Cytokine profiling in healthy children shows association of age with cytokine concentrations

Abstract

Cytokine-based diagnostic assays are increasingly used in research and clinical practice. Assays developed for adults such as the interferon-gamma release assay for tuberculosis show inferior performance in children. Limited evidence suggests that release of cytokines is influenced by age but normal ranges of cytokines in children are lacking. Whole blood of healthy children (0-12 years) undergoing elective/diagnostic procedures was stimulated with SEB, PHA, Candida albicans for 24 hours or left unstimulated. Concentrations of eight cytokines were measured by multiplex bead-based immunoassays and associations with age and other factors quantified by regression analysis. 271 children (median age 5.2 years) were included. In unstimulated samples IL-1ra, IP-10 and TNF-α concentrations decreased by up to -60% with age. Following antigen stimulation, an age-associated increase (ranging from +90% to +500%) was observed for all cytokines except IL-1ra (significant for IL-4, IFN-γ and TNF-α). Inter-individual variability in cytokine concentrations was large with a coefficient of variation ranging from 42% to 1412%. Despite inter-individual variation age was identified as a strong influencing factor of cytokine concentrations. Age-specific normal values need to be considered for cytokine-based diagnostic purposes. These results are relevant for development of novel cytokine-based diagnostic assays and for optimal dosing of therapeutic agents targeting cytokines.

Figure 1

Observed cytokine concentrations over age on a semi-logarithmic scale (normal-scale for age and a log10-scale for cytokines). Eg concentration of 10⁻² pg/mL equals a concentration of 0.01 pg/mL, 10⁰ equals 1 pg/mL, 10² equals 100 pg/mL (note that the depicted range of concentration differs between stimulatory antigens used for better readability). Dark grey circles: concentrations measured within the range calibration curve of the analytical method. Light grey circles: concentrations beyond the calibration curve limits, i.e. below or above the limit of quantification. Black line: non-parametric regression line (locally weighted smoothing).

Figure 2

Association of cytokine levels with age, body weight (z-score body weight for age), vitamin D concentrations and sex. The color indicates the general direction of association (red = increase, blue = decrease). The depicted numerical percentage (%) change per indicated unit was estimated from censored linear regression on log-transformed cytokine concentrations and is depicted for significant associations only (regression coefficient with a p-value < 0.05). X: no regression analysis performed since >50% of data below limit of quantification.

Figure 3

Cytokine for age percentile; solid line represents the 50^th and dotted lines the 5^th and 95^th percentile, respectively. Data are presented on a semi-logarithmic scale (log10-scale for cytokines, normal-scale for age). For cytokines 10⁻² pg/mL equals a concentration of 0.01 pg/mL, 10⁰ equals 1 pg/mL, 10² equals 100 pg/mL. Note that the depicted range of concentration differs between stimulants for better readability. For IL-2, IL-4, IL-6 and IL-10 percentiles for unstimulated samples were not quantifiable.