Intestinal Epithelial Cell Apoptosis, Immunoregulatory Molecules, and Necrotizing Enterocolitis
Journal of Clinical and Cellular Immunology

Journal of Clinical and Cellular Immunology
Open Access

ISSN: 2155-9899

Review Article - (2012) Volume 0, Issue 0

Intestinal Epithelial Cell Apoptosis, Immunoregulatory Molecules, and Necrotizing Enterocolitis

Tamas Jilling*
NorthShore University HealthSystem Research Institute and Northwestern University Feinberg School of Medicine, 2650 Ridge Ave, Evanston, IL 60201, USA
*Corresponding Author: Tamas Jilling, MD, The Ellrodt-Schweighauser Family Chair of Perinatal Research, Evanston Northwestern Healthcare Research Institute, Research Associate Professor, Northwestern University Feinberg School of Medicine, Evanston Hospital, Department Of Pediatrics, 2650 Ridge Ave, Evanston, IL 60201, USA Email:


Necrotizing enterocolitis is one of the most severe, life-threatening consequences of premature birth, affecting 5-15% of premature neonates with birth weights <1,500 grams. Many lines of evidence suggest a role for the dysregulation of enterocyte apoptosis in NEC pathogenesis. In addition to apoptosis, the roles of several inflammatory mediators such as platelet-activating factor, IL-8, TNFα and endotoxin have been shown to be pathogenic. Receptors for these ligands and downstream cellular signaling pathways, such as mitochondrial injury-induced caspase activation and NFĸB-mediated transcriptional regulation are thought to be involved in the mechanisms of mucosal injury in NEC. In this review, we attempt to summarize the role of enterocyte apoptosis in NEC along with an analysis of the connection between inflammatory signaling and apoptosis in this disease.


The nineteenth century French physiologist Claude Bernard, who is considered to be the father of modern physiology, eloquently defined the concept of homeostasis:

“The living body, though it has need of the surrounding environment, is nevertheless relatively independent of it. This independence, which the organism has of its external environment, derives from the fact that in the living being, the tissues are in fact withdrawn from direct external influences and are protected by a veritable internal environment which is constituted, in particular, by the fluids circulating in the body” [1].

One of the most important requirements to maintain this homeostasis is the integrity of epithelial tissues, which constitute the barrier and transport facility between the highly regulated internal milieu and the variable outside world. As it is detailed through various chapters of this book, the intestinal epithelium is a dynamic and complex structure and it is in charge of simultaneously forming a barrier and mediating interactions between the internal milieu of the body and the intestinal content, which is a direct extension of the external environment. This interface is extremely intricate due to the number of transport processes that are required for food digestion, absorption of nutrients and regulation of intestinal luminal environment; the presence of vast quantity and diversity of microbes that inhabit the intestinal lumen; and the diverse immune and immune regulatory processes that take place concurrently. This complexity is compounded by the fact that enterocytes have one of the highest turnover rates of all cell types in the human body, with the entire intestinal epithelial lining being renewed every few days. In the early neonatal period, this rapid enterocyte turnover coincides with fast growth and with the adaptation of the intestine to interactions with the extrauterine environment and to the stress of food intake.

In the early neonatal period, the adjustment from being exposed to the very stable amniotic fluid to processing and absorbing food involves large scale changes in gene expression of transporters, enzymes, and receptors, as well as an adaptation of splanchnic circulation to the rapidly increasing energy consumption of the active intestine and to the need for carrying the absorbed nutrients to the systemic circulation. Another part of this adaptation is the colonization of the lamina propria with cells of the adaptive immune system, which enables the host to mount proper immune reactions to invading pathogens. Yet another part of this process is the adjustment of the innate immune system’s reactivity, allowing the host to accommodate the colonization of probiotic bacteria and to reduce reactivity to low levels of bacterial cell wall components or other microbial constituents that may be present in food normally. All of these adaptation processes have important implications for intestinal diseases occurring in the perinatal period. To orchestrate the processing and absorption of nutrients and the aforementioned various dramatic changes, a number of mediators that are not present in the intestinal microenvironment prior to birth must be released in an orderly fashion after oral feeding begins. The colonization of the lamina propria with lymphocytes, granulocytes and monocytes increases the number of highly reactive cell types that are capable of producing cytokines and chemokines which may have profound effects on epithelial physiology and viability. The proper adjustment of innate immune signaling is essential to preempting any unnecessary inflammatory signaling by the epithelium in reaction to normal intestinal luminal content which, in turn, may trigger additional inflammatory signaling by inflammatory cells that are establishing their presence in the lamina propria.

While the aforementioned growth and adaptation processes progress with high fidelity and without major difficulties in mature neonates, in the premature infant these mechanisms appear to be less than perfect, as up to 15% of premature newborns weighing less than 1500 grams suffer from a catastrophic collapse of enteric integrity following the beginning of oral feeding and are affected by a disease termed necrotizing enterocolitis (NEC). Although our understanding of the exact mechanisms that are responsible for the collapse of mucosal integrity is just emerging, there are a number of mediators and cellular processes that have been identified as major players in pathology.

The risk factors and clinical features of NEC

NEC is a devastating gastrointestinal disease affecting premature infants, and despite recent advances in neonatology, it remains a leading cause of morbidity and mortality in this high-risk population [2]. The disease incidence varies worldwide, but estimates in babies born weighing less than 1500 grams range from 10% in the U.S. to 14% in Argentina and 28% in Hong Kong. In one report, the incidence of death from NEC was just below that from sudden infant death syndrome, a leading cause of death in infants [3]. The number one predictor of risk for NEC is prematurity. There is a very clear and inverse relationship between gestational age and NEC incidence as well as birth weight and NEC incidence [4]. Compromised circulation and oxygenation likely plays a role, since NEC is observed in full term infants only if disordered circulation occurs as a result of major cardiac malformations, open heart surgery, polycythemia, double-exchange transfusion or birth asphyxia and the incidence of NEC in premature neonates who had congenital heart disease is significantly higher than in infants without heart disease [5]. Breast feeding provides some protection from NEC; therefore, formula feeding is considered to be a risk factor. Aberrant bacterial colonization or exposure to pathogens is thought to predispose to NEC, but this area is not very well characterized in detail [6]. Typically, NEC presents several days after initiation of oral feeding with symptoms of distended abdomen, reduced peristalsis and bloody stool, and the disease often progresses to include systemic signs of inflammation. The definitive diagnosis of NEC is made based on the signs of pneumatosis intestinalis and/or gas in the portal circulation on abdominal x-ray. Currently, the mainstay of initial treatment is nonspecific supportive care, and includes the management of systemic inflammation and sepsis. Surgical intervention is provided for signs of intestinal perforation and/or worsening local and systemic signs of disease, and might include a conservative approach with bedside drainage or an exploratory laparotomy with resection of necrotic bowel. There is no clear consensus regarding the benefits of conservative or more invasive surgical management as recent studies have identified similar morbidity and mortality rates amongst these two options. Notably, surgical intervention does not seem to influence mortality in this complex disorder.

