Calcium signaling and related ion channels in neutrophil recruitment and function

Abstract

The recruitment of neutrophils to sites of inflammation, their battle against invading microorganisms through phagocytosis and the release of antimicrobial agents is a highly coordinated and tightly regulated process that involves the interplay of many different receptors, ion channels, and signaling pathways. Changes in intracellular calcium levels, caused by cytosolic Ca²⁺ store depletion and the influx of extracellular Ca²⁺ via ion channels play a critical role in synchronizing neutrophil activation and function. In this review, we provide an overview of how Ca²⁺ signaling is initiated in neutrophils and how changes in intracellular Ca²⁺ levels modulate neutrophil function.

Neutrophil recruitment cascade

Neutrophils (polymorphonuclear leukocytes, PMNs) are a central component of the innate immune system and are among the first cells to be recruited to sites of infection and inflammation¹. In doing so, the cells engage in a well-defined multistep cascade of adhesion and activation events. Initially, free flowing PMNs are recruited from the blood stream via selectin-glyco-ligand interactions, triggering rolling of the cells along inflamed postcapillary venules². Engagement via selectins expressed on the inflamed endothelium with P-selectin glycoprotein ligand 1 (PSGL-1), L-selectin (in humans³) and other sialylated ligands initiates the activation of β₂-integrins and subsequent deceleration of rolling neutrophils^4,5. Additionally, PMNs ligate immobilized chemokines presented on inflamed endothelial cells, thereby inducing the high affinity conformation of β₂-integrins lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1), which leads to firm adhesion of neutrophils on the inflamed endothelium⁶. Post arrest modifications, including β_2-integrin-clustering and anchoring to the cytoskeleton, results in neutrophil spreading, adhesion strengthening and intraluminal crawling⁷. Crawling of neutrophils along the inflamed vessel wall is a critical step for their localization at appropriate sites for extravasation (diapedesis). Similar to the intravascular adhesion steps, emigration of neutrophils into inflamed tissue also proceeds in a cascade like fashion including transmigration across the endothelial layer into the subendothelial space, followed by penetration of the vascular basement membrane⁸. Within tissue, neutrophils migrate along chemokine and cytokine gradients to the actual site of tissue insult, where they respond to invading pathogens by engaging in phagocytosis, degranulation and secretion of proteolytic enzymes and reactive oxygen species.

Ca²⁺ signaling/SOCE in neutrophils

The neutrophil recruitment cascade and its underlying molecular mechanisms have been the subject of intensive research over the last decade. Changes in intracellular Ca²⁺ levels are a hallmark in the activation process of neutrophils⁹. Hence, versatile regulation of Ca²⁺ fluxes during neutrophil recruitment and antimicrobial responses is a finely tuned process in terms of temporal and spatial organization⁹. Under resting conditions, levels of cytosolic Ca²⁺ in neutrophils (0.1 μM) are 10 000-fold lower than in the extracellular milieu ¹⁰. Upon stimulation, cytosolic Ca²⁺ levels rapidly increase by the release of Ca²⁺ from intracellular stores and/or by influx of extracellular Ca²⁺.

