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Thread: Monocyte, engulfed platelets, Platelet Satellitism picture - blood histology atlas

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    Default Monocyte, engulfed platelets, Platelet Satellitism picture - blood histology atlas

    Introduction
    Although platelets are best known as the primary mediators of hemostasis in humans and other mammals, they also play important roles in a multitude of other physiological functions (Klinger and Jelkmann, 2002). Platelets are rapidly deployed to sites of injury or infection and release proteins that kill certain bacteria and fungi. Platelets also modulate inflammatory processes by interacting with leukocytes and by secreting cytokines, chemokines, and other inflammatory mediators (Elzey et al., 2005; Semple and Freedman, 2010). In addition, their function has been linked with various pathological conditions, including atherosclerosis, arthritis and immune thrombocytopenic purpura (ITP) (Semple and Freedman, 2010; Cines and Blanchette, 2002; Gasparyan, 2010). Our demonstration that human platelets can bind and endocytose IgG complexes in vitro (Worth et al., 2006) suggested that platelets also participate in the clearance of IgG-containing complexes.

    Immune complexes (IC) are present in the circulation of healthy individuals and the formation of such complexes is part of a normal immune process. Efficient clearance of IgG complexes can be critical because their deposition in tissues and organs can set off reactions that lead to inflammation and tissue damage (Jancar and Snchez Crespo, 2005; Mayadas et al., 2009). For example, in some pathological conditions, including autoimmune diseases such as systemic lupus erythematosus and autoimmune glomerulonephritis significant amounts of immune complexes are formed and deposited in the kidney and other tissues, causing severe injury (Niederer et al., 2010; Bagavant and Fu, 2009).

    FcγRIIA is the only Fcγ receptor expressed on platelets (Cassel et al., 1993; King et al., 1990). On professional human phagocytes such as monocytes and neutrophils, FcγRIIA plays an important role in the clearance of IgG immune complexes and the phagocytosis of IgG coated particles (McKenzie and Schreiber, 1994). Apart from the recognition that FcγRIIA is important for platelet activation by von Willebrand factor (Canobbio et al., 2001), the role of FcγRIIA in platelet function has not been well defined.

    It is known that platelets from patients suffering from certain autoimmune and thrombocytopenic disorders have high levels of surface bound IgG. Platelets from normal donors also bind IgG immune complexes in vivo, albeit at lower levels (Romero-Guzmn et al., 2000; George, 1990; Christopoulos et al., 1993; Court et al., 1987). We present evidence that platelets of normal individuals can serve as vehicles for delivery of immune complexes for destruction by phagocytes and that circulating neutrophils/ monocytes play a role in this process. We also demonstrate that in addition to the ability to ingest small IgG particulates (heat aggregated IgG, Worth et al., 2006), human platelets can internalize larger IgG coated particles by a process similar in some respects to phagocytosis in leukocytes.

    Reagents
    PE labeled F(ab)2 fractions of goat anti-rat IgG, goat anti-human IgG, goat anti-mouse IgG and FITC labeled human IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Mouse anti-human CD61 was obtained from BD Biosciences, (San Jose, CA). Fab fractions of mAb anti-human FcγRII (IV.3) were prepared in our laboratory. Monomeric human IgG (mono-IgG) was prepared by ultracentrifugation of human IgG (MP Biomedicals, OH) at 80K for 15 min. Heat aggregated human IgG (HA-IgG) was prepared by heating human IgG (10mg/ml) in PBS at 62C for 20 min, followed by centrifugation at 12,000 rpm for 10 min to remove insoluble aggregates. The final IgG complex was used at 100 μg/ml as assessed by absorbance at 280 nm.
    Monocyte, engulfed platelets, Platelet Satellitism attachment.php?s=f9d43a1117ee6a318ffda7114b3a727f&attachmentid=1437&d=1439745863

    Animals and cell lines
    FcγRIIA transgenic mice were provided by Dr. Steven E. McKenzie (Thomas Jefferson University, Philadelphia, PA). All protocols were performed in accordance with National Institutes of Health guidelines and with the approval by the University of Pennsylvania Animal Use Committee. COS cells stably expressing FcγRIIA cell line (COSIIA) was constructed in our laboratory as previously described (Huang et al., 2004).

