ATP Evokes Ca²⁺ Responses and CXCL5 Secretion via P2X₄ Receptor Activation in Human Monocyte-Derived Macrophages

Adenosine 5′-triphosphate is a danger-associated molecular pattern (DAMP) released during cellular stress and cell death at sites of inflammation and injury (1–3). The extracellular ATP DAMP signal can be sensed by leukocytes and mediates physiological responses via activation of cell surface P2 receptors: ligand-gated cation channel P2X (P2X_1–7) receptors and G-protein coupled P2Y (P2Y_{1,2,4,6,11–14}) receptors (4). Although the main agonist for P2X receptors is ATP, there are five major native ligands for P2Y receptors: ATP (P2Y₂, P2Y₁₁), ADP (P2Y₁, P2Y₁₂, and P2Y₁₃), UTP (P2Y₂, P2Y₄), UDP (P2Y₆), and UDP-glucose (P2Y₁₄) (5). The activation of ligand-gated P2X receptors leads to a direct increase in intracellular Ca²⁺ level. P2Y receptors, however, involve a more complex downstream signaling pathway with activation of G_q-coupled receptors (P2Y_1,2,4,6,11) resulting in activation of PLC/IP₃ pathway leading to increased Ca²⁺ concentration through release of stored endoplasmic reticulum Ca²⁺ and capacitative influx pathways, and activation of G_i-coupled receptors (P2Y_12–14) resulting in inhibition of adenylate cyclase and reduction in cAMP level (6).

P2X₁, P2X₄, and P2X₇ are commonly coexpressed by leukocytes (7, 8). P2X₁ and P2X₄ are maximally activated by low micromolar ATP levels (9), whereas the activation threshold for P2X₇ is at a much higher ATP concentration (>500 μM) (10–13). Although the role of P2X₇ receptor activation in leukocytes has received significant attention (14–19), the cellular role of P2X₄ is less well understood. Some evidence in mouse macrophage has emerged for the involvement of P2X₄ in the release of inflammatory mediators like PGE₂ (20) and in P2X₇-mediated autophagy (15). More recently, it was reported that P2X₇-mediated inflammation in mouse macrophage cell line is regulated by coexpression with P2X₄ through facilitation of IL-1β release (14). Interaction between P2X₄ and P2X₇ receptors has also been described as an important determinant of macrophage phenotypic function and their role in clearance of apoptotic cells following tissue damage (21). Despite these efforts, a functional role of the P2X₄ receptor in human macrophage remained elusive.

In the context of inflammation, macrophages possess three key roles, which include Ag presentation, phagocytosis, and immunomodulation through the production of cytokines, chemokines, and growth factors (22). Cytokines and chemokines are potent signaling molecules that are produced by many cell types, primarily those of the immune system, and have key roles in mediating cell-to-cell communication (23). Other roles of these proteins include the regulation of local and systemic inflammation to chemotaxis, tissue repair, cellular proliferation, and metabolism (23). In humans, CXCL5/ENA-78 is a proinflammatory chemokine that regulates CXCR2-dependent neutrophil trafficking. It is believed that platelets serve as the main source of CXCL5 under homeostatic conditions; however, during severe infection, CXCL5 is produced predominantly by lung epithelial cells (24, 25). In addition to this, a study has illustrated high levels of expression and secretion of the chemokine CXCL5 in the tissue-resident macrophage population of white adipose tissue (26). Despite being secreted by various cells, it has not been reported whether human macrophages are able to secrete CXCL5 upon sensing DAMP signals such as ATP, and to what end purinergic receptors play in this mechanism.

Here, we studied the functional role of P2X₄ receptor in human primary monocyte-derived macrophages (MDMs) as a surrogate cell model of primary tissue macrophages. Although studies have revealed some differentially expressed genes (27, 28) and proteins (29, 30), MDMs differentiated through treatment with GM-CSF have been typically considered to be phenotypically and “behaviorally” similar to human lung macrophages (31–33). In the lung, MDMs are recruited following inflammation in a CCR2-dependent manner (34) and have been shown to exacerbate pulmonary fibrosis in mice (35–41). In addition to this, it has been shown that increased CXC chemokine levels may serve as important pathogenetic mediators of pulmonary fibrosis (42, 43).

