Inflammatory effects of phthalates in neonatal neutrophils

Abstract

Hospitalized infants are exposed to numerous devices containing the plasticizer di-(2-ethylhexyl) phthalate. Urinary levels of the phthalate metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are markedly elevated in premature infants. Phthalates inactivate peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a nuclear transcription factor that mediates the resolution of inflammation, a process impaired in neonates. We speculate that this increases their susceptibility to MEHP, and this was analyzed. MEHP inhibited neutrophil apoptosis; neonatal cells were more sensitive than adult cells. In neonatal, but not in adult neutrophils, MEHP also inhibited chemotaxis, stimulated oxidative metabolism, and up-regulated expression of NADPH oxidase-1. In both adult and neonatal neutrophils, MEHP stimulated IL-1beta and VEGF production, whereas IL-8 production was stimulated only in adult cells. In contrast, MEHP-inhibited production of MIP-1beta by adult cells, and Regulated on Activation Normal T Cell Expressed and Secreted (RANTES) by neonatal neutrophils. The effects of MEHP on apoptosis and oxidative metabolism in neonatal cells were reversed by the PPAR-gamma agonist, troglitazone. Whereas troglitazone had no effect on MEHP-induced alterations in inflammatory protein or chemokine production, constitutive IL-8 and MIP-1beta production was reduced in adult neutrophils, and RANTES and MIP-1beta in neonatal cells. These findings suggest that neonatal neutrophils are more sensitive to phthalate-mediated inhibition of PPAR-gamma, which may be related to decreased anti-inflammatory signaling.

Figure 1. Effects of MEHP on neutrophil apoptosis

Adult (■) and neonatal (□) neutrophils were treated with MEHP (0–500 µM), TgT (10 µM), or MEHP (500 µM) + TgT for 24 h and then analyzed for apoptosis by flow cytometry using BD FACSArray quadrant and two-dimensional histogram statistics based on relative fluorescence. Each bar represents the mean ± SE (n=3–8). *Significantly different (p < 0.05) from adult; ^†Significantly different (p < 0.05) from control.

Figure 2. Effects of MEHP on neutrophil chemotaxis

Chemotaxis of adult (■) and neonatal (□) neutrophils was assayed using microwell chambers. Cells were treated with MEHP (500 µM), TgT (2 µM), MEHP + TgT, or control (Ctl) for 1 h prior to measurement of chemotaxis toward fMLP or medium control. Each bar represents the mean ± SE (n = 3). *Significantly different (p<0.05) from adult; ^†Significantly different (p<0.05) from Ctl/Ctl; ^‡Significantly different (p<0.05) from Ctl/fMLP; ^§Significantly different (p<0.05) from MEHP/fMLP.

Figure 3. MEHP stimulates hydrogen peroxide production by neonatal neutrophils

Adult (panel A) and neonatal (panel B) neutrophils (5 × 10⁴ cells/well) were incubated with Amplex Red (25 µM) and horseradish peroxidise, in the absence or presence of MEHP (500 µM), and TgT or control. H₂O₂ production was quantified at 1 min intervals for 30 min. Each point represents the mean ± SE (n = 6). MEHP significantly increased H₂O₂ production in neonatal cells at times >13 min with no effect on adult cells; TgT significantly attenuated this response in control and MEHP treated neonatal cells at all time points. Control,●; MEHP,○; TgT,▼; TgT + MEHP, Δ

Figure 4. Effects of MEHP on expression of NOX1, SOD, and catalase in neutrophils

Adult (■) and neonatal (□) neutrophils were incubated with MEHP (500 µM), or medium control (Ctl) for 4 h. mRNA expression of the (A) NOX1, (B) SOD, and (C) catalase genes were quantified by real-time PCR. Results were normalized to GAPDH expression. Each bar represents the mean ± SE (n=15). *Significantly different (P<0.05) from adult; ^†Significantly different (P<0.05) from Ctl.

Figure 5. Effects of MEHP on inflammatory mediator production

Adult (■) and neonatal (□) neutrophils were incubated in the presence or absence of MEHP (500 µM) and/or TgT (10 µM) for 24 h. The inflammatory mediators (A) IL-8, (B) IL-1β, (C) RANTES, (D) IL-6, (E) MIP-1β, and (F) VEGF were measured in culture supernatants using cytometric bead array analysis. Each bar represents the mean ± SE (n=10). *Significantly different (P<0.05) from adult; ^†Significantly different (P<0.05) from control; ^‡Significantly different from MEHP.