Date Published: July 12, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Taylor W. Starnes, Anna Huttenlocher.
The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo.
“Certain of the lower animals, transparent enough to be observed alive, clearly show in their midst a host of small cells with moving extensions. In these animals the smallest lesion brings an accumulation of these elements at the point of damage. In small transparent larvae, it can easily be shown that the moving cells, reunited at the damage point do often close over foreign bodies .” Ilya Mechnikov, one of the fathers of immunology, spoke these words at his Nobel Prize lecture in 1908. More than one hundred years after his seminal studies using transparent starfish larvae to illuminate a role for phagocytosis in immunity, we are again exploiting the power of transparent larvae for research on the immune system. Studies of neutrophils in both humans and mammalian model systems have brought great advances in our knowledge of their functions; however, zebrafish, a small tropical fish with transparent larvae, have demonstrated that direct observation of neutrophils in live animals can provide important insights that would have otherwise faced significant technical challenges using mice.
An important feature of any model organism is the ability to infer similarity of function with the species of interest, typically humans. The high conservation of immune cell lineages and effector functions indicates the suitability of zebrafish as a model through which we can better understand the human immune system. Zebrafish have many immune cell lineages in common with humans: neutrophils [8, 11, 12], macrophages [11, 13–15], T cells , B cells , mast cells , eosinophils [11, 19], and basophils . However, the 2–4 days-post-fertilization larvae used for most zebrafish neutrophil research do not have T or B cells . Particularly important for the study of neutrophil reverse migration is the conservation of function within the innate immune system. Like human neutrophils, zebrafish neutrophils are the first responders to inflammatory stimuli, where they are able to phagocytose bacteria and degranulate [15, 20, 21]. Further support for the conservation of neutrophil functions is in the recapitulation of neutrophil phenotypes in zebrafish models of Wiskott-Aldrich syndrome (WAS), warts-hypogammaglobulinemia-infections-myelokathexis (WHIM) syndrome, and leukocyte adhesion deficiency-(LAD-) like syndrome [22–24]. Many other immune effector functions are present in both fish and mammals, and these have been expertly reviewed elsewhere [25–28].
The use of tissue-specific expression with powerful imaging tools has facilitated the application of a cell biology toolkit to zebrafish inflammation research and increased our fundamental knowledge of neutrophil motility and wound recruitment. The first studies utilizing fluorescent neutrophils in zebrafish larvae demonstrated that neutrophils rapidly respond to mechanical wound-induced stimuli, which raised two fundamental questions: (1) What are the intracellular signals that promote directional migration and (2) What are the signals recruiting neutrophils to wounds? Advances in zebrafish imaging strategies have helped to answer both of these questions.
Prior to the observation of neutrophil reverse migration, the previous paradigm of neutrophil responses, based on mammalian studies, was that neutrophils underwent apoptosis after responding to an inflammatory stimulus [5, 6]. The process of macrophage clearance of apoptotic neutrophils in tissues has been well established [4, 7]. However, a previous study using an experimental rat model of nephritis showed that intravascular neutrophils do not necessarily undergo apoptosis but can leave a site of inflammation through glomerular capillaries, suggesting that alternative mechanisms may mediate resolution of neutrophil-mediated inflammation . In support of this idea, in vivo imaging of zebrafish neutrophils was the first direct demonstration that reverse migration was responsible for clearance of neutrophils from the interstitium of wounded tissues . Indeed, studies of neutrophil reverse migration in zebrafish larvae have found that neutrophil apoptosis at a wound site is a rare event, occurring in less than 3% of the responding neutrophils . Mathias et al. were able to demonstrate this reverse migratory behavior by tracking wound-responsive neutrophils in the transgenic (Tg) zebrafish line, Tg(mpx : GFP), in which GFP is expressed specifically in neutrophils. This study further demonstrated that neutrophils undergoing both forward and reverse migration to a wound had nearly equivalent velocity and directionality, implying that each was a robust, active process . Using zebrafish, other groups have also observed neutrophil reverse migration under similar experimental conditions [38, 48, 49].
Recent work in zebrafish has implicated hypoxia-inducible factor-1α (Hif-1α) in neutrophil reverse migration. Pharmacologic stabilization of Hif-1α or the expression of a dominant active Hif-1α impaired resolution of inflammation by neutrophil reverse migration . While this is a promising first step, it does not appear that Hif-1α is the dominant factor regulating reverse migration.
While current studies have not demonstrated a definite role for reverse migrated neutrophils, the recent findings of several groups have challenged the idea that neutrophils are short-lived cells with narrowly defined functions. Because reported neutrophil half-lives were less than 12 hours and there was no knowledge of neutrophil reverse migration, neutrophils were not thought to have immunomodulatory roles other than through the cytokines and effectors that they produced at sites of inflammation. However, recent reports have challenged the short lifespan of neutrophils. Although controversial, a recent study used 2H2O labeling to determine that the in vivo half-life of human neutrophils was 3.8 days (total lifespan: approximately 5.4 days) [68–71]. Others have also reported neutrophil lifetimes that were longer than 24 hours. Neutrophils that underwent reverse transendothelial migration, trafficked to lymph nodes, or were cocultured with TNF-α/IL-17 stimulated synovial fibroblasts had their expected lifetimes extended [51, 52, 72]. Zebrafish neutrophils that underwent reverse migration could also be found for at least 2 days after they had left a wound . While evidence supports the existence of reverse migration and prolonged neutrophil life in vivo, data supporting either a proinflammatory or anti-inflammatory role for reverse migrated neutrophils remain plausible.
The last five years have yielded exciting developments in the study of neutrophil biology, including the process of reverse migration, which are rapidly changing the view that neutrophils are short-lived cells with narrowly defined effector functions. It seems that in at least some circumstances neutrophils can regulate T cell activity and present antigen in the context of MHC II, functions which were previously ascribed to macrophages and dendritic cells, the professional antigen presenting cells [52–55]. While evidence in support of the existence of reverse migration in mammals continues to grow, the mechanisms driving reverse migration and the functions of reverse migrated neutrophils remain to be further defined.