Dapsone Inhibits IL-8 Secretion From Human Bronchial Epithelial Cells Stimulated With Lipopolysaccharide and Resolves Airway Inflammation in the Ferret

CHEST

Original Research OBSTRUCTIVE LUNG DISEASES

Dapsone Inhibits IL-8 Secretion From Human Bronchial Epithelial Cells Stimulated With Lipopolysaccharide and Resolves Airway Inflammation in the Ferret Soichiro Kanoh, MD, PhD; Tsuyoshi Tanabe, MD, PhD; and Bruce K. Rubin, MD, FCCP

Background: IL-8 is an important activator and chemoattractant for neutrophils that is produced by normal human bronchial epithelial (NHBE) cells through mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) p65 pathways. Dapsone, a synthetic sulfone, is widely used to treat chronic neutrophil dermatoses. We investigated the effects of dapsone on polarized IL-8 secretion from lipopolysaccharide (LPS)-stimulated NHBE cells and further evaluated its ability to decrease LPS-induced inflammation in the ferret airway. Methods: NHBE cells were grown at air-liquid interface (ALI) to ciliated differentiation. Baseline and endotoxin (LPS)-stimulated IL-8 secretion was measured by enzyme-linked immunosorbent assay at air and basal sides with and without dapsone. Western blotting was used to determine signaling pathways. In vivo, ferrets were exposed to intratracheal LPS over a period of 5 days. Once inflammation was established, oral or nebulized dapsone was administered for 5 days. Intraepithelial neutrophil accumulation was analyzed histologically, and mucociliary transport was measured on the excised trachea. Results: Dapsone, 1 mg/mL, did not influence unstimulated (basal) IL-8 secretion. Apical LPS stimulation induced both apical and basolateral IL-8, but basolateral LPS increased only basolateral IL-8. Dapsone inhibited polarized IL-8 secretion from ALI-conditioned cells. Dapsone also decreased LPS-induced IL-8 mRNA level. LPS led to phosphorylation of extracellular signalregulated kinase 1/2, but not p38 MAPK or c-Jun NH2-terminal kinase. LPS also induced NF-kB p65 phosphorylation, an effect that was inhibited by dapsone. Both oral and aerosol dapsone decreased LPS-induced intraepithelial neutrophil accumulation, but only treatment with aerosol dapsone restored mucociliary transport to normal. Conclusions: Dapsone, given either systemically or as an aerosol, may be useful in treating neutrophilic airway inflammation. CHEST 2011; 140(4):980–990 Abbreviations: ALI 5 air-liquid interface; AP 5 apical; BL 5 basolateral; CCK-8 5 cell counting kit-8; CF 5 cystic fibrosis; DEX 5 dexamethasone; DMSO 5 dimethylsulfoxide; DPB 5 diffuse panbronchiolitis; ERK 5 extracellular-regulated protein kinase; ETT 5 endotrachial tube; GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase; HRP 5 horseradish peroxidase; JNK 5 c-Jun NH2-terminal protein kinase; LPS 5 lipopolysaccharide; MAPK 5 mitogen-activated protein kinase; MCT 5 mucociliary transport; MEK 5 mitogen-activated protein kinase/extracellular-regulated protein kinase kinase; NF-kB 5 nuclear factor-kB; NHBE 5 normal human bronchial epithelial; PD98059 5 29-amino-39-methoxyflavone; SAPK 5 stress-activated protein kinase; TLR4 5 toll-like receptor 4

tract is lined with epithelial cells Thethatrespiratory separate the internal milieu of the host from

the outside world. Airway epithelia not only are a mechanical barrier to external stimuli and microbes but also are actively involved in innate and acquired immune responses and airway inflammation.1 In response to bacterial invasion, mucociliary clearance is

stimulated, and inflammatory mediators and cytokines are secreted as a defense, but these also can damage the airway.2 Among epithelial-derived pleiotropic cytokines, IL-8, a member of the cysteine-X-cysteine chemokine family, acts as one of the most potent neutrophil chemoattractants. Neutrophil-dominated inflammation is a characteristic of COPD, diffuse

