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Blood, Vol. 110, Issue 9, 3407-3416, November 1, 2007
Reticulocyte-secreted exosomes bind natural IgM antibodies: involvement of a ROS-activatable endosomal phospholipase iPLA2
Blood Blanc et al.
110: 3407
Supplemental materials for: Blanc et al
Files in this Data Supplement:
- Figure S1. IgM antibodies bind to exosomes following their release (JPG, 108 KB)
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(A) Binding of IgM to apoptotic cells was carried out as in figure 7, after pre-incubation of FCS (100 µl) with microplates coated with phosphatidylcholine (PC) or lysophosphatidylcholine (LPC). (B) Reticulocytes and erythrocytes were assessed for IgM binding from FCS. (Tinted patterns: absence of primary antibody; dotted lines: specific binding).
- Figure S2. Red cells maturation in rats rendered anemic by repeat phlebotomy (JPG, 91.8 KB)
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Two immature red cells populations at different maturation stages were purified by Percoll gradient from the blood of a rat rendered anemic by repeated bleeding. RNA levels were monitored using Thiazole orange as described in the “Materials and Methods” (A), mitochondrial membrane potential was monitored using MitoTracker CMXRos (B) and intracellular ROS was assayed using the hydroethidine dye (C). These assays were performed on immature (left panels) and more mature (right panels) red cells.
- Figure S3. IgM binding and C3 deposition on exosomes from a phlebotomized rat (JPG, 109 KB)
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Latex beads were coated with exosomes isolated after in vitro reticulocyte maturation or directly from the plasma of an anemic animal. The complexes were then analyzed by flow cytometry for the presence of IgM and C3 using the appropriate antibodies. Shaded histograms: absence of primary antibodies.
- Figure S4. Proposed model for the clearance of reticulocyte-secreted exosomes (JPG, 80.4 KB)
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Based on the example of Transferrin Receptor (TfR) sorting into exosomes, we propose a mechanism in which the endosomal iPLA2 can be activated (iPLA2*) by caspase 3 cleavage and ROS liberated during mitoptosis. This iPLA2 is then secreted in association with exosomes. At this point, the phospholipase can generate lysophosphatidylcholine (LPC) leading to the recruitment of IgM antibodies and C3 component on the surface of the vesicles, favoring the elimination of exosomes via a mechanism similar to that involved in the clearance of apoptotic cells.
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