- Email: [email protected]
3 pathway
Cytokine 86 (2016) 53–63
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Cytokine secreted by S100A9 via TLR4 in monocytes delays neutrophil apoptosis by inhibition of caspase 9/3 pathway Na Rae Lee a,1, Beom Seok Park b,c,1, Seong Yeol Kim c, Ayoung Gu c, Da Hye Kim c, Ji-Sook Lee d,⇑, In Sik Kim a,c,⇑ a
Department of Biomedical Laboratory Science, School of Medicine, Eulji University, Daejeon 34824, Republic of Korea Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Republic of Korea Department of Senior Healthcare, BK21 Plus Program, Graduate School, Eulji University, Daejeon 34824, Republic of Korea d Department of Clinical Laboratory Science, Wonkwang Health Science University, Iksan 54538, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 25 March 2016 Received in revised form 5 July 2016 Accepted 8 July 2016 Available online 25 July 2016 Keywords: Allergy Cytokine Neutrophil apoptosis Monocytes S100A9
a b s t r a c t Dysregulation of neutrophil apoptosis causes pathogenesis and aggravation of allergy. S100A9 exists as one of the proteins in the neutrophils, triggering inflammatory responses by activating the immune cells. In this study, we investigated whether S100A9 affects constitutive neutrophil apoptosis by activating the monocytes in normal and allergic subjects. Supernatant from human monocytic THP-1 cells after treatment with S100A9 suppressed normal neutrophil apoptosis by inhibiting the activations of caspase 9 and caspase 3. S100A9 upregulated the release of MCP-1, IL-6, and IL-8 in THP-1 cells. An increase in cytokine was suppressed by CLI-095, a Toll-like receptor (TLR) 4 inhibitor, PP2, a Src inhibitor, rottlerin, a PKCd inhibitor, MAP kinase inhibitors, including PD98059, SB202190, and SP600125, and BAY-11-7085, an NF-jB inhibitor. Src, PKCd, ERK1/2, p38 MAPK, and JNK were phosphorylated by S100A9. The phosphorylation of Src and PKCd was suppressed by CLI-095, and the activation of ERK1/2, p38 MAPK, and JNK was inhibited by CLI-095, PP2, and rottlerin. S100A9 induced NF-jB activity, and the activation was suppressed by CLI-095, PP2, rottlerin, and MAPK kinase inhibitors. In normal and allergic subjects, supernatant from normal and allergic monocytes after stimulation with S100A9 suppressed normal and allergic neutrophil apoptosis, respectively; MCP-1, IL-6, and IL-8 in the supernatant was increased by S100A9. The cytokine secretion induced by S100A9 is related to TLR4, Src, PKCd, ERK1/2, p38 MAPK, JNK, and NF-jB. Taken together, S100A9 induces anti-apoptotic effect on normal and allergic neutrophils by increasing cytokine secretion of monocytes. These findings may help us to better understand neutrophil apoptosis regulated by S100A9 and pathogenesis of allergic diseases. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Cell death of neutrophils is an important step in the resolution of inflammation and infection [1–3]. Neutrophil apoptosis is regulated by various stimulators, which includes pro-apoptotic stimulators, such as Fas ligand and TNF-a, and contains anti-apoptotic cytokines, such as IL-6, IL-8, and GM-CSF [4,5]. Our recent report demonstrated that MCP-1 is a new anti-apoptotic molecule in
⇑ Corresponding authors at: Department of Clinical Laboratory Science, Wonkwang Health Science University, 501, Iksandaero, Iksan 54538, Republic of Korea (J.-S. Lee). Department of Biomedical Laboratory Science, School of Medicine, Eulji University, 77, Gyeryoung-ro 771 beon-gil, Jung-Gu, Daejeon 34824, Republic of Korea (I.S. Kim). E-mail addresses: [email protected] (J.-S. Lee), [email protected] (I.S. Kim). 1 Authors contributed equally to this work. http://dx.doi.org/10.1016/j.cyto.2016.07.005 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
neutrophil apoptosis [6]. Intracellular mechanisms of neutrophil apoptosis involve caspase, Bcl family proteins, and reactive oxygen species (ROS) [5,7]. A delay in neutrophil apoptosis induces longterm existence of neutrophils in healthy tissues, which destroys the tissue. Neutrophil-mediated tissue injury is associated with allergic diseases, such as asthma and allergic rhinitis [8–11]. S100A9 is a type of S100 family proteins, and constitutively expressed in the neutrophils and monocytes [12]. S100A9 acts as damage-associated molecular pattern (DAMP) via Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), and triggers abnormal inflammatory responses that result in asthma, chronic obstructive pulmonary disease, colitis, rheumatoid arthritis, Alzheimer’s disease, and tumor [13,14]. It has been reported that S100A9 increases cytokine secretion. S100A9 induces the expression of IP-10 in monocytes/macrophages, accelerating IL-8 secretion induced by GM-CSF in the neutrophils [15,16].
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In this work, we studied that S100A9 induces cytokine secretion in monocytes, which alters spontaneous apoptosis of normal and allergic neutrophils.
caspase 9 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-ERK1/2 was obtained from Cell Signaling Technology (Beverly, MA, USA).
2. Materials and methods
2.2. Normal subjects and allergic subjects
2.1. Materials
Twelve allergy patients (seven allergic asthma and five allergic rhinitis subjects) were recruited from Eulji University Hospital. Allergic patients between 15 and 52 years of age (average = 35.9 years) had mild to severe symptoms of the disease. Allergic status was based on the presence of positive results of a skin prick test (P2+), multiple allergen simultaneous test (MAST) (Pclass 2), or measurement of specific HDM IgE using the Pharmacia Unicap 100 system to common allergens. Levels of total IgE of normal and allergic subjects using an ADVIA Centaur immunoassay (Siemens Medical Solutions Diagnostics, Erfurt, Germany) were 68.5 IU/ml and 565.3 IU/ml, respectively. Additionally, ten normal subjects between 18 and 30 years of age (average = 24.4 years) were recruited as controls. The normal subjects had normal lung
RPMI 1640, penicillin and streptomycin solution, fetal bovine serum (FBS), and TRIzol reagent were obtained from Life Technologies Inc. (Gaithersburg, MD). CLI-095, an inhibitor of Toll-like receptor (TLR) 4 (TLR4i) was purchased from Invivogen (San Diego, CA, USA). Src inhibitor (PP2), PKCd inhibitor (rottlerin), a classic PKC inhibitor (Ro-31-8425), MEK inhibitor (PD98059), p38 MAPK inhibitor (SB202190), JNK inhibitor (SP600125), and NF-jB inhibitor (BAY-11-7085) were products of Calbiochem (San Diego, CA, USA). Anti-phospho-Src, anti-Src, anti-phospho-PKCd, anti-PKCd, anti-ERK2, anti-phospho-p38 MAPK, anti-p38 MAPK, antiphospho-JNK, anti-JNK, anti-cleaved caspase 3, and anti-cleaved
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Fig. 1. Spontaneous neutrophil apoptosis is delayed by secreted molecules due to S1000A9 in THP-1 cells. (A) THP-1 cells were incubated with and without 1 lg/ml of S100A9 for 6 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of normal subjects. Neutrophils apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄⁄p < 0.01 indicates a significant difference between the control and S100A9-treated groups or between the control supernatant and supernatant-treated groups. (B) Neutrophils isolated from normal subjects were incubated with the supernatant (Sup) or the S100A9-treated supernatant of THP-1 cells. Caspase 9 and caspase 3 were detected by Western blotting. The membrane was stripped and reprobed with anti-ERK2 antibodies as an internal control (upper panel). Densitometric data (n = 3) are presented relative to the negative control, which was set at l (lower panel).
