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Respiratory Research
Published in: Respiratory Research 1/2009

Open Access 01-12-2009 | Research

Mitogen-activated protein kinases and NFκB are involved in SP-A-enhanced responses of macrophages to mycobacteria

Authors: Joseph P Lopez, David J Vigerust, Virginia L Shepherd

Published in: Respiratory Research | Issue 1/2009

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Abstract

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 NFκB 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 μg of SP-A to 5 × 105 BCG for 30 min at 37°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 NFκB-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 IκB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively.

Conclusion

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 NFκB.
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Metadata
Title
Mitogen-activated protein kinases and NFκB are involved in SP-A-enhanced responses of macrophages to mycobacteria
Authors
Joseph P Lopez
David J Vigerust
Virginia L Shepherd
Publication date
01-12-2009
Publisher
BioMed Central
Published in
Respiratory Research / Issue 1/2009
Electronic ISSN: 1465-993X
DOI
https://doi.org/10.1186/1465-9921-10-60

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