Neutrophilic inflammation in the lung protects against infectious disease, and usually resolves spontaneously after removal of the inflammatory stimulus. However, much lung disease is caused by a failure of resolution of neutrophilic inflammation. Our laboratory is seeking an understanding of the biochemical basis of inflammation resolution, using the zebrafish model system. Zebrafish larvae are transparent, allowing visualization of GFP (green fluorescent protein)-labelled leucocytes during inflammation in vivo, and they can be readily manipulated by a range of forward and reverse genetic techniques. This combination of advantages makes zebrafish a powerful tool for the study of in vivo inflammatory processes. Using this model, we have visualized the process of inflammation resolution in vivo, and identified a role for apoptosis in this process. In addition, we have performed a forward genetic screen for mutants with defective resolution of inflammation, and reverse genetic experiments examining the influence of candidate genes on inflammation resolution. We have established a platform for screening for compounds with anti-inflammatory activity, which has yielded a number of interesting leads. Looking forward to succeed in the future, we are working at combining mutants, transgenes and pharmacological agents to dissect the biochemical basis of inflammation resolution, and to identify compounds that might be used to treat patients with respiratory disease.

Introduction

Diseases of unresolved neutrophilic inflammation are a part of everyday life in respiratory medicine. The molecular events controlling neutrophilic inflammation and its resolution remain unknown, and it is this ‘biochemical basis’ of neutrophilic respiratory disease that the work in our laboratory seeks to address.

The evolutionary origins of inflammatory disease

Multicellular organisms, including ourselves, have evolved over hundreds of millions of years to survive in a range of demanding environments. An ever-present and highly important element of those environments is the microbiological flora to which we are exposed. Since the earliest multicellular animals, we have had to compete with unicellular organisms for available resources. An ‘arms race’ between the potential pathogen and host has arisen, which has driven the development of highly efficient immune systems. These have allowed multicellular animals to thrive despite the ubiquitous presence of unicellular organisms. The first immune systems were relatively simple: for example, wandering phagocytic cells with host defence function have been present since the earliest multicellular organisms [1]. Such a function is preserved in the neutrophils and macrophages of the mammalian immune system today. Similarly, many invertebrate species possess antibacterial peptides [2], and recognition systems for PAMPs (pathogen-associated molecular patterns) [3], whose relatives help us to defend ourselves against pathogens on a daily basis. It seems a general principle that new levels of complexity and control have been built on existing foundations, while those foundations have been largely preserved.

An immune system shaped by such powerful forces over millions of years is highly capable, and maintains our ability to pass on our genes to future generations. For millions of years, this system has protected us from infection, enough for human civilization to flourish. The greatest threats to human health have until recently been infectious diseases, and such diseases have severely limited human lifespan for most of our time on this planet. Thus the evolutionary pressure has favoured robust antimicrobial strategies. This has led to the evolution of specialized cells of the immune system, with specialized antibacterial products released from those cells on activation, protecting against infection. Any unintended damage to the host by these products is balanced by protection against infectious disease. With the improvements in infection control over the last 50–100 years, we can see a reduction in the number of deaths from infectious disease and also a dramatic extension of our lifespan. In the developed world, the balance has therefore shifted: tissue damage and death from disorders of immune overactivation now outweigh deaths from infectious disease.

The role of neutrophilic inflammation in lung disease

Many such inflammatory disorders affect the lung, and in many of these, unresolved neutrophilic inflammation is a major contributor to the tissue damage that is the hallmark of such conditions. COPD (chronic obstructive pulmonary disease) is one of the most important causes of death in the developed world. In COPD, it is now widely accepted that the action of neutrophil proteases is responsible for the destruction of elastic tissue and the resulting emphysema [4]. In contrast, asthma is generally regarded as predominantly an eosinophilic disease. There is mounting evidence, however, that in a subset of patients with asthma (including those with severe asthma) the inflammation is predominantly neutrophilic, and in this group conventional steroid-based anti-inflammatory regimens are ineffective [5]. In IPF (idiopathic pulmonary fibrosis), neutrophil numbers in the lung correlate with survival [6], suggesting at least an association of neutrophilic inflammation with progressive fibrosis in the lung. Finally, the predominant inflammatory cell in ARDS (acute respiratory distress syndrome) is the neutrophil, with levels of the neutrophil chemoattractant IL (interleukin) -8 related to outcomes suggesting that an increase in the number of neutrophils leads to more tissue damage and poorer outcomes [7]. Taken together, these diseases are a huge drain on health care resources, and contribute significantly to the burden of illness in the developed world.

