Fig 1.
Extracellular acidification delays neutrophil apoptosis and reduces caspase 3 activation.
(A) Neutrophils apoptosis is delayed in the acid medium. Freshly prepared neutrophils were cultured in the medium of different pH for indicated periods of time. Cell death was assessed by FACS analysis with annexin V and PI staining. At least three separate experiments were carried out with a minimum of 10,000 cells counted per data point. (B) Light microscopy of a neutrophil population with Wright staining aged for 24h (×1,000). (C) FACS analysis of neutrophil apoptosis. Purified neutrophils were labeled with FITC-annexin V and PI after cultured for indicated time and detected by flow cytometer. (D,E) pH changed from acid to neutral at either the 4th hour(D) or the 12th hour(E) of cell culture will accelerate the neutrophil death. Neutrophils in pH 6.0 were collected and resuspended in the medium of pH 7.4 at the indicated time point for a further culture to 24h. Cell apoptosis was detected by FACS. (F)The cleavage of caspase 3 was decreased when neutrophils were put into the acid medium for 24 hours. Neutrophils were cultured in the medium of pH 6.0 to7.4 for 24h. Protein extracts were resolved on SDS/PAGE. Pro and cleaved caspase 3 were detected by western blot using anti-caspase 3 antibody. (G)The cleaved caspase 3 was observed by immunofluorescence microscope. Immunofluorescence staining was carried out on the neutrophils cutured for 24h. Green stands for the cleaved caspase 3, red for cytoskeleton and blue for nucleus. Scale bar, 7μm. Data are from three independent experiments(A,D,E) or are representative of three experiments(B, C,F,G,). (***,p<0.001, **, p<0.01).
Fig 2.
Superoxide production of neutrophils was inhibited in acid environment.
(A, E)Fluorescent probe DCFH-DA. 5×105 cells were labeled with DCFH-DA and stimulated by 1μM fMLP for 10 min. Fluorescence intensity were read by the Microplate Reader(A) and images were captured using Leica laser scanning confocal microscope (E). (B)Luminol chemiluminescence assay of neutrophils ROS production. Purified neutrophils in different pH medium were stimulated by 1μM fMLP in the presence of lunimol (50μM) and horseradish peroxidase(HRP, 4U/ml). ROS production was monitored in a luminometer at 37°C. Cells pre-treated with 25μM DPI were as negative control. (C, D) Quantification of F-actin of neutrophils in acid and neutral medium. At the 3th minute of fMLP(10nM) stimulation, cells were fixed and stained with rhodamine-phalloidin(Red) and DAPI(Blue), the fluorescence intensity of rhodamine-phalloidin was read by the Microplate Reader(D). Pictures were taken by fluorescence microscope(C). (F)Extracellular acid increase the Akt phosphorylation of neutrophils Purified neutrophils were cultured in the medium of pH 6.0 to7.4 for 2h, Protein extracts were resolved on SDS/PAGE. Total and phosphorylated Akt were detected by Western blot using anti-Akt and anti-phospho-Akt (Ser473) antibodies. (G) The glutathionylation of actin was down regulated in neutrohils in acid environment. Neutropihls cultured for 4h were collected and lysed by RIPA. Anti-actin antibody and protein A/G agrose beads were used to pull down and anti-glutathione antibody were used in the western blot to detect the glutathionylated actin. Data are from three independent experiments(A,D) or are representative of three experiments(B,C, E,F,G). (***,p<0.001, *,p<0.05).
Fig 3.
Neutrophils in acid medium were more sensitive to the fMLP-induced polarization.
(A) fMLP (concentration ranged from 0nM to 10μM,) induced ruffling in neutrophils cultured in medium of pH 6.0 and pH 7.4. Images were captured every 5 sec for 10 min. (B, C)At 3 min and 5 min of fMLP stimulation, percentage of polarization cells were calculated in each group of different fMLP concentration. Data are from three independent experiments(B, C) or are representative of three experiments(A). (***, p<0.001, **, p<0.01, *, p<0.05).
Fig 4.
Extracellular acidification altered neutrophil chemotaxis.
(A) The screening of neutrophils chemotaxis process. 3000 cells were plated into the TAXIScan-FL device and exposed to shallow chemoattractant gradient generated by addition of 1μl fMLP (1μM). (B) Cell tracks of migrating neutrophils were traced from captured images by the Tracking Tool software (Gradientech). More than 30 cells were picked randomly and analysed. (C, D, E) The directionality and speed were auto-analyzed by the Tracking Tool software (Gradientech). (F) Representative images of migrating neutrophils, black arrowsheads specify pseudopodia. Data are representative of three experiments (**, p<0.01).
Fig 5.
Extracellular acid enhance neutrophil endocytosis but suppress the bacteria killing ability.
(A,B,C,D) Neutrophils endocytose FITC-Zymosan. 2×106 neutrophils were mixed with 1×107 opsonized FITC-Zymosan and co-incubated at 37°C for 30 min. Pictures were captured by a fluorescence microscope(600×). (A) Percentage of neutrophils happened to phagocytose. More than 100 cells were counted from random fields of each group. (B) Phagocytosis index was expressed as the number of the internalized particles per 100 neutrophils. (C) Binding index was expressed as the number of the binding particles per 100 neutrophils. (D) Fluorescence images of neutrophil phagocytosis. (E,F,G)Killing assay using neutrophils incubated with E.Coli.2×106 neutrophils were mixed with 1×107 opsonized E.Coli and co-incubated at 37°C for 30min. (E) Cells containing E.Coli were lysed, diluted and spread on the LB agar. CFUs of this group stand for the bacteria loading. Another two groups of neutrophils containing E.Coli were further incubated in the medium of pH 6.0 and pH 7.4 separately. After incubated for another 30 min, cells were lysed, diluted and spread on the LB agar. (F) The number of CFUs of each group were listed in the table. (G). Data are from three independent experiments(A,B,C) or are representative of three experiments(D,E,F,G,H,I). (***,p<0.001, **, p<0.01, *,p<0.05).