The location and mechanisms of leukocyte sequestration in the pulmonary circulation have been investigated by using high-magnification in vivo videomicroscopy to record the passage of unlabeled native leukocytes through canine pulmonary capillaries. Of 650 leukocytes traversing capillary networks, 46 +/- 6% (SE) of the leukocytes passed through without stopping, 42 +/- 9% stopped in segments between junctions, and 12 +/- 4% stopped in junctions. Leukocytes rolling along arteriolar walls were nearly spherical, as 94% had aspect ratios (major axis divided by minor axis) < or = 1.25. To pass through the capillary bed, the leukocytes deformed into elongated shapes. Many leukocytes remained elongated after entering the venules (53% had aspect ratios > or = 1.25). Venular rolling was blocked by fucoidin (blocking both L- and P-selectin) but not by anti-P-selectin antibodies alone, indicating that rolling leukocytes adhered to the venular endothelium by L-selectin. These observations demonstrate that leukocytes deform to transit the capillary bed, that they stop more frequently in segments than in junctions, and that rolling leukocytes in the venular marginated pool adhere via L-selectin.

Although the lung is known to be a major site of neutrophil margination, the anatomic location of these sequestered cells within the lung is controversial. To determine the site of margination and the kinetics of neutrophil transit through the pulmonary microvasculature, we infused fluorescein isothiocyanate-labeled canine neutrophils into the pulmonary arteries of 10 anesthetized normal dogs and made fluorescence videomicroscopic observations of the subpleural pulmonary microcirculation through a window inserted into the chest wall. The site of fluorescent neutrophil sequestration was exclusively in the pulmonary capillaries with a total of 951 labeled cells impeded in the capillary bed for a minimum of 2 s. No cells were delayed in the arterioles or venules. Transit times of individual neutrophils varied over a wide range from less than 2 s to greater than 20 min with an exponential distribution skewed toward rapid transit times. These observations indicate that neutrophil margination occurs in the pulmonary capillaries with neutrophils impeded for variable periods of time on each pass through the lung. The resulting wide distribution of transit times may determine the dynamic equilibrium between circulating and marginated neutrophils.

Hypoxia has been shown to cause an increased number of pulmonary capillaries to be perfused. Changes in cardiac output and left atrial pressure have been previously ruled out as causes of this capillary recruitment. Increased pulmonary vein pressure and increased pulmonary artery pressure remain as two potential mechanisms. To differentiate between these two possible causes, we measured pulmonary artery and vein pressures with directly placed catheters and capillary recruitment with in vivo microscopy. During isocapnic hypoxia pulmonary artery pressure doubled, observed capillary recruitment increased fivefold, and pulmonary vein pressure remained constant. When the vasodilator prostaglandin E1 was infused during hypoxia, pulmonary artery pressure and capillary recruitment fell to control values and pulmonary vein pressure remained constant. Since capillary recruitment correlated with pulmonary artery pressure in each dog, but not with pulmonary vein pressure, we conclude that arterial, not venous, constriction is the probable cause of this recruitment.

In vivo microscopic observations of pulmonary capillaries are limited to subpleural networks that are less dense than interior networks. In addition to the density difference, subpleural and interior capillary diameters may differ, although there are conflicting data on this point. We measured the diameters of subpleural and interior capillaries in rats and dogs. Subpleural diameters were 30% larger in rats and 20% larger in dogs. Because diameter and density differences might cause differences in recruitment between subpleural and interior networks, we measured subpleural and interior recruitment by counting the number of red blood cells per 10 microns of alveolar wall in histological cross sections of rapidly frozen rat lungs. Lung inflation pressures of 4, 12, and 25 cmH2O created a wide range of capillary recruitment in different groups of animals. Red blood cell counts for interior and subpleural capillaries moved in parallel and progressively increased as inflation pressures were reduced. These data demonstrate that recruitment in subpleural capillaries accurately reflect recruitment in interior capillaries and validate the use of in vivo microscopic observations of subpleural capillaries to investigate pulmonary capillary recruitment in general.

We have previously shown that airway hypoxia causes pulmonary capillary recruitment and raises diffusing capacity for carbon monoxide. This study was designed to determine whether these events were caused by an increase in pulmonary vascular resistance, which redistributed blood flow toward the top of the lung, or by an increase in cardiac output. We measured capillary recruitment at the top of the dog lung by in vivo microscopy, gas exchange surface area of the whole lung by diffusing capacity for carbon monoxide, and blood flow distribution by radioactive microspheres. During airway hypoxia recruitment occurred, diffusing capacity increased, and blood flow was redistributed upward. When a vasodilator was infused while holding hypoxia constant, these effects were reversed; i. e., capillary "derecruitment" occurred, diffusing capacity decreased, and blood flow was redistributed back toward the bottom of the lung. The vasodilator was infused at a rate that left hypoxic cardiac output unchanged. These data show that widespread capillary recruitment during hypoxia is caused by increased vascular resistance and the resulting upward blood flow redistribution.