Entry of extracellular Ca²⁺ into immune cells, including neutrophils, is primarily mediated through Ca²⁺ release-activated Ca²⁺ (CRAC) channels, located in the plasma membrane (PM). The process of Ca²⁺ influx is termed store operated calcium entry (SOCE) (Figure 1A). In neutrophils, SOCE can be initiated by several ligand receptor interactions including the engagement of G-protein coupled receptors (GPCRs)¹¹ and Fcγ-receptors (FcγRs)¹², the activation of β₂-integrins^13,14 or the interaction of E-selectin with PSGL-1 and L-selectin^4,15,16. Downstream signaling involves activation of phospholipase C (PLC). GPCRs mainly trigger the activation of the PLCβ subfamily members 2 and 3, while FcγRs, E- and P-selectin ligands, and β₂-integrins predominantly activate PLCγ1 and PLCγ2, respectively^4,17. PLCβ2/3 and PLCγ1/2 convert phosphatidylinositol 4,5 bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol-1,4,5 triphosphate (IP₃ or InsP3). The IP₃ receptor (IP₃R) is localized in the membrane of the endoplasmic reticulum (ER) and the activation of this non-selective Ca²⁺ channel by IP₃ triggers the release of ER-stored Ca²⁺ into the cytoplasm¹⁸. The drop in Ca²⁺ level in the ER is sensed by stromal interaction molecule 1 (STIM1), an ER transmembrane protein which then translocates to specific regions of the ER close to the PM (PM-ER junctions). At these sites, STIM1 binds to and activates the PM associated Ca²⁺ channel denoted CRAC modulator 1 (CRACM1), also termed Orai1, the predominant CRAC channel in neutrophils^16,19. Stimulation of Orai1 leads to Ca²⁺ influx²⁰ and concomitant increase of cytosolic Ca²⁺ concentration. FcγR activation is thought to exhibit an alternative mechanism to induce the release of Ca²⁺ from ER stores in a PLCγ (and therefore IP₃) independent manner ^by activation of phospholipase D (PLD) via phosphatidylinositol 3-kinase (PI3K) and subsequent generation of sphingosine 1 phosphate (S1P) by sphingosine kinase (SK). Blockade of either SK or PI3K, was shown to reduce Ca²⁺ flux in neutrophils upon FcγRIIA and FcγRIIIB activation²¹. A role of S1P in calcium signaling was also demonstrated by Itagaki and Hauser²² who showed that S1P formation is dependent on ER store depletion. Furthermore, the authors found that inhibition of SK results in a reduction of Ca²⁺ entry. Recently, it was shown for glioblastoma cells that a member of the transient receptor potential (TRP) channel family (TRPC1; see also below) might be involved in S1P mediated calcium signaling²³. However, the exact mechanism by which S1P mediates changes in intracellular Ca²⁺ and whether this signaling cascade cooperates with other activation pathways is currently unknown and requires further investigation.

Figure 1. Calcium signaling cascade in neutrophils.

(A) Store operated calcium entry (SOCE) can be initiated either via activation of Fcγ-receptors (FcγRs), β₂-integrins, the engagement of P-selectin glycoprotein ligand 1 (PSGL-1) and L-selectin with E-selectin expressed on inflamed endothelium, or via G-protein coupled receptors (GPCRs). Activation of GPCRs leads to dissociation of the G-protein subunits α and βγ and subsequent activation of phospholipase (PLC) β. FcγRs, β₂-integrins and PSGL-1/L-selectin signaling in turn, activate PLCγ involving spleen tyrosine kinase (Syk). Both, activated PLCβ and PLCγ convert phosphatidylinositol 4,5 bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol-1,4,5 triphosphate (IP₃). IP₃ binds to and opens the IP₃ receptor (IP₃R) in the membrane of the endoplasmic reticulum (ER) which results in Ca²⁺ flux out of the ER into the cytoplasm via IP₃R. Upon store depletion, the Ca²⁺ sensor stromal interaction molecule 1 (STIM1) translocates to PM-ER-junctions and activates Orai1, the predominant Ca²⁺ release activated Ca²⁺ (CRAC) channel in neutrophils. This allows the entry of extracellular Ca²⁺ into the neutrophil, exerting a variety of functions, including (B) phagocytosis, ROS production, secretion and degranulation of vesicles, activation of β₂-integrins and cytoskeletal rearrangement leading to polarization and migration.

