Sulfhydryl Modification Induces Calcium Entry through IP3-Sensitive Store-Operated Pathway in Activation-Dependent Human Neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT1MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.


Introduction
As the first line of host defense against invasion of pathogenic microbes, neutrophils play a crucial role in a variety of inflammatory responses [1,2]. In generally, researchers have known that there are two main states for neutrophils: resting and activation. The activation of the human neutrophils leads to a spectrum of responses, including aggregation, stimulation of the respiratory burst and degradation of microbes. During the transition of neutrophils from resting to activation state, the increase of cytosolic calcium concentration ([Ca 2+ ] c ), due to the calcium release and calcium entry, has been considered as the major intracellular modification factor [3,4]. As a second messenger, [Ca 2+ ] c executes profound effects across a wide range of physiological functions and signaling transductions in neutrophil immunity responses, including the initiation of cytoskeletal changes, degranulation, adhesion, apoptosis, and oxidative burst [5]. In particular, store-operated calcium entry (SOCE), a critical mechanism of [Ca 2+ ] c regulation in non-excitable cells [6][7][8], plays a crucial role in modulating the immune responses of neutrophils [9][10][11].
Sulfhydryl groups, a key component of proteins, can be modified by different agents through oxidation, reduction, or alkylation. These modifications play a significant role in various signaling transductions though affecting the activity and structure of different kinds of enzymes, receptors, and ion channel proteins [12,13]. For instance, N-ethylmaleimide (NEM), a irreversible sulfhydryl-alkylating agent, has long been used for the chemical modification of sulfhydryl groups of cysteine residues in various cells types [14][15][16]. More recently, studies have indicated that nitric oxide (NO) conveys a large part of ubiquitous influence via S-nitrosylation of sulfhydryl groups for providing redox-based physiological regulation [17]. S-Nitrosylation of proteins, a modification of cysteine residues by NO [18], first demonstrated by Stamler [19], performs an important regulatory role in cell apoptosis [20], ion channel activation [21], mitochondrial caspases modulation.
For resting rat neutrophils, Wang et al demonstrated that alkylating agent NEM and NO donor 5-amino-3-(3,4-dichlorophenyl)-1,2,3,4-oxatriazolium (GEA3162) just stimulated calcium entry through a non-store-operated pathway via direct sulfhydryl modification [22][23][24]. In addition, Gill et al reported that NEM and membrane-permeant NO donors could activate non-storeoperated calcium entry through sulfhydryl modification in DDT 1 MF-2 cells [25][26][27]. However, in phorbol 12-myristate 13acetate (PMA)-activated human neutrophils, we have previously shown that NO donor sodium nitroprusside (SNP) induced calcium release from inositol 1,4,5-trisphosphate (IP 3 ) receptorsensitive stores via S-nitrosylation of sulfhydryl groups [28]. These reports indicated that sulfhydryl modification had different effects on [Ca 2+ ] c of activated neutrophils as compared to resting neutrophils. The present study investigated the calcium entry mechanism induced by the alkylation agent NEM and the exogenous NO donor SNP in PMA-activated human neutrophils. We asked whether the effects of NEM and SNP on [Ca 2+ ] c were due to the activation of neutrophils. Data showed that NEM or SNP-triggered [Ca 2+ ] c increase was due to a IP 3 receptor-sensitive calcium release and SOCE via sulfhydryl modification in PMAinduced activation-dependent human neutrophils.

