Eosinophil Survival and Activation Sulfonylureas Inhibit Cytokine

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Sulfonylureas Inhibit Cytokine-Induced Eosinophil Survival and Activation Jennifer L. Bankers-Fulbright, Gail M. Kephart, David A. Loegering, Annabel L. Bradford, Shinji Okada, Hirohito Kita and This information is current as Gerald J. Gleich of September 26, 2021. J Immunol 1998; 160:5546-5553; ; http://www.jimmunol.org/content/160/11/5546 Downloaded from References This article cites 46 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/160/11/5546.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Sulfonylureas Inhibit Cytokine-Induced Eosinophil Survival and Activation1

Jennifer L. Bankers-Fulbright, Gail M. Kephart, David A. Loegering, Annabel L. Bradford, Shinji Okada, Hirohito Kita, and Gerald J. Gleich2

Eosinophils play a key role in the pathogenesis of asthma and other allergic inﬂammatory diseases. We have previously shown that treatment of eosinophils with lidocaine preferentially inhibits IL-5-induced survival. This inhibition cannot be overcome by increasing concentrations of IL-5 and is not due to the blocking of Na؉ channels by lidocaine. Here we report that one class of K؉ channel blockers, the sulfonylureas, inhibits eosinophil survival in a manner similar to lidocaine. The sulfonylurea glyburide inhibits eosinophil survival even at high concentrations of IL-5. In contrast, increasing concentrations of IL-3 or granulocyte- macrophage CSF overcome glyburide inhibition. Glyburide also blocks cytokine-induced eosinophil superoxide production. Sim- Downloaded from ilar results were seen with the sulfonylureas tolbutamide and glipizide. Interestingly, the effects of glyburide are not antagonized by the ATP-sensitive K؉ channel openers cromakalim, pinacidil, or diazoxide. Although Scatchard analysis of [3H]glyburide binding to eosinophil membranes indicated that the high afﬁnity sulfonylurea receptor (SUR1) is not present on eosinophils, human eosinophils do express mRNA homologous to the sulfonylurea receptor family, in keeping with the presence of a sulfonylurea receptor. Finally, coculture of eosinophils with combinations of glyburide, lidocaine, and dexamethasone resulted in synergistic inhibition of cytokine-mediated eosinophil survival and superoxide production. These results have intriguing clinical http://www.jimmunol.org/ implications for the treatment of eosinophil-associated diseases. The Journal of Immunology, 1998, 160: 5546–5553.

uman asthma is characterized by reversible bronchial eosinophil granule proteins into sensitive tissues, such as the lung hyperreactivity and infiltration of inflammatory cells, es- (7–11). If, however, cytokines are present (as they are in bronchial H pecially eosinophils, into the lung (1, 2). In recent years lavage fluid from patients with asthma (12)) eosinophil survival is it has become obvious that treating the underlying inflammation is prolonged, granule proteins are released (13), and eosinophil critical to maintaining good airway function (3). The most potent apoptosis is substantially delayed (12). We reported previously agents currently used for controlling inflammation are the glu- that lidocaine treatment of eosinophils in vitro ablates cytokine- cocorticoids. However, the adverse effects of long term treatment induced survival in a manner similar to dexamethasone (14–16). by guest on September 26, 2021 with oral glucocorticoids have stimulated efforts to identify effec- Other topical anesthetics tested blocked eosinophil survival as tive anti-inflammatory substitutes (4, 5). We have previously re- well, but their potencies in the survival assay did not correlate with ported the clinical efficacy of the topical anesthetic lidocaine in the their potencies as anesthetics (17). Additionally, Naϩ channel treatment of asthma in an open label trial (6). Treatment of glu- blockers that should mimic the anesthetic effect of lidocaine had no cocorticoid-dependent patients with nebulized lidocaine allowed effect on cytokine-stimulated eosinophil survival in vitro (17, 18). for the complete elimination of steroid treatment in 13 of 20 pa- These results support the hypothesis that the effect of lidocaine on tients, although exacerbations still required oral steroids. This eosinophil survival is a novel function of the drug. study suggests that lidocaine functions as an anti-inflammatory Several reports in the literature indicate that lidocaine at high agent and is thus able to replace long term oral steroid treatment. concentrations blocks Kϩ channels (19–21). Using three classes of Lidocaine, like the glucocorticoids, may partially exert anti- Kϩ channel blockers, we investigated the effect of Kϩ channel inflammatory effects by directly inhibiting eosinophil survival and regulation on eosinophil survival. Here we report that the sulfo- activation in the lung. In the absence of exogenous cytokines, such nylureas, one class of Kϩ channel blockers, inhibit eosinophil sur- as IL-5, IL-3, or GM-CSF,3 eosinophils undergo apoptosis (7–9). vival and activation in vitro. This mode of cell death with consequent engulfment by macrophages appears to be critical for preventing the release of toxic Materials and Methods Materials HIT T15 cells were obtained from American Type Culture Collection Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, MN (Rockville, MD). Human rIL-5 was a gift from Schering Plough (Ken- 55905 ilworth, NJ). IL-3 and GM-CSF were purchased from R&D Systems (Min- Received for publication August 22, 1997. Accepted for publication January 29, 1998. neapolis, MN). Tolbutamide, glipizide, apamin, tetraethylammonium chlo- The costs of publication of this article were defrayed in part by the payment of page ride (TEA), diazoxide, and pinacidil were purchased from Research charges. This article must therefore be hereby marked advertisement in accordance Biochemicals International (Natick, MA). Unless otherwise indicated, all with 18 U.S.C. Section 1734 solely to indicate this fact. other materials were purchased from Sigma (St. Louis, MO). 1 This work was supported in part by National Institutes of Health Grants AI07047, AI34486, and AI34577 and by the Mayo Foundation. Eosinophil purification 2 Address correspondence and reprint requests to Dr. Gerald J. Gleich, Department of Eosinophils were purified from normal human subjects and patients, usu- Immunology, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. ally allergic, with slightly elevated numbers of eosinophils using a mag- 3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; TEA, netic bead separation protocol previously described (22, 23). Briefly, blood tetraethylammonium chloride; PI, propidium iodide; SUR, sulfonylurea receptor; was layered on Percoll (density ϭ 1.085 g/ml) and centrifuged at 900 ϫ g RPA, ribonuclease protection assay. to remove PBMC and hypodense (activated) eosinophils. Erythrocytes