Local regulation of intracellular Ca²⁺ concentrations is a prerequisite for eliciting the broad machinery of innate immune defense processes in neutrophils for instance, phagocytosis, production of reactive oxygen species (ROS) and degranulation (reviewed in²⁴). Furthermore, β₂-integrin activation¹⁵, cytoskeletal rearrangement²⁵ and migration of neutrophils²⁶ within the tissue, are all Ca²⁺ signaling dependent processes (Figure 1B). The importance of STIM1 and Orai1 for SOCE in neutrophils has been validated using mouse mutant models^27–30 and knockdown in neutrophil-like cell lines (i.e. HL-60 cells)^16,31 and has been reviewed in detail in³². Additional evidence comes from patients with mutations in either Orai1 or STIM1 who suffer from severe combined immunodeficiency-like diseases and autoimmune disorders, accompanied by an increased susceptibility to infections, sepsis and neutropenia^33,34. Surprisingly, neutrophils from patients with loss of function mutations in either STIM1 or Orai1 displayed no differences in ROS production, static adhesion, chemotaxis and IL-8 production and only a modest reduction of SOCE, and less sustained Ca²⁺ flux, respectively³⁵. This indicates that the severe autoimmune disorders in these patients are not primary neutrophil dependent functions. One possible explanation might be the expression of Orai1 and STIM1 isoforms in neutrophils which contribute to SOCE and may compensate for a loss of function in Orai1 and STIM1, respectively. One potential candidate would be STIM2 which is an additional Ca²⁺ sensor expressed in neutrophils. Of note, STIM2 exerts additional functions in neutrophil activation, distinct from classical Ca²⁺ sensing. This was shown in STIM2 deficient neutrophils, which exhibit reduced cytokine production during inflammatory responses³⁶. However, production of reactive oxygen species (ROS), degranulation and phagocytosis, which are impaired in STIM1 deficient PMNs, were normal in the absence of STIM2^28,29. Interestingly, both, STIM1 and STIM2 seem to be dispensable for neutrophil migration in the mouse^27,36. The distinct and redundant functions of the two isoforms STIM1 and STIM2 have been summarized recently³⁷. Evidence for the expression of the CRAC channel homologs Orai2 and Orai3 exists for HL-60 cells^31,38 and most likely for human neutrophils³⁵. Whereas Steinckwich and colleagues³¹ reported no significant differences in global cytosolic Ca²⁺ levels during phagocytosis in Orai2 and Orai3 knock down HL-60 cells, Diez-Bello et al.³⁸ observed altered intracellular Ca²⁺ levels in HL-60 cells lacking ORAI2. Further functional data are necessary to better understand the functions of the ORAI1 and STIM1 isoforms and to decipher the differing compensatory properties in mouse and human neutrophils.

Temporal and spatial resolution

A fine balance exists between the protective antimicrobial functions of neutrophils and potential tissue damage associated with their unchecked recruitment during exuberant inflammation. Neutrophils have evolved mechano-regulatory mechanisms by which tensile forces exerted on cells in blood flow that act on selectin and integrin bonds, are converted to biochemical signals during the transition from rolling to cell arrest and transmigration (Figure 2). Recently, it was reported that an allosteric shift from low or intermediate LFA-1 to a high affinity state enables bond formation with intracellular adhesion molecules (ICAMs) that effectively functions like a clutch in transmission of sufficient tension to the transient LFA-1/Kindlin-3 linkage to elicit Orai1 mediated Ca²⁺ flux.^14,15 Engagement of β₂-integrins to their target ligands on inflamed endothelium is essential in activation of calcium dependent kinases and to achieve maximum cytosolic calcium influx via SOCE that activates the opening of intracellular Ca²⁺ release channels ³⁹. As introduced above, the canonical pathway downstream from ligation of GPCR involves activation of PLC-β following release of the G_βγ subunit⁴⁰. PLC cleaves membrane lipid PiP₂ into DAG and IP₃, resulting in Ca²⁺ flux and activation of protein kinase C (PKC)⁴¹. In conjunction with PKC, calcium itself activates adhesion receptors, whose ligation in turn amplifies downstream immune responses⁴². Thus, inside-out and outside-in signals cooperate in a synchronous manner in the emptying of cytosolic calcium stores, which in turn drives CRAC influx from the extracellular milieu. Regulation of the CRAC current has been carefully documented in T-cell immune synapse formation, where STIM1 and STIM2 act as the sensors of calcium store depletion and together with Orai1 play a central role in regulating local calcium transients that orient the polymerization of cellular actin during migration^43,44. A key reason why CRAC current has not been widely studied in human neutrophils (hPMNs) is that they are much shorter-lived than T-cells and are not readily amenable to genetic manipulation. HL-60 cells are a myeloid cell line that are differentiated to neutrophils following culture with DMSO, and retain most features of primary hPMNs including inflammatory recruitment. Employing siRNA on differentiated HL-60 it has been shown that Orai1 is a primary mediator of membrane Ca²⁺ flux during neutrophil recruitment on inflamed endothelium¹⁶. More recently, it was reported that knockdown of Kindlin-3 elicited a similar phenotype of impaired neutrophil arrest in shear flow due in part to lack of Ca²⁺ flux and high affinity integrin clustering⁴⁵. The picture that has emerged is that during arrest, neutrophils engage and exert force on a complex of LFA-1/ICAM-1/Kindlin-3 allowing the cell to ‘sense’ mechanical cues at the site of adhesive contact with inflamed endothelium. In this manner, the magnitude of shear stress directly regulates calcium flux locally through application of tensile force on LFA-1/ICAM-1 bonds, which strategically are positioned at the base of the uropod and pseudopod on polarized migrating neutrophils^15,46 This provides intrinsic regulation on the number of PMNs that arrest and transmigrate under conditions of low flow such as during tissue ischemia.

