Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome.
BACKGROUND
Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown.


OBJECTIVE
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), I(125) 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.


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.
Sixt
Stephan Urs
SU
Klinik für Anästhesiologie und Intensivmedizin, Universitätsklinikum Essen, Hufelandstrasse 55, Essen, Germany. anaesthesixt@gmx.de
Adamzik
Michael
M
Spyrka
Daniel
D
Saul
Boris
B
Hakenbeck
Jan
J
Wohlschlaeger
Jeremias
J
Costabel
Ulrich
U
Kloss
Alexander
A
Giesebrecht
Jan
J
Dahlmann
Burkhardt
B
Peters
Jürgen
J
D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D018450:Disease Progression; D004797:Enzyme-Linked Immunosorbent Assay; D045604:Extracellular Fluid / Q000201:enzymology*; D005260:Female; D005500:Follow-Up Studies; D005858:Germany; D006801:Humans; D015994:Incidence; D008297:Male; D016253:Microscopy, Immunoelectron; D008875:Middle Aged; D011379:Prognosis; D011446:Prospective Studies; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000201:enzymology* / Q000648:ultrastructure; D012128:Respiratory Distress Syndrome / Q000201:enzymology* / Q000453:epidemiology / Q000473:pathology; D012307:Risk Factors
D015415:Biomarkers; D046988:Proteasome Endopeptidase Complex