Lipopeptide Pam3Cys4 Synergizes N-Formyl-Met-Leu-Phe (fMLP)-induced Calcium Transients in Mouse Neutrophils

Abstract

N-Formyl-Met-Leu-Phe (fMLP), a mimic of N-formyl oligopeptides that are released from bacteria, is a potent leukocyte chemotactic factor. It induces intracellular calcium ([Ca²⁺]i) transient that is important for various neutrophil biological functions, e.g., adhesion, ROS and cytokine productions. Toll-like receptors (TLRs), an essential part of host innate immunity, regulate neutrophil activities, but their role in [Ca²⁺]i signaling is less clear. In the current study, we examined the effect of several TLR ligands, including Pam3Cys4 (TLR1/2), lipopolysaccharide (LPS, TLR4), and lipoteichoic acid (LTA, TLR2/6), on calcium signaling and on the fMLP-induced [Ca²⁺]i transients in mouse neutrophils loaded with Fura-2/AM. We found that unlike fMLP, the three TLR ligands tested did not elicit any detectable Ca²⁺ flux. However, Pam3Cys4, but not LPS or LTA, markedly synergized the fMLP-induced [Ca²⁺]i transients, and had no effect on the host component keratinocyte-derived cytokine (KC)- or C5a-induced calcium flux. The effect of Pam3Cys4 on the fMLP-induced [Ca²⁺]i transients is by enhancing extracellular Ca²⁺ influx, not intracellular Ca²⁺ release. Surprisingly, deletion of TLR2 or MyD88 in neutrophils had no impact on the Pam3Cys4’s effect, suggesting a TLR2-MyD88-independent mechanism. Finally, using the pan PKC activator and inhibitor, we demonstrated that PKC negatively regulated fMLP-induced [Ca²⁺]i transients and that inhibition of PKC did not prohibit Pam3Cys4’s synergistic effect on the fMLP-induced calcium influx. In conclusion, the current study identified a novel synergistic effect of Pam3Cys4 on fMLP-induced [Ca²⁺]i transients, a process important for many neutrophil biological functions.

INTRODUCTION

Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their activation may also cause tissue injury and contribute to autoimmune and inflammatory diseases (1, 2). Neutrophils express many cell surface receptors responsible for pathogen recognition and inflammatory responses. These include G-protein-coupled chemokine and chemoattractant receptors, various cytokine receptors, as well as Toll-like receptors (3).

N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP), a N-formylated tripeptide, is the prototypical representative of the N-formylated oligopeptides that are released from bacteria and possess potent chemoattractant property. fMLP attracts circulating leukocytes, such as neutrophils, and activates host innate immunity by binding to its G protein-coupled receptors (GPCRs) (4). Once activated, GPCRs lead to phospholipase C activation and inositol 1,4,5-triphosphate (IP3) production. IP3 binds to its receptor in the endoplasmic reticulum (ER) and results in rapid Ca²⁺ release from the ER stores, which subsequently results in Ca²⁺ influx across the plasma membrane by mechanisms generally referred to as store-operated calcium entry (5–7). Of importance, cellular Ca²⁺ hemostasis has profound impact on a variety of neutrophil functions, such as initiation of cytoskeletal changes, adhesion molecule presentation, cytokine production, oxidative burst, granule release and formation of neutrophil extracellular traps (NETs) (8–10). These neutrophil functions are very important for the host innate immune response to pathogen invasion as well as in the pathogenesis of sepsis.

Toll-like receptors (TLRs) play an essential role in pathogen recognition (11). Neutrophil TLR activation results in chemotaxis, increased phagocytosis, priming of superoxide generation, and production of a number of cytokines and chemokines (12, 13). For example, TLR recognition of some bacterial components, such as Pam3Cys4 (a TLR2 agonist), flagellin (a TLR5 agonist) and zymosan (a TLR2 agonist) can lead to a priming effect in neutrophils for subsequent fMLP-induced superoxide generation (12). However, whether or not these TLR ligands regulate fMLP-induced intracellular calcium transients in neutrophils remain unclear.