In the current study, several key questions were investigated: 1) the identification of purinergic receptors (P2X and P2Y) expressed in human MDMs, 2) characterization of purinergic receptors contributing to ATP-evoked intracellular Ca²⁺ ([Ca²⁺]_i) response in macrophages, and finally, 3) elucidating a functional role of P2X₄ receptor in human macrophages, particularly its roles in cytokine/chemokine production.

The following reagents were used: PSB-12062 and ATP (Sigma-Aldrich), A438079 (Abcam, Cambridge, U.K.), 5-BDBD, MRS2578, BX430, ivermectin (IVM), Ro0437626, NF340, MRS2211, Arc-118925xx, MRS2500, U-73122 (all from Tocris), recombinant human GM-CSF (rhuGM-CSF) (PeproTech), and Fura-2 AM (TefLabs).

Generation of overexpressing cells were performed in house. Blank 1321N1 astrocytoma cells (2.5 × 10⁵) were transduced with hP2X₄ mCherry or hP2X₇ mCherry lentiviral particles using spinoculation method (1600 × g; 60 min; 22°C). Supernatant was slowly aspirated and cells were gently resuspended in cell culture media to allow recovery in a T75 flask. Success of transduction was confirmed by visualization under fluorescence microscopy. To obtain highly expressing cells, transduced 1321N1 were sorted using BD FACSAria. All concentration responses and inhibition curves were performed in cells with low passages (P1–P4 cells) to ensure maximal response.

Peripheral venous blood was collected from healthy human volunteers through the National Health Service Blood and Transplant (Addenbrooke’s Hospital, Cambridge University Hospital, Cambridge, U.K.). Blood was layered on top of Histopaque-1077 (Sigma-Aldrich, Haverhill, U.K.) for centrifugation at 1000 × g for 25 min. Buffy coat layers were collected and PBMCs were counted. Cells were allowed to adhere onto T75 flasks (Corning, U.K.) for 2 h and cultured in RPMI 1640 with 2 mM l-glutamine, 2.5% heat-inactivated autologous serum, and 50 IU/ml penicillin and 50 μg/ml streptomycin at 37°C for 6 d with 10 ng/ml rhuGM-CSF (PeproTech).

Cells were loaded for 1 h with Fura-2 AM and treated with antagonists for 30 min. Measurements were made on a 96-well plate reader (FlexStation III; Molecular Devices). Change in [Ca²⁺]_i concentration is indicated as ratio of Fura-2 AM emission intensities at 340 and 380 nm (F ratio). SBS buffer contained (millimolars): 130 NaCl, 5 KCl, 1.2 MgCl₂, 1.5 CaCl₂, 8 d-glucose, 10 HEPES pH 7.4. Ca²⁺-free SBS was prepared by excluding CaCl₂ and supplemented with 2 mM EGTA. Loading of cells with Fura-2 AM was performed in SBS buffer supplemented with 0.01% (w/v) pluronic acid.

MDMs were detached using TrypLE Express (Thermo Fisher). Cells (1 ml at 0.5 × 10⁶/ml) were stimulated with either vehicle, antagonists, or modulators for 30 min followed by stimulation with 100 μM ATP for various time points (6, 9, 18, 24, 32, or 48 h). Supernatants were collected at different time points and cells were lysed using TRI Reagent (Sigma-Aldrich) for RNA extraction.

Cells (100 μl at 1 × 10⁶/ml) were incubated for 10 min at room temperature (RT) with Fc block (BD Biosciences) and immunostained with anti-human CD14 PE or hIgG isotype control PE (both from BD Biosciences) before acquisition on the Cytoflex instrument (Beckman Coulter). Positive staining was assessed based on gating of negative control (isotype control). Analyses were performed on CytExpert software.

