Phagocytosis Of Erythrocytes Research Articles

Cripto-1 modulates macrophage cytokine secretion and phagocytic activity via NF-κB signaling.

Cripto-1 is an oncogenic protein belonging to the epidermal growth factor–Cripto-1/FRL-1/Cryptic family. It has important roles in tumor formation and metastasis, but its effects on the immune system are unclear. In the present study, we investigated the effects of Cripto-1 overexpression on macrophage activities and examined the underlying mechanisms. A cell line stably overexpressing Cripto-1 was developed. The culture supernatant from this cell line was collected and used to condition macrophages (RAW264.7, THP-1, and primary mouse macrophages) for various times. Exposure to this supernatant significantly increased the mRNA and protein expression levels of the anti-inflammatory cytokine interleukin (IL)-10 and of three pro-inflammatory cytokines (tumor necrosis factor-α, IL-6, and IL-1β), but did not affect the expression of transforming growth factor-β, another anti-inflammatory cytokine. Exposure to this supernatant also enhanced macrophage phagocytosis of chicken erythrocytes and yeast cells. Similar effects were observed in macrophages stimulated with purified Cripto-1 protein. Mechanistic experiments revealed that Cripto-1 activated nuclear factor (NF)-κB signaling by inducing IκB kinase phosphorylation and p65 nuclear translocation. Pretreatment with ammonium pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, inhibited Cripto-1-induced cytokine secretion and phagocytosis of macrophages. Taken together, our present findings suggest that Cripto-1 enhances macrophage phagocytic activity and upregulates the production of anti- and pro-inflammatory cytokines via the NF-κB signaling pathway.

Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria

AbstractThe clinical management of paroxysmal nocturnal hemoglobinuria (PNH), a rare but life-threatening hematologic disease, has fundamentally improved with the introduction of a therapeutic that prevents complement-mediated intravascular hemolysis. However, a considerable fraction of PNH patients show insufficient treatment response and remain transfusion dependent. Because the current treatment only prevents C5-induced lysis but not upstream C3 activation, it has been speculated that ongoing opsonization with C3 fragments leads to recognition and phagocytosis of PNH erythrocytes by immune cells. Here, for the first time, we provide experimental evidence for such extravascular hemolysis and demonstrate that PNH erythrocytes from anti–C5-treated patients are phagocytosed by activated monocytes in vitro. Importantly, we show that this uptake can be mediated by the end-stage opsonin C3dg, which is not traditionally considered a phagocytic marker, via interaction with complement receptor 3 (CR3). Interaction studies confirmed that C3dg itself can act as a ligand for the binding domain of CR3. The degree of C3dg-mediated erythrophagocytosis in samples from different PNH patients correlated well with the individual level of C3dg opsonization. This finding may guide future treatment options for PNH but also has potential implications for the description and management of other complement-mediated diseases.

Abstract 114: CX3CR1-null Microglia Fail to Transition to an M2 Phenotype after Intracerebral Hemorrhage

Introduction: Intracerebral hemorrhage (ICH) results in the activation of microglia. Microglia can polarize into either a pro-inflammatory (M1), or a reparative (M2) phenotype; the effect of microglial polarization after ICH is unknown. Microglia express the chemokine receptor CX3CR1, which has been shown to regulate microglial neurotoxicity. CX3CR1 +/GFP mice have a functional CX3CR1 and are frequently used to identify microglia. We previously reported that mice with CX3CR1-null microglia do not show functional recovery 14 days after ICH. We hypothesize that 1) microglia transition from M1 to M2 phenotypes in the acute to subacute period after ICH and 2) CX3CR1-null microglia fail to make this transition, inhibiting recovery after ICH. Methods: ICH was modeled by injecting 20ul of WT blood into the right striatum. Microglia were sorted from male CX3CR1 +/GFP mice 0.5, 1, 3, 7, and 14 days after ICH in order to specifically study microglia phenotypes. RNA was extracted and qRT-PCR was performed to analyze changes in gene expression. To study microglial CX3CR1 function, C57BL/6 (WT) and CX3CR1 GFP/GFP (CX3CR1-null) mice were irradiated and reconstituted with WT bone marrow (CD45.1) to generate bone marrow chimeras (CD45.1->WT or CD45.1->CX3CR1-null). Brains were harvested for flow cytometry 14 days after ICH. Results: At 12 hours after ICH, microglia have high IL-6 gene expression (M1). However, M2 markers arginase-1, TGF-β and SOCS3 increase over time (Fig. 1), suggesting microglia transition from M1 to M2 over the first 2 weeks after ICH. By flow cytometry, CX3CR1-null microglia had significantly less SIRPα, a receptor involved in phagocytosis of erythrocytes, and CD206, an M2 marker, than WT microglia 14 days after ICH. Conclusions: Our data show WT microglia transition from an M1 to an M2 phenotype after ICH. Our results also suggest microglial CX3CR1 is necessary for transition toward an M2 phenotype after ICH and this transition is required for recovery after ICH.

Chronic HIV infection impairs nonopsonic phagocytosis of malaria parasites.

Malaria-specific immune responses are altered in HIV/malaria-coinfected individuals and are associated with higher parasite burdens and more severe clinical disease. Monocyte/macrophage phagocytosis is a major mechanism of malaria parasite clearance. We hypothesized that phagocytosis of malaria-parasitized erythrocytes is impaired in coinfected individuals and could contribute to the increased parasite burdens observed. We show that nonopsonic phagocytosis of Plasmodium falciparum parasitized erythrocytes is impaired in monocytes isolated from HIV-infected individuals. The observed defects in phagocytic capacity were rescued after 6 months of antiretroviral therapy, demonstrating the importance of HIV treatment and immune reconstitution in the context of coinfection.

The effects of panaxadiol saponins on megakaryocytic maturation and immune function in a mouse model of immune thrombocytopenia

We have identified a biologically active component, panaxadiol saponins component (PDS-C), from Chinese ginseng herb extract. Panaxadiol saponins component contains five ginsenoside monomers with total purity of 92.44%. In this study, the BALB/c mouse model with immune thrombocytopenia (ITP) was established by injection of antiplatelet antibody every other day for 5 total times; the peripheral blood platelet counts steadily decreased to 20%-30% of normal levels and remained decreased for about 10 days. The antiplatelet antibody was derived from the sera of guinea pigs immunized with the platelets of BALB/c mice. Mice with ITP were treated with PDS-C at a low, a moderate, or a high dose for 10 consecutive days. We observed that the peripheral blood platelet counts of ITP mice were significantly higher than that of ITP controls (untreated) after treatment of PDS-C in a dose-dependent manner. Treatment with PDS-C also increased the mature megakaryocytes in the bone marrow of treated ITP animals with a concomitant decease of immature megakaryocyte precursors. Furthermore, macrophage phagocytosis of exogenous erythrocytes in the intra-abdominal cavity of ITP mice was inhibited by PDS-C treatment, indicating that PDS-C also could modulate immune function and may possibly prevent phagocytosis of antibody-coated platelets. Altogether, our findings suggest that PDS-C may have a dual role, promoting proliferation and differentiation of megakaryocytes, as well as modulating immune function, and it may therefore be very helpful in the treatment of ITP.

Crystal structure of calcium binding protein-5 from Entamoeba histolytica and its involvement in initiation of phagocytosis of human erythrocytes.

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca2+ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Å resolution in the Ca2+-bound state, which shows an unconventional mode of Ca2+ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca2+ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role.

Lead modulation of macrophages causes multiorgan detrimental health effects.

