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.     

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.     

Effect of hypoxia on erythrocyte deformability in different species

The contribution of erythrocyte deformability to the pulmonary vascular resistance during hypoxia in different animal species has not been examined. We hypothesized that the increase in pulmonary vascular resistance during hypoxia was partially due to erythrocytes (RBC's) becoming less deformable during hypoxia, and therefore their transit in the capillaries becomes restricted. To test this, we measured an index of deformability of RBC's from six animal species (dog, pig, cat, rabbit, hamster, rat) during normoxic and hypoxic condition, and compared the changes in deformability with the pulmonary hypoxic pressor response (HPR) which has been reported in the same species. Deformability was indexed as the resistance that a Hemafil polycarbonate membrane (Nucleopore filter, 4.7 micron pores) offers to a 10% suspension of RBC's. The RBC suspension was either normoxic (PO2 = 150 torr) or hypoxic (PO2 = 50 torr). We found that hypoxia decreased RBC deformability; the largest decrease occurred in rat RBC's, a small but significant decrease was observed in the RBC's of cats, rabbits and hamsters, but no change was detected in RBC's of dogs or pigs. In general, such changes in deformability do not correlate well with the HPR in intact or in isolated lungs, for example the pig, had the largest HPR but the smallest change in RBC deformability. In some species, however, there was a positive correlation between RBC deformability and HPR, for example rats, rabbits and cats are usually better responders than dogs and hamsters, similarly the deformability of RBC's in rats, rabbits and cats were also more influenced by hypoxia than RBC's from dogs. The limiting factors in this relationship are the artificial conditions which were used to measure deformability and HPR, both may be different than in the intact conditions. Nevertheless the present data show that erythrocytes of some species can become less flexible during hypoxia, and hence may impede the transit in the capillaries. Therefore we propose that the hypoxic pressor response in the pulmonary vasculature may be partly due to smooth muscle contraction (vasoconstriction) and partly due to a decrease in erythrocyte deformability (capillary obstruction). Both components are likely to be species dependent.     

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.     

Pulmonary neutrophil kinetics after thrombin-induced intravascular coagulation

Previous studies have indicated that neutrophils are required for the development of increased lung vascular permeability after thrombin-induced pulmonary microembolization. In this study, we examined neutrophil kinetics and uptake in the sheep lung before and after lung vascular injury. Sheep neutrophils were isolated by a Percoll-gradient method and labeled with indium-111 oxine. A maximum lung activity of 40% of the injected indium-111 neutrophil activity was attained 8-12 min after the injection. The calculated half-lives of both circulating and pulmonary neutrophils were 700 min. The rate of washout of labeled neutrophils from the lungs was the same as the loss of the peripheral blood activity, indicating removal of neutrophils from the lung and blood by a common pathway (e.g., liver and spleen). Intravenous infusion of alpha-thrombin resulted in an immediate uptake of neutrophils of 14% above the base-line activity. The increased uptake was associated with an immediate decrease in the blood activity, indicating sequestration of the neutrophils in the pulmonary circulation. The neutrophil uptake after alpha-thrombin was transient, reaching a maximum 15 min after infusion. Neutrophil uptake did not occur with alpha-thrombin (which lacks the fibrinogen recognition site), suggesting that the uptake was secondary to intravascular coagulation. An increase in the pulmonary blood volume cannot explain the increased neutrophil sequestration because pulmonary blood volume determined by [99mTc]pertechnetate-labeled erythrocytes did not increase after the alpha-thrombin infusion. Therefore, alpha-thrombin results in a transient neutrophil sequestration in the lung, and the response is secondary to the intravascular coagulation induced by the alpha-thrombin.     

