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Optimized Dialysis and Protease Inhibition of Sputum Dithiothreitol Supernatants

¹ Clinical Studies Unit, National Heart and Lung Institute, Imperial College, London, United Kingdom; ² Department of Respiratory Medicine, St. Mary's Hospital, London, United Kingdom; ³ Department of Respiratory Paediatrics, and ⁴ Department of Nephrology, Royal Free Hospital, London, United Kingdom; and⁵ Department of Leukocyte Biology, and ⁶ Department of Thoracic Medicine, Imperial College, London, United Kingdom

Correspondence and requests for reprints should be addressed to Trevor T. Hansel, M.D., Ph.D., NHLI Clinical Studies Unit, Royal Brompton Hospital, Fulham Road, London SW3 6HP, UK. E-mail: t.hansel{at}imperial.ac.uk

Rationale: Dithiothreitol (DTT) is commonly used to liquefy induced sputum samples before assessment of cytology, but causes reduction of disulfide bonds and denaturation of proteins.

Objectives: 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.

Measurements and Main 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-–inducible protein-10), CXCL8 (IL-8), and CCL3 (macrophage inflammatory protein-1); 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-, 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.

Key Words: sputum cytokines • dithiothreitol • dialysis • protease inhibitors

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Analysis of chemokines and cytokines in sputum is a controversial and technically difficult area. Dithiothreitol is used to liquefy conventionally processed sputum and its effects on analysis of sputum mediators require assessment and correction. What This Study Adds to the Field The technique of optimized dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines.