Surfactant protein-D attenuates the lipopolysaccharide-induced inflammation in human intestinal cells overexpressing toll-like receptor 4.
OBJECTIVE
Necrotizing enterocolitis (NEC) is a devastating inflammatory disease of preterm infants that may depend on overexpression of toll-like receptor-4 (TLR4) in the immature intestine. Surfactant protein (SP)-D is a member of the collectin family and plays an important role in innate immunity, particularly in the airways. Although SP-D also exists in the intestines, little is known about its function. This study investigated whether SP-D can attenuate the inflammatory response of TLR4-overexpressing embryonal intestinal cells.


METHODS
All experimental procedures were performed using the human intestinal cell line INT407 originally derived from human embryonal intestines. Platelet-activating factor (PAF), reported to be elevated in NEC patients, was used to induce TLR4 overexpression in the human embryonal intestinal cell line INT407. TLR4 expression was measured using quantitative real-time PCR. Inflammatory responses to PAF (5 µM), the TLR4 agonist lipopolysaccharide (LPS, 100 ng/ml), PAF + LPS, and PAF + LPS following SP-D pretreatment (20 µg/ml) were assessed by enzyme-linked immunosorbent assay (ELISA) of interleukin-8 (IL-8) release (in pg/ml).


RESULTS
Expression of TLR4 mRNA (mean ± SD) was upregulated by PAF (369 % ± 28 %, p < 0.001). Stimulation with PAF + LPS resulted in higher IL-8 release (1959.3 ± 52.3) than control (141.2 ± 12.4), LPS (167.3 ± 65.8), or PAF (1527.2 ± 129.4) treatment (p < 0.05). Release in response to PAF + LPS (1590.1 ± 319.3) was attenuated by SP-D pretreatment (1161.6 ± 131.6; p < 0.05).


CONCLUSIONS
SP-D attenuates LPS-induced IL-8 production in TLR4-overexpressing intestinal cells, suggesting that SP-D may have a protective effect in the development of NEC in preterm infants.
Department of Pediatric Surgery, Hyogo College of Medicine, Nishinomiya, Japan.|Department of Pediatric Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, 5650871, Japan.|Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan.
Ryuta
R
Saka
Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan.|Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Japan.
Tetsu
T
Wakimoto
Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan.
Fumiko
F
Nishiumi
Department of Pediatric Surgery, Hyogo College of Medicine, Nishinomiya, Japan.
Takashi
T
Sasaki
Department of Pediatric Surgery, Hyogo College of Medicine, Nishinomiya, Japan.
Satoko
S
Nose
Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan.
Masahiro
M
Fukuzawa
Department of Pediatric Surgery, Hyogo College of Medicine, Nishinomiya, Japan.
Takaharu
T
Oue
Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan.
Itaru
I
Yanagihara
Department of Pediatric Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, 5650871, Japan. okuyama@pedsurg.med.osaka-u.ac.jp.
Hiroomi
H
Okuyama
D018929:Cell Culture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D008070:Lipopolysaccharides; D037663:Pulmonary Surfactant-Associated Protein D / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D051197:Toll-Like Receptor 4 / Q000187:drug effects / Q000378:metabolism*
D008070:Lipopolysaccharides; D037663:Pulmonary Surfactant-Associated Protein D; D051197:Toll-Like Receptor 4
Lipopolysaccharide; Necrotizing enterocolitis; Platelet-activating factor; Surfactant protein; Toll-like receptor-4