Total RNA was isolated from cells using TRI Reagent (Sigma-Aldrich) and contaminating genomic DNA was eliminated using DNA-free kit (Ambion). cDNA was synthesized from 0.5 μg of total RNA using SuperScript II reverse transcriptase kit (Invitrogen). TaqMan primer probe sets for human CXCL5 (Hs01099660_g1), P2X₁ (Hs00175686_m1), P2X₂ (Hs04176268_g1), P2X₃ (Hs01125554_m1), P2X₄ (Hs00602442_m1), P2X₅ (Hs01112471_m1), P2X₆ (Hs01003997_m1), P2X₇ (Hs00175721_m1), P2Y₁ (Hs00704965_s1), P2Y₂ (Hs04176264_s1), P2Y₄ (Hs00267404_s1), P2Y₆ (Hs00366312_m1), P2Y₁₁ (Hs01038858_m1), P2Y₁₂ (Hs01881698_s1), P2Y₁₃ (Hs03043902_s1), P2Y₁₄ (Hs01848195_s1), GAPDH (Hs02758991_g1), and RPLP0 (Hs99999902_m1) were obtained predesigned from Applied Biosystems. Real-time RT-PCR was performed in a 7500 Fast Real-Time PCR instrument (Applied Biosystems). Target gene expression was normalized to GAPDH endogenous control and relative quantification was done by the ΔΔCT method.

Human Cytokines and Chemokines RT² Profiler PCR Array (Qiagen) was employed to screen 84 genes central to immune response according to the manufacturer’s instructions. Real-time PCR was performed on 7500 fast instrument (Applied Biosystems). Values were corrected against the geometric mean of five housekeeping genes (ACTB, B2M, GAPDH, RPLP0, and HPRT1) and analyzed using the ΔΔ crossing time method. Data were represented as heat map generated by Matrix2png and recalculated as log₂ fold change whereby the value 0 denotes no change in expression, positive value denotes upregulation, and negative value denotes downregulation.

The amount of proteins secreted in the supernatants of stimulated cells was quantified using either the Milliplex MAP Kit Human Th17 Magnetic Bead Panel 96-well plate assay (IFN-γ, IL-17A, IL-6, IL-23, IL-17E, IL-27, IL-12(p70), and IL-33) or Milliplex MAP Kit Human Cytokine/Chemokine Magnetic Bead Panel 96-well plate assay (IL-10, IL-8, RANTES, Eotaxin, IL-13, IL-4, IL-5, IL-9, and MDC) (both from Millipore, U.K.) according to the manufacturer’s instructions.

The amount of CXCL5 protein secreted in supernatants of stimulated cells was quantified using huCXCL5 ELISA kit (BioLegend, Cambridge, U.K.) according to manufacturer’s instructions.

Cells (1 ml at 2.5 × 10⁴/ml) were fixed with 4% paraformaldehyde (15 min, RT) and permeabilized with 0.25% Triton X-100 (10 min, RT). Cells were blocked with 1% BSA (30 min, RT) and incubated overnight at 4°C with the following primary Abs: rabbit polyclonal P2X_4,7 and P2Y_11,13 (Alomone, Israel), goat polyclonal P2X₁,₅ and P2Y₁₂, and rabbit polyclonal P2Y₁,₂ (Santa-Cruz Biotechnology, USA). Cells were stained with secondary Ab goat anti-rabbit (Life Technologies) or rabbit anti-goat (Abcam) Alexa Fluor 488. Nuclear staining was performed with Vectashield Antifade containing DAPI (Vector Laboratories, U.K.). Cell imaging was performed using laser-scanning confocal microscope Zeiss LSM510 META (Zeiss).

Data were analyzed using Origin Pro 9.0 software (Origin Laboratory). Concentration-response curves were fitted assuming a Hill coefficient of 1. Peak response was defined as the highest point of magnitude following agonist stimulation (represented by F ratio) and measurement of sustained phase was calculated using: 1) area under the curve recorded between 0 and 230.4 s and 2) decay kinetics (τ) value. Hypothesis testing for experiments with paired datasets were performed using paired Student t test. Data are expressed as mean ± SEM.

Human primary MDMs were obtained by the differentiation of monocytes following 6 d stimulation with rhuGM-CSF. Morphological characteristics of these MDMs were confirmed through bright field image system, where the majority of the population had a round and granular “fried-egg” structural feature (Fig. 1A), a main hallmark of GM-CSF–derived MDMs (GM-MDMs) (44–47). Forward and side scatter plot from flow cytometry illustrated that the MDM population makes up ∼69.8 ± 5.1% of the entire culture population (P2 gate, Fig. 1B, left panel). Further characterization of these cells using FACS analysis illustrated that only 70.5 ± 4.6% of the MDM population are CD14⁺ (Fig. 1B, right panel). This is not surprising as CD14 is a known surface marker of monocytes that is downregulated by GM-CSF upon differentiation toward macrophages at both the mRNA and protein level (48, 49).

