0031-399819 1/3001-0118$03.00/0 PEDIATRIC RESEARCH Vol. 30, No. 1, 1991 Copyright O 199 1 International Pediatric Research Foundation, Inc. Printed in U.S.A. The Role of C3 in Mediating Binding and Ingestion of Group B Streptococcus Serotype I11 by Murine Macrophagesl GARY J. NOEL, STEVEN L. KATZ, AND PAUL J. EDELSON Department of Pediatrics, Division of Pediatric Infectious Diseases and Immunology, Cornell University Medical College, New York, New York 10021 ABSTRACT. To understand how complement effects role with encapsulated pneumococci and Haemophilus influen- phagocytosis of type I11 group B streptococcus, we as- zae has underscored that complement-mediated phagocytosis is sessed the specific role of C3 in mediating binding and important in defending against these pathogens (2-6). These ingestion of these bacteria by macrophages. Phagocytosis studies are consistent with the hypothesis that clearance of com- of bacteria by resident mouse peritoneal macrophages was plement-opsonized bacteria from the circulation by mononuclear measured under conditions in which C3 deposition on bac- phagocytes composing the reticuloendothelial system is essential teria was inhibited or after blockade of C3-ligands or of in defending against bacteremia and in preventing disseminated complement receptor type three (CR3) with specific anti- infection. This complement-mediated host defense is likely to be bodies. C3 depletion, incubation with F(abf)2 fragments of particularly important in the nonimmune patient who must clear antibody to C3, or blockade of CR3 completely inhibited bacteria from the blood in the presence of little or no antibacterial the binding of bacteria that was seen in the presence of antibody. nonimmune serum. Immune serum increased the number Complement opsonization is generally considered to be im- of associated organisms 6-fold compared to that seen with portant in defending against group B streptococcal infection. In nonimmune serum. With this serum, 82% of organisms vitro studies measuring neutrophil chemiluminescenceor neutro- were ingested. C3 depletion or CR3 blockage had a modest phil-directed bacterial killing suggest that complement enhances effect, but this interaction could be ablated completely only antibody-mediatedphagocytosis (7-1 5). Although the newborn's after Fc receptors were blocked. Using varied concentra- unique susceptibility for disseminated group B streptococcal tions of an IgG2a MAb against type I11 capsular antigen, infection has been associated with a relative complement defi- it was possible to show that small amounts of antibody ciency (16, 17), the precise role for complement in defending the incapable of mediating bacterial binding by itself directed newborn against group B streptococci is not known. Based on an interaction that also depended upon C3. Phagocytosis work with other encapsulated bacteria, however, it is likely that of group B streptococci by macrophages in the presence of complement serves as an important opsonin, mediating phago- little or no antibody requires complement and C3 opsoni- cyte-bacteria interactions in the presence and in the absence of zation specifically. C3-dependent binding may be important type-specific antibody. in determining mononuclear phagocyte-dependent clear- In the work presented in this report, we examined the role of ance of these pathogens from blood, particularly in patients complement in determining the binding and ingestion of type with little or no type-specific serum antibody. (Pediatr Res 111 group B streptococci by resident peritoneal mouse macro- 30: 118-123,1991) phages. We characterized the degree of binding and ingestion in fresh serum and examined the specific roles of C3 and CR3 in Abbreviations this interaction. Because the newborn is likely to depend on host defense mechanisms that occur in the presence of little or no CR3, complement receptor type 3 type-specific antibody (18, 19), this study was additionally di- Ni-S, nonimmune serum rected at examining complement's role in phagocytosis that Im-S, immune serum occurs in Ni-S and serum containing varied concentrations of CoVF, cobra venom factor type-specific antibody. Macrophages were studied because it is PB, phagocytosis buffer likely that interaction of encapsulated bacteria with mononuclear i.p., intraperitoneal phagocytes determines clearance of these pathogens from the HBSS, Hanks' balanced salt solution bloodstream (14, 20, 21). MATERIALS AND METHODS Organisms. An isolate of group B streptococcus serotype I11 Serum complement proteins play a critical role in host defense was provided by Dr. Jose I. Santos (Hospital Infantil de Mexico, against a variety of microbes (1). Recent work examining this Mexico, DCpartemento FCdiral). This isolate has been previously characterized and studied (22). Streptococcal group of this isolate Received November 9, 1990; accepted February 26, 199 1. Correspondence: Gary J. Noel, M.D., N-834, The New York Hospital-Cornell was confirmed before testing using FITC conjugated antibody to Medical Center, 525 