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2.3. Isolation of neutrophils and monocytes Human neutrophils and monocytes were isolated from the heparinized peripheral blood of normal and allergic subjects using Ficoll-Hypaque gradient centrifugation. A CD16 microbeads magnetic cell sorting kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for isolation of neutrophil from granulocytes. A monocyte isolation kit II (Miltenyi Biotec) was used for isolation of monocytes from peripheral mononuclear cells. Isolated neutrophils and monocytes were washed by phosphate buffered saline (PBS) buffer after removal of erythrocytes by hypotonic lysis. Purity of neutrophils and monocytes was greater than 97%. 2.4. Cell culture The human monocytic cell line THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA). THP-1 cells,
A **
MCP-1 (pg/ml)
5000
**
3000 **
**
2000
2.5. Production of recombinant S100A9 proteins Recombinant S100A9 protein was produced as previously reported [17]. Briefly, total RNA of human neutrophils was extracted using TRIzol reagent and first strand cDNA was synthesized. S100A9-1 (50 -ttccatatgatgacttgcaaaatgtcgca), and S100A9-2 (50 -ccgctcgagactgtggtctta-gggggtgc) were used for cDNA synthesis of S100A9. The cDNA was cloned into pET28 expression vector (Merck Millipore, Darmstadt, Germany), respectively. Recombinant His-Tag S100A9 was induced and then purified using a nickel column. The purified protein was identified by western blotting using anti-S100A9 antibodies. 2.6. Enzyme-linked immunosorbent assay (ELISA) The concentrations of MCP-1, IL-6, and IL-8 in a cell supernatant were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) using OptEIATM Set human IL-6, IL-8 and MCP-1 (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions.
0 1h 3h 6h 9h 12h 24h 48h
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4000
neutrophils and monocytes were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 lg/ml).
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function, no history of asthma and allergic rhinitis, and did not require medication. This study was approved by the Institutional Review Board of Eulji University for normal volunteers and by the Institutional Review Board of Eulji University Hospital for allergic patients. All participants in this study gave their written informed consent.
S100A9
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Fig. 2. S100A9 induces the secretion of MCP-1, IL-6, and IL-8 in THP-1 cells. (A) THP-1 cells were incubated in the absence or presence of 1 lg/ml S100A9 for the indicated time. (B) THP-1 cells were incubated in the indicated concentration of S100A9 for 6 h. The supernatant was collected and analyzed by ELISA. Data are expressed as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9-treated groups.
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The blots were developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech.).
2.7. Western blotting Cells were lysed in cytosolic lysis buffer (TransLab, Daejeon, Korea). The lysate was centrifuged and then the supernatant was collected. Protein concentration of the lysate was evaluated by protein assay kit (Thermo scientific, Waltham, MA, USA). Protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose filters. The blots were incubated with primary antibodies including anti-phospho-Src, anti-phospho-PKCd, anti-phosphoERK, anti-phospho-p38 MAPK, anti-phospho-JNK, anti-caspase 3 or anti-caspase 9 antibodies, after which the blots were incubated with secondary antibodies including goat anti-rabbit antibodies.
2000
500
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1600 1200
The DNA-binding activity of NF-jB was assessed using EZDetectTM transcription factor kits for NF-jB p65 (PIERCE, Rockford, IL), following the manufacturer’s instructions. DNA binding specificity was assessed using wild type or mutant NF-jB oligonucleotides. Chemiluminescent detection was performed using a luminometer.
% IL-6 release
% MCP-1 release
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2.8. NF-jB p65 transcription factor assay
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Fig. 3. S100A9 increases the secretion of MCP-1, IL-6, and IL-8 through TLR4, Src, PKCd, MAPK, and NF-jB activation in THP-1 cells. (A and B) THP-1 cells were pre-treated for 1 h with and without the indicated concentration of TLR4i (A), 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB), 20 lM SP600125 (SP) and 10 lM BAY11-7085 (BAY) (B), after which the cells were incubated for 6 h in the absence and presence of S100A9 (1 lg/ml). The supernatant was collected and analyzed by ELISA. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9treated groups or between the S100A9-treated and inhibitor-treated groups. (C) THP-1 cells were incubated with S100A9 (1 lg/ml) for the indicated time. (D and E) THP-1 cells were pre-treated for 1 h with and without 2 lM TLR4i (D), 20 lM PP2 and 5 lM rottlerin (E), after which the cells were incubated with S100A9 (1 lg/ml) for 30 min. After harvested cells were lysed, phosphorylation of Src, PKCd, ERK, p38 MAPK, and JNK in the lysates was detected by Western blotting (upper panel). Densitometric data (n = 3) are presented relative to the negative control, which was set at 1 (lower panel). (F) THP-1 cells were incubated with S100A9 (1 lg/ml) for the indicated time. (G) THP-1 cells were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB) and 20 lM SP600125 (SP), after which the cells were incubated with S100A9 (1 lg/ml) for 1 h. After harvested cells were lysed, NF-jB in the lysates was detected by luciferase assay. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9-treated groups or between the S100A9-treated and inhibitor-treated groups.
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C
Time
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p-JNK JNK p-p38 MAPK p38 MAPK Fig. 3 (continued)
2.9. Production of monocyte supernatant
2.10. Detection of apoptosis
THP-1 cells and monocytes were incubated with and without 1 lg/ml of S100A9 for 6 h and 48 h, respectively. The supernatant was collected and diluted by RPMI 1640 medium (1:1 ratio). The diluted supernatant was added to the fresh neutrophils isolated from normal and allergic subjects, and incubated for 24 h.
An annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Diego, CA, USA) was used for detection of neutrophil apoptosis. Isolated neutrophils were treated with DP, then incubated with FITC-labeled annexin V and propidium iodide (PI) for 15 min at room temperature. Apoptotic neutrophils were analyzed using a FACSCalibur flow cytometer
N.R. Lee et al. / Cytokine 86 (2016) 53–63
+
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Luciferase activity (Fold)
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Fig. 3 (continued)
with the CellQuest software (BD bioscience) and reported as the percentage of cells showing annexin V+/PI and annexin V+/PI+. 2.11. Statistical analysis Data were represented as the means ± S.E.M. Statistical differences were analyzed using a paired t-test for two-group comparisons and one-way ANOVA for comparison of more than two groups. All analyses were conducted using the SPSS statistical software package (Version 10.0, Chicago, IL), and a p value less than 0.05 was considered significant. 3. Results 3.1. Spontaneous neutrophil apoptosis is delayed by secreted molecules due to S1000A9 in THP-1 cells We first examined the effect of monocyte activation induced by S100A9 on neutrophil apoptosis. Supernatant was collected from THP-1 cells after exposure to S100A9, and its anti-apoptotic effect was evaluated. S100A9 directly inhibited neutrophil apoptosis (Fig. 1A). The supernatant treated with S100A9 was more effective with regard to the inhibition of normal neutrophils apoptosis compared with the direct effect of S100A9. The anti-apoptotic effect of the supernatant was mediated by the inhibition of caspase 9 and caspase 3 activations, post spontaneous apoptosis by neutrophil isolation (Fig. 1B). These results indicate that the action of S100A9 in the monocytes regulates neutrophil apoptosis, and suggests that this process is involved in the caspase 9/3 pathway. 3.2. S100A9 induces the secretion of MCP-1, IL-6, and IL-8 through TLR4, Src, PKCd, MAPK, and NF-jB in THP-1 cells Since the supernatant stimulated with S100A9 inhibited neutrophil apoptosis, we examined that S100A9 increases cytokines,
