In Vivo Role of p38 Mitogen-Activated Protein Kinase in Mediating the Anti-inflammatory Effects of CpG Oligodeoxynucleotide in Murine Asthma This information is current as of September 23, 2021. Barun K. Choudhury, James S. Wild, Rafeul Alam, Dennis M. Klinman, Istvan Boldogh, Nilesh Dharajiya, William J. Mileski and Sanjiv Sur J Immunol 2002; 169:5955-5961; ; doi: 10.4049/jimmunol.169.10.5955 Downloaded from http://www.jimmunol.org/content/169/10/5955 References This article cites 37 articles, 21 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/169/10/5955.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology In Vivo Role of p38 Mitogen-Activated Protein Kinase in Mediating the Anti-inflammatory Effects of CpG Oligodeoxynucleotide in Murine Asthma1 Barun K. Choudhury,*† James S. Wild,*† Rafeul Alam,*† Dennis M. Klinman,¶ Istvan Boldogh,‡ Nilesh Dharajiya,*† William J. Mileski,§ and Sanjiv Sur2*† DNA containing unmethylated CpG motifs is intrinsically immunostimulatory, inducing the production of a variety of cytokines and chemokines by immune cells. The strong Th1 response triggered by CpG oligodeoxynucleotide (ODN) inhibits the develop- ment of Th2-mediated allergic asthma in mice. This work documents that CpG ODN-induced IL-12 production plays a critical role in this process, because intrapulmonary CpG ODN inhibits allergic inflammation in wild-type but not IL-12؊/؊ mice. CpG ODN rapidly localized to alveolar macrophages (AM), thereby triggering the phosphorylation of p38 mitogen-activated protein kinase Downloaded from (MAP kinase). AM cultured with CpG but not control ODN up-regulated IL-12 p40 expression and release, and these effects were blocked by the highly specific p38 MAP kinase inhibitor SB202190. Intrapulmonary administration of this inhibitor blocked the ability of CpG ODN to produce IL-12 in the lungs and reversed the anti-inflammatory effects of CpG ODN on allergic lung inflammation. These findings indicate that IL-12 production by AM is stimulated by intrapulmonary CpG ODN administration through a p38 MAP kinase-dependent process, and IL-12 is a key cytokine that mediates CpG ODN-induced protection against allergic lung inflammation. The Journal of Immunology, 2002, 169: 5955–5961. http://www.jimmunol.org/ sthma is an inflammatory disease of the airways that induce the proinflammatory and Th1-associated cytokines, such as affects 5–10% of the general population. Airway eosin- IL-12 (11–13). Investigators have reported that administering CpG A ophilia and Th2 cytokine production are characteristic of ODN before sensitization or challenge by a variety of allergens asthma (1–3). In contrast to the permissive role of Th2 cytokines inhibits the development of allergic inflammation (14, 15). We in asthma, IFN-␥, a product of Th1 cells, potently inhibits eosin- extended these observations by demonstrating that intrapulmonary ophilic lung inflammation (4, 5). IL-12 is a cytokine that potenti- administration of CpG ODN provided long term protection against ates differentiation of naive T cells into Th1 effector cells while allergic inflammation in a murine model of asthma and docu- inhibiting their differentiation into Th2-secreting cells (6, 7). We mented that the inhibitory effects of CpG ODN were mediated by guest on September 23, 2021 and others have shown that IL-12 inhibits eosinophil recruitment, through IFN-␥ (16). decreases IgE levels, and suppresses bronchial hyperresponsive- The mitogen-activated protein kinases (MAP kinases), p38 and ness in murine models of allergic asthma when it is given system- c-Jun N-terminal kinase (JNK) are activated by a variety of envi- ically within 4–72 h of allergen challenge (8–10). ronmental stresses, and some evidence suggests that MAP kinases It is well documented that synthetic oligodeoxynucleotides play a role in T cell activation and cytokine production. The role (ODN)3 containing CpG motifs potently stimulate the mammalian of the MAP kinase JNK2 has been investigated in Th1 differenti- immune system. These CpG ODN rapidly stimulate T cells, B ation, and IL-12 fails to induce differentiation of JNK2-deficient cells, NK cells, and macrophages to proliferate, secrete Abs, and CD4ϩ T cells into effector Th1 cells (17, 18). These observations suggest that the p38 and JNK MAP kinase-signaling pathways play an important role in the induction of Th1 immune responses. *National Institutes of Health Asthma and Allergic Diseases Research Center and the CpG have been shown to activate MAP kinases and regulate cy- Departments