Blood Cells, Molecules and Diseases 55 (2015) 127–133

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Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps Rui Yamaguchi, Jin Kawata, Toshitaka Yamamoto, Yasuji Ishimaru, Arisa Sakamoto, Tomomichi Ono, Shinji Narahara, Hiroyuki Sugiuchi, Eiji Hirose, Yasuo Yamaguchi ⁎ Graduate School of Medical Science, Kumamoto Health Science University, Kumamoto, Japan

a r t i c l e

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Article history: Submitted 5 February 2015 Accepted 29 May 2015 Available online 31 May 2015 Keywords: Interferon-gamma Myeloperoxidase Neutrophil elastase Mannose receptor p38 Mitogen-activated protein kinase

a b s t r a c t Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell–cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFNgamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signalregulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Neutrophils have long been thought of as cells involved in innate immunity protecting against microbial invasion. Serine proteases such as cathepsin G, human leukocyte elastase, and proteinase 3 are major proteins found in neutrophil granules. These proteases are commonly thought to be involved in the intralysosomal degradation of engulfed cell debris or microorganisms. Proteolysis has also been suggested to be a fundamental mechanism regulating the activities of various components of the cytokine network [1]. Neutrophil serine proteases act as alternative processing enzymes for the activation of pro-inflammatory cytokines such as interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha [2], IL-18 [3], and matrix metalloproteinase-9 [4]. Thus, these proteases are involved in the regulation of cytokine bioactivity and availability. Neutrophil proteases also modulate other mechanisms regulating inflammation such as Abbreviations: ELISA, enzyme-linked immunosorbent assay; HNE, human neutrophil elastase; IL, interleukin; IFN, interferon; MAPK, mitogen-activated protein kinase; NETs, neutrophil extracellular traps; PMA, phorbol myristate acetate; PAR-2, proteaseactivated receptor-2; PBMCs, peripheral blood mononuclear cells; RT-PCR, reverse transcription polymerase chain reaction; Th1, T helper type 1; TNF, tumor necrosis factor. ⁎ Corresponding author at: Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan. E-mail address: [email protected] (Y. Yamaguchi).

http://dx.doi.org/10.1016/j.bcmd.2015.05.012 1079-9796/© 2015 Elsevier Inc. All rights reserved.

inactivation of the anti-inflammatory mediators progranulin [5] and IL-6 [6]. Thus, these serine proteases have a marked influence on innate immune responses [7]. The neutrophil granule enzyme myeloperoxidase plays an important role in antimicrobial responses and is also required for formation of neutrophil extracellular traps [8]. The major immunoregulatory cytokines include interferon (IFN)gamma, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-15, and IL-18. These cytokines can dramatically alter both the strength of the immune response and its character. IFN-gamma is a major activator of macrophages [9] and enhances their ability to kill microorganisms [10]. IFN-gamma also upregulates HLA class II antigen expression [11] and promotes T helper type 1 (Th1) differentiation [12], while downregulating the proliferation of Th2 cells. Moreover, IFN-gamma regulates the production of immunoglobulin E and some immunoglobulin G subclasses in humans [13]. In the innate immune response, IFN-gamma is predominantly produced by natural killer cells and natural killer T cells, while it is produced by Th1 CD4 and CD8 cytotoxic T cells in the antigen-specific immune response. Thus, IFN-gamma has a crucial role in immunity against intracellular pathogens and also in control of tumors [14]. Invading pathogens are attacked by both the innate and adaptive arms of the immune system. Chemokines play a critical role in leukocyte trafficking in inflammatory conditions and are key players in the links between innate and adaptive immunity [15]. The chemokine system

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Table 1 Reagents used in this study. Reagent

Action

U73122 TMB-8 PDTC PD98059 SB203580

Phospholipase C inhibitor Intracellular calcium antagonist NF-kB inhibitor Extracellular signal-regulated kinase inhibitor p38 Mitogen-activated protein kinase inhibitor (p38alpha and p38beta inhibitor) p38 Mitogen-activated protein kinase inhibitor (p38gamma and p38delta inhibitor)

