CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG (2-10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgG1 and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

The effect of extracellular nucleotides on cytokine production and phagocytosis of macrophages

Bacterial sepsis is a severe clinical condition, leading to severe sepsis, septic shock, and death. The complex pathophysiology of sepsis is not yet fully understood. Cytokines, released by immune cells such as macrophages, play an important role in the pathophysiology of sepsis. Kupffer cells are the largest population of macrophages in the body. Purinergic signaling, mediated by different nucleosides and nucleotides, and purinergic receptors, has been shown to have various effects on cytokine release, inflammatory processes and the immune system. In our work with in vitro experiments we studied the effect of extracellular nucleotides on the release of TNFα by primary murine Kupffer cells, and the effect of extracellular nucleotides on the phagocytosis of murine RAW 264.7 and human U-937 cell culture macrophages. Secretion of TNFα was measured using ELISA, phagocytosis of bio particles was measured using a plate reader phagocytosis assay and flow cytometry. Our experiments show, that extracellular LPS stimulate release of TNFα in murine Kupffer cells and that extracellular nucleotides inhibit this effect in a dose dependent matter. Our other experiments show phagocytosis of fluorescence labeled bio particles by both macrophage cell lines RAW 264.7 and U-937 in a dose dependent manner. The experiments could not show an effect of extracellular nucleotides on phagocytosis of cell culture macrophages.

The granulocyte orphan receptor CEACAM4 is able to trigger phagocytosis of bacteria.

Human granulocytes express several glycoproteins of the CEACAM family. One family member, CEACAM3, operates as a single-chain phagocytic receptor, initiating the detection, internalization, and destruction of a limited set of gram-negative bacteria. In contrast, the function of CEACAM4, a closely related protein, is completely unknown. This is mainly a result of a lack of a specific ligand for CEACAM4. By generating chimeric proteins containing the extracellular bacteria-binding domain of CEACAM3 and the transmembrane and cytoplasmic part of CEACAM4 (CEACAM3/4) we demonstrate that this chimeric receptor can trigger efficient phagocytosis of attached particles. Uptake of CEACAM3/4-bound bacteria requires the intact ITAM of CEACAM4, and this motif is phosphorylated by Src family PTKs upon receptor clustering. Furthermore, SH2 domains derived from Src PTKs, PI3K, and the adapter molecule Nck are recruited and associate directly with the phosphorylated CEACAM4 ITAM. Deletion of this sequence motif or inhibition of Src PTKs blocks CEACAM4-mediated uptake. Together, our results suggest that this orphan receptor of the CEACAM family has phagocytic function and prompt efforts to identify CEACAM4 ligands.

DETERMINANTS OF THE SELECTIVE PHAGOCYTOSIS OF CARCINOGENIC PARTICULATE NICKEL COMPOUNDS

Certain inorganic nickel compounds such as crystalline NiS and Ni(,3)S(,2) are potent inducers of carcinogenesis and in vitro cell transformation, while several closely-related compounds such as amorphous NiS are essentially devoid of genotoxic activity. The phenomenon of selectivity of phagocytosis among such particulate nickel compounds has been hypothesized to account for their widely varying toxicological potency, yet the determinants of this selectivity have not been well characterized. Extracellular medium composition, particle dissolution, and particle surface charge were examined as potential determinants of selective phagocytosis for the carcinogenic crystalline and noncarcinogenic amorphous modifications of NiS. Selectivity and avidity of uptake of crystalline NiS by CHO cells was not dependent upon serum: phagocytosis of crystalline, but not amorphous NiS proceeded readily in a minimal salts/glucose medium at 37(DEGREES)C. The evolution of phagocytosis-inhibiting Ni(II) from the surface of amorphous NiS particles did not demonstrably contribute to the lower uptake of these noncarcinogenic particles despite their somewhat greater dissolution rate than the readily phagocytosed crystalline NiS particles. Significant differences in surface charge were noted between crystalline and amorphous NiS, the former being more negative in charge in distilled water suspension. Exposure of amorphous NiS particles to the vigorously reducing environment of a LiAlH(,4) solution under an inert atmosphere resulted in the particles' acquisition of a more negative surface charge. Amorphous NiS particles thus treated were phagocytosed by CHO cells to an extent similar to that of untreated crystalline NiS particles and likewise were shown to induce morphological transformation of primary Syrian hamster embryo cells with a similar potency. The potentiation of uptake characteristic of LiAlH(,4)-treated amorphous NiS was lost gradually upon storage of particles in ambient oxygenated atmosphere and was lost rapidly by apparent particle surface oxidation in aerated distilled water suspensions aged for up to 7 days. Concomitant with this loss of uptake there occurred a loss of negative surface charge. These results suggest the predominant role of particle surface charge rather than adsorbed serum components or particle dissolution as a determinant of selective phagocytosis among particulate nickel compounds. ^

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires αvβ5 integrin, rather than αvβ3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both αvβ3 and αvβ5 integrins, only αvβ3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of αvβ5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, αvβ5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas αvβ3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, αvβ5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for αvβ5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of αvβ5 integrin with internalized ROS. Control experiments showed that blocking αvβ3 function with antibodies did not inhibit ROS phagocytosis and that αvβ3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor αvβ5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-αvβ5 complex. αvβ5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.

