Quantitation of the Third Component of Human Complement Attached

Vol. 26, No. 3

INFECTION AND IMMUNITY, Dec. 1979, p. 808-812 0019-9567/79/12-0808/07$02.00/0

Quantitation of the Third Component of Human Complement Attached to the Surface of Opsonized Bacteria: OpsoninDeficient Sera and Phagocytosis-Resistant Strains HENRI A. VERBRUGH,'* WILLEMIEN C. VAN DIJK,' MARIJKE E. VAN ERNE,1 ROEL PETERS, PHILLIP K. PETERSON,2 AND JAN VERHOEF' Laboratory ofMicrobiology, State University of Utrecht School ofMedicine, 3511 GG Utrecht, The Netherlands,' and Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota 554552 Received for publication 18 September 1979

The role of the third component of human complement (03) in the opsonization of bacteria in nonimmune human sera was evaluated. The amount of C3 that becomes attached to the surface of bacteria upon incubation in serum was measured in a quantitative fluorescent immunoassay using fluorescein-conjugated monospecific antiserum to human C3. The intensity of the fluorescence from opsonized bacteria was found to be directly proportional to the absolute amount of C3 fixed, and this enabled the detection of as few as 300 molecules of bound C3 per bacterium. In normal serum the rate of C3 fixation was closely correlated with an increase in opsonization of the bacteria for human PMNs. Both C3 fixation and opsonization were maximal after 15 min of incubation. C3 fixation was also observed, albeit at a significantly slower rate, in human serum with a nonfunctional classical pathway but an intact alternative complement pathway and in serum deficient in immunoglobulins. Again, the kinetics of C3 fixation correlated with bacterial opsonization. Using a total of 21 strains of several bacterial species, including Staphylococcus aureus and Escherichia coli, encapsulation of bacteria was found to interfere with the process of C3 fixation in normal human serum, rendering these organisms resistant to subsequent phagocytosis by human polymorphonuclear leukocytes.

Recognition of invading bacterial pathogens by human phagocytes requires the participation of serum factors (opsonins) including immunoglobulins and the complement system. Deficiencies in this phagocytosis-promoting activity of serum have been associated with an increased susceptibility to bacterial infections in a variety of diseases such as hypogammaglobulinemia (18), active systemic lupus erythematosus (14), congenital complement deficiencies (2, 15), sic-

ing them more virulent than unencapsulated strains (13, 17, 23, 25). In the present report, the kinetics of bacterial C3 fixation in human serum were studied using a quantitative fluorescent immunoassay employing fluorescein-conjugated antiserum monospecific for human C3. Opsonic recognition of bacteria by human polymorphonuclear leukocytes (PMNs) was shown to be closely related to the amount of C3 bound to their surfaces. Opsonic defects in serum from patients could be detected with this method. Furthermore, it was shown that encapsulated strains of several bacterial species failed to bind significant amounts of C3 in nonimmune human serum and were inefficiently phagocytized by human PMNs.

kle cell anemia (12), and cystic fibrosis (3). In patients with gram-negative bacteremia, a decrease in the opsonic activity of serum has been correlated with an increase in mortality (1). The phagocytosis-enhancing capacity of serum from nonimmune individuals depends largely upon the heat-labile complement system (15, 24, 26, 30). Activation of complement (C) via both the MATERIALS AND METHODS classical and the alternative pathways can result Bacterial strains and cultural conditions. S. in the fixation of opsonically active C3b molecules to the surface of bacteria (10, 16, 27). aureus Cowan I, Wood 46, H, and Ev (29, 30) and S. aureus M (encapsulated) and M variant (unencapsuHowever, certain bacteria such as encapsulated lated), kindly provided by M. A. Melly, Vanderbilt strains of Staphylococcus aureus, Escherichia University School of Medicine, Nashville, Tenn. (17), coli, and pneumococci are able to resist phago- were used in this study. cytosis in the presence of normal serum, renderFive encapsulated strains of E. coli containing 808

VOL. 26, 1979

KINETICS OF BACTERIAL C3 FIXATION

polysaccharide K antigens and five strains without the K antigen, reported previously (28), were also studied. Clinical isolates of Salmonella typhi (Vi antigen positive), Klebsiella pneumoniae (mucoid colonies), and Proteus mirabilis were likewise tested. All strains were maintained on blood agar plates at 40C. In studies of bacterial C3 fixation, organisms were freshly grown in 5 ml of Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) for 18 h at 370C with shaking. After incubation, the bacteria were washed three times with sterile isotonic phosphate-buffered saline (pH 7.4) and adjusted to a concentration of 5 x 108 colony-forming units per ml of phosphate-buffered saline by using a spectrophotometric method confirmed by pour-plate colony counts (30). Radioactive labeling of bacteria. In studies of bacterial opsonization, organisms were radioactively labeled by adding 0.02 mCi of [methyl-3H]thymidine (specific activity, S Ci/mmol; The Radiochemical Centre, Amersham, England) to the medium as previously described (28, 30). Serum sources. Normal human sera were obtained from 20 healthy donors and were stored at -70'C individually and as a serum pool. Each serum contained normal levels of total hemolytic complement (range, 80 to 125%) and C3 (range, 0.8 to 1.6 mg/mn). Also quantitative immunoglobulins were within normal ranges. In addition, sera were obtained from each of the following patients: (i) a 12-year-old patient with a primary X-linked immunoglobulin deficiency (Bruton type, with 0.05 mg of immunoglobulin G per ml of serum and undetectable immunoglobulins M, A, E, and D; total hemolytic complement and C3 were within normal limits); (ii) a 6-year-old male with Mediterranean-type kala azar of 1 year duration (this hyperglobulinemic serum had a total hemolytic complement of 74% and contained 0.4 mg of C3 per ml); (iii) a 16-year-old female with active systemic lupus erythematosus (total hemolytic complement, 45%, and C3, 0.3 mg/ml; quantitative immunoglobulins within normal limits); (iv) a 19-year-old female with systemic lupus erythematosus and an undetectable serum total hemolytic complement (