Species variation in the mechanism of killing of inhaled pneumococci.

Detailed studies in murine models show inhaled staphylococci are killed mainly by alveolar macrophages. Recently, using histology and lung lavage to determine the site of killing of inhaled 59Fe-labeled pneumococci in rats, we found unexpectedly that most of the organisms were killed extracellularly. In the present studies we compared clearance of inhaled 59Fe-pneumococci in rats, guinea pigs, and rabbits to determine if extracellular killing of pneumococci is species-dependent. Absolute clearance rates were measured using Andersen plates. lung lavage and differential centrifugation were used to measure leukocyte-associated and free 59Fe-pneumococci and nonsedimentable 59Fe. Clearance was rapid in all species but was fastest in rabbits, which killed pneumococci almost as quickly as they were deposited (p less than 0.025 versus rats and guinea pigs). At 1-1/2 hours after pneumococcal deposition in rats, when clearance had reached 92%, alveolar macrophages contained only 31% of the total 59Fe while 56% was in the nonsedimentable, extracellular fraction. At 1-1/2 hours in guinea pigs, when clearance was 96% complete, macrophages contained 51% of the 59Fe and 34% was nonsedimentable (p less than 0.002 versus rats). In rabbits at 1-1/2 hours, macrophages had 94% of the 59Fe and only 4% was nonsedimentable (p less than 0.001 versus the other species). In no species were opsonic antibodies detected in sera or concentrated lavage. In vitro, rabbit and guinea pig alveolar macrophages killed pneumococci opsonized with both specific antibody and fresh serum, while rat macrophages had little activity (p less than 0.001). We conclude that the role of alveolar macrophages in killing inhaled pneumococci varies in different species.

Impaired antipneumococcal activity of bronchoalveolar lining material of neonatal rats.

Pulmonary clearance of inhaled pneumococci is markedly impaired in neonatal rats compared with that in adult rats. To determine whether this impairment is due to a deficiency of extracellular bactericidal factors, the antipneumococcal activity of free fatty acids (FFA) in lung surfactant and the levels of lysozyme and transferrin in lavage fluids were quantified. Surfactant from adult rats averaged 68 U of antipneumococcal activity per g (dry weight) of lung, compared with less than 0.25 U for rats less than 1 week old (P less than 0.001). The kinds of FFA in surfactant of neonatal and adult rats were essentially identical, and the antipneumococcal activity of highly purified FFA from surfactant of neonatal and adult rats was also the same. However, the quantity of FFA in surfactant varied significantly with age, and rats less than 3 weeks old had much lower levels of surfactant FFA than did adults (P less than 0.001). In addition, lavage fluids from neonatal rats inhibited the antipneumococcal activity of surfactant FFA more than lavage fluids from adults did (P less than 0.02). This inhibitory activity did not appear to be due to protein binding. Lavage fluids from neonates showed an age-related deficiency of lysozyme (P less than 0.001), but lysozyme appeared to play no role in pneumococcal killing by the surfactant fraction of lavage fluids in vitro. Transferrin levels in lavage fluids were similar for neonates and adults. It was concluded that lung surfactant from neonatal rats was deficient in antipneumococcal activity, due mostly to low levels of FFA and to a lesser degree to increased levels of inhibitor(s) in lavage fluids.

Impaired pulmonary clearance of pneumococci in neonatal rats.

Lung infections are a common cause of morbidity and mortality in neonates. To evaluate neonatal lung defenses against pneumococci, we challenged rats with aerosols of encapsulated pneumococci in an airborne infection apparatus. Whereas adult rats cleared greater than 95% of inhaled type 1 or type 25 pneumococci within 4 h, pneumococci proliferated in the lungs of newborn rats and reached 200-600% of the baseline value by 4 h and 1000-1700% by 24 h. As neonatal rats matured, their ability to clear inhaled pneumococci improved, but compared with adults some impairment in clearance was present until approximately 4 wk of age. Newborn rats had significantly fewer resident alveolar macrophages per g of lung tissue than did adults (p less than 0.001). Although the number of resident macrophages increased with time, a significant deficit in alveolar macrophages persisted for the first 3 wk of life (p less than 0.01). Aerosols of pneumococci caused an influx of granulocytes into the lungs of adult rats within 4 h, compared with 24 h for neonatal rats. Even at 24 h after pneumococcal challenge, newborn rats had significantly fewer granulocytes per g of lung tissue (p less than 0.05) than did adults, although 7-day-old rats had reached an adult level by this time. Significant (p less than 0.05) increases in granulocyte chemotactic activity were observed in lavage fluids of adult, but not newborn, rats after pneumococcal challenge. Thus, impaired clearance of pneumococcal aerosols by neonatal rats was associated with an age-dependent deficiency in numbers of resident alveolar macrophages and impaired generation of chemotactic activity and recruitment of granulocytes to the lung.

Effect of rat surfactant lipids on complement and Fc receptors of macrophages.

Murine resident alveolar macrophages have very low numbers of complement receptors detectable by rosetting with erythrocyte complexes. These macrophages are relatively inactive in tests of complement- or antibody-mediated bacterial phagocytosis in vitro. It is not known whether these characteristics are intrinsic or are environmentally modulated. We found previously that rat bronchoalveolar lavage fluid or isolated rat surfactant lipid causes a unique and marked reduction in the ability of peritoneal macrophages to form rosettes with immunoglobulin G or complement containing erythrocyte complexes in vitro. In this study the antirosetting activity of rat surfactant was found to be due to its neutral lipid component and, specifically, to the free fatty acid (FFA) fraction of the neutral lipids. Rat surfactant contained a higher level of FFA than has been reported for canine, guinea pig, or human surfactant. Studies with pure FFA showed that activity in blocking macrophage Fc and complement receptors correlated with increasing unsaturation and chain length. A mixture of eight commercially available FFAs, representing the most abundant FFA in rat surfactant and consisting mostly of saturated FFA, had much less effect on rat macrophage receptors than the naturally occurring FFA mixture. These findings suggest that long-chain-unsaturated FFAs in rat surfactant are the most important for the antireceptor activity. The antireceptor activity of rat surfactant or FFA on peritoneal macrophage receptors in vitro and known intrinsic properties of murine alveolar macrophages could not be precisely correlated, suggesting that impaired binding of immune complexes by murine alveolar macrophages and a high level of FFAs in rat surfactant may be independent rather than causally related phenomena.