TG or FMLP induces SOCE in PMA-activated neutrophils
PMA, a potent protein kinase C (PKC) activator, can activate NADPH oxidase by stimulating PKC and subsequently induce respiratory burst in neutrophils [29,30]. In our experiments, resting human neutrophils were activated by incubation with 2 mM PMA for 5 min at 37uC, which caused adhesion to the glass surface resulting in observable flattening of the cells with differential interference contrast (DIC) microscopy ( Fig. 1A and B). To determine whether the mechanism of SOCE was in use after PMA activation, we measured the [Ca 2+ ] c responses to thapsigargin (TG) [31,32] and N-formyl-methionyl-leucyl-phenylalanine (FMLP) [33,34], two powerful activators of SOCE in neutrophils. Figure 1 showed the effects of TG and FMLP on [Ca 2+ ] c of PMA-pretreated neutrophils. Addition of 2 mM TG induced a sharp rising of [Ca 2+ ] c due to calcium store release in Ca 2+ -containing medium, followed by a second extended rising of [Ca 2+ ] c (Fig. 1C). The latter peak represented extracellular calcium entry and was abolished when the medium was replaced with nominally Ca 2+ -free HBSS as shown in Figure 1C and D. Moreover, readdition of 1 mM external calcium evidently resulted in a repeat of calcium entry ( Fig. 1C and D), indicating the familiar response for SOCE. As illustrated in Figure 1G and H, FMLP (2 mM) showed the same effects on [Ca 2+ ] c in the presence or absence of external calcium in PMA-activated neutrophils. In addition, when 75 mM 2-Aminoethoxydiphenyl borate (2-APB) was added during the entry phase, it rapidly and obviously abolished calcium entry (Fig. 1E). When 75 mM 2-APB was added together with TG, it had little effect on the calcium release but completely inhibited the calcium entry component of [Ca 2+ ] c in Ca 2+ -containing buffer (Fig. 1F). It is well known that 2-APB is a potent membrane-permeant antagonist of IP 3 receptor [35][36][37][38] and a reliable blocker of SOCE [9,38,39]. Taken together, these results clearly indicated that the SOCE mechanism still occurred in PMA-activated neutrophils.

NEM or SNP induces SOCE via sulfhydryl modification in PMA-activated neutrophils
As shown in Figure 2A and B, NEM (100 mM) or SNP (500 mM) induced a rapid [Ca 2+ ] c increase followed by a second extended rising of on [Ca 2+ ] c in PMA-preincubated neutrophils within Ca 2+ -containing HBSS. When cells were pretreated with NEM (500 mM), the effect of SNP on [Ca 2+ ] c was significantly abolished (gray line in Fig. 2B). Our previous work [28] also demonstrated that application of 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz (b-1,4) oxazin-1-one (NS2028), a specific inhibitor of soluble guanylate cyclase, had no effect on SNP-induced the rising of [Ca 2+ ] c in PMA-activated neutrophils. These results showed that two kinds of sulfhydryl modification, alkylation by NEM and Snitrosylation by SNP, could cause obvious [Ca 2+ ] c increase in PMA-activated neutrophils.
Moreover, in the absence of external Ca 2+ , the increase of [Ca 2+ ] c was still observed with the latter peak being completely blocked, indicating that calcium stores release and calcium entry were co-existing (Fig. 2C). To further explore the relationship between calcium stores release and calcium entry, [Ca 2+ ] c were triggered with NEM (100 mM) or SNP (500 mM) in the absence of Ca 2+ followed by readdition of external Ca 2+ in PMA-activated neutrophils. It was found that subsequent addition of external Ca 2+ resulted in a further calcium entry event after stimulation by NEM or SNP, demonstrating calcium entry should be storedepletion-dependent ( Fig. 2D and E). Besides, addition of 1 mM external calcium had no effect on [Ca 2+ ] c of Ca 2+ -free PMAactivated neutrophils before application of stimulation (Fig. 2F). In summary, NEM and SNP could induce believable SOCE via sulfhydryl modification in PMA-activated neutrophils.

The effect of NEM or SNP on [Ca 2+ ] c of resting human neutrophils
To further determine whether SOCE induced by NEM and SNP was due to the activation of neutrophils, experiments were performed with resting human neutrophils in suspension (1610 6 / mL) using spectrofluorometer. It was found that NEM (100 mM) or SNP (500 mM) did not caused [Ca 2+ ] c increase with Ca 2+containing buffer in resting neutrophils as shown in Figure 3A. However, FMLP (2 mM), a classic activator of calcium mobilization in neutrophils [33,34], induced evident rising of [Ca 2+ ] c in resting neutrophils served as a positive control (Fig. 3B). Considered together, these results suggested that NEM and SNPinduced SOCE via sulfhydryl modification was dependent on activation of neutrophils.

