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First published online 3 January 2006
doi: 10.1242/jcs.02741

Journal of Cell Science 119, 314-325 (2006)
Published by The Company of Biologists 2006
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CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells

Heidi Schabath, Steffen Runz, Safwan Joumaa and Peter Altevogt*

Tumor Immunology Programme, D010, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany



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  		Fig. 1. Cell migration of CD19+ bone marrow and spleen-derived cells from CD24–/– and CD24+/+ mice. (A) CD19+ B-lymphocytic cells from the bone marrow and spleen of CD24–/– and control C57BL/6 mice were isolated using magnetic beads. An aliquot of cells was used for FACS analysis of CD24 and CD45 expression. A CD24 isotype control antibody was also used. (B,C) Chemotactic cell migration of CD24–/– versus CD24+/+ B lymphocytes from the bone marrow (B) and spleen (C) in response to SDF-1. *P≤0.035; **P≤0.02.

 


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  		Fig. 2. CD24 alters SDF-1-induced chemotaxis in pre-B cell lines. (A) Cytofluorographic analysis of N232.18, 18H18– (CD24–/–) and 18H18+ (CD24+/+) cells with monoclonal antibodies to CD24 (mAb 79) followed by PE-conjugated goat anti-rat IgG and chemokine receptor CXCR4 with biotinylated mAb 2B11 followed by streptavidin-PE. (B) Chemotactic cell migration of N232.18 and 18H18– (CD24–/–) versus 18H18+ (CD24+/+) cells in response to SDF-1. *P≤0.05; **P≤0.03. (C) Chemotactic cell migration of N232.18 (CD24–/–) versus 18H18+ (CD24+/+) towards chemokines IP-10, MIP-3{alpha}, BLC and SDF-1. *P≤0.033. (D) Detection of CXCR4 receptor by western blot analysis. Cell lysates from the indicated cell lines were tested. Calnexin was detected as a loading control. (E) Binding of biotinylated SDF-1 to CD24–/– (N232.18 and 18H18–) and CD24+/+ (18H18+) cells. Cells were stained with biotinylated SDF-1 followed by streptavidin-FITC and analysed by FACS.

 


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  		Fig. 3. CD24 affects cellular cholesterol levels in pre-B cells. (A) Cellular cholesterol levels in the indicated cell lines were determined using the Amplex Red colorimetric method. Equal numbers of cells were used. (B) Non-esterified cholesterol levels were measured by staining with the cholesterol-binding dye Filipin and FACS analysis.

 


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  		Fig. 4. Cellular cholesterol levels affect chemotactic cell migration. (A) CD24+/+ 18H18+ cells were loaded with cholesterol by incubation with 30 µg/ml soluble cholesterol and tested for chemotactic cell migration in response to SDF-1. CD24–/– cells N232.18 and 18H18– were treated with fluvastatin for 72 hours and then tested for chemotactic cell migration in response to SDF-1. Data are presented as the percentage migration (± s.e.m.) compared with untreated control cells. (B) SDF-1-triggered ERK-phosphorylation in CD24–/– (N232.18 and 18H18–) and CD24+/+ (18H18+) cells. Cells were exposed for the indicated length of time (in minutes) to 100 ng/ml SDF-1 then lysed. An equal volume of lysate was mixed with sample buffer and analysed by SDS-PAGE and western blotting using a phospho-ERK-specific antibody and ECL detection. For a loading control, blots were reprobed with an ERK-1-specific antibody. (C) CD24–/– (18H18–) cells were treated with fluvastatin for 72 hours and then ERK phosphorylation in response to SDF-1 was tested. Note that ERK phosphorylation in 18H18– cells was abolished after fluvastatin treatment.

 


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  		Fig. 5. CD24high and CD24low variants of MDA-MB-231 breast carcinoma cells. (A) Phenotype of MDA-MB-231 sub-lines. CD24high-expressing cells were generated by stable overexpression of CD24 and FACS selection. Both sub-lines were transduced with a retroviral CXCR4-GFP expression construct. The transduced cells were sorted for CXCR4 expression. (B) Migration of MDA-MB-231 CD24high versus CD24low breast carcinoma cells (either non-transduced or CXCR4-GFP tranduced) in response to SDF-1. Note the overall elevated level of cell migration in CXCR4-GFP-overexpressing cells (**P≤0.033; *P≤0.05; ***P≤0.02). (C) Migration of MDA-MB-231 CD24high CXCR4-GFP cells after transfection with CD24-specific or control siRNA. FACS analysis indicates reduced CD24 expression 72 hours after CD24 siRNA transfection compared with the control siRNA (*P≤0.013).

 


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  		Fig. 6. CD24 prevents growth of MDA-MB-231 tumours in NOD/SCID mice. (A) Tumour volume of subcutaneously injected MDA-MB-231 CD24high and CD24low cells after 37 days (n=7; *P≤0.0039). (B) Tumour volume of subcutaneously injected MDA-MB-231 CD24high CXCR4-GFP and MDA-MB-231 CD24low CXCR4-GFP cells after 37 days (n=8; * P≤0.0019). (C) Cell numbers of MDA-MB-231 CD24high and CD24low cells after incubation with different SDF-1 concentrations for 24 hours (*P≤0.019; **P≤0.007).

 


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  		Fig. 7. CD24 affects the localization of CXCR4 in lipid rafts. (A) Distribution of CD24 in membrane rafts isolated from CD24+/+ 18H18+ cells. Gradient fractions were probed with mAb M1.69 to mouse CD24 followed by peroxidase-conjugated secondary antibody and ECL detection. (B) Distribution of CXCR4 in raft and non-raft fractions of the indicated pre-B-cell lines (upper panels). Note the presence of CXCR4 only in raft fraction 2 of CD24–/– cells (*). Distribution of Fyn in raft and non-raft fractions of the indicated pre-B cell lines (lower panels). Note the presence of Fyn in raft fraction 2 of all cell lines. (C) Distribution of CXCR4 in raft and non-raft fractions of CD24high and CD24low MDA-MB-231 CXCR4-GFP cells. Note the presence of CXCR4 in raft fraction 2 of only CD24low cells as indicated with (*) and the equal presence of Fyn in both cell lines.

 


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  		Fig. 8. Model of CD24 activity and CXCR4 function. In CD24–/– cells (N232.18 and 18H18– as well as in MDA-MB-231 CD24low cells) the CXCR4 receptor is more abundant in membrane rafts. As a result of this residence, CXCR4 signalling in response to SDF-1 triggers cell motility via ERK phosphorylation. In CD24+/+ cells (18H18+ and MDA-MB-231 CD24high cells), CXCR4 is excluded from lipid rafts and cannot transmit signals in response to SDF-1. This prevents ERK phosphorylation, blocks cell motility and attenuates tumour growth.

 

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© The Company of Biologists Ltd 2006