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 10(6) colony forming units (cfu) were incubated at 37 degrees C for 2-4 hours with 10(6) 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 x 10(3) cfu were incubated with 2 x 10(6) neutrophils and 80% autologous serum at 37 degrees 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.

The cardiac glycoside digoxin disrupts host defense in experimental pneumococcal pneumonia by impairing neutrophil mobilization.

Normal CD-1 mice were administered digoxin (4 micrograms/kg/24 h) and infected with type 3 Streptococcus pneumoniae in order to assess the effects of the cardiac glycoside on the chemotactic responsiveness of peripheral blood neutrophils and the mobilization of granulocytes from storage pools. The chemotactic responses to autologous zymosan-activated serum (C5a) by neutrophils obtained from uninfected digoxin-treated and control animals were similar; comparable observations were made with circulating granulocytes isolated from animals at 24 or 48 h after intratracheal challenge with 5 x 10(5) colony-forming units (cfu) of bacteria. However, at 4 and 6 h after intratracheal pneumococcal challenge, the number of immature neutrophils in the peripheral blood was significantly lower in the glycoside-treated animals versus controls; at 24 and 48 h, these differences were not apparent. Following the intravenous inoculation of pneumococci, the number of circulating immature neutrophils was also found to be significantly lower at 4 and 24 h in animals given the cardiac glycoside versus controls. We conclude that digoxin disrupts host defense in experimental pneumococcal pneumonia and bacteremia by impairing the mobilization of neutrophils.

The release of neutrophil chemoattractant activity by bronchoalveolar macrophages from adult and senescent mice.

To assess the effects of advanced age on the release of neutrophil chemoattractant activity by resident bronchoalveolar macrophages (BAM), cells from three strains of pathogen- and disease-free mice were secured by lung lavage and stimulated in vitro with unopsonized zymosan or the calcium ionophore, A23187. Chemoattractant release by BAM from adult (5-8 mos) and senescent (19-26 mos) C57BL/6 and DBA/2 mice in response to both stimulants was comparable; however, the generation of chemoattractant activity by BAM from senescent (18-20 mos) BALB/c mice was greater than that observed with cells from younger (4-6 mos) animals with both zymosan and A23187. In the presence of 50 microM piriprost potassium, a 5-lipoxygenase inhibitor, the release of chemoattractant activity and leukotriene B4 (LTB4) in response to zymosan and A23187 by BAM from both groups of C57BL/6 mice was significantly impaired. With BAM from BALB/c mice, 100 microM piriprost potassium was required to produce changes in A23187-stimulated chemoattractant and LTB4 release; of note, the generation of LTB4 in response to A23187 by BAM from aged BALB/c mice was significantly greater than that observed with cells from the younger animals under all conditions studied. Finally, with BAM from DBA/2 mice, 50 microM piriprost potassium significantly reduced chemoattractant activity in both groups of animals, but the lipoxygenase inhibitor had no effect on LTB4 production. Thus, although these studies revealed substantial age and strain-related differences in the release of neutrophil chemoattractant activity by murine BAM, they did not demonstrate deficiencies that might enhance the susceptibility of the senescent host to infection of the lower respiratory tract.

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 microgram/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.

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.