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TREM-2 ratio in acute lung injury
Regulatory Peptides 167 (2011) 56–64
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Vasoactive intestinal peptide re-balances TREM-1/TREM-2 ratio in acute lung injury Guo-Ying Sun a, Cha-Xiang Guan a,⁎, Yong Zhou a, Yong-Ping Liu b, Shu-Fen Li a, Hui-Fang Zhou a, Chun-Yan Tang a, Xiang Fang c a b c
Department of Physiology, Central South University Xiangya Medical School, Changsha, Hunan 410078, China Department of Physiology, Hunan University of Traditional Chinese Medicine, Changsha, Hunan 410208, China Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA
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Article history: Received 31 July 2010 Received in revised form 21 November 2010 Accepted 25 November 2010 Available online 2 December 2010 Keywords: Vasoactive intestinal peptide TREM-1 TREM-2 Macrophage Acute lung injury
a b s t r a c t Vasoactive intestinal peptide (VIP) is one of the most plentiful neuropeptides in the lung and it has antiinflammatory effects in the respiratory system. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) regulate immune responses to lipopolysaccharide (LPS). In the present study, we tested the expressions of TREM-1 and TREM-2 in various pulmonary cell lines and/or tissue using an animal model of LPS-induced acute lung injury (ALI), and determined the effects of VIP on expression of the TREM-1 and TREM-2 in lung tissues and cells from ALI mice. We found 1) expression of the TREM-1 mRNA from lung tissues of ALI was significantly increased, whereas the expression of TREM-2 mRNA was decreased in these tissues; 2) TREM-1 mRNA was only expressed in macrophages, while TREM-2 mRNA was detected in HBECs, lung fibroblasts, lung adenocarcinoma cells and macrophages; 3) the ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced lung tissues and macrophages; 4) VIP inhibited expression of the TREM-1 mRNA in a time- and dose-dependent manner in lung cells from LPS-induced ALI mice; however, it increased expression of the TREM-2 mRNA. As a result of these effects, VIP normalized the ratio of TREM-1 to TREM-2 mRNA in these cells. Our results suggest that VIP might exert its anti-inflammatory effect through a mechanism involved in regulation of expression of the TREM-1 and TREM-2 in LPS-induced ALI. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Vasoactive intestinal peptide (VIP) is a bioactive neuropeptide involved in a wide range of biological functions in the respiratory system. It causes relaxation of airway smooth muscles [1], regulates smooth muscle proliferation of airway [2], and secretion of exocrine and endocrine glands [3]. VIP also regulates immune responses in different conditions [4]. Because of its numerous biological effects, modulation of VIP has been considered as a potential therapeutic target for the treatment of many diseases such as rheumatism [5] and diabetes [6]. The patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) have high mortality rate (~40%), and these acute respiratory disorders are still considered as major threats to human health in the current medicine [7]. It has been shown previously that VIP possesses an anti-inflammatory property in respiratory diseases such as asthma. It is important to determine its role in the pathological processes in ALI. Abbreviations: VIP, vasoactive intestinal peptide; TREM-1, triggering receptors expressed on myeloid cells-1; TREM-2, triggering receptors expressed on myeloid cells-2; LPS, lipopolysaccharide; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; TLR, toll-like receptor; NLR, nod-like receptor; RF, respiratory frequency; Cdyn, dynamic compliance; TV, tidal volume; TPR, total pulmonary resistance. ⁎ Corresponding author. Tel.: + 86 731 82355051; fax: + 86 731 82355056. E-mail address: [email protected] (C.-X. Guan).
ALI is characterized by cell injuries in pulmonary capillary endothelial, alveolar epithelial cells, and increased vascular permeability due to inflammatory reactions. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) are newly identified inflammatory molecules [8]. TREM-1 possesses biological activities similar to activation of Toll-like receptor (TLR) and Nod-like receptor (NLR). It stimulates cellular immune signaling and produces excessive inflammatory responses [9]. TREM-2 inhibits the reactions induced by TLR in macrophages, and provides protective effects on TREM-1 mediated inflammation [10]. It is unknown whether TREM-1 and TREM-2 are expressed in lung tissues and pulmonary cells, particularly in these tissues/cells from ALI. It is remained to be investigated whether VIP can affect the expression of TREM-1 and TREM-2. In the present study, we tested the hypothesis that VIP plays an important role in the regulation of expression of TREM-1/TREM-2 in lipopolysaccharide (LPS) stimulated lung tissues and pulmonary cells. 2. Materials and methods 2.1. Materials DMEM (High Glucose) medium, RPMI 1640 medium and TRIzol were the products of Invitrogen Life Technologies (California, USA).
0167-0115/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2010.11.008
G.-Y. Sun et al. / Regulatory Peptides 167 (2011) 56–64
LPS, VIP and D-P-C1 Phe(6)-Leu(17)-VIP were purchased from Sigma (St.Louis, MO, USA). TREM-1 antibody and FITC-labeled anti-mouse TREM-1 antibody were from Santa Cruz (Dover, USA). EDTA and Nuclease-Free Water were from Amresco Company (Ohio, USA). Calf serum was bought from Sijiqing Company (Hangzhou, China). Reverse transcription (RT) kit and Taq DNA polymerase were purchased from Fermentas (Ohio, USA). All primers were synthesized by Shanghai Bio-engineering Company (Shanghai, China). Biowest agarose was from Agarose Bead Technologies (Madrid, Spain). DNA maker was from Tiangen Biotech (Beijing, China).
