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 m icrocirculation, in which arterioles, venules, and capillaries are exc eedingly intricate and densely convoluted, we recently developed a rea l-time confocal laser luminescence microscope with a high-speed analys is component. The system has the capacity to yield confocal images of rapidly moving cells at a rate of 1000 frames/second and at sufficient ly high degrees of magnification. Applying this novel method to isolat ed perfused rat lungs, we estimated the endothelial distributions of c onstitutively expressed intercellular adhesion molecule-1 (ICAM-1) and P-selectin and also studied leukocyte hemodynamic behavior in the pul monary microvasculature under conditions in which ICAM-1, P-selectin, and L-selectin were inhibited, respectively, by 1A29 (monoclonal antib ody to rat ICAM-1), ARP2-4 (monoclonal antibody to rat P-selectin), an d fucoidin (competitive inhibitor of both P- and L-selectin). The resu lts were compared with those obtained with a nonconfocal microscope us ing conventional epiluminescence. Intertwined microvessel networks in the lung were clearly distinguishable in confocal images but not in co nventional nonconfocal views. ICAM-1 was perceptibly expressed along v enular and capillary but not arteriolar endothelium, whereas P-selecti n was undetectable in all microvessels examined. Leukocytes were not f irmly adhered to venular or arteriolar endothelial cells. Leukocyte ro lling was recognized more frequently along arteriolar walls than along venular walls and was suppressed in arterioles by L-selectin inhibiti on but not by either ICAM-1 or P-selectin inhibition. In capillaries, transient and sustained arrest of leukocytes occurred at physiologic s hear rates. Inhibition of ICAM-1 or P-selectin had no remarkable effec t upon either transient or sustained entrapment of leukocytes in capil laries. In conclusion, physiologic and biologic characteristics of pul monary microvessels appear to be quite different from those of the sys temic microcirculation.