including MCP-1, IL-6, and IL-8, which are associated with the survivability of neutrophils. As shown in Fig. 2, S100A9 began to increase the secretion of MCP-1, IL-6, and IL-8 at 3 h, which peaked at 6 h after the stimulation of S100A9. S100A9 elevated the cytokine secretion in a dose-dependent manner at 6 h. Next, we examined how the signal mechanism of S100A9 induces cytokine secretion by evaluating the alteration of secretion after an inhibitor treatment. As shown in Fig. 3A and B, TLR4i inhibited the expressions of MCP-1, IL-6, and IL-8 in a dose-dependent manner; other signal inhibitors, such as PP2, rottlerin, PD98059, SP600125, and BAY-11-7085 blocked the increased cytokine expression. SB202190 inhibited the expression of MCP-1 and IL-6, except IL8 expression. S100A9 induces the phosphorylation of Src, PKCd, ERK, p38 MAPK, and JNK in a time-dependent manner (Fig. 3C). The phosphorylation of Src and PKCd was suppressed by TLR4i, and the activation of ERK, p38 MAPK, and JNK was inhibited by TLR4i, PP2, and rottlerin (Fig. 3D and E). In addition, S100A9 activated NF-jB in a time-dependent manner, and the activation was suppressed by TLR4i, PP2, rottlerin, PD98059, SB202190, and SP600125 (Fig. 3F and G). 3.3. The secretion of MCP-1, IL-6, and IL-8 enhanced by S100A9 is involved in TLR4, Src, PKCd, MAPKs, and NF-jB in normal monocytes, and the cytokine secretion suppresses normal neutrophil apoptosis Since cytokine secretion induced by S100A9 in THP-1 cells affects neutrophil apoptosis, we investigated in detail regarding the action of S100A9 in normal monocytes and neutrophils. S100A9 significantly increased the release of MCP-1, IL-6, and IL-8 in normal monocytes in a time-dependent manner, and the increase peaked at 48 h (p < 0.05). This was in contrast to the peak time of cytokine secretion induced by S100A9 in THP-1 cells, which was at 6 h (Fig. 4A). These results indicate that the use of primary cells and confirmation of the cell line are needed in an experimental research. TLR4i, PP2, rottlerin, PD98059, SB202190, SP600125,
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A 0 1h 3h 6h 9h 12h 24h 48h
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Fig. 4. The secretion of MCP-1, IL-6, and IL-8 enhanced by S100A9 is involved in TLR4, Src, PKCd, MAPKs, and NF-jB in normal monocytes, and the cytokine secretion suppresses normal neutrophil apoptosis. (A) Normal monocytes isolated from normal subjects (n = 3) were incubated in the absence or presence of 1 lg/ml S100A9 for the indicated time. (B) Normal monocytes were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB), 20 lM SP600125 (SP) and 10 lM BAY-11-7085 (BAY), after which the cells were incubated for 48 h in the absence and presence of S100A9 (1 lg/ml). The supernatant was collected and analyzed by ELISA. (C) Normal monocytes were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB) and 20 lM SP600125 (SP), after which the cells were incubated with S100A9 (1 lg/ml) for 1 h. After harvested cells were lysed, NF-jB in the lysates was detected by luciferase assay. Data are expressed as the means ± S.E.M. ⁄p < 0.05 indicates a significant difference between the control and S100A9-treated groups or between the S100A9treated and inhibitor-treated groups. (D) Normal monocytes were incubated with and without 1 lg/ml of S100A9 for 48 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of normal subjects. Neutrophils apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄⁄p < 0.01 indicates a significant difference between the control and S100A9-treated groups or between the control supernatant and supernatant-treated groups. (E) Neutrophils isolated from normal subjects were incubated with the supernatant (Sup) or the S100A9treated supernatant of normal monocytes. Caspase 9 and caspase 3 were detected by Western blotting. The membrane was stripped and reprobed with anti-ERK2 antibodies as an internal control (upper panel). Densitometric data (n = 3) are presented relative to the negative control, which was set at 1 (lower panel).
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E Time
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Fig. 4 (continued)
and BAY-11-7085 significantly blocked the increased expression of MCP-1, IL-6, and IL-8 due to S100A9 in normal monocytes (p < 0.05) (Fig. 4B). S100A9 induced NF-jB activation and the activation was significantly blocked by TLR4i, PP2, rottlerin, PD98059, SB202190, and SP600125 (p < 0.05) (Fig. 4C). The supernatant was collected from normal monocytes at 48 h after the S100A9 treatment, and it treated normal neutrophils. The supernatant was effective with regard to the inhibition of normal neutrophils apoptosis compared with the effect of the control supernatant (Fig. 4D). The anti-apoptotic activity of the supernatant is mediated by the inhibition of caspase 9 and caspase 3 (Fig. 4E). 3.4. The cytokine secretion such as MCP-1, IL-6, and IL-8 due to S100A9 in allergic monocytes inhibits allergic neutrophil apoptosis We investigated whether the effect of S100A9 on cytokine release and apoptosis is influenced by an allergic disease. The secretion of MCP-1, IL-6, and IL-8 elevated in allergic monocytes due to S100A9, and the elevation was inhibited by TLR4i, PP2, rottlerin, PD98059, SB202190, SP600125, and BAY-11-7085 (Fig. 5A). NF-jB activation was triggered by S100A9, and it was significantly inhibited by TLR4i, PP2, rottlerin, PD98059, SB202190, and SP600125 (p < 0.01) (Fig. 5B). Cytokine release induced by S100A9 inhibited the apoptosis of allergic neutrophils (Fig. 5C). In a comparison between normal and allergic subjects, MCP-1 expression increased by S100A9 and IL-6 expression with no treatment in allergic monocytes is higher than in normal monocytes (Fig. 6A). IL-8 expression with no treatment in allergic monocytes is lower than in normal monocytes. As shown in Fig. 6B, the control supernatant of allergic monocytes inhibited neurophil apoptosis of allergic subjects, which was in contrast to that of normal monocytes. S100A9-treated supernatant of allergic monocytes is more effective on neutrophil apoptosis than on normal monocytes. These results indicate that there are different responses to S100A9 in the blood cells of normal and allergic subjects. 4. Discussion S100A9 is included in the S100 family protein and induces various inflammatory responses, such as cytokine secretion,
chemotaxis, and cell viability [18]. The regulation of neutrophil apoptosis is an important step in the pathogenesis of allergic diseases [11,19–21]. In this study, we demonstrated that S100A9 has an indirect anti-apoptotic activity mediated by the monocytes, as well as direct inhibition on neutrophil apoptosis (Figs. 1A, 4D and 5C). Although S100A9 is a potential mediator in the pathogenesis of allergies, a recent report demonstrated that S100A9 plays an important role as a scavenger of oxidants against oxidative stress, which induces the aggravation of allergic diseases [22,23]. There is an abundant amount of S100A9 in the body, and it may induce pro- or anti-inflammatory responses, depending on the cause or status of the diseases. Our results show that S100A9 has pro-inflammatory activity because it increases the cytokine secretion of monocytes, prolonging neutrophil survival. It has been reported that S100A9 is useful biomarkers in local inflammation by using molecular imaging [24]. We think that the elevation of S100A9 is used in a biomarker of allergic diseases, particularly neutrophil-associated diseases, and we are conducting an ongoing study on S100A9 in allergic disease-like animal models and patients with allergic disease. TLR4 is known as a receptor of S100A9 and important in the innate immunity against infection, autoimmune diseases, and tumor [25]. Recent reports demonstrated that TLR4 is associated with a pathogenic mechanism of allergic diseases [26–28]. TLR4 agonists may be useful in allergen immunotherapy [26]. Our results show that TLR4 is an essential receptor in a proinflammatory response of monocytes induced by S100A9 (Figs. 3A, 4B and 5A). In contrast, hyaluronan, a TLR4 agonist, inhibits LPS-treated tissue injury via TLR4-mediated signal pathway [29]. These results indicate that TLR4 is an anti-inflammatory receptor. A previous report demonstrated that hyaluonan induces lung injury due to the ozone, via a TLR4 activation [30]. Based the above reports and our results, TLR4 is evaluated as a major inflammatory receptor; however it can function as an antiinflammatory receptor, depending on the situation, such as the kind of stimulator binding to TLR4, inflammation status, and stage of diseases. S100A9 induces the secretion of MCP-1, IL-6, and IL-8 via TLR4, and the TLR4-mediated signal involves in Src, PKCd, MAPKs including ERK1/2, p38 MAPK and JNK, and NF-jB. There is a limitation in this study. S100A9 binds to RAGE, as well as TLR4. A recent study reported on the role of RAGE in
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Fig. 5. The cytokine secretion such as MCP-1, IL-6, and IL-8 due to S100A9 in allergic monocytes inhibits allergic neutrophil apoptosis. (A) Allergic monocytes (n = 3) were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB), 20 lM SP600125 (SP) and 10 lM BAY-11-7085 (BAY), after which the cells were incubated for 48 h in the absence and presence of S100A9 (1 lg/ml). The supernatant was collected and analyzed by ELISA. (B) Allergic monocytes were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB) and 20 lM SP600125 (SP), after which the cells were incubated with S100A9 (1 lg/ml) for 1 h. After harvested cells were lysed, NF-jB in the lysates was detected by luciferase assay. Data are expressed as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9-treated groups or between the S100A9-treated and inhibitortreated groups. (C) Allergic monocytes were incubated with and without 1 lg/ml of S100A9 for 48 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of allergic subjects. Neutrophils apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄⁄p < 0.01 indicates a significant difference between the control and S100A9-treated groups or between the control supernatant and supernatant-treated groups.
