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Purpose There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. Methods THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. Results We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. Conclusions The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages., Author(s): Laura R. Sadofsky [sup.1] , Yvette A. Hayman [sup.2] , Jesse Vance [sup.3] , Jorge L. Cervantes [sup.3] , Simon D. Fraser [sup.2] , Holly N. Wilkinson [sup.1] , [...]

The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation, and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for remaining apoptotic debris in regulating the immunologic response to and growth of solid tumors. Here, we investigated the role of macrophage LC3-associated phagocytosis (LAP) within the BM microenvironment of AML. Depletion of BM macrophages (BMMs) increased AML growth in vivo. We show that LAP is the predominate method of BMM phagocytosis of dead and dying cells in the AML microenvironment. Targeted inhibition of LAP led to the accumulation of apoptotic cells (ACs) and apoptotic bodies (ABs), resulting in accelerated leukemia growth. Mechanistically, LAP of AML-derived ABs by BMMs resulted in stimulator of IFN genes (STING) pathway activation. We found that AML-derived mitochondrial damage-associated molecular patterns were processed by BMMs via LAP. Moreover, depletion of mitochondrial DNA (mtDNA) in AML-derived ABs showed that it was this mtDNA that was responsible for the induction of STING signaling in BMMs. Phenotypically, we found that STING activation suppressed AML growth through a mechanism related to increased phagocytosis. In summary, we report that macrophage LAP of apoptotic debris in the AML BM microenvironment suppressed tumor growth., Introduction Acute myeloid leukemia (AML) is a genetically, biologically, and clinically heterogeneous set of diseases that share in common the malignant proliferation of clonal hematopoietic stem and progenitor cells (HSPCs) [...]

In amoeboid cells, food particles are engulfed only after receptors on the phagocytic cell's membrane bind to ligands on a particle's surface. Ciliates also feed via phagocytosis but, instead of enveloping particles, some ciliates take them up through a complex, permanent, funnel-shaped, feeding apparatus. It is unclear whether receptor-ligand interactions are needed to trigger the process. If ciliates were shown to feed selectively on certain particles over others, based on the particles' surface properties, then receptor-ligand interactions would likely play a role in phagocytosis. The literature includes few reports of such selectivity. To further investigate this issue, we chose to study feeding preference in the ciliate Tetrahymena pyriformis. We fed Tetrahymena mixtures of orange and green, fluorescent, 3 [micro]m, polystyrene beads at two concentrations. One of the two types of beads was coated with bovine serum albumin. Authors were blinded to experimental conditions. We found no evidence of a preference for coated or uncoated beads at either concentration. We also found no trend toward the development of selective feeding as cells acquired more beads over time. Although we cannot rule out the possibility that Tetrahymena feeds selectively, we did not find convincing evidence of such selectivity when T. pyriformis is given a choice between uncoated beads and those coated with albumin. Our results failed to demonstrate a role for molecular recognition when Tetrahymena engages in phagocytosis. Keywords: Alveolata, alveolate, Ciliophora, filter-feeding, INTRODUCTION Phagocytosis is best understood as it occurs in amoeboid cells that serve as model organisms (Cardelli 2001; Alberts et al. 2002, p. 747). The process is triggered by molecular [...]

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal roles, including immune defence, tissue homeostasis, and remodeling. Accordingly, phagocytes display a high level of plasticity in their capacity to recognize, engulf, and process targets that differ in composition and morphology. Here, we review how phagocytosis adapts to its multiple roles and discuss in particular the effect of target morphology in phagocytic uptake and phagosome maturation.Key words: phagocytosis, target morphology, phagocytic plasticity, phagosomal maturation, phagocytic cup.La phagocytose est un processus conserve durant l'evolution. Chez les protozoaires, la phagocytose remplit des fonctions alimentaires alors que chez les metazoaires, la phagocytose s'est diversifiee pour remplir de multiples roles dans l'organisme, dont la defense immunitaire, l'homeostasie des tissus et le remodelage. Consequemment, les phagocytes montrent un haut degre de plasticite dans leur capacite a reconnaitre, engloutir et transformer des particules cibles qui varient quant a leur composition et leur morphologie. Les auteurs presentent ici l'adaptation d'un phagocyte a ses multiples roles et discutent plus particulierement de l'effet de la morphologie d'une particule cible dans la captation phagocytaire et la maturation du phagosome. [Traduit par la Redaction]Mots-cles : phagocytose, morphologie de la cible, plasticite phagocytaire, maturation du phagosome, coupe phagocytaire., Overview of phagocytosis and phagosome maturationProtozoa and certain cells within Metazoa have the ability to selectively internalize particles and degrade them intracellularly through a process known as phagocytosis. In this [...]