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panbronchiolitis (DPB), and cystic fibrosis (CF). IL-8 is produced by airway epithelial cells.2 Increased IL-8 in sputum and BAL fluid is associated with the severity of DPB and CF,3,4 and there is increased IL-8 gene expression in the bronchial epithelium of subjects with severe asthma and COPD.5,6 Proinflammatory cytokines, bacterial flagellin, and lipopolysaccharide (LPS) can increase IL-8 production by normal human bronchial epithelial (NHBE) cells.7-9 Among the many agents present in organic dusts, LPS is a major inducer of the inflammatory reaction.10 LPS binds to toll-like receptor 4 (TLR4), which activates intracellular signaling pathways, including the nuclear factor-kB (NK-kB) pathway, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase (MAPK) pathways.11 Three MAPK pathways contribute to IL-8 gene expression: the extracellular-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPK cascades. The relative degree of activation of each pathway and the functional consequences differ among cell types and experimental systems.12 Macrolide antibiotics decrease neutrophils and IL-8 concentration in BAL of subjects with DPB3 and sputum IL-8 concentration in CF.13 Macrolides can inhibit IL-8 release from airway epithelial cells in culture through inactivation of ERK or NK-kB.8,14,15 Dapsone, a synthetic sulfone, has been used to treat leprosy, Pneumocystis jiroveci pneumonia, and malaria. Dapsone also is recognized as an antiinflammatory drug and has been used systemically and topically to treat skin diseases like dermatitis herpetiformis, which is characterized by neutrophildominated inflammation.16 We speculated that dapsone would inhibit IL-8 secretion by stimulated airway cells; therefore, we studied the effect of dapsone on IL-8 secretion from NHBE cells stimulated with LPS and further investigated the signaling pathways involved. We then evaluated the effectiveness of dapsone in decreasing airway neutrophil recruitment and preserving mucociliary clearance when administered orally or as an aerosol to ferrets with airways that had been exposed to (ie, inflamed by) LPS. Manuscript received November 11, 2010; revision accepted March 2, 2011. Affiliations: From the Department of Pediatrics, Virginia Commonwealth University School of Medicine, Richmond, VA. Funding/Support: This research was funded by Virginia Commonwealth University and National Defense Medical College (Japan) internal finds. Correspondence to: Bruce K. Rubin, MD, FCCP, Office of the Chairman, Department of Pediatrics, 1001 E Marshall St, Virginia Commonwealth University School of Medicine, Richmond, VA 23298; e-mail: [email protected] © 2011 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-2908 www.chestpubs.org

Materials and Methods Reagents Dapsone (4,49-diaminodiphenyl sulfone), LPS (Escherichia coli serotype 0111:B4), and all other reagents were purchased from Sigma-Aldrich Corp (St Louis, Missouri), unless otherwise indicated. PD98059 (29-amino-39-methoxyflavone), an MAPK/ERK kinase (MEK) inhibitor (an upstream kinase of ERK1/2) was obtained from Calbiochem (La Jolla, California). Phospho- and nonphospho-specific ERK1/2, anti-p38 MAPK, anti-stressactivated protein kinase (SAPK)/JNK, and phospho-specific NF-kB p65 (Ser536) as well as anti-rabbit-IgG horseradish peroxidase (HRP) antibodies were purchased from Cell Signaling Technology, Inc (Beverly, Massachusetts). Dimethylsulfoxide (DMSO) was used as a solvent of dapsone, and the final concentration did not exceed 0.01% (v/v). Preliminary in vitro experiments showed that 0.01% DMSO medium had no significant effect on cell viability and IL-8 secretion for up to 72 h (data not shown). NHBE Cell Culture NHBE cells (Lonza Walkersville, Inc; Walkersville, Maryland) were plated at 3,500 cells/cm2 in culture dishes in bronchial epithelial cell growth medium supplemented with the singlequot kit (Lonza Walkersville, Inc) without antibiotics and cultured at 37°C in a 5% CO2 incubator. We used endotoxin-free media (, 0.005 endotoxin units/mL) and second-passage cells for all experiments. Cells were grown to confluence for 6 days. Cultures without antibiotics were then transferred to six-well or 35-mm dishes coated with type 1 rat-tail collagen and seeded at 3,500 cells/cm2. The medium was changed every 24 h. To avoid influence of growth factors on cell signaling and IL-8 secretion, cells were cultured in supplement-free bronchial epithelial cell basal medium for 24 h before stimulation. We evaluated cell response at the time of cell confluence rather than normalized to the relative number of cells because cell maturation could affect cell signaling and cytokine secretion, and at confluence, all cells are at similar growth stages. For NHBE cell differentiation, cells were plated at 2.0 3 105 cells/cm2 onto polycarbonate inserts of 6.5-mm diameter, 0.4-mm pore size, and 10-mm thickness (Transwell Clear; Corning Costar; Cambridge, Massachusetts); coated with type 1 rat-tail collagen; and cultured with serum-free Dulbecco modified eagle medium:nutrient mixture F-12 medium containing 1.0% ITS-A (Invitrogen Corp; Carlsbad, California), 0.5 ng/mL recombinant human epidermal growth factor (Invitrogen Corp), 10 ng/mL triiodothyronine (MP Biomedicals LLC; Solon, Ohio), 0.5 mg/mL hydrocortisone (MP Biomedicals LLC), 1.0 3 1027 M all-trans retinoic acid (Sigma-Aldrich Corp), 2.0 mg/mL bovine serum albumin (Sigma-Aldrich Corp), and 30 mg/mL bovine pituitary extract (Invitrogen Corp). After achieving confluence, the apical medium was removed, and cells were cultured with an airliquid interface (ALI) method. The culture medium was changed every 48 h, and cells were maintained at 37°C in 5% CO2 for 10 to 14 days.17 Cytotoxicity Assay To determine the number of viable cells, formazan dye generation was measured using cell counting kit-8 (CCK-8) (Dojindo, KK; Kumamoto, Japan). Cells treated with CCK-8 assay solution were incubated for 2 h, and the absorbance at 450 nm was measured with a microplate reader. Data were expressed as percent of control cells that were not exposed to dapsone. CHEST / 140 / 4 / OCTOBER, 2011

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Measurement of IL-8 Secretion

Animal Study

Culture supernatants were collected and centrifuged for 5 min at 200g and stored at 220°C until assayed. IL-8 was measured by enzyme-linked immunosorbent assay (Beckman Coulter, Inc; Brea, California) according to the manufacturer’s instructions. Concentrations in each sample were obtained by interpolation from standard curves and calculated as the mean of the results at the sample dilution.

