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The Journal of Immunology, 2004, 173: 6433-6439.
Copyright © 2004 by The American Association of Immunologists

Group IB Secretory Phospholipase A2 Stimulates CXC Chemokine Ligand 8 Production via ERK and NF-{kappa}B in Human Neutrophils1

Eun Jin Jo2,*,{dagger}, Ha-Young Lee2,*,{dagger}, Youl-Nam Lee*, Jung Im Kim*,{dagger}, Hyun-Kyu Kang*, Dae-Won Park{ddagger}, Suk-Hwan Baek{ddagger}, Jong-Young Kwak*,{dagger} and Yoe-Sik Bae3,*,{dagger}

* Medical Research Center for Cancer Molecular Therapy and {dagger} Department of Biochemistry, College of Medicine, Dong-A University, Busan, Korea; and {ddagger} Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the level of group IB secretory phospholipase A2 (sPLA2-IB) has been reported to be up-regulated during inflammatory response, the role of sPLA2-IB on the regulation of inflammation and immune responses has not been fully elucidated. In this study, we found that sPLA2-IB stimulates the expression and secretion of CXCL8 without affecting other proinflammatory cytokines, such as IL-1{beta} or TNF {alpha} in human neutrophils. The induction of CXCL8 secretion by sPLA2-IB occurs at both the transcription and translational levels and correlates with activation of NF-{kappa}B. Moreover, the NF-{kappa}B inhibitors pyrrolidinedithiocarbamate, dexamethasone, or sulfasalazine were found to prevent CXCL8 production by sPLA2-IB in human neutrophils. In addition, the signaling events induced by sPLA2-IB included activation of the MAPK ERK and an increase in intracellular Ca2+, which are both required for CXCL8 production. The exogenous addition of sPLA2-IB did not induce arachidonic acid release from human neutrophils, and the inactivation of sPLA2-IB by EGTA did not affect CXCL8 production by sPLA2-IB in human neutrophils. Taken together, we suggest that sPLA2-IB plays a role in the modulation of inflammatory and immune responses via the sPLA2 receptor, by inducing CXCL8 in human neutrophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Secretory phospholipase A2 (sPLA2)4 enzymes may be classified into three groups, groups I, II, and V (1). Of these, group IB sPLA2 (sPLA2-IB) is called pancreatic PLA2, because it is produced by pancreatic acinar cells and functions in the digestion of dietary lipids within the intestinal lumen (1, 2). It has also been reported that sPLA2-IB is expressed in other tissues, including lung, spleen, kidney, and ovary (3, 4, 5). Previously, it was suggested that sPLA2-IB is involved in various cellular physiological responses, such as cell proliferation, cell contraction, and cellular lipid mediator release (6, 7). In addition, it has been suggested to be involved in pathological responses, such as in acute lung injury and endotoxic shock (8). The M-type cell surface receptor was reported to be a target of sPLA2-IB (9). Initially, M-type sPLA2 receptors were identified in skeletal muscle cells (10). Several other tissue types, including, liver, lung, spleen, and pancreas, are also now known to express M-type sPLA2 receptor, which is believed to have an important role in many biological functions (9). However, the involvement of sPLA2-IB in regulation of immune response has not been comprehensively investigated.

Human neutrophils perform a crucial role in the modulation of host immune response, by producing several kinds of cytokines, which regulate the functions of other immune cells such as T and B lymphocytes (11). CXCL8 (IL-8) is an important cytokine and is released by human neutrophils (12, 13, 14), which have also been reported to express the M-type sPLA2 receptor (15). Moreover, the stimulation of human neutrophils with sPLA2-IB has been reported to cause elastase release and neutrophil adhesion (15). However, the physiologic roles of sPLA2-IB as a functional modulator of human neutrophils have not been investigated.

