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Alveolar Extracellular 20S Proteasome in Patients with Acute Respiratory Distress Syndrome

¹ Klinik für Anästhesiologie und Intensivmedizin, Universität Duisburg-Essen, ² Institut für Pathologie und Neuropathologie, and ³ Abteilung für Pneumologie und Allergologie, Ruhrlandklinik, Universitätsklinikum Essen, Essen, Germany; ⁴ Institut für Biochemie/CCM, and ⁵ Institut für Medizinische Physik und Biophysik/CCM, Charité-Universitätsmedizin-Berlin, Berlin, Germany

Correspondence and requests for reprints should be addressed to S. U. Sixt, M.D., Klinik für Anästhesiologie und Intensivmedizin, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. E-mail: anaesthesixt{at}gmx.de

Rationale: Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown.

Objectives: To test whether the 20S proteasome is present and functional in the alveolar space in patients with ARDS.

Methods: Proteasome antigenic concentration in bronchoalveolar lavage (BAL) supernatants was measured by ELISA in patients with ARDS (n = 64), acute lung injury (ALI) (n = 8), sarcoidosis (n = 13), and in healthy subjects (n = 8). Cleavage of specific fluorogenic substrates (±epoxomicin), I¹²⁵ albumin degradation rate, and gel filtration were used to quantify and characterize proteasomal activity. The presence of proteasomes was confirmed independently by electron microscopic techniques.

Measurements and Main Results: Proteasome concentrations in patients with ARDS were markedly increased (1,069 ± 1,194 ng/ml) in comparison to healthy subjects (60.8 ± 49.8; P < 0.001), ALI (154 ± 43; P = 0.006), and sarcoidosis (97.6 ± 42.2; P = 0.037). All fluorogenic substrates were hydrolyzed (Suc-LLVY-AMC, 3.6 ± 8.8 pkat/mg; BZ-VGR-AMC, 1.8 ± 3.1; Suc-LLE-AMC, 1 ± 1.7) by BAL supernatants of patients with ARDS, with inhibition by epoxomicin (P = 0.0001), and the majority of proteolytic activity was detected in BAL supernatant. Maximum hydrolyzing activity occurred at 660 kD and 20S proteasome was seen microscopically after purification and being released by pneumocytes type II. Proteasomal activity and albumin degradation rate in patients with ARDS were approximately 17-fold lower than in healthy subjects. Proteasomal activity in normal BAL was inhibited by BAL aliquots from patients with ARDS but not by denatured BAL, and returned to normal by purification.

Conclusions: For the first time, we identified extracellular, biologically active 20S proteasome in the alveolar space of patients with ARDS in concentrations much higher than in normal subjects or in those with ALI.

Key Words: acute lung injury • bronchoalveolar lavage • albumin degradation • circulating proteasome • proteolysis

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Acute respiratory distress syndrome (ARDS) is characterized by lung endothelial and epithelial injury, resulting in the accumulation of protein-rich edema fluid within the alveolus. Mechanisms contributing to clearance of proteinaceous debris from the airspace remain poorly understood. What This Study Adds to the Field Elevated levels of biologically active 20S proteasome, a multicatalytic proteinase, are found in the bronchoalveolar lavage fluid of patients with ARDS, as compared with healthy subjects and patients with other inflammatory lung diseases. Extracellular 20S proteasome activity is reduced due to the presence of an inhibitor found in the BAL fluid of patients with ARDS.