INFECTION AND IMMUNITY, Dec. 1993, p. 5035-5043 Vol. 61, No. 12 0019-9567/93/125035-09$02.00/0 Copyright C) 1993, American Society for Microbiology Alpha-2-Macroglobulin Functions as an Inhibitor of Fibrinolytic, Clotting, and Neutrophilic Proteinases in Sepsis: Studies Using a Baboon Model J. P. DE BOER," A. A. CREASEY,2 A. CHANG,3 J. J. ABBINK,lt D. ROEM,1 A. J. M. EERENBERG,1 C. E. HACK,`* AND F. B. TAYLOR, JR.3 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University ofAmsterdam, Amsterdam, The Netherlands'; Chiron Corporation, Emeryville, California 946082; and Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 731043 Received 24 May 1993/Returned for modification 21 July 1993/Accepted 23 September 1993 Alpha-2-macroglobulin (ca2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of ac2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated ac2M (ica2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in ica2M significantly correlated with the increase of thrombin-antithrombin IH, plasmin-ar2-antiplasmin, and, to a lesser extent, with that of elastase-ad- antitrypsin complexes, raising the question of involvement offibrinolytic, clotting, and neutrophilic proteinases in the inactivation of a2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with ar2M. Changes in ca2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that ar2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that a2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased av2M levels in clinical sepsis. There is abundant evidence for a role of cytokines in the property of proteinases bound to a.2M has been used to pathogenesis of sepsis (5, 7, 12, 19). In addition to these identify the proteinases in complex with ot2M (25-27). mediators, the generation of proteinases may play an impor- Plasma levels of a2M are reduced in patients with sepsis tant role in the development of sepsis (8, 14, 29). Excessive (14, 15, 34, 41, 51) and associated with a fatal outcome in activity of proteinases in vivo is counteracted by inhibitors, some studies (14, 51) but not in others (1). The formation and including serine-proteinase inhibitors and alpha-2-macro- subsequent clearance of at2M-proteinase complexes may globulin (a2M). This latter protein inhibits proteinases of all explain the decrease of at2M levels in sepsis (33, 45, 46). four catalytic classes (4, 23, 49). Proteinases inhibited by However, increased plasma levels of a2M-proteinase com- a2M include the coagulation proteinases thrombin and factor plexes occur only infrequently in patients with sepsis (3). To Xa (13, 16), the fibrinolytic enzymes urokinase-type and clarify the role of a2M in sepsis, we have studied the state of tissue-type plasminogen activators as well as plasmin (24, cc2M in a baboon model for sepsis by using a recently 31, 36, 49), kallikrein of the contact system (22, 35, 50, 60), developed assay to quantify the total amount of inactivated the neutrophilic proteinases elaAase, cathepsin G, and col- a2M (i.e., chemically inactivated a2M and a2M in complex lagenase (49, 62), and several bacterial proteinases (40). with a proteinase) in plasma. Our results show that, in this Thus, a2M may play an important role in regulating the animal model for sepsis, a2M is inactivated because of the hemostatic and inflammatory reactions that occur in sepsis. formation of complexes with proteinases. The inhibition of proteinases by a2M is due to a unique mechanism: the proteinases cleave a2M at the bait region, which contains a number of peptidyl bonds that are easily MATERIALS AND METHODS hydrolyzed by various proteinases (4, 23). Cleavage of the bait regions then induces the exposure of internal thiolester Preparation of Escherichua coli organisms. Pathogenic E. bonds, which are subsequently hydrolyzed (52, 53). This coli organisms type B were isolated from stool specimens at latter process coincides with a change in conformation of Children's Memorial Hospital, Oklahoma City, Okla., as ot2M, which results in entrapment of the proteinase (4, 20, described previously (30). The serotype of this strain is 21). A variable amount of the proteinase entrapped is not 086K61H; the lipopolysaccharide phenotype is part rough. bound to a2M via its active site and, therefore, retains some The strain produces no hemolysin. After growth in tryptic activity against small synthetic substrates (4, 42, 49). This soybean agar, the microorganisms were lyophilized and stored at 4°C. Shortly before the experiment, the bacteria were reconstituted, washed, and