Interleukin-4 rapidly inhibits calcium transients in response to carbachol in bovine airway smooth muscle cells

Abstract

To assess interleukin (IL)-4 effects on calcium signaling, bovine airway smooth-muscle (ASM) cells were loaded with fura-2 and cytosolic calcium ([Ca(2+)](i)) was measured in single cells by digital microscopy. Human recombinant IL-4 (50 ng/ml) caused small increases in [Ca(2+)](i). For single cells, carbachol-stimulated calcium transients were compared before (S1) and after (S2) exposure to IL-4 or IL-13. When cells were treated with IL-4 (50 ng/ml) for 20 min, the S2/S1 ratio was 0.17 +/- 0.04 (n = 7) even though IL-4 had been washed from the chamber for 10 min before the S2 response. In contrast, controls not treated with IL-4 had S2/S1 of 0.70 +/- 0.04 (n = 13, P < 0.01). Lower concentrations of IL-4 variably decreased transients and IL-13 had no effect. In other experiments, 5 min of IL-4 did not immediately decrease transients but did after a 25-min delay. Goat antihuman IL-4 antibody abolished the effect of IL-4. IL-4 (50 ng/ml) also inhibited responses to caffeine (S2/S1: 0.30 +/- 0.04 and 0.54 +/- 0.06 for IL-4-treated versus control). We conclude that IL-4 rapidly inhibited calcium transients. Because caffeine-stimulated transients were inhibited, IL-4 may act, at least in part, by depleting calcium stores. IL-4 inhibition of cholinergic signaling may be important for modulating ASM responses during inflammation.

Figure 1

Effect of IL-4 on [Ca²⁺]_i in bovine airway smooth muscle cells. Cells loaded with fura 2 were exposed to human, recombinant IL-4 (50 ng/ml) and changes in [Ca²⁺]_i were recorded in single cells. The maximum response to IL-4 occurred within one minute. Data are mean±SEM, n=35. * indicates significantly different from basal (p<0.05).

Figure 2

S2/S1 ratio. For control cells (open squares) not exposed to IL-4, each cell was stimulated with carbachol (10⁻⁵ M) and the maximum change in [Ca²⁺]_i during the resulting transient was recorded and defined as S1. After washing with PSS for 45 min, the same cell was stimulated a second time with carbachol and the magnitude of the resulting transient was recorded and defined as S2. The figure shows that the magnitude of the S1 response did predict the magnitude of the S2 response in that same cell. For each control cell, an S2/S1 ratio was calculated as an index of cell responsiveness and averaged 0.70±0.04. Other cells (closed squares) were treated identically except that thapsigargin was present during the recovery period between S1 and S2 stimuli.

Figure 3

Effects of IL-4 and IL-13 on responses to carbachol. (A) Following the S1 stimulus with carbachol (10⁻⁵ M), the cells were washed for 15 min and then exposed to IL-4 (5,25,50 ng/ml) (n= 5−7) or IL-13 (50 ng/ml)(n=7) for 20 min. Finally, cells were washed for 10 min before being stimulated a second time with carbachol (S2). Mean data are presented. * indicates significantly different than control (p<0.01). (B) Data from panel A are presented to show the individual responses of control cells (open squares) versus those cells exposed to IL-4 (50 ng/ml) (open circles). Closed symbols show mean values for control (squares) and IL-4-treated cells (circles).

Figure 4

Time-dependence of IL-4 effect on S2/S1 ratios. Following the S1 stimulus with carbachol (10⁻⁵ M) and a 15 min wash, cells were exposed to IL-4 (50 ng/ml) for 5 min. In one group of cells (n=6, left side of figure), the second stimulus (S2) with carbachol (10⁻⁵M) followed immediately after the 5 min exposure to IL-4. In that case, IL-4 had no apparent effect on the S2/S1 ratio compared to time-matched controls. For other cells (n=6, right side of figure), the S2 stimulus with carbachol (10⁻⁵M) was applied after the cells had been exposed to IL-4 for 5 min, but then washed for 25 min before S2 testing. In this case, the S2/S1 ratio was significantly decreased compared to time-matched controls. * indicates significantly different from control (p<0.01). The presence of goat anti-human IL-4 neutralizing antibody inhibited the effect that IL-4 had on S2/S1 (n=4).

Figure 5

Effects of IL-4 on responses to caffeine. Following the S1 stimulus with carbachol (10⁻⁵ M) and a 15 min wash, cells were exposed to IL-4 (50 ng/ml) for 20 min. Cells then were washed for 10 min before being stimulated with caffeine (10 mM)(S2). Time-matched controls were not exposed to IL-4. Data are the mean±SEM, n= 5. * indicates significantly different from control (p<0.01).

Figure 6

Effect of SR filling state on IL-4 inhibition of transients. Following the S1 stimulus with carbachol (10⁻⁵ M), cells were treated in two different ways. One group of cells was washed immediately, for 2 min, with buffer containing IL-4 (50 ng/ml) and then the S2 stimulus with carbachol was applied. The other group of cells was washed with buffer alone for 10 min to allow refilling of intracellular stores and then the cells were exposed to IL-4 for 2 min before the S2 stimulus with carbachol. For both groups, controls were identically treated cells not exposed to IL-4. Data are the mean±SEM, n=4−7. * indicates significantly different from control (p<0.05).