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  • Final Report Summary - LUNG INFLAMMATION (An investigation into the differential mechanisms and regulation of leukocyte clearance from the human lung across two distinct barriers: the bronchial epithelium versus the alveolar epithelium)
FP7

LUNG INFLAMMATION Sintesi della relazione

Project ID: 326928
Finanziato nell'ambito di: FP7-PEOPLE
Paese: United Kingdom

Final Report Summary - LUNG INFLAMMATION (An investigation into the differential mechanisms and regulation of leukocyte clearance from the human lung across two distinct barriers: the bronchial epithelium versus the alveolar epithelium)

Lung disease is the biggest single cause of morbidity and mortality worldwide. Many lung diseases including chronic obstructive pulmonary disease, pneumonia, influenza, cystic fibrosis and pulmonary fibrosis are characterised by lung inflammation. White blood cells traffic continuously throughout the body for immune surveillance, but excessive leukocyte recruitment can result in lung inflammation and damage, and it is essential that these cells are cleared efficiently once infection has been controlled. Although much is known of how leukocytes enter the lung, much less is known about how they leave, or are cleared from, the lung during resolution of inflammation.
This research project was therefore to identify the mechanisms involved in neutrophil trafficking across the alveolar and the bronchial epithelia. The aim was to investigate the adhesion molecules involved and examine their expression in normal and inflammatory conditions.
Over the course of the fellowship Dr Rebeyrol established a novel in vitro model of neutrophil trans-epithelial migration (TEpM) in normal human bronchial epithelial cells (16HBE14o-) and human alveolar epithelial cells (A549). Briefly, the epithelial cells were seeded on the bottom of a transwell insert and cultured at air-liquid interface (ALI) for 10 days. Neutrophils isolated from healthy volunteers were allowed to migrate for 3h towards a chemoattractant gradient of bacterial peptide fMLP. At the end of the incubation the neutrophils that had migration into the bottom well were collected and analysed by flow cytometry. The adhesion molecules on neutrophils involved in the neutrophil TEpM across the bronchial and alveolar epithelium were investigated using antibodies against candidate molecules such as lymphocyte function-associated antigen-1 (LFA-1; αLβ2-integrin, CD11a/CD18) and macrophage antigen-1 (MAC-1; αMβ2-integrin, CD11b/CD18) and other β2 integrins on the neutrophils. The results show that migration of activated neutrophils across the bronchial epithelium was significantly inhibited by blocking surface-expressed cell adhesion molecules with anti-CD11b (50% inhibition) and anti-CD11a (more than 75% inhibition) antibodies. However, neutrophil TEpM across the alveolar epithelium was significantly inhibited only by blocking surface-expressed cell adhesion molecules with anti-CD11b antibody (50% inhibition) but not with anti-CD11a antibody.
This assay required extensive optimisation but resulted in an elegant, accurate and reproducible system. This has generated a lot of interest and led to collaborations with other groups at UCL.
Dr Rebeyrol has extended this work in vivo to examine the role of integrins and adhesion molecules in leukocytes recruitment to the lung and alveolar airspace, using a combination of control mice, gene deficient mice and blocking antibodies in the mouse LPS model of inflammation. The role of ICAM-2 was investigated using ICAM-2-deficient mice and littermate control C57BL/6J (bred at UCL animal facility). Wild type (WT) and knock-out (KO) mice (matched for sex and age) received PBS or LPS intranasally. Lungs were harvested at 24h and cell counts have been performed on the broncho-alveolar lavage fluid (BALf) and the whole lung homogenates. Although LPS induced neutrophil recruitment into the lung, there was no difference between the 2 groups in terms of leukocyte recruitment. This study shows the surprising conclusion that ICAM-2 is not involved in neutrophil migration in the lung, in a LPS model of inflammation.
In another set of experiments, an ICAM-1 blocking antibody (clone KAT-1) or an isotype control was used in WT and ICAM-2 KO mice in order to investigate if ICAM-1 was important and/or if there was any compensation between these two adhesion molecules in terms of leukocyte migration. The results again showed no difference between WT and ICAM-2 KO even when ICAM-1 is blocked.
As previous studies demonstrated differences in neutrophil transmigration into the lung between LPS and Streptococcus pneumoniae models of lung injury, Dr Rebeyrol went on to examine the effect of Streptococcus pneumoniae. In particular, as ICAM-2 is involved in barrier integrity, she wanted to investigate whether the ICAM-2 deficient mice would show difference in bacteraemia. No significant difference was shown in neutrophils and macrophages counts in the BALf, and neutrophils, alveolar macrophages, monocytes-derived macrophages and monocytes in the lungs between the WT and ICAM-2 KO mice. Moreover no difference was found either in bacterial counts in the BALf or in the whole lung homogenates between KO mice versus WT. No bacteria were detected in the blood in any group. This again gave the interesting result that ICAM-2 is not involved in host defence against Streptococcus pneumoniae infection.
Conclusions: This work has used an elegant in vitro and in vivo approach to challenge the current understanding of leukocyte migration into the lung. In particular, the work has highlighted a redundancy of the adhesion molecules ICAM-1 and ICAM-2 that were thought to play a key role in this process. The implications if these findings are extensive and may explain why some therapeutic approaches that target these adhesion molecules have not been translated into effective treatments for lung inflammation. These novel findings have established a programme of work that will be continued to investigate novel approaches in the approach to lung inflammation.

Informazioni correlate

Contatto

Malgorzata Kielbasa, (European Contracts Executive)
Tel.: +4420310853064
Fax: +442078132849
E-mail
Numero di registrazione: 182246 / Ultimo aggiornamento: 2016-05-13
Fonte d'informazione: SESAM