Neutrophils in reexpansion pulmonary edema

This study investigated the possible contribution of neutrophils to development of reexpansion pulmonary edema (RPE) in rabbits. Rabbits' right lungs were collapsed for 7 days and then reexpanded with negative intrathoracic pressure for 2 h before study, a model that creates unilateral edema in the reexpanded lungs but not in contralateral left lungs. Two hours after lung reexpansion, significant increases in lavage albumin concentration (17-fold), percent neutrophils (14-fold), and total number of neutrophils (7-fold) recovered occurred in the reexpanded lung but not in the left. After 2 h of reexpansion increased leukotriene B4 was detected in lavage supernatant from right lungs (335 +/- 33 pg/ml) compared with the left (110 +/- 12 pg/mg, P less than 0.01), and right lung lavage acid phosphatase activity similarly increased (6.67 +/- 0.35 U/l) compared with left (4.73 +/- 0.60 U/l, P less than 0.05). Neutropenia induced by nitrogen mustard (17 +/- 14 greater than neutrophils/microliters) did not prevent RPE, because reexpanded lungs from six neutropenic rabbits were edematous (wet-to-dry lung weight ratio 6.34 +/- 0.43) compared with their contralateral lungs (4.97 +/- 0.04, P less than 0.01). An elevated albumin concentration in reexpanded lung lavage from neutropenic rabbits (8-fold) confirmed an increase in permeability. Neutrophil depletion before reexpansion did not prevent unilateral edema, although neutrophils were absent from lung sections and alveolar lavage fluid from neutropenic rabbits.     

Effects of lung inflation and blood flow on capillary transit time in isolated rabbit lungs.

In a previous study, direct measurements of pulmonary capillary transit time by fluorescence video microscopy in anesthetized rabbits showed that chest inflation increased capillary transit time and decreased cardiac output. In isolated perfused rabbit lungs we measured the effect of lung volume, left atrial pressure (Pla), and blood flow on capillary transit time. At constant blood flow and constant transpulmonary pressure, a bolus of fluorescent dye was injected into the pulmonary artery and the passage of the dye through the subpleural microcirculation was recorded via the video microscope on videotape. During playback of the video signals, the light emitted from an arteriole and adjacent venule was measured using a video photoanalyzer. Capillary transit time was the difference between the mean time values of the arteriolar and venular dye dilution curves. We measured capillary transit time in three groups of lungs. In group 1, with airway pressure (Paw) at 5 cmH2O, transit time was measured at blood flow of approximately 80, approximately 40, and approximately 20 ml.min-1.kg-1. At each blood flow level, Pla was varied from 0 (Pla less than Paw, zone 2) to 11 cmH2O (Pla greater than Paw, zone 3). In group 2, at constant Paw of 15 cmH2O, Pla was varied from 0 (zone 2) to 22 cmH2O (zone 3) at the same three blood flow levels. In group 3, at each of the three blood flow levels, Paw was varied from 5 to 15 cmH2O while Pla was maintained at 0 cmH2O (zone 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endotoxin-pretreated neutrophils increase pulmonary vascular permeability in dogs

Endotoxin causes pulmonary vascular neutrophil sequestration and injures the lung. Whether this is primarily due to a direct effect of endotoxin on the endothelium or is mediated by an action on the neutrophil is unclear. Canine neutrophils, isolated on plasma-Percoll gradients in vitro, were incubated with Salmonella enteriditis endotoxin, washed, and injected via wedged pulmonary arterial catheters into discrete lung zones of anesthetized dogs, whereas untreated neutrophils were administered into contralateral control lung zones. 113mIn-transferrin was administered intravenously 2 h before the animals were killed. Morphometry and extravascular protein accumulation were assessed at 4 h. Endotoxin treatment of neutrophils ex vivo induced a two- to three-fold increase in neutrophils in these lung zones (0.096 +/- 0.012 vs. 0.05 +/- 0.002 neutrophils/alveolar septal intercept, P less than 0.05). Extravascular-to-intravascular protein ratios in zones receiving endotoxin-treated neutrophils were significantly increased compared with control zones (0.146 +/- 0.02 vs. 0.079 +/- 0.02, P less than 0.05). Because complement fragments increase injury to endothelium in vitro, exogenous C5 fragments were administered to other dogs before administration of neutrophils but failed to significantly increase the extravascular protein signal (0.154 +/- 0.03 vs. 0.124 +/- 0.04). In summary, endotoxin treatment of neutrophils leads to neutrophil sequestration and increased pulmonary extravascular protein accumulation. C5 fragments failed to further enhance the protein accumulation. These data are consistent with an effect of endotoxin on the neutrophil to initiate neutrophil-endothelial interaction and subsequent lung injury.     