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2.3. Culture and activation of lung cell lines The human bronchial epithelial cell (HBEC) line, 16HBE140- was generously provided by Dr. Gruenert from the University of California, San Francisco. The mouse macrophages cell line (RAW264.7), mouse embryonic fibroblast cell line (NIH 3T3), and human lung adenocarcinoma cell line (A549) were purchased from Cell Center, Xiangya School of Medicine, Central South University. The cells were cultured in DMEM (High Glucose) medium at 37 °C with 5% CO2. The medium was replaced by serum-free medium for 8 h, when the cell reached confluence of 80%.
2.2. Animals Thirty male Kunming mice (18 g to 23 g, from department of experimental animals of the Central South University), were housed in an SPF animal room maintained at constant temperature (24 °C ± 2 °C) and under a 12-h light/dark cycle with free access to food and water. All animal studies were approved by the Central South University Animal Studies Committee.
2.4. Verification of LPS-induced pulmonary injury and activation of macrophages Kunming mice were sensitized to LPS by immunization. The mice received i.p. injections of lipopolysaccharide (LPS, 15 mg/kg). Buxco was used to monitor the changes in respiratory function parameters in ALI mice. HE staining of lung tissues from these mice was
Fig. 1. The pathological (panel A) and respiratory functional changes (panel B) in LPS-stimulated mice. The mice were treated with LPS (15 mg/kg, i.p.). After 6 h of treatment, the respiratory functions were determined by Buxco, and the pathological changes were observed by Hematoxylin–eosin (HE) staining under microscope. All data are represented as the mean ± SD, n = 10 in each group. **P b 0.01 vs. control group.
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performed for pathological characterization. The amount of TNF-α protein released into the medium in LPS-stimulated macrophages was detected by ELISA. 2.5. Detection of TREM protein by flow cytometry The cells were incubated with VIP in the presence or absence of different inhibitors for 6 h. After incubation, the cells were washed twice with PBS, and unlabeled first antibody was added into the 100 μl cell suspension in each tube. All tubes were then incubated at 4 °C, and washed by PBS after 30 min. The cell suspension was centrifuged at 2000 g for 5 min, and the cells incubated for additional 30 min at 4 °C with fluorescein isothiocyanate (FITC) labeled secondary antibody. The cells were washed again, and the cells were re-suspended with 1% paraformaldehyde, and then the protein was determined by flow cytometry (FC). 2.6. Determination of TREM-1 and TREM-2 mRNA Total RNA was prepared by using TRIzol, and 2 μg of total RNA was used for detection of mRNA using a reverse transcription-PCR (RT-PCR). The following primers sequences were used for RT-PCR amplification: TREM-1 (338 bp) forward: 5′-CTGCTGTGCGTGTTCTTT-3′, TREM-1 reverse: 5′-TCATTCGGAGGATGGTAA-3′, TREM-2(260 bp) forward: 5′GGAACCGTCACCATCACT-3′, TREM-2 reverse: 5′-GCCAGGAGGAGAAGAATG-3′, and GAPDH (580 bp) forward: 5′-AAGCCCATCACCATCTTC CA-3′, GAPDH reverse: 5′-CCTGCTTCACCACCTTCTTG-3′. PCR was performed under the conditions of an initial 5 min at 94 °C followed by 35 cycles (30 s at 94 °C 30 s at 52 °C for TREM-1 or 30 s at 58 °C for TREM-2 and GAPDH, 1 min at 94 °C with a final 5 min at 72 °C. PCR products after electrophoresis were imaged by using Quantity One. 2.7. Statistical analyses The data was expressed as mean ± standard deviation (SD) and analyzed with SPSS 13.0 statistical package. The repeated measures analysis of variance (ANOVA) test was used to compare the differences among multiple groups. Tamhane test was used to compare the difference between two groups. P b 0.05 was considered as statistically significant. 3. Results 3.1. The structure of lung and respiratory functions were impaired in LPS-stimulated mice We first examined the pathological and pulmonary function changes in LPS-treated mice. The alveolar spaces of the lung from LPS-treated mice were filled with white blood cells and alveolar macrophages, and the alveolar septum of lung were also destructed (Fig. 1A). The pulmonary function test by Buxco showed respiratory frequency (RF) and dynamic compliance (Cdyn) were decreased by ~20% and ~ 67% respectively in LPS-treated mice as compared with those of control mice. However, the tidal volume (TV) and total pulmonary resistance (TPR) were increased by ~ 15% and ~ 70% respectively in the LPS-treated mice as compared with those of control (Fig. 1B). These pathological and functional changes are consistent with acute lung injury.
Fig. 2. Effects of LPS on expressions of the TREM-1 (panel A) and TREM-2 mRNA (panel B) and ratio of TREM-1 mRNA to TREM-2 mRNA (panel C) in lung tissues of mice. The mice were treated with LPS as described in legends of Fig. 1. The expressions of TREM-1 and TREM-2 mRNA in the lungs of mice were detected by RT-PCR. All data are represented as the mean± SD n = 10 in each group. *P b 0.05, **P b 0.01 vs. control group.