Biology of the neutrophil granulocyte

Leucocytes are fascinating cells, and there are a number of features that are unique to neutrophils. Humans make neutrophils at a rate of over a million every minute, increasing dramatically at times of infection [8]. These neutrophils circulate for a limited time before being removed from the circulation, either by homing of senescent neutrophils back to the bone marrow [9], or by uptake of apoptotic cells by macrophages in the liver and spleen [10]. Neutrophils have the shortest lifespan of any cell, and die by apoptosis with a half-life of approx. 8 h [10], both in vitro [11] and in vivo [12,13]. The reason for this seemingly wasteful overproduction of neutrophils is not known, but explanations include the ability to rapidly and dramatically increase neutrophil number during times of need, while reducing the risks of malignant transformation, which are inherent in any system characterized by high levels of cell production. The biochemical processes executing neutrophil apoptosis appear to be the same as those of apoptosis in other cells. Neutrophil apoptosis, like that seen in other cell types, appears to be caspase-dependent, although data are somewhat contradictory [14]. However, the control points of this process, and in particular the regulatory steps that distinguish the uniquely rapid initiation of neutrophil apoptosis from that seen in all other cell types, are unknown.

Neutrophils that encounter a site of inflammation may be recruited into that site by changes in the local endothelium, induced by inflammatory cytokines and chemokines. These molecular changes induce reciprocal changes on the neutrophil surface, and lead sequentially to rolling, firm adhesion and migration through the endothelium (diapedesis) [15]. In the tissues, neutrophils will eventually die by apoptosis, and this process is essential for preventing tissue injury, as it ‘turns off’ the inflammatory functions of the neutrophil, rendering it unable to actively degranulate, and unable to release the toxic contents of its many granules into the tissues [16]. In addition, it keeps these granules intact within the cell membrane, preventing their inadvertent release. The process of apoptosis marks the neutrophil for phagocytosis by macrophages, which in turn renders those macrophages less willing to participate in the inflammatory process by virtue of altering their cytokine expression profile to a pro-resolution phenotype [17]. This entire process is central to the resolution of inflammation, and removes neutrophils in an anti-inflammatory way.

Once a neutrophil is committed to participation in the local immune response to infection or injury at inflammatory sites, it is exposed to a range of environmental agents that act to extend its lifespan [1820]. This extension of neutrophil lifespan might increase the antibacterial potency of the neutrophil, and indeed might render it less susceptible to manipulation of its apoptotic programme by bacterial exotoxins. Existing evidence suggests that the extension of neutrophil lifespan at inflammatory sites is due to the differential effects of survival signals received by those neutrophils, rather than any fundamental changes in neutrophil phenotype [21], and it seems likely that this is responsible for much of the tissue injury seen in a variety of inflammatory diseases, including those discussed above.

Key survival signals for neutrophils fall into three categories: host-derived, pathogen-derived or physical properties of the inflammatory environment. It is clear that the ability to detect pathogens, and to respond to them by delaying local rates of apoptosis, may have a profound effect on local antibacterial capabilities, and as such would have been under selective evolutionary pressure. What is less clear is that the other two categories may have evolved to fulfil the same functions. Hypoxia is an obvious distinguishing physical property of an infected tissue. Bacteria utilize oxygen, and the formation of abscesses creates oxygen-deplete environments in which neutrophils must not only survive, but function efficiently [22]. In this context it is perhaps not surprising that hypoxia is a profound stimulus for neutrophil survival [23]. The other physical parameter altered in inflammatory sites is temperature. The effects of increased local temperatures on neutrophil function are less well studied, but it appears that increases in temperature during febrile episodes accelerate neutrophil apoptosis [24].

