Neutrophil transit times through pulmonary capillaries: the effects of capillary geometry and fMLP-stimulation

The deformations of neutrophils as they pass through the pulmonary microcirculation affect their transit time, their tendency to contact and interact with the endothelial surface, and potentially their degree of activation. Here we model the cell as a viscoelastic Maxwell material bounded by constant surface tension and simulate indentation experiments to quantify the effects of (N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulation on its mechanical properties (elastic shear modulus and viscosity). We then simulate neutrophil transit through individual pulmonary capillary segments to determine the relative effects of capillary geometry and fMLP-stimulation on transit time. Indentation results indicate that neutrophil viscosity and shear modulus increase by factors of 3.4, for 10(-9) M fMLP, and 7.3, for 10(-6) M fMLP, over nonstimulated cell values, determined to be 30.8 Pa.s and 185 Pa, respectively. Capillary flow results indicate that capillary entrance radius of curvature has a significant effect on cell transit time, in addition to minimum capillary radius and neutrophil stimulation level. The relative effects of capillary geometry and fMLP on neutrophil transit time are presented as a simple dimensionless expression and their physiological significance is discussed.     

Computational modeling of RBC and neutrophil transit through the pulmonary capillaries.

A computational model of the pulmonary microcirculation is developed and used to examine blood flow from arteriole to venule through a realistically complex alveolar capillary bed. Distributions of flow, hematocrit, and pressure are presented, showing the existence of preferential pathways through the system and of large segment-to-segment differences in all parameters, confirming and extending previous work. Red blood cell (RBC) and neutrophil transit are also analyzed, the latter drawing from previous studies of leukocyte aspiration into micropipettes. Transit time distributions are in good agreement with in vivo experiments, in particular showing that neutrophils are dramatically slowed relative to the flow of RBCs because of the need to contract and elongate to fit through narrower capillaries. Predicted neutrophil transit times depend on how the effective capillary diameter is defined. Transient blockage by a neutrophil can increase the local pressure drop across a segment by 100--300%, leading to temporal variations in flow and pressure as seen by videomicroscopy. All of these effects are modulated by changes in transpulmonary pressure and arteriolar pressure, although RBCs, neutrophils, and rigid microspheres all behave differently.     

Vertical gradient of pulmonary capillary transit times

The key determinants of alveolar capillary perfusion are transit times and the extent of recruitment. Capillaries are known to be heavily recruited in the dependent lung, but there are no direct data that bear on how capillary transit times might be affected by gravity. We directly determined mean capillary transit times on the surface of the upper, middle, and lower lung by measuring the passage of fluorescent dye through the capillaries using in vivo television microscopy. In anesthetized dogs, mean capillary transit times averaged 12.3 s in the upper lung, 3.1 s in the midlung, and 1.6 s in the lower lung. This near order of magnitude variation in speed of blood transit establishes that there is a vertical gradient of capillary transit times in the lung. As expected, dependent capillary networks were nearly fully recruited, whereas relatively few capillaries were perfused in the upper lung. The lengthy transit times and sparsely perfused capillary beds in the upper lung combine to provide a substantial part of pulmonary gas exchange reserve.     

Physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries

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.     

Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties.

Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.     

Distribution of pulmonary capillary transit times in recruited networks

When pulmonary blood flow is elevated, hypoxemia can occur in the fastest-moving erythrocytes if their transit times through the capillaries fall below the minimum time for complete oxygenation. This desaturation is more likely to occur if the distribution of capillary transit times about the mean is large. Increasing cardiac output is known to decrease mean pulmonary capillary transit time, but the effect on the distribution of transit times has not been reported. We measured the mean and variance of transit times in single pulmonary capillary networks in the dependent lung of anesthetized dogs by in vivo videofluorescence microscopy of a fluorescein dye bolus passing from an arteriole to a venule. When cardiac output increased from 2.9 to 9.9 l/min, mean capillary transit time decreased from 2.0 to 0.8 s. Because transit time variance decreased proportionately (relative dispersion remained constant), increasing cardiac output did not alter the heterogeneity of local capillary transit times in the lower lung where the capillary bed was nearly fully recruited.     

In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability".