The environmental toxicant lead (Pb) has detrimental effects on a number of organ systems, including the immune system. Pb exposure decreases host immune defenses against numerous microorganisms and cancer. Although Pb effects on humoral and cell-mediated immunity as well as on erythrocyte, neural, and renal pathophysiology have been well documented, there are few reports regarding Pb's impact on innate immunity, which can affect multiorgan processes. This review focuses on Pb modulation of a key innate immune cell, the macrophage. The impact of Pb on macrophages in different organs, on immature versus mature macrophages, and on low versus high Pb concentrations is discussed. Pb decreases phagocytosis and chemotaxis of macrophages and affects nitric oxide production and eicosanoid metabolism in mature macrophages. Pretreatment of macrophages with Pb increases TNF-α secretion after in vitro stimulation with lipopolysaccharide; however, Pb exposure decreases in vivo intracellular pathogen killing. More recent evidence from mouse studies indicates that even low, environmentally relevant, blood concentrations of Pb result in increased phagocytosis of erythrocytes and decreased expression of interferon-gamma-inducible GTPases, p65-GBP, and p47-IRG, which are necessary for intracellular pathogen killing. Taking into account the effects of Pb on macrophages, the review describes posited mechanisms to account for Pb-altered health effects; Pb effects on heme levels may play a key role as well as Pb's preferential induction of helper type-2 T (Th2) cells and M2 macrophages, which is related to oxidative stress. The discussion links old findings with new, thereby adding new insight into the effects of Pb on macrophages and the resultant compromised immunity and health.

Phagocytic activity of LPS tolerant macrophages

Endotoxin tolerance is defined as a reduced capacity of the host to respond to LPS activation following a first exposure to this stimulus. It affects all leukocytes and regarding macrophages, most studies focus on the reduced ability of these cells to secrete pro-inflammatory cytokines. Therefore, we evaluated other macrophages functions (fungicidal capacity, reactive oxygen species production and antigen presentation) in cells from tolerant mice. We have performed a tolerance model in our laboratory that does not stimulate directly the place from where the cells will be removed (peritoneal cavity). Mouse received subcutaneous injections of LPS in the scruff for 5 days and we analyze the capacity of peritoneal macrophages to phagocyte using three different receptors: Fc, C3b and mannose receptors. We found a reduction in the phagocytosis of erythrocytes and Candida albicans related to the Fc and mannose receptors. These differences can be due to a macrophage reprogramming, as demonstrated by altered expression of cytokines and chemokines. Despite this reduction in phagocytosis capacity, macrophages from tolerant animals exhibited enhanced hydrogen peroxide production and expression of antigen presentation molecules, suggesting that their ability to combat an infection is improved. In summary, our data indicates that LPS tolerance drives macrophages from a predominant release of proinflammatory mediators that amplify inflammation and host damage toward a better killing and antigen presentation state.