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 5547 were lysed by osmotic shock, and neutrophils were removed from the tated, resuspended in loading buffer, and separated on a 10% polyacryl- eosinophils by separation using a steel wool column (type CS) and anti- amide gel. The gel was transferred to a positively charged membrane (Am- CD16-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA). Eosin- bion), and hybridized biotinylated probe was detected using the BrightStar ophil purity exceeded 95% as determined by Randolph’s stain. The primary BioDetect Kit (Ambion) and Kodak autoradiographic film (Eastman contaminating cells were neutrophils. Kodak, Rochester, NY). Survival assays Purified eosinophils were washed with HybriCare medium (American Statistical analysis Type Culture Collection) supplemented with 2 mM glutamine, 50 ␮g/ml gentamicin sulfate, and 2.5 to 10% ␣ calf serum (HyClone, Logan, UT). Statistical analyses were performed using two-tailed, paired Student’s t Eosinophils were resuspended at 0.5 ϫ 106 cells/ml in supplemented Hy- test. Statistical analysis of dose-dependent glyburide apoptosis induction briCare and were incubated with drugs and cytokines in 96-well plates (data not shown) was calculated using the Spearman rank correlation co- (Costar, Cambridge, MA) as indicated in the text. Drugs that were not efficient. directly soluble in media were first dissolved in DMSO and diluted to the appropriate concentrations in media. Unless indicated, the final DMSO concentration in the well never exceeded 0.5%, which was shown in con- Results trol experiments to have no effect on eosinophil survival. The final con- ϩ centration of eosinophils was 5 ϫ 104/well in a total volume of 200 ␮l. Effects of K channel blockers on human eosinophil survival

Plates were incubated at 37°C in 5% CO2 for 4 days. At that time, each ϩ ϫ To test the hypothesis that K channels are involved in eosinophil well was transferred into 12- 75-mm polystyrene tubes (Falcon, Becton ϩ Dickinson Labware, Lincoln Park, NJ), and 200 ␮l of propidium iodide survival, we compared the activities of three different K channel

(PI; final concentration, 0.5 ␮g/ml) were added to each tube to stain dead blockers (glyburide, TEA, and apamin) with the activity of lido- Downloaded from cells. Stained cells were analyzed using a FACScan flow cytometer and PC caine on IL-5-stimulated eosinophils. Glyburide is a blocker of LYSIS software (Becton Dickinson Immunocytometry Systems, San Jose, ϩ ϩ CA). Eosinophil survival is plotted as relative percent survival (percentage ATP-sensitive K channels, TEA blocks K channels and is a 2ϩ of the control) according to the following equation: relative eosinophil nicotinic cholinergic receptor antagonist, and apamin blocks Ca - survival ϭ (% survival/% survival at maximum cytokine concentration) ϫ activated Kϩ channels (28, 29). As shown in Figure 1, TEA and Х 100. Eosinophils cultured with IL-5 (1000 pg/ml; 22 pM) usually apamin had no significant effect on IL-5-induced eosinophil sur- showed Ͼ90% survival, whereas cells cultured in control medium usually Ϫ ϫ 4 http://www.jimmunol.org/ had Ͻ30% spontaneous survival. vival. Only glyburide (1 10 M) inhibited IL-5-mediated eosinophil survival as effectively as lidocaine. To determine whether Apoptosis assay this was a dose-response effect, doses of glyburide between 10Ϫ5 Ϫ Eosinophil apoptosis was assayed using flow cytometry and the DNA- and 10 4 M were tested. As shown in Figure 2, glyburide exhib- binding fluorophores Hoechst 33342 (Molecular Probes, Eugene, OR) and ited a dose-response effect, which was especially evident at lower PI as previously described (24, 25). Following 48 h of stimulation, eosinophils were stained with 10 ␮M Hoechst and 32 ␮M PI and analyzed on IL-5 concentrations. However, only the inhibition mediated by Ϫ4 a Becton Dickinson FACSVantage flow cytometer (San Jose, CA) with UV 10 M glyburide was statistically significant. laser excitation at 357 nm. Dead cells fluoresced red and blue, live cells had One of the characteristics of lidocaine inhibition of eosinophil low intensity blue fluorescence only, and apoptotic cells had high intensity

