Anthony L. Esposito

An assessment of the respiratory burst and bactericidal activity of alveolar macrophages from adult and senescent mice.

To assess the effects of advanced age on the nonspecific antimicrobial activity of resident alveolar macrophages (AM), superoxide anion (O2 ‐) release and the phagocytic and bactericidal capacity of cells from three genetically distinct murine strains were evaluated. In initial experiments, resident AM, obtained by bronchoalveolar lavage of pathogen‐free adult female CD‐1 mice and studied in suspension, were found to produce O2 ‐ spontaneously and in response to phorbol myristate acetate (PMA) snd unopsonized zymosan. Maximum quantities of O2 ‐ were released following stimulation with 1 μg/ml PMA and by a particle‐to‐cell ratio of 100:1 with zymosan; responses to the agonists peaked between 60 and 90 min. Resident AM obtained from pathogen and disease‐free senescent (18‐26‐month‐old) female C57BL/6, BALB/c, and DBA/2 mice released significantly more O2 ‐ in response to both PMA and zymosan than did cells secured from adult (4‐8‐month‐old) animals. In vivo, the capacity of AM from adult and senescent animals to phagocytose Streptococcus pneumoniae (unencapsulated strain) and Staphylococcus aureus was comparable, and although the cells from the senescent mice tended to be more efficient in their ability to kill internalized bacteria, statistically significant differences between the two groups were not observed. The results of these studies indicate that the enhanced susceptibility of the senescent host to infection of the lower respiratory tract cannot be attributed to age‐related changes in the nonspecific antimicrobial activity of resident AM.

An assessment of the factors contributing to the killing of type 3 Streptococcus pneumoniae by human polymorphonuclear leukocytes in vitro

To characterize the factors that contribute to the killing of type 3 S. pneumoniae, human neutrophils were obtained from healthy donors and incubated with viable organisms. In contrast to prior observations with other pneumococcal serotypes, killing was not detected when 106 colony forming units (cfu) were incubated at 37°C for 2–4 hours with 106 neutrophils in the presence of 20–80% fresh autologous serum; further, pneumococcidal activity was not found when preopsonized bacteria and primed neutrophils were employed in the standard assay. However, when the bacterium to cell ratio was reduced to 1:100 and 1:1000, microbicidal action was detected; a 10‐fold reduction in the number of viable bacteria was observed when 2 times 103 cfu were incubated with 2 times 106 neutrophils and 80% autologous serum at 37°C for 4 hours. To assess the effects of serum factors on killing, bactericidal assays were performed in the presence of normal human serum (NHS), heat‐inactivated human serum (HIHS) and absorbed human serum (AHS); heating reduced and absorption eliminated the capacity of serum to support killing. Studies performed with mutanolysin, an enzyme that lyses type 3 pneumococci, demonstrated that the effects of HIHS and AHS on bactericidal activity were highly correlated with alterations in the ability of the sera to support phagocytosis. Studies of neutrophil activation revealed changes in the production of superoxide anion that correlated well with phagocytosis and killing; however, the results of assays of leukotriene B4 generation and degranulation (beta‐glucuronidase and lactoferrin release) were more variable. In mixing experiments, the capacity of HIHS to support killing was normalized with NHS; however, the ability of AHS to promote killing was not restored with HIHS or NHS. Thus, these studies demonstrate the relatively limited capacity of human serum to support the killing of type 3 pneumococci, and they emphasize the importance of killing assays in assessing interactions between the bacterium and neutrophils.

In vitro Assessment of Chemotaxis by Peripheral Blood Neutrophils from Adult and Senescent C57BL/6 Mice: Correlation with in vivo Responses to Pulmonary Infection with Type 3 Streptococcus pneumoniae

