Reticulocyte-secreted exosomes bind natural IgM antibodies: involvement of a ROS-activatable endosomal phospholipase iPLA2

Abstract

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. It has been suggested that reticulocytes remodel the plasma membrane of the immature red cell during erythropoiesis by specifically eliminating various proteins. We report here that exosome release is associated with a physiologic cascade induced by the expression of a 15-lipoxygenase at the reticulocyte stage. We found that the phospholipase iPLA2 specifically associated with the endosomal and exosomal membranes could be activated by reactive oxygen species (ROSs) produced during mitochondria degeneration induced by 15-lipoxygenase. Since iPLA2 has recently been demonstrated to participate in the clearance of apoptotic cells, we investigated its role in vesicle removal. We found that exosomes isolated directly from the blood of an anemic rat or released during in vitro maturation of rat reticulocytes bind IgM antibodies on their surface, in contrast to immature and mature red cells. These natural IgM antibodies recognize lysophosphatidylcholine and are able to specifically bind to apoptotic cells. Finally, evidence of C3 deposition on the exosome surface leads us to hypothesize that this cascade may favor the clearance of exosomes by cells once released into the bloodstream, via a mechanism similar to that involved in the elimination of apoptotic cells. IntroductionExosomes are membrane vesicles secreted by various cells, especially hematopoietic and epithelial cells. They correspond to intralumenal vesicles of multivesicular endosomes (MVEs) released in the extracellular medium by fusion of the MVE with the plasma membrane. The physiologic function of exosomes is not completely known. For lymphocytes and epithelial cells, secreted exosomes have been described as devices allowing antigenic information transfer and spreading of the immunologic response. Exosomes either stimulate an immunologic response or induce tolerance to the antigen, depending on the context of peptide presentation by major histocompatibility complexes (MHCs) on the exosomal surface. 1,2 The response appears to require the transfer of exosomes to specialized immunologic cells such as dendritic cells and might thus be involved in a relatively local transfer. [3][4][5] In the case of reticulocytes, cells that release high amounts of vesicles into the bloodstream, 6 exosome secretion may be viewed differently since these cells do not have any immunologic ability. The function commonly attributed to these particles is the removal of obsolete or unwanted molecules from the surface of mature red cells. Transferrin receptor (TfR) and integrin ␣4␤1 are 2 examples of proteins cleared from the red cell plasma membrane during reticulocyte maturation. 7,8 Exosome secretion would therefore be a crucial event leading to membrane remodeling during this last stage of erythropoiesis, since the lysosomal degradation capacity of the reticulocyte is trivial. One can imagine that the released vesicles are then digested by specialized cells, but contrary to e...

Exosomes as intercellular signalosomes and pharmacological effectors

“…Therefore an efficient exosome clerarance mechanism appears necessary to maintain a steady-state content of these vesicles in the body. So far, clearance of reticulocyte exosomes has been shown to involve a similar mechanism to that involved in elimination of apoptotic cells and regulated by the activity of a calcium-independent phospholipase A2 (iPLA2) [84]. Exosomes are enriched in a phospholipid, namely the lyso-phosphatidylcholine (LPC) [8] which can originate from an iPLA2 activity, which itself is activated by reactive oxygen species [84].…”

“…So far, clearance of reticulocyte exosomes has been shown to involve a similar mechanism to that involved in elimination of apoptotic cells and regulated by the activity of a calcium-independent phospholipase A2 (iPLA2) [84]. Exosomes are enriched in a phospholipid, namely the lyso-phosphatidylcholine (LPC) [8] which can originate from an iPLA2 activity, which itself is activated by reactive oxygen species [84]. LPC constitutes a "eat-me signal" for apoptotic cells, allowing the clearance of the reticulocyte exosomes [84].…”

“…Exosomes are enriched in a phospholipid, namely the lyso-phosphatidylcholine (LPC) [8] which can originate from an iPLA2 activity, which itself is activated by reactive oxygen species [84]. LPC constitutes a "eat-me signal" for apoptotic cells, allowing the clearance of the reticulocyte exosomes [84]. Also exosome surface proteins such as galectin-5 has been shown to modulate reticulocyte exosomes uptake by macrophages [85].…”

Le signaleat-medes exosomes de réticulocytes

“…Des travaux publiés récemment montrent un autre point de similitude entre apoptose et sécrétion d'exosomes à partir de réticulocytes [5]. Les corps apoptotiques résultant de la mort programmée des cellules sont rapidement éliminés par phagocytose sans occasionner de lésion inflammatoire.…”

“…De plus, Tim4, identifié récemment comme le récepteur de la PS impliqué dans la phagocytose des cellules apoptotiques, est capable de lier des exosomes [10]. Il a maintenant été montré que les exosomes de réticu-locytes recrutent à leur surface des anticorps naturels de classe IgM spécifiques de la LPC [5]. Comme pour les cellules apoptotiques, la production de LPC dans les exosomes de réticulocytes met en jeu une iPLA2 sensible à un inhibiteur spéci-fique : la bromo-enolactone (BEL).…”