Vitronectin protects alveolar macrophages from silica toxicity.

Silicosis is an interstitial lung disease caused by the inhalation of crystalline silicon dioxide. Current concepts suggest that a crucial step in the development of silicosis is silica-induced injury of alveolar macrophages (AM). The adhesive protein vitronectin is a natural constituent of the lung, in which its function is largely unexplored. This study investigated a possible role for vitronectin in protecting AM from silica exposure. In this study, the concentration of vitronectin was shown to be increased in the bronchoalveolar lavage fluid of silica-treated rats. Vitronectin affinity for silica was shown both in vitro and in vivo by immunostaining. Vitronectin reduced silica-induced injury to cultured AM as determined with the (51)Cr release assay. Vitronectin reduced silica-induced free radical production as determined with a cell-free thiobarbituric acid assay. Additionally, vitronectin reduced the silica-induced respiratory burst in AM as determined with chemiluminescence. This study suggests that vitronectin may protect AM during the initial exposure to silica.

Bleomycin-mediated pulmonary toxicity: evidence for a p53-mediated response.

Bleomycin damages DNA and causes lung injury and fibrosis. To determine whether bleomycin is associated with the appearance of DNA damage-inducible proteins, C3H mice received either 0.4 mg bleomycin or normal saline intratracheally and were killed 1 to 14 d later. The lungs were examined for expression of p53, p21(WAF1/PiCl), and proliferating cell nuclear antigen (PCNA) using immunohistochemistry and Western blotting. p53-positive cells first appeared at 5 d after treatment and peaked at 7 d; PCNA-positive cells appeared at 1 d after treatment and peaked at 7 d; and p21-positive cells appeared at 5 d and peaked at 9 d. Western blot analysis confirmed that bleomycin upregulated the DNA damage-inducible proteins in a similar fashion. This is the first evidence that bleomycin causes a p53-dependent response associated with acute injury in the lung.

L- and P-selectin and CD11/CD18 in intracapillary neutrophil sequestration in rabbit lungs.

Infusion of complement fragments induces rapid sequestration of neutrophils within pulmonary capillaries. This study examined the mechanisms through which this sequestration occurs, as well as the effect of complement fragments on the expression of L-selectin and CD11/CD18 using ultrastructural immunohistochemistry. Studies using anti-P-selectin antibodies, fucoidin, L-selectin-depleted neutrophils, and anti-CD18 antibodies showed that selectins and CD18 were not required for neutrophil sequestration. However, maintaining the sequestered neutrophils within the pulmonary capillaries required both L-selectin and CD11/CD18. Neutrophils in the pulmonary capillaries of rabbits given complement fragments expressed 72% less L-selectin and 98% more CD11/CD18 than did those in rabbits given saline. Shedding of L-selectin occurred preferentially from the microvillar processes of the plasma membrane rather than from the flat intervening regions. About 28% of L-selectin still remained on intracapillary neutrophil membranes after 15 min and was likely available for binding. Shedding of L-selectin appeared slower in vivo than in vitro. These studies indicate that neutrophil sequestration induced by complement fragments requires at least two sequential steps, one that does not require recognized adhesion molecules followed by a second that requires L-selectin and CD11/ CD18.

Complement fragment-induced release of neutrophils from bone marrow and sequestration within pulmonary capillaries in rabbits.

Infusion of complement fragments induces rapid sequestration of neutrophils within the pulmonary capillaries. This study examined the contributions of the bone marrow (BM) and the liver to the accumulation of neutrophils within the lungs. Complement fragments induced the release of neutrophils from the BM within 7 minutes of infusion, and these neutrophils sequestered in the lungs immediately upon reaching the pulmonary capillaries. Neutrophils expressing high levels of L-selectin were preferentially retained within the pulmonary microvasculature. By 30 minutes after the infusion was stopped, the circulating neutrophil counts had increased, primarily because of release from the BM. The number of neutrophils sequestered in the lung had decreased by only 27%, and the number of neutrophils in the liver increased by 223%. These studies indicate that complement fragments induce the release of neutrophils from the BM far more rapidly than previously described. These newly released neutrophils immediately sequester within the lung, increasing the number of neutrophils available to injure the lung many fold beyond the number that were circulating before infusion. The preferential retention of L-selectin-expressing neutrophils likely reflects the requirement for L-selectin-mediated adhesion in maintaining sequestered neutrophils within the pulmonary microvasculature. The number of circulating neutrophils reflects a balance between pulmonary sequestration, rapid release from the BM, and uptake by the liver and other organs.