In the present study, we investigated the ability of several TLR ligands, including Pam3Cys4, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), in activating Ca²⁺ transients and its regulatory effect on fMLP-induced cytosolic Ca²⁺ response in neutrophils. We report that while TLR ligands does not activate Ca²⁺ transients in neutrophils, Pam3Cys4, not LPS or LTA, exhibits a synergistic ability to markedly enhance the fMLP-induced Ca²⁺ transients.

MATERIALS AND METHODS

Animals

Wild-type (WT) C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in an animal facility at Massachusetts General Hospital for at least one week before experiments. TLR2^−/− or MyD88^−/− mice in C57BL/6J background were generated by Takeuchi (14) or Kawai and colleagues (15), respectively. All mice used in the study were gender- and age-matched, 8–12 week-old and weighed between 20–30 g. All animals were housed in a pathogen-free, temperature-controlled, and air-conditioned facility with 12-h/12-h light/dark cycles and fed with the same bacteria-free diet (Prolab Isopro RMH 3000, LabDiet, Brentwood, MO) and water. The animal protocols used in the study were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital (Boston, MA). The experiments were performed in compliance with the guideline from the National Institutes of Health (Bethesda, MD). Simple randomization method was used to assign animals to various experimental conditions.

Reagents

Fura-2/acetoxymethyl ester (AM) was purchased from Molecular Probes (Eugene, OR). Pam3Cys4 and staurosporine were purchased from Enzo Life Science (Farmingdale, NY). Phorbol-12-myristate-13-acetate (PMA), fMLP, LPS (Escherichia coli 0111:B4), and lipoteichoic acid were from Sigma-Aldrich (St Louis, MO). Digitonin was from Biosynth (Napcr-ville, IL). C5a and keratinocyte-derived cytokine (KC) were purchased from R&D (Minneapolis, MN) and Peprotech (Rocky Hill, NJ), respectively.

Neutrophil isolation

Bone marrow cells were removed from both femur and tibia of mice as described previously (16). Cells were washed once with cold PBS and layered onto a Histopaque gradient (1,077 and 1,119) (Sigma-Aldrich, St. Louis, MO) and centrifuged at 700 g (without brake) for 30 min. Neutrophils were concentrated at the interface between 1077 and 1199. As we reported previously (16), the neutrophil purity was > 90% as determined by flow cytometry after Gr-1 and Ly-6G antibodies staining.

Fura-2-AM loading

Neutrophils were suspended in HBSS/BSA/Ca²⁺ buffer (pH 7.4, 20 mM HEPES, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 1% BSA) at a density of 2×10⁶ cells/ml and incubated with 1.5 µM Fura-2-AM for 60 min at 37°C. After washed twice with HBSS/BSA/Ca²⁺ buffer, cells were divided into 1×10⁶ cells/200 µl aliquots and placed on ice in dark until experiments. Right before each experiment, individual aliquots were incubated at 37°C for 5 min and then resuspended in 0.8 ml pre-warmed HBSS/BSA/Ca²⁺ buffer. In some experiments, cells were washed and resuspended in Ca²⁺-free HBSS/BSA buffer.

Quantification of [Ca²⁺]i

[Ca²⁺]i was measured by measuring Fura-2-AM fluorescence at 510 nm, using 340/380 nm dual-wavelength excitation in a fluorescent SpectraMax M5 Microplate Reader (Molecular Devices, Sunnyvale, CA). The interval time of fluorescence recording is 7 s. Cuvette temperatures were kept at 37°C. Calibration was performed at the end of each experiment by addition of 100 µM digitonin for the maximum fluorescence ratio and then 30 mM EGTA for the minimum fluorescence ratio. [Ca²⁺]i was then calculated from the 340/380 nm excitation fluorescence ratio (K_d = 220 nM) as described previously (17, 18). In our studies, dye leakage was small and had no influence on [Ca²⁺]i calculations. The different groups of samples were measured alternately to minimize the impact of the incubation time prior to fluorescent measurements. In each sample, the first 50 s recording was used as the unstimulated control and the total acquisition time was less than 10 min as indicated in each experiment.