BIRB796

has been recognized an essential regulator of dendritic cell and lymphocyte trafficking, which are involved in transforming innate immune responses into adaptive responses [16]. Because IFN-gamma induces chemokine production, it enhances adaptive immunity [17]. 2. Material and methods 2.1. Ethics statement All human materials such as peripheral blood used in this study were obtained from nonsmoking healthy volunteers who gave informed consent. The study protocol was approved by the Institutional Review Board of Kumamoto Health Science University. 2.2. Reagents Human neutrophil elastase (HNE) with an activity of 200 U/L was purchased from SERVA Electrophoresis (Heidelberg, Germany). Human neutrophils were activated by exposure to phorbol 12-myristate 13-acetate (Merck Millipore, Bedford, MA) to induce the formation of neutrophil extracellular traps (NETs). Neutrophil granular proteins such as HNE, cathepsin G (Cosmo Bio Co., Tokyo, Japan), proteinase 3 (Cosmo Bio Co.), and myeloperoxidase (Athens Research and Technology, Athens, GA) were utilized with the micropatterned Transwell system (Corning Incorporated Corning, NY) to investigate cytokine production by monocytes. SB203580 (Wako, Kanagawa, Japan), PD98059 (Wako), BIRB796 (Axon Medchem, Groningen, Netherlands), PDTC (BioVision, Mountain View, CA), TMB-8 (Sigma-Aldrich, Oakville, Ontario, Canada), and U73122 (Merck Millipore) were employed to investigate the intracellular signal transduction pathways involved in IFN-gamma production. All

reagent solutions were negative for endotoxin according to the Endospecy test [18]. The actions of these reagents are summarized in Table 1. 2.3. Isolation of adherent monocytes from peripheral blood mononuclear cells Lymphocyte medium for thawing (BBLYMPH1) was obtained from Zen-Bio, Inc. (Research Triangle Park, NC). Lymphoprep was obtained from Nycomed (Oslo, Norway). Peripheral blood mononuclear cells (PBMCs) were isolated as described previously [19]. Briefly, heparinized blood samples were obtained from nonsmoking healthy volunteers and were diluted 1:1 with pyrogen-free saline. PBMCs were isolated immediately after collection using Lymphoprep gradients, after which the cells were suspended in BBLYMPH1 and incubated for 3 h. For isolation of monocytes by adherence, cells were distributed into 12-well plates (Corning Inc. Costar, NY, USA) at 1 × 106 cells per well and allowed to adhere for 2 h at 37 °C in a 5% CO2 incubator, followed by washing 3 times with warm phosphate-buffered saline (PBS) to remove nonadherent cells. Then monocytes were cultured in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS) and 10 × 103 μg/L gentamicin at 37 °C in humidified air with 5% CO2. Adherent monocytes were recovered with a cell scraper and their purity was evaluated by staining with CD14-phycoerythrin (PE) mouse anti-human monoclonal antibody (Life Technologies, Staley Road, Grand Island, NY) and flow cytometric (FACS) analysis. Recovery of monocytes was also evaluated by trypan blue staining and counting under a Zeiss microscope (Jena, Germany). Only CD14+ monocytes with N85% purity were used for the present experiments. Monocytes were resuspended in RPMI-1640 medium (Sigma-Aldrich, Oakville, Ontario, Canada) supplemented with 25 mM HEPES (Sigma-Aldrich), 100 mM/L L-glutamine (Sigma-Aldrich), 100 × 103 U/L penicillin, and 100 × 103 μg/L streptomycin (Sigma-Aldrich), and then were stimulated with HNE for 6 h. 2.4. Isolation of human neutrophils Human neutrophils were isolated from the peripheral blood of healthy nonsmoking volunteers as previously described [20]. Briefly, a suspension containing neutrophils was prepared by dextran sedimentation, Ficoll-Paque centrifugation, and hypotonic lysis of residual

Fig. 1. Cytokine mRNA expression by monocytes exposed to NETs. RT-PCR showed that PMA alone did not upregulate expression of IL-10, IL-12, and IFN-gamma by monocytes, while NETs from PMA-stimulated human neutrophils upregulated IL-10 and IFN-gamma mRNA expression. HNE also upregulated IL-10 and IFN-gamma mRNA expression. The relative density of the bands normalized to β-actin is shown on the right. PMA: phorbol 12-myristate 13-acetate, HNE: neutrophil elastase, and RT-PCR: reverse transcription polymerase chain reaction. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; N.S., not significant. 1. Neutrophil elastase (85 × 103 μM/mL). 2. Phorbol myristate acetate (200 × 103 mM/L). 3. Phorbol myristate acetate (200 × 103 mM/L) + Human neutrophils (2 × 105 cells).

