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.     

Role of interstitium in clearance of alveolar fluid in normal and injured lungs

We investigated the contribution of the pulmonary interstitial space to the removal of alveolar fluid and solute. We prepared anesthetized sheep for the collection of lung lymph. A balloon-tipped catheter was advanced into a lower lung lobe, and 20 ml Ringer lactate solution (RL) were instilled in one group. Other groups received 20 ml RL with 4 mg/ml Evans blue dye (EB) or 10 micrograms/kg phorbol myristate acetate (PMA) or both. Instillation of 20 ml RL and EB resulted in an increase in lymph flow over RL alone, presumably by an osmotic mechanism. After 4 h, small perivascular fluid cuffs, which contained little EB, were present, and 1.9% of the instilled EB was removed by the lymphatics. An average of 9.2 ml of excess water remained in the lung. Instillation of RL, EB, and PMA resulted in an increase in lymph flow and large perivascular fluid cuffs, which contained large amounts of EB. Lymphatic removal of the instilled EB accounted for 1.2% of the total amount instilled. An average of 19.1 ml water was present in the lung after 4 h. We conclude that alveolar instillation of PMA results in epithelial and endothelial membrane injury and that when lung injury is present interstitial fluid reservoirs may be important sites of alveolar fluid accumulation and important routes of fluid removal from the air space.     

Role of neutrophils in xanthine/xanthine oxidase-induced oxidant injury in isolated rabbit lungs.

Reactive oxygen species have been shown to play an important role in the pathogenesis of lung injury. This study was designed to clarify the role of intrapulmonary neutrophils in the development of xanthine/xanthine oxidase (X/XO)-induced lung injury in isolated buffer-perfused rabbit lungs. We measured microvascular fluid filtration coefficient (K(f)) and wet-to-dry weight ratio to assess lung injury. X/XO induced a significant increase in K(f) and wet-to-dry weight ratio in neutrophil-replete lungs, whereas the lung injury was attenuated in neutrophil-depleted lungs. A neutrophil elastase inhibitor, ONO-5046, also attenuated the lung injury. In addition, X/XO induced a transient pulmonary arterial pressure (P(pa)) increase. The thromboxane inhibitor OKY-046 attenuated the P(pa) increase but did not alter the increase in permeability. Neutrophil depletion reduced the K(f) increase but had no effect on the P(pa) increase. These results suggest that intrapulmonary neutrophils activated by X/XO play a major role in development of the lung injury, that neutrophil elastase is involved in the injury, and that the X/XO-induced vasoconstriction is independent of intrapulmonary neutrophils.     

Analysis of stress distribution in the alveolar septa of normal and simulated emphysematic lungs.

The alveolar septum consists of a skeleton of fine collagen and elastin fibers, which are interlaced with a capillary network. Its mechanical characteristics play an important role in the overall performance of the lung. An alveolar sac model was developed for numerical analysis of the internal stress distribution and septal displacements within the alveoli of both normal and emphysematic saline-filled lungs. A scanning electron micrograph of the parenchyma was digitized to yield a geometric replica of a typical two-dimensional alveolar sac. The stress-strain relationship of the alveolar tissue was adopted from experimental data. The model was solved by using commercial finite-element software for quasi-static loading of alveolar pressure. Investigation of the state of stresses and displacements in a healthy lung simulation yielded values that compared well with experimentally reported data. Alteration of the mechanical characteristics of the alveolar septa to simulate elastin destruction in the emphysematic model induced significant stress concentrations (e.g., at a lung volume of 60% total capacity, tensions at certain parts in an emphysematic lung were up to 6 times higher than those in a normal lung). The combination of highly elevated stress sites together with the cyclic loading of breathing may explain the observed progressive damage to elastin fibers in emphysematic patients.     

