Neutrophil microdomains: linkingheterocellular interactions with vascular injury

Abstract

Purpose of review

Neutrophil infiltration is an important feature in inflammatory scenarios. Before these cells infiltrate tissues, however, they contribute to crucial intravascular events in which neutrophil microdomains mediate heterotypic interactions with endothelial cells, red blood cells and/or platelets. In vascular diseases, this can result in exacerbated neutrophil activation, subsequent vascular injury and obstruction of microcirculatory blood flow. This review discusses recent advances in elucidating these neutrophil domains and their associated functions in cell adhesion.

Recent findings

Neutrophil recruitment is mediated by sequential interactions with the endothelium, termed rolling, adhesion and extravasation. Evidence points to novel signaling pathways induced during the rolling phase resulting in the transition to leukocyte adhesion, which appear to contribute to chemokine mediated activation. In addition, specific neutrophil microdomains are important for interactions with other hematopoietic cells inducing reductions in microvascular flow and injury.

Summary

Neutrophils integrate signals received from the endothelium to act as linkers between the vessel wall and a variety of vascular components (i.e. endothelial cells, platelets, RBCs) in acute and chronic inflammatory conditions to mediate interactions that can result in vascular injury and vasoocclusion.

Introduction

Neutrophils are key players of the innate immune response as they are the initial cells that fight invading pathogen and keep the infection from spreading while an adaptive immune response is mounted. To achieve this, these cells need to get recruited very rapidly to the specific site of inflammation to destroy pathogens while keeping collateral tissue damage to a minimum as activated neutrophils are equipped with powerful proteolytic enzymes and damaging reactive oxygen species (ROS). When neutrophil activation is elevated and remains unchecked (e.g. in cases of chronic or acute inflammatory conditions), these ROS can lead to detrimental effects due to the destruction of the microenvironment, providing acute insult and exacerbating chronic infection. To reach sites of inflammation, neutrophils use cell adhesion receptors that are often clustered in microdomains on the plasma membrane. These domains can restructure to serve different functions during the adhesion process. The initial concept of the leukocyte adhesion cascade (leukocyte rolling, adhesion and extravasation) is constantly getting more refined by new evidence that sheds light on events that mediate the transitions between the individual steps [1–3]. Recent evidence suggests that during their initial rolling step along the endothelium-lined vessel wall neutrophils already integrate important signals for downstream events, such as adhesion to endothelial cells and activation. In addition, these domains can interact with other blood components to exert (patho-) physiological events while still inside the vascular lumen, which can dramatically alter blood flow.

Microdomains in neutrophil rolling, polarization and adhesion

Circulating neutrophils undergo the canonical events of the leukocyte adhesion cascade that lead to their tissue recruitment. At sites of inflammation (or under homeostatic conditions in the skin, for example), neutrophils tether and roll along activated endothelium, slow down, and finally adhere to endothelial cells.