Twenty adult male ferrets (weight, 1.3-2.0 kg) were obtained from Marshall Farms (Rose, New York). Ferrets were anesthetized with 40 mg/kg ketamine and 5 mg/kg xylazine and exposed to endotoxin for 30 min once daily for 5 days by intubating with a 3.0 uncuffed endotracheal tube (ETT) coated with 10 mg of LPS mixed in 300 mL water-soluble K-Y Brand Jelly (Johnson & Johnson; New Brunswick, New Jersey).19 As control subjects, K-Y Brand Jelly without LPS was used in two ferrets. Dapsone was prepared in a vehicle of 5% (v/v) ethanol/5% (v/v) DMSO/0.5% (w/v) methylcellulose solution for oral administration at a final concentration of 3 mg/mL. To administer in nebulized form, dapsone was dissolved in 0.67% (v/v) DMSO/saline at a concentration of 0.5 mg/mL. Eighteen LPS-treated ferrets were randomized to receive 5-day dapsone treatment starting on day 4 (after 3 days of LPS) and continued for 5 days. For oral administration, 2 mg/kg dapsone (n 5 4) or vehicle alone (n 5 4) was given daily through a nasogastric tube. This dose is equivalent to the oral dose used to treat persons with skin disease. For aerosol inhalation, ferrets were shallowly intubated with an ETT, which was placed 1 cm below the larynx. Ferrets were treated with nebulized 0.5 mg/mL dapsone (n 5 5) or 0.67% DMSO/saline (vehicle) (n 5 5) for 15 min once daily using a jet nebulizer (PariMaster; Pari Respiratory Equipment, Inc; Midlothian, Virginia). Ferrets (body weight, 1.5 kg) have 0.3- to 0.4-mL total lung lining fluid volume. Therefore, the dose delivered to the lung, is estimated to be 0.1 to 1.0 mg in this system, given reported nebulization efficiency. On day 9, dapsone-treated and vehicle-control animals were killed and the tracheas removed. Each tracheal segment was fixed in 10% formalin, embedded in paraffin, and processed for histologic analysis using a light microscope (CKX41; Olympus, Corporation; Tokyo, Japan) and by photography using a digital camera system (AxioCam ICc1; Carl Zeiss, Inc; Thornwood, New York). The tissue was cut in 4-mm thicknesses, and the slides were stained with hematoxylin and eosin. To measure the accumulation of inflammatory cells and overall severity of inflammation, the total number of intraepithelial neutrophils was counted over 150 mm in eight random sites per specimen from four different sections and averaged. We also used an excised tracheal segment to measure tracheal mucociliary transport (MCT) velocity. A tracheal segment

Immunoblotting After stimulation, the plated cells were washed with cold phosphate-buffered solution and then lysed on ice in a modified radio immunoprecipitation buffer (1% Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris pH 7.5, 5 mM sodium pyrophosphate, 1 mM NaVO4, 5 mM NaF, 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride) for 15 min and then scraped from the dishes. DNA was sheared by passing the lysate though a 27-gauge needle, and insoluble material was removed by centrifugation at 20,000g for 15 min at 4°C. The protein concentration of the resulting supernatant was quantified by the detergent compatible protein assay (Bio-Rad Laboratories, Inc; Hercules, California). Equal amounts of protein extracts were loaded on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis mini gel and transferred to a nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked with blocking buffer (150 mM NaCl, 20 mM Tris, and 0.1% Tween 20; pH 7.6) containing 5% nonfat dry milk at 4°C overnight. Subsequently, membranes were rinsed and incubated with the primary antibody phospho (p)-p44/42 MAPK (Thr202/Tyr204) (diluted 1:2,000), p-p38 MAPK (Thr180/Tyr182) (diluted 1:1,000), p-SAPK/JNK (Thr183/Tyr185) (diluted 1:1,000), or p-NF-kB p65 rabbit polyclonal IgG (diluted 1:1,000) (Cell Signaling Technology) for 2 h at room temperature. The membranes were then incubated at room temperature for 1 h with the anti-rabbit IgG HRP secondary antibody (diluted 1:2,000). Subsequently, the blot membranes were developed with LumiGLO chemiluminescent substrate peroxide (Cell Signaling Technology). Membranes were stripped with a stripping buffer (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mM Tris/HCl pH 6.7) for 30 min at 30°C. The blots were reprobed with anti-p44/42 MAPK, anti-p38 MAPK, or anti-SAPK/JNK antibody (diluted 1:1,000 for each) followed by anti-rabbit-IgG HRP secondary antibody (diluted 1:2,000). To correct small differences in loading for NF-kB p65, blots were stripped and reprobed with anti-human b-actin antibody (diluted 1:5,000) followed by anti-mouse IgG HRP secondary antibody (diluted 1:5,000). Western blot images were scanned and analyzed on National Institutes of Health Image J software (National Institutes of Health; Bethesda, Maryland).18 Real-Time Quantitative Polymerase Chain Reaction For the relative quantification of IL-8 mRNA expression, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. EvaGreen (Biotium, Inc; Hayward, California) was used as a DNA intercalator dye to monitor amplified DNA quantification, and real-time quantitative polymerase chain reaction curves were analyzed by CFX Manager software (Bio-Rad Laboratories) in order to obtain threshold cycle values for each sample. Quantification was based on a standard curve. The following primers were used: IL-8 forward, 59-CTGCGCCAACACAGAAATTA-39; IL-8 reverse, 59-ACTTCTCCACAACCCTCTGC-39; GAPDH forward, 59-TGAACGGGAAGCTCACTGG-39; GAPDH reverse, 59-TCCACCACCCTGTTGCTGTA-39.