Finally, most naturally occurring tissue injuries are accompanied by a breach in the protecting epithelium, and exposure to the environment. In this context, a prompt neutrophilic response to tissue injury would have the benefit of anticipating the impending microbiological contamination, even if bacteria are not present at high enough levels to directly or immediately trigger the immune response. Thus certain cell products retained within cells in health, such as ATP, profoundly influence aspects of neutrophil function including rates of apoptosis [25,26]. In addition, cytokines secreted by the immune system to modulate the immune response may do so in part by regulating the survival of neutrophils. Well characterized examples of this include G-CSF (granulocyte colony-stimulating factor) and GM-CSF (granulocyte/macrophage colony-stimulating factor) as well as ILs, including IL-1 [18,19]. Some of these effects may be indirect [27], but in a whole animal system the effects are the same.

Induction of neutrophil apoptosis as a therapeutic strategy

Neutrophil-directed therapies might allow us to redress this imbalance between host-defence functions of neutrophils, and their tissue-injuring properties. Adriano Rossi and colleagues in Edinburgh have previously established the precedent for this, with their work on the CDK (cyclin-dependent kinase) inhibitor, roscovitine [28]. They have elegantly demonstrated that inflammation in the lung (bleomycin-induced pulmonary fibrosis and carageenin-induced pleurisy), and in the joints (serum transfer arthritis) can be ameliorated by the induction of neutrophil apoptosis by roscovitine. However, any non-selective induction of neutrophil apoptosis carries a risk of immune suppression. It would therefore be preferable to specifically target the delayed neutrophil apoptosis seen at sites of inflammation. Survival signals received by the neutrophils delay neutrophil apoptosis, and potentially increase tissue damage at those sites. Specifically removing the influence of these survival signals would allow therapeutic removal of inflammatory neutrophils, while leaving unstimulated circulating neutrophils unaffected. However, understanding how these survival factors influence the intracellular signalling processes that determine neutrophil survival is, we believe, the central question in neutrophil biology.

The biochemical basis of inflammatory respiratory disease

There are many control points in inflammation, and many of these are important in determining the fate of an inflammatory process, and thus determining whether inflammation resolves, and at what rate. At a molecular level these control points define the biochemical basis of inflammatory respiratory disease. One key control point is the regulation of neutrophil apoptosis. If this process becomes dysregulated, neutrophils will persist at inflammatory sites, perpetuating tissue damage. Thus termination of neutrophil pro-inflammatory functions by apoptosis is essential for the resolution of inflammation. If we perceive inflammation as a mechanism for the efficient delivery of neutrophils into an inflammatory site, then it might therefore be argued that the resolution of inflammation is about efficiently removing neutrophils from the site of inflammation. Apoptosis in general is characterized by a series of stereotypic, energy-dependent and genetically determined biochemical steps. These include activation of a cascade of proteolysis within the cell orchestrated by the caspase family of cysteine proteases and leading to cleavage of a diverse set of proteins essential for cell function. These include structural proteins such as actin [29], DNA repair enzymes such as PARP [poly(ADP-ribose) polymerase], and inhibitors of other essential functions, such as iCAD (inhibitor of caspase-activated DNase), the inhibitor of the enzyme that cleaves DNA into characteristic and non-functional fragments (recently reviewed in [30]). Interestingly, different members of the caspase family are also responsible for the cleavage of ILs including IL-1, -17 and -33 [31]. Caspase activity is initiated in a number of caspase-activating platforms, including the inflammasome (for cytokine processing caspases), the DISC (death-inducing signalling complex) and the apoptosome (recently reviewed in [32]). The DISC activates caspases in response to death receptor ligation, and the apoptosome in response to cytochrome c release from mitochondria. These two mechanisms of initiation of apoptosis explain most other forms of apoptosis induction in other cell types. There is evidence that under certain circumstances both routes can be the cause of neutrophil apoptosis [21,33,34]. However, which of these mechanisms (or indeed a third unknown mechanism) is responsible for the uniquely rapid death of neutrophils is not known.

