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Keywords:

  • Chemokines;
  • Endothelium;
  • Intravital microscopy;
  • Neutrophil

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Chemokines are proinflammatory mediators that regulate leukocyte trafficking at different steps of the leukocyte recruitment cascade. Studies using new imaging approaches and new mouse models are giving new insights into the role of chemokines in neutrophil migration at sites of inflammation. Conventional rolling and adhesion paradigms as well as previously unappreciated functions of signaling pathways triggering leukocyte adhesion, intralumenal crawling and transendothelial migration are compiled and described here. In this review we will summarize recent work in this field, highlighting in vivo imaging studies that examine the behavior of neutrophils in response to chemokines.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Chemokines are proinflammatory mediators that, among other functions, regulate leukocyte trafficking. They are produced in response to infectious and other inflammatory stimuli by a number of different cell types 1. Chemokines comprise a large family of low-molecular-weight cytokines with, usually, four conserved cysteine residues linked by disulfide bonds 1 and are classified based on the arrangement of their amino terminal cysteine residues into four subfamilies (CXC, CC, C, and CX3C) 1. The CXC subfamily is further subdivided into those that have a glutamate-leucine-arginine (ELR) motif before the CXC motif (ELR-CXC chemokines) and those that do not. Chemokines transmit their signals through interaction with heptahelical G-protein-coupled receptors (GPCRs) 2. With respect to neutrophil recruitment, ELR-CXC chemokines are selective for promoting neutrophil migration 1. The best described ELR-CXCL chemokines in mice include CXCL1 (keratinocyte-derived chemokine or KC), CXCL2 (macrophage inflammation protein (MIP)-2), CXCL5 (LPS-inducible CXC chemokine or LIX), and CXCL7 (neutrophil-activating peptide-2 or NAP-2) 3, 4. In the mouse, the ELR-CXCL chemokines bind to the chemokine receptor CXCR2 1. However, some CC chemokines, mainly CCL3 or MIP-1α and CCL4 or MIP-1β, can also direct neutrophil recruitment in mice through interaction with the CCR1 receptor 5. Neutrophils also express functional CCR2 at times of chronical inflammation; CCR2 mainly interacts with CCL2 also called monocyte chemoattractant protein-1 or MCP-1 6.

Neutrophils are thought to be the first cells that are rapidly recruited to inflamed sites during an innate immune response to tissue damage and/or infection. The use of in vivo imaging of inflamed microvasculatures by intravital microscopy has revealed a multi-step paradigm of leukocyte recruitment 7–9. Neutrophils first tether to and then roll along the endothelium before firmly adhering 7–9. Once arrested, the cells leave the vasculature and migrate to distinct sites of inflammation along gradients of chemoattractants. We will restrict this review to studies that have focused on in vivo aspects of chemokine-induced neutrophil recruitment with specific emphasis on imaging techniques such as intravital microscopy.

Leukocyte tethering and rolling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Endothelial cell activation results in the expression of selectins (E- and P-selectins) which facilitate the initial tethering and rolling of neutrophils in the postcapillary venules of the peripheral vasculature. Interaction with counter receptors on neutrophils such as the P-selectin glycoprotein ligand 1 (PSGL-1) is well characterized 10.

Rolling for the most part occurs independently of chemokines and is instead due to histamine, oxidants, thrombin, and cytokines like TNF-α and IL-1β. Two imaging studies, one in the cremaster and the other in the colonic microcirculation have suggested that CXCL1, CXCL2, CCL3, and CCL2 can induce increases in rolling through mast cell activation 11, 12. Indeed, activated mast cells can release histamine and within minutes endothelial Weibel-Palade bodies (storage granules of endothelial cells) fuse with the endothelial plasma membrane, increasing surface expression of P-selectin which is pre-formed and stored in these granules. Pre-formed IL-8 can simultaneously also be released from Weibel-Palade bodies 13. However, other groups have claimed that chemokines do not induce mast cell degranulation 14 regardless of the expression of different chemokine receptors such as CXCR2 or CCR1 on mast cell surface. Therefore, the potential role of chemokines on neutrophil rolling remains controversial.

