|Year : 2018 | Volume
| Issue : 2 | Page : 146-151
Involvement of Gas6 protein in mouse model of lipopolysaccharide-induced lung inflammation
William R Surin1, Surbhi Mundra1, Chellakkan S Blesson2, Ramachandra S Gudde3
1 Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, Karnataka, India
2 Division for Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, USA
3 Central Animal Facility, Indian Institute of Science, Bengaluru, Karnataka, India
|Date of Web Publication||14-Jun-2018|
Dr. William R Surin
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru - 560 012, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Gas6 is a product of growth arrest-specific gene 6 and is mostly expressed by the growth-arrested organs and is implicated in thrombosis, various inflammation, and age-related diseases. However, its role in chronic obstructive pulmonary disease has not been investigated yet. Methods: We developed lipopolysaccharide-induced mouse model of chronic lung inflammation to study its involvement. Therefore, male C57BL/6 mice (20–25 g) were grouped into control and LPS group; each group having 15 animals each. LPS group was nebulized with LPS (1 mg/ml) for 45 min, once a week for 8 weeks to induce chronic inflammatory conditions in the lungs by use of nebulizer. The mice model was validated by assessing infiltration of various immune cells (T-cells, B-cells, and neutrophils) into the lungs and pro-inflammatory and anti-inflammatory cytokine profile by cytokine bead array kit by flow cytometry. Lung deterioration was assessed by lung histology. The level of gas6 protein from the lung homogenate was measured by quantikine enzyme-linked immunosorbent assay kit. Furthermore, lung homogenate was analyzed by Western blotting for expression profile of gas6 protein. Results: There was significant increase in the level of T-cells (19±8% vs 65±11%), B-cells (24±7% vs 64±10%), and neutrophils (22±9% vs 57±10%) and significant increase in the level of tumor necrosis factor (200 ± 17 pg/ml vs. 1222 ± 152 pg/ml) and IL-6 (106 ± 13 pg/ml vs. 448 ± 122 pg/ml). Lung deterioration was observed in LPS group. We observed significant increase in the level of gas6 protein in lung homogenate in LPS group (0.16 ± 0.1 ng/ml vs. 4.2 ± 0.1 ng/ml). Furthermore, we found significant increase in gas6 protein in the lung homogenate of LPS-treated group by Western blotting. Conclusion: The current study establishes the involvement gas6 protein in lung inflammation.
Keywords: Chronic obstructive pulmonary disease, cytokine, gas6, inflammation, lung
|How to cite this article:|
Surin WR, Mundra S, Blesson CS, Gudde RS. Involvement of Gas6 protein in mouse model of lipopolysaccharide-induced lung inflammation. Biomed Biotechnol Res J 2018;2:146-51
|How to cite this URL:|
Surin WR, Mundra S, Blesson CS, Gudde RS. Involvement of Gas6 protein in mouse model of lipopolysaccharide-induced lung inflammation. Biomed Biotechnol Res J [serial online] 2018 [cited 2020 Mar 31];2:146-51. Available from: http://www.bmbtrj.org/text.asp?2018/2/2/146/234455
| Introduction|| |
Gas6 protein is the product of growth arrest-specific gene 6 and is mostly expressed by the growth-arrested organs and cells such as platelets, fibroblasts, lungs, and central nervous systems. It is a 75kda protein and belongs to the vitamin K-dependent family and is closely related to the protein S and to a steroid hormone transport protein called sex hormone-binding protein. It is the ligand for Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) families of receptor protein tyrosine kinases. These receptors have been shown to enhance platelet activation and thrombotic response. Studies on gas6−/− mice have shown that gas6−/− mice are protected against thrombotic challenge. Further, it is interesting that these animals did not show any bleeding diathesis and gas6 knock out did not affect their state of viability. Moreover, the role of gas6 protein in inflammatory disease like atherosclerosis have been reported in recent studies. Further, its role has been shown in airway allergic diseases in mice and asthma recently.
