Eosinophils are a subset of granulocytes that play a vital role in the inflammatory processes associated with a number of airway diseases including asthma [1
], eosinophilic granulomatosis with polyangiitis (EGPA) [2
], and hypereosinophilic syndrome (HES) [3
]. As the positive correlation between eosinophil accumulation in pulmonary tissues and disease severity has been ascertained [4
], the past decade has seen the development of several biologics that target interleukin-5 (IL-5), a TH
2 cytokine largely responsible for the mobilization, recruitment, and survival of eosinophils at sites of inflammation [5
]. Although some of these anti-IL-5 clinical trials have reported effective reduction and/or depletion of eosinophils from the blood and airways of severe asthmatic patients [5
], it has become a point of interest to investigate other factors such as epithelial cell-derived cytokines (IL-25, TSLP) and IL-13-secreting type 2 innate lymphoid cells (ILC2
) that may also contribute significantly to eosinophil migration [5
]. It is vital to untangle the series of signaling events that lead to specific actin cytoskeleton reorganization, which allow for (1) cellular adhesion to the endothelium and (2) transmigration through the extracellular matrix into the surrounding tissues by utilizing membrane protrusions such as pseudopodia.
In the current model of eosinophil extravasation, inactive integrins expressed on the surface of circulating eosinophils will undergo a conformational change to a highly active state upon exposure to chemokines (e.g., eotaxin) near the bronchial vascular endothelium [9
]. In particular, the very late antigen-4 (VLA-4, α4β1) integrin on the eosinophilic cell surface will bind to the vascular cell adhesion molecule-1 (VCAM-1) counter ligand, which is upregulated in the asthmatic lung [10
]. Furthermore, in vivo pre-activation and/or priming of the cells mediated by increased IL-5 levels in asthmatics result in intermediate-activated integrin conformations displayed on the surface [9
], resulting in augmented integrin activation; extravasation from circulation; and an overaccumulation of eosinophil populations in respiratory tissues, oftentimes leading to subsequent airway inflammation and obstruction.
Our previous study identified a novel phenomenon in eosinophils, which we coined the perfusion-induced calcium response (PICR), in which the perfusion of media without any pharmacological agents over adhered eosinophils from peripheral blood elicited an integrin-mediated release of intracellular calcium [Ca2+
from internal stores. The calcium flux was observed concurrently with changes in eosinophil morphology (flattening, membrane protrusions) [13
], suggesting the following: (1) the signaling pathways involved in actin skeleton restructuring may be regulated by [Ca2+
mobilization, and (2) mechanical stimuli of fluid shear stress detected by integrin mechanosensors on the eosinophil surface may play a pivotal role in eosinophil migration from the vasculature. Indeed, eosinophils in vivo would experience varying magnitudes of shear stress corresponding to their strength of adhesion to the vascular endothelium, in turn triggering the PICR under optimal environmental conditions. In a subsequent study, we observed, in response to physiologically-relevant fluid shear stress levels, a temporal link between [Ca2+
release and increased cell surface contact area with the fibronectin-coated substratum via cell flattening [14
]. Unfortunately, at the time, we were neither able to clearly elucidate the correlated/causative nature nor the directionality of this relationship in eosinophils. The present study utilizes improved real-time confocal microscopy techniques and refined analytic methods to investigate the effects of pharmacological inhibitors to calcium and actin cytoskeleton signaling pathways on the PICR in eosinophils. Improving upon the limited knowledge regarding eosinophil trafficking processes may assist in the future development of novel treatments and therapies of eosinophilia-driven pathologies such as asthma.
