Virulence factors from Pseudomonas aeruginosa increase lung epithelial permeability.

Pseudomonas aeruginosa infection frequently complicates lung injury and can be fatal in immunocompromised or debilitated individuals. Previous studies from our laboratory indicate that elastase from P. aeruginosa increases epithelial permeability by disrupting tight junctions between epithelial cells. Because the inflammatory reaction of the host is a prominent feature of bacterial infection, we reasoned that additional virulence factors from this organism could extend and augment the initial pulmonary injury by prompting accumulation of neutrophils. To test this hypothesis, we compared responses of guinea pigs to aerosols of elastase (PE) and lipopolysaccharide (LPS) from P. aeruginosa. After each treatment, we measured epithelial permeability and accumulation of neutrophils, interleukin 8 (IL-8), and beta-glucuronidase in epithelial lining fluid (ELF). We found that PE increased epithelial permeability, as measured by both the clearance of aerosolized radiolabeled albumin from the air spaces and the concentration of plasma albumin in epithelial lining fluid, but it was less effective than LPS at recruiting neutrophils into the lungs. In contrast, LPS had no significant effect on epithelium, but it increased the concentration of neutrophils, IL-8, and beta-glucuronidase in ELF. Increased epithelial permeability induced by PE does not cause lung inflammation, but it may facilitate the LPS-induced influx of neutrophils.

Peptide inhibitor of interleukin-8 (IL-8) reduces staphylococcal enterotoxin-A (SEA) induced neutrophil trafficking to the lung.

Staphylococcal enterotoxin A (SEA) is a superantigen, produced by some strains of Staphylococcus aureus (S. aureus), which can cause a variety of clinical manifestations ranging from food poisoning to shock. SEA can also stimulate human alveolar macrophages to produce interleukin-8 (IL-8), a member of the alpha-chemokine subfamily that activates and is chemotactic for neutrophils. In these studies we showed that in rabbits, intravenous SEA significantly decreased the circulating white blood cell population from a baseline value of 6409 +/- 2027 x 10(3) cells/ml to 1943 +/- 862 x 10(3) cells/ml in 7 h. There was a concommitent increase in IL-8 in the circulating plasma (baseline: 60 +/- 34 pg/ml, 7 h post SEA: 109 +/- 64 pg/ml). The increase in circulating IL-8 was accompanied by a much greater increase in the IL-8 concentration of the epithelial lining fluid (ELF) where the IL-8 increased from 0.05 +/- 0.08 ng/ml (control) to 13.8 +/- 9.3 ng/ml (SEA treated). The increase in IL-8 concentration in the alveolar spaces was paralleled by an increase in both the percentage of neutrophils (1.4 +/- 0.9% (control) to 26.0 +/- 10.8% (SEA treated)) and total number of neutrophils (0.04 +/- 0.02 x 10(6)/ml (control) to 4.8 +/- 3.3 10(6)/ml (SEA treated) in the airspaces, and the numbers of neutrophils in the ELF correlated with the IL-8 concentration r = 0.62, p = 0.006). When antileukinate, a hexapeptide which inhibits the binding of IL-8 to neutrophils, was administered to animals receiving SEA, the IL-8 concentration in the ELF (14.8 +/- 10.7 ng/ml) was not significantly different from the concentration of IL-8 in those animals receiving SEA alone). However, both the percentage of neutrophils (9.5 +/- 3.2%), and the total number of neutrophils (1.3 +/- 1.0 x 10(6)/ml) in the ELF following SEA and antileukinate administration was significantly lower than in animals which only received SEA (p < 0.05). The findings suggest that SEA released into the circulation during a Staphylococcal infection can cause an inflammatory reaction in the lung. Since this reaction is at least partially mediated by IL-8, antileukinate may have pharmacologic potential in reducing the inflammatory reaction.

A modified bronchoalveolar lavage procedure that allows measurement of lung epithelial lining fluid volume.