Data and statistical analysis

The [Ca²⁺]i data of each sample was calculated as the Area Under the Curve (AUC) of the [Ca²⁺]i response above the mean of the baseline (pink area) as illustrated in Fig. 1. AUC represents the total net [Ca²⁺]i flux and is considered more representative of the cellular calcium response than the peak or maximal [Ca²⁺]i. It reduces the effect of artifacts on assessments and avoids the use of curve-smoothing programs. Statistical analysis was performed using Graphpad Prism 5 software (Graphpad, La Jolla, CA). Unless stated otherwise, the distributions of the continuous variables were expressed as the mean ± SD. The statistical significance of the difference between two groups was measured by two-tailed unpaired Students’ t test. Comparisons of more than two groups of data were performed by ANOVA with a post-hoc test as indicated. The null hypothesis was rejected for P < 0.05.

Figure 1. Diagram of the main experiment protocol and data analysis.

After Fura-2-AM loading, mouse neutrophils were treated with 1 µg/ml of P3C at 50 s, followed by 1 µM of fMLP at 150 s. [Ca²⁺]i signal was continuously recorded for up to 420 seconds and quantified as detailed in the Materials and Methods. The Area Under the Curve (AUC) above baseline (lower dashed line) (area in pink) was acquired for the final data analysis of [Ca²⁺]i. P3C: Pam3Cys4; fMLP: N-Formyl-Met-Leu-Phe.

RESULTS

TLR2 and TLR4 ligands do not induce calcium influx in neutrophils

To determine whether or not TLR activation leads to Ca²⁺ transients, mouse neutrophils were loaded with Fura-2/AM and treated with the specific TLR agonists, Pam3Cys4 (for TLR1/2), LPS (for TLR4), and LTA (for TLR2/6). All of the TLR ligands, at 1 µg/ml, failed to generate Ca²⁺ flux (data not shown). Higher dose of Pam3Cys4 (10 – 15 µg/ml) reportedly induces calcium flux in both airway epithelial cell and platelet cell (19, 20). But we found that neither 10 µg/ml nor 20 µg/ml of Pam3Cys4 induced Ca²⁺ transients in neutrophils (data not shown)

Pam3Cys4, but not LPS or LTA, enhances fMLP-induced calcium transients in neutrophils

To test whether or not TLR ligands regulate fMLP-induced Ca²⁺ flux, we pre-treated neutrophils with or without various TLR ligands prior to fMLP stimulation. As indicated in Fig. 2A, in the absence of TLR ligand pre-treatment, fMLP, at 0.5 µM and 1 µM, induced a dose-dependent increase in [Ca²⁺]i, both the peak and the AUC of [Ca²⁺]i (Fig. 2A). In comparison, pre-treatment with Pam3Cys4 at 1 µg/ml markedly enhanced the fMLP-induced intracellular calcium transients as measured by the AUC of [Ca²⁺]i (90% increase at 0.5 µM fMLP, P<0.001, and 50% increase at 1 µM fMLP, P<0.001, respectively). Moreover, the synergistic effect of Pam3Cys4 on fMLP-induced calcium flux was dose-dependent with 50% increase in [Ca²⁺]i at 1 µg/ml and 90% at 5 µg/ml, respectively (Fig. 2B). In contrast, the same dose of LPS or LTA (1µg/ml) had no effect on the fMLP-induced calcium flux (Fig. 2C).

Figure 2. Pam3Cys4 synergizes fMLP-induced Ca²⁺ flux in mouse neutrophils.