Bedside evaluation of the resistance of large and medium pulmonary veins in various lung diseases.

To evaluate the contribution of large and medium pulmonary veins to the total pulmonary vascular resistance in various human lung diseases, we compared in 64 patients the pulmonary arterial proximal wedge pressure (Ppw), obtained when the balloon of a 7F pulmonary artery catheter was inflated with 1.5 ml air, with the distal wedge pressure (Pdw), obtained after the tip of the catheter was advanced until wedged in a small artery without balloon inflation. Ppw, reflecting the pressure in a large pulmonary vein, approximates the left atrial pressure, whereas Pdw reflects the pressure in a smaller pulmonary vein. Pdw was greater than Ppw in all 64 patients. The Pdw-Ppw gradient was 1.1 +/- 0.5 mmHg in nine patients with normal lungs and was significantly higher in 13 patients with chronic congestive heart failure (3.8 +/- 0.8 mmHg, P less than 0.01) and in 22 patients with adult respiratory distress syndrome (3.8 +/- 0.8 mmHg; P less than 0.01), but not in 20 patients with chronic obstructive pulmonary disease (1.8 +/- 0.7 mmHg). The distribution of the pulmonary vascular resistance was clearly different among the four groups. The fraction of the total pulmonary vascular resistance attributable to large and medium pulmonary veins was significantly increased (P less than 0.01) in adult respiratory distress syndrome (27.5 +/- 12%) and cardiac patients (27.5 +/- 9%) compared with patients with chronic obstructive pulmonary disease (13 +/- 5%) and normal lungs (13.5 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of radiolabeled neutrophils in swine

The kinetics of radiolabeled neutrophils (PMNs) as they pass through the lungs of swine were evaluated and compared with those in rabbits (J. Appl. Physiol. 63: 1806-1815, 1987) and dogs (J. Appl. Physiol. 63: 1253-1261, 1987; 65: 1217-1225, 1988) previously reported from our laboratory. 111In-labeled PMNs (111In-PMNs) and 99mTc-labeled erythrocytes were simultaneously injected into the right atrium, and the 111In-PMN percent extraction on the first passage through the lung was determined by the indicator-dilution technique. After 10 min of circulation the distribution of 111In-PMNs in selected organs was determined. The extraction of 111In-PMNs in swine was 88 +/- 3%, which was significantly greater than that of rabbits (78 +/- 3%) or dogs (72 +/- 2%). The recovery of the 111In-PMNs in the lungs of swine was 60 +/- 7%, which was two to three times higher than the recovery in lungs of rabbits or dogs. These results show that radiolabeled PMNs injected intravenously are less able to pass through the pulmonary vasculature and are retained much more within the lung in swine than in rabbits or dogs. This difference could be the result of the presence of pulmonary intravascular macrophages in the lungs of swine.     

Reduced erythrocyte deformability alters pulmonary hemodynamics

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) to assess the effect of altered RBC deformability on pulmonary hemodynamics. RBC suspensions were prepared using cells previously incubated in isosmolar phosphate-buffered saline with or without 0.0125 or 0.01875% glutaraldehyde. Washed RBCs were resuspended in isosmolar 4% albumin saline solution. Isolated rat lungs were perfused with control and stiffened cells by the use of a perfusion system that allowed rapid switching between suspensions. Pressure-flow (P/Q) curves were constructed by measuring pulmonary arterial pressure (Ppa) over a range of flow rates. In a second set of experiments, P/Q curves were generated for perfusion with control and stiffened cells (0.0125% glutaraldehyde) before and after vasoconstriction with a synthetic prostaglandin analogue (U 46619). RBC deformability was quantified in all experiments by determination of filtration time of a dilute cell suspension through a 4.7 microns Nuclepore filter. Incubation with 0.0125 or 0.01875% glutaraldehyde produced a 6 or 21% decrease in RBC deformability, respectively. These decreases in deformability were associated with significant increases in Ppa at each flow rate. The increases in Ppa correlated significantly with the degree of RBC stiffening. With 0.0125% glutaraldehyde, the P/Q curve was shifted upward without a change in slope, whereas incubation with 0.01875% glutaraldehyde resulted in a significant increase in slope. Vasoconstriction and perfusion with stiffened RBCs had additive effects on Ppa. These findings suggest that decreases in RBC deformability cause physiologically significant elevations in hemodynamic resistance in the pulmonary circuit independent of vasoactivity.     