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erythrocytes. The purity of the neutrophil fraction was typically N93%, as determined by FACS analysis of CD15, CD14, and CD10 expression.

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protein in whole-cell lysates were measured by ELISA (Abcam Inc., Cambridge, MA) with an IL-10 monoclonal antibody and an IFNgamma monoclonal antibody.

2.5. Transwell assay for neutrophil extracellular traps (NETs) 2.9. Effects of myeloperoxidase on IFN-gamma production by monocytes The micropatterned Transwell system (Corning Incorporated Corning, NY) was used to evaluate the effect of NET formation stimulated by phorbol 12-myristate 13-acetate (PMA) on cytokine mRNA expression by monocytes. To examine the contribution of soluble factors independently of cell contact, we used inserts (with a pore size of 0.4 μm) in the Transwell system to prevent cell–cell contact while allowing diffusion of soluble factors. Human neutrophils (2 × 105 cells) with or without PMA (200 × 103 mM/L) stimulation were placed in the Transwell insert and monocytes (1 × 106 cells) were added to the bottom chamber. In addition, the effects of human neutrophil elastase were examined by placing elastase (85 × 103 μM/L) in the Transwell inserts and monocytes in the bottom chamber.

Myeloperoxidase (0, 10, 50, 100, and 200 × 103 mU/L) was placed in the Transwell inserts and monocytes (1 × 106) were added to the bottom chambers. After incubation for 6 h, IFN-gamma protein levels in whole-cell lysates were determined by ELISA (Abcam Inc.). 2.10. Effect of intracellular signaling pathways on IFN-gamma production Monocytes (1 × 106 cells per well) were distributed into 12-well plates (Corning Inc. Costar, NY, USA). Then SB203580 (10 × 103 μM/L), PD98059 (10 × 103 μM/L), PDTC (10 × 103 μM/L), TMB-8