The mechanisms of pain induction by inflammation have been extensively studied. However, the mechanisms of pain resolution are not fully understood. Here, we report that GPR37, expressed by macrophages (MOs) but not microglia, contributes to the resolution of inflammatory pain. Neuroprotectin D1 (NPD1) and prosaptide TX14 increase intracellular [Ca.sup.2+] (i[Ca.sup.2+]) levels in GPR37-transfected HEK293 cells. NPD1 and TX14 also bind to GPR37 and cause GPR37- dependent i[Ca.sup.2+] increases in peritoneal M[PHI]s. Activation of GPR37 by NPD1 and TX14 triggers M[PHI] phagocytosis of zymosan particles via calcium signaling. Hind paw injection of pH-sensitive zymosan particles not only induces inflammatory pain and infiltration of neutrophils and M[PHI]s, but also causes GPR37 upregulation in M[PHI]s, phagocytosis of zymosan particles and neutrophils by M[PHI]s in inflamed paws, and resolution of inflammatory pain in WT mice. Mice lacking Gpr37 display deficits in M[PHI] phagocytic activity and delayed resolution of inflammatory pain. Gpr37-deficient M[PHI]s also show dysregulations of proinflammatory and antiinflammatory cytokines. M[PHI] depletion delays the resolution of inflammatory pain. Adoptive transfer of WT but not Gpr37-deficient M[PHI]s promotes the resolution of inflammatory pain. Our findings reveal a previously unrecognized role of GPR37 in regulating M[PHI] phagocytosis and inflammatory pain resolution., Introduction Inflammation is characterized by 5 cardinal signs: rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). As a cardinal feature of inflammation, pain [...]

The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved. Using intravital microscopy, we found that antibody-dependent phagocytosis (ADPh) by macrophages is a prominent mechanism for removal of tumor cells from the circulation in a murine tumor cell opsonization model. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells). In the absence of mAbs, Kupffer cells sampled tumor cells; however, this sampling was not sufficient for elimination. By contrast, antitumor mAb treatment resulted in rapid phagocytosis of tumor cells by Kupffer cells that was dependent on the high-affinity IgG-binding Fc receptor (FcγRI) and the low-affinity IgG-binding Fc receptor (FcγRIV). Uptake and intracellular degradation were independent of reactive oxygen or nitrogen species production. Importantly, ADPh prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy were lost after Kupffer cell depletion. Our data indicate that macrophages play a prominent role in mAb-mediated eradication of tumor cells. These findings may help to optimize mAb therapeutic strategies for patients with cancer by helping us to aim to enhance macrophage recruitment and activity., Introduction Therapeutic monoclonal antibodies (mAbs), which can be designed to specifically interact with tumor-associated antigens, represent a promising novel category of drugs for targeting malignancies in addition to chemotherapy or [...]

Anti-CD20 Ab therapy has proven successful for treating B cell malignancies and a number of autoimmune diseases. However, how anti-CD20 Abs operate in vivo to mediate B cell depletion is [...]

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, [...]

