Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury

Abstract

The signals required to direct PMN into the different compartments of the lung have not been fully elucidated. In a murine model of LPS-induced lung injury, we investigated the sequential recruitment of PMN into the pulmonary vasculature, lung interstitium, and alveolar space. Mice were exposed to aerosolized LPS and bronchoalveolar lavage fluid (BAL), and lungs were harvested at different time points. We developed a flow cytometry-based technique to assess in vivo trafficking of PMN in the intravascular and extravascular lung compartments. Aerosolized LPS induced consistent PMN migration into all lung compartments. We found that sequestration in the pulmonary vasculature occurred within the first hour. Transendothelial migration into the interstitial space started 1 h after LPS exposure and increased continuously until a plateau was reached between 12 and 24 h. Transepithelial migration into the alveolar air space was delayed, as the first PMN did not appear until 2 h after LPS, reaching a peak at 24 h. Transendothelial migration and transepithelial migration were inhibited by pertussis toxin, indicating involvement of G␣i-coupled receptors. These findings confirm LPS-induced migration of PMN into the lung. For the first time, distinct transmigration steps into the different lung compartments are characterized in vivo. polymorphonuclear leukocytes; pulmonary circulation; chemokines; lipopolysaccharide; flow cytometry; pertussis toxin ACUTE LUNG INJURY (ALI) and acute respiratory distress syndrome (ARDS) are characterized by a disturbance of the alveolar-capillary barrier associated with several clinical disorders. There is no specific therapy, and the mortality of this disease is still high. Our current understanding of the molecular mechanisms of ALI/ARDS has recently been described as "embryonic at best" (27).Migration of activated polymorphonuclear leukocytes (PMN) plays a key role in development of ALI and ARDS (1). Here, we investigate the sequential migration steps from blood to air space (intravascular sequestration, transendothelial migration, transepithelial migration).A variety of stimuli induce PMN migration into the lung. Endotoxin of gram-negative bacteria [lipopolysaccharide (LPS)] induces a range of inflammatory responses. Toll-like receptor-4 (TLR4) is the most important cellular receptor for LPS. LPS stimulates the response to chemoattractants and increases PMN migration at sites of inflammation (14). TLR4 is essential for LPS-induced PMN migration into the lung as shown by the absence of a response in TLR4-deficient mice (3). In the lung, the response to LPS is regulated by radioresistant cells, most likely endothelial cells (2) or alveolar macrophages (28).The administration of LPS alone might not reflect the whole complexity of the human disease, because it does not consider preexisting diseases, fluid resuscitation, or mechanical ventilation (36). However, infections with gram-negative bacteria and exposure to their predominant pathogenic component play a key role in both development and ou...

Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation

“…Platelet depletion significantly (P < 0.05) prevented the deterioration of gas exchange following HCl application ( Figure 1A). To investigate the PMN recruitment pattern, including intravascular accumulation, transendothelial migration, and transepithelial migration, PMNs in lung homogenates were identified by flow cytometry (11). Two hours after acid instillation, HCl-treated mice showed a significant accumulation of PMNs in the intravascular and interstitial compartments compared with control mice (data not shown).…”

“…In order to distinguish between the localization of intravascular and interstitial PMNs, we used a recently developed flow cytometry-based method to determine pulmonary PMN extravasation (11). In brief, Alexa Fluor 633-labeled Gr-1 Abs (clone RB6-8C5, purified from the supernatant of the Gr-1 hybridoma [ATCC] at the biomolecular facility of the University of Virginia; Alexa Fluor 633 Protein Labeling Kit [Invitrogen]) to murine PMNs, which label all intravascular but not interstitial or alveolar PMNs (11), were injected i.v.…”

“…PMN activation, sequestration, and emigration into the lung proceed in a sequence of overlapping events (11). In contrast with the well-characterized multistep adhesion cascade of leukocyte recruitment in systemic microcirculation, the molecular requirements of leukocyte recruitment into the lung are incompletely defined.…”

The increase in surface CXCR4 expression on lung extravascular neutrophils and its effects on neutrophils during endotoxin-induced lung injury

“…27 An Alexa Fluor 647-conjugated anti-mouse Ly-6G (Gr-1) antibody (1 mg) was injected intravenously and allowed to circulate for 5 min. After 5 min, the mice were killed.…”

Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo

“…This prompted us to test the hypothesis that stimulation of Fpr1 and Fpr2 induces cross-desensitization in vitro and inhibits neutrophil migration in vivo in mice exposed to lipopolysaccharide (LPS) aerosol, which is a well-described mouse model for studying neutrophil migration into the airway. 21 In this study, we found that neutrophils stimulated with Cpd43 lost their chemotactic responses to chemoattractants in vitro, and that oral administration of Cpd43 to mice inhibited LPS-induced neutrophil migration into the airways. These results are consistent with the idea that the agonist for mouse Fpr1 and Fpr2 induced crossdesensitization in neutrophils and attenuated neutrophil migration into the airways.…”