Leukocytes within the circulation are in dynamic equilibrium with a marginated pool, thought to reside mainly within the pulmonary capillaries. The size discrepancy between the mean diameter of circulating leukocytes (6-8 microns) and that of the pulmonary capillaries (approximately 5.5 microns) forces the cells to deform in order to transit the capillary bed. Consequently, we investigated the hypothesis that the biophysical properties of cell size and deformability determined differential leukocyte retention in the lung. Comparison of the filtration properties of human neutrophils, lymphocytes, monocytes, platelets, and erythrocytes through polycarbonate filters (5-micron pore diameter) revealed that the largest leukocytes (neutrophils and monocytes) were retained to the greatest extent and the smaller cells (lymphocytes and platelets) the least. Undifferentiated HL-60 cells, of greater diameter than their differentiated counterparts, were also retained to a greater extent, confirming that cell size was one important determinant of retention in these model capillaries. However, compared with neutrophils, which are of similar diameter, monocytes were retained to a greater extent, suggesting that monocytes might be less deformable than neutrophils. To test this hypothesis, deformability was measured directly using the cell poker. Monocytes were found to be the stiffest, neutrophils the softest, and lymphocytes intermediate. Glutaraldehyde treatment of neutrophils markedly increased their stiffness and decreased their ability to transit the pores of the filters in vitro and the pulmonary microvasculature of rabbits without changing their adhesive properties or size. These observations support the hypothesis that biophysical properties of leukocytes (size and deformability) determine in part their ability to transit the pulmonary capillaries and may determine the magnitude of their marginated pools.

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.

Platelet-activating factor caused rapid pulmonary vasoconstriction and edema in isolated lungs perfused with albumin-free salt solution devoid of formed blood elements. These effects may be due in part to the action of leukotrienes D4 and C4, which were identified by bioassay and high-pressure liquid chromatography in the lung effluent after stimulation by platelet-activating factor. These findings help illuminate some of the deleterious effects that platelet-activating factor elicits in anaphylactic reactions and possibly in other forms of lung injury.

Diverse stimuli induce macrophages to become nonspecifically cytotoxic for transformed cells in vitro (1). Bacterial infections of mice by Bacillus CalmetteGu~rin (BCG) 1 (2), protozoal infestations by Toxoplasma gondii or Besnoitia jellisoni (3), certain viral infections (4), and intraperitoneal inoculations of bacterial lipopolysaccharides (LPS) (5) produce macrophages that injure transformed cells in vitro. In addition, macrophages obtained directly from tumors nonspecifically lyse transformed target cells (6-9). Taken together, these data suggest a central role for stimulated macrophages both in host control of tumor cell proliferation and in the pathogenesis of the chronic inflammatory process. However, the mechanisms involved in the stimulation of macrophages, the generation of the lytic state, and the mediation of target cell injury are not understood. Recently, we and others have shown that cytolytic properties can be induced in peritoneal macrophages by using LPS (10, 11) thereby offering the prospect of examining how macrophages become stimulated, then injure tissues. The studies reported here investigate the nature of the interaction between LPS and murine macrophages that results in the generation of their capacity to kill tumor cells in a nonspecific manner. Materials and Methods Preparations of LPS.The heptose-deficient mutant from Salmonella minnesota (R595) was grown in trypticase soy broth (Baltimore Biological Laboratories, Cockeysville, Md.) under aerated conditions. Cells were harvested by centrifugation and washed twice with 0.85% sodium chloride. LPS from R595 was extracted and purified in our laboratory by Dr. D. C. Morrison using the phenol-petroleum ether-chloroform procedure of Galanos et al. (12). The water insoluble LPS extract was then sonicated and dissolved in 0.1% triethylamine. After extensive dialysis, the LPS preparation of R595 was stored at -70°C until required. LPS from Escherichia coli serotype 0111:B4 (ATCC 12015) was extracted by the phenol-water method of Westphal and Jann (13).Media and Cell Lines. Eagle's minimal essential medium (MEM) was supplemented

Despite extensive investigation, the pathogenesis of the adult respiratory distress syndrome (ARDS) remains uncertain. As yet, there is no clear explanation of why some patients at risk for ARDS develop the syndrome, whereas others do not. Neutrophils and complement fragments have been implicated in the acute lung injury, but it is clear from published data that evidence of complement activation alone predicts neither the development nor the severity of ARDS. We investigated whether the combination of endotoxin, a leukocyte-priming agent, and complement fragments, leukocyte-stimulating agents, was associated with the development of ARDS. Ninety-eight patients were identified as being either at risk for the development of ARDS or having ARDS, and serial blood samples were obtained. There was no correlation between C5 fragments and the development of ARDS. C3 fragment levels were increased in 89% of the patients with ARDS, but they were also increased in 62% of patients at risk. Endotoxin was detected in 74% of the plasma samples obtained from patients at risk who subsequent developed ARDS and in 64% of the plasma samples obtained from the patients with ARDS. In contrast, only 22% of the plasma samples obtained from the patients at risk who did not develop ARDS had measurable endotoxin. We suggest that the combination of endotoxin and complement fragments may be one mechanism involved in the development of ARDS.