In response to infection, “frontline” leukocytes migrate to neutralize and eliminate potentially injurious stimuli. Activated neutrophils, with their lifespan extended by various factors,1,2 accumulate in lung tissue to neutralize and eliminate potentially injurious stimuli in patients with acute lung injury (ALI).3,4 However, the activated neutrophils per se also damage the surrounding normal tissues and prolong the inflammatory response by releasing large quantities of superoxide and chemical mediators. A number of clinical conditions—such as acute respiratory distress syndrome (ARDS), septicemia with multiorgan failure, ischemia/reperfusion injury, and rheumatoid arthritis—have all been linked to inappropriate neutrophil-mediated tissue damage.5 Timely resolution of inflammation is important to maintain the homeostasis of the tissues. Proresolution strategies are followed with great interest in the treatment of inflammation. Apoptosis of neutrophils and their subsequent ingestion by phagocytes6 have been reported to be key processes in the inflammation resolution.7,8
Endothelial cells are strategically positioned to regulate neutrophil accumulation in inflamed tissues. Activated endothelial cells help recruit neutrophils through the release of chemoattractants and by expressing leukocyte adhesion receptors, such as selectins and immunoglobulin G superfamily members on their surface. Activated endothelial cells have also been implicated in the delay of apoptosis of neutrophils,9 and thus potentially increase the number of leukocytes in inflammatory tissues.10
Angiopoietin1 (Ang1) acts on the tyrosine kinase with immunoglobulin-like loop and epidermal growth factor homology domains 2 (Tie2) receptor, found primarily on endothelial cells. Ang1 pretreatment protects the endothelium from excessive activation,11 prevents plasma leakage induced by vascular endothelial growth factor, and strongly supports the anti-inflammatory effect in ALI.12 However, Ang1 is down-regulated in ALI. Previous studies focused primarily on the role of Ang1 at the initiation stage. It is not known whether Ang1 impacts resolution of inflammation.
The aim of this study was to evaluate the impact of Ang1 pretreatment on resolution of inflammation in endotoxin-induced ALI mice. To effectively increase the level of Ang1, we administered IV adenoviral-GFP-Ang1 (Ad-GFP-Ang1) into the tail vein. Our results indicate that Ang1 overexpression promotes the resolution of inflammation of ALI through accelerating the apoptosis of neutrophils and its phagocytosis by macrophage cells.
Recombinant adenoviral expressing human Ang1 was constructed with the Admax Vector System (Microbix Biosystems, Mississauga, Ontario, Canada) from Genchem Biotechnology Inc. (Shanghai, China). Full-length cDNAs encoding human Ang1 were cloned into shuttle vector AdCMV5GFP. Viral titer was 1.5 × 1012 plaque-forming unit (PFU) per milliliter using the Spearman–Karber method.
Epithelial Cell Culture and Human Ang1 Expression in Vitro
The human epithelial lung cell line A549 was purchased from the American Type Culture Collection (Rockville, MD) and was grown in RPMI-1640 supplemented with 10% fetal calf serum and antibiotics. Cell viability was systematically evaluated before and after each treatment, and mortality never exceeded 5%.
A549 cells were infected with either Ad-GFP or Ad-GFP-Ang1 at a multiplicity of infection of 10. and 0.1 mL supernatant was collected at 24, 48, 72, and 96 hours after transfection to analyze the Ang1 protein with the Ang1 ELISA kit (R&D, Minneapolis, MN).
Mouse Model of ALI
Pathogen-free male BALB/c mice weighing 20 to 25 g were used (purchased from the Laboratory Animal section, Tongji Medical College, Wuhan, China). The mice were fed a standard diet and water ad libitum. This experiment was conducted according to the guidelines of animal study in China, and the study protocol was approved by the Laboratory Study Review Board of Tongji Medical College, Huazhong University of Science and Technology.
ALI was induced by intratracheal instillation of lipopolysaccharide (LPS, Escherichia coli serotype O55:B5; Alpha Chemical and Plastics Co., Hollister, MO) at a dose of 3 mg · kg−1 according to methods previously described,13 immediately followed by 3 insufflations of 0.6 mL of air and by rotation of the animals to attempt to homogeneously distribute LPS in the lungs.
