The effect of dithiothreitol on VLA-4 detection in peripheral blood and induced sputum cells.
BACKGROUND
Induced sputum has been shown to be a reliable technique for investigating airway inflammation non-invasively. Flow cytometry could provide useful information in this area. However, the viscosity of the sample entails the use of a mucolytic agent. Dithiothreitol (DDT) is the most frequently used agent although it could affect detection of different inflammatory markers.


METHODS
To measure the effect of DDT on the detection of certain adhesion molecules in eosinophils and lymphocytes, sputum was induced from seven non-smoking asthmatic and non-asthmatic subjects treated with 0.1 M DDT. The samples were analyzed by flow cytometry. Whole blood samples from the same subjects were also processed with DTT and analyzed by flow cytometry.


RESULTS
Very late activated antigen-4 (VLA-4) levels on eosinophils in intracellular and surface staining were much lower than expected. VLA-4 on lymphocytes was also altered but less so than on eosinophils. VLA-4 levels were also decreased on blood cells after DTT treatment. No abnormalities were found in the detection of CD29 on eosinophils and the beta7-chain in lymphocytes.


CONCLUSIONS
Flow cytometry could be used as a complementary method to induced sputum in the investigation of airway inflammation. However, DTT could interfere with the detection of some inflammatory markers, as is the case with VLA-4.
Department of Allergy. Hospital Clínico San Carlos. Madrid. Spain. fajavdom@hotmail.com
J
J
Domínguez-Ortega
F
F
León
A
A
Alonso-Llamazares
E
E
Roldán
T
T
Robledo
P
P
Agustín
A
A
Bootello
C
C
Martínez-Cócera
D000328:Adult; D016477:Artifacts; D001249:Asthma / Q000175:diagnosis* / Q000473:pathology; D004229:Dithiothreitol / Q000494:pharmacology*; D004804:Eosinophils / Q000737:chemistry*; D005100:Expectorants / Q000494:pharmacology*; D005188:False Negative Reactions; D005434:Flow Cytometry; D006801:Humans; D007249:Inflammation; D039441:Integrin alpha4 / Q000032:analysis*; D039041:Integrin alpha4beta1 / Q000032:analysis*; D019012:Integrin beta1 / Q000032:analysis; D013183:Sputum / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects
D005100:Expectorants; D039041:Integrin alpha4beta1; D019012:Integrin beta1; D039441:Integrin alpha4; D004229:Dithiothreitol