Chronic obstructive pulmonary disease (COPD) is a major inflammatory airway disorder characterized by the airway obstruction. It is slow, progressive, and irreversible. It is associated with abnormal inflammatory response of lungs to noxious particles and gases. It includes two disease conditions mainly emphysema and chronic bronchitis. Emphysema is characterized by enlargement of airspace, destruction of lung parenchyma, and chronic bronchitis. These conditions have inflammatory components characterized by the infiltration of neutrophils and macrophages into the lungs. There is mounting evidence of the involvement of gas6 protein in various inflammatory diseases such as autoimmune encephalitis, sepsis, and cancer., COPD is an inflammatory disease associated with airways and lungs; we undertook this study to investigate the involvement of gas6 protein in COPD.
| Methods|| |
Mice and treatment
Male mice C57BL/6 (20–25 g) were procured from the Central Animal Facility, of the Institute. They were housed in temperature-controlled room with 12/12 light dark room with relative humidity (55% ± 10%). The mice were provided with chow pellet and water ad libitum. All the animals were housed in a well-controlled animal facility in accordance with the guideline for the care and use of laboratory animals with the prior approval of the Institute Animal Ethics Committee (CAF/Ethics/194/2010). Mice were grouped into control and LPS-treated group (n = 15 in each group). The animals were kept in a nebulization chamber made of plexiglass (20 cm × 20 cm × 30 cm) as described by Corbel et al. 2002 with slight modifications. The nebulization unit (Aeroneb Lab Nebulizer Unit, Small VMD, Kent Scientific, USA) was attached to the plexiglass chamber. LPS (1 mg/ml) dissolved in 0.9% NaCl, (Lipopolysaccharides from Escherichia More Details coli 055:B5) was nebulized for 45 min, once a week, for 8 weeks to induce chronic inflammation in lungs.
Collection of bronchoalveolar lavage fluid and flow cytometry analysis
The mice were slightly sedated with anesthetic ether followed by cervical dislocation. The mice were sterilized with 70% ethanol to clean the hairs on their skin. The skin was carefully excised to expose the trachea. Slight incision was made on the trachea to insert the canula. The lung was lavaged four times with 0.2 ml of PBS. The lavage fluid was collected and centrifuged at 400 g for 10 minutes at 4°C to obtain pellets. The lavage fluid was collected and centrifuged to obtain pellets. 1 × 107 cells were taken and labeled with anti-mouse CD3e FITC, anti-mouse CD45R PE-B220, and anti-mouse Ly-6G (Gr-1) PE-Cy7 to detect T-cell, B-cells, and neutrophils, respectively, as per the manufacturer's instructions (eBioscience, Inc. San Diego, USA). The samples were analyzed in a flow cytometer (BD FACS Canto™ II, BD Biosciences, USA) and 10,000 events were acquired. The data were analyzed by FACSDiva Software (Version 6.1.3). The respective cell type was quantified based on the respective antibody binding and percent was determined out of total 10,000 events acquired. The data were analyzed by FACSDiva Software (Version 6.1.3). Each cell type was quantified and expressed as percentage and comparisons were made between control and LPS-treated group.
Inflammatory cytokine analysis
At the end of the treatment period, mice were euthanized and lung from each mouse was collected. Each lung was homogenized individually in lysis buffer, in the presence of protease inhibitors cocktail (Sigma Aldrich, USA). These lysates were then centrifuged at 2500 g for 10 min at 4°C and the supernatant was collected. These supernatants were used to determine the level of various pro-inflammatory and anti-inflammatory cytokines by Cytometry Bead Array, (Mouse inflammation CBA Kit, Becton Dickinson, San Jose, CA, USA) by flow cytometry (BD FACS Canto™ II) as per the manufacturer's instructions.