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3.2. Effects of LPS on TREM-1 and TREM-2 mRNA expressions in mice lung We next determined whether the expressions of TREM-1 and TREM-2 mRNA were altered in the lung tissues of LPS-treated mice. RT-PCR analysis showed that expression of the TREM-1 mRNA in
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lungs of LPS-treated mice was significantly higher than that of the normal mice (Fig. 2A); in contrast to that, expression of the TREM-2 mRNA in lungs of ALI mice was significantly lower than that of the normal mice (Fig.2B). The ratio of TREM-1 to TREM-2 mRNA in lung tissues of ALI mice (0.5667 ± 0.1939) was higher than the ratio in the lungs of normal mice (0.1383 ± 0.0224) (P b 0.01) (Fig. 2C).
Fig. 3. Expressions of the TREM-1 (panels A) and TREM-2 (panel B) and the ratio of TREM-1/TREM-2 (panel C) in LPS-induced pulmonary cells. The cells were treated with LPS (1 μg/ml) for 6 h. Expressions of the TREM-1 and TREM-2 mRNA were detected by RT-PCR and flow cytometry (FC). All data are represented as the mean ± SD, n = 5 in each group. **P b 0.01 vs. Control group. CON: control; 3T3: lung fibroblasts; A549: lung adenocarcinoma cells; HBECs: bronchial epithelial cells; RAW: macrophages.
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Fig. 3 (continued).
3.3. Activation of macrophages To determine whether the macrophages were activated in LPStreated mice, we measured the production of TNF-α, an inflammatory cytokine. ELISA showed that the amount of TNF-α protein produced by LPS-induced macrophages was much higher as compared with that from inactivated macrophages [20.58 ± 1.67 pg/ml (LPS group) vs. 8.84 ± 0.33 pg/ml (control), P b 0.01].
3.4. Expressions of TREM-1 and TREM-2 and TREM-1/TREM-2 ratio in LPS-induced pulmonary cells To identify the major sources of cells that might express TREM-1 and TREM-2 mRNA, we then determined the differences in expressions of the TREM-1 and TREM-2 mRNA in various lung cells. RT-PCR analysis and FC showed that TREM-1 was expressed in macrophages, and the expression of TREM-1 was increased in LPS-treated
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macrophages. However, we were not able to detect the expression of TREM-1 mRNA in 3 T3, A549, and HBECs (Fig. 3A). Expression of TREM-2 mRNA was detected in different cell lines of lung including bronchial epithelial cells, fibroblasts, lung adenocarcinoma cells and macrophages. However, the expression of TREM-2 mRNA was decreased in LPS-induced macrophages and bronchial epithelial cells (Fig.3B). The ratio of TREM-1 mRNA to TREM-2 mRNA in LPSinduced macrophages (0.2469 ± 0.1264) was higher as compared with this ratio from normal macrophages (0.0007 ± 0.0002) (P b 0.01) (Fig.3C).
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3.5. Effects of VIP on TREM-1 and TREM-2 expressions and TREM-1/TREM-2 ratio in LPS-treated lung cells We further determined effects of VIP on expressions of the TREM-1 and TREM-2 mRNA in LPS-treated lung cells. The detection of TREM-1 was only performed in LPS-treated macrophages because it is a main source of TREM-1 expression. Our results showed that VIP inhibited expression of the TREM-1 mRNA in LPS-treated macrophages when the cells were pretreated with VIP for various times (2 h, 4 h, 6 h, 12 h) or at different concentrations (VIP 10− 10 M, 10− 9 M, 10− 8 M,
Fig. 4. Effects of VIP on expressions of the TREM-1 (panels A, B) and TREM-2 mRNA (panel C) and ratio of TREM-1/TREM-2 (panel D) in LPS-induced pulmonary cells. The cells were pretreated with various concentrations of VIP (10− 10, 10− 9, 10− 8, 10− 7 and 10− 6 mol/L) for 30 min and followed by the addition of LPS (1 μg/ml) for additional 0, 2, 4, 6, 12 and 24 h. TREM-1 and TREM-2 expression were detected by RT-PCR. All data are represented as the mean ± SD, n = 5 in each group. #P b 0.05, ## P b 0.01 vs. LV group; ▲P b 0.05 vs. 0 h group; △P b 0.05, △△ P b 0.01 vs. LV10 group; ※P b 0.05 vs. Control group; * P b 0.05, ** P b 0.01 vs. LPS group. LV: LPS + VIP.
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Fig. 4 (continued).
10− 7 M, 10− 6 M) (Fig. 4A–B). Furthermore, we found that VIP increased expression of the TREM-2 mRNA in bronchial epithelial cells, lung fibroblasts, lung adenocarcinoma cells and macrophages (Fig. 4C). The ratio of TREM-1 mRNA to TREM-2 mRNA in LPS-induced macrophages with VIP pretreatment (0.1764 ± 0.0875) was lower than that in LPS-induced macrophages without VIP pretreatment (0.2469 ± 0.1264) (P b 0.05) (Fig.4D). 3.6. Effects of VIP receptor antagonist on TREM expressions in LPS-treated lung cells To test whether the effects of VIP on TREM expressions are mediated by activation of the VIP receptor, we determined the effects of the VIP receptor antagonist on TREM expressions in LPS-treated
lung cells. The VIP receptor antagonist inhibited the effect of VIP on expression of the TREM-1 mRNA (Fig. 5A) and TREM-1 protein (Fig. 5B) in LPS-treated macrophages. 4. Discussion ARDS is a serious acute reaction to various types of injuries to the lung. ARDS is characterized by inflammation of the lung parenchyma and it causes significant mortality around the world. It is estimated that there are ~ 190,000 cases of ALI/ARDS, and it accounts for ~74,500 deaths each year in the United States [11]. Acute inflammation with neutrophil infiltration is a typical pathologic change at the early stage of ALI/ARDS, it is important to understand the underling mechanisms responsible for excessive inflammation in ALI/ARDS.
