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%) .

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.

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.

Neutrophil emigration in response to acid aspiration does not require the adhesion complex, CD11/ CD18. This study examined the role of CD11b/CD18 using the anti-CD11b F(ab')2, 1B6, in focal HCI-induced intracapillary neutrophil sequestration and edema formation within rat lungs, as well as the effect of pretreatment with endotoxin on this injury. The results show that at the site of aspiration pneumonia, anti-CD11b F(ab')2 did not inhibit neutrophil sequestration or edema formation, either with or without endotoxin pretreatment. In the contralateral lung, focal HCI aspiration induced neutrophil sequestration that was inhibited by the anti-CD11b F(ab')2, but no edema formation. The combined effect of endotoxin pretreatment and HCI aspiration induced CD11b/CD18-independent edema formation in the contralateral lung. These data indicate that CD11b/CD18-independent pathways mediate neutrophil sequestration and edema formation at that pneumonic site with or without pretreatment with endotoxin. CD11b/CD18 mediates neutrophil sequestration at distant sites when no endotoxin is present, although this CD11b/CD18-dependent sequestration is not association with edema formation. The combined effects of endotoxin and HCI aspiration induce edema formation at distant sites that could not be prevented by inhibiting the function of the CD11b/CD18 prior to aspiration.

To investigate the requirements for adhesion molecules in neutrophil emigration during peritonitis, mice received intraperitoneal injections of Streptococcus pneumoniae while the functions of multiple adhesion molecules were blocked. Emigration after 4 h was compromised by antibodies against ICAM-1 or genetic deficiency of ICAM-1. Anti-CD11a/CD18 antibodies decreased emigration in ICAM-1 mutant mice, suggesting that ICAM-1 independent emigration requires CD11/CD18 complexes. In contrast, mice mutant in ICAM-1 plus E-selectin showed no defect in emigration, suggesting that E-selectin commits neutrophils to an ICAM-1-dependent pathway during streptococcal peritonitis. However, in mutant mice lacking the three endothelial adhesion molecules E-selectin, P-selectin, and ICAM-1, emigration after 4 h was significantly compromised. Thus, P-selectin is essential to ICAM-1-and E-selectin-independent acute peritoneal inflammation. After 24 h of peritonitis, there were no differences between WT and E-selectin/P-selectin/ ICAM-1 mutant mice, demonstrating that these endothelial adhesion molecules are not essential to neutrophil emigration during later stages of peritonitis. J. Leukoc. Biol. 64: 291-297; 1998.

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.

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.

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.