Stiffened erythrocytes augment the pulmonary hemodynamic response to hypoxia

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) during normoxic and hypoxic ventilation to assess the effect of reduced RBC deformability on the hypoxic pressor response. RBC suspensions were prepared with cells previously incubated in isotonic phosphate-buffered saline with or without 0.0125% glutaraldehyde. The washed RBCs were resuspended in isotonic bicarbonate-buffered saline (with 4% albumin) to hematocrits of approximately 35%. The lungs were perfused with control and experimental cell suspensions in succession while pulmonary arterial pressure was measured during normoxic (21% O2) and hypoxic (3% O2) ventilation. On the attainment of a peak hypoxic pressor response, flow rate was changed so that pressure-flow curves could be constructed for each suspension. RBC deformability was quantified by a filtration technique using 4.7-microns-pore filters. Glutaraldehyde treatment produced a 10% decrease in RBC deformability (P less than 0.05). Over the range of flow rates, Ppa was increased by 15-17% (P less than 0.05) and 26-31% (P less than 0.05) during normoxic and hypoxic ventilation, respectively, when stiffened cells were suspended in the perfusate. The magnitude of the hypoxic pressor response was 50-54% greater with stiffened cells over the three flow rates. In a separate set of experiments, normoxic and hypoxic arterial blood samples from conscious unrestrained rats were used to investigate the effects of acute hypoxia on RBC deformability. Deformability was measured with the same filtration technique. There was no difference in the deformability of hypoxic compared with normoxic RBCs. We conclude that the presence of stiffened RBCs enhances the hemodynamic response to hypoxia but acute hypoxia does not affect RBC deformability.     

Impaired deformability of copper-deficient neutrophils

We have previously shown that dietary copper deficiency augments neutrophil accumulation in the lung microvasculature. The current study was designed to determine whether a diet deficient in copper promotes neutrophil chemoattraction within the lung vasculature or if it alters the mechanical properties of the neutrophil, thus restricting passage through the microvessels. Sprague-Dawley rats were fed purified diets that were either copper adequate (6.3 microg Cu/g diet) or copper deficient (0.3 microg Cu/g diet) for 4 weeks. To assess neutrophil chemoattraction, bronchoalveolar lavage fluid was assayed for the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2) by enzyme-linked immunosorbent assay. Neutrophil deformability was determined by measuring the pressure required to pass isolated neutrophils through a 5-microm polycarbonate filter. The MIP-2 concentration was not significantly different between the dietary groups (Cu adequate, 435.4 +/- 11.9 pg/ml; Cu deficient, 425.6 +/- 14.8 pg/ml). However, compared with controls, more pressure was needed to push Cu-deficient neutrophils through the filter (Cu adequate, 0.150 +/- 0.032 mm Hg/sec; Cu deficient, 0.284 +/- 0.037 mm Hg/sec). Staining of the filamentous actin (F-actin) with FITC-Phalloidin showed greater F-actin polymerization and shape change in the Cu-deficient group. These results suggest that dietary copper deficiency reduces the deformability of neutrophils by promoting F-actin polymerization. Because most neutrophils must deform during passage from arterioles to venules in the lungs, we propose that copper-deficient neutrophils accumulate in the lung because they are less deformable.     

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.     

