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- Functions as an Inhibitor of Fibrinolytic, Clotting, and Neutrophilic Proteinases in : 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 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 -antithrombin IH, -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 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 -proteinase inhibitors and alpha-2-macro- subsequent clearance of at2M-proteinase complexes may (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 -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), 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 , 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 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 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 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. coli. incubated with 50-,u plasma samples as described previously These two animals were euthanized at T+480 min. (2, 3). The Sepharose was washed five times with saline and, All experimental procedures described in this manuscript finally, once with the appropriate substrate buffer (50 mM were performed as described in the NIH guidelines for the Tris-100 mM NaCl at pH 7.8 for S2302, at pH 7.4 for S2251, use of experimental animals. and at pH 8.4 for S2238; and 0.1 M HEPES [N-2-hydroxy- Reagents. CNBr-activated Sepharose 4B and zinc chelat- ethylpiperazine-N'-2-ethanesulfonic acid]-0.5 M NaCl-10% ing Sepharose were purchased from Pharmacia Fine Chem- [vol/vol] dimethyl sulfoxide [pH 7.5] for M4765 and S7388). icals (Uppsala, Sweden), hexadimethrine bromide (i.e., The Sepharose suspensions were brought to 100 pI and Polybrene) was from Janssen Chimica (Beerse, Belgium), incubated with 100 pI of substrate solution in the appropriate and Tween 20 and dimethyl sulfoxide were from J. T. Baker buffer (final concentration, 2 mM) under continuous shaking. Chemicals Co. (Phillipsburg, N.J.). Purified human neutro- After 240 min, substrate hydrolysis was stopped by the phil elastase and cathepsin G were purchased from Elastine addition of 200 pI of 50% acetic acid. The Sepharose was Products Co. Inc. (Pacific, Mo.). Human thrombin was a pelleted, 200 ptl of each supernatant was transferred into kind gift from K. Mertens of the Department of Coagulation enzyme-linked immunosorbent (ELISA) plates, and the A405 of the Red Cross Blood Transfusion Service, Amsterdam, was measured in microtiter plates (Titertek; Flow Laborato- The Netherlands, human kallikrein was generously provided ries, Inglewood, Calif.). Finally, the amount of pNA re- by G. Tans from the Department of Biochemistry of the leased from the chromogenic substrate was calculated (e = University of Limburg, Maastricht, the Netherlands. Human 10,000 mol- liter-l). Results were expressed as micromoles plasmin and the chromogenic substrates H-D-Pro-Phe-Arg- of substrate hydrolyzed per hour. Preformed complexes of p-nitroanilide (H-D-Pro-Phe-Arg-pNA; S2302), H-D-Val- a2M and elastase, kallikrein, plasmin, thrombin, and cathe- Leu-Lys-pNA(S2251), and H-D-Phe-Pip-Arg-pNA(S2238) psin G were prepared by incubating these proteinases at a were purchased from Kabi Vitrum (Stockholm, Sweden); concentration of 1 puM with a twice-molar excess of ao2M for the substrates M4765 (MeO-Suc-Ala-Ala-Pro-Val-pNA) and 90 min at room temperature. Thereafter, 50 pI of serial S7388 (N-Suc-Ala-Ala-Pro-Phe-pNA) were from Sigma dilutions of each a2M-proteinase complex mixture was Chemical Co. (St. Louis, Mo.). Monospecific rabbit anti- tested in the chromogenic assay for a2M-proteinase com- serum against human ao2M (KH-08-P01) was obtained from plexes as described above. the Department of Immune Reagents, Red Cross Blood (iv) Other assays. Plasmin-ot2-antiplasmin complexes were Transfusion Service. measured with a radioimmunoassay that originally was de- Purification of human a2M. Human a2M was purified from veloped for use in human studies (38) and modified for use in VOL. 61, 1993 ALPHA-2-MACROGLOBULIN IN BABOON SEPSIS 5037