Rolling

The initial capture and tethering of neutrophils to the vessel wall is mediated by P-selectin glycoprotein ligand (PSGL)-1, E-selectin ligand (ESL)-1 and L-selectin [4,5], molecules that are located on the tips (PSGL-1, L-selectin) and sides (ESL-1), respectively, of neutrophil microvilli [6–8]. These membrane protrusions increase the overall neutrophil surface thus facilitating interactions with the vessel wall. In addition, the different topographical localizations ensure a sequential coordinated segregation of initial contacts with microvilli followed by contacts with molecules located on the cell body. Neutrophil PSGL-1 interacts with both endothelial E- and P-selectin, while ESL-1 binds to E-selectin. L-selectin can bind to a variety of endothelial molecules, the most important of which are GlyCAM-1 and PSGL-1 [4,5]. Leukocyte rolling allows the cells to get in closer and more prolonged contact with the endothelium where chemokines presented within the glycocalyx have long been described to be the single most important mediator of leukocyte activation and adhesion via G-protein-coupled receptors (GPCRs) [9,10]. Evidence, however, suggests that signaling events in the leukocyte can already occur during the rolling phase to induce activation of leukocyte integrins, the crucial step for adhesion, therefore mediating the transition between steps [11] (Figure 1). Specifically, E-selectin interactions with PSGL-1 induce sequential intracellular signaling events in neutrophils via the Src kinase Fgr and the spleen tyrosine kinase Syk resulting in partial activation of the integrin LFA-1 with reduction in leukocyte rolling velocities [12,13]. In addition, ESL-1 is emerging as an important mediator in promoting the transition from neutrophil tethering to slow rolling and flattening, by cooperating with both microvillar PSGL-1 in mediating tethering and CD44, located on the cell body, to regulate rolling velocity [14]. The presence of ESL-1 on the lateral sides of microvilli between the tips and the cell body appears to facilitate this transition. CD44 engagement, together with PSGL-1 and ESL-1 another physiologically relevant ligand for E-selectin [15], induces p38 MAPK signaling, which leads to the redistribution of PSGL-1 and L-selectin to the major pole on slow rolling leukocytes [14]. In addition to PSGL-1-induced E-selectin signaling, interactions of PSGL-1 with P-selectin have recently also been shown to activate Src kinases via the adaptor protein Nef-associated factor 1 (Naf-1). This in turn induces phosphatidylinositol 3-kinase (PI3K) activity/and or recruitment and activation of the integrins αLβ2 (also known as leukocyte function antigen, LFA-1) andαMβ2 (also known as Mac-1) [16]. It has become apparent that the redistribution of microdomains on the cell surface is closely associated with intracellular signaling components that must also cluster in a sequential fashion [17,18]. In addition, these signaling components are closely linked to the cytoskeleton to induce subsequent neutrophil polarization for cellular migration [19].

Figure 1.

Neutrophil microdomains mediating intravascular heterotypic interactions.

Interactions of PSGL-1 and ESL-1 on neutrophil microvilli interact with endothelial cell P-and E-selectin to induce activity of members of the Src kinasefamilyand partial LFA-1 and Mac-1 integrin activation. Partly activated integrins in conjunction with CD44 and ESL-1 mediate slow rolling on ICAM-1 and E-selectin, resulting in p38 kinase activity and leukocyte polarization of molecules to the major pole in rolling cells. Levels of intracellular calcium riseand proximity of chemokinereceptors tochemokines presented on the endothelial cell surface leads to full integrin activation and LFA-1-mediated firm arrest. This induces cytoskeletal rearrangement and leukocyte polarization to form a leading and trailing edge. Calcium microdomains appear in the trailing edge, a domain to which L-selectin and PSGL-1 cluster, in a process partly achieved by flotilins located in lipid rafts. PSGL-1 in the uropod can induce interactions with free flowing leukocytes. LFA-1 outside-in signaling leads toMac-1 activation mediated via Vav-1.Additionally, Mac-1 can be activated via E-selectin-mediated binding to ESL-1. Mac-1 activity at the leading edge in turn allows the neutrophil to crawl within the vascular lumen and mediate interactions with RBCs and platelets through yet unidentified receptors.

