RESUMO
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(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.
Assuntos
Apoptose/fisiologia , Doença Granulomatosa Crônica/metabolismo , Macrófagos/fisiologia , NADPH Oxidases/metabolismo , Neutrófilos/fisiologia , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Criança , Feminino , Doença Granulomatosa Crônica/genética , Humanos , Macrófagos/citologia , Masculino , Neutrófilos/citologia , Oxirredução , FosfolipídeosRESUMO
Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.
Assuntos
Anticorpos , Apoptose , Macrófagos/efeitos dos fármacos , Proteínas Opsonizantes , Fagocitose/imunologia , Silicatos/farmacologia , Anticorpos/imunologia , Apoptose/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Tomografia com Microscopia Eletrônica , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Humanos , Células Jurkat , Macrófagos/imunologia , Microscopia Eletrônica de Varredura , Neutrófilos/imunologia , Neutrófilos/patologia , Proteínas Opsonizantes/imunologia , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Silicatos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Difração de Raios XRESUMO
Single-walled carbon nanotubes (SWCNT) are being produced in increasing quantities and the application of these materials in a large number of new technologies and consumer products necessitates studies of their potential impact on human health and the environment. To determine whether SWCNT affect viability or function of macrophages, important components of the innate immune system, we performed in vitro studies using primary human monocyte-derived macrophages (HMDM). Our findings show that SWCNT with a low content of metal impurities do not exert direct cytotoxic effects on HMDM. However, SWCNT suppressed chemotaxis of primary human monocytes in a standard chemotaxis assay. Moreover, macrophage engulfment of apoptotic target cells was significantly impaired following pre-incubation of HMDM with SWCNT at non-cytotoxic concentrations. These results are in line with previous studies showing that ultrafine carbon particles and carbon nanotubes may impair alveolar macrophage ingestion of microorganisms, and suggest that tissue homeostasis may be compromised by SWCNT due to suppressive effects on macrophages.
Assuntos
Apoptose , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Material Particulado/toxicidade , Fagocitose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Humanos , Células Jurkat , Macrófagos/imunologia , Fatores de TempoRESUMO
Macrophage clearance of dying cells is of crucial importance to maintain tissue homeostasis. Here, we show that brief treatment (15min) of Jurkat T cells with agonistic anti-Fas antibodies or recombinant Fas ligand results in efficient phagocytosis by human monocyte-derived macrophages prior to the occurrence of common biomarkers of apoptosis. Similar findings were obtained when using primary human T cells. Macrophage engulfment of pre-apoptotic target cells was suppressed in the absence of serum. Moreover, pre-apoptotic cells secreted annexin I and administration of Boc1, a formyl peptide receptor/lipoxin receptor antagonist markedly attenuated their engulfment. Finally, pre-apoptotic Jurkat cells induced lower macrophage production of tumor necrosis factor-alpha and higher production of interleukin-10 in comparison to apoptotic target cells.
Assuntos
Apoptose , Macrófagos/imunologia , Fagocitose , Linfócitos T/imunologia , Receptor fas/antagonistas & inibidores , Anexina A1/fisiologia , Anticorpos/imunologia , Anticorpos/farmacologia , Caspases/metabolismo , Citocinas/biossíntese , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Humanos , Células Jurkat , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Linfócitos T/citologia , Receptor fas/imunologiaRESUMO
Palladium (Pd) nanoparticles are recognized as components of airborne automotive pollution produced by abrasion of catalyst materials in the car exhaust system. Here we produced dispersions of hydrophilic spherical Pd nanoparticles (Pd-NP) of uniform shape and size (10.4 ± 2.7 nm) in one step by Bradley's reaction (solvothermal decomposition in an alcohol or ketone solvent) as a model particle for experimental studies of the Pd particles in air pollution. The same approach provided mixtures of Pd-NP and nanoparticles of non-redox-active metal oxides, such as Al(2)O(3). Particle aggregation in applied media was studied by DLS and nanoparticle tracking analysis. The putative health effects of the produced Pd nanoparticles and nanocomposite mixtures were evaluated in vitro, using human primary bronchial epithelial cells (PBEC) and a human alveolar carcinoma cell line (A549). Viability of these cells was tracked by vital dye exclusion, and apoptosis was also assessed. In addition, we monitored the release of IL-8 and PGE(2) in response to noncytotoxic doses of the nanoparticles. Our studies demonstrate cellular uptake of Pd nanoparticles only in PBEC, as determined by TEM, with pronounced and dose-dependent effects on cellular secretion of soluble biomarkers in both cell types and a decreased responsiveness of human epithelial cells to the pro-inflammatory cytokine TNF-α. When cells were incubated with higher doses of the Pd nanoparticles, apoptosis induction and caspase activation were apparent in PBEC but not in A549 cells. These studies demonstrate the feasibility of using engineered Pd nanoparticles to assess the health effects of airborne automotive pollution.
Assuntos
Engenharia/métodos , Saúde , Nanopartículas Metálicas , Modelos Biológicos , Paládio/toxicidade , Material Particulado/toxicidade , Emissões de Veículos/toxicidade , Transporte Biológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Paládio/química , Paládio/metabolismo , SoluçõesRESUMO
Macrophage recognition and disposal of neutrophils are important steps in the resolution of inflammation. Externalization of phosphatidylserine (PS) on the cell surface serves as a common recognition signal for macrophages and is associated with the apoptosis program in neutrophils. Here, we report that macrophage-differentiated PLB-985 cells induce rapid, caspase-independent PS externalization in human neutrophils. A similar degree of PS externalization was seen when neutrophils were cocultured with gp91(phox)-deficient PLB-985 macrophages, thus demonstrating that macrophage-induced PS externalization was NADPH oxidase-independent. Macrophage-induced PS externalization required cell-to-cell contact and kinase activation and was shown to correlate with neutrophil degranulation. Of note, the degree of engulfment of such PS-positive neutrophils by activated human monocyte-derived macrophages was considerably lower than for neutrophils undergoing constitutive apoptosis, indicating that PS externalization alone is not sufficient for macrophage disposal of neutrophils. However, addition of recombinant milk fat globule epidermal growth factor 8, a PS-binding protein, restored engulfment of the macrophage-cocultured target cells. Finally, neutrophils undergoing spontaneous apoptosis but not macrophage-cocultured neutrophils displayed surface expression and release of annexin I, and the addition of N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe (Boc1), a formyl peptide receptor/lipoxin receptor antagonist, suppressed clearance of apoptotic neutrophils. Conditioned medium from apoptotic neutrophils also promoted the engulfment of macrophage-cocultured neutrophils, and Boc1 blocked this process. Taken together, these studies highlight a novel pathway of PS externalization in primary human neutrophils and also provide evidence for an auxiliary function of annexin I in macrophage clearance of neutrophils.
Assuntos
Caspases , Macrófagos/imunologia , Neutrófilos/metabolismo , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio , Anexina A1/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos , NADPH Oxidases , FagocitoseRESUMO
Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid "eat-me" signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.
Assuntos
Nanotubos de Carbono/química , Fagócitos/metabolismo , Fosfatidilserinas/química , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Técnicas In Vitro , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanotubos de Carbono/toxicidade , Fosfatidilserinas/metabolismo , RatosRESUMO
Previous studies have suggested that 1,25(OH)2D3, the active form of vitamin D3, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D3 has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH)2D3 induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in this model. Moreover, 1,25(OH)2D3 failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D3.