Salima Sadallah

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-α and IL-10 by macrophages activated with LPS or zymosan A. In addition, PLT-Ect induced the immediate release of TGF-β from macrophages, a release that was not modified by LPS or zymosan A. Macrophages had a reduced TNF-α 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.

Erythrocyte-derived ectosomes have immunosuppressive properties

Several clinical studies have suggested that blood transfusions are immunosuppressive. Whereas there have been reports describing immunosuppression induced by leukocytes or fragments thereof, the possibility that microparticles, released by erythrocytes during storage, are also involved was not investigated. We present evidence here that such microparticles have all the properties of ectosomes including size, the presence of a lipid membrane, and the specific sorting of proteins. These erythrocyte‐derived ectosomes (E‐ecto) fixed C1q, which was followed by activation of the classical pathway of complement with binding of C3 fragments. Similarly to ectosomes released by PMN, they express phosphatidylserine on their surface membrane, suggesting that they may react with and down‐regulate cells of the immune system. In vitro, they were taken up by macrophages, and they significantly inhibited the activation of these macrophages by zymosan A and LPS, as shown by a significant drop in TNF‐α and IL‐8 release (respectively, 80% and 76% inhibitions). In addition, the effect of E‐ecto was not transient but lasted for at least 24 h. In sum, E‐ecto may interfere with the innate immune system/inflammatory reaction. Therefore, E‐ecto transfused with erythrocytes may account for some of the immunosuppressive properties attributed to blood transfusions.

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-β1. 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-β1 was independent of the MerTK pathway and did not require a Ca(2+) flux. TGF-β1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-β1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-β1 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-β1 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-β1 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-β1, and each might influence the immunomodulatory effects of macrophages.

Glomerulonephritis in a patient with Complement factor I deficiency

Complement factor I deficiency is known to be associated with recurrent pyogenic infections. The patient described here had recurrent attacks of otitis, sinusitis, and bronchopneumonia since childhood. At the age of 24 years, he had an acute episode of systemic vasculitis with purpura, but no nephritis. A factor I deficiency was diagnosed when he was 36 years old. Because of the uncontrolled activation of the alternative pathway of complement, several other components were depleted, in particular C3, which explained the predisposition for pyogenic infections. A progressive loss of renal function accompanied by proteinuria and hematuria started after the age of 40 years. Renal biopsy showed a focal segmental glomerulonephritis (GN) with glomerular deposits of immunoglobulins and complement C3 and C4 fragments. The glomerular podocytes showed an almost complete loss of complement receptor 1 (CR1; CD35). The expression of CR1 was very low on erythrocytes, as well. Thus, CR1, the most efficient cell-bound cofactor for the inactivation of C4b/C3b by factor I, appears to be consumed when factor I is missing. Although this is the first report of factor I deficiency associated with GN, it is unlikely that the development of the nephritis was fortuitous because GN has been found in many other diseases characterized by uncontrolled activation of the alternative pathway.

Clinical Value of Autoantibodies Against C1q in Children With Glomerulonephritis

OBJECTIVE. Autoantibodies against C1q (anti-C1q) have been found in a number of autoimmune and renal diseases. They are best described in adult patients with systemic lupus erythematosus, where a strong correlation between the occurrence of anti-C1q and severe lupus nephritis (LN) has been observed. However, the role of anti-C1q in children with systemic lupus erythematosus has not yet been determined. Furthermore, the clinical importance of anti-C1q in other forms of glomerulonephritis remains to be elucidated. The aim of this study was to investigate anti-C1q in children with different forms of glomerulonephritis including LN. METHODS. We prospectively investigated 112 children with different forms of newly diagnosed glomerulonephritis for the presence of anti-C1q by an enzyme-linked immunosorbent assay and compared them with healthy controls. Associations between anti-C1q and disease manifestations at the time of the measurements and during follow-up were investigated. RESULTS. Twenty-one of 112 patients were positive for anti-C1q compared with 0 of 40 healthy controls. Anti-C1q was associated with activity in LN and with disease severity in patients with acute poststreptococcal glomerulonephritis (APSGN). In LN, 7 of 12 patients were found to be anti-C1q positive. Six of these 7 had active disease at the time of the serum sampling compared with 1 of 5 of the anti-C1q-negative children. In children with APSGN, 8 of 24 were positive for anti-C1q. Anti-C1q-positive APSGN patients had significantly higher proteinuria and more often hypertension than those without anti-C1q. All 4 patients in which APSGN did not resolve spontaneously were anti-C1q positive. CONCLUSIONS. Anti-C1q is associated with active LN in children. In addition, children with anti-C1q-positive APSGN have more severe disease than those who are anti-C1q negative. These data suggest APSGN is another disease in which anti-C1q has a pathogenic role.

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.

