The counter-intuitive role of the neutrophil in the acute respiratory distress syndrome



Arlette Vassallo, Alex J Wood, Julien Subburayalu, Charlotte Summers, Edwin R Chilvers

Department of Medicine, University of Cambridge, Cambridge, UK






Correspondence: 
Professor ER Chilvers FRCP, PhD, ScD, FMedSci
Department of Medicine, Box 157 Addenbrookes Hospital, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0QQ
e-mail:  erc24@cam.ac.uk, telephone: (+44) 1223 331531


Acknowledgements: 
The research in the authors laboratories is funded by the MRC, British Lung Foundation, Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Gates-Cambridge Scholarship Programme, Addenbrooke’s Charitable Trust, NIH Oxford-Cambridge Scholarship Programme, Cancer Research UK, Wolfson Foundation and non-commercial grants from MedImmune, Bristol Myer Squibs and GlaxoSmithKline.  We are grateful to Mr Philip Ball for help with the illustrations.


Short title:  
Neutrophils in ARDS


Introduction

‘I was really shocked.  I thought my immune system was there to protect me….’
[Quote from a patient recovering from sepsis-induced ARDS]

The first description of the acute respiratory distress syndrome (ARDS) was a case series published just over fifty years ago.1 It reported 12 patients with refractory hypoxaemia, diffuse pulmonary infiltrates, and a reduction in lung compliance.  Several important observations elicited from this series have stood the test of time, notably the similarity in clinical and pathological findings despite the variety of original insults, the importance of peak end-expiratory pressure (PEEP) in patient management, and the limitations of pharmacological treatments.  

ARDS is a heterogeneous clinical condition that remains common in the critically ill patient population and carries a mortality of 35 – 55%.2 There are no pathognomonic histological features of ARDS, despite the widely described diffuse alveolar damage, and the interpretation of clinical findings are highly variable.3 The definition of ARDS identifies three categories of worsening severity based on worsening oxygenation. The limitations of this approach are a reflection of paucity of knowledge of the biological processes underpinning the clinical phenotypes.4  However, significant advances are being made in our understanding of the pathobiology of this complex and economically burdensome syndrome.5  Interestingly, the identification of ARDS endotypes using latent class analysis may provide novel insights into differential responses to standard therapies such as fluid resuscitation, PEEP and statins.6-9 This review will focus on the role of the neutrophil in the initiation, progression and resolution of ARDS.

Perhaps the most ‘counter-intuitive’ paradox in all human immunology is how a system designed seemingly so perfectly to protect against infection, tissue injury and ‘non-self’ can, at times, inflict such devastation.  Unsurprisingly the innate immune system is considered to play a central role in the pathobiology of ARDS, and neutrophils being first responders are amongst the earliest and most abundant immune cells to be recruited to the lung.  With pneumonia and sepsis being the most common precipitants of ARDS, it appears that what often starts as an entirely appropriate anti-bacterial response rapidly escalates to a pattern of unrestrained immunological self-harm and tissue injury.

The homing of large numbers of neutrophils to the lung occurs largely through systemic priming or priming at a remote site; this results in increased neutrophil stiffness and mechanical entrapment within the narrow pulmonary capillary network; in the presence of a second usually epithelial insult, trans-endothelial migration of these cells to the interstitial and airspace compartments then follows.  

There is growing interest in the role of neutrophil subsets (or polarisation states) in the context of both ARDS and sepsis.  Relevant examples, to be discussed in more detail below, include the appearance of a set of CD62Ldim/CD16brightCD11bbright/CD54bright mature neutrophils, which are seen in severe injury or following experimental lipopolysaccharide (LPS) challenge and have the capacity to suppress T cell activation,10 the ability of activated C5a to reduce the phagocytic capacity of a significant proportion of circulating neutrophils,11 and the observation that a small number of sub-endothelial neutrophils synthesise and express intracellular adhesion molecule-1hi (ICAM-1hi neutrophils) and can return to the circulation via a process called reverse-trans endothelial migration (rTEM), home to the lung, and contribute to lung injury.12,13