Figure 2. Calcium signaling functions to synchronize events in neutrophil recruitment.

Neutrophil rolling via PSGL-1, L-selectin, and E-selectin causes downstream activation of IP₃R and subsequent release of ER stored Ca²⁺, which in turn shifts LFA-1 from a low to an intermediate and high affinity state. This shift leads to deceleration of the rolling neutrophil via LFA-1-ICAM-1 interaction. The activation of GPCRs by chemokines further increases intracellular Ca²⁺ levels via IP₃R. Additionally, GPCR signaling via DAG activates additional LFA-1 which increases the number of LFA-1/ICAM-1 bonds, resulting in total arrest of the neutrophil (inside-out signaling). High affinity LFA-1 binding to ICAM-1 further enhances rise in intracellular Ca²⁺ concentrations (outside-in signaling), amplified by shear forces. Upon force transduction, Kindlin-3 binds to β₂-integrin tails, recruits Orai1 and thus forms a complex which ensures increase in Ca²⁺ levels directly at focal adhesion spots, a process again highly dependent on shear forces to orient cell polarization and migration. This is followed by clustering of LFA-1 and the recruitment of Talin1 which links the focal adhesion spots to the cytoskeleton, enabling intraluminal crawling of the neutrophil.

Role of adaptor molecules in local regulation of Ca²⁺ flux

Absence of Kindlin-3 severely impairs β₂-integrin function on leukocytes, resulting in leukocyte adhesion deficiency (LAD)-III in mouse and human⁴⁷. As a direct consequence, Kindlin-3 deficiency in LAD-III patients results in severe bleeding and resistance to arterial thrombosis, as well as immunodeficiency⁴⁸. Mechanistically, Kindlin-3 binds by its FERM F3 subdomain directly to regions of β-integrin tails distinct from Talin1, which in turn triggers integrin activation⁴⁹. Kindlin-3 is required for the formation of micro-clusters (e.g. ~10–100 receptors) of high affinity LFA-1. Neutrophils engage and exert force on these receptors during bond formation with ICAM-1 that in turn enables the cell to ‘sense’ mechanical cues and transform them into localized sites of mechanotransduction at appropriate inflammatory sites on endothelial cells. It is currently unknown how force transmitted from the outside-in induces binding between the β-integrin tail and membrane bound Kindlin-3. This interaction precedes and is necessary to engage the CRAC machinery (e.g. STIM1 and STIM2) and catalyze the subsequent association with Orai1 adjacent to the endoplasmic reticulum for SOCE⁵⁰. A clasp formed by a salt-bridge between the integrin α and β tails is crucial for maintaining integrins in the bent, inactive conformation. Intracellular proteins that interact with integrin tails, such as Talin1 and Kindlin-3 can induce conformational activation of the integrin by disrupting the integrin clasp⁵¹. Following inside-out signaling via GPCRs a separation of the clasp results in triggering extension of ectodomains and a conformational change to the activated and extended high affinity ligand binding site of LFA-1. In this manner, we propose that kindlins serve as coactivators, as they cooperate with Talin1 and Orai1 to activate integrins⁵². Further, we envision a mechanism by which engagement of high-affinity LFA-1 acts synergistically with chemokine receptor signaling to effectively amplify release of intracellular Ca^{2+ 45}. Kindlin-3 association with high-affinity LFA-1 is necessary for this amplification in Ca²⁺ release in the presence of shear flow. We speculate that Kindlin-3 functions as a mechanosensor, since it binds constitutively with LFA-1 but its interaction increases by several-fold with an upshift to high-affinity as tensile force is applied to high-affinity bond clusters⁹. We and others have reported that silencing expression of Kindlin-3 or Orai1 impairs LFA-1 clustering at adhesive contacts, which correlates with a significant decrease in adhesion strengthening⁴⁵. In contrast, silencing expression of Talin-1 does not alter high-affinity LFA-1 clustering and only slightly diminishes stable adhesion at high shear stress. This suggests that Talin-1 does not play an eminent role in clustering of LFA-1. What remains unknown is how force acting on LFA-1 bond clusters results in tight engagement between LFA-1 and Kindlin-3 and how it engages locally with CRAC channels in modulation of local Ca²⁺ release. It has been reported that as little as 5% of physiologic Kindlin-3 levels in hematopoietic cells are essential for the retention in bone marrow and sufficient for embryonic and postnatal development, but exposure to stress such as infection at this low level of Kindlin-3 significantly impaired the capacity for PMN adhesion and host response^47,53. Thus, a ratio of two LFA-1 to each Kindlin-3 appears to be the optimal configuration for homeostatic regulation of adhesive function and to prevent spontaneous bleeding and microbial infections. We propose that the local engagement of adhesion receptors and regulation of cytosolic Ca²⁺ are rate limiting steps in mediating where and when neutrophils are activated to arrest and initiate a migratory phenotype, events that serve as a gatekeeper in neutrophil recruitment to sites of inflammation.