A key regulatory step for most forms of apoptosis is the regulation of cytochrome c release from mitochondria by the Bcl-2 family of proteins. These proteins can be either pro-or anti-apoptotic, and members of each class interact in incompletely defined ways to regulate cytochrome c release and hence cell survival. Several members of this class are present in neutrophils, although the key player appears to be Mcl-1 (myeloid cell leukaemia sequence 1) [35]. This anti-apoptotic protein is rapidly turned over, and its levels appear to be increased by survival signals such as GM-CSF and LPS (lipopolysaccharide) [36]. When Mcl-1 is knocked out in murine myeloid cells, macrophage survival is unaffected, whereas neutrophils are almost completely eliminated, underscoring its importance in maintaining neutrophil viability [37]. It is therefore a good candidate for an important regulator of neutrophil survival.

Despite being of utmost importance in determining the outcome of inflammatory disease, the biochemical basis of survival signal action on the apoptotic pathway is poorly understood. Although some authorities have implicated Mcl-1 as the final arbiter of neutrophil survival [35], whether this is universally true, and indeed why Mcl-1 levels should be of such critical and unique importance in neutrophils, remains unclear.

Models for the study of inflammatory processes

Most of the work to elucidate the molecular controls of neutrophil apoptosis has been performed in purified human peripheral blood neutrophils. Thus much work relies on studies using inhibitors with various degrees of substrate specificity and off-target effects, or on population studies where effects in individual cells are not observed directly. In such studies, rates of apoptosis, levels of protein expression or enzyme activation are compared between populations with different levels of apoptosis. Induction of mechanistic insights from such observations is fraught with difficulty. In particular, it can be difficult to untangle the order in which biochemical events occur, from the order in which they cross variable thresholds for detection in the population of cells studied. Human neutrophils are not readily amenable to genetic manipulation, and where they have been manipulated, rates of apoptosis have been delayed [38].

Mammalian gene manipulation studies have been extremely fruitful; for example, the observation that Mcl-1 is essential for neutrophil viability comes from such studies in the mouse [37]. However, this system works best with well-defined and readily testable hypotheses that justify the time and cost implications of this approach. Although unbiased approaches in the mouse are also well established, with ENU (N-ethyl-N-nitrosourea) mutagenesis screens yielding much important information on the vertebrate immune system [39], as yet these are too time consuming and laborious to extend to studies of neutrophil survival or inflammation resolution. Finally, while mammalian models can be manipulated to allow in vivo imaging of neutrophil recruitment, the resolution phases of inflammation have proved more elusive.

The utility of non-mammalian models has been well established in many other fields of biology. Indeed, for the study of apoptosis, the nematode worm Caenorhabditis elegans has proved invaluable [40]. However, for studies of cellular immunity this model is unsuitable, owing to a lack of professional phagocytes. The fruitfly Drosophila melanogaster has particular advantages for the genetic study of more advanced processes, and indeed possesses haemocytes, which are clearly myeloid cell equivalents and participate in inflammatory processes after injury or infection in the fly [41]. This model lacks cells with the specific features of neutrophils, and exactly where this model can be used to illuminate vertebrate biology remains to be determined.

Zebrafish models of the inflammatory response

In search of a simple vertebrate model for the study of a range of physiological processes, many scientists are now turning to the zebrafish Danio rerio. As a vertebrate, zebrafish offer advantages over invertebrate models, particularly in terms of similarity to mammalian models. High homology is seen at both a cellular level and at a molecular level between zebrafish and humans [42]. This is particularly important in the immune system where functional specialization is rapidly evolving. At a molecular level, proteins involved in host defence have often diverged further from their counterparts in other organisms when compared with essential proteins necessary for development, for example. However, functional homology seems to be relatively well preserved [42].

At a cellular level, zebrafish have analogous cells to neutrophils (sometimes termed heterophils in the zebrafish), macrophages, eosinophils, mast cells, T-cells and B-cells. The exact functional correspondences are not determined for eosinophils and mast cells as yet, and T- and B-cells are not functional until approx. 4 weeks post-fertilization [42]. This implies that the zebrafish larva has to survive until that time solely on the strength of its innate immune system. Zebrafish have evolved in slow moving or stagnant water in tropical environments, and it is hard to imagine that this is an environment sheltered from microbiological challenges. There may be a reflection of this in the zebrafish genome: there is an expansion of a number of innate immune regulatory gene families in the zebrafish. The TLR (Toll-like receptor) family contains at least 24 members in the zebrafish [43], and the NLR (Nod-like receptor) family contains an additional division entirely restricted to fish [44]. However, the degree of functional conservation is the subject of ongoing study.