Role of the pulmonary endothelium and epithelium in ARDS

The pulmonary endothelium is an expansive and highly specialised endothelial surface, which in health is believed to play a predominantly anti-inflammatory or ‘lung-protective’ role.  The integrity of the pulmonary capillary endothelium is clearly essential in preventing microvascular leak and oedema formation and the inadvertent entrapment and migration of immune cells.  The pulmonary endothelial surface becomes activated in most critical illness states mainly in response to systemic and/or locally produced cytokines such as TNF, IFN, IL-6, CXCL10 and CCL2 (Figure 1).  Murine models of influenza underscore the importance of the pulmonary endothelium for immune cell sequestration and cytokine production and highlight the role of the endothelial sphingosine-1-phosphate receptor in both of these events.14  Likewise, inhalation of LPS, a highly lipophilic and hence diffusible molecule, results in the loss of vascular endothelial (VE)-cadherin at inter-endothelial cell junctions, and consequently pulmonary oedema formation and extravasation of leukocytes.15,16  Pulmonary endothelial cells also express several leukocyte adhesion molecules, including ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) that are up-regulated in acute lung injury and serve to further propagate leukocyte migration to the injured lung.17  Hence the pulmonary capillary bed, which receives the entire cardiac output, and through which all circulating neutrophils transit on average once per minute, sits squarely in the firing line for local and systemic vascular insults and appears uniquely vulnerable to neutrophil entrapment and neutrophil-mediated damage.

Alveolar epithelial injury is likewise a well-recognised and established hallmark of ARDS (Figure 1).  One of many vital functions of this large and contiguous surface is that of fluid homeostasis and clearance, the driving force of which is active trans-epithelial sodium transport.  Ionic transportation of sodium and chloride ions occurs through multiple channels including the amiloride-sensitive epithelial sodium channel (ENaC) expressed on type II alveolar cells, aquaporins 1 and 4, and the cystic fibrosis transmembrane regulator (CFTR) with fluid shifts following the osmotic gradient.  The process of alveolar fluid clearance is severely impaired in ARDS, with worsening function associated with poorer clinical outcomes.18,19  The pulmonary epithelium is also responsible (in the presence of increased permeability and oedema) for driving a pro-coagulant environment, where the constitutively expressed tissue factor (TF) present on lung epithelial cells and macrophages comes into contact with and binds factor VIIa triggering a coagulation cascade.  TF expression on pulmonary epithelial cells is up-regulated in response to several circulating cytokines.20 The resultant hypercoagulable state results in excess alveolar fibrin deposition, which in turn promotes ongoing inflammation and fibrosis, loss of surfactant function, and alveolar atelectasis. 

Neutrophil subsets in ARDS

The idea of leucocyte heterogeneity is now well established and relevant to all major cell types involved in innate and adaptive immunity.21,22 For many years, circulating and tissue neutrophils were thought of as being homogeneous, short-lived and transcriptionally quiescent.  This view was based on the fact that these cells have only a brief intra-vascular half-life23,24 and following arrival at an inflamed site, either die via a process of constitutive or phagocytosis-induced apoptosis, or are eliminated from the body via the urinary, gastrointestinal or respiratory route.  However, emerging evidence indicates that few of these assumptions are entirely true and that significant neutrophil heterogeneity, longevity and plasticity exists and that this ‘new biology’ impacts greatly on the genesis and resolution of ARDS. 

