Determination of optimal incubation time for the production of acute phase cytokines ex vivo by peripheral blood mononuclear cells from renal transplant recipients

Levels of acute phase cytokines secreted ex vivo by peripheral blood mononuclear cells (PBMCs) have been shown to be associated with clinical conditions or histologic lesions in renal transplant recipients. One of the limiting factors for the potential use of this assay as a diagnostic tool is the incubation time needed to measure adequate cytokine levels. Here, we validated that shorter time periods than the usual 48 h are sufficient for the production of acute phase cytokines. Cytokine levels were measured with the Luminex platform. We observed that, in contrast to cytokines associated with adaptive immunity, cytokines such as IL-1β, IL-6 and TNF-α are measurable as early as 2 h following incubation at a concentration of 1.5 million PBMC/150 μL. Levels obtained in the 2 h cultures have good correlations with the levels obtained after 48 h of culture for IL-1β and TNF-α (R=0.79, P=0.004 and R=0.92, P<0.001 respectively). We conclude that same-day incubation of PBMCs and measurement of these cytokines following blood collection in transplant recipients is feasible. It provides a rationale for further studies using shorter incubation times for ex vivo cellular assay measuring acute phase cytokine levels.