survival is a relative specificity for IL-5 (17). To determine by guest on September 26, 2021 blue fluorescence. whether glyburide exhibits a similar preference, we examined the Superoxide assay ability of glyburide to inhibit eosinophil survival mediated by IL-3 Ninety-six-well plates were blocked with 50 ␮l of 1% human serum albu- and GM-CSF. As shown in Figure 3, eosinophil survival was in- minfor2hat37°C. Before adding cells, blocked wells were washed twice hibited by glyburide at low IL-3 and GM-CSF concentrations, but with 200 ␮l of saline. Purified eosinophils were washed with HBSS me- the inhibition was completely overcome by increased concentra- ϫ 5 dium (10 mM HEPES, pH 7.4) and resuspended to 5 10 cells/ml in tions of these cytokines. This pattern of inhibition was essentially cytochrome c solution (2.4 mg/ml cytochrome c in HBSS medium). Eo- sinophils were plated at a final concentration of 5 ϫ 104 cells/well in 200 identical with that seen with lidocaine (compare left and right col- ␮l and were pretreated with drugs for 30 min at 37°C before cytokines umns of Fig. 3). Glyburide and lidocaine both preferentially inhibit were added. Immediately after cytokines were added, the absorbance at IL-5-induced survival compared with IL-3- or GM-CSF-mediated 550 nm was analyzed in a Thermomax plate reader (Molecular Devices, Sunnyvale, CA) every 15 to 30 min for 2 h. Conversion of absorbance to survival, and the inhibition could not be overcome by increased nanomoles of cytochrome c reduced per 1 ϫ 106 eosinophils used the concentrations of IL-5 (up to 10,000 pg/ml; data not shown). Nei- Ϫ ϭ following equation: 19.1(absorbance absorbancetime 0)/0.05 nmol cy- ther lidocaine nor glyburide mediated a general toxic effect on the 6 tochrome c reduced/10 cells (23). cells, since eosinophils stimulated with IL-3 or GM-CSF survived Glyburide binding assay in the presence of the drugs. Additionally, the decreased eosinophil survival seen in the glyburide-treated cells correlated with in- Cell membranes were prepared as previously described (26), and 20 ␮gof membrane protein was incubated in 0.1 ml of 50 mM 3-(N-morpholino) creased apoptosis, as detected by staining cells with Hoechst propanesulfonic acid, pH 7.4, with 0.05–50 nM [3H]glyburide (Amersham, 33342 and PI (data not shown). This effect was statistically sig- Arlington Heights, IL) for2hatroom temperature. Bound radioligand was Ͻ ϭ Ͻ nificant ( p 0.05) and dose dependent (rs 1.0; p 0.05). separated from free using a Skatron cell harvester (Skatron Instruments, Additionally, the concentrations of glyburide required to increase Sterling, VA) with double glass filters. The filters were dried and counted in 2 ml of scintillation fluid using a beta counter. Specific picomoles of apoptosis were essentially identical with the concentrations re- [3H]glyburide bound per milligram of protein were determined by sub- quired to inhibit eosinophil survival. tracting nonspecific binding in the presence of excess unlabeled glyburide. Glyburide, also called glybenclamide, belongs to the sulfonyl- ϩ RNase protection assay urea family of compounds. Sulfonylureas block ATP-sensitive K channels by binding to an accessory regulating receptor, the SUR Biotinylated probes for human actin or the NBF2 region of human sulfonylurea receptor (SUR) (27) were made with the BrightStar BIOTINscript (30). If glyburide inhibits eosinophil survival by blocking an ATP- ϩ Kit (Ambion, Austin, TX). Guanidine thiocyanate lysates of purified hu- sensitive K channel, then other sulfonylureas should have similar man eosinophils were prepared and analyzed with the Direct Protect Lysate activities. To test this, we examined the effects of two other sul- Ribonuclease Protection Assay Kit (Ambion). Briefly, the lysates were fonylureas: tolbutamide and glipizide. As shown in Figure 4, tol- incubated with biotinylated probes and treated with RNase to degrade any remaining single-stranded probe. After the RNase was inactivated with butamide and glipizide inhibited eosinophil survival as well as did sarcosyl and proteinase K, the RNase-protected fragments were precipi- glyburide. High concentrations (1000 pg/ml) of IL-3 or GM-CSF 5548 SULFONYLUREAS INHIBIT EOSINOPHIL SURVIVAL

FIGURE 2. Glyburide shows dose-dependent inhibition of eosinophil survival. Puriﬁed eosinophils were cultured for 4 days with various concentrations of IL-5. Survival was assayed as described in Materials and Methods and is plotted as a percentage of eosinophil survival at 1000 pg/ml

