Xiao Di Tan

Neonatal Necrotizing Enterocolitis: Clinical Considerations and Pathogenetic Concepts

Necrotizing enterocolitis (NEC), a disease affecting predominantly premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. Although several predisposing factors have been identified, such as prematurity, enteral feeding, and infection, its pathogenesis remains elusive. In the past 20 years, we have established several animal models of NEC in rats and found several endogenous mediators, especially platelet-activating factor (PAF), which may play a pivotal role in NEC. Injection of PAF induces intestinal necrosis, and PAF antagonists prevent the bowel injury induced by bacterial endotoxin, hypoxia, or challenge with tumor necrosis factor-a (TNF) plus endotoxin in adult rats. The same is true for lesions induced by hypoxia and enteral feeding in neonatal animals. Human patients with NEC show high levels of PAF and decreased plasma PAF-acetylhydrolase, the enzyme degrading PAF. The initial event in our experimental models of NEC is probably polymorphonuclear leukocyte (PMN) activation and adhesion to venules in the intestine, which initiates a local inflammatory reaction involving proinflammatory mediators including TNF, complement, prostaglandins, and leukotriene C4. Subsequent norepinephrine release and mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. Bacterial products (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and endotoxin synergizes with PAF to amplify the inflammation. Reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final pathway for tissue injury. Protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase, and indigenous probiotics such as Bifidobacteria infantis. The former maintains intestinal perfusion and the integrity of the mucosal barrier, and the latter keep virulent bacteria in check. The development of tissue injury depends on the balance between injurious and protective mechanisms.

Milk fat globule–EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium

Milk fat globule-EGF factor 8 (MFG-E8)/lactadherin participates in several cell surface-mediated regulatory events. Although its mRNA is present in the gut, the physiological roles of MFG-E8 in the intestinal mucosa have not been explored. Here we show that MFG-E8 was expressed in intestinal lamina propria macrophages from mice. Using a wound-healing assay, MFG-E8 was shown to promote the migration of intestinal epithelial cells through a PKCepsilon-dependent mechanism. MFG-E8 bound to phosphatidylserine and triggered reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Depleting MFG-E8 in mice by administration of anti-MFG-E8 antibody or targeted deletion of the MFG-E8 gene resulted in a slowing of enterocyte migration along the crypt-villus axis and focal mucosal injury. Moreover, in septic mice, intestinal MFG-E8 expression was downregulated, which correlated with intestinal injury, interrupted enterocyte migration, and impaired restitution. Treatment with recombinant MFG-E8 restored enterocyte migration, whereas deletion of MFG-E8 impeded mucosal healing in mice with sepsis. These results suggest that a decrease in intestinal MFG-E8 impairs intestinal mucosal repair in sepsis. Together, our data indicate that MFG-E8 plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing and suggest that recombinant MFG-E8 may be beneficial for the treatment of bowel injuries.

Platelet-activating factor, tumor necrosis factor, hypoxia and necrotizing enterocolitis

The pathogenesis of necrotizing enterocolitis (NEC) is poorly understood. We have established several animal models of NEC by using a combination of various stimuli and stress, including endotoxin, PAF, TNF, and hypoxia. We discuss the mechanism of their actions and the possible roles of these factors in the pathogenesis of human NEC.

Intestinal NF-κB is activated, mainly as p50 homodimers, by platelet-activating factor

Abstract NF-κB, a transcription factor, upregulates gene transcription of many inflammatory mediators. Here, we examined the activity of NF-κB in the rat small intestine, and how it may be affected by platelet-activating factor (PAF), an important mediator for intestinal injury and inflammation. Ileal nuclear extracts from sham-operated and PAF (1.5 μg/kg)-injected rats were prepared for the assessment of NF-κB DNA-binding activity, and the identification of NF-κB subunits. The experiment was also performed on neutrophil-depleted rats to examine whether the PAF effect is neutrophil-dependent. Cellular NF-κB was localized by immunohistochemistry. We found that: (a) NF-κB is constitutively active in rat small intestine; (b) PAF at a dose below that causing shock and bowel necrosis enhances DNA-binding activity of NF-κB within 30 min after injection; activated NF-κB contains predominantly p50 subunits; (c) immunohistochemistry showed that PAF induced translocation of p50 into the nucleus of cells of the lamina propria, as well as of the epithelium; and (d) the effect of PAF is abrogated by neutrophil depletion, suggesting a role of neutrophils in NF-κB activation. Our study suggests that NF-κB is weakly active constitutively in the intestine, and inflammatory stimuli such as PAF activate NF-κB and enhance its DNA-binding activity in the intestine, which contains predominantly p50 subunits.

