Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils

Abstract

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.

Fig. 1.

Effect of extracellular lactate (20 mM) or low extracellular pH (pH_e) on neutrophil intracellular pH (pH_i). A: neutrophils were suspended in normal buffer pH 7.4 (Ctl) or with lactate buffers (pH 7.4, 6.7, 6.0) without (thick lines) or with (thin lines) 10 μM 5-(N,N-hexamethylene)amiloride (HMA). The graph is representative of at least 4 similar experiments. B: BCECF labeled neutrophils were exposed to solutions containing different concentrations of NH₄Cl ([NH₄Cl]), pH 7.4, for 30 min, then washed two times at room temperature (RT) with pH 7.4 control buffer + 10 μM HMA to thoroughly remove NH₄Cl. Cells were resuspended in the same control buffer for measurement. □, control experiments obtained from cells preincubated in 30 mM NH₄Cl, then washed two times at RT with pH 7.4 control buffer without HMA. The pH_i was determined at the indicated times (n = 6).

Fig. 2.

Effect of extracellular lactacidosis on L-selectin shedding in human neutrophils. A and B: L-selectin shedding in control pH 7.4 without (A; w/o) or with (B) lactate. C and D: effect of 10 μM HMA in pH 6.7 lactate buffer. E and F: effect of 10 μM HMA in pH 6.0 lactate buffer. Graphs are flow cytometry data for 10⁴ cells showing the distribution of FITC-labeled surface L-selectin molecules and are representative of 2–4 similar experiments. G: cell viability under different acidic conditions assessed as annexin V-positive polymorphonuclear neutrophils at 2 h. Data are means ± SE of at least 3 separate experiments for each point. *P < 0.05 vs. pH 7.4 lactate, **P < 0.03 vs. pH 7.4 lactate.

Fig. 3.

Effect of pH_i on L-selectin shedding in human neutrophils. Different [NH₄Cl] in the prepulse for 30 min at 37°C, followed by two washes at RT and a 30-min incubation at 37°C in control pH 7.4 + 10 μM HMA to inhibit the Na⁺/H⁺ exchanger, were used to lower the pH_i (determined using BCECF), and the corresponding L-selectin surface expression was determined. Continuous line is the fit of a titration curve with a pK of 6.3.

Fig. 4.

Summary of effects of different Na⁺/H⁺ exchanger (NHE) inhibitors on L-selectin shedding induced by a 30 mM NH₄Cl prepulse. Cells were exposed to NH₄Cl for 30 min at 37°C followed by two washes at RT and a 30-min incubation at 37°C in pH 7.4 control buffer with 10 μM HMA, 5 μM cariporide, or 20 μM 5-(N,N-dimethyl)amiloride (DMA). Control samples were either incubated for 30 min at 37°C, washed two times at RT and incubated at 37°C for 30 min in pH 7.4 control buffer (black column), or subjected to NH₄Cl prepulse, washed two times with pH 7.4 control buffer and subsequent incubation in pH 7.4 buffer for 30 min (empty column). Thus ∼50 min elapsed from the beginning of the first wash after the prepulse to the placing of samples on ice for labeling. Number of experiments in each condition with duplicate samples was 14 (controls), 13 (HMA), 3 (cariporide), and 2 (DMA).

Fig. 5.

Summary of effects of low pH_i and of different NHE exchanger inhibitors on Mac-1 (CD11b) and CD18 induced by lactate pH 6 or by a 30 mM NH₄Cl prepulse. A: effect of HMA, cariporide, and DMA on expression of CD11b. Control samples were handled as in Fig. 4. Approximately 50 min elapsed (20-min washing plus 30-min incubation) from the beginning of the first wash after the prepulse to the placing of samples on ice for labeling. Number of experiments with duplicate samples for each condition was 10 (HMA), 3 (cariporide), and 2 (DMA). Differences were not statistically significant except for the lactate pH 6.0 result. B: enhancement of CD18 surface expression by lactacidosis and HMA (number of experiments with duplicate samples for each condition was 3). *Statistically significant difference from control.

Fig. 6.

Effects of metalloprotease inhibitors on L-selectin shedding (A) and β₂-integrin (B) surface expression seen with low pH_i. L-selectin shedding and β₂-integrin surface expression were induced by a 30 mM NH₄Cl prepulse for 30 min in the presence or absence of 30 μM RO-31-9790 (RO) or 20 μM KD-IX-73-4 (KD), followed by two washes at RT and a 30-min incubation in pH 7.4 control buffer + 10 μM HMA with or without each metalloprotease inhibitor. Control samples were handled as in Fig. 4. L-selectin data are for n = 13 experiments (control) and n = 3 experiments (inhibitors). β₂-Integrin data are for n = 10 experiments (control) and n = 3 experiments (inhibitors) with duplicate samples in each experiment.

Fig. 7.

Effects of p38 MAP kinase inhibitors on L-selectin surface expression. A: human neutrophils were exposed to a 30 mM NH₄Cl prepulse with or without 2 μM SB-203580 or 2 μM SB-239063, followed by two washes at RT and a 30-min incubation in pH 7.4 control buffer + 10 μM HMA with or without each p38 MAPK inhibitor. Control samples were handled as in Fig. 4. L-selectin levels were assessed on the basis of mean fluorescence intensity compared with the pH 7.4 control. Data are representative of n = 5 (SB-203580) and n = 3 (SB-239063) different experiments with duplicate samples in each experiment. B: intracellular acidification enhanced p38 MAP kinase activity. Top, Western blot using protein (24 μg in each lane) isolated from neutrophils subjected to NH₄Cl prepulse (1, density = 21.9) or 10 μM HMA applied after the wash period (2, density = 55.7). Bottom, Western blot using protein (22.5 μg in each lane) isolated from neutrophils exposed to NH₄Cl prepulse and then treated 10 μM HMA applied after the wash period (1, density = 74.3) or from neutrophils exposed to 2 μM SB-203580 during NH₄Cl prepulse and then to 10 μM HMA ± 2 μM SB-203580 applied after the wash period (2, density = 46.1).