The Science Journal of the American Association for Respiratory Care

1998 OPEN FORUM Abstracts

TRACKING OF ALVEOLAR MACROPHAGE MEMBRANE RECYCLING DURING PHAGOCYTOSIS

Brook R. Ballard, BS RRT, Andrea L. May, BS CRTT, Matthew A. Roth, BS RRT, Neal D. Morgan, BS CRTT, Patricia A. Smith, BS, and Douglas G. Perry, PhD. Respiratory Therapy Program, School of Allied Health Sciences, and Division of Pulmonary and Critical Care Medicine, Indiana University School of Medicine, Indianapolis IN.

Background: Pulmonary alveolar macrophages (AMs) provide the primary defense in the lung against foreign organisms and inhaled particulates. One of the immunological roles of the AM is to clear these from the lung. This cellular clearance is accomplished by phagocytosis, which involves AM recognition, internalization, and digestion of these foreign targets. Membrane recycling has been shown to occur in endocytosis and cell migration; it is possible that this process also occurs during phagocytosis. The purpose of this study is to visualize and quantify membrane recycling during phagocytosis, and correlate membrane turnover with phagocytic activity. Methods: Rat AMs were obtained by whole lung lavage. AM plasma membrane was labeled with 10 mg/ml FITC-lipophilic dextran. Fluorescence of the external label was quenched with 25 mM NaHCO_{3} titrated to pH 5.0. Internalization of membrane was measured as fluorescence intensity of the dequenched label. AM membrane recycling was observed using a Zeiss/Meridian confocal microscope while employing AM interaction with an immunoreactive substrate as a two-dimensional model of phagocytosis. For data collection, AMs were randomly selected by panning the field. Fluorescence and phase contrast images of AMs were acquired every 5 minutes for one hour. Membrane turnover was quantified as persistence (total label area over time) and as relative label size (ratio of label perimeter to area over time), and phagocytic activity was quantified as pleomorphism (shape factor residuals over time). Results: AMs demonstrated phagocytic activity under the conditions of this experiment: Pleomorphism ranged from 0.01 to 0.35 mean squared residuals (MSR). Membrane turnover increased for any given level of phagocytic activity: Persistence, which is inversely proportional to membrane turnover, had a moderate negative correlation with phagocytic activity (r^{2} = 0.60). Relative label size, which is also inversely proportional to membrane turnover, had a strong negative correlation (r^{2} = 0.92). Conclusions: Membrane turnover increased as phagocytic activity increased. These results suggest that membrane recycling occurs during phagocytosis. This may be the mechanism by which membrane material is replenished at the leading edges of filopodia and pseudopodia during phagocytosis.

Supported by NIH HL50128 (D.G.P.).

The 44th International Respiratory Congress Abstracts-On-DiskĀ®, November 7 - 10, 1998, Atlanta, Georgia.

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