Imaging inflammation in vivo

One of the main advantages of the zebrafish model is the ability to view physiological processes such as inflammation in vivo. Early studies in zebrafish larvae identified immune cells with phenotypic features similar to mammalian phagocytes. Early experiments in our laboratory imaged the recruitment of inflammatory cells to a tail fin wound in larval zebrafish (Figure 1 and Supplementary Movie S1 at http://www.biochemsoctrans.org/bst/037/bst0370830add.htm). Inflammatory leucocytes can be clearly seen on DIC (differential interference contrast) imaging, and their behaviour can be readily assessed. Unfortunately, DIC imaging does not allow identification of the different types of immune cells. However, lineage-specific labels can be readily employed in the zebrafish by the use of tissue-specific transgenic approaches. The first myeloid cell transgenics were Pu.1:GFP (where GFP is green fluorescent protein) lines, which labelled primitive myeloid cells, allowing for the first time the in vivo assessment of myeloid cell development and function, including recruitment to sites of tissue injury [45,46]. Our group and others have now developed a range of tools labelling myeloid populations in the zebrafish. Of these, two are restricted specifically to neutrophils, Tg(mpx:eGFP)i114 [47] and CLGY463 [48], and others have overlap in other immune cell types: Tg(LysC::GFP/dsRed) [49] and Tg(zMPO:GFP)uw [50]. For many research questions, immune cell specificity is not a major issue. However, when asking questions about the nature of neutrophil behaviour during inflammation in vivo, compared with the behaviour of other phagocytes, the specificity of the label can assume significance. It would appear that the Tg(mpx:eGFP)i114 line is the only neutrophil-specific line that labels the whole neutrophil population (C.A. Loynes and S.A. Renshaw, unpublished work).

Three images from a DIC time series showing firm adhesion and paracellular endothelial transmigration of a zebrafish leucocyte after tail fin injury

Figure 1
Three images from a DIC time series showing firm adhesion and paracellular endothelial transmigration of a zebrafish leucocyte after tail fin injury

The complete time course from which these images are taken can be seen in Supplementary Movie S1 at http://www.biochemsoctrans.org/bst/037/bst0370830add.htm.

Figure 1
Three images from a DIC time series showing firm adhesion and paracellular endothelial transmigration of a zebrafish leucocyte after tail fin injury

The complete time course from which these images are taken can be seen in Supplementary Movie S1 at http://www.biochemsoctrans.org/bst/037/bst0370830add.htm.

Using these models, the fate of individual cells can be tracked and the behaviour of populations observed. The data generated by such imaging systems are a valuable resource for understanding neutrophil function.

Reverse genetics in the zebrafish

Zebrafish researchers routinely use morpholino-modified antisense oligonucleotides (‘morpholinos’) to block access to mRNAs of interest by transcription or splicing protein complexes [51]. This results in a block of translation or aberrant splicing, and can lead to reduced protein expression. However, the longevity of these morpholinos is variable, and many do not last beyond day 3. This has limited the applicability of morpholino knockdown for the study of neutrophil behaviour, since neutrophilic inflammation is not fully developed until day 3 of development.

Overexpression of proteins of interest, or their dominant-negative forms, overcomes many of the problems associated with morpholino injection. With the adoption of technologies increasing the efficiency of transgenesis (our method of choice is the Tol2 system [52]) and with the use of Gal4/UAS (upstream activating sequence) systems, this will probably become the method of choice for genetic manipulation of all but the youngest zebrafish. In addition, the zebrafish community eagerly awaits the broad applicability of strategies for the tissue-specific overexpression of siRNA (small interfering RNA) constructs.

