- [Show abstract] [Hide abstract] ABSTRACT: Platelet-activating factor (PAF), an endogenous proinflammatory phospholipid, when injected intravascularly to rats and mice, causes shock, acute bowel injury, and a rapid activation of NF-kappaB p50-p50 with upregulation of the chemokine CXCL2 in the intestine. In this study, we investigate the mechanism of NF-kappaB activation and the role of the NF-kappaB p50 subunit in PAF-induced shock and acute bowel injury. NF-kappaB p50-deficient mice and wild-type mice were anesthetized and tracheotomized, and their carotid artery was cannulated for blood pressure monitoring, blood sampling, and PAF administration. For determination of bowel injury, shock, and survival, PAF (2.2 microg/kg, intra-arterially, i.a.) was injected. Two hours later, animals were euthanized, and their small intestines were removed for histological examination. For biochemical studies, PAF (1.5 microg/kg i.a.) was administered and the small intestine removed after 15-60 min. We found that PAF induced an increase in p105 processing within 30 min, but there were no changes in the levels of the NF-kappaB inhibitory proteins IkappaBalpha and beta. NF-kappaB p50-deficient mice were protected against PAF-induced mortality, shock, intestinal hypoperfusion, and injury compared with wild-type animals. We also found that p50-deficient mice had decreased gene expression of CXCL2 and TNF and a decrease in CXCL2 protein production compared with wild-type mice. Our study suggests that PAF increases the processing of NF-kappaB p105 into p50, with upregulation of proinflammatory cytokines, which leads to PAF-induced systemic inflammatory response and acute bowel injury.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-κB (NF-κB). It remains unknown, however, whether NF-κB mediates injury in neonatal NEC. We therefore examined the activation status of NF-κB perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-κB is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-κB remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-κB inhibitor protein IκBα and IκBβ at d 2. To determine the importance of elevated NF-κB activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IκB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-κB activity. Our findings therefore lead us to conclude that selective NF-κB inhibition represents a promising therapeutic strategy for NEC.Abbreviations: EMSA, electrophoretic mobility shift assay; IκB, inhibitor protein κB; IKK, IκB kinase; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NBD, NEMO-binding domain; NEC, necrotizing enterocolitis; NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; PAF, platelet-activating factor
- [Show abstract] [Hide abstract] ABSTRACT: Backgroud: Necrotizing enterocolitis (NEC), a disease of premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. Its known predisposing factors include prematurity, enteral feeding, and infection, but the pathogenesis is still obscure. Data sources and Results: Several animal models of NEC have been established in adult and neonatal rats and mice. Endogenous mediators, especially platelet-activating factor (PAF), play a pivotal role in NEC. Injection of PAF results in intestinal necrosis, and PAF antagonists prevent the bowel injury induced by endotoxin, hypoxia, or tumor necrosis factor-α (TNF) in adult rats. PAF antagonists or PAF-acetylhydrolase, the enzyme degrading PAF, also ameliorate the pathology of the neonatal model induced by hypoxia and enteral feeding. Human patients with NEC have elevated levels of plasma PAF and decreased PAF-acetylhydrolase. However, to exert its injurious action, PAF requires the presence of commensal bacteria in the intestine. The initial event in our models of NEC is probably polymorphonuclear leukocytes (PMN) activation and adhesion to venules in the intestine, probably in response to local PAF release. Other molecules and mediators taking part in the process include NF-κB, TNF, chemokines (especially MIP-2/CXCL2), and the complement system, leading to a local inflammatory reaction. Subsequent release of vasoconstrictors and the consequent mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. Toxins from commensal bacteria (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and synergize with PAF and TNF to amplify the inflammation. Reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final effector for tissue injury. Protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase (which maintains intestinal perfusion and the integrity of the mucosal barrier), and the innate immune response, enhanced by indigenous probiotics such as Bifidobacteria and Lactobacilli.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: CXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-kappaB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-kappaB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF- and LPS- induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-kappaB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein kappaB kinase (IKK) activation, a major upstream kinase mediating NF-kappaB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-kappaB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.