The modifications to the cytoskeletal architecture due to stimuli ranging from mechanical fluid shear stress to ligand binding chemokines occur in a systematic fashion to ensure proper spatial and temporal conditions are met for migration-related behavior. Yet, the molecular processes of eosinophil extravasation detailing the activation of its integrin receptors and the subsequent outside-in signaling transduction cascades have not been fully unraveled. Although many groups are investigating the migrational responses triggered and guided by molecular stimuli (chemotactic and chemokinetic ligands), we are pioneering a role for physical stimuli (shear stress) in triggering the extravasation of activated eosinophils from the circulation and into the vessel wall. That is, shear stress elicits a complex and carefully choreographed sequence of morphological changes, which all take place within 10 min, and sets the stage for the migrational response, which occurs over the next many hours. Furthermore, we are finding that the immediate triggering event is connected to intracellular calcium fluxes. In the present study, we disrupt both actin cytoskeleton and calcium pathways with pharmacological agents to probe the eosinophil response to physiologically relevant shear stresses experienced by the cell once it adheres to the vessel wall.
To link the two crucial steps in classically studied leukocyte extravasation of adhesion and membrane protrusion, we utilized fibronectin as our substrate coating. It is well documented that firm cell adhesion to the endothelium and the extracellular matrix is primarily mediated through integrin engagement. The VLA-4 integrin on the eosinophil surface not only binds to VCAM-1 upregulated on the endothelium in disease models [19
], but also readily adheres to fibronectin [20
], allowing our experimental model to better simulate in vivo endothelium environments. Integrin activation has been shown to activate Rac1, which in turn signals to the Arp2/3 complex [21
], a seven-subunit protein aggregate necessary for actin nucleation and actin filament polymerization for numerous essential cell functions including vesicle trafficking, migration, and membrane protrusions (lamellipodia and pseudopodia) [21
]. Rac1 promotes the linkage between the Arp2/3 complex with vinculin, an adaptor protein that physically links the transmembrane integrin receptor to the actin cytoskeleton [24
]. This connection may be further stimulated by PIP2
, which induces a conformational change in vinculin to expose additional binding sites for the Arp2/3 complex [25
itself is also involved in the activated Gq
/11 pathway, which results in IP3
-mediated calcium release. As such, we were motivated to determine the inhibition of the integrin/Rac1/Arp2/3 pathway, a central component to both cellular adhesion and motility, with the compound CK-666, which would interfere with the eosinophil PICR. We also utilized the compound Y-27632 as a cell-permeable Rho-associated protein kinase (ROCK)-selective inhibitor to reduce actin filament stabilization [26
]. The GTPase Rho emerged in the late 20th century as a major facilitator of actin skeleton rearrangement necessary for actin stress fiber formation and cell migration through focal adhesions [26
]. ROCK was found to be a key downstream Rho effector molecule specifically responsible for stress fiber formation, and itself inhibits actin filament depolymerization [26
In our study, both CK-666- and Y-27632-treated eosinophils displayed stunted capabilities with regards to environment exploration and cell motility. The inability of the Arp2/3 complex to transiently bind to vinculin and integrin receptor aggregates because of CK-666 inhibition was markedly reflected in its smaller area ratio (Figure 1
B) and lack of membrane ruffling (Figure 4
D). However, the CK-666-treated eosinophils boasted a robust and rapid calcium spike (Figure 2
B,C), suggesting that intracellular calcium release signaling remained upstream or independent from actin rearrangement. It is interesting to note, however, the perfusion of Y-27632 did not significantly affect the eosinophils’ response to fluid shear stress outside of pseudopodia formation (Figure 4
C) and distance traveled (Figure 5
A). In fact, eosinophils treated with Y-27632 exhibited all of the hallmark features of the fluid shear stress response indicated by the lack of significant differences in area ratio, fluorescence ratio, and high positive correlations between calcium flux, elongation, and directional shift timepoints. These results indicate that pseudopodia formation requires long-term stability of actin polymerization relative to membrane ruffling, and is a necessary checkpoint for substantial eosinophil motility.
However, calcium signaling inhibitor data support our theory that eosinophil PICR is responsible for triggering actin cytoskeleton rearrangement. The compound ryanodine functions as a full antagonist to ryanodine receptors (RyRs) at micromolar concentrations [27
]. Although stimulated RyRs are predominantly known to trigger [Ca2+
release from the sarcoplasmic reticulum in skeletal muscle cells to drive contraction [28
], it was recently discovered that RyRs are also expressed on the endoplasmic reticulum of other cell types including immune cells [29
]. Subsequent to [Ca2+
release in cells, the Ca2+
-ATPases on the plasma membrane and endomembrane are responsible for restoring calcium homeostasis by pumping Ca2+
into the extracellular space and back into intracellular stores, respectively [30
]. Therefore, treating cells with the ER Ca2+
-ATPase inhibitor, CPA, would deplete calcium stores over time.
Although the CPA-treated eosinophils displayed fluorescence ratios of a smaller magnitude, it did not influence the latency of the calcium flux post-perfusion (Figure 2
C). The CPA data strongly suggest that cycling intracellular calcium ions between the cytosol and ER storage is integral to migration-related actin rearrangement. In essence, as a consequence of calcium ion reuptake inhibition, the smaller PICR in this treatment group resulted in weak temporal association with both cellular elongation and directional changes in movement. On the other hand, we surmised that the reason ryanodine-treated eosinophils did not abolish the PICR (Figure 2
A), despite taking a significantly longer time achieving it (Figure 2
C), was due to the involvement of alternative calcium signaling pathways such as the PLC-IP3
The acquisition of spatial asymmetry in cells is critical for the onset of cellular migration. In essence, polarized cell morphology with distinguished front and back ends allows the cell to continue the formation of actin polymerization at the leading region with filaments released from adhesions at the rear [31
]. In order for sustained migration to occur, the membrane protrusions must be firmly adhered to the substrate. If the pseudopodia fail to stabilize adhesions, they will retract towards the cell body [33
]. On the other hand, although membrane ruffles lack adhesion sites and cannot promote displacement [34
], it has been suggested the ruffles play an indirect role in promoting migration. Membrane ruffling is associated with macropinocytosis, a transient endocytic process that internalizes extracellular components in addition to parts of the cell membrane and their surface receptors [34
]. Specifically, the ruffles may be responsible for redistributing the β1 integrins towards newly formed adhesion sites at the leading edge and the establishment of the front-rear-axis of the cell. The necessity for both pseudopodia and membrane ruffling formation in cell ruffling is evident in the ryanodine treatment group. Although these eosinophils had the most comparable degree of distance traveled relative to the control group because of their higher ratio of pseudopodia formation, the complete lack of membrane ruffling appears to be limiting their potential.
The calcium-spike is a very rapid, all-or-none, pan-cellular event; these are all good and useful features of a finely tuned trigger mechanism. Our data suggest that the morphological changes (flattening, ruffling, and pseudopod-formation) can still occur after disruption of this trigger, but their latency is greatly increased. This might offer up a new avenue for treatment strategy for eosinophilic inflammation. That is, in the physiological setting, circulating eosinophils become primed by agents (such as IL-5), then roll along the vessel wall until they are stimulated by a chemoattractant signal (such as eotaxin) to extravasate. The markedly increased shear stress produced during the rolling phase triggers the PICR, which in turn commits the cells to adhere strongly and accelerate extravasation. If that PICR could be disrupted, the primed cells might otherwise continue to roll and eventually be swept away back into the systemic circulation, thus reducing inflammation at that site. In conclusion, we theorize that adequate strength of the PICR ‘jump-starts’ the extravasation process in vivo and plays a significant role in the temporal aspect of migration.
The study of mechanosensors on circulating leukocytes is fairly novel, and the majority of shear stress research remains dominantly limited to non-migratory cells such as the endothelium. However, it is crucial that we begin to differentiate between the role of mechanical stimuli for the various cell types. For example, the β1 integrins highly expressed on endothelial cells (ECs) were recently found to sense unidirectional flow and promote cellular alignment for vascular homeostasis, suggesting a role distinct from the β1 integrins on eosinophils [35
]. Furthermore, some studies have shown that mechanical stress on ECs triggered the influx of calcium from extracellular sources [36
], whereas our study indicates the increases in cytosolic calcium are from internal stores. In conclusion, investigating the distinct role of integrin mechanosensors in eosinophils and their role in mobilization may have future therapeutic implications for asthma and other eosinophilia-driven conditions.