Leukocyte polarization

During the transition from rolling to adhesion the neutrophil polarizes to form a leading and trailing edge, termed pseudopod and uropod, respectively. Polarization is intrinsic for cell movement and several molecules play key roles in this process. Cell polarity in general is induced by the small GTPase Cdc42 in conjunction with Par proteins and the atypical protein kinase C (aPKC) [20]. The front of the cell is determined by PI3K activity and its generated second messenger phosphatidolinositol trisphosphate (PIP₃) [21,22], as well as the small GTPases Rac and Cdc42 [23,24], which together initiate F-actin remodeling to form the pseudopod. In contrast, the PI3K antagonist PTEN localizes to sides of the cell and the uropod [25] while Rho/ROCK GTPases regulate actomyosin contraction and uropod detachment [26,27]. Another important second messenger for inducing cell polarity is calcium (Ca²⁺) [28]. Ca²⁺ fluxing after E-selectin engagement has been recently observed to occur just before firm arrest and pseudopod formation [29] and Ca²⁺ microdomains have been reported in the uropod of polarized neutrophils [30]. However, the functional relevance of these events remains currently unknown. During neutrophil polarization the spatial distribution of PSGL-1 changes and the molecule distributes to the uropod of the leukocyte [14,31]. The relevance for PSGL-1/L-selectin concentration at the uropod may be to mediate secondary tethers with leukocytes [32,33]. Cytoskeletal mechanisms are responsible for the redistribution of neutrophil microdomains via actin polymerization and microtubli reorganization [19]. Transmembrane adhesion molecules can be anchored to the cytoskeleton either directly or indirectly via adaptor proteins such as ezrin/radixin/moesin (ERM) [34]. In addition, recent evidence has shown that flotilins – present in lipid rafts – can organize microdomains in the uropod [35]. These molecules associate with PSGL-1 and subsequently enforce its actin-mediated localization to the trailing edge in a manner that does not rely on its cytoplasmic domain [36]. Under static in vitro conditions the distribution of CD44 also localizes to the trailing edge [37,38] whereas under flow conditions in vivo the molecule exhibits a uniform distribution with higher concentrations in proximity to the endothelium [14], suggesting shear-stress dependent differences in localization.

Adhesion and intraluminal crawling

Activating signals coming from molecules involved in neutrophil rolling in conjunction with signals delivered through chemokines/chemoattractants and their receptors lead to conformational changes in the structure of the integrins crucial for the adhesion phase of neutrophil recruitment. The latter is known as inside-out signaling, and on the neutrophils, involves mainly LFA-1 and Mac-1. When partly-activated, these integrins can support slow rolling [13,39], while full activation results in higher affinity to endothelial cell receptors and firm arrest. Inside-out signaling not only renders the integrin in a more adhesive state, this conformational transformation also allows integrins to receive signals, a process termed outside-in signaling. In addition to conformational changes, which increase integrin affinity, integrins can associate laterally to form nano- and microclusters, which increase the overall number of ligand interactions and avidity [40,41]. Both LFA-1 and Mac-1 are homogenously distributed over the cell body but Mac-1 activity is mainly localized to the pseudopod of the leukocyte [42]. LFA-1 affinity has been reported to be particularly high at the lamellipodium and the cell center [43,44]; it is, however, also important for uropod attachment/dissociation suggesting the possibility of sequential transmission of activity from the front to the back thus priming for the transendothelial migration step [45]. LFA-1 interactions with intercellular adhesion molecule (ICAM)-1 are crucial for the initial adhesive phase and adhesion strengthening, which is immediately followed by Mac-1/ICAM-1-dependent crawling [46,47]. The latter event presumably occurs to probe for suitable sites for the subsequent transendothelial migration [31,46–50], a process that also requires extensive redistribution of neutrophil microdomains discussed elsewhere in this issue. Mac-1/ICAM-1-mediated crawling has recently been proposed to rely on prior LFA-1-induced outside-in signaling mediated via Vav1 [47], a guanin nucleotide exchange factor (GEF) for Rho GTPases, inducing cytoskeletal remodeling [51,52].

Microdomains in mediating interactions with RBC and platelets

Neutrophil adhesion is a prerequisite for transendothelial migration but the duration of leukocyte adherence in the microvasculature varies greatly. Monocytes have been reported to crawl to survey the vasculature [53]. Although a similar behavior for neutrophils is likely, whether it is the case has not been studied in detail. During endothelial cell adhesion neutrophils reside in a pivotal position where they can integrate and link signals between activated endothelial cells and free flowing blood components. As a general phenomenon, adherent leukocytes protrude into the vascular lumen and therefore affect blood flow, especially in small diameter venules. During inflammation, neutrophils are being recruited en masse, producing flow obstructions. Indeed, the presence of adherent leukocytes in the microcirculation has emerged as a crucial factor in mediating vasooclusion in sickle cell disease (SCD), as P- and E-selectin deficiency in a humanized mouse model of the disease protectsfrom this event [54].