Figure 1. Effect of dapsone on normal human bronchial epithelial (NHBE) cell viability. NHBE cells were grown in the presence of 1 mg/mL dapsone for 24 or 72 h, and cell viability was evaluated using cell counting kit-8 (CCK-8). Dapsone did not decrease cell viability. Data are expressed as percentage of control and presented as mean ⫾ SE; n 5 3. Cont 5 cells not exposed to dapsone.

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was placed on a piece of gauze saturated with Ringer solution in a chamber in which the relative humidity was maintained at 95% to 100% and the temperature was maintained at 22°C to 24°C. MCT was measured by focusing a tracheal segment under a microscope with a gradicule (grid line) eyepiece and recording the transport time of the leading edge of very fine shavings of plastic that were placed on the tracheal epithelium. The time to transport the particle 3 mm was used to calculate the MCT (mm/min). Measurements were repeated five times for each tracheal segment. This study was approved by the Animal Care and Use Committee of Virginia Commonwealth University. Statistics Results are expressed as mean ⫾ SE or mean ⫾ SD, as appropriate. Statistical analysis were performed with StatView, version 5 (SAS Institute Inc; Cary, North Carolina) statistical software. Comparisons between two groups were made by unpaired Student t test. Multiple comparisons were made by one-way analysis of variance using Fisher protected least significant difference test. A P , .05 was considered significant.

Results Effect of Dapsone on NHBE Cell Viability To confirm that the total number of cultured NHBE cells was not influenced by dapsone treatment, the viability of cells was evaluated using CCK-8. NHBE cells seeded onto 96-well plates (3,000 cells/well) were cultured at 37°C for 72 h (ⵑ 70% confluence). Dapsone at concentrations of 0.3, 1, or 10 mg/mL was added for 24 or 72 h. As shown in Figure 1, the total viable cell number was similar to that of the nontreated control group over 72 h. Effect of Dapsone on LPS-Induced IL-8 Secretion

Figure 2. Effect of dapsone on IL-8 secretion from NHBE cells in culture. Growth factors were withdrawn from the culture medium 24 h before LPS or dapsone exposure, and supernatants were harvested 24 h after LPS stimulation. A, LPS 10 mg/mL significantly increased IL-8, and dapsone 0.3, 1, or 10 mg/mL suppressed this effect. B, Dapsone 1 mg/mL did not influence basal IL-8 secretion at 24, 48, and 72 h. C, Dapsone 1 mg/mL inhibited www.chestpubs.org

To evaluate the effect of dapsone on IL-8 secretion from NHBE cells, cell supernatants were harvested 24 h after stimulation with LPS in the presence and absence of dapsone, which had been added at the same time as the LPS. We chose to assess cell response at the time of cell confluence rather than normalize to the relative number of cells because cell maturation could potentially affect cell signaling and cytokine secretion, and at confluence, all cells are at similar growth stages. LPS at 10 mg/mL significantly increased IL-8 secretion from NHBE cells (P , .001), but dapsone at 0.3, 1, or 10 mg/mL suppressed this (P , .01 for each) (Fig 2A). Dapsone at 1 mg/mL did not affect basal IL-8 secretion for 48 and 72 h (Fig 2B). In the presence of dapsone 1 mg/mL, LPS-induced IL-8 secretion was significantly and persistently decreased for up to 72 h (Fig 2C). LPS-induced IL-8 secretion to the control level at 24 and 72 h. Data are presented as mean ⫾ SE; n 5 6. *P , .05. ***P , .001 compared with Cont. #P , .05. ##P , .01 compared with LPS alone. LPS 5 lipopolysaccharide. See Figure 1 legend for expansion of other abbreviations. CHEST / 140 / 4 / OCTOBER, 2011