Neutrophils are normally delayed in transit through the lung microcirculation, relative to the passage of erythrocytes. This sequestration contributes to a pulmonary pool of neutrophils that may relate to the relative inability of neutrophils to deform compared with erythrocytes when in transit in the pulmonary capillaries. A micropore membrane was used to model the human pulmonary microcirculation, in which cell deformability was measured as the pressure developed during filtration of the cells through the membrane at a constant flow. We demonstrated a significant correlation between in vitro deformability and in vivo lung sequestration of indium-111-labeled neutrophils in 10 normal subjects (r = 0.69, P less than 0.02). In eight patients with stable chronic obstructive pulmonary disease, this relationship was not significant (r = -0.2, P greater than 0.05). Furthermore, in a subject with microscopic pulmonary telangiectasia known to allow significant passage of 30-microns microspheres, neutrophils passed through the lungs without delay. Moreover, neutrophils from patients studied acutely with an exacerbation of chronic obstructive pulmonary disease were temporarily less deformable (P less than 0.01). These studies confirm that cell deformability is an important determinant of the normal neutrophil sequestration within the lungs. Changes in cell deformability may alter the extent of this sequestration.     

Electrical resistance of a capillary endothelium

The electrical resistance of consecutive segments of capillaries has been determined by a method in which the microvessels were treated as a leaky, infinite cable. A two-dimensional analytical model to describe the potential field in response to intracapillary current injection was formulated. The model allowed determination of the electrical resistance from four sets of data: the capillary radius, the capillary length constant, the length constant in the mesentery perpendicular to the capillary, and the relative potential drop across the capillary wall. Of particular importance were the mesothelial membranes covering the mesenteric capillaries with resistances several times higher than that of the capillary endothelium. 27 frog mesenteric capillaries were characterized. The average resistance of the endothelium was 1.85 omega cm2, which compares well with earlier determinations of the ionic permeability of such capillaries. However, heterogeneity with respect to resistance was observed, that of 10 arterial capillaries being 3.0 omega cm2 as compared with 0.95 omega cm2 for 17 mid- and venous capillaries. The average in situ length constant was 99 micrometers for the arterial capillaries and 57 micrometers for the mid- and venous capillaries. It is likely that the ions that carry the current must move paracellularly, through junctions that are leaky to small solutes.     

Stress failure in pulmonary capillaries

In the mammalian lung, alveolar gas and blood are separated by an extremely thin membrane, despite the fact that mechanical failure could be catastrophic for gas exchange. We raised the pulmonary capillary pressure in anesthetized rabbits until stress failure occurred. At capillary transmural pressures greater than or equal to 40 mmHg, disruption of the capillary endothelium and alveolar epithelium was seen in some locations. The three principal forces acting on the capillary wall were analyzed. 1) Circumferential wall tension caused by the transmural pressure. This is approximately 25 dyn/cm (25 mN/m) at failure where the radius of curvature of the capillary is 5 microns. This tension is small, being comparable with the tension in the alveolar wall associated with lung elastic recoil. 2) Surface tension of the alveolar lining layer. This contributes support to the capillaries that bulge into the alveolar spaces at these high pressures. When protein leakage into the alveolar spaces occurs because of stress failure, the increase in surface tension caused by surfactant inhibition could be a powerful force preventing further failure. 3) Tension of the tissue elements in the alveolar wall associated with lung inflation. This may be negligible at normal lung volumes but considerable at high volumes. Whereas circumferential wall tension is low, capillary wall stress at failure is very high at approximately 8 x 10(5) dyn/cm2 (8 x 10(4) N/m2) where the thickness is only 0.3 microns. This is approximately the same as the wall stress of the normal aorta, which is predominantly composed of collagen and elastin. The strength of the thin part of the capillary wall is probably attributable to the collagen IV of the basement membranes. The safety factor is apparently small when the capillary pressure is raised during heavy exercise. Stress failure causes increased permeability with protein leakage, or frank hemorrhage, and probably has a role in several types of lung disease.     

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.     

Marginated pool of neutrophils in rabbit lungs

The size and location of the marginated pool of neutrophils (PMNs) in rabbit lungs were evaluated, and the rate of exchange of the PMNs with the circulating pool was determined. 99mTc-labeled erythrocytes (99mTc-RBCs) and 125I-labeled macroaggregated albumin (125I-MAA) were used to determine RBC transit times in the pulmonary circulation. Radiolabeled PMNs were studied on their first passage through the lungs. After 10 min of circulation, the lungs were fixed, gamma counted, and prepared for morphometric and autoradiographic studies; 74 +/- 3% of the PMNs was retained in the lungs on the first passage, and 23 +/- 2% was within the pulmonary marginated pool 10 min later. The regional PMN retention and the rate of exchange between the marginated and circulating PMN pools in the lung were directly related to RBC transit time. The radiolabeled PMNs distributed similarly to the unlabeled cells within the microvasculature and had a similar exchange rate between the marginated and circulating pools (1.4 +/- 0.2%/s using labeled cells and 1.5 +/- 0.5%/s using unlabeled cells). The marginated pool was located primarily within alveolar capillaries and contained two to three times as many PMNs as the total circulating pool.     

