• innate immunity
• lung transplantation
• macrophage biology
• neutrophil biology

Ischaemia reperfusion injury at the time of lung transplantation may lead to primary graft dysfunction (PGD).1 Occurring within the first 72 hours following allograft reperfusion, PGD is characterised by hypoxaemia and alveolar infiltrates in the transplanted organ and occurs in up to 30% of patients.2 The development of PGD carries significant early mortality of up to 50% in 30 days.3 Those who survive the early postoperative period have a higher propensity to develop bronchiolitis obliterans syndrome,4 and long-term mortality is also higher at 1, 5 and 10 years.5

Several risk factors have been identified for the development of PGD, including a donor history of smoking, higher FiO₂ at the time of reperfusion, elevated pulmonary arterial pressures, large volume blood transfusion and raised recipient body mass index.6 This observation has led to the development of ex vivo lung perfusion. Here. lungs are ventilated and perfused with an acellular buffer. The use of this technique has allowed longer total ischaemic time prior to transplantation without an increase in incidence of PGD.7 However, incomplete understanding of the immune mechanisms leading to ischaemia reperfusion injury is a major limitation to preventing disease onset. There are no disease-specific therapeutics available for the condition, nor to prevent progression to PGD. Improved understanding of the immune responses underlying ischaemia reperfusion injury may allow development of new therapeutic strategies.

Dysregulation of the innate immune response is central to ischaemia reperfusion pathobiology. Neutrophils are a recognised early protagonist of inflammation,8 with extravasation mediated by preceding monocyte recruitment.9 10 Monocytes are mononuclear phagocytes, and in a most simplistic description belong to two principal subsets. Classical monocytes (Ly-6C^HighCCR2+ in mice) are migratory leucocytes in response to injury or inflammation.11 They may phagocytose pathogens, present antigens via MHCII or secrete chemokines to recruit other cells to the site of inflammation. In the lung, classical monocytes may traffic into the interstitium and differentiate to form alveolar macrophages or dendritic cells. Alveolar macrophages are a crucial mediator of the early inflammatory response and deletion of these cells is protective in experimental ischaemia reperfusion injury.12 Non-classical, endothelial patrolling monocytes (Ly-6C^LowCX3Cr1^High in mice) have been shown to adhere to vascular endothelium, slowly crawling via cell adhesion molecule interactions.13 These monocytes are also capable of migration and further differentiation.

It is recognised that circulating monocytes can be transferred from donor to recipient during transplantation.14 15 These cells remain present in lung explants following 10 L perfusion, and donor monocytes remain detectable in the recipient’s circulation many months following transplantation. However, the role of these ‘intravascular passenger’ monocytes in ischaemia reperfusion injury has not been previously appreciated. In Thorax, Tatham et al demonstrate a specific role of donor lung-marginated intravascular monocytes in ischaemia reperfusion injury during lung transplantation.16

In a mouse model of ex vivo perfusion, a dual intravascular and intra-alveolar antibody delivery technique followed by tissue dissociation and flow cytometry allowed the authors to identify and enumerate intravascular monocytes and interstitial macrophages. Intriguingly, despite intravascular perfusion for 15 min, approximately half of Ly-6C^High and Ly-6C^Low intravascular monocytes were retained within the lung. The activation status of these retained monocytes during ischaemia reperfusion was determined by modelling 2 hours of warm normoxic ischaemia followed by 2 hours of reperfusion incorporating three open-circuit washout periods in comparison with reperfusion only. L-selectin (CD62L) is expressed by Ly-6C^High monocytes that have recently left bone marrow and is required for trafficking to lymph nodes and tissue during inflammation.17 On contact with vascular endothelium, CD62L is downregulated and as part of monocyte maturation/differentiation, markers, including the costimulatory molecule CD86, are increased. Tatham and colleagues demonstrated that following ischaemia reperfusion there was activation of intravascular Ly-6C^High monocytes with reduced L-selectin and increased CD86 expression.16 Ly-6C^Low monocytes which do not express high levels of L-selectin also demonstrated increased CD86 expression following ischaemia reperfusion. Together this demonstrated activation of the donor intravascular monocyte pool.

Tatham and colleagues then selectively depleted intravascular monocytes using intravenous liposomal clodronate injection 24 hours prior to experimental lung perfusion in order to determine the effect of these vascular passenger cells on ischaemia reperfusion injury.16 This method significantly reduced Ly-6C^High and Ly-6C^Low monocytes in the vascular compartment, while vascular neutrophils, interstitial macrophages and alveolar macrophage frequencies were unaltered. Wet:dry lung ratios and bronchoalevolar lavage protein concentrations, both biomarkers of pulmonary inflammation, were significantly reduced in liposomal clodronate-treated mice following ischaemic reperfusion. Consistent with this, MIP-2 (CXCL2), a murine functional homologue of the powerful neutrophil chemoattractant interleukin 8 (CXCL8)18 and shown to recruit neutrophils in experimental PGD,10 was significantly reduced post reperfusion in the monocyte-depleted group.16 MCP-1 (CCL2) is an important chemokine for monocyte adhesion to vascular endothelium during trafficking to inflammatory sites.19 Consistent with lack of consumption by monocytes,20 this chemokine was significantly increased in the liposomal clodronate group.16 The authors also demonstrated a reduction in the epithelial cell injury marker RAGE in the monocyte-deplete mice, highlighting a potential early role for monocytes in ischaemia reperfusion, and hence PGD development.

Finally, Tatham and colleagues analysed the presence of donor monocytes and granulocytes in lung tissue from a small cohort of 13 human lungs prior to implantation.16 Consistent with their murine observations, despite anterograde perfusion and retrograde perfusion of 5 L of perfusate, high numbers of donor monocytes and neutrophils were still found in tissue. Electron microscopy confirmed the presence of monocytes and neutrophils in human lung capillaries, with possible cell-to-cell interactions between leucocytes and endothelial cells. Following transplantation, total monocyte numbers and intensity of CD86 staining were shown to be inversely correlated with PaO₂:FiO₂ ratios at 72 hours and 48 hours, respectively. Those who went on to develop Grade 2 or 3 PGD at 48 hours had higher expression of the monocyte activation markers CD86 and TREM-1.

The experimental data presented by Tatham et al 16 suggest a previously unappreciated role of ‘passenger’ intravascular donor monocytes in PGD following lung transplantation. While limited human data are presented in this manuscript, high numbers of monocytes appear to be retained in the human lung following standard perfusion protocols. Strengthening the findings of this paper is a recent confirmatory study of the importance of donor intravascular monocytes for neutrophil chemotaxis during PGD.10 The ability of these cells to withstand perfusion implicates high-affinity adhesive interactions between monocytes and endothelial cells. If mechanical removal of these cells prior to implantation is not clinically feasible, further elucidating the regulation of monocyte adhesion and trafficking, including the role of chemokine ligand/receptor pairs, and integrin/cell adhesion molecule interactions is of high importance in order to identify potential disease-selective therapeutic strategies. Future studies will also be required to examine whether ex vivo perfusion may provide a novel means of safely delivering therapy to the donor organ prior to transplantation, whether this approach may avoid systemic toxicity and if donor intravascular monocyte-targeted therapy may reduce the incidence of PGD or modify the natural history of this devastating condition.