The Journal of Immunology Transepithelial Migration of Neutrophils in Response to Leukotriene B4 Is Mediated by a Reactive Oxygen Species-Extracellular Signal-Regulated Kinase-Linked Cascade1 Chang-Hoon Woo,* Min-Hyuk Yoo,† Hye-Jin You,† Sung-Hoon Cho,† Yeung-Chul Mun,‡ Chu-Myong Seong,‡ and Jae-Hong Kim2* The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B4 (LTB4) is known to be a principal che- moattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB4 induces transepithelial migration of neutrophils. We found that LTB4 induces concentration-dependent transmigration of DMSO-differ- entiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB4 receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB4-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB4 signaling to transep- ithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB4 led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB4 was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB4 receptor antagonist. The Journal of Immunology, 2003, 170: 6273–6279. 3 eukotriene B4 (LTB4) is a key mediator of inflammatory LTB4 contributes to inflammatory responses. In particular, the in- processes, immune responses, and host defense against flux of neutrophils into the airway is a common feature of several L infection (1, 2) and is known to stimulate chemotaxis, inflammatory diseases, including acute bronchitis, adult respira- degranulation, release of lysosomal enzymes, and reactive oxygen tory distress syndrome (ARDS), and chronic obstructive pulmo- species (ROS) generation (3, 4). Although the precise signaling nary disease (COPD) (8, 9), as well as severe asthma (10, 11). pathway along which the biological activities of LTB are trans- Once leukocytes are beyond the endothelial barrier, chemotactic by guest on October 1, 2021. Copyright 2003 Pageant Media Ltd. 4 duced remains largely unknown, it appears to act via two G pro- signals frequently lead to their accumulation near mucosal epithe- tein-coupled receptors: LTB4 receptor (BLT1), a high affinity re- lial cells and in the lumen of the airway. Airway epithelial cells ceptor first isolated from HL-60 cells, and BLT2, a low affinity may thus be important regulators not only of retention and acti- receptor that shares ϳ45% amino acid identity with BLT1 (5–7). vation of leukocytes, but also of their passage into the airway Neutrophils are the primary targets of LTB4, and their recruit- itself. It has therefore been suggested that LTB4-induced transep- ment to sites of inflammation is one of the principal ways in which ithelial migration of neutrophils plays a crucial role in the patho- genesis of ARDS, COPD, and severe asthma. Yet despite this im- plication, the signaling pathway via which LTB4 mediates *Graduate School of Biotechnology, Korea University, Anam-dong, Seoul, Korea; http://classic.jimmunol.org transepithelial migration of neutrophils remains largely unknown. †Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, Korea; and ‡Department of Oncology/Hematology, Ewha Woman’s Uni- In the present study, therefore, we investigated the signaling versity, College of Medicine, Seoul, Korea pathway by which LTB4 induces transepithelial migration of neu- Received for publication November 15, 2002. Accepted for publication April trophil-like granulocytes (differentiated HL-60 neutrophils (dHL- 14, 2003. 60)) differentiated from HL-60 cells, which have been shown to be The costs of publication of this article were defrayed in part by the payment of page a valid model system for the analysis of human neutrophilic migration charges. This article must therefore be hereby marked advertisement in accordance and chemotaxis (12), and by human polymorphonuclear neutrophils Downloaded from with 18 U.S.C. Section 1734 solely to indicate this fact. (PMNs) isolated from healthy volunteers. Our results indicate that 1 This work was supported by grants from the Basic Research Program (RO2-2002- 000-00029-0), the Science Research Center program (Aging and Apoptosis Research LTB4-induced transepithelial migration is accomplished via an ROS- Center), 2002 from the Korea Science and Engineering Foundation, and the Frontier extracellular signal-regulated kinase (ERK)-linked pathway. In addi- 21 