Lipopolysaccharide activates nuclear factor κB in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor

We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor κB (NF‐κB) activation in the intestine and the roles of endogenous platelet‐activating factor (PAF), tumour necrosis factor‐α (TNF) and neutrophils. We also compared the time course of NF‐κB activation in response to PAF and LPS. Ileal nuclear extracts from LPS (8 mg kg−1, IV)‐injected rats were assayed for NF‐κB‐DNA‐binding activity and identification of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti‐TNF antibody, or anti‐neutrophil antiserum. NF‐κB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 μg kg−1, IV) for comparison. LPS activates intestinal NF‐κB, both as p50‐p50 and p50‐p65 dimers within 15 min, and the effect peaks at 2 h. The effect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF‐κB was immunolocalized within epithelial and lamina propria cells. LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti‐TNF antibody, or anti‐neutrophil antiserum (P<0.05). LPS activates intestinal NF‐κB in vivo and neutrophil activation is involved in its action. The LPS effect is mediated by both endogenous PAF and TNF.

PAF and TNF increase the precursor of NF-kappa B p50 mRNA in mouse intestine: quantitative analysis by competitive PCR

NF-kappa B, a nuclear transcription factor, is involved in the regulation of inflammatory cytokines. We have previously reported that PAF and TNF induce intestinal injury in rats and mice and the interaction of TNF and PAF probably plays a central role in its pathogenesis. In the present study, we developed a competitive PCR method to quantitate the transcripts of NF-kappa B p50/p105 gene, and investigated the effects of PAF and TNF on p50/p105 gene expression in the small intestine of C3H/HeN mice - p105 is the precursor of the p50 subunit of NF-kB. We found that NF-kappa B p50/p105 gene is constitutively expressed in the normal small intestine in small quantities (7.05 +/- 1.04 attomol/micrograms total RNA). PAF at a dose (1 microgram/kg) causing no systemic changes (e.g., hypotension, hemoconcentration), markedly increased intestinal p50/p105 transcripts within 30 min. TNF, at dose (1 mg/kg) also insufficient to induce systemic changes, increased intestinal p50/p105 gene expression, although its effect was much slower than PAF. The effect of TNF was not blocked by WEB 2086, a PAF antagonist. Our results indicate that both PAF and TNF stimulate the expression of NF-kappa B p50/p105 in vivo. However, the mechanisms of their respective actions are probably different.

Endotoxin, but not platelet-activating factor, activates nuclear factor-κB and increases IκBα and IκBβ turnover in enterocytes

Bacterial endotoxin (lipopolysaccharide; LPS) and platelet‐activating factor (PAF) are important triggers of bowel inflammation and injury. We have previously shown that LPS activates the transcription factor nuclear factor (NF)‐κB in the intestine, which up‐regulates many pro‐inflammatory genes. This effect partly depends on neutrophils and endogenous PAF. However, whether LPS and PAF directly activate NF‐κB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF‐κB activation in IEC‐6 (a non‐transformed rat small intestinal crypt cell line) cells, by electrophoresis mobility shift assay and supershift, and found that LPS, but not PAF, activates NF‐κB mostly as p50–p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF‐κB‐dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF‐κB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IκBα and IκBβ. We found that TNF causes rapid degradation of IκBα and IκBβ. In contrast, LPS did not change the levels of IκBα and IκBβ up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IκBα and IκBβ, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IκBα and IκBβ and therefore activates NF‐κΒ via at least two mechanisms: initially, through an IκB‐independent mechanism, and later, via an increased turnover of the inhibitor IκB. NF‐κΒ activation precedes the gene expression of iNOS (assayed by reverse transcription–polymerase chain reaction), suggesting that LPS up‐regulates iNOS via this transcription factor.

Roles of nitric oxide synthases in platelet-activating factor-induced intestinal necrosis in rats.