Iron Metabolism In Macrophages In Non Transfusion Dependent Thalassemia

Background β-thalassemia is a disease characterized by alteration of β-globin chains production. The phenotype is associated with development of anemia, ineffective erythropoiesis (IE), and iron overload. Cellular iron homeostasis in macrophages is regulated at multiple steps and by numerous genes. Macrophages can acquire iron by Transferrin receptor 1-mediated uptake of transferrin-bound iron, acquisition of molecular iron via the divalent metal transporter 1 (Dmt1) and phagocytosis of senescent erythrocytes with subsequent recycling of iron. There is only one well-characterized pathway by which iron can exit cells, ferroportin-1 (Fpn1), which is expressed on the cell surface of macrophages and acts as the exclusive trans-membrane export protein for ferrous (Fe2+) iron. Hepcidin, a mainly liver-derived peptide induced by iron and cytokines and master regulator of body iron homeostasis, exerts its regulatory effects via binding to Fpn1, which is thought to be the hepcidin receptor. This interaction results in Fpn1 internalization, proteasomal degradation and blockage of iron export. Little is known about what happens at the level of macrophages in thalassemic patients and how they face the high iron concentration. The aim of this study was to characterize the differential expression of the genes involved in iron homeostasis and changes in iron trafficking in fully differentiated unpolarized (M0) human macrophages in Non Transfusion Dependent Thalassemia (NTDT) patients. Methods Monocytes were purified using positive selection with CD14-coated magnetic beads (Miltenyi Biotec) from peripheral blood of 7 NTDT patients and 7 healthy normal controls. Monocytes were cultured for 6 days in RPMI containing 10% FBS and 25 ng/ml GM-CSF and differentiated in mature macrophages. The expression of specific macrophages surface proteins was analyzed by flow cytometry. We used immunohistochemistry to evaluate differentiation and iron retention of macrophages. For basic morphology characterization, formalin-fixed cells were stained using hematoxylin and eosin staining. Iron was detected with DAB-enhanced Perls' staining (Prussian blue reaction). Total mRNA was extracted from cultured cells and gene expression profile was analyzed by real-time PCR using Taqman-probes technologies. Results The purity of the resulting cells suspension was tested by fluorescent-activated cell sorting analysis and was beyond 98%. The cell morphology of macrophages showed no differences between control and thalassemic patients. Using immunohistochemistry iron was not detected in human cultured macrophages of thalassemia patients and controls. We characterized the expression of genes related to iron homeostasis. We analyzed the gene expression levels of SLC40A1 (Ferroportin) , SLC11A2 (Dmt1), HAMP (Hepcidin) that are directly involved in macrophage iron traffic and these results show no statistical differences between patients and controls. Conclusions Due to the heterogeneity of the cellular morphology of macrophages there was no significant difference between the macrophages morphology from NTDT and controls. Iron was not present in M0 magrophages. The gene expression levels of ferroportin, Dmt1 and Hepcidin in mature macrophages grown in regular culture medium without other stimuli were comparable between controls and patients. This culture system does not reflect the in vivo iron metabolism of thalassemic patients due to possibly monocytes inability to internalize and accumulate iron. Different stimuli are necessary to simulate the in vivo condition to differentiate the macrophages. Cells of the monocyte-macrophage lineage are characterized by marked phenotypical and functional heterogeneity. Classical activation by microbial agents and/or Th1 cytokines is associated with the production of oxygen radicals and the pro-inflammatory cytokines involved in cytotoxicity and microbial killing (M1 polarization), but macrophages can also follow a different activation pathway after stimulation with the Th2 cytokines IL-4 or IL-13 (M2 polarization). We will therefore investigate whether iron homeostasis is regulated differently in M1 and M2 macrophages and possibly provide a better understanding of the changes in iron metabolism that take place under thalassemia condition. Disclosures: No relevant conflicts of interest to declare.

Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36

Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of β-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and β-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where β-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.

Hemophagocytosis‐mediated keratinization in oral carcinoma in situ and squamous cell carcinoma: A possible histopathogenesis of keratin pearls

Although the histopathogenetic process of keratin pearls is still poorly understood, acceleration of keratinization in squamous cell carcinoma (SCC) cells may represent one possible therapeutic avenue. Based on our histopathological observations, we have hypothesized that SCC cells are keratinized by phagocytosis of extravasated erythrocytes. To confirm this hypothesis, we firstly examined immature keratin pearls in oral carcinoma in situ (CIS) and mature ones in SCC by immunohistochemistry. Concentric dyskeratotic cells in CIS keratin pearls became positive for keratin (K) 10, K17, heme oxygenase-1 (HO-1), or protease activated receptor-2 (PAR-2), a candidate regulator for hemophagocytosis. When ZK-1 cells, an SCC cell system, were incubated with human peripheral blood erythrocytes, or with crude and purified hemoglobins (Hbs), their erythro-hemophagocytotic activities were confirmed by immunofluorescence. Immunofluorescence signals for K10, K17, and HO-1 were enhanced due to hemophagocytosis in time-dependent manners. mRNA expression levels for the three molecules were most enhanced by purified Hb, followed by crude Hb and erythrocytes. K17/K10 mRNA expression levels were more elevated when PAR-2 was activated in ZK-1 cells. The results indicated that immature and mature keratin pearls in CIS and SCC were generated by oxidative stresses derived from erythro-hemophagocytosis, which might mediate HO-1 expression and be regulated by PAR-2. Thus, hemorrhage from the rupture of blood vessels can be one of the triggers for keratin pearl formation in oral CIS and SCC.

