Quantitative flow cytometric analysis of opsonophagocytosis and killing of nonencapsulated Haemophilus influenzae by human polymorphonuclear leukocytes

Abstract

Since nonencapsulated Haemophilus influenzae persists in the lower respiratory tracts of patients with chronic bronchitis despite the presence of specific antibodies, complement, and polymorphonuclear leukocytes (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsulated H. influenzae isolated from the sputa of chronic bronchitis patients was labeled with fluorescein isothiocyanate and incubated with human PMNs in the presence of complement and antibodies for 30 min at 37 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined by flow cytometry. Fluorescence quenching with ethidium bromide was used to discriminate between intracellular and extracellular bacteria. Opsonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the presence of complement and human pooled sera containing high titers of strain-specific antibodies. Opsonophagocytosis of six other H. influenzae 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 bacteria associated with PMNs was internalized. Opsonophagocytosis of heat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viable bacteria of the same strain (P < 0.05). This result suggests that the accessibility of epitopes on H. influenzae for opsonizing antibodies is better on killed than on viable bacteria. We conclude that viable nonencapsulated H. influenzae is poorly opsonophagocytized in the presence of strain-specific antibodies and complement.

Immune selection for antigenic drift of major outer membrane protein P2 of Haemophilus influenzae during persistence in subcutaneous tissue cages in rabbits

“…This discrepancy between the results of ELISAs and bactericidal assays was previously described by Groeneveld et al (10). Selection can also be mediated by phagocytosis (18), but H. influenzae is poorly opsonophagocytized (24), and opsonophagocytosis is more inefficient in foreign body-associated infections (27,28).…”

“…We conclude that MOMP P2 variants of H. influenzae persisting in subcutaneous tissue cages in rabbits are selected under pressure of the variant-specific serum bactericidal activity elicited by vaccination with the homologous strain. The selection process is slow, probably because of inefficiency of antibody-mediated defense mechanisms, especially opsonophagocytosis (24). Often the antigenic variant and the original strain both persisted in the tissue cages for some time.…”

Fine mapping of outer membrane protein P2 antigenic sites which vary during persistent infection by Haemophilus influenzae

“…During infection by H. influenzae strain-specific opsonizing antibodies also develop (19,26). Opsonophagocytosis of nonencapsulated H. influenzae is strain specific (26) and has been described to be poor (33). In addition, in subcutaneous tissue cages phagocytosis was shown to be impaired (35).…”

Opsonic antibodies to outer membrane protein P2 of nonencapsulated Haemophilus influenza are strain specific

“…Flow cytometry. Samples were analyzed with the FACScan model (Becton and Dickinson, Heidelberg, Germany) with computer-assisted evaluation of data (FACScan software), on the basis of the method described elsewhere (24). Data were acquired by using an instrument status with a linear data mode for forward scatter and side scatter and a logarithmic mode for fluorescence at 515 to 545 nm (FL1).…”