Increase in alveolar antioxidant levels in hyperoxic and anoxic ventilated rabbit lungs during ischemia

Increases in free radicals are believed to play a central role in the development of pulmonary ischemia/reperfusion (I-R) injury, leading to microvascular leakage and deterioration of pulmonary surfactant. Continued ventilation during ischemia offers significant protection against I-R injury, but the impact of alveolar oxygen supply both on lung injury and on radical generation is still unclear. We investigated the influence of hyperoxic (95% O2) and anoxic (0% O2) ventilation during ischemia on alveolar antioxidant status and surfactant properties in isolated rabbit lungs. Normoxic and hyperoxic ventilated, buffer-perfused lungs (n = 5 or 6) and native lungs (n = 6) served as controls. As compared with controls, biophysical and biochemical surfactant properties were not altered in anoxic as well as hyperoxic ventilated ischemic (2, 3, and 4 h) lungs. Assessment of several antioxidants (reduced glutathione (GSH), alpha-tocopherol (vitamin E), retinol (vitamin A), ascorbic acid (vitamin C), uric acid, and plasmalogens (1-O-alkenyl-2-acyl-phospholipids)) in bronchoalveolar lavage fluid (BALF) revealed a significant increase in antioxidant compounds under anoxic and hyperoxic ventilation, with maximum levels occuring after 3 h of ischemia. For example, GSH increased to 5.1 +/- 0.8 microM (mean +/- SE, p <.001) after 3 h of anoxic ventilated ischemia and to 2.7 +/- 0.2 microM (p <.01) after hyperoxic ventilated ischemia compared with native controls (1.3 +/- 0.2 microM), but did not significantly change under anoxic and hyperoxic ventilation alone. In parallel, under ischemic conditions, oxidized glutathione (GSSG) increased during hyperoxic (3 h: 0.81 +/- 0.04 microM, p <.001), but remained unchanged during anoxic (3 h: 0.31 +/- 0.04 microM) ventilation compared with native controls (0.22 +/- 0.02 microM), whereas F2-isoprostanes were elevated under both hyperoxic (3 h: 63 +/- 15 pM, p <.01) and anoxic (3 h: 50 +/- 9 pM, p <.01) ventilation compared with native controls (16 +/- 4 pM). We conclude that oxidative stress is increased in the lung alveolar lining layer during ischemia, during both anoxic and hyperoxic ventilation. This is paralleled by an increase rather than a decrease in alveolar antioxidant levels, suggested to reflect an adaptive response to oxidative stress during ischemia.     

Phagocytosis of Candida albicans by rabbit alveolar macrophages and guinea pig neutrophils.

Studies of host-parasite relationships at the cellular level, using Candida albicans and rabbit alveolar macrophages or guinea pig neutrophils are presented. Guinea pig neutrophils killed the intracellular candida cells presumed by myeloperoxidase-halide-hydrogen peroxide system. In contrast, rabbit alveolar macrophages did not kill the intracellular candida cells although their phagocytic rate was almost comparable to that of neutrophils. Phagocytizing macrophages were eventually destroyed by the intracellular proliferation of candida cells and formation of germ tubes and pseudomycelia. No significant improvement of candidacidal activity was observed with macrophages from normal and immunized rabbits in immune serum. The mode of phagocytosis by macrophages and neutrophils were also studied under the scanning electron microscope.     

Morphologic detection and functional assessment of reconstituted normal alveolar macrophages in the lungs of SCID mice

Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for approximately 50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense.     

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 +/- 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 +/- 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.     

The effects of neutrophils and phospholipase A2 on transvascular albumin flux in isolated rabbit lungs

