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Quantitative recovery of pulmonary intravascular macrophages from sheep lungs
R A Rogers1, D R Tasat, A E Warner
1Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115.
Abstract:
Pulmonary intravascular macrophages (PIMs) adhere to the endothelium of lung capillaries and sequester circulating particles and pathogens from the blood. Iron oxide (gamma Fe2O3) 5 mg/kg, administered intravenously, specifically labeled PIMs in situ within the living sheep. Attempts to isolate gamma Fe2O3-labeled PIMs using vascular perfusion (VP) procedures yielded few cells. To improve recovery of PIMs, a proteolytic lung digestion (PLD) procedure was developed. Following PLD, gamma Fe2O3-containing PIMs were recovered by magnets and the amount of gamma Fe2O3 present measured by fluxgate magnetometry. Proteolytic lung digestion recovered 34% of the total gamma Fe2O3 in lung samples and yielded 2 x 10(5) PIMs/g lung with 95% viability. In contrast, VP recovered only 3% of the total gamma Fe2O3 in the lung; furthermore, less than 2% of the recovered gamma Fe2O3 was cell associated. Proteolytic lung digestion followed by magnetic separation is an effective way to recover viable sheep PIMs for in vitro study.
Insights
A new method using proteolytic lung digestion effectively isolates viable pulmonary intravascular macrophages (PIMs) labeled with iron oxide for research. This technique significantly improves PIM recovery compared to traditional vascular perfusion methods.
Area of Science:
- Immunology
- Cell Biology
- Pulmonary Research
Background:
- Pulmonary intravascular macrophages (PIMs) are crucial for sequestering blood-borne particles and pathogens within lung capillaries.
- Current methods for isolating PIMs, such as vascular perfusion (VP), are inefficient, yielding few cells.
Purpose of the Study:
- To develop and validate an improved method for recovering viable PIMs from sheep lungs.
- To compare the efficacy of a novel proteolytic lung digestion (PLD) procedure against VP for PIM isolation.
Main Methods:
- Intravenous administration of iron oxide (gamma Fe2O3) to label PIMs in vivo.
- Isolation of labeled PIMs using either vascular perfusion (VP) or a newly developed proteolytic lung digestion (PLD) procedure.
- Magnetic separation of iron oxide-labeled PIMs following PLD, with quantification by fluxgate magnetometry.
Main Results:
- PLD recovered 34% of total lung iron oxide, yielding 2 x 10(5) PIMs/g lung with 95% viability.
- VP recovered only 3% of total lung iron oxide, with less than 2% being cell-associated.
- PLD followed by magnetic separation proved significantly more effective for PIM recovery.
Conclusions:
- Proteolytic lung digestion is a superior method for recovering viable pulmonary intravascular macrophages.
- This technique enables effective isolation of PIMs for subsequent in vitro studies.
- The findings offer a valuable tool for pulmonary and immunological research.