baboons (10). In this assay, MAb AAP-11, which is directed against a neoepitope on complexed ot2-antiplasmin, is used o MA-NHP as a catching antibody, and polyclonal radiolabeled rabbit * MA-NBP antibodies against human plasmin(ogen) is used as a detect- o NHP ing antibody. Baboon plasma in which a maximal amount of r- * NBP was generated by the addition of o - plasmin-a2-antiplasmin .04-I'- 40F a methylamine and urokinase (10, 38) was used as a standard. Elastase-al-antitrypsin complexes were measured by a eQ. radioimmunoassay as described previously (43). In this Z$ ._- assay, polyclonal rabbit antibodies against human elastase .14 4 and a radiolabeled MAb against complexed al-antitrypsin .jo were used as catching and detecting antibodies, respec- -_ 20F tively. Results were expressed in nanomoles per liter by c'JIn reference to a standard curve of normal baboon plasma in U which 66.6 nmol of elastase-al-antitrypsin complexes per liter was generated by incubation of 1 volume of purified human elastase (10 ,ug/ml in phosphate-buffered saline [PBS]) with 4 volumes of normal baboon plasma for 15 min 10 at room temperature (11). Thrombin-antithrombin III com- dilution of sample plexes were measured with a commercial ELISA obtained FIG. 1. Radioimmunoassay for detection of ioa2M in baboons. from Behringwerke AG (10). Serial dilutions of MA-NBP, or methylamine-treated human plasma Analysis of data. In the lethal group of animals, three (MA-NHP) were tested as described in Materials and Methods. The samples at T+180, one at T+240, and two at T+360 min, and symbols represent the mean and standard deviation of three titration in the sublethal group, six samples at T+240, eight at T+360, curves. Values obtained for normal baboon plasma (NBP) and and seven at T+1,440 min were available for the present normal human plasma (NHP), both tested at a 1-to-20 dilution, are investigation. At all other time points, 10 and 4 samples were also shown. The results are expressed as a percentage of the available in the sublethal and lethal groups, respectively. '"I-anti-a2M antibodies that had bound. The parameters which were assessed in the baboons were expressed as means + standard errors of the means. Most of the parameters studied were not normally distributed. the lethal and sublethal groups, respectively [see references Therefore, the Kruskall-Wallis test was used to assess 10 and 11]). significant changes in parameters during the observation Detection of iar2M in baboon plasma. Initially, we estab- period. Subsequently, the Wilcoxon-Mann-Whitney rank lished whether iat2M in baboon plasma could be detected by sum test was used to assess the differences between levels at the Ml assay, which was developed to measure human ica2M individual time points and baseline levels and to determine (3). Serial dilutions of MA-NBP (3) and of untreated normal the significance of the differences between the animals baboon plasma were tested in this assay. Similarly, human challenged with a lethal and with a sublethal dose of E. coli. plasma incubated with or without methylamine was also A difference was considered significant, by using a two- tested as a control. Although the assay for baboon ia2M was tailed probability test, at a P of <0.05 and highly significant approximately 10-fold less sensitive than that for human at a P of <0.005. Spearman's rank correlation analysis was ia2M, the dose-response curve of MA-NBP was very repro- used to assess the correlation between the parameters. ducible (Fig. 1). In addition, the results obtained with normal baboon plasma showed that the assay was sufficiently sen- RESULTS sitive to assess levels of ia2M in baboons. The amount of iat2M was expressed as a percentage of that in MA-NBP, Course of sepsis in baboons challenged with a lethal or which was arbitrarily set at 100%. ia2M in normal baboon sublethal dose ofE. coli. Clinical and biochemical parameters plasma appeared to be 0.5% of that in MA-NBP, i.e., of the animals used for this study have been described comparable to the levels found in human plasma (3). elsewhere (10, 11). Briefly, after the challenge with E. coli, Total and inactivated ai2M in septic baboons. During