Fcγ Receptors I and III on Splenic Macrophages Mediate GPIIb/IIIa Autoantibody-Dependent Phagocytosis of Platelets in Human Immune Thrombocytopenia

Autoantibody-opsonized platelets in immune thrombocytopenia (ITP) are thought to be destroyed primarily by macrophage Fc gamma receptor (FcγR)-mediated phagocytosis in the spleen. Blockade of splenic macrophage FcγRs has been proposed as a therapeutic mechanism for ITP intervention. Unfortunately, the contribution of specific FcγRs to disease in ITP remains unknown. Our objective was to determine which FcγRs are responsible for the phagocytosis of ITP autoantibody-opsonized platelets by splenic macrophages. Splenic macrophages were purified by CD14 positive selection from spleens of splenectomized ITP patients, and were treated with blocking antibodies to FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII. Blocking antibodies were deglycosylated to prevent non-specific blocking effects by their Fc region. Two separate ITP sera confirmed positive for anti-GPIIb/IIIa autoantibodies by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay were used to opsonize healthy donor human platelets. Phagocytosis was determined by confocal microscopy and non-phagocytosed (external) platelets were differentiated by an anti-platelet antibody stain following macrophage fixation. Human ITP splenic macrophages were found to express FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII, and expression was not significantly different compared to healthy (trauma) controls (n=5). The two anti-GPIIb/IIIa-positive ITP sera induced a mean 3.7- and 4.2-fold increase of platelet uptake by ITP splenic macrophages relative to normal human serum controls (n=3 each, p<0.001). Blockade of all FcγRs significantly reduced phagocytosis of serum-opsonized platelets for both ITP sera as compared to an IgG control (p<0.001) down to background (non-opsonized) levels. Using single blocking antibodies, inhibition of FcγRI reduced splenic macrophage phagocytosis of platelets by 45% and 37% for the two respective ITP sera as compared to IgG controls (n=3 for each patient sera, p<0.01), while inhibition of FcγRIII reduced phagocytosis of platelets by 43% and 44% (n=3 each, p<0.05). Blockade of FcγRIIa or FcγRIIa/b/c only marginally inhibited splenic macrophage phagocytosis and was not significant. An Fab-like FcγRIII-blocking antibody with a mutant Fc region completely deficient for FcγR binding demonstrated equal FcγRIII blockade compared to the deglycosylated anti-FcγRIII antibody. In comparison to ITP sera-opsonized platelets, FcγRI had a greater involvement in ITP splenic macrophage phagocytosis of anti-D-opsonized human erythrocytes as FcγRI blockade inhibited phagocytosis by 70% (n=5, p<0.001), while FcγRIII blockade inhibited phagocytosis by only 30% (p<0.001) and FcγRIIa or FcγRIIa/b/c blockade had no significant effect. This work demonstrates that FcγRI and FcγRIII are the primary phagocytic receptors on splenic macrophages for anti-GPIIb/IIIa autoantibody-opsonized platelets in human ITP and suggests that FcγRI and FcγRIII are the best targets for FcγR blockade as a potential therapeutic maneuver in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.

Abstract 007: TRPM7-kinase Modulates Renal and Splenic Macrophage and T-lymphocyte Infiltration in Aldosterone-salt-induced Hypertension