Leukocyte activation and adhesion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Rolling neutrophils can detect chemokines immobilized by glycosaminoglycans (GAGs) or heparan sulfate (HS), in particular on the lumenal membrane of endothelial cells (Fig. 1) 15–17. The involvement of chemokines on neutrophil arrest was widely established by the use of neutralizing antibodies against the chemokine or the chemokine receptor being investigated. Either functional blockade of CXCL1 and/or CXCL2, CXCL5 or CXCR2, effectively decreased neutrophil adhesion in the synovial, mesenteric or colonic microvasculature 18–22. In addition to GAGs, the emergence of alternative, “atypical” chemokine receptors (ACRs) increased even further the interactive complexity within the chemokine system. ACRs are heptaspanning membrane receptors homologous to chemokine GPCRs but unable to couple to G-proteins 23. Among them, the Duffy antigen receptor for chemokines (DARC) as well as HS participate in transcytosing chemokines from the tissue to the lumenal surfaces of endothelial cells 9, 15, 17, 24. Therefore, GAG and ACR activity may establish a gradient that is responsible in several in vivo settings for the directionality of cell responses to chemokines. Accordingly, DARC expression supports optimal chemokine-induced leukocyte migration in vitro and in vivo 24. Furthermore, endothelial cells themselves can also produce chemokines and present them to leukocytes on the luminal cell surface 13.

thumbnail image

Figure 1. Role of neutrophil-active chemokines in the different steps of the neutrophil recruitment cascade. Neutrophil mobilization from the bone marrow during inflammation requires the interaction between chemokines and their G-protein-coupled receptors (GPCRs). In the microvasculature, endothelial cell activation results in the expression of selectins which facilitate the initial tethering and rolling of neutrophils; however, the role of chemokines on neutrophil rolling remains controversial. Rolling neutrophils can detect chemokines immobilized by glycosaminoglycans (GAGs) or by heptaspanning membrane receptors, such as the DARC, that are homologous to chemokine GPCRs but unable to couple to G-proteins. Activation of neutrophils through chemokine-receptor engagement on rolling neutrophils generates inside-out signals that activate integrins, leading to firm adhesion. Then, neutrophils crawl in search of optimal sites for transendothelial migration probably guided by intralumenal chemokine gradients, an event supported by the neutrophil integrin Mac-1. Neutrophils leave the microvasculature by transendothelial migration between endothelial cells (paracellular route) or directly through endothelial cells (transcellular route). Once inside tissues, neutrophils migrate to the site of inflammation along gradients of chemokines. The boxed region in the blood vessel (microvasculature) is shown in greater (cellular and molecular) detail underneath the description of the stages involved in neutrophil trafficking.

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Through their engagement to cognate GPCRs “inside-out” signals are triggered which lead to the activation of leukocyte integrins (β2 and/or α4 integrins) 25, 26. Integrin activation results in clustering and a conformational change from a bent and inactive conformation to a fully extended conformation with high binding affinity 25, 26. Activated integrins bind with relatively high affinity to their counter endothelial ligands including intercellular adhesion molecule-1 (ICAM-1) for β2-integrins, and vascular cell adhesion molecule- (VCAM-1) for α4-integrins, resulting in firm adhesion and arrest (Fig. 1) 25, 26. However, within the vasculature mature neutrophils do not express α4 integrins and therefore do not interact with VCAM-1 27. Integrins are not only responsible for the attachment of neutrophils to the endothelium, they are also able to transfer signals from the extracellular domain into the cell (outside-in signaling) 28. These signals strengthen adhesion and induce crawling after leukocytes flatten and extend pseudopods across the endothelial surface 9, 28, 29.

