Bleomycin increases neutrophil adhesion to human vascular endothelial cells independently of upregulation of ICAM-1 and E-selectin

ABSTRACT

Aim of the Study: Bleomycin-induced lung disease is a serious complication of therapy characterized by alveolar injury, cytokine release, inflammatory cell recruitment, and eventually pulmonary fibrosis. The mechanisms underlying bleomycin-induced pulmonary fibrosis may be relevant to other progressive scarring diseases of the lungs. Pulmonary vascular endothelial cells are critically involved in immune cell extravasation at sites of injury through adhesion molecule expression and cytokine release. We sought to determine the effects of bleomycin on adhesion molecule expression and cytokine release by pulmonary vascular endothelial cells, and their functional relevance to inflammatory cell recruitment. Materials and Methods: The effects of pharmacologically relevant concentrations of bleomycin on adhesion molecule expression and cytokine release by human vascular endothelial cells in vitro were studied by flow cytometry, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. A flow chamber model was used to assess the functional consequences on adhesion of flowing human neutrophils to endothelial cell monolayers. Results: Bleomycin increased intercellular adhesion molecule 1 (ICAM-1; CD54), vascular cell adhesion molecule (VCAM-1; CD106), and E-selectin (CD62E) expression, and increased monocyte chemoattractant protein (MCP-1) and interleukin (IL-8) release by endothelial cells. Increases in protein expression were accompanied by increased mRNA transcription. In contrast, there was no direct effect of bleomycin on the profibrotic cytokines transforming growth factor-beta (TGF-β), platelet-derived growth factor-BB (PDGF-BB), or endothelin-1. Under flow conditions, endothelial cells exposed to bleomycin supported increased neutrophil adhesion which was independent of ICAM-1 or E-selectin. Conclusion: Our findings demonstrate that bleomycin promotes endothelial-mediated inflammation and neutrophil adhesion. These mechanisms may contribute to the development of pulmonary fibrosis by supporting immune cell recruitment in the lungs.

KEYWORDS:

• bleomycin
• cell adhesion
• endothelium
• neutrophil
• pulmonary fibrosis

Acknowledgments

We are grateful to the following colleagues for their advice and technical assistance: Prof. Alyn Morice, Dr. Camille Ettelaie, Dr. Yu Pei Xiao, Dr. Kevin Morgan, Simon Fraser, Rob Bennett, Chris Crow, and lab members in Prof. Khalid Naseem's platelet biology group.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The lead and corresponding authors confirm that no commercial relationship exist in the form of financial support or personal financial interests in relation to this paper.

Funding

This work was funded by a University of Hull studentship (JDW).

Notes on contributors

JDW designed the study and performed the experiments; LRS supervised the experiments; MGC designed the study; JG designed the study and supervised the experiments; and SPH designed the study and supervised the experiments. All authors contributed to writing the manuscript.