Cytokine expression in normal, atopic, and asthmatic subjects using the combination of sputum induction and the polymerase chain reaction.
BACKGROUND
The importance of cytokines in the asthmatic inflammatory response is becoming apparent. The aim of this study was to determine whether the non-invasive method of induced sputum combined with the polymerase chain reaction would allow the detection of messenger RNA (mRNA) encoding a range of cytokines on a qualitative basis.


METHODS
Four groups were studied comprising 10 normal subjects, six atopic, 10 mild and five moderately severe asthmatic subjects. Sputum was induced by the inhalation of nebulised 3.5% saline and total RNA extracted from the expectorated cells. Expression of cytokine message within induced sputum was examined by reverse transcription and the polymerase chain reaction (PCR) using primers specific for a range of cytokines (IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, RANTES, TNF alpha, IFN alpha, IFN gamma). Presence or absence of the signal was determined at 35 and 70 cycles of PCR by electrophoretic size fractionation on ethidium bromide stained agarose gels.


RESULTS
Cytokine message was detectable in sputum by this method. All samples showed a positive result for actin control. Analysis of signal for the cytokines in all subjects showed that, at 70 cycles, IL-1, IL-5, IL-8, and TNF alpha were detected in more subjects than would be expected by chance. IL-5 mRNA was detected in more of the asthmatic patients (moderate 80%, mild 40%) than in the atopic subjects (33%), who in turn showed expression of this cytokine in more individuals than nonatopic subjects (10%).


CONCLUSIONS
The combination of sputum induction and PCR appears to be a useful, non-invasive tool to explore the chronic inflammation of asthma and possibly other lung disorders. It should enable differences between normal and asthmatic subjects to be identified for future confirmation by quantitative techniques.
D000328:Adult; D001249:Asthma / Q000188:drug therapy / Q000378:metabolism*; D001483:Base Sequence; D016207:Cytokines / Q000378:metabolism*; D005260:Female; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000378:metabolism*; D007375:Interleukin-1 / Q000378:metabolism; D015848:Interleukin-5 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D013183:Sputum / Q000737:chemistry*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D007375:Interleukin-1; D015848:Interleukin-5; D016209:Interleukin-8; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha