Immature anti-inflammatory response in neonates

Abstract

The inflammatory response plays a major role in the induction of several neonatal diseases. We hypothesize that an imbalance between the pro- and anti-inflammatory response is crucial for the previously shown enhanced production of proinflammatory cytokines in term and preterm infants during infection. To test this hypothesis, we compared the capacity to produce the main anti-inflammatory cytokines IL-10 and TGF-beta in term infants, preterm infants and adults at different levels of synthesis by quantitative real time reverse-transcribed PCR, flow cytometry, as well as enzyme-linked immunoassay. Term and preterm infants showed a profoundly diminished IL-10 mRNA-expression and IL-10 production after stimulation. In addition, the amount of TGF-beta-positive lymphocytes was significantly less in neonates than adults. Furthermore, there was a considerably lower inhibition of production of IL-1alpha, IL-6, IL-8 and TNF-alpha by the use of recombinant IL-10 in term and preterm infants compared with adults. These results demonstrate not only a diminished anti-inflammatory capacity but also a reduced response to anti-inflammatory stimuli in term and preterm infants. From these data we conclude that neonates display an immature compensatory anti-inflammatory response syndrome (CARS) which may predispose preterm infants to harmful effects of proinflammatory cytokines resulting in severe organ sequelae during infection.

Fig. 1

Kinetics of IL-10 production a, IL-10 mRNA expression determined by QRT-PCR The relative amount of IL-10 mRNA/β-Actin was significantly lower in neonates compared with adults (n = 6, respectively) after stimulation with LPS for 18 h. Differences are significant at a level of *P < 0·01. b, Intracytoplasmic IL-10 detection by flow cytometry The percentage of IL-10/CD14-positive monocytes was considerably lower in neonates (n = 5) than adults (n = 5) after stimulation with LPS for 10, 18 and 24 h. Differences are significant at a level of *P < 0·05 or **P < 0·01. c, Determination of IL-10 in culture supernatant by enzyme-linked immunoassay The amount of IL-10 in culture supernatant was strikingly lower in both preterm infants and term infants than adults (n = 6, respectively) after stimulation with LPS for 18 and 24 h. Differences are significant at a level of *P <0·02.

Fig. 2

Intracytoplasmic TGF-β detection in neonatal and adult CD3-positive lymphocytes by flow cytometry. After 24 h of stimulation with PMA and ionomycin the amount of TGF-β-positive lymphocytes was significantly lower in neonates (n = 11) than adults (n = 25) (P < 0·0001). Data are presented as a vertical point plot, the median is indicated by a line.

Fig. 3

Inhibition of cytokine synthesis by interleukin-10. (a) Adults; (b) Term infants; (c) Preterm infants. Cells were incubated with recombinant IL-10 in whole blood culture one hour before stimulation with LPS. Stimulated cells which were incubated without recombinant IL-10 were set as 100%. There was a dose-related inhibition of cytokine-positive cells in all investigated groups. However, the inhibition of IL-1α-, IL-6-, IL-8- and TNF-α-positive monocytes was significantly lower in term infants and preterm infants compared with adults (n = 6, respectively) using 0·1 ng/ml or 1 ng/ml IL-10 (data are given in result section).

Fig. 4

Inhibition of cytokine synthesis by TGF-β. Cells were incubated with recombinant TGF-β in whole blood culture one hour before stimulation with LPS. Stimulated cells which were incubated without recombinant TGF-β were set as 100%. (a) Recombinant TGF-β induced only a modest dose-related inhibition of IL-6, IL-8 and TNF-α-producing monocytes in adults (n = 5). (b) There was no response to TGF-β in IL-8- and TNF-α-producing monocytes and only a slight decrease in IL-1α- and IL-6-producing monocytes in neonates (n = 5; P < 0·05).