Expression of purinergic receptors (P2X and P2Y) in MDMs were investigated at the mRNA and protein level. Quantitative RT-PCR (qRT-PCR) analysis identified mRNA expression of all P2X genes with the exception of P2X₂, P2X₃, and P2X₆, and all P2Y genes with the exception of P2Y₁₂ and P2Y₁₄ (Fig. 1C). Exclusion for genes was defined so that genes amplified above cycle threshold 35 were determined as “absent.” Using immunocytochemistry, protein expression of purinergic receptors in MDMs was also studied. P2X₁, P2X₄, P2X₅, P2X_7, P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, and P2Y₁₃ were all expressed in MDMs at the protein level. Meanwhile, no positive staining was observed for P2Y₁₂ receptor in the MDMs.

ATP evoked a concentration-dependent increase in [Ca²⁺]_i response in both the presence and absence of extracellular Ca²⁺ (EC₅₀ = 11.4 ± 2.9 μM in presence of extracellular Ca²⁺ versus EC₅₀ = 9.77 ± 2.4 μM in absence of extracellular Ca²⁺, Fig. 2A). Responses to ATP (100 μM) were biphasic: an initial rapid [Ca²⁺]_i response which peaked and was followed by a modestly decaying sustained phase which returned to baseline level (Fig. 2B). Throughout the study, 100 μM ATP was used as the agonist concentration, as our intracellular Ca²⁺ measurement data in overexpressing cells illustrated maximal activation of P2X₄ and no activation of P2X₇ (Supplemental Fig. 1A, 1E).

Preincubation of MDMs with 10 μM PLC inhibitor (U-73122) almost completely abolished the rapid peak in ATP-evoked [Ca²⁺]_i response (90.0 ± 0.96% inhibition, n = 3, p < 0.005, Supplemental Fig. 2E), indicating that the majority of the ATP-evoked [Ca²⁺]_i response in MDMs are dependent on PLC activation. To identify and characterize which receptors are crucial for ATP-evoked [Ca²⁺]i response in MDMs, various P2Y receptor antagonists were tested. Pretreatment of MDMs with P2Y₁ (MRS2500, 1 μM), P2Y₂ (ARC-118925xx, 10 μM), or P2Y₆ (MRS2578, 10 μM) antagonists had no significant effect on the amplitude of ATP-evoked [Ca²⁺]_i response (Supplemental Fig. 2F–K). The amplitude of the ATP-evoked [Ca²⁺]_i response in MDMs was mediated by coactivation P2Y₁₁ and P2Y₁₃, as pretreatment of MDMs with selective P2Y₁₁ antagonist (NF340, 10 μM) caused a 21.4 ± 5.1% inhibition (n = 3, p < 0.05, Fig. 2C, 2F) and P2Y₁₃ antagonist (MRS2211, 10 μM) caused a significant inhibition of 30.4 ± 2.1% (n = 3, p < 0.001, Fig. 2D, 2F). In the presence of both antagonists, ATP-evoked [Ca²⁺]_i response was significantly inhibited by 45.9 ± 4.3% (n = 3, p < 0.001, Fig. 2E, 2F).

