Quantifying rat pulmonary intravascular mononuclear phagocytes

Abstract

Cells of the mononuclear phagocyte system (MPS) protect the host by clearing effete and foreign particulates from the circulation. The current study was designed to identify, quantify, harvest, and provide a partial functional characterization of the systemic host-defense cell located in the pulmonary microvasculature of the rat. Critical colloid doses of test particulates (monastral blue B [MBB] or polystyrene beads) were infused intra-arterially into anesthetized rats so that phagocytically active pulmonary intravascular phagocytes could be identified. Morphologic characterization of in situ phagocytes was performed using electron microscopy. The number of active phagocytes was then determined using tissue samples processed for light microscopy. Finally, sequential perfusion of the pulmonary vasculature with buffer, chelating agent, and collagenase allowed elution and preliminary functional characterization of the pulmonary intravascular mononuclear phagocyte (PIMP). Electron microscopy demonstrated that both mononuclear phagocytes and neutrophils contributed to pulmonary sequestration of circulating particulates. Light microscopy showed that the microvasculature of each alveolus contained 0.50+/-0.19 active mononuclear phagocytes and 0.14+/-0.12 active neutrophils. A chelation/collagenase elution technique was then used to harvest the PIMP. Histologic evaluation of the postperfusion lungs indicated that 80% of the active phagocytes were removed by the technique. In total, the elution fluids contained 2.63+/-1.04 x 10(7) cells, with 1.60+/-0.78 x 10(7), 0.49+/-0.17 x 10(7), and 0.54+/-0.26 x 10(7) of those cells being mononuclear phagocytes, neutrophils, and lymphocytes, respectively. Functionally, the mononuclear phagocyte population exhibited a spectrum of phagocytic activities, with 51.5+/-19.5% of the cells being inactive, 33.9+/-13.4% exhibiting moderate phagocytic activity, and 14.6+/-9.8% demonstrating intensive phagocytic capacity. The current study provides the first quantified demonstration that mononuclear phagocytes are primarily responsible for sequestering blood-borne foreign particulates in the pulmonary circulation of the rat. Approximately 2 x 10(7) PIMP existed in the lungs of 300 gram rats. The functionally heterogeneous mononuclear phagocytes exhibited phagocytic capacities ranging from avidly phagocytic (14.6+/-9.8%) through moderately active (33.9+/-13.4%) to inactive. The lung microvasculature's large pool of inactive mononuclear phagocytes may provide a recruitable mechanism to allow significant increases in clearance of circulating particulates. A resident pool of activatable mononuclear phagocytes might explain previous clinical observations of increased particulate localization in the lung microvasculature of septic patients.

Intravascular donor monocytes play a central role in lung transplant ischaemia-reperfusion injury

“…Sequestration of leukocytes within the pulmonary microvasculature, in particular monocytes, during ex vivo perfusion of rodent lungs has previously been reported, 20 27 and the traditional method to define lung tissue (interstitial) leukocytes as cells remaining after lung perfusion is now recognised as a flawed approach: 28 near-complete removal of these marginated pools would require draconian perfusion methods (eg, using EDTA or enzyme-containing perfusate). 20 27 Crucially, intravascular monocytes have also been observed in transplant lungs by histological examination. 13 This is consistent with our novel EM data, which clearly confirm that monocytes are located in the pulmonary microvasculature, and indicate a likely interaction with the endothelium.…”

Mobilization and Margination of Bone Marrow Gr-1high Monocytes during Subclinical Endotoxemia Predisposes the Lungs toward Acute Injury

“…The pulmonary artery and left atrium were cannulated, and the lungs were perfused at a constant flow of 50 ml/kg and left atrial pressure of 2.5 mmHg, and ventilated with 21% O 2 and 5% CO 2 in N 2 . The perfusate used was RPMI-1640 without phenol red (Invitrogen) with pH adjusted to 7.35-7.45, supplemented with 4% BSA (Sigma, Poole, UK) and 5 mM EDTA to facilitate release of marginated monocytes (39). After perfusion for 40 min at 37°C, the lungs were harvested and single cells suspensions prepared for analysis by flow cytometry.…”