Kinetics of Assembly andDecay of Complement Components on

INFECTION AND IMMUNITY, Aug. 1985, p. 402-406 0019-9567/85/080402-05$02.00/0 Copyright C) 1985, American Society for Microbiology

Vol. 49, No. 2

Kinetics of Assembly and Decay of Complement Components on Escherichia coli O111:B4 Preparation of Stable Intermediates GIANDOMENICO ROTTINI,* FRANCESCO TEDESCO, MARINA BASAGLIA, LUCIA RONCELLI, PIERLUIGI PATRIARCA

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Istituto di Patologia Generale, University of Trieste, Trieste, Italy Received 28 January 1985/Accepted 29 April 1985

The preparation of bacterial intermediates bearing complement components at various steps of the complement sequence was investigated by suspending immunoglobulin M-opsonized Escherichia coli Oll1:B4 cells in complement-deficient sera at different temperatures and ionic strengths. The optimal conditions for the formation of the intermediates at T,n were found to be an ionic strength of 0.091 , and a temperature of 37°C, except for BAC142, which could be formed equally well at room temperature. In contrast to all the other intermediates, which, once formed at Tmax, were stable in the presence of the whole serum, BAC142 decayed with a half-life of 10 min due to the lability of bound C2. Washing with a buffer of either 0.091 or 0.046 ,u did not affect the bacterial intermediates, with the exception of BAC1-3 formed either in the presence of CS-deficient serum or with purified C3 added to BAC142. All the intermediates were found to be stable after incubation in 0.091-,u buffer for 30 min at 37°C.

In .previous studies we provided evidence for the contribution of surface-bound complement components to the intracellular killing of Escherichia coli O111:B4 by human polymorphonuclear leukocytes (PMN) (15, 21). This novel effect of the human complement system was found to depend on the components of the complement sequence assembled on the surface of the bacteria, since bound C5 and C8 promoted killing, whereas C3 and C7 were ineffective in this regard (21; G. D. Rottini, F. Tedesco, L. Roncelli, and P. Patriarca, Eur. J. Clin. Invest. 14:33, 1984). For these investigations, bacterial intermediates (BACs) bearing components in functionally active form were employed as targets to test the killing effect of intact PMN and their granule extracts. In the course of these studies problems with the stability of the BACs under the conditions of the bactericidal assay were often encountered. As the BACs varied in their susceptibility to PMN-dependent killing, it was essential to maintain them in a stable form throughout the experimental procedure to eliminate the risk of a susceptible intermediate being transformed into a nonsusceptible type. The aim of the present paper is to analyze the optimal conditions for the preparation of stable BACs. These conditions may differ from those required to obtain the corresponding erythrocyte intermediates in view of the more complex interactions known to occur between the surface of the bacteria and the activated complement components (9, 18). The kinetics of assembly of the components in the various steps of the complement reaction, the effect of the washing procedure with buffers of different ionic strengths, and the degree of decay of the BACs under the conditions of the experimental assay will also be considered.

0.179 ,u. When buffers of lower ionic strength were required, KRP buffer was mixed with a solution of 9% sucrose containing 0.64 mM Mg2' and 0.16 mM Ca2' at a ratio of 1:1 or 1:3 (vol/vol) to obtain buffers of 0.091 and 0.046 L, respectively. Bacteria. The strain of E. coli O111:B4 employed in this study was obtained from the stock collection of the Institute of Microbiology, University of Trieste. The bacteria were grown in nutrient broth (Difco Laboratories, Detroit, Mich.) for 6 h at 37°C and then on nutrient agar (Difco) overnight. These culture conditions were chosen to obtain viable bacteria in the stationary phase, thus avoiding dilution in the surface-bound complement components as a result of bacterial multiplication. The methods for purifying the immunoglobulin M (IgM) fraction from the rabbit antiserum against E. coli O111:B4 (Behringwerke, Marburg, West Germany) and for the preparation of IgM-opsonized bacteria (BA) have been reported previously (21). Complement-deficient sera. The following human sera with selected deficiencies of complement components were used throughout this study. C3-deficient serum was obtained from a healthy young girl, to be described elsewhere, who was found to have about 1% of the normal content of C3 due to the presence of C3 nephritic factor in the serum. The C5-deficient serum was a generous gift from P. Densen (University of Iowa Hospitals and Clinics, Iowa City, Iowa) and was obtained from a patient with recurrent meningococcal infections. The patients providing the C6- and C8deficient sera have been reported previously (19, 22). All sera had a selected complement deficiency with normal or slightly reduced levels of all other components. The deficient was