The effect of 2-APB on NEM or SNP-induced SOCE in PMA-activated neutrophils
To further study these calcium stores release and calcium entry responsible for sulfhydryl modification-induced SOCE in PMAactivated neutrophils, we measured [Ca 2+ ] c responses to the potent IP 3 receptor [35][36][37][38] and SOCE [9,38,39] blocker 2-APB in Ca 2+containing medium. It was found that addition of NEM (100 mM) caused no increase in [Ca 2+ ] c elevation when cells were preincubated with 150 mM 2-APB as shown in Figure  The effect of FMLP or U73122 on sulfhydryl modificationinduced SOCE in PMA-activated neutrophils The classic G protein-coupled receptors (GPCRs)-G protein-Phospholipase C (PLC)-IP 3 signaling pathway is well-known in the terms of calcium mobilization for neutrophils. FMLP, a classics activator of neutrophils, could result in SOCE on neutrophils through this GPCRs-G protein-PLC-IP 3 signaling pathway [33,34,40]. Therefore, to investigate whether the upstream signaling pathway of sulfhydryl modification-induced SOCE was similar to that of FMLP, PMA-activated neutrophils were stimulated by NEM, SNP and FMLP within Ca 2+ -containing medium in different chronological order. Data showed that FMLP (2 mM) had little capacity for inducing NEM (100 mM) or SNP (500 mM)-pretreated [Ca 2+ ] c change in PMA-activated human neutrophils ( Fig. 5A and B). This inhibitory effect proposed that sulfhydryl modification may induce SOCE through a GPCRs-G protein-PLC signaling pathway similar to that of FMLP. On the contrary, addition of NEM or SNP after FMLP activation still caused a rapid rising of [Ca 2+ ] c ( Fig. 5C and D). Comparing with results in Figure 5A and B, it indicated that the pathway of GPCRs-G protein-PLC maybe not the only means for sulfhydryl modification-induced calcium mobilization.

