Our results show that SP-A and SP-D, the pulmonary collectins, contribute to clearance of a nonmucoid strain of P. aeruginosa
from the lungs in mice. Nonmucoid strains of P. aeruginosa
are a major cause of nosocomial pneumonia in intensive care units and are involved in the early stages of CF (15
). In the noncompromised host, P. aeruginosa
is cleared from the lungs by innate immune mechanisms that involve the mucociliary apparatus, phagocytic cells, and antimicrobial molecules. This study indicates that the pulmonary collectins modulate innate immunity against P. aeruginosa in vivo
Previous studies have shown that SP-A−/− mice had decreased clearance of bacteria from their lungs after intratracheal infection (11
). In contrast, in prior studies, SP-D−/− mice cleared gram-positive and gram-negative bacteria from their lungs as efficiently as WT mice (13
). The present study confirms the clearance defect observed in SP-A−/− mice but shows that SP-D−/− mice have decreased pulmonary clearance of P. aeruginosa
at an early time point (). Furthermore, SP-AD−/− mice have decreased clearance of P. aeruginosa
compared with SP-A−/− or SP-D−/− mice. The differences between our study and previous reports may be related to differences in age and strain of mice and differences in species and strain of bacteria. Previous studies performed a bacterial challenge in collectin-deficient mice at an age of 35–42 d (11
). At this age, ongoing pulmonary inflammation and macrophage activation in SP-D−/− mice could have modified their response to infection. In the present study, mice were infected at an age of 21 d to minimize the potential effect of their lung phenotype on their response to infection.
One of the most well characterized functions of SP-A and SP-D is their ability to act as opsonins and enhance the uptake of micro-organisms by phagocytic cells (3
). In vitro
, previous experiments have shown that SP-A and SP-D bind some but not all strains of P. aeruginosa
and increase their phagocytosis by AM (2
). SP-A can also increase phagocytosis by stimulating macrophages directly without binding to the bacteria (6
). In the present study, SP-A−/− and SP-D−/− mice had decreased phagocytosis of PAK-GFP by AM compared with WT mice (). SP-AD−/− mice had decreased phagocytosis of PAK-GFP by AM compared with WT and SP-A−/− mice. Therefore, our results show that SP-A and SP-D stimulate phagocytosis of P. aeruginosa
by AM in vivo
SP-A and SP-D can modulate the inflammatory response to pathogens by regulating the production of cytokines and the influx of inflammatory cells in the lungs (1
). This was confirmed in our study, but SP-A−/− and SP-D−/− mice differed in their inflammatory response. We found that after infection with P. aeruginosa
, SP-D−/− mice had increased pulmonary inflammation compared with WT mice. On qualitative histology, SP-D−/− mice had increased pulmonary infiltration by inflammatory cells (). SP-D−/− mice also had higher levels of MPO activity () and higher levels of MIP-2 and IL-6 () in their lungs compared with WT mice. This increased production of MIP-2 could have contributed to the increased influx of neutrophils observed in SP-D−/− mice. In SP-A−/− mice, on histology, there was also an increased pulmonary infiltration after infection with P. aeruginosa
. However, SP-A−/− mice did not have a statistically significant increase in infiltration of the lungs by neutrophils, as measured by MPO activity at 48 h. Pulmonary levels of MIP-2 and IL-6 tended to be higher in SP-A−/− mice compared with WT mice, but the difference was not statistically significant. Previous studies have shown that SP-A and SP-D can upregulate or downregulate cytokine production by macrophages in vitro
). The nature of these effects depends on various factors, including the type of pathogen or stimulus, the type and the state of activation of the cell, and the cellular receptor that is engaged. Our results show that SP-D−/− but not SP-A−/− mice had a significantly increased pulmonary inflammatory response after infection with a nonmucoid strain of P. aeruginosa
, despite a bacterial load that tended to be higher in SP-A−/− than in SP-D−/− mice. These data suggest that, in addition to promoting bacterial clearance, SP-D downregulates the pulmonary inflammatory response.
