PEPTIDOGLYCAN OF STAPHYLOCCUS AUREUS INDUCES ENHANCED LEVELS OF MATRIX METALLOPROTEINASE-9 IN HUMAN BLOOD ORIGINATING FROM NEUTROPHILS Yun Yong Wang,* Anders E. Myhre,* Solveig J. Pettersen,* Maria K. Dahle,* Simon J. Foster, ‡ Christoph Thiemermann, § Kristin Bjørnland,* ,† Ansgar O. Aasen,* and Jacob E. Wang* *University of Oslo, Family Division Rikshospitalet, Institute for Surgical Research and † Surgical Department, Rikshospitalet University Hospital, Oslo, 0027, Norway; ‡ University of Sheffield, Sheffield, S70 2TN, United Kingdom; and § The William Harvey Research Institute, Queen Mary University of London, London, ECTM 6BQ United Kingdom Received 7 Feb 2005; first review completed 26 Mar 2005; accepted in final form 13 Jun 2005 ABSTRACT—Enhanced plasma levels of matrix metalloproteinase 9 (MMP-9) detected in patients with severe sepsis are thought to contribute to the development of organ dysfunction in endotoxemia. We have recently reported that peptidoglycan, the major wall component of gram-positive bacteria, increases MMP-9 levels in lung and liver and organ injury in the rat. Thus far, it is unclear whether MMP-9 is part of the septic response to peptidoglycan in human blood. The aim of the present study was to examine the regulation of MMP-9 by peptidoglycan in human leukocytes. The addition of peptidoglycan to whole human blood caused enhanced levels of MMP-9 after 1 h of incubation (306 vs. 75 ng/mL, P # 0.05) and onward, as measured by enzyme-linked immunoabsorbant assay. In neutrophil cultures, MMP-9 values increased significantly after 30 min of incubation with peptidoglycan (242 vs. 121 ng/mL, P # 0.05), whereas muramyl dipeptide had no effect. In contrast, adherent monocytes released insignificant amounts of MMP-9. To examine whether the released MMP-9 resulted from de novo synthesis, intracellular and secreted MMP-9 was measured during stimulation of neutrophils. The total MMP-9 values (the sum of intracellular and secreted MMP-9) before and after stimulation were mainly unaltered. The enhanced MMP-9 levels induced by peptidoglycan was attenuated by inhibitors of p38 mitogen-activated protein kinases (MAPK), (SB202190, 25 mM) and ERK1/2 (PD98059, 25 mM) and inhibitors of Src Tyrosine kinase (PP2, 5 mM) and PI3-K (LY294002, 25 mM). KEYWORDS—Matrix metalloproteinases, peptidoglycan, gram-positive infection, neutrophil, monocyte INTRODUCTION Today, gram-positive bacteria are a leading cause of community-acquired and nosocomial bacteremia, a condition associated with high morbidity and mortality. Peptidoglycan is the major component of gram-positive bacteria and is also found in gram-negative bacteria. A number of endotoxic prop- erties have been assigned to peptidoglycan (1, 2). Most notably, it has recently been documented that peptidoglycan causes organ injury in vivo, associated with induction of systemic inflammation and activation of NF-kB and AP-1 in the liver (3, 4). Matrix metalloproteinases (MMPs) are a class of enzymes with major functions in the degradation and remodeling of all components of the extracellular matrix. Recent studies have shown that MMP-9 plays a pivotal role in innate immunity and inflammation (5). Elevated pro-MMP-9 and activated forms of MMP-9 have been detected in the plasma of patients with sepsis, and MMP-9 levels correlate with the severity of disease (6, 7). Furthermore, concentrations of MMP-9 in healthy volun- teers increased after intravenous injection of lipopolysaccha- ride (LPS) (8). In human blood, the increased MMP-9 induced by LPS has been shown to occur after degranulation of neutrophils (7). The regulation of MMP-9 by peptidoglycan has been scarcely studied. However, we recently demonstrated that peptidoglycan induced enhanced MMP-9 levels in the rat liver, lung, and blood (9). In the present study, we have examined the effect of pepti- doglycan on the release of MMP-9 in human blood and in isolated leukocyte cultures. MATERIALS AND METHODS Purification of peptidoglycan Peptidoglycan was isolated from Staphylococcus aureus as previously described (10). Peptidoglycan extracts were subjected to SDS-PAGE with no evidence of any spurious protein. The peptidoglycan was also enzymatically digested, and it gave the expected reverse-phase/high-pressure liquid chromatography muropeptide profile with no spurious products. The peptidoglycan aggregates were dispersed by soni- cation (3000 Hz, 3 3 10 s) on ice before administration to increase their ability to interact with cells. The LPS was derived from Escherichia coli (serotype B6:026; Sigma, St. Louis, MO). Muramyl dipeptide (MDP) was from Sigma. Incubation of whole human blood ex vivo A whole human blood model was used as previously described (11). In brief, venous blood from healthy volunteers was anticoagulated with heparin (25 U/mL blood; Leo, Ballerup, Denmark) and was incubated in plastic syringes (Monovette, Sarstedt, Germany) at 37°C with slow rotation in the presence of peptidoglycan (3 mg/mL blood) or LPS (10 ng/mL blood) or saline, respectively. At 0, 1, 4, 6, 12, and 24 h, plasma was obtained by centrifugation at 7000g for 3 min and was stored at –20°C. Cultures of human neutrophils and monocytes Polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Polymorphprep; Axis- shield Poc AS, Oslo, Norway) according to the manufacturer’s instruction. Briefly, heparinized whole human blood was laid over an equal amount of Polymorphprep. The samples were centrifuged at 450 to 500g for 30 min in a swing-out rotor at room Address reprint requests to Jacob E. Wang, PhD, Institute for Surgical Research, Rikshospitalet University Hospital, Oslo, 0027, Norway. E-mail: j.e.wang@medisin.uio.no. DOI: 10.1097/01.shk.0000174935.13786.6c Copyright Ó 2005 by the Shock Society 214 SHOCK, Vol. 24, No. 3, pp. 214–218, 2005