1. by Feng Guo, Debra Weih, Elke Meier, and Falk Weih
Constitutive alternative NF-κB signaling promotes marginal zone B-cell development but disrupts the marginal sinus and induces HEV-like structures in the spleen
by Feng Guo, Debra Weih, Elke Meier, and Falk Weih
Blood
Volume 110(7):
October 1, 2007
©2007 by American Society of Hematology

2. Reduced percentage of immature B cells in spleens from p100−/− mice.
Reduced percentage of immature B cells in spleens from p100−/− mice. Splenocytes from 17- to 20-day-old wild-type, p100−/−, and nfkb2−/− mice were stained for CD19 and the early B-cell marker 493 (top row) or CD19 and IgM (bottom row) and analyzed by flow cytometry (lymphocyte gate). Numbers indicate percentages from indicated regions. Representative data from 3 to 4 experiments are shown.
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology

3. Mice lacking p100 have markedly increased numbers of MZ B cells.
Mice lacking p100 have markedly increased numbers of MZ B cells. (A) Splenocytes from wild-type, p100−/−, and nfkb2−/− mice were stained for CD23 and CD21 and analyzed by flow cytometry (lymphocyte gate). Percentages indicate CD23−CD21+ MZ B cells. (B) CD23− (top row) and CD23+ (bottom row) splenocyte subpopulations from wild-type, p100−/−, and nfkb2−/− mice were analyzed for CD21 and IgM expression. Percentages of newly formed T1 B cells (CD23−CD21−IgMhi), T2 B cells (CD23+CD21+IgMhi), MZ B cells (CD23−CD21hiIgMhi), and FO B cells (CD23+CD21intIgMlo) are shown (black regions). Among the T2 B-cell subpopulation, percentages of presumed MZ precursor B cells (MZPB; CD23+CD21hiIgMhi) are indicated (red regions). Representative data from 3 to 4 experiments are shown. (C) Absolute numbers of B-cell subpopulations per 106 splenocytes from wild-type, p100−/−, and nfkb2−/− mice are shown. Error bars indicate standard deviation (SD) from at least 5 mice per genotype. (D) MZ B cells from p100−/− mice show better survival than wild-type controls. Splenocytes were cultured for 48 hours in media or stimulated with LPS or anti-CD40 mAb. Percentages of Annexin-V+ apoptotic cells in MZ and FO B-cell subpopulations are shown. Error bars indicate SD from 3 experiments. Significant differences are indicated (Student t test, *P < .05; **P < .01; ***P < .002). (E) Spleens from p100−/− mice exhibit an enlarged MZ B-cell population adjacent to MOMA-1+ macrophages. Spleen sections from age-matched wild-type and p100−/− mice were stained with anti-IgM for B cells (Vector Blue) and MOMA-1 for MMMs (DAB brown). Objective: × 40. Images were acquired through an AX70 Olympus microscope (Olympus, Hamburg, Germany) with a 40×/0.75 objective by an Olympus DP70 digital camera running analySISB Soft Imaging System software (Olympus) and processed with Adobe Photoshop 8.0 software (Adobe, San Jose, CA).
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology

4. Accumulation of MZ B cells in p100−/− mice is cell intrinsic.
Accumulation of MZ B cells in p100−/− mice is cell intrinsic. (A) Lethally irradiated C57BL/6 recipients were reconstituted with either wild-type (wt → B6) or p100−/− bone marrow cells (p100−/− → B6). At 6 weeks after transplantation, splenocytes from chimeras were stained for CD23, CD21, and IgM and analyzed for MZ B cells (CD23−CD21hiIgMhi). Only CD23− lymphocytes are shown. (B) MZ B-cell accumulation in p100−/− mice is dependent on relB. Splenocytes from wild-type, p100−/−relB−/+, and p100−/−relB−/− mice were analyzed for MZ B cells as described in panel A.
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology

5. Deregulated chemokine expression in p100−/− spleens.
Deregulated chemokine expression in p100−/− spleens. Total RNA from wild-type and p100−/− spleens was analyzed for chemokine expression by QRT-PCR. Relative mRNA expression corrected for β-actin levels is shown. Error bars indicate SD from 3 to 4 experiments. Significant differences are indicated (Student t test, **P < .01; ***P < .002).
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology

6. Impaired LPS-induced relocation of p100−/− MZ B cells.
Impaired LPS-induced relocation of p100−/− MZ B cells. (A) Wild-type or p100−/− mice were injected with PBS or LPS as indicated. After 4 hours, spleens were removed and frozen sections were stained for IgM (blue) and MOMA-1 (brown). Note that a significant number of B cells remained outside the MOMA-1+ ring in spleens from LPS-treated p100−/− mice. Images were acquired as in Figure 2E with a 20×/0.70 objective. (B) Impaired migration of p100−/− MZ B cells to CXCL13. B-cell chemotaxis to CXCL13 and S1P was analyzed in Transwell assays. Error bars indicate SD from 3 to 4 experiments. Significant differences are indicated (Student t test, **P < .01; ***P < .002).
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology

7. Increased adhesion of p100−/− MZ B cells to VCAM-1.
Increased adhesion of p100−/− MZ B cells to VCAM-1. (A) Adhesion of MZ B cells to plate-bound recombinant Fc-VCAM-1 was measured as described in “Cell culture, detection of apoptotic cells, B-cell migration, and adhesion asays.” (B) Increased mRNA expression of cell-adhesion molecules in spleens from p100−/− mice. Total RNA from wild-type and p100−/− spleens was analyzed for VCAM-1, ICAM-1, and MAdCAM-1 expression by QRT-PCR. Relative mRNA expression corrected for β-actin levels is shown. Error bars indicate SD from 3 to 4 experiments. Significant differences are indicated (Student t test, *P < .05; **P < .01). Immunohistochemical staining of spleen sections from wild-type and p100−/− mice for (C) VCAM-1 (DAB brown), (D) MZMs (MARCO+; DAB brown), and (E) MAdCAM-1 (MECA-367+; Vector Blue). Sections in panels C and D were counterstained with hematoxylin. (F) Immunohistochemical staining of spleen sections (MARCO+, DAB brown; MECA-367+, Vector Blue) from lethally irradiated C57BL/6 mice that were reconstituted with wild-type (left panel) or p100−/− bone marrow (right panel). Images were acquired as in Figure 2E, with 20×/0.70 objective (C), 10×/0.40 objective (D,E), and 40×/0.75 objective (F).
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology

8. Lack of p100 results in the induction of ectopic HEV-like structures in the splenic red pulp.
Lack of p100 results in the induction of ectopic HEV-like structures in the splenic red pulp. (A) QRT-PCR analysis of GlyCAM-1, β3GlcNAcT-3 (core 1), Core2GlcNAcT-I (core 2), GlcNAc6ST-1, GlcNAc6ST-2, and FucT-VII in spleens from wild-type and p100−/− mice. Note the log scale of relative mRNA expression. Error bars are SD. Significant differences are indicated (Student t test, ***P < .002). (B) Immunohistochemical staining of wild-type and p100−/− spleen sections for MZMs (anti-MARCO; DAB brown) and MECA-79 (Vector Blue). (C) 2-color immunofluorescence of p100−/− spleen sections stained with MECA-367 (M367; FITC green) and MECA-79 mAbs (M79; Texas red). Most MECA-367+ structures exhibited coexpression of PNAd (merge; yellow fluorescence). Insets, mesenteric LN HEVs from p100−/− mice. (D) 2-color immunofluorescence of p100−/− spleen sections stained with MECA-79 (M79; FITC green) and anti-CCL21 (SLC; Texas red). Most MECA-79+ structures exhibited coexpression of CCL21 (merge; yellow fluorescence). (E) Spleen sections from p100−/− mice (middle panel) and control littermates (left panel) that were intravenously injected with FITC-labeled E. coli were stained with the IBL-11 mAb (red) to indicate the white pulp. The right panel shows FITC-E. coli in p100−/− spleen sections stained with MECA-367 (Vector Blue) to indicate ectopic HEV-like structures. Dotted lines mark the boundary of the white pulp. Images were acquired as in Figure 2E, with 20×/0.70 objective (B), 40×/0.75 objective (C,D); 20×/0.70 objective (panel E left and middle), 40×/0.75 objective (panel E right).
Feng Guo et al. Blood 2007;110:
©2007 by American Society of Hematology