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Effect of Dapsone in ALI-Conditioned NHBE Cells To confirm whether the IL-8-inhibiting effect of dapsone is seen in differentiated cells, we used NHBE cells cultured under ALI conditions for 14 days. For cells at ALI, samples were collected from both apical (AP) and basolateral (BL) chambers of cells grown on filters. To collect samples from the AP chamber, 150 mL of Hanks balanced salt solution (Lonza Walkersville) was added to the AP side. Apical IL-8 concentrations were expressed as values of fourfold dilution to be equal to the BL medium volume of 600 mL. NHBE cells were stimulated with LPS from the AP side (AP-LPS) or BL side (BL-LPS) for 24 h, and IL-8 secretion levels in both chambers were measured. Dapsone was added only to the BL medium. As shown in Figure 3A, AP IL-8 level was significantly increased by AP-LPS (P , .001), and dapsone inhibited this response (P , .05). Likewise, BL IL-8

was significantly increased by AP-LPS or BL-LPS (P , .001 for each) (Fig 3B), and dapsone inhibited both the AP and the BL response (P , .001 for AP-LPS, P , .01 for BL-LPS). To compare with corticosteroids, we examined the effect of dexamethasone (DEX) on LPS-induced IL-8 secretion. DEX suppressed basal IL-8 level in both AP-LPS and BL-LPS (P , .05 for each) (Fig 3C-3D). DEX also significantly inhibited AP-LPS- and BL-LPS-induced increases in IL-8 (P , .05 for each). Effect of Dapsone on LPS-Induced IL-8 mRNA Expression To examine the action of dapsone on IL-8 mRNA, RNA was extracted from the cells after 4-h stimulation of LPS, dapsone, DEX, or their combination and prepared for real-time quantitative polymerase chain reaction. As shown in Figure 4, 1 mg/mL dapsone did not influence the basal IL-8 mRNA level,

Figure 3. Effect of dapsone and DEX on LPS-induced AP (A, C) and BL (B, D) IL-8 secretion from NHBE cells cultured under an air-liquid interface condition. NHBE cells were incubated in medium with and without dapsone 1 mg/mL (A, B) or DEX 0.1 mg/mL (C, D) and stimulated with LPS 10 mg/mL for 24 h from the AP or BL side. A, AP-LPS significantly increased AP IL-8 secretion, an effect that was inhibited by dapsone. B, AP-LPS and BL-LPS significantly increased BL IL-8 secretion, an effect that was inhibited by dapsone. Data are presented as mean ⫾ SE; n 5 6 for dapsone. ***P , .001 compared with control. #P , .05. ###P , .001 compared with LPS alone. C, AP-LPS significantly increased apical IL-8 secretion. DEX inhibited LPS-induced IL-8 secretion as well as basal IL-8 level. D, AP-LPS and BL-LPS significantly increased BL IL-8 secretion, an effect that was inhibited by DEX. DEX also inhibited basal IL-8 level. Data are presented as mean ⫾ SE; n 5 4 for DEX. *P , .05. **P , .001 compared with control. #P , .05 compared with LPS alone. AP 5 apical; BL 5 basolateral; DEX 5 dexamethasone. See Figure 1 and 2 legends for expansion of other abbreviations. 984

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Effect of Dapsone on LPS-Induced Phosphorylation of NF-kB p65 Because LPS simulates TLR4 and then induces IL-8 through the NF-kB pathway,11 we examined the effect of dapsone on NF-kB p65 phosphorylation in NHBE cells. LPS 10 mg/mL significantly increased NF-kB p65 phosphorylation at 15 min (P , .01), and this effect persisted for up to 2 h (P , .05) (Fig 7A). Dapsone at 1 mg/mL significantly inhibited LPSinduced NF-kB p65 phosphorylation to the control level (P , .01 for 15 and 30 min, P , .05 for 1 and 2 h). Further, this inhibitory effect was dose dependent (Fig 7B) (P 5 .16 for dapsone 0.3 mg/mL and P , .01 for dapsone 1 and 10 mg/mL). Figure 4. Effect of dapsone on LPS-induced IL-8 mRNA expression. Growth factors were withdrawn from the culture medium 24 h before LPS, dapsone, or DEX exposure. NHBE cells were stimulated with LPS 10 mg/mL, dapsone 0.3 to 10 mg/mL, DEX 0.1 mg/mL, or their combination for 4 h, and IL-8 mRNA expression was evaluated using real-time quantitative polymerase chain reaction. Dapsone 1 mg/mL did not influence basal IL-8 mRNA level, but DEX reduced this. LPS 10 mg/mL increased IL-8 mRNA expression more than fivefold, an effect that was inhibited by dapsone 1 and 10 mg/mL and by DEX 0.1 mg/mL. Data are expressed as fold change compared with control subjects and presented as mean ⫾ SE; n 5 4. *P , .05. ***P , .001 compared with control subjects. #P , .05. ##P , .01 compared with LPS alone. GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase. See Figure 1 to 3 legends for expansion of other abbreviations.

but 0.1 mg/mL DEX decreased this by ⵑ 40% (P , .05). LPS 10 mg/mL increased IL-8 mRNA level more than fivefold of control (P , .001). Dapsone at 1 mg/mL and 10 mg/mL significantly inhibited LPS-induced IL-8 mRNA overexpression (P , .05 for each). DEX at 0.1 mg/mL also inhibited this (P , .01). Effect of Dapsone on LPS-Induced Phosphorylation of MAPKs MAPK signaling are important pathways in the synthesis of IL-8.7,20 We evaluated the effect of dapsone on LPS-induced phosphorylation of ERK1/2, p38, and JNK in NHBE cells. LPS at 10 mg/mL significantly phosphorylated ERK1/2 at 1, 4, and 24 h (P , .05 for each) but not p38 and JNK (Fig 5). Dapsone 1 mg/mL inhibited LPS-induced ERK1/2 phosphorylation at 1 h (P , .05), although this effect disappeared after 4 h. We then assessed the effect of PD98059, a selective cell-permeable MEK inhibitor. As shown in Figure 6, LPS dose dependently increased ERK1/2 phosphorylation and IL-8 secretion, and 20 mM 29-amino-39-methoxyflavone (PD98059) abolished 10 mg/mL LPS-induced ERK1/2 phosphorylation. However, this concentration of PD98059 did not inhibit IL-8 secretion. www.chestpubs.org