2.6. Transwell assay of neutrophil granule proteins To examine effects of neutrophil granule proteins on cytokine production by monocytes, granule proteins such as myeloperoxidase (200 × 103 mU/L), proteinase 3 (1 × 103 μg/L), and cathepsin G (1 × 103 μg/L) were placed in the Transwell inserts and monocytes (1 × 106 cells) were added to the bottom chamber. 2.7. Extraction of RNA and reverse transcription polymerase chain reaction (RT-PCR) Monocytes from the bottom chamber (1 × 106 cells) were extracted with 1 ml of Isogen RNA kit (Nippon Gene, Toyama, Japan). Total RNA was isolated and precipitated according to the manufacturer's instructions, after which 200 ng of total RNA was reverse-transcribed using Oligo dT primer with PrimeScript™ RT reagent Kit (Takara Bio, Shiga, Japan). Then the reverse-transcribed RNA was amplified by PCR using a TaKaRa PCR thermal cycler (Takara Bio). The primer sequences used were as follows: IL-4: forward, 5′- TGCTGCCTCCAAGAACACAACTG-3′, and reverse, 5′- CATGATCGTCTTTAGCCTTTCCA-3′;IL-6: forward, 5′GTACCCCCAGGAGAAGATTC-3′ and reverse 5′-CAAACTGCATAGCCACTT TC-3′;IL-8: forward, 5′-GCTTTCTGATGGAAGAGAGC-3′ and reverse, 5′GGCACAGTGGAACAAGGACT-3′;IL-10: forward, 5′-ATGCCCCAAGCTGA GAACCAAGAC-3′ and reverse, 5′-TCTCAAGGGGCTGGGTCAGCTATC CCA-3′; IL-12: forward, 5′-TCACAAAGGAGGCGAGGTTC-3′ and reverse, 5′-TGAACGGCATCCACCATGAC-3′;TNF-alpha: forward, 5′-TCGGGCCA ATGCCCTCCTGGCCAA-3′ and reverse, 5′-CTGGGATGCTCTTCGACCTC3′;IFN-gamma: forward, 5′-ATAATGCAGAGCCAAATTGTCTC-3′ and reverse, 5′-CAGCTGTATTGGTCGATTGC-3′;beta-actin: forward, 5′-GTGG GGCGCCCCAGGCACCA-3′ and reverse, 5′-CTCCTTAATGTCACGCACGA TTTC-3′. The PCR conditions were as follows: IL-4, 35 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s); IL-6, 35 cycles (94 °C for 60 s, 60 °C for 60 s, and 72 °C for 30 s); IL-8, 35 cycles (94 °C for 60 s, 60 °C for 60 s, and 72 °C for 30 s); IL-10, 35 cycles (94 °C for 60 s, 60 °C for 60 s, and 72 °C for 30 s); IL-12, 35 cycles (94 °C for 60 s, 60 °C for 60 s, and 72 °C for 30 s); TNF-alpha, 35 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s); IFN-gamma, 35 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s); and beta-actin, 35 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s). PCR products were analyzed on agarose gels. 2.8. Enzyme-linked immunosorbent assay (ELISA) for IL-10 and IFNgamma HNE (85 × 103 μM/L), myeloperoxidase (200 × 103 mU/L), cathepsin G (1 × 103 μg/L), and proteinase 3 (1 × 103 μg/L) were placed in the Transwell inserts and monocytes (1 × 106) were added to the bottom chambers. After incubation for 6 h, the levels of IL-10 and IFN-gamma

Fig. 2. IL-10 expression by monocytes exposed to NETs. a. Effect of NETs on IL-10 mRNA expression by monocytes demonstrated by RT-PCR and densitometry of the PCR products. NETs from PMA-stimulated human neutrophils enhanced IL-10 mRNA expression, as did myeloperoxidase and cathepsin G. In contrast, proteinase-3 was less effective for promoting IL-10 mRNA expression than NETs. The relative density of the bands normalized to βactin is shown. PMA: phorbol 12-myristate 13-acetate, NETs: neutrophil extracellular traps, RT-PCR: reverse transcription polymerase chain reaction. 1. Myeloperoxidase (200 × 103 mU/L). 2. Proteinase-3 (1 × 103 μg/L). 3. Cathepsin G (1 × 103 μg/L). 4. PMA (200 × 103 mM/L) + neutrophils (2 × 105 cells). b. The Transwell assay showed that cathepsin G and myeloperoxidase both increased IL-10 protein production by monocytes. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; **P b .05; N.S., not significant. 1. Human neutrophil elastase (0 × 103 μM/L). 2. Human neutrophil elastase (85 × 103 μM/L). 3. Myeloperoxidase (200 × 103 mU/L). 4. Proteinase-3 (1 × 103 μg/L). 5. Cathepsin G (1 × 103 μg/L).

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(10 × 103 μM/L) and U73122 (1 × 103 μM/L) were employed to investigate the intracellular signal transduction pathways involved in IFNgamma production by cells stimulated with myeloperoxidase (50 × 103 mU/L). In addition, BIRB796 (5, 10, 20 × 103 μM/L) was utilized to investigate the role of p38 gamma/delta cellular signaling in IFN-gamma production. Monocytes were stimulated by incubation for 6 h in the presence of myeloperoxidase with or without these agents, after which IFN-gamma protein was measured in whole-cell lysates by ELISA (Abcam Inc.). 2.11. Statistical analysis Results are expressed as the mean ± SE. Analysis of variance and the t-test of independent mean values were used to assess differences between multiple groups and differences between two groups, respectively. When the F ratio was significant, mean values were compared by a