The role of intestinal ischemia

The newborn intestinal vasculature exhibits very low resistance, primarily due to an increased baseline and stimulus-induced production of endothelial-derived nitric oxide [7]. This low baseline vascular resistance limits the ability of the neonatal splanchnic vasculature to adapt to systemic decrease of blood supply or oxygen. Furthermore, due to the role of endothelium in maintaining this low vascular resistance, any endothelial dysfunction may lead to severe vasoconstriction. It has been shown that in human neonatal intestinal microvasculature there is severe vasoconstriction in areas of necrosis and that in these submucosal arterioles there is a defective endothelium-mediated autoregulation in response to pressure changes [8]. This is compounded by an exaggerated endothelin-1-dependent vasoconstrictive response [9]. Tissue ischemia is a potent inducer of inflammatory molecules, including platelet-activating factor [10,11].

The role of platelet activating factor homeostasis

Studies from our lab and others have shown that PAF plays an important role in the pathophysiology of intestinal inflammation and NEC in adult rats; for example: 1) exogenous PAF given intravenously results in ischemic bowel necrosis [12], 2) endotoxin, hypoxia, or TNFinduced intestinal injury can be prevented by PAF receptor antagonists [13-15], 3) endotoxin and hypoxia stress increases intestinal PAF content [13]. Additional experiments have evaluated the importance of PAF in neonatal rats using the typical risk factors of NEC, including asphyxia and formula feeding [16]. In this model, we have shown that PAF receptor blockade reduces the incidence of NEC, and that the PAF-degrading enzyme PAF-AH given with enteral feeding interferes with the initiation of NEC [17,18]. Breast milk, which is thought to reduce the risk for NEC, contains significant quantities of this enzyme. Importantly, both PAFR antagonist and PAF-AH protected animals from NEC when they were administered luminally, i.e.; mixed into the formula end without absorbing into the systemic circulation. These data strongly suggest that luminal PAF content and PAFR in the luminal plasma membranes of enterocytes might have important pathological significance.

PAF receptor in enterocytes

The PAFR was originally cloned from guinea pig lung [19], then subsequently from a number of other species. Based on its general architecture PAFR belongs to the seven transmembrane domain, G protein coupled receptors (GPCRs). PAFR is expressed on the surfaces of a broad range of cells, including various leukocytes [20,21], endothelial cells [22], neurons [23] and epithelial cells lining the airways [24] and the GI tract [25]. PAFR is expressed at the highest level in intestinal epithelial cells [26,27], yet its physiological function in these cells is poorly characterized. We have found that the PAFR is localized and functions exclusively in the apical plasma membrane in cultured colonic epithelial cells [25]. PAFR activation by mucosal PAF elicits Cl- transport in colonocytes [25], apoptosis in small intestinal epithelial cells [28] and intracellular acidification in both colonocytes and small intestinal epithelial cells [29]. Strikingly, PAFR can be activated on the mucosal surfaces of colonic epithelial cells only at three orders of magnitude higher PAF concentrations than in non-polarized cells [25]. These findings correlate with reports indicating a similar low affinity activation in airway epithelial cells [24]. The data suggest that the exclusive apical localization of PAFR has a discrete physiological significance, and that the investigation of PAFR targeting and function in epithelial cells has the potential to reveal details that cannot be obtained from non-polarized cells. It will be essential to characterize the molecular determinants of targeting, trafficking and function of the PAFR in polarized epithelial cells. The PAFR belongs to one of the largest receptor families known in biology, the seven transmembrane domain G protein coupled receptors (GPCR). Therefore, knowledge accumulated about the PAFR in intestinal epithelial biology will have relevance to signaling via many others in this large family.

GPCRs in intestinal epithelial cells

Cultured intestinal epithelial cells express a number of GPCRs, such as the VIP receptor [30], secretin receptor [31], β adrenergic receptor [32], angiotensin II and lysophosphatidic acid receptors [33], muscarinic acetylcholine receptor [34], PAF receptor [26], proteinaseactivated receptors 1 and 2 [35,36], purinergic receptors [37] and the thromboxane receptor (our unpublished data), to name a few. Activation of many of these receptors profoundly affects enterocyte proliferation, differentiation and cell death among their regulatory effects on many other cellular functions. The various GPCRs elicit their effects on enterocytes via multiple distinct signaling mechanisms. Many of these signaling mechanisms were characterized based on the pharmacology of agonist-induced epithelial transport properties. For instance, VIP acts primarily through Gαs and elicits a sustained Cl- secretory current via generation of cAMP [38], while purinergic receptor activation results in an increase of intracellular free calcium via G(i/o6) [39], which is a poor direct activator of Cl- currents, but results in a large potentiation of cAMP-induced secretory current. Furthermore, while we have overwhelming evidence that PAFR activation is a potent pro-apoptotic signal for enterocytes [28,29,40], activation of other GPCR-s, such as the lysophosphatidic acid receptor is antiapoptotic [41]. Given that both Gs and G(i/o)-dependent signaling has implications on epithelial proliferation and cell death, GPCR signaling is an important aspect of NEC pathogenesis. The best characterized GPCR in NEC pathogenesis is the PAFR, but many other epithelial GPCRs and their signaling mechanisms warrant further investigation in this matter.

PAFR signaling

Activation of PAF-receptor leads to stimulation of several signal transduction pathways culminating in physiological or pathological regulation of vasoconstriction and/or vasodilatation, leukocyte stimulation and migration, synthesis and activation of cell adhesion molecules, increased capillary permeability, production of reactive oxygen and nitrogen species, and alterations in intestinal mucosal permeability [42,43]. To elicit signaling, the PAFR is capable of linking to both Gs or G(i/o) [44], but it is more commonly found to link to Gi [45,46]. Downstream from G protein activation, PAFR activation results in phosphatidylinositol turnover [47], phosphorylation of signaling proteins, such as β-catenin [48], VE-cadherin [49], ERK1/2 [50], Tyk2 [51], elevation of intracellular free [Ca++] [52], protein kinase C activation [53], and subsequent activation of signal transducers and activators of transcription (STATs) [54,55] and NF?B [56-58]. We identified three major downstream consequences of these PAFRactivation- evoked signaling steps in intestinal epithelial cells that may have significance in NEC pathogenesis. These are 1) regulation of Clchannels [25], 2) regulation of gene expression (our unpublished data) and 3) regulation of apoptosis [28].