OBJECTIVE To examine the role of constitutive and inducible nitric oxide synthases (cNOS and iNOS) in platelet-activating factor (PAF)-induced shock and intestinal injury. DESIGN Prospective, randomized, controlled experimental study. SETTING Hospital research laboratory. SUBJECTS Young adult male Sprague-Dawley rats were anesthetized and studied. INTERVENTIONS Rats were injected with PAF, either alone or after the following pretreatments: a) selective iNOS inhibitors aminoguanidine or S-methylisothiourea; b) 3-morpholinosydnonimine, a NO donor; c) S-methylisothiourea + 3-morpholinosydnonimine; and d) antineutrophil antibody (to deplete neutrophils). MEASUREMENTS AND MAIN RESULTS Blood pressure, hematocrit, white blood cell counts, intestinal injury, and intestinal cNOS and iNOS activities were assessed. We found that: a) cNOS is the predominant NOS in the intestine and its activity is inversely correlated to the level of tissue injury; b) there is a time-dependent increase in cNOS activity in sham-operated animals, which was abolished by PAF; c) Western blotting and immunohistochemistry showed iNOS present in the normal intestine, localizing mainly in crypt cells; d) iNOS inhibitors attenuated PAF-induced injury in animals with high cNOS activity, but had no protective effect in animals with low cNOS activity; e) 3-morpholinosydnonimine, alone or together with S-methylisothiourea, alleviated PAF-induced injury; and f) neutrophil depletion blocked the suppressive effect of PAF on cNOS and prevented injury. CONCLUSIONS We conclude that cNOS and iNOS play different roles in PAF-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of iNOS inhibitors.

Milk fat globule-EGF factor 8 is a critical protein for healing of dextran sodium sulfate-induced acute colitis in mice

Milk fat globule-EGF factor 8 (MFG-E8) has been shown to play an important role in maintaining the integrity of the intestinal mucosa and to accelerate healing of the mucosa in septic mice. Herein, we (a) analyzed the expression of MFG-E8 in the gut of wild-type (WT) C57BL/6 (MFG-E8+/+) mice with and without dextran sulfate sodium (DSS)-induced colitis, (b) characterized the pathological changes in intestinal mucosa of MFG-E8+/+ and MFG-E8-/- mice with DSS-induced colitis and (c) examined the therapeutic role of MFG-E8 in inflammatory bowel disease by using DSS-induced colitis model. Our data documented that there was an increase in colonic and rectal MFG-E8 expression in MFG-E8+/+ mice during the development of DSS colitis. MFG-E8 levels in both tissues decreased to below baseline during the recovery phase in mice with colitis. Changes in MFG-E8 gene expression correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in MFG-E8+/+ mice. MFG-E8-/- mice developed more severe crypt-epithelial injury than MFG-E8+/+ mice during exposure to DSS with delayed healing of intestinal epithelium during the recovery phase of DSS colitis. Administration of MFG-E8 during the recovery phase ameliorated colitis and promoted mucosal repair in both MFG-E8-/- and MFG-E8+/+ mice, indicating that lack of MFG-E8 causes increased susceptibility to colitis and delayed mucosal healing. These data suggest that MGF-E8 is an essential protective factor for gut epithelial homeostasis, and exogenous administration of MFG-E8 may represent a novel therapeutic target in inflammatory bowel disease.

Lysozyme-Modified Probiotic Components Protect Rats against Polymicrobial Sepsis: Role of Macrophages and Cathelicidin-Related Innate Immunity

Severe sepsis is associated with dysfunction of the macrophage/monocyte, an important cellular effector of the innate immune system. Previous investigations suggested that probiotic components effectively enhance effector cell functions of the immune system in vivo. In this study, we produced bacteria-free, lysozyme-modified probiotic components (LzMPC) by treating the probiotic bacteria, Lactobacillus sp., with lysozyme. We showed that oral delivery of LzMPC effectively protected rats against lethality from polymicrobial sepsis induced by cecal ligation and puncture. We found that orally administrated LzMPC was engulfed by cells such as macrophages in the liver after crossing the intestinal barrier. Moreover, LzMPC-induced protection was associated with an increase in bacterial clearance in the liver. In vitro, LzMPC up-regulated the expression of cathelicidin-related antimicrobial peptide (CRAMP) in macrophages and enhanced bactericidal activity of these cells. Furthermore, we demonstrated that surgical stress or cecal ligation and puncture caused a decrease in CRAMP expression in the liver, whereas enteral administration of LzMPC restored CRAMP gene expression in these animals. Using a neutralizing Ab, we showed that protection against sepsis by LzMPC treatment required endogenous CRAMP. In addition, macrophages from LzMPC-treated rats had an enhanced capacity of cytokine production in response to LPS or LzMPC stimulation. Together, our data suggest that the protective effect of LzMPC in sepsis is related to an enhanced cathelicidin-related innate immunity in macrophages. Therefore, LzMPC, a novel probiotic product, is a potent immunomodulator for macrophages and may be beneficial for the treatment of sepsis.