Disease-specific ex vivo stimulation of whole blood for cytokine production: applications in the study of tuberculosis

Abstract

As a simple method for field studies to assess the cytokine-status of patients with tuberculosis (TB), the use of whole blood instead of isolated cells has advantages, especially since the risk of contamination is minimal. Therefore, cytokine production in whole blood cultures was determined using non-specific and disease-specific stimuli. Heparinized blood from healthy volunteers was either incubated in closed vacutainer tubes or in tissue culture wells after dilution in culture medium. Dose-response and kinetics were investigated for the production of TNFα, IL-1β, IL-1ra, IL-10 and IFNγ. Patients with TB and healthy individuals were examined for IFNγ production in whole blood. In the absence of a stimulus, the production of cytokines is negligible in whole blood cultures. LPS induces the production of TNFα, IL-1β, IL-1ra and IL-10; PHA induces the production of IFNγ and IL-10. Live BCG induces the production of proinflammatory cytokines, irrespective of tuberculin skin status. In contrast, PPD and MTB-culture filtrate induce production of IFNγ in skin-test positive and not in skin-test negative healthy subjects. Five out of 13 patients with TB had a low antigen-specific IFNγ production, suggestive of a minimal or absent specific T-cell response. For most purposes, cultures in closed vacutainer tubes are optimal. If one wishes to focus on T-cell cytokines or if only small volumes of blood are available, dilution of whole blood in culture medium before incubation in tissue culture wells may be preferable.