The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis in Dictyostelium discoideum

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: Evidence that oxidation-specific epitopes mediate macrophage recognition

Apoptosis is recognized as important for normal cellular homeostasis in multicellular organisms. Although there have been great advances in our knowledge of the molecular events regulating apoptosis, much less is known about the receptors on phagocytes responsible for apoptotic cell recognition and phagocytosis or the ligands on apoptotic cells mediating such recognition. The observations that apoptotic cells are under increased oxidative stress and that oxidized low-density lipoprotein (OxLDL) competes with apoptotic cells for macrophage binding suggested the hypothesis that both OxLDL and apoptotic cells share oxidatively modified moieties on their surfaces that serve as ligands for macrophage recognition. To test this hypothesis, we used murine monoclonal autoantibodies that bind to oxidation-specific epitopes on OxLDL. In particular, antibodies EO6 and EO3 recognize oxidized phospholipids, including 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine (POVPC), and antibodies EO12 and EO14 recognize malondialdehyde-lysine, as in malondialdehyde-LDL. Using FACS analysis, we demonstrated that each of these EO antibodies bound to apoptotic cells but not to normal cells, whereas control IgM antibodies did not. Confocal microscopy demonstrated cell-surface expression of the oxidation-specific epitopes on apoptotic cells. Furthermore, each of these antibodies inhibited the phagocytosis of apoptotic cells by elicited peritoneal macrophages, as did OxLDL. In addition, an adduct of POVPC with BSA also effectively prevented phagocytosis. These data demonstrate that apoptotic cells express oxidation-specific epitopes—including oxidized phospholipids—on their cell surface, and that these serve as ligands for recognition and phagocytosis by elicited macrophages.

Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.

Numerous immature thymocytes undergo apoptosis and are rapidly engulfed by phagocytic thymic macrophages. The macrophage surface receptors involved in apoptotic thymocyte recognition are unknown. We have examined the role of the class A macrophage scavenger receptor (SR-A) in the engulfment of apoptotic thymocytes. Uptake of steroid-treated apoptotic thymocytes by thymic and inflammatory-elicited SR-A positive macrophages is partially inhibited by an anti-SR-A mAb and more completely by a range of scavenger receptor ligands. Thymic macrophages from mice with targeted disruption of the SR-A gene show a 50% reduction in phagocytosis of apoptotic thymocytes in vitro. These data suggest that SR-A may play a role in the clearance of dying cells in the thymus.

Clustered syk tyrosine kinase domains trigger phagocytosis.

Phagocytosis is a phylogenetically primitive mechanism adapted by specialized cells of the immune system to ingest particulate pathogens. Recent evidence suggests that the program of specific cytoskeletal rearrangements that underlies phagocytosis may share elements with the antigen receptor signaling pathway in lymphocytes. Tyrosine phosphorylation, necessary for both lymphocyte effector function and phagocytosis, is thought to allow cytoskeletal elements to couple to the intracellular domains of antigen and Fc receptor subunits. We show here that the intracellular domains of the receptors are not inherently required for cytoskeletal coupling. Chimeric transmembrane proteins bearing syk but not src family tyrosine kinase domains are capable of autonomously triggering phagocytosis and redistribution of filamentous actin in COS cells. These responses cannot be initiated by a receptor chimera bearing a point mutation in the syk catalytic domain, and the kinase domain alone is sufficient for initiating cytoskeletal coupling.

Phagocytosis of Photoreceptor Outer Segments by Transplanted Human Neural Stem Cells as a Neuroprotective Mechanism in Retinal Degeneration

Purpose. Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. Methods. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). Results. The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor–bipolar–horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. Conclusions. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Acanthamoebal surface properties and the modulation of phagocytosis

The surface nature of Acanthamoeba trophozoites and cysts was investigated with respect to cell surface charge, hydrophobicity and surface carbohydrate composition. Particulate microelectrophoresis revealed a marked negative charge for both morphological forms, though less for cyst surfaces. Hydrophobicity was determined by adhesion to n-hexadecane and indicated a relatively low hydrophobic nature of both forms, though less so for cysts. Surface carbohydrate composition was studied by the use of fluorescent lectins and flow cytometry, using a ligand-receptor approach for further in depth analysis of binding of particular lectins. These studies showed trophozoite and cyst surfaces to be rich in N-acetylglucosamine, N-acteylneuraminic acid, mannose and glucose, with the addition of N-acetylgalactosamine on cysts. The importance of such surface properties was investigated with respect to phagocytosis of polystyrene latex microspheres, of different surface types and size. Investigations into the optimum conditions of uptake of beads indicated a preference for a medium devoid of nutrients, such as saline, though temperature was not a factor. An amoebal predilection for beads of lower charge and greater hydrophobicity was demonstrated. Furthermore, a preference for the largest bead size used (2.0 m) was observed. The influence of either Con A or mannose or glucose on bead association was apparently limited. The fate of foreign DNA ingested by Acanthamoeba appeared to indicate that such DNA was destroyed, as it could not be detected following extraction procedures and PCR amplification.

Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.

Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.