Effects Of SRAGE On LPS-induced Acute Lung Injury In Mice

IntroductionAcute lung injury (ALI) is a critical illness with high mortaliy. Despite variouspharmacologic interventions developed in the last two decades aiming at specifictargets, such as cytokine cascades, the therapeutic strategy for this syndrome has notbeen established. High mobility group box 1 (HMGB1) is a late inflammatory mediatorof sepsis or lipopolysaccharide (LPS) lethality. Anti-HMGB1 antibodies conferredsignificant protection against LPS-induced lung injury even when the antibodies wereadministered after the early cytokine responses had resolved. These findings broadenthe treatment window of lung injury.Receptor for advanced glycation end-products (RAGE) has been implicated inmediating the cytokine activity of HMGB1. Its ligands include advanced glycation endproducts, amyloidβ-peptide, S100 proteins as well as HMGB1. RAGE-ligandinteraction results in rapid and sustained cellular activation and gene transcription,diverse intracellular signals activated such as ERK1/2(p44/p42),p38 and SAPK/JNKMAP kinases, rho-GTPases, phosphoinositol-3-kinase, and the JAK/STAT, in mostcases culminating in the activation of nuclear factor-kappaB (NF-κB). This cellularactivation is related to amplified and sustained inflammatory processes or tissue injury.RAGE knockout mice were recently reported to be resistant to septic shock induced bycecal ligation and puncture, suggesting that RAGE potentially plays a role in systemicacute inflammation.Recently, several carboxyl-terminal truncated isoforms of RAGE such as solubleRAGE (sRAGE) and endogenous secretory RAGE (esRAGE) were identified in thelung of both humans and mice. Because these isoforms lack a transmembrane domain, they are secreted and act as decoy receptors. Indeed, sRAGE has been shown to preventor reverse RAGE signal in experimental models of diabetic atherosclerosis, woundhealing, Delayed-Type Hypersensitivity, rheumatoid arthritis and chronic colitis.However, its putative protective roles on ALI have not been previously characterized.Here, we conducted a study to examine the participation of RAGE and itsisoforms in the pathogenesis of LPS-induced lung injury in mice. For this purpose, thekinetic changes of the mRNA were observed and protein expression of RAGE and itsisoforms were measured in the lungs and BAL fluid after intratracheal LPS challenge.Our second objective was to test the hypothesis that blockade of ligand-RAGEinteraction with recombinant sRAGE mitigates the inflammatory response andconsequent tissue damage and affords protective roles in LPS-induced lung injury.Finally, we tried to explore the protective mechanisms of sRAGE in relation toinflammation and apoptosis which will highlight the perspective of pharmacologicalintervention in infection-associated lung jury.Materials and methods(1) To build the mouse models of LPS-induced ALI. C57BL/6J mice, 8-11weeksof age, 18-22g in weight, were anesthetized with intraperitoneally injected ketamine(100 mg/kg) and xylazine (10 mg/kg) before the trachea was surgically exposed by amidline incision and 3.0 mg/kg E. coli LPS (serotype B:55) was instilled into the leftlung by intratracheal puncturation with a 24 G canula inserted in the left bronchus. LPSwas given as a solution of 1.2 mg LPS/ml PBS and same volume of phosphate-bufferedsaline (PBS) was used as vehicle.(2) The lungs were sampled from mice sacrificed at designated time points andbronchoalveolar lavage fluid (BALF) was collected 24h after LPS intratrachealinstillation. Western blot was performed to determine the protein expression of RAGEand its variants in the lung homogenate and BALF. Quantitative real-time polymerasechain reaction (PCR) were employed to evaluate the kinetic changes of total RAGE andesRAGE mRNA in the lung using primers amplifying total RAGE and esRAGE respectively.(3) Mice of sRAGE group were injected intraperitoneally with recombinant mousesRAGE (100μg /mouse diluted in 100μl PBS) and LPS group were injectedintraperitoneally with PBS (100μl/mouse) 1h after the intratracheal instillation of LPS.PBS group received intraperitoneal injection of 100μl PBS 1h after PBS intratrachealadministration. The animals were sacrificed under deep anesthesia (pentobarbital 250mg/kg intraperitoneally) at 24 h after the instillation of LPS or PBS and subjected tobronchoalveolar lavage for leukocyte counting and cytokine measurement (Bio-Plextechnique) and lung sampling for histopathology under H.E. stain.