Introduction

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ATP-binding cassette (ABC) transporters are a large family of proteins that mediate the transport of a wide range of substances across biological membranes (Higgins, 1995). ABC proteins are defined by the presence of the ABC unit, which contains two conserved peptide motifs (Walker A and Walker B) that are able to bind ATP (Klein et al., 1999). As membrane transporters, the ABC proteins also contain membrane-embedded transmembrane domains. The minimal structural requirement for an active ABC protein is to have two transmembrane domains and two ABC units (Klein et al., 1999). More than 100 ABC proteins have now been cloned in a variety of species, including bacteria and plants, as well as mammals (Higgins, 1995). The best characterized ABC proteins are the sulfonylurea receptors (SURs) 1 and 2, cystic fibrosis conductance regulator (CFTR), multidrug resistance protein (MDR), and Tangier disease protein ABC1. In addition to their structural similarity, SURs, CFTR, MDR, and ABC1 are also similar in that their activity is selectively inhibited by the sulfonylurea drug glibenclamide.

Recent data indicate that various members of the ABC protein family are present in immune cells. For example, MDR, a plasma-membrane glycoprotein that confers multidrug resistance on tumor cells, is expressed in cells of the immune system, including macrophages and lymphocytes (Hughes et al., 1983). CFTR has been found in both human macrophages and neutrophils (Yoshimura et al., 1993). Another member of the ABC family, the TAP-1/TAP2 peptide transporter, is involved in antigen presentation (Marusina and Monaco, 1996). A novel member of ABC proteins, ABC1, has recently been shown to be expressed by cells of the monocyte/macrophage lineage (Luciani and Chimini, 1996; Langmann et al., 1999). ABC1 is required for engulfment of cells undergoing apoptosis by macrophages and it is involved in the translocation of phospholipids and cholesterol to apo-AI (Luciani and Chimini, 1996; Hamon et al., 2000). Genetic deficiency of ABC1 in humans causes Tangier disease, which is characterized by accumulation of phospholipids in the immune system with enlarged yellow tonsils and hepatosplenomegaly (Bodzioch et al., 1999; Orsó et al., 2000). ABC1 was also recently implicated in interleukin (IL)-1 processing and release (Hamon et al., 1997; Andrei et al., 1999), because the release of IL-1 in response to extracellular ATP is inhibited by glibenclamide and other ABC1 inhibitors (Hamon et al., 1997), such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and sulfobromophthalein (BSP).

IL-12 p40 is part of two heterodimeric cytokines that are secreted mainly by activated antigen-presenting cells and play a key role in determining the nature of the immune response to exogenous or endogenous antigens. IL-12 is a composite of IL-12 p40 and IL-12 p35 (Trinchieri, 1995), whereas IL-12 p40 engages p19 to form IL-23 (Oppmann et al., 2000). Both IL-12 and IL-23 enhance the proliferation, cytotoxicity, and production of interferon (IFN)- by T lymphocytes and natural killer cells (Trinchieri, 1995; Oppmann et al., 2000), which is essential for the clearance of bacterial infections (Trinchieri, 1995; Oppmann et al., 2000). Mice that are genetically deficient in IL-12 p40 are highly susceptible to infection with various intracellular pathogens (Mattner et al., 1996). On the other hand, IL-12 p40 is an important pathogenetic factor in autoimmune disease. This is demonstrated by the fact that although IL-12 p40-deficient mice are resistant to collagen-induced arthritis (McIntyre et al., 1996), transgenic overexpression of IL-12 p40 exacerbates the course of this disease (Parks et al., 1998).

Because, as described above, IL-12 p40 plays a crucial role in orchestrating the immune response, it is important to investigate the cellular mechanisms that regulate the production of this cytokine. In this report, we demonstrate that pharmacological inhibition of ABC proteins suppresses the production of IL-12 p40.

	Materials and Methods

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Mice. Male BALB/c mice (8 weeks) were purchased from Charles River Laboratories, Inc. (Wilmington, MA).

