1995 OPEN FORUM Abstracts
Adaptor Ap-2, A Coated Pit Protein Complex, Is Required For Alveolar Macrophage Phagocytosis
Douglas G. Perry, PhD, RRT, and Gena Daugherty, BS Respiratory Therapy Program, School of Allied Health Sciences, and Division of Pulmonary and Critical Care Medicine, Indiana University School of Medicine, Indianapolis IN 46202.
Introduction: Clathrin-coated pits (CCPs) are found on the cytoplasmic membrane surface of alveolar macrophages (AMs). CCPs are involved in receptor/membrane recycling during cell migration and may also play a role in phagocytosis. We previously demonstrated that clathrin is directly involved in phagocytosis (Perry DG and Martin WJ, 1994: Am J Resp Crit Care Med 149:A238). To determine whether another CCP component, adaptor AP-2, is expressed by AMs, we lo-calized AP-2 by immunocytochemistry. To test whether AP-2 is also involved in phagocytosis, we inhibited AP-2 activity using liposomes to deliver anti-AP-2 monoclonal antibody (mAb) to rat AMs and measured subsequent phagocytic activity by fluorometry.
Methods: Rat AMs were obtained by lavage, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton, incubated with 1:40 anti-AP-2 mAb, rinsed, and incubated with 1:100 FITC-lgG. Control cells did not receive anti-AP-2 mAb. Liposomes were prepared by aqueous reconstitution with 1:40 anti-AP-2 mAb; control liposomes contained nonspecific antibody. AMs labeled with the fluorophore Dil were incubated with the liposomes, then challenged for phagocytosis with fluorescent liposomes. Phagocytosis was measured with a dual-beam fluorometer.
Results: AP-2 is present in rat AMs, with distribution largely limited to the cell membrane, where clathrin-mediated retrieval of surface receptors occurs. AMs treated with anti-AP-2 liposomes had significantly lower phagocytosis than control cells (4.81 ± 0.23 vs. 9.54 ± 0.76 liposomes/cell, respectively; p < 0.001). Conclusion: These findings further support the hypothesis that CCPs are directly involved in phagocytosis. Supported in full by NIH grant HL50128 (D.G.P.).
SEE ORIGINAL GRAPH