The phenotypes of SP-A−/− and SP-D−/− mice have been reported previously. Although uninfected SP-A−/− mice are largely indistinguishable from WT mice except for the absence of a surfactant fraction called tubular myelin (27
), SP-D−/− mice develop patchy pulmonary inflammation, airspace remodeling, and surfactant accumulation (18
). These apparently noninfectious changes start early in life but are pronounced by 6 wk of age (28
). The mice used for this study were 3 wk of age. At this age, pulmonary cytokine levels, MPO activity, and lung histology were similar in WT mice compared with SP-D−/− mice. In SP-D−/−mice, the number of AM increases progressively after birth. These macrophages progressively enlarge and accumulate intracellular lipids. Our results show that AM from SP-D−/− mice had decreased phagocytosis of P. aeruginosa
compared with AM from WT mice in vitro
(). This defect in phagocytosis observed in SP-D−/− mice was partially rescued by the administration of rSP-D. Taken together, these data suggest that the decrease in clearance of P. aeruginosa
observed in SP-A−/− and SP-D−/− mice is due to the absence of the collectins. However, functional changes in AM of SP-D−/− mice may have contributed to decreased bacterial clearance.
SP-A and SP-D levels were measured 6 and 48 h after infection in the lungs of WT mice (). Compared with uninfected mice, there was a dramatic decrease in SP-A levels as early as 6 h after infection. There was a small but significant decrease in SP-D levels at 6 and 48 h. At least four different mechanisms could explain this decrease in SP-A and SP-D levels. First, several reports indicate that P. aeruginosa
can secrete proteases that degrade SP-A and SP-D in vitro
). In our experiments, degradation of SP-A and SP-D by bacterial enzymes could have occurred in vivo
. However, we did not detect any immunoreactive degradation products for SP-A or SP-D with the polyclonal antibodies used. Second, SP-A has a relatively short half-life (30
). Acute lung injury and damage to alveolar type II cells and Clara cells by the bacteria could have resulted in decreased production of SP-A and SP-D. Third, other investigators have found that phagocytes and epithelial cells can degrade lung collectins (31
) and that clearance of SP-D by neutrophils was increased after inflammation induced by lipopolysaccharide (33
). Finally, as in previous studies, we measured SP-A and SP-D in the supernatant of lung homogenates after centrifugation (34
). The fraction of SP-A and SP-D that was bound to the surface of the bacteria and to various cell types removed during centrifugation was not measured. This could have contributed to an underestimation of the collectin levels in the lungs of infected mice. In summary, our data show that despite having decreased collectin levels after acute pulmonary infection with P. aeruginosa
, WT mice cleared bacteria more efficiently than SP-A−/− and SP-D−/− mice.
Figure 7. SP-A and SP-D levels. SP-A and SP-D levels were assessed in lung homogenates of WT mice by Western blotting. At 6 and 48 h after infection, there was a dramatic decrease in SP-A levels and a smaller but significant decrease in SP-D levels compared with (more ...)
The concentrations of pulmonary collectins required to clear bacteria in vivo
are unknown. Low levels of pulmonary collectins have been associated with several human diseases, including CF (16
), pneumonia (35
), and chronic lung disease of prematurity (37
). Infection and inflammation contribute to the pathogenesis of CF and chronic lung disease of prematurity. Therefore, SP-A and SP-D may play an important role in the defense against bacterial infections in humans.
In conclusion, this study shows that SP-A and SP-D enhance pulmonary clearance of P. aeruginosa by stimulating phagocytosis by AM and by modulating the inflammatory response in the lungs. This is the first report of a bacterial challenge in mice deficient in both SP-A and SP-D. More bacteria remained in the lungs in SP-AD−/− mice compared with SP-A−/− and SP-D−/− mice (). In addition, markers of inflammation, including the histologic appearance of the lungs, tended to be more pronounced in SP-AD−/− compared with SP-A−/− or SP-D−/− mice (, , and ). Therefore, our results are consistent with a model where SP-A and SP-D have overlapping but not identical functions in vivo. These findings may be clinically important in human diseases associated with decreased levels of SP-A and SP-D.