Abstract
BACKGROUND:
Myeloid cell (TREM-1) is an important mediator of the signal transduction pathway in inflammatory response. In this study, a mouse model of acute lung injury (ALI) by intraperitoneal injection of lipopolysaccharide (LPS) was established to observe the expression pattern of TREM-1 in lung tissue and the role of TREM-1 in pulmonary inflammatory response to ALI.
METHODS:
Thirty BALB/C mice were randomly divided into a normal control group (n=6) and an ALI group (n=24). The model of ALI was made by intraperitonal injection of LPS in dose of 10 mg/kg. Specimens from peripheral blood and lung tissue were collected 6, 12, 24 and 48 hours after LPS injection. RT-PCR was used to detect TREM-1 mRNA, and ELISA was employed for detection of TREM-1 protein and TNF-a protein, and HE staining was performed for the pathological Smith lung scoring under a light microscope.
RESULTS:
The expressions of TREM-1 mRNA in lung tissue and blood of the ALI group 6, 12, 24, and 48 hours after injection of LPS were higher than those in the control group. The levels of TREM-1 protein and the levels of TNF-a protein in lung tissue of the ALI group 6, 12, 24, and 48 hours after LPS injection were higher than those of the control group; the level of TREM-1 protein peaked 12 hours after LPS injection, but it was not significantly correlated with the expression of TREM-1 mRNA (P=0.14); the TNF-a concentration was positively correlated with TREM-1 levels in lung tissue and with Smith pathological score (r=0.795, P=0.001:r=0.499, P=0.034), but not with the expression of TREM-1 mRNA (P=0.176).
CONCLUSIONS:
The expression of TREM-1 mRNA in lung tissue of mice with ALI is elevated, and the expression of TREM-1 mRNA is related to the level of TNF-a and the severity of inflammatory response to ALI. The expressions of the TREM-1 gene are not consistent with the levels of TREM-1 protein, suggesting a new functional protein involved in immune regulation.
KEY WORDS: Acute lung injury, Triggering receptor-1, Myeloid cell, Expression, Tumor necrosis factor, Pathological scoring
INTRODUCTION
Triggering receptor expressed on myeloid cell-1 (TREM-1) was first reported by Bouchon et al[1] in 2001, which is the membrane receptor expressed on the surface of neutrophils and other myeloid cells.[2] TREM-1 may trigger and amplify the inflammatory response. It is an important mediator of the signal transduction pathway in the inflammatory response.[3-5] In this study, a mouse model of acute lung injury (ALI) was established by intraperitoneal injection of lipopolysaccharide (LPS) to observe the expression pattern of TREM-1 in lung tissue and the role of TREM-1 in pulmonary inflammatory response to ALI.
METHODS
Thirty male BALB/C mice for SPF (Animal Model Institute of Nanjing University), weighing 18-22 g, were randomly divided into a normal control group (n=6) and an ALI group (n=24). The ALI group was given an intraperitoneal injection of LPS at a dose of 10 mg/kg (E coli O111: B4, Sigma Company, USA) to copy ALI model.[6]
Experimental methods
Lung tissue and blood samples were collected at 6, 12, 24 and 48 hours after modeling. Pentobarbital sodium (30 mg/kg) was injected intraperitoneally for anesthesia. After removal of eyeballs to get peripheral blood, and the chest was opened to collect lung tissue. The right lung was solidified with 10% neutral formaldehyde solution, and the left lung and blood samples were saved in liquid nitrogen (-196 °C).
Pathological observation and scoring
Lung tissue of 4-6 μm was embedded in paraffin and HE stained. The changes of lung tissue was observed with an optical microscope. Smith scoring was done in the lung tissue, and half quantitative evaluation was performed for the severity of lung injury.[7]
Expression of TREM-1 mRNA detected by RT-PCR
Total RNA was extracted from peripheral blood mononuclear cells using Trizol reagent, and it was reversely transcribed into cDNA M-MLV by reverse transcriptase (Promega Corporation, USA). β-actin served as an internal control. According to the GeneBank database reference cDNA series, TREM-1 primers (Nanjing Ji Ao Biotechnology Limited Company, China) were synthesized as follows: The upstream primer: 5’-TCCTCCGAATGACCCTGTTG-3’; downstream primer: 5’-GAACACATCTGAAGAACCCTTGGT- 3’; the target gene fragment length 340bp, probe: FAM-TTCCATCCTGTCCGCCTGGTGGT-BHQ1. β-actin upstream primer: 5’-GAGACCTTCAACACCCCAGC-3’; downstream: 5’-CACAGAGTACTTGCGCTCAG-3’; the target gene fragment length 654bp; probe: FAM-TTCCATCCTGTCCGCCTGGTGGT -BHQ1. PCR reaction conditions were 94 °C 10 minutes, 94 °C 60 seconds, 55 °C 34 seconds, and 40 cycles. After reaction, the relative mRNA expression was calculated using the PCR7500 analysis system.
Concentrations of TREM-1, TNF-α detected by ELISA
The reagent boxes were supplied from the United States R&D Company, Shenzhen Jingmei Biological Engineering Limited Company, and operated strictly in accordance with the instructions. At a wave length of 450 nm and 492 nm, a microplate reader was used for measurement of OD, and the actual content was calculated according to standard concentration curve.
Statistical analysis
All data were presented as mean±SD. Statistical comparisons were made with the SPSS 13.0 statistical software. Analysis of variance and Spearman’s rank-order correlation coefficient analysis were used. P<0.05 was considered statistically significant.
RESULTS
Pathologic changes in lung tissues
Under the naked eye, lung volume increased, edema, and focal point hemorrhage could be found in the surface of the lung tissue in the ALI group. Pathologic slices showed that under a light microscope the lung was well expanded without bleeding in the control group (Figure 1 A); thare were a large number of inflammatory cells in widened interstitial lung with alveolar collapse, alveolar exudation, pulmonary capillary congestion, and hemorrhage (Figure 1 B-E).