Prior to fMLP stimulation, mouse neutrophils were treated with the indicated TLR ligands. The equal volume of HBSS was used as the control. A: The effect of Pam3Cys4 (1 µg/ml) on [Ca²⁺]i transients induced by 0.5 and 1.0 µM of fMLP in neutrophils. B: The dose-dependent effect of Pam3Cys4 on fMLP-induced [Ca²⁺]i transients. C: The effect of P3C (1 µg/ml), LPS (1 µg/ml), or LTA (1 µg/ml) on fMLP-induced [Ca²⁺]i transients. Left column: representative [Ca²⁺]i transient recording; Right column: final quantitative [Ca²⁺]i transient data. Each error bar represents mean ± SD; n = 3–4 measurements from a representative experiment. * P<0.05, ** P<0.01, *** P<0.001. The experiments were repeated three times with separate cell preparations. P3C: Pam3Cys4; fMLP: N-Formyl-Met-Leu-Phe.

TLR2-MyD88 signaling does not mediate the synergistic effect of Pam3Cys4 on fMLP-induced calcium transients

Since Pam3Cys4 signals through TLR1/2 and MyD88, we tested whether or not Pam3Cys4 synergizes the fMLP-induced Ca²⁺ flux through TLR2-MyD88 signaling. To our surprise, Pam3Cys4 exhibited a similar synergic enhancing effect on fMLP-induced [Ca²⁺]i transients in neutrophils deficient of TLR2 or MyD88 (60% and 50% increase, respectively) (Fig. 3), suggesting that the synergistic Ca²⁺-promoting effect of Pam3Cys4 is independent of TLR2-MyD88 signaling.

Figure 3. Pam3Cys4 synergizes fMLP-induced Ca²⁺ flux in mouse neutrophils of WT, TLR2^−/−, or MyD88^−/− mice.

Bone marrow neutrophil isolated from WT or TLR2^−/− or MyD88^−/− mice were treated with Pam3Cys4 (1 µg/ml) followed by the stimulation of fMLP (1 µM). A–C: Representative [Ca²⁺]i transient recording in cells from WT (A), TLR2^−/− (B), or MyD88^−/− (C); D: Final quantitative [Ca²⁺]i data. Each error bar represents mean ± SD; n = 3–4 measurements from a representative experiment. *** P<0.001. The experiments were repeated three times with separate cell preparations. P3C: Pam3Cys4; fMLP: N-Formyl-Met-Leu-Phe; WT: Wild type; KO: knockout.

Pam3Cys4 promotes fMLP-induced extracellular Ca²⁺ influx

To determine the mechanism by which Pam3Cys4 enhances fMLP-induced Ca²⁺ transients, we tested whether Pam3Cys4 synergized fMLP-induced [Ca²⁺]i transients by enhancing extracellular Ca²⁺ influx or intracellular Ca²⁺ release from the ER. As shown in Fig. 4A–B, in the absence of extracellular Ca²⁺, fMLP induced a modest response in [Ca²⁺]i, which most likely represented Ca²⁺ released from intracellular storages. Surprisingly, Pam3Cys4 pre-treatment had no impact on the fMLP-induced intracellular Ca²⁺ release. Upon the addition of 1.8 mM CaCl₂ to the extracellular space in the assay buffer, fMLP-treated cells had a second and larger rise in [Ca²⁺]i, which was most likely the result of extracellular Ca²⁺ influx. Importantly, Pam3Cys4 pre-treatment markedly enhanced the fMLP-induced extracellular Ca²⁺ influx (Fig. 4).

Figure 4. The effect of Pam3Cys4 on fMLP-induced intracellular Ca²⁺ release and extracellular Ca²⁺ influx.

Mouse neutrophils were incubated in Ca²⁺-free buffer and treated with Pam3Cys4 (1 µg/ml) or equal volume of HBSS followed by treatment of fMLP (1 µM). After 150 seconds, 1.8 mM CaCl₂ was added into the assay buffer. A: Representative [Ca²⁺]i transient recording. B: Final quantitative [Ca²⁺]i data. Each error bar represents mean ± SD; n = 3–4 measurements from a representative experiment., ** P<0.01, *** P<0.001. The experiments were repeated three times with separate cell preparations. P3C: Pam3Cys4; fMLP: N-Formyl-Met-Leu-Phe.