sera were used in the assays at final dilutions at which no direct bactericidal activity was observed except in the presence of the missing components, the addition of which produced maximal killing of BA. Complement reagents. Partially purified human C3 and C5 were purchased from Cordis Laboratories, Miami, Fla. Highly purified C8 and C9 were isolated as described by Steckel et al. (17) and Biesecker and Muller-Eberhard (1), respectively. A reagent providing functionally active C6, C7, C8, and C9 was prepared by treating pooled human sera with

MATERIALS AND METHODS

Buffers. Krebs-Ringer phosphate (KRP) buffer, pH 7.4, containing 16.6 mM sodium phosphate, 119 mM NaCl, 4.9 mM KCI, 0.44 mM MgCl2, and 0.16 mM CaCl2 was used. The ratio of NaH2PO4 to Na2HPO4 in the KRP buffer was 1:5 (vol/vol), and the ionic strength of the KRP buffer, calculated on the basis of the molar concentration of the ionized species, *

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KSCN and hydrazine hydrate, following the procedure described for guinea pig serum by Dalmasso and MullerEberhard (4). Natural antibodies against E. coli 0111:B4 were removed from human serum by absorption as previously described (15). Preparation of BACs. The procedure used to investigate both the kinetics of assembly of the complement components on BA and the stability of the BACs is shown in Fig. 1. The time required for the optimal binding of complement components to the bacteria (Tmax) was established by measuring the killing of BA, which had been incubated in the presence of complement-deficient sera for various lengths of time, after the addition of the remaining components necessary to complete the complement sequence. The stability of the BACs was ascertained by evaluating the percentage of killing of the BACs formed at Tmax with the complementdeficient sera, washed and subsequently incubated in buffer for various lengths of time before addition of the remaining complement components. Bactericidal assay. The susceptibility of the BA to complement-dependent killing was estimated by incubating 5 x 105 BACs with the corresponding revealing system that provided the missing component(s) to a total volume of 150 ,ul at 37°C for 30 min. The revealing system for the various intermediates contained 15 50% hemolytic complement (CH50) units of the required components and 10 ,u of the reagent containing C6 through C9 when needed. The CH50 units of C3 and C5 were those indicated by the manufacturer (Cordis Laboratories). The CH50 units of C8 and C9 indicated the amount of purified component that was able to lyse 50% of the cells in a test system containing 1.25 x 107 sensitized erythrocytes to a total volume of 250 RI, as previously reported (20). The number of killed bacteria was evaluated by the method of dilution and counting of CFU in nutrient agar plates, using the formula [100 (CFU with reagent/CFU with KRP)] x 100. -

RESULTS Kinetics of formation of BAC142, BAC1-3, BAC1-5, and BAC1-7. This first series of experiments was performed to establish the Tma required for-the highest number of bacteria to reach the desired intermediate state. To this end, mixtures of BA and various complement-deficient sera were incubated at different temperatures and at an ionic strength of 0.091 ,u. This ionic strength was chosen because preliminary experiments had shown that it allows the optimal complement-dependent killing of BA by absorbed human serum. Samples of bacterial suspension were collected at various time intervals and tested for surface-bound complement

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components by measuring the killing of bacteria after incubation with a reagent that provided all the remaining components. The assembly of C3 convertase on BA, as judged from BAC142 formation, proceeded at a fast rate at 37°C as well as at 30°C and at room temperature (RT), being in all

