Role of MyD88 in diminished tumor necrosis factor alpha production by newborn mononuclear cells in response to lipopolysaccharide

Abstract

Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes.

FIG. 1.

Plastic adherent newborn monocytes and MNC produce less TNF-α than adult monocytes. (A and B) The TNF-α protein concentrations in the supernatants of adherent monocytes of adults and newborns was measured after incubation in autologous plasma and LPS (1 ng/ml) for the times indicated. (C) MNC were incubated for 3 h with LPS (at 0.1 and 10 ng/ml) in the presence or absence of adult plasma (5%). The TNF-α concentration was measured by using an ELISA (A and C) or bioassay (B) of TNF-α as described in the text and is expressed as the mean ± the SD (n = 4). ✽, P < 0.05; ✽✽, P < 0.01 (adult versus newborn).

FIG. 2.

TNF-α mRNA levels are similar in MNC from adults and newborns. Adult and newborn monocytes were incubated with LPS (0.1 ng/ml) in the presence or absence of adult plasma (5%) for 3 h. Total cellular RNA was extracted and used in reverse transcription-PCR with specific primer sets for TNF-α and β-actin. A representative result of three independent experiments is shown.

FIG. 3.

LPS-stimulated newborn monocytes have lower amounts of TLR-4 and CD14 than adult cells. (A) Leukocyte-rich plasma from adults and newborns was left untreated (Control) or stimulated with 5 ng of LPS/ml for 30 min at 37°C and then stained with a mouse anti-TLR-4 MAb and then an FITC-conjugated goat anti-mouse IgG before being assessed by FACS. The mean ± the SD fluorescent intensity for adult and newborn monocytes is shown. (B) Adult venous blood or full term newborn cord blood were incubated with 0, 0.1, or 10 ng of LPS/ml for 30 min at 37°C, after which the samples were stained with anti-CD14 MAb (MY4) or an isotype control antibody. The cells were then incubated with FITC-conjugated rabbit anti-mouse IgG for 30 min at 4°C and then assayed by FACS. The results are expressed as mean ± the SD fluorescent intensity for adult (•) and newborn (○) monocytes. ✽, P < 0.05; ✽✽, P < 0.01 (adult versus newborn).

FIG. 4.

Activation of MAP kinases and IκBα in adult and newborn monocytes after LPS stimulation. Adherent monocytes were incubated with 0, 0.1, or 10 ng of LPS/ml for 20 min before lysis. Lysate proteins were analyzed by Western blotting with antibodies specific for phosphorylated ERK1/2 (A), p38 (C), or IκBα (E) or for IκBα protein (F). The equal loading of samples was confirmed by stripping and reblotting the membranes with anti-ERK2 (B) or p38 (D) protein antibodies. Figure 4 is representative of four experiments with comparable results.

FIG. 5.

Activation of MAP kinases in adult and newborn MNC after LPS stimulation. Nonadherent MNC were incubated with (+) or without (−) 0.1 ng of LPS/ml for 20 min before lysis. (A) Lysate proteins were analyzed by Western blotting for phosphorylated p38. (B) The membrane was stripped and reblotted for phosphorylated ERK1/2, and then we repeated this stripping-reblotting for β-actin (C). Figure 5 is representative of three experiments with comparable results.

FIG. 6.

Expression of MyD88 and TLR-4 in adult and newborn monocytes. (A) Adherent monocytes were harvested and lysed as described in the text. Proteins were analyzed by Western blotting with a rabbit anti-MyD88 antibody. (B and C) The same membranes were stripped and reblotted with a rabbit anti-TLR-4 antibody (B) and then repeatedly with the stripping-reblotting cycle for β-actin (C). A pooled result of the densitometry analysis of MyD88 bands in panel A is shown in panel D as means ± the SD of arbitrary units normalized by the density of β-actin bands (n = 7). ✽, P = 0.0003 (paired t test).