Regulation of epithelial ion transport by PAF

Since many GPCRs, including the the VIP receptor [38], secretin receptor [59], β adrenergic receptor [60], muscarinic acetylcholine receptor [61], protease-activated receptors [35], and purinergic receptors [37] regulate ion transport in enterocytes it is very feasible that PAF does the same. In order to verify this expectation we investigated PAF-induced transepithelial ion transport and have found that indeed PAF activates a transepithelial ion flux in HT29- Cl19A polarized colonic epithelial monolayers [25]. We also found that the PAFRs localize to the apical plasma membrane of polarized colonocytes, suggesting interesting implications for the luminal origin of PAF in NEC pathology. In our more recent studies we have found that in addition to regulating transepithelial ion flux, PAFR activation may lead to intracellular acidification due to release of HCO3 - through CLC-3 chloride channels [29]. These findings may be significant because several caspases and apoptosis-related nucleases have acidic pH optima [62] and cytoplasmic acidification has been shown to promote apoptosis [63]. To support this notion, we have found that over-expression of the Na+/H+ exchanger NHE1, or knocking down CLC-3 using shRNA results in inhibition of PAF-induced apoptosis in enterocytes [29].

Regulation of epithelial gene expression by PAF

Many GPCRs that are expressed in epithelial cells are known to regulate gene expression in various cell types. PAFR signaling specifically, is known to regulate gene expression as activation of the PAFR leads to expression of immediate, early oncogenes in rat fibroblasts [64], HEC-1A endometrial carcinoma cells [53], and in A-431 human epithelial carcinoma cells [65]. More importantly from the point of view of enteral health, PAFR activation leads to intestinal TNFα, PLA2-II, PAFR gene expression in an in vivo, PAF-perfusioninduced bowel necrosis model [66-68]. As discussed above, signaling via the PAFR results in the activation of transcription factors such as STAT [55] and NF?B [58,59]. These transcription factors are likely to play major roles in PAF-induced gene expression regulation. We have found that PAF induces expression of PAFR, PLA2-II and toll-like receptor 4 (TLR4) and 2 (TLR2) in enterocytes (our unpublished data). While the upregulation of PAFR and PLA2-II is evidently important because it suggest that there is an autoregulatory positive feedback loop for PAF-induced cellular effects, the upregulation of TLR-s warrants further discussion.

The role of TLR-s and bacterial colonization in NEC

Toll-like receptors have been identified as the class of pattern recognition receptors mediating signaling upon the host’s encounter with microorganisms. There are now over 10 human TLR-s identified; the most prominent example being TLR4, which recognizes lipopolysaccharide, an endotoxin that is present in the cell wall of gram negative bacteria [69]. As one of the predominant pathways following the initial signaling steps of TLR4 activation is I?B ubiquitination and degradation, enabling NF?B translocation to the nucleus and activation of mRNA transcription, primarily of pro-inflammatory cytokines [70]. Data from neonatal rodents and human fetal tissue explants suggest that this pro-inflammatory pathway is prone to excessive activation in the neonatal period, even more so in premature rodents and humans [71,72]. The reason for this hypersensitivity is only partly understood but recent findings have shed some light on the underlying mechanisms. Unlike in the mature, healthy intestinal epithelial cell, where TLR4 is poorly expressed, in the neonate that has experienced asphyxia and formula feeding, TLR4 is up-regulated on the luminal side of the intestinal epithelium, thereby allowing for gram negative bacteria or their cell wall components to activate TLR4 signaling [73]. Another line of evidence suggests that I?B expression in epithelial cells is lower in fetal and premature neonatal intestine and activation of TLR signaling results in higher levels of I?B phosphorylation in these premature cells than in more mature enterocytes [72]. These findings together suggest that a combination of higher receptor expression, diminished inhibitory capacity by I?B and more active signaling contribute to the exaggerated NF?B activation and inflammatory mediator production in the premature gut. The pathological significance of TLR4-dependent and NF?B-mediated proinflammatory signaling in NEC is underscored by the observation in two independent studies that TLR4 mutant mice are protected from experimental NEC and additional findings that NF?B inhibitors reduce the risk for experimental NEC [71,73].

Enterocyte apoptosis in NEC

The layer of enterocytes forms a dynamic barrier between the balanced milieu intérieur [1] and the intestinal luminal content. It has been postulated that a collapse of this barrier is an important step in NEC pathogenesis [74]. One obvious mechanism that may damage the integrity of this barrier is the death of enterocytes on a massive scale. Investigation of human intestinal samples that was obtained during surgery for NEC revealed that there is an abundance of apoptotic nuclei in the epithelium of intestines affected by NEC [75]. To determine whether this observation is a mere coincidence, or whether apoptosis may indeed play a pathological role in NEC, we investigated the role of apoptosis in a well established animal model of NEC. In addition to confirming the findings in the human specimens that NEC is accompanied by profuse enterocyte apoptosis, these animal studies have shown that apoptosis occurs prior to gross histological damage and, more importantly, inhibition of apoptosis by chemical caspase inhibitors preempted the development of experimental NEC [76]. Since this early observation, several studies have shown that the development of experimental NEC is halted by various growth factors that cause enterocyte cell survival, such as EGF [77], IGF-I [78] and hb-EGF [79], or by a blocking antibody to TNFα [80] which is a proapoptotic molecule for enterocytes in vitro [81].

The role of defective repair mechanisms in NEC

Given that there are repair mechanisms to correct the defects in the epithelial lining that are caused by apoptosis, it has been postulated that these repair mechanism are likely to be deficient in premature neonates, otherwise the damage caused by apoptotic death could be bypassed. An evidence for such defective repair mechanisms was found when the role of TLR4 was evaluated in a neonatal murine model of NEC. It was found that in experimental NEC, in addition to increased apoptosis as was shown earlier, there was a reduced epithelial cell proliferation and defective migration along the crypt villus axis; these defects required intact TLR4 signaling and were accompanied with an increased phosphorylation of focal adhesion kinase (FAK) on S722, i.e., a phosphorylation state of FAK that inhibits epithelial cell migration [82]. In vitro, TLR4 activation resulted in FAK phosphorylation and inhibited enterocyte migration.

Mechanisms leading to large scale enterocyte apoptosis

We are only beginning to understand the mechanisms of NEC but there is a convergence of evidence indicating a role for premature and exaggerated enterocyte apoptosis in the disease. To understand the underlying mechanisms that may be responsible for the large scale apoptosis of enterocytes, multiple studies have investigated the mediators and mechanisms regulating enterocyte apoptosis. Cell survival is regulated by a balance of survival and death signals. Cell death is initiated by either a loss of survival signal or by the activation of an active cell death signal. Survival signal for enterocytes comes from three principal sources: 1) cell to extracellular matrix attachment {Strater, 1996 #5668}, 2) homotypic cell adhesion {Ireland, 2004 #5669} and 3) growth factor receptor activation {Clark, 2005 #5413}. Cell death signals may come from either the loss of any of the above 3 survival signals or by evoking a number of cell death signals, including: 1) nutrient deprivation {Lemasters, 2005 #5670}, 2) DNA damage {Okudela, 1999 #5671}, 3) activation of a death receptor pathway {Tang, 2004 #5672}, 4) mitochondrial damage {Lu, 2004 #5013} or 5) signaling that may uncouple any of the survival signals.