- [Show abstract] [Hide abstract] ABSTRACT: We reported previously that neuronal nitric oxide synthase (nNOS) is the predominant NOS in rat small intestine and is down-regulated by platelet-activating factor (PAF). The severity of the bowel injury induced by PAF is inversely related to its suppressing effect on nNOS. Here, we investigated whether intestinal perfusion is regulated by nNOS and whether tetrahydrobiopterin, a co-factor and stabilizer of nNOS, reverses PAF-induced intestinal hypoperfusion and injury. Animal laboratory. We first examined nNOS regulation of splanchnic blood flow by measuring the perfusion of the heart, lung, ileum, and kidney in rats after a nNOS inhibitor. We then examined the protective effect of tetrahydrobiopterin on PAF-induced bowel injury, mesenteric hypoperfusion, and systemic inflammation. Adult male Sprague-Dawley rats. In part 1 of the experiment, rats were given 7-nitroindazole (a specific nNOS inhibitor, 50 mg.kg.day). In part 2 of the experiment, rats were treated with tetrahydrobiopterin (20 mg/kg) 5 mins before and 30 mins after PAF challenge (2.2 microg/kg, intravenously) Perfusion of the heart, lung, ileum, and kidney was measured at 1 and 4 days after 7-nitroindazole, using fluorescent microspheres. Intestinal injury and inflammation (myeloperoxidase content), blood perfusion, calcium dependent-NOS activity, and systemic inflammation (hypotension and hematocrit increase) were assessed 1 hr after PAF with and without tetrahydrobiopterin treatment. In part 1 of the experiment, 7-nitroindazole induced a long-lasting reduction of blood perfusion and inducible NOS expression selectively in the ileum but not in nonsplanchnic organs such as heart, lungs, and kidneys. In part 2, tetrahydrobiopterin protected against PAF-induced intestinal necrosis, hypoperfusion, neutrophil influx, and NOS suppression. It also reversed hypotension and hemoconcentration. Sepiapterin (2 mg/kg, stable tetrahydrobiopterin precursor) also attenuated PAF-induced intestinal injury. We conclude that nNOS selectively regulates intestinal perfusion. Tetrahydrobiopterin prevents PAF-induced intestinal injury, probably by stabilizing nNOS and maintaining intestinal perfusion.
- [Show abstract] [Hide abstract] ABSTRACT: Platelet-activating factor (PAF) is a potent endogenous mediator of bowel inflammation. It activates neutrophils that are needed to initiate the inflammatory response. Macrophage inflammatory protein-2 (MIP-2), a critical C-X-C chemokine secreted by macrophages and epithelial cells, is a potent chemoattractant for neutrophils. Whereas MIP-2 has been previously shown to mediate the injury in various organs, its role in acute intestinal injury has never been assessed. In this study, we first investigated the effect of PAF on MIP-2 expression in the intestine. Anesthetized young adult male Sprague-Dawley rats were injected intravenously with either PAF (1.5 microg/kg) or saline. Sixty minutes later, ileal MIP-2 gene expression was determined by semiquantitative RT-PCR, and plasma and ileal MIP-2 protein was determined by ELISA. In a second step, we assessed the role of MIP-2 in PAF-induced bowel injury. Rats were pretreated with rabbit anti-rat MIP-2 antibodies or control IgG for 90 min and then injected intravenously with PAF (2.5 microg/kg) for 90 min. We found that, in the rat intestine, 1) MIP-2 mRNA was only minimally expressed constitutively in sham-operated animals; 2) MIP-2 mRNA was significantly upregulated in response to PAF; 3) MIP-2 protein plasma levels and local production of MIP-2 in the ileum were markedly induced by PAF; 4) the administration of anti-rat MIP-2 IgG, but not control rabbit IgG, markedly reduced PAF-induced bowel injury (injury scores of 0.19 +/- 0.09 vs. 1.12 +/- 0.43, P < 0.05), hypotension, and leukopenia but did not reduce PAF-induced hemoconcentration. Thus we conclude that MIP-2 mediates PAF-induced intestinal injury.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: 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)-kappaB 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-kappaB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF-kappaB 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-kappaB mostly as p50-p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF-kappaB-dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF-kappaB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IkappaBalpha and IkappaBbeta. We found that TNF causes rapid degradation of IkappaBalpha and IkappaBbeta. In contrast, LPS did not change the levels of IkappaBalpha and IkappaBbeta up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IkappaBalpha and IkappaBbeta, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IkappaBalpha and IkappaBbeta and therefore activates NF-kappaBeta via at least two mechanisms: initially, through an IkappaB-independent mechanism, and later, via an increased turnover of the inhibitor IkappaB. NF-kappaBeta activation precedes the gene expression of iNOS (assayed by reverse transcription-polymerase chain reaction), suggesting that LPS up-regulates iNOS via this transcription factor.
