Exposure of hospitalized newborns to phthalates is a major health concern because large quantities are infused intravenously with procedures such as blood transfusions (27
). DEHP exposure may also occur via mechanical ventilator tubes and lipid infusions, resulting in exposures more than three orders of magnitude higher in neonates relative to non-hospitalized infants and children (3
). We hypothesized that MEHP exerts pro-inflammatory activity in neutrophils, and that this may increase susceptibility to chronic diseases. To test this, we compared the effects of MEHP on inflammatory activity of adult and neonatal neutrophils. Because serum phthalate levels in hospitalized neonates have not been reported, MEHP concentrations used in this study were calculated based on measurements in adults after prolonged exposure. Platelet apheresis can provide up to approximately 2 mg of DEHP (33
). Extrapolating to a continuous intravenous infusion in a 1 kg infant with a blood volume of 100 ml, and assuming rapid metabolism to MEHP, this would yield a serum concentration of approximately 1 mM. While the rate of metabolism of MEHP in neonates is not known, it has a half-life of 6.3 hr in mature animals (34
). Therefore, doses of 100 to 500 µM are expected to be within the magnitude of levels to which neutrophils may be exposed in the blood of NICU neonates.
Increased susceptibility of newborns to bronchopulmonary dysplasia and other inflammatory diseases is thought to be due to impaired resolution of inflammation and clearance of neutrophils by apoptosis (35
). Consistent with this, we have previously reported that apoptosis is reduced in neonatal neutrophils relative to adult cells; moreover, this is related to decreased responsiveness to Fas ligand and anti-inflammatory eicosanoids (14
). Findings in the present studies that MEHP suppresses apoptosis, and that this effect is greater in neonatal, when compared to adult neutrophils, suggest that exposure of neonates to phthalates may further impair the clearance of neutrophils, exacerbating inflammatory conditions in these patients.
Neutrophils accumulate in tissues in response to chemokines generated at sites of infection or injury. IL-8 is a potent neutrophil chemoattractant; it also up-regulates expression of cell adhesion molecules (37
). The present studies demonstrate that MEHP augmented fMLP-induced chemotaxis in adult, but not neonatal cells. This correlated with increased production of IL-8 by adult neutrophils. MEHP has also been shown to induce calcium flux and up-regulate expression of the integrin, CD11b, which are important in cell motility (10
). It has previously been reported that calcium mobilization and expression of CD11b are developmentally impaired in neonatal neutrophils (25
). These defects, together with the inability of MEHP to stimulate IL-8 production in neonatal cells, may contribute to the inhibitory effects of MEHP on chemotactic responses in these cells.
Inflammatory cytokines and bacterial-derived products trigger the generation of reactive oxygen species (40
). The present studies show that both adult and neonatal neutrophils constitutively generate significant quantities of H2
. However, the rate of production and total amount of H2
generated were greater in neonatal cells, which is consistent with our previous observations (18
). MEHP was found to stimulate H2
production by neonatal, but not adult neutrophils. These findings are in accord with reports that phthalates induce oxidative metabolism in neonatal neutrophils (41
). We also found that MEHP up-regulated NOX1 expression, but only in neonatal cells. NOX1 is the major enzymatic mediator of superoxide anion generation in neutrophils (42
). Increased expression of NOX1 by neonatal neutrophils is consistent with previous reports that in response to inflammatory stimuli, these cells produce greater amounts of superoxide anion relative to adult cells (43
). Of note is our observation that constitutive expression of NOX1 was lower in neonatal relative to adult cells. These findings are in accord with reports that constitutive production of superoxide anion is low or undetectable in these cells (44
). Interestingly, constitutive expression of SOD and catalase was elevated in neonatal neutrophils, when compared to adult cells, which supports the idea that these antioxidants are key to protecting neonates from reactive oxygen species generated in fetal and maternal circulation (45
). In contrast to its stimulatory effects on NOX1 and oxidative metabolism in neonatal neutrophils, MEHP had no effect on expression of SOD or catalase. MEHP-induced increases in NOX1 and production of reactive oxygen species occur in the absence of increases in antioxidant production. Consistent with the idea that oxidative activity is increased in neonates, the proportion of phthalates excreted as mono (2-ethyl-5-carboxypentyl) phthalate, an oxidative metabolite of DEHP, is markedly increased in neonates when compared to adults (31
). Increased reactive oxygen species may also contribute to increased susceptibility of neonates to oxidant-induced tissue injury, characterized by sustained inflammation leading to cytotoxicity, apoptosis, and fibrosis.