The release of mature neutrophils from the bone marrow is regulated by the CXCL12/CXCR4 and IL-6/IL-6R axis.25  In the circulation, the neutrophils partition between two discrete pools; a freely circulating blood pool, and a marginated blood pool, which exists within the vasculature of discrete organs, largely the spleen, liver and bone marrow.26  Traditionally, the lungs were also considered to contain a significant pool of marginated granulocytes.27  However, the lack of a neutrophil signal in routine radiolabelled leucocyte scintigraphy and more recent gamma scintigraphy data from our laboratory using carefully prepared autologous radio-labelled cells, suggests that the size of any marginated pool of neutrophils within the lung may have been over-estimated.28  These seemingly conflicting views are most likely explained by the inadvertent ex-vivo activation of the neutrophils used in the earlier studies as deliberate (ex-vivo) priming with agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) results in immediate (i.e. ‘first-pass’) and near-complete retention of neutrophils within the pulmonary capillary bed.28 

Neutrophil subsets have also been observed in a large number of experimental and disease states, including ARDS.  In a human model of intravascular endotoxemia employing low-dose LPS, phenotypic and functional heterogeneity of circulating neutrophils is observed as early as 3-6 hours.  Immature CD16low neutrophils appear, which have reduced capacity to generate reactive oxygen species (ROS), reduced interaction with opsonised bacteria, and reduced cell surface expression of immune receptors.29  A further neutrophil subset characterised as CD16hi/CD11bhi/CD62Llow also appears, with a distinctive immunosuppressive phenotype and the ability to suppression T cell proliferation via a Mac-1-dependent hydrogen peroxidase pathway.10  These findings support the counter-intuitive notion of critical illness and ARDS triggering both hyper-inflammatory (e.g. systemic neutrophil priming, with enhanced CD11b expression and NADPH oxidase responses) and immune-paretic states (e.g. reduced phagocytic capacity, acquired T cell suppressive activity and delayed apoptotic susceptibility), which happen in parallel.  This results in a dysregulated and potentially damaging inflammatory response, with the dual outcomes of tissue injury and heightened susceptibility to infection.30 A very similar paradox is seen across multiple disease states including children with severe viral infection.31 

Neutrophils transmigrate across the endothelium and into tissues along a chemokine and/or lipid mediator gradient.  It has been a long-standing view that neutrophils then undergo apoptosis, before being phagocytosed (or ‘efferocytosed’) and removed by tissue-resident or influxing macrophages.  However, while apoptotic neutrophil debris and evidence of apoptotic cell engulfment is clearly identifiable in the lung following an acute inflammatory insult32 this does not appear to be the only cell disposal route, and the capacity for tissue neutrophils to traffic back to the bone marrow and lymph node compartments is now well recognised.  Likewise, as noted, there is also evidence of a specific phenomenon referred to as reverse trans-endothelial migration (rTEM), a process whereby neutrophils migrate from the sub-endothelial space back into the circulation; these neutrophils are characterised as ICAM-1hi/CXCR1low and display an enhanced capacity for ROS generation, as well as increased longevity and phagocytic capacity.33  In murine models, these cells are associated with worse lung injury scores, suggesting they may play an important role in ARDS.12  Further neutrophil subsets have been identified including the highly NETosis-prone ‘low-density’ neutrophils (LDNs) evident in a range of autoimmune vasculitis conditions,34 and a fascinating set of tumour-associated neutrophils (TANs) and pro-angiogenic neutrophils(PANs).35

Hence, while the relevance of these differing neutrophil subsets to lung injury and ARDS in particular are uncertain, these varying states have a marked impact on the homing, pro- and anti-inflammatory properties of neutrophils, and it is clear that these cells exhibit a greater degree of heterogeneity than previously recognised.  This may go some way to explain the rather complex effects of neutrophils in critical illness and lung injury.  Understanding of the drivers and signalling events underlying the genesis of neutrophil subsets and a fuller understanding of the functional effects of such switching should enable us to improve our predictive modelling and offer the possibility of new and more refined targeted therapies. 