S100A9-mediated responses [31]. The concentration of MCP-1, IL6, and IL-8 increased by S100A9 is not optimal to show the full anti-apoptotic activity on neutrophil apoptosis. In other words, the supernatant after exposure to S100A9 may contain unknown neutrophil survival factors besides MCP-1, IL-6, and IL-8. We will study the association of RAGE with cytokine secretion and identify other unknown cytokines induced by S100A9. Cytokine secretion due to S100A9 induces inflammation, and it can amplify the secondary inflammatory responses. S100A9 increases the IL-6 expression in peripheral blood mononuclear cells, and elevates IL-1b, TNF-a, and IL-6 in THP-1 cells and den-
dritic cells [14,32]. Also, S100A9 induces the release of IL-6 and IL-8 in periodontal ligament cells [33]. Our results demonstrated that S100A9 induces MCP-1, IL-6, and IL-8 (Figs. 2A, 4A and 5A). IL-6 functions as a mediator of synthesis of acute phase protein and neutrophil development in bone marrow, and MCP-1 and IL8 increases the chemotaxis and activation of neutrophils [34]. Cytokine release induced by S100A9 in monocytes delays neutrophil apoptosis, which may also increase the pro-inflammatory responses, such as cell migration and production of inflammatory proteins. As indicated by the above reports and suggested by our results, S100A9 plays an important role in inflammatory diseases,
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Fig. 6. Comparison of cytokine secretion enhanced by S100A9 and anti-apoptotic effect of S100A9-treated supernatant in THP-1 cells, normal and allergic monocytes. (A) Data are presented by classifying the results (6 h, 48 h, 48 h) from Figs. 2A, 4A and 5A, depending on THP-1 cells, norma1, and allergic subjects. (B) Data are presented by classifying the results from Figs. 1A, 4D, and 5C, depending on THP-1 cells, normal, and allergic subjects. ⁄p < 0.05 indicates a significant difference between the allergy and THP-1 or normal groups.
such as allergy, as a main inflammatory protein as well as an inflammatory beginner. In conclusion, S100A9 induces the interactive network between the monocyte and neutrophil through cytokine secretion and apoptosis (Fig. 7). These findings may contribute to unveiling the
S100A9
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Fig. 7. The proposed anti-apoptotic pathway of neutrophils by cytokines of monocytes released by S100A9. The secretion of cytokines due to S100A9 associates with the TLR4/Src/PKCd/MAPKs/NF-jB pathway. The secreted cytokines inhibit neutrophil apoptosis by decreasing caspase 9 and caspase 3 activations.
pathogenesis of inflammatory diseases, such as allergic diseases, by providing detailed understanding of the actions of S100A9 and TLR4. References [1] A.D. Kennedy, F.R. DeLeo, Neutrophil apoptosis and the resolution of infection, Immunol. Res. 43 (1–3) (2009) 25–61, http://dx.doi.org/10.1007/s12026-0088049-6. [2] H.U. Simon, Cell death in allergic diseases, Apoptosis 14 (4) (2009) 439–446, http://dx.doi.org/10.1007/s10495-008-0299-1. [3] J.M. McCracken, L.A. Allen, Regulation of human neutrophil apoptosis and lifespan in health and disease, J. Cell Death 7 (2014) 15–23, http://dx.doi.org/ 10.4137/JCD.S11038. [4] H.U. Simon, Neutrophil apoptosis pathways and their modifications in inflammation, Immunol. Rev. 193 (2003) 101–110, http://dx.doi.org/10.1034/ j.1600-065X.2003.00038.x. [5] H.R. Luo, F. Loison, Constitutive neutrophil apoptosis: mechanisms and regulation, Am. J. Hematol. 83 (4) (2008) 288–295, http://dx.doi.org/10.1002/ ajh.21078. [6] E.J. Yang, E. Choi, J. Ko, D.H. Kim, J.S. Lee, I.S. Kim, Differential effect of CCL2 on constitutive neutrophil apoptosis between normal and asthmatic subjects, J. Cell. Physiol. 227 (6) (2012) 2567–2577, http://dx.doi.org/10.1002/jcp.22995. [7] M.P. Murphy, E. Caraher, Mcl-1 is vital for neutrophil survival, Immunol. Res. 62 (2) (2015) 225–233, http://dx.doi.org/10.1007/s12026-015-8655-z. [8] D. Scheel-Toellner, K.Q. Wang, P.R. Webb, S.H. Wong, R. Craddock, L.K. Assi, et al., Early events in spontaneous neutrophil apoptosis, Biochem. Soc. Trans. 32 (3) (2004) 461–464, http://dx.doi.org/10.1042/bst0320461. [9] J. Monteseirín, Neutrophils and asthma, J. Investig. Allergol. Clin. Immunol. 19 (5) (2009) 340–354. [10] I.S. Kim, M.J. Kim, H. do Kim, E. Choi, J.S. Lee, Different anti-apoptotic effects of normal and asthmatic serum on normal eosinophil apoptosis depending on house dust mite-specific IgE, Mol. Biol. Rep. 40 (10) (2013) 5875–5881, http:// dx.doi.org/10.1007/s11033-013-2695-z. [11] E.H. Kim, J.S. Lee, N.R. Lee, S.Y. Baek, E.J. Kim, S.J. Lee, et al., Regulation of constitutive neutrophil apoptosis due to house dust mite allergen in normal and allergic Rhinitis subjects, PLoS ONE 9 (9) (2014) e105814, http://dx.doi. org/10.1371/journal.pone.0105814. [12] J.1. Goyette, C.L. Geczy, Inflammation-associated S100 proteins: new mechanisms that regulate function, Amino Acids 41 (4) (2011) 821–842, http://dx.doi.org/10.1007/s00726-010-0528-0. [13] C. Gebhardt, J. Németh, P. Angel, J. Hess, S100A8 and S100A9 in inflammation and cancer, Biochem. Pharmacol. 72 (11) (2006) 1622–1631, http://dx.doi.org/ 10.1016/j.bcp.2006.05.017. [14] B. Chen, A.L. Miller, M. Rebelatto, Y. Brewah, D.C. Rowe, L. Clarke, et al., S100A9 induced inflammatory responses are mediated by distinct damage associated molecular patterns (DAMP) receptors in vitro and in vivo, PLoS ONE 10 (2) (2015) e0115828, http://dx.doi.org/10.1371/journal.pone.0115828. [15] J.C. Simard, C. Noël, P.A. Tessier, D. Girard, Human S100A9 potentiates IL-8 production in response to GM-CSF or fMLP via activation of a different set of
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Cytokine secreted by S100A9 via TLR4 in monocytes delays neutrophil apoptosis by inhibition of caspase 9/3 pathway Na Rae Lee a,1, Beom Seok Park b,c,1, Seong Yeol Kim c, Ayoung Gu c, Da Hye Kim c, Ji-Sook Lee d,⇑, In Sik Kim a,c,⇑ a
Department of Biomedical Laboratory Science, School of Medicine, Eulji University, Daejeon 34824, Republic of Korea Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Republic of Korea Department of Senior Healthcare, BK21 Plus Program, Graduate School, Eulji University, Daejeon 34824, Republic of Korea d Department of Clinical Laboratory Science, Wonkwang Health Science University, Iksan 54538, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 25 March 2016 Received in revised form 5 July 2016 Accepted 8 July 2016 Available online 25 July 2016 Keywords: Allergy Cytokine Neutrophil apoptosis Monocytes S100A9
a b s t r a c t Dysregulation of neutrophil apoptosis causes pathogenesis and aggravation of allergy. S100A9 exists as one of the proteins in the neutrophils, triggering inflammatory responses by activating the immune cells. In this study, we investigated whether S100A9 affects constitutive neutrophil apoptosis by activating the monocytes in normal and allergic subjects. Supernatant from human monocytic THP-1 cells after treatment with S100A9 suppressed normal neutrophil apoptosis by inhibiting the activations of caspase 9 and caspase 3. S100A9 upregulated the release of MCP-1, IL-6, and IL-8 in THP-1 cells. An increase in cytokine was suppressed by CLI-095, a Toll-like receptor (TLR) 4 inhibitor, PP2, a Src inhibitor, rottlerin, a PKCd inhibitor, MAP kinase inhibitors, including PD98059, SB202190, and SP600125, and BAY-11-7085, an NF-jB inhibitor. Src, PKCd, ERK1/2, p38 MAPK, and JNK were phosphorylated by S100A9. The phosphorylation of Src and PKCd was suppressed by CLI-095, and the activation of ERK1/2, p38 MAPK, and JNK was inhibited by CLI-095, PP2, and rottlerin. S100A9 induced NF-jB activity, and the activation was suppressed by CLI-095, PP2, rottlerin, and MAPK kinase inhibitors. In normal and allergic subjects, supernatant from normal and allergic monocytes after stimulation with S100A9 suppressed normal and allergic neutrophil apoptosis, respectively; MCP-1, IL-6, and IL-8 in the supernatant was increased by S100A9. The cytokine secretion induced by S100A9 is related to TLR4, Src, PKCd, ERK1/2, p38 MAPK, JNK, and NF-jB. Taken together, S100A9 induces anti-apoptotic effect on normal and allergic neutrophils by increasing cytokine secretion of monocytes. These findings may help us to better understand neutrophil apoptosis regulated by S100A9 and pathogenesis of allergic diseases. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Cell death of neutrophils is an important step in the resolution of inflammation and infection [1–3]. Neutrophil apoptosis is regulated by various stimulators, which includes pro-apoptotic stimulators, such as Fas ligand and TNF-a, and contains anti-apoptotic cytokines, such as IL-6, IL-8, and GM-CSF [4,5]. Our recent report demonstrated that MCP-1 is a new anti-apoptotic molecule in