Eighty-four mice were randomly assigned to either the GFP group (n = 42) or Ang1 group (n = 42). To ensure optimal gene delivery, we administered 109 pfu of adenovirus-GFP vector (GFP group) or adenovirus-GFP-Ang1 in 100-μL solutions (Ang1 group) into the tail vein. After 24 hours, LPS was intratracheally instilled at a dose of 3 mg · kg−1 to evoke ALI. Six mice were killed for sample collection at each indicated time point (4, 12, 24, 48, 72, and 96 hours after LPS instillation). An additional 6 mice in each group were killed before LPS instillation as control (0-hour point).
Blood was collected via the lateral canthus approach before the mice were killed. Bronchoalveolar lavage (BAL) was performed 3 times with 0.8 mL sterile saline each time. Cells in BAL fluid (BALF) were obtained for analyses described below. Total protein in cell-free BALF was assayed as an index of lung injury and capillary leakage. Protein quantification was accomplished with the BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL). The right lower lobe of the lungs was fixed in buffered formalin and processed for histologic analysis. The left lobes of lungs and the right livers were removed and flash frozen for further analysis.
Differential Leukocyte Counts and Fluorescence-Activated Cell-Sorting (FACS) Analysis
Cells in BALF were removed by centrifugation (400 g, 4°C, 6 minutes). The cells were counted with a hemocytometer14 and differentiated after centrifugation for 6 minutes at 400 g by Wright's staining on a glass slide. For determining polymorphonuclear leukocyte (PMN) apoptosis, cells were labeled with APC-conjugated antiannexin-V Ab (5 μg Ab/106 cells; Bender MedSystems, Inc., Vienna, Austria) and PE-conjugated antimouse Gr-1 (Ly-6G) Ab (0.03 μg/106 cells; eBioscience, Inc., San Diego, CA) for 20 minutes. The annexin-V+Gr-1+ PMN population was determined by FACS.
To determine macrophage phagocytosis of apoptotic PMN, we blocked cells with antimouse CD16/32 blocking Ab (0.5 μg/106 cells; eBioscience, Inc., San Diego, CA) for 5 minutes, stained with APC-conjugated antimouse F4/80 (2 μg/106 cells; eBioscience, Inc., San Diego, CA) for 20 minutes, and then fixed and made permeable with the reagent combination Fix & Perm Kit (An der Grub, Vienna, Austria) according to the manufacturer's recommendations. The permeable cells were then stained with PE-conjugated antimouse Ly-6G (0.03 μg/106 cells; eBioscience, Inc., San Diego, CA). The F4/80+ Gr-1+ cell population was determined by FACS.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from lungs and livers using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed (Qiagen, Tokyo, Japan). Quantitative polymerase chain reaction (PCR) was performed using SYBR GreenI PCR Master Mix and the ABI PRISM 7900HT (Applied Biosystems, Inc., Foster City, CA). The data of the mRNA expression are reported as fold change.
The PCR primer sequences for human Ang1 (GenBank Accession, NM_001146) and β-actin (GenBank Accession, AK088691) as a housekeeping gene were designed using Primer 5.0 software. The forward primers are 5′-AAGGGAACCGAGCCTATTCACAG-3′, 5′-CCGTAAAGACCTCTATGCCAACA-3′; the reverse primers are 5′-GCATCAAACCACCATCCTCCTG-3′, and 5′-CTGCTGGAAGGTGGACAGTGAG-3′, respectively, for human Ang-1 and β-actin. The size of the fragment amplified was 211 bp from human Ang1c DNA and 196 bp from β-actin cDNA.
Western Blot and ELISA for Protein Analysis
For Western blot analysis, lung and liver tissues were homogenized in the presence of protease inhibitors to obtain extracts of lung proteins. Protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA). Samples (50-μg proteins) were mixed with sample buffer, separated by 10% SDS-PAGE and electroblotted to a nitrocellulose membrane. The membrane was blocked for 1 hour at room temperature with blocking solution (5% nonfat milk in Tris buffered saline with Tween). Blots were then incubated overnight at 4°C with primary anti-Ang1 antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with a horseradish peroxidase conjugated secondary antibody for 1 hour at room temperature. Immunoreactive proteins were visualized using enhanced chemoluminescence with ECL reagent (Amersham Biosciences, Piscataway, NJ) treatment and exposed to radiograph film.