In this study, we investigated whether sPLA2-IB induces a primary cytokine response in neutrophils, and interestingly, we found that sPLA2-IB stimulates CXCL8 production in human neutrophils. We then further investigated the signaling pathways involved in sPLA2-IB-induced CXCL8 production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

RPMI 1640 medium was purchased from Invitrogen Life Technologies (Carlsbad, CA). Dialyzed FBS and supplemented bovine calf serum were from HyClone (Logan, UT). sPLA2-IB (from porcine pancreas), Histopaque-1077, L-glutamine, antibiotic-antimycotic solution (10,000 UI/ml penicillin, 10 mg/ml streptomycin, and 25 µg/ml amphotericin B), LPS (from Escherichia coli strain 055:B5), and cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO). Actinomycin D (ActD), pyrrolidine dithiocarbamate (PDTC), 2'-amino-3'-methoxyflavone (PD98059), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) were from Calbiochem (San Diego, CA). [5, 6, 8, 9, 11, 12, 14, 15-[3H](N)]-Arachidonic acid and 1-palmitoyl-2-[1-14C]linoleoyl L-3-phosphatidylcholine were from PerkinElmer (Boston, MA). Rabbit anti-human Abs to ERK2 (catalogue 9102), phospho-ERKs (catalogue 9101), and phospho-p38 kinase (catalogue 9211) were from Cell Signaling Technology (Beverly, MA), and HRP-conjugated Abs to mouse and rabbit IgG were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

Isolation of human neutrophils

Peripheral blood was collected from healthy donors, and human neutrophils were isolated by dextran sedimentation, hypotonic lysis of erythrocytes, and by using a lymphocyte separation medium gradient, as described previously (16). Donors had not taken anti-inflammatory drugs for at least 3 wk before sampling and were free of systemic illnesses, such as asthma or allergic rhinitis. Informed consent was obtained from all volunteers, and the study was approved by the local institutional review board at Dong-A University Hospital. Venous blood was collected in sodium citrate solution (3.8%). The cellular portion was mixed with a solution of 3% dextran in 0.9% NaCl solution and kept at 25°C for 45 min. The neutrophil-rich upper layer of the suspension was then collected and centrifuged (250 x g, 10 min, 4°C). Residual erythrocytes were removed by hypotonic lysis, and the pellet obtained was suspended in ice-cold PBS. The suspension was centrifuged (250 x g, 45 min) on Histopaque solution at 4°C. Isolated neutrophils were maintained in RPMI 1640 medium supplemented with 5% FBS at 37°C. Neutrophils were shown to be >95% pure by microscopy. Cell viability was determined by trypan blue dye exclusion assay, and >98% of neutrophils were viable. Isolated human neutrophils were used promptly.

Cytokine assay

Cytokine measurement was performed, as previously described (17). Neutrophils (3 x 106 cells/0.3 ml) were placed in RPMI 1640 medium containing 5% FBS in 24-well plates and kept in a 5% CO2 incubator at 37°C. After stimulation, cell-free supernatants were collected, centrifuged, and measured for CXCL8, IL-1{beta}, and TNF-{alpha} by ELISA (BD Pharmingen, San Diego, CA), according to the instruction of the vendor.

RT-PCR analysis

Neutrophils (1 x 106 cells) were stimulated with 1 µM sPLA2-IB in a total volume of 0.2 ml for the indicated times. mRNA was isolated by using a QIAshredder and an RNeasy kit (Qiagen, Hilden, Germany). mRNA, Moloney murine leukemia virus reverse transcriptase, and pd(N)6 primers (Invitrogen Life Technologies) were used to obtain cDNA. The primers used for the RT-PCR analysis have been reported previously (18). The sequences of the primer used were as follows. CXCL8 (273-bp product): sense, 5'-ATGACTTCCAAGCTGGCCG-3'; antisense, 5'-CTCAGCCCTCTTCAAAAACTT-3'. GAPDH (246-bp product): sense, 5'-GATGACATCAAGAAGGTGGTGAA-3'; antisense, 5'-GTCTTACTCCTTGGAGGCCATGT-3'. We ran 30 PCR cycles at 94°C (denaturation, 1 min), 62°C (annealing, 1 min), and 72°C (extension, 1 min). PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.

