Nuclear factor kappa B activation in human cord blood mononuclear cells

Abstract

The immunologic signals participating in immune responses early in life have not been completely elucidated. Regarding the characterization of neonatal cells, little is known concerning the activity of transcription factor nuclear factor kappa B (NF-kappaB), which regulates inflammatory genes and cytokine production. The aim of this study was to characterize NF-kappaB activation in cord blood mononuclear cells (CBMC). We analyzed the potential association of NF-kappaB activity with lymphocyte proliferation and influences on cytokine secretion in the early immune system. To determine the contribution of a disease whereby inheritance may impact neonatal immunity, we assessed the influence of maternal allergic disease on NF-kappaB regulation and cytokine secretion. CBMC from healthy newborns were isolated and stimulated with mitogen (n = 28). Nuclear extracts were analyzed by electrophoretic mobility shift assay, cytokine secretion by ELISA. FISH analysis excluded relevant maternal contamination of CBMC. All samples showed a positive lymphoproliferative response, and NF-kappaB activity was both increased and decreased after mitogen stimulation. Increased NF-kappaB activation was significantly associated with decreased TNF-alpha secretion (median 6.1 versus 50.3 pg/mL) in unstimulated CBMC. Mitogen stimulation resulted in increased NF-kappaB activity with a trend to increased IL-13 production. Maternal allergic disease was associated with higher TNF-alpha (median 982 versus 173 pg/mL) and IL-13 secretion (median 1328 versus 1120 pg/mL) after mitogen stimulation. Together, NF-kappaB activity is differentially activated in cord blood and associated with a distinct cytokine pattern. Whether differential NF-kappaB activity in cord blood is related to the subsequent development of immune diseases requires further investigation.

Figure 1

Specific DNA binding activities of NF-κB in human CBMC. (A) Kinetic analysis of NF-κB activation after stimulation with PHA for 20, 40, 60, and 120 min in CBMC (indicated as +20, +40, +60, +120). Nuclear extracts from PHA-stimulated CBMC (lanes 1–8) were incubated with radio-labeled NF-κB. NF-κB–specific bands were confirmed by supershift experiments using polyclonal antibodies against p65 (lanes 2, 4, 6, 8). One representative cord blood sample is shown. (B and C) Example of a neonate with increased NF-κB DNA binding (A) and a neonate with decreased NF-κB DNA binding (B). Nuclear extracts from unstimulated (lane 1) and PHA-stimulated CBMC (lane 2) were incubated with radio-labeled NF-κB. NF-κB specific bands were confirmed by supershift experiments using polyclonal antibodies against p65 (lane 3). (A–C) Nuclear protein extracts were prepared from CBMC unstimulated (lane 1 in B, C) or stimulated with PHA (lanes 2 and in B, C) as described in “Methods.” Nuclear extracts were incubated with radio-labeled consensus sequences for NF-κB and analyzed by EMSA. Bottom arrow indicates NF-κB DNA binding. Top arrow indicates supershift with p65 antibody. Density of bands was analyzed by ImageQuant software and a ratio of density from stimulated over unstimulated nuclear extracts was created as described in “Methods”; n = 28 total subjects.

Figure 2

Differential NF-κB DNA binding after PHA stimulation of CBMC. NF-κB-DNA binding was measured by densitometry. The ratio of the density of stimulated over unstimulated nuclear extracts was calculated as described in “Methods.” Three groups were created: decreased for a ratio below 0.87 (n = 15), no change for a ratio ≥0.87 and ≤1.15 (n = 2), and increased for a ratio above 1.15 (n = 11).

Figure 3

Variation of lymphocyte proliferation independent of NF-κB activation. Lymphocyte proliferation was similar in CBMC with increased compared with decreased NF-κB activity. Lymphocyte proliferation was determined in the presence or absence of PHA stimulation for 3 d by ³H-thymidine uptake as described in “Methods.” A ratio of counts per minute from stimulated over unstimulated cells was created. A positive lymphoproliferative response to PHA was defined as a SI > 3. Horizontal lines represent the median.

Figure 4

Increased NF-κB activation is associated with decreased TNF-α secretion at baseline and increased IL-13 secretion after PHA stimulation. Significantly lower TNF-α secretion was detected in CBMC with increased NF-κB activation in unstimulated samples (A, inset) Inset in A indicates cytokine secretion of TNF-α in unstimulated cells with logarithmic scaled y axis for TNF-α levels. A trend of increased IL-13 secretion in CBMC was observed for increased NF-κB activation after 60 h of PHA stimulation (B). Cytokine secretion of TNF-α and IL-13 in mitogen-stimulated CBMC was compared with secretion in unstimulated samples. Data are shown as box and whiskers plots (median, whiskers: 5% and 95% quantile) with outliers. Pairwise comparison between “decrease” and “increase” in NF-κB production was analyzed for each time point by Mann-Whitney U test.