Volume 122, Issue 7, Pages 1853-1868 (June 2002) Abnormal organogenesis of Peyer's patches in mice deficient for NF-κB1, NF-κB2, and Bcl-3  Stephan Paxian, Heidrun Merkle, Marc Riemann, Monika Wilda, Guido Adler, Horst Hameister, Susanne Liptay, Klaus Pfeffer, Roland M. Schmid  Gastroenterology  Volume 122, Issue 7, Pages 1853-1868 (June 2002) DOI: 10.1053/gast.2002.33651 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Generation of NF-κB2−/− mice. (A) Structure of the targeting vector, genomic organization, and the mutated locus of NF-κB2 after homologous recombination. Black boxes indicate exons 1a to 23. In the targeting vector, the PGK-neo cassette replaced exons 1b to 9, and the herpes simplex virus (HSV)-tk gene was added to the 3' side. The location of the DNA probe used for Southern analysis is shown (3' probe). (B) Southern blot analysis of tail DNA derived from wild-type (wt), NF-κB2+/−, and NF-κB2−/− littermates born to NF-κB2+/− parents. DNA was digested with DraI/EcoRI restriction enzymes, resulting in a 5.6-kb fragment for the wild-type allele and a 4.2-kb fragment for the targeted allele when hybridized with the 3' probe. (C) Supershift analysis of NF-κB2−/− spleen nuclear extracts for p52. Ab, antibody. (D) Western blot analysis of NF-κB1−/−, NF-κB2−/−, and Bcl-3−/− spleen whole-cell protein extracts for Bcl-3, p105/p50, and p100/p52. Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 A comparison of the number of PP in wild-type mice and in NF-κB1−/−, NF-κB2−/−, and Bcl-3−/− mice. In NF-κB2−/− mice, PP could not be identified macroscopically. NF-κB1−/− and Bcl-3−/− mice showed a reduced number of PP in the whole small intestine. Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Whole-mount preparations of the small intestine stained with IL-7Rα messenger RNA to detect B-cell follicles in PP structures. (A) Wild-type; note the sharp demarcated follicles. (B) Barely discriminable configurations were detected in NF-κB2−/− mice without follicles. (C) NF-κB1–deficient mice show a maximum of 2 undersized, but clearly demarcated, follicular structures. (D) Bcl-3 mutant mice display only a weak follicular staining without clear demarcations (original magnification 10×). Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Structural defects of PP of mice deficient for Bcl-3, NF-κB1, or NF-κB2. Paraffin sections of spleens were stained with H&E. Clear follicular structures compared with wild-type littermates (A) could not be detected in NF-κB2 (B), NF-κB1 (C), or Bcl-3 mutant mice (D). NF-κB2−/− PP were small in size and displayed no dome area and no clear demarcations to the gut mucosa but showed infiltration of leukocytes extending from the PP to the neighboring tissue (original magnification 100×). Arrowheads indicate follicles; asterisks indicate subepithelial dome region. Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Defective follicular organization in PP of NF-κB2–, NF-κB1–, and Bcl-3–deficient mice compared with wild-type controls. Immunocytochemical analyses were performed on cryostat sections. Bound antibodies were visualized with diaminobenzidine, and sections were counterstained with hematoxylin. B lymphocytes were identified by staining with anti-B220 (A) and anti-IgM (B). PP from wild-type mice show regularly distributed B-cell areas with formations of B-cell follicles and several well-formed GCs. Strongly enlarged B-cell follicles are present in PP from NF-κB1–deficient mice that contain only small numbers of GCs. Less identifiable B cells with no clear detectable follicular structures could be found in Bcl-3 mutant mice. Disorganized, very small B-cell accumulations terminal to PP structures could be identified in NF-κB2−/− mice. T cells stained with anti-CD3 were found to form distinct T cell–rich areas in wild-type mice (C). Compared with B cells in NF-κB1–deficient mice, T-cell areas were underrepresented and small in size. In contrast to wild-type and NF-κB1 mutant mice, PP of Bcl-3−/− and NF-κB2−/− mice mainly consisted of T cells (A and C, original magnification 100×; B, original magnification 200×). Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 NF-κB2 and Bcl-3 mutants failed to form GC, whereas these were reduced in number and size in NF-κB1–deficient mice. GCs were identified by staining with peanut agglutinin (PNA) expressed by B cells exclusively located in GCs (A). For visualization of follicular dendritic cells (FDC), sections were stained with FDC-M1 antibody (B). In PP of wild-type mice and NF-κB1 mutants, large numbers of FDC-M1–positive cells were distributed within the B-cell follicles. In PP of Bcl-3–deficient mice, FDC were regularly located within the smaller B-cell area but were reduced in numbers. In contrast, FDC were scattered in the narrow B-cell area adjacent to the PP of NF-κB2 mice (arrowheads). (Original magnification 200×). Asterisks indicate mucosal margin. Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) In wild-type and NF-κB1−/− mice, CD11b− CD8− DCs (brown) are preferentially located beneath the follicle-associated dome epithelium, and CD11b+ myeloid DCs (dark brown) are mainly dispersed in the SED and at the border to the B-cell follicle. In Bcl-3−/− mice, CD11b−CD8− DCs are recognizably reduced in number, and myeloid DCs show a disordered distribution in the SED and form a closer network in the adjacent B-cell region. Myeloid DCs and CD11b− CD8− DCs are significantly reduced in NF-κB2−/− mice and are mainly restricted to the small B-cell areas. (B) Analysis of lymphoid DCs by double labeling with CD11c (brown) and CD8 (red) showed that this DC subset (pink/brown) was preferentially located in the T-cell regions of wild-type and all mutant mice. In contrast to NF-κB1–deficient mice, Bcl-3−/− and, to a lesser extent, also NF-κB2−/− mice, showed an increase of CD8+ T cells (red) in the T-cell area. (C) Staining with NLDC, specific for DEC-205 expressed on lymphoid and activated myeloid DCs, confirmed these results. (D) Because the induction of a T helper type 2 and 3 response by myeloid DCs after encounter of food antigen is thought to take place in the SED, we analyzed the distribution of CD4+ T cells in the PP of the different mutant mice by double staining with anti-CD4 (brown) and B220 (red). Whereas substantial numbers of CD4+ T cells are located in the SED in close contact to the FAE in wild-type, NF-κB1−/−, and Bcl-3−/− mice, only a low number of CD4+ T cells are found in the proximity of the epithelium of B cell–infiltrated villi pertinent to the PP of NF-κB2–deficient mice. In all mutant mice, the majority of T cells in the IFR represent CD4+ T lymphocytes, but small germinal centers containing substantial amounts of CD4+ T cells comparable to wild-type mice can be detected only in PP of NF-κB1−/− mice. (A, wt), (B), and (D, wt, NF-κB1−/−, Bcl-3−/−) (original magnification 100×); (A, NF-κB2−/−, NF-κB1−/−, Bcl-3−/−), (C), and (D, NF-κB2−/−) (original magnification 200×). Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Reverse-transcription–PCR of genes of NF-κB2−/− mice, known as NF-κB regulated, or genes known to be required for PP development or homing of lymphocyte populations. Messenger RNA was quantitated in spleen (A) and small intestine (B) by using real-time PCR as described in Materials and Methods. Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 GALT-specific expression of MAdCAM-1 in PP of adult mice. Cryosections of mouse small intestine were incubated with biotinylated rat anti-mouse MAdCAM-1 and detected with streptavidin-horseradish peroxidase. MAdCAM-1 is expressed in the PP HEV and is less distinctive in the follicular DCs of wild-type mice mainly located in the GC and SED (A). (B) NF-κB2–deficient mice show increased numbers of HEV with an altered distribution but lack MAdCAM-1 expressing follicular DCs. (C) NF-κB1−/− mice show slightly reduced numbers of HEV but a generous reduction of MAdCAM-1–positive DCs in GC and small GC. (D) Bcl-3–deficient mice display increased numbers of HEV but only small numbers of MAdCAM-1–expressing DCs located in the SED, but not within the B-cell follicle (original magnification 100×). Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 Bcl-3 and NF-κB2, but not NF-κB1, are required for a proper expression of the chemokines MIP-3α and BLC in PP. Immunohistochemical analysis of MIP-3α (brown) and B cells (red) reveals that in PP of wild-type mice, MIP-3α is mainly produced by epithelial cells of the FAE covering the SED, some stromal cells underlining the FAE, and presumably DCs dispersed in the SED (A). Although in p52-deficient mice and the majority of Bcl-3–deficient mice the expression of MIP-3α is completely abolished within the PP and is restricted to a few epithelial cells within the epithelium covering the SED-like extravasations infiltrated with significant numbers of B cells, MIP-3α may be detected in a substantial number of epithelial cells but only a tiny number of cells within rather large SED regions in PP of Bcl-3−/− mice. Note the only moderate reduction of MIP-3α–secreting cells in the FAE and within the SED of NF-κB1–deficient mice. High numbers of BLC-expressing cells are dispersed within the B-cell follicle and GC of wild-type and NF-κB1−/− mice but cannot be detected within the PP of NF-κB2–deficient mice and the majority of Bcl-3 mutant mice (B). (C) Whereas wild-type, NF-κB1, and Bcl-3 mutants display a strong anti-TECK reactivity in the epithelial cells, expression of this chemokine is substantially reduced in NF-κB2–deficient mice. (A, wt) and (C) original magnification 100×; (A, NF-κB2−/−, NF-κB1−/−, Bcl-3−/−), and (B) original magnification 200×. Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 11 All mutant mice show an increase of M cells, which is mostly pronounced in NF-κB2−/− mice. M cells are stained by exploitation of their selective binding capacity of the lectin Ulex europaeus according to the protocol depicted in Materials and Methods. Although only a slight increase of M cells could be observed in NF-κB1 (C), Bcl-3–deficient mice (D) showed a 2–3-fold enhancement and NF-κB2−/− (B) showed a 3–5-fold enhancement of the total number of M cells compared with wild-type controls (A). In contrast to the other mutant mice, in which M cells were still clearly restricted to the FAE covering a demarcated SED region, NF-κB2–deficient mice displayed a confused distribution of these cells along the whole epithelium covering the PP (original magnification 200×). Gastroenterology 2002 122, 1853-1868DOI: (10.1053/gast.2002.33651) Copyright © 2002 American Gastroenterological Association Terms and Conditions