Discussion
There are a variety of agonists can activate resting neutrophils in vitro, including PMA, FMLP, leukotriene B 4 , interleukin-8 and  platelet-activating factor [1,43,44]. PMA, a diacylglycerol (DAG) analog, specifically activates PKC which in turn activates NADPH oxidase, resulting in stimulation of the respiratory burst of neutrophils [29,30]. Although PMA is not a physiological neutrophil activator, it was widely used as a model stimulus to study inflammatory responses of neutrophils in vitro experiments [45][46][47][48]. There are two main reasons for us to select PMA as an activator of neutrophils. First, PMA activates neutrophils without an apparent rising of [Ca 2+ ] c [29,49,50] and maintains stability of [Ca 2+ ] c basal level before stimulation, as shown in all our experiments of [Ca 2+ ] c measurement. Second, the activation of the human neutrophils by PMA leads to depolarization of the plasma membrane [29,51] as seen in Figure 1B, as well as inhibiting spontaneous polarization-induced random migration. Both these attributes ease measurement of [Ca 2+ ] c in single cell level with fluorescence microscopy. On the contrary, FMLP, a physiological neutrophil activator, not only caused strong calcium mobilization but also induced obvious migration of neutrophils, thereby complicating measurements of [Ca 2+ ] c change in single cell level.
Our studies using TG and FMLP firstly demonstrated that the mechanism of SOCE still exist in PMA-activated neutrophils (Fig. 1). TG, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2+ ATPases [32], causes calcium stores depletion and calcium entry via SOCE pathway in many types of cells including neutrophils [31]. FMLP, a chemotactic tripeptide, is well known as a potent and classic SOCE activator in neutrophils [33,34]. We subsequently found that NEM or SNP induced significant rising of [Ca 2+ ] c in PMA-activated neutrophils no matter whether calcium was in the buffer or not ( Fig. 2A, 2B, 2C). SNP had no effect on [Ca 2+ ] c when PMA-activated neutrophils were pretreated with NEM (Fig. 2B). Readdition of external Ca 2+ led to a further calcium entry event after activation of NEM or SNP (Fig. 2D and E), indicating calcium entry should be store-depletion-dependent. Besides, we found that addition of 1 mM external calcium did not induce [Ca 2+ ] c increase in Ca 2+ -free PMA-activated neutrophils without any stimulation, and subsequently adding NEM (100 mM) to achieve an apparent SOCE observed (Fig. 2F). Moreover, we found that NEM or SNP did not induced change of [Ca 2+ ] c in resting human neutrophils which were performed in suspension as shown in Figure 3. In conclusion, these results indicated that NME and SNP could induce a believable response for SOCE via sulfhydryl modification in PMAinduced activation-dependent human neutrophils.
2-APB, a specific inhibitor of IP 3 receptor [35][36][37][38], abolished NEM-induced calcium release in a concentration-dependent manner ( Fig. 4A and B). 2-APB also had the same effect on SNPinduced [Ca 2+ ] c elevation in PMA-activated neutrophils (data not shown). Besides, much research has demonstrated that 2-APB was a reliable blocker of SOCE in recent years [9,38,39]. Gill et al reported that 2-APB showed obvious inhibitory effect on SOCE at 75 mM [52]. In our experiments, application of 2-APB (75 mM) completely inhibited TG, NEM and SNP-induced SOCE in PMAactivated human neutrophils (Fig. 1E, F and Fig. 4). Moreover, we found that ruthenium red, a specific inhibitor of ryanodine receptorsensitive stores [53], had no effect on NEM or SNP-induced calcium release PMA-activated neutrophils (data not shown). Considered together, these data further indicated that NEM and SNP resulted in calcium entry through IP 3 receptor-sensitive stores-operated pathway in PMA-activated human neutrophils.
In addition, it is easy to find that we use a relatively high an order (2 mM) of magnitude higher than usual-PMA-dose to activate neutrophils from the resting state. It should be important to see whether the same phenomenon occurs at a submicromolar concentration. First, we found that 200 nM PMA (30 min at 37uC) could result in neutrophils obvious spreading on glass. Under this condition, NEM (100 mM) or SNP (500 mM) caused obvious [Ca 2+ ] c increase (data not shown). However, further result showed that neutrophils could not spread on glass evidently when neutrophils were preincubated with 200 nM for 5 min at 37uC. Subsequent application of NEM or SNP had no obvious effect on [Ca 2+ ] c (data not shown). It could be proposed that the activation ability of PMA on neutrophils were concentration and activation time-dependent. Lower concentration of PMA used, more activation time needed. The degree of neutrophils adhesion on glass substrate may have effect on sulfhydryl modification-induced [Ca 2+ ] c change.
Having known this, we sought to offer an explanation as to the upstream signaling pathway of NEM and SNP induced-SOCE. For this aim, we firstly studied the effect of FMLP on NEM or SNP-induced SOCE in PMA-activated neutrophils. It is well known [33,34,42] that FMLP, a classic activator of neutrophils, can cause stimulation of GPCRs on neutrophils, with consequent activation of PLC and generation of IP 3 and DAG by breakdown of membrane phosphatidyl inositol 4,5-bisphosphate. Finally, IP 3 interacts with Ca 2+ -mobilizing receptors, resulting in IP 3 receptorsensitive calcium stores release and SOCE. Our data showed that NEM or SNP blocked calcium signaling induced by subsequent stimulation of FMLP (Fig. 5A and B). In contrast, FMLP failed to inhibit calcium elevation caused by subsequent activation by NEM or SNP in PMA-activated neutrophils ( Fig. 5C and D). Together, these data proposed that NEM or SNP-induced calcium mobilization in PMA-activated neutrophils may involve the same signaling pathway as that of FMLP, namely the GPCRs-G protein-PLC-IP 3 pathway. However, this may not be the only pathway for NEM or SNP-induced SOCE.
Furthermore, it was found that demonstrated that U73122 completely blocked NEM or SNP-induced calcium mobilization in Ca 2+ -containing medium (Fig. 6A and B). U73122 is a putative and specific PLC inhibitor which has become an important tool in establishing the link between PLC activation and intracellular calcium signaling [40,41]. However, we must admit that the inhibitory mechanism of U73122 on NEM or SNP-induced SOCE seems to be problematic because U73122 is a sterol derivative of NEM. Therefore, such data just indicated possible roles for PLC in NEM or SNP-induced calcium responses. In addition, we directly observe an interesting phenomenon that U73122 caused morphological contraction of PMA-induced spreading of neutrophils in glass substrate observed by DIC microscopy (Fig. 6C and D), which is in accordance with observations by Smith et al who reported that U73122 could cause a inhibition of PMA-triggered neutrophils adhesion on fibronectin and fetal bovine serum-coated plates [54]. This result also suggested that sulfhydryl modification-induced [Ca 2+ ] c elevation may associate with the degree of neutrophils adhesion on glass substrate similar to that of PMA.
Interestingly, Wang et al showed that NO donor GEA3162 and NEM could induce calcium entry through a non-store-operated mechanism via direct sulfhydryl modification in resting rat neutrophils which were performed in suspension [22][23][24]. Gill et al also reported that NO donors and NEM caused non-storeoperated calcium entry through sulfhydryl modification in DDT 1 MF-2 cells [25][26][27]. It is obvious that activation of neutrophils by PMA adherent to a glass substrate give a different result of sulfhydryl modification-induced calcium mobilization compared to resting neutrophils. Other studies reported that neutrophils would give different physiological or psychological responses to stimuli depending on their being in the activation or resting state [44,55]. We analysed that activation of neutrophil respiratory burst may be implicated in the different regulation of [Ca 2+ ] c induced by sulfhydryl modification. To address this hypothesis, diphenylene iodonium (DPI), a putative inhibitor of the respiratory burstgenerating NADPH oxidase, was used in the present work. The results provided evidence that DPI had a inhibitory effect on NEMinduced SOCE in PMA-treated neutrophils (data not shown). Our previous report also demonstrated that DPI could block SNPinduced calcium release via S-Nitrosylation [28]. Meanwhile, U73122 completely inhibited NEM and SNP-induced SOCE in PMA-activated neutrophils (Fig. 6A and B). It was reported that U73122 could inhibit PMA-elicited adhesion-dependent respiratory burst [54]. Moreover, We also found that NEM and SNP did not induce obvious [Ca 2+ ] c increase when neutrophils was preincubated with PMA just at submicromolar concentration for little time at 37uC (data not shown). These evidence suggested the activation degree of respiratory burst may contribute to generation of sulfhydryl modification-induced SOCE in neutrophils. However, further study is still needed to elucidate the relationship between activation of respiratory burst-generating NADPH oxidase and sulfhydryl modification.
In summary, we clearly demonstrated that NEM, a thiolalkylating agent, and SNP, a donor of exogenous NO, induced obvious calcium entry through IP 3 -sensitive store-operated pathway via sulfhydryl modification in PMA-induced activationdependent human neutrophils. Since SOCE plays a crucial role in neutrophils, this study on investigating sulfhydryl modificationinduced SOCE may help contribute towards understanding mechanisms of [Ca 2+ ] c regulation surrounding neutrophil inflammatory reactions.

