Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37 °C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia.     

A progressive reduction in autophagic capacity contributes to induction of replicative senescence in Hs68 cells

Autophagy has been implicated in delayed aging and extended longevity. Here, we aimed to study the possible effects of autophagy during the progression of replicative senescence, which is one of the major features of aging. Human foreskin fibroblasts, Hs68 cells, at an initial passage of 15 were serially cultured for several months until they reached cellular senescence. A decrease in cell proliferation was observed during the progression of senescence. Induction of replicative senescence in aged cells (at passage 40) was confirmed by senescence-associated β-galactosidase (SA-β-gal) activity that represents a sensitive and reliable marker for quantifying senescent cells. We detected a significantly increased percentage (%) of SA-β-gal-positive cells at passage 40 (63%) when compared with the younger SA-β-gal-positive cells at passage 15 (0.5%). Notably, the gradual decrease in basal autophagy coincided with replicative senescence induction. However, despite decreased basal autophagic activity in senescent cells, autophagy inducers could induce autophagy in senescent cells. RT-PCR analysis of 11 autophagy-related genes revealed that the decreased basal autophagy in senescent cells might be due to the downregulation of autophagy-regulatory proteins, but not autophagy machinery components. Moreover, the senescence phenotype was not induced in the cells in which rapamycin was added to the culture to continuously induce autophagy from passage 29 until passage 40. Together, our findings suggest that reduced basal autophagy levels due to downregulation of autophagy-regulatory proteins may be the mechanism underlying replicative senescence in Hs68 cells.     

The induction of host cell autophagy triggers defense mechanisms against Trypanosoma cruzi infection in vitro

Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.     

THIS ARTICLE HAS BEEN RETRACTED Antifungal antibiotic hamycin increases susceptibility of Candida albicans to phagocytosis by murine macrophages

Hamycin is an antifungal antibiotic produced by Streptomyces pimprina Thirum. In the present study, the effect of hamycin on (a) the phagocytosis of Candida albicans by murine peritoneal macrophages and (b) the cell surface hydrophobicity (CSH) of C. albicans was investigated. Addition of hamycin to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by macrophages. Pretreatment of Candida cells with hamycin increased their vulnerability to killing by macrophages. Examination of physico-chemical properties of Candida cell surface showed a significant decrease in the CSH. These findings suggest that the binding of hamycin to Candida cells induces biochemical/physico-chemical alterations of the surface, so that it becomes more susceptible to phagocytosis by murine macrophages.     

Spore diffusate isolated from some strains of Aspergillus fumigatus inhibits phagocytosis by murine alveolar macrophages

Aspergillus fumigatus is a ubiquitous fungus that grows in decaying organic matter. It can cause disease in both immunodeficient and immunocompetent patients by using virulence factors to escape the host defenses. Some of these factors, such as a diffusate, released from the spores of A. fumigatus, have previously been described. This diffusate was demonstrated to inhibit oxidative burst and phagocytosis of coated red blood cells. The present study has shown that this substance can inhibit the phagocytosis of A. fumigatus spores by murine alveolar macrophages (MALU) and evaluated the action of this substance. We quantified phagocytosis by MALU cells with and without diffusate and evaluated the inhibition of phagocytosis by testing diffusates from different strains. We conclude that the spore diffusate of some strains of A. fumigatus can reversibly decrease the ability of alveolar macrophages to ingest A. fumigatus spores.     

Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.     

Phagocytosis of apoptotic and necrotic thymocytes is inhibited by PAF-receptor antagonists and affects LPS-induced COX-2 expression in murine macrophages

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.     

Trypanosoma brucei: recognition in vitro of two developmental forms by murine macrophages

In vitro, murine macrophages attach to the midgut form of Trypanosoma brucei in the absence of exogenous proteins. Recognition appears to be specific and saturable. Attachment is mediated by a non-Fc, non-C3 receptor on the macrophage plasma membrane. The attachment mechanism is protease sensitive, temperature sensitive, requires the presence of divalent cations, and is functional in fetal calf serum-free medium. The midgut form is also lysed in normal rabbit serum by activating the alternative complement pathway. The form isolated from the blood is neither lysed in normal serum nor is it spontaneously recognized by the macrophage, in vitro. Partial trypsinization of the bloodstream form, however, results in both the triggering of alternative complement pathway lysis and spontaneous uptake into macrophages. Murine macrophages attach to the midgut form of T. brucei by a receptor on the macrophage plasma membrane which is capable of recognizing particulate activators of the alternative complement pathway.     

