Lipoxin A4 is synthesized by neutrophils late during the inflammatory process and evidence suggests that it is involved in triggering the resolution of the response (24
). This is due, in part, to the ability of lipoxin A4 to promote the clearance of senescent neutrophils by macrophages, and to block adhesion and respiratory burst activity (1
). Spontaneous apoptosis was reduced in neonatal neutrophils relative to adults, and LPS suppressed apoptosis in both cell types. We also found that, while lipoxin A4 augmented apoptosis in LPS-treated adult cells, neonatal neutrophils were not responsive. While some previous investigators have reported that synthetic analogues of lipoxin do not affect neutrophil survival (13
), our finding in LPS-treated adult cells is consistent with previous reports that aspirin-triggered lipoxin and it analogs induce apoptosis in neutrophils exposed to inflammatory stimuli (17
). The induction of neutrophil apoptosis by lipoxin A4 under inflammatory conditions may be of biologic importance in reducing oxidant-mediated cytotoxicity. Our findings that neonatal cells are hyporesponsive are consistent with increased susceptibility of neonates to inflammatory diseases and suggest that clearance of neonatal neutrophils from sites of infection is not dependent on lipoxin A4.
Neutrophils accumulate in tissues in response to chemotactic factors generated at sites of infection or injury. Inflammatory cytokines and bacterial-derived products also trigger the generation of reactive oxygen intermediates, which are key in bacterial killing and may also play a role in tissue injury (33
). The present studies demonstrate that lipoxin A4 suppressed chemotaxis and respiratory burst activity in adult neutrophils, which is in accord with its anti-inflammatory effects. However, lipoxin A4-induced inhibition of chemotaxis and respiratory burst activity was significantly attenuated or absent in neonatal neutrophils. These findings are consistent with our results on apoptosis and suggest that the ability of lipoxin A4 to abrogate neutrophilic extravasation and limit tissue injury is impaired in neonates.
The biologic activities of lipoxin A4 are mediated, in part, via the membrane-bound G-protein coupled receptor, FPRL1 (also known as ALXR). Despite impaired responsiveness of neonatal neutrophils to lipoxin A4, these cells expressed greater levels of FPRL1 protein than adult cells. These data indicate that expression of FPRL1 is not a rate-limiting step in the response to lipoxin A4 in neonatal neutrophils. It is possible that over-expression of FPRL1 in neonatal neutrophils is a compensatory response to reduced lipoxin A4-mediated activation of downstream signaling pathways. In this regard, we have previously demonstrated that the activities of phosphoinositol-3 kinase and caspase 3, two signaling molecules up regulated following FPRL1 activation, are impaired in neonatal relative to adult neutrophils (5
PPAR-γ is a nuclear transcription factor that down regulates inflammation (34
). In the lung, PPAR-γ agonists have been shown to reduce neutrophil accumulation during endotoxemia (35
), and to down regulate Cox-2, ICAM-1 and p-selectin expression (36
). PPAR-γ activation triggers expression of target genes such as NGAL, which binds to and sequesters inflammatory mediators, including bacterial formyl peptides and leukotriene B4, and stimulates neutrophil apoptosis (37
). Previous studies have implicated 15-Lox and the lipoxin precursor, 15S-HETE, in the activation of PPAR-γ (27
). Consistent with this, we found that lipoxin A4 up regulated expression of PPAR-γ, as well as NGAL in adult neutrophils. In contrast, no effects were observed in neutrophils from neonates. Moreover, constitutive expression of PPAR-γ was significantly reduced in neonatal neutrophils, when compared to adult cells. These data suggest a potential mechanism underlying persistent activation of neonatal neutrophils at inflammatory sites.
Previous studies have shown that PGE2, which is produced early in the inflammatory response (1−2 hr), functions as a “primer” to induce of pathways important in down regulating the response, including 5-Lox and 15-Lox, which catalyze the generation of anti-inflammatory eicosanoids (24
). The present studies demonstrate that exposure of adult neutrophils to PGE2 primed these cells to respond to lipoxin A4, resulting in increased expression of PPAR-γ. In contrast, in neonatal cells pretreated with PGE2, there appear to be developmental defects in responsiveness to lipoxin A4. Thus in neonatal cells, LXA4 reduced expression of PPAR-γ, as well as NGAL. Moreover, lipoxin A4 alone had no significant effect on the cells. Interestingly, although PGE2 down regulated expression of 5-Lox and 15-Lox in both adult and neonatal neutrophils, PGE2 primed neutrophils to respond to LPS, a potent inducer of Lox expression during infection. However, this was only evident in adult cells. Hyporesponsiveness to lipoxin A4 and LPS in neonatal neutrophils, primed by exposure to PGE2 to exhibit the late-inflammatory phenotype, may lead to prolonged activity of pro-inflammatory eicosanoids, and exacerbate tissue injury. Of interest is our observation that culturing neutrophils from both adults and neonates resulted in increased constitutive expression of PPAR-γ, NGAL, and Lox. This may be attributed to changes in cellular responsiveness associated with adherence to culture dishes (41
The present studies show that the anti-inflammatory effects of lipoxin A4 in neutrophils are reduced in neonatal relative to adult cells. This may be due, in part, to defective activation of PPAR-γ in response to lipoxin A4 during the late phase of the inflammatory response. Impaired activity of lipoxygenases may further contribute to this effect. Previous studies have demonstrated that neutrophil hyporesponsiveness to lipoxin A4 may play a role in the pathogenesis of specific chronic inflammatory diseases in children, such as periodontitis (42
). Similarly, developmental impairment of specific lipoxin A4-mediated responses may increase the susceptibility of neonates to prolonged neutrophilic inflammation. Our findings suggest that novel therapeutic approaches using lipoxin A4 or its analogs may ameliorate inflammatory diseases in neonates.