Reagents and Drugs. Lipopolysaccharide (LPS; Escherichia coli serotype 055:B5), DIDS, BSP, agarose, thioglycollate medium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Glibenclamide (N-p-[2-(5-chloro-2-methoxybenzamido)ethyl]benzene-sulfonyl-N'- cyclohexylurea), glipizide, tolbutamide, pinacidil, diazoxide, and minoxidil were obtained from Sigma/RBI (Natick, MA). 2,2'-Iminodibenzoic acid (DPC) was purchased from Aldrich Chemical (Milwaukee, WI). Glibenclamide, pinacidil, diazoxide, glipizide, minoxidil, DIDS, and BSP were dissolved in DMSO, with a 0.5% final DMSO concentration in the medium. RPMI-1640, F-12K medium, fetal bovine serum, and penicillin-streptomycin were obtained from Invitrogen (Carlsbad, CA).

Cell Lines. The J774, RAW 264, and NIT-1 cell lines were obtained from American Type Culture Collection (Manassas, VA). The mouse macrophage cell lines J774 and RAW 264 were grown in RPMI-1640 or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO₂. The mouse insulinoma cell line NIT-1 was cultured in F-12K medium supplemented with 10% heat inactivated fetal bovine serum and 100 U/ml penicillin, and 100 µg/ml streptomycin.

Preparation of Peritoneal Macrophages. Mice were injected intraperitoneally with 2 ml of 2% thioglycollate and peritoneal cells were harvested 3 to 4 days later. The cells were plated on 96-well plastic plates at 1 million cells/ml and incubated in RPMI-1640 for 2 h at 37°C in a humidified 5% CO₂ incubator. Nonadherent cells were removed by rinsing the plates three times with 5% dextrose in PBS.

Treatment of J774 Cells and Peritoneal Macrophages. Cells in 96-well plates were treated with various concentrations of ABC inhibitors 30 min before the addition of 10 µg/ml LPS and 100 U/ml IFN- or 10 µg/ml LPS. Twenty-four hours after stimulation with LPS or LPS/IFN-, supernatants were taken for IL-12 p40, tumor necrosis factor (TNF)-, and nitric oxide determination. For the determination of intracellular IL-12 p40 and TNF-, J774 macrophages in 12-well plates were pretreated with glibenclamide followed by LPS/IFN- stimulation 30 min later. After an additional 24-h incubation, the supernatants were removed and the cells were lysed using 200 µl of modified radioimmunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, 1% Nonidet P-40, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄). IL-12 p40 and TNF- levels in cell supernatants or cell lysates were determined by ELISA as described below.

Cytokine Assays. Cytokine concentrations were determined by ELISA kits that are specific against murine IL-12 p40 or TNF-. Levels of IL-12 p40 and TNF- were measured using ELISA kits purchased from Genzyme (Boston, MA). Plates were read at 450 nm by a Spectramax 250 microplate reader from Molecular Devices (Sunnyvale, CA). The detection limit was 10 pg/ml. Assays were performed according to the manufacturer's instructions.