Preparation of nuclear extracts

Nuclear extract of human neutrophils was prepared, as described previously (19). Briefly, human neutrophils (1 x 106 cells) were incubated with sPLA2-IB for several times, as indicated. Cells were harvested in PBS containing 2% FBS, washed twice with PBS, and resuspended in 400 µl of buffer (10 mM HEPES, pH 7.9, 5 mM MgCl2, 10 mM KCl, 1 mM ZnCl2, 0.2 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 0.5 mM DTT, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A). After the cells were incubated on ice for 10 min and then lysed by the addition of 50 µl of 10% Nonidet P-40 (1.1% final concentration), the nuclei were harvested by centrifugation. The nuclear pellets were resuspended in 60 µl of extraction buffer (10 mM HEPES, pH 7.9, 5 mM MgCl2, 300 mM NaCl, 1 mM ZnCl2, 0.2 mM EGTA, 25% glycerol, 1 mM Na3VO4, 10 mM NaF, 0.5 mM DTT, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A) and incubated for 15 min on ice. Nuclear debris was removed by centrifugation (13,000 rpm for 10 min), and the nuclear protein extract was used for gel-shift analysis. Protein concentration was determined by the Bradford method.

EMSA

Gel-shift analysis of nuclear extracts was performed using oligonucleotides containing the consensus sequence for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG-3'; Santa Cruz Biotechnology, Santa Cruz, CA) end labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Promega, Madison, WI), as described previously (20). Typical binding reactions consisted of 10 µg of nuclear extract, 1 ng of DNA probe, 2 µg/ml poly[d(I-C)] in a buffer containing 20 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol, and were incubated at 30°C for 20 min. Binding reactions were separated on 6% Tris-glycine nondenaturing polyacrylamide gels in a 2x Tris-glycine buffer system. The gels were transferred to Whatman paper (Tewksbury, MA), dried, and subjected to autoradiography.

Stimulation of cells with sPLA2-IB for Western blot analysis

Freshly isolated human neutrophils (2 x 106) were stimulated with the indicated concentrations of sPLA2-IB for the predetermined lengths of time. After stimulation, the cells were washed with serum-free RPMI 1640 medium and lysed in lysis buffer (20 mM HEPES, pH 7.2, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF). Detergent-insoluble materials were pelleted by centrifugation (12,000 x g, 15 min, at 4°C), and the soluble supernatant fraction was removed and either stored at –80°C or used immediately. Protein concentrations in the lysates were determined using Bradford protein assay reagent.

Electrophoresis and immunoblot analysis

Protein samples were prepared for electrophoresis and then separated using a 10% SDS-polyacrylamide gel and the buffer system described previously (21). Following the electrophoresis, the proteins were blotted onto nitrocellulose membrane, which was blocked by incubating with TBST containing 5% nonfat dried milk. The membranes were then incubated with anti-phospho-ERK Ab, anti-phospho-p38 kinase Ab, or anti-ERK Ab, and washed with TBST. Ag-Ab complexes were visualized after incubating the membrane with 1/5000 diluted goat anti-rabbit IgG or goat anti-mouse IgG Ab coupled to HRP using the ECL detection system.

Ca2+ measurement

Intracellular calcium concentration ([Ca2+]i) was determined by Grynkiewicz’s method using fura 2-AM (21). Briefly, prepared cells were incubated with 3 µM fura 2-AM at 37°C for 50 min in fresh serum-free RPMI 1640 medium with continuous stirring. A total of 2 x 106 cells was aliquoted for each assay into Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM HEPES, pH 7.3, 10 mM glucose, 2.2 mM CaCl2, and 0.2 mM EGTA). Fluorescence was measured at 500 nm at excitation wavelengths of 340 and 380 nm.

Measurement of PLA2 activity in cells

Isolated human neutrophils (107 cells/ml) were prelabeled with 0.5 µCi/ml [3H]arachidonic acid in RPMI 1640 medium containing 10% FBS for 2 h at 37°C in a humidified incubator supplied with 95% air and 5% CO2, as described before (22). The labeled cells were then washed twice with serum-free RPMI 1640 medium and incubated in RPMI 1640 medium containing 0.1% fatty acid-free BSA for 15 min at 37°C. After discarding the medium, the cells were stimulated with 1 µM sPLA2-IB for 30 min. Radioactivity in the medium was measured using a liquid scintillation counter.

PLA2 activity assay

PLA2 activities were measured by the acylhydrolysis of 1-palmitoyl-2-[1-14C]linoleoyl L-3-phosphatidylcholine, as described before (23, 24). Each reaction mixture was incubated with the enzyme and substrate (total 20,000 cpm) in a reaction buffer containing 100 mM Tris (pH 7.4) and 4 mM CaCl2 for 30 min at 37°C. The reaction was terminated by extracting released [14C]fatty acid, as described previously (23, 24). Results are calculated as cpm free fatty acid hydrolyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The stimulation of human neutrophils with sPLA2-IB induces CXCL8 production

To investigate the effect of sPLA2-IB on primary cytokines in human neutrophils, freshly isolated human neutrophils were stimulated with various concentrations of sPLA2-IB for 24 h. As shown in Fig. 1A, sPLA2-IB induced CXCL8 production in a concentration-dependent manner, showing maximal activity at 1 µM. Moreover, CXCL8 production by 1 µM sPLA2-IB was similar to LPS-induced CXCL8 production. However, sPLA2-IB did not affect the production of other cytokines such as IL-1{beta} or TNF-{alpha} (Fig. 1A). LPS, used as a positive control, induced the productions of these two proinflammatory cytokines (Fig. 1A). Time dependency of sPLA2-IB-induced CXCL8 production was also investigated, and its production was apparent from 2 h after stimulation and increased until 24 h after stimulation (Fig. 1B).



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  		FIGURE 1. sPLA2-IB stimulates CXCL8 production in human neutrophils. A, Freshly isolated human neutrophils were stimulated with several concentrations of sPLA2-IB or LPS (10 ng/ml) for 24 h. Secreted cytokine levels were determined by ELISA. B, Neutrophils were stimulated with 1 µM sPLA2-IB for various times. Secreted CXCL8 was measured by ELISA. All data are presented as means ± SEM of three independent experiments, each performed in triplicate.

 
sPLA2-IB-induced CXCL8 production is transcriptional and translational activation mediated

To investigate the mechanism involved in sPLA2-IB-induced CXCL8 production, we pretreated neutrophils with the transcription inhibitor ActD or the protein synthesis inhibitor CHX. When human neutrophils were pretreated with ActD or CHX before the addition of sPLA2-IB, sPLA2-IB-induced CXCL8 production was almost completely inhibited (Fig. 2A). We also examined the effect of sPLA2-IB on the accumulation of the CXCL8 mRNA transcript by RT-PCR. As shown in Fig. 2B, the stimulation of human neutrophils with 1 µM sPLA2-IB caused CXCL8 mRNA transcript accumulation in a time-dependent manner. The CXCL8 mRNA transcript was significantly increased after stimulation of cells for 1–4 h with sPLA2-IB (Fig. 2B). These results indicate that sPLA2-IB-induced CXCL8 production requires transcriptional activation and de novo protein synthesis.



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  		FIGURE 2. sPLA2-IB-induced CXCL8 secretion requires transcription and de novo protein synthesis. A, Neutrophils were preincubated for 1 h with or without ActD (10 µg/ml) or CHX (100 µM), and then stimulated with 1 µM sPLA2-IB for 4 h. Secreted CXCL8 was measured by ELISA. All data are presented as means ± SEM of three independent experiments, each performed in triplicate. B, Neutrophils, stimulated with 1 µM sPLA2-IB for 0, 1, 2, or 4 h, or incubated in the absence of sPLA2-IB for 4 h, were harvested for RNA preparation. RT-PCR was performed using specific primers for human CXCL8 and GAPDH. PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide. The data obtained from one representative experiment performed in quadruplicate are shown.

 
sPLA2-IB stimulates NF-{kappa}B activity, and this results in CXCL8 production in human neutrophils

