Role of CD47 as a Marker of Self on Red Blood Cells
Tóm tắt
Từ khóa
Tài liệu tham khảo
Yokoyama W. M., Curr. Biol. 7, 110 (1995).
Colonna M., Navarro F., López-Botet M., Curr. Top. Microbiol. Immunol. 244, 115 (1999).
Campbell I. G., Freemont P. S., Foulkes W., Trowsdale J., Cancer Res. 52, 5416 (1992).
Male and female CD47 − / − C57BL/6J mice backcrossed to C57BL/6J (Jackson Laboratory) for 16 or more generations and their heterozygous and homozygous littermates were from our breeding colony. All experiments described were also reproduced with isogenic 129sv/eg mice with very similar results (not shown). Rag1 − / − mice (32) on a C57BL/6J background were a gift from D. Chaplin (Washington University St. Louis MO). C3 − / − mice (33) on a mixed 129/C56BL/6 background were a gift from H. Molina (Washington University St. Louis MO). Animals were kept in accordance with NIH and local guidelines and maintained in a specific pathogen-free barrier facility.
RBCs were stained with the fluorescent dyes 5/6-carboxyfluorescein diacetate succimidyl ester (CFSE; Molecular Probes Eugene OR) or PKH-26 (Sigma) and resuspended in sterile pyrogen-free 0.9% NaCl to 30% (v/v). Recipients were given 200 μl of the stained RBCs and the clearance of fluorescent RBCs was measured by flow cytometry (Epics XL; Coulter Hialeah FL) of 5-μl blood samples collected from a tail vein at the time points indicated. The fraction of fluorescent RBCs of the total number of recipient RBCs studied (100 000 per sample) was determined. Levels at 5 15 and 30 min were virtually identical. The data were normalized to the fraction at 30 min after injection (donor RBCs constituted 4 to 6% of the total number of RBCs at this time point). Results were independent of the dye used. Control experiments with unlabeled CD47 − / − RBCs in wild-type recipients and wild-type RBCs in CD47 − / − recipients (detection by staining for CD47 and flow cytometry) gave similar results.
Dichloromethylene diphosphonate (clodronate; a gift from Roche Diagnostics/Boehringer Mannheim GmbH) liposomes was prepared as described (18). Clodronate (200 μl) or NaCl liposomes were given to recipient mice by tail-vein injection at 48 and 24 hours before RBC transfer. Immunohistochemical analyses revealed a complete macrophage depletion in recipient spleens 48 hours after a single injection of clodronate liposomes.
Spleen specimens were taken from recipients at 20 hours after the administration of PKH26-stained CD47 − / − RBCs. Spleens were embedded in O.C.T compound (Sakura Finetek U.S.A. Torrance CA) frozen in liquid nitrogen and stored at −80°C. Cryostat sections (8 μm) were incubated with antibodies to F4/80 [red pulp macrophages (34)] or MOMA-1 [marginal metallophilic macrophages (35)]. After incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies F4/80 + and MOMA-1 + cells were visualized with Tyramide Signal Amplification (NEN Life Science Products Boston MA).
Wild-type spleens were digested with collagenase D (Roche Diagnostics Indianapolis IN) [400 U/ml in Hanks balanced salt solution (HBSS) without Ca 2+ or Mg 2+ ] for 30 min at 37°C. After RBC lysis and extensive washing the cells were resuspended to 5 × 10 7 /ml in ice-cold HBSS with 1 mM Ca 2+ 1 mM Mg 2+ 1% human serum albumin (HSA). Macrophages were allowed to adhere for 60 min at 37°C in plastic eight-well Lab-Tek chamber slides (200 μl/well; Nalgene Nunc International Naperville IL). Anti-SIRPα P84 (5 μg) (9) or the isotype-matched control (anti-murine CD14; clone rmC5-3; Pharmingen) or no antibody was present during the last 15 min. Subsequently 50 μl of PKH26-labeled wild-type or CD47 − / − RBCs were added to each well for another 60 min. Nonadherent cells were washed off with warm phosphate-buffered saline (PBS)−0.1% HSA and noningested RBCs were lysed with ACK lysis buffer. Phagocytosis index (the number of RBCs phagocytosed per 100 adherent macrophages) was determined by fluorescence microscopy (Zeiss Axioscope).
Bone marrow–derived macrophages were prepared as described (36). Mature macrophages (1.5 × 10 6 ) were plated on 60-mm bacteriological plastic Petri dishes (Valmark; Midwest Scientific St. Louis MO) for 16 hours before experiments. The cells were rinsed once with PBS and 2 ml of cold serum-free Iscove's modified DMEM (IMDM) was added to each dish. After 2 min at 4°C 2 × 10 6 wild-type or CD47 − / − RBCs were added then allowed to settle for 3 min at 4°C after which sodium pervanadate (Sigma) was added to 2 mM and cells were transferred to a water bath at 37°C. At the times indicated the medium was quickly aspirated and cells were lysed in cold lysis buffer [50 mM tris-HCl (pH 7.5) 150 mM NaCl 1% NP-40 (Pierce Rockford IL) 1 mM diisopropyl aprotinin (10 μg/ml) 2 mM sodium pervanadate]. Lysates were cleared by centrifugation and incubated for 2 hours at 4°C with anti-SIRP monoclonal antibody (mAb) P84 bound to 20 μl of goat anti-rat Sepharose (ICN Costa Mesa CA) for 2 hours at 4°C. After two washes in lysis buffer immunoprecipitated proteins were eluted with reducing SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer separated on 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Bedford MA). Tyrosine phosphorylation was detected by Western blot with anti-phosphotyrosine mAb 4G10 followed by HRP-conjugated secondary antibody and enhanced chemoluminescence as described (36).
Reviewed in
F. P. Lindberg et al. unpublished data.
L. G. Poels et al. J. Natl. Cancer Inst. 76 781 (1986).
Kraal G., Janse M., Immunology 58, 665 (1986).
T. Roach et al. Curr. Biol. 7 408 (1997).
We thank T. Steinberg W. Yokoyama J. Atkinson and E. Brown for critically reading the manuscript; D. Chaplin H. Molina and P. Leenen for reagents; and E. Ford for technical assistance. Supported by grants from the NIH (GM57573-01) the American Diabetes Association the Washington University–Monsanto Research Agreement a pilot grant from the Howard Hughes Medical Institute and the Medical Research Service of the Depatment of Veterans Affairs. P.-A.O is supported by a postdoctoral fellowship from the Umeå University–Washington University exchange program and by the Swedish Medical Research Council.