the marked changes in mean systemic arterial pressure occurred first 6 h after the start of the E. coli infusion, the levels of (decreases from 102 ± 3.7 to 81 + 5.7 mm Hg at T+120 min total a2M did not change significantly from baseline values in the sublethal group and from 105 + 4.8 to 61 + 5.0 mm Hg both in the animals with a lethal sepsis and those with a in the lethal group), leukocyte counts (decreases from 7.1 + nonlethal sepsis (Fig. 2A). At 24 h, however, total a2M 0.9 to 2.4 + 0.68 cells per mm' at T+120 min in the sublethal levels had increased to 1.62 times the baseline value in the group and from 8.1 + 1.24 to 1.6 + 0.16 per mm3 in the lethal sublethal group. No data from the lethal group of baboons group), and counts (decreases from 289 + 22 to 154 were available at this time point, since all animals of this ± 24 cells per mm3 at T+360 min in the sublethal group and group had died between 6 and 10 h. from 380 ± 37 to 187 ± 29 cells per mm3 in the lethal group). In contrast to total a2M, levels of ia2M differed markedly In the sublethal group, these parameters returned gradually between both groups of animals. During the first 6 h, ica2M to almost baseline values, whereas in the lethal group they levels in the sublethal group increased gradually from a mean did not (10). The more severe course after the lethal E. coli baseline value of 0.42% + 0.06% of the level in MA-NBP to challenge was also reflected in the changes of activation 1.07% + 0.09% at T+360 min (Fig. 2B), whereas at 24 h, parameters of the coagulation, fibrinolytic, and complement these levels had decreased to 0.66% + 0.11%. In the lethal systems (i.e., compared with baseline values, thrombin- group, the increase of ia2M levels was more pronounced, antithrombin III complexes increased 425- and 33-fold, plas- reaching a maximum value of 9.1% + 1.9% of the level of min-a2-antiplasmin complex levels increased 38- and 20- MA-NBP at T+360 min (Fig. 2B). This increase differed fold, and C5b-9 complex levels increased 12- and 3-fold in significantly from that in the sublethal group from T+ 120 min 5038 DE BOER ET AL. INFECT. IMMUN.

analysis) the levels of ia2M with those of plasmin-a2- antiplasmin, thrombin-antithrombin III, and elastase-al- antitrypsin complexes. All three complexes correlated sig- 1.5 nificantly with ia2M (Table 1). In particular, there was a 4-A strong correlation between ia2M and thrombin-antithrombin III (Fig. 3A) and between ia2M and plasmin-a2-antiplasmin 1.0 (Fig. 3B) complexes in the lethal group. Identification of the proteinases responsible for the inacti- C1 Fisublethall vation of ac2M in septic baboons. The results mentioned _0.5- above suggested that clotting and fibrinolytic proteinases were involved in the inactivation of at2M in the baboons. co However, we considered it necessary to obtain more direct evidence that at least part of ia2M had arisen from the 0.0 . . . . . interaction with fibrinolytic, clotting, and neutrophilic pro- teinases. We used the method developed by Harpel et al. 0 (25-27) and modified by Abbink et al. (2, 3) to identify which z10B- proteinases had formed complexes with ax2M in the septic baboons. ia2M species were purified from baboon plasma by absorption onto MAb Ml-Sepharose and incubated with the U A Isub~~~leth;l chromogenic substrates for plasmin (S2251), kallikrein 5 (S2302), thrombin (S2238), elastase (M4765), and cathepsin G (S7388) as described in Materials and Methods. The increase in ia2M in the sublethal group was too small to allow a further characterization by these assays (Fig. 4). However, in plasma samples from the lethal group, several a2M-proteinase complexes could be detected by using the o 60 120 180 240 300 360 chromogenic substrates. Levels of complexes between a2M time (minutes) and the neutrophilic proteinases (elastase and cathepsin G) FIG. 2. Mean levels (and standard errors of the mean) of total increased immediately after the start of the E. coli infusion, a2M (A) and inactivated a2M (B) in baboons that received intrave- reaching peak values at T+360 min (i.e., 5.08 ± 0.42 and 6.08 nously a lethal or a sublethal dose of E. coli. The differences in ia2M + 0.25 [mean + standard error of the mean] ,uM substrate between both groups at 120, 180, and 360 min were significant. conversion per h for a2M-elastase and a2M-cathepsin G complexes, respectively). The course of a2M-elastase com- plexes is shown in Fig. 4B. That of ot2M-cathepsin G was and thereafter. Thus, a significant inactivation of a2M oc- similar. Complexes of ot2M with plasmin, kallikrein, and curred in the animals challenged with a lethal dose of E. coli. values at of 19.21 + constituted thrombin reached peak T+120 min The inactivation in this group approximately 12.81, 31.17 + 15.86, and 47.00 + 15.90 ,uM/h, respectively. 10% of the total amount of a2M. The rate of a2M-plasmin complex formation is shown in Fig. Correlation between levels of ica2M and activation parame- 4A. Those of a2M-thrombin and ac2M-kallikrein were simi- ters of neutrophils, coagulation, and in septic lar. The known overlap of specificity of the chromogenic baboons. In previous studies (10, 11), we observed increased substrates (18), however, prompted further experiments to levels of thrombin-antithrombin III, plasmin-ot2-antiplas- identify more precisely which proteinases had formed com- min, and elastase-al-antitrypsin complexes in the plasma plexes with ao2M. Initially, we tested the specificity of the samples obtained from the same animals described in this chromogenic substrates to be used by incubating them with article. These findings indicated the formation or release of performed purified human a2M-proteinase complexes (Table activated proteinases of both the fibrinolytic and clotting 2). Complexes of a2M and thrombin, plasmin, and kallikrein systems and of neutrophils. To investigate whether the all hydrolyzed to some extent the chromogenic substrates inactivation of a2M was related to the action of these specific for each of these proteinases, whereas they did not proteinases, we correlated (by Spearman's rank correlation convert the substrates for elastase and cathepsin G. Con- versely, a2M-elastase and -cathepsin G complexes slightly hydrolyzed their mutual substrates, whereas they did not TABLE 1. Spearman's rank correlation analysis of ia2M levels react at all with the other substrates used (Table 2). Distinc- with plasmin-a2-antiplasmin, thrombin-antithrombin III, and two com- elastase-cal-antitrypsin complexes in baboons challenged tion between the neutrophil-derived proteinases in with a sublethal or lethal dose of E. coli plex with a2M could be made by using the chromogenic substrate S7388, since this substrate appeared to be rather Correlation of iax2M level in: specific for a2M-cathepsin G complexes (Table 2). Thus, the with dem- Total group of Lethal group of Sublethal group of experiments purified a2M-proteinase complexes Complex' baboons (n = 51)b baboons (n = 22) baboons (n = 29) onstrated that the chromogenic substrates were not entirely monospecific. Nevertheless, we used the results of these r P r P r P experiments to estimate the proteinase(s) in complex with PAP 0.5780 8.9x 10-6 0.7483 6.2 x 10-5 0.0121 0.95 a2M in the lethal group of baboons by assuming that the TAT 0.8421 9.7 x 10-15 0.9176 1.9 x 10-9 0.7778 6.7 x 10-7 ratio of hydrolysis of chromogenic substrates for each ot2M- ELAT 0.7965 4.6 x 10-12 0.6968 3.1 x 10-4 0.6515 1.7 x 10-4 proteinase complex would be constant and therefore spe- was that a PAP, plasmin-a2-antiplasmin; TAT, thrombin-antithrombin III; ELAT, cific. Thus, the hydrolysis of S2302 plotted against elastase-al-antitrypsin. of S2251 for both purified a2M-plasmin as well as a2M- bn, number of observations. kallikrein complexes. The values obtained with complexed VOL. 61, 1993 ALPHA-2-MACROGLOBULIN IN BABOON SEPSIS 5039

102 . 0L A B -l 103 I* *I* 0 * tn 0 : 0 co lo0 - * 0L) 0 *-9 U) * 0 0 0 x 102 ~E -0 .8 0 0 * 0 100 * 0 01.t I- lo,