Leukocyte crawling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Once neutrophils are adhered, they crawl intralumenally before emigrating (Fig. 1). Using confocal fluorescence microscopy in vivo, Phillipson et al. 30 reported that adherent leukocytes actively crawl along the lumenal surface of inflamed vessels in search of optimal sites for transendothelial migration. While initial adhesion was dependent on the αLβ2 integrin (LFA-1), subsequent intraluminal crawling was mediated by the αMβ2 integrin (Mac-1). Inhibition of crawling delayed but did not eliminate emigration in vivo. Subsequent studies have attempted to determine whether neutrophils crawl along the endothelial surface till they get a specific location for transmigration. Most studies agree that leukocyte transmigration primarily occurs via endothelial junctions (intersection of the borders of adjacent endothelial cells). However, some evidence suggests little association between junctions and location of crawling neutrophils 31. In an attempt to mimic pathophysiological conditions, a further study employed an extravascular CXCL2-containing gel to generate a proper chemokine gradient 32. The localized extravascular chemokine release induced directed neutrophil crawling and accelerated their recruitment into the area where the CXCL2-containing gel was placed. In other words, neutrophil emigration followed a chemotactic gradient (they moved towards increased chemokine concentrations). In fact, when the chemokine-containing gel was placed on the right side of a vessel, neutrophils adhered and transmigrate through the right side. Inasmuch, immunofluorescence studies revealed that intralumenal chemokine immobilization occurred exclusively in venules and heparan sulfate was required for its presentation to neutrophils. In the absence of this GAG, random neutrophil crawling was detected with no selective directionality which was accompanied by a decreased number of emigrated cells.

Furthermore, a recent study has shown chemokine expression in the liver sinusoids poststerile thermal injury 33. In this case neutrophils crawled along a gradient of chemokines (CXCL2 and CXCL1) in a CXCR2-dependent manner towards the injury site. This was the first study that observed and quantified a chemokine gradient in vivo. Interestingly, this gradient ended abruptly ∼150 μm from the injury; perhaps due to the loss of GAG expression on the vessels in close proximity to the thermal injury. However, the neutrophils continued to migrate into the focus of necrosis independently of the CXCR2 signals. Further investigation revealed that this final stretch of the neutrophils' journey was guided by FPR1-dependent danger signals emanating from the necrotic cells (likely mitochondrial N-formyl-peptides) 33, highlighting that multiple gradients can form in blood vessels to help direct neutrophils to afflicted sites. Nevertheless, a chemokine presence on the endothelium is not enough to initiate leukocyte diapedesis and a chemotactic gradient over the vessel wall with a higher extravascular concentration is probably necessary. Whether intralumenal chemokine receptor desensitization is required prior to transmigration or whether a yet undescribed hierarchy in the chemokine family regulates neutrophil extravasation remain unknown.

Leukocyte emigration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

The molecular mechanism of emigration is the least well understood and probably the most complex aspect of neutrophil trafficking, and involves platelet/endothelial cell adhesion molecule-1 (PECAM-1 or CD31), junctional adhesion molecule (JAM), CD99, endothelial cell-selective adhesion molecule (ESAM) and intercellular adhesion molecules (ICAMs) and integrins 9. Neutrophil emigration through the endothelial monolayer may occur at either intercellular junctions (paracellular), or directly through the body of intact endothelial cells (transcellular route) (Fig. 1) 9. Mac-1-dependent intralumenal crawling guides cells to preferentially transmigrate at junctional sites, with transcellular migration accounting for at most ∼20% of emigrated cells 30. In contrast, when Mac-1 is inhibited, a dramatic increase in neutrophil emigration through the endothelial cells (transcellular migration) is observed 30. More recently, it has been suggested that neutrophils preferentially use tricellular junctional regions (enriched in ICAM-1) as “portals” for their transmigration 31.