Inhalation of Staphylococcus aureus enterotoxin A induces IFN-gamma and CD8 T cell-dependent airway and interstitial lung pathology in mice

Staphylococcus aureus, a primary source of bacterial superantigen (SAg), is known to colonize the human respiratory tract and has been implicated in airway inflammation. Studies have documented a role for SAgs in respiratory disorders, such as nasal polyps, chronic obstructive pulmonary disease, chronic rhinosinusitis, and asthma. However, cellular and molecular mediators involved in SAg-mediated pulmonary disease have not been clearly identified. In this study, we investigated the effect of intranasal staphylococcal enterotoxin A (SEA) exposure on murine lung. The pathological features in the lung resulting from SEA exposure had characteristics of both obstructive and restrictive pulmonary disorders. There was also an increase in bronchoalveolar lavage protein concentration and cellularity following SEA challenge. Massive CD8(+)Vbeta3(+) T cell accumulation observed in the lung was dependent on CD4 T cell help, both for recruitment and for IFN-gamma synthesis. The primary source of IFN-gamma synthesis was CD8 T cells, and depletion of these cells abrogated disease. IFN-gamma deficiency also prevented SEA-mediated disease, and this was by enhancing early recruitment of neutrophils as detected in the bronchoalveolar lavage. Thus, IFN-gamma appeared to selectively aid the recruitment of T cells to the lungs while preventing the neutrophil accumulation. Therefore, our results show that IFN-gamma-producing CD8 T cells mediated pulmonary alveolitis and inflammation, which were dependent upon CD4 T cells for their recruitment to the lung.     

Effect of chronic hyperoxic exposure on duroquinone reduction in adult rat lungs

NAD(P)H:quinone oxidoreductase 1 (NQO1) plays a dominant role in the reduction of the quinone compound 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) to durohydroquinone (DQH2) on passage through the rat lung. Exposure of adult rats to 85% O2 for > or =7 days stimulates adaptation to the otherwise lethal effects of >95% O2. The objective of this study was to examine whether exposure of adult rats to hyperoxia affected lung NQO1 activity as measured by the rate of DQ reduction on passage through the lung. We measured DQH2 appearance in the venous effluent during DQ infusion at different concentrations into the pulmonary artery of isolated perfused lungs from rats exposed to room air or to 85% O2. We also evaluated the effect of hyperoxia on vascular transit time distribution and measured NQO1 activity and protein in lung homogenate. The results demonstrate that exposure to 85% O2 for 21 days increases lung capacity to reduce DQ to DQH2 and that NQO1 is the dominant DQ reductase in normoxic and hyperoxic lungs. Kinetic analysis revealed that 21-day hyperoxia exposure increased the maximum rate of pulmonary DQ reduction, Vmax, and the apparent Michaelis-Menten constant for DQ reduction, Kma. The increase in Vmax suggests a hyperoxia-induced increase in NQO1 activity of lung cells accessible to DQ from the vascular region, consistent qualitatively but not quantitatively with an increase in lung homogenate NQO1 activity in 21-day hyperoxic lungs. The increase in Kma could be accounted for by approximately 40% increase in vascular transit time heterogeneity in 21-day hyperoxic lungs.     

Effects of protamine, heparinase, and hyaluronidase on endothelial permeability and surface charge

We undertook studies in the isolated perfused rat lung to determine 1) the effects of endothelial charge neutralization with the polycation protamine sulfate on microvascular permeability, lung water, and anionic ferritin binding to the endothelium and 2) the role of heparan sulfate and hyaluronate, negatively charged cell surface glycosaminoglycans, on permeability. Capillary permeability was determined by tissue 125I-albumin accumulation in isolated perfused rat lungs. In control lungs the 5-min albumin uptake was 0.50 +/- 0.05 cm3.s-1.g dry tissue-1 X 10(-3). It was increased by 132 +/- 7.8% (P less than 0.001) by protamine (0.08 mg/ml) and 65 +/- 12% (P less than 0.01) by heparinase (5 U/ml), whereas hyaluronidase (25 NFU/ml) was without effect. In control lungs total water was 4.83 +/- 0.15 ml g/dry tissue. Protamine increased lung water 12 +/- 2% (P less than 0.05). Heparinase caused a 9 +/- 3% increase (P less than 0.05), and hyaluronidase had no effect. Electron microscopy demonstrated that protamine increased anionic ferritin binding to the surface of endothelial cells. We conclude that protamine sulfate neutralization of negative charge in the pulmonary microcirculation leads to increased microvascular permeability. Heparin sulfate may be responsible for this charge effect.     