Fig. 4. IFN-gamma production by monocytes stimulated with myeloperoxidase (MPO). MPO (0, 10, 50, 100, and 200 × 103 mU/L) was placed in the Transwell inserts and monocytes (1 × 106) were introduced into the bottom chamber. After incubation for 6 h, IFNgamma protein levels in whole-cell lysates were determined by ELISA. MPO upregulated IFN-gamma production in a dose-dependent manner. ELISA: enzyme-linked immunosorbent assay. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; **P b .05; N.S., not significant. 1. MPO (0 × 103 mU/L). 2. MOP (10 × 103 mU/L). 3. MPO (50 × 103 mU/L). 4. MPO (100 × 103 mU/L). 5. MPO (200 × 103 mU/L).

post hoc Bonferroni test. A probability (P) value b 0.05 was considered to indicate a significant difference in all analyses. 3. Results The Transwell assay showed that exposure to HNE increased the expression of mRNA for IL-4, IL-6, IL-8, IL-10, IL-12, TNF-alpha, and IFN-

Fig. 3. IFN-gamma expression by monocytes exposed to NETs. a. RT-PCR and densitometry of the PCR products demonstrated that NETs from PMA-stimulated human neutrophils enhanced IFN-gamma mRNA expression, as did myeloperoxidase. The Transwell assay showed that proteinase-3 and cathepsin G also increased IFN-gamma mRNA expression, but myeloperoxidase had a significantly stronger effect than these compounds. The relative density of the bands normalized to β-actin is shown. 1. Myeloperoxidase (200 × 103 mU/L). 2. Proteinase-3 (1 × 103 μg/L). 3. Cathepsin G (1 × 103 μg/L). 4. PMA (200 × 103 mM/L) + neutrophils (2 × 105 cells). b. The Transwell assay showed that myeloperoxidase caused a significantly larger increase of IFN-gamma protein production by monocytes than neutrophil elastase, proteinase-3, or cathepsin G. NETs: neutrophil extracellular traps, RT-PCR: reverse transcription polymerase chain reaction. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; **P b .05. 1. Human neutrophil elastase (0 × 103 μM/L). 2. Human neutrophil elastase (85 × 103 μM/L). 3. Myeloperoxidase (200 × 103 mU/L). 4. Proteinase-3 (1 × 103 μg/L). 5. Cathepsin G (1 × 103 μg/L).

Fig. 5. Effect of SB203580, PD98059, PDTC, TMB-8, and U73122 on IFN-gamma production by monocytes. Neither SB203580 nor PD98059 influenced IFN-gamma production. However, pretreatment of monocytes with either PDTC or TMB-8 significantly decreased IFN-gamma production. SB203580: p38 mitogen-activated protein kinase inhibitor, PD98059: extracellular signal-regulated kinase inhibitor, PDTC: NF-kB inhibitor, TMB-8: intracellular calcium antagonist, U73122: phospholipase C inhibitor, MPO: myeloperoxidase. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; **P b .05; N.S., not significant. 1. MPO (50 × 103 mU/L). 2. SB203580 (10 × 103 μM/L) + MPO (50 × 10 3 mU/L). 3. PD98059 (10 × 10 3 μM) + MPO (50 × 10 3 mU/L). 4. PDTC (10 × 10 3 μM/L) + MPO (50 × 10 3 mU/L). 5. TMB-8 (10 × 103 μM/L) + MPO (50 × 103 mU/L). 6. U73122 (1 × 103 μM/L) + MPO (50 × 103 mU/L).