To investigate whether P2X receptors could also contribute to the ATP-evoked [Ca²⁺]_i response in MDMs, several P2X antagonists and modulators were tested. Pretreatment of MDMs with a selective P2X₁ receptor antagonist (Ro0437626, 30 μM) did not have any inhibitory effect; in fact, it potentiated the amplitude of the ATP-evoked [Ca²⁺]_i response by 31.3 ± 11.4% (n = 4, p < 0.01, Supplemental Fig. 2A, 2B). Selective antagonism of P2X₇ receptor with (A438079, 5 μM) had no significant effect on the [Ca²⁺]_i response (Supplemental Fig. 2C, 2D). Finally, the contribution of P2X₄ receptor toward ATP-evoked [Ca²⁺]_i response was tested using several pharmacological tools: a positive allosteric modulator, IVM (50), and selective antagonists, PSB-12062 (51) and BX430 (52). Pretreatment of MDMs with IVM (3 μM) significantly potentiated the amplitude of [Ca²⁺]_i response (18.2 ± 4.58% potentiation, n = 12, p < 0.01, Fig. 3A) and significantly increased the net calcium movement as quantified by area under the curve (198.81 ± 27.91% increase, n = 12, p < 0.01, Fig. 3B). Pretreatment of MDMs with PSB-12062 (10 μM) caused a very minor effect on the amplitude of the [Ca²⁺]_i response (7.7 ± 2.7% inhibition, n = 12, p < 0.05, Fig. 3C) but had a more significant effect in inhibiting the duration of [Ca²⁺]_i response in response to ATP (38.97 ± 7.49% inhibition, n = 12, p < 0.01, Fig. 3D). Pretreatment of MDMs with newly identified selective antagonist BX430 produced a similar effect to PSB-12062, whereby 5 μM BX430 had a minor effect on the peak magnitude of ATP-evoked Ca²⁺ response (Fig. 3E), although significantly inhibiting net calcium movement (57.72 ± 12.65% inhibition, n = 5, p < 0.05, Fig. 3F). The coapplication of P2X₄ antagonists (10 μM PSB-12062 or 5 μM BX430) was performed in the presence of both P2Y₁₁ antagonist (10 μM NF340) and P2Y₁₃ antagonist (10 μM MRS2211) to further illustrate the contribution of these receptors to ATP-evoked Ca²⁺ response in human MDMs. In the presence of P2Y₁₁ and P2Y₁₃ receptor antagonists, 5 μM BX430, but not 10 μM PSB-12062, resulted in a further inhibition of peak magnitude of the ATP-evoked Ca²⁺ response (45.88 ± 4.29% inhibition without BX430 versus 51.73 ± 2.85% with BX430, n = 4, p < 0.0.5, Fig. 3G, Supplemental Table I). To provide further evidence that activation of P2X₄ receptor, but not P2Y₁₁ or P2Y₁₃ receptor, contributed toward the duration and shape of the Ca²⁺ response, decay kinetics of Ca²⁺ response in the presence of these antagonists were quantified. P2X₄ receptor antagonists (PSB-12062 or BX430) significantly increased response decay kinetics (Supplemental Table I). Therefore, although P2Y₁₁ and P2Y₁₃ receptor activation contributes to the amplitude of the ATP-evoked [Ca²⁺]_i response in MDMs, the activity of P2X₄ receptor contributes to the duration of response.

To investigate the potential role of P2X₄ in cytokine and chemokine expression in MDMs, an RT² profiler PCR array that screened for 84 different genes was performed, following stimulation with 100 μM ATP in the presence or absence of 10 μM PSB-12062 for 6 h. MDMs were also stimulated with 100 ng/ml LPS for 6 h, as a positive control. A heat map summarizing the gene expression data for 74 different genes is illustrated in Fig. 4A. Eight genes (BMP4, CXCL10, CXCL11, CXCL12, IL-2, IL-17A, TNFRSF11B, and XCL1) were excluded from the heat map as they were found at undetectable levels in all conditions (unstimulated, 100 μM ATP and 100 ng/ml LPS). Interestingly, the majority of the genes investigated were found to be constitutively expressed in MDMs (data not shown), although stimulation of MDMs with 100 μM ATP resulted in the induction of various genes, with CXCL2, CXCL5, IL-12α, IL-12β, OSM, PPBP, and TGF-β2 being positively induced (Fig. 4B). As a positive control, 100 ng/ml LPS significantly upregulated the mRNA expression of not <35 genes as indicated in red in Fig. 4A. In addition to this, the effect of 100 μM ATP stimulation was investigated at the protein level for several cytokines and chemokines, although no significance was observed (Supplemental Fig. 3). Out of the proteins investigated, one chemokine (eotaxin) and 11 cytokines (IL-13, IL-9, IL-4, IL-5, IFN-γ, IL-12p70, IL-17A, IL-33, IL-23A, IL-6, and IL-17E) were found to be below the detectable range (data not shown).