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Fig. 5. Effects of the VIP receptor antagonist on expression of the TREM-1 in LPS-treated lung cells (panels A, B). TREM-1 expression was detected by RT-PCR and flow cytometry (FC). All data are represented as the mean ± SD, n = 5 in each group. ## P b 0.01 vs. LV group; ※※ P b 0.01 vs. Control group; * P b 0.05, ** P b 0.01 vs. LPS group. LV: LPS + VIP; VR: VIP receptor antagonist (10− 5 mol/L), LVVR: LPS + VIP + VIP receptor antagonist.
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VIP is a well-characterized endogenous anti-inflammatory neuropeptide that is abundant in the lung tissue. VIP has local regulatory effect in the columnar epithelia of the allergic lung [12], produces bronchodilation [13] and inhibits smooth muscle cell proliferation [2]. It exerts anti-inflammatory effect in experimental asthmatic rats [14]. Our previous findings also show that VIP enhances the repair of bronchial epithelial cells [15,16]. Recently, the role of triggering receptor expressed on myeloid cells (TREM) in the regulation of inflammatory processes has been identified. TREM-1 is thought to be an amplifier of the immune responses, while TREM-2 is believed to be a protective negative regulator against inflammation [17,18]. It is unknown whether TREM-1 and TREM-2 are expressed in lung tissues and/or cells in the absence or presence of LPS stimulation. Furthermore, it is not clear whether anti-inflammatory effect of VIP is related to its ability to regulate the expression of the TREM-1 and TREM-2. In this study, we found that the expression of the TREM-1 in lung tissues of ALI mice was significantly higher than that of normal mice; whereas the expression of the TREM-2 in lungs of ALI mice was significantly lower than that of normal mice. As a result, the ratio of TREM-1 to TREM-2 in lungs of ALI mice was higher than that in lungs of normal mice. It seems that the expression of TREM-1 and TREM-2 is regulated in an opposite direction under condition of LPS-induced ALI. It is possible that there is a dynamic equilibrium of TREM-1/TREM-2 ratio, and the loss of the balance of TREM-1/TREM-2 plays an important role in the inflammatory processes in ALI. The difference in the expressions of TREM-1 and TREM-2 among different cells might be related to their distinct roles in regulation of inflammation in innate immunity and bacterial infection. It is interesting to note that the expressions of the TREM-1 and TREM-2 were cell specific. We found that TREM-1 was only expressed in macrophages, while TREM-2 was expressed in variety types of cells in lung, such as bronchial epithelial cells, fibroblasts, lung adenocarcinoma cells and macrophages. Although the distribution and biological activities of the TREM-1 and TREM-2 in the respiratory system need to be further investigated, our results indicate that relative balance between TREM-1 and TREM-2 (low ratio of TREM-1 to TREM-2) might be essential for maintaining microenvironment stability of normal lung tissue. Alveolar macrophages (AM) contribute to the immunological defense by releasing inflammatory mediators [19]. Activated macrophages increase the production of pro-inflammatory cytokines including TNF-α which activates the NF-κB cascades [20–22]. Consistent with this notion, the production of TNF-α was significantly increased in LPS-treated macrophages. Both TREM-1 and TREM-2 are expressed in activated macrophages suggesting a role of TREM-1 and TREM-2 in activation of macrophages of ALI. The observations that VIP down-regulated the expression of TREM-1 and up-regulated expression of TREM-2 suggest a novel mechanism involved in the regulation inflammatory processes in LPSinduced ALI. The effects of VIP on expressions of the TREM-1 and TREM-2 were in a time- and dose-dependent manner. The ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced macrophages, and this effect was reversed in the presence of VIP. These results indicate that VIP can function as an endogenous regulator in balancing the expressions of TREM-1 and TREM-2. It is well-known that VIP produces an anti-inflammatory effect through modulating the expression and function of pro-inflammatory factors by inhibiting NF-κB activation [23,24], and down-regulating the expression and function of Toll-like receptors (TLRs) [25–27]. It also has been shown that TREM-1 and TREM-2 expressions were regulated at transcriptional level by NF-κB [28,29]. Furthermore, functional genomics of TREM-1 and TREM-2 were related to TLR signaling [30,31]. It is possible that VIP regulates the activation of NF-κB by the VIP receptor mediated intracellular signaling pathways, and consequently it modulates the expression of TREM-1 and TREM-2, and ultimately affects the outcome of ALI. These possibilities remain to be investigated.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30870915) and Hunan Provincial Innovation Foundation for Postgraduate (NO. CX2010B096) and Central South University Open Sharing Foundation for the valuable equipment (No. Zkj2009013).We thanks Dr. Gruenert from the University of California, San Francisco, for providing an immortalized human BEC line 16HBE140-.