An analysis of the sluicing gate in pulmonary blood flow

For pulmonary blood flow in zone 2 condition, in which the blood pressure in the venule (pven) is lower than the alveolar gas pressure (pA), the blood exiting from the capillary sheet and entering a venule must go through a sluicing gate. The sluicing gate exists because the venule remains patent while the capillaries will collapse when the static pressure of blood falls below the alveolar gas pressure. In the original theory of sheet flow the effect of the tension in the interalveolar septa on the flow through the sluicing gate was ignored. Since the tension multiplied by the curvature of the membrane is equivalent to a lateral pressure tending to open the gate, and since the curvature of the capillary wall is high in the gate region, this effect may be important. The present analysis improves the original theory and demonstrates that the effect of membrane tension is to cause flow to increase when the venous pressure continues to decrease. The shape of the sluicing gate resembles that of a venturi tube, and can be determined by an iterative integration of the differential equations. The result forms an important link in the theory of pulmonary blood flow in zone 2 condition.     

Alveolar vessel behavior in the zone 2 lung inferred from indicator-dilution data

To gain insight into the changes occurring in alveolar vessels when alveolar pressure exceeds venous pressure at the downstream end of the alveolar vessels (zone 2), we compared the uptake of serotonin and the extravascular volume accessible to 3HOH (Qev) under zone 2 and 3 conditions in isolated dog lung lobes. We also examined the influence of occluding some of the small pulmonary arteries with 58- to 548-micron-diam beads on the serotonin uptake and Qev. We found that, with the bead embolization, both the serotonin uptake and the Qev were reduced, whereas the change from zone 3 to 2 reduced serotonin uptake but did not change Qev. A plausible explanation for these observations is that the beads occluded vessels that were relatively large compared with those in which significant transvascular 3HOH exchange and serotonin uptake take place. Perfusion ceased in the collection of capillaries normally served by the obstructed arteries. Thus the extravascular water and the serotonin uptake sites downstream from the obstructions were not accessible to the indicators during the short time interval of the indicator passage through the lung. On the other hand, the change from zone 3 to zone 2 resulted in the collapse of small individual capillary segments within the alveolar vessel bed. Since the serotonin does not readily diffuse from the vessels through the tissue, it could not reach the endothelial cells of the collapsed capillaries. However, since the distances for diffusion between collapsed capillaries and neighboring perfused capillaries were small, the more highly diffusible 3HOH had access to the same Qev under both zone 2 and 3 conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of raised alveolar pressure on leukocyte retention in the human lung

To determine whether an increase in alveolar pressure delays the passage of leukocytes (WBCs) through the lung by compressing the lung capillaries, we measured the concentration of WBC across the lung in response to a forced expiratory maneuver. In 20 human subjects, blood was sampled from catheters placed in the pulmonary artery (PA) and left ventricle (LV) before, during, and after a forced expiratory maneuver held for greater than or equal to 20 s against an occluded airway. Pressures were recorded at the mouth and from both catheters. A significant fall in LV WBC (P less than 0.01) but not in PA WBC occurred during or immediately after the maneuver in 18 subjects, with a mean maximum decrease of 26 +/- 12% (SD) from base line (range 9-46%). Between 1 and 3 min after the maneuver, there was an increase in LV and PA WBC (P less than 0.01) above base line. The neutrophil and lymphocyte counts showed similar changes, but erythrocyte and platelet counts remained unchanged. The degree of fall in LV WBC correlated closely (r = 0.68, P less than 0.01) with the changes from lung zone 3 to zone 2 and 1 conditions, as determined from the pressure changes. We conclude that WBCs are retained in the lung during a forced expiratory maneuver because of alveolar capillary compression.     

Capillary perfusion patterns in single alveolar walls.

We studied capillary perfusion patterns in single alveolar walls through a transparent thoracic window implanted in pentobarbital-anesthetized dogs. The capillaries were maximally opened by brief inflation of a balloon in the left atrium to raise pressure. After the balloon was deflated and pulmonary hemodynamics returned to zone 2 baseline conditions, the capillaries that remained perfused in the observed field were videotaped with the use of in vivo microscopy. The cycle of elevated pressure and baseline observation was repeated three times. Perfusion of different capillaries during each of the observations would imply that the capillaries had characteristics that permitted flow to switch between segments. Perfusion of a specific set of pathways through the network each time would demonstrate that flowing blood sought a unique and repeatable combination of segments, presumably with the least total pathway resistance. We found that the same capillary segments were perfused 79% of the time, a strong indication that a reproducible combination of individual segmental resistances determined the predominant pattern of pulmonary capillary perfusion.     