Phagocytosis of apoptotic cells is critical to resolution of inflammation. High mobility group box 1 protein (HMGB1), a mediator of inflammation, has been shown to diminish phagocytosis through binding to phosphatidylserine (PS) exposed on the surface of apoptotic neutrophils. However, it is currently unknown whether HMGB1 also modulates the activity of receptors involved in PS recognition on the surface of phagocytes. In the present studies, we found that preincubation of macrophages with HMGB1 decreased their ability to engulf apoptotic neutrophils or thymocytes. Preincubation of macrophages with HMGB1 prevented the enhancement of efferocytosis resulting from exposure to milk fat globule EGF factor 8 (MFG-E8), an opsonin that bridges PS and [[alpha].sub.v][[beta].sub.3] as well as [[alpha].sub.v][[beta].sub.5]-integrins on the surface of phagocytes. The inhibitory effect of HMGB1 on the phagocytic activity of macrophages was prevented by preincubation of HMGB1 with soluble [[alpha].sub.v][[beta].sub.3], but not with soluble [[alpha].sub.v][[beta].sub.5]. HMGB1 colocalized with the [[beta].sub.3]-integrin on the cell membrane of macrophages and bound to soluble [[alpha].sub.v][[beta].sub.3], but not to soluble [[alpha].sub.v][[beta].sub.5]. HMGB1 suppressed the interaction between MFG-E8 and [[alpha].sub.v][[beta].sub.3]. HMGB1 also inhibited intracellular signaling events, including ERK phosphorylation and Rac-1 activation, which are activated in macrophages during phagocytosis of apoptotic cells. These results demonstrate that HMGB1 blocks [[alpha].sub.v][[beta].sub.3]-dependent recognition and uptake of apoptotic cells. phagocytosis; milk fat globule-epidermal growth factor 8; apoptotic neutrophils; high mobility group box 1 protein doi: 10.1152/ajpcell.00152.2010.

During Fc[gamma] receptor (FcR)-mediated phagocytosis by macrophages, cytoplasm advances over IgG-coated particles by the sequential ligation of FcR in plasma membranes. If FcR signaling was strictly autonomous, then the signals generated during phagocytosis should be proportional to the number of ligated receptors. By measuring FcR-dependent responses to beads coated with various densities of IgG, this study identified nonlinear signaling that organizes an all or none response during particle ingestion, Phagocytosis of beads with IgG at low density either stalled after making small, actin-rich cups or proceeded to completion at the same rate as phagocytosis of high-density IgG beads. Signals were measured by quantifying the recruitment of YFP-labeled probes to phagocytic cup membranes. Although the magnitude of early signals correlated with IgG density, later signals showed an all or none response, which was regulated by the concentrations of 3' phosphoinositides in phagocytic cup membranes. Thus, 3' phosphoinositides, shown previously to be required for phagocytosis, function in a feedback regulatory mechanism affecting late but not early signals. This indicates a mechanism for the coordination of cell movements initiated by receptor signaling. fluorescence | macrophage | microscopy | phosphatidylinositol 3-kinase | phosphoinositides doi/ 10.1073/pnas.1008248107

Together with the development of genetically encoded fluorescent probes, digital imaging has provided great impetus to the study of cell signaling by providing enhanced sensitivity and much-improved spatial and temporal resolution. We have used phagocytosis as a paradigm of signal transduction, taking advantage of the generous size of phagosomes and of their comparatively leisurely rate of formation. Aided by the design of specific probes, we demonstrated a highly localized and elegantly choreographed sequence of changes in the level of several phosphoinositides and were able to also monitor the fate of phosphatidylserine. The net changes in the content of these anionic phospholipids are accompanied by marked alterations in the surface charge of the membrane of nascent phagosomes. These, in turn, cause the relocation of proteins that associate with the membrane by electrostatic interactions. Our studies suggest that anionic lipids control protein targeting not only through stereospecific recognition by specialized domains but also by electrostatic association mediated by polycationic motifs. The 'electrostatic switch' can be turned on or off by altering the charge of the protein ligand (e.g., by phosphorylation) or, alternatively, by modifying the lipid composition of the target membrane. phosphoinositide; phosphatidylserine; phagosome; macrophage doi: 10.1152/ajpcell.00342.2010.

Chemokines and chemokine receptors are key evolutionary innovations of vertebrates. They are involved in morphogenetic processes and play an important role in the immune system. Based on an analysis of the chemokine receptor gene family in teleost genomes, and the expression patterns of chemokine receptor genes during embryogenesis and the wounding response in young larvae of Oryzias latipes, we identified the chemokine receptor cxcr3a as a marker of innate immune cells. Cells expressing cxcr3a were characterized in fish transgenic for a cxcr3a:gfp reporter. In embryos and larvae, cxcr3a-expressing cells are motile in healthy and damaged tissues, and phagocytic; the majority of these cells has the morphology of tissue macrophages, whereas a small fraction has a dendritic phenotype. In adults, cxcr3a-positive cells continue to specifically express myeloid-associate markers and 'genes related to antigen uptake and presentation. By light microscopy and ultrastructural analysis, the majority of cxcr3a-expressing ceils has a dendritic phenotype, whereas the remainder resembles macrophage-like cells. After challenge of adult fish with bacteria or CpG oligonucleotides, phagocytosing cxcr3a-positive cells in the blood up-regulated il12p40 genes, compatible with their function as part of the mononuclear phagocytic system. Our results identify a marker of teleost mononuclear phagocytic cells and suggest a surprising degree of morphological and functional similarity between the innate immune systems of lower and higher vertebrates. dendritic cell | chemokine receptor | immune system | evolution | il12p40 doi/ 10.1073/pnas.1000467107