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Contents lists available at ScienceDirect
Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Vasoactive intestinal peptide re-balances TREM-1/TREM-2 ratio in acute lung injury Guo-Ying Sun a, Cha-Xiang Guan a,⁎, Yong Zhou a, Yong-Ping Liu b, Shu-Fen Li a, Hui-Fang Zhou a, Chun-Yan Tang a, Xiang Fang c a b c
Department of Physiology, Central South University Xiangya Medical School, Changsha, Hunan 410078, China Department of Physiology, Hunan University of Traditional Chinese Medicine, Changsha, Hunan 410208, China Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA
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Article history: Received 31 July 2010 Received in revised form 21 November 2010 Accepted 25 November 2010 Available online 2 December 2010 Keywords: Vasoactive intestinal peptide TREM-1 TREM-2 Macrophage Acute lung injury
a b s t r a c t Vasoactive intestinal peptide (VIP) is one of the most plentiful neuropeptides in the lung and it has antiinflammatory effects in the respiratory system. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) regulate immune responses to lipopolysaccharide (LPS). In the present study, we tested the expressions of TREM-1 and TREM-2 in various pulmonary cell lines and/or tissue using an animal model of LPS-induced acute lung injury (ALI), and determined the effects of VIP on expression of the TREM-1 and TREM-2 in lung tissues and cells from ALI mice. We found 1) expression of the TREM-1 mRNA from lung tissues of ALI was significantly increased, whereas the expression of TREM-2 mRNA was decreased in these tissues; 2) TREM-1 mRNA was only expressed in macrophages, while TREM-2 mRNA was detected in HBECs, lung fibroblasts, lung adenocarcinoma cells and macrophages; 3) the ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced lung tissues and macrophages; 4) VIP inhibited expression of the TREM-1 mRNA in a time- and dose-dependent manner in lung cells from LPS-induced ALI mice; however, it increased expression of the TREM-2 mRNA. As a result of these effects, VIP normalized the ratio of TREM-1 to TREM-2 mRNA in these cells. Our results suggest that VIP might exert its anti-inflammatory effect through a mechanism involved in regulation of expression of the TREM-1 and TREM-2 in LPS-induced ALI. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Vasoactive intestinal peptide (VIP) is a bioactive neuropeptide involved in a wide range of biological functions in the respiratory system. It causes relaxation of airway smooth muscles [1], regulates smooth muscle proliferation of airway [2], and secretion of exocrine and endocrine glands [3]. VIP also regulates immune responses in different conditions [4]. Because of its numerous biological effects, modulation of VIP has been considered as a potential therapeutic target for the treatment of many diseases such as rheumatism [5] and diabetes [6]. The patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) have high mortality rate (~40%), and these acute respiratory disorders are still considered as major threats to human health in the current medicine [7]. It has been shown previously that VIP possesses an anti-inflammatory property in respiratory diseases such as asthma. It is important to determine its role in the pathological processes in ALI. Abbreviations: VIP, vasoactive intestinal peptide; TREM-1, triggering receptors expressed on myeloid cells-1; TREM-2, triggering receptors expressed on myeloid cells-2; LPS, lipopolysaccharide; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; TLR, toll-like receptor; NLR, nod-like receptor; RF, respiratory frequency; Cdyn, dynamic compliance; TV, tidal volume; TPR, total pulmonary resistance. ⁎ Corresponding author. Tel.: + 86 731 82355051; fax: + 86 731 82355056. E-mail address: [email protected] (C.-X. Guan).
ALI is characterized by cell injuries in pulmonary capillary endothelial, alveolar epithelial cells, and increased vascular permeability due to inflammatory reactions. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) are newly identified inflammatory molecules [8]. TREM-1 possesses biological activities similar to activation of Toll-like receptor (TLR) and Nod-like receptor (NLR). It stimulates cellular immune signaling and produces excessive inflammatory responses [9]. TREM-2 inhibits the reactions induced by TLR in macrophages, and provides protective effects on TREM-1 mediated inflammation [10]. It is unknown whether TREM-1 and TREM-2 are expressed in lung tissues and pulmonary cells, particularly in these tissues/cells from ALI. It is remained to be investigated whether VIP can affect the expression of TREM-1 and TREM-2. In the present study, we tested the hypothesis that VIP plays an important role in the regulation of expression of TREM-1/TREM-2 in lipopolysaccharide (LPS) stimulated lung tissues and pulmonary cells. 2. Materials and methods 2.1. Materials DMEM (High Glucose) medium, RPMI 1640 medium and TRIzol were the products of Invitrogen Life Technologies (California, USA).
0167-0115/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2010.11.008
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LPS, VIP and D-P-C1 Phe(6)-Leu(17)-VIP were purchased from Sigma (St.Louis, MO, USA). TREM-1 antibody and FITC-labeled anti-mouse TREM-1 antibody were from Santa Cruz (Dover, USA). EDTA and Nuclease-Free Water were from Amresco Company (Ohio, USA). Calf serum was bought from Sijiqing Company (Hangzhou, China). Reverse transcription (RT) kit and Taq DNA polymerase were purchased from Fermentas (Ohio, USA). All primers were synthesized by Shanghai Bio-engineering Company (Shanghai, China). Biowest agarose was from Agarose Bead Technologies (Madrid, Spain). DNA maker was from Tiangen Biotech (Beijing, China).