To identify potential candidates, several criteria were applied. These criteria included genes that: 1) were induced by stimulation of ATP, 2) were unaffected by pretreatment of antagonist (10 μM PSB-12062) alone, and 3) had their mRNA expression altered when the antagonist was present together with the agonist. Taking these criteria together allowed the identification of CXCL5 as a candidate gene for further study in the MDMs. ATP induced a time-dependent increase in mRNA expression of CXCL5 in MDMs across four different time points (6, 9, 18, and 24 h) with expression peaking at 24-h time point (23.5 ± 5.0 fold upregulation, n = 4, Fig. 4C). Having identified a role for P2X₄ receptor in modulating CXCL5 mRNA expression, we further investigated its effects at the protein level. Similar to mRNA expression, stimulation of MDMs with ATP also caused a time-dependent increase in CXCL5 protein secretion as quantified by ELISA across four different time points (0, 24, 32, and 48 h) with secretion peaking at 48-h time point (686.9 ± 148.3 pg/ml, n = 7, p < 0.01, Fig. 4D). The level of secreted CXCL5 protein was only detectable at higher time points, as at 6 and 9 h no significant secretion was yielded (data not shown).

In the presence of ATP, pretreatment of MDMs with PSB-12062 caused a significant reduction in ATP-induced CXCL5 mRNA expression at lower time points (6 h = 40.4 ± 4.9% reduction, n = 5, p < 0.01; 9 h = 34.8 ± 7.2% reduction, n = 4, p < 0.05; Fig. 5A). This inhibition was found to be translated to the protein level as pretreatment of MDMs with PSB-12062 also caused a significant reduction in ATP-induced CXCL5 secretion at higher time points (32 h = 22.9 ± 8.5% reduction, n = 6, p < 0.01; 48 h = 30.70 ± 6.29% reduction, n = 7, p < 0.001; Fig. 5B, 5C). To ensure the selectivity of the compound, we also investigated the effect of lower concentrations of PSB-12062 on MDM cells. PSB-12062 caused a concentration-dependent inhibition in ATP-induced CXCL5 secretion at 48 h (1 μM = 12.0 ± 4.6% inhibition, n = 3, p < 0.05; 5 μM = 26.4 ± 7.2% inhibition, n = 4, p < 0.05; Fig. 5D). Similar to the inhibitory effect observed in the presence of PSB-12062, pretreatment of MDMs with 5 μM BX430 caused a significant inhibition in ATP-induced CXCL5 secretion at 48 h (20.07 ± 2.05% inhibition, n = 7, p < 0.01, Fig. 5E).

In addition to this, the effect of IVM was also tested on MDMs prior to stimulation with ATP. Pretreatment of MDMs with IVM caused a significant increase in the amount of CXCL5 protein secretion at 48-h time point (26.48 ± 10.70% potentiation, n = 6, p < 0.05, Fig. 5F). In an effort to understand the mechanism by which P2X₄ receptor stimulates CXCL5 secretion, we performed experiments in the absence of extracellular Ca²⁺ to investigate a dependency upon Ca²⁺ influx. In these experiments, the absence of extracellular Ca²⁺ caused a significant reduction in ATP-induced CXCL5 secretion (41.80 ± 5.36% inhibition, 48 h, n = 7, p < 0.001, Fig. 5G) versus control experiments in the presence of extracellular Ca²⁺. Furthermore, the inhibitory action of PSB-12062 and BX430 on ATP-induced CXCL5 secretion was lost in the absence of extracellular Ca²⁺ (Fig. 5H). Together, these data illustrated a role of P2X₄ receptor activation in the modulation of ATP-induced CXCL5 mRNA expression and secretion.

To investigate the potential involvement of other P2 receptors, the effect of P2X₇, P2Y₁₁, and P2Y₁₃ receptor antagonists on ATP-mediated secretion of CXCL5 was studied. Pretreatment of MDMs with A438079 (P2X₇, Fig. 6A), NF340 (P2Y₁₁, Fig. 6B), or MRS2211 (Fig. 6C) had no significant inhibitory effect on ATP-evoked CXCL5 protein secretion. When applied together, 10 μM NF340 and 10 μM MRS2211 also had no significant effect on ATP-induced CXCL5 secretion (Fig. 6D). These data illustrate that P2X₇, P2Y₁₁, and P2Y₁₃ receptors are unlikely to play a role in modulating ATP-induced CXCL5 secretion in human MDMs.