The expression of the CXCL8 gene was reported to require the activation of NF-{kappa}B (25, 26). In the present study, we examined the effect of sPLA2-IB on NF-{kappa}B activity in human neutrophils. To investigate the involvement of NF-{kappa}B in the induction of CXCL8 by sPLA2-IB, we performed EMSA. Accordingly, human neutrophils were stimulated for several lengths of time with sPLA2-IB. As shown by Fig. 3A, two faint DNA-protein complexes were identified in nuclear extracts of unstimulated neutrophils, and the intensities of these complexes increased after exposing the neutrophils to sPLA2-IB. The intensities of these bands increased time dependently after treating cells with sPLA2-IB, and showed maximal activity 30 min after stimulation (Fig. 3A). In addition, sPLA2-IB-induced NF-{kappa}B-specific DNA-protein complex formation was inhibited in the presence of a 100-fold excess of unlabeled NF-{kappa}B probe (Fig. 3A).



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  		FIGURE 3. sPLA2-IB-stimulated CXCL8 production is NF-{kappa}B dependent. A, Neutrophils were stimulated with 1 µM sPLA2-IB for different times. Nuclear proteins were extracted and an EMSA was performed. The NF-{kappa}B/DNA complex was detected by using a 32P-labeled NF-{kappa}B oligonucleotide probe. An unlabeled NF-{kappa}B probe (in 100x excess) was used as a competitor to determine the specificity of DNA binding (lane 6). Data shown are from one representative experiment performed in triplicate. B, Neutrophils were preincubated with or without 10 µM sulpasalazine, 10 µM dexamethasone, or 30 µM PDTC at 37°C for 1 h and then stimulated with 1 µM sPLA2-IB for 24 h. The amount of CXCL8 secreted was determined by ELISA. Data are presented as means ± SEM of three independent experiments performed in duplicate.

 
To investigate the role of NF-{kappa}B on sPLA2-IB-induced CXCL8 production, we pretreated neutrophils with several types of NF-{kappa}B inhibitor (27, 28), i.e., sulfasalazine, dexamethasone, or PDTC, before adding sPLA2-IB. As shown in Fig. 3B, sulfasalazine, dexamethasone, or PDTC blocked sPLA2-IB-induced CXCL8 production by 63, 62, and 80%, respectively. These results indicate that NF-{kappa}B activation is essentially required for sPLA2-IB-induced CXCL8 production in human neutrophils.

ERK activity is required for sPLA2-IB-induced CXCL8 production in human neutrophils

MAPK has been reported to mediate extracellular signals to the nucleus in several cell types (29). In this study, we examined whether sPLA2-IB stimulates MAPKs by using Western blot analysis with anti-phospho-specific Abs against each enzyme. When human neutrophils were stimulated with 1 µM sPLA2-IB for different times, the phosphorylation level of ERK was transiently increased, peaking after 5–10 min (Fig. 4A) and sustained for 30 min (Fig. 4A). However, another important MAPK, p38 kinase, was not activated (Fig. 4A). We also examined the concentration dependency of sPLA2-IB-induced ERK activation. When human neutrophils were stimulated with various concentrations of sPLA2-IB, ERK was activated in a concentration-dependent manner (Fig. 4A), significantly at 100 nM and maximally at 1 µM (Fig. 4A).



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  		FIGURE 4. Role of ERK on sPLA2-IB-induced CXCL8 production in neutrophils. A, Neutrophils were stimulated with 1 µM sPLA2-IB for different times (left panel), and with various concentrations for 5 min (right panel). Each sample (30 µg of protein) was subjected to 10% SDS-PAGE, and phosphorylated ERK or phosphorylated p38 kinase levels were determined by immunoblot analysis using anti-phospho-ERK Ab or anti-phospho-p38 kinase Ab (A). The results shown are representative of at least three independent experiments (A). B, The cells were preincubated with vehicle, 50 µM PD98059 (60 min), or 20 µM SB203580 (15 min) before being treated with 1 µM sPLA2-IB for 24 h. The amount of CXCL8 secreted was measured by ELISA. All data are presented as means ± SEM of three independent experiments performed in triplicate.

