Leukocyte kinetics in the pulmonary microcirculation: observations using real-time confocal luminescence microscopy coupled with high-speed video analysis.
To quantitatively assess blood cell kinetics in the intact pulmonary microcirculation, in which arterioles, venules, and capillaries are exceedingly intricate and densely convoluted, we recently developed a real-time confocal laser luminescence microscope with a high-speed analysis component. The system has the capacity to yield confocal images of rapidly moving cells at a rate of 1000 frames/second and at sufficiently high degrees of magnification. Applying this novel method to isolated perfused rat lungs, we estimated the endothelial distributions of constitutively expressed intercellular adhesion molecule-1 (ICAM-1) and P-selectin and also studied leukocyte hemodynamic behavior in the pulmonary microvasculature under conditions in which ICAM-1, P-selectin, and L-selectin were inhibited, respectively, by 1A29 (monoclonal antibody to rat ICAM-1), ARP2-4 (monoclonal antibody to rat P-selectin), and fucoidin (competitive inhibitor of both P- and L-selectin). The results were compared with those obtained with a nonconfocal microscope using conventional epiluminescence. Intertwined microvessel networks in the lung were clearly distinguishable in confocal images but not in conventional nonconfocal views. ICAM-1 was perceptibly expressed along venular and capillary but not arteriolar endothelium, whereas P-selectin was undetectable in all microvessels examined. Leukocytes were not firmly adhered to venular or arteriolar endothelial cells. Leukocyte rolling was recognized more frequently along arteriolar walls than along venular walls and was suppressed in arterioles by L-selectin inhibition but not by either ICAM-1 or P-selectin inhibition. In capillaries, transient and sustained arrest of leukocytes occurred at physiologic shear rates. Inhibition of ICAM-1 or P-selectin had no remarkable effect upon either transient or sustained entrapment of leukocytes in capillaries. In conclusion, physiologic and biologic characteristics of pulmonary microvessels appear to be quite different from those of the systemic microcirculation.