Neutrophil margination, sequestration, and emigration in the lungs of L-selectin-deficient mice.

These studies tested the hypothesis that L-selectin plays a role in neutrophil traffic in the lungs, particularly in neutrophil margination, sequestration, and emigration, using L-selectin-deficient mice. No defect in neutrophil margination within either capillaries or arterioles and venules was observed in uninflamed lungs of L-selectin-deficient mice. The initial rapid sequestration of neutrophils within the pulmonary capillaries 1 min after intravascular injection of complement fragments was not prevented. In contrast, L-selectin did contribute to the prolonged neutrophil sequestration (> or = 5 min). Interestingly, neutrophil accumulation within noncapillary microvessels required L-selectin at both 1 and 5 min after complement injection. During bacterial pneumonias, L-selectin played a role in neutrophil accumulation within noncapillary microvessels in response to either Escherichia coli or Streptococcus pneumoniae and within capillaries in response to E. coli but not S. pneumoniae. However, L-selectin was not required for emigration of neutrophils or edema in response to either organism. These studies demonstrate a role for L-selectin in the prolonged sequestration of neutrophils in response to intravascular complement fragments, in the intracapillary accumulation of neutrophils during E. coli-induced pneumonia, and in the accumulation of neutrophils within noncapillary microvessels when induced by either intravascular complement fragments or

The roles of CD11/CD18 and ICAM-1 in acute Pseudomonas aeruginosa-induced pneumonia in mice.

Neutrophil accumulation in response to Pseudomonas aeruginosa in the lungs is mediated through CD11/CD18. This study determined the roles of CD11a, CD11b, and intercellular adhesion molecule (ICAM)-1 in P. aeruginosa-induced pneumonia and compared the function of ICAM-1 using Abs or ICAM-1 mutant mice. Anesthetized BALB/c mice pretreated with either Abs against CD11a, CD11b, ICAM-1, or rat IgG received intratracheal instillation of P. aeruginosa for 4 h. In other studies, ICAM-1 mutant and wild-type mice received either anti-ICAM-1 Ab or rat IgG followed by instillation of P. aeruginosa. The data show that Abs against CD11a, CD11b, and ICAM-1 in BALB/c mice inhibited neutrophil emigration by 79, 81, and 56%, respectively. ICAM-1 mutant mice showed no inhibition of neutrophil emigration compared with wild-type mice. Pretreatment with anti-ICAM-1 Ab inhibited neutrophil emigration in wild-type (129/SvxC57) mice by 67% but had no effect in ICAM-1 mutant mice, suggesting that the Ab was acting specifically through recognition of its Ag. We conclude that CD11a and CD11b are required for neutrophil emigration. The observed function of ICAM-1 varies depending on the method by which it is inhibited. Abs may overestimate function by altering other cellular functions or mutant mice may develop alternative pathways of emigration.

Neutrophil emigration in the lungs.