Histological analysis of lung
After euthanizing the mice, lungs were instilled intratracheally with 15 ml of 10% neutral buffered formalin and were collected and stored in 10% neutral buffered formalin at 4°C till further use. Then, the lung tissues were dehydrated, embedded in paraffin, and 5 μm sections were cut using microtome (Leica, Germany) and were stained with hematoxylin and eosin staining. The histological sections were studied under the microscope.
Estimation of gas6 protein by enzyme-linked immunosorbent assay
The level of gas6 protein from the lung homogenate was measured using Quantikine enzyme-linked immunosorbent assay (ELISA) kit as per the manufacturer's instructions (R and D Systems, Inc, Minneapolis, USA). Briefly, 50 μl of RD1-43 assay diluent was added to each well of the microplate followed by 50 μl of standard, control, or sample according to the protocol. These constituents were mixed by gentle tapping of the frame for a minute and the plate was incubated for 3 h at 2–8°C. Then, wells were aspirated and washed five times with the wash buffer. The plate was dried by tapping it against clean paper towels. The microplate was incubated for an hour at 2–8°C with mouse gas6 conjugate. Then, the wells were again washed with wash buffer for five times and were dried by tapping against paper towels. Then, 100 μl of substrate solution was added to each well and was incubated for 30 min at room temperature in the dark. Then, again 100 μl of stop solution was added to the wells. It was mixed by tapping the frame of the plate gently, and the absorbance of the wells was taken using a 96 well plate reader (Infinite® 200Pro, Tecan, Switzerland) at 450 nm with correction wavelength of 540/570 nm within 30 min of adding stop solution.
Protein gel and Western blotting
Mice lung tissues were homogenized using glass tissue homogenizer (Cole Parmer, UK) in the lysis buffer. It was centrifuged at 2500 g for 10 min at 4°C to collect the supernatant and protein was estimated by Bradford reagent. Proteins (100 μg) in each well were separated on 10% PAGE and were transferred to PVDF membrane. Gas6 on the membrane were visualized using anti-mouse gas6 IgG (Anti-GAS6 antibody [RM0084-6J2] Abcam, Cambridge, USA).
All the values have been expressed as the means ± SEM. All statistical analyses were performed with a one-way ANOVA, followed by a Tukey post hoc test (P < 0.05 was considered significant). Microcal software version 6.0 was used for data analysis (Northampton, USA).
| Results|| |
Flow cytometry analysis of bronchoalveolar lavage fluid
Bronchoalveolar lavage (BAL) fluid was collected from the control and LPS-treated animals and analyzed to determine the level of T-cells, B-cells, and neutrophils. Significant increase in the level of T-cells was observed in the BAL fluid obtained from LPS-nebulized animals, 65±11% in LPS vs 19±8% in control (P < 0.01) [Figure 1]a, [Figure 1]b, [Figure 1]c. We also assessed the level of B-cells in BAL fluid. We also observed that B-cell numbers were significantly higher in LPS-nebulized mice in comparison to controls (64±10% in LPS vs 24±7% in control, P < 0.01) [Figure 1]d, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f. Further, there was significant increase in neutrophil in LPS-nebulized mice in comparison to controls; 57±10% in LPS vs 22±9% in control, (P < 0.05) [Figure 2]a, [Figure 2]b, [Figure 2]c. Significant increase in the percentage of T-cells, B-cells, and of neutrophils in the LPS-treated group suggests the onset of inflammation in lung milieu.
