Surfactant protein D enhances phagocytosis and killing of unencapsulated phase variants of Klebsiella pneumoniae

Abstract

Pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin (collectin) that is secreted into the alveoli and distal airways of the lung. We have studied the interactions of SP-D and alveolar macrophages with Klebsiella pneumoniae, a common cause of nosocomial pneumonia. SP-D does not agglutinate encapsulated K. pneumoniae but selectively agglutinates spontaneous, unencapsulated phase variants, such as Klebsiella strain K50-3OF, through interactions with their lipopolysaccharides (LPS). These effects are calcium dependent and inhibited with maltose but not lactose, consistent with involvement of the SP-D carbohydrate recognition domain. Precoating of K50-3OF with SP-D enhances the phagocytosis and killing of these organisms by rat alveolar macrophages in cell culture and stimulates the production of nitric oxide by the NR-8383 rat alveolar macrophage cell line. SP-D similarly enhances the NO response to K50-3OF LPS adsorbed to Latex beads under conditions where soluble LPS or SP-D, or soluble complexes of SP-D and LPS, do not stimulate NO production. Our studies demonstrate that interactions of SP-D with exposed arrays of Klebsiella LPS on a particulate surface can enhance the host defense activities of alveolar macrophages and suggest that activation of macrophages by SP-D requires binding to microorganisms or other particulate ligands. Because unencapsulated phase variants are likely to be responsible for the initial stages of tissue invasion and infection, we speculate that SP-D-mediated agglutination and/or opsonization of K. pneumoniae is an important defense mechanism for this respiratory pathogen in otherwise healthy individuals.

FIG. 1

SP-D-induced agglutination of unencapsulated K. pneumoniae. The time course of the decrease in absorbance for mixtures of Klebsiella strain K50-3OF and RrSP-D (10 μg/ml) in the presence of calcium (open squares), absence of calcium (solid squares), or competing maltose in the presence of calcium (circles) was measured as described in Materials and Methods. Each point is the average of three experiments; each determination was performed in triplicate. The standard deviation is indicated by error bars.

FIG. 2

Inhibition of SP-D-induced agglutination of K. pneumoniae and E. coli with LPS. The percent inhibition of LPS from Klebsiella strain K50-3OF (squares), E. coli Y1088 (diamonds) and Klebsiella strain K21a (circles) was calculated from the change of the transmission of SP-D-induced aggregates of K50-3OF and of Y1088 strains in the presence of indicated concentrations of LPS (see Fig. 1). Each point is the mean of three experiments; each determination was performed in triplicate. The standard deviation did not exceed 20% of the mean for any of the data points.

FIG. 3

SP-D-induced agglutination of latex beads coated with LPS. Latex beads coated with LPS from Klebsiella strain K50-3OF and with LTA from S. pyogenes were exposed to the indicated concentration of SP-D in HBS supplemented with 10 mM CaCl₂, with or without 50 mM maltose. After 45 min of rotation end-over-end, the mixture was allowed to stand in spectrophotometer holder and the optical density at 660 nm was recorded after 15 min. Each point is the mean of three experiments; each determination was performed in triplicate. The standard deviation is indicated by error bars. Values of both LTA and LPS coated latex with maltose, however, were significantly different from those of LPS coated latex at corresponding SP-D concentrations of 0.8 to 3.3 μg/ml (P < 0.01 at the highest concentration).

FIG. 4

Binding of SP-D to LPS extracted from Klebsiella strain K50-3OF. The Klebsiella strain K50-3OF LPS used for the agglutination studies was examined by silver staining (Ag) as described in Materials and Methods. Consistent with our previous studies, the LPS showed a predominance of rough forms with a faint ladder of larger species. Direct binding of SP-D to the LPS was visualized by lectin blotting (Blot) as described. The strongest band corresponds to the rough forms identified on the silver stain; however, there was also detectable labeling of the larger species. The spot approximately one-third of the distance from the top of the blot is an artifact.

FIG. 5

Tetrazolium dye reduction assay of bacterial killing. Monolayers of rat AM were exposed to SP-D-coated (filled bars) or uncoated (open bars) Klebsiella strain K50-3OF for 30 min. The macrophages were washed free of nonbound bacteria and incubated in medium. At the indicated time and temperature 0.1% Tween was added to selectively lyse the phagocytic cells. Viable bacteria were allowed to grow for 3 h at 37°C, at which time the tetrazolium dye was added, and the absorbency was recorded after 10 min. Each point is the mean of three experiments; each determination was performed in triplicate. The standard deviation is indicated by error bars.

FIG. 6

NO production by NR-8383 rat AM. Macrophages were stimulated with: 10⁵ CFU/ml bacteria (bar 1), SP-D-coated bacteria (bar 2), SP-D-coated K50-3OF Klebsiella in buffer depleted of calcium with EDTA (10 mM) (bar 3), or SP-D-coated bacteria in the presence of maltose (bar 4). Control cultures included macrophages incubated with sonicated bacteria in the absence of SP-D (bar 5), sonicates of organisms coated with 7 μg of SP-D per ml (bar 6), sonicates of organisms coated with 14 μg of SP-D per ml (bar 7), or medium alone. The amount of nitrite released in quadruplicate cultures of macrophages stimulated by the indicated agents was determined. The data represent the means of three experiments. Because the absolute amount of nitrite produced varied between experiments, the results are presented as the percent of the nitrite released by macrophages triggered with 0.1 μg of LPS per ml, which stimulated the production of a mean ± standard deviation of 33 ± 12 μM per well. Comparisons of the means were made using the Student t test, and a P value of <0.05 was considered significant. The differences between the values of cultures depleted of calcium (bar 3) or samples containing SP-D-coated bacteria in the presence of maltose (bar 4) and the samples containing SP-D-coated bacteria (bar 2) were significant. There was no statistical difference (P > 0.1) between the means of the control cultures (bars 5, 6, and 7) and those of cultures containing bacteria alone (bar 1). However, the differences between the values of each of the control mixtures or of the mixtures containing uncoated bacteria and those of SP-D-coated bacteria were highly significant (P < 0.01).