High Salt Induces a Delayed Activation of Human Neutrophils

Abstract

High salt (NaCl) concentrations are found in a number of tissues under physiological and pathological conditions. Here, we analyzed the effects induced by high salt on the function of human neutrophils. The culture of neutrophils in medium supplemented with high salt (50 mM NaCl) for short periods (30-120 min) inhibited the ability of conventional agonists to induce the production of IL-8 and the activation of respiratory burst. By contrast, exposure to high salt for longer periods (6-18 h) resulted in the activation of neutrophils revealed by the production of high levels of IL-8, the activation of the respiratory burst, and a marked synergistic effect on the production of TNF-α induced by LPS. Increasing osmolarity of the culture medium by the addition of sorbitol or mannitol (100 mM) was shown to be completely unable to stimulate neutrophil responses, suggesting that high sodium but not an increased osmolarity mediates the activation on neutrophils responses. A similar biphasic effect was observed when the function of monocytes was analyzed. Short term exposure to high salt suppressed IL-8 and TNF-α production induced by LPS while culture for longer periods triggered the production of IL-8 but not TNF-α in the absence of LPS stimulation. Contradictory results have been published regarding how high salt modulates neutrophil function. Our results suggest that the modulation of neutrophil function by high salt is strongly dependent on the exposure time.

Keywords: IL-8 (CXCL8); inflammation; innate immunity; oxygen reactive intermediates; sodium chloride.

Figure 1

High salt modulates IL-8 production by neutrophils in a biphasic manner. (A) Neutrophils (2 x 10⁶/ml) were cultured for 18 h at 37°C in culture medium supplemented, or not, with NaCl 50 mM or LPS (100 ng/ml). IL-8 levels were determined by ELISA in culture supernatants. The mean ± SE from 14 experiments is shown. (B) Neutrophils were cultured for different times in culture medium supplemented, or not, with NaCl (50 mM) and/or LPS (100 ng/ml), and IL-8 levels were determined by ELISA in culture supernatants. Results are expressed as the mean ± SE from 3 experiments. (C) Neutrophils were cultured for 18 h in culture medium supplemented or not with different concentrations of NaCl. Then, IL-8 levels were determined by ELISA in culture supernatants. Results are expressed as the mean ± SE from 5 experiments. (D) Production of IL-8 was evaluated by intracellular staining and flow cytometry after 8 h of culture in culture medium supplemented, or not, with NaCl (25 mM or 50 mM). During the last 4 h of incubation, all samples were treated with the Golgi transport inhibitor BFA (1 μg/ml). Left, a representative experiment is shown. Right, the mean ± SE from 4 experiments is shown. (E) Production of IL-8 was evaluated by intracellular staining and confocal microscopy after 8 h of culture in culture medium supplemented. or not, with NaCl 50 mM or LPS (100 ng/ml). A representative experiment (n=3) is shown. (F) Neutrophils were cultured for 18 h at 37°C in culture medium supplemented or not with sorbitol or mannitol (100 mM) to increase culture medium osmolarity to values of 400 mOsm. Then, IL-8 production was measured in cell supernatants by ELISA. The mean ± SE from 3 experiments is shown. (G) Neutrophils were cultured for 30 min in culture medium supplemented, or not, with sorbitol or mannitol (100 mM) and/or LPS (100 ng/ml), and IL-8 levels were determined by ELISA in culture supernatants. Results are expressed as the mean ± SE from 6 experiments. (H) Neutrophils were cultured for 18 h at 37°C in culture medium supplemented or not with NaCl 50 mM, in the absence or presence of the p38 MAPK inhibitors SB202190 or SB353022 (10 μM). Then, IL-8 production was measured in cell supernatants by ELISA. The mean ± SE from 6 experiments is shown. (I) Neutrophils (5 x 10⁶/ml) were cultured for 18 h at 37°C in culture medium supplemented or not with NaCl 50 mM, in the absence or presence of LPS (100 ng/ml). Then, the production of IL-1α, IL-6, IL-10, and TNF-α was measured in cell supernatants by ELISA. The mean ± SE from 5-7 experiments is shown. (J) Neutrophils were cultured for different times in culture medium supplemented or not with NaCl 50 mM, in the absence or presence of LPS (100 ng/ml). Then, the production of TNF-α was measured in cell supernatants by ELISA. The mean ± SE from 5 experiments is shown. *P <.05, **P <.01, and ***P<.001.

Figure 2

High salt induces IL-8 production in whole blood samples. (A) Whole blood samples (200 μl) supplemented or not with NaCl (25 mM and 50 mM) were incubated for 6 h at 37°C. Then, IL-8 in the plasma fraction was evaluated by ELISA. Results are expressed as the mean ± SE from 5 experiments. (B) Gating strategy used to evaluate the production of IL-8 by neutrophils and monocytes in whole blood samples. (C) Whole blood samples (200 μl) supplemented or not with NaCl (25 mM or 50 mM) or LPS (100 ng/ml) were cultured for 8 h (the last 4 h of incubation was performed in the presence of the Golgi transport inhibitor BFA 10 μg/ml), and IL-8 production was analyzed by intracellular staining and flow cytometry in the gate of neutrophils and monocytes. A representative experiment (n=3) is shown. *P <.05, and ***P <.001.