Hydraulic conductance of lung endothelial phenotypes and Starling safety factors against edema

Recent permeability studies comparing endothelial cell phenotypes derived from alveolar and extra-alveolar vessels have significant implications for interpreting the mechanisms of fluid homeostasis in the intact lung. These studies indicate that confluent monolayers of rat pulmonary microvascular endothelial cells had a hydraulic conductance (L(p)) that was only 5% and a transendothelial flux rate for 72-kDa dextran only 9% of values determined for rat pulmonary artery endothelial cell monolayers. On the basis of previous studies partitioning the filtration coefficients between alveolar and extra-alveolar vascular segments in rat lungs and previous studies of lymph albumin fluxes and permeability, the contribution of the alveolar capillary segment to total albumin flux in lymph was estimated to be less than 10%. In addition, the Starling safety factors against the edema calculated for the alveolar capillaries are quite different from those estimated for whole lung. Estimates of the edema safety factor due to increased filtration across the alveolar capillary wall based on the low L(p) indicate it is quantitatively the greatest safety factor, although it would be a minor safety factor for extra-alveolar vessels. Also, a markedly higher effective protein osmotic absorptive force for plasma proteins must occur in the capillaries relative to extra-alveolar vessels. The lower L(p) for alveolar capillaries also has implications for the sequence of hydrostatic edema formation, and it also may have a role in preventing exercise-induced alveolar flooding.     

Effect of positive airway pressure on capillary transit time in rabbit lung

We used fluorescence videomicroscopy to measure the passage of fluorescent dye through the subpleural microcirculation of the lung. With the rabbit in the left lateral decubitus position, the subpleural microcirculation was viewed either through a transparent parietal pleural window located in the superior part of the chest or directly with the chest open. There was no physical contact with the chest or lung. The rabbit was anesthetized, paralyzed, and mechanically ventilated with 100% O2. The dye was injected into the right ventricle during a 2-min apneic period to eliminate lung movement due to ventilation. The video signal of the passage of the dye was analyzed frame by frame by use of digital image processing to compensate for cardiogenic oscillations of the lung surface. Gray scale levels of an arteriole and adjacent venule were measured every 1/30 s. Capillary transit time was determined from the difference between the concentration-weighted mean time values of the arteriolar and venular dye dilution curves. We studied the effect of airway pressure (0-20 cmH2O) on transit time. Cardiac output was measured at different airway pressures by the thermal dilution technique. Capillary transit time averaged 0.60 s at functional residual capacity. Right ventricular-to-arteriolar transit time was four times as large as the capillary transit time. An increase in airway pressure from 0-5 to 20 cmH2O resulted in a fourfold increase in both capillary and arterial transit times and a threefold decrease in cardiac output.     

Comparison of direct and indirect measurements of pulmonary capillary transit times

Using in vivo microscopy, we made direct measurements of pulmonary capillary transit time by determining the time required for fluorescent dye to pass from an arteriole to a venule on the dependent surface of the dog lung. Concurrently, in the same animals, pulmonary capillary transit time was measured indirectly in the entire lung using the diffusing capacity method (capillary blood volume divided by cardiac output). Transit times by each method were the same in a group of five dogs [direct: 1.75 +/- 0.27 (SE) s; indirect: 1.85 +/- 0.33 s; P = 0.7]. The similarity of these transit times is important, because the widely used indirect determinations based on diffusing capacity are now shown to coincide with direct measurements and also because it demonstrates that measurements of capillary transit times on the surface of the dependent lung bear a useful relationship to measurements on the capillaries in the rest of the lung.     

Capillary recruitment and transit time in the rat lung

Presson, Robert G., Jr., Thomas M. Todoran, Bracken J. DeWitt, Ivan F. McMurtry, and Wiltz W. Wagner, Jr.Capillary recruitment and transit time in the rat lung.J. Appl. Physiol. 83(2): 543-549, 1997.Increasing pulmonary blood flow and the associated rise incapillary perfusion pressure cause capillary recruitment. The resultingincrease in capillary volume limits the decrease in capillary transittime. We hypothesize that small species with relatively high restingmetabolic rates are more likely to utilize a larger fraction ofgas-exchange reserve at rest. Without reserve, we anticipate thatcapillary transit time will decrease rapidly as pulmonary blood flowrises. To test this hypothesis, we measured capillary recruitment andtransit time in isolated rat lungs. As flow increased, transit timedecreased, and capillaries were recruited. The decrease in transit timewas limited by an increase in the homogeneity of the transit time distribution and an increased capillary volume due, in part, to recruitment. The recruitable capillaries, however, were nearly completely perfused at flow rates and pressures that were less thanbasal for the intact animal. This suggests that a limited reserve ofrecruitable capillaries in the lungs of species with high restingmetabolic rates may contribute to their inability to raiseO₂ consumption manyfold abovebasal values.