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2.3. Culture and activation of lung cell lines The human bronchial epithelial cell (HBEC) line, 16HBE140- was generously provided by Dr. Gruenert from the University of California, San Francisco. The mouse macrophages cell line (RAW264.7), mouse embryonic fibroblast cell line (NIH 3T3), and human lung adenocarcinoma cell line (A549) were purchased from Cell Center, Xiangya School of Medicine, Central South University. The cells were cultured in DMEM (High Glucose) medium at 37 °C with 5% CO2. The medium was replaced by serum-free medium for 8 h, when the cell reached confluence of 80%.
2.2. Animals Thirty male Kunming mice (18 g to 23 g, from department of experimental animals of the Central South University), were housed in an SPF animal room maintained at constant temperature (24 °C ± 2 °C) and under a 12-h light/dark cycle with free access to food and water. All animal studies were approved by the Central South University Animal Studies Committee.
2.4. Verification of LPS-induced pulmonary injury and activation of macrophages Kunming mice were sensitized to LPS by immunization. The mice received i.p. injections of lipopolysaccharide (LPS, 15 mg/kg). Buxco was used to monitor the changes in respiratory function parameters in ALI mice. HE staining of lung tissues from these mice was
Fig. 1. The pathological (panel A) and respiratory functional changes (panel B) in LPS-stimulated mice. The mice were treated with LPS (15 mg/kg, i.p.). After 6 h of treatment, the respiratory functions were determined by Buxco, and the pathological changes were observed by Hematoxylin–eosin (HE) staining under microscope. All data are represented as the mean ± SD, n = 10 in each group. **P b 0.01 vs. control group.
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performed for pathological characterization. The amount of TNF-α protein released into the medium in LPS-stimulated macrophages was detected by ELISA. 2.5. Detection of TREM protein by flow cytometry The cells were incubated with VIP in the presence or absence of different inhibitors for 6 h. After incubation, the cells were washed twice with PBS, and unlabeled first antibody was added into the 100 μl cell suspension in each tube. All tubes were then incubated at 4 °C, and washed by PBS after 30 min. The cell suspension was centrifuged at 2000 g for 5 min, and the cells incubated for additional 30 min at 4 °C with fluorescein isothiocyanate (FITC) labeled secondary antibody. The cells were washed again, and the cells were re-suspended with 1% paraformaldehyde, and then the protein was determined by flow cytometry (FC). 2.6. Determination of TREM-1 and TREM-2 mRNA Total RNA was prepared by using TRIzol, and 2 μg of total RNA was used for detection of mRNA using a reverse transcription-PCR (RT-PCR). The following primers sequences were used for RT-PCR amplification: TREM-1 (338 bp) forward: 5′-CTGCTGTGCGTGTTCTTT-3′, TREM-1 reverse: 5′-TCATTCGGAGGATGGTAA-3′, TREM-2(260 bp) forward: 5′GGAACCGTCACCATCACT-3′, TREM-2 reverse: 5′-GCCAGGAGGAGAAGAATG-3′, and GAPDH (580 bp) forward: 5′-AAGCCCATCACCATCTTC CA-3′, GAPDH reverse: 5′-CCTGCTTCACCACCTTCTTG-3′. PCR was performed under the conditions of an initial 5 min at 94 °C followed by 35 cycles (30 s at 94 °C 30 s at 52 °C for TREM-1 or 30 s at 58 °C for TREM-2 and GAPDH, 1 min at 94 °C with a final 5 min at 72 °C. PCR products after electrophoresis were imaged by using Quantity One. 2.7. Statistical analyses The data was expressed as mean ± standard deviation (SD) and analyzed with SPSS 13.0 statistical package. The repeated measures analysis of variance (ANOVA) test was used to compare the differences among multiple groups. Tamhane test was used to compare the difference between two groups. P b 0.05 was considered as statistically significant. 3. Results 3.1. The structure of lung and respiratory functions were impaired in LPS-stimulated mice We first examined the pathological and pulmonary function changes in LPS-treated mice. The alveolar spaces of the lung from LPS-treated mice were filled with white blood cells and alveolar macrophages, and the alveolar septum of lung were also destructed (Fig. 1A). The pulmonary function test by Buxco showed respiratory frequency (RF) and dynamic compliance (Cdyn) were decreased by ~20% and ~ 67% respectively in LPS-treated mice as compared with those of control mice. However, the tidal volume (TV) and total pulmonary resistance (TPR) were increased by ~ 15% and ~ 70% respectively in the LPS-treated mice as compared with those of control (Fig. 1B). These pathological and functional changes are consistent with acute lung injury.
Fig. 2. Effects of LPS on expressions of the TREM-1 (panel A) and TREM-2 mRNA (panel B) and ratio of TREM-1 mRNA to TREM-2 mRNA (panel C) in lung tissues of mice. The mice were treated with LPS as described in legends of Fig. 1. The expressions of TREM-1 and TREM-2 mRNA in the lungs of mice were detected by RT-PCR. All data are represented as the mean± SD n = 10 in each group. *P b 0.05, **P b 0.01 vs. control group.
