Mitogen-activated protein kinases and NFkappaB are involved in SP-A-enhanced responses of macrophages to mycobacteria.
BACKGROUND
Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFkappaB in mediating the enhanced signaling in response to BCG in the presence of SP-A.


METHODS
Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 microg of SP-A to 5 x 105 BCG for 30 min at 37 degrees C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined.


RESULTS
Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFkappaB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IkappaB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively.


CONCLUSIONS
These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFkappaB.
Lopez
Joseph P
JP
Department of Veterans' Affairs Medical Center, Nashville, TN, USA. joe.lopez@vanderbilt.edu
Vigerust
David J
DJ
Shepherd
Virginia L
VL
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D009163:Mycobacterium bovis; D016328:NF-kappa B / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis; D010587:Phagocytosis / Q000187:drug effects; D037662:Pulmonary Surfactant-Associated Protein A / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017828:Rifabutin / Q000031:analogs & derivatives
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D016328:NF-kappa B; D037662:Pulmonary Surfactant-Associated Protein A; D017828:Rifabutin; D009569:Nitric Oxide; C020754:herbimycin; D020928:Mitogen-Activated Protein Kinases