inoL_ in-1 10 -1 10 10' 10-1 100 10' ia2m (% of MA-NBP) ia2m (% of MA-NBP) FIG. 3. Correlation between ia2M and thrombin-antithrombin III (TAT) complexes (A) and between ia2M and plasmin-at2-antiplasmin (PAP) complexes (B) in baboons challenged intravenously with a lethal dose of E. coli. Spearman's rank coefficients (r values) of the correlation between ia2M and TAT and between ia2M and PAP complexes were 0.9176 (P = 1.9 x 10-9) and 0.7483 (P = 6.2 x 10-5), respectively (n = 22). a2M in the baboons were then plotted in the same diagram. MA-NBP standard at 2 h and of 28.9 nmol/liter at 4 h, As shown in Fig. SA, values of ia2M from the baboons, in respectively (Fig. 6B). Similar changes in thrombin-an- particular at the higher levels, fell around the line of the tithrombin III, plasmin-a2-antiplasmin, ia2M, and elastase- purified a2M-plasmin complexes, suggesting that the hydro- al-antitrypsin complexes, although less pronounced, were lysis of S2302 in the baboons was due in part to the presence observed in another baboon that received a challenge, of a2M-plasmin complexes. The hydrolysis of S2251 and of consisting of 6.5 x 1010 heat-inactivated S. aureus organisms S2238 was plotted in a similar graph (Fig. 5B), which shows per kg (data not shown). Thus, the course of these parame- that almost all baboon data were between the lines of ters in the baboons challenged with S. aureus was similar to purified a2M-plasmin and a2M-thrombin complexes, indi- that in the animals that had received E. coli. cating that plasmin as well as thrombin complexes contrib- uted to ia2M in the lethal group of baboons. Taken together, DISCUSSION these experiments suggested that, in the lethal group of baboons, circulating ia2M consisted at least in part of In this study, we demonstrate that in a well-established a2M-plasmin, a2M-thrombin, a2M-cathepsin G, and a2M- model for sepsis (30, 55, 58), i.e., baboons challenged with elastase complexes. live E. coli, a2M is inactivated by the formation of com- a2M in baboons challenged with S. aureus. We had the plexes with proteinases. In addition, similar results were opportunity to study the changes of at2M in two baboons obtained in two animals challenged with S. aureus, suggest- challenged with S. aureus. In one baboon challenged with ing that the observed inactivation of a2M is not a particular 7.3 x 1010 CFU/kg of body weight, plasmin-a2-antiplasmin feature of E. coli sepsis. complexes increased to a peak level of 66% of the urokinase- A proteinase that is inhibited by a2M becomes physically treated MA-NBP standard (i.e., normal baboon plasma in entrapped in the inhibitor but still retains some of its activity which a maximal amount of plasmin-a2-antiplasmin com- against small synthetic substrates (4, 42, 49). Entrapment of plexes was generated [10]) at 2 h, whereas thrombin-an- the proteinase by a2M may prevent its detection in the tithrombin III complexes rose to 628 ,ug/liter at 8 h (Fig. 6A). complex by polyclonal or monoclonal antibodies. Therefore, Thus, like the animals challenged with E. coli (10), the we used the method developed by Harpel et al. (25-27) and baboon which had received S. aureus developed a procoag- modified by Abbink et al. (2, 3) to identify the proteinases ulant state 4 h after the challenge, which was characterized that had formed complexes with a2M in the septic baboons. by increasing thrombin-antithrombin III complexes and de- Conversion of chromogenic substrates was not observed creasing plasmin-a2-antiplasmin complexes. Inactivated with ia2M purified from plasma samples obtained before the a2M and elastase-al-antitrypsin complexes also increased challenge or obtained from animals challenged with a suble- in this animal, reaching peak levels of 19% of that in the thal dose of E. coli. In contrast, ia2M purified from plasma

TABLE 2. Hydrolysis of chromogenic substrates (final concentration, 2 mM) by 50 Ill of preformed a2M-proteinase complexes (1 ,uM) absorbed onto MAb Ml-Sepharose Chromogenic Hydrolysis (Lmol/h) by: substrate a2M-thrombin a2M-plasmin a2M-kallikrein a2M-elastase a2M-cathepsin G S2238 (thrombin) 2,805 1,355 1,932 92 76 S2251 (plasmin) 229 847 344 12 12 S2302 (kallikrein) 1,582 1,452 2,518 66 78 M4765 (elastase) NDa ND ND 1,248 520 S7388 (cathepsin G) 16 ND ND 5 153 a ND, not detected. 5040 DE BOER ET AL. INFECT. IMMUN.