Tim-4 is a phosphatidylserine (PS) receptor that is expressed on various macrophage subsets. It mediates phagocytosis of apoptotic cells by peritoneal macrophages. The in vivo functions of Tim-4 in phagocytosis and immune responses, however, are still unclear. In this study, we show that Tim-4 quickly forms punctate caps on contact with apoptotic cells, in contrast to its normal diffused expression on the surface of phagocytes. Despite its expression in marginal zone and tingible body macrophages, Tim-4 deficiency only minimally affects outcomes of several acute immune challenges, including the trapping of apoptotic cells in the marginal zone, the clearance apoptotic cells by tingible body macrophages, and the formation of germinal centers and elicitation of antibody responses against sheep red blood cells (SRBCs). In addition, Tim-[4.sup.-/-] resident peritoneal macrophages (rPMs) phagocytose necrotic cells and other opsonized targets normally. However, their ability to bind and engulf apoptotic cells is significantly compromised both in vitro and in vivo. Most importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Resting rPMs produce higher TNF-[alpha] in culture. Their response to LPS, on the contrary, is dampened. Our data support an indispensible role of Tim-4 in maintaining the homeostasis of rPMs. phagocytosis | apoptotic cells | receptor dynamics | TNF-[alpha] doi/10.1073/pnas.0910929107

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal. animal model; bile duct; collagen; collagenase; cirrhosis; extracellular matrix; gelatinase; hepatic stellate cell; Kupffer cell; integrin [alpha]v[beta]6; liver fibrosis resolution; matrix metalloproteinase; phagocyte; PDGF; repair; transforming growth factor-[beta]; tissue inhibitor of matrix metalloproteinases; zymography; urokinase plasminogen activator receptor-1 doi:10.1152/ajpgi.00394.2009.

Organic dust exposure in agricultural animal environments results in airway diseases. Dendritic cells (DCs) orchestrate inflammatory immune response in the airways, but little is known about how organic dust affects differentiation and maturation of monocyte-derived immature and mature DCs (iDCs, mDCs). Peripheral blood monocytes were differentiated in vitro into iDCs with granulocyte-macrophage colony stimulating factor + IL-4 (6 days) with and without swine facility organic dust extract (ODE, 0.1%). Unlike control iDCs, ODE-conditioned iDCs maintained key monocyte properties (increased mCD14, increased phagocytic ability) while expressing DC features [increased mCD83, HLA-DR, CD80, CD86, diminished cytokine (TNF-[alpha], IL-6) responsiveness]. At day 6, iDCs were cultured for an additional 48 h (days 7 and 8) with lipopolysaccharide (LPS) to induce mDCs. ODE-conditioned mDCs maintained high expression of [mCD14.sup.+] and elevated phagocytosis while their DC features weakened as evidenced by decreased CD11c, CD83, HLA-DR, CD86, and CCR7 expression and reduced lymphocyte-stimulating capacity. Similar results were observed when monocytes were exposed to ODE for only the first 48 h and with ODE depleted of endotoxin. Control iDCs exposed to ODE during the final 2 days of iDC maturation (days 7 and 8) did not differ from control (no ODE) iDCs in surface marker expression and phagocytic ability, but exhibited enhanced lymphocyte-stimulating capacity. Dust exposure alters monocyte differentiation to iDCs and prevents maturation of iDC to mDCs. The first 48 h of monocyte differentiation appears to be the susceptible period to exposure. Environmental exposures present during early monocyte differentiation may impact the critical balance of DCs in the lung. phagocytosis; lymphocyte proliferation; innate cell surface molecules; cytokines; swine doi: 10.1152/ajplung.00107.2009

Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target. efferocytosis; Rho GTPase; inflammation; epithelial cells doi: 10.1152/ajplung.00030.2009

Efficient removal of apoptotic cells is essential for resolution of inflammation. Failure to clear dying cells can exacerbate lung injury and lead to persistent inflammation and autoimmunity. Here we show that TNF[alpha] blocks apoptotic cell clearance by alveolar macrophages and leads to proinflammatory responses in the lung. Compared with mice treated with intratracheal TNF[alpha] or exogenous apoptotic cells, mice treated with the combination of TNF[alpha] plus apoptotic cells demonstrated reduced apoptotic cell clearance from the lungs and increased recruitment of inflammatory leukocytes to the air spaces. Treatment with intratracheal TNF[alpha] had no effect on the removal of exogenous apoptotic cells from the lungs of TNF[alpha] receptor-1 (p55) and -2 (p75) double mutant mice and no effect on leukocyte recruitment. Bronchoalveolar lavage from mice treated with TNF[alpha] plus apoptotic cells contained increased levels of proinflammatory cytokines IL-6, KC, and MCP-1, but exhibited no change in levels of anti-inflammatory cytokines IL-10 and TGF-[beta]. Administration of TNF[alpha] plus apoptotic cells during LPS-induced lung injury augmented neutrophil accumulation and proinflammatory cytokine production. These findings suggest that the presence of TNF[alpha] in the lung can alter the response of phagocytes to apoptotic cells leading to inflammatory cell recruitment and proinflammatory mediator production. apoptosis; phagocytosis doi: 10.1152/ajplung.90569.2008.

Resolution of inflammation requires clearance of activated neutrophils by macrophages in a manner that prevents injury to adjacent tissues. Surface changes, including phosphatidylserine (PS) exposure, may target neutrophils for phagocytosis. In this study, we show that externalization of PS is defective in phorbol myristate acetate (PMA)-activated neutrophils obtained from chronic granulomatous disease (CGD) patients with mutations in components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Moreover, activated neutrophils from CGD patients failed to undergo clearance upon cocultivation with macrophages from normal donors. In line with these results, treatment of donor neutrophils with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, blocked PMA-induced PS oxidation and externalization and prevented their engulfment by macrophages. Furthermore, primary macrophages from CGD patients or human [gp91.sup.phox]-deficient PLB-985 cells differentiated into macrophage-like cells were defective for engulfment of apoptotic target cells. Pretreatment of normal macrophages with DPI also suppressed the subsequent ingestion of PS-positive target cells. Together, these data demonstrate that NADPH oxidase plays an important role in the process of macrophage disposal of target cells (programmed cell clearance). Thus we speculate that the lack of a functional NADPH oxidase results in impaired neutrophil clearance and the exaggerated inflammation that is characteristic for CGD. inflammation; apoptosis; phagocytosis; phosphatidylserine

Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPlases), the streptococcal lipoprotein rotamase A (SIrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that SIrA has PPlase activity, and that the enzyme plays a role in pneumococcal virulence. Here, we investigated the contribution of PpmA to pneumococcal pathogenesis. Pneumococcal mutants of D39 and TIGR4 lacking the gene encoding PpmA were less capable of persisting in the nasopharynx of mice, demonstrating the contribution of PpmA to pneumococcal colonization. This observation was partially confirmed in vitro, as the pneumococcal mutants NCTC10319AppmA and TIGR4[DELTA]cps[DELTA]ppmA, but not D39[DELTA]cps[DELTA]ppmA, were impaired in adherence to Detroit 562 pharyngeal cells. This suggests that the contribution of PpmA to pneumococcal colonization is not solely the result of its role in adherence to epithelial cells. Deficiency in PpmA did not result in reduced binding to various extracellular matrix and serum proteins. Similar to SIrA, we observed that PpmA was involved in immune evasion. Uptake of PpmA-deficient D39[DELTA]cps and NCTC10319 by human polymorphonuclear leukocytes was significantly enhanced compared to the isogenic wild-types. In addition, ingestion of D39[DELTA]ppmA, but not that of either NCTC10319[DELTA]ppmA or TIGR4[DELTA]ppmA, by murine macrophage cell line J774 was also enhanced, whereas intracellular killing remained unaffected. We conclude that PpmA contributes to the early stages of infection, i.e. colonization. The contribution of PpmA to virulence can be explained by its strain-specific role in adherence to epithelial cells and contribution to the evasion of phagocytosis.