 
To determine the role of each MAPK on sPLA2-IB-induced CXCL8 production, we preincubated human neutrophils with PD98059, a selective MEK inhibitor, or with SB203580, a selective p38 kinase inhibitor, before sPLA2-IB treatment. PD98059 completely inhibited CXCL8 production by sPLA2-IB (Fig. 4B), whereas SB203580 had no effect. These results demonstrate that MEK- but not p38 kinase-dependent MAPK activity is essentially required for sPLA2-IB-induced CXCL8 production.

Intracellular Ca2+ increase is required for sPLA2-IB-induced CXCL8 secretion

It has been reported previously that Ca2+ mobilization is required for the secretion of several molecules, including cytokines (30, 31). To characterize the signaling pathway associated with CXCL8 secretion by sPLA2-IB, we measured the effect of sPLA2-IB on [Ca2+]i mobilization. When fura 2-loaded human neutrophils were stimulated with 1 µM sPLA2-IB, a rapid increase in [Ca2+]i was observed (Fig. 5A). Moreover, the preincubation of neutrophils with BAPTA-AM, a Ca2+ chelating agent, completely blocked sPLA2-IB-induced Ca2+ mobilization (Fig. 5A). To examine the role of Ca2+ mobilization on sPLA2-IB-induced CXCL8 secretion, we preincubated human neutrophils with BAPTA-AM, an intracellular Ca2+ chelating agent, before adding sPLA2-IB. As shown in Fig. 5B, BAPTA-AM completely blocked sPLA2-IB-induced CXCL8 secretion, suggesting that Ca2+ mobilization has a critical role in CXCL8 secretion.



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  		FIGURE 5. Intracellular Ca2+ elevation is required for CXCL8 secretion. A, Fura 2-AM-labeled neutrophils were stimulated with sPLA2-IB (1 µM) with or without BAPTA-AM (20 µM). The relative intracellular Ca2+ concentration is expressed as fluorescence ratio (340:380 nm). B, Secretion of CXCL8 by 1 µM sPLA2-IB-stimulated neutrophils was determined with or without BAPTA-AM. Data are presented as means ± SEM of three independent experiments performed in duplicate.

 
sPLA2-IB-induced CXCL8 secretion is receptor mediated

A previous report has demonstrated that calcium is required for the proper activation of sPLA2-IB (32). To investigate whether sPLA2-IB stimulates CXCL8 production via enzymatic action, we carefully investigated the effect of sPLA2-IB on arachidonic acid release in human neutrophils. However, the stimulation of [3H]arachidonic acid-labeled human neutrophils with 1 µM sPLA2-IB for 30 min did not affect arachidonic acid release (Table I). The result suggests that sPLA2-IB can stimulate human neutrophils in an enzymatic activity-independent manner. Moreover, formyl-methionyl-leucyl-phenylalanine (1 µM), a positive control, was found to significantly stimulate arachidonic acid release from [3H]arachidonic acid-labeled human neutrophils (data not shown). We also tested the enzymatic activity of sPLA2-IB in vitro. A total of 1 µM sPLA2-IB completely hydrolyzed the substrate releasing free fatty acid (Table I). Furthermore, the addition of 5 mM EGTA before the PLA2 activity assay almost completely inhibited the enzymatic activity of sPLA2-IB, which is consistent with previous reports (33, 34). We also examined the effect of preincubation with EGTA on sPLA2-IB-induced CXCL8 production. As shown in Fig. 6A, the preincubation of neutrophils with 5 mM EGTA before sPLA2 addition did not elicit any significant change on sPLA2-IB-induced CXCL8 production, thus supporting our notion that sPLA2-IB-induced CXCL8 production is not mediated by the enzymatic activity of sPLA2.


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  		Table I. Enzymatic activity of sPLA2-IB with [3H]arachidonic acid-labeled neutrophilsa or in vitrob

 


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  		FIGURE 6. The sPLA2 receptor is involved in sPLA2-IB-induced CXCL8 production in neutrophils. A, Cells were stimulated with 1 µM sPLA2-IB with or without 5 mM EGTA for 24 h. Secreted CXCL8 was quantified by ELISA. All data are presented as means ± SEM of three independent experiments performed in triplicate. B, Neutrophils were stimulated with 1 µM sPLA2-IB for various times or stimulated with various concentrations of sPLA2-IB for 5 min in the presence of 5 mM EGTA. Each sample (30 µg of protein) was subjected to 10% SDS-PAGE, and phosphorylated ERK was determined by immunoblotting using anti-phospho-ERK Ab (B). The results shown are representative of at least three independent experiments (B).