Neutrophil recruitment and trafficking

Endothelial activation occurs in response to circulating and locally produced cytokines, and an array of pathogen- and damage-associated molecular patterns (PAMPs/DAMPs).  This results in up-regulation of a variety of adhesion molecules including P- and E-selectin and this stimulates neutrophils tethering and rolling.36  Neutrophils can also form lymphocyte function associated antigen 1 (LFA-1) coated slings that bind to ICAM-1 (CD54) and support rolling at high-shear forces; the relevance of this latter event to lung migration is however far from certain.37,38  Endothelial rolling is thought to promote neutrophil priming, resulting in up-regulation of CD11b (Mac-1) and shedding of CD62L (L-selectin) and subsequent neutrophil arrest on the endothelium.  In the systemic vasculature this is followed by phosphorylation of the VE-cadherin complex and Rho GTPase activity resulting in cytoskeletal conformational change, disassembly of the endothelial junctions, and the development of inter-cellular gaps allowing for neutrophil trans-endothelial migration (see Figure 2).39

It is important to realise however that this classic canonical pathway of neutrophil recruitment may not hold true in the lung.  The size and geometry of the very unique capillary network that makes up the pulmonary microcirculation necessitates that neutrophils must deform significantly (akin to that seen in the red cell) in order to pass through what is a complex series of uniquely tight (5 µm) capillary segments;40 this geometry is not replicated in other microvascular beds and it is clear that neutrophil entrapment and migration is somewhat different in this setting.  For example, in many infection models, neutrophil migration to the lung is entirely β2-integrin-independent.41

CXCL5 is a potent cytokine produced by type II alveolar epithelial cells that signals via CXCR2 and drives neutrophil emigration.  Hence high CXCL5 concentrations in bronchoalveolar lavage fluid (BALF) correlate well with the number of neutrophils in the injured lung.  Furthermore, following lung injury CXCL5-deficient mice have reduced trafficking of neutrophils to the lung.  By contrast, in a murine model of E. coli pneumonia, CXCL5 deficient mice have increased neutrophil trafficking and better microbial clearance than wild type animals.  This again demonstrates the complexity and context-dependency of the innate immune response.42-44 

It has already been noted that much of the initial neutrophil sequestration that occurs in the lung may be mechanical and driven by priming-mediated changes in the actin cytoskeleton.45  A further proposed mechanism for neutrophil migration involves the activation of transient receptor potential vanilloid 4 (TRPV4) channels causing an increase in membrane endothelial permeability and oedema formation.  In murine models of inhalational lung injury TRPV4 deficiency attenuates inflammatory cell infiltration.  However, administration of a therapeutic TRPV4 inhibitor after the establishment of lung injury has not proved to be beneficial.46,47 

Overall therefore, the mechanisms by which neutrophils emigrate into the alveoli remain poorly understood.  Further elucidation of this process will undoubtedly yield novel targets in our search for improved ARDS treatments.  


Neutrophil effector functions

Given the potential for neutrophils to induce tissue damage, it is entirely intuitive that these cells should possess a ‘safety switch’ to preclude inappropriate full activation as this leads to unrestrained de-granulation and major extracellular superoxide anion generation.  The requirement for these cells to be initially primed before they can display their full activation potential thus makes sense.  Priming changes the repertoire of a wide range of cell adhesion molecules, initiates f-actin polymerisation and cellular polarisation resulting in a stiff and less deformable cell, delays apoptosis, and up-regulates subsequent agonist-induced degranulation and NADPH oxidase responses.48,49  Priming also causes a large proportion of these cells to be sequestered in the lung,50,51 which can be shown quite vividly in humans using radio-labelled neutrophils primed ex-vivo.28  What is less intuitive is the finding that priming reduces subsequent direction migration (chemotaxis) and for certain agents such as activated C5a, impairs the cell’s capacity for bacterial phagocytosis.

Conventionally, priming was thought to be irreversible.  Early data from our laboratory however, demonstrated that neutrophils studied in-vitro had the capacity to ‘de-prime’ and regain their rounded non-polarised shape and down-regulate CD11b and the capacity to generate extracellular superoxide anions.  Perhaps even more surprising was the demonstration that neutrophils primed with the rapid priming agent platelet-activation factor (PAF) could in fact participate in a full cycle of priming, de-priming and re-priming (Figure 3), again suggesting that priming and activation are highly dynamic fluid and ‘non-fixed’ states.52  This cycling also appears to occur in-vivo as we have shown that neutrophils primed ex-vivo with PAF but then allowed time to de-prime prior to reinjection have a near normal first pass pulmonary  transit time compared to their fully primed counterparts and likewise that cell primed ex-vivo with GM-CSF while initially get stuck in the pulmonary capillary network eventually move out of the lung and partition relatively normally between the circulating and marginated intra-vascular neutrophils pools.28 