Figure 5. Temporal effect of dapsone on LPS-induced mitogenactivated protein kinase activation over 24 h. Growth factors were withdrawn from the culture medium 24 h before LPS or dapsone exposure. Threonine and tyrosine phosphorylation of ERK1/2, p38, and JNK was measured by Western blotting. The band intensity was calculated with National Institutes of Health Image J software. LPS 10 mg/mL increased the ratio of p-ERK1/2/ERK1/2, but not p-p38/p38. P-JNK was not detected. Dapsone 1 mg/mL inhibited LPS-induced ERK1/2 phosphorylation at 1 h, but not at 4 and 24 h. Data are presented as mean ⫾ SE from more than three independent experiments. *P , .05 compared with control subjects (LPS 2 and dapsone 2 at each time). #P , .05 compared with LPS alone. ERK 5 extracellular-regulated protein kinase; JNK 5 c-Jun NH2-terminal protein kinase; p 5 phospho; SAPK 5 stress-activated protein kinase. See Figure 2 legend for expansion of other abbreviation. CHEST / 140 / 4 / OCTOBER, 2011

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ferret with an LPS-inflamed airway and treated with nebulized vehicle did not complete the study because of death on day 6, and this animal was excluded from further analysis. As shown in Figure 9A, LPS exposure induced marked neutrophil accumulation in the ferret tracheal epithelium. Dapsone treatment reduced intraepithelial neutrophil number (Fig 9B). Orally administered dapsone tended to inhibit neutrophil recruitment (P 5 .3) (Fig 9C), and nebulized dapsone significantly inhibited neutrophil accumulation (P , .05) (Fig 9D). MCT on Excised Tracheal Segments MCT was timed over a 3-mm segment. LPS dramatically decreased MCT to 1 to 3 mm/min (normal, ⵑ 7 mm/min). Oral dapsone increased MCT, but the increase was not significant (P 5 .09). However, aerosol dapsone preserved MCT at near-normal velocity (6 mm/min, P 5 .007) compared with LPS control as shown in Figure 10. Discussion

Figure 6. Effect of PD98059 (a mitogen-activated protein kinase/ERK inhibitor) on LPS-induced ERK1/2 phosphorylation (A) and IL-8 secretion (B). Growth factors were withdrawn from the culture medium 24 h before LPS exposure. PD98059 20 mM was added 1 h before LPS stimulation. A, LPS increased ERK1/2 phosphorylation in a dose-dependent manner at 4 h, an effect that was abolished by PD98059. Data are presented as mean ⫾ SE from four independent experiments. *P , .05. **P , .01 compared with Cont. #P , .05 compared with LPS alone. B, PD98059 did not inhibit LPS-induced IL-8 secretion. Data are presented as mean ⫾ SE; n 5 6. ***P , .001 compared with Cont. PD98059 5 29-amino-39-methoxyflavone. See Figure 1, 2, and 5 legends for expansion of other abbreviations.

Effect of Tracheal LPS With and Without Dapsone on Ferret Activity and Weight There was no measureable effect of LPS with or without dapsone treatment on ferret activity or appetite and no difference in weight over 9 days when comparing these two groups (Fig 8). Intraepithelial Neutrophil Accumulation in LPS-Inflamed Ferret Trachea To evaluate the in vivo effect of dapsone, we used topical LPS coated onto an ETT to recruit neutrophils and inflame the trachea of anesthetized and spontaneously breathing ferrets.19 Control ferrets intubated with an ETT coated with only a water-soluble jelly (used as the LPS vehicle in the other group) showed few epithelial neutrophils (, 3/150 mm). One

We have shown that dapsone inhibits IL-8 secretion from NHBE cells stimulated with LPS. Dapsone is used to treat dermatologic disorders, most notably those with neutrophil infiltrates.16 It has been postulated that dapsone impairs neutrophil chemotaxis and function at the sites of inflammation, apparently without increased risk of opportunistic infections.21 This is consistent with immunomodulation, but not immunosuppression.14 Dapsone inhibits local production of toxic reactive oxygen species, myeloperoxidase, and elastase,22-24 but this seems unlikely as a principal mode of action because the clinical response to dapsone is characterized by decreasing the neutrophil numbers.25 Other investigators have shown that dapsone may impair neutrophil chemotaxis by interfering with activation of adhesion molecule CD11b/CD18 in vitro.26,27 However, this seems to require a higher concentration of dapsone than therapeutic levels measured in vivo. The concentration of dapsone we used in the present study is within the ranges required for therapeutic serum levels of 0.5 to 5 mg/mL.16 We found that the lower concentration of dapsone of 0.3 mg/mL inhibited LPS-induced IL-8 secretion, and this effect was not due to cell toxicity as measured by the number of viable cells. Schmidt et al28 reported that dapsone, in a therapeutic concentration, inhibited the bullous pemphigoid IgG-mediated IL-8 release from cultured normal human epidermal keratinocytes and that dapsone did not depress basal IL-8 level. These data are similar to the present results using NHBE cells. It also has been