instances complete within 2 min (Fig. 2). The activity of stirface-bound C3 convertase, however, decayed rapidly at both 37 and 30°C, with a half-life of ca. 10 min, whereas it remained stable at RT for at least 40 min. A longer time was needed for the binding to BA of components that were activated beyond C42, for which Tmax ranged from ca. 12 min for BAC1-3 formation to 25 min for BAC1-5 and BAC1-7 formation. No major differences were found in the rate of formation of BAC1-3, BAC1-5, and BAC1-7 at either 37 or 30°C, whereas the number of bacteria reaching these intermediate states at RT was definitely lower even after prolonged incubation. BAC1-3, BAC1-5, and BAC1-8 could also be prepared by incubating BAC142, BAC1-3, or BAC1-7, prepared at Tma, as described above, with purified C3, C5, or C8, respectively. This enabled us to study the kinetics of the binding of the individual components. It was found that the assembly of C8 on BAC1-7 occurred in less than 2 min at 37°C, whereas longer times were required for the binding of C3 to BAC142, about 9 min, and for the binding of C5 to BAC1-3, about 15 min (data not shown). Effect of washing on preparation of the BACs. The possibility that the washing procedure might affect the stability of the BACs as result of elution or decay of the active components from the surface of the bacteria was investigated by using two wash buffers, of 0.091 and 0.046 IL. BA were incubated with the complement-deficient sera, and at Tmax samples of the bacterial suspensions were examined for the surface-bound complement components after two washes with one of the two buffers (Fig. 3). The BACs were not modified by washing, except for BAC1-3 which was significantly unstable with the 0.091- but not the 0.046-,u buffer. This instability of BAC1-3 was even more marked when the intermediate was prepared from BAC142 and purified C3, since the number of bacteria in the state of BAC1-3 decreased significantly after washes with either the 0.091- or 0.046-p. buffer (data not shown). Effect of incubation on stability of BACs. Having established the optimal conditions for preparing the BACs, it was important to investigate their stability under the subsequent conditions of experimental use. To this end, the various BACs were prepared by incubating BA with the appropriate complement-deficient serum for the required Tmax and then washed with the 0.046-,u buffer. The BA were subsequently

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FIG. 2. Kinetics of assembly of complement components on the surface of BA. BA (3 x 107) were incubated with the complementdeficient sera at a final dilution of 1:30 for the C3-deficient serum (A), 1:40 for the CS-deficient serum (B), 1:30 for the C6-deficient serum (C), and 1:20 for the C8-deficient serum (D). The mixture was brought to a final volume of 300 pJ with 0.091-,u KRP buffer and incubated at either 37°C (0), 300C (U), or RT (A). At various time intervals 25-plI samples were removed and diluted with cold buffer to achieve at least a 1:500 dilution of the original amount of complement-deficient serum. The number of bacteria in the intermediate state was evaluated by incubating 100 1tl of the bacterial suspension with the revealing system to a total volume of 150 pul for 30 min at 370C.

incubated at 37°C in the presence of either the 0.091- or 0.179-,u buffer. Samples were then collected at various time intervals and mixed with the revealing reagent as described above. The results (Fig. 4) clearly indicate that the BACs were fairly stable in the 0.091-p. buffer, and only a slight decrease in BAC1-3, about 20%, was observed toward the end of the 30-min incubation time. In contrast, incubation with the 0.179-p. buffer led to a more marked decrease of BAC1-3 and BAC1-5. DISCUSSION The preparation of cellular intermediates bearing complement components at various steps of the complement sequence can be greatly facilitated by the use of complementdeficient sera, which avoids the laborious procedures of isolating the components in highly purified form. Nonetheless, it is not always easy to obtain stable intermediates,

particularly when the active comnponents are assembled on targets with complex structures such as the bacterial cell wall (9, 18). The results of our study demonstrate that stable intermediates can be prepared with E. coli O111:B4 by adjusting the conditions for the reaction of BA with complement-deficient sera for time and temperature of incubation and by using buffers of well-defined ionic strength. The rapid assembly of the C3 convertase on the surface of BA, which occurred in less than 2 min and independently of the incubation temperature, confirmed previous results obtained with sensitized sheep erythrocytes (12). Likewise, the decay of BAC142 with a half-life of about 10 min resembles that seen on the erythrocytes (2) and is similarly due to the lability of C2, since the decayed intermediate could be killed by guinea pig C4-deficient serum (data not shown). An intrinsic regulation of the C3 convertase that results in the dissociation of C2 from the C42 complex does not explain