Other ion channels involved in Ca²⁺ signaling in neutrophils

Although the primary mechanism of calcium flux into the cytosol of neutrophils is predominantly mediated via IP₃ dependent Ca²⁺ release out of ER stores and subsequent SOCE upon activation of the CRAC channel Orai1, there are additional ion channels involved in calcium signaling in neutrophils (Table 1). The literature within this broad field of Ca²⁺ signaling is rather controversial, as human and murine (mPMNs) neutrophils and even primary human neutrophils and HL-60 cells do not always display the same expression pattern of ion channels on their surface^19,54,55. This makes it difficult to directly translate the different findings between species, cell lines and primary cells.

Table 1.

Known ion channels and sensors being expressed and involved in calcium signaling in neutrophils, their permeability and directionality. ↓: influx ↑: efflux; h=human; m= mouse (h̶/m̶= not expressed)

Channels	Permeability	Directionality	Expression in neutrophils (Human/Mouse)	Reference
Orai1	Ca2+	↓	h,m	16,19
Orai2	Ca2+	↓	h	35
Orai3	Ca2+	↓	h	35
STIM1	Ca2+-sensor	-	h,m	28,90
STIM2	Ca2+-sensor	-	h,m	36,90
TRP Channels
TRPC1	non-selective; Ca2+, Na+	↓	h, m	57,61,91
TRPC3	non-selective; Ca2+, Na+	↓	h, m̶	55,57,91
TRPC4	non-selective; Ca2+, Na+	↓	h	57,91
TRPC6	non-selective; Ca2+, Na+	↓	h, m	54,55,91
TRPM2	non-selective; Na+, K+, Ca2+	↓	h, m	54,65,91,92
TRPM4	non-selective; Na+, K+, Cs+	↓	m	55,91,92
TRPV1	non-selective; Ca2+	↓	h,	91,93
TRPV2	non-selective; Ca2+, Na+	↓	h,	91,93
TRPV4	non-selective; Ca2+	↓	h, m	55,68,91
TRPV5	non-selective; mainly Ca2+	↓	h,	91,93
TRPV6	non-selective; mainly Ca2+	↓	h,	91,93
Potassium channels
KV1.3	K+	↑	h	73
KCa3.1	K+	↑	h, m	71
P2X receptors
P2X1R	non-selective; Ca2+, Na+	↓	h, m	74,76,77
P2X3R	non-selective; Ca2+, Na+	↓	h̶, m	76,77
P2X4R	non-selective; Ca2+, Na+	↓	h	74,75,77
P2X5R	non-selective; Ca2+, Na+	↓	h	74,77
P2X7R	non-selective; Ca2+, Na+, K+	Ca2+, Na+: ↓ K+: ↑	h, m	77,81,82
Proton channel
VSOP/Hv1	H+	↑	h, m	87

TRP-channels

The superfamily of transient receptor potential (TRP) channels is a group of non-selective ion channels consisting of six different subfamilies, namely the anykyrin (TRPA), the canonical (TRPC), the melastatin (TRPM), the mucolipin (TRPML), the polycystin (TRPP) and the vanilloid (TRPV) families. Human and murine PMNs only express members of the TRPC, TRPM and TRPV channels, which we will introduce within this section.