Neutrophil emigration into the lung occurs in response to inflammatory mediators in the interstitium and the airspace. Emigration through the pulmonary microvasculature occurs through two pathways, one that requires CD11/CD18 and ICAM-1 and one that does not: Which pathway is utilized is determined by the stimulus. The ability of a stimulus to upregulate ICAM-1 by inducing the production of pro-inflammatory cytokines including TNF-alpha appears to determine the selection of the CD11/CD18, ICAM-1, ICAM-1-dependent pathway Recently, a third pathway has been identified that requires CD11/CD18 but not ICAM-1. The ligand for this pathway, as well as the ligands for CD11/CD18, ICAM-1-independent adhesion have not been identified. During recurrent pneumonia, the adhesion molecules required for emigration are different than those utilize during acute inflammation in previously normal lung tissue. For example, studies investigating the role of CD11/CD18 in recurrent pneumonia induced by P. aeruginosa, a stimulus which elicits CD11/CD18-dependent emigration initially, showed that when the organisms are instilled at the same site 7 days after the initial instillation, most emigration occurs through CD11/CD18-independent mechanisms. These studies suggest that when an acute stimulus is placed at a site of resolving inflammation, new pathways of adhesion are recruited. Whether these molecules are the same ones mediating acute CD11/CD18-independent adhesion remains to be determined. In summary, neutrophil emigration in the lung can occur through several adhesion pathways, which pathway is utilized can change during the inflammatory process, and cytokines participate in the selection of the pathway.

The role of P-selectin and ICAM-1 in acute lung injury as determined using blocking antibodies and mutant mice.

Cobra venom factor (CVF) induces lung injury through oxidant- and neutrophil-dependent mechanisms. Adhesion molecules, particularly L-selectin, P-selectin, CD11/CD18, and ICAM-1, are required for full expression of injury in rats. This study compared the roles of P-selectin and ICAM-1 using either mutant mice or blocking Abs. Mice deficient in either P-selectin, ICAM-1, or both adhesion molecules were compared with wild-type mice. Wild-type and single mutant mice were given Abs against murine P-selectin or ICAM-1. CVF was injected i.v., and neutrophil sequestration and extravascular albumin were measured 30 min later. Neither P-selectin, ICAM-1, nor P-selectin/ICAM-1 double mutants showed a reduction in neutrophil sequestration or lung injury when compared with wild-type mice. Anti-P-selectin Abs inhibited both sequestration and injury in wild-type mice by 57% and 60%, respectively, but had no effect in P-selectin mutants. Similar results were found using anti-ICAM-1 Ab in wild-type mice (78% inhibition of sequestration and 88% inhibition of injury) and ICAM-1 mutant mice (no reduction). These results suggest that the apparent role of these molecules in CVF-induced lung injury depends on the method used to block function. When studied using blocking Abs, both P-selectin and ICAM-1 were required for neutrophil sequestration and lung injury, while neither played a role singly or together when studied using mice with genetic deletions. Abs may inhibit neutrophil sequestration and lung injury through mechanisms other than simply adhesion blockade, or mutant mice may utilize alternative adhesion pathways.

Changes in neutrophil actin and shape during sequestration induced by complement fragments in rabbits.

Complement fragment-induced sequestration of neutrophils within the lungs may be mediated by stimulus-induced decreases in the deformability of neutrophils, prolonging their lung capillary transit times. As changes in deformability often occur through changes in cytoskeletal proteins, this study determined whether the distribution of actin within intracapillary neutrophils was altered by intravascular complement fragments and whether sequestered neutrophils were less deformed. Ultrathin cryosections of lung tissue from rabbits given an infusion of complement fragments or saline were immunolabeled with anti-actin antibodies. The number of gold particles/microvillus and the density of gold particles/microgram 2 cytoplasm in the submembrane and the central region of intracapillary neutrophils was quantitated. Neutrophil shape was evaluated using laser confocal microscopy. In control rabbits, the ratio of submembrane/central gold was always greater than one and most neutrophils were elongated, 97% having shape factors > 1.10. The ratio of submembrane/central gold was greater in complement-treated rabbits (5.1 +/- 0.9) than controls (2.6 +/- 0.4; P < 0.026). The number of gold particles/microvillus was also increased in complement-treated rabbits (3.9 +/- 0.5) compared with controls (2.3 +/- 0.5; P < 0.045). Neutrophils were more often spherical when rabbits received complement fragments for 1.5 minutes than in control lungs or after 15-minute infusions. These data suggest that complement fragments induce a rapid redistribution of actin from the central to the submembrane region and the microvilli and result in more round neutrophils. This redistribution may decrease the deformability of neutrophils by altering the stiffness of the submembrane region and/or by preventing the microvilli from flattening.

Infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin double mutant mice.

During the initial phase of the inflammatory response, leukocytes marginate and roll along the endothelial surface, a process mediated largely by the selectins and their ligands. Mice with mutations in individual selectins show no spontaneous disease and have mild or negligible deficiencies of inflammatory responses. In contrast, we find that mice with null mutations in both endothelial selectins (P and E) develop a phenotype of leukocyte adhesion deficiency characterized by mucocutaneous infections, plasma cell proliferation, hypergammaglobulinemia, severe deficiencies of leukocyte rolling in cremaster venules with or without addition of TNF-alpha, and an absence of neutrophil emigration at 4 h in response to intraperitoneal Streptococcus pneumoniae peritonitis. These mice provide strong evidence for the functional importance of selectins in vivo.

Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

Role of CD 11/CD 18 in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia in rabbits.

This study examined CD11/CD18-mediated adhesion in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia. Neutrophil emigration during acute pneumonia was studied in anti-CD18 antibody or murine-IgG-pretreated rabbits 4 hours after intrabronchial instillation of P. aeruginosa. To examine emigration in recurrent pneumonias, rabbits given P. aeruginosa on day 0 received anti-CD18 antibody or IgG on day 7. A second instillate was placed either at the initial site or in a separate lobe, and emigration into alveolar spaces was quantitated morphometrically after 4 hours. The results show that CD11/CD18 was required for neutrophil emigration in acute pneumonias and in recurrent pneumonias that occurred at a site distant from the initial infection. However, when the recurrent pneumonia occurred in the previously inflamed site, CD11/CD18 was not required. When the same number of organisms were instilled on days 0 and 7, emigration was reduced to 15 to 20 percent of the number that migrated initially and only CD18-independent adhesion pathways were used. Increasing the concentration of organisms threefold increased emigration through both CD18-dependent and CD18-independent pathways. These data indicate that P. aeruginosa induces CD11/CD18-dependent emigration during acute pneumonia and recurrent pneumonia at previously uninflamed sites. However, adhesion pathways are altered in regions of chronic inflammation, and a greater proportion of neutrophil emigration occurs through CD11/CD18-independent pathways.

TNF-alpha, IL-8, soluble ICAM-1, and neutrophils in sputum of cystic fibrosis patients.

The cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and intercellular adhesion molecule-1 (ICAM-1) have important roles in regulating neutrophil migration and the inflammatory response. To determine whether the concentration of these cytokines and soluble ICAM-1 (sICAM-1) in sputum was increased in patients with cystic fibrosis during acute exacerbations, we conducted (1) a cross-sectional study of 40 patients, 22 who were clinically well and 18 with acute pulmonary exacerbations; and (2) an 11 months longitudinal study of 16 patients. Significant differences in clinical scores, pulmonary function, and sputum neutrophil density were found between the acutely ill and the well group. There was a strong linear relationship (P < 0.0005) between TNF-alpha and IL-8 concentrations in sputum, but no association between clinical status and cytokine concentrations. The concentration of sICAM-1 was lower in acutely ill compared with well patients in the cross-sectional study. Recovery of exogenous IL-8 added to sputum was complete, while recovery of TNF-alpha averaged 70%. Recovery of exogenous sICAM-1 was only 43%, and the recoveries were lower in sputum samples from acutely ill patients than those from stable patients (P = 0.018). These data indicate that in cystic fibrosis patients, sputum concentrations of TNF-alpha and IL-8 are not increased during acute exacerbations of pulmonary inflammation.

Allogeneic bronchoalveolar lavage cells induce the histology of acute lung allograft rejection, and deposition of IgG2a in recipient murine lungs.