SCD results from a point mutation in the gene encoding β-globin, rendering the molecule less soluble (particularly in deoxygenating conditions) and leading to its polymerization and membrane protrusions [55,56]. This induces the characteristic sickle shape of affected red blood cells (SS RBCs), which are less deformable and may induce endothelial cell activation [57,58]. In addition, perturbations in the sickle plasma membranes exposes molecules such as phosphatidyl serine and sulfated glycolipids [59,60], which may activate cells within the vasculature, i.e. endothelial cells, leukocytes and platelets [61]. This results in a systemic state of vascular inflammation and granulocytosis [62,63], which is characterized by recurring painful crises due to local vascular occlusive events that can lead to life-threatening acute chest syndromes and organ failure [64]. In addition to obstructing blood flow by simply attaching to venular endothelium, adherent neutrophils can also interact with activated RBCs and platelets to form conglomerates that can exacerbate vasoocclusion [65,66].

Mechanisms of heterotypic interactions

Recent evidence obtained by high-speed multichannnel fluorescence intravital microscopy (MFIM) has identified the leading edge of the neutrophil as the site for mediating RBC interactions, and both leading and trailing edges for platelet capture [31,67]. Under inflammatory scenarios such as SCD, these interactions are greatly increased and are then almost exclusively mediated via the pseudopod for both RBCs and platelets. The interactions with the pseudopod are induced by the engagement of E-selectin with ESL-1, resulting in neutrophil inside-out signaling and clustering of activated Mac-1 to the pseudopod where it interacts with currently unidentified receptors on RBCs and platelets [67]. In the SCD model, neutrophil interactions with RBCs are increased and are responsible for lethal vasooclusion [65–67]. Although the responsible receptors on RBCs remain to be determined, potential candidates include complement [68]and ICAM-4 (also known as Landsteiner-Wiener glycoprotein) [69].

Interestingly, the same mechanism of clustered Mac-1 activity in neutrophil microdomains is also involved in mediating neutrophil interactions with platelets in another model of microvascular inflammation, namely transfusion-related acute lung injury (TRALI), resulting in neutrophil activation, ROS generation and vascular leakage [67]. Although the initial events in neutrophil activation in this model are due to Fcγ-receptor mediated neutrophil recruitment [70,71], Mac-1 activation is ultimately also dependent on ESL-1. Whether these platelet interactions are different from those observed for neutrophil-mediated clearance, which depends on P-selectin-PSGL-1 engagement, remains to be investigated [72]. These data emphasize the importance and general relevance of neutrophil Mac-1 in thrombo-inflammatory diseases.

Conclusions

Cell adhesion molecules located in neutrophil microdomains are involved in mediating heterotypic interactions with the endothelium, RBCs and platelets. During neutrophil recruitment to the venular wall microdomains continuously redistribute on the cell surface, which leads to sequential interactions of molecules with counter-receptors on the interacting cell. Since the main cell surface adhesion receptors for neutrophil-endothelial cell adhesion have been identified, current research focuses on how the transition between the individual steps of rolling, adhesion and transmigration is mediated. Clearly, in vivo imaging has provided a great tool to visualize the clustering and distribution of adhesion molecules during heterotypic neutrophil interactions. New imaging techniques using genetically modified animals exhibiting fluorescent adhesion receptors and the use of fluorescently conjugated antibodies will yield exciting more detail, particular in how intracellular and membrane components interact to induce polarization, adhesion and intraluminal interactions of neutrophils.