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gamma by monocytes. When PMA alone was placed in the upper compartment of the Transwell chamber, it induced the expression of IL-4, IL6, IL-8, and TNF-alpha mRNA by monocytes, but expression of IL-10, IL12, and IFN-gamma mRNA was not detected. When formation of NETs was induced by adding neutrophils and PMA to the upper compartment, this enhanced the expression of IL-10 and IFN-gamma mRNA by monocytes, but did not upregulate IL-12 mRNA (Fig. 1). The Transwell assay was also performed with human neutrophil granule proteins in the upper compartment, including myeloperoxidase, proteinase-3, and cathepsin G. Myeloperoxidase and cathepsin G both significantly increased IL-10 mRNA expression by monocytes compared with proteinase-3 and PMA plus neutrophils (Fig. 2a). When HNE was placed in the upper chamber, it weakly stimulated IL-10 mRNA expression by monocytes. On the other hand, myeloperoxidase and cathepsin G induced significantly more IFN-gamma production by monocytes than HNE or proteinase-3 (Fig. 2b). The Transwell assay also revealed that expression of IFN-gamma mRNA by monocytes was increased when formation of NETs was induced by exposing neutrophils to PMA in the upper compartment, as well as when myeloperoxidase, proteinase-3, or cathepsin G was added to the upper compartment (Fig. 3a). Myeloperoxidase significantly increased IFN-gamma protein production by monocytes compared with HNE, proteinase-3, and cathepsin G (Fig. 3b). As shown in Fig. 4, the stimulatory effect of myeloperoxidase on IFNgamma protein production by monocytes was dose-dependent. This effect of myeloperoxidase on IFN-gamma production was reduced by an intracellular calcium antagonist (TMB-8) and by an inhibitor of NF-kB (PDTC), but not by an extracellular signal-regulated kinase inhibitor (PD98059), a p38 mitogen-activated protein kinase inhibitor (SB203580), or a phospholipase C inhibitor (U73122) (Fig. 5). RT-PCR

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showed that pretreatment of monocytes with a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma mRNA expression in monocytes after stimulation with myeloperoxidase (50 × 103 mU/L), while a combined p38alpha and p38beta inhibitor (SB203580) did not have this effect. Although SB203580 did not reduce IFN-gamma production, exposure to higher concentrations of BIRB796 significantly decreased IFN-gamma protein levels (Fig. 6a and b). 4. Discussion This study demonstrated that neutrophil myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. We utilized the Transwell assay to investigate the effect of NETs, which were obtained by using PMA to stimulate neutrophils. However, PMA itself induces cytokine production by monocytes. Nuclear factor kappa-B (NF-kB) [21,22] and protein kinase C [23] have long been considered to mediate a prototypical proinflammatory signaling pathway, and PMA is a specific activator of both protein kinase C and NF-κB [24–26]. PMA induces the phosphorylation of extracellular signal-regulated kinase (ERK), which is dependent on the protein kinase C alpha pathway [27, 28]. In the present study, adding PMA alone in the Transwell assay upregulated the expression of IL-4, IL-6, IL-8, and TNF-alpha mRNA by monocytes. Indeed, production of proinflammatory cytokines (but not anti-inflammatory cytokines) by PMA-treated THP-1 cells and monocyte-derived macrophages has already been reported [29]. The present study also demonstrated that expression of IL-10, IL-12, and IFN-gamma mRNA by monocytes was not detected after stimulation with PMA alone. Unlike PMA, the present study demonstrated that human neutrophil granule proteins (including HNE, myeloperoxidase, proteinase-3, and

Fig. 6. Effect of SB203580 and BIRB796 on IFN-gamma expression by monocytes. a. Effect of SB203580 or BIRB796 on IFN-gamma mRNA expression by monocytes demonstrated by RT-PCR and densitometry of the PCR products. Pretreatment of monocytes with BIRB796 significantly decreased IFN-gamma mRNA levels after stimulation with myeloperoxidase (MPO), but not SB20350. SB203580: combined p38alpha and p38beta inhibitor, BIRB796: combined p38gamma and p38delta inhibitor, RT-PCR: reverse transcription polymerase chain reaction. The relative density of the bands normalized to β-actin is shown. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; **P b .05; N.S., not significant. b. Pretreatment of monocytes with SB203580 did not influence IFN-gamma production, but BIRB796 (5 × 103 μM/L) significantly decreased IFN-gamma production compared with SB203580. Higher concentrations of BIRB796 (10 and 20 × 103 μM/L) had a significantly greater effect on IFN-gamma production by monocytes. Data were obtained by using samples from three volunteers in each group and are represented as the mean ± SE. *P b .01; **P b .05; N.S., not significant. 1. MPO (50 × 103 mU/L). 2. SB203580 (20 × 103 μM/L) + MPO (50 × 103 mU/L). 3. BIRB796 (5 × 103 μM/L) + MPO (50 × 103 mU/L). 4. BIRB796 (10 × 103 μM/L) + MPO (50 × 103 mU/L). 5. BIRB796 (20 × 103 μM/L) + MPO (50 × 103 mU/L).

