Optimized dialysis and protease inhibition of sputum dithiothreitol supernatants.
BACKGROUND
Dithiothreitol (DTT) is commonly used to liquefy induced sputum samples before assessment of cytology, but causes reduction of disulfide bonds and denaturation of proteins.


OBJECTIVE
To process sputum supernatants containing DTT to enable quantification of cytokines and chemokines.


METHODS
A standard solution of 22 pooled chemokines and cytokines was incubated with DTT at the concentrations used during sputum liquefaction and then dialyzed under 20 different denaturant and redox conditions.


RESULTS
After incubation of the standard solution with DTT there was loss of detectable protein mediators on immunoassay, but optimized dialysis permitted recovery of chemokines to 96 +/- 4% and cytokines to 91 +/- 6%. Optimized dialysis of DTT supernatants from subjects with asthma covering a range of severities (n = 35) was performed in the presence of a cocktail of protease inhibitors and demonstrated significantly elevated levels of the chemokine CXCL10 (IFN-gamma-inducible protein-10), CXCL8 (IL-8), and CCL3 (macrophage inflammatory protein-1alpha); with lower but significantly elevated levels of CCL2 (monocyte chemotactic protein-1), CCL11 (eotaxin), and CCL5 (regulated on activation, normal T-cell expressed and secreted) in severe asthma. In sputum from subjects with severe asthma there were also significantly elevated levels of IL-4, IL-5, IL-13, tumor necrosis factor-alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-12(p40).


CONCLUSIONS
The technique of optimized dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines. The detection of elevated levels of particular sputum chemokines and cytokines in individual patients may provide a rationale for specific therapies.
Erin
Edward M
EM
NHLI Clinical Studies Unit, Royal Brompton Hospital, Fulham Road, London SW3 6HP, UK.
Jenkins
Gavin R
GR
Kon
Onn Min
OM
Zacharasiewicz
Angela S
AS
Nicholson
Grant C
GC
Neighbour
Helen
H
Tennant
Rachel C
RC
Tan
Andrew J
AJ
Leaker
Brian R
BR
Bush
Andrew
A
Jose
Peter J
PJ
Barnes
Peter J
PJ
Hansel
Trevor T
TT
D000328:Adult; D000368:Aged; D001249:Asthma / Q000276:immunology*; D016022:Case-Control Studies; D002452:Cell Count; D018925:Chemokines / Q000032:analysis*; D003430:Cross-Sectional Studies; D016207:Cytokines / Q000032:analysis*; D003956:Dialysis / Q000295:instrumentation / Q000379:methods*; D004229:Dithiothreitol; D005260:Female; D005541:Forced Expiratory Volume; D006801:Humans; D008297:Male; D008875:Middle Aged; D011480:Protease Inhibitors; D012710:Serum Albumin, Bovine; D012720:Severity of Illness Index; D013048:Specimen Handling / Q000379:methods; D013183:Sputum / Q000737:chemistry*
D018925:Chemokines; D016207:Cytokines; D011480:Protease Inhibitors; D012710:Serum Albumin, Bovine; D004229:Dithiothreitol