LPS Responsiveness and Neutrophil Chemotaxis In Vivo Require PMN MMP-8 Activity Angus M. Tester 1. , Jennifer H. Cox 1. , Andrea R. Connor 1 , Amanda E. Starr 1 , Richard A. Dean 1 , Xose S. Puente 2 , Carlos Lo ´ pez-Otı ´n 2 , Christopher M. Overall 1 * 1 University of British Columbia Centre for Blood Research, Departments of Oral Biological and Medical Sciences, and Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada, 2 Department Bioquimica y Biologia Molecular, Instituto Universitario de Oncologia, Universidad de Oviedo, Oviedo, Spain We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP- 8 cleaves LPS-induced CXC chemokine (LIX) at Ser 4 ,Val 5 and Lys 79 ,Arg 80 . LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8- cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4,5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg 5 -Ser 6 and at Val 7 -Leu 8 in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue. Citation: Tester AM, Cox JH, Connor AR, Starr AE, Dean RA, et al (2007) LPS Responsiveness and Neutrophil Chemotaxis In Vivo Require PMN MMP-8 Activity. PLoS ONE 2(3): e312. doi:10.1371/journal.pone.0000312 INTRODUCTION Polymorphonuclear neutrophils (PMNs) are crucial inflammatory leukocytes in host protection from infection, where their primary role is in phagocytosis and killing of bacteria, fungi and protozoa, and in wound debridement and healing [1,2]. Given these critical roles of PMNs, it has long been recognised that neutropenic patients are at greater risk of infection [3], and that is often observed after intensive cancer chemotherapy [4,5]. Proteolysis of phagosome contents and damaged extracellular matrix are key PMN actions in inflammation. Cell migration, crossing basement membrane and connective tissue matrix barriers are other aspects of PMN function traditionally thought to require proteolytic activity [6]. Additionally, PMNs are a source of chemotactic factors that guide the recruitment of specific and non-specific immune effector cells [7] and so these first line defence cells play key roles in innate and acquired immunity. Of the two major chemokine subfamilies that provide di- rectional cues for leukocyte migration and activation [8], the CXC chemokines predominantly influence PMNs and T-lymphocytes whereas the CC chemokines are active on monocytes, basophils and eosinophils [9]. The expression of CXC chemokines is rapidly upregulated during acute inflammatory responses, such as that initiated by the endotoxin lipopolysaccaride (LPS) [10–13]. A subset of the CXC chemokines are characterised by an ELR (glutamic acid-leucine-arginine) sequence proximal to the con- served CXC motif. ELR is essential for binding CXC-receptors (CXCR) 1 and 2 [14] leading to PMN activation, degranulation and release of proteases [15]. The murine ELR + CXC chemokines act through a single receptor that is homologous to human CXCR2 [16]. In humans there are seven ELR + CXC chemokines; CXCL8/interleukin-8 (IL-8); CXCL7/neutrophil-activating pep- tide-2 (NAP-2); CXCL6/granulocyte chemotactic protein-2 (GCP-2); CXCL5/epithelial cell-derived neutrophil activating peptide-78 (ENA-78); and CXCL1, -2 and -3 (also known as growth-related oncogenes (GRO) a,-b, and -c). Only CXCL8/IL- 8, the most potent of these chemokines, and CXCL6/GCP-2 bind CXCR1, whereas all members signal through the closely related receptor CXCR2 [14]. Mice lack a homologue of CXCL8/IL-8 having only four ELR + CXC chemokines: LPS-induced CXC chemokine (LIX), the most abundant and potent of the murine chemokines and regarded as the orthologue of CXCL8 [17]; keratinocyte-derived chemokine (KC); macrophage inflammatory protein-2 (MIP-2); and dendritic cell inflammatory protein-1 (DCIP-1). Physiological N-terminal cleavage of chemokines modifies their bioactivity—either enhancing activity of the ELR + CXC chemokines [15] or generating potent receptor antagonists from the CC chemokines CCL2, -7, -8 and -13 (also known as macrophage chemotactic proteins 1 to 4) [18,19]. Although several Academic Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received January 24, 2007; Accepted March 2, 2007; Published March 21, 2007 Copyright: ß 2007 Tester et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Michael Smith Foundation for Health Research (JHC), Natural Sciences and Engineering Research Council of Canada (JHC and AES), CIHR Strategic Training Program STP-53877 (JHC and AES). CMO is a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology, and has research grants from the Canadian Institutes of Health Research, the National Cancer Institute of Canada, and a Centre Grant from the Michael Smith Research Foundation (UBC Centre for Blood Research). Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: chris.overall@ubc.ca . These authors contributed equally to this work. PLoS ONE | www.plosone.org 1 March 2007 | Issue 3 | e312