In the macrophage system, DAMP signals (such as ATP) work as extracellular signaling molecules that are essential for intercellular communication through activation of P2 receptors (53). Although various P2 receptors have been shown to be expressed in macrophages, it is unclear which P2 receptors are responsible for ATP-evoked [Ca²⁺]_i in human MDMs. Through qRT-PCR analysis and immunocytochemistry, the current study illustrates that human MDMs express four P2X receptors, P2X₁, P2X₄, P2X₅, and P2X₇, and all P2Y receptors, with the exception of P2Y₁₂ and P2Y₁₄. Our data corroborate a previous finding which reported the presence of all P2 receptors except P2Y₁₂, P2X₂, P2X₃, and P2X₆ on human alveolar macrophages (54). Next, Ca²⁺ experiments were used as an indication that functional P2X and P2Y receptors were present on human MDM cells. Through intracellular Ca²⁺ measurements of Fura-2 AM loaded cells, we have shown that ATP-induced [Ca²⁺]_i response in human MDMs exhibited a biphasic kinetic. The first component of the [Ca²⁺]_i response showed features of a metabotropic mechanism, lacking dependence on extracellular calcium and high sensitivity toward PLC inhibition. In addition to this, the activation of P2Y₁₁ and P2Y₁₃ receptors appeared to be essential for the amplitude of the ATP-evoked [Ca²⁺]_i response in human MDMs. Treatment of MDMs with antagonists for P2Y₁, P2Y₂, and P2Y₆ did not have any effect on the amplitude and decay phase of the ATP-evoked Ca²⁺ response in macrophages, suggestive of their limited role. This was an interesting observation as P2Y₂ and P2Y₆ appear to have been found functional through intracellular Ca²⁺ measurements in monocytes and PBMCs (55, 56). The second delayed phase of ATP-evoked [Ca²⁺]_i response exhibited characteristics of an ionotropic response with P2X₄ receptor, but not P2X₁ or P2X₇ receptors, contributing to this response. Selective P2X₄ receptor antagonists, PSB-12062 and BX430, significantly accelerated the decay kinetics of the Ca²⁺ response, causing a consequential reduction in area under the curve and reducing the time it takes for the ATP-evoked Ca²⁺ decay response to return to the baseline. Although Nörenberg et al. identified that IVM was also able to potentiate currents of human P2X₇ receptor in human MDMs, it was clarified that the effect it has on delaying decay of currents is specific for P2X₄ receptor (50). In our MDM system, we were able to illustrate that IVM potentiated not only the amplitude of the [Ca²⁺]_i response but also delay of the decay kinetics. The effect of IVM in increasing the duration of [Ca²⁺]_i are therefore inconsistent with a role for P2X₇; furthermore 100 μM ATP is significantly below the activation threshold for P2X₇ (13). The effect of IVM is consistent with positive allosteric modulation of P2X₄ (9, 57).

Transcriptional profiler array study identified that in human MDMs, 100 μM ATP induced the expression of various cytokine and chemokine genes that are relevant to the immune system, with CXCL5 being a major candidate that was studied further. To the best of our knowledge, there has been no reported evidence that human MDMs are able to synthesize and secrete CXCL5 in response to ATP stimulation. CXCL5, a ligand of CXCR2 receptor, is typically expressed in inflammatory cells in various tissues (58). They belong to the CXC family of chemoattractant molecules specialized in the modulation of neutrophil chemotaxis and chemokine scavenging (59, 60). Here, we showed that human MDMs are not only able to transcriptionally express mRNA of CXCL5 genes but also synthesize and secrete CXCL5 protein, in a time-dependent manner. This expression and secretion of CXCL5 was found to be modulated by the activation of P2X₄, as illustrated by the effect of IVM and PSB-12062. However, as the PSB-12062 effect was only observed at higher time points (after 32 h), it is likely that rather than regulating the secretion of CXCL5, P2X₄ activation modulates the synthesis of ATP-induced CXCL5. In addition to this, we also tested the possible involvement of other purinergic receptors on ATP-induced CXCL5 secretion. The lack of effect of selective antagonists for P2X₇, P2Y₁₁, and P2Y₁₃ receptors indicate that these receptors are not involved in modulating the secretion of CXCL5 in human MDMs, despite their potential involvement in ATP-evoked [Ca²⁺]_i response. Earlier studies by a group found that elevation in [Ca²⁺]_i is required, but not necessarily sufficient for the release of certain cytokines and chemokines in brain macrophages (microglia) (61). The lack of involvement of P2Y₁₁ and P2Y₁₃ receptors in mediating ATP-driven CXCL5 secretion led to the hypothesis that it may involve a process independent of calcium level. However, when the level of ATP-induced CXCL5 secretion was investigated in conditions where extracellular Ca²⁺ was removed, we found that there was a significant inhibition in secreted CXCL5 level and to our surprise, to a similar level obtained when P2X₄ receptor was blocked with either PSB-12062 or BX430. More interestingly, in the absence of extracellular Ca²⁺, the inhibitory action of P2X₄ antagonists was lost. Altogether, these observations demonstrate that ATP-induced CXCL5 secretion in human MDMs is dependent on Ca²⁺ influx via the P2X₄ receptor.