Growth factors and NEC

In the neonatal period, an important source of survival signals for enterocytes derives from human milk. There is evidence to indicate that preterm infants who are fed maternal milk are significantly less likely to develop NEC than those infants fed commercial infant formula [83]. Breast milk contains several growth factors that are known to promote epithelial survival such as epidermal growth factor (EGF) [84], heparinbinding EGF-like growth factor (hb-EGF) [85], hepatocyte growth factor (HGF) [86], insulin-like growth factor (IGF) [87], transforming growth factor-beta (TGF-β) [88], erythropoietin [89] and vascular endothelial growth factor (VEGF) [90]. Several of these factors have recently been found to be potentially protective against development of NEC when given as formula supplementation in animal studies [79,91] and all of these growth factors have been shown to promote enterocyte survival either in vivo or in vitro [78,86,92-95]. Commercial formulas are devoid of these growth factors and, therefore, enterocytes of formula-fed infants are deprived of the exogenous supplementation of trophic and survival signals imparted by these molecules. However, NEC is extremely rare in full term infants even if they receive 100% of their nutrition from formula instead of breast milk. This is not necessarily surprising, since there are endogenous sources for all of these growth factors and there is evidence that several pro-apoptotic mechanisms are exaggerated in premature newborns. Nevertheless, a better understanding of endogenous sources for these growth factors and the developmental regulation of their expression in endogenous sources may be important. For instance, EGF and IGF is produced in the salivary gland [96,97] and a recent study has shown a gestationalage dependent increase in salivary EGF output while revealing a correlation between salivary EGF release kinetics and the incidence of NEC [98]. Similar information regarding developmental regulation of endogenous sources of other milk-derived epithelial-protective growth factors is not yet available, but should be investigated. However, it is well established that there are several mechanisms in addition to growth factor withdrawal that may actively signal cell death and several of these appear to play roles in NEC pathogenesis.

Pro-apoptotic signaling in NEC

Some GPCRs have been shown to signal cell survival in enterocytes, such as cholinergic receptors [99] and the lysophosphatidic acid (LPA) receptor [41], and some have shown to induce apoptosis, such as proteinase-activated receptor [100] and PAFR [28]. Since PAF has been shown to have pathogenic significance in NEC, we investigated the mechanisms of PAF induced apoptosis in IEC-6 cells. We have found that PAF induces apoptosis in enterocytes via a sequence of events that involves Bax translocation to mitochondria and the collapse of mitochondrial membrane potential within 30 minutes of exposure to PAF, followed by caspase activation that maximizes by 6 hrs of exposure, which is followed by DNA fragmentation plateauing at 12-16 hrs after PAF treatment [28]. In the same study we have found that heterologous over-expression of Bcl-2, a molecular antagonist of Bax, prevented PAF-induced collapse of mitochondrial membrane potential and apoptosis in these cells, indicating that mitochondrial damage is important in the mechanism of PAF-induced apoptosis, and suggesting that understanding the expression profiles of the Bcl family of apoptosis regulators during intestinal development may be important for our understanding of NEC pathogenesis. In light of our earlier observation that wide-spread enterocyte apoptosis precedes and accompanies gross tissue necrosis in an animal model of NEC and that caspase inhibition prevents the development of experimental NEC [76], our in vitro evidence for PAF-induced enterocyte apoptosis is a significant observation to aid our mechanistic understanding of NEC pathogenesis. Additionally, we have shown that PAF does not only directly regulate apoptosis, but that signaling via the PAFR induces TLR4 expression in enterocytes (unpublished data). TLR4, the receptor for LPS, has been shown to take part in NEC pathogenesis [73,82], causes enterocyte apoptosis [101] and defective enterocyte migration [82]. Furthermore, TLR4 activation leads to induction of inflammatory molecules such as TNFα [102] and nitric oxide (NO) [103]; both of these molecules have been shown to be involved in NEC pathogenesis [15,81,104] and have been shown to be pro-apoptotic for enterocytes [81,105]. It is notable that other GPCRs that are closely related to PAFR may impart either pro or anti-apoptotic signals on enterocytes as discussed above [41,99,100] and the PAFR can cause anti-apoptotic signaling in other cell types that can actively promote inflammation such as PMN [106] and lymphocytes [108]. These findings suggest that we are only beginning to skim the surface of the intricate mechanisms of GPCR-mediated cell death and survival and that other GPCRs may play significant roles in NEC.

Nutrition, PAFR signaling, apoptosis and NEC

A seemingly unlikely convergence of two parallel research areas may offer valuable insights into NEC pathogenesis and potentially into clinically useful NEC prevention strategies. Several years ago, a clinical study that was geared towards evaluating the efficacy of polyunsaturated fatty acid (PUFA) supplementation to premature neonatal formula to improve long term neurodevelopmental outcomes has found, as an incidental finding, that PUFA supplementation reduced the incidence of NEC [108]. In order to verify these findings and to further understand the mechanisms that may be involved in this unexpected beneficial effect of PUFA, in two subsequent studies we have shown that PUFA supplementation to formula dramatically reduced the incidence of NEC in rodent models of NEC [109,110]. Importantly, both in the human study a in our animal model experiments, both n-3 and n-6 PUFA appeared to be protective, which is quite different from some other models where n-3 PUFA is beneficial via antagonizing undesirable pro-inflammatory prostaglandin and leukotriene production from n-6 PUFA precursors [111]. In the mean time, our laboratory was heavily involved in investigating pro-apoptotic signaling mechanisms via PAFR activation. We have found that the earliest step that we can identify in this signaling cascade is a PAFR activation-induced inhibition of the phosphatidylinositol 3 kinase (PI3K) [40]. Due to accumulating evidence indicating that PUFA can inhibit signaling via GPCR-s via a unique mechanism based on an effect on protein acylation and due to the role of PUFA in both human and experimental NEC, in the same study we investigated the effect of polyunsaturated fatty acids on PAFR signaling. We have found that, paralleling our in vivo data, both n-3 and n-6 PUFA antagonized PAFR activation-induced signaling and apoptosis, this effect was independent of prostaglandin synthesis and was mimicked by a synthetic inhibitor of palmitoylation 2Br-palmitate [40]. Palmitoylation is a posttranslational modification on many proteins and it is a covalent attachment of a fatty acyl chain to cysteine residues that are surrounded by basic and aromatic amino acids [112]. Typically, the fatty acyl chain that is involved in such a reaction is palmitate, as the most common saturated fatty acid in cells. The reaction takes place between fatty acyl coenzyme A (CoA) and appropriate cysteine residues in peptide chains even in the absence of enzymes, but there are enzymes known to facilitate both incorporation and hydrolysis of this bond and their expression levels have implications on apoptotic signaling [113,114]. The attachment of saturated fatty acyl chains endow proteins with new characteristics, such as converting cytoplasmic proteins to membrane-bound entities and targeting proteins to cholesterol-rich membrane microdomains, commonly referred to as lipid rafts [115,116]. Palmitoylation is excessively common in the family of GPCR-s and has been thought to be important in the formation of receptor-signal-transduction complexes, by targeting GPCR-s, G proteins and kinases that execute G-protein-dependent signaling to lipid rafts while enhancing signaling efficiency, by creating proximity between molecules that need to interact for signaling to take place [116,117]. It has been shown that PUFA can displace palmitate in normally palmitoylated proteins in a competitive manner and that this displacement of palmitate results in diminished signaling [115,118]. In our most recent study, we were able to document that both n-3 and n-6 PUFA can displace palmitate in C317 of the C terminus cytoplasmic tail of the PAFR (unpublished observation). These data indicate that PUFA are potent modulators of PAFR signaling via a mechanism that is independent of their effect on prostaglandin and leukotriene biosynthesis, and that they may be effective in the prevention of NEC where signaling via the PAFR plays a major role.


