Abstract
BackgroundChemokine CX3CL1 possesses unique properties, including combined adhesive and chemotactic functions. Human amniotic epithelial cells (HAEC) show expression of CX3CL1 receptor (CX3CR1) and produce CX3CL1 in response to both physiologic and pathologic stimuli. Chorioamnionitis (ChA) is a common complication of pregnancy and labour. ChA is often accompanied by local hypoxia because of the high oxygen consumption at the site of inflammation. We examined comparatively (ChA-complicated vs. normal pregnancy) CX3CR1 expression and the effects of hypoxia, lipopolysaccharide (LPS), and CX3CR1 blockade on CX3CL1 production in HAEC cultured in vitro.MethodsHAEC have been isolated using trypsinization, and cultured under normoxia (20% O2) vs. hypoxia (5% O2). According to the experimental design, LPS (1 μg/ml) and neutralizing anti-CX3CR1 antibodies were added at respective time points. Mean CX3CL1 concentration in the supernatant samples were determined by ELISA. Expression of immunostained CX3CR1 was analyzed using quantitative morphometry.ResultsWe have found that the mean levels of CX3CL1 and CX3CR1 expression were remarkably (p < 0.05) higher in ChA, compared to normal pregnancy. Significantly increased expression of CX3CR1 was observed in ChA during both normoxia and hypoxia. Hypoxia exposure produced decrease in the mean concentration of CX3CL1 in both groups, however this reduction was stronger in normal pregnancy. In normoxia, LPS-evoked rise in the mean concentration of CX3CL1 was higher (p < 0.05) in normal pregnancy. This response was positively correlated with CX3CR1 expression. Blockade of CX3CR1 canceled the secretory response to LPS in all groups.ConclusionsChA-complicated pregnancy up-regulates CX3CR1 in HAEC cultured in vitro with simultaneous increase in CX3CL1 production. Hypoxia-resistant production of CX3CL1 may be responsible for ChA-related complications of pregnancy and labor.
Highlights
Chemokine CX3CL1 possesses unique properties, including combined adhesive and chemotactic functions
The immunoenzymatic assay procedure (ELISA) used in this study revealed significant (p < 0.05) differences in the mean CX3CL1 concentration between the two main groups as well as between the subgroups, including altered secretory responses of Human amniotic epithelial cells (HAEC) to LPS challenge
After in vitro establishment of the HAEC cultures (5-6 days following enzymatic digestion of the amnion samples), the mean levels of CX3CL1 were significantly (p < 0.05) higher in ChA compared to normal pregnancy specimens and amounted to 38.75 ± 7.48 and 39.99 ± 7.11 vs. 12.31 ± 3.28 and 13.49 ± 2.65
Summary
Chemokine CX3CL1 possesses unique properties, including combined adhesive and chemotactic functions. We examined comparatively (ChA-complicated vs normal pregnancy) CX3CR1 expression and the effects of hypoxia, lipopolysaccharide (LPS), and CX3CR1 blockade on CX3CL1 production in HAEC cultured in vitro. Located on human chromosome 16, CX3CL1 is the only CX3C (delta) subfamily member, with three amino acid residues located between the first two cysteine residues in the molecular formula. It has been previously shown that the main roles of membrane-bound CX3CL1 include promotion of leukocyte binding and adhesion, as well as activation of target cells. The soluble chemokine domain of human CX3CL1 is chemotactic for T cells and monocytes [8]. Its dual role as an adhesive particle and a chemoattractant makes CX3CL1 unique. Most stimuli that influence cell homeostasis potentially induce CX3CL1 secretion [9]
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