|Figure 1: Infiltration of various B-cells and T-cells to the lungs following LPS exposure by flow cytometer. (a) Cells alone control, (b) B-cells in lungs of control group, (c) B-cells in lungs LPS-treated group. (d) Cells alone control, (e) T-cells in lungs of control group, (f) T-cells in lungs of LPS-treated group|
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|Figure 2: Dot plots of neutrophil infiltration into lungs following long-term LPS exposure (a) cell alone (b) control group (c) LPS-treated group|
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Analysis of cytokine profile by flow cytometry
The lung homogenates of the mice were analyzed for the level of various pro-inflammatory and anti-inflammatory cytokines in the lung homogenate by flow cytometry. There was significant increase in the level of tumor necrosis factor (TNF) (200 ± 17 pg/ml in control group vs. 1222 ± 152 pg/ml in LPS-treated group, P < 0.01) and IL-6 (106 ± 13 pg/ml in control group vs. 448 ± 122 pg/ml in LPS-treated group, P < 0.05) in LPS-nebulized animals as compared to control animals [Figure 3]. However, no significant increase was observed for INF-γ (1.6±0.2 pg/ml in control group vs 3.2±3.2 pg/ml in LPS treated group, P = 0.63), IL-10 (7.7±2.3 pg/ml in control group vs 1.6±2.8 pg/ml in LPS treated group, P=0.16), MCP-1 (306±15 pg/ml in control group vs 212±36 pg/ml in LPS treated group, P = 0.07) and IL-12p70 (2.6±2.7 pg/ml in control group vs 4±4.8pg/ml in LPS treated group, P = 0.08) cytokines in LPS treated group as compared to control.
|Figure 3: The level of various pro- and anti-inflammatory cytokines following long-term nebulization of LPS (for *: P <0.05; for **: P <0.01) (n ≥ 4 from 3 animals from each group)|
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Histological analysis of lungs
Normal architecture was seen in the lung sections from the control animals [Figure 4]a. In case of LPS-nebulized groups, edematous fluid was observed throughout the lung tissue, lung architecture was swollen slightly and vacuolar degeneration was observed throughout the tissue [Figure 4]b. This observation confirmed the lung deterioration following long-term LPS exposure as observed in COPD.
|Figure 4: The lung histology of control (a) and long-term LPS-treated (b) mice|
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Analysis of gas6 protein in lung homogenate
Lung homogenates were assessed for the level of gas6 protein by ELISA. The gas6 protein in the lung homogenate of control group was 0.16 ± 0.1 ng/ml, whereas it was 4.2 ± 0.1 ng/ml in the LPS-treated group suggesting a significant increase in long-term LPS-exposed mice (n = 3, P < 0.01) [Figure 5]. This suggests that LPS-induced inflammation increases the level of gas6 protein in the lung.
|Figure 5: Effect on gas6 protein level in lung tissues homogenate following long-term LPS exposure. (n ≥ 4 for each group from 3 animals from each group)|
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Protein gel and Western blotting
Further, we assessed the level of gas6 protein in lung homogenate by Western blotting. We observed signifi cant increase in the level of gas6 protein in the lung homogenate of LPS-exposed mice [Figure 6]. However, no increase was observed in tubulin protein. Densitometric analyses were performed using ImageJ software.
|Figure 6: Expression level of gas6 protein in control and long term LPS treated animals. Tubulin was used as a loading control. (a) representative blot (b) densitometric analysis and (c) fold change. (n ≥ 4 for each group from 3 animals from each group, for *:P < 0.05)|
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| Discussion|| |
This is the first report on the involvement of gas6 protein in inflammatory respiratory diseases like COPD. Chronic inflammatory lung disease is characterized by infiltration of various inflammatory cells such as eosinophils, mast cells, neutrophils, and lymphocytes into the airways. On LPS exposure to lungs, inflammatory cells such as macrophages, neutrophils, and lymphocytes migrate to the lungs. Neutrophils constitute the major percentage of inflammatory cells in COPD., Therefore, We wanted to evaluate the level or percentage of various immune cells in the bronchoalveolar fluid of mice following long-term exposure of LPS. Lymphocytes release various anti- and pro-inflammatory cytokines. There is increased infiltration of inflammatory cells such as eosinophils, neutrophils, mast cells, and lymphocytes during chronic lung inflammations. Further, there is increased release of pro-inflammatory cytokines. LPS-induced lung inflammation simulates the inflammatory responses in lung disease such as COPD and asthma.,,
Various studies show that CD8+ T-cells plays important role in airway remodeling in the pathogenesis of COPD. It has been reported that there is an increase in the level of CD8+ T-cells in smokers with symptoms of chronic bronchitis and chronic airflow limitation as compared to normal and healthy subjects. CD8+ T-cells required for the inflammation in the lungs of cigarette smoke-induced emphysema in mice. Infiltration of neutrophils to lungs following long-term LPS exposure leads to release of neutrophils elastase which leads to degradation of lungs. The significant increase in the level of B-cells, T-cells, and neutrophils confirms the generation of chronic inflammation initiated by long-term LPS exposure.,
TNF-alpha is stored in the storage granules of mast cells and is released from mast cells and secreted from macrophages during inflammatory conditions. TNF-alpha is the early pro-inflammatory cytokines and triggers the activation of other cytokines such as IL-6 and IL-8. In COPD, there is gradual decrease in the lung function and it leads to gradual destruction of lung parenchyma. Further, our observations corroborate the increase in the level of inflammatory cytokines following long-term LPS exposure. However, it is not clear whether the increase is a protective mechanism in response to inflammation.