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Figure 7. Effect of dapsone on LPS-induced NF-kB p65 phosphorylation. Growth factors were withdrawn from the culture medium 24 h before LPS or dapsone exposure. The threonine phosphorylation of NF-kB p65 was measured by Western blotting. The band intensity was calculated with National Institutes of Health Image J software. A, LPS 10 mg/mL significantly phosphorylated NF-kB p65 up to 2 h, and this was inhibited by dapsone 1 mg/mL. B, Dapsone inhibited LPS-induced NF-kB p65 phosphorylation, and this was dose dependent. Data are presented as mean ⫾ SE from more than three independent experiments. *P , .05. **P , .01 compared with control subjects (LPS 2, dapsone 2, at each time). #P , .05. ##P , .01 compared with LPS alone. NF-kB 5 nuclear factor-kB. See Figure 2 legend for expansion of other abbreviation.

reported that dapsone decreases production of tumor necrosis factor a and IL-8 in peripheral blood mononuclear cells following LPS stimulation.29 Airway epithelia are functionally polarized, and there is evidence that epithelial cells can secrete cytokines in a bidirectional manner.30,31 To better understand the mode of action of dapsone in a differentiated and polarized airway epithelial model, NHBE cells were cultured at an ALI.32 In the presence of dapsone, IL-8 secretion induced by LPS stimulation was significantly reduced, whereas constitutive www.chestpubs.org

IL-8 release was not inhibited by dapsone. Interestingly, when stimulated apically (as would occur during an airway infection), NHBE cells secreted IL-8 toward AP as well as toward BL sides, whereas when stimulated basolaterally, they secreted only toward the BL side. Airway epithelial cells grown on filters and stimulated by Staphylococcus aureus at the AP side caused T-cell chemotaxis toward the AP- but not the basal-side supernatant.33 The presence of an apically oriented chemoattractant gradient may be necessary to drive immune cells like neutrophils CHEST / 140 / 4 / OCTOBER, 2011

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We then examined the effect of dapsone on NF-kB activation. NF-kB can induce gene expression of inflammatory mediators and cytokines in airway epithelial cells,7,19 and LPS can activate NF-kB through TLR4.11 The basic NF-kB complex is a dimer of two members of the Rel family of proteins: p50 and p65 (RelA). Both subunits contact DNA, but only p65 contains a transactivation domain within the C-terminal region that directly interacts with the transcription apparatus. NF-kB p65 is activated by phosphorylation, which enhances its transcriptional activity, and is associated with nuclear translocation.36 The present study shows that LPS 10 mg/mL elicited significant NF-kB p65 phosphorylation from 15 min to 2 h Figure 8. Treatment schedule and effect of tracheal LPS with and without dapsone on ferret weight. Ferrets stimulated with LPS for 5 days received 5-day dapsone treatment () or vehicle alone (). There was no difference in weight over 9 days when comparing these two groups. Data are presented as mean ⫾ SD; n 5 8 for vehicle and n 5 9 for dapsone group at each time point. See Figure 2 legend for expansion of abbreviation.

and lymphocytes across epithelial surfaces.34 Additionally, we tested the effect of DEX, a potent synthetic corticosteroid, in our cultured cell system. DEX is reported to inhibit IL-8 release from NHBE cells,8 and we confirmed that DEX inhibited IL-8 mRNA expression more than dapsone. However, unlike dapsone, DEX suppressed basal IL-8 level as well as an LPS-induced increase, suggesting that DEX is immunosuppressive, whereas dapsone is immunomodulatory. The intracellular signaling pathways by which dapsone inhibits IL-8 have not been well characterized. Because MAPK regulates IL-8 gene expression in airway epithelial cells,7,8,15 we evaluated the effect of dapsone on these pathways. We found that LPS dose dependently increased ERK1/2 phosphorylation but not JNK and p38 in NHBE cells, and elevated p-ERK1/2 level continued for at least 24 h. We have previously shown that sustained activation of ERK1/2 is required for basal IL-8 secretion from unstimulated NHBE cells and that a specific MEK inhibitor, PD98059, suppressed IL-8, whereas a pharmacologic inhibitor of JNK or p38 did not.8 Baines et al35 showed that pretreatment with PD98059 inhibits LPS-induced phosphorylation of ERK1/2 at 1 and 2 h. In the current study, the inhibitory effect of PD98059 was confirmed even at 4 h. However, PD98059 did not decrease LPS-induced IL-8 secretion. Dapsone 1 mg/mL inhibited p-ERK1/2 at 1 h but not after 4 h. Taken together, it is unlikely that ERK1/2 inhibition alone will suppress IL-8 in LPSstimulated cells and, therefore, that temporal ERK1/2 inhibition by dapsone does not account for the effect on IL-8.