High Incidence of Transiently Appearing Complement-Sensitive Bone Marrow Precursor Cells in Patients with Severe Aplastic Anemia - A Possible Role of High Endogenous IL-2 in Their Suppression

In a prospective long-term study on the incidence of paroxysmal nocturnal hemoglobinuria (PNH), 115 consecutive patients with severe aplastic anemia (SAA), 97 treated with antilymphocyte globulin (ALG) and 18 with bone marrow transplantation (BMT), were observed over a period of 4–18 years and tested for the presence of complement-sensitive hematopoietic precursor cells with the bone marrow (BM) sucrose test. Sixteen (14%) of the ALG-treated patients developed clinical signs of PNH between 0.5 and 8 years after treatment. Complement-sensitive BM precursors were found in 89% of the SAA patients at some time during their disease, but in none of 18 normal donors. At diagnosis, their proportion was significantly higher in patients who later developed PNH than in patients who later achieved disease-free complete remission (CR). After ALG, the abnormal population was found in both groups, but it was gradually replaced by normal precursors in remission patients. After BMT, the complement-sensitive population decreased to very low numbers in patients with a stable graft, but increased again in 3 patients upon graft rejection. Mimicking the PNH defect by enzymatic removal of glycosyl-phosphatidylinositol (GPI)-linked proteins from CD34+ cells resulted in their complement sensitivity, suggesting that the BM sucrose test identifies precursor cells carrying the PNH defect. In 66 patients, white blood cells (WBC) in peripheral blood (PB) were examined for GPI-deficient populations by flow cytometry (FACS). Ten patients with signs of clinical or laboratory PNH had over 25% complement-sensitive precursor cells in the BM and a GPI-deficient WBC population in the PB. Of 56 SAA patients without PNH, 8 had an abnormal population detectable with both tests, 26 only with the BM sucrose test, 4 only with PB FACS analysis, and in 18, no abnormal cells were detected with either test. In search for parameters which might explain why in some patients the abnormal population expands, while it regresses or disappears in others, we tested the release of IL-2 as a parameter of immune competence. At diagnosis, IL-2 release was approximately 50% of normal in patients who later developed PNH, while it was double the normal value in patients who later achieved CR. We conclude that the majority of SAA patients transiently harbor complement-sensitive precursor cells in the BM. Patients with more than 25% abnormal BM precursors and low endogenous IL-2 release are at risk of progression to clinical PNH.

A C3 convertase assay for nephritic factor functional activity

C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase which stabilizes this otherwise inherently labile neoenzyme and induces a continuous activation of the alternative pathway with C3 depletion. NeF is found in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF activity is usually detected in plasma by hemolytic tests. In order to obtain reproducible data for the functional activity of purified C3NeF IgG a solid phase assay was developed. C3 convertase was generated on immobilized C3b by incubation with factors B and D in the presence of Ni(2+). Convertase sites were left to decay in the presence of normal IgG or NeF IgG. Residual convertase activity was measured by adding 125I-C3 and capturing nascent 125I-C3b on the plate surface via covalently coupled NH2-Glu-Tyr dipeptide. In the presence of factor H during C3 convertase decay, a dose dependent stabilizing activity was shown for NeF IgG including NeF IgG purified from urine. A second format of the assay was developed in which C3 convertase was assembled on C3b(2)-IgG complexes in the presence of Mg(2+). Since these complexes are more efficient as convertase precursors the signal was five-fold higher than with C3b. Convertase decay, on the other hand, was not influenced by the nature of the precursor and in both systems the stabilizing activity of NeF IgG was similar.

Soluble complement receptor 1 is increased in patients with leukemia and after administration of granulocyte colony-stimulating factor.

Complement receptor type 1 is expressed by erythrocytes and most leukocytes. A soluble form is shed from the leukocytes and found in plasma (sCR1). sCR1 is a powerful inhibitor of complement. We report an increased sCR1 in the plasma of leukemia patients, up to levels producing measurable complement inhibition. Half of the 180 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) had sCR1 levels above the normal range. The highest levels were observed in T‐ALL (17 patients). The complement function of a T‐ALL serum was improved by blocking sCR1 with a specific mAb (3D9). Measurements in 16 peripheral stem cell donors before and after granulocyte colony‐stimulating factor (G‐CSF) administration showed an increase in sCR1 (before, 43.8 ± 15.4; at day 5, 118.3 ± 44.7 ng/mL; P < 0.0001). This increase paralleled the increase in total leukocyte counts and was concomitant with de novo leukocyte mRNA CR1 expression in all three individuals tested. Whether pharmacological intervention may be used to up‐regulate sCR1 so as to inhibit complement in vivo should be further investigated. J. Leukoc. Biol. 65: 94–101; 1999.

Platelet-Derived Ectosomes Reduce NK Cell Function.

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-β1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti–TGF-β1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-β1–mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti–TGF-β1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-β1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell–derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-β1–dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.