The immunologic and histologic changes associated with lung allograft rejection are believed to result from the presentation of donor lung alloantigens to recipient lymphocytes resulting in up-regulated Th1 lymphocyte activity. The ability of allogeneic lung immune cells to induce the pathologic and immunologic changes associated with acute lung allograft rejection are unknown. The current study determined whether allogeneic (C57BL/6, I-a(b)) bronchoalveolar lavage (BAL) cells (> or = 97% macrophages), when instilled into the lungs of recipient BALB/c mice (I-a(d)), induced the histology and immunology associated with acute lung allograft rejection. BALB/c mice received BAL cells from either C57BL/6 mice (allogeneic instillate) or BALB/c mice (autologous instillate) or PBS (control) by nasal insufflation weekly for 4 wk. Allogeneic BAL cells resulted in a lymphocytic bronchitis and vasculitis analogous to grade 1 to 2 lung allograft rejection. The mice given allogeneic instillates had a greater percentage of lymphocytes in the BAL fluid than those given autologous instillates. After instillation of allogeneic BAL cells, the Th1 cytokines, IL-2 and IFN-gamma (IFN-gamma), were produced locally in greater quantities and more frequently than Th2 cytokine IL-10. IL-4, another Th2 cytokine, was not detected. The local production of IgG1 and IgG2a, which are dependent on IL-4 and IFN-gamma, respectively, were increased. However, only IgG2a was deposited in the perivascular and peribronchiolar tissues. These data show that installation of allogeneic BAL cells into the airways of recipient mice induced up-regulated Th1 lymphocyte activity and caused the histologic changes associated with lung allograft rejection.

Lymphocyte accumulation during Pseudomonas aeruginosa-induced pneumonia in rodents does not require CD11a and intercellular adhesion molecule-1.

During Pseudomonas aeruginosa-induced pneumonia in rodents, the acute infiltrate of neutrophils is followed by accumulation of lymphocytes in the perivascular connective tissue. The roles of the adhesion molecules CD11a/CD18 and intercellular adhesion molecule-1 (ICAM-1) in this accumulation of lymphocytes were investigated. The numbers of lymphocytes in P. aeruginosa-induced pneumonia were compared in animals treated with blocking antibodies to either CD11a, ICAM-1, IgG, or no antibody. In other experiments, the lymphocyte accumulation during P. aeruginosa-induced pneumonia in ICAM-1 mutant mice was compared with that in wild-type mice. In rats, both a murine anti-rat CD11a antibody and nonspecific murine IgG partially inhibited the lymphocyte accumulation by 30 to 40% compared with animals that received no antibodies. In mice, blocking antibodies to either CD11a or ICAM-1 did not decrease the lymphocyte accumulation compared with mice given IgG or no antibody. Further, there was no attenuation of the lymphocyte accumulation induced by P. aeruginosa in the ICAM-1 mutant mice compared with wild-type mice, either in the total number of lymphocytes or the number of CD4+, CD8+, or B cells. We conclude that neither CD11a/CD18 nor ICAM-1 are required for lymphocyte accumulation during P. aeruginosa-induced pneumonia in rodents. The partial inhibition of the lymphocyte accumulation in both the anti-CD11a- and IgG-treated rats may be due to nonspecific effects of foreign proteins on cellular functions.

P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli.

Neutrophil emigration during an inflammatory response is mediated through interactions between adhesion molecules on endothelial cells and neutrophils. P-Selectin mediates rolling or slowing of neutrophils, while intercellular adhesion molecule-1 (ICAM-1) contributes to the firm adhesion and emigration of neutrophils. Removing the function of either molecule partially prevents neutrophil emigration. To analyze further the role of P-selectin and ICAM-1, we have generated a line of mice with mutations in both of these molecules. While mice with either mutation alone show a 60-70% reduction in acute neutrophil emigration into the peritoneum during Streptococcus pneumoniae-induced peritonitis, double mutant mice show a complete loss of neutrophil emigration. In contrast, neutrophil emigration into the alveolar spaces during acute S. pneumoniae-induced pneumonia is normal in double mutant mice. These data demonstrate organ-specific differences, since emigration into the peritoneum requires both adhesion molecules while emigration into the lung requires neither. In the peritoneum, P-selectin-independent and ICAM-1-independent adhesive mechanisms permit reduced emigration when one of these molecules is deficient, but P-selectin-independent mechanisms cannot lead to ICAM-1-independent firm adhesion and emigration.