- [Show abstract] [Hide abstract] ABSTRACT: We previously showed that group II phospholipase A2 (PLA2-II), a secretory, bactericidal, and proinflammatory protein in intestinal crypts, is upregulated after lipopolysaccharide (LPS) and platelet-activating factor (PAF) challenge. Here we examined whether germ-free environment (GF) or antibiotic treatment (ABX) affects the pathophysiological responses and intestinal PLA2-II activity after PAF (1.5 microg/kg) or LPS (8 mg/kg) injection. We found that LPS and PAF induced hypotension and mild intestinal injury in conventionally fed (CN) rats; these changes were milder in ABX rats, whereas GF rats showed no intestinal injury. PLA2-II enzyme activity was detected in normal rat small intestine; the basal level was not diminished in ABX or GF rats. PAF and LPS caused an increase in PLA2-II activity, which was abrogated in GF and ABX rats. Recolonization of GF rats by enteral contamination restituted their PLA2-II response to PAF and LPS and susceptibility to bowel injury. We conclude that PAF- and LPS-induced increases in PLA2-II activity are dependent on gut bacteria, and ABX and GF rats are less susceptible to LPS-induced injury than CN rats.
- [Show abstract] [Hide abstract] ABSTRACT: The transcription factor family CCAAT/enhancer binding proteins (C/EBP) is involved in inflammation via the regulation of the gene expression of various pro-inflammatory cytokines and proteins. PAF and endotoxin (lipopolysaccharide, LPS) are known agents causing intestinal inflammation and injury. In this study, we examined the binding activity of C/EBP isoforms in rat small intestine in response to PAF (1.5 μg kg−1, i.v.) or LPS (5 mg kg−1, i.v.). We found that C/EBP is constitutively active in normal small intestine, mainly as C/EBP-α and β (C/EBP-β>α). Both C/EBP-α and β are localized in the intestinal epithelial cells: C/EBP-α mainly in the crypts, and C/EBP-β in both villi and crypts, as well as in some lamina propria cells. Only minute amounts of C/EBP-δ were found. PAF rapidly upregulates the binding activity of C/EBP-α and β within 30 min. The increase in C/EBP-α is prominent in the crypt cells, whereas the change of C/EBP-β is more widespread. LPS also increases the binding activity of C/EBP-α and β, and the response is slower than PAF. PAF synergizes with LPS to markedly activate all three subunits. The increase in C/EBP-α is transient, whereas the other two have a sustained elevation until 120 min. After challenge with PAF (but not LPS), small amounts of nuclear factor -κB (NF-κB) p50 and p65 subunits are found in the C/EBP-DNA binding complex, indicating cross-dimerization of the two transcription families. Pretreatment of rats with pyrrolidine dithiocarbamate (PDTC) suppresses LPS-, but not PAF-, induced NF-κB and C/EBP binding activity, and significantly increases the C/EBP-δ subunit in LPS- or PAF-induced C/EBP complex. These results suggest that PAF and LPS activate intestinal C/EBP in vivo, probably via different pathways. British Journal of Pharmacology (2001) 133, 713–721; doi:10.1038/sj.bjp.0704102
- [Show abstract] [Hide abstract] ABSTRACT: We previously reported that neuronal nitric oxide synthase (nNOS) is the predominant NOS in the intestine. Inducible NOS (iNOS), an enzyme involved in the inflammatory response, is regulated by cytokines via the transcriptional factor NF-kappaB. We examined a new mechanism of intestinal iNOS regulation with respect to the role of nNOS and its effect on NF-kappaB. Young Sprague-Dawley rats were treated for 4 days with 1) saline, 2) 7-nitroindazole (7-NI, specific nNOS inhibitor), 3) 7-NI + pyrrolidine dithiocarbamate (PDTC, NF-kappaB inhibitor), or 4) PDTC. Intestinal iNOS mRNA, NF-kappaB activity, and the tissue content of the regulatory IkappaBalpha were examined. We found that 7-NI-treated animals had higher intestinal NF-kappaB (p50-p65) activity, lower IkappaBalpha content, and increased intestinal iNOS mRNA, iNOS protein, and iNOS activity compared with controls. All of these changes were abolished when PDTC was given together with 7-NI. PDTC alone had no effect. 7-NI induces a delayed increase in intestinal myeloperoxidase activity (after elevation in NF-kappaB and iNOS), which could be abrogated by PDTC. We conclude that in normal rat small intestine, nNOS suppresses the gene expression of iNOS through NF-kappaB down-regulation and that nNOS suppression leads to IkappaBalpha degradation, NF-kappaB activation, and iNOS expression.