Until recently we were very uncertain whether de-priming was an active or passive event and occurred in response to all priming stimuli, or what the mechanism for this recovery event might be.  However using a number of biophysical techniques involving optical stretchers and a novel microcirculation mimetic, all of which induce physiologically-relevant mechanical deformations in neutrophils , we have shown that these neutrophils can de-polarise extremely rapidly (< 1 min) following mechanical stimulation and that this occurs in associated with a parallel fall in CD11b expression.53  Although speculative, it is tempting to propose that one function of the unique central position and architecture of the pulmonary capillary network might be to ‘deliberately’ capture systemically or remotely primed neutrophils and help to mechanically de-priming these cells; loss of this protective function or the presence of a concurrent secondary alveolar insult (such as infection, ventilator-induced injury, acid inhalation etc.) may then be all that is required to develop ARDS.28,54

Activated neutrophils release thread-like structures called neutrophil extra cellular traps (NETs) composed of citrullinated histones, chromatin DNA and granular proteins.  These structures trap and kill bacteria extracellularly.55 The processes by which NET formation occurs can be broadly divided into two: early or vital NET formation and late or suicidal NETosis.  Vital NET production is thought to occur in response to pro-inflammatory cytokines or pathogens, mediated through toll-like receptors, and to be NADPH oxidase-independent56 whereas suicidal NETosis is classically seen following phorbol myristate acetate (PMA) stimulation.  The NADPH oxidase plays an important role in mobilising myeloperoxidase into the nucleus resulting in chromatin de-condensation and triggers a pathway that eventually results in the expulsion of extracellular traps and cell death.57  Determining the true role for NETosis in both protective and pathogenic settings has been challenging but clearly NET formation can facilitate bacterial capture and killing.  It has also been postulated that NETosis occurs only within a subset of neutrophils specifically targeted for demise.58  Likewise NETs have been shown to induce tissue factor (TF) and factor XII-mediated coagulation and some consider the formation of micro-thrombi to be an important evolutionary, adaptive response to contain bacteria and reduce spread.59

NETs have been found to be elevated in several models of lung injury, and postulated to mediate tissue injury.60-62  It is also possible that NET formation is simply a down-stream consequence of epithelial injury and the loss of surfactant protein D, which is known to be involved in NET clearance.63  Whether NET formation is a major player in ARDS remains to be determined.

The role of neutrophils in the resolution of ARDS

So far the implication of this review is that neutrophils are mainly damage-inducing cells and a major driver of ARDS; indeed the clear correlation between circulating neutrophil elastase and ARDS severity would certainly support this.  However, tissue damage is not the only consequence of neutrophil accumulation and, as in the setting of bacterial pneumonia where the alveolar space, interstitium, and microvasculature are flooded with neutrophils yet the lung can repair fully, ARDS can and does resolve and we are now learning that neutrophils can play a role in this process also.

Neutrophil apoptosis has already been referred to as a powerful mechanism for the ‘safe’ disposal of effete neutrophils and this event has been widely reviewed.  However, work from the Serhan laboratory has revealed the additional importance of a group of ‘specialized pro-resolving mediators’ (SPMs) including resolvins, protectins, maresins, and lipoxins, which appear to be major players in the resolution of inflammation.64  One particularly fascinating observation is that these SPMs are not only produced by tissue and alveolar macrophages, but also by apoptotic neutrophils, which thus serve to promote their own removal from the inflamed site.64 Lipoxin A4 as an exemplar SPM not only stimulates monocyte migration but also promotes the phagocytosis of apoptotic neutrophils and inhibits CXCL8 release.