Organic dust exposure in agricultural environments results in an inflammatory response that attenuates over time, but repetitive exposures can result in chronic respiratory disease. Animal models to study these mechanisms are limited. This study investigated the effects of single vs. repetitive dust-induced airway inflammation in mice by intranasal exposure method. Mice were exposed to swine facility dust extract (DE) or saline once and once daily for 1 and 2 wk. Dust exposure resulted in increased bronchoalveolar lavage fluid neutrophils and macrophages after single and repetitive exposures. Lavage fluid TNFot, IL-6, keratinocyte chemoattractant, and macrophage inflammatory protein-2 were significantly increased after single and repetitive dust exposures, but were dampened in 2-wk dust-exposed mice compared with single exposure. Dust exposure induced PKCot and -e activation in isolated tracheal epithelial cells but were dampened with repetitive exposures. Ex vivo stimulation of alveolar macrophages from 2-wk animals demonstrated reduced cytokine responsiveness and phagocytic ability. Significant lung pathology occurred with development of mixed mononuclear cellular aggregates (T and B lymphocytes, phagocytes) after repetitive dust exposure, a novel observation. Airway hyperresponsiveness to methacholine occurred after single dust exposure but resolved after 2 wk. Collectively, intranasal exposure to DE results in significant lung inflammatory and pathological responses marked by a modulated innate immune response to single and repetitive dust exposures that is associated with PKC activity. antigen presenting cell; phagocytosis; cytokines; aggregate; airway hyperresponsiveness

Surgical stress and anesthesia result in systemic immunosuppression. Propofol, a commonly used anesthetic agent, alters immune cell functions. Previously, we demonstrated that extracellular pressure increases macrophage phagocytosis. We hypothesized that propofol might influence pressure-induced macrophage phagocytosis in monocytes from patients undergoing surgery. Pressure (20 mmHg above ambient pressure) augmented phagocytosis in monocytes from non-propofol-anesthetized patients but reduced phagocytosis in monocytes from propofol-anesthetized patients. In vitro, propofol stimulated phagocytosis but reversed pressure-induced phagocytosis in THP-1 macrophages and monocytes from healthy volunteers. The [GABA.sub.A] receptor antagonists picrotoxin and SR-95531 did not affect basal THP-1 phagocytosis or prevent pressure-stimulated phagocytosis. However, picrotoxin and SR-95531 negated the inhibitory effect of pressure in propofol-treated cells without altering propofol-induced phagocytosis. Phosphorylation of the adaptor protein p130cas was inversely related to phagocytosis: it was inhibited by pressure or propofol but increased by pressure + propofol compared with propofol alone. Reduction of p130cas by small interfering RNA in THP-1 macrophages increased basal phagocytosis and prevented pressure and propofol effects. In conclusion, propofol may alter macrophage responses to pressure via the GABAA receptor and p130cas, whereas pressure also acts via p130cas but independently of [GABA.sub.A] receptors, p130cas may be an important target for modulation of macrophage function in anesthetized patients. anesthesia; mechanotransduction; monocyte

During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic proteins like c-Src to maturing phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in phagosome maturation and microbial killing.

Promoting the degradation of Hsp90 client proteins by inhibiting Hsp90, an important protein chaperone, has been shown to be a promising new anticancer strategy. In this study, we show that an oxazoline analogue of apratoxin A (oz-apraA), a cyclodepsipeptide isolated from a marine cyanobacterium, promotes the degradation of Hsp90 clients through chaperone-mediated autophagy (CMA). We identify a KFERQ-like motif as a conserved pentapeptide sequence in the kinase domain of epidermal growth factor receptor (EGFR) necessary for recognition as a CMA substrate. Mutation of this motif prevents EGFR degradation by CMA and promotes the degradation of EGFR through the proteasomal pathway in oz-apraA--treated cells. Oz-apraA binds to Hsc70/Hsp70. We propose that apratoxin A inhibits Hsp90 function by stabilizing the interaction of Hsp90 client proteins with Hsc70/Hsp70 and thus prevents their interactions with Hsp90. Our study provides the first examples for the ability of CMA to mediate degradation of membrane receptors and cross talks of CMA and proteasomal degradation mechanisms.