Figure 3

High salt modulates hydrogen peroxide production by neutrophils. (A, B) Neutrophils (2 x 10⁶/ml) were incubated at 37°C for 30 min (A) or 8 h (B) in culture medium supplemented, or not, with NaCl (50 mM). Cells were then labeled with dihydrorhodamine-123 and stimulated, or not, with fMLP (0.5 μM). After 20 min of incubation at 37°C, production of hydrogen peroxide was measured by flow cytometry. Representative experiments are shown. (C) Production of hydrogen peroxide was evaluated by flow cytometry in neutrophils cultured for different times with or without NaCl (50 mM), fMLP (0.5 μM) or NaCl (50 mM) plus fMLP (0.5 μM). Results are expressed as the mean ± SE from 14, 3, 17, and 3 experiments, for time points of 30 min, 4 h, 8 h and 18 h, respectively. (D) Neutrophils were incubated by 8 h in culture medium supplemented with 25 or 50 mM NaCl and the production of hydrogen peroxide was then evaluated. A representative experiment is shown. (E) Neutrophils were incubated for 8 h in culture medium supplemented with 50 mM NaCl, Sorbitol or Mannitol (100 mM) (final osmolarity of the culture) supplemented medium = 400 mOsm). Then, the production of hydrogen peroxide was evaluated. Results are expressed as the mean ± SE of 5 experiments. (F) Neutrophils were cultured for 30 min in culture medium supplemented, or not, with sorbitol or mannitol (100 mM) and/or fMLP (0.5 μM). Then, the production of hydrogen peroxide was evaluated. Results are expressed as the mean ± SE from 9 experiments. (G, H) Production of hydrogen peroxide was evaluated by flow cytometry in neutrophils cultured for 8 h in culture medium supplemented with 50 mM NaCl, in the absence or presence of the p38 MAPK inhibitor SB202190 (10 μM) (G) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 uM) (H). Representative experiments and the mean ± SE from 4 experiments are shown. **P <.01, and ***P<0.01.

Figure 4

Effect of high salt on neutrophil degranulation induced by LPS and the production of IL-8 and hydrogen peroxide induced by Zymosan. (A, B) Neutrophils (2 x 10⁶/ml) were incubated at 37°C for 30 min (A) or 8 h (B) in culture medium supplemented, or not, with NaCl (50 mM) and/or LPS (100 ug/ml), and the expression of CD11b was then analyzed by flow cytometry. Representative experiments and the mean ± SE from 4-13 experiments are shown. (C) Neutrophils (5 x 10⁶/ml) were incubated at 37°C for 30 min or 8 h in culture medium supplemented or not with NaCl (50 mM) and/or LPS (100 ng/ml). Supernatants were then harvested and gelatinase activity was assessed as described under Materials and Methods. A representative experiment and the mean ± SE of 4 experiments are shown. The results are expressed in relative units having assigned the value of 1 to controls (neutrophils cultured in control medium). (D) Neutrophils (2 x 10⁶/ml) were incubated at 37°C for different times in culture medium supplemented, or not with NaCl (50 mM) and/or Zymosan (50 μg/ml). Then, the presence of IL-8 in cell supernatants and the production of hydrogen peroxide were evaluated by ELISA and flow cytometry, respectively. Results are expressed as the mean ± SE of 3 experiments. (E, F) Neutrophils were cultured for 30 min in culture medium supplemented, or not, with sorbitol or mannitol (100 mM) and/or Zymosan (50 μg/ml). Then, the presence of IL-8 in cell supernatants (E) and the production of hydrogen peroxide (F) was evaluated by ELISA and flow cytometry, respectively. Results are expressed as the mean ± SE from 6 experiments. *P <.05, **P <.01, and ***P <.001.

Figure 5

Effect of high salt on neutrophil phagocytosis and the production of IL-8 and TNF-α by monocytes. (A) Neutrophils were labeled with PKH26 and incubated (2 x 10⁶ neutrophils/ml) for 30 min in culture medium supplemented, or not, with NaCl (50 mM). Then, CFSE-labeled yeast (C.albicans) were added (neutrophil/yeast ratio of 1:1), in the absence or presence of fresh autologous serum (10% v/v). After 40 min of incubation at 37°C, yeast phagocytosis was evaluated in the gate of neutrophils (PKH26 positive cells) by flow cytometry using trypan blue to quench the fluorescence of the yeast attached (but not internalized) by neutrophils. A representative experiment and the mean ± SE from 5 experiments are shown. (B) Phagocytosis of C. albicans was evaluated as described in A, in neutrophils preincubated for 8 h in culture medium supplemented, or not, with NaCl (50 mM). Results represent the mean ± SE of 4 experiments. (C) Neutrophils (2 x 10⁶ neutrophils/ml) were cultured for 30 min or 8 h in medium supplemented, or not, with NaCl (50 mM). Then, fluorescent-labeled Zymozan particles (50 μg/ml) were added. After 1 h of incubation at 37°C, phagocytosis of Zymosan particles was evaluated by flow cytometry. Results are expressed as the mean ± SE from 4 experiments. (D) Neutrophils (2 x 10⁶/ml) were incubated at 37°C for 30 min, 8 h or 18 h in medium supplemented, or not, with NaCl (50mM). Then cells were labeled with annexin-V FITC and propidium iodide, and apoptosis was evaluated by flow cytometry. A representative experiment is shown. (E, F) Neutrophils (2 x 10⁶/ml) were incubated at 37°C for 30 min, 8 h or 18 h in medium supplemented, or not, with NaCl (50mM), sorbitol or mannitol (100 m), in the absence (E) or presence of Zymosan (F). Then cells were labeled with annexin-V FITC and propidium iodide, and apoptosis was evaluated by flow cytometry. Results are expressed as the mean ± SE from 7-17 experiments. (G, H) Isolated monocytes (1 x 10⁶/ml) were incubated at 37°C for 2 or 18 h in culture culture medium supplemented, or not, with NaCl (50 mM) and/or LPS (100 ng/ml). Then, the production of IL-8 (G) and TNF-α (H) was evaluated in cell supernatants by ELISA. The mean ± SE from 4-8 experiments are shown. *P <.05, **P <.01, and ***P <.001.