The mouse granulocyte macrophage colony-stimulating factor (GMCSF) in BALF and Ang1 levels in serum were respectively assayed following the manufacturer's instructions of GMCSF ELISA kit (Bender MedSystems, Inc., Vienna, Austria) and Ang1 ELISA kit (R&D, Minneapolis, MN).
Results are expressed as mean ± SD. Data were analyzed using the SPSS 13 software (SPSS, Inc., Chicago, IL). Results grouped by time point and pretreatment method were analyzed using a 2-way analysis of variance (ANOVA). Values that were considered significantly different from each other by ANOVA were further analyzed using a post hoc Tukey t test. Data having only 2 groups were analyzed using a Student t test. The criterion for significant differences was set at P < 0.05 for all studies.
Expression of Ang1 in Vitro and in Vivo
Ang1 was successfully detected in the medium of the cells infected by Ad-Ang1 but not in medium of cells infected with Ad-GFP (Fig. 1). As with other adenoviral vectors,15 IV administration of Ad-Ang1 led to specific gene uptake and expression in the liver (Fig. 2, A and B). Meanwhile, the Ang1 level in the serum reduced to a minimum at 48 hours after LPS exposure and then slowly returned in the GFP group. Pretreatment with Ad-Ang1 increased the angiopoietin1 protein in the serum in comparison with that in the GFP group of the same time point (Fig. 2C).
Effect of Ang1 on LPS-Induced ALI
In comparison with the lung tissues just pretreated with Ad-GFP (Fig. 3A) or Ad-Ang1 (Fig. 3B), lung tissues of mice after endotoxin administration were associated with alveolar interstitial thickening, alveolar collapse, and cellular infiltration in both the GFP group (Fig. 3C) and the Ang1 group (Fig. 3D). The morphologic changes were less pronounced after LPS challenge when pretreated with Ad- Ang1 in comparison with that pretreated with Ad-GFP.
Although total protein in BALF was increased in mice after exposure to LPS in both the GFP group and the Ang1 group, the total protein in BALF in animals receiving Ang1 pretreatment was significantly lower than that in mice receiving GFP pretreatment (P = 0.001, Fig. 4).
Ang1 Overexpression Promoted Resolution of Inflammation
BALF recovery (1.92 ± 0.11 mL) was similar between groups. Cells in BALF were collected during a 96-hour period. Our results showed that the number of leukocytes and the PMN continuously increased and plateaued at 48 hours and then gradually receded from the tissues after LPS challenge in both the GFP group and the Ang1 group. However, the infiltration of leukocytes and PMN in the Ang1 group was markedly inhibited in comparison with the GFP group (P = 0.001, Fig. 5A).
The time course of PMN followed a similar trend, peaking at 48 hours after LPS challenge in both the GFP group (Fig. 5B) and the Ang1 group (Fig. 5C). From 48 hours (Tmax) to 77 hours (T50), the number of PMNs in the BALF decreased from 12.87 × 106 (Ψmax; maximal PMN number) to 6.44 × 106 (R50; essentially 50% reduction of PMN) in the GFP group. This period of neutrophilic loss is termed the resolution interval (Ri).16 In mice pretreated with Ad-GFP-Ang1, Ri was 17 hours (i.e., 48 to 65 hours). It was apparent that Ang1 pretreatment significantly decreased Ψmax and shortened the Ri from 29 hours to 18 hours when the resolution indices were calculated (Table 1).
Ang1 Overexpression Accelerated PMN Apoptosis and Their Removal by Macrophages
Cells in BALF collected at 48 hours and 96 hours after LPS instillation were labeled with APC-annexin-V and PE-conjugated anti-Gr-1 Ab, a specific cell surface marker for mouse PMN. The apoptotic ratio at 48 and 96 hours after LPS exposure was low. It was lower at 96 hours than at 48 hours in both the GFP group and the Ang1 group. Ang1 pretreatment significantly increased annexin-V+ Gr-1+ cells (1.45 ± 0.06%, n = 6) by 131% at 48 hours and by 430% at 96 hours (0.69 ± 0.10%, n = 6) in comparison with that in the GFP group (P < 0.001, Fig. 6A).