⇑ Corresponding authors at: Department of Clinical Laboratory Science, Wonkwang Health Science University, 501, Iksandaero, Iksan 54538, Republic of Korea (J.-S. Lee). Department of Biomedical Laboratory Science, School of Medicine, Eulji University, 77, Gyeryoung-ro 771 beon-gil, Jung-Gu, Daejeon 34824, Republic of Korea (I.S. Kim). E-mail addresses: [email protected] (J.-S. Lee), [email protected] (I.S. Kim). 1 Authors contributed equally to this work. http://dx.doi.org/10.1016/j.cyto.2016.07.005 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
neutrophil apoptosis [6]. Intracellular mechanisms of neutrophil apoptosis involve caspase, Bcl family proteins, and reactive oxygen species (ROS) [5,7]. A delay in neutrophil apoptosis induces longterm existence of neutrophils in healthy tissues, which destroys the tissue. Neutrophil-mediated tissue injury is associated with allergic diseases, such as asthma and allergic rhinitis [8–11]. S100A9 is a type of S100 family proteins, and constitutively expressed in the neutrophils and monocytes [12]. S100A9 acts as damage-associated molecular pattern (DAMP) via Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), and triggers abnormal inflammatory responses that result in asthma, chronic obstructive pulmonary disease, colitis, rheumatoid arthritis, Alzheimer’s disease, and tumor [13,14]. It has been reported that S100A9 increases cytokine secretion. S100A9 induces the expression of IP-10 in monocytes/macrophages, accelerating IL-8 secretion induced by GM-CSF in the neutrophils [15,16].
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In this work, we studied that S100A9 induces cytokine secretion in monocytes, which alters spontaneous apoptosis of normal and allergic neutrophils.
caspase 9 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-ERK1/2 was obtained from Cell Signaling Technology (Beverly, MA, USA).
2. Materials and methods
2.2. Normal subjects and allergic subjects
2.1. Materials
Twelve allergy patients (seven allergic asthma and five allergic rhinitis subjects) were recruited from Eulji University Hospital. Allergic patients between 15 and 52 years of age (average = 35.9 years) had mild to severe symptoms of the disease. Allergic status was based on the presence of positive results of a skin prick test (P2+), multiple allergen simultaneous test (MAST) (Pclass 2), or measurement of specific HDM IgE using the Pharmacia Unicap 100 system to common allergens. Levels of total IgE of normal and allergic subjects using an ADVIA Centaur immunoassay (Siemens Medical Solutions Diagnostics, Erfurt, Germany) were 68.5 IU/ml and 565.3 IU/ml, respectively. Additionally, ten normal subjects between 18 and 30 years of age (average = 24.4 years) were recruited as controls. The normal subjects had normal lung
RPMI 1640, penicillin and streptomycin solution, fetal bovine serum (FBS), and TRIzol reagent were obtained from Life Technologies Inc. (Gaithersburg, MD). CLI-095, an inhibitor of Toll-like receptor (TLR) 4 (TLR4i) was purchased from Invivogen (San Diego, CA, USA). Src inhibitor (PP2), PKCd inhibitor (rottlerin), a classic PKC inhibitor (Ro-31-8425), MEK inhibitor (PD98059), p38 MAPK inhibitor (SB202190), JNK inhibitor (SP600125), and NF-jB inhibitor (BAY-11-7085) were products of Calbiochem (San Diego, CA, USA). Anti-phospho-Src, anti-Src, anti-phospho-PKCd, anti-PKCd, anti-ERK2, anti-phospho-p38 MAPK, anti-p38 MAPK, antiphospho-JNK, anti-JNK, anti-cleaved caspase 3, and anti-cleaved
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Fig. 1. Spontaneous neutrophil apoptosis is delayed by secreted molecules due to S1000A9 in THP-1 cells. (A) THP-1 cells were incubated with and without 1 lg/ml of S100A9 for 6 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of normal subjects. Neutrophils apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄⁄p < 0.01 indicates a significant difference between the control and S100A9-treated groups or between the control supernatant and supernatant-treated groups. (B) Neutrophils isolated from normal subjects were incubated with the supernatant (Sup) or the S100A9-treated supernatant of THP-1 cells. Caspase 9 and caspase 3 were detected by Western blotting. The membrane was stripped and reprobed with anti-ERK2 antibodies as an internal control (upper panel). Densitometric data (n = 3) are presented relative to the negative control, which was set at l (lower panel).
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2.3. Isolation of neutrophils and monocytes Human neutrophils and monocytes were isolated from the heparinized peripheral blood of normal and allergic subjects using Ficoll-Hypaque gradient centrifugation. A CD16 microbeads magnetic cell sorting kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for isolation of neutrophil from granulocytes. A monocyte isolation kit II (Miltenyi Biotec) was used for isolation of monocytes from peripheral mononuclear cells. Isolated neutrophils and monocytes were washed by phosphate buffered saline (PBS) buffer after removal of erythrocytes by hypotonic lysis. Purity of neutrophils and monocytes was greater than 97%. 2.4. Cell culture The human monocytic cell line THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA). THP-1 cells,
A **
MCP-1 (pg/ml)
5000
**
3000 **
**
2000
2.5. Production of recombinant S100A9 proteins Recombinant S100A9 protein was produced as previously reported [17]. Briefly, total RNA of human neutrophils was extracted using TRIzol reagent and first strand cDNA was synthesized. S100A9-1 (50 -ttccatatgatgacttgcaaaatgtcgca), and S100A9-2 (50 -ccgctcgagactgtggtctta-gggggtgc) were used for cDNA synthesis of S100A9. The cDNA was cloned into pET28 expression vector (Merck Millipore, Darmstadt, Germany), respectively. Recombinant His-Tag S100A9 was induced and then purified using a nickel column. The purified protein was identified by western blotting using anti-S100A9 antibodies. 2.6. Enzyme-linked immunosorbent assay (ELISA) The concentrations of MCP-1, IL-6, and IL-8 in a cell supernatant were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) using OptEIATM Set human IL-6, IL-8 and MCP-1 (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions.
0 1h 3h 6h 9h 12h 24h 48h
**
4000
neutrophils and monocytes were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 lg/ml).
1000
**
400
**
**
**
**
*
150 **
100 50 Con
S100A9
500 IL-8 (pg/ml)
** **
200
0
0 Con
**
250 IL-6 (pg/ml)
function, no history of asthma and allergic rhinitis, and did not require medication. This study was approved by the Institutional Review Board of Eulji University for normal volunteers and by the Institutional Review Board of Eulji University Hospital for allergic patients. All participants in this study gave their written informed consent.