We replaced the standard serial bronchoalveolar lavage technique with a new "rewash" lavage procedure to allow estimation of the volume and protein concentration of the epithelial lining fluid (ELF) in anesthetized sheep. A bronchoscope 6.0 mm in diameter wedged in an airway was used to lavage a segment of lung with four cycles of instillation and aspiration of the lavage solution containing a radioactive tracer (technetium pertechnetate, 99mTcO4-). Errors caused by the fall in concentration of the tracer during the lavage were minimized by extrapolating the tracer concentration back to time zero when the lavage solution had mixed with the ELF, but had not had time to be affected by loss of the tracer or influx of fluid from the interstitium. In control sheep, the ELF of these lavaged segments had a mean volume of 1.6 +/- 1.0 ml and a mean protein concentration that was 26 +/- 19% of the protein concentration measured in the plasma. Increasing the left atrial pressure 19 +/- 5 cm H2O to cause "cardiac lung edema" had no significant effect on the ELF volume, but it increased the mean protein concentration to 57 +/- 30% of the plasma value (p less than 0.01). Lung injury caused by intravenous oleic acid caused lung edema, increased the mean ELF volume to 6.8 +/- 2.2 ml, and increased the mean ELF protein concentration to 86 +/- 26% of the plasma value (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary lymphatic clearance of 99mTc-DTPA from air spaces during lung inflation and lung injury.

A total of 22 sheep with lymphatic cannulas were used to determine if 99mTc-labeled diethylenetriaminepentaacetic acid (DTPA) clears directly from the air spaces of the lungs into the lymph vessels. Each sheep was anesthetized and ventilated with an aerosol of the DTPA for 2-5 min, and the DTPA activities in the lymph and plasma were measured every 15 min for 2 h. After the first 45 min, the average ratio of the DTPA in the lymph to that in the plasma (L/P) was 1.03 +/- 0.06 (SD) in the six control experiments and 1.11 +/- 0.05 in the six experiments in which the lungs were inflated with a positive end-expired pressure of 10 cmH2O throughout the study. Direct movement of the DTPA from the air spaces into the lymph was not necessary to account for the DTPA clearance in these experiments because the L/P ratio was not significantly different from 1.0. Eight additional sheep received intravenous infusions of air at 0.2 ml.kg-1.min-1 for 2 h to induce lung injury before depositing the DTPA. In these sheep L/P was 1.53 +/- 0.28, which was significantly higher than the value measured in the control group (P less than 0.01). We considered the possibility that the increased L/P ratio in these sheep could be due to alterations in the distribution of the blood flow to the tissue, but the L/P ratio in four sheep whose distribution of blood flow was altered by inflation of a balloon in the right pulmonary artery was 1.05 +/- 0.10, the same as the control value.(ABSTRACT TRUNCATED AT 250 WORDS)

Uneven gas mixing during rebreathing assessed by simultaneously measuring dead space.

To evaluate the rate of gas mixing in human lungs during rebreathing maneuvers used to measure pulmonary tissue volume (Vt) and pulmonary capillary blood flow (Qc), we devised a method to determine the dead space during rebreathing (VRD). Required measurements are initial concentration of a foreign inert insoluble gas in the rebreathing bag, first mixed expired concentration, equilibrated concentration, volume inspired, and volume of the first expired breath. In subjects breathing rapidly at 30 breaths/min with inspired volumes in excess of 2 liters, VRD had values three or more times greater than the predicted anatomical dead space (VD). Breath holding after the first inspiration progressively diminished VRD so that after 10-15 s, it approximately equaled predicted VD. VRD measured with helium was smaller than VRD measured with sulfur hexafluoride. The reported degree of uneven ventilation from gravitational forces in normal humans can account for only about one-third of the difference between VRD and VD. These findings support the concept that mixing by diffusion between peripheral parallel airways is incomplete at normal breathing rates in humans and can result in errors as high as 25% in Vt and Qc.

Effect of lung denervation on development of pulmonary edema.

The objective of this study was to determine the relationship of an intact nervous system to the lung in the development of pulmonary edema. After unilateral denervation of a lung by autotransplantation, eight dogs were subjected to a period of increased intracranial pressure with air. A significant increase in pulmonary artery pressure and a 20% right-to-left shunt developed in all animals. The innervated lung of the animals subjected to increased intracranial pressure developed higher wet/dry lung weight ratios and 51Cr platelet counts compared to denervated lungs and to the five control innervated lungs. Thus intrapulmonary platelet sequestration and pulmonary edema occurred only in the intact innervated lung. These findings support the thesis that pulmonary edema following elevation of the intracranial pressure with air is the result of neurally induced alterations in pulmonary hemodynamics and platelet sequestration that could possibly lead to an increase in pulmonary vascular permeability.