0031-3998/94/3504-0389$03.00/0 PEDIATRIC RESEARCH Vol. 35, No.4, 1994 Copyright © 1994 International Pediatric Research Foundation. Inc. Printed in U.S.A. Supplemental Complement Component C9 Enhances the Capacity of Neonatal Serum to Kill Multiple Isolates of Pathogenic Escherichia coli' HERBERT A. LASSITER, JESSICA L. WILSON, RICHARD C. FELDHOFF, JOHN M. HOFFPAUIR, AND KATHLEEN M. KLUEBER Division of Neonatology. Department ofPediatrics {H.AL. JL W.{. the Department ofAnatomical Sciences and Neurobiology {J.M.H.. K.M.K.]. and the Department of Biochemistry {R.C.F.]. University ofLouisville School of Medicine. Louisv ille. Kentucky 40292 ABSTRACf. Previous studies demonstrated that, com­ BPBS·, PBS with 1 mM MgCh and 0.5% BSA pared with adult serum, neonatal serum contained a dimin­ BPBS··, PBS with 1 mM MgCh, 0.5% BSA, and 0.15 ished concentration of complement component C9 and that mMCaCh supplemental C9 enhanced the capacity of neonatal serum to kill an isolate of Escherichia coli. Therefore, experi­ ments were designed to determine the mechanisms by which supplemental C9 enhances the bactericidal capacity of neonatal serum and to determine whether supplemental Escherichia coli is the second most common bacterium iso­ C9 enhances the capacity of neonatal serum to kill several lated from the blood of septic neonates (I, 2). Newborn infants different pathogenic strains of E. coli.A radiobinding assay acquire E. colisepsis for reasons that are not completely under­ and immunogold electron microscopy using a monoclonal stood. In the adult host, complement-mediated cytolysis (bac­ anti-C9 antibody revealed that, compared with 40% adult teriolysis) has been proposed as a mechanism of defense against serum, neonatal serum deposited a diminished quantity of invasion by gram-negative bacteria (3-5). However, compared C9 onto E. coli 07w:Kl:NM. Supplemental C9 (75 mgfL) with serum from adults, serum from human neonates kills E. significantly enhanced the quantity of C9 deposited by the coli inefficiently (6, 7) , The ninth component of complement neonatal serum. Treatment with 10 mM MgEGTA (a (C9) is a circulating glycoprotein that is required for efficient mixture of 100 mM MgCh and 100 mM EGTA that blocks complement-mediated cytolysis of E. coli(8-10). The concentra­ activation of the classic complement pathway but leaves tion of C9 is diminished in the sera of human neonates (II, 12). the alternative pathway intact) abolished the capacity of Moreover, supplemental C9 enhanced the capacity of sera from neonatal serum to deposit C9 and to kill the bacteria. newborn infants to kill a pathogenic isolate of E. coli(12). Supplemental C9 enhanced the capacity of neonatal serum The mechanism by which supplemental C9 increases the bac­ to kill eight different blood isolates of E. coli. Therefore, tericidal capacity of neonatal serum is not known. Also, whether supplemental C9 enhanced the capacity of neonatal serum supplemental C9 affects the bactericidal activity of neonatal to kill E. coli by increasing the total quantity of C9 depos­ serum against other isolates of E. coli is not known. Therefore, ited via activation of the classic complement pathway. experiments were designed to determine the effect of supple­ Neonatal serum contained sufficient quantities of classic mental C9 on the kinetics of bacterial killing by neonatal serum, pathway components, other than C9, to deposit the supple­ the deposition of C9 onto the surface of E. coli, and the capacity mental C9 onto E. coli and to enhance bacterial killing. of neonatal serum to kill several different isolates of pathogenic The bactericidal activity of neonatal serum against multiple E. coli. isolates of pathogenic E. coli was increased after C9 sup­ plementation. We speculate that C9 deficiency may beone MATERIALS AND METHODS of the defects in antibacterial host defense that predisposes neonates to the acquisition of E. coli sepsis. (Pediatr Res Buffers and reagents. The following buffers and reagents were 35:389-396,1994) used: Dulbecco PBS (Media Tech, Washington, DC), pH 7.4; BPBS+; BPBS++; 100mM MgEGTA (Sigma, St. Louis, MO), pH 7.4; and tryptic soy broth (Difco Laboratories, Detroit, MI). Abbreviations Buffers used in the radiobinding assays and the immunogold MgEGTA, 100 mM MgCh and 100 mM EGTA assays contained 0.02% sodium azide (Sigma). Buffers used in minimum concentration of adult serum required to the bactericidal assays contained no bacteriostatic agents. kill 90% of an isolate of Escherichia coli during 90 min Bacteria. Eight isolates of E. coliwere obtained from the blood of incubation of eight septic neonates who were patients at the Neonatal Intensive Care Unit of Kosair Children's Hospital, Louisville, Received June I. 1993: accepted November 19. 1993. KY. The serotype of each organism was determined at the E. Correspondence and reprint requests : Herbert A. Lassiter. M.D.