S100A9
**
300 200
**
100 0 Con
S100A9 300
MCP-1 (pg/ml)
5000 **
4000
**
3000 **
2000 1000
*
200 150 * 100 50
0 S100A9 ( g/ml)
*
250 IL-6 (pg/ml)
B
0 -
0.2
0.5
1
2
S100A9 ( g/ml)
-
0.2
0.5
1
2
IL-8 (pg/ml)
500 **
400
**
300
**
200 ** 100 0
S100A9 ( g/ml)
-
0.2
0.5
1
2
Fig. 2. S100A9 induces the secretion of MCP-1, IL-6, and IL-8 in THP-1 cells. (A) THP-1 cells were incubated in the absence or presence of 1 lg/ml S100A9 for the indicated time. (B) THP-1 cells were incubated in the indicated concentration of S100A9 for 6 h. The supernatant was collected and analyzed by ELISA. Data are expressed as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9-treated groups.
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The blots were developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech.).
2.7. Western blotting Cells were lysed in cytosolic lysis buffer (TransLab, Daejeon, Korea). The lysate was centrifuged and then the supernatant was collected. Protein concentration of the lysate was evaluated by protein assay kit (Thermo scientific, Waltham, MA, USA). Protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose filters. The blots were incubated with primary antibodies including anti-phospho-Src, anti-phospho-PKCd, anti-phosphoERK, anti-phospho-p38 MAPK, anti-phospho-JNK, anti-caspase 3 or anti-caspase 9 antibodies, after which the blots were incubated with secondary antibodies including goat anti-rabbit antibodies.
2000
500
**
1600 1200
The DNA-binding activity of NF-jB was assessed using EZDetectTM transcription factor kits for NF-jB p65 (PIERCE, Rockford, IL), following the manufacturer’s instructions. DNA binding specificity was assessed using wild type or mutant NF-jB oligonucleotides. Chemiluminescent detection was performed using a luminometer.
% IL-6 release
% MCP-1 release
A
2.8. NF-jB p65 transcription factor assay
*
**
800
**
**
**
400
**
400
**
**
300
** **
200
**
100 0
0 TLR4i ( M)
-
-
0.1
0.2
0.5
1
2
TLR4i ( M)
-
-
0.1
0.2
0.5
1
2
S100A9
-
+
+
+
+
+
+
S100A9
-
+
+
+
+
+
+
*
* *
**
% IL-8 release
1000
**
800 600 400 200 0
TLR4i ( M)
-
S100A9
-
0.1
0.2
0.5
1
2
+
+
+
+
+
+
**
2000 1600 1200
**
800
**
400
**
**
**
**
% IL-6 release
% MCP-1 release
B
-
0 Inhibitor
-
-
PP2
Rt
PD
S100A9
-
+
+
+
+
**
*
% IL-8 release
1000
600 500
** *
400 300 200 100 0
**
**
**
** **
SB
SP
BAY
Inhibitor
-
-
PP2
Rt
PD
SB
SP
BAY
+
+
+
S100A9
-
+
+
+
+
+
+
+
**
800 600
**
**
400
**
200 0
Inhibitor
-
-
PP2
Rt
PD
SB
SP
S100A9
-
+
+
+
+
+
+
BAY +
Fig. 3. S100A9 increases the secretion of MCP-1, IL-6, and IL-8 through TLR4, Src, PKCd, MAPK, and NF-jB activation in THP-1 cells. (A and B) THP-1 cells were pre-treated for 1 h with and without the indicated concentration of TLR4i (A), 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB), 20 lM SP600125 (SP) and 10 lM BAY11-7085 (BAY) (B), after which the cells were incubated for 6 h in the absence and presence of S100A9 (1 lg/ml). The supernatant was collected and analyzed by ELISA. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9treated groups or between the S100A9-treated and inhibitor-treated groups. (C) THP-1 cells were incubated with S100A9 (1 lg/ml) for the indicated time. (D and E) THP-1 cells were pre-treated for 1 h with and without 2 lM TLR4i (D), 20 lM PP2 and 5 lM rottlerin (E), after which the cells were incubated with S100A9 (1 lg/ml) for 30 min. After harvested cells were lysed, phosphorylation of Src, PKCd, ERK, p38 MAPK, and JNK in the lysates was detected by Western blotting (upper panel). Densitometric data (n = 3) are presented relative to the negative control, which was set at 1 (lower panel). (F) THP-1 cells were incubated with S100A9 (1 lg/ml) for the indicated time. (G) THP-1 cells were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB) and 20 lM SP600125 (SP), after which the cells were incubated with S100A9 (1 lg/ml) for 1 h. After harvested cells were lysed, NF-jB in the lysates was detected by luciferase assay. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9-treated groups or between the S100A9-treated and inhibitor-treated groups.
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C
Time
0
5min 15min 30min 1h
2h
p-Src Src p-PKC PKC p-ERK ERK2 p-p38 p38 p-JNK
0 min 5 min 15min
JNK
30min 1h 2h
Relative level
8 6 4 2 0 p-PKC
p-Src
-
S100A9
-
Inhibitor
+
D
p-p38
p-JNK
p-ERK
TLR4i
Con S100A9 S100A9+TLR4i
+
p-Src Src
PKC p-ERK
Relative level
p-PKC
3 2 1 0 p-Src
ERK2
p-PK C
p-ERK
p-JNK
p-p38
p-JNK JNK p-p38 MAPK p38 MAPK Fig. 3 (continued)
2.9. Production of monocyte supernatant
2.10. Detection of apoptosis
THP-1 cells and monocytes were incubated with and without 1 lg/ml of S100A9 for 6 h and 48 h, respectively. The supernatant was collected and diluted by RPMI 1640 medium (1:1 ratio). The diluted supernatant was added to the fresh neutrophils isolated from normal and allergic subjects, and incubated for 24 h.
An annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Diego, CA, USA) was used for detection of neutrophil apoptosis. Isolated neutrophils were treated with DP, then incubated with FITC-labeled annexin V and propidium iodide (PI) for 15 min at room temperature. Apoptotic neutrophils were analyzed using a FACSCalibur flow cytometer
N.R. Lee et al. / Cytokine 86 (2016) 53–63
+
S100A9
-
Inhibitor
PP2
+
+
Rt
-
PP2
+
Rt
Inhibitor
+
S100A9
-
+
p-ERK
p-JNK
p-p38
ERK2
JNK
p38
Relative level
4
-
S100A9
PP2
Rt
+
E Inhibitor
-
58
+
+
Con S100A9 S100A9+PP2 S100A9+Rt
3 2 1 0 p-ERK
p-JNK
p-p38
G Luciferase activity (Fold)
Luciferase activity (Fold)
F 20 **
15
**
10
*
5 0 0
5min 15min 30min
1h
2h
4h
S100A9
16 **
12 8
**
*
**
*
**
**
4
0 Inhibitor
-
-
TLR4i
PP2
Rt
PD
SB
SP
S100A9
-
+
+
+
+
+
+
+
Fig. 3 (continued)
with the CellQuest software (BD bioscience) and reported as the percentage of cells showing annexin V+/PI and annexin V+/PI+. 2.11. Statistical analysis Data were represented as the means ± S.E.M. Statistical differences were analyzed using a paired t-test for two-group comparisons and one-way ANOVA for comparison of more than two groups. All analyses were conducted using the SPSS statistical software package (Version 10.0, Chicago, IL), and a p value less than 0.05 was considered significant. 3. Results 3.1. Spontaneous neutrophil apoptosis is delayed by secreted molecules due to S1000A9 in THP-1 cells We first examined the effect of monocyte activation induced by S100A9 on neutrophil apoptosis. Supernatant was collected from THP-1 cells after exposure to S100A9, and its anti-apoptotic effect was evaluated. S100A9 directly inhibited neutrophil apoptosis (Fig. 1A). The supernatant treated with S100A9 was more effective with regard to the inhibition of normal neutrophils apoptosis compared with the direct effect of S100A9. The anti-apoptotic effect of the supernatant was mediated by the inhibition of caspase 9 and caspase 3 activations, post spontaneous apoptosis by neutrophil isolation (Fig. 1B). These results indicate that the action of S100A9 in the monocytes regulates neutrophil apoptosis, and suggests that this process is involved in the caspase 9/3 pathway. 3.2. S100A9 induces the secretion of MCP-1, IL-6, and IL-8 through TLR4, Src, PKCd, MAPK, and NF-jB in THP-1 cells Since the supernatant stimulated with S100A9 inhibited neutrophil apoptosis, we examined that S100A9 increases cytokines,
including MCP-1, IL-6, and IL-8, which are associated with the survivability of neutrophils. As shown in Fig. 2, S100A9 began to increase the secretion of MCP-1, IL-6, and IL-8 at 3 h, which peaked at 6 h after the stimulation of S100A9. S100A9 elevated the cytokine secretion in a dose-dependent manner at 6 h. Next, we examined how the signal mechanism of S100A9 induces cytokine secretion by evaluating the alteration of secretion after an inhibitor treatment. As shown in Fig. 3A and B, TLR4i inhibited the expressions of MCP-1, IL-6, and IL-8 in a dose-dependent manner; other signal inhibitors, such as PP2, rottlerin, PD98059, SP600125, and BAY-11-7085 blocked the increased cytokine expression. SB202190 inhibited the expression of MCP-1 and IL-6, except IL8 expression. S100A9 induces the phosphorylation of Src, PKCd, ERK, p38 MAPK, and JNK in a time-dependent manner (Fig. 3C). The phosphorylation of Src and PKCd was suppressed by TLR4i, and the activation of ERK, p38 MAPK, and JNK was inhibited by TLR4i, PP2, and rottlerin (Fig. 3D and E). In addition, S100A9 activated NF-jB in a time-dependent manner, and the activation was suppressed by TLR4i, PP2, rottlerin, PD98059, SB202190, and SP600125 (Fig. 3F and G). 3.3. The secretion of MCP-1, IL-6, and IL-8 enhanced by S100A9 is involved in TLR4, Src, PKCd, MAPKs, and NF-jB in normal monocytes, and the cytokine secretion suppresses normal neutrophil apoptosis Since cytokine secretion induced by S100A9 in THP-1 cells affects neutrophil apoptosis, we investigated in detail regarding the action of S100A9 in normal monocytes and neutrophils. S100A9 significantly increased the release of MCP-1, IL-6, and IL-8 in normal monocytes in a time-dependent manner, and the increase peaked at 48 h (p < 0.05). This was in contrast to the peak time of cytokine secretion induced by S100A9 in THP-1 cells, which was at 6 h (Fig. 4A). These results indicate that the use of primary cells and confirmation of the cell line are needed in an experimental research. TLR4i, PP2, rottlerin, PD98059, SB202190, SP600125,
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N.R. Lee et al. / Cytokine 86 (2016) 53–63
A 0 1h 3h 6h 9h 12h 24h 48h
MCP-1 (ng/ml)
15 12 9 6 3
300 250 Il-6 (ng/ml)
18
200 *
100 50
0
0 Con
S100A9
Con
S100A9
*
300 *
250 Il-8 (ng/ml)
*
150
200 150 (n=3)
100 50 0 Con **
500 % MCP-1 release
400 300
C
200
Luciferase activity (Fold)
B
S100A9
**
100
**
**
**
**
**
**
0 Inhibitor
-
-
TLR4i
PP2
Rt
PD
SB
SP
BAY
S100A9
-
+
+
+
+
+
+
+
+
6 * 5 4
*
3 2
*
*
* *
*
1
% IL-6 release
0
400
**
*
300 **
*
**
200 **
**
100 0 -
-
TLR4i
PP2
Rt
PD
SB
SP
BAY
S100A9
-
+
+
+
+
+
+
+
+
** * **
**
**
150 100
**
**
50
-
TLR4i
PP2
Rt
PD
SB
SP
S100A9
-
+
+
+
+
+
+
+
D
120 100 80 60 40 20 0
% Apoptosis
% IL-8 release
300
200
-
**
Inhibitor
250
Inhibitor
**
0
** **
3
Con
S100A9
Con
S100A9
Mon Sup
Inhibitor
-
-
TLR4i
PP2
Rt
PD
SB
SP
BAY
S100A9
-
+
+
+
+
+
+
+
+
Fig. 4. The secretion of MCP-1, IL-6, and IL-8 enhanced by S100A9 is involved in TLR4, Src, PKCd, MAPKs, and NF-jB in normal monocytes, and the cytokine secretion suppresses normal neutrophil apoptosis. (A) Normal monocytes isolated from normal subjects (n = 3) were incubated in the absence or presence of 1 lg/ml S100A9 for the indicated time. (B) Normal monocytes were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB), 20 lM SP600125 (SP) and 10 lM BAY-11-7085 (BAY), after which the cells were incubated for 48 h in the absence and presence of S100A9 (1 lg/ml). The supernatant was collected and analyzed by ELISA. (C) Normal monocytes were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB) and 20 lM SP600125 (SP), after which the cells were incubated with S100A9 (1 lg/ml) for 1 h. After harvested cells were lysed, NF-jB in the lysates was detected by luciferase assay. Data are expressed as the means ± S.E.M. ⁄p < 0.05 indicates a significant difference between the control and S100A9-treated groups or between the S100A9treated and inhibitor-treated groups. (D) Normal monocytes were incubated with and without 1 lg/ml of S100A9 for 48 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of normal subjects. Neutrophils apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄⁄p < 0.01 indicates a significant difference between the control and S100A9-treated groups or between the control supernatant and supernatant-treated groups. (E) Neutrophils isolated from normal subjects were incubated with the supernatant (Sup) or the S100A9treated supernatant of normal monocytes. Caspase 9 and caspase 3 were detected by Western blotting. The membrane was stripped and reprobed with anti-ERK2 antibodies as an internal control (upper panel). Densitometric data (n = 3) are presented relative to the negative control, which was set at 1 (lower panel).