 
We also checked the effect of EGTA on sPLA2-IB-induced ERK phosphorylation in human neutrophils. As shown by Fig. 6B, sPLA2-IB stimulated ERK phosphorylation in a time-dependent manner in the presence of 5 mM EGTA, showing maximal activity at 10 min after stimulation. Moreover, this sPLA2-IB-induced ERK phosphorylation was apparent at 0.1–2 µM sPLA2 (Fig. 6B). These results correlated well with the finding that sPLA2-IB induced ERK phosphorylation in the absence of EGTA (Fig. 4A). Taken together, sPLA2-IB-induced ERK phosphorylation and subsequent CXCL8 production do not seem to be enzymatic activity dependent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the effect of sPLA2-IB on the production of cytokines from human neutrophils. We observed that sPLA2-IB induced a concentration-dependent production of CXCL8, but not of TNF-{alpha} or IL-1{beta} in these cells. Moreover, this effect of sPLA2-IB on CXCL8 was associated with the accumulation of CXCL8-specific mRNA, suggesting that sPLA2-IB induces CXCL8 production at the transcriptional level. We also found that sPLA2-IB stimulates CXCL8 production via NF-{kappa}B activation downstream of sPLA2 receptor activation. Thus, this study provides first evidence that sPLA2-IB activates the production of the proinflammatory cytokine (CXCL8) in human neutrophils.

sPLA2 is regarded as a proinflammatory mediator, as it is highly elevated in the circulation and locally in tissues in various pathologic conditions such as sepsis. Previous reports have suggested that the proinflammatory effect of sPLA2 is thought to proceed via the generation of arachidonic acid as an eicosanoid precursor (35, 36). Baek and colleagues (37) suggested a role for sPLA2 in the potentiation of inducible NO synthase and NF-{kappa}B-regulated gene expression, which are involved in LPS signal transduction. Recently, sPLA2 was reported to induce cytokine release from blood and synovial monocytes (34). Moreover, several sPLA2 groups (groups IA, IB, IIA, and III) were found to stimulate the release of IL-6 and TNF-{alpha} from blood monocytes at the transcriptional level (34), suggesting that sPLA2 release during inflammatory disease has a proinflammatory effect. The present study shows that sPLA2-IB specifically stimulates CXCL8 production (Fig. 1A). Of the various members of the chemokine family, CXCL8 potently targets leukocytes expressing the CXCL8 receptors CXCR1 and CXCR2 (38). In addition, CXCL8 is a major neutrophil-activating factor and chemotactic, and is produced by several human cell types (11). Moreover, the production of CXCL8 at the site of an inflammation can activate neutrophils in an autocrine mode, and because monocytes and a subpopulation of T cells express CXCR1 or CXCR2, CXCL8 may also recruit and activate the cells locally, resulting in a modulation of cell-mediated immune response (39). Bearing in mind that CXCL8 plays a crucial role in the modulation of several immune responses, and that sPLA2-IB stimulates CXCL8 production in neutrophils, it is evident that sPLA2-IB should be regarded as a target molecule for the development of immunomodulators of CXCL8-related diseases.

Previous reports have demonstrated that elevated sPLA2 levels are associated with several neutrophil-mediated syndromes, such as acute lung injury and arthritis (40, 41, 42). Recently, it has been suggested that the sPLA2 receptor is involved in elastase release and neutrophil adhesion (15). Silliman et al. (15) demonstrated that human neutrophils express M-type sPLA2 receptor by using a polyclonal Ab to the human M-type sPLA2 receptor. In our study, we demonstrated that sPLA2-IB stimulates CXCL8 production in human neutrophils. On the intracellular signaling pathways induced by sPLA2-IB, Silliman et al. (15) showed that sPLA2-IB stimulated not ERK, but p38 kinase, which performs a crucial role in the elastase release. However, in our study, we found that ERK phosphorylation was dramatically induced by sPLA2-IB in human neutrophils, and ERK activity is critically involved in the sPLA2-IB-induced CXCL8 production in human neutrophils (Fig. 4). We could not detect any significant increase of p38 kinase phosphorylation (Fig. 4). At this point, it is unclear what caused this differential effect on the MAPK activity by sPLA2-IB in human neutrophils. Intracellular signaling pathways induced by the activation of receptor(s) for sPLA2-IB should be further investigated.