RNA Isolation and RT-PCR. Total RNA was isolated from mouse heart, spleen, and kidney, as well as from the J774, RAW264, and NIT-1 cells by using TRIzol Reagent (Invitrogen). Reverse transcription of the RNA was performed using 50 U/µl MuLV reverse transcriptase from PerkinElmer (Foster City, CA). RNA (5 µg) was transcribed in a 20-µl reaction containing 10.7 ml of RNA, 2 µl of 10× PCR buffer, 2 µl of 10 mM dNTP mix, 2 µl of 25 mM MgCl₂, 2 µl of 100 mM dithiothreitol, 0.5 µl of RNase inhibitor (20 U/µl; PerkinElmer), 0.5 µl of 50 µM oligo d(T)₁₆ (with the exception of SUR2 and CFTR, where the antisense primer for PCR amplification was used), and 0.3 µl of reverse transcriptase. The reaction mix was incubated at 42°C for 15 min for reverse transcription. Thereafter, the reverse transcriptase was inactivated at 99°C for 5 min. RT-generated DNA (1-5 µl) was amplified using Expand high-fidelity PCR system (Roche Molecular Biochemicals, Indianapolis, IN). The reaction buffer (25 µl) contained 1 to 5 µl cDNA, water, 2.5 µl of PCR buffer, 1.5 µl of 25 mM MgCl₂, 1 µl of 10 mM dNTP mix, 0.5 µl of 10 µM oligonucleotide primer (each), and 0.2 µl of enzyme. cDNA was amplified using the following primers and conditions: SUR1, 5'-ATTAACCTGAGAGGGGCGAT-3' (sense) and 5'-GAGGTGTAGACAGCGAAGGC-3' (antisense), an initial denaturation at 94°C × 5 min, 35 cycles of 94°C × 30 s, 58°C × 45 s, 72°C × 45 s, and a final dwell at 72°C × 7 min; SUR2A/B, 5'-TGCGACATTTGTGACACATG-3' (sense) and 5'-CGTAAGCCACAGAATACCTGC-3' (antisense), an initial denaturation at 94°C × 5 min, 35 cycles of 94°C × 30 s, 58°C × 45 s, 72°C × 45 s, and a final dwell at 72°C × 7 min; Kir6.1, 5'-GCAAACCCGAGTCTTCTAGG-3' (sense) and 5'-GCAGACGTGAATGACCTGAC-3' (antisense), an initial denaturation at 94°C × 5 min, 35 cycles of 94°C × 30 s, 56°C × 30 s, 72°C × 45 s, and a final dwell at 72°C × 7 min; Kir6.2, 5'-CTGGCCATCCTCATTCTC-3' (sense) and 5'-GATGCCCGTGGTTTCTAC-3' (antisense), an initial denaturation at 94°C × 5 min, 38 cycles of 94°C × 30 s, 57°C × 30 s, 72°C × 45 s, and a final dwell at 72°C × 7 min; ABC1, 5'-GGAGTCTAGTCCTCTTTCTC-3' (sense) and 5'-CCATGAATCGAGATATCGTC-3' (antisense), an initial denaturation at 94°C × 5 min, 38 cycles of 94°C × 30 s, 58°C × 45 s, 72°C × 45 s, and a final dwell at 72°C × 7 min; CFTR (Marvao et al., 1998), 5'-CAGTCATCTCTGCCTTGTGGGA-3' (sense) and 5'-CGAACTGAAGCTCGGACGTAGACT-3' (antisense), an initial denaturation at 94°C × 5 min, 35 cycles of 94°C × 30 s, 60°C × 30 s, 72°C × 45 s, and a final dwell at 72°C × 7 min; and -actin, 5'-GAGACCTTCAACACCC-3' (sense) and 5'-GTGGTGGTGAAGCTGTAGCC-3' (antisense), an initial denaturation at 94°C × 5 min, 30 cycles of 94°C × 30 s, 58°C × 45 s, 72°C × 45 s, and a final dwell at 72°C × 7 min. With the exception of Kir6.2, in the absence of the reverse transcription reaction, no bands were detected after the amplification. Because the Kir6.2 primers amplified a product even without reverse transcription, in the case of Kir6.2 RT-PCR, the RNA was treated with a DNA removal kit from Ambion (Austin, TX). The expected PCR products were SUR1, 470 bp; SUR2A/B, 570/600 bp; Kir6.1, 477 bp; Kir6.2, 420 bp; ABC1, 422 bp; CFTR, 600 bp; and -actin, 230 bp. PCR products were resolved on a 1.5% agarose gel and stained with ethidium bromide.