TRPC channels are non-selective cation channels for Ca²⁺ and Na⁺, displaying a strong tendency to form heterodimers⁵⁶. Four members of TRPC channels are described in neutrophils, namely TRPC1, TRPC3, TRPC4, and TRPC6 which is the best studied TRPC channel in this cell type^{19,54,55,57–59}. Knockdown of TRPC6 in HL-60 cells reduced NADPH oxidase activity and therefore ROS production, demonstrating its contribution to changes in intracellular Ca²⁺ levels¹⁹. Subsequently, the use of TRPC6 deficient mPMNs demonstrated that TRPC6 plays a role in CXCL2 mediated Ca²⁺ influx⁵⁵. However, TRPC6 dependent changes in cytosolic Ca²⁺ levels are stimulus dependent, as formyl-methionyl-leucyl-phenylalanine (fMLF) was unable to initiate this process of Ca²⁺ entry⁵⁵. Further, knockout of TRPC6 resulted in delayed Ca²⁺ responses and altered cytoskeletal dynamics in CXCL2 stimulated neutrophils. In 2013, Lindemann et al.⁵⁹ confirmed the relevance of TRPC6 in chemokine receptor mediated actin remodeling and subsequent chemotaxis in neutrophils. TRPC6^−/− mPMNs were unable to efficiently transmigrate into the inflamed peritoneal cavity in a mouse peritonitis model and additionally, knockout mPMNs displayed a reduction in intracellular Ca²⁺peaks following CXCL1 stimulation compared to wildtype control cells. It is known that TRPC6 (together with TRPC3) is activated independently of ER Ca²⁺ store depletion directly via DAG⁶⁰, the second cleavage product of PIP₂ conversion into IP₃. This mechanism has been termed receptor operated calcium entry (ROCE)⁵⁹. Hence, the findings in the context of TRPC6 function in neutrophils reveals an alternative process of Ca²⁺ influx through the PM, which functions independent of the classical IP₃/IP₃R/STIM1-axis.

TRPC3 is another member of the TRPC family expressed on human, but not on mPMNs^55,57. Similar to TRPC6, it was reported to be involved in the regulation of ROS production in neutrophils¹⁹.

TRPC1 may primarily function as a regulator of neutrophil migration and chemotaxis. TRPC1^−/− mPMNs were described to display reduced migratory and chemotactic properties following fMLF stimulation⁶¹. Surprisingly, Ca²⁺ influx was not diminished in TRPC1^−/− mPMNs, but rather increased. However, absence of TPRC1 resulted in abnormal subcellular Ca²⁺ distribution with higher Ca²⁺ levels at the uropod, leading to reduced migratory capacity of the neutrophils. The authors suggested that TRPC1 may regulate the activity of other members of the signaling cascade, rather than being directly involved in SOCE or ROCE. These findings are in line with a study from Storch et al.⁶², who showed that loss of TRPC1 resulted in an increase in Ca²⁺ influx in neuronal cells. Only one publication describes a putative role of TPRC1 in SOCE¹⁹, where silencing of TRPC1 in HL-60 cells led to reduced intracellular Ca²⁺ levels and NADPH oxidase activation upon fMLF stimulation.

Members of the TRPM family are monovalent-cation channels, characterized by a variable selectivity for divalent ions⁵⁶. TRPM2, for instance is permeable for Ca^{2+ 56} and is the only member of the TRPM family known to be expressed on human and murine neutrophils^54,63. TRPM2 may play an important role as a stress sensor, translating metabolic and oxidative signals into Ca²⁺ flux⁶⁴. This channel is activated by ADP ribose (ADPR), cyclic ADPR (cADPR), adenosine monophosphate (AMP), nicotinic acid adenine dinucleotide phosphate (NAADP) and by hydrogen peroxide (H₂O₂)⁶⁴. Additionally, TRMP2 may be involved in the Ca²⁺ signaling downstream of formyl-peptide receptors (FPRs), but not downstream of chemokine receptors, since impaired Ca²⁺ flux was shown in TRPM2^−/− mPMNs following fMLF, but not CXCL2 stimulation⁶⁵. Further evidence for a specific role of TRPM2 in fMLF induced signaling comes from the observation that in hPMNs, TRPM2 acts exclusively downstream of FPRs⁶⁶. Moreover, TRPM2 may restrain neutrophil migration, as the channel is internalized together with FPR1 at the site of infection, hence permitting and initiating neutrophil mediated host defense, namely phagocytosis and secretion of antimicrobial agents⁶³.

Expression of TRPM4 on mRNA level has been shown via qPCR^55,67. However, no functional role in the context of Ca²⁺ signaling could be attributed to TRPM4 in neutrophils. Serafini and colleagues⁶⁷ did not detect any differences in mobilization and function of TRPM4^−/− neutrophils in a mouse model for sepsis and in intracellular Ca²⁺ levels in these cells activated with E.coli.