sPLA2-IB, in the present study, released fatty acid from its substrate in vitro (Table I). Because arachidonic acid released from sPLA2-IB substrates can modulate the cellular activity of human neutrophils, it remains to be clarified whether the CXCL8 production induced by sPLA2-IB may be secondary to the release of arachidonic acid in human neutrophils. However, three pieces of evidences indicate that the possible engagement of sPLA2-IB enzymatic activity in sPLA2-IB-induced CXCL8 production in human neutrophils should be ruled out. First, the stimulation of human neutrophils with 1 µM sPLA2-IB for 30 min did not affect arachidonic acid release (Table I). Previously, Bezzine et al. (33) demonstrated that exogenously added sPLA2-IB did not effectively release arachidonic acid from mammalian cells, and Triggiani et al. (34) also demonstrated that sPLA2-IB does not induce arachidonic acid release from blood monocytes, which is consistent with our findings. Second, according to our result (Table I) and a previous report, chelation of Ca2+ with 5 mM EGTA inhibits sPLA2-IB activity by ~95% (15), thus demonstrating that Ca2+ is required for the enzymatic activity of sPLA2-IB. Moreover, the present study shows that sPLA2-induced CXCL8 production is unaffected by preincubating human neutrophils with 5 mM EGTA (Fig. 6A), and that ERK phosphorylation by sPLA2-IB is not inhibited by 5 mM EGTA (Fig. 6B). Third, we investigated whether arachidonic acid metabolites are involved in sPLA2-IB-induced CXCL8 production in human neutrophils. The role of the 5-lipoxygenase pathway on sPLA2-IB-induced CXCL8 production in human neutrophils was examined using three different 5-lipoxygenase-selective inhibitors, AA-861, MK-886, and NDGA (43). Preincubation of human neutrophils with AA-861 (1 and 10 µM), MK-886 (0.1 and 1 µM), or NDGA (1 and 10 µM) before adding 1 µM sPLA2-IB did not significantly affect sPLA2-IB-induced CXCL8 production (data not shown). We also checked the effect of the cyclooxygenase-2 inhibitor, NS398 (44), on CXCL8 production by sPLA2-IB in human neutrophils, and found that the preincubation of neutrophils with NS398 (1 and 5 µM) before adding sPLA2-IB had no effect on CXCL8 production by sPLA2-IB (data not shown). Based on the findings of previous reports and our results, the involvement of the M-type sPLA2 receptor on sPLA2-IB-induced CXCL8 production in human neutrophils requires further investigation, to determine the potential therapeutic benefits of targeting the M-type sPLA2 receptor as an immunomodulator of CXCL8-related (patho)physiological responses.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Korea Science and Engineering Foundation through the Medical Science and Engineering Research Center for Cancer Molecular Therapy at Dong-A University, and by the National R&D Program for Fusion Strategy of Advanced Technologies of Most, Korea. Back

2 E.J.J. and H.-Y.L. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Yoe-Sik Bae, Medical Research Center for Cancer Molecular Therapy and Department of Biochemistry, College of Medicine, Dong-A University, Busan 602-714, Korea. E-mail address: yoesik{at}donga.ac.kr Back

4 Abbreviations used in this paper: sPLA2, secretory phospholipase A2; ActD, actinomycin D; [Ca2+]i, intracellular calcium concentration; CHX, cycloheximide; PD98059, 2'-amino-3'-methoxyflavone; PDTC, pyrrolidine dithiocarbamate; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; sPLA2-IB, group IB sPLA2. Back

Received for publication May 17, 2004. Accepted for publication September 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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