Further, during the pathogenesis of COPD, the inflammatory reaction is characterized by increased infiltration of neutrophils into the BAL. However, it is not known whether the increased expression of gas6 protein is due to increased infiltration of neutrophils. Further, there are increased pro-inflammatory mediators, such as IL-8 and TNF-α. Further, it is not clear whether the increased buildup of pro-inflammatory cytokines in lungs milieu leads to increased infiltration of neutrophils. Further, it is not known whether neutrophils contain any gas6 protein. It has been reported that gas6 attenuates neutrophil migration and lung injury during sepsis. Therefore, the increased expression of gas6 protein in the lung may be a protective mechanism to attenuate neutrophil infiltration to lungs. Furthermore, toll-like receptor (TLR) activation suppresses the expression of Gas6, which facilitates TLR-mediated inflammatory responses. It seems that LPS-induced lung inflammation increases the expression of gas6 protein which in turn attenuates neutrophil infiltration to lungs, decreases TNF alpha, and IL-6 expression in mice. Increased expression of gas6 seems to be a protective mechanism against inflammation and injury as in neuronal inflammation.,
Other aspects of the mechanism of action of gas6 protein may be through modulation of TLR-dependent inflammasome signaling. We have shown that there is an increased expression of inflammatory cytokines during the chronic inflammations which may activate inflammasome complex. It also seems that gas6 plays role in immune dysregulation. It has been observed that Gas6/Axl pathway is activated in chronic liver diseases associated with inflammation. Further, induction of gas6 protein has been observed CCl4-induced liver injury. Understanding the signaling associated with gas6 and its receptors may help in preventing lung inflammation and COPD. Recently, it has been reported that upregulation of gas6 in cerebrospinal fluid (CSF) may be part of a defensive response aimed at counteracting AD progression., This confirms that the increase in gas6 protein during long-term LPS administration is a protective mechanism of lungs against inflammatory injury. Gas6 and its receptors have been implicated in various types of cancer. Tyro-3, Axl, and Mer and have been detected in various types of cancer., Further gas6 binding to these receptors promotes proliferation and survival of cancer cells in vitro.
Further, gas6 protein has been shown to be involved in immune modulation in vitro and in vivo. Some of these effects are probably mediated through the involvement of monocytes/macrophages. Alciato et al. (2010) have demonstrated that gas6 inhibits TNF-α and IL-6 secretion in LPS-stimulated U937 cells and monocytes/macrophages. Further, Mer activation leads to increased Akt phosphorylation in monocytes and macrophages is responsible for the reduction of cytokine expression. They have shown that GSK3 β phosphorylation and consequent inhibition of nuclear factor (NF)-κB nuclear translocation on gas6 stimulation. Therefore, GAS6 modulates macrophage cytokine secretion, triggering an “anti-inflammatory pathway” involving PI3K/Akt/GSK3 β and NF-κB. Further, it has been shown that exogenous gas6 attenuate inflammations induced by silica on macrophages and gas6 significantly suppressed silica-induced TNF-α, IL-1 β, and IL-6 levels in macrophages.
| Conclusion|| |
We have shown for the first time the involvement of gas6 protein mouse model of lipopolysaccharide-induced COPD. However, further investigation will be carried out in the future to understand the signaling pathways involved in increased expression of gas6 in COPD. It may pave the way for the development of therapeutics targeting gas6 signaling in COPD.