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cathepsin G) increased IFN-gamma production by monocytes. Among these proteins, only myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Therefore, we investigated the mechanism underlying IFN-gamma production by monocytes stimulated with myeloperoxidase. Myeloperoxidase is a peroxidase enzyme that is most abundantly expressed in neutrophil granules. It participates in innate immune defense mechanisms through formation of microbicidal reactive oxygen species and diffusible radicals. The process of NET generation, called netosis, is a specific type of cell death that differs from necrosis and apoptosis. Intravascular NETs capture bacteria from the bloodstream during sepsis and may also modulate immunoregulatory responses. It has been demonstrated that myeloperoxidase is required for NET formation and innate immunity, with persons who have complete myeloperoxidase deficiency failing to form NETs [8]. It has been reported that enzymatically active myeloperoxidase is released from PMAstimulated neutrophils after 2–4 h, concomitant with NET formation [30]. Therefore, IFN-gamma mRNA and protein levels were determined 6 h after the stimulation of NET formation in this study. Interestingly, it was found that myeloperoxidase markedly enhanced IFN-gamma production by monocytes in a dose-dependent manner. Nuclear factor-kappaB (NF-kB) plays a key role in inflammation. Pyrrolidine dithiocarbamate (PDTC) reduces the production of inflammatory cytokines via the NF-κB pathway. This study demonstrated that pretreatment of monocytes with PDTC attenuated IFNgamma production after stimulation with myeloperoxidase. Because myeloperoxidase catalyzes the formation of oxygen radicals, it plays an important role in inflammation and oxidative stress. Indeed, it has been shown that myeloperoxidase, lactoperoxidase, and eosinophil peroxidase have a central role in oxidative damage associated with inflammatory disorders by utilizing hydrogen peroxide and halides/pseudo halides to generate the corresponding hypohalous acid [31]. NETs have been reported to contain oxidative enzymes, including myeloperoxidase, NADPH oxidase, and nitric oxide synthase [32]. Therefore, the inhibitory effect of PDTC on IFN-gamma production by monocytes may be related to both its role as a low molecular weight thiol antioxidant and its potent inhibition of NF-kB. IFN-gamma production by monocytes stimulated with myeloperoxidase was also attenuated by the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8). TMB-8 blocks both Ca2 + channels and Na+ channels and reduces membrane K+ conductance. Sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase plays a critical role in Ca2 + homeostasis. Myeloperoxidase-derived oxidants increase cytosolic Ca 2 + levels in human coronary artery endothelial cells, so myeloperoxidase-mediated modulation of intracellular Ca 2 + may exacerbate endothelial dysfunction [33]. The mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that regulate diverse cellular programs by relaying extracellular signals. Conventional MAPKs comprise the extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun amino (N)-terminal kinases 1/2/3 (JNK1/2/3), p38 isoforms (alpha, beta, gamma, and delta), and ERK5 [34]. PD98059 was identified as a MEK1/2 inhibitor and this molecule also inhibit MEK5 [35]. However, PD98059 did not affect IFN-gamma production by monocytes in this study. The stressactivated protein kinase (SAPK) p38 isoforms are also MAPK family members, comprising p38alpha, p38beta, SAPK3/p38gamma, and SAPK4/ p38delta [36]. Because p38 MAPK may regulate cytokine production, we investigated the effect of a p38 MAPK inhibitor (SB203580) on the IFNgamma production by monocytes. This study demonstrated that SB203580 did not affect IFN-gamma production. SB203580 has been shown to inhibit p38alpha and p38beta, but not the p38gamma and p38delta isoforms [37]. Next, a p38 MAPK inhibitor (BIRB796) was utilized to investigate intracellular signal transduction. BIRB796 is a diaryl urea compound (1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2morpholin-4-yl-ethoxy)naphthalen-1-yl] urea) that is not only a potent