Western Blot Analysis. Peritoneal macrophages in six-well plates were pretreated with glibenclamide or vehicle, and 30 min later the cells were stimulated with 10 µg/ml LPS for 15 min (Haskó et al., 2000a,b). After washing with PBS, the cells were lysed by the addition of radioimmunoprecipitation buffer. The lysates were transferred to Eppendorf tubes, centrifuged at 15,000g, and the supernatant was recovered. Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). A sample (25-40 µg) was separated on 8 to 16% Tris-glycine gel (Novex, San Diego, CA) and transferred to a nitrocellulose membrane. The blot was conducted according to the ECL Western blotting protocol (Amersham Biosciences, Inc., Piscataway, NJ). The membranes were probed with anti-phospho-mitogen-activated protein kinase (MAPK; p42/p44, ERK1/2), anti-phospho-c-Jun N-terminal protein kinase (JNK) (Promega, Madison, WI), anti-phospho-p38 MAP kinase (p38 MAPK; New England Biolabs, Beverly, MA), or an anti-inhibitory factor B (IB; Upstate Biotechnology, Lake Placid, NY) Ab and subsequently incubated with a secondary horseradish peroxidase-conjugated donkey anti-rabbit Ab (Roche Molecular Biochemicals). Bands were detected using ECL Western blotting detection reagent (Amersham Biosciences, Inc.).

Measurement of Nitrite Concentration. Nitrite production, an indicator of nitric oxide synthesis, was measured as previously described (Haskó et al., 1996) by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100-µl samples of medium. The optical density at 550 nm was measured using a Spectramax 250 microplate reader (Molecular Devices). The measurements of nitrite were performed using reagents free of nitrite: no basal or background nitrite levels were detected.

Measurement of Mitochondrial Respiration. Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondria-dependent reduction of MTT to formazan (Haskó et al., 1996). Cells in 96-well plates were incubated with 0.5 mg/ml MTT for 60 min at 37°C. Culture medium was removed by aspiration, and the cells were solubilized in 100 µl of DMSO. The extent of reduction of MTT to formazan within cells was quantitated by measurement of absorbance at 550 nm by using a Spectramax 250 microplate reader.

Detection of Surface I-A^d by Flow Cytometry. Peritoneal macrophages were plated on 12-well plates and treated with glibenclamide (dissolved in 0.5% DMSO) in the presence or absence of IFN- (100 U/ml; R & D Systems, Minneapolis, MN) for 48 h. Cells were removed by scraping into 0.5 ml of Versene (Invitrogen) and washed in PBS. After washing, the cells were resuspended in PBS containing 10% mouse serum and Fc Block (rat anti-mouse CD16/CD32; BD PharMingen, San Diego, CA) and then stained with a fluorescein isothiocyanate-conjugated anti-I-A^d (BD PharMingen). The cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Statistical Evaluation. Values in the figures, tables, and text are expressed as mean ± S.E.M. of n observations. Statistical analysis of the data was performed by Student's t test or one-way analysis of variance followed by Dunnett's test, as appropriate.

	Results

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ABC Protein Inhibitors Suppress IL-12 p40 Production by both J774 Macrophages and Thioglycollate-Elicited Mouse Peritoneal Macrophages. To determine whether ABC proteins are involved in the modulation of IL-12 p40 production, we pretreated LPS/IFN--stimulated J774 macrophages with glibenclamide and measured IL-12 levels from the supernatants taken 24 h after the LPS/IFN- challenge. The results of these experiments showed that glibenclamide inhibited the production of IL-12 p40 in a concentration-dependent manner (Fig. 1A). Glibenclamide did not affect cell viability at the concentrations tested as determined with the MTT assay (Fig. 1C). We next investigated whether the effect of glibenclamide could be reproduced using primary cells (peritoneal macrophages) instead of the J774 macrophage cell line. Figure 1B shows that glibenclamide suppressed the production of IL-12 p40 also in peritoneal macrophages. Cell viability was not affected by glibenclamide in these cells (data not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Glibenclamide suppresses IL-12 p40 production in both J774 cells (A) and peritoneal macrophages (B). The cells were pretreated with various concentrations of glibenclamide 30 min before LPS/IFN- or LPS (in the peritoneal cells) stimulation, and IL-12 p40 concentrations were measured from the supernatants collected 24 h after stimulation. Glibenclamide does not affect cell viability of the J774 cells as determined using the MTT assay (C). Data are expressed as the mean ± S.E.M. of six wells. *, p < 0.05; **, p < 0.01.