Programs (Proteomics to J.H.K. and Stem Cell to C.M.S.) from the Korea Ministry tion, the physiological relevance of this pathway was validated in vivo of Science and Technology. by demonstrating that ROS generation and ERK activation are both 2 Address correspondence and reprint requests to Dr. Jae-Hong Kim, Graduate School of Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul 136-701, required for transmigration of peripheral blood neutrophils across the Korea. E-mail address: [email protected] airway epithelium of BALB/c mice. 3 Abbreviations used in this paper: LTB4, leukotriene B4; ARDS, adult respiratory distress syndrome; BAL, bronchoalveolar lavage; BLT, LTB4 receptor; COPD, Materials and Methods chronic obstructive pulmonary disease; cPLA2, cytosolic phospholipase A2; DCFDA, 2Ј,7Ј-dichlorofluorescin diacetate; dHL-60, differentiated HL-60 neutrophils; DPI, di- Chemicals phenylene iodonium; ERK, extracellular signal-regulated kinase; NAC, N-acetylcys- teine; PMN, polymorphonuclear neutrophil; PI 3-kinase, phosphatidylinositol 3-ki- 2Ј,7Ј-Dichlorofluorescin diacetate (DCFDA) was purchased from Molec- nase; PKC, protein kinase C; ROS, reactive oxygen species. ular Probes (Eugene, OR). LTB4APA, LY1718, genistein, and AACOCF3 Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 6274 LTB4-INDUCED TRANSEPITHELIAL MIGRATION VIA ROS-DEPENDENT PATHWAY were obtained from Biomol (Plymouth Meeting, PA). LTB4 and U75302 dene difluoride membranes using a wet transfer unit (NOVEX, San Diego, were purchased from Cayman Chemical Co. (Ann Arbor, MI). BSA, CA;for2hat100V).Themembranes were then blocked for 1 h with TBS DMSO, PMA, GF109203X, wortmannin, diphenylene iodonium (DPI), containing 0.05% (v/v) Tween 20 plus 5% (w/v) nonfat dry milk and in- and N-acetylcysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, cubated, first for 2 h with the primary Ab (1/2000 dilutions of anti- MO). FBS, DMEM, phenol red-free DMEM, gentamicin, and nonessential phospho-ERK or anti-p38) in TBS containing 0.05% (v/v) Tween 20 plus amino acids were purchased from Life Technologies (Gaithersburg, MD). 3% (w/v) BSA, and then for 1 h with HRP-conjugated secondary Ab. The All other chemicals were obtained from standard sources and were molec- bands were developed using an ECL kit and were visualized on XAR-5 ular biology grade or higher. film (Eastman Kodak, Rochester, NY). Cell culture and differentiation RNA isolation and RT-PCR Human promyelocytic HL-60 cells obtained from American Type Culture Total cellular RNA was extracted from dHL-60 cells using RNAzol B Collection (Manassas, VA; CCL-240) were grown in RPMI 1640 medium (Tel-Test, Friendswood, TX), dissolved in diethylpyrocarbonate-treated supplemented with 10% heat-inactivated FBS, 0.1 mM nonessential amino water, and quantified by UV scanning. One microgram of the extracted acids, 50 U/ml penicillin, and 50 ␮g/ml streptomycin at 37°C under a RNA was then reverse transcribed by incubating it for 60 min at 37°Cin humidified 95%/5% (v/v) mixture of air and CO2. To induce cells to un- 20 ␮l in buffer containing 10 mM Tris (pH 8.3); 50 mM KCl; 5 mM dergo differentiation along the neutrophil lineage, aliquots of HL-60 cell MgCl2; 1 mM each of dATP, dCTP, dGTP, and dTTP; and oligo(dT) suspension (5 ϫ 105 cells/ml) were seeded onto dishes and grown for 5 primers. Hot-start PCR was used to increase the specificity of the ampli- days in the absence or the presence of 1.25% DMSO. Cell differentiation fication with specific primers for human BLT1 (sense, 5Ј-TATGTCTGCG was then evaluated using four criteria (13–15): 1) morphological changes GAGTCAGCATGTACGC; antisense, 5Ј-CCTGTAGCCGACGCCCTAT (nuclear segmentation and granules formation) were analyzed microscop- GTCCG), human BLT2 (sense, 5Ј-AGCCTGGAGACTCTGACCGCTTT ically using H&E cytochemical staining; 2) cell proliferation was assayed CG; antisense, 5Ј-GACGTAGAGCACCGGGTTGACGCTA), and human with 0.4% trypan blue exclusion; 3) the capacity to reduce nitro blue tet- GAPDH (15). The PCR protocol entailed 25 cycles of denaturation at 94°C razolium, a property associated with myeloid cell maturation, was deter- for 30 s and elongation at 68°C for 2 min in a Mygenie Thurmal Block mined spectrophotometrically at 540 nm; and