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IL-4 inhibits calcium transients in bovine trachealis cells by a ryanodine receptor-dependent mechanism

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

¹Correspondence: Department of Medicine, LRB 319, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605, USA. E-mail: mark.madison{at}umassmed.edu

SPECIFIC AIMS

1. Interleukin (IL) -4 effects on SR calcium mobilization
Brief treatment of bovine airway smooth muscle cells with the inflammatory cytokine IL-4, but not IL-13, inhibits the magnitude of calcium transients stimulated by carbachol or caffeine, suggesting that IL-4 could have important effects on the calcium signaling that regulates contraction or other cellular functions. To account for this rapid inhibitory effect of IL-4, the first aim of this study was to determine whether IL-4 blocks calcium release from the sarcoplasmic reticulum (SR) or, instead, decreases the amount of calcium in the SR available for release.

2. Role of ryanodine receptor (RyR) calcium release channels
The second aim of the study was to determine whether the inhibitory effect of IL-4 on calcium transients depended on cyclic ADP-ribose (cADPR) and RyR calcium release channels.

PRINCIPAL FINDINGS

1. Treatment with IL-4, but not IL-13, inhibits the magnitude of ionomycin-stimulated calcium transients
Bovine trachealis cells were loaded with fura 2-AM and cytosolic calcium concentrations ([Ca²⁺]_i) were measured in single cells by digital microscopy. In the absence of extracellular calcium, the calcium ionophore ionomycin caused a calcium transient by mobilizing SR calcium stores. As expected, this response to ionomycin was abolished by pretreating cells with cyclopiazonic acid, a calcium pump inhibitor that depletes SR calcium stores. In the main experiments, stimulation (S1) of cells with carbachol caused a rapid, transient increase in [Ca²⁺]_i that served as a control for each cell. After each cell was washed and allowed to recover, stimulation (S2) of the same cell with ionomycin again caused a calcium transient that was similar to the magnitude of the S1 response (S2/S1=1.03±0.29). However, after only 20 min of pretreatment with IL-4 (50 ng/mL), ionomycin no longer stimulated large S2 responses (S2/S1=0.50±0.16, P=0.02, n=6) (Fig. 1 ). In contrast to IL-4, similar pretreatment with the cytokine IL-13 had no effect on S2 responses.

View larger version (15K): [in this window] [in a new window]   Figure 1. IL-4 inhibits calcium transients in response to ionomycin. Bovine trachealis cells were loaded with fura 2-AM and single cells were imaged ratiometrically. In the presence of extracellular calcium, cells were stimulated with carbachol (10 µM, cch) for 2 min and the resulting transient was designated S1. Cch was washed from the cells and cells were perfused by vehicle control or IL-4 (50 ng/mL) for 20 min. In the absence of extracellular calcium, cells were stimulated with ionomycin (10 µM) for 2 min and the resulting transient recorded (S2). A) Representative traces for an IL-4 treated cell are shown and, for schematic purposes, time is not to scale. B) S2/S1 ratios for individual cells are compared for vehicle (open square) vs. IL-4 treated (closed squares) cells. Mean ± SE for control (open triangle) and IL-4 treated (closed triangle) cells are shown.

2. The inhibitory effect of IL-4 on calcium transients is blocked by high concentrations of ryanodine but not by an antagonist of cADPR
Consistent with the presence of RyR calcium release channels in airway smooth muscle, rabbit anti-RyR identified a protein of high molecular mass (565 K_d), and caffeine caused a rapid, transient increase in [Ca²⁺]_i. As expected, a relatively high concentration of ryanodine (200 µM) inhibited calcium transients stimulated by caffeine. The same high concentration of ryanodine had no effect on the calcium transients stimulated by ionomycin alone but did block the inhibitory effect of IL-4 on calcium transients to ionomycin (S2/S1=1.01±0.11, n=5) (Fig. 2 ). In contrast, an antagonist of cADPR binding to the RyR calcium release channel, 8-bromo- (8Br-) cADPR, had no effect on the ability of IL-4 to inhibit calcium transients (S2/S1=0.48±0.13, n=5).

View larger version (33K): [in this window] [in a new window]   Figure 2. Ryanodine inhibits the effect of IL-4 on transients. After the S1 response to carbachol, cells were washed 10 min, then exposed to vehicle or IL-4 for 20 min in the absence and presence of ryanodine (200 µM) or 8-bromo-cADPR (100 µM). The S2 response to ionomycin was recorded and the S2/S1 ratio calculated as an index of cell responsiveness. Ryanodine, but not 8-bromo-cADPR, inhibited the effects that IL-4 has on calcium transients. *P < 0.05, n = 5–7.

CONCLUSIONS AND SIGNIFICANCE

Evidence suggests that the T helper-2 cytokines IL-4 and IL-13 have important roles in the pathogenesis of asthma. For bovine airway smooth muscle cells, this study shows that IL-4 significantly inhibits the magnitude of calcium transients caused by SR calcium release in response to ionomycin and that this effect of IL-4 depends on the function of RyR calcium release channels. IL-13, a T helper-2 cytokine that often shares many biological effects with IL-4, did not inhibit calcium transients.