Lung endothelial and epithelial permeability after platelet-activating factor

We investigated whether platelet-activating factor (PAF) increased epithelial or endothelial permeability in isolated-perfused rabbit lungs. PAF was either injected into the pulmonary artery or instilled into the airway of lungs perfused with Tyrode's solution containing 1% bovine serum albumin. The effect of adding neutrophils or platelets to the perfusate was also tested. Perfusion was maintained 20-40 min after adding PAF and then a fluid filtration coefficient (Kf) was determined to assess vascular permeability. At the end of each experiment, one lung was lavaged, and the lavagate protein concentration (BALP) was determined. Wet weight-to-dry weight ratios (W/D) were determined on the other lung. PAF added to the vascular space increased peak pulmonary arterial pressure (Ppa) from 13.5 +/- 3.1 (mean +/- SE) to 24.2 +/- 3.3 cmH2O (P less than 0.05). The effect was amplified by platelets [Ppa to 70.8 +/- 8.0 cmH2O (P less than 0.05)] but not by neutrophils [Ppa to 22.0 +/- 1.4 cmH2O (P less than 0.05)]. Minimal changes in Ppa were observed after instilling PAF into the airway. The Kf, W/D, and BALP of untreated lungs were not increased by injecting PAF into the vasculature or into the air space. The effect of PAF on Kf, W/D, and BALP was unaltered by adding platelets or neutrophils to the perfusate. PAF increases intravascular pressure (at a constant rate of perfusion) but does not increase epithelial or endothelial permeability in isolated-perfused rabbit lungs.     

Impaired pulmonary conversion of angiotensin I to angiotensin II in rats exposed to chronic hypoxia

The effects of exposing rats to hypoxia at normal atmospheric pressure for periods of 21-24 days on intrapulmonary conversion of angiotensin I (ANG I) to angiotensin II (ANG II) were examined using an isolated rat lung preparation perfused at constant flow. 125I-ANG I (160 fmol) was injected alone and with graded doses (0.1, 1.0, and 100 nmol) of unlabeled ANG I into the pulmonary artery, and the effluent was collected for measurement of ANG I, ANG II, and metabolites. At low doses of injected ANG I (125I-ANG I alone or with 0.1 or 1.0 nmol unlabeled ANG I), the percent conversion of ANG I to ANG II was 67.5 +/- 2.1 (SE), 65.1 +/- 2.0, and 62.5 +/- 1.6 in 21-day hypoxia-exposed animals and 83.8 +/- 2.7, 81.4 +/- 3.9, and 79.6 +/- 2.3 (P less than 0.01) in control rats maintained under normoxic conditions. At the highest dose (100 nmol) of injected ANG I, percent conversion was reduced in both hypoxic and control groups to 46.8 +/- 5.0 and 64.0 +/- 6.0, respectively (P less than 0.05). Mean transit times of labeled material through the pulmonary circulation were not significantly different in hypoxic vs. normoxic lungs at any ANG I load, suggesting that the decreased conversion seen in hypoxic lungs was not related to altered kinetics of substrate exposure. Thus chronic hypoxia is associated with significant inhibition of transpulmonary ANG I conversion that is independent of perfusate flow. We postulate that this phenomenon is due to alterations at the endothelial membrane level.     

Monokine-induced acute lung injury in rabbits

Interleukin-1 (IL-1) mediates components of the acute phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied in unanesthetized rabbits the effects of intravenous divided dose infusions of a murine monokine preparation containing IL-1 activity, on circulating granulocytes, their sequestration within the pulmonary microvasculature, pulmonary edema formation, and changes in pulmonary vascular permeability. Monokine administration induced significant (P less than 0.01) granulocytopenia as well as a significant (P less than 0.001) increase in mean alveolar septal wall granulocytes per high power field (HPF) compared with saline-injected controls. Infusions of the monokine preparation significantly (P less than 0.005) increased lung wet-to-dry weight ratios as well as significantly (P less than 0.025) increased pulmonary extravasation of radiolabeled albumin. Electron microscopic analysis of lung sections obtained from monokine-infused animals demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural tracer. We conclude that a monokine preparation containing IL-1 activity can induce profound granulocytopenia, pulmonary leukostasis, and acute pulmonary vascular endothelial injury.