East 68th Street, New York, NY 10021. group B antigen (Difco Laboratories Inc., Detroit, MI). The LD,, Supported in part by U.S. Public Health Grant A119888-041 and BRS grant of this strain after i.p. inoculation in 5-d-old infant rats was 5 x support 2S07 RR05398-27. G.J.N. is a recipient of a Clinical Associate Physician lo3 colony forming units. Organisms were stored at -70°C in Award and is supported in part by U.S. Public Health Grants 1 MO 1 RR06020 1-0 1 and A130063-01. skim milk broth (Difco). Shavings of frozen broth were inocu- ' Presented in part by S.L.K. as his Medical Research Thesis at Cornell University lated onto trypticase soy agar with 5% sheep blood (BBL Micro- Medical College, May 1989. biology Systems, Cockeysville, MD) and incubated overnight at .18 C3 MEDIATES GBS-MACROPHAGE INTERACTION 119 37°C. Colonies were scraped from plates and incubated for 4-5 Monolayers were adhered to glass coverslips placed in the bottom h at 37°C in 1 mL of Todd-Hewitt Broth (Difco) containing 0.02 of 24-well Falcon tissue culture plates. The number of cells mCi of tritiated thymidine (sp act 20 Ci/mmol; New England adherent to coverslips ranged from 8 x 104 to 1.3 x 10'. This Nuclear, Boston, MA). Labeled organisms were washed three was determined for each day's experiment by counting cells in times in PB [HBSS (Gibco, Grand Island, NY) containing 0.2% each of five representative fields with an inverted microscope glucose, 0.02% gelatin]. Label uptake was determined for each and extrapolating to total coverslip surface area. experiment and ranged between 400 and 2 100 colony forming Binding and ingestion of organisms to macrophages was de- units/cpm. termined by a radiobinding assay similar to that previously Serum. Serum was obtained from CDF-1 mice as previously described by us (3). Between 8 x lo6 to 2 x lo7, radiolabeled described (6). Ni-S was obtained from adult CDF-1 mice (20-25 organisms were added to macrophage monolayers in the absence g; Charles River Breeding Laboratories, Wilmington, MA). or presence of serum or antibody in PB in a total volume of 0.3 Im-S was obtained from adult mice 2 wk after a second i.p. mL. After incubation, monolayers were washed four times in PB inoculation of group B streptococcus type 111. Serum was ob- to remove unbound bacteria. Coverslips were then removed and tained from CoVF (Cordis Laboratories, Miami, FL)-treated, placed in 3.5 mL of Aquassure (New England Nuclear) and nonimmune mice 20 h after a 5-U CoVF i.p. inoculation. Serum counted. The number of organisms associated with macrophages obtained from these animals had no detectable C3 fixation titer was calculated by dividing the number of bacteria associated (s1:2) and had less than 20 mg/dL of C3 as determined by a with each coverslip (counts per coverslip x colony forming units radial immunodiffusion method (6). Serum was heat-inactivated per count) by the number of macrophages per coverslip. Exper- by incubation for 60 rnin at 56°C. This serum had no functional iments were done in duplicate and the results from each day's complement activity as measured by C3 fixation to rabbit red experiment were averaged. Binding directly to coverslips was blood cells (6). assessed by measuring counts associated with coverslips incu- Concentration of serum antibody directed against type I11 bated with labeled organisms in the absence or presence of serum. group B streptococcus was determined by a whole organism Background binding was consistently calculated to be less than ELISA similar to that previously described (23). Serum was 0.4 organisms/macrophage (40counts/coverslip). diluted in PBS (Gibco) containing 0.5% BSA to yield a final Glutaraldehyde-fixed, Giemsa-stained monolayers were di- - serum concentration of 25 to 0.01%, and was incubated with rectly examined using phase contrast microscopy in some exper- approximately lo9 organisms in 1.5-mL Eppendorf polypropyl- iments and were done in parallel to radiobinding assays. This ene micro tubes (Sarstedt, Princeton, NJ) at 37°C for 30 min. examination demonstrated that over 98% of bacteria were asso- Organisms were washed three times in cold PBS and resuspended ciated with macrophages (Fig. 1). in 100 pL of a 1:200 dilution of horseradish peroxidase-conju- Binding versus ingestion of bacteria. Ingestion of organisms gated goat antibody to mouse IgG and IgM (Jackson laboratories, was assessed by methods similar to those used to measure inges- Bar Harbor, ME) and incubated for 30 rnin at 4°C. Organisms tion of H. inyuenzae by macrophages (3). After incubation with were then washed three times, resuspended in 1 mL of 0.075% bacteria, washed monolayers were fixed in either 100% methanol 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) or 3.3% neutral buffered formalin (Sigma Chemical Co.). Fixed containing 0.03% H202.The reaction was allowed to proceed for monolayers were then serially incubated with a 1:200 dilution of 15 min before the OD of the supernatant was measured at 460 an IgM MAb to type I11 capsule (SIIIV18C2 ascites fluid, de- nm.