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that [[Delta].sup.9]-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2[alpha] (eIF2[alpha]) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers., Introduction Macro-autophagy, hereafter referred to as "autophagy," is a highly conserved cellular process in which cytoplasmic materials--including organelles--are sequestered into double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation [...]

Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and [p210.sup.BCR/ABL]-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular [Ca.sup.2+], suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML., Introduction Chronic myeloid leukemia (CML) is a malignancy arising from transformation of the hematopoietic stem cell, which typically evolves through 3 distinct disease stages: an indolent chronic phase (CP), characterized [...]

Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits ([beta]', [beta], or [alpha]) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.

In cross-presentation by dendritic cells (DCs), internalized proteins are retrotranslocated into the cytosol, degraded by the proteasome, and the generated antigenic peptides bind to MHC class I molecules for presentation on the cell surface. Endoplasmic reticulum (ER) contribution to phagosomal membranes is thought to provide antigen access to the ER-associated degradation (ERAD) machinery, allowing cytosolic dislocation. Because the ERAD pathway is present in all cell types and exogenous antigens encounter an ER-containing compartment during phagocytosis, we postulated that forcing phagocytosis in cell types other than DCs would render them competent for cross-presentation. Indeed, FcR[gamma]IIA expression endowed 293T cells with the capacity for both phagocytosis and ERAD-mediated cross-presentation of an antigen provided as an immune complex. The acquisition of this ability by nonprofessional antigen-presenting cells suggests that a function potentially available in all cell types has been adapted by DCs for presentation of exogenous antigens by MHC class I molecules.

Autophagy is a highly conserved process that maintains homeostasis by clearing damaged organelles and long-lived proteins. The consequences of deficiency in autophagy manifest in a variety of pathological states including neurodegenerative diseases, inflammatory disorders, and cancer. Here, we studied the role of autophagy in the homeostatic regulation of innate antiviral defense. Single-stranded RNA viruses are recognized by the members of the RIG-I-like receptors (RLRs) in the cytosol. RLRs signal through IPS-1, resulting in the production of the key antiviral cytokines, type I IFNs. Autophagy-defective [Atg5.sup.-/-] cells exhibited enhanced RLR signaling, increased IFN secretion, and resistance to infection by vesicular stomatitis virus. In the absence of autophagy, cells accumulated dysfunctional mitochondria, as well as mitochondria-associated IPS-1. Reactive oxygen species (ROS) associated with the dysfunctional mitochondria were largely responsible for the enhanced RLR signaling in [Atg5.sup.-/-] cells, as antioxidant treatment blocked the excess RLR signaling. In addition, autophagy-independent increase in mitochondrial ROS by treatment of ceils with rotenone was sufficient to amplify RLR signaling in WT cells. These data indicate that autophagy contributes to homeostatic regulation of innate antiviral defense through the clearance of dysfunctional mitochondria, and revealed that ROS associated with mitochondria play a key role in potentiating RLR signaling. innate immunity | interferon | reactive oxygen species | virus infection | mitochondria

Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.

Protein degradation in eukaryotic cells is mediated primarily by the ubiquitin-proteasome system and autophagy. Turnover of protein aggregates and other cytoplasmic components, including organelles, is another function attributed to autophagy. The ubiquitin-proteasome system and autophagy are essential for normal cell function but under certain pathological conditions can be overwhelmed, which can lead to adverse effects in cells. In this review we will focus primarily on the insulin-producing pancreatic [beta]-cell. Pancreatic [beta]-cells respond to glucose levels by both producing and secreting insulin. The inability of [beta]-cells to secrete sufficient insulin is a major contributory factor in the development of type 2 diabetes. The aim of this review is to examine some of the crucial roles of the ubiquitin-proteasome system and autophagy in normal pancreatic [beta]-cell function and how these pathways may become dysfunctional under pathological conditions associated with metabolic syndromes. autophagy; insulin secretion; ubiquitin proteasome; endoplasmic reticulum stress

