Phagocytosis

Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. Alveolar macrophages (AMs) were obtained from bronchoalveolar lavage from healthy donors or COPD patients and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line, in which the CD68 promoter ensures macrophage specific expression of human Mcl-1 (CD68.hMcl-1), was used to model the molecular aspects of COPD. COPD AM had elevated levels of Mcl-1, an anti-apoptotic Bcl-2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AM phenocopied the microbicidal defect since transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived...

Young and adult Wistar rats were submitted to total splenectomy and compared to animals not submitted to any surgical manipulation in order to evaluate the phagocytic function of spleen. The animals were infected with Escherichia coli labeled with technetium-99m and killed 20 minutes later. Liver, lung, spleen and a blood clot sample were taken. No significant differences were found in the percentage of bacterial radioactivity uptake in mononuclear phagocyte system (MPS) organs in young and adult splenectomized rats. However, phagocytosis index by macrophages of MPS organs was smaller in splenectomized animals than in control group. Splenectomized rats were associated with a higher blood bacterial radioactivity uptake than animals of the control group (p<0.0001) due to a larger bacterial remnant in the bloodstream. This finding suggested that some failure in the MPS occurred in the absence of the spleen, demonstrating the need to develop alternative surgical techniques for total ...

Autogenous splenic implant seems to be the only alternative for preservation of splenic tissue after total splenectomy. This work was carried out to analyze the morphologic regeneration of autotransplanted splenic tissue in Wistar rats and to determine the bacterial phagocytic function of their macrophages. We utilized an experimental model with thirty-two rats, of both sexes, submitted to total splenectomy combined with autotransplantation in greater omentum of slices of the whole spleen mass. The animals were divided into two groups: I - young rats weighing 100 to 150 g; and II - adult rats weighing 250 to 300 g. Sixteen weeks later animals were intravenously inoculated with a suspension of Escherichia coli AB1157. Twenty minutes after inoculation, the animals were sacrificed and the splenic autotransplants were removed for morphological study. There was regeneration of autotransplanted splenic tissue in all animals. A similar morphological aspect among all animals was observed, w...

is a remarkably infectious facultative intracellular bacterium of macrophages that causes tularemia. Early evasion of host immune responses contributes to the success of as a pathogen. entry into human monocytes and macrophages is mediated by the major phagocytic receptor, complement receptor 3 (CR3, CD11b/CD18). We recently determined that despite a significant increase in macrophage uptake following C3 opsonization of the virulent Type A spp. Schu S4, this phagocytic pathway results in limited pro-inflammatory cytokine production. Notably, MAP kinase/ERK activation is suppressed immediately during C3-opsonized Schu S4-CR3 phagocytosis. A mathematical model of CR3-TLR2 crosstalk predicted early involvement of Ras GTPase-activating protein (RasGAP) in immune suppression by CR3. Here, we link CR3-mediated uptake of opsonized Schu S4 by human monocytes and macrophages with inhibition of early signal 1 inflammasome activation, evidenced by limited caspase-1 cleavage and IL-18 release. ...

Mycobacterium tuberculosis imposes a large global health burden as the airborne agent of tuberculosis. Mycobacterium tuberculosis has been flourishing in human populations for millennia and is therefore highly adapted to the lung environment. Alveolar macrophages, a major host cell niche for M. tuberculosis, are not only phagocytose inhaled microbes and particulate matter but are also crucial in catabolizing lung surfactant, a lipid-protein complex that lines the alveolar spaces. Because macrophage host defense properties can be regulated by surfactant and M. tuberculosis can use host lipids as a carbon source during infection, we sought to determine the receptor(s) involved in surfactant lipid uptake by human macrophages and whether the presence of those lipids within macrophages prior to infection with M. tuberculosis enhances bacterial growth. We show that preformed scavenger receptor CD36 is redistributed to the cell membrane following exposure to surfactant lipids and surfactan...

Disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with high mobility and mortality. However, the molecular basis of host defense against disseminated candidiasis remains elusive, and treatment options are limited. Here we report that the E3 ubiquitin ligase CBLB directs polyubiquitination of dectin-1 and dectin-2, two key pattern-recognition receptors for sensing Candida albicans, and their downstream kinase SYK, thus inhibiting dectin-1- and dectin-2-mediated innate immune responses. CBLB deficiency or inactivation protects mice from systemic infection with a lethal dose of C. albicans, and deficiency of dectin-1, dectin-2, or both in Cblb(-/-) mice abrogates this protection. Notably, silencing the Cblb gene in vivo protects mice from lethal systemic C. albicans infection. Our data reveal that CBLB is crucial for homeostatic control of innate immune responses mediated by dectin-1 and dectin-2. Our data also indicate that CBLB...