Effect of pentoxifylline on changes in neutrophil sequestration and emigration in the lungs.

The response of neutrophils to inflammatory stimuli includes sequestration, adhesion, and migration. Pentoxifylline protects against many neutrophil-mediated lung injuries. This study investigated whether pentoxifylline prevented changes in neutrophil kinetics induced by infusion of complement fragments or neutrophil emigration induced by Streptococcus pneumoniae. Complement fragments were infused in New Zealand White rabbits treated with pentoxifylline or saline, and the circulating neutrophil counts in the arterial and venous blood samples were measured. Neutrophil emigration was induced by intrabronchial instillation of S. pneumoniae and quantitated morphometrically. The results show that, at doses achievable in vivo, pentoxifylline did not prevent either the CD18-dependent or -independent phase of complement-mediated neutrophil sequestration within the pulmonary microvasculature or the release of neutrophils from the bone marrow. Pentoxifylline also did not alter either the deformability of unstimulated leukocytes or stimulus-induced decreases in deformability. Finally, neutrophil emigration into the alveolar space was neither attenuated nor accentuated by pentoxifylline. These data suggest that, in vivo, pentoxifylline does not protect against lung injury by inhibiting neutrophil sequestration or emigration and may act to alter the generation of mediators that affect neutrophil behavior, rather than acting directly on neutrophils.

The role of cytoskeletal proteins in neutrophil emigration during pneumonia in rabbits.

The cytoskeletal proteins, actin and tubulin, are critical in modulating many aspects of the structural, mechanical, and biochemical properties of cells. This study determined if rearrangements of microtubules or filamentous actin were necessary for neutrophil margination within the pulmonary microvasculature or emigration into the alveolar spaces in response to Streptococcus pneumoniae. Microtubule assembly was inhibited using colchicine, and F-actin depolymerization was inhabited using phalloidin. Anesthetized rabbits received an intrabronchial instillation of S. pneumoniae either after intravenous pretreatment with colchicine (1 mg/kg every 2 h) or combined with TRITC-phalloidin (2 microM in instillate). Four hours later, the lungs were fixed and removed. The results show that the intravenous injection of colchicine caused a rapid decrease in circulating neutrophil counts, most likely caused by sequestration within the pulmonary microvasculature, that gradually recovered. In the pneumonic region, colchicine inhibited neutrophil emigration by 74 +/- 5%, but it did not prevent the stimulus-induced increase in margination. Phalloidin inhibited neutrophil emigration by 83 +/- 4%. These studies suggested that microtubule reassembly occurs during neutrophil transit through the normal pulmonary microvasculature and that it is required for migration but not sequestration during pneumonia. Rearrangement of actin filaments in lung cells but not neutrophils are required for neutrophil emigration induced by S. pneumoniae.

Contributions of capillary pathway size and neutrophil deformability to neutrophil transit through rabbit lungs.

Neutrophil margination within the pulmonary capillary is due to a delay in their transit compared with that of red blood cells (RBC). This delay has been attributed to the large fraction of capillary segments that are narrower than spherical neutrophils and differences between the time required for deformation of neutrophils and that required for deformation of RBC. This study investigated the characteristics of neutrophil deformation in vivo and the perfusion patterns of segments within capillary pathways. Studies comparing the extraction of neutrophils with that of nondeformable microspheres in one transit through the pulmonary circulation suggest that neutrophils can undergo a rapid deformation from 6.4 to 5.0-5.1 microns, whereas larger deformations require a delay. Effective diameters of the perfused capillary pathways were larger than expected for a random distribution of capillary segment diameters within these pathways. The longer transit times of neutrophils in the upper regions of the lung were associated with a greater fraction of pathways containing narrow segments. These studies suggest that neutrophil deformability and capillary pathway diameters are important in determining the size of the marginated pool of neutrophils within the pulmonary capillaries.