(4) To evaluate the influence of sRAGE on NF-κB activation induced by LPS,nuclear extracts were prepared from the lung tissue in the 3 goups of miceabove-mentioned at 4 h after LPS or PBS instillation and DNA binding activity ofNF-κB P65 in nuclear extract was determined using a BD^TM TransFactorChemiluninescent kit. Pulmonary apoptosis was determined by terminal-deoxynucleotidyl transferase mediated-dUTP nick end labeling (TUNEL) assay inparaffin-embedded slides 24 h after intratracheal administration. Slides were visualizedwith a laser confocal microscopy. The numbers of TUNEL positive cells werecompared between the three groups.(5) Statistical analysis. Data are expressed as means±SE. Student t test orone-way ANOVA with a Least Squares Differences test for Post Hoc analysis wasperformed with SPSS 14.0 statistical analysis software, and a difference was acceptedas significant if the P value was＜0.05.Results1,The expression of RAGE and its varients in LPS-challenged lungof miceWestern blot assay showed that the antibody against specific RAGE extracellulardomain recognized three isoforms of RAGE sized approximately 38, 48, and 54 kD in lung homogenate in untreated mice (0 h). No significant concemtration changes ofthese isoforms were noticed 6, 12, and 24 h after LPS instillation. In contrast, only oneisoform of RAGE (48 kD) was detected in BALF. The PBS instillation caused a weakexpression of 48 kD isoform in the BALF, while significantly increased levels of thesame isoform were detected in LPS group 24 h after intratrachealadministration(P＜0.05).Quantitative real-time PCR determination revealed that intrapulmonary RAGEmRNA remarkablly decreased at 6 h after LPS challenge and presented atime-dependent down-regulation afterwards. Significant differences were noticed inmice at 6, 12, and 24 h compared with untreated controls (0 h) (P＜0.01). As foresRAGE transcripts, no significant change was found in lungs at different time points(P＞0.05).2,The effects of sRAGE intervention on LPS-induced acute lunginjuryThe mice intratracheally instilled with LPS revealed significantly increasednumbers of both the total leukocytes and the neutrophils compared with those instilledwith PBS (P＜0.01). Mice treated with sRAGE 1 h after LPS administration revealedsignificantly lower numbers of both the total cells and the neutrophils in BALF thanthose given PBS after LPS challenge (P＜0.05).LPS instillation induced an increase at 24 h in all the 8 cytokines (IL-1β, IL-6,IL-10, TNF-α, MIP-1α, MIP-1β, MCP-1 and KC) as compared with the PBS-treatedanimals (P＜0.01). sRAGE treatment 1 h after LPS challenge significantly attenuatedthe upregulation of TNF-α, MIP-1αand MIP-1βcaused by LPS (P＜0.05), but nochange in other cytokine levels was found between the mice treated with sRAGE andPBS in the presence of LPS(P＞0.05).We examined lung specimens stained with hematoxylin-eosin under lightmicroscopy and found that 24 h after intratracheal challenge, PBS group showed intactalveolar structure, no obvious neutrophil recruitment and interstitial edema were noticed. LPS-instilled mice demonstrated disorder of alveolar construction, numerousneutrophil and some exudation in the alveolar space with thickening of the alveolarsepta and areas of atelectasis, sRAGE markedly attenuated neutrophils infiltration andpathological changes caused by LPS instillation.3,The effects of sRAGE on NF-κB activation and apoptosis inLPS-challenged lungAs compared with PBS group, LPS group demonstrated a significant increase inNF-κB p65 DNA binding at 4 h after intratracheal instillation (P＜0.01). Treatment withsRAGE 1h after LPS challenge prevented LPS-induced NF-κB activation (P＜0.05).The binding specificity between DNA and NF-κB was confirmed by competitionassays.TUNEL assay indicated that 24 h after intratracheal administration, PBS groupshowed few Positive TUNEL cells in the alveolar septa with nuclei stained as brightgreen spots. In contrast, LPS group demonstrated much more TUNEL-positive cells(P＜0.05) which were significantly diminished by sRAGE treatment (P＜0.05).Conclusion(1) RAGE proteins are abundantly expressed in normal lungs of mice. A 48kDsoluble isoform of RAGE is released into the alveolar space in response to LPS andcarboxyl terminal truncation by proteolysis from full-length RAGE may be aresponsible mechanism.(2) Treatment with mice with recombinant sRAGE confers protective roles againstLPS-induced acute lung injury by attenuating increases in neutrophil infiltration,production of inflammatory cytokines and pathological changes in the lungs.(3) sRAGE suppresses LPS-induced NF-κB activation and apoptosis in mice. Itserves protective roles against LPS-induced lung injury through dual mechanisms ofboth anti-inflammation and anti-apoptosis in the lung.