Figure 1.
Pathological changes of lung tissue in the control group and ALI group (HE, original magnification×100). A: control group; B: ALI group at 6 hours; C: ALI group at 12 hours; D: ALI group at 24 hours; E: ALI group at 48 hours.
TREM-1 concentrations in lung tissues
The concentrations of TREM-1 in lung tissue of the ALI group at 6, 12, 24 and 48 hours (997.8±114.62), (1579.70±45.92), (1123.9±108.2) and (429.8±89.96) pg/ml respectively) were higher than those of the control group (279.22±4.63) pg/ml, (P=0.024, 0.007, 0.011 and 0.04), and peaked at 12 hours (Table 1).
Table 1.
Comparison of TREM-1,TNF-α and Smith scores between control group and ALI group at different time points
Expression of TREM-1mRNA in lung tissues and blood
The expressions of TREM-1 mRNA in lung tissue of the ALI group at 6, 12, 24 and 48 hours (6.61±0.08, 34.71±0.83, 61.85±14.05 and 56.46±8.89 respectively) were higher than those of the control group (1.00±0.00) (P=0.017, 0.009, 0.002 and 0.003). The expressions in blood (14.01±3.24, 47.07±0.98, 8.18±0.43 and 8.06±0.05) were also higher than those of the control group (1.00±0.00) (P=0.010, 0.004, 0.011 and 0.011). The expressions of TREM-1mRNA in blood at 6 and 12 hours were higher than those in lung tissues (P=0.036 and 0.018), while at the 24 and 48 hours were lower than those in lung tissues (P=0.003 and 0.001) (Table 1).
Concentrations of TNF-α in lung tissues
The concentrations of TNF-α in lung tissue of the ALI group at 6, 12, 24 and 48 hours (313.16±39.50), (491.91±96.65), (388.48±29.84) and (282.5±52.76) pg/ml respectively, were significantly above those of the control group (256.6±28.31) pg/ml (P=0.037, 0.019, 0.032 and 0.043), and peaked at 12 hours (Table 1). The expression of TREM-1mRNA in lung tissue was not correlated with the concentration of TNF-α (P=0.176), but TREM-1 and TNF-α concentrations were positively correlated (r=0.795, P=0.001), and both changed in the same regular pattern.
Smith pathological scores of lung injury
Smith pathologic scores of the ALI group at each time point were significantly higher than those of the control group (P=0.003, 0.007, 0.009 and 0.006). The most serious lung injury was seen at 6 hours after modeling (Table 1), with infiltration of a large number of inflammatory cells, interstitial pulmonary edema, exudation, capillary stasis, alveolar collapse, and other pathological changes. And Smith pathological scores were positively correlated with the expression of TREM-1 mRNA and the concentration of TREM-1 (r=0.377 and 0.499, P=0.047 and 0.034).
DISCUSSION
TREM-1, a receptor expressed in neutrophils and the membrane surface of other myeloid cells, can trigger and amplify the inflammatory response.[1-4] Lung is often the first organ with inflammatory response leading to the occurrence of ALI.[5,8] As an important mediator of inflammatory responses, the expression of TREM-1 in the lung and the activity in the lung inflammatory response should be investigated.
In this experiment, the expression of TREM-1 mRNA in the lung began to increase 6 hours after LPS injection, peaked at 24 hours and maintained at a high level. It was proved that TREM-1 can express in the lung and can be found in the early stage of lung inflammatory response. The present experiment also showed that the peak expression of TREM-1 mRNA was earlier in blood than in lung tissue, which was correlated with intraperitoneal LPS injection-induced exogenous ALI. The changes of TNF-α in the lung tissue of ALI mice were due to the effect of TREM-1, thus TREM-1 was confirmed to be related to the inflammatory response to ALI. LPS-induced TREM-1mRNA rapid expression in blood[9] stimulated a large number of neutrophils, macrophages and other myeloid cells in lung tissue, and prompted continuous expression of TREM-1 mRNA. TREM-1 can up-regulate the expressions of TNF-α, IL-1b, GM-CSF and other cytokines of downstream, inhibit the expression of inflammatory factor IL-10, and start the inflammatory response cascade chain.[10-12] Therefore TREM-1 may play an important role in ALI-induced lung inflammation.
The gene expression of TREM-1 was inconsistent with the protein level in our study, the role of TREM-1 in lung inflammation in addition to the regulation of the level of transcription, but was possibly interactive with other functional proteins. Routsi et al[13] found that TREM-1mRNA shear variations generated soluble protein, which is a soluble triggering receptor expressed on myeloid cells -1 (sTREM-1).[14-16] Moreover TNF-α in lung tissue peaked at 12 hours and did not increase with the increasing expression of TREM-1 mRNA possibly because sTREM-1 is involved in the immune regulation and production of TNF-α. sTREM-1 combines with the unknown TREM-1 ligand[17-19] in the blood and inhibits the collection of ligand and TREM-1 in cell membrane, thus preventing inflammatory signal transduction.[20-22]
In summary, the expression of TREM-1 increases in lung tissue with ALI, promoting the release of inflammatory factors and the involving lung inflammation. For TREM-1, the gene expression and its protein levels are inconsistent. sTREM-1, a new functional protein, may play an important role in immune regulation, so it will indicate a new target for treating ALI.
Footnotes
Funding: This study was supported by a grant from the Board of Health of Nanjing (YKK08071).
Ethical approval: Not needed.
Conflicts of interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject.
Contributors: Liu N proposed the study and wrote the first draft. All authors contributed to design and interpretation of the study and to further drafts.
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