    Isolation of human and mouse platelets
    Platelet-rich plasma was prepared from heparinized venous blood of healthy volunteers by centrifugation of the blood at 900 rpm (175g) at room temperature. Platelets were isolated by gel filtration on Sepharose 2B. A similar protocol was used to isolate platelets from WT and FcγRIIA TG mice.

    Internalization of IgG coated beads
    Streptavidin conjugated polystyrene beads (0.1, 0.5 and 1.0 μm) (Bangs Laboratories, Inc, Fishers, IN) were coated with biotin/anti-biotin mAb according to the manufacturer's instructions. The IgG-coated beads were allowed to bind (45 min) to platelets at 4C. One set of cells was maintained at 4C as a binding control. Other cells were warmed to 37C for 20 min to allow internalization of bound IgG-coated beads and then returned to 4C to stop internalization. Goat anti-mouse IgG conjugated to phycoerythrin (PE) was added to label remaining externally bound IC polystyrene beads and cell fluorescence was analyzed by flow cytometry. The cells maintained at 4C retain a large amount of IC on the surface and display bright fluorescence. However, cells incubated at 37C that have internalized some of the surface bound IgG-coated beads display less fluorescence. Internalization is defined as the % loss of surface expression (fluorescent label). For each condition, one set of cells was pre-incubated with cytochalesin D (CytD) and CytD was also present during the uptake assay.

    Isolation of human monocytes and neutrophils
    Heparinized venous blood collected from healthy volunteers was overlaid on Ficoll-Hypaque (LSM; Organon-Teknika, West Chester, PA) and centrifuged at 2200 rpm for 30 minutes. Pelleted granulocytes were cleared of residual erythrocytes by brief hypertonic lysis and resuspended in RPMI. Mononuclear cells from the interface were washed, suspended in RPMI medium (GIBCO, Grand Island, NY) plus 10% heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT), and incubated at 37C on serum-coated flasks for 45 minutes to adhere monocytes. Nonadherent cells were removed by washing with RPMI.

    Internalization of human platelets by human monocytes and neutrophils: Analysis by flow cytometry
    Gel filtered human platelets were incubated with HA-IgG for 30 min at 0C. The IgG-coated platelets were washed and then incubated with human monocytes or neutrophils on ice for 45 min. One aliquot was maintained at 0C for an additional 30 min for use as a binding control. The other aliquot was rapidly warmed and incubated at 37C for 30 min to allow internalization of the HA-IgG platelet complexes. Both sets of cells were washed with ice-cold PBS and further incubated for 30 min on ice with 3.5 μg/ml phycoerythrin (PE) conjugated F(ab)2 goat anti-human IgG to label surface bound HA-IgG platelet complexes. Cells were washed again with ice-cold PBS before analysis by flow cytometry. The mean fluorescence (MF) of cells maintained on ice throughout the procedure was used as the binding reference (no phagocytosis) (Van de Winkel and Capel, 1993). In other experiments, monocytes were exposed to platelets labeled with FITC-conjugated HA-IgG. Under these conditions, FITC fluorescence represents both surface-bound and intracellular HA-IgG platelet complexes.

    Electron microscopy
    Monocytes, isolated as described above, were washed and fixed with 2% gluteraldehyde at 4C overnight. The samples were then washed extensively with cacodylate buffer, fixed with osmium tetroxide, dehydrated, embedded in Spurr's resin and sectioned (Worth, 2006). The sections were viewed at the EM core facility at University of Pennsylvania.

    References:
    Human Platelet FcγRIIA and Phagocytes in Immune-Complex Clearance











    Last edited by Medical Photos; 08-16-2015 at 05:24 PM.

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