Abstract
Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.
Highlights
Leukocyte migration is of paramount importance for immune responses
Ventral fin wounding is followed by rapid neutrophil mobilization from the caudal hematopoietic tissue (CHT) into the ventral fin and clustering at the wound margin, within 30-60 min (Figure 1)
We visualized the distribution of two chemokine receptors, Cxcr[1] and Cxcr[2], which are expressed by zebrafish neutrophils[24] and recognize Cxcl8a and Cxcl8b14, using spinning-disk confocal microscopy
Summary
Leukocyte migration is of paramount importance for immune responses. Immune cells are prototypical migratory cells, which are remarkably capable of traversing tissues and blood vessels and sensing a range of chemical guidance cues to migrate directionally towards microbes or other host cells of importance. Generation of transgenic mouse models, in which leukocytes express a fluorescent receptor or mutant receptors with informative trafficking defects5 , 6 , entails considerable investment of time and resources Even in these instances, the imaging resolution and contrast for imaging receptor dynamics in the live animal can be limited and studies have used immunohistochemistry on fixed tissue sections[5 ]. We provide a side-protocol to test chemokine receptor responses to candidate ligands, which is useful when trying to establish ligand recognition patterns in uncharacterized receptors These techniques can be used in combination with further genetic manipulations, such as inhibition of endogenous chemokine expression or generation of mutant receptors with altered ligand-induced trafficking, to interrogate how specific signaling dynamics affect leukocyte behavior in vivo. Allow larvae to hatch naturally and use at 3 dpf when neutrophils are abundant[21 ]
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