We next determined whether Ang1 impacts macrophage ingestion of PMNs. We performed a phagocytosis-based analysis. Macrophages collected at 48 hours and 96 hours were labeled with the APC-conjugated anti-F4/80 Ab. This was followed by permeabilization, allowing labeling of ingested PMN with PE-conjugated anti-Gr-1 Ab. Cells with positive staining of both F4/80 and Gr-1 were then monitored by FACS analysis.
Cells collected from mice in the Ang1 group at 48 hours after LPS challenge showed significantly increased F4/80+ Gr-1+ cells (11.41 ± 2.56%, n = 6) when compared with those pretreated with Ad-GFP (2.54 ± 0.17%, n = 6) (P = 0.023, Fig. 6 B). Of interest, phagocytosis increased to 42.6 ± 2.86%, n = 6, of the Ang1 group and 20.62 ± 9.84%, n = 6, of the GFP group at the 96-hour time point. The difference between the 2 groups was significant (P = 0.003, Fig. 6B).
GMCSF Levels in BALF
The GMCSF in BALF of the Ang1 group reduced from 71.26 ± 8.30 pg · mL−1 before LPS challenge to 4.97 ± 1.75 pg · mL−1 at 48 hours after LPS instillation, and then gradually increased to 27.50 ± 2.25 pg · mL−1 at 96 hours after LPS challenge. This was significantly higher than the GFP group analyzed with 2-way ANOVA (n = 6 for each time point, P = 0.001, Fig. 7).
A systems approach to mapping the resolution of acute inflammation demonstrated that resolution is an active process17 as well as a new terrain of cellular and molecular processes directed toward returning the tissue to homeostasis.16,18 The consistent association between PMNs and lung injury in humans and animal models, and the propensity of PMNs and their products to cause tissue injury in experimental systems, led to the conclusion that PMNs have an important causative role in ALI.19 A study of the metabolic activity of lung PMNs showed that the most metabolically active PMNs are in the alveolar spaces, and not in the microvasculature or the interstitium, suggesting that the major activation events occur in the alveolar spaces in response to bacteria.20 Using this differential-temporal and quantitative systems approach to analyze the resolution of inflammation in ALI, we identified, for the first time, that Ang1 pretreatment promotes resolution of inflammation through accelerating the apoptosis of neutrophils and its phagocytosis by macrophage cells. These data underscore the protective role of Ang1 in terms of the resolution of inflammation in ALI.
We used a validated mouse model 13 that exhibits some physiological and biological similarities with human ALI.14,21 The model was characterized by a complex sequence of events, including organ-specific leukocyte recruitment, production of proinflammatory cytokines, and end-organ injury or death.22 In our mode of ALI, inflammatory cells migrated into the lung tissues associated with alveolar interstitial thickening, and alveolar collapse. Meanwhile, the expression of Ang1 reduced significantly after the LPS challenge. The gene therapy method was used to effectively increase the serum Ang1 because it overcomes the issue of short protein half-time when recombinant Ang1 is injected IV. Our results showed that the Ad-Ang1 pretreatment increased the expression of Ang1 in vitro and in vivo. Huang et al.11 found that the exogenous Ang1 produced by Ad-Ang1 could specifically bind to the TIE2 receptor as the endogenous Ang1 protein. Additionally, our results confirmed that IV administration of Ad-Ang1 to the mice led to specific uptake and expression of Ang1 in the liver and increased the level of Ang1 protein in serum.12
The Ang1 pretreatment in this study reduced the protein exudation in BALF and supports the protection of Ang1 on the endothelial/epithelial cells from injury. Ang1 interacts with the endothelial cell-specific tyrosine kinase receptor Tie-2 to remodel primitive vessels and stabilize mature vessels.23 It has been reported that the endothelial monolayer plays a critical role in many aspects of the pathogenesis of ALI and ARDS.24 Endothelial activation/injury with subsequent loss of integrity is a prerequisite for development of interstitial edema and accumulation of inflammatory cells.25,26 Although the activation of endothelial cells in inflammation facilitates the infiltration of leukocytes into the inflammatory site to promote the clearance of local pathogenic microorganisms,27,28 the activation of endothelial cells delays the apoptosis of the leukocytes in the inflammatory tissues.9,29
Our results also demonstrate the anti-inflammatory and proresolution effects of Ang1. PMN apoptosis and their subsequent removal by macrophages are essential components of resolution of inflammation.30 To quantitatively analyze the effect of Ang1 on the resolution of inflammation, we used the resolution indices introduced by Bannenberg et al.16 These indices included the following: Ψmax, the maximal neutrophil numbers that are presented during the inflammation response; Tmax, the time when Ψmax occurs; and the resolution interval (Ri), the time interval from Tmax to T50 when neutrophil numbers reach half Ψmax. Quantitative analysis of the resolution of inflammation showed that pretreatment with Ad-Ang1 reduced Ψmax and shortened the resolution interval (Ri).