View this table: [in this window] [in a new window]   TABLE 1 Effect of DIDS, BSP, and DPC on IL-12 p40 production in J774 macrophages DIDS and BSP suppress and DPC enhances IL-12 p40 production in J774 macrophages stimulated with 10 µg/ml LPS and 100 U/ml IFN-. Data are expressed as the mean ± S.E.M. of six wells.

Molecular Characterization of ABC Proteins in Macrophages. To further investigate which ABC proteins may be involved in the regulation of IL-12 p40 production, we conducted a series of RT-PCRs to determine which ABC protein mRNAs are expressed in macrophages.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   RT-PCR analysis of CFTR mRNA [top; mouse kidney (lane 1), J774 cells (lane 2), peritoneal macrophages (lane 3), and mouse spleen (lane 4)]. The bottom panel demonstrates mRNA expression of SUR1, SUR2 A and B, ABC1, and -actin in the mouse heart (lane 1), NIT-1 cells (lane 2), the J774 macrophage cell line (lane 3), the RAW 264 macrophage cell line (lane 4), and the mouse spleen (lane 5). This figure is representative of three separate experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   RT-PCR analysis of Kir6.1 and Kir6.2 mRNA expression in the mouse heart (lane 1), NIT-1 cells (lane 2), the J774 macrophage cell line (lane 3), the RAW 264 macrophage cell line (lane 4), and the mouse spleen (lane 5). This figure is representative of three separate experiments.

Glibenclamide Prevents Intracellular Accumulation of IL-12 p40 but not TNF-. Next, we asked the question whether glibenclamide acts by decreasing the accumulation of intracellular IL-12 p40 or it affects the release of IL-12 p40. The results of this experiment showed that treatment of the cells with LPS/IFN- induced the appearance of both intracellular and extracellular IL-12 p40, which were both suppressed to the same extent by glibenclamide pretreatment (Fig. 4A). On the other hand, glibenclamide did not influence the release of both intracellular and extracellular TNF- (Fig. 4B).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   A, glibenclamide (glib) inhibits both the extracellular and intracellular accumulation of IL-12 p40. B, glibenclamide does not alter the production of TNF-. C, glibenclamide decreases nitric oxide formation. J774 macrophages were pretreated with 100 µM glibenclamide and 30 min later the cells were exposed to LPS/IFN- for another 24 h. At the end of the incubation period, supernatants were collected and the adherent cells were lysed for the determination of intracellular IL-12 p40 and TNF-. IL-12 p40 and TNF- levels were determined by ELISA. Nitric oxide production was measured from the cell supernatant by using the Griess method. Data are expressed as the mean ± S.E.M. of eight wells. **, p < 0.01.

Glibenclamide Suppresses Nitric Oxide Production by LPS-IFN--Stimulated J774 Macrophages. To examine whether the glibenclamide suppression of macrophage inflammatory mediator production was confined to IL-12 p40, we tested the effect of glibenclamide on nitric oxide production. Figure 4C demonstrates that similar to IL-12 p40, the formation of nitric oxide was attenuated by glibenclamide.

Glibenclamide Fails to Alter LPS-Induced IB Degradation, MAPK, and JNK Activation. P42/44 MAPK, p38 MAPK, and JNK are important intracellular components of cellular responses to LPS and IFN- (Firestein and Manning, 1999). Therefore, we tested whether the inhibitory effect of glibenclamide on IL-12 p40 production was due to an interference with these pathways. Figure 5 shows that the activation of these enzymes by LPS was not influenced by pretreatment with glibenclamide.