of †Internal Medicine, ‡Microbiology and Immunology, and §Surgery, University of Texas Medical Branch, Galveston, TX 77555; and ¶Section of Retro- tokine production from B cell lines and monocytic cell lines (19– viral Research, Food and Drug Administration, Bethesda, MD 20892 21). On the basis of these in vitro studies suggesting an important Received for publication February 19, 2002. Accepted for publication September role of MAP kinases in the production of Th1-associated cyto- 16, 2002. kines, we examined their role in mediating the anti-inflammatory The costs of publication of this article were defrayed in part by the payment of page effects of CpG ODN in vivo. Our results suggest that p38 MAP charges. This article must therefore be hereby marked advertisement in accordance kinase is critical for mediating the anti-inflammatory effects of with 18 U.S.C. Section 1734 solely to indicate this fact. CpG ODN in murine asthma. 1 This work was supported by National Institutes of Health Grants PO1 AI46004 (to S.S.) and KO8 AI01539 (to S.S.), Sealy Center for Environmental Health and Med- icine Pilot Project (to S.S.), National Institute of Environmental Health Sciences Materials and Methods Center Cell Biology Core Grant E506676 (to I.B.), and McLaughlin Foundation at Animals and immunization University of Texas Medical Branch (to J.S.W.). 2 Address correspondence and reprint requests to Dr. Sanjiv Sur, Department of In- Experiments were performed on 6- to 8 wk-old female BALB/c mice that ternal Medicine, Division of Allergy and Immunology, University of Texas Medical were purchased from Harlan Sprague Dawley (Indianapolis, IN). Some Ϫ/Ϫ Branch, Medical Research Building 8.104, Galveston, TX 77555-0762. E-mail ad- experiments were conducted on wild-type (WT) and IL-12 BALB/c dress: [email protected] mice purchased from The Jackson Laboratory (Bar Harbor, ME). All mice 3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; AM, alveolar mac- were housed in a pathogen-free environment throughout the experiment at rophage; BAL, bronchoalveolar lavage; i.n., intranasal; PM, peritoneal macrophage; University of Texas Medical Branch (Galveston, TX). The Institutional RW, ragweed pollen extract; MAP kinase, mitogen-activated protein kinase; WT, Animal Care and Use Committee of the Academic Medical Center ap- wild type; ERK, extracellular regulated kinase; c-JNK, Jun N-terminal kinase. proved all animal experiments. Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 5956 p38 MAP KINASE IN VIVO AND ANTI-INFLAMMATORY EFFECTS OF CpG ODN CpG and control GpC ODN quences of labeled CpG and GpC ODN were identical with those listed above. At various time points after intrapulmonary administration, a BAL Two immunostimulatory ODN containing CpG motifs (GCTAGACGT was performed. A cytocentrifuge preparation was analyzed by krypton- TAGCGT and TCAACGTT) were synthesized using a phosphorothioate argon laser two-color confocal microscopy (Olympus, Salt Lake, UT). backbone as previously described (16). Control ODN were of the same sequence except the CpG motif was inverted to GpC (GCTAGAGCT IL-12 expression in alveolar macrophages (AM) TAGCGT and TCAAGCTT). All ODN were produced on the same syn- thesizer and were column purified. These ODN contained undetectable AM 99% pure were isolated from BAL fluids of five naive BALB/c mice ␮ ␮ levels of endotoxin (Ͻ0.02 U/kg, as determined using a Limulus amebo- and pooled. These cells were cultured with PBS, 2 M CpG ODN, or 2 M cyte lysate analysis kit (QCL-1000; BioWhittaker, Walkersville, MD). For control ODN (GpC) for 2 h and then lysed to extract total RNA. In some in vivo experiments, all ODN were administered intranasally (i.n.) at a dose experiments, AM were pretreated for 1 h with various concentrations of ␮ of 35 ␮g/animal in 100 ␮l PBS. SB202190 and then cultured with 2 M CpG ODN for 2 h. The mRNA expression level of IL-12 p40 was determined by either relative RT-PCR or In vivo experiments real time quantitative RT-PCR performed in TaqMan ABI Prism. In both types of PCR, the IL-12 p40 primer sequences were as follows: sense, The first set of in vivo experiments (Figs. 1A and 2A) were designed to 5Ј-ACA TCT ACC GAA GTC CAA TGC A-3Ј; antisense, 5Ј-GGA ATT determine the role of CpG ODN in mediating IL-12 production in naive GTA ATA GCG ATC CTG AGC-3Ј. For TaqMan, the internal probe se- BALB/c mice. Either control GpC or CpG were given i.n. After 48 h, quence was: 5Ј-6FAM-TGC ACG CAG ACA TTC CCG CCT-TAMRA- bronchoalveolar lavage (BAL) was performed.