ABSTRACTOritavancin, a lipoglycopeptide antibiotic in development, accumulates to high levels in the lysosomes of eukaryotic cells. We examined specific functions of macrophages (phagocytic capacity, lysosomal integrity, metabolic activity, and production of reactive oxygen species [ROS]) in correlation with the cellular accumulation of the drug, using J774 mouse macrophages and THP-1 human monocytes differentiated into macrophages using phorbol 12-myristate 13-acetate. Oritavancin did not affectPseudomonas aeruginosaphagocytosis, lysosomal integrity, or metabolic activity in cells incubated for 3 h with extracellular concentrations ranging from 5 to 50 μg/ml. At extracellular concentrations of ≥25 μg/ml, oritavancin reduced latex bead phagocytosis by approximately 50% and doubled ROS production in J774 macrophages only. This may result from the fact that the cellular accumulation of oritavancin was 15 times higher in J774 cells than in activated THP-1 cells at 3 h. Human pharmacoki...

P-glycoprotein (P-gp; MDR1), a major efflux transporter, recognizes various antibiotics and is present in macrophages. We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized Staphylococcus aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill equation pattern) in THP-1 and MDCK cells with (i) 50% effective drug extracellular concentration (EC 50 ; relative potency) and static concentrations at 9 to 10 times the MIC and (ii) maximal efficacy ( E max ; CFU decrease at infinite extracellular drug concentration) at 1.6 to 2 log compared to that of the postphagocytosis inoculum. Verapamil (100 μM) and elacridar (GF 120918; 0.5 μM), two known inhibitors of P-gp, decreased daptomycin...

The role of messengers derived from hydrolysis of phosphoinositides and other phospholipids, of the basal level of [Ca2+]i and of the increase in [Ca2+]i in phagocytosis and respiratory burst was investigated, using normal neutrophils and neutrophils Ca2(+)-depleted by pretreatment with Quin2/AM and EGTA. 1) Phagocytosis and respiratory burst in control neutrophils challenged with yeast opsonized with IgG or C3b/bi were associated with a stimulation of the production of inositol phosphates, diacylglycerol, phosphatidic acid, arachidonic acid, and rise in [Ca2+]i. 2) In Ca2(+)-depleted neutrophils (basal [Ca2+]i 10 to 20 nM) the phagocytosis of yeast-IgG was similar to that in control neutrophils, the respiratory burst was slightly depressed (-30%), while the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, and phosphatidic and arachidonic acid did not occur. 3) In Ca2(+)-depleted neutrophils the phagocytosis of yeast-C3b/bi was slightly lower than that in c...

Autoreactive thymocytes are eliminated during negative selection in the thymus, a process important for establishing self-tolerance. Thymic phagocytes serve to remove dead thymocytes, but whether they play additional roles during negative selection remains unclear. Here, we demonstrate that phagocytosis promotes negative selection, and that negative selection is more efficient when the phagocyte also presents the negative selecting peptide. Our findings support a two-step model for negative selection in which thymocytes initiate the death process following strong TCR signaling, but ultimately depend upon phagocytosis for their timely death. Thus, the phagocytic capability of cells that present self-peptides is a key determinant of thymocyte fate.

Platelet-activating factors (PAF), a phospholipid mediator of anaphylaxis, is also known to be released in vitro from both phagocytic polymorphonuclear neutrophils (PMN) and monocytes in response to a variety of stimuli. The fact that human myeloid cells of the HL-60 line can be made to differentiate in vitro into macrophage-like cells by 12- O-tetradodecanoylphorbol-13-acetate (TPA) prompted us to investigate the generation and release of PAF during this transformation. Both passive release of PAF at pH 9.5, and active release, following phagocytosis of C3b- and C3d-opsonized yeast spores, and stimulation with C5a anaphylatoxin from untreated and TPA-treated HL-60 cells, PMN, and plastic-adherent normal human monocytes were studied. It was found that after 3 days of TPA treatment, HL-60 cells released PAF following phagocytosis of C3b- and C3d-opsonized yeast spores. Inhibition of PAF release by a selective inhibitor of phospholipase A2 and labeling of PAF with sodium 14C-acetate i...

Dendritic cells (DC) are powerful antigen‐presenting cells that have drawn many attentions due to the recent development of anti‐cancer vaccines. Clinical grade production of monocyte‐derived DC (Mo‐DC) is extensively studied, and many efforts are made to develop and improve clinical standard operating procedures. Most of the parameters involved, such as the cytokines and maturation agents, have been widely assessed. However, very few are investigated about how culture medium and additional protein components affect DC yield, viability and maturation. Thus, our study aimed to compare the impact of standard culture medium on Mo‐DC differentiation and maturation. Commercially available media for hematopoietic cell culture as well as different protein supplementations, that is foetal calf serum (FCS), autologous plasma (AP), human serum (HS) and human serum albumin (HSA) were tested. Culture yields, cell viability and DC maturation were investigated. Differentiation yields were similar...