Costimulatory molecule expression on stimulated basophils.

<p>CD40 (A), CD80 (B), CD86 (C) and MHC Class II (D) expression on IL-3, IFN-γ and GM-CSF stimulated basophils of 2 atopic HDM-allergic (i,ii) and 1 non-atopic (iii) donor. CpG-stimulated B cells served as a positive control for costimulatory molecule detection (iv). Gates were determined using corresponding isotype controls.</p

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

MHC Class II expression on stimulated basophils.

<p>Representative dot plots showing MHC Class II expression on basophils freshly isolated (A), and after 72 hours culture with medium alone (B) or IL-3 (10 ng/ml), IFN-γ and GM-CSF (100 ng/ml) (C). Corresponding isotype control (D). Percentage of MHC Class II positive basophils for individual atopic HDM-allergic (open shapes) and non-atopic (closed shapes) donors after 72 hours culture with various stimuli (E). Differences between groups were calculated with One-way ANOVA and Dunett's Multiple comparison post-test with IL-3 as the control group for complete data sets of the 6 donors, * p&lt;0.05, ** p&lt;0.01.</p

T cell proliferation assay.

<p>(i) Percentage of MHC Class II positive basophils after 72 hours culture with IL-3, IFN-γ and GM-CSF prior to co-culture with T cells. HDM extract (10 µg/ml) was also added to the basophil stimulation culture for A. (ii) Percentage of CD25⁺ CFSE^low proliferating CD3⁺CD4⁺ T cells after culture of T cells alone or with autologous basophils or monocytes in the presence of 10 µg/ml HDM (A, non-atopic donor; similar results for 2 HDM-allergic donors) for 72 hours or 10 µg/ml peptide (B) for 120 hours. T cell: APC ratio indicated below. (C) Representative dot plot showing gating strategy for identification of proliferating CD3⁺CD4⁺ T cells.</p

Gene expression of MHC Class II components and costimulatory molecules.

<p>RT-PCR of RNA from freshly isolated basophils (1), MHC Class II negative (2) and MHC Class II positive basophils (3) after 72 hours of culture with IL-3, IFN-γ, GM-CSF and HDM extract, compared with CD123⁺ IgE^low pDC (4), CD19⁺ B cells from freshly isolated PBMC (5) and CD19⁺ B cells from CpG stimulated PBMC (6) as positive controls, and a RT negative control (7). Data for a non-atopic donor are shown, representative of 5 separate experiments.</p

Isolated basophil purity and viability.

<p>Representative dot plots showing proportion of basophils in PBMC and after isolation assessed by antibodies specific for (A) IgE and CD123, (B) FcεRI and CD203c, (C) FcεRI and Lineage-1. Example of basophil viability after culture for 72 hours with (D) IL-3 (10 ng/ml) or (E) medium alone. (F) Percentage of viable basophils (7AAD⁻, Annexin V⁻) after 72 hours of culture with a panel of cytokines and TLR ligands. Representative of 2 separate experiments.</p

CXCR7, CXCR4, and CXCL12 expression in mature glomeruli.

<p>(A–C) Brightfield micrographs of cresyl violet stained mature glomeruli (E16.5) after hybridization with ³⁵S- labeled probes for CXCR7, CXCR4, and CXCL12. (<b>A,B</b>) Signals for CXCR7 are restricted to the podocyte layer whereas CXCR4 is restricted to the center of the glomerulus. (<b>C</b>) CXCL12 is detected both in the podocytes and in the area of the vascular pole (vp, arrowhead). (D–E) Mature glomeruli after dual in situ hybridization with ³⁵S labeled probes for CXCR4 (D), CXCL12 (E), and a DIG-labeled probe for podocyte marker WT1 (D,E). (<b>D</b>) The darkfield micrograph reveals CXCR4 labeling (white signals) close to the vascular pole but not in WT1 stained podocytes of the visceral blade of Bowman's capsule. (<b>E</b>) The brightfield image shows labeling for CXCL12 mRNA (black grains) in the WT1 positive podocyte layer and in the WT1 negative area of the vascular pole (vp, arrowhead). (F–G) Confocal images of dual immunofluorescent stainings for GFP/CXCR4 (F) and GFP/podocin (G) in E16.5 BAC transgenic mice expressing EGFP under the control of the CXCR7 promoter. (<b>F</b>) CXCR4 immunoreactivity is present in the glomerular tuft (arrow), presumptive arterioles (arrowheads), and at the luminar membrane of tubular epithelial cells (asteriscs). Some tubules are co-positive for CXCR7-GFP and CXCR4 (filled asteristics), others display exclusively CXCR4 protein signals (open asteristics). In the glomerulus, signals for CXCR4 and CXCR7-GFP do not overlap. (<b>G</b>) Podocytes labeled by the selective marker podocin (red) are CXCR7-GFP positive. Scale bars represent 10 µm (C,G′″) and 20 µm (F).</p

Differential expression of CXCR7 and CXCR4 in nephrogenic mesenchyme, ureteric bud, and forming glomeruli.