Recent advances have enabled 3-dimensional reconstructions of biological structures in vivo, ranging in size and complexity from single proteins to multicellular structures. In particular, tomography and confocal microscopy have been exploited to capture detailed 3-dimensional conformations of membranes in cellular processes ranging from viral budding and organelle maintenance to phagocytosis. Despite the wealth of membrane structures available, there is as yet no generic, quantitative method for their interpretation. We propose that by modeling these observed biomembrane shapes as fluid lipid bilayers in mechanical equilibrium, the externally applied forces as well as the pressure, tension, and spontaneous curvature can be computed directly from the shape alone. To illustrate the potential power of this technique, we apply an axial force with optical tweezers to vesicles and explicitly demonstrate that the applied force is equal to the force computed from the membrane conformation. lipid bilayers | membrane mechanics | optical trapping | membrane tethering

Autophagy mediates the cellular response to nutrient deprivation, protein aggregation, and pathogen invasion in human. Dysfunction of autophagy has been implicated in multiple human diseases including cancer. The identification of novel autophagy factors in mammalian cells will provide critical mechanistic insights into how this complicated cellular pathway responds to a broad range of challenges. Here, we report the cloning of an autophagy-specific protein that we called Barkor (Beclin 1-associated autophagy-related key regulator) through direct interaction with Beclin 1 in the human phosphatidylinositol 3-kinase class III complex. Barkor shares 18% sequence identity and 32% sequence similarity with yeast Atg14. Elimination of Barkor expression by RNA interference compromises starvation- and rapamycin-induced LC3 lipidation and autophagosome formation. Overexpression of Barkor leads to autophagy activation and increased number and enlarged volume of autophagosomes. Tellingly, Barkor is also required for suppression of the autophagy-mediated intracellular survival of Salmonella typhimurium in mammalian cells. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and the complex formation of Barkor and Beclin1 is required for their localizations to autophagosomes. Therefore, we define a regulatory signaling pathway mediated by Barkor that positively controls autophagy through Beclin 1 and represents a potential target for drug development in the treatment of human diseases implicated in autophagic dysfunction. Atg14 | autophagosome | LC3 | Salmonella | UVRAG

Autophagy--a process of "self-eating" that involves enzymatic digestion and recycling of cellular constituents in response to stress--contributes to both cancer cell death and survival. In this issue of the JCI, Lu et al. report that controlled induction of tumor suppressor gene aplasia Ras homolog member I (ARHI) results in autophagic cell death of human ovarian cancer cells in vitro (see the related article beginning on page 3917). However, within xenograft tumors in mice, multiple factors within the tumor microenvironment switched ARHI-induced autophagy to a mechanism of tumor cell survival, leading to tumor dormancy. Since ARHI expression is suppressed in the majority of breast and ovarian cancers but is high in premalignant lesions, ARHI-induced autophagy could be manipulated for therapeutic benefit., Cells that encounter a variety of stresses undergo an evolutionarily conserved process of self-digestion termed autophagy. The importance of this intracellular damage response for pathophysiology has been established across multiple [...]

Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages. LC3 | phagocytosis

Chaperone-mediated autophagy (CMA), a selective mechanism for degradation of cytosolic proteins in lysosomes, contributes to the removal of altered proteins as part of the cellular quality-control systems (1,2). We have previously found that CMA activity declines in aged organisms and have proposed that this failure in cellular clearance could contribute to the accumulation of altered proteins, the abnormal cellular homeostasis and, eventually, the functional loss characteristic of aged organisms. To determine whether these negative features of aging can be prevented by maintaining efficient autophagic activity until late in life, in this work we have corrected the CMA defect in aged rodents. We have generated a double transgenic mouse model in which the amount of the lysosomal receptor for CMA, previously shown to decrease in abundance with age (3), can be modulated. We have analyzed in this model the consequences of preventing the age-dependent decrease in receptor abundance in aged rodents at the cellular and organ levels. We show here that CMA activity is maintained until advanced ages if the decrease in the receptor abundance is prevented and that preservation of autophagic activity is associated with lower intracellular accumulation of damaged proteins, better ability to handle protein damage and improved organ function., Autophagy is a cellular process that mediates the degradation of intracellular components in lysosomes, thus contributing to maintenance of cellular homeostasis, intracellular clearance of damaged structures and adaptation to environmental [...]

The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type Ill effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type Ill secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell. host-pathogen | type III secretion | apoptosis | effector | inflammatory