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3.2. Effects of LPS on TREM-1 and TREM-2 mRNA expressions in mice lung We next determined whether the expressions of TREM-1 and TREM-2 mRNA were altered in the lung tissues of LPS-treated mice. RT-PCR analysis showed that expression of the TREM-1 mRNA in
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lungs of LPS-treated mice was significantly higher than that of the normal mice (Fig. 2A); in contrast to that, expression of the TREM-2 mRNA in lungs of ALI mice was significantly lower than that of the normal mice (Fig.2B). The ratio of TREM-1 to TREM-2 mRNA in lung tissues of ALI mice (0.5667 ± 0.1939) was higher than the ratio in the lungs of normal mice (0.1383 ± 0.0224) (P b 0.01) (Fig. 2C).
Fig. 3. Expressions of the TREM-1 (panels A) and TREM-2 (panel B) and the ratio of TREM-1/TREM-2 (panel C) in LPS-induced pulmonary cells. The cells were treated with LPS (1 μg/ml) for 6 h. Expressions of the TREM-1 and TREM-2 mRNA were detected by RT-PCR and flow cytometry (FC). All data are represented as the mean ± SD, n = 5 in each group. **P b 0.01 vs. Control group. CON: control; 3T3: lung fibroblasts; A549: lung adenocarcinoma cells; HBECs: bronchial epithelial cells; RAW: macrophages.
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Fig. 3 (continued).
3.3. Activation of macrophages To determine whether the macrophages were activated in LPStreated mice, we measured the production of TNF-α, an inflammatory cytokine. ELISA showed that the amount of TNF-α protein produced by LPS-induced macrophages was much higher as compared with that from inactivated macrophages [20.58 ± 1.67 pg/ml (LPS group) vs. 8.84 ± 0.33 pg/ml (control), P b 0.01].
3.4. Expressions of TREM-1 and TREM-2 and TREM-1/TREM-2 ratio in LPS-induced pulmonary cells To identify the major sources of cells that might express TREM-1 and TREM-2 mRNA, we then determined the differences in expressions of the TREM-1 and TREM-2 mRNA in various lung cells. RT-PCR analysis and FC showed that TREM-1 was expressed in macrophages, and the expression of TREM-1 was increased in LPS-treated
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macrophages. However, we were not able to detect the expression of TREM-1 mRNA in 3 T3, A549, and HBECs (Fig. 3A). Expression of TREM-2 mRNA was detected in different cell lines of lung including bronchial epithelial cells, fibroblasts, lung adenocarcinoma cells and macrophages. However, the expression of TREM-2 mRNA was decreased in LPS-induced macrophages and bronchial epithelial cells (Fig.3B). The ratio of TREM-1 mRNA to TREM-2 mRNA in LPSinduced macrophages (0.2469 ± 0.1264) was higher as compared with this ratio from normal macrophages (0.0007 ± 0.0002) (P b 0.01) (Fig.3C).
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3.5. Effects of VIP on TREM-1 and TREM-2 expressions and TREM-1/TREM-2 ratio in LPS-treated lung cells We further determined effects of VIP on expressions of the TREM-1 and TREM-2 mRNA in LPS-treated lung cells. The detection of TREM-1 was only performed in LPS-treated macrophages because it is a main source of TREM-1 expression. Our results showed that VIP inhibited expression of the TREM-1 mRNA in LPS-treated macrophages when the cells were pretreated with VIP for various times (2 h, 4 h, 6 h, 12 h) or at different concentrations (VIP 10− 10 M, 10− 9 M, 10− 8 M,
Fig. 4. Effects of VIP on expressions of the TREM-1 (panels A, B) and TREM-2 mRNA (panel C) and ratio of TREM-1/TREM-2 (panel D) in LPS-induced pulmonary cells. The cells were pretreated with various concentrations of VIP (10− 10, 10− 9, 10− 8, 10− 7 and 10− 6 mol/L) for 30 min and followed by the addition of LPS (1 μg/ml) for additional 0, 2, 4, 6, 12 and 24 h. TREM-1 and TREM-2 expression were detected by RT-PCR. All data are represented as the mean ± SD, n = 5 in each group. #P b 0.05, ## P b 0.01 vs. LV group; ▲P b 0.05 vs. 0 h group; △P b 0.05, △△ P b 0.01 vs. LV10 group; ※P b 0.05 vs. Control group; * P b 0.05, ** P b 0.01 vs. LPS group. LV: LPS + VIP.
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Fig. 4 (continued).
10− 7 M, 10− 6 M) (Fig. 4A–B). Furthermore, we found that VIP increased expression of the TREM-2 mRNA in bronchial epithelial cells, lung fibroblasts, lung adenocarcinoma cells and macrophages (Fig. 4C). The ratio of TREM-1 mRNA to TREM-2 mRNA in LPS-induced macrophages with VIP pretreatment (0.1764 ± 0.0875) was lower than that in LPS-induced macrophages without VIP pretreatment (0.2469 ± 0.1264) (P b 0.05) (Fig.4D). 3.6. Effects of VIP receptor antagonist on TREM expressions in LPS-treated lung cells To test whether the effects of VIP on TREM expressions are mediated by activation of the VIP receptor, we determined the effects of the VIP receptor antagonist on TREM expressions in LPS-treated
lung cells. The VIP receptor antagonist inhibited the effect of VIP on expression of the TREM-1 mRNA (Fig. 5A) and TREM-1 protein (Fig. 5B) in LPS-treated macrophages. 4. Discussion ARDS is a serious acute reaction to various types of injuries to the lung. ARDS is characterized by inflammation of the lung parenchyma and it causes significant mortality around the world. It is estimated that there are ~ 190,000 cases of ALI/ARDS, and it accounts for ~74,500 deaths each year in the United States [11]. Acute inflammation with neutrophil infiltration is a typical pathologic change at the early stage of ALI/ARDS, it is important to understand the underling mechanisms responsible for excessive inflammation in ALI/ARDS.
