Ectosomes as immunomodulators

Considerable progress has been made in recognizing microvesicles as important mediators of intercellular communication rather than irrelevant cell debris. Microvesicles released by budding directly from the cell membrane surface (i.e., ectocytosis) either spontaneously or in response to various stimuli are called shed vesicles or ectosomes. Ectosomes are rightside-out vesicles with cytosolic content, and they expose phosphatidylserine in the outer leaflet of their membrane. Depending on their cellular origin, ectosomes have been associated with a broad spectrum of biological activities. In the light of recent findings, we now know that ectosomes derived from polymorphonuclear leukocytes, erythrocytes, platelets, and tumor cells have profound effects on the innate immune system, as well as on the induction of the adaptive immunity, globally reprogramming cells such as macrophages or dendritic cells toward an immunosuppressive and possibly tolerogenic phenotype. Although the effects observed in the circulation are mainly procoagulant and pro-inflammatory, ectosomes might be anti-inflammatory/immunosuppressive in local inflammatio

High prevalence of anti-C1q antibodies in biopsy-proven active lupus nephritis

Background. Anti-C1q antibodies (anti-C1q) have been shown to correlate positively with systemic lupus erythematosus (SLE) nephritis. Several clinical studies indicated a high negative predictive value, suggesting that active lupus nephritis is rarely seen in patients with no anti-C1q. However, the true prevalence of anti-C1q at the time of active lupus nephritis has not been well established. The aim of this study was to determine prospectively the prevalence of anti-C1q in proven active lupus nephritis at the time of the renal biopsy. Methods. In this prospective multi-centre study, we investigated adult SLE patients undergoing renal biopsy for suspected active lupus nephritis. Serum samples were taken at the time of the biopsy and analysed for the presence of anti-C1q in a standardized way. The activity of lupus nephritis was classified according to the renal histology. Biopsies were also analysed for the presence of glomerular IgG, C1q and C3 deposition. Results. A total of 38 patients fulfilling at least 4/11 American College of Rheumatology (ACR) criteria for the diagnosis of SLE were included. Out of this, 36 patients had proliferative (class II, III or IV) and two had class V lupus nephritis. All but one patient with proliferative lupus nephritis were positive for anti-C1q (97.2%) compared with the 35% of control SLE patients with inactive lupus nephritis and 25% of SLE patients without lupus nephritis ever. All patients were positive for glomerular C1q (36/36) and 37/38 patients had glomerular IgG deposits. Anti-C1q strongly decreased during successful treatment. Conclusions. Anti-C1q have a very high prevalence in biopsy-proven active lupus nephritis, thus a negative test result almost excludes active nephritis. The data support the hypothesis of a pathogenic role of anti-C1q in lupus nephriti

Ectosomes as immunomodulators

Considerable progress has been made in recognizing microvesicles as important mediators of intercellular communication rather than irrelevant cell debris. Microvesicles released by budding directly from the cell membrane surface (i.e., ectocytosis) either spontaneously or in response to various stimuli are called shed vesicles or ectosomes. Ectosomes are rightside-out vesicles with cytosolic content, and they expose phosphatidylserine in the outer leaflet of their membrane. Depending on their cellular origin, ectosomes have been associated with a broad spectrum of biological activities. In the light of recent findings, we now know that ectosomes derived from polymorphonuclear leukocytes, erythrocytes, platelets, and tumor cells have profound effects on the innate immune system, as well as on the induction of the adaptive immunity, globally reprogramming cells such as macrophages or dendritic cells toward an immunosuppressive and possibly tolerogenic phenotype. Although the effects observed in the circulation are mainly procoagulant and pro-inflammatory, ectosomes might be anti-inflammatory/immunosuppressive in local inflammation

Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells

Microparticles (MP) shed by platelets (PLT) during storage have procoagulant activities, but little is known about their properties to modify inflammation or immunity. In this study, we studied the capacity of MP present in PLT concentrates to alter the function of macrophages and dendritic cells (DC). The size of the purified MP was between 100 and 1000 nm, and they expressed phosphatidylserine; surface proteins of PLT (CD61, CD36, CD47), including complement inhibitors (CD55, CD59), but not CD63; and proteins acquired from plasma (C1q, C3 fragments, factor H). These characteristics suggest that the MP shed by PLT are formed by budding from the cell surface, corresponding to ectosomes. The purified PLT ectosomes (PLT-Ect) reduced the release of TNF-? and IL-10 by macrophages activated with LPS or zymosan A. In addition, PLT-Ect induced the immediate release of TGF-? from macrophages, a release that was not modified by LPS or zymosan A. Macrophages had a reduced TNF-? release even 24 h after their exposure to PLT-Ect, suggesting that PLT-Ect induced a modification of the differentiation of macrophages. Similarly, the conventional 6-d differentiation of monocytes to immature DC by IL-4 and GM-CSF was modified by the presence of PLT-Ect during the first 2 d. Immature DC expressed less HLA-DP DQ DR and CD80 and lost part of their phagocytic activity, and their LPS-induced maturation was downmodulated when exposed to PLT-Ect. These data indicate that PLT-Ect shed by stored PLT have intrinsic properties that modify macrophage and DC differentiation toward less reactive states

Ectosomes released by platelets induce differentiation of CD4+T cells into T regulatory cells

Accumulating evidence suggests an immune-modulatory role for platelets (PLT) and PLT-derived microvesicles. In particular, ectosomes, i.e. vesicles budding from PLT surface, have been shown to exert immunosuppressive activities on phagocytes. Here we investigated the effects mediated by PLT-derived ectosomes (PLT-Ecto) on CD4+ T cells. Exposure of activated CD4+ T cells to PLT-Ecto decreased their release of IFNgamma, TNFalpha and IL-6, and increased the production of TGF-beta1. Concomitantly, PLT-Ecto-exposed CD4+ T cells displayed increased frequencies of CD25high Foxp3+ cells. These phenomena were dose-dependent and PLT-Ecto specific, since they were not observed in the presence of polymorphonuclear- and erythrocyte-derived ectosomes. Analysis of specific T cell subsets revealed that PLT-Ecto induced differentiation of naive T cells into Foxp3+ cells, but had no effect on pre-differentiated Foxp3+ regulatory T cells (Tregs). Importantly, PLT-Ecto-induced Foxp3+ cells were as effective as peripheral blood Tregs in suppressing CD8+ T cell proliferation. PLT-Ecto-mediated effects were partly dependent on PLT-derived TGF-beta1, as they were to some extent inhibited by PLT-Ecto pretreatment with TGF-beta1-neutralising antibodies. Interestingly, ectosome-derived TGF-beta1 levels correlated with Foxp3+ T cell frequencies in blood of healthy donors. In conclusion, PLT-Ecto induce differentiation of CD4+ T cells towards functional Tregs. This may represent a mechanism by which PLT-Ecto enhance peripheral tolerance

Ectosomes of polymorphonuclear neutrophils activate multiple signaling pathways in macrophages

Ectosomes are vesicles shed directly from the cell surface. Human polymorphonuclear neutrophils release ectosomes (PMN-Ect) upon their activation. PMN-Ect expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, and down-modulate the inflammatory response of human macrophages and dendritic cells exposed to TLR-2 and -4 ligands. This down-modulation is mediated by PS via the engagement and activation of the Mer receptor tyrosine kinase (MerTK). In the present study, we demonstrate that exposure of macrophages to PMN-Ect activates directly 2 additional pathways, an immediate Ca(2+) flux and a rapid release of TGF-beta1. As expected, the Ca(2+) flux was necessary for the activation of TLR-2 pathway with the release of cytokines. However, MerTK blockade with antibodies did not modify the Ca(2+) flux, indicating an independent activation of Ca(2+) by PMN-Ect. Striking was that the rapid release of TGF-beta1 was independent of the MerTK pathway and did not require a Ca(2+) flux. TGF-beta1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-beta1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-beta1 by macrophages was seen only for PMN-Ect and not for PS-liposomes or erythrocyte ectosomes, which express PS. However, blocking the PS of PMN-Ect inhibited TGF-beta1 release, suggesting that PS expression was necessary although not sufficient for this release. Interestingly, the effects of PMN-Ect pre-exposure were lasting for 24h with the macrophages being less receptive to TLR-2 activation and TGF-beta1 stores remaining low. In sum, PMN-Ect induce several signaling pathways in resting and stimulated macrophages, which include independently the MerTK pathway, Ca(2+) flux and the release of stored TGF-beta1, and each might influence the immunomodulatory effects of macrophages