IL-5. Shown are the mean Ϯ SD of three independent experiments using Downloaded from eosinophils from both normal and allergic donors. Eosinophils were cocultured with IL-5 and medium control (⅜) or glyburide (f indicates 1 ϫ 10Ϫ5 M, Œ indicates 3.3 ϫ 10Ϫ5 M, ࡗ indicates 6.7 ϫ 10Ϫ5 M, ⅷ indicates 1 ϫ 10Ϫ4 M). The asterisk indicates p Ͻ 0.05 (compared with 1000 pg/ml IL-5 alone). http://www.jimmunol.org/

We were unable to inhibit the effects of glyburide by cromakalim pretreatment despite repeated efforts (n ϭ 8; data not shown). However, we observed the potentiation of eosinophil survival in the presence of IL-5. As shown in Figure 5, the IL-5-mediated survival in these experiments was markedly less than usual; eosinophil absolute survival is normally Ͼ90% after 4-day stimulation with 1000 pg/ml IL-5. The reason for the low survival in these by guest on September 26, 2021 experiments was a high (1%) final concentration of DMSO in the medium from preparation of the cromakalim stock. However, this decreased survival response to IL-5 in the presence of 1% DMSO allowed us to observe the phenomenon of cromakalim “rescue” of eosinophils. As shown in Figure 5, this rescue was dramatic and FIGURE 1. Glyburide, an ATP-sensitive Kϩ channel blocker, inhibits restored the survival of IL-5-treated eosinophils to normal. This eosinophil survival. Purified eosinophils were cultured for 4 days with rescue was also observed when eosinophils were treated with the increasing concentrations of IL-5 in the presence of different inhibitors. Kϩ channel openers pinacidil and diazoxide and was a dose-de- Survival was assayed as described in Materials and Methods and is plotted pendent effect (data not shown). as a percentage of eosinophil survival at 1000 pg/ml IL-5. Each panel shows the mean Ϯ SD of at least three independent experiments using cells Glyburide inhibits human eosinophil superoxide production from both normal and allergic patients. Eosinophils were treated with IL-5 in the presence of medium control (⅜), 1 ϫ 10Ϫ3 M lidocaine (f), or Eosinophil activation and degranulation, not their mere presence, various concentrations of drugs. Top, Glyburide (Œ indicates 10Ϫ6 M, ⅷ are probably critical in initiating the tissue damage associated with indicates 10Ϫ5 M, ખ indicates 10Ϫ4 M). Center, TEA (Œ indicates 10Ϫ5 M, eosinophilic inflammation (1). Activation of eosinophils in vitro ⅷ indicates 10Ϫ4 M, ખ indicates 10Ϫ3 M). Bottom, Apamin (Œ indicates can be detected by measuring superoxide production in response to 10Ϫ7 M, ⅷ indicates 10Ϫ6 M, ખ indicates 10Ϫ5 M). The asterisk indicates stimuli. We have previously shown that lidocaine significantly in- p Ͻ 0.05 (compared with 1000 pg/ml IL-5 alone). hibits cytokine-mediated eosinophil superoxide production (17). To determine whether glyburide also inhibits eosinophil superoxide production, we cultured eosinophils with IL-5, IL-3, or GM- overcame the inhibitory effects of the sulfonylureas, whereas eo- CSF in the presence of glyburide. As shown in Figure 6, all three sinophils treated with 1000 pg/ml IL-5 in the presence of gly- cytokines stimulated superoxide production by eosinophils. How- buride, tolbutamide, or glipizide showed a 20 to 30% average de- ever, superoxide production initiated by the cytokines was dramat- crease in survival. Thus, glyburide is representative of the general ically inhibited by glyburide, and excess cytokine did not over- effectiveness of sulfonylureas in inhibiting eosinophil survival. come this inhibition. Interestingly, no preferential inhibition of

ϩ IL-5 stimulation was seen in the superoxide assay compared to that K channel openers rescue stressed eosinophils in the survival assay. Instead, glyburide inhibited superoxide gen- If blocking an ATP-sensitive Kϩ channel results in inhibition of eration induced by all cytokines, including that generated by plate- eosinophil survival, then an ATP-sensitive Kϩ channel opener let-activating factor (data not shown). As in the survival assay, should block the effects of glyburide and/or potentiate survival pretreatment of eosinophils with cromakalim did not block the (31). To test this, we examined the effects of the ATP-sensitive Kϩ glyburide effect under normal (low DMSO) conditions (data not channel opener cromakalim on IL-5-mediated eosinophil survival. shown). The Journal of Immunology 5549

FIGURE 3. Glyburide preferentially inhibits IL-5-mediated eosinophil survival. Puriﬁed eosinophils were cultured for 4 days with various concentrations of IL-5, IL-3, or GM-CSF. Eo- sinophil survival was analyzed as described in Materials and Methods and is plotted as a percentage of eosinophil survival at maximal cytokine concentrations. Each panel shows the mean Ϯ SD of at least four independent experiments using eosinophils from both normal and allergic patients. When not visible, the SD was smaller than the symbol. Eosinophils were Downloaded from cocultured with cytokines and medium control (⅜), 1 ϫ 10Ϫ3 M lidocaine (f; left), or 1 ϫ 10Ϫ4 M glyburide (Œ; right). A single asterisk indicates p Ͻ 0.05; a double asterisk indicates p Ͻ 0.005 (compared with 1000 pg/ml cytokine alone). http://www.jimmunol.org/