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E Time
Mono sup – S100A9 treatment
Con Mono sup 0
2h
4h
8h
12h
24h
0
48h
2h
4h
8h
12h
24h
48h
Casepase 3 Casepase 9 ERK2
Relative Casepase 9 level
Relative Casepase 3 level
Con
15 12 9 6 3 0 0h
2h
4h
8h
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S100A9
6 5 4 3 2 1 0 0h
24h
2h
4h
8h
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Fig. 4 (continued)
and BAY-11-7085 significantly blocked the increased expression of MCP-1, IL-6, and IL-8 due to S100A9 in normal monocytes (p < 0.05) (Fig. 4B). S100A9 induced NF-jB activation and the activation was significantly blocked by TLR4i, PP2, rottlerin, PD98059, SB202190, and SP600125 (p < 0.05) (Fig. 4C). The supernatant was collected from normal monocytes at 48 h after the S100A9 treatment, and it treated normal neutrophils. The supernatant was effective with regard to the inhibition of normal neutrophils apoptosis compared with the effect of the control supernatant (Fig. 4D). The anti-apoptotic activity of the supernatant is mediated by the inhibition of caspase 9 and caspase 3 (Fig. 4E). 3.4. The cytokine secretion such as MCP-1, IL-6, and IL-8 due to S100A9 in allergic monocytes inhibits allergic neutrophil apoptosis We investigated whether the effect of S100A9 on cytokine release and apoptosis is influenced by an allergic disease. The secretion of MCP-1, IL-6, and IL-8 elevated in allergic monocytes due to S100A9, and the elevation was inhibited by TLR4i, PP2, rottlerin, PD98059, SB202190, SP600125, and BAY-11-7085 (Fig. 5A). NF-jB activation was triggered by S100A9, and it was significantly inhibited by TLR4i, PP2, rottlerin, PD98059, SB202190, and SP600125 (p < 0.01) (Fig. 5B). Cytokine release induced by S100A9 inhibited the apoptosis of allergic neutrophils (Fig. 5C). In a comparison between normal and allergic subjects, MCP-1 expression increased by S100A9 and IL-6 expression with no treatment in allergic monocytes is higher than in normal monocytes (Fig. 6A). IL-8 expression with no treatment in allergic monocytes is lower than in normal monocytes. As shown in Fig. 6B, the control supernatant of allergic monocytes inhibited neurophil apoptosis of allergic subjects, which was in contrast to that of normal monocytes. S100A9-treated supernatant of allergic monocytes is more effective on neutrophil apoptosis than on normal monocytes. These results indicate that there are different responses to S100A9 in the blood cells of normal and allergic subjects. 4. Discussion S100A9 is included in the S100 family protein and induces various inflammatory responses, such as cytokine secretion,
chemotaxis, and cell viability [18]. The regulation of neutrophil apoptosis is an important step in the pathogenesis of allergic diseases [11,19–21]. In this study, we demonstrated that S100A9 has an indirect anti-apoptotic activity mediated by the monocytes, as well as direct inhibition on neutrophil apoptosis (Figs. 1A, 4D and 5C). Although S100A9 is a potential mediator in the pathogenesis of allergies, a recent report demonstrated that S100A9 plays an important role as a scavenger of oxidants against oxidative stress, which induces the aggravation of allergic diseases [22,23]. There is an abundant amount of S100A9 in the body, and it may induce pro- or anti-inflammatory responses, depending on the cause or status of the diseases. Our results show that S100A9 has pro-inflammatory activity because it increases the cytokine secretion of monocytes, prolonging neutrophil survival. It has been reported that S100A9 is useful biomarkers in local inflammation by using molecular imaging [24]. We think that the elevation of S100A9 is used in a biomarker of allergic diseases, particularly neutrophil-associated diseases, and we are conducting an ongoing study on S100A9 in allergic disease-like animal models and patients with allergic disease. TLR4 is known as a receptor of S100A9 and important in the innate immunity against infection, autoimmune diseases, and tumor [25]. Recent reports demonstrated that TLR4 is associated with a pathogenic mechanism of allergic diseases [26–28]. TLR4 agonists may be useful in allergen immunotherapy [26]. Our results show that TLR4 is an essential receptor in a proinflammatory response of monocytes induced by S100A9 (Figs. 3A, 4B and 5A). In contrast, hyaluronan, a TLR4 agonist, inhibits LPS-treated tissue injury via TLR4-mediated signal pathway [29]. These results indicate that TLR4 is an anti-inflammatory receptor. A previous report demonstrated that hyaluonan induces lung injury due to the ozone, via a TLR4 activation [30]. Based the above reports and our results, TLR4 is evaluated as a major inflammatory receptor; however it can function as an antiinflammatory receptor, depending on the situation, such as the kind of stimulator binding to TLR4, inflammation status, and stage of diseases. S100A9 induces the secretion of MCP-1, IL-6, and IL-8 via TLR4, and the TLR4-mediated signal involves in Src, PKCd, MAPKs including ERK1/2, p38 MAPK and JNK, and NF-jB. There is a limitation in this study. S100A9 binds to RAGE, as well as TLR4. A recent study reported on the role of RAGE in
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600 % MCP-1 release
A
**
500 400
*
300 **
200
** **
100
**
**
0 -
-
TLR4i
PP2
Rt
PD
SB
SP
BAY
S100A9
-
+
+
+
+
+
+
+
+
*
*
% IL-6 release
Inhibitor
400
**
300
*
200
** ** **
0 -
-
S100A9
-
+
% IL-8 release
*
100
Inhibitor
PP2
Rt
PD
SB
SP
BAY
+
+
+
+
+
+
+
*
*
**
*
TLR4i
**
400
*
300 200
**
100
**
0
Inhibitor
-
-
TLR4i
PP2
Rt
PD
SB
SP
BAY
S100A9
-
+
+
+
+
+
+
+
+
C
5
**
4 3
**
**
2
**
**
**
**
1 0
% Apoptosis
Luciferase activity (Fold)
B
**
120 100 80 60 40
** ** **
20 0
3 Con
Inhibitor
-
-
S100A9
-
+
TLR4i PP2 +
+
Rt
PD
SB
SP
+
+
+
+
S100A9
Con
S100A9
Mon Sup
Fig. 5. The cytokine secretion such as MCP-1, IL-6, and IL-8 due to S100A9 in allergic monocytes inhibits allergic neutrophil apoptosis. (A) Allergic monocytes (n = 3) were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB), 20 lM SP600125 (SP) and 10 lM BAY-11-7085 (BAY), after which the cells were incubated for 48 h in the absence and presence of S100A9 (1 lg/ml). The supernatant was collected and analyzed by ELISA. (B) Allergic monocytes were pre-treated for 1 h with and without 2 lM TLR4i, 20 lM PP2, 5 lM rottlerin, 20 lM PD98059 (PD), 20 lM SB202190 (SB) and 20 lM SP600125 (SP), after which the cells were incubated with S100A9 (1 lg/ml) for 1 h. After harvested cells were lysed, NF-jB in the lysates was detected by luciferase assay. Data are expressed as the means ± S.E.M. ⁄p < 0.05 and ⁄⁄p < 0.01 indicate a significant difference between the control and S100A9-treated groups or between the S100A9-treated and inhibitortreated groups. (C) Allergic monocytes were incubated with and without 1 lg/ml of S100A9 for 48 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of allergic subjects. Neutrophils apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% as the means ± S.E.M. ⁄⁄p < 0.01 indicates a significant difference between the control and S100A9-treated groups or between the control supernatant and supernatant-treated groups.
S100A9-mediated responses [31]. The concentration of MCP-1, IL6, and IL-8 increased by S100A9 is not optimal to show the full anti-apoptotic activity on neutrophil apoptosis. In other words, the supernatant after exposure to S100A9 may contain unknown neutrophil survival factors besides MCP-1, IL-6, and IL-8. We will study the association of RAGE with cytokine secretion and identify other unknown cytokines induced by S100A9. Cytokine secretion due to S100A9 induces inflammation, and it can amplify the secondary inflammatory responses. S100A9 increases the IL-6 expression in peripheral blood mononuclear cells, and elevates IL-1b, TNF-a, and IL-6 in THP-1 cells and den-
dritic cells [14,32]. Also, S100A9 induces the release of IL-6 and IL-8 in periodontal ligament cells [33]. Our results demonstrated that S100A9 induces MCP-1, IL-6, and IL-8 (Figs. 2A, 4A and 5A). IL-6 functions as a mediator of synthesis of acute phase protein and neutrophil development in bone marrow, and MCP-1 and IL8 increases the chemotaxis and activation of neutrophils [34]. Cytokine release induced by S100A9 in monocytes delays neutrophil apoptosis, which may also increase the pro-inflammatory responses, such as cell migration and production of inflammatory proteins. As indicated by the above reports and suggested by our results, S100A9 plays an important role in inflammatory diseases,
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350
Normal
20
Allergy
15 10
250 200 150 100
5
50
0
0
Con
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% Apoptosis
B
300 IL-8 (ng/ml)
25
IL-6 (ng/ml)
A MCP-1 (ng/ml)
300
THP-1
250 200 150 * 100 50 0
Con
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120 100
Con
S100A9
THP-1 Normal
80 60 40
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3 * *
20 0 Con
S100A9
S100A9
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Fig. 6. Comparison of cytokine secretion enhanced by S100A9 and anti-apoptotic effect of S100A9-treated supernatant in THP-1 cells, normal and allergic monocytes. (A) Data are presented by classifying the results (6 h, 48 h, 48 h) from Figs. 2A, 4A and 5A, depending on THP-1 cells, norma1, and allergic subjects. (B) Data are presented by classifying the results from Figs. 1A, 4D, and 5C, depending on THP-1 cells, normal, and allergic subjects. ⁄p < 0.05 indicates a significant difference between the allergy and THP-1 or normal groups.
such as allergy, as a main inflammatory protein as well as an inflammatory beginner. In conclusion, S100A9 induces the interactive network between the monocyte and neutrophil through cytokine secretion and apoptosis (Fig. 7). These findings may contribute to unveiling the
S100A9
TLR4
MD2
Src, PKC
MAPKs (ERK, p38 MAPK, JNK)
Monocyte NF-
MCP-1
IL-6 IL-8 Cytokine receptor
Neutrophil Caspase 9
Caspase 3
Fig. 7. The proposed anti-apoptotic pathway of neutrophils by cytokines of monocytes released by S100A9. The secretion of cytokines due to S100A9 associates with the TLR4/Src/PKCd/MAPKs/NF-jB pathway. The secreted cytokines inhibit neutrophil apoptosis by decreasing caspase 9 and caspase 3 activations.
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