- [Show abstract] [Hide abstract] ABSTRACT: Ab-based therapies have undergone a renaissance in recent years, but infusion-related reactions are a significant clinical problem. Administration of certain mAbs to Swiss Webster mice infected with Cryptococcus neoformans can result in acute lethal toxicity (ALT) characterized by cardiovascular collapse. The ability of a mAb to produce ALT is isotype dependent and occurs with IgG1 but not IgG3. To investigate this phenomenon, we measured spleen and liver cytokine responses and platelet-activating factor (PAF) content in mice given C. neoformans glucuronoxylomannan (GXM) followed by specific Ab of IgG1 or IgG3 isotype. We found no evidence to suggest that the differences in IgG1 and IgG3 toxicity were due to differences in chemokine or cytokine response. In contrast, liver and spleen tissue PAF content was significantly greater in mice IgG1. Furthermore, our results show differences in the response to IgG1- and IgG3-GXM complexes regarding: 1) macrophage-inflammatory protein-1alpha and monocyte chemoattractant protein-1 regulation, 2) splenic and hepatic PAF content, and 3) hepatic PAF content in infected mice. IgG1-associated ALT appears to be the result of greater production of PAF in response to IgG1-GXM complex formation. The results are consistent with the view that IgG1 and IgG3 interact with different Fc receptors. Our findings strongly suggest that the mechanism for Ab-mediated ALT is different from the cytokine release syndrome described after administration of other therapeutic mAbs.
- [Show abstract] [Hide abstract] ABSTRACT: 1. Platelet-activating factor (PAF), an inflammatory mediator, plays an important role in mediating intestinal injury. However, it remains unclear whether PAF has a function in the intestine. The production of PAF by normal intestine and by unstimulated intestinal epithelial cell lines suggests that PAF may have a regulatory function in the normal bowel. 2. In this study we investigated the role of PAF in modulating intestinal mucosal permeability in rats. Lumen-to-blood transit of FD-4 (dextran 4400), (an index of intestinal permeability), was assessed in sham-operated rats and rats injected with PAF (1.25 microg kg(-1), i.v., a dose insufficient to induce intestinal injury). 3. PAF-induced villus cytoskeletal changes were examined by staining the intestine for F-actin. The effect of PAF on tyrosine phosphorylation of the junctional protein E-cadherin was examined by immunoprecipitation. Some rats were pretreated with AG1288 (a tyrosine kinase inhibitor) before PAF injection, and mucosal permeability change was assessed. 4. To investigate the role of endogenous PAF upon mucosal permeability, we studied the effect of PAF antagonists on (intraluminal) glucose-induced increase in mucosal permeability. 5. We found that low dose PAF: (a) alters the cytoskeletal structure of intestinal epithelium, (b) causes the influx of FD4 from intestinal lumen to systemic circulation, (c) induces tyrosine phosphorylation of E-cadherin and cadherin-associated proteins. Glucose-induced mucosal permeability increase is abolished by using two structurally different PAF antagonists. 6. These results suggest that endogenous PAF modulates macromolecular movement across the intestinal mucosal barrier, probably via tyrosine phosphorylation of E-cadherin and cytoskeletal alteration of enterocytes.
- [Show abstract] [Hide abstract] ABSTRACT: 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. British Journal of Pharmacology (2000) 129, 307–314; doi:10.1038/sj.bjp.0703055
- [Show abstract] [Hide abstract] ABSTRACT: Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca(2+)-dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT-PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine.
- [Show abstract] [Hide abstract] ABSTRACT: Platelet-activating factor (PAF) is a potent mediator involved in bowel injury. We investigated PAF receptor transcription and its mRNA localization in the small intestine of normal (conventionally fed) and germ-free rats, by competitive polymerase chain reaction (PCR) and in situ hybridization. A dose of PAF (1.5 microg/kg, i.v.) insufficient to cause gross bowel injury was injected into rats. Some rats were pretreated with dexamethasone (1 mg/kg). We found: (1) PAF receptor (PAF-R) mRNA localized predominantly in lamina propria eosinophils and in epithelial cells; (2) PAF increased PAF-receptor signals in the epithelial cells; (3) Dexamethasone depleted eosinophils in the intestine and markedly decreased PAF-receptor transcripts; the response to PAF was also weaker than control rats; (4) Germ-free rats had less PAF-R mRNA than normal rats, and showed a weaker response to PAF than conventionally fed rats. Thus, we conclude: (1) PAF receptor mRNA is constitutively expressed in the epithelium and in lamina propria eosinophils in the intestine. (2) PAF-R transcription is up-regulated by PAF and gut flora, mostly in the epithelium. (3) PAF-R transcription is down-regulated by glucocorticoids, mainly as a result of eosinophil depletion. These results suggest a functional role for PAF receptors both in host defence and the inflammatory response in the small intestine.
Alabama A & M UniversityHuntsville, Alabama, United States
Northwestern UniversityEvanston, Illinois, United States
Washington University in St. LouisSan Luis, Missouri, United States