Specific functions of lysosomal proteases in endocytic and autophagic pathways

Endolysosomal vesicles form a highly dynamic multifunctional cellular compartment that contains multiple highly potent proteolytic enzymes. Originally these proteases have been assigned to cooperate solely in executing the unselective ‘bulk proteolysis’ within the acidic milieu of the lysosome. Although to some degree this notion still holds true, evidence is accumulating for specific and regulatory functions of individual ‘acidic’ proteases in many cellular processes linked to the endosomal/lysosomal compartment. Here we summarize and discuss the functions of individual endolysosomal proteases in such diverse processes as the termination of growth factor signaling, lipoprotein particle degradation, infection, antigen presentation, and autophagy. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase

Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl^−/− macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl^−/− macrophages. Hence, phagocytosis was also decreased in vivo when Atgl^−/− mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Inhibition by rapamycin of the lipoteichoic acid-induced granulocyte-colony stimulating factor expression in mouse macrophages

Granulocyte-colony stimulating factor (G-CSF) is a cytokine which involves in anti-inflammation and inflammation as well. Rapamycin is an inhibitor of mTOR which also plays a role in innate immunity. This study investigated the effect of rapamycin on the lipoteichoic acid (LTA)-induced expression of G-CSF in macrophages and its underlying mechanism. Our data show that LTA induced G-CSF expression in RAW264.7 and bone marrow-derived macrophages and that this effect was inhibited by rapamycin. Analysis of the G-CSF 5′ flanking sequence revealed that the −283 to +35 fragment, which contains CSF and octamer elements, was required for maximal promoter activity in response to LTA stimulation. Western blot analyses of proteins that bind to the CSF and octamer element show that LTA increased protein levels of NF-κB, C/EBPβ and Oct-2, and that rapamycin inhibited the LTA-induced increase in Oct-2 protein levels, but not the others. Knockdown of Oct-2 by RNA interference resulted in a decrease in LTA-induced G-CSF mRNA levels. Moreover, forced expression of Oct-2 by transfection with the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LTA-induced increase in G-CSF mRNA levels and promoter activity. This study demonstrates that rapamycin reduces G-CSF expression in LTA-treated macrophages by inhibiting Oct-2 expression.     

A Salmonella enterica conjugative plasmid impairs autophagic flux in infected macrophages

pR_ST98 was originally isolated from Salmonella enterica serovar typhi and could be transferred among enteric bacilli by conjugation. Our previous studies indicated that it could intervene in autophagy of host cells, while the mechanism remained undefined. Here, we explored how pR_ST98 influenced the autophagic flux of murine macrophage-like cell line (J774A.1). S. enterica serovar typhimurium wild type strain (χ3306), harboring a 100 kb virulence plasmid, was used as a positive control. pR_ST98 was transferred into χ3306 virulence plasmid cured strain (χ3337) to create the transconjugant strain (χ3337/pR_ST98). The bacterial strains incubated with J774A.1 revealed that survival rate of intracellular bacteria carrying pR_ST98 was higher than that of plasmid free strain; presence of pR_ST98 decreased the number of autophagy vacuoles, LC3 positive and p62 positive bacteria, and also the level of LC3-II and degradation of p62 in macrophages. After intervention with autophagy inhibitor chloroquine, the amount of LC3-II and autophagy vacuoles were still lower in macrophages infected with strains carrying pR_ST98. Our study suggested that pR_ST98 could block or delay the formation of autophagosome in the earlier autophagy process, but couldn't affect the function of autolysosome. This finding provided novel insights into the role of enteric conjugation plasmid in bacterial pathogenesis.     

Micromechanics of filopodia mediated capture of pathogens by macrophages

The biological function of filopodia has been extensively studied while only little work has been done on their mechanical properties. In the present study, we apply magnetic microbeads to explore the capturing and initial step of phagocytosis of pathogens by macrophages through filopodia. Microbeads were covered by the bacterial coat protein invasin which is known to trigger the invasion of the intestine by the bacteria Yersinia enterocolitica. These mimetics of bacteria were placed in the vicinity of J774 mouse macrophages exhibiting long filopodia. The specific adhesion of beads to the tip of a filopodium induced the retraction of the protrusion resulting in the dragging of the bead towards the cell body. The dynamics of the retraction process was analyzed by following the in-plane motion of the bead. We estimated the minimal force developed by filopodia and compared the results with previous magnetic tweezer studies of mechanical force induced growth of protrusions (Vonna et al. 2003). We show that very thin filopodia can generate astonishingly large retraction forces over large distances (>10 μm) and can act as an efficient mechanical tool to detach pathogens adhering on surfaces.     

Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection

Abstract Phagosome-lysosome fusion in murine macrophages infected with S. typhimurium LT2 or S. typhi 1079 was investigated. Fusion of phagosome containing S. typhimurium LT2 with lysosome was markedly impaired, whereas S. typhi 1079 did not inhibit phagosome-lysosome fusion in murine macrophages. A similar inhibition of fusion was observed with LPS-deficient mutants of S. typhimurium LT2, suggesting that O-antigens do not contribute to the inhibition of fusion. Phagosome-lysosome fusion in macrophages after ingestion of UV-killed S. typhimurium LT2 was much greater than that of live bacteria. Furthermore, treatment of S. typhimurium LT2 with streptomycin, an inhibitor of bacterial protein synthesis, caused an increase in the extent of phagosome-lysosome fusion. Therefore protein synthesis in live bacteria is probably required for the inhibition of phagosome-lysosome fusion. These results suggest that phagosome-lysosome fusion in murine macrophages is impaired by some product(s) of viable S. typhimurium LT2.     

Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.     

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of ¹²⁵I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronection receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.