ABSTRACT
Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.
Subject(s)
Macrophages/metabolism , Mycobacterium tuberculosis/immunology , Nitric Oxide/metabolism , Proteolipids/metabolism , Pulmonary Surfactants/metabolism , Animals , Antibodies/pharmacology , BCG Vaccine/immunology , Benzothiazoles , Blotting, Western , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Female , Macrophages/cytology , Macrophages/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Nitric Oxide/pharmacology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Proteolipids/pharmacology , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Proteins , Pulmonary Surfactants/pharmacology , Rats , Rats, Sprague-Dawley , Thiazines/antagonists & inhibitors , Thiazines/metabolism , Thiazoles/antagonists & inhibitors , Thiazoles/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Uracil/metabolism , omega-N-Methylarginine/pharmacologyABSTRACT
Surfactant-associated protein A (SP-A) is a C-type lectin that is involved in surfactant metabolism as well as host defense functions in the lung. We have recently identified a receptor on macrophages [specific 210-kDa SP-A receptor (SPR210)] that binds SP-A. In the current study we have investigated the role of SP-A in mediating uptake of bacillus Calmette-Guérin (BCG) by rat macrophages and human monocytes and have examined the role of the macrophage SPR210 in this process. 125I-labeled SP-A bound BCG in a Ca(2+)-, carbohydrate-, and dose-dependent manner. To examine association of SP-A-BCG complexes with macrophages, BCG were opsonized with SP-A and were incubated with rat bone marrow-derived macrophages (RBMM), rat alveolar macrophages (RAM), or human monocytes at a 1-to-1 ratio for 4 h. The cells were washed, fixed in formalin, and stained with auramine-rhodamine. Cell-associated organisms were enumerated by fluorescent microscopy. The percentage of cells with one or more associated BCG was increased by SP-A from 27% of RBMM with BCG alone to 54% with SP-A-BCG complexes; 1-16% in RAM; and 39-67% in human monocytes. This enhanced uptake was dependent on the dose of SP-A, with maximal increases seen with 10 micrograms/ml. Electron microscopic analysis supported the conclusion that organisms were ingested by and not simply bound to the macrophages. Inclusion of SPR210 antibodies blocked association of SP-A-BCG complexes, suggesting a role for SPR210 in mediating the interaction of SP-A-BCG with the macrophages. This was further supported by the finding that modulation of SPR210 activity resulted in altered SP-A-BCG uptake. These results demonstrate that SP-A binds to BCG and that uptake of these SP-A-BCG complexes is mediated in part by the SPR210 on rat macrophages and human monocytes.
Subject(s)
Macrophages/physiology , Mycobacterium bovis/physiology , Phagocytosis , Proteolipids/pharmacology , Pulmonary Surfactants/pharmacology , Receptors, Cell Surface/physiology , Animals , Bone Marrow Cells , Female , Humans , Macrophages, Alveolar/physiology , Monocytes/physiology , Mycobacterium bovis/metabolism , Proteolipids/metabolism , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Proteins , Pulmonary Surfactants/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
During the past few years, many promising new agents for the treatment of sepsis have been studied to varying degrees in vitro as well as in vivo in animals and humans. Although there is a relative plethora of animal data, full-scale clinical trials of size sufficient to yield clear answers are rare. Many of the agents appear to hold promise based on preliminary data in animals or from small human studies, and some are undergoing multicenter clinical investigation. At present, however, none of the agents discussed clearly has shown survival benefit when administered to patients with sepsis. Certainly, none can be recommended as standard therapy, and others such as glucocorticoids should be avoided. Nevertheless, the pharmacotherapy of sepsis remains an area of intense research, and ongoing clinical trials as well as continuing basic research into the pathophysiologic mechanisms of sepsis yet may yield a well-studied drug that offers survival benefit to patients with sepsis.