Neutrophils undergoing apoptosis are vital mechanisms for the resolution of inflammation and immune system homeostasis. In this study, we found that the apoptosis of neutrophils was low at both the 48-hour and 96-hour time points after LPS instillation. The phagocytosis of apoptotic PMNs by macrophages played an important role in this phenomenon, because the phagocytosis ratio at the 96-hour time point was significantly higher than that at the 48-hour time point. Spontaneous neutrophil apoptosis may be regulated by many proteins, molecules, or both, including LPS, leukotriene B4, interleukin-8, and GMCSF.31 An in vitro experiment has confirmed that delayed neutrophil apoptosis by activated endothelium is in part attributed to the GMCSF,10 which acts by stimulating PI 3-kinase and extracellular signal-regulated kinase-dependent pathways.32 It has also been proven that normal human PMN were significantly less apoptotic when incubated in BALF of ARDS patients with this phenomenon partially blocked by immunodepletion of GCSF and GMCSF.33 Of interest, we found that although the apoptosis ratio in the Ang1 group was higher than that in the GFP group, the concentration of GMCSF in the BALF was not downregulated by Ad-Ang1 pretreatment. On the contrary, it was higher than that in the GFP group. These results imply that the promotion of neutrophil apoptosis by Ang1 pretreatment may be regulated by molecules other than GMCSF. Together with the study that Ang1 pretreatment increased survival by mitigating endotoxin-induced ALI,11 this supports the result that higher levels of GMCSF in the air spaces were associated with improved outcome in ARDS patients.34
The modulation of the constitutive apoptotic program in the neutrophil correlates with the expression and engagement of surface adhesion molecules.35 Pretreatment with Ad-Ang1 significantly accelerates the apoptosis of PMNs, perhaps because of Ang1 reducing the endothelium-derived molecules.24 Alternatively, the inhibition of nuclear factor (NF)-κB activation by Ang136 may be responsible for this result. The activation of NF-κB delays neutrophil apoptosis in ALI through increasing expression of antiapoptotic proteins.37
We should emphasize that although some similarities with human ALI, such as neutrophilic inflammatory response with increased intrapulmonary cytokines, are recognized in the ALI model induced by LPS instillation, the changes in alveolar–capillary permeability are mild and the ALI could be induced by various other factors. Whether Ang1 could accelerate inflammation resolution in other ALI models or whether it could be given after endotoxin instillation remains to be investigated.
In summary, this study provides, for the first time, a quantitative analysis on resolution of inflammation in ALI with the resolution indices. Our data demonstrate a moderate protection of Ang1 in experimental ALI in terms of resolution of inflammation. Ang1 overexpression promoted the resolution of inflammation of ALI by accelerating the apoptosis of neutrophils and phagocytosis by macrophage cells. Data from this study, together with previous studies, indicate that increased survival with Ang1 pretreatment in ALI may be due to both the anti-inflammatory and proresolution properties of Ang1. The Ang1–Tie2 signaling pathway may be a potential strategy to improve the outcome after ALI and other inflammation-related diseases.
Name: You-Nian Xu, MD.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Attestation: Younian Xu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Zhao Zhang, MD.
Contribution: This author helped design the study and conduct the study.
Attestation: Zhao Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Pu Ma, MD.
Contribution: This author helped conduct the study.
Attestation: Pu Ma has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Shi-Hai Zhang, MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Shihai Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
We are indebted to Dr. Jinghui Zhang of the Laboratory of the Gastrointestinal Department of Union Hospital, Dr. Shunchang Zhou of the Laboratory Animal Section of the Tongji Medical College, and the team of the Immunology Laboratory of the Tongji Medical College, Central Laboratory and Cytology Laboratory of Union Hospital for technical assistance.
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© 2011 International Anesthesia Research Society
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