Lidocaine, glyburide, and dexamethasone synergize to inhibit sinophil lysates, we used a biotinylated RNA probe for the con- by guest on September 26, 2021 eosinophil survival and activation served NBF2 region of SUR1 (27) in a ribonuclease protection Lidocaine and glyburide directly inhibit cytokine-mediated eosin- assay (RPA). The NBF2 region is conserved in both SUR1 and ophil survival and activation, probably through mechanisms dis- SUR2, and is presumably common to the entire family of SUR tinct from those of dexamethasone (16). This follows because glu- proteins (32). Binding of the SUR probe should result in a pro- cocorticoid inhibition of IL-5-stimulated survival is overcome at a tected mRNA fragment of 80 bp. As shown in Figure 8, eosinophil concentration of 1000 pg/ml IL-5, whereas this is not the case with lysates from three separate donors expressed mRNA that hybrid- lidocaine or glyburide. Thus, these drugs may have additive or ized with the SUR probe (lanes 6, 8, and 10), indicating that the even synergistic effects when used in combination. To test this, we eosinophils are actively transcribing a gene that is homologous to incubated eosinophils with IL-5 in the presence of lidocaine, dexa- the NBF2 region of human SUR1. methasone, glyburide, or combinations of these drugs. As shown in To characterize this putative SUR on human eosinophils, we Table I, eosinophils treated with lidocaine, dexamethasone, or gly- performed binding assays using [3H]glyburide as a ligand. As a buride showed decreased survival at 1000 pg/ml IL-5. All combi- positive control we used the HIT T15 hamster cell line, previously nations showed synergistic activity in the survival assay. Most shown to express a functional SUR1 (27, 33). Scatchard analysis strikingly, the combination of lidocaine and glyburide essentially ϭ of equilibrium binding data revealed high affinity (Kd 0.42 nM) abolished the delay of apoptosis induced by IL-5. binding sites on HIT cell membranes (data not shown), in agree- Eosinophil superoxide production is also inhibited by lidocaine ment with published values. However, in three separate experi- (17) and by glyburide (Fig. 6), whereas dexamethasone has no ments, human eosinophil membranes did not show specific effect (data not shown). Treatment of IL-5-stimulated eosinophils [3H]glyburide binding, and in the one experiment showing specific with lidocaine and glyburide results in the synergistic inhibition of binding, Scatchard analyses of equilibrium binding data showed a superoxide production, as shown in Figure 7. Combinations of poor correlation (coefficient Ͻ0.5) between the bound/free ratio lidocaine or glyburide with dexamethasone did not increase inhi- and picomoles of ligand bound (data not shown). Additionally, bition more than either drug alone (data not shown). This latter result was expected, as dexamethasone does not inhibit eosinophil independent photoaffinity labeling experiments performed on pre- superoxide production. Thus, the combination of lidocaine and pared eosinophil membranes failed to show surface labeling with glyburide synergistically inhibits IL-5-induced superoxide glyburide (data not shown; L. Aguilar-Bryan, unpublished obser- production. vations). These data indicate that high affinity SUR1 is not present on human eosinophils. This is consistent with the concentrations of Eosinophils express a SUR glyburide necessary to exert an effect on eosinophils. The inability The effects of the sulfonylureas suggest that a SUR is present on to detect specific binding in this assay suggests that the SUR on eosinophils. To detect message for a SUR family member in eo- eosinophils has a low affinity for glyburide, similar to SUR2. 5550 SULFONYLUREAS INHIBIT EOSINOPHIL SURVIVAL

FIGURE 5. Cromakalim potentiates survival of stressed eosinophils. Puriﬁed human eosinophils were cultured with increasing concentrations of IL-5 in the presence of medium control (micromolar) or varying concen- Ϫ Ϫ trations of cromakalim (f indicates 10 7 M, Œ indicates 10 6 M, ⅷ in- Downloaded from dicates 10Ϫ5 M). All cultures had a ﬁnal DMSO concentration of 1%, which “stressed” the eosinophils and was responsible for the low survival at 1000 pg/ml IL-5. Shown are the mean Ϯ SD of three independent experiments using eosinophils from both normal and allergic donors. The asterisk indicates p Ͻ 0.05 (compared with 1000 pg/ml IL-5 stimulation alone in the presence of 1% DMSO). http://www.jimmunol.org/

glyburide and lidocaine to synergize with dexamethasone in the survival assay suggests that the actions of the drugs are compli- mentary and work through independent mechanisms. One can speculate that glyburide and lidocaine target a channel(s), whereas glucocorticoids work at the nuclear level to alter gene transcription (37).