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triggers the alternative pathway. This in turn promotes the assembly of the C3 convertase of the alternative pathway, which is known to be resistant to the action of the C3b inactivator when present on the bacterial cell wall (Fearon, J. Immunol. 120:1772, 1978). The formation of the more stable C3 convertase of the alternative pathway may also explain the relative stability of BAC1-3 prepared with the C5-deficient serum compared with that of BAC1-3 obtained with BAC142 and purified C3. The stability of the trimolecular complex C567 on BA confirmed similar findings previously reported for sheep erythrocytes (11). In contrast, the observation of stable BAC1-5 was somewhat unexpected in view of the reported instability of this intermediate on sheep erythrocytes, which is apparently due to the lability of bound C2 (3). This instability, however, may well depend on the target, as Rother et al. (14) have provided evidence for a stable intermediate carring C5 on chicken erythrocytes. In conclusion, stable BACs form better at 37°C, except for BAC142, for which the temperature of choice is RT. In addition, the BACs remained more stable when washed in the 0.046-,u buffer after their formation at Tmax and when incubated at 37°C in 0.091-,u buffer. Some BACs proved to be more stable than the corresponding BACs on sheep erythrocytes, as was the case for BAC1-3 and BAC1-5. This

FIG. 3. Effect of the washing procedure on the stability of the BACs. BA were incubated with the complement-deficient sera as indicated in the legend to Fig. 2, except for BAC1-8, which was obtained by incubating 107 BAC1-7 with 100 CH50 units of purified C8. At T x samples of the bacterial suspension were diluted and washed twice with 50 volumes of either 0.091-,u (open bars) or 0.046-p,(hatched bars) KRP buffer. The percentage of intermediates remaining after the washing process was evaluated as described in the legend to Fig. 2.

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the decay of BAC142, because this intermediate, once formed at Tma,, and subsequently washed, remained stable after further incubation at 37°C. For the same reason, a decay-accelerating factor that might be present in the bacterial cell wall, similar to those found in the stromata of both human and guinea pig erythrocytes (5, 7, 8, 13), is unlikely to be responsible for the lability of bound C2. An alternative possibility, though not proven in our case, is that the C4-binding protein present in whole serum might displace C2 from the C42 complex by binding to C4b (6). Whatever the reason for the decay of BAC142 at both 37 and 30°C, we took advantage of the stability of BAC142 formed at RT and chose this temperature for the preparation of the intermedi-

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The formation of BAC1-3 and BAC1-5 was temperature dependent and occurred predominantly at 37 and 30°C and, to a much lower extent, at RT. Unlike BAC142, however, BAC1-3 formed in the presence of serum was stable at 37°C for up to 40 min of incubation. This result, similar to that obtained by Joiner et al. (10) with 125I-labeled C3 bound to presensitized E. coli O111:B4 cells, contrasts with the wellknown instability of C3 on other target cells, such as sheep erythrocytes, which is due to the action of the C3b inactivator (16). It may be possible that the C5 convertase of the classical pathway is protected by the bacterial cell wall in a manner similar to that of the C5 convertase of the alternative pathway (D. T. Fearon, J. Immunol. 120:1772, 1978). However, the lability of the C42 complex, which is part of the C5 convertase, on the bacterial surface does not support this hypothesis. It is more likely that the binding of C3 to BA induced by the C3 convertase of the classical pathway

TIME (MIN) FIG. 4. Effect of incubation of BACs at 37°C with buffers of different ionic strength. The BACs were washed at Tmax twice with 0.046-p. KRP buffer and then incubated with either 0.091-p. (A) or 0.179-p. (B) buffer. Samples were removed at various time intervals, and the number of intermediates was evaluated as described in the legend to Fig. 2.