TRPM7 is a Mg2 + channel linked to a kinase domain important in cell proliferation and survival. We demonstrated that cells deficient in TRPM7-kinase domain are prone to aldosterone (aldo)-induced oxidative stress and inflammation. Here, we investigated whether TRPM7-kinase plays a role in inflammatory responses in aldo-induced hypertension. Wild-type (WT) or heterozygote TRPM7-kinase domain (TRPM7+/-) mice were infused with aldo (600μg/Kg/day - osmotic minipumps) and 1% NaCl in the drinking water (aldo-salt) for 4 weeks. Inflammatory responses were evaluated by examining T cells (CD4+ and CD8+) and macrophages (M1- and M2-phenotype) infiltration in kidneys and spleens, using flow cytometry. Gene and protein expression was assessed by real-time PCR and immunoblot respectively. ROS was evaluated by lucigenin chemiluminescence. Aldo-salt increased kidney mass and urinary levels of albumin, Ca 2+ , Mg 2+ and K + , similarly in WT and TRPM7+/- (p<0.05 vs controls). Kidneys from TRPM7+/- mice presented a higher total number of inflammatory infiltrated cells (0.87x10 6 vs WT 0.22x10 6 cells/g), TCD4+ cells (27% vs WT 19%), and macrophages (47% vs WT 31%) (p<0.05 vs controls). Kidneys from WT aldo-salt showed increased ROS production (1.7-fold) and Nox2 (2-fold) protein expression (p<0.05 vs control) and presented similar cell infiltration to TRPM7+/- mice. Kidneys from TRPM7+/- aldo-salt show increased M2-macrophages CD206+ (4.3% vs WT 2.3%) and mRNA expression for the anti-inflammatory cytokine IL-10 (55% vs WT), whereas decreased the expression of the pro-inflammatory TNFα (30% vs WT) and Nox2 protein (1.8-fold). Spleen mass was increased by 40% only in WT aldo-salt. Spleens from untreated TRPM7+/- showed increased infiltrated inflammatory cells (3.7x10 8 vs WT 2.0x10 8 cells/mg). Splenic macrophages were higher in untreated TRPM7+/- (8.8% vs WT 5.9%) and presented an increase in CD206 M2-marker (16% vs WT 7%), higher TCD4+ (68% vs WT 50%) and TCD8+ (26% vs WT 17%), values that were similar to WT aldo-salt. Our data provide insights into the importance of the TRPM7-kinase domain in the immune system activation, which when down regulated provokes an increase in inflammatory cell infiltration in kidneys and spleens in aldo-salt-induced hypertension.

Effects of Unleaded Petroleum on the Macrophage Aggregates (MA) formation in Red King tilapia (Oreochromis sp.) Fingerlings

The Philippines is one of the major producers of tilapia, the most cultured fish and widely consumed in the world. Although fishes in general is said to be adapted to various stressful conditions, the effect on several cellular immune parameters may be of interest to determine the capacity of the organism to withstand stressors. In this paper, the effect of unleaded petroleum on the splenic macrophage aggregate (MA) formation was studied. This was done to have an overview of the immune response of Tilapia or fishes in general when an oil spill, which almost occur annually at different parts of the world, happen. Histological analysis assessed the area occupied by splenic MA 24 hours after introduction of unleaded petroleum to the aquatic system. To determine whether Mabuhay balls, a technology that claims to be beneficial in terms of improving water quality, was added to one tank (T1) to be able to compare it with another tank (T2). There is a strong statistically significant difference between the groups at day1 (p=0.000) opposite the result of day 6 (p=0.155). Thus, unleaded petroleum increased MA formation, a sign that may indicate a high immune activity as an initial positive response to stress. Mabuhay ball have lessen the mortality but has no effect on splenic MA formation.

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)+ macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE−/− mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.

Functional enhancement of platelet activation and aggregation by erythrocytes: role of red cells in thrombosis

Platelets expose phosphatidylserine (PS), a component of the prothrombinase complex, on the outer surface of the plasma membrane when activated. [ref 1] The prothrombinase complex catalyzes the conversion of prothrombin to thrombin, and it has been demonstrated that an increase in PS exposure is correlated with an increase in thrombin generation by platelets. [refs 2,3] Similarly, erythrocyte (RBC) activation, or eryptosis, is also characterized by PS exposure on the plasma membrane. [ref 4] Although PS exposure on RBCs is considered a signal for splenic macrophage destruction, eryptosis may allow RBCs to contribute to thrombosis.[ref 4] The aims of this study were to determine whether the addition of RBCs to platelets increased functional platelet aggregation and coagulation properties. A ratio of 4 RBCs to 1 platelet (4:1) was evaluated for aggregation and coagulation compared to platelet control. Platelet aggregation and coagulation properties were evaluated with impedance aggregometry and thromboelastography, respectively. The 4:1 experimental group had significant increases in aggregation and coagulation relative to the platelet control. These results indicate that RBCs increase platelet aggregation and coagulation properties. This suggests that RBCs play a role in diseases traditionally thought of as associated solely via dysregulated platelet activation.