•Division of Neonatology. Department of Pediatrics. Un iversity of Louisville School of Medi­ coli Reference Center of Pennsylvania State University, Univer­ cine, Louisville. KY 40292. sity Park, PA (Table I). The bacteria were grown overnight and Supported by grants from the Division of Neonatology. Department of Pediat­ stored in 5-mL aliquots of tryptic soy broth at - 70"C. Before an rics, University of Louisville School of Medicine. and the Alliant Community Trust experiment, 35 mL of fresh broth were added, and the bacteria Fund. Louisville. Kentucky. were incubated at 37"C for 3 to 4 h to bring the organisms to log I Presented in part at the 61st annual meeting of the Society for Pediatric Research, Baltimore , MD, May 1992 and at the 62nd annual meeting of the Society phase. The bacteria were then washed three times in BPBS" or for Pediatric Research , Washington, DC. May 1993. BPBS+ and adjusted by absorbance at 600 nm to a concentration 389 390 LASSITER ET AL. Table I. Characteristics ofE. coli isolates" 5.,-----------------------, Isolate LC (%) Serotype 90 I 20 07w:KI:NM ll.i 4 2 30 O(-):H(-) "0 3 40 07w:K I:8w 0­ M 4 60 O(-):KI:H(-) III 5 30 OI :KI:NM ,.: 6 30 O(-):KI:NM 7 40 O{-):H(-) en 2 8 50 07:KI:NM o E * Each isolate was obtained from the blood of a septic neonate. ep 1 Cl Not 140 C Delacled • NEONATAL SERUII AdS AdS NooS NooSCg NooSCg II NEONATAL SERUII • ct 120 + + § • ADULT SERUII MgEGTA MgEGTA 100 I&i Fig. 3. Effect of supplemental C9 on the deposition of C9 onto E. -' 0( 80 coliby neonatal serum. E. coli07w:KI:NM were incubated with pooled adult serum (AdS), AdS treated with MgEGTA, pooled neonatal serum a: :;:) 10 (NeoS), NeoS supplemented with C9 (NeoSC9), and C9-supplemented en NeoS treated with MgEGTA . As determined by radiobinding assay, the 40 results are depicted as the mean (+ SE) Quantity of 12' 1-labeled anti-C9 antibodies specifically bound to the bacteria (n = 3). *, p < 0.0 I vsadult 20 serum ; .., p < 0.01 vs pooled neonatal serum. C9 deposition onto E. coli, mediated by the classic complement pathway, was inefficient in the 15 30 45 10 75 10 pooled neonatal serum but was enhanced by supplemental C9. TIME (MINUTES) The serum from each neonate contained lessthan 0.1 g/L of IgA Fig. I. Effect of supplemental C9 on the bactericidal activity of and less than 0.2 gjL of IgM, as determined by single radial neonatal serum. E. coli 07w:KI:NM were incubated in pooled neonatal immunodiffusion (Endplate IgA and Endplate IgM, Kallestad, serum (diamonds), pooled neonatal serum supplemented with C9 Austin, TX) (13). Most experiments used serum that was pooled (squares), and pooled adult serum (circles). As determined by bactericidal from the blood of the 20 neonates. Sera were also obtained and assay, the values are depicted as the mean bacterial survival ± SE. *, p < pooled from five healthy adult volunteers. The serum was sepa­ 0.05 vs adult serum and p < 0.05 vs C9-supplemented neonatal serum. rated from the clotted blood by centrifugation and passed The onset of bacterial killing was delayed in neonatal serum compared through a filter to ensure the removal of bacterial con­ with adult serum. However, in neonatal serum, supplemental C9 accel­ taminants. Individual sera and the pooled serum were stored at erated the onset of bacterial killing. - 70·C in sterile vials. Complement component C9. Purified C9 (Lot no. A40TT2390 I) was purchased from Quidel Corporation (San I Diego, CA) as a solution containing 1000 of C9jmL of PBS without bacteriostatic agents. This C9 preparation was isolated I&i 5 by the method of Biesecker and Muller-Eberhard (14) and was ·0 0- subjected by the manufacturer to an antiimpurities solid-phase M 4 1ft immunoaffinity column containing the IgG fraction isolated ,.: from monospecific goat antihuman C3, -C5, -C6, -C7, and -C8 i 3 to insure the complete removal of these complement proteins. fj 2 SDS-PAGE and hemolytic assays by the manufacturer demon­ strated that more than 95% of the protein in the solution was • 1 pure C9 and that the solution was completely free ofC3, C5, C6, ( C7, and C8 functional activities. ell To confirm the purity of the C9 preparation used in these C 10 studies, various assayswere also performed in our laboratory. By Fig. 2. Deposition of C9 onto E. coli by neonatal sera. E. coli nephelometry (Beckman Array Protein System, Beckman Instru­ 07w:KI:NM were incubated with pooled adult serum (AdS) and with ments, Brea, CA), the solution did not contain detectable IgA, sera from 10 healthy neonates (numbers 1-10). As determined by ra­ IgM, or IgG. Because neonatal serum normally contains dimin­ diobinding assay, the results are depicted as the mean (+ SE) Quantity of ished concentrations of various complement components, such '2sl_labeledanti-C9 ant ibodies specifically bound to the bacteria (n = 4).