Sulforaphane promotes immune responses in a WEHI-3-induced leukemia mouse model through enhanced phagocytosis of macrophages and natural killer cell activities in vivo
Yung‐Luen ShihLung‐Yuan WuChing‐Hsiao LeeYung‐Liang ChenShu‐Ching HsuehHsu‐Feng LuNien‐Chieh LiaoJing‐Gung Chung
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Sulforaphane (SFN) is an isothiocyanate, inducing cytotoxic effects in various human cancer cells, including leukemia cells through cell cycle arrest and apoptosis. However, the effect of SFN on the immune responses in a leukemia mouse model remains to be investigated. The present study investigated whether SFN has an effect on the immune responses in a WEHI‑3‑induced leukemia mouse model in vivo. Normal BALB/c mice were injected with WEHI‑3 cells to generate the leukemia mouse model, and were subsequently treated with placebo or SFN (0, 285, 570 and 1,140 mg/kg) for 3 weeks. Following treatment, all mice were weighted and blood samples were collected. In addition, liver and spleen samples were isolated to determine cell markers, phagocytosis and natural killer (NK) cell activities, and cell proliferation was examined using flow cytometry. The results indicated that SFN treatment had no significant effect on the spleen weight, however it decreased liver and body weight. Furthermore, SFN treatment increased the percentage levels of CD3 (T cells) and CD19 (B cell maker), however had no effect on the levels of CD11b (monocytes) or Mac‑3 (macrophages), compared with the WEHI‑3 control groups. The administration of SFN increased the phagocytosis of macrophages from peripheral blood mononuclear cells and peritoneal cavity, and increased the activity of NK cells from splenocytes. Administration of SFN promoted T and B cell proliferation following stimulation with concanavalin A and lipopolysaccharide, respectively.Objective To explore the effect of corticotropin-releasing hormone(CRH) on phagocytosis of rat enterocoelia macrophage.Methods The chicken-red cells were added in the media of rat enterocoelia macrophage cultivated by CRH after 1、2、4、8、16 hours.Then the effect of CRH on phagocytosis of rat enterocoelia macrophage was evaluated by the chicken-red cells phagocytosis index and phagocytosis rate.Results Comparing with the control groups,the phagocytosis index and phagocytosis rate of experimental groups both improved significantly in each time point(P0.05).Among the experimental groups,the phagocytosis index and phagocytosis rate were the highest and the phagocytosis was at the top in the 2 hours group.Conclusion CRH could markedly improve the phagocytosis of rat enterocoelia macrophage.
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Phagocytosis plays diverse roles in biology, but our understanding of the purpose, interplay, and cell signaling mechanisms associated with different modes of phagocytosis is limited, without being able to capture and visualize each step in this rapid process from the beginning to end. A new study by Walbaum et al. uses stunning time-lapse 3D imaging of the engulfment of erythrocytes by macrophages via sinking, ruffling, and cup formation, unequivocally confirming a visionary 44-year-old theory derived from still electron microscopy photos that phagocytosis mediated by complement receptor CR3 occurs via a sinking mechanism and antibody-mediated phagocytosis occurs via phagocytic cup formation. The article also challenges the dogma, showing that phagocytic cup formation is not unique to antibody receptor phagocytosis, rather CR3 plays a complex role in different modes of phagocytosis. For example, inhibition of antibody-mediated phagocytosis leads to a compensatory upregulation of CR3-mediated sinking phagocytosis. These findings animate, in vivid colors, processes previously only captured as stills, exposing interactions between different phagocytic mechanisms and altering our basic understanding of this important process.
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We analysed kinetics of phagocytosis by using the whole blood and fluorescent microspheres with flow cytometry. The rate of phagocytosis (y) was determined by the T/C (target particles-to-cell) ratio (x) (y=K1×log (x) +K2 K1, K2: constant) . We defined the Phagocytosis 50 (Ph 50) that indicated the value of T/C ratio in which 50% of phagocytes injested the particles. The Ph 50 might be the new general parameter of phagocytosis independent of several conditions.The Ph 50 in cases of systemic lupus erythematosus (SLE) were statistically higher than in normal controls, which indicated the decreased phagocytic activity in SLE.