Inflammatory signaling and enterocyte apoptosis play important roles in NEC pathophysiology. Based on results from animal models, human sample analysis and based on available data from in vitro experimentation, we hypothesize (Figure 1) that the premature infant, when exposed to risk factors for NEC: 1) exhibits a propensity to produce and react to platelet-activating factor, 2) consequently, PAFR activation on enterocytes leads to abnormally increased TLR4 expression, which in turn, together with bacterial colonization 3) results in a hyperactive innate immune system that is skewed toward a pro-inflammatory response in the intestinal epithelium; these factors lead to intestinal epithelial cell apoptosis, mucosal barrier dysfunction, and necrosis, ultimately resulting in NEC in a subset of patients. There may be several other contributing factors to these alterations, including a dysregulation of splanchnic microcirculation, decreased input of exogenous growth factors due to formula feeding, a deficient production of endogenous growth factors due to prematurity and potential signaling via other pro-apoptotic mechanisms. A better understanding of these mechanisms by future studies and elucidation of the mechanisms of enterocyte apoptosis may lead to new preventive or therapeutic approaches for NEC. Nevertheless, the findings discussed throughout this chapter reaffirm the genius of Claude Bernard, who made his seminal discoveries on the importance of the regulated internal milieu of the human body and they show that understanding the miracles and wonders of a single cell layer that is 15-30 μm thick and is responsible to separate and connect our regulated internal fluids with the widely changing external environment is one of the keys that we need to find to further human health.


Figure 1: The role of inflammatory signaling and apoptosis in NEC.


