The Journal of Immunology Induction of Vascular Leakage and Blood Pressure Lowering through Kinin Release by a Serine Proteinase from Aeromonas sobria1 Takahisa Imamura,2* Hidemoto Kobayashi,‡ Rasel Khan,‡ Hidetoshi Nitta,*† and Keinosuke Okamoto‡ Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B2 receptor-, and partially in a histamine-H1 receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B2 receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra- arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B2 receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease. The Journal of Immunology, 2006, 177: 8723–8729. eromonas species are facultative anaerobic Gram-nega- Aeromonas infections. Understanding the mechanism of Aeromo- tive rods that are ubiquitous, waterborne bacilli (1), most nas septic shock is crucial in the development of therapeutic treat- A commonly implicated as causative agents of gastroenter- ments. Recently, overactivation of the immune system by the ex- itis (2–5). Aeromonas hydrophila, Aeromonas caviae, and Aero- cessive release of proinflammatory cytokines, e.g., TNF-␣, mainly monas sobria recovered predominantly from clinical samples have from monocytes/macrophages stimulated by bacterial lipopolysac- been reported to be involved in a wide range of extraintestinal and charides through Toll-like receptors, a class of pattern recognition systemic infections, including septicemia, wound infections, men- molecules, have been suggested to lead to septic shock in Gram- ingitis, peritonitis, and hepatobiliary disease (6). The majority of negative bacteria infections (11). However, the fact that impair- patients inflicted with Aeromonas septicemia are infants under 2 ment of the host immune system increases the incidence of years of age and immunocompromised adults with multiple un- Aeromonas septic shock (6) may indicate involvement of a mech- derlying medical complications, mostly malignancy, hepatobiliary anism(s) other than overactivation of the immune system in shock disease, or diabetes. Mortality rates for these patients generally induction. range from 25 to 50% (7–9). Patients with severe wound infection The plasma kallikrein/kinin system is initiated by activation of (myonecrosis) caused by this microorganism also develop sepsis, coagulation factor XII on a negatively charged surface, with acti- and Ͼ90% of the patients succumb to their infections (7, 10). vated factor XII converting plasma prekallikrein to kallikrein, re- Septic shock is a major cause of death of these patients, but no leasing bradykinin (BK)3 from high m.w. kininogen (HK) (12). mechanism leading to this fatal complication has been shown in The plasma levels of the kallikrein/kinin system components are low in sepsis patients (13–16), indicating the activation and sub- sequent consumption of these components. BK, the final product, † *Department of Molecular Pathology and Department of Gastroenterological binds to BK-B2 receptor on vascular endothelial cells (17) and Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, induces vascular leakage (VL), leading to hypotension due to a Kumamoto, Japan; and ‡Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan decrease in circulating blood volume. Indeed, the activation of the plasma kallikrein/kinin system in an animal model causes lethal Received for publication February 28, 2006. Accepted for publication September 28, 2006. hypotension (18, 19). Hence, activation of the kallikrein/kinin sys- The costs of publication of this article were defrayed in part by the payment of page tem, and/or direct kinin release from kininogens, seems to con- charges. This article must therefore be hereby marked advertisement in accordance tribute to septic shock. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by a grant from the Japanese Ministry of Education and Science (Grant 16590319; to T.I.). 2 Address correspondence and reprint requests to Dr. Takahisa Imamura, Department 3 Abbreviations used in this paper: BK, bradykinin; HK, high m.w. kininogen; VL, of Molecular Pathology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto vascular leakage; LK, low m.w. kininogen; ASP, Aeromonas sobria serine proteinase; University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail address: taka@kaiju. BP, blood pressure; SBTI, soybean trypsin inhibitor; DFP, diisopropyl fluorophos- medic.kumamoto-u.ac.jp phate; ScpA, staphopain A. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 8724 ASP AND VL Aeromonas species release a number of putative virulence fac- leased in the solution was measured (fluorescence at 440 nm with ex- tors including hemolysins, enterotoxins, and proteinases (20). We citation at 380 nm) using a fluorescence spectrophotometer (model no. have shown that bacterial proteinases induce VL through activa- 650-40; Hitachi). tion of the plasma kallikrein/kinin system, and/or direct kinin re- Treatment of plasmas and kininogens with ASP lease from HK and low m.w. kininogen (LK), which represents another kinin source in plasma (21–23). A. sobria is predominantly Forty-five microliters of human plasma supplemented with 1 mM 1,10- phenanthroline for kininase inhibition was incubated with 5 ␮lofASP isolated in patient’s blood (24), and is more pathogenic than A. at various concentrations and 37°C for 5 min, followed by the addition hydrophila (25). Therefore, to study the mechanism of septic of 50 ␮l of 10 mM Tris-HCl (pH 7.3) containing 150 mM NaCl (TBS) shock by Aeromonas infection, we examined the A. sobria serine supplemented with 1 mM 1,10-phenanthroline. Ten microliters of ASP ␮ ␮ ␮ proteinase (ASP) for VL and blood pressure (BP)-lowering activ- and 90 l of TBS containing HK (80 g/ml) or LK (130 g/ml) were incubated at 37°C for 5 min. One ␮l of 100 mM diisopropyl fluoro- ities, and investigated an involvement of the plasma kallikrein/ phosphate (DFP) was added to plasma or kininogen samples after in- kinin system in these activities. cubating with ASP to completely inhibit ASP (27). DFP specifically phosphorylates the active-site serine residue of serine proteases, inac- Materials and Methods tivating its enzymatic activity. Materials VL assay Human HK, factor XII, and prekallikrein were purchased from Enzyme This experiment was performed and approved according to the criteria of Research Laboratories, and human LK was obtained from Athens Research animal experiments of the Kumamoto University Animal Experiment Technology. Evans blue was obtained from Merck. Soybean trypsin inhib- Committee. Guinea pigs (350ϳ450 g body weight, both sexes) were anes- itor (SBTI) was purchased from Sigma-Aldrich. BK, benzyloxycarbonyl- thetized with an i.m. injection of ketamine (80 mg/kg body weight). Thirty L-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), mg/kg body weight of Evans blue (2.5% solution in 0.6% saline) was then and t-butyloxycarbonyl-L-glutaminylglycyl-L-arginine-4-methylcoumaryl- administered i.v., followed by an intradermal injection of 0.1 ml of test 7-amide (Boc-Gln-Gly-Arg-MCA) were obtained from the Peptide Insti- samples (dissolved in TBS) into the clipped flank of the guinea pig. Bluing tute. BK B2 receptor antagonist, HOE140 was obtained from Hoechst. of injection sites starts within a few minutes and terminates by 10 min. Polyclonal anti-human kininogen goat IgG and HRP-conjugated rabbit IgG After 10 min, the guinea pig was euthanized by bleeding under ether an- against goat IgG were purchased from R&D Systems and Nichirei, respec- esthesia and blue dye-leaked skin tissues were excised, and incubated in 3 tively. The ProteoSeek albumin/IgG removal kit was obtained from Pierce. ml of formamide at 60°C for 48 h. VL activity was determined by quan- Other chemicals were purchased from Wako Pure Chemicals. Normal hu- titatively measuring the extracted Evans blue by absorbance at 620 nm as man plasma was prepared by centrifugation of a mixture of 9 vol of freshly described previously (30). Activity was expressed in terms of micrograms drawn blood from healthy volunteers and 1 vol of 3.8% (w/v) sodium of dye extracted. The activity of the buffer was subtracted from the activity citrate. of each sample. In the case of VL activity production from human plasma, the activity of plasma incubated with the buffer, followed by DFP addition, Purification of ASP was subtracted from the activity of each sample. HOE140 (10 nm/kg body ASP was initially purified from the culture supernatant of a clinically iso- weight) or diphenhydramine (30 mg/kg body weight) dissolved in 200 ␮l lated strain of A. sobria. To obtain sufficient ASP for the experiments of TBS was injected s.c.