phils. The nature of the proteinases that were responsible for conversion of the substrates for plasmin, thrombin, and kallikrein was more difficult to establish, since these sub- strates are all hydrolyzed to a variable extent by these three serine proteinases, as was also observed for preformed complexes with ao2M (Table 2). We think that at least thrombin and plasmin contributed to the inactivation of at2M for the following reasons. First, thrombin-antithrombin III complexes as well as plasmin-a2-antiplasmin complexes correlated strongly with total ioa2M (Fig. 3). Second, by 2sublethal using a radioimmunoassay for a2M-plasmin complexes (39), 0 we found immunochemical evidence for the presence of these complexes in plasma harvested from animals receiving 0 a lethal dose of E. coli (data not shown). Attempts to further characterize the a2M-proteinase complexes by SDS-poly- acrylamide gel electrophoresis and immunoblotting, how- ever, failed because of the limited number of plasma samples available for analysis. The observed conversion of the chromogenic substrate S2302 by ia2M in the baboons at first glance suggested involvement of kallikrein in the inactivation of a2M. How- l f I len halthal ever, comparison of the conversion of the substrates S2302 and S2251 by preformed ox2M-kallikrein and a2M-plasmin complexes with that by a2M complexes from the baboons (Fig. 5) suggested that most of the conversion of S2302 by ia2M was due to plasmin in complex with a2M. Pixley et al. (47) have shown that a2M-kallikrein complexes, measured sulta doeo . cl. . . . by an immunochemical assay (35), also increase in baboons challenged with a lethal dose of E. coli, and therefore, kallikrein very likely also contributes to the inactivation of a2M in these animals. In agreement herewith, conversion of EI.co.hydolyzedrseveralth chromogenic substrates (fig.4),mi S2302 and S2251 by ia2M in baboon plasma samples with that51thi ia2M atr lelasti art consisteb ofa2M-proteinase m moderately increased levels was between that by purified coplexes.prfe yasrto noM-ehrs rmpam kallikrein- and plasmin-a2M complexes (Fig. 5A), suggesting the presence of both plasmin- and kallikrein-at2M complexes in these samples. Our results do not allow definite conclusions regarding the TE. conihdolzdsversiolochromogenic substratesM75(eigas-) proportion of icx2M that had arisen by interaction with host proteinases. However, assuming that the concentration of a2M in the blood of humans and baboons is similar, i.e., 20 tase) and S7388 (cathepsin G) by preformed at2M complexes to 50 ,uM, and that the chromogenic substrates used are appeared to be rather specific for elastase-at2M and cathep- equally well converted by baboon proteinases as they are by sin G-al2M complexes (Table 2). This led us to conclude that their human counterparts, we could estimate (by comparing part of a2M in the septic baboons was inactivated by forming the conversion rate of the substrates by the preformed complexes with proteinases derived from activated neutro- human at2M complexes [Table 2] with that by ia2M in the

140

1-1 120 A I |alikreina2m| t- 1- 12 Jj rn 100 0 _Em0 r=] ,r_ 0 80 I - C\) cscM 60 , cs U] as) 00 .2U CM 0tn 40 , CM L- u) U c 20i 0) O 1z .-. .-. . I 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 conversion S2251 (imol/hr) conversion S2251 (timol/hr) FIG. 5. Plot of the conversion of the chromogenic substrates S2251 versus that of S2302 (A) or that of S2238 (B) by a2M-proteinase complexes in the group of baboons challenged with a lethal dose (n = 22). For comparison, results obtained with purified a2M-kallikrein, oa2M-plasmin, and a2M-thrombin complexes are also shown. VOL. 61, 1993 ALPHA-2-MACROGLOBULIN IN BABOON SEPSIS 5041