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most likely reflects structural differences between the two targets and possibly results from different types of interactions between the complement components and the cell membranes (9, 18). The investigation on the preparation of BACs, besides providing information on the relationship between the complement components and the bacterial surface, is a necessary prerequisite to understand the contribution of surface-bound complement components to the bactericidal effect of intact PMN and PMN extract (15, 21; Rottini et al., Eur. J. Clin. Invest. 14:33, 1984). This study is in progress in our laboratory. ACKNOWLEDGMENTS This work was supported by grant 83.00678.52 from the National Research Council of Italy (targeted project on the control of infectious diseases) and by grants from the Ministry of Public Education of Italy (MPI 40% and 60%). LITERATURE CITED 1. Biesecker, G., and H. J. Muller-Eberhard. 1980. The ninth component of human complement: purification and physicochemical characterization. J. Immunol. 124:1291-1296. 2. Borsos, T., H. J. Rapp, and M. M. Mayer. 1961. Studies on the second component of complement. I. The reaction between EAC'14 and C'2: evidence on the single site mechanism of immune hemolysis and determination of C'2 on a molecular basis. J. Immunol. 87:310-325. 3. Cooper, N. R., and H. J. Muller-Eberhard. 1970. Reaction mechanism of human C5 in immune hemolysis. J. Exp. Med. 132:775-793. 4. Dalmasso, A. P., and H. J. Muller-Eberhard. 1966. Hemolytic activity of lipoprotein-depleted serum and effect of certain anions on complement. J. Immunol. 97:680-685. 5. Fearon, D. T. 1980. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte and monocyte. J. Exp. Med. 152:20-30. 6. Gigli, I., T. Fujita, and V. Nussenzweig. 1979. Modulation of the classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator. Proc. Natl. Acad. Sci. U.S.A. 76:6596-6600. 7. Hoffmann, E. M. 1969. Inhibition of complement by a substance isolated from human erythrocytes. I. Extraction from human erythrocyte stromata. Immunochemistry 6:391-403. 8. Hoffmann, E. M. 1969. Inhibition of complement by a substance isolated from human erythrocytes. II. Studies on the site and mechanism of action. Immunochemistry 6:405-419. 9. Joiner, K. A., E. J. Brown, and M. M. Frank. 1984. Complement and bacteria: chemistry and biology in host defence.

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Annu. Rev. Immunol. 2:461-491. 10. Joiner, K. A., R. C. Goldman, C. Hammer, L. Leive, and M. M. Frank. 1983. Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli O111:B4 by the alternative complement pathway without increasing C5b-9 deposition. J. Immunol. 131:25632569. 11. Lachmann, P. J., and R. A. Thompson. 1970. Reactive lysis: the complement-mediated lysis of unsensitized cells. II. The characterization of activated reactor as C56 and the participation of C8 and C9. J. Exp. Med. 131:643-657. 12. Mayer, M. M. 1971. Complement and complement fixation, p. 181-184. In E. A. Kabat and M. M. Mayer (ed.), Experimental immunochemistry. Charles C Thomas, Publisher, Springfield, Ill. 13. Nicholson-Weller, A., J. Burge, and K. F. Austen. 1981. Purification from guinea pig erythrocyte stroma of a decayaccelerating factor for the classical C3 convertase, C4b,2a. J. Immunol. 127:2035-2039. 14. Rother, U., G. Till, and R. Thumb. 1973. Studies on the reaction of the sixth component of rabbit complement. Z. Immun Forsch 146:260-273. 15. Rottini, G. D., F. Cian, F. Tedesco, G. De Nicola, and P. Patriarca. 1979. Effect of antibodies and complement on the interaction between Escherichia coli O111:B4 and polymorphonuclear leukocytes. Infection 7:160-165. 16. Ruddy, S., and K. F. Austen. 1969. C3 inactivator of man. I. Hemolytic measurement by the inactivation of cell-bound C3. J. Immunol. 102:533-543. 17. Steckel, E. W., R. G. York, J. B. Monahan, and J. M. Sodetz. 1980. The eighth component of human complement. Purification and physicochemical characterization of its unusual subunit structure. J. Biol. Chem. 255:11997-12005. 18. Taylor, P. W., and H. P. Kroll. 1984. Interaction of human complement proteins with serum-sensitive and serum-resistant strains of Escherichia coli. Mol. Immunol. 21:609-620. 19. Tedesco, F., M. Bardare, A. M. Giovanetti, and G. Sirchia. 1980. A familial dysfunction of the eighth component of complement (C8). Clin. Immunol. Immunopathol. 16:180-191. 20. Tedesco, F., P. Densen, M. A. Villa, B. H. Petersen, and G. Sirchia. 1983. Two types of dysfunctional eighth component of complement (C8) molecules in C8 deficiency in man. Reconstitution of normal C8 from the mixture of two abnormal C8 molecules. J. Clin. Invest. 71:183-191. 21. Tedesco, F., G. D. Rottini, and P. Patriarca. 1981. Modulating effect of the late-acting components of the complement system on the bactericidal activity of human polymorphonuclear leukocytes on E. coli 0111:B4. J. Immunol. 127:1910-1915. 22. Tedesco, F., C. M. Silvani, M. Agelli, A. M. Giovanetti, and S. Bombardieri. 1981. A lupus-like syndrome in a patient with deficiency of the sixth component of complement. Arthritis Rheum. 24:1438-1440.