  1. Bernard C, Dastre A (1878) Leçons sur les phénomènes de la vie communs aux animaux et aux végétaux. J-B Baillière, Paris.
  2. Caplan MS, Jilling T (2001) New concepts in necrotizing enterocolitis. Curr Opin Pediatr 13: 111-115.
  3. Ryder RW, Shelton JD, Guinan ME (1980) Necrotizing enterocolitis: a prospective multicenter investigation. Am J Epidemiol 112: 113-123.
  4. Lemons JA, Bauer CR, Oh W, Korones SB, Papile LA, et al. (2001) Very low birth weight outcomes of the National Institute of Child health and human development neonatal research network, January 1995 through December 1996. NICHD Neonatal Research Network. Pediatrics 107: E1
  5. Dees E, Lin H, Cotton RB, Graham TP, Dodd DA (2000) Outcome of preterm infants with congenital heart disease. J Pediatr 137: 653-659
  6. Schwiertz A, Gruhl B, Lobnitz M, Michel P, Radke M, et al. (2003) Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, full-term infants. Pediatr Res 54: 393-399.
  7. Nankervis CA, Nowicki PT (1995) Role of nitric oxide in regulation of vascular resistance in postnatal intestine. Am J Physiol 268: G949-958.
  8. Nowicki PT, Caniano DA, Hammond S, Giannone PJ, Besner GE, et al. (2007) Endothelial nitric oxide synthase in human intestine resected for necrotizing enterocolitis. J Pediatr 150: 40-45.
  9. Nowicki PT, Dunaway DJ, Nankervis CA, Giannone PJ, Reber KM, et al. (2005) Endothelin-1 in human intestine resected for necrotizing enterocolitis. J Pediatr 146: 805-810.
  10. Shi LC, Wang HY, Friedman E (1998) Involvement of platelet-activating factor in cell death induced under ischemia/postischemia-like conditions in an immortalized hippocampal cell line. J Neurochem 70: 1035-1044.
  11. Noel AA, Hobson RW 2nd, Duran WN (1996) Platelet-activating factor and nitric oxide mediate microvascular permeability in ischemia-reperfusion injury. Microvasc Res 52: 210-220.
  12. Hsueh W, Gonzalez-Crussi F, Arro yave JL (1986) Platelet-activating factor-induced ischemic bowel necrosis. An investigation of secondary mediators in its pathogenesis. Am J Pathol 122: 231-239.
  13. Caplan MS, Kelly A, Hsueh W (1992) Endotoxin and hypoxia-induced intestinal necrosis in rats: the role of platelet activating factor. Pediatr Res 31: 428-434.
  14. Caplan MS, Sun XM, Hsueh W (1990) Hypoxia causes ischemic bowel necrosis in rats: the role of platelet-activating factor (PAF-acether). Gastroenterology 99: 979-986.
  15. Caplan MS, Sun XM, Hseuh W, Hageman JR (1990) Role of platelet activating factor and tumor necrosis factor-alpha in neonatal necrotizing enterocolitis. J Pediatr 116: 960-964.
  16. Caplan MS, Hedlund E, Adler L, Hsueh W (1994) Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis. Pediatric Pathol 14: 1017-1028.
  17. Caplan MS, Lickerman M, Adler L, Dietsch GN, Yu A (1997) The role of recombinant platelet-activating factor acetylhydrolase in a neonatal rat model of necrotizing enterocolitis. Pediatr Res 42: 779-783.
  18. Caplan MS, Hedlund E, Adler L, Lickerman M, Hsueh W (1997) The platelet-activating factor receptor antagonist WEB 2170 prevents neonatal necrotizing enterocolitis in rats. J Pediatr Gastroenterol Nutr 24: 296-301.
  19. Honda Z, Nakamura M, Miki I, Minami M, Watanabe T, et al. (1991) Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung. Nature 349: 342-346.
  20. Nakamura M, Honda Z, Izumi T, Sakanaka C, Mutoh H, et al. (1991) Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J Biol Chem 266: 20400-20405.
  21. Muller E, Dagenais P, Alami N, Rola-Pleszczynski M (1993) Identification and functional characterization of platelet-activating factor receptors in human leukocyte populations using polyclonal anti-peptide antibody. Proc Natl Acad Sci U S A 90: 5818-5822.
  22. Flickinger BD, Olson MS (1999) Localization of the platelet-activating factor receptor to rat pancreatic microvascular endothelial cells. Am J Pathol 154: 1353-1358.
  23. Perry SW, Hamilton JA, Tjoelker LW, Dbaibo G, Dzenko KA, et al. (1998) Platelet-activating factor receptor activation. An initiator step in HIV-1 neuropathogenesis. J Biol Chem 273: 17660-17664.
  24. Tamaoki J, Sakai N, Isono K, Kanemura T, Yamawaki I, et al. (1991) Effects of platelet-activating factor on bioelectric properties of cultured tracheal and bronchial epithelia. J Allergy Clin Immunol 87: 1042-1049.
  25. Claud EC, Li D, Xiao Y, Caplan MS, Jilling T (2002) Platelet-activating factor regulates chloride transport in colonic epithelial cell monolayers. Pediatr Res 52: 155-162.
  26. Merendino N, Dwinell MB, Varki N, Eckmann L, Kagnoff MF (1999) Human intestinal epithelial cells express receptors for platelet-activating factor. Am J Physiol 277: G810-818.
  27. Wang H, Tan X, Chang H, Gonzalez-Crussi F, Remick DG, et al. (1997) Regulation of platelet-activating factor receptor gene expression in vivo by endotoxin, platelet-activating factor and endogenous tumour necrosis factor. Biochem J 322: 603-608.
  28. Lu J, Caplan MS, Saraf AP, Li D, Adler L, et al. (2004) Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 286: G340-350
  29. Claud EC, Lu J, Wang XQ, Abe M, Petrof EO, et al. (2008) Platelet-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells. Am J Physiol 294: G1191-1200.
  30. Sreedharan SP, Huang JX, Cheung MC, Goetzl EJ (1995) Structure, expression, and chromosomal localization of the type I human vasoactive intestinal peptide receptor gene. Proc Natl Acad Sci U S A 92: 2939-2943.
  31. Virgolini I, Yang Q, Li S, Angelberger P, Neuhold N, et al. (1994) Cross-competition between vasoactive intestinal peptide and somatostatin for binding to tumor cell membrane receptors. Cancer Res 54: 690-700.
  32. Devedjian JC, Schaak S, Gamet L, Denis-Pouxviel C, Paris H (1996) Regulation of alpha 2A-adrenergic receptor expression in the human colon carcinoma cell line HT29: SCFA-induced enterocytic differentiation results in an inhibition of alpha 2C10 gene transcription. Proc Assoc Am Physicians 108: 334-344.
  33. Jiang X, Sinnett-Smith J, Rozengurt E (2007) Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells. Cell Signal 19: 1000-1010.
  34. Cummins MM, O'Mullane LM, Barden JA, Cook DI, Poronnik P (2000) Purinergic responses in HT29 colonic epithelial cells are mediated by G protein alpha -subunits. Cell Calcium 27: 247-255.
  35. Buresi MC, Schleihauf E, Vergnolle N, Buret A, Wallace JL, et al. (2001) Protease-activated receptor-1 stimulates Ca(2+)-dependent Cl(-) secretion in human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 281: G323-332.
  36. Jarry A, Dorso L, Gratio V, Forgue-Lafitte ME, Laburthe M, et al. (2007) PAR-2 activation increases human intestinal mucin secretion through EGFR transactivation. Biochem Biophys Res Commun 364: 689-694.
  37. Inoue CN, Woo JS, Schwiebert EM, Morita T, Hanaoka K, et al. (1997) Role of purinergic receptors in chloride secretion in Caco-2 cells. Am J Physiol 272: C1862-1870.
  38. Bertelsen LS, Barrett KE, Keely SJ (2004) Gs protein-coupled receptor agonists induce transactivation of the epidermal growth factor receptor in T84 cells: implications for epithelial secretory responses. J Biol Chem 279: 6271-6279.
  39. Muller T, Bayer H, Myrtek D, Ferrari D, Sorichter S, et al. (2005) The P2Y14 receptor of airway epithelial cells: coupling to intracellular Ca2+ and IL-8 secretion. Am J Respir Cell Mol Biol 33: 601-609.
  40. Lu J, Caplan MS, Li D, Jilling T (2008) Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 294: G1181-1190.