Finally, neutrophils have been reported to express TRPV channels, non-selective cation channels, which exhibit only a moderate permeability to Ca²⁺ (with the exception of TRPV5 and TRPV6) and which sense and react to chemical compounds like capsaicin and physical environmental cues like temperature or changes in osmotic pressure⁵⁶. Whereas TRPV4 is expressed on both, human and murine PMNs^55,68, TRPV1, TRPV2, TRPV5 and TRPV6 were up to now only found on hPMNs⁵⁴. TRPV4^−/− mPMNs, as well as hPMNs pretreated with a TRPV4 inhibitor (HC-067047), exhibited a reduction in Ca²⁺ flux upon stimulation with platelet activating factor (PAF), resulting in reduced ROS production⁶⁸. In a mouse model for acute lung injury (ALI), TRPV4 deficient cells were not recruited efficiently to inflamed lung tissue, pointing out a critical role of this Ca²⁺ channel in leukocyte migration and activation⁶⁸.

Very little is known about the role of TRPV1 in neutrophil function. This receptor (also known as capsaicin receptor) is permeable to Ca²⁺ and can be activated by capsaicin or high temperatures in the noxious range⁶⁹. Nevertheless, no Ca²⁺ flux was detectable in hPMNs treated with capsaicin⁵⁴. Besides the presence of TRPV2, TRPV5 TPRV6 on hPMNs, their respective roles in neutrophil function remains unknown. Further studies are needed to elucidate whether TRPV1, TRPV2, TRPV5 and/or TPRV6 are involved in Ca²⁺ dependent neutrophil activation.

Potassium channels

K⁺ efflux has been shown to be important for sustained Ca²⁺ influx in immune cells via Ca²⁺ permeable channels. It preserves the depolarization of the PM by maintaining the electrochemical gradient to drive further Ca²⁺ influx¹⁸. Using patch clamp technology, Krause and Welsh⁷⁰ observed two distinct K⁺ currents in hPMNs: one of them a voltage dependent K⁺ current and a second class that relies on elevation of cytoplasmic Ca²⁺ concentration. Recently, the latter K⁺ current was related to the presence of K_Ca3.1 (also known as IKCa1)⁷¹. Interestingly, inhibition of K_Ca3.1 via the specific inhibitor TRAM-34 did not alter increased intracellular Ca²⁺ levels in response to fMLF. This observation was surprising, as an influence of K⁺ efflux via K_Ca3.1 on intracellular Ca²⁺ levels had been shown for other cell types (eg. Jurkat cells)⁷². However, pharmacological blockade of this channel resulted in functional deficiencies of hPMNs. Likewise, K_Ca3.1^−/− neutrophils displayed a reduction of their migratory behavior in a transwell migration assay compared to control cells⁷¹. In line with these observations, recruitment of K_Ca3.1^−/− mPMNs was reduced in an acute lung injury model⁷¹. One candidate for the voltage dependent K⁺ current in neutrophils might be K_V1.3, which was shown to be expressed on hPMNs⁷³. However, functional data on the role of K_V1.3 in Ca²⁺ signaling in PMNs are still missing.

P2X receptors

P2X receptors are ATP-gated non-selective cation channels. Two members, P2X1R and P2X7R are expressed on human and mouse neutrophils, whereas P2X4R and P2X5R may be expressed on hPMNs^74,75 and P2X3R on mPMNs⁷⁶. However, functional data is not available. Extracellular ATP levels are elevated at sites of inflammation and influence intracellular Ca²⁺ levels via two different pathways. ATP can either bind and activate GPCRs (P2Y receptors) and affect Ca²⁺ signaling via SOCE or directly via P2X receptors⁷⁷. P2X1R is expressed on human and murine neutrophils^74,76 and its activation allows Na⁺ and Ca²⁺ to enter the cell⁷⁷. Lecut et al. showed that P2X1R engagement leads to downstream activation of RhoA and promotes neutrophil migration⁷⁶. In vivo, loss of P2X1R resulted in impaired migratory behavior and reduced extravasation of mPMNs into the inflamed peritoneal cavity. This observation was confirmed by Maître and colleagues, where P2X1^−/− mice showed a defect in LPS induced neutrophil emigration from cremaster venules into inflamed tissue⁷⁸. Furthermore, these mice displayed diminished responses in a model of acute systemic inflammation provoked by LPS⁷⁸. In contrast to these reports, more recently Lecut et al.⁷⁹ found an increased susceptibility to LPS induced endotoxemia and an increase in ROS production in P2X1^−/− mice. They concluded that P2X1R may have a protective role in endotoxemia by negatively regulating systemic neutrophil activation. Wang et al.⁸⁰ assumed that P2X1R may be involved in stopping chemotactic migration of neutrophils during LPS induced autocrine ATP signaling, thereby providing a signal to neutrophils at the inflammatory site to exert their antimicrobial functions.