CD47 functions as a removal marker on aged erythrocytes

CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor signal regulatory protein alpha (SIRPα) expressed by macrophages. Thus, the CD47‐SIRPα interaction constitutes a negative signal for erythrocyte phagocytosis. However, we recently reported that CD47 does not only function as a ‘don't eat me’ signal for uptake but can also act as an ‘eat me’ signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47‐ SIRPα interactions.Furthermore, we provide evidence that experimental ageing of erythrocytes induces a conformational change in CD47 that switches the molecule from an inhibitory signal into an activating one. We also demonstrate that aged erythrocytes have the capacity to bind the CD47‐binding partner thrombospondin‐1 (TSP‐1) and that treatment of aged erythrocytes with a TSP‐1‐derived peptide enabled their phagocytosis by human red pulp macrophages. Finally, CD47 on erythrocytes that had been stored for prolonged time was shown to undergo a conformational change and bind TSP‐1.These findings reveal a more complex role for CD47‐ SIRPα interactions in erythrocyte removal, with CD47 acting as a molecular switch for controlling erythrocyte phagocytosis.

FcγRIII (CD16) equips immature 6-sulfo LacNAc–expressing dendritic cells (slanDCs) with a unique capacity to handle IgG-complexed antigens

AbstractBinding and uptake of immune complexes (ICs) via low-affinity Fcγ receptors (FcγRs) on dendritic cells (DCs) is well known as a booster of immune responses. It can be helpful when stimulating immunity against pathogenic microbes but may be harmful when antibodies form complexes with autologous antigens. To date, no human DC subtype specialized in handling ICs has been identified. By incubating human blood mononuclear cells with ICs and studying their cellular binding, we identified 6-sulfo LacNAc-expressing DCs (slanDCs) as having an outstanding capacity to bind ICs compared with other myeloid DCs, plasmacytoid DCs, or monocytes. Using selective blocking of different (FcγRs), we identified CD16 (FcγRIII) as the major IC-binding structure on slanDCs. In addition, CD16 proved critical for phagocytosis of IgG-coated erythrocytes, and CD16-targeted antigen led to a more efficient proliferation of CD4+ T cells than CD32 (FcγRII)-targeted antigen. Interestingly, these CD16-mediated functions are short-lived and restricted to the immature stage of slanDCs in blood. We show that CD16 is rapidly shed from the surface of maturing slanDCs, resulting from the combined action of the metalloproteinases ADAM10 and ADAM17. In conclusion, these data provide strong evidence that slanDCs play an important role in IC-driven immune responses.

P2X receptor‐dependent erythrocyte damage by α‐hemolysin from Escherichia coli triggers phagocytosis by THP‐1 cells

The pore‐forming exotoxin α‐hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+‐sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of both of these channels potentiate the HlyA‐induced haemolysis. This means that Ca2+‐ dependent activation of KCa3.1 and TMEM16A protects the cells against early haemolysis. Simultaneous to the HlyA‐induced shrinkage the erythrocytes show increased exposure of phosphatidyl‐serine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis.We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP‐1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP‐1 cells. The HlyA‐induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS‐exposure in human erythrocytes exposed to both ionomycin and HlyA. Moreover, we show that the P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA‐induced PS‐exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA‐insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis

Resistance to Malaria by Enhanced Phagocytosis of Erythrocytes in LMP7-deficient Mice

General cellular functions of proteasomes occur through protein degradation, whereas the specific function of immunoproteasomes is the optimization of antigen processing associated with MHC class I. We and others previously reported that deficiency in subunits of immunoproteasomes impaired the activation of antigen-specific CD8+ T cells, resulting in higher susceptibility to tumor and infections. We demonstrated that CD8+ T cells contributed to protection against malaria parasites. In this study, we evaluated the role of immunoproteasomes in the course of infection with rodent malaria parasites. Unexpectedly, Plasmodium yoelii infection of mice deficient in LMP7, a catalytic subunit of immunoproteasomes, showed lower parasite growth in the early phase of infection and lower lethality compared with control mice. The protective characteristics of LMP7-deficient mice were not associated with enhanced immune responses, as the mutant mice showed comparable or diminished activation of innate and acquired immunity. The remarkable difference was observed in erythrocytes instead of immune responses. Parasitized red blood cells (pRBCs) purified from LMP7-deficient mice were more susceptible to phagocytosis by macrophages compared with those from wild-type mice. The susceptibility of pRBC to phagocytosis appeared to correlate with deformity of the membrane structures that were only observed after infection. Our results suggest that RBCs of LMP7-deficient mice were more likely to deform in response to infection with malaria parasites, presumably resulting in higher susceptibility to phagocytosis and in the partial resistance to malaria.