The authors acknowledge the invaluable support from members of the Central Animal Facility and FACS Facility during the study.
Financial support and sponsorship
Financial support to William R Surin by a grant from Science and Engineering Research Board, Govt. of India (SR/FT/LS-182/2009) is gratefully acknowledged.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Prieto AL, Weber JL, Tracy S, Heeb MJ, Lai C. Gas6, a ligand for the receptor protein-tyrosine kinase tyro-3, is widely expressed in the central nervous system. Brain Res 1999;816:646-61.
Manfioletti G, Brancolini C, Avanzi G, Schneider C. The protein encoded by a growth arrest-specific gene (gas6) is a new member of the Vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol 1993;13:4976-85.
Ohashi K, Honda S, Ichinomiya N, Nakamura T, Mizuno K. Molecular cloning and in situ
localization in the brain of rat sky receptor tyrosine kinase. J Biochem 1995;117:1267-75.
Gould WR, Baxi SM, Schroeder R, Peng YW, Leadley RJ, Peterson JT, et al.
Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic responses. J Thromb Haemost 2005;3:733-41.
Angelillo-Scherrer A, de Frutos P, Aparicio C, Melis E, Savi P, Lupu F, et al.
Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis. Nat Med 2001;7:215-21.
Lutgens E, Tjwa M, Garcia de Frutos P, Wijnands E, Beckers L, Dahlbäck B, et al.
Genetic loss of gas6 induces plaque stability in experimental atherosclerosis. J Pathol 2008;216:55-63.
Shibata T, Ismailoglu UB, Kittan NA, Moreira AP, Coelho AL, Chupp GL, et al.
Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice. Am J Respir Cell Mol Biol 2014;51:615-25.
Fabbri L, Pauwels RA, Hurd SS; GOLD Scientific Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary updated 2003. COPD 2004;1:105-41.
Barnes PJ. Cellular and molecular mechanisms of chronic obstructive pulmonary disease. Clin Chest Med 2014;35:71-86.
Wu KS, Hung YJ, Lee CH, Hsiao FC, Hsieh PS. The involvement of GAS6 signaling in the development of obesity and associated inflammation. Int J Endocrinol 2015;2015:202513.
Gruber RC, Ray AK, Johndrow CT, Guzik H, Burek D, de Frutos PG, et al.
Targeted GAS6 delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis. J Neurosci 2014;34:16320-35.
Corbel M, Germain N, Lanchou J, Molet S, R e Silva PM, Martins MA, et al.
The selective phosphodiesterase 4 inhibitor RP 73-401 reduced matrix metalloproteinase 9 activity and transforming growth factor-beta release during acute lung injury in mice: The role of the balance between tumor necrosis factor-alpha and interleukin-10. J Pharmacol Exp Ther 2002;301:258-65.
Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153:530-4.
Ronchi MC, Piragino C, Rosi E, Amendola M, Duranti R, Scano G, et al.
Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD. Thorax 1996;51:1000-4.
Schuster M, Tschernig T, Krug N, Pabst R. Lymphocytes migrate from the blood into the bronchoalveolar lavage and lung parenchyma in the asthma model of the brown Norway rat. Am J Respir Crit Care Med 2000;161:558-66.
Barnes PJ, Shapiro SD, Pauwels RA. Chronic obstructive pulmonary disease: Molecular and cellular mechanisms. Eur Respir J 2003;22:672-88.
Gharaee-Kermani M, Ullenbruch M, Phan SH. Animal models of pulmonary fibrosis. Methods Mol Med 2005;117:251-9.