View larger version (82K): [in this window] [in a new window]   Fig. 5.   Lack of effect of glibenclamide (glib) on LPS-induced degradation of IB and the activation of p38, p42/44 MAPK, and JNK. Peritoneal macrophages were pretreated with vehicle (cont) or 100 µM glibenclamide for 30 min followed by an LPS challenge for 15 min. The degradation of IB, and MAPK and JNK activation were determined using Western blotting.

Glibenclamide Suppresses IFN--Induced Up-Regulation of Surface I-A^d Molecules in Peritoneal Macrophages. To further examine the effect of glibenclamide on macrophage activation, we measured surface expression of major histocompatibility complex (MHC) II molecules in response to IFN-. I-A^d expression was decreased, in a concentration-dependent manner, by treatment with glibenclamide (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Glibenclamide (glib) suppresses IFN--induced up-regulation of MHCII molecules in peritoneal macrophages. Peritoneal macrophages were plated on 12-well plates and treated with 100 µM glibenclamide in the presence or absence of IFN- for 48 h. MHCII expression was analyzed with a FACSCalibur flow cytometer. This figure is representative of two separate experiments.

	Discussion

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The results of the present study demonstrate that the inhibition of ABC proteins suppresses the production of IL-12 p40 but not TNF- by activated macrophages. The suppression of macrophage activation by the inhibition of ABC proteins is not limited to LPS/IFN--induced processes, because IFN--induced MHCII up-regulation is also attenuated by the blockade of ABC protein function. Furthermore, previous studies have shown that the blockade of ABC proteins also impairs the production of IL-1 in macrophages induced with ATP (Hamon et al., 1997; Andrei et al., 1999). It appears that the mechanism by which ABC protein inhibition suppresses the production of IL-12 p40 is different from how ABC protein inhibition attenuates IL-1 production. The suppression of ATP-induced IL-1 production by the blockade of ABC proteins is due to an inhibitory effect on the translocation of pro-IL-1 from the cytosol to IL-1-containing exocytotic vesicles, and thereby the secretion of IL-1 (Andrei et al., 1999). However, we did not expect such a mechanism to be responsible for the inhibitory effect of glibenclamide on IL-12 p40 production, because the secretion of IL-12 p40 occurs independently of exocytotic vesicles (Trinchieri, 1995). This assumption was confirmed by our data, which demonstrate that glibenclamide does not affect the release of IL-12 p40, but this agent rather decreases the intracellular accumulation of IL-12 p40.