Role of PKC in the Pam3Cys4-induced synergistic effect on [Ca²⁺]i

Previous study has demonstrated that fMLP-induced Ca²⁺ response is negatively regulated by PKC (21). As indicated in Fig. 5A, in the absence of Pam3Cys3, staurosporine, a PKC inhibitor, enhanced fMLP-induced [Ca²⁺]i transients in a dose-dependent and bi-phasic manner. At the concentrations between 0 – 0.5 µM, staurosporine induced an dose-dependent increase in [Ca²⁺]i transients, but then a dose-dependent decrease at the concentrations between 0.5 – 2 µM. To test whether or not PKC is involved in the synergistic effect of Pam3Cys4, PKC inhibitor (staurosporine) or activator (PMA) was used to test the impact of PKC inhibition or activation on Pam3Cys4-induced synergistic effect. As illustrated in Fig. 5B, activation of PKC by PMA not only completely inhibited fMLP-induced Ca²⁺ transients, but also eliminated the Pam3Cys4-elicited synergistic effect with fMLP. In contrast, inhibition of PKC by staurosporine markedly enhanced calcium response induced by fMLP. In the presence of PKC, Pam3Cys4 treatment was still able to induce additional increase in fMLP-induced Ca²⁺ influx.

Figure 5. The role of PKC in the Pam3Cys4-induced synergistic effect.

A: Dose response of the PKC inhibitor staurosporine on fMLP-induced [Ca²⁺]i transients. Mouse neutrophils were treated with staurosporine at 0.1, 0.5, 1.0 or 2.0 µM, or an equal amount of DMSO (0.5%) at 200 s before fMLP (1 µM) stimulation. B: The role of PKC in the Pam3Cys4-induced synergistic effect. Mouse neutrophils were treated with the PKC activator (PMA, 200 nM) or inhibitor (Staurosporine, 500 nM) or an equal amount of DMSO was added at 50 s. After 100 s, Pam3Cys4 (1 µg/ml) or an equal volume of HBSS (Control) was added and followed by the stimulation of fMLP (1 µM). Left: Representative [Ca²⁺]i transient recording. Right: Final quantitative [Ca²⁺]i transient data. Each error bar represents mean ± SD; n = 3–4 measurements from a representative experiment. ** P<0.01, *** P<0.001. The experiments were repeated three times with separate cell preparations. PMA: Phorbol-12-myristate-13-acetate; fMLP: N-Formyl-Met-Leu-Phe; Strauro: Staurosporine; P3C: Pam3Cys4.

Pam3Cys4 had no impact on KC or C5a-induced calcium transients in mouse neutrophil

To test whether Pam3Cys4 has similar effect on other chemokine-induced Ca²⁺ flux, we investigated KC or C5a, two chemoattractants known for their roles as Ca²⁺ flux triggers (22–24). As shown in Fig. 6, at 100 s after Pam3Cys4 pre-treatment, mouse neutrophils were stimulated with KC (50 nM), C5a (10 nM), or fMLP (1 µM). Interestingly, the [Ca²⁺]i flux recordings in response to KC or C5a were markedly different from that in fMLP-treated group (Fig. 6). Most importantly, Pam3Cys4 pre-treatment had no effect on KC or C5a-induced Ca²⁺ flux.

Figure 6. Pam3Cys4 has no impact on KC- or C5a-induced Ca²⁺ transients in mouse neutrophils.

Mouse neutrophils were treated with Pam3Cys4 (1 µg/ml) or HBSS 100 s prior to treatment with KC (50 nM), C5a (10 nM) or fMLP (1 µM). A: Representative [Ca²⁺]i transients. B: Final quantitative data. Each error bar represents mean ± SD; n = 3–4 measurements from a representative experiment. *** P<0.001. The experiments were repeated three times with separate cell preparations. KC: keratinocyte-derived cytokine; P3C: Pam3Cys4; fMLP: N-Formyl-Met-Leu-Phe.

