Tumor Necrosis Factor Expression is Ameliorated After Exposure to an Acidic Environment

LPS-induced TNF-α expression is reduced in MΦs by pretreatment at a pH of 6.0. Cells (5 × 10⁵) were pretreated in HEPES-supplemented RPMI 1640 medium at pH 6.0 or 7.4 for 1 or 3 h at 37°C. After pretreatment, the cells were returned to neutral pH (7.4) and stimulated with LPS (100 ng/mL) for 1.5 h. TNF-α mRNA levels were measured by qRT-PCR (A) or protein in the extracellular medium by ELISA and normalized by the number of viable cells in each well measured by the MTT method (B). (A) All LPS-treated samples displayed higher TNF-α mRNA levels than their non-treated counterparts (n = 4, ^† P < 0.05). A reduction of 57% was observed in TNF-α mRNA levels after exposure to pH 6 for 3 h with respect to pH 7.4 (n = 4, ∗P < 0.05 pH 6, 3 h versus pH 7.4). (B) Protein levels were reduced in 53% at pH 6.0 compared with those pretreated at pH 7.4 (n = 3, ∗P < 0.05).

The reduction in LPS-induced production of TNF-α in cells pretreated in an acidic medium persists after return to a neutral pH. Cells (2 × 10⁵) were pretreated in HEPES-supplemented RPMI 1640 medium at pH 6.0 or 7.4 for 3 h at 37°C. After pretreatment, the cells were returned to neutral pH (7.4) medium. At different points in their recovery from pretreatment (t = 0, 15, 30, 45, and 60 min), they were exposed to LPS (100 ng/mL) for 1.5 h. TNF-α levels were measured in the extracellular medium by ELISA and normalized by the number of viable cells in each well measured by the MTT method. There was a significant reduction in the amount of TNF-α by those cells pretreated at pH 6.0 until 45 min of recovery (n = 3,∗P < 0.05 compared with control conditions, t = 0).

Cell surface levels of CD14 and Tlr4 did not change after exposure to low pH. J774 cells were incubated for 1 or 3 h at pH 6.0 or 7.4 at 37°C. Cells were then harvested, washed, and Fcγ III and II receptors were blocked using purified anti-CD16/32 antibodies. Afterward, cells were stained with FITC-conjugated anti-mouse CD14 and APC-conjugated anti-mouse Toll-like receptor 4/MD-2 antibodies for 30 min at 4°C. Data acquisition was performed with a BD FACSCanto flow cytometer and analyzed using the FlowJo software. There was no change in the cell surface expression of CD14 and Tlr4/MD-2 after exposure to pH 6.0 for 1 and 3 h compared with pH 7.4 (n = 4).

Pretreatment at a pH of 6.0 inhibits NFκB nuclear translocation after incubation with LPS. Cells (0.5 × 10⁶) on a glass cover slide were pretreated at pH 6.0 or 7.4 for 3 h at 37°C. Cells were then returned to neutral pH (7.4) and stimulated with LPS (100 ng/mL) for 25 min. Cells were fixed with 4% paraformaldehyde, permeabilized with acetone and immunostained for NFκB or IK-B. Nuclei were stained with DAPI. (A) Control samples (without LPS stimulation); (B) samples after LPS stimulation.

Pretreatment at pH 6.0 inhibits IκBα phosphorylation after incubation with LPS. Cells (6 × 10⁶) were pretreated at pH 6.0 or 7.4 for 3 h at 37°C. Cells were then returned to neutral pH (7.4) medium and incubated with LPS (100 ng/mL) for 10 min. Then, cells were lysed for protein analysis. The phosphorylated form of IκBα (A) or IKKβ (B) was determined by Western blotting. Equal loading was determined by measuring actin levels by Western blotting. The histogram represents relative degree of LPS-stimulated phosphorylation. (A) There was a significant decrease in the phosphorylation of IκBα in those cells pretreated at pH 6.0 compared with pH 7.4. (n = 3,∗P < 0.05). (B) There was no change in the phosphorylation of IKKβ in those cells pretreated at pH 6.0 compared with pH 7.4 (n = 3). Representative Western blots of phosphorylated IκBα (A) or IKKβ (B) are presented.

Incubation in acidic medium reduces the internal pH of J774 MΦs. Cells (2 × 10⁵ cells/well) were plated in a 96-well plate and incubated overnight (16 h) at 37°C in neutral pH (7.4) medium. (A) The medium was removed and replaced with HEPES-buffered RPMI at pH 6.0 or pH 7.4 containing 1 μM BCECF. (B) The medium was removed and replaced with HEPES-supplemented RPMI 1640 medium at a pH of 6.0 or 7.4 for 3 h at 37°C. After pretreatment, the cells were returned to neutral pH (7.4) medium containing 1 μM BCECF. The fluorescence of the internal BCECF was measured in a fluorometer at excitation 450 and 500 nm and emission 530 nm every 15 min for 60 min. The internal pH was determined by comparing to a calibration curve (n = 4).

Pharmacologic reduction of the internal pH of J774 cells to pH 6.0 inhibits Iκbα phosphorylation after incubation with LPS. Cells (6 × 10⁶) were incubated with HEPES-supplemented RPMI 1640 medium at a pH of 6.0 or 7.4 containing 0.1 μM nigericin, 125 mM KCl for 1 h 37°C. The internal pH was clamped with the addition of 5 mg/mL fatty-acid-free serum albumin in neutral RPMI medium. The cells were washed twice with neutral PBS, rested in neutral pH for 30 min, and then incubated with LPS (100 ng/mL) for 10 min. Then, cells were lysed for protein analysis. The phosphorylated form of IκBα (A) or IKKβ (B) was determined by Western blot. The histogram represents relative degree of LPS-stimulated phosphorylation. (A) There was a significant reduction in the phosphorylation of IκBα in those cells pretreated at pH 6.0 compared to pH 7.4. (n = 3,∗P < 0.05). (B) There was no change in the phosphorylation of IKKβ in those cells pretreated at pH 6.0 compared to pH 7.4. Representative Western blots of phosphorylated IκBα (A) or IKKβ (B) are presented.