  41. Deng W, Wang DA, Gosmanova E, Johnson LR, Tigyi G (2003) LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway. Am J Physiol Gastrointest Liver Physiol, 284: G821-829.
  42. Benveniste J (1988) Paf-acether, an ether phospho-lipid with biological activity. Prog Clin Biol Res 282: 73-85.
  43. Venable ME, Zimmerman GA, McIntyre TM, Prescott SM (1993) Platelet-activating factor: a phospholipid autacoid with diverse actions. J Lipid Res 34: 691-702.
  44. Levistre R, Masliah J, Bereziat G (1993) Stimulatory and inhibitory guanine-nucleotide-binding regulatory protein involvement in stimulation of arachidonic-acid release by N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor from guinea-pig alveolar macrophages. Differential receptor/G-protein interaction assessed by pertussis and cholera toxins. Eur J Biochem 213: 295-303.
  45. Wang HY, Yue TL, Feuerstein G, Friedman E (1994) Platelet-activating factor: diminished acetylcholine release from rat brain slices is mediated by a Gi protein. J Neurochem 63: 1720-1725.
  46. Teixeira MM, Giembycz MA, Lindsay MA, Hellewell PG (1997) Pertussis toxin shows distinct early signalling events in platelet-activating factor-, leukotriene B4-, and C5a-induced eosinophil homotypic aggregation in vitro and recruitment in vivo. Blood 89: 4566-4573.
  47. Ishii I, Izumi T, Tsukamoto H, Umeyama H, Ui M, et al. (1997) Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants. J Biol Chem 272: 7846-7854.
  48. Boccellino M, Camussi G, Giovane A, Ferro L, Calderaro V, et al. (2005) Platelet-activating factor regulates cadherin-catenin adhesion system expression and beta-catenin phosphorylation during Kaposi's sarcoma cell motility. Am J Pathol 166: 1515-1522.
  49. Hudry-Clergeon H, Stengel D, Ninio E, Vilgrain I (2005) Platelet-activating factor increases VE-cadherin tyrosine phosphorylation in mouse endothelial cells and its association with the PtdIns3'-kinase. FASEB J 19: 512-520.
  50. Hidalgo MA, Ojeda F, Eyre P, LaBranche TP, Smith C, et al. (2004) Platelet-activating factor increases pH(i) in bovine neutrophils through the PI3K-ERK1/2 pathway. Br J Pharmacol 141: 311-321.
  51. Lukashova V, Chen Z, Duhe RJ, Rola-Pleszczynski M, Stankova J (2003) Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus. J Immunol 171: 3794-3800.
  52. Wang X, Bae JH, Kim SU, McLarnon JG (1999) Platelet-activating factor induced Ca(2+) signaling in human microglia. Brain Res 842: 159-165.
  53. Bonaccorsi L, Luconi M, Maggi M, Muratori M, Forti G, et al. (1997) Protein tyrosine kinase, mitogen-activated protein kinase and protein kinase C are involved in the mitogenic signaling of platelet-activating factor (PAF) in HEC-1A cells. Biochim Biophys Acta 1355: 155-166.
  54. Deo DD, Axelrad TW, Robert EG, Marcheselli V, Bazan NG, et al. (2002) Phosphorylation of STAT-3 in response to basic fibroblast growth factor occurs through a mechanism involving platelet-activating factor, JAK-2, and Src in human umbilical vein endothelial cells. Evidence for a dual kinase mechanism. J Biol Chem 277: 21237-21245.
  55. Lukashova V, Asselin C, Krolewski JJ, Rola-Pleszczynski M, Stankova J (2001) G-protein-independent activation of Tyk2 by the platelet-activating factor receptor. J Biol Chem 276: 24113-24121.
  56. Heon Seo K, Ko HM, Kim HA, Choi JH, Jun Park S, et al. (2006) Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation. Cancer Res 66: 4681-4686.
  57. Kravchenko VV, Pan Z, Han J, Herbert JM, Ulevitch RJ, et al. (1995) Platelet-activating factor induces NF-kappa B activation through a G protein-coupled pathway. J Biol Chem 270: 14928-14934.
  58. Venkatesha RT, Ahamed J, Nuesch C, Zaidi AK, Ali H (2004) Platelet-activating factor-induced chemokine gene expression requires NF-kappaB activation and Ca2+/calcineurin signaling pathways. Inhibition by receptor phosphorylation and beta-arrestin recruitment. J Biol Chem 279: 44606-44612.
  59. Fukuda M, Ohara A, Bamba T, Saek Y (2000) Activation of transepithelial ion transport by secretin in human intestinal Caco-2 cells. Jpn J Physiol 50: 215-225.
  60. del Castillo JR, Arevalo JC, Burguillos L, Sulbaran-Carrasco MC (1999) beta-adrenergic agonists stimulate Na+-K+-Cl- cotransport by inducing intracellular Ca2+ liberation in crypt cells. Am J Physiol 277: G563-571.
  61. Huflejt ME, Blum RA, Miller SG, Moore HP, Machen TE (1994) Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells. J Clin Invest 93: 1900-1910.
  62. Barry MA, Eastman A (1993) Identification of deoxyribonuclease II as an endonuclease involved in apoptosis. Arch Biochem Biophys 300: 440-450.
  63. Gottlieb RA, Dosanjh A (1996) Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. Proc Natl Acad Sci U S A 93: 3587-3591.
  64. Kume K, Shimizu T (1997) Platelet-activating factor (PAF) induces growth stimulation, inhibition, and suppression of oncogenic transformation in NRK cells overexpressing the PAF receptor. J Biol Chem 272: 22898-22904.
  65. Tripathi YB, Lim RW, Fernandez-Gallardo S, Kandala JC, Guntaka RV, et al. (1992) Biochem J 286: 527-533.
  66. Huang L, Tan X, Crawford SE, Hsueh W (1994) Platelet-activating factor and endotoxin induce tumour necrosis factor gene expression in rat intestine and liver. Immunology 83: 65-69.
  67. Tan XD, Wang H, Gonzalez-Crussi FX, Chang H, Gonzalez-Crussi F, et al. (1996) Platelet activating factor and endotoxin increase the enzyme activity and gene expression of type II phospholipase A2 in the rat intestine. Role of polymorphonuclear leukocytes. J Immunol 156: 2985-2990.
  68. Wang H, Tan X, Chang H, Gonzalez-Crussi F, Remick DG, et al. (1997) Regulation of platelet-activating factor receptor gene expression in vivo by endotoxin, platelet-activating factor and endogenous tumour necrosis factor. Biochem J 322: 603-608.
  69. Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, et al. (1999) Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med 189: 615-625.
  70. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F (1999) Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 274: 10689-10692.
  71. De Plaen IG, Liu SX, Tian R, Neequaye I, May MJ, et al. (2007) Inhibition of nuclear factor-kappaB ameliorates bowel injury and prolongs survival in a neonatal rat model of necrotizing enterocolitis. Pediatr Res 61: 716-721.
  72. Claud EC, Lu L, Anton PM, Savidge T, Walker WA, et al. (2004) Developmentally regulated IkappaB expression in intestinal epithelium and susceptibility to flagellin-induced inflammation. Proc Natl Acad Sci U S A 101: 7404-7408.
  73. Jilling T, Simon D, Lu J, Meng FJ, Li D, et al. (2006) The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis. J Immunol 177: 3273-3282.
  74. Deitch EA (1994) Role of bacterial translocation in necrotizing enterocolitis. Acta Paediatr Suppl 396: 33-36.
  75. Ford H, Watkins S, Reblock K, Rowe M (1997) The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis. J Pediatr Surg 32: 275-282.
  76. Jilling T, Lu J, Jackson M, Caplan MS (2004) Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis. Pediatr Res 55: 622-629.
  77. Clark JA, Lane RH, Maclennan NK, Holubec H, Dvorakova K, et al. (2005) Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 288: G755-762.
  78. Ozen S, Akisu M, Baka M, Yalaz M, Sozmen EY, et al. (2005) Insulin-like growth factor attenuates apoptosis and mucosal damage in hypoxia/reoxygenation-induced intestinal injury. Biol Neonate 87: 91-96.