To damage the airway epithelium and promote bronchial hyper- by guest on September 26, 2021 reactivity, eosinophils must not only be present, but also activated (38, 39). Glyburide and lidocaine are effective at blocking cytokine-induced superoxide production. Interestingly, the drugs show no specificity for IL-5 stimulation (as they do in the survival assay), but inhibit equally well superoxide induced by IL-3 and GM- FIGURE 4. Sulfonylureas preferentially inhibit IL-5-mediated eosinophil survival. Purified eosinophils were cultured for 4 days with various CSF. The combination of lidocaine and glyburide in the superox- concentrations of IL-5, IL-3, or GM-CSF in the presence of medium con- ide assay resulted in synergistic inhibition of eosinophil activation, trol (⅜), 1 ϫ 10Ϫ4 M glyburide (f), 1 ϫ 10Ϫ3 M tolbutamide (Œ), or 1 ϫ suggesting that in this assay lidocaine and glyburide have unique 10Ϫ3 M glipizide (ⅷ). Eosinophil survival was analyzed as described in mechanisms of action. It is interesting that glucocorticoids do not Materials and Methods and is plotted as a percentage of the eosinophil inhibit eosinophil superoxide production, suggesting that gly- survival at maximal cytokine concentrations. Each panel shows the buride (and lidocaine) may be more effective in the prevention of mean Ϯ SD of five independent experiments using eosinophils from both eosinophil-mediated tissue damage. Ͻ normal and allergic donors. The asterisk indicates p 0.05 (compared with Similar functions of glyburide and lidocaine suggest that the 1000 pg/ml cytokine treatment alone). sulfonylureas and lidocaine may be working through similar mechanisms. Our proposed model is that lidocaine exerts its effects by directly blocking a Kϩ channel, comparable to its blocking of Naϩ Discussion channels (18). Similarly, sulfonylureas block ATP-sensitive Kϩ Apoptosis of lung eosinophils is correlated with the resolution of channels by binding to the associated SUR on the cell surface. airway inflammation in asthma (10, 11, 34). Glucocorticoid ther- Thus, both drugs may exert their effects by blocking Kϩ channels, apy presumably is effective in part by directly inhibiting cytokine- but through different mechanisms. This model is consistent with mediated eosinophil survival, resulting in eosinophil apoptosis and our results showing that lidocaine and glyburide have the same clearing by macrophages (16). Here we report that sulfonylureas, effects in biologic assays using eosinophils. However, our results including glyburide, tolbutamide, and glipizide, mimic glucocor- showing synergy of glyburide and lidocaine are more difficult to ticoids by inhibiting cytokine-mediated survival of eosinophils ob- explain in light of this model, since synergistic compounds typi- tained from both normal and allergic patients. However, the sul- cally act through different mechanisms. It is quite possible that fonylureas differ from the glucocorticoids because the effects of the glyburide and lidocaine, in addition to the mechanisms stated sulfonylureas, unlike those of the glucocorticoids, cannot be over- above, exert their effects on eosinophils through alternative means come with high concentrations of IL-5, a key mediator of eosin- as well. This is evidenced by the ability of glyburide, but not ophil survival and activation in vitro and in vivo (12, 35, 36). This lidocaine (17), to block platelet-activating factor-induced superox- preferential inhibition of IL-5-induced signals is also observed ide production. Thus, the similar biologic effects of lidocaine and when eosinophils are treated with lidocaine (14, 17). The ability of glyburide could be explained by their shared targeting of a Kϩ The Journal of Immunology 5551