Data on the presence of P2X7R on neutrophils is still conflicting, but emerging evidence suggests that hPMNs⁸¹ and mPMNs⁸² both express P2X7R. Activation of P2X7R via binding of extracellular ATP leads to rapid influx of Na⁺ and Ca²⁺, as well as K⁺ efflux via this channel⁷⁷. This in turn was shown to activate the NLRP3 inflammasome and to initiate neutrophil recruitment⁸³ and IL-1β secretion⁸². It is broadly accepted that activation of the NLRP3 inflammasome is associated with K⁺ efflux in myeloid cells^84,85.

Proton channel VSOP/Hv1

The proton channel murine voltage-sensing domain only protein (VSOP) and its human homologue Hv1 have been shown to inhibit azurophilic granule release in neutrophils. VSOP/Hv1^−/− deficient neutrophils displayed an enhanced release of MPO and neutrophil elastase compared to WT cells when activated with PMA⁸⁶. Furthermore, VSOP/Hv1 was described to play an important role in NADPH oxidase mediated phagosomal ROS production in neutrophils by counterbalancing the oxidase induced cytosolic acidification and cell depolarization^87,88. This compensatory effect has been shown to influence Ca²⁺ influx as well, since VSOP/Hv1^−/− mPMNs exhibited a reduction in intracellular Ca²⁺ levels upon activation with PMA/fMIVIL (a pentapeptide from Listeria monocytogenes), leading to migration defects in these mice⁸⁹.

Conclusion

Neutrophil recruitment to vascular sites of inflammation is a cascade-like molecular event that is initiated by selectin and chemokine bond formation superposed to activate the transition from cell rolling to integrin dependent arrest and transmigration into the parenchyma. Uninterrupted PMN recruitment is a robust and synchronous process that by nature’s design is faithfully reproduced a million-fold in healthy individuals during the innate immune response to inflammatory stimuli. Deletion or disruption of any single receptor-ligand interaction in this multistep process results in defective emigration and diminished accumulation of neutrophils to the site of injury or infection. Calcium signaling is an integral part of the integrin and chemokine signaling pathways that cooperate to synchronize the recruitment process. Although Orai1 expressed on circulating neutrophils is the predominant SOCE channel, it is clear that it cooperates in a dynamic sense with additional ion channels such as TRP, P2X, Potassium, and proton channels to maintain a balance in the electrochemical potential across the plasma membrane. As discussed, genetic deletion or pharmacological inhibition of individual channels disrupts a diverse set of functions from degranulation and ROS production to activation of β₂-integrin mediated cell adhesion and migratory functions. Calcium influx via CRAC channels provide a means by which neutrophils initiate cell activation within the plane of adhesive contact, where focal clusters of integrin bonds form and trigger IP₃-gated channels downstream of PLC to activate SOCE and synchronize the transition from cell rolling to arrest and shape polarization. Individuals with a point mutation in the Orai1 or STIM1 gene suffer from severe immune deficiencies with frequent infections due to insufficient immune cell recruitment to sites of inflammation as well as defects in hair, skin and sweat gland morphogenesis. Although, these observations point to an involvement of neutrophils in the development of diseases, PMNs of these patients show only moderate changes in intracellular Ca²⁺ levels. However, physiological shear stress is necessary to observe significant Ca²⁺ flux and with this integrin mediated outside-in signaling in neutrophils. Since the studies on neutrophil function in patients with Orai1 or STIM1 mutation were performed in the absence of shear, this, together with the assumed compensatory capacities of Orai and STIM isoforms might explain the discrepancy between preserved neutrophil and impaired immune function. Nevertheless, over the past 80 million years, human neutrophils have acquired even more ion channels than mouse neutrophils to regulate their activity. Key questions that remain are why neutrophils require so many ion channels and what are the detailed mechanisms by which neutrophils use their channels to carry out their relatively short-lived innate immune functions.