Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages

Surfactant protein (SP)A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species. Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls. In addition, purified preparations of SPs often contain endotoxin. The goals of this study were I) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-beta-D-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels. Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin. There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages. SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production. These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination.