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Published on: June 8, 2012
Oxidative responses of rabbit alveolar macrophages: comparative priming activities of MIF/MAF, sera, and serum
H Hayakawa1, K Umehara, Q N Myrvik
1Department of Microbiology and Immunology, Bowman Gray School of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Abstract:
The comparative abilities of various reagents to prime rabbit alveolar macrophages (AM) to produce reactive oxygen intermediates (ROI) in a chemiluminescent (CL) assay were investigated. It was noted that AM from normal rabbits cultured in a serum-free medium for 18 hr exhibited a "spontaneous" priming response following a challenge with phorbol myristate acetate (PMA); however, "spontaneous" priming was not evident when the AM were cultured for only 3 hr. It was further established that pretreatment of normal AM for 3 or 18 hr with MIF/MAF preparations (serum-free), fetal bovine serum (FBS), or bovine serum albumin (BSA) exhibited marked increases in their CL responses following challenge with PMA. When FBS was used in the culture medium, the priming activity of MIF/MAF was masked because of the high CL responses of controls due to the priming effects of FBS. BSA at concentrations approximately equivalent to the amount in FBS also displayed marked priming activity. Bacterial products (lipopolysaccharide and muramyl dipeptide), latex particles, rabbit IgG, PMA, and opsonized as well as nonopsonized zymosan and bacteria (BCG and Staphylococcus epidermidis) were inactive as priming agents. In comparison, AM from BCG-immune rabbits that were primed in vivo yielded a very large CL response when challenged with PMA. Opsonized zymosan and bacteria produced twofold increases in the CL responses in BCG-immune AM compared to nonopsonized preparations. The marked priming effect of serum on AM cultured for even a short period (3 hr) indicates that normal AM undergo marked changes in culture that complicate the interpretation of AM function when AM are cultured in vitro in media containing serum.
Insights
Fetal bovine serum and bovine serum albumin can prime alveolar macrophages (AM) for reactive oxygen intermediate production. However, serum components can mask other priming agents and complicate in vitro studies of AM function.
Area of Science:
- Immunology
- Cell Biology
Background:
- Alveolar macrophages (AM) play a crucial role in lung immunity.
- Understanding factors that modulate AM function, such as priming, is essential for immune research.
Purpose of the Study:
- To investigate the comparative abilities of various reagents to prime rabbit AM for reactive oxygen intermediate (ROI) production.
- To assess the impact of culture conditions and serum components on AM priming.
Main Methods:
- Chemiluminescent (CL) assay to measure ROI production by AM.
- Culturing rabbit AM with different priming agents including serum-free preparations, fetal bovine serum (FBS), and bovine serum albumin (BSA).
- Challenging primed AM with phorbol myristate acetate (PMA) and evaluating CL responses.
Main Results:
- AM cultured in serum-free medium showed spontaneous priming after 18 hours but not 3 hours.
- Pretreatment with MIF/MAF, FBS, or BSA significantly increased CL responses.
- FBS masked the priming activity of MIF/MAF due to its own potent priming effect.
- BSA demonstrated significant priming activity at concentrations similar to FBS.
- Bacterial products, latex particles, IgG, PMA, and zymosan/bacteria were inactive as priming agents.
- BCG-immune AM primed in vivo showed a substantial CL response to PMA.
Conclusions:
- Serum components, particularly FBS and BSA, are potent priming agents for rabbit AM.
- The presence of serum in culture media can significantly alter AM responses and complicate the interpretation of in vitro functional assays.
- In vivo priming in BCG-immune rabbits leads to a heightened response.