differences in the clearance of ia2M or to the shorter 800 z intervals between blood sampling, i.e., 1 h in the animal model versus 6 h in the patients (3). In addition, the intensity 600 of the trigger may have also contributed to the generation of 50 0 iot2M. The activation of the coagulation, fibrinolytic, and 0 complement systems and of neutrophils as well as the 4,~~~~~~~~~~~~~~~~~~~ concomitant changes in organ functions and hemodynamic 25- parameters occurs within hours after the challenge with E. 2063 coli (10, 11, 47). In contrast, in patients with sepsis, these processes tend to develop more slowly, evolving over days. 0. Most studies on the role of coagulation, fibrinolysis, and neutrophils in animal models for sepsis have been done in 30 animals challenged with E. coli or endotoxin. For example, there is no study documenting a procoagulant state, such as or in z elast-ailAT th that induced by endotoxin (54, 61) live E. coli (10),

20 0 gram-positive sepsis. The observations in the baboons chal- 0~~~~~~~~~~~~~~~~~ lenged with live S. aureus (Fig. 6) or heat-inactivated S. 9 Ka m ~~~~~~~15 aureus (data not shown) suggest that such a procoagulant state also occurs in gram-positive sepsis. The inactivation 3 6C 10 pattern of a2M in these animals was also remarkably similar 3 ~~~5- to that observed in the animals challenged with E. coli. These observations as well as a recent study in which rabbits 0 60 120 180 240 300 360 420 480 were challenged with Staphylococcus epidermidis (63) dem- time (minutes) onstrate that, with respect to pathogenic mechanisms, gram- FIG. 6. Course of thrombin-antithrombin III (TAT), plasmin- negative and gram-positive sepsis are similar conditions. oa2M-antiplasmin (PAP), and elastase-al-antitrypsin complexes In conclusion, we show that in baboons challenged with E. (elast-ctlAT) and of ia2M in a baboon challenged intravenously with coli and S. aureus, a2M is inactivated by forming complexes 7.3 x 1010 CFU of live S. aureus per kg of body weight. with proteinases derived from activated coagulation, fibrino- lytic and contact cascades, and activated neutrophils. These results suggest that a2M regulates the activities of these baboon plasma samples [see, for example, Fig. 4]) that the proteinases during the inflammatory response that occurs in highest concentrations of ia2M in the baboons receiving the sepsis. lethal dose of E. coli was approximately 2 ,M and that up to 0.2 ,uM, i.e., 10% of the total amount of ia2M, could be explained by the presence of complexes of a2M and the ACKNOWLEDGMENTS endogenous proteinases plasmin, thrombin, kallikrein, elas- We thank Wim Schaasberg and Ed Nieuwenhuis for their assis- tase, and cathepsin G. An explanation for this discrepancy tance in the statistical analysis of our data. between the total levels of ia2M and the levels of a2M This study was financially supported in part by grant 900-512-121 complexes actually measured in the baboon samples may be from the Netherlands Organization for Scientific Research. that proteinases other than those mentioned contributed to the inactivation as well. For example, recent studies suggest REFERENCES that activated also may form complexes with aL2M (28, 32). This proteinase is probably activated in the septic 1. Aasen, A. O., N. Smith Ericksen, M. J. Gallimore, and E. Amundsen. 1980. Studies on components of the plasma kal- baboons (56, 59). To what extent activated protein C con- likrein-kinin system in plasma samples from normal individuals tributes to the inactivation of aL2M remains to be established. and patients with septic shock. Adv. Shock Res. 4:1-10. Another explanation for the discrepancy between levels of 2. Abbink, J. J., A. 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