Fig. 3.

Macrophages are the sources of CRT and secrete CRT to label the target cells. a Expression of CRT measured by qRT-PCR in neutrophils and macrophages 8 h after thioglycollate. Macrophages and neutrophils were collected from MRP8-Bcl2 mice as described in “Experimental Procedures”. CRT mRNA level in macrophages were dramatically higher in macrophages as compared to neutrophils. *P < 0.05 (t-test) for expression of CRT between macrophages and neutrophils. Error bars represent standard deviation. b, c Immunofluorescent staining of CRT in mouse macrophages (b) and neutrophils (c). CRT is undetectable in neutrophils but abundant in macrophages. CRT localized to perinuclear regions, vesicles and cell surface of macrophages. Macrophages and neutrophils were collected from MRP8-Bcl2 mice. d ELISA assay showing the amount of CRT in medium (RPMI) with and without macrophages. n = 3. Error bars represent standard deviation. Macrophages were able to secrete CRT to the extracellular medium. e Expression levels of cell surface CRT on neutrophils cultured alone or with macrophages in a 0.4-μm Boyden chamber overnight, assayed by flow cytometry. Co-culture with macrophages led to a significant increase of cell surface CRT on neutrophils. n = 3. **P < 0.01 (t-test) for expression levels of cell surface CRT on neutrophils cultured alone or with macrophages. Error bars represent standard deviation. f A schematic showing the Click-iT assay to examine transfer of CRT from macrophages to neutrophils during co-culture. Macrophages proteins were labeled with a methionine analog AHA (l-azidohomoalanine) receptive for click chemistry. The proteins secreted by macrophages were all labeled with AHA. Because neutrophils and macrophages were cultured in a way independent of contact, the AHA-labeled proteins detected on neutrophils originate from the macrophages. g Neutrophils were cultured alone or together with AHA-treated macrophages in a 0.4-μm Boyden chamber. Cells were collected and CRT was immunoprecipitated and then subjected to click-chemistry adding in biotin to receptive methionine analogs. Samples were subjected to SDS-PAGE and then western blot for the presence of biotin. Detection of AHA-labeled CRT on neutrophils indicated that macrophage-secreted CRT to label neutrophils and this was independent of the contact between these two cell types