  79. Feng J, El-Assal ON, Besner GE (2006) Heparin-binding epidermal growth factor-like growth factor reduces intestinal apoptosis in neonatal rats with necrotizing enterocolitis. J Pediatr Surg 41: 742-747.
  80. Halpern MD, Clark JA, Saunders TA, Doelle SM, Hosseini DM, et al. (2006) Reduction of experimental necrotizing enterocolitis with anti-TNF-alpha. Am J Physiol Gastrointest Liver Physiol 290: G757-764.
  81. Ruemmele FM, Dionne S, Levy E, Seidman EG (1999) TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death. Biochem Biophys Res Commun 260: 159-166.
  82. Leaphart CL, Cavallo J, Gribar SC, Cetin S, Li J, et al. (2007) A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair. J Immunol 179: 4808-4820.
  83. Lucas A, Cole TJ (1990) Breast milk and neonatal necrotising enterocolitis. Lancet 336:1519-1523.
  84. Dvorak B, Fituch CC, Williams CS, Hurst NM, Schanler RJ (2004) Concentrations of epidermal growth factor and transforming growth factor-alpha in preterm milk. Adv Exp Med Biol 554: 407-409
  85. Michalsky MP, Lara-Marquez M, Chun L, Besner GE (2002) Heparin-binding EGF-like growth factor is present in human amniotic fluid and breast milk. J Pediatr Surg 37: 1-6.
  86. Itoh H, Itakura A, Kurauchi O, Okamura M, Nakamura H, et al. (2002) Hepatocyte growth factor in human breast milk acts as a trophic factor. Horm Metab Res 34: 16-20.
  87. Socha P, Janas R, Dobrzanska A, Koletzko B, Broekaert I, et al. (2005) Insulin like growth factor regulation of body mass in breastfed and milk formula fed infants. Data from the E.U. Childhood Obesity Programme. Adv Exp Med Biol 569: 159-163.
  88. Saito S, Yoshida M, Ichijo M, Ishizaka S, Tsujii T (1993) Transforming growth factor-beta (TGF-beta) in human milk. Clin Exp Immunol 94: 220-224.
  89. Ledbetter DJ, Juul SE (2000) Erythropoietin and the incidence of necrotizing enterocolitis in infants with very low birth weight. J Pediatr Surg 35: 178-181
  90. Siafakas CG, Anatolitou F, Fusunyan RD, Walker WA, Sanderson IR (1999) Vascular endothelial growth factor (VEGF) is present in human breast milk and its receptor is present on intestinal epithelial cells. Pediatr Res 45: 652-657.
  91. Dvorak B, Halpern MD, Holubec H, Williams CS, McWilliam DL, et al. (2002) Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model. Am J Physiol Gastrointest Liver Physiol 282: G156-164.
  92. Sheng G, Guo J, Warner BW ( 2007) Epidermal growth factor receptor signaling modulates apoptosis via p38alpha MAPK-dependent activation of Bax in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 293: G599-606.
  93. Nakajima T, Ueda T, Takeyama Y, Yasuda T, Shinzeki M, et al. (2007) Protective effects of vascular endothelial growth factor on intestinal epithelial apoptosis and bacterial translocation in experimental severe acute pancreatitis. Pancreas 34: 410-416.
  94. Jeschke MG, Bolder U, Finnerty CC, Przkora R, Muller U, et al. (2005) The effect of hepatocyte growth factor on gut mucosal apoptosis and proliferation, and cellular mediators after severe trauma. Surgery 138: 482-489.
  95. Kuenzler KA, Pearson PY, Schwartz MZ (2002) Hepatocyte growth factor pretreatment reduces apoptosis and mucosal damage after intestinal ischemia-reperfusion. J Pediatr Surg 37: 1093-1097.
  96. Thesleff I, Viinikka L, Saxen L, Lehtonen E, Perheentupa J (1988) The parotid gland is the main source of human salivary epidermal growth factor. Life Sci 43: 13-18.
  97. Hansson HA, Tunhall S (1988) Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. Immunohistochemical evidence. Acta Physiol Scand 134: 383-389.
  98. Warner BB, Ryan AL, Seeger K, Leonard AC, Erwin CR, (2007) Ontogeny of salivary epidermal growth factor and necrotizing enterocolitis. J Pediatr 150:358-363.
  99. Toledo A, Yamaguchi J, Wang JY, Bass BL, Turner DJ, et al. (2004) Taurodeoxycholate stimulates intestinal cell proliferation and protects against apoptotic cell death through activation of NF-kappaB. Digestive diseases and sciences 49: 1664-1671.
  100. Chin AC, Vergnolle N, MacNaughton WK, Wallace JL, Hollenberg MD, et al. (2003) Proteinase-activated receptor 1 activation induces epithelial apoptosis and increases intestinal permeability. Proc Natl Acad Sci U S A 100: 11104-11109.
  101. Chin AC, Flynn AN, Fedwick JP, Buret AG (2006) The role of caspase-3 in lipopolysaccharide-mediated disruption of intestinal epithelial tight junctions. Can J Physiol Pharmacol 84: 1043-1050.
  102. Ruemmele FM, Beaulieu JF, Dionne S, Levy E, Seidman EG, et al. (2002) Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor alpha. Gut 51: 842-848.
  103. Forsythe RM, Xu DZ, Lu Q, Deitch EA( 2002) Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion. Shock 17:180-184.
  104. Nadler EP, Dickinson E, Knisely A, Zhang XR, Boyle P, et al. (2000) Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis. J Surg Res 92: 71-77.
  105. Nishikawa M, Takeda K, Sato EF, Kuroki T, Inoue M (1998) Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells. Am J Physiol 274: G797-801.
  106. Biffl WL, Moore EE, Moore FA, Barnett CC Jr (1996) Interleukin-6 delays neutrophil apoptosis via a mechanism involving platelet-activating factor. J Trauma 40: 575-578.
  107. Toledano BJ, Bastien Y, Noya F, Baruchel S, Mazer B (1997) Platelet-activating factor abrogates apoptosis induced by cross-linking of the surface IgM receptor in a human B lymphoblastoid cell line. J Immunol 158: 3705-3715.
  108. Carlson SE, Montalto MB, Ponder DL, Werkman SH, Korones SB (1998) Lower incidence of necrotizing enterocolitis in infants fed a preterm formula with egg phospholipids. Pediatr Res 44: 491-498.
  109. Caplan MS, Russell T, Xiao Y, Amer M, Kaup S, et al. (2001) Effect of polyunsaturated fatty acid (PUFA) supplementation on intestinal inflammation and necrotizing enterocolitis (NEC) in a neonatal rat model. Pediatr Res 49: 647-652.
  110. Lu J, Jilling T, Li D, Caplan MS (2007) Polyunsaturated fatty acid supplementation alters proinflammatory gene expression and reduces the incidence of necrotizing enterocolitis in a neonatal rat model. Pediatr Res 61: 427-432.
  111. James MJ, Gibson RA, Cleland LG (2000) Dietary polyunsaturated fatty acids and inflammatory mediator production. Am J Clin Nutr 71: 343S-348S.
  112. Qanbar R, Bouvier M (2003) Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function. Pharmacol Ther 97: 1-33.
  113. Cho S, Dawson G (2000) Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt-caspase pathway in neuroblastoma cells. J Neurochem 74: 1478-1488.
  114. Cho S, Dawson PE, Dawson G (2000) Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA-N-5 neuroblastoma cells. J Neurosci Res 62: 234-240.
  115. Webb Y, Hermida-Matsumoto L, Resh MD (2000) Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275: 261-270.
  116. Moffett S, Brown DA, Linder ME (2000) Lipid-dependent targeting of G proteins into rafts. J Biol Chem 275: 2191-2198.
  117. Kraft K, Olbrich H, Majoul I, Mack M, Proudfoot A, et al. (2001) Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. J Biol Chem 276: 34408-34418.
  118. Liang X, Nazarian A, Erdjument-Bromage H, Bornmann W, Tempst P, et al. (2001) Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction. J Biol Chem 276: 30987-30994.
Citation: Jilling T, Lu J,Caplan MS (2012) Intestinal Epithelial Cell Apoptosis, Immunoregulatory Molecules, and Necrotizing Enterocolitis. J Clin Cell Immunol S3:007.

Copyright: © 2012 Jilling T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.