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A4775 - CXCR4 Does Not Contribute to Pulmonary Sequestration of Primed Neutrophils in Humans
Author Block: J. Pillay1, N. C. Tregay2, N. Farahi3, L. S. C. Lok4, C. Mitrofan4, R. Simmonds1, C. Summers5, L. Koenderman6, E. R. Chilvers7; 1Respiratory Medicine, University of Cambridge, Cambridge, United Kingdom, 2U, University of Cambridge, Cambridgeshire, United Kingdom, 3Univ of Cambridge, Cambridge CB2 2QQ, United Kingdom, 4University of Cambridge, Cambridge, United Kingdom, 5Department of Medicine, University of Cambridge, Cambridge, United Kingdom, 6Department of Respiratory Medicine, University Medical Centre Utrecht, Utrecht, Netherlands, 7Univ of Cambridge, Cambridge, United Kingdom.
RATIONALE: Primed neutrophils lodge in the pulmonary circulation, this is the first step in a cascade leading to neutrophil extravasation or capillary damage and contributes to a range of pulmonary inflammatory diseases, ranging from COPD to ARDS. The mechanism of pulmonary sequestration of neutrophils in humans is incompletely understood. Recently several important studies in mice have suggested a role of CXCR4 in this process.
METHODS: Expression and function of CXCR4 was studied using flow cytometry. Interactions with HUVEC under flow conditions were observed using a custom setup with HUVEC confluently grown on IBIDI-slides.In this setup HUVEC was pre-incubated with CXCL12 for 30 minutes and then washed. HUVEC was then perfused with medium containing BSA, followed by neutrophils with or without inhibitors. Images were taken after 6 and 12 minutes after neutrophil perfusion to study rolling, adhered or transmigrated neutrophils. In vivo pulmonary neutrophil sequestration was measured by dynamic PET scanning after reinfusing GM-CSF (1ng/ml) primed and 99-technetium labelled neutrophils into healthy volunteers. In this placebo-controlled single-blinded experimental medicine study the intervention-group received the small molecule CXCR4-inhibitor plerixafor.
RESULTS: We show that human neutrophils weakly express CXCR4, however robust functional responses are seen using physiological concentrations. These responses consist of calcium fluxes to 10-100nM of CXCL12 and transient adhesion without rolling under flow conditions to HUVEC preincubated with CXCL12. This interaction was dependent on CXCR4 and required neutrophil LFA-1 and ICAM-1 on HUVEC.In vivo, robust pulmonary sequestration was seen in both groups with a ratio of 63.47% ± 4.33 (mean ± SEM) vs 65.59%± 3.51 (plerixafor vs placebo) of pulmonary signal present at 40 minutes. In addition recovery of neutrophils from the peripheral blood at 40 minutes was similar 6.93% ± 3.69 (mean ± SEM) vs 6.10% ± 1.94 (plerixafor vs placebo).
CONCLUSIONS: Human neutrophils express functional CXCR4, however in contrast to murine studies, this chemokine-receptor does not play a role in pulmonary neutrophil sequestration in humans.
Funding: Janesh Pillay received a grant from the Dutch Lung Foundation 5.2.14.058JO