Inhibition of Allergic Inflammation in the Airways Using Aerosolized Antisense to Syk Kinase 1 Grant R. Stenton, 2 * Marina Ulanova, 2 * Rene ´ E. De ´ry,* Shaheed Merani,* Moo-Kyung Kim, ¶ Mark Gilchrist,* Lakshmi Puttagunta, † Sorin Musat-Marcu, § Deborah James, ‡ Alan D. Schreiber, ¶ and A. Dean Befus 3 * Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of FcR and FcR, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of  2 integrins,  4 integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach. The Journal of Immunology, 2002, 169: 1028 –1036. S timulation of FcR and FcR leads to downstream signal- ing events, gene transcription, mediator release, and in some cases phagocytosis. In leukocytes, cross-linking of FcR results in the activation of Src and Syk protein tyrosine ki- nases. These kinases associate with immunoreceptor tyrosine- based activation motifs, which serve as specific recognition se- quences in the intracellular domain of FcR (1–3). Syk plays an essential role in activation of immune cells and lymphocyte de- velopment. In mast cells, this molecule is involved in regulation of multiple intracellular signaling pathways, leading to release of al- lergic mediators. Important downstream targets of Syk include phospholipase C1, the activation of which results in Ca 2 mo- bilization and eventually in NF-AT activation. Syk also induces activation of the mitogen-activated protein kinase cascade and generation of phosphatidylinositide 3-phosphate, which in turn regulate transcription factors necessary for cytokine gene expres- sion (4). In addition to being expressed in macrophages (5, 6) and mast cells (7–9), Syk (p72 Syk ) is expressed in eosinophils (10), neu- trophils (11), T cells (12, 13), and B cells (13, 14). Recently, it has been shown that Syk is also expressed in nonhemopoietic cells: hu- man fibroblasts (15), breast epithelium (16), and rat hepatocytes (17). Matsuda et al. (18) demonstrated that treatment of peripheral blood monocytes with Syk antisense oligodeoxynucleotides (ASO) 4 inhibits Syk expression when compared with cells treated with scrambled ASO (Scr ASO). This inhibition correlates with the suppression of FcR-mediated phagocytosis, suggesting that Syk plays a critical role in FcR-mediated cellular signaling and func- tion in monocytes/macrophages. We have recently demonstrated that aerosolized Syk ASO in vivo suppresses Syk expression, the release of NO and TNF from macrophages, and pulmonary inflammation in an infection model of airway inflammation (19). In the current studies we used this short-term gene therapy approach to treat allergic inflammation in the airways in a Brown Norway rat model of OVA-induced asthma. Materials and Methods Animals Male Brown Norway rats (Harlan Sprague Dawley, Indianapolis, IN) and male Sprague Dawley rats (Charles River Breeding Laboratories, St.-Con- stant, Quebec, Canada) were housed in the Health Sciences Laboratory Animal Service (University of Alberta, Edmonton, Alberta, Canada) in filter-top cages. The animals were rested for a minimum of 1 wk before experimentation. They were exposed to 12-h light/dark cycles and given food and water ad libitum. The Brown Norway rats were sensitized to OVA i.p. as described pre- viously (20) and used on day 21 following sensitization. The Sprague Dawley rats were infected by s.c. injection of larvae of Nippostrongylus brasiliensis (Nb) and were studied 4 wk later (21) when the inflammation Departments of *Medicine, † Laboratory Medicine and Pathology, and ‡ Pharmacy, University of Alberta, and § HistoBest Inc., Edmonton, Alberta, Canada; and ¶ Uni- versity of Pennsylvania School of Medicine, Philadelphia, PA 19104 Received for publication January 17, 2002. Accepted for publication May 10, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Canadian Institutes for Health Research grant (to A.D.B.), National Institutes of Health Grant HL-27068 (to A.D.S.), and post-doctoral fellowships from the Canadian Lung Association/Medical Research Council of Can- ada/GlaxoWellcome (to G.R.S.) and the Canadian Society of Allergy and Clinical Immunology/Merck Frosst (to M.U.). 2 G.R.S. and M.U. are joint first authors of this paper. 3 Address correspondence and reprint requests to Dr. A. Dean Befus, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. E-mail ad- dress: dean.befus@ualberta.ca 4 Abbreviations used in this paper: ASO, antisense oligodeoxynucleotide; Scr ASO, scrambled ASO; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPE, dio- leoyl-phosphatidyl-ethanol-amine; BAL, bronchoalveolar lavage; Nb, Nippostrongy- lus brasiliensis; PMN, polymorphonuclear neutrophil; WE, worm equivalent. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00