FIGURE 7. Lidocaine and glyburide synergistically block IL-5-induced superoxide production. Purified eosinophils were treated with IL-5 in the Ϫ Ϫ presence of medium control (⅜), 3 ϫ 10 3 M lidocaine (f), 1 ϫ 10 4 M Downloaded from glyburide (Œ), or a combination of these drugs (ⅷ; solid line). The broken line indicates the predicted additive inhibition of the combination of glyburide and lidocaine. Superoxide production was measured as described in Materials and Methods. Background readings at time zero were subtracted from each time point, and superoxide production was plotted as nanomoles of cytochrome c reduced per million eosinophils vs time. Shown are the mean Ϯ SD of five experiments using eosinophils from both normal and http://www.jimmunol.org/ allergic donors. The single asterisk indicates p Ͻ 0.01; the double asterisk indicates p Ͻ 0.005 (compared with stimulation with IL-5 alone at FIGURE 6. Glyburide inhibits cytokine mediated superoxide produc- 180 min). tion by eosinophils. Purified eosinophils were treated with IL-5, IL-3, or GM-CSF in the presence of medium control (⅜)or1ϫ 10Ϫ4 M glyburide (Œ). Superoxide production was measured as described in Materials and Methods. Background readings at time zero were subtracted from each time of survival, but not inhibition of superoxide generation. Although point, and superoxide production was plotted as nanomoles of superoxide the receptors for GM-CSF, IL-3, and IL-5 share a common ␤-chain Ϯ produced per million eosinophils vs time. Each panel shows the mean that is thought to be responsible for signal transduction, several by guest on September 26, 2021 SD of at least four independent experiments using eosinophils from both reports have indicated a signaling role for the specific ␣-chains of normal and allergic donors. The single asterisk indicates p Ͻ 0.005; the the receptors (40–44). Thus, one could postulate that the signaling double asterisk indicates p Ͻ 0.001 (compared with cytokine stimulation events responsible for prolonging eosinophil survival are influ- alone at 105 min). enced by the ␣-chain, whereas the signals required for superoxide production depend primarily on ␤-chain activation. This hypoth- channel, whereas the ability of the two drugs to synergize could be esis could explain why IL-5-induced survival is more sensitive to attributed to the individual effects of each drug on other channels glyburide (and lidocaine) than that induced by IL-3 or GM-CSF, or signaling receptors. and why superoxide production initiated by all three cytokines is It is intriguing that glyburide shows different specificities in the equally well inhibited. Support for this view comes from a recent eosinophil survival and activation assays. High concentrations of report describing the biologic activity of an IL-5 protein in which ␤ GM-CSF and IL-3, unlike IL-5, can overcome glyburide inhibition an important -chain contact site contained a charge reversal, thus affecting ␤-chain activation and subsequent signal transduction (45). The mutant IL-5 still induced eosinophil survival, but did not Table I. Lidocaine, dexamethasone, and glyburide synergize to inhibit induce activation (as measured by adhesion) and, in fact, func- IL-5-mediated eosinophil survivala tioned as a specific antagonist in that assay. A SUR family has recently been identified, and members of the ϩ Expected Actual family associate with and regulate the inwardly rectifying K Survival Survival channel, Kir6.2 (32). The two identified members, SUR1 and Treatment (% Control)b (% Control) b SUR2, are structurally related but functionally distinct, in that the Media control 100 Ϯ 0 SUR2/Kir6.2 combination is less sensitive to glyburide than is the 10Ϫ3 M lidocaine 79 Ϯ 5 Ϫ4 SUR1/Kir6.2 complex. Based on the concentration of glyburide 10 M glyburide 44 Ϯ 13 Ϫ4 10Ϫ6 M dexamethasone 97 Ϯ 2 necessary to inhibit eosinophil survival in our experiments (10 M), we speculate that glyburide is not acting through the classic Ϫ3 ϩ Ϫ4 Ϯ Ϯ 10 M lidocaine 10 M glyburide 23 12 8 6** SUR1 and may instead function through an analogous family 10Ϫ3 M lidocaine ϩ 10Ϫ6 M dexamethasone 76 Ϯ 635Ϯ 20* 10Ϫ4 M glyburide ϩ 10Ϫ6 M dexamethasone 41 Ϯ 13 21 Ϯ 13** member with a lower affinity for glyburide. This hypothesis is supported by the results from our binding and RPA studies. The a Eosinophil survival was assayed as described in Materials and Methods and is RPA results support the conclusion that eosinophils express a SUR listed as a percentage of the eosinophil survival at 1000 pg/ml IL-5 in the absence of drug, defined as 100%. All treatments are in the presence of 1000 pg/ml IL-5. family member, because mRNA isolated from human eosinophil b Expected survival values represent the predicted additive effect of the drugs lysates hybridizes with a probe derived from the conserved NBF2 based on their individual effects. Actual survival values are the experimental values actually obtained when drug combinations were used. region of SUR1. However, the results from our binding assays *p Ͻ 0.05, n ϭ 10; **p Ͻ 0.01, n ϭ 10. indicate that the affinity of the eosinophil SUR for glyburide is 5552 SULFONYLUREAS INHIBIT EOSINOPHIL SURVIVAL

eosinophilic inflammation (48) and causes a high incidence of side effects (50). Although we were unable to block the action of lidocaine or glyburide with 1 ϫ 10Ϫ5 M cromakalim, pinacidil, or diazoxide pretreatment (data not shown), we were able to potentiate the IL-5-induced survival of eosinophils stressed by high concentrations of DMSO. The latter data suggest that there is a cromakalim-responsive Kϩ channel on human eosinophils. However, because we could not block the action of glyburide with the openers, this is probably distinct from the channel opened by glyburide. These data suggest that glyburide either is binding to a novel SUR that is unresponsive to the openers or, alternatively, is targeting a receptor other than a SUR. Other ion channels, such as the cystic fibrosis transmembrane regulator, are inhibited by the sulfonylureas (51). We are confident that the eosinophil SUR is not SUR1, based on both its lack of responsiveness to cromakalim (under low DMSO conditions) and the high (10Ϫ4 M) concentration of glyburide required to inhibit eosinophil survival. Thus, eosinophils may express SUR2 or a novel SUR family member with distinct Downloaded from characteristics. The observations presented here have intriguing clinical implications for the treatment of asthma and other eosinophil-associated FIGURE 8. Eosinophils express mRNA for a SUR family member. The diseases. The results suggest that ion channels on eosinophils, es- ϩ presence of SUR and actin mRNA in the eosinophil lysates was determined pecially ATP-sensitive K channels, may be targets for future Ϫ by RPA. Samples were prepared as described in Materials and Methods. drug development. Achieving a concentration of 10 4 M glyburide Hybridized biotinylated probe was detected by the BrightStar BioDetect in an asthmatic lung is most likely possible, but may precipitate http://www.jimmunol.org/ Kit. Lanes 1 and 3 show the actin and SUR probes alone, respectively. unwanted side effects. The synergy of the sulfonylureas with glu- Lanes 2 and 4 show that the probes are completely digested in the presence cocorticoids and lidocaine could allow the use of lower concen- of RNase, indicating that there is no residual signal. Lanes 5, 7, and 9 show trations if necessary. Also, the risk of adverse reactions resulting eosinophil lysates from three donors probed with the actin probe to dem- from sulfonylurea treatment would be minimized by local (rather onstrate the integrity of the mRNA. Lanes 6, 8, and 10 are lysates from the than systemic) application. Because lidocaine has been shown to same three donors probed with the SUR1 NBF2 probe. Lane 11 is blank. Biotinylated RNA size markers are shown in lane 12. Expected sizes of the be effective in an open label clinical trial (6), the sulfonylureas protected fragments are 127 bp for actin and 80 bp for SUR and are indi- may similarly represent a potential class of anti-inflammatory