In this study, addition of phospholipase A2 (PLA2) to salt-perfused isolated rabbit lungs containing rabbit polymorphonuclear leukocytes leads to an increase in pulmonary capillary permeability. We add 1.5 X 10(8) polymorphonuclear leukocytes to the perfusate. Next, indomethacin is added to the perfusate and 40 units of PLA2 are infused into the pulmonary arterial inflow of the lungs. At the end of the study, a lung sample is removed for measurement of transvascular albumin flux using I125-albumin as a measure of the permeability-surface area product. Control studies demonstrate no increase in transvascular albumin flux. Addition of a dual cyclooxygenase and lipoxygenase inhibitor, BW755C, to the perfusate prevents the increase in transvascular albumin flux. We conclude that PLA2 interacts with polymorphonuclear leukocytes to increase protein permeability. Since PLA2 can release endogenous arachidonic acid and platelet-activating factor from cells, this suggests that release of such products may contribute to an increase in pulmonary capillary permeability from polymorphonuclear leukocytes. The ability of BW755C to prevent the increase suggests the possibility that lipoxygenase products contribute.     

A morphometric examination of type II alveolar epithelial cells in normal and isolated-perfused dog lungs

The volume densities of type II alveolar cell cytoplasmic organelles and alveolar surface densities were estimated by established stereologic procedures. The morphometric measurements were obtained from normal dog lungs (in situ) and isolated dog lungs perfused for 30-minute, 1-hour, and 2-hour periods. The type II cell lamellar body volume densities and the alveolar surface densities progressively decreased as the times of perfusion were increased. The volume densities of the granular and agranular endoplasmic reticulum progressively increased during the periods of perfusion. These morphometric parameters from lungs in situ and isolated lungs suggest that changes occur in pulmonary surfactant synthesis and activity during perfusion. It is further postulated that progressive increases in the rates of surfactant removal and/or inactivation during perfusion may contribute to spontaneous edema in lungs isolated for periods exceeding two hours. The morphologic and physiologic integrity of isolated perfused lung preparations, widely used as models of lungs in vivo, in situ requires further evaluation.     

Vascular resistance and Kf in normal and PMA-injured rabbit lungs: effects of adenosine

The effects of adenosine (ADO) on pulmonary vascular resistance (PVR) distribution, vascular compliance (C), and permeability were determined in normal and PMA-injured isolated rabbit lungs perfused with a 1:1 mixture of 6% albumin in Krebs-Henseleit buffer and autologous blood. ADO or vehicle was continuously infused into the reservoir at 1,4, or 5 mumol/min after a 1-mumol bolus of ADO or vehicle. The capillary filtration coefficient (Kf) and arterial, venous, and double occlusion pressures were measured at baseline and 30 min after phorbol myristate acetate (PMA; 4 x 10(-8) M) or vehicle. Perfusate differential and total leukocyte counts as well as adenine nucleotides, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) concentrations were determined at each measurement period. ADO was recovered as hypoxanthine and inosine in the perfusate. ADO alone did not alter PVR, C, Kf, or TxB2 but reduced 6-keto-PGF1 alpha levels. PMA induced an increase in Kf (0.024 +/- 0.002 to 0.040 +/- 0.006 g.cmH2O-1.min-1, P less than 0.05) that was completely blocked by 4 or 5 mumol/min ADO. PVR increased by 63 +/- 11% after PMA, primarily in the arteries and arterial and venous microvessels. The postcapillary resistance increase was blunted by 4 mumol/min ADO; 5 mumol/min ADO prevented the PVR increase in all segments. ADO did not affect the initial adherence of neutrophils in the lung or the PMA-induced 87 +/- 2% decrease in circulating leukocytes (greater than 98% lymphocytes) or threefold increase in TxB2 levels. These results suggest that protection by ADO is not mediated by the altering of cyclooxygenase products or by leukocyte adherence.     

Lipids in the lungs and alveolar surfactant in anaphylactic shock

The process of anaphylactoid response of rats to introduction of egg protein is associated with a decrease of the pulmonary surfactant surface activity. The factors of metabolic surfactant inactivation are as follows: protein accumulation, the disturbance of lipids transport between pulmonary cells and alveolar surface, change in fatty-acidic composition of surfactant phospholipids. The isolation of arachidonic acid from surfactant phospholipids in anaphylactoid shock is an evidence for the participation of the pulmonary surface-active phase in the process of biosynthesis of the lipid mediators in respiratory organs.