Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis

Abstract

Background: The early signaling events in the development of necrotizing enterocolitis (NEC) remain undefined. We have recently shown that the endotoxin (lipopolysaccharide [LPS]) receptor toll-like receptor 4 (TLR4) on enterocytes is critical in the pathogenesis of experimental NEC. Given that the membrane receptor CD14 is known to facilitate the activation of TLR4, we now hypothesize that endotoxemia induces an early upregulation of CD14 in enterocytes and that this participates in the early intestinal inflammatory response in the development of NEC.

Methods: IEC-6 enterocytes were treated with LPS (50 microg/mL), and the subcellular localization of CD14 and TLR4 was assessed by confocal microscopy. C57/Bl6 or CD14-/- mice were treated with LPS (5 mg/kg), whereas experimental NEC was induced using a combination of gavage formula feeding and intermittent hypoxia. CD14 expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse transcriptase-polymerase chain reaction, and interleukin 6 was quantified by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction.

Results: Exposure of IEC-6 enterocytes to LPS led to an initial, transient increase in CD14 expression. The early increase in CD14 expression was associated with internalization of CD14 to a perinuclear compartment where increased colocalization with TLR4 was noted. The in vivo significance of these findings is suggested as treatment of mice with LPS led to an early increase in CD14 expression in the intestinal mucosa, whereas the persistent endotoxemia of experimental NEC was associated with decreased CD14 expression within enterocytes.

Conclusions: LPS signaling in the enterocyte is marked by an early, transient increase in expression of CD14 and redistribution of the receptor. This process may contribute to the early activation of the intestinal inflammatory response that is observed in the development of NEC.

Figure 1. CD14 expression exhibits a transient response to LPS stimulation in IEC-6 cells

(A) IEC-6 cells were stimulated with LPS (50 µg/ml) for 0 to 24 hours. SDS-PAGE and band density analysis of lysates demonstrates a peak of CD14 protein at 6 hours after treatment followed by a gradual return to near baseline levels by 24 hours. (B) IEC-6 cells stimulated with LPS demonstrate an increase in phosphorylation of the MAPK p38 and ERK after 20 minutes of stimulation. (C) Analysis of the media of LPS-stimulated IEC-6 cells by ELISA demonstrates a significant increase in IL-6 levels at 6 hours after stimulation (1.0 +/− 0.1 vs. 7.1 +/− 2.6 fold increase, *p<0.05). IL-6 levels return to baseline by 24 hours (7.1 +/− 2.6 vs. 1.2 +/− 0.2 fold increase, *p<0.05).

Figure 2. CD14 is internalized along with TLR4 in IEC-6 cells stimulated with LPS

IEC-6 cells were stimulated with media alone or LPS (50 µg/ml) for 1 hour and prepared for confocal immunofluorescence microscopy. A–C: Untreated cells; D–F – LPS-treated cells. Shown are the expression of CD14 (panels A, D), TLR4 (B, E) or colcoalization (C, F), as calculated using ImageJ software.

Figure 3. Endotoxemia stimulates an early CD14-dependent inflammatory response in mice

(A) C57Bl/6 mice were injected with LPS (5mg/kg) and sacrificed at 3 hours. RT-PCR analysis of ileal mucosa mRNA demonstrates increased expression of CD14 and IL-6 as compared to control mice. (B) SDS-PAGE of ileal mucosa from WT mice exposed to LPS for 3 hours similarly demonstrates increased CD14 expression as compared to saline-treated mice. (C) SDS-PAGE of ileal mucosa from WT mice exposed to LPS for 3 hours demonstrates increased phosphorylation of p38 and ERK as compared to saline-treated mice. (D) C57Bl/6 mice were sacrificed at 0, 3 and 14 hours after injection with LPS. Analysis of serum IL-6 by ELISA demonstrates a significant increase in levels from 0 to 3 hours (5.5 +/− 4.5 vs. 8656 +/− 1541.6, *p<0.05) followed by a significant return to baseline by 14 hours (8656 +/− 1541.6 vs. 27.6 +/− 18.2, *p<0.05). (E) C57Bl/6 mice and CD14−/− mice were sacrificed at 3 hours after injection with LPS. CD14−/− mice demonstrated a significant decrease in IL-6 levels as compared to WT mice (14445.5 +/− 137.6 vs. 568.6 +/− 83.5, *p<0.05). (F) The phosphorylation of MAPK in the ileal mucosa of CD14−/− mice compared to WT mice sacrificed 3 hours after injection with LPS is markedly decreased.

Figure 4. The transient response of CD14 expression to LPS is implicated in the development of necrotizing enterocolitis

C57Bl/6 mice were subjected to an experimental model of NEC and sacrificed at 5 days. (A) Semi-quantitative RT-PCR analysis of ileal mucosa mRNA and (B) western blot analysis of ileal mucosal scrapings demonstrate decreased expression of CD14 as compared to control mice.