the CCR6–/– mice to mount mucosal immune responses to oral antigens.10 We observed no differences in the proportion of the PP cellular population that were dendritic cells (MHCII+, CD11c+) when comparing wild-type and CCR6–/– mice. Our findings are consistent with a recent study using similar methods that found a modest increase in the population of PP cells that are CD11c+ in the CCR6–/– mice.11 In contrast, previous reports demonstrated an absent or diminished CD11b+ dendritic cell population in the PP subepithelial dome of CCR6–/– mice.10,13 Our study did not evaluate dendritic cell subpopulations or the positioning of these subpopulations within the PP. When our findings are evaluated in the context of the significantly decreased total PP cellular population in the CCR6–/– mice, it becomes apparent that PP dendritic cell numbers are decreased in CCR6–/– mice and, therefore, are consistent with a diminished PP CD11b+ DC subpopulation as well as potential defects in positioning of these subpopulations within the PP in the absence of CCR6. Overall, our observations indicate that CCR6 deficiency affects the B-lymphocyte population in both forms of organized intestinal lymphoid structures and do not preclude deficiencies in other cellular populations in either structure.
The findings presented here are consistent with previous observations regarding CCR6 expression and function in B lymphocytes. CCR6 expression is acquired by B-2 B lymphocytes as they mature and exit the bone marrow and is largely restricted to mature, follicular (B-2) B lymphocytes and to a lesser extent a small subset of germinal center and marginal zone B lymphocytes.32 In addition CCR6 expression on follicular B lymphocytes is down-regulated by B-cell receptor ligation.33 Our observation that ILF B lymphocytes are largely CCR6+ is consistent with these findings, as ILF B lymphocytes are exclusively B-2 B lymphocytes and do not have a requirement for antigen activation for their localization to ILFs.1,2,25 We observed a hierarchy of CCR6 expression with ILF B lymphocytes having greater CCR6 expression when compared with PP B lymphocytes, and PP B lymphocytes have greater CCR6 expression than the splenic B lymphocytes. This observation is also consistent with previous studies of CCR6 expression by B lymphocytes, as the PPs and the spleen have more developed germinal centers that contain a population of CCR6-negative B lymphocytes, and the spleen also contains a population of CCR6-negative marginal zone B lymphocytes.32 Although we have consistently observed this hierarchy of CCR6 expression on ILF and PP B lymphocytes, the chemotaxis of freshly isolated ILF or PP B lymphocytes in response to CCL20 has been equivalent to or less than that seen in splenic B lymphocytes (data not shown). Possible explanations for this apparent inconsistency include differences in the local environment of the PPs and ILFs that would desensitize B lymphocytes to CCR6-dependent stimuli, including elevated local expression of CCL20, as we have demonstrated here. In the context of these previous observations regarding the restricted patterns of CCR6 expression on B lymphocytes and our observations documenting that ILFs contain polyclonal follicular B-2 B lymphocytes that can be antigen-naïve,25,34 we interpret the findings of this study to indicate that CCL20 and CCR6 play a role in recruiting antigen naïve (follicular) B-2 B lymphocytes to the sites of ILF formation.
Two LT-dependent steps have been identified in ILF development, differing in the cellular source of LT. Non-B, non-T lymphocytes can deliver the early signals necessary for the formation of iILFs, these events may be mediated by the lineage c-kit+ cells within CPs.8 The transition of immature ILFs into mature ILFs requires LT-sufficient B lymphocytes, and these LT-sufficient B lymphocytes can be antigen-naïve.2,25 Chemokines play a critical role in the development of organized lymphoid structures, with CXCL13 and its receptor CXCR5 playing an essential role in the development of B-cell follicles in the spleen, lymph nodes, and PPs.35 CXCL13 not only recruits CXCR5+ B lymphocytes in this process, bit it also induces the expression of LT by antigen-naïve B lymphocytes, thus inducing the production of additional CXCL13 by LTßR-expressing stromal cells and driving the development of a follicle containing antigen-naïve B lymphocytes.35 Given that CCR6 deficiency affected ILF development to a greater degree than PP development, we questioned whether CCL20 might be playing a similar role to CXCL13 in ILF formation. In support of this role, CCL20 is produced following LTßR ligation in epithelial cells.36 Despite these associations, we did not observe the induction of LT expression in B lymphocytes following stimulation with CCL20 (data not shown). An alternative role for CCR6 in ILF development is suggested by prior studies noting that CCR6+ B lymphocytes are also CXCR5+32 ; therefore these two chemokines may act synergistically, with the recruitment of CCR6+ B lymphocytes facilitating the formation of the CXCL13-driven positive feedback loop driving the development of ILFs in response to CCL20 inducing inflammatory stimuli.
PPs and ILFs are distinct members of gastrointestinal-associated lymphoid tissues. Studies to date indicate that these structures can function in similar manners and initiate noninflammatory adaptive immune responses to luminal antigens.3,4,34 A primary distinction between these structures is the way in which they are formed, with the formation of PPs being developmentally driven and the development of ILFs occurring after birth and being influenced by luminal stimuli.1,2,26 We observed that CCR6 deficiency affects B-lymphocyte populations in intestinal lymphoid structures and resulted in hypoplastic PPs and largely absent ILFs. CCL20 is considered an inflammatory chemokine, and it is reasonable to assume that CCR6 deficiency alters ILF development, which is more affected by inflammatory stimuli, to a greater degree than PP development, which is largely developmentally driven. This assumption is supported by our findings of unchanged numbers of PPs and a more than 10-fold decrease in the numbers of ILFs in the absence of CCR6. Our observations indicate that CCR6 expression by B lymphocytes is essential for the formation of ILFs and suggests that CCL20 production by CPs in response to inflammatory stimuli may be an event facilitating the recruitment of B lymphocytes and the subsequent transition into ILFs.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported in part by National Institutes of Health grants DK-64798 (to R.D.N.) and DK-64730 (to I.R.W.), the Broad Medical Research Program IBD-0042 (to R.D.N.), the Crohn’s and Colitis Foundation of America (to R.D.N.), and by Washington University School of Medicine Digestive Diseases Research Core Center grant P30-DK52574.
Accepted for publication December 28, 2006.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