DISCUSSION

In this study, we investigated the effect of various TLR ligands on intracellular calcium flux and on the chemoattractant fMLP-induced calcium transients in mouse neutrophils. We found that the three TLR ligands tested, Pam3Cys4, LPS, and LTA did not induce any detectable changes in [Ca²⁺]i. Pam3Cys4, a TLR1/2 ligand, markedly synergized the fMLP-induced calcium flux, while other TLR ligands, such as LPS (TLR4 ligand), LTA (TLR2/6 ligand), did not. Interestingly, this synergistic effect of Pam3Cys4 appears specific on fMLP, a mimic of bacterial wall component, as it had no effect on [Ca²⁺]i transients induced by the host components such as KC or C5a. We further identified that Pam3Cys4 specifically promoted the extracellular Ca²⁺ influx, without affecting intracellular ER Ca²⁺ release in response to fMLP. Finally, we demonstrated that the synergistic effect of Pam3Cys4 on fMLP-induced Ca²⁺ influx was completely inhibited by PKC activation, but remained intact even when PKC was inhibited.

Pam3Cys4 is a synthetic mimic of the N-terminal portions of lipopeptides that are present in the cell walls of both Gram-positive and Gram-negative bacteria and has been commonly identified as a TLR1/2 agonist. During lipoprotein biosynthesis, the two-step biosynthetic pathway in Gram-positive bacteria leads to diacylated mature lipoprotein. In Gram-negative and some Gram-positive bacteria, the diacylated lipoprotein can be further acylated at the N-terminal cysteine residue by lipoprotein N-acyl transferase (Lnt) to form triacylated lipoprotein. While it is true that Lnt is widely present in Gram-negative bacteria, its homologues have been identified in all classes of high GC Gram-positive bacteria and mycobacteria (25). Previous studies have shown that Pam3Cys4 induces calcium flux in the airway epithelial cells (19) and regulates Ca²⁺ mobilization in platelets (20). A more recent study suggests that TLR4 ligand LPS-induced extracellular ATP release was dependent on calcium mobilization in macrophages (26). However, in the current study, we did not detect any calcium response following TLR ligand treatment in mouse neutrophils, even at higher doses (such as Pam3Cys4 at 10 or 20 µg/ml). While we do not have a clear explanation, we speculate that different cell types and species may be the cause.

A few studies have looked at the blood concentrations of lipoprotein during bacterial infection and sepsis. In a mouse model of polymicrobial sepsis, Hellman and colleagues found that the levels of a bacterial lipoprotein (PAL) in the plasma are at least 128 ng/ml (27). Presumably, the concentration of the bacterial lipoprotein is much higher at bacterial infection site. Moreover, they found that bacteria could shed a large amount of its lipoprotein components into the blood. Four hours after exposed to E. coli, human sera contain the bacterial lipoprotein at concentrations between 2.2 – 3.96 µg/ml (28). Based on these reports, it seems reasonable to speculate that the concentrations of lipoprotein in the circulation during polymicrobial infection can reach a range between sub-µg to µg/ml and we have chosen 1.0 µg/ml of Pam3cys4 for the current studies.

Although Pam3Cys4 has been traditionally regarded as a TLR1/2 agonist, our results clearly suggest that Pam3Cys4 has biological effects that are independent of TLR2 signaling. Pam3Cys4 at 1 µg/ml, a dose also used in other functional tests (29, 30), synergized fMLP-induced calcium transients in neutrophils deficient of TLR2 or MyD88. Like the current study, our previous study shows that Pam3Cys4 induces ROS generation in mouse neutrophils and that TLR2 deficiency only attenuates the ROS production by approximately 30% (29). Moreover, Nguyen, et al. report that Pam3Cys4 enhances the binding of respiratory syncytial virus to the target cells in the absence of TLR2 (31). These data are consistent with the conclusion that Pam3Cys4 is able to exhibit its various biological effects via TLR2-dependent and independent mechanisms.