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Fig. 5. Effects of the VIP receptor antagonist on expression of the TREM-1 in LPS-treated lung cells (panels A, B). TREM-1 expression was detected by RT-PCR and flow cytometry (FC). All data are represented as the mean ± SD, n = 5 in each group. ## P b 0.01 vs. LV group; ※※ P b 0.01 vs. Control group; * P b 0.05, ** P b 0.01 vs. LPS group. LV: LPS + VIP; VR: VIP receptor antagonist (10− 5 mol/L), LVVR: LPS + VIP + VIP receptor antagonist.
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VIP is a well-characterized endogenous anti-inflammatory neuropeptide that is abundant in the lung tissue. VIP has local regulatory effect in the columnar epithelia of the allergic lung [12], produces bronchodilation [13] and inhibits smooth muscle cell proliferation [2]. It exerts anti-inflammatory effect in experimental asthmatic rats [14]. Our previous findings also show that VIP enhances the repair of bronchial epithelial cells [15,16]. Recently, the role of triggering receptor expressed on myeloid cells (TREM) in the regulation of inflammatory processes has been identified. TREM-1 is thought to be an amplifier of the immune responses, while TREM-2 is believed to be a protective negative regulator against inflammation [17,18]. It is unknown whether TREM-1 and TREM-2 are expressed in lung tissues and/or cells in the absence or presence of LPS stimulation. Furthermore, it is not clear whether anti-inflammatory effect of VIP is related to its ability to regulate the expression of the TREM-1 and TREM-2. In this study, we found that the expression of the TREM-1 in lung tissues of ALI mice was significantly higher than that of normal mice; whereas the expression of the TREM-2 in lungs of ALI mice was significantly lower than that of normal mice. As a result, the ratio of TREM-1 to TREM-2 in lungs of ALI mice was higher than that in lungs of normal mice. It seems that the expression of TREM-1 and TREM-2 is regulated in an opposite direction under condition of LPS-induced ALI. It is possible that there is a dynamic equilibrium of TREM-1/TREM-2 ratio, and the loss of the balance of TREM-1/TREM-2 plays an important role in the inflammatory processes in ALI. The difference in the expressions of TREM-1 and TREM-2 among different cells might be related to their distinct roles in regulation of inflammation in innate immunity and bacterial infection. It is interesting to note that the expressions of the TREM-1 and TREM-2 were cell specific. We found that TREM-1 was only expressed in macrophages, while TREM-2 was expressed in variety types of cells in lung, such as bronchial epithelial cells, fibroblasts, lung adenocarcinoma cells and macrophages. Although the distribution and biological activities of the TREM-1 and TREM-2 in the respiratory system need to be further investigated, our results indicate that relative balance between TREM-1 and TREM-2 (low ratio of TREM-1 to TREM-2) might be essential for maintaining microenvironment stability of normal lung tissue. Alveolar macrophages (AM) contribute to the immunological defense by releasing inflammatory mediators [19]. Activated macrophages increase the production of pro-inflammatory cytokines including TNF-α which activates the NF-κB cascades [20–22]. Consistent with this notion, the production of TNF-α was significantly increased in LPS-treated macrophages. Both TREM-1 and TREM-2 are expressed in activated macrophages suggesting a role of TREM-1 and TREM-2 in activation of macrophages of ALI. The observations that VIP down-regulated the expression of TREM-1 and up-regulated expression of TREM-2 suggest a novel mechanism involved in the regulation inflammatory processes in LPSinduced ALI. The effects of VIP on expressions of the TREM-1 and TREM-2 were in a time- and dose-dependent manner. The ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced macrophages, and this effect was reversed in the presence of VIP. These results indicate that VIP can function as an endogenous regulator in balancing the expressions of TREM-1 and TREM-2. It is well-known that VIP produces an anti-inflammatory effect through modulating the expression and function of pro-inflammatory factors by inhibiting NF-κB activation [23,24], and down-regulating the expression and function of Toll-like receptors (TLRs) [25–27]. It also has been shown that TREM-1 and TREM-2 expressions were regulated at transcriptional level by NF-κB [28,29]. Furthermore, functional genomics of TREM-1 and TREM-2 were related to TLR signaling [30,31]. It is possible that VIP regulates the activation of NF-κB by the VIP receptor mediated intracellular signaling pathways, and consequently it modulates the expression of TREM-1 and TREM-2, and ultimately affects the outcome of ALI. These possibilities remain to be investigated.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30870915) and Hunan Provincial Innovation Foundation for Postgraduate (NO. CX2010B096) and Central South University Open Sharing Foundation for the valuable equipment (No. Zkj2009013).We thanks Dr. Gruenert from the University of California, San Francisco, for providing an immortalized human BEC line 16HBE140-.
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