It was not until very recently that the mechanism involved downstream of CXCL5 was revealed in greater detail. CXCL5 has been shown to activate ERK, JNK, p38, and MAPK signaling pathways, all of which are key players in tumor growth and metastasis (62). In the context of inflammatory diseases, CXCL5 has been shown to offer an atheroprotective role by enhancing cholesterol efflux capacity of macrophages and regulating foam cell formation (63). More recent studies revealed that tissue-resident CCR2⁺ MDMs are essential mediators of neutrophil recruitment into ischemic myocardial tissue. This process is mediated by a TLR9/MyD88/CXCL5 pathway with CXCL5 playing a critical role in guiding neutrophil crawling, as shown through photon imaging system (64). In this present study, we are able to consider that upon stimulation with DAMP signal, human MDMs synthesize and secrete CXCL5, which can then in turn activate neutrophils and recruit them to sites of injury. Although the mechanism by which P2X₄ receptor activation modulates expression and secretion of CXCL5 in human MDMs has not been investigated here, several speculations can be made. Studies by Song et al. (65) illustrated that proinflammatory chemokine CXCL5 was found to be rapidly upregulated by local presence of IL-1β and that its action was potentiated by MMP-2 and MMP-9, working synergistically to initiate neutrophil recruitment. It is possible that P2X₄ receptor activation modulates ATP-induced CXCL5 secretion by regulating IL-1β levels. Recent structural and functional studies provided evidence of possible interactions of P2X₄ receptor with P2X₇ receptor in macrophages, which was followed by a study illustrating that the expression of P2X₄ receptor is required for P2X₇ receptor–dependent IL-1β release in mouse bone marrow–derived dendritic cells (66, 67). In addition to this, a study by Zineh et al. (68) illustrated that treatment of HUVECs with atorvastatin reduced IL-1β–induced CXCL5 levels in a concentration-dependent manner. Whether this effect involves P2X₄ activity is unclear; however, treatment of human monocytes with fluvastatin has resulted in suppression of P2X₄ activity through depletion of cholesterol levels (69). Further studies will undoubtedly be required to unravel the mechanism by which P2X₄ regulates CXCL5 synthesis and secretion in human macrophages. Of further interest is the recent identification of CXCL5 as mediator in inflammatory pain arising from UVB irradiation of skin (sunburn) (70).

In conclusion, our data show that both P2X and P2Y receptors are responsible for ATP-evoked [Ca²⁺]_i response in human MDMs. P2Y₁₁ and P2Y₁₃ receptor activation underlies the amplitude of ATP-evoked [Ca²⁺]_i response, whereas P2X₄ receptor activity is responsible for the duration of Ca²⁺ response. Additionally, we have elucidated a potential role for P2X₄ receptor as a modulator of ATP-induced CXCL5 expression and secretion in human MDMs, which may be important in the recruitment of neutrophils. Although additional work will be required to uncover the mechanism of P2X₄-CXCL5 interaction in human macrophages, the current study exposed a novel role of P2X₄ in human macrophages and its contribution toward the innate immune system.

The authors have no financial conflicts of interest.