Cytokine responses correlate differentially with age in infancy and early childhood

Abstract

The functional differentiation of immune cells at early age plays a central role in immune physiology, e.g. for the sufficient eradication of pathogens. However, imbalances in effector cell responses may also have an impact in the pathophysiology of childhood diseases such as atopy and autoimmune disorders. As information on immune cell responses in infancy and early childhood is scarce, we conducted an observational, cross-sectional study in healthy newborns (n = 18), infants and young children (n = 54) aged 1-96 months and adult controls (n = 19) to assess cytokine mRNA and protein expression upon phorbol 12-myristate 13-actate/ionomycin stimulation and LPS-induced IL-12 expression in monocytes. The intracellular expression of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha (R = 0.748, P < 0.0001; R = 0.784, P < 0.0001, respectively) and interleukin (IL)-2 protein expression (R = 0.384, P = 0.008) was demonstrated to increase progressively with age. While a correlation between IL-4 protein expression and age was noted (R = 0.342, P = 0.007), the levels of IL-5 and IL-10 protein expression tended to be regulated on an individual basis during infancy and early childhood. An age correlation was also observed for intracellular IL-12 expression (R = 0.331, P = 0.009) in monocytes. These findings are valuable for further assessment of normal variations and maturation processes in immune cell responses and for the clinical-therapeutic monitoring of immunological status in various childhood diseases.

Fig. 1

Strong age correlation of intracellular interferon production in infancy and early childhood. Whole blood cultures were stimulated for 5 h with 3 µg/ml phorbol myristate acetate (PMA) and 3 µM ionomycin to induce IFN-γ (a: preterm infants, n = 30; newborns, n = 26; infants and children n = 54; adults, n = 18) and TNF-α synthesis (b: preterm infants, n = 14; newborns, n = 13, infants and children, n = 54; adults, n = 19). Data are expressed as percentage of cytokine-positive cells and plotted as individual points on the graph. All data were included to estimate the non-parametric correlation between age and cytokine expression using Spearman's rho test (R, rank order correlation coefficient; two-sided level of significance is indicated as P-value; SPSS version 11·0); (c: correlation of assessment methods for investigation of TNF-α expression (cytokine production in supernatants vs. percentage of TNF-α producing T cells; Spearman's rho test).

Fig. 2

Th1 and Th2 cytokine production are correlated differentially with age in infancy and early childhood. Whole blood cultures were stimulated for 24 h with 3 µ g/ml phorbol myristate acetate (PMA) and 3 µM ionomycin to induce cytokine protein secretion. Data are expressed as protein secretion (pg/ml) and plotted as individual points on the graph (newborns, n = 10, infants and children, n = 41; adults, n = 12). All data were included to estimate the non-parametric correlation between age and cytokine production (a–e) or cytokine/cytokine production (f, g) using the Spearman's rho test (R, rank order correlation coefficient; two-sided level of significance is indicated as P-value, P < 0·05 was regarded as significant; SPSS version 11·0).

Fig. 3

Cytokine mRNA expression levels increase with age in early infancy. Whole blood cultures were stimulated for 24 h with 3 µg/ml phorbol myristate acetate (PMA) and 3 µM ionomycin to induce cytokine mRNA expression. mRNA levels were determined in duplicate by real-time reverse transcription-polymerase chain reaction (RT-PCR) and normalized with respect to β-actin mRNA levels (cytokine mRNA expression/10⁶β-actin mRNA copies). Following age strata were applied to this experiment: newborns (1–28 days old; n = 10), 0–6 months (29–182 days old; n = 10), 7–12 months (183–364 days old; n = 5), 13–24 months old (n = 6), 25–48 months old (n = 7), 49–96 months old (n = 9), and data are plotted as individual points on the graph with mean levels indicated as horizontal lines.

Fig. 4

Age correlation of monocytic interleukin (IL)-12 expression in early childhood. (a) Intracellular IL-12 expression kinetics after lipopolysaccharide (LPS) stimulation. Whole blood cultures were preincubated with 10 ng/ml interferon (IFN)-γ for 2 h and stimulated with 10 ng/ml LPS to induce IL-12 expression as indicated. The IL-12 expression in newborns (n = 5; mean ± s.d.) was lower compared to adults (n = 5; mean ± s.d.) throughout the LPS-stimulation kinetics. (b) Whole blood cultures were preincubated with 10 ng/ml IFN-γ for 2 h stimulated for 24 h with 10 ng/ml LPS to induce IL-12 expression in newborns, infants and children (n = 61). Data are expressed as a percentage of cytokine-positive cells and plotted as individual points on the graph. All data were included to estimate the non-parametric correlation between age and cytokine expression using Spearman's rho test (R, rank order correlation coefficient; two-sided level of significance is indicated as P-value; SPSS version 11·0).