Jun 24, 1982 - Counterimmunoelectrophoresis was carried out in 0.5% agarose in barbital buffer for. 30 min (Model 077-922 electrophoresis apparatus; Hy-.
INFECTION AND IMMUNITY, Jan. 1983, p. 297-304 0019-9567/83/010297-08$02.00/0 Copyright C 1983, American Society for Microbiology
Vol. 39, No. 1
Opsonizing and Bactericidal Effects of Normal Human Serum on Nontypable Haemophilus influenzae DANIEL M. MUSHER,1.2,3* MICKEY HAGUE-PARK,' ROBERT E. BAUGHN,1 3'4 RICHARD J. WALLACE, JR.,23 AND BENJAMIN COWLEY1 Infectious Disease Section, Veterans Administration Medical Center, Houston, Texas 77211,1 and the Departments of Medicine,2 Microbiology and Immunology,3 and Dermatology,4 Baylor College of Medicine, Houston, Texas 77030
Received 24 June 1982/Accepted 14 October 1982
The observation that nontypable (NT) Haemophilus influenzae causes serious infection in adults has stimulated interest in mechanisms that may protect the human host against NT H. influenzae infection. Incubating NT H. influenzae with normal human serum (NHS) caused dose- and time-dependent killing that varied with the individual NHS and NT H. influenzae. Adsorption of NHS with NT H. influenzae removed bactericidal activity against the adsorbing isolate but not necessarily that against others, suggesting antigenic diversity and supporting recent studies that show different outer membrane protein profiles among NT H. influenzae. Heating NHS to 56°C for 30 min abolished bactericidal activity; this activity was not restored by complement-rich guinea pig serum or NT H. influenzae-adsorbed NHS. This is analogous to the "third factor" needed for intraleukocytic killing of pneumococci. Optimal opsonization of NT H. influenzae for phagocytosis by human polymorphonuclear leukocytes required antibody and complement, but other serum factors also played a role. Bactericidal activity generally, but not uniformly, correlated with opsonizing activity of individual NHS. Humoral factors may be important in host defenses against NT H. influenzae infection; their emergence during convalescence warrants further
study.
Although Haemophilus influenzae type b re- zae for phagocytosis by normal human polymormains the predominant pathogenic H. influenzae phonuclear leukocytes (PMN). for infants and children (27) and is far more This work was presented in part at the Southvirulent than other typable or nontypable H. ern Society for Clinical Investigation, New Orinfluenzae in animal models (18, 21), recent leans, January 1982, and at the Annual Meeting clinical evidence has implicated nontypable of the American Society for Microbiology, At(NT) H. influenzae as a surprisingly important lanta, March 1982. pathogen in adults (5, 26, 31; R. J. Wallace, Jr., C. J. Baker, F. J. Quinones, D. G. Hollis, R. E. MATERIALS AND METHODS Weaver, and K. Wiss, Rev. Infect. Dis., in
press). One study (31) of H. influenzae isolated
from blood cultures showed that NT H. influenzae were more frequently identified than H. influenzae type b as a cause of bacteremia in a variety of adult infections including pneumonia, empyema, meningitis, postpartum sepsis, and sepsis without a detectable focus; in all, 64% of
bacteremic H. influenzae infections in adults
were due to NT H. influenzae. These clinical observations have provided us with the impetus to investigate mechanisms of host defense against infection by NT H. influenzae. In the present paper, we describe results pertaining to the bactericidal effect of normal human serum (NHS) on NT H. influenzae and the capacity of NHS to opsonize NT H. influen-
Bacteria. NT H. influenzae isolated from sputum or from blood cultures were obtained from the Clinical Microbiology Laboratory, Veterans Administration Medical Center, Houston, Tex., after their identity had been verified by morphology, Gram-staining characteristics, and requirements for hemin and NAD. A total of 21 isolates were studied, 16 from sputum and 5 from blood cultures. Isolates were subcultured once at 37°C in a candle jar on chocolate agar, suspended in Mueller-Hinton broth containing 15% glycerol, and frozen without further passage at -70°C. Bacteria used in bactericidal and opsonophagocytic studies and for adsorption were grown in Mueller-Hinton broth supplemented with 10 ,ug each of hemin and NAD (Sigma Chemical Co., St. Louis, Mo.) per ml at 37°C; incubation was for about 18 h unless stated to the contrary. Bacteria were washed three times in phosphate-buffered saline (PBS; pH 7.2) with centrifuga-
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MUSHER ET AL.
0.3 ,ul of [3H]thymidine (specific activity, 80 Ci/mmol; New England Nuclear Corp., Boston, Mass.) per ml. Sera. Sera obtained from blood of 10 normal, healthy adults were processed promptly and stored in 1-ml samples, either individually or pooled. Serum from a patient with common variable immune deficiency (CVIDS) was kindly provided by Susan Gardner, Texas Children's Hospital, Houston; this serum contained 156 mg of immunoglobulin G (IgG), 14 mg of IgM, 9 mg of IgA, and
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