Additional comparative studies revealed that constitutive production of IL-1β, IL-6, IL-8, MIP-1β, RANTES, and VEGF was significantly reduced in neonatal neutrophils when compared to adult cells. These findings are in accord with previous reports that the generation of cyokines and chemokines is developmentally impaired in neonatal cells (46
). Whereas in adult neutrophils, MEHP suppressed the production of MIP-1β, in neonatal cells, it suppressed RANTES production. MIP-1β and RANTES are CC chemokines that act primarily on monocytes and macrophages (47
). Down regulation of these mediators by MEHP suggests that the pro-inflammatory effects of MEHP are mainly directed towards neutrophils. This is supported by our findings that MEHP stimulated IL-8 production in adult cells. The fact that this was not evident in neonatal cells is in accord with our findings that MEHP blocks chemotaxis in these cells. MEHP was also found to up-regulate IL-1β and VEGF production in both cell types. IL-1β is a marker of neutrophil activation during chronic inflammatory disease (48
), and VEGF has been shown to mediate neutrophil adhesion and migration (49
). Increased production of these mediators may represent an important mechanism contributing to MEHP-induced neutrophilic inflammation in both adults and neonates.
PPAR-γ is a nuclear transcription factor important in down-regulating the production of pro-inflammatory cytokines and reactive nitrogen species during the resolution phase of inflammation (50
). PPAR-γ agonists have been shown to reduce neutrophil-mediated lung and liver injury during endotoxemia (51
). Previous studies have shown that phthalates bind to PPAR-γ, suppressing its activity (23
). The PPAR-γ agonist TgT attenuated MEHP-mediated suppression of apoptosis and stimulation of oxidative metabolism by neonatal neutrophils. These data suggest that MEHP modulates these activities in neonates by inhibiting anti-inflammatory signaling via PPAR-γ. In contrast, TgT had no effect on MEHP-induced alterations in production of inflammatory proteins or chemokines by adult or neonatal neutrophils, indicating that these effects are mediated by PPAR-γ-independent pathways. Interestingly, TgT suppressed the effects of MEHP on chemotaxis in adult neutrophils, suggesting that that the role of PPAR-γ signaling in chemotaxis is developmentally regulated. Developmental alterations in PPAR-γ signaling are also supported by our observation that adult and neonatal neutrophils display differential sensitivity to the effects of TgT alone. For example, TgT stimulated apoptosis and inhibited IL-8 production in adult, but not neonatal cells. It may be that PPAR-γ plays a role in regulating neutrophil longevity and migration in adult neutrophils, and that these pathways are impaired in neonatal cells. It has been reported that prostaglandin J2 and other eicosanoids may be endogenous ligands for PPAR-γ, but the role of these mediators in neonatal disease is not known (52
Clinical case reports indicate that phthalates may contribute to neonatal inflammatory disease. For example, high levels of DEHP have been reported in the gastrointestinal tissue of infants who succumbed to necrotizing enterocolitis (54
), and phthalate exposure has been implicated in chronic lung disease in premature infants (55
). The present studies demonstrate that MEHP induces oxidative metabolism and up regulates expression of NOX1 in neonatal neutrophils. This is associated with reduced apoptosis and chemotaxis. Taken together, these data suggest that neonatal neutrophils are more sensitive to phthalate-mediated inhibition of PPAR-γ signaling, which may be related to decreased basal anti-inflammatory signaling via this pathway. Understanding the inflammatory effects of phthalates in neonates may support efforts to limit or discontinue the use of phthalate-containing medical devices in neonates.