QUANTITATIVE FLOW CYTOMETRIC ANALYSIS OF OPSONOPHAGOCYTOSIS AND KILLING OF NONENCAPSULATED HAEMOPHILUS-INFLUENZAE BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Since nonencapsulated Haemophilus influenzae persists in the lower res piratory tracts of patients with chronic bronchitis despite the presen ce of specific antibodies, complement, and polymorphonuclear leukocyte s (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsu lated H. influenzae isolated from the sputa of chronic bronchitis pati ents was labeled with fluorescein isothiocyanate and incubated, with h uman PMNs in the presence of complement and antibodies for 30 min at 3 7 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined b y flow cytometry. Fluorescence quenching with ethidium bromide was use d to discriminate between intracellular and extracellular bacteria. Op sonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the pre sence of complement and human pooled sera containing high titers of st rain-specific antibodies. Opsonophagocytosis of six other H. influenza e strains was also poor. Under the same conditions, opsonophagocytosis of Staphylococcus aureus was 90% +/- 5%, and opsonophagocytosis of C. albicans was 55% +/- 23%. About half of the number of H. influenzae b acteria associated with PMNs was internalized. Opsonophagocytosis of h eat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viab le bacteria of the same strain (p < 0.05). This result suggests that t he accessibility of epitopes on H. influenzae for opsonizing antibodie s in better on killed than on viable bacteria. We concluded that viabl e nonencapsulated H. influenzae is poorly opsonophagocytized in the pr esence of strain-specific antibodies and complement.