inhibitor of p38alpha and p38beta, but also inhibits p38gamma and p38delta at higher concentrations [38,39]. The present study demonstrated that higher concentrations of BIRB796 significantly decreased IFNgamma production by monocytes stimulated with myeloperoxidase, indicating that myeloperoxidase partly stimulates IFN-gamma production via the p38gamma/delta pathway. The mannose receptor is a 175 kDa type I transmembrane protein that is a member of the C-type lectin family and contains multiple carbohydrate recognition domains. Mannose receptor CD206 was initially identified as part of a mechanism protecting tissues against inflammatory damage through removal of mannosyl glycoconjugate ligands, including lysosomal hydrolases. Natural ligands that are cleared by the mannose receptor include lysosomal hydrolases, tissue plasminogen activator, myeloperoxidase, and thyroglobulin. In fact, it was reported that rat bone marrow-derived macrophages internalized 75% of [125I]myeloperoxidase through a mannose-specific process [40]. Surface expression of CD206 by CD14+ monocytes has been demonstrated in healthy volunteers [41] and CD206 expression increases during differentiation into macrophages [42]. In conclusion, myeloperoxidase was found to stimulate IFN-gamma production by monocytes via the CD206/Ca2+/p38gamma/delta pathway in the presence of NETs. Role of the funding source The funding source had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Conflict of interest declaration The authors declare that there are no conflicts of interest. Acknowledgement This study was supported by a Kumamoto Health Science University special fellowship grant (No. 24-A-02). References [1] U. Bank, S. Ansorge, More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control, J. Leukoc. Biol. 69 (2001) 197–206. [2] C. Coeshott, C. Ohnemus, A. Pilyavskaya, S. Ross, M. Wieczorek, H. Kroona, A.H. Leimer, J. Cheronis, Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6261–6266. [3] S.E. Robertson, J.D. Young, S. Kitson, A. Pitt, J. Evans, J. Roes, D. Karaoglu, L. Santora, T. Ghayur, F.Y. Liew, J.A. Gracie, I.B. McInnes, Expression and alternative processing of IL-18 in human neutrophils, Eur. J. Immunol. 36 (2006) 722–731. [4] T.C. Lin, C.Y. Li, C.S. Tsai, C.H. Ku, C.T. Wu, C.S. Wong, S.T. Ho, Neutrophil-mediated secretion and activation of matrix metalloproteinase-9 during cardiac surgery with cardiopulmonary bypass, Anesth. Analg. 100 (2005) 1554–1560. [5] K. Kessenbrock, L. Fröhlich, M. Sixt, T. Lämmermann, H. Pfister, A. Bateman, A. Belaaouaj, J. Ring, M. Ollert, R. Fässler, D.E. Jenne, Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin, J. Clin. Invest. 118 (2008) 2438–2447. [6] E.P. McGreal, P.L. Davies, W. Powell, S. Rose-John, O.B. Spiller, I. Doull, S.A. Jones, S. Kotecha, Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis, Biochim. Biophys. Acta 1802 (2010) 649–658. [7] U. Meyer-Hoffert, O. Wiedow, Neutrophil serine proteases: mediators of innate immune responses, Curr. Opin. Hematol. 18 (2011) 19–24. [8] K.D. Metzler, T.A. Fuchs, W.M. Nauseef, D. Reumaux, J. Roesler, I. Schulze, V. Wahn, V. Papayannopoulos, A. Zychlinsky, Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity, Blood 117 (2011) 953–959. [9] D.M. Paulnock, K.P. Demick, S.P. Coller, Analysis of interferon-gamma-dependent and -independent pathways of macrophage activation, J. Leukoc. Biol. 67 (2000) 677–682. [10] M. Goldberg, L.S. Belkowski, B.R. Bloom, Regulation of macrophage function by interferon-gamma. Somatic cell genetic approaches in murine macrophage cell lines to mechanisms of growth inhibition, the oxidative burst, and expression of the chronic granulomatous disease gene, J. Clin. Invest. 85 (1990) 563–569.

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