Effects of homogenization of induced sputum by dithiothreitol on polymorphonuclear cells.
Induced sputum represents a useful and non-invasive tool to isolate different cells from the airways. Complete homogenization of sputum is important for dispersion of cells and is usually achieved by use of dithiothreitol (DTT). However, it is not known if DTT will influence the viability and functionality of cells obtained by induced sputum. In the present study, induced sputum was processed by DTT or by PBS treatment. The obtained neutrophils were compared with neutrophils obtained from peripheral blood and from bronchoalveolar lavage fluid (BAL). These isolated neutrophils were treated in a similar way as the sputum neutrophils with DTT or PBS. All isolated cells were used for chemiluminescence tests and for the measurement of elastase and myeloperoxidase release after stimulation with fMLP. The results showed that the maximum chemiluminescence response was always significantly lower after DTT treatment: blood, 16.68 +/-1.89 vs. 2.62 +/-0.43 mV, P<0.0001; sputum, 2.96 +/-0.30 vs. 1.09 +/-0.01 mV, P<0.01; BAL, 25.47 +/-0.88 vs. 8.22+/-0.20 mV, P<0.0001. Both spontaneous and fMLP-induced release of elastase and myeloperoxidase (MPO) was in most cases enhanced after DTT-treatment (P-values range from 0.24 to <0.01). We conclude that the use of DTT to homogenize sputum for dispersion of cells is harmful to cell functions and these cells are hampered for the evaluation of their normal functional characteristics.
van Overveld
F J
FJ
Department of Respiratory Medicine, Antwerp University, Antwerp, Belgium. frans.vanoverveld@pmintl.com
Demkow
U
U
Górecka
D
D
Skopinska-Rozewska
E
E
de Backer
W A
WA
Zielinski
J
J
D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology*; D005100:Expectorants / Q000494:pharmacology*; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D009195:Peroxidase / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013183:Sputum / Q000166:cytology*; D013997:Time Factors
D005100:Expectorants; D017382:Reactive Oxygen Species; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009195:Peroxidase; D019272:Leukocyte Elastase; D004229:Dithiothreitol