characterized as described by Hinshaw et al. (30). * Corresponding author. Animals. A mixed breed of Papio c. cynocephalus and t Present address: Department of Lung Diseases, University Papio c. anubis baboons (Charles River Breeding Laborato- Hospital, Leiden, The Netherlands. ries Inc., Wilmington, Del.) were housed at the University of 5035 5036 DE BOER ET AL. INFECT. IMMUN. Oklahoma Health Sciences Center Animal Facility at the human plasma by a modification of the method of Kurecki et Oklahoma City Zoo. The animals were tuberculosis free, al. (37). After differential (NH4)2SO4 precipitation, the ao2M weighed 5 to 17 kg, and had leukocyte concentrations of preparation was loaded onto a zinc-chelated Sepharose 5,000 to 10,000 cells per RI and hematocrits exceeding 36%. column (2.5 by 11 cm). The column was washed with 0.02 M They were observed for a minimum of 30 days to ensure sodium phosphate-0.15 M NaCl (pH 6.0) at a flow rate of 50 adequate equilibration prior to experimentation. During re- ml/h until the A280 was 0.04. Bound proteins were eluted covery from shock, the baboons were observed daily and with 0.02 M NaOAc-0.15 M NaCl (pH 5.0). Fractions medically treated as deemed appropriate. Surviving animals containing protein were pooled and concentrated. The puri- were euthanized with sodium pentobarbital after a minimum fied a2M migrated as a homogeneous band with an Mr of of 7 days. The clinical and laboratory data of the animals 184,000 on a sodium dodecyl sulfate (SDS)-4 to 15% poly- used for this study have been described elsewhere (10). acrylamide gel under reducing conditions. The preparation Experimental procedures. Experimental and infusion pro- was stored at 4°C in the presence of 0.02% sodium azide. cedures on the baboons were performed as described previ- Assays. (i) Quantification of total a2M. Antigenic levels of ously (9, 56, 57). Briefly, baboons were fasted overnight aL2M in baboon plasma were determined with a Behring prior to each experiment and given water ad libitum. After nephelometric analyzer (Behringwerke AG, Marburg, Ger- immobilization and proper anesthesia with sodium pentobar- many) with antiserum against human a2M (Behringwerke bital, the baboons were challenged with a 2-h intravenous AG) and as described in the manufacturer's instructions. infusion of either a lethal dose of E. coli (4 baboons), i.e., 4 Results were expressed as levels relative to the baseline. x 1010 CFU/kg of body weight, or a sublethal dose of E. coli Prechallenge levels of each individual baboon were arbi- (10 baboons), i.e., 0.4 x 101u CFU/kg. The animals receiving trarily set at 1.0. the lethal dose died between 6 and 10 h. During the experi- (ii) Radioimmunoassay for ca2M. Inactivated ao2M (iao2M) ments, heart rate, mean systemic arterial pressure, respira- in baboon plasma was measured by the Ml assay as de- tion rate, and rectal temperature were monitored hourly for scribed by Abbink et al. (2, 3). Briefly, monoclonal antibody 6 h and daily for 7 days. Hematologic parameters were (MAb) Ml, which binds icx2M (i.e., chemically inactivated assessed in blood samples collected at T+0, +30, +60, and/or complexed a2M) but not native ot2M, was coupled to +120, +180, +240, +300, +360, and +1,440 min as de- Sepharose and incubated with 50 ,ul of baboon plasma for 4 scribed previously (56). In addition, at each of these time h at room temperature by head-over-head rotation. Bound points, 2.5-ml blood samples were collected in 10 mM EDTA iat2M was then quantified by a subsequent incubation with and 0.05% (wtlvol) Polybrene (final concentrations) as de- 1`I-anti-ac2M antibodies as described previously (2, 3). scribed previously (3, 44). After centrifugation, the resulting Levels of iao2M in baboon plasma were expressed as per- plasma samples were stored in aliquots at -70°C and used in centages of that in normal baboon plasma in which a2M was the present study. completely inactivated by incubation with methylamine One baboon was challenged with 7.3 x 1010 live CFU of chloride (final concentration, 0.2 M; methylanine-treated Staphylococcus aureus per kg of body weight, and another normal baboon plasma [MA-NBP]) similarly as described for baboon was challenged with 6.5 x 1010 heat-inactivated human plasma (3). CFU of S. aureus per kg. The infusion procedure, monitor- (iii) Chromogenic assay for a2M-proteinase complexes in ing of the animals, and sampling of the blood were identical plasma. The MAb Ml-Sepharose suspension (0.5 ml) was to those procedures for the animals challenged with E.