One of the most important findings of this study is that the effect of glibenclamide is independent of SURs. SURs are the regulatory part of ATP-gated potassium (K_ATP) channels. So far, three different types of SURs have been cloned: SUR1, SUR2A, and SUR2B (Tucker and Ashcroft, 1998). The other subunit of the K_ATP channels is a protein belonging to the inwardly rectifying potassium channel superfamily, designated Kir6.X. Kirs constitute the K⁺ channel (pore) in K_ATP channels. Although in most cases, SURs confer ATP and sulfonylurea sensitivity to K_ATP channels, glibenclamide has been shown to bind and inhibit the Kir6 subunit (Tucker and Ashcroft, 1998). Until recently, glibenclamide was thought to be a selective inhibitor of K_ATP channels. K_ATP channels occur in a variety of cell types, where their role is to couple cell metabolism to K⁺ fluxes and electrical activity (Babenko et al., 1998). In pancreatic -cells, where their physiological role is best understood, they are primarily involved in the regulation of insulin secretion. Under resting conditions, pancreatic K_ATP channels are open, whereas elevation of blood glucose concentration raises intracellular ATP levels, which results in the closure of the these channels (Tucker and Ashcroft, 1998). The subsequent membrane depolarization opens voltage-gated Ca²⁺ channels, bringing about an increase in intracellular Ca²⁺ levels and insulin secretion. This mechanism is mimicked by sulfonylurea and other type K_ATP channel blockers, which establishes these drugs as the mainstay of therapy in noninsulin-dependent diabetes mellitus (Edwards and Weston, 1993). K_ATP channels are also involved in the regulation of vascular tone (Quayle et al., 1997), ischemic preconditioning (Yao and Gross, 1994), and central nervous system function (Tucker and Ashcroft, 1998). Because K_ATP channels are found in a variety of cell types and nonselective inhibition of K⁺ channels suppresses the LPS-induced production of TNF- (Maruyama et al., 1994) and LPS/IFN--induced IL-12 p40 production (G. Haskó, unpublished observation), we hypothesized that K_ATP channels may be the target of the suppressive effect of glibenclamide on IL-12 p40 production. However, our data demonstrating the absence of SURs or Kir6 proteins in macrophages rule out a role for these channels in the modulation of cytokine production. Although an inward rectifier current has been described in J774 macrophages, this current is not sensitive to changes in intracellular ATP levels (Randriamampita and Trautmann, 1987), which confirms our observations showing a lack of K_ATP channels in these cells. It is important to note at this point that besides macrophage function, eosinophil activation is also suppressed by glibenclamide (Bankers-Fulbright et al., 1998). The target of glibenclamide's action in eosinophils is not known; however, eosinophils express mRNA, which contains the conserved nucleotide binding domain characteristic of ABC proteins (Bankers-Fulbright et al., 1998).

Recent evidence indicates that glibenclamide also inhibits both MDR and CFTR, as well as ABC1 (Schultz et al., 1996; Ishida-Takahashi et al., 1998). The involvement of MDR in the inhibitory effect of glibenclamide on IL-12 p40 production is unlikely, because verapamil, a selective MDR inhibitor (Hamon et al., 1997), failed to mimic the effect of glibenclamide on IL-12 p40 production. Similarly, verapamil did not affect the release of IL-1 in ATP-stimulated macrophages (Hamon et al., 1997). CFTR does not appear to be involved in the effect of glibenclamide, because we were unable to detect CFTR mRNA in the macrophages and the CFTR blocker DPC did not decrease the production of IL-12 p40. Interestingly, CFTR-deficient epithelial cells failed to express the chemokines regulated on activation, normal T cell expressed and secreted, IL-8, and monocyte chemoattractant protein-1 in response to TNF- stimulation (Schwiebert et al., 1999). Furthermore, dysregulation of cytokine production was also observed in CFTR-deficient lymphocytes, where the production of the anti-inflammatory cytokine IL-10 was enhanced, and the release of the proinflammatory cytokine IFN- was decreased (Moss et al., 2000). These observations suggest that CFTR regulates cytokine production in certain cell types; however, this is not the case in mouse macrophages. On the other hand, it is possible that CFTR may regulate cytokine production in human monocytes/macrophages, because these cells have been shown to express CFTR (Yoshimura et al., 1993). The observations that ABC1 is expressed in macrophages, and that glibenclamide and the other ABC1 blockers DIDS and BSP suppress ABC1 activity in macrophages (Hamon et al., 1997) suggest that ABC1 could play a role in the inhibitory effect of these agents on IL-12 p40 production. However, it is important to emphasize that similar to glibenclamide, neither DIDS nor BSP is a selective inhibitor of ABC1. For example, DIDS is a well known purinoceptor antagonist (Ralevic and Burnstock, 1998), and BSP also inhibits the organic anion transporter organic anion transporting polypeptide-1 (van Montfoort et al., 1999). Clearly, further studies will be required to exactly pinpoint the targets of the anti-inflammatory effect of glibenclamide, DIDS, and BSP.

In summary, this article demonstrates that macrophage activation is inhibited by the blockade of ABC proteins by glibenclamide as well as other ABC protein inhibitors. Because glibenclamide is widely used as an antidiabetic agent, the effects of this drug on the immune system need to be considered.