These new findings suggest a mechanism by which IL-4 inhibits calcium transients in response to both carbachol and caffeine. Since carbachol and caffeine mobilize calcium from the SR by distinct mechanisms, an IL-4 effect on both agents would be consistent with IL-4 depleting SR calcium stores. However, another possibility is that IL-4 simultaneously inhibits both inositol trisphosphate receptor (IP₃R) -mediated and RyR-mediated calcium release from the SR. To distinguish these possibilities, we tested whether IL-4 inhibits transients elicited by ionomycin, a calcium ionophore that mobilizes SR calcium by mechanisms independent of IP₃R and RyR. The data show that pretreatment with IL-4 did inhibit responses to ionomycin and, therefore, that IL-4 does not act by inhibiting IP₃R and RyR simultaneously. Instead the results are most consistent with IL-4 decreasing calcium in SR stores. That is, because the calcium transients elicited by ionomycin depend on the mobilization of calcium from intracellular stores, these new findings suggest that, over a relatively brief interval, IL-4 significantly decreases intracellular calcium stores by causing a net release of calcium from the SR.

The calcium content of the SR is determined by a dynamic balance between rates of uptake and leak of calcium from the SR. Theoretically, IL-4 could decrease calcium in the SR by inhibiting calcium uptake via sarcoendoplasmic reticulum calcium ATPase (SERCA) and/or by stimulating the release of calcium from the SR. In an earlier study of these same cells, IL-4 inhibited calcium transients in response to carbachol even in the presence of a high concentration of cyclopiazonic acid, an inhibitor of SERCA. This finding suggested that IL-4 does not decrease SR calcium content by inhibiting calcium uptake and, therefore, must instead be increasing the rate of release of calcium from the SR. Subsequent experiments showed that xestospongin C, an inhibitor of IP₃R, did not block the effects of IL-4. Therefore, in the current study, it was hypothesized that IL-4 depletes SR calcium by stimulating release of calcium ions through RyR calcium release channels that are present in these cells as evidenced by Western blot analysis and the presence of calcium transients in response to caffeine.

Although low concentrations of ryanodine are known to cause release of SR calcium through RyR, high concentrations of ryanodine block calcium release through RyR. To assess whether RyR function is necessary for IL-4 to decrease SR calcium stores, concentrations of ryanodine high enough to inhibit responses to caffeine were identified and applied to the cells during IL-4 exposure. In the presence of ryanodine (200 µM), IL-4 no longer inhibited transients in response to ionomycin. The aggregate data suggest that IL-4 causes a net loss of SR calcium and that this depends on the release of calcium ions through RyR calcium release channels.

RyR are highly regulated channels modulated by cytosolic calcium, luminal calcium, ATP/Mg²⁺, redox status, phosphorylation, cADPR, and associated proteins such as calmodulin, calsequestrin, and FK-506 binding protein. Although the mechanism by which cADPR regulates RyR has not been determined, cADPR has been shown to cause calcium release in many cell types including airway smooth muscle. To assess whether cADPR plays a role in linking IL-4 to RyR function, we tested the effects of 8Br-cADPR, a cell permeable antagonist of cADPR at the RyR calcium release channel. This cADPR antagonist did not block the inhibitory effect of IL-4 on calcium transients. Therefore, although the effect of IL-4 on SR calcium depends on functional RyR calcium release channels, it does not appear to depend on cADPR signaling.

The phosphatidylinositol 3-kinase (PI3K) antagonists wortmannin and deguelin both inhibited the effect of IL-4 on calcium transients. Because the current experiments showed that IL-4 causes a net loss of SR calcium by mechanisms dependent on RyR calcium release channels, the simplest model for our findings is that IL-4 stimulates signaling pathways involving PI3K to increase the release of calcium through RyR, thereby causing a net loss of calcium from the SR (Fig. 3 ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic for IL-4 effects on calcium stores in the SR. Over 20–30 min, IL-4, but not IL-13, decreases calcium in SR stores enough that calcium transients in response to muscarinic agonists, caffeine, or a calcium ionophore are all decreased. IL-4 decreases SR calcium by increasing leak of calcium from the SR through RyR calcium release channels. These channels are blocked by high concentrations of ryanodine. Because IL-4 by itself did not cause detectable increases in global cytosolic calcium, the schematic suggests that calcium ions are released into poorly imaged, near-plasma membrane regions of the cytosol, then pumped from the cell by calcium pumps. Items in boldface text highlight features addressed in the current study. An earlier study showed that the effects of IL-4 on calcium were inhibited by the PI3K antagonist wortmannin but not inhibited by antagonists of IP3R or SERCA.

Several important issues remain unresolved and are areas of active investigation. First, the pathways linking PI3K activity to the release of SR calcium through RyR calcium release channels are not known and could be multiple for this highly regulated channel. Second, although the results show RyR function is required for IL-4 to decrease SR calcium, that does not necessarily mean that IL-4 signaling pathways directly modulate RyR channel function in these experiments. For example, it is theoretically possible that basal leak of calcium ions through RyR is not directly modulated by IL-4 signaling, but still required for enough SR calcium depletion to occur and be detectable in our experimental protocols. Third, the basis for the different effects of IL-4 and IL-13 on calcium is not known but the findings are consistent with prior studies of airway smooth muscle cells showing other differential effects for these two cytokines. Fourth, the functional implications of IL-4 causing release of calcium from the SR and decreasing SR calcium stores are not known but both effects could have implications for a variety of cell functions.

In summary, as it did for carbachol and caffeine, a relatively brief exposure of cells to IL-4 inhibited the magnitude of calcium transients elicited by ionomycin. Moreover, the inhibitory effect of IL-4 was antagonized by the presence of high concentrations of ryanodine that blocked RyR calcium release channels. Therefore, for bovine airway smooth muscle cells, IL-4, but not IL-13, causes calcium release from the SR that decreases SR calcium stores and this depends on the presence of functional RyR calcium release channels.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4031fje;

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