Neutrophils also help recruit peripheral blood monocytes to the site of inflammation,65 where they undergo differentiation and polarization into highly plastic macrophages.66 These cells play a sentinel role in clearing aged neutrophils before any release of their phlogistic cargo takes place.67,68  Neutrophils themselves are also capable of phagocytosing (cannibalising) other apoptotic neutrophils, however the magnitude of this effect relative to macrophage efferocytosis is uncertain.  Macrophage engulfment of apoptotic neutrophils induces a pivotal immune-resolving reprogramming in these cells, which is thought to initiate the resolution phase and prevent organ fibrosis.65,69  This switch in the macrophage triggers the release of IL-4 that in turn induces 15-lipoxygenase type I, a pivotal enzyme for SPM production in both macrophages and the apoptotic neutrophil.65 The broader importance of IL-4 in restoring tissue homeostasis is now widely recognised70,71  as this molecule has also been found to limit hypoxic neutrophil survival, hence displaying a positive feedback loop of macrophage-driven and SPM-facilitated apoptotic neutrophil removal.66  Other functions of SPMs have been proposed including rescuing mice from lethal Citrobacter rodentium infection.72

There is little doubt that a lot more will be discovered regarding the beneficial roles of neutrophils in tissue homeostasis and repair and the next decade of research promises to reveal much in this area; we have clearly underplayed the role of the neutrophil in inflammatory resolution.  One hint in that direction is the recent work of Wang and colleagues who show in a fully repairing thermal liver injury model that neutrophils penetrate the damaged site and dismantle injured vessels and create channels for new vascular regrowth and the findings of Lin and co-workers who provide evidence that a subset of specialised non-inflammatory neutrophils drive vascularization.73,74 If this pertains to vascular repair in the lung following ARDS then we have much to learn and look forward to.  If neutrophils flip from a pro-inflammatory role to that of driving inflammatory resolution then therapeutically we will have to be extremely nimble in targeting these cells and recognising early when this transition point happens. 
Murine models remain a vital part of our research armamentarium, providing us with tightly-regulated conditions in clean phenotypes, allowing us to understand pathophysiological processes in an unfettered manner.  There remains, however, a large divide in translating these important findings to humans and it is clear that studies undertaken in human models are necessary to advance further our understanding of ARDS.75

Conclusion

Neutrophils are increasingly recognised as complex and multi-faceted cells that play important, albeit paradoxical roles, in initiating and maintaining inflammation as well as driving resolution.  Neutrophil depletion is associated with worse outcomes, yet excessive degranulation and ROS release can perpetuate host tissue injury.  Understanding the trafficking of neutrophils in the pulmonary vascular bed and the plasticity of these cells including their ability to de-prime will be key to defining future therapies.  The presence of neutrophil subsets or polarisation starts similarly offers the potential for manipulation in favour of an immune-regulatory and anti-inflammatory phenotype. 

Figure legends

Figure 1
The early phase pathological changes in the acute respiratory distress syndrome 
Alveolar epithelial injury results in the release of pro-inflammatory chemokines and promotes the migration and accumulation of neutrophils in the alveolar space.  Neutrophil activation propagates injury through pro-inflammatory mediators including neutrophil elastase, reactive oxygen species generation and neutrophil extracellular trap formation. Barrier injury results in oedema and increased gap formation.

Figure 2 
The classical pathway for neutrophil trans-endothelial migration (TEM) in the systemic circulation 
Activated endothelium encourages neutrophil rolling, resulting in priming with subsequent CD62L shedding and up-regulation of CD11b.  The neutrophils arrest and migrate through gap junctions in the endothelium down a chemoattractant gradient.  A subset of ICAM1hi neutrophils have been reported to have the capability to undergo reverse trans-endothelial migration (rTEM).

Figure 3 
Neutrophil priming results in well-defined phenotypical and functional changes 
While neutrophil de-priming appears to return this cell to its basal state, important differences persist.  Hence while unstimulated resting neutrophils are CD62Lhigh/CD11bdim, deprimed neutrophils are CD62Ldim/CD11bdim.  The functional properties of de-primed neutrophils remain poorly described. 



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