Absence of IL-1 Receptor Antagonist Impaired Wound Healing along with Aberrant NF-B Activation and a Reciprocal Suppression of TGF- Signal Pathway 1 Yuko Ishida,* Toshikazu Kondo, 2 * Akihiko Kimura,* Kouji Matsushima, † and Naofumi Mukaida ‡ Although enhanced expression of IL-1 family proteins, including IL-1, IL-1, and IL-1 receptor antagonist (IL-1ra) during wound healing has been observed, the pathophysiological roles of these factors, particularly IL-1ra, still remain elusive. We explored skin wound-healing processes in IL-1ra-deficient mice. Compared to wild-type (WT) mice, IL-1ra-deficient mice exhib- ited impaired wound healing, as evidenced by attenuated collagen deposition and delayed neovascularization. In contrast, neu- trophil recruitment was significantly exaggerated, with the augmented expression of IL-1s, TNF-, and CXC chemokines, MIP-2 and KC, in IL-1ra-deficient mice compared with WT mice. Because the transcription of these proinflammatory cytokines and CXC chemokines requires the activation of NF-B, a major target of IL-1- and TNF--mediated signal pathway, we examined the activation states of NF-B. Nuclear translocation of NF-B p65 was significantly enhanced and prolonged in IL-1ra-deficient mice, compared to that in WT mice. The cross-talk between NF-B and TGF--mediated signals has been proposed based on in vitro observations. Indeed, compared to WT mice, the amounts of total and phosphorylated Smad2 and Smad3 were decreased with a reciprocal increase in the amount of Smad7 in skin wound sites of IL-1ra-deficient mice. Moreover, the gene expression of vascular endothelial growth factor, a target gene of TGF-1, was decreased in IL-1ra-deficient mice. Thus, the absence of IL-1ra may suppress TGF--mediated signaling pathway, which is crucial for collagen deposition and vascular endothelial growth factor- mediated neovascularization in wound healing. The Journal of Immunology, 2006, 176: 5598 –5606. W ound healing is a complicated but well-orchestrated biological event composed of three phases: inflam- mation, proliferation, and maturation (1, 2). In the initial inflammatory phase, various types of leukocytes such as neutrophils and macrophages are recruited at the wound sites. These cells eradicate microbes and provide cytokines and growth factors, which have profound effects on the subsequent prolifera- tive phase (1, 2). This phase is characterized by granulation tissue formation and neovascularization, the processes that are governed by fibroblasts and endothelial cells. IL-1, a pleiotropic inflammatory cytokine, is produced by var- ious kinds of cells such as neutrophils, macrophages, and fibro- blasts. Two distinct gene products, IL-1 and IL-1, have similar biological activities after binding to a common IL-1 receptor and are implicated as essential mediators of tissue destruction in var- ious inflammatory diseases such as septic shock and rheumatoid arthritis (3, 4). IL-1 receptor antagonist (IL-1ra) 3 is a member of the IL-1 family and exhibits an identical -pleated sheet structure as IL-1s (5, 6). Due to its similar structure, IL-1ra can also bind to IL-1R at the same sites with a similar affinity as IL-1s (7), but fails to associate with IL-1R accessory protein, which is indispensable for the biological activities of IL-1s (8, 9). Thus, IL-1ra antago- nizes IL-1s at the receptor levels. Moreover, IL-1ra is aberrantly produced in various diseases, such as rheumatoid arthritis and in- fectious diseases, to negatively regulate the bioactivities of IL-1s (7, 10). This notion was supported by phenotypic changes in mice lacking IL-1ra (IL-1ra KO mice). IL-1ra KO mice on a BALB/c background exhibited spontaneously polyarthritis resembling rheumatoid arthritis (11) and developed exaggerated bacteria-in- duced intrahepatic granuloma formation (12). Several lines of evidence indicate aberrant expression of IL-1s and IL-1ra at wound sites (13, 14). Given the ability of IL-1s to induce the expression of various growth factors and chemokines (15), it is reasonable to assume that IL-1s have roles in the wound- healing process. Nevertheless, it still remains controversial on the roles of IL-1s in wound healing. Topical application of IL-1 ac- celerated epidermal wound healing (16). In contrast, IL-1R KO mice exhibited retarded oral but not dermal wound healing (17), and short-term but not long-term blockade of IL-1 can promote periodontal wound healing (18). Moreover, to date, the biological role of IL-1ra in skin wound healing has not yet been investigated. Thus, we examined the biological roles of the IL-1 system, par- ticularly IL-1ra, by the use of IL-1ra KO mice. *Department of Forensic Medicine, Wakayama Medical University, Wakayama, Ja- pan; † Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Tokyo, Japan; and ‡ Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan Received for publication October 4, 2005. Accepted for publication February 14, 2006. 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 Grants-in-Aids from the Ministry of Education, Cul- ture, Science, and Technology of the Japanese government. 2 Address correspondence and reprint requests to Dr. Toshikazu Kondo, Department of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan. E-mail address: kondot@wakayama-med.ac.jp 3 Abbreviations used in this paper: IL-1ra, IL-1 receptor antagonist; Hyp, hydoxypro- line; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; pAb, polyclonal Ab; MPO, myeloperoxidase; P-Smad, phosphorylated Smad; SMA, -smooth muscle actin; WT, wild type; Col I, collagen type I. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00