Next, neutrophils have to transmigrate through the vascular basement membrane which may involve proteolytic cleavage of extracellular matrix (ECM) components. The role of proteases in cell migration through basement membranes has been the subject of intense interest for many years and remains a contentious issue 34. The potential role of enzymatically active neutrophil elastase (NE), which can be expressed on the cell surface of stimulated neutrophils, was found to be involved in the in vivo neutrophil transmigration through the venular basement membrane in the cremasteric microvasculature 35. More recently, it was demonstrated that the existence of regions of low matrix protein deposition in the venular basement membrane of multiple organs acted as “gates” for transmigrating neutrophils in response to different inflammatory stimuli including chemokines 36.

Outside the vasculature, leukocytes move along a chemical gradient (haptotaxis) in the extracellular matrix toward the chemokine source 37. Indeed, the use of perivenular microinjection of CCL3 has provided new insights into the study of the interstitial migration of neutrophils 38. In this experimental setting, Rho kinase inhibition not only attenuated the directional movement and polarization of emigrated leukocytes but also blocked their motility.

Chemokine-mediated signaling in the neutrophil recruitment cascade

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Binding of selectins to their ligands allows activating signals to be transmitted through adjacent GPCRs and directly through selectin ligation 9. Activation of the GPCR in neutrophils leads to dissociation of the Gα-subunit from the Gβγ-complex. The elimination of the Gαi2-subunit leads to almost complete loss of chemokine-induced arrest in the cremasteric postcapillary venules and in vitro 39.

Downstream signalling pathways have been proposed to be involved in the chemokine-induced neutrophil arrest and probably the best studied is the phosphatidylinositol 3-kinase (PI3K) pathway 4. Chemotaxing cells polarize, a leading edge (pseudopod) facing the source of a chemoattractant, and a trailing tail (uropod) are formed. It has been proposed that upon detecting a chemotactic stimulus, cells will activate PI3K in such a way that PI3K is active along the region of the cell facing the chemoattractant, resulting in the production and accumulation of phosphatidylinositol triphosphate (PIP3). This is followed by the accumulation of proteins containing PIP3-binding domains, thus recruiting the proteins required to form the pseudopod of the migrating cell. At the same time, enzymes which mediate the breakdown of PIP3 are active on the sides and the uropod, thus limiting PI3K activity to the front of the cell 4. In regard to this, whereas Smith et al. 40 showed a crucial role for PI3Kγ in mediating CXCL1-induced neutrophil adhesion which was triggered by CXCL1-CXCR2 engagement, Liu et al found a minor effect of PI3Kγ on adhesion in response to either CXCL1 or CXCL2 41. In the latter study, however, leukocyte emigration was severely impaired in PI3Kγ-KO mice in the early response (first 90 min) to chemokines. In more prolonged responses (for 4−5 h) chemokine-induced neutrophil migration was almost entirely PI3Kγ independent but largely dependent on PI3Kδ, suggesting that each isoform mediates non-overlapping and temporally distinct events 41.

More recently, the use of mice with a myeloid-specific deletion of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) revealed enhanced neutrophil “invasivity” or emigration capability in response to CXCL2 indicating that PTEN acts as a negative regulator in neutrophil transendothelial migration. These effects were due to increased PIP3 production since inhibition of PI3K or Src kinase abolished these responses 42. A different study reported PTEN to be important in recruitment of neutrophils in response to chemokines like CXCL2 (MIP-2) that used PI3K, but not in response to FMLP that activates p38 MAPK 43. Furthermore, neutrophils are more responsive to FMLP than to MIP2, a hierarchy not evident in PTEN-deficient neutrophils 43.

Spleen tyrosine kinase (Syk) is another key mediator of immunoreceptor signaling in immune cells. Syk activation further leads to PI3K signaling. However, the role of Syk in the in vivo migration of neutrophils have led to conflicting results. While a recent study have shown that Syk deletion in adult mice has no effect on the migration of neutrophils in mast cell-driven animal models of allergy and asthma 44, earlier reports have suggested a role of Syk in the migration of neutrophils 45.