Ethics Statement
The study was reviewed and approved by the Ethics Committee of Nankai University. All participants gave written informed consent.

Preparation of human neutrophils
Neutrophils were obtained from the blood of normal healthy adults by step-density gradient centrifugation over Histopaque 1077 and 1119 solutions at 5006g for 15 min. The cells were .96% viable as assessed by trypan blue exclusion and had a purity of .95% as demonstrated by Hoechst 33342 nuclear staining. Cells were suspended in HBSS (145 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes and 10 mM glucose, PH adjusted to 7.4 with NaOH 1 mM) and then kept in an ice bath before use.

Microscopy
For all imaging experiments, cells in chamber (volume, 1 mL) were observed microscopically at 37uC by using a temperaturecontrolled stage. Fluorescence was performed with a fluorescent microscope (Axio observer D1, Carl Zeiss, Germany) with a 100 W mercury lamp. A shutter (VS25S2ZM1, Uniblitz, USA) was equipped behind the mercury lamp to control the exposure time and the interval time for fluorescence imaging. Fluorescence signaling was detected by an electron multiplying charge coupled device (EMCCD) (DU-897D-CS0-BV, Andor, U.K., <280uC) which was connected to the left exit side of microscope. The EMCCD was controlled by MetaMorph software version 7.1 (Universal Imaging Corp., USA). DIC and fluorescence images were collected by this microscopy system.

Measurement of [Ca 2+ ] c
Human neutrophils were incubated with calcium-sensitive probe Fluo-3 AM 4 mM for 30 min at 37uC in HBSS. For single cell calcium imaging, [Ca 2+ ] c was measured by the micro-imaging system as shown above. Fluo-3 AM-loaded cells were excited by a mercury lamp with a 485/20 nm excitation filter, and fluorescence was collected by a fluar oil objective with a 510 nm long-pass dichroic mirror and a 540/50 emission filter. Each fluorescence image was acquired for 50 msec with a 2 s interval between frames. The obtained images were quantitatively analyzed for changes of fluorescence intensities within region of interest.
[Ca 2+ ] c change was represented by relative fluorescence intensity (F/F 0 , intensity after stimulation/basal intensity before stimulation). At least 30 individual cells were selected from three independent experiments, with one characteristic [Ca 2+ ] c trace plotted to represent .10 similar traces. Besides, Ca 2+ -free HBSS was prepared by substituting MgCl 2 for CaCl 2 at the same concentration, with 2 mM EGTA. For [Ca 2+ ] c measurement in suspension, Fluo-3 AM-loaded neutrophils were suspended in HBSS to 1610 6 cells/mL. Fluorescence was monitored with a spectrofluorometer (FSL920, Edinburgh, U.K.) at 530/30 nm with excitation at 488 nm.

Statistical analysis
All data were presented as means 6 standard error of the mean (S.E.M.). The statistical comparison between groups was carried out using Student's t-test. P,0.05 was considered statistically significant.