To assess the effects of advanced age on the ability of circulating neutrophils to respond to biologically relevant chemoattractants, cells were isolated from the peripheral blood of pathogen and disease-free C57BL/6 mice and evaluated in a microchemotaxis chamber. The responses of granulocytes obtained from senescent mice (26-28 months) to the chemotactic peptide, FMLP, and to leukotriene B4 were similar to those found with cells from the younger animals (8-10 months). In contrast, the migration of neutrophils in response to sonicated type 3 Streptococcus pneumoniae was significantly greater with cells from the older animals. Similarly, the chemotactic response of neutrophils to zymosan-activated serum was greater with cells and serum from the senescent animals; however, the enhanced chemotaxis exhibited by granulocytes from the aged mice was a consequence of serum factors. Following the deposition of viable type 3 S. pneumoniae into the lower respiratory tract, the neutrophil influx at 24 h after challenge was significantly greater in the senescent mice; however, age-related differences in survival rates and LD50 were not detected. Thus, in the C57BL/6 mouse, senescence is not associated with deficiencies in the response of neutrophils in vitro to chemoattractants that contribute to lung host defense against the pneumococcus; further, in this murine strain, advanced age does not result in an attenuation of the pulmonary inflammatory reaction to infection with type 3 S. pneumoniae.

Protease Inhibitor Eglin-C Affects Superoxide Anion Release but not Bacterial Killing by Human Polymorphonuclear Leukocytes

In order to assess the influence of the protease inhibitor eglin-c on superoxide anion (O-2) release by human polymorphonuclear leukocytes (PMN), cells were secured from normal donors and stimulated with phorbol myristate acetate (PMA), opsonized zymosan, or n-formyl-methionyl-leucyl-phenylalanine (FMLP). In the presence of 100 micrograms/ml eglin-c, the activation time was prolonged and the maximum linear rate of O-2 formation was depressed following stimulation with PMA; a concentration of 1000 micrograms/ml eglin-c was required to produce a similar effect with opsonized zymosan. Eglin-c did not influence the activation time following stimulation with FMLP, but at 2000 micrograms/ml, the protease inhibitor attenuated the rate of O-2 production in response to the chemotactic peptide. In the presence of cytochalasin B, the inhibitory effect of eglin-c on O-2 release following stimulation with FMLP became more pronounced. In spite of these alterations in O-2 formation, the protease inhibitor did not impair the bactericidal activity of PMN against Staphylococcus aureus. Therefore, we conclude that although eglin-c can disrupt the activation and the activity of the superoxide-generating system of human PMN, the effect is stimulus dependent and is not associated with an alteration in the microbicidal capacity of neutrophils against S. aureus.

The effects of capsular polysaccharide on the capacity of serum to support the killing of type 3 Streptococcus pneumoniae by human neutrophils.

To assess the effects of purified capsular polysaccharide from type 3 S. pneumoniae (PPS‐3) on the capacity of serum to support pneumococcal killing by human neutrophils, varying concentrations of PPS‐3 (0.01–100 μg/ml) were incubated (4 °C) with pooled serum for 30 min, and the resulting preparation, termed absorbed serum, was evaluated in bactericidal and phagocytic assays. The ability of serum to promote the killing of type 3 S. pneumoniae was significantly reduced at 1.0 μg/ml PPS‐3; similarly, serum absorbed with 1.0 μg/ml PPS‐2 failed to support the killing of type 2 S. penumoniae. However, the impact of these penumococcal polysaccharides was serotype specific, since the killing of type 3 S. pneumoniae was not impaired in serum absorbed with PPS‐2, and the killing of type 2 S. pneumoniae was not attenuated in serum treated with PPS‐3. The failure of serum absorbed with PPS‐3 to promote the killing of type 3 S. pneumoniae was primarily a consequence of impaired bacterial ingestion; the reduction in phagocytosis was associated with parallel changes in superoxide anion release. The defects in phagocytosis and killing induced by PPS‐3 were not associated with alterations in classical or alternative complement pathway activity; however, they were highly correlated with changes in serum antibody levels to the polysaccharide. The addition of polyclonal human IgG to serum treated with PPS‐3 did not restore its capacity to support killing; however, preopsonization of the bacterium with the IgG preparation did partially correct the deficiency. Finally, neutrophils preincubated with serum containing 10.0 μg/ml PPS‐3 exhibited an impaired bactericidal activity against type 3 S. pneumoniae. Thus, these studies demonstrate that the presence of PPS‐3 decreases the capacity of serum to support the killing of type 3 S. pneumoniae by absorbing immunoglobulins and by generating factors that interfere with neutrophil function.