AAT – A Novel Cartilage‐Protective Mediator

Abstract

IntroductionThe resolution of inflammation is a process that ensures repair and restoration of tissue physiology. Using integrated approach with proteomic analyses and bio‐assays with human chondrocytes and experimental arthritis, we identified α1‐Antitrypsin(AAT), the chief neutrophil elastase(NE) inhibitor, as a novel chondroprotective protein produced in resolving exudates.MethodsResolving rat pleural exudates (24h post‐carrageenan injection) were run for mass spectrometry using ion‐trap mass analyzer (MS/MS LTQ Orbitrap XL). Human C28/I2 chondrocytes were grown in high‐density micromass(MM)1 incubated with AAT with/without catabolic stimuli interleukin(IL)‐1β (20ng/ml) or osteoarthritic synovial fluids (OASF; 1:100) for 48h. Glycosaminoglycans (GAG) deposition was assessed using Alcian Blue(AB) staining. Gene expression was quantified by qPCR. Data are expressed as 2−ΔΔCt equation–relative amount of target genes, normalized to GAPDH and vehicle, with expression set to 1.0 (N=3). Inflammatory arthritis was induced though K/BxN serum transfer model2. Knee joint structure and cartilage integrity were determined 48h after intra‐articular(i.a.) or intra‐peritoneal(i.p.) injection of AAT (n=8) by the GAG‐sensitive toluidine blue staining of knee sections. Knee inflammation (diameter; n=14), changes in leukocyte kinetics (number of rolling and adherent leukocytes; intravital microscopy; n=8), blood perfusion (Laser Speckle Contrast Analysis; n=8), and pain (von Frey algesiometry; n=8) were evoked by i.a. injection of NE (5μg)3 with/without AAT(100ng) and quantified 4h post‐injection. One‐way ANOVA, or student's T‐test were applied for stats.ResultsProteomics identified AAT in pro‐resolving (24h post‐carrageenan) exudates. Exogenous AAT abrogated the effect of IL‐1β on MMP13 (p<0.05). Incubation of chondrocyte MM with OASF augmented IL6 and MMP13 mRNA, with concomitant down‐regulation of COL2A1 and ACAN gene products: tested at 3–10 μg/ml, AAT significantly attenuated these effects (p<0.01). GAG deposition confirmed the chondroprotection afforded by AAT: ~80% reduction by OASF and reversal to control levels with AAT (p<0.01).In the model of arthritis, knee joint cartilage was eroded by ~65% (GAG content; p<0.05). Intra‐articular injection of AAT(100ng) recovered cartilage integrity by 52% compared to vehicle‐injected contralateral joints (p<0.05). Systemic AAT (10μg i.p.) reduced cartilage destruction by ~30%, compared to vehicle‐injected animals (p<0.05).NE induced acute inflammation and pain in knee joints of mice: it caused a marked increase in knee diameter, synovial microcirculation, vascular response with increased leukocyte kinetics and a decrease in hindpaw withdrawal threshold (p<0.01). Co‐administration of NE and AAT(100ng) significantly counteracted NE‐induced knee inflammation (p<0.01) and potently reverted the NE‐induced pain (p<0.01).ConclusionUsing resolving exudates as a bio‐source, we identified the neutrophil elastase inhibitor AAT as a novel tissue‐protective protein with a broad spectrum of activities, thus paving the way for new strategies to prevent joint pain and repair/restore compromised cartilage functions.