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Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
Published on: June 8, 2012
Binding of polymyxin B to rat alveolar macrophages
G K Bysani1, D C Stokes, M Fishman
1Cardiopulmonary-Critical Care Division, St. Jude Children's Research Hospital, Memphis.
Abstract:
The specific binding of radiolabeled polymyxin B (PmB) to rat alveolar macrophages was investigated. PmB retained its ability to inhibit lipopolysaccharide-induced tumor necrosis factor production by macrophages as long as one of five amino groups on PmB was unbound. Binding was saturable and temperature- and time-dependent, reaching steady state by 30 min at 37 degrees C and by 18 h at 4 degrees C. Macrophages had approximately 1.6 X 10(7) (Kd = 0.28 nM) PmB binding sites per cell. Lipid A had no appreciable effect on the number of sites. Binding did not occur to rat platelets, L929 fibroblast cells, a rat thymoma cell line, or precursor monocytic and myeloid cell lines. Precursor cells activated with 12-O-tetradecanoylphorbol-13-acetate acquired binding similar to that seen in alveolar macrophages, but L929 fibroblasts did not. Binding sites were sensitive to trypsin but not to phospholipase C. PmB may interact with specific binding sites involved in lipopolysaccharide-induced activation, production, or release of tumor necrosis factor by macrophages, inhibiting the effects of lipopolysaccharide on macrophages.
Insights
Polymyxin B (PmB) specifically binds to rat alveolar macrophages, inhibiting lipopolysaccharide-induced tumor necrosis factor. This binding is saturable and dependent on temperature and time, suggesting a targeted interaction.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Polymyxin B (PmB) is an antibiotic known for its activity against Gram-negative bacteria.
- Lipopolysaccharide (LPS) is a key component of Gram-negative bacterial outer membranes that triggers inflammatory responses.
- Tumor necrosis factor (TNF) is a pro-inflammatory cytokine implicated in various immune responses and diseases.
Purpose of the Study:
- To investigate the specific binding of radiolabeled Polymyxin B (PmB) to rat alveolar macrophages.
- To understand the characteristics and potential biological significance of PmB binding to macrophages.
Main Methods:
- Radiolabeling of Polymyxin B for binding assays.
- Saturation binding studies at different temperatures and time points.
- Testing binding specificity across various cell types (macrophages, platelets, fibroblasts, cell lines).
- Enzymatic sensitivity assays (trypsin, phospholipase C).
Main Results:
- Polymyxin B (PmB) demonstrated specific, saturable, temperature- and time-dependent binding to rat alveolar macrophages.
- Macrophages possess approximately 1.6 x 10^7 PmB binding sites per cell with a Kd of 0.28 nM.
- Binding was observed on activated precursor cells but not on platelets, fibroblasts, or non-activated precursor cells, indicating specificity.
- Binding sites were sensitive to trypsin but not phospholipase C.
Conclusions:
- Polymyxin B (PmB) binds to specific sites on rat alveolar macrophages, suggesting a targeted mechanism of action.
- These binding sites are likely involved in the inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production.
- PmB's interaction with macrophages may modulate LPS-mediated inflammatory responses.