fMLP induces [Ca²⁺]i transients from two sources: intracellular ER Ca²⁺ release or extracellular Ca²⁺ influx. In Ca²⁺-free buffer, fMLP stimulation led to a moderate rise in [Ca²⁺]i, which indicates the intracellular Ca²⁺ release. The addition of Ca²⁺ to the extracellular solution caused a stronger and longer Ca²⁺ response, which represents the extracellular Ca²⁺ influx. Our data showed that Pam3Cys4 treatment markedly enhanced fMLP-induced extracellular Ca²⁺ influx, but had no impact on the intracellular ER Ca²⁺ release.

The PKC family consists of at least 10 members and is divided into three subfamilies based on the primary structures: conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC) (32). The role of PKC in regulating calcium influx has been intensely investigated in different cell types, such as smooth muscle cells and pulmonary endothelial cells (33, 34). PKC regulates store-operated Ca²⁺ entry (SOCE) or receptor-operated Ca²⁺ entry (ROCE) by phosphorylation of Ca²⁺ channel proteins of the Transient Receptor Potential (TRP) superfamily (such as TRPC1, 3–7) or Orai1 (35–39). To investigate whether Pam3Cys4 synergizes fMLP-induced calcium influx through PKC, we pre-treated neutrophils with the pan PKC activator or inhibitor. We found that activation of PKC by PMA not only markedly decreased fMLP-induced calcium flux, but also diminished the synergistic effect of Pam3Cys4. On the other hand, the PKC inhibition by staurosporine markedly increased fMLP-induced Ca²⁺ transients. Of note, in the presence of staurosporine, Pam3Cys4 further enhanced the fMLP-induced Ca²⁺ transients. Together, these data demonstrate that fMLP-induced extracellular Ca²⁺ influx is negatively regulated by PKC activity. The fact that Pam3Cys4 maintained its ability to enhance the fMLP-induced [Ca²⁺]i transients when PKC was fully inhibited by staurosporine suggests that other PKC-independent mechanisms may contribute to the synergistic effect of Pam3Cys4.

It has been well established that intracellular Ca²⁺ signals are triggered by various chemoattractants produced by either pathogen such as fMLP, or host such as C5a, KC, and IL-8 (22–24). To demonstrate the specificity of Pam3Cys4 on fMLP-induced calcium response, we tested the role of Pam3Cys4 on Ca²⁺ transients induced by KC and C5a. Surprisingly, while C5a has been proven to induce calcium flux through PKC regulation (40), Pam3Cys4 had no impact on either C5a- or KC-induced calcium response. These data suggest that the synergistic effect of Pam3Cys4 is indeed specific for the pathogen component fMLP.

The current study was performed in vitro in isolated neutrophils. While the in vitro cell system offers an advantage of delineating the synergistic effect among specific bacterial and host components, the in vivo relevance of the current finding that the two bacterial components synergize to enhance neutrophil Ca²⁺ transients will need to be tested in animal models of bacterial infection and sepsis. Nevertheless, given the importance of intracellular Ca²⁺ in regulating diverse neutrophil functions, the current study is significant and relevant to our understanding of how pathogen components work together to control neutrophil activation such as Ca²⁺ hemostasis.

In summary, the current study demonstrates that Pam3Cys4, a TLR1/2 ligand and synthetic mimic of bacterial lipopeptide, synergizes the bacterial chemoattractant fMLP-induced calcium influx in mouse neutrophils. This effect appears specific on fMLP and independent of TLR2→MyD88 signaling. Moreover, while PKC is well known for its inhibitory effect on fMLP-induced Ca²⁺ transients, the synergistic effect of Pam3Cys4 seems PKC-independent. Thus, our study identifies a novel biological effect of Pam3Cys4 in regulating intracellular Ca²⁺ hemostasis, a process essential for many neutrophil biological functions.