P2X Receptor-Dependent Erythrocyte Damage by α-Hemolysin from Escherichia coli Triggers Phagocytosis by THP-1 Cells

The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis.

Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration and Phagocytosis of Plasmodium-Infected Erythrocytes

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE.

Hemophagocytic syndrome in a cat with multiple myeloma

An 11-year-old, castrated male, Domestic Medium Hair cat was presented to the University of Florida Small Animal Hospital with a 2-week history of upper respiratory infection and increased serum globulins, as reported by the referring veterinarian. Physical examination was unremarkable other than melanosis of the left iris, with no evidence of ocular, nasal, or respiratory disease. Laboratory abnormalities included moderate nonregenerative anemia, mild leukopenia, mild hyperfibrinogenemia, severe hyperglobulinemia, mild hypoalbuminemia, and hypocholesterolemia. Abdominal radiographs and ultrasonographic examination revealed mild splenomegaly with no other abnormalities. Thoracic radiographs revealed no abnormalities. Cytologic evaluation of fine-needle aspirates from the spleen, liver, and bone marrow revealed numerous plasma cells and many vacuolated macrophages exhibiting marked phagocytosis of mature erythrocytes and platelets, occasionally metarubricytes and leukocytes, and rarely plasma cells. The cytologic interpretation was multiple myeloma and associated hemophagocytic syndrome (HPS). Serum protein electrophoresis revealed a monoclonal gammopathy, providing further evidence for a multiple myeloma. To the authors' knowledge, this is the first report of HPS secondary to neoplasia in a cat.

Abl Family Kinases Regulate FcγR-Mediated Phagocytosis in Murine Macrophages

Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

Bilirubin, the Gold Within

When heme-containing proteins such as hemoglobin and cytochrome P450 are degraded, their prosthetic groups cannot be metabolized and have to be excreted. Elimination of heme could be considered straightforward detoxification, but a large body of evidence shows that this pathway plays a role in various physiological processes as well. Article see p 2556 The first step in the catabolism of heme is cleavage of the porphyrin ring by heme oxygenase (HO); this reaction yields biliverdin, carbon monoxide, and iron. Two HO isoforms mediate this process, constitutively expressed HO-2 and inducible HO-1. Many cell types express HO, with high expression present in cells of the reticuloendothelial system involved in the degradation of erythrocytes.1 The first step in heme catabolism has attracted considerable interest because HO activity is involved in the regulation of processes such as arthrosclerosis, inflammation, and diabetes mellitus.1 This regulation is mediated by the products of HO action on heme, with the potent signaling molecule carbon monoxide likely playing a major role.1 The other product of HO action on heme is the green and water-soluble compound biliverdin. In mammals, levels of biliverdin are low because it is quickly converted to the hydrophobic yellow compound bilirubin by ubiquitously expressed biliverdin reductase. However, other animal species, such as fish,2 do not completely metabolize all biliverdin to bilirubin and excrete significant amounts of the green pigment. Because bilirubin is poorly water soluble, mammals require efficient conjugation with glucuronic acid by UDP glucuronyltransferase before bilirubin glucuronides can be excreted into bile. This aspect of heme metabolism has always puzzled scientists. Why is bilirubin the sole end product of heme degradation in mammals? Bilirubin is toxic, and the absence of bilirubin UDP glucuronyltransferase (UGT1A1) in patients with Crigler-Najjar syndrome causes high unconjugated bilirubin levels and subsequent severe brain damage. In …