Unbiased genetic screens

One of the main strengths of the zebrafish is the ability to perform forward genetic screens without the complexity and costs associated with murine screens. Novel genes involved in a variety of processes (including organogenesis, physiology and disease) have been uncovered by mutational analysis of mouse, zebrafish, fly, rat, worm and plant (Arabidopsis thaliana). In 1981, George Streisenger reported that the zebrafish would be an ideal candidate for mutational studies, random mutagenesis and mutant screens [53]. A year earlier, the Nobel Prize-winning achievements of Nüsslein-Volhard and Wieschaus were published showing the production and subsequent recovery of mutant fruit flies [54]. Streisinger's pioneering genetic work was continued at the University of Oregon, helping the zebrafish to become the vertebrate model system of choice for high-throughput, genome-wide, random mutagenesis screens for genes involved in development [55]. ENU was shown to be the most effective instigator of random point mutations within the zebrafish genome without lethality [5658]. Zebrafish are relatively resistant to ENU toxicity. Therefore, compared with other vertebrate models, ENU treatment of zebrafish generates higher levels of mutagenesis and high locus-specific hit rates [5961]. Experiences from mouse mutagenesis screens allowed for the development of protocols suitable for the zebrafish [61]. These screens have identified a range of mutants, which have defined roles for many proteins in all organ systems. We were fortunate to have participated in the Tübingen screen in 2005/6, and have identified a range of mutants with delayed inflammation resolution. Detailed characterization and mapping of these mutants are currently under way. An example of a mutant phenotype is shown in Figure 2.

Wild-type and mutant zebrafish (QE029-03) from the ENU mutagenesis screen for delayed inflammation resolution

Figure 2
Wild-type and mutant zebrafish (QE029-03) from the ENU mutagenesis screen for delayed inflammation resolution

Mutants were crossed with the mpx:gfp line, and bred to homozygosity. At 3 days post-fertilization, tail fin transection was performed under Tricaine anaesthesia, and the fish was allowed to recover. Images were taken at 5 days post-fertilization, and show persistent tissue neutrophilia in the mutant strain. Wild-type strain matched controls are shown for comparison.

Figure 2
Wild-type and mutant zebrafish (QE029-03) from the ENU mutagenesis screen for delayed inflammation resolution

Mutants were crossed with the mpx:gfp line, and bred to homozygosity. At 3 days post-fertilization, tail fin transection was performed under Tricaine anaesthesia, and the fish was allowed to recover. Images were taken at 5 days post-fertilization, and show persistent tissue neutrophilia in the mutant strain. Wild-type strain matched controls are shown for comparison.

Other groups have screened libraries of insertional mutants for neutrophil phenotypes, and the group of Anne Huttenlocher has been highly successful in identifying mutants with increased neutrophil numbers. To date, they have published two of these mutants. The first such mutant, in the Hai1 gene, results in damage to the epithelium and is associated with an increased retention of neutrophils at the site of epithelial injury [62]. The second, in Fad24, leads to muscle damage, and is again associated with increased tissue neutrophilia [63]. These unique models of spontaneous inflammation elegantly demonstrate the power of zebrafish forward genetics.

Future screens might capitalize on advances in transposon-mediated mutagenesis to mobilize reporters and transgenes around the genome [64]. Clearly the most important aspect of any screen is the quality of the assay. Assay designs must be robust, and should avoid selection of mutants with non-specific phenotypes.

‘Chemical genetic’ screens

While genetic screens might help elucidate the molecular events underpinning regulation of the inflammatory response, parallel compound screens can add additional dimensions, along with the potential to identify early lead compounds for drug discovery programmes. There are, to date, no published drug screens for compounds modifying neutrophil function in vivo. Our own screening programme is under way, and interesting leads are emerging. These will require prompt translation into human and murine models, and it is our hope that these techniques may one day lead to new therapies for inflammatory disease.

This ‘whole animal’-based compound discovery strategy has key differences when compared with traditional biochemical drug discovery programmes. Whereas traditional approaches start with a target, and look in vitro for inhibitors of that target, this approach begins with a physiological process (inflammation resolution) and looks for compounds accelerating that process. These compounds may act in a number of ways, and indeed may target several pathways at once. While this may have advantages in terms of therapies (many known anti-inflammatory therapies, including corticosteroids, work in this way), it can be problematic when optimizing lead compounds and getting regulatory approval for these compounds. Fortunately, these compound screens are not performed in isolation, and the parallel genetic screens and candidate gene approaches described above will define a range of phenotypes, which will greatly aid identification of the relevant pathways. Once the compounds have been ‘pinned’ to a particular pathway, their molecular targets can be identified in more traditional ways.

