21.06 CCR2⁺ Monocytes Mobilized from Spleen Cause Neutrophil Extravasation During Lung Reperfusion Injury

R. Fernandez¹, M. Akbarpour¹, S. F. Chiu¹, H. Sun¹, A. Misharin¹, G. S. Budinger¹, A. Bharat^{1  1}Northwestern University,Thoracic Surgery,Chicago, IL, USA

Introduction: Lung ischemia reperfusion injury (LIRI) is the primary cause of graft failure and mortality after lung transplant. LIRI is mediated by neutrophils. Neutrophil depletion ameliorates LIRI but is not clinically applicable due to their importance in pathogen clearance. Accordingly, we focused on elucidating trafficking of neutrophils. We identified a novel subset of bone marrow derived classical monocytes (CM) which, upon mobilization from the spleen, mediates neutrophil extravasation during LIRI in mice and humans.

Methods: LIRI was induced in mice by hilar clamping for 60 mins followed by 2 hours of reperfusion. Splenectomy and heterotopic spleen transplant were performed using standard techniques. Multipanel flow cytometry was used to quantify myeloid cell populations in tissues. Human lung samples were taken from lung grafts before and after reperfusion. Student’s t-tests and ANOVA were used for statistical analysis.

Results:LIRI induced an influx of CCR2⁺CM into the lung (29.3±4.7 vs. 3.9±0.7 cells/mg lung; p<0.001) which was associated with neutrophil extravasation into the alveoli compared to a resting state (16.0±3.1 vs. 2.3±0.3% extravasated, p<0.001). Depletion of all intravascular monocytes with clodronate-liposomes suppressed neutrophil extravasation compared to control (4.5±0.9 vs. 9.1±1.6%; p=0.03). Specific depletion of CM with anti-CCR2 antibody abrogated neutrophil extravasation compared to isotype control (15.4±1.4 vs. 20.7±1.8%; p=0.04). LIRI in Nr4a1^-/- mice, which lack only non-classical monocytes, showed no difference in neutrophil extravasation. Splenectomy impaired neutrophil extravasation (6.8±0.6 vs. 16.6±2.2%; p=0.001) and reduced CM lung trafficking after LIRI (13.5±3.2 vs. 53.8±14.0 cells/mg lung; p=0.01). Heterotopic spleen transplant after native splenectomy restored CM trafficking (Figure 1A) (195.3±73.7 vs. 13.5±3.2 cells/mg lung; p=0.008) and neutrophil extravasation (Figure 1B) (14.1±1.2 vs. 6.8±0.6%; p<0.001) compared to spleen-lacking mice. Recipient bone marrow derived CCR2⁺CM repopulated the heterotopic spleen grafts and were recruited to the lungs during LIRI. Reconstitution with CCR2⁺CM did not restore neutrophil extravasation in splenectomized mice. Human lung allografts demonstrated over 2-fold increase in CCR2⁺CM influx immediately after reperfusion which showed linear correlation with neutrophil recruitment (p<0.001).

Conclusion:CCR2⁺ CM are responsible for neutrophil extravasation after LIRI but must pass through the spleen to mediate their function. As CCR2⁺CM are short-lived and replenished rapidly by the bone marrow, transient depletion using anti-CCR2 antibodies can ameliorate LIRI without affecting host pathogen response.