Extracellular Acidification Acts as a Key Modulator of Neutrophil Apoptosis and Functions

<div><p>In human pathological conditions, the acidification of local environment is a frequent feature, such as tumor and inflammation. As the pH of microenvironment alters, the functions of immune cells are about to change. It makes the extracellular acidification a key modulator of innate immunity. Here we detected the impact of extracellular acidification on neutrophil apoptosis and functions, including cell death, respiratory burst, migration and phagocytosis. As a result, we found that under the acid environment, neutrophil apoptosis delayed, respiratory burst inhibited, polarization augmented, chemotaxis differed, endocytosis enhanced and bacteria killing suppressed. These findings suggested that extracellular acidification acts as a key regulator of neutrophil apoptosis and functions.</p></div

Neutrophils in acid medium were more sensitive to the fMLP-induced polarization.

<p>(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). (***, <i>p</i><0.001, **, <i>p</i><0.01, *, <i>p</i><0.05).</p

Superoxide production of neutrophils was inhibited in acid environment.

<p>(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 3^th 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). (***,<i>p</i><0.001, *,<i>p</i><0.05).</p

Extracellular acid enhance neutrophil endocytosis but suppress the bacteria killing ability.

<p>(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 <i>E</i>.<i>Coli</i>.2×10⁶ neutrophils were mixed with 1×10⁷ opsonized <i>E</i>.<i>Coli</i> and co-incubated at 37°C for 30min. (E) Cells containing <i>E</i>.<i>Coli</i> were lysed, diluted and spread on the LB agar. CFUs of this group stand for the bacteria loading. Another two groups of neutrophils containing <i>E</i>.<i>Coli</i> 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)</p><p></p><p></p><p>killing percentage<mo>=</mo></p><p><mo>(</mo></p><p><mn>1</mn><mo>-</mo></p><p></p><p>CFUs of pH 6.0/7.4</p><p>bacteria loading</p><p></p><p></p><mo>)</mo><p></p><mo>×</mo><mn>100</mn><mi>%</mi><p></p><p></p><p></p>. Data are from three independent experiments(A,B,C) or are representative of three experiments(D,E,F,G,H,I). (***,<i>p</i><0.001, **, <i>p</i><0.01, *,<i>p</i><0.05).<p></p