Cytometry
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The effect of phagocytosis-stimulating factor (PSF) on phagocytosis was studied in detail. PSF did not affect the Fc receptor-mediated phagocytosis, whereas PSF enhanced the ingestion step, but not attachment step, of C3b receptor-mediated phagocytosis by polymorphonuclear neutrophils (PMNs), suggesting that PSF may specifically modulate the C3b receptor function of PMNs. PSF generated from guinea pig PMNs enhanced phagocytosis by rabbit PMNs, and rabbit PMN-produced PSF accelerated the phagocytosis by guinea pig PMNs, indicating that PSF is not specific for animal species. The effects of PSF on some PMN functions, such as O2- generation, chemotaxis, adherence, and enzyme release, were also studied. Only O2- generation from PMNs was significantly increased in the initial phase of phagocytosis, and this stimulation of O2- generation was completely parallel with the stimulation of phagocytosis by PSF. Resting PMNs hardly generated the superoxide anions by PSF treatment, suggesting that PSF does not affect the O2- forming system directly.
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ABSTRACT We have studied the ability of human polymorphonuclear leukocytes (PMN) to phagocytose Capnocytophaga ochracea in three-dimensional fibrin meshworks. Phagocytosis was assessed in three systems: (1) the PMN and bacteria were mixed together with plasma and clotted; 60±13% phagocytosis occurred after 60 min; (2) PMN were overlaid on clots containing bacteria; the PMN migrated into the clot and after 60 min 52±7% phagocytosis was seen; (3) PMN had to migrate from within one clot into a second containing bacteria; phagocytosis after 60 min was 54±3 %. In the clots, PMN released lysozyme but this was not significantly enhanced by phagocytosis. These findings indicate that PMN are capable of phagocytosing in each of the threedimensional systems tested and that they are capable of both migration into and subsequent phagocytosis in a model that more closely mimics the in vivo structure in which PMN would normally perform.
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Generation of a Phagocytosis-Stimulating Factor by Polymorphonuclear Neutrophils during Phagocytosis
Polymorphonuclear neutrophils (PMNs) generated a phagocytosis-stimulating factor (PSF) intracellularly during phagocytosis of opsonized zymosan or latex particles. The generation of PSF by PMNs reached the maximum at 60 min after the start of phagocytosis. When PSF was washed out after preincubation with PMNs or opsonized zymosan particles, no enhancement of phagocytosis by PMNs was observed, suggesting that the continuous existence of PSF in the incubation medium is required for stimulation of phagocytosis.
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Divalent
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Abstract. Phagocytosis was investigated using human peripheral monocytes and erythrocytes sensitized with known amounts of subclass‐specific IgG anti‐Rh antibodies. The erythrocyte‐bound IgG was quantitated by a radiometric antiglobulin test. This evaluation revealed the following: (1) there is a relationship between phagocytosis and the number of erythrocyte‐bound IgG molecules; (2) phagocytosis is IgG subclass‐dependent, since a similar degree of phagocytosis is observed with fewer IgG3 than IgGl molecules and also the minimum number of IgG3 molecules for phagocytosis is 150–640, whilst for IgGl the minimum is 1,230‐4,020; (3) the minimum levels of sensitization for phagocytosis should be detectable by the serological antiglobuiin test; (4) the phagocytosis assay is no more sensitive than the monocyte rosette assay for the detection of anti‐Rh alloantibodies, and (5) phagocytosis of adherent erythrocytes observed by video‐enhanced microscopy indicated that erythrocytes may adhere to monocytes for a considerable time before phagocytosis, but that phagocytosis itself was rapid.
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