Routes of neutrophil disposal in the zebrafish model

Studies of inflammation resolution have identified three disposal routes for the removal of unwanted neutrophils from sites of inflammation: (i) neutrophils may be lost into exudates into the gut, onto the skin or into the alveolar lumen and expectorated; (ii) neutrophils may die by apoptosis and be taken up by macrophages and be thus removed (this is thought to be the predominant route for disposal of neutrophils in most inflammatory settings); and (iii) neutrophils may undergo migration away from the site of inflammation and may in certain circumstances re-enter the circulation. The latter pathway is still controversial, and direct evidence from mammalian systems is lacking. However, there is indirect evidence that neutrophils with a phenotype identical with reverse transmigrated cells are present in the blood of patients with rheumatoid arthritis [65]. In the zebrafish it is evident on simple inspection that neutrophils move in two directions between the site of injury and the rest of the fish, and there are reports of cells re-entering the circulation [50,66]. The ultimate fate of such cells is not yet known, and the potential function of such trafficking away from inflammatory sites remains a matter for conjecture.

Direct visualization of neutrophil apoptosis occurring in vivo during inflammation resolution has been more difficult to obtain. Apoptotic cells in general persist for a very brief period, and even during times of high cell loss, visible rates of apoptosis are low, often less than 5% [67]. Small changes in the observable rate of apoptosis can lead to profound changes in cell number. The ability to visualize apoptotic neutrophils in a model such as the zebrafish would require an order of magnitude higher than that seen in the type of experimental system where relatively few leucocytes are seen to be recruited to the site of injury [50,68]. The relative contribution of each process will differ in different models of inflammation, and there may be differences between the zebrafish model and mammalian systems. Our own work shows that apoptotic neutrophils are identifiable during inflammation resolution at levels comparable with other models of apoptotic cell loss (J.S. Martin and S.A. Renshaw, unpublished work) and that inhibitors of neutrophil apoptosis delay inflammation resolution [47]. This suggests that apoptosis contributes to some form of inflammation resolution in the zebrafish, although more sophisticated methods will be required to demonstrate the relative contribution of the different mechanisms available for the removal of neutrophils from inflammatory sites.

Conclusion

Zebrafish hold huge promise for adding to our knowledge of neutrophil biology and the regulation of inflammation. The model is now coming of age: hypothesis testing is under way for many of the candidate regulators of inflammation resolution, and in parallel a unique combination of unbiased screens is under way. These unbiased approaches combine genetic screens and compound screens and should identify new pathways involved in inflammation resolution, and new compounds to modify these pathways. This may in time lead to new treatments for inflammatory diseases.

Biochemical Basis of Respiratory Disease: Biochemical Society Focused Meeting held at AstraZeneca, Loughborough, U.K., 5–6 March 2009. Organized and Edited by Colin Bingle (Sheffield, U.K.) and Alan Wallace (AstraZeneca, U.K.).

Abbreviations

     
  • COPD

    chronic obstructive pulmonary disease

  •  
  • DIC

    differential interference contrast

  •  
  • DISC

    death-inducing signalling complex

  •  
  • GFP

    green fluorescent protein

  •  
  • GM-CSF

    granulocyte/macrophage colony-stimulating factor

  •  
  • ENU

    N-ethyl-N-nitrosourea

  •  
  • IL

    interleukin

  •  
  • Mcl-1

    myeloid cell leukaemia sequence 1

We thank Catherine Loynes for her expert technical assistance and Professor Moira Whyte, Professor Philip Ingham and Professor Paul Hellewell for their expert advice on establishing the models discussed.

Funding

The work performed in our laboratory was funded by Medical Research Council Clinician Scientist and Senior Clinical Fellowships [grant numbers G108/595 and G0701932] to S.A.R. and by Medical Research Council Centre Grants [grant numbers G0400100 and G0700091]. J.S.M. was supported by an Medical Research Council studentship. The genetic screen was funded by the Zebrafish Models for Human Development and Disease, Framework 6 Grant.

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Supplementary data