<p>(A–D) A digoxigenin (DIG) labeled WT1 antisense probe was used as a marker for mesenchymal and mesenchyme derived nephrogenic structures at E14.5 and co-hybridized with ³⁵S labeled probes for CXCR7 or CXCR4. (<b>A</b>) Strong CXCR7 signals (black grains) are present in a T-shaped WT1 negative early ureteric bud tip (eub) which is associated with a CXCR7/WT1 co-positive pretubular aggregate (pa; black grains/brown staining). Weaker CXCR7 expression is found in a late ureteric bud tip (lub) which is associated with CXCR7/WT1 co-positive renal vesicles (rv). Note weak CXCR7 mRNA expression in the cap mesenchyme (cm) and strong CXCR7 gene activity above the cm (brown staining) at the renal capsule (rc). (<b>B,B′,C,C′</b>) Bright- and darkfield views of a comma-shaped body (cb in B) and S-shaped body (sb in C) after hybridization with a DIG labeled WT1 probe and a ³⁵S labeled CXCR7 riboprobe (black grains in B,C; white grains in B′,C′). Both WT1 positive structures exhibit clear CXCR7 antisense mRNA signals. (<b>D,D′</b>) Bright- and darkfield micrographs showing WT1-positive renal tissue (D) and radiosignals of CXCR4 riboprobe (D′). Strong CXCR4 gene expression is detected in a WT1 negative early ureteric bud tip (eub in D, dotted line in D′). Weak CXCR4 labeling is seen in a late ureteric bud tip (lub) which is associated with a WT1 positive/CXCR4 negative renal vesicle (rv). Note that CXCR4 mRNA is also present in WT1 positive cap mesenchyme (cm). WT1 positive epithelial cells of S-Shaped bodies display no CXCR4 mRNA expression. The vascular cleft of S-shaped bodies (arrows in D′) as well as putative arterioles (arrowhead in D′) are CXCR4 positive. (<b>E</b>,<b>F</b>) GFP immunostaining in sections from BAC transgenic mice expressing EGFP under the control of the CXCR7 promotor (G) or CXCR4 promotor (H). Calbindin was co-stained as an ureteric bud marker. (<b>G</b>) CXCR7-GFP is highly expressed in the renal capsule (rc) as well as in comma- and S-shaped bodies (cb, sb) associated with a calbindin positive late ureteric bud (lub). CXCR7-GFP is weak expressed in cap mesenchyme (cm) and not present in the late ureteric bud. (<b>H</b>) Strong CXCR4-GFP signals are detected only in cap mesenchyme (cm). All scale bars equal 20 µm.</p

Histological analysis of CXCR7-deficient kidneys.

<p>Kidney sections of E16.5 wildtype (A–C) and CXCR7 deficient littermates (D–F) were stained with periodic acid-Schiff (PAS) reagents. (<b>A,D</b>) Macroscopic images demonstrate that the overall kidney morphology is normal in the CXCR7 knockout embryo. (<b>B,C,E,F</b>) Microscopic views of pretubular aggregates (pa), renal vesicles (rv), ureteric buds (ub) (B,E), and S-shaped bodies (C,F) do not reveal any abnormalities in the CXCR7 knockout kidney. Scale bars equal 200 µm (D) and 20 µm (B,C).</p

Transmission electron microscopic analysis of the glomeruli.

<p>Glomerular capillaries at 3.300× magnification (<b>A</b>,<b>B</b>) and details of the filtration barrier at 21.600× magnification (<b>C</b>,<b>D</b>; framing in A,B). CXCR7^+/+ (C) and CXCR7^−/− (D) showed no differences in general morphology and glomerular basement membrane (gbm) attachment of podocytes (pod). However endothelial cells (en) seemed to be detached from the gbm (arrows). Scale bars correspond to 2 µm (A,B) and 0.3 µm (C,D).</p