In addition to PI3K, the Gβγ-subunit activates phospholipase-C (PLC β2 and β3). PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce inositol-triphosphate (IP3) and diacylglycerol (DAG). IP3 mobilizes Ca2+ from nonmitochondrial stores. Ca2+ and DAG bind to and activate CalDAG-GEFI, which subsequently activates Rap1 and β2-integrins 46, 47. The CXCR2-induced neutrophil arrest was found to be totally CalDAG-GEFI dependent 46, whereas LFA-1 activation following E-selectin engagement was only partially CalDAG-GEFI-dependent. Therefore, these findings suggest that the GPCR signaling pathway merges with the E-selectin-mediated pathway at the stage of CalDAG-GEFI.

Moreover, inhibition of p38 MAPK activity was also found to limit neutrophil transmigration across the endothelium and subsequent neutrophil chemotaxis through the interstitium induced by CXCL1; however, this was likely related to inhibition of p38 MAPK in the endothelium and not the leukocytes 48.

Chemokine-induced neutrophil mobilization

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

Another key event of early cellular immunity is the mobilization of neutrophils from the BM. A recent and elegant study used intravital 2-photon microscopy to visualize the mobilization of neutrophils from the tibial BM to the circulation 49. The systemic administration of a single dose of granulocyte-colony stimulating factor (G-CSF) rapidly induced the motility and entry of neutrophils into blood vessels within the tibial BM. This effect was CXCR2-dependent and both CXCL1 and CXCL2 were expressed by megakaryocytes and endothelial cells. In situ production of CXCL1 was strongly enhanced by thrombopoietin (TPO). Thus, neutrophil release from the BM, and their mobilization seem to be in part a CXCR2-binding chemokine-mediated event (Fig. 1). Another study has also proposed that CXCR4 and CXCR2 signaling antagonistically regulate neutrophil release from the BM 50. In this context, CXCR4 is constitutively present in BM and both attenuation of CXCR4 signaling and induction of CXCR2 signaling leads to entry of neutrophils into the circulation. Thus, under basal conditions, the balance of chemokine production favors neutrophil retention in the BM whereas stress conditions, cause the expression and release of inflammatory cytokines, promoting neutrophil release from the BM.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

In vivo imaging has become an increasingly important and powerful tool in the analysis of spatio-temporal dynamics of directed neutrophil migration. Moreover, different aspects of such migration that were unable to be predicted by in vitro systems could be identified thanks to the advances in this technology, notably the chemokine-directed intravascular neutrophil crawling and the directional movement and polarization of emigrated leukocytes towards an extravascular chemokine gradient. Nevertheless, how diapedesis is initialized, the passage of neutrophils through the basement membrane and surrounding pericytes before entering into the tissues or how neutrophil chemotax in the extracellular matrix towards the chemokine source are all avenues for future imaging research.

Furthermore, previously established in vitro signaling pathways require further confirmation in vivo using imaging. Moreover, little is known regarding the role of chemokine receptor heterodimerization in vivo. Finally, the co-evolvement of atypical chemokine receptors simultaneously with chemokine GPCRs may indicate an important non-redundant nature and the requirement of both of these receptor types for optimal chemokine function in vivo, but this possibility has still to be evaluated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

The work in the authors' laboratory is supported by Canadian Institutes for Health Research operating grants and group grant, as well as the Canadian Foundation for Innovation. P. K. is an Alberta Heritage Foundation for Medical Research (AIHS) Scientist and the Snyder Chair in Critical Care Medicine. M.-J. S. is supported by a grant from the Spanish Ministry of Education (PR2010-0477).

Conflict of interest: The authors declare no financial or commercial of interest.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Leukocyte tethering and rolling
  5. Leukocyte activation and adhesion
  6. Leukocyte crawling
  7. Leukocyte emigration
  8. Chemokine-mediated signaling in the neutrophil recruitment cascade
  9. Chemokine-induced neutrophil mobilization
  10. Concluding remarks
  11. Acknowledgements
  12. References
  13. Supporting Information

See related review on leukocyte traffic: http://dx.doi.org/10.1002/eji.201142223

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