Figure 9. Effect of dapsone treatment on LPS-induced neutrophil accumulation in ferret airways. Ferrets were intubated with an LPS (10 mg)-coated endotracheal tube for 30 min once daily for 5 days, and dapsone was administered po or in nebulized form from day 4 to day 8. Tracheas were removed on day 9, and histologic analyses were performed. The total number of intraepithelial neutrophil was counted over 150 mm in eight random sites per specimen from four different sections and averaged. A and B, Light microscopy of LPS-exposed ferret tracheal epithelium treated with (B) or without (A) dapsone. Infiltrated neutrophils (arrowheads) into the epithelium were observed (hematoxylin-eosin, bar 5 50 mm). C, Oral dapsone decreased intraepithelial neutrophil number, but not significantly. Data are presented as mean ⫾ SD; n 5 4. D, Nebulized dapsone significantly inhibited neutrophil accumulation. Data are presented as mean ⫾ SD; n 5 4 for vehicle and n 5 5 for dapsone. *P , .05 compared with vehicle (LPS 1, dapsone 2). See Figure 2 legend for expansion of abbreviation.

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Figure 10. Effect of dapsone treatment on LPS-induced inhibition of MCT timed over a 3-mm segment. A, Oral dapsone increased MCT, but not significantly (P 5 .09). Data are presented as mean ⫾ SD; n 5 4. B, Nebulized dapsone significantly increased MCT (P 5 .007) to normal levels. Data are presented as mean ⫾ SD; n 5 5. **P , .01 compared with vehicle (LPS 1, dapsone 2). MCT 5 mucociliary transport. See Figure 2 legend for expansion of other abbreviation.

after stimulation in NHBE cells. Similar kinetics in response to LPS has been shown in murine intestinal myofibroblasts in which phosphorylation of NF-kB p65 was detected within 30 min of LPS treatment and slowly decreased over 4 h.12 In the present study, we found that dapsone 1 mg/mL inhibited LPS-induced NF-kB p65 phosphorylation over 2 h to basal levels, suggesting that dapsone may inhibit induction of IL-8 secretion by blocking NF-kB p65 phosphorylation. Additionally, the dose-dependent inhibitory effect on phospho-NF-kB p65 was consistent with results of an experiment in IL-8 mRNA expression (Figs 4, 7B). Accordingly, the action of dapsone is due, at least in part, to downregulating IL-8 at gene transcription. However, although dapsone 0.3 mg/mL did not significantly inhibit NF-kB p65 activation, the same concentration of dapsone strongly inhibited IL-8 release. More than 1 mg/mL of dapsone was needed to inhibit LPS-induced IL-8 mRNA expression. Schmidt et al28 speculated that dapsone inhibits IL-8 release from normal human epidermal keratinocytes at the posttranscriptional level without affecting mRNA concentration. These immunomodulatory effects were similar to those of macrolides, such as erythromycin, clarithromycin, and azithromycin.14 Clarithromycin modulates ERK1/2 phosphorylation in NHBE cells,8 and azithromycin inhibits NF-kB activity in a CF cell line,37 thus regulating IL-8. It is likely that dapsone also modulates airway inflammation through modulation of IL-8. After inducing tracheal inflammation in ferrets using topically applied LPS, we evaluated the effects of 5 days of oral or aerosol dapsone on neutrophil inflammation and MCT, an integrated measure of epithelial integrity. Oral dapsone decreased intraepithelial neutrophil accumulation, but this was not statistically significant. Recognizing that dapsone is www.chestpubs.org

effective when used topically as a cream to treat neutrophilic dermatoses, we evaluated the potential for a lower dose of aerosol dapsone to inhibit LPS-induced tracheal inflammation. Nebulized dapsone significantly inhibited airway neutrophil infiltration and preserved or restored MCT despite exposure to LPS. Although it is difficult to calculate the precise dose to the airway, even if 10% of the total dose is delivered efficiently to the trachea,38 the dapsone delivered was only 0.3 mg. This dose is ⵑ 0.1 the systemic dose administered orally in a 1.5-kg ferret. Together, these data show that dapsone inhibits IL-8 in human airway cells and neutrophil recruitment in the LPS-inflamed ferret trachea in vivo while preserving MCT. Aerosol dapsone could be a promising therapy to treat chronic inflammatory airway diseases, such as CF, chronic bronchitis, or severe asthma. Acknowledgments Author contributions: Dr Rubin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Dr Kanoh: contributed to the study design, performance of the experiments, and writing of the manuscript. Dr Tanabe: contributed to the performance of the studies, analysis of the research data, and writing and revision of the manuscript. Dr Rubin: contributed, as the principal investigator, to the study concept and design, analysis of the results, and editing of the manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Drs Kanoh, Tanabe, and Rubin have filed a patent disclosure for the use of dapsone to treat airway diseases through Virginia Commonwealth University. Role of sponsors: The sponsors provided only academic salary support and had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. Other contributions: We thank Melissa A. Yopp, BS, for technical assistance. We also thank Gil Yosipovitch, MD, of Wake Forest University for insightful discussion and ideas.

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