cated by the arrows. Shown is a representative experiment (n ϭ 2). asthma drugs for the treatment of asthma and eosinophil-associ- by guest on September 26, 2021 ated diseases. much less than that of SUR1. This is consistent with the eosinophil Acknowledgments glyburide target being SUR2-like, as the affinity of SUR2 for gly- We thank Dr. Lydia Aguilar-Bryan for her confirmation of binding studies buride hampers detectable surface binding (L. Aguilar-Bryan, un- and helpful advice, Ms. Linda H. Arneson for excellent secretarial assis- published observations). tance, and Ms. Cheryl Adolphson and Mr. James Fulbright for editorial Although we could not detect SUR1 on eosinophil membranes, assistance. the presence of a SUR homologous transcript is worth emphasiz- ing. In the absence of specific Abs to confirm the existence of a References low affinity SUR protein, we rely on the response of the cell to 1. Seminario, M. C., and G. J. Gleich. 1994. The role of eosinophils in the patho- sulfonylureas to determine whether the protein is expressed and genesis of asthma. Curr. Opin. Immunol. 6:860. 2. Thomas, L. H., and J. A. Warner. 1996. The eosinophil and its role in asthma. functional. By definition, a cell responsive to sulfonylureas ex- Gen. Pharmacol. 27:593. presses a sulfonylurea receptor, although the affinity of that recep- 3. Barnes, P. J. 1993. Anti-inflammatory therapy for asthma. Annu. Rev. Med. 44: 229. tor may be low. We believe that this is the case for the putative 4. Greenberger, P. A. 1992. Corticosteroids in asthma: rationale, use, and problems. SUR expressed on eosinophils. The family of SUR proteins may Chest 101:418S. be quite large, as shown by the recent discovery that the ATP- 5. Szefler, S. J., A. K. Kamada, D. Hughes, A. M. Brenner, and E. W. Gelfand. 1992. Alternative treatments for asthma: assessing the need. J. Asthma 29:91. binding cassette family of proteins (of which the SUR are mem- 6. Hunt, L. W., H. A. Swedlund, and G. J. Gleich. 1996. Effect of nebulized lido- bers) comprises 5% of the Escherichia coli genome (46). This caine on severe glucocorticoid-dependent asthma. Mayo Clin. Proc. 71:361. suggests that we have only scratched the surface in our identifi- 7. Stern, M., L. Meagher, J. Savill, and C. Haslett. 1992. Apoptosis in human eosinophils: programmed cell death in the eosinophil leads to phagocytosis by mac- cation of the human homologues. Additionally, even the two iden- rophages and is modulated by IL-5. J. Immunol. 148:3543. tified human SUR proteins have different ligand binding charac- 8. Owen, W. F., Jr., M. E. Rothenberg, D. S. Silberstein, J. C. Gasson, R. L. Stevens, K. F. Austen, and R. J. Soberman. 1987. Regulation of human eosinophil via- teristics (27, 32), indicating that there may be a wide range of SUR bility, density, and function by granulocyte/macrophage colony-stimulating fac- specificities. tor in the presence of 3T3 fibroblasts. J. Exp. Med. 166:129. If glyburide is exerting its effects on eosinophil survival and 9. Rothenberg, M. E., W. F. Owen Jr., D. S. Silberstein, J. Woods, R. J. Soberman, K. F. Austen, and R. L. Stevens. 1988. Human eosinophils have prolonged sur- activation by binding to a conventional SUR, then we should be vival, enhanced functional properties, and become hypodense when exposed to ϩ able to reverse that inhibition with an ATP-sensitive K channel human interleukin-3. J. Clin. Invest. 81:1986. opener, such as cromakalim (31). Interestingly, the ATP-sensitive 10. Tsuyuki, S., C. Bertrad, F. Erard, A. Trifilieff, J. Tsuyuki, M. Wesp, ϩ G. P. Anderson, and A. J. Coyle. 1995. Activation of the Fas receptor on lung K channel opener cromakalim has been tested in clinical trials as eosinophils leads to apoptosis and the resolution of eosinophilic inflammation of an oral asthma drug (47–50). The proposed mechanism of cro- the airways. J. Clin. Invest. 96:2924. makalim is relaxation of airway smooth muscle via the opening of 11. Wooley, K. L., P. G. Gibson, K. Carty, A. J. Wilson, S. H. Twaddell, and ϩ M. J. Woolley. 1996. Eosinophil apoptosis and the resolution of airway inflam- plasmalemmal K channels (47). Cromakalim has no effect on mation in asthma. Am. J. Respir. Crit. Care Med. 154:237. The Journal of Immunology 5553

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