Hsueh W, Sun X, Rioja LN, Gonzalez-Crussi F. The role of the complement system in shock and tissue injury induced by tumour necrosis factor and endotoxin. Immunology 1990;70:309-14.
Puljic R, Benediktus E, Plater-Zyberk C, Baeuerle PA, Szelenyi S, Brune K, et al.
Lipopolysaccharide-induced lung inflammation is inhibited by neutralization of GM-CSF. Eur J Pharmacol 2007;557:230-5.
Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, et al.
CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;157:822-6.
Nadel JA. Role of neutrophil elastase in hypersecretion during COPD exacerbations, and proposed therapies. Chest 2000;117:386S-9S.
Lapperre TS, Postma DS, Gosman MM, Snoeck-Stroband JB, ten Hacken NH, Hiemstra PS, et al.
Relation between duration of smoking cessation and bronchial inflammation in COPD. Thora×2006;61:115-21.
Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, et al.
The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004;350:2645-53.
Ohkawara Y, Yamauchi K, Tanno Y, Tamura G, Ohtani H, Nagura H, et al.
Human lung mast cells and pulmonary macrophages produce tumor necrosis factor-alpha in sensitized lung tissue after IgE receptor triggering. Am J Respir Cell Mol Biol 1992;7:385-92.
Drost EM, MacNee W. Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: Effects on neutrophil deformability, adhesion receptor expression, and chemotaxis. Eur J Immunol 2002;32:393-403.
Giangola MD, Yang WL, Rajayer SR, Nicastro J, Coppa GF, Wang P, et al.
Growth arrest-specific protein 6 attenuates neutrophil migration and acute lung injury in sepsis. Shock 2013;40:485-91.
Feng X, Deng T, Zhang Y, Su S, Wei C, Han D, et al.
Lipopolysaccharide inhibits macrophage phagocytosis of apoptotic neutrophils by regulating the production of tumour necrosis factor α and growth arrest-specific gene 6. Immunology 2011;132:287-95.
Sainaghi PP, Collimedaglia L, Alciato F, Molinari R, Sola D, Ranza E, et al.
Growth arrest specific gene 6 protein concentration in cerebrospinal fluid correlates with relapse severity in multiple sclerosis. Mediators Inflamm 2013;2013:406483.
Sainaghi PP, Bellan M, Lombino F, Alciato F, Carecchio M, Galimberti D, et al.
Growth arrest specific 6 concentration is increased in the cerebrospinal fluid of patients with Alzheimer's disease. J Alzheimers Dis 2017;55:59-65.
Bárcena C, Stefanovic M, Tutusaus A, Joannas L, Menéndez A, García-Ruiz C, et al.
Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation. J Hepatol 2015;63:670-8.
Dransfield I, Farnworth S. Axl and Mer receptor tyrosine kinases: Distinct and nonoverlapping roles in inflammation and cancer? Adv Exp Med Biol 2016;930:113-32.
Schmitz R, Valls AF, Yerbes R, von Richter S, Kahlert C, Loges S, et al.
TAM receptors tyro3 and Mer as novel targets in colorectal cancer. Oncotarget 2016;7:56355-70.
Shiozawa Y, Pedersen EA, Patel LR, Ziegler AM, Havens AM, Jung Y, et al.
GAS6/AXL axis regulates prostate cancer invasion, proliferation, and survival in the bone marrow niche. Neoplasia 2010;12:116-27.
Alciato F, Sainaghi PP, Sola D, Castello L, Avanzi GC. TNF-alpha, IL-6, and IL-1 expression is inhibited by GAS6 in monocytes/macrophages. J Leukoc Biol 2010;87:869-75.
Shen Y, Cui X, Rong Y, Zhang Z, Xiao L, Zhou T, et al.
Exogenous gas6 attenuates silica-induced inflammation on differentiated THP-1 macrophages. Environ Toxicol Pharmacol 2016;45:222-6.
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