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Results for “Intestinal Barrier Function”

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PubMed1983

[Vesicular peroxidase transport by the epithelial cells of the small intestine in the adult rat].

Electron cytochemistry has shown that after administering horse radish peroxidase to the gastrointestinal tract of adult rats it penetrates the epithelial barrier of the small intestine and is transported functionally intact into the circulation. Peroxidase was largely absorbed on the apex of the villi, entered the enterocytes by pinocytosis and was transported via the cytoplasm within the pinocytotic vesicles. Peroxidase was not detected in cytosole or endoplasmic reticulum of the enterocytes. It is assumed that the disclosed functional heterogeneity of absorption depends on adsorption selectivity of the microvilli of the enterocyte apical membrane. It is concluded that the pinocytotic mechanism also functions in adult mammals, by which foreign protein is transported to the circulation, causing allergic reactions.

Animals↗

PubMed2006

Role of intestinal epithelial cells in the innate immune defence of the pig intestine.

The intestinal epithelium serves as a dynamic barrier, which in the course of its normal function, maintains regulated uptake of nutrients and water while excluding potential pathogens. Over the past decade many studies have also revealed the immunological importance of intestinal epithelial cells (IEC). IEC have developed a variety of mechanisms to reduce the risk of infection by invasive pathogens or damage by toxic compounds. The effective maintenance of a physical barrier function is dependent on the establishment of well-organised intercellular junctions and a constant state of regeneration/renewal of the epithelium. IEC also participate in the innate immune responsiveness of the intestine by their ability to secrete mucus and antimicrobial peptides. IEC are also able to secrete cytokines and to respond to exogenous chemokines. This review summarises the current knowledge of the innate immune mechanisms developed by porcine IEC.

Animals↗

PubMed1996

Regulation by prostaglandin E2 of interleukin release by T lymphocytes in mucosa.

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E2 in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E2 is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E2 modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E2's regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E2 concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E2, when present in as low a concentration as 10(-10) M. PGE2 also inhibited (by > or = 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E2 dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [3H] thymidine uptake studies suggested this was not due to a prostaglandin E2-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E2, e.g., high concentrations (10(-6) M) of prostaglandin E2 inhibited IL-3 release by only 41%. We conclude that prostaglandin E2 in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there.

Animals↗

PubMed2004

Inhibition of oxidant-induced nuclear factor-kappaB activation and inhibitory-kappaBalpha degradation and instability of F-actin cytoskeletal dynamics and barrier function by epidermal growth factor: key role of phospholipase-gamma isoform.

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-gamma (PLC-gamma), which prevents NF-kappaB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 microg) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H(2)O(2)). Stably overexpressing PLC-gamma (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma (-99%) substantially prevented all measures of EGF protection against NF-kappaB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappaB; 2) Activation of PLC-gamma by itself is protective against NF-kappaB activation; 3) the ability to modulate the dynamics of NF-kappaB/I-kappa Balpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.

Actins↗

PubMed2006

Inflammatory bowel disease and the apical junctional complex.

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the underlying interstitium. This intestinal barrier is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised in a number of intestinal diseases, including inflammatory bowel disease (IBD). In vitro studies have demonstrated that proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), that are increased in the intestinal mucosa of patients with IBD can induce a leaky mucosal barrier. There is a growing evidence that the increased permeability and altered AJC structure observed in IBD are mediated by internalization of junctional proteins. This review summarizes barrier defects observed in IBD and addresses mechanisms by which proinflammatory cytokines, such as IFN-gamma and TNF-alpha, modulate AJC structure and epithelial barrier function.

Adherens Junctions↗

PubMed2006

Disruption of tight junctions and induction of proinflammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni.

Campylobacter jejuni is a leading cause of human enterocolitis and is associated with postinfectious complications, including irritable bowel syndrome and Guillain-Barré syndrome. However, the pathogenesis of C. jejuni infection remains poorly understood. Paracellular pathways in intestinal epithelial cells are gated by intercellular junctions (tight junctions and adherens junctions), providing a functional barrier between luminal microbes and host immune cells in the lamina propria. Here we describe alterations in tight junctions in intestinal epithelial monolayers following C. jejuni infection. Apical infection of polarized T84 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of the tight junctional transmembrane protein occludin from an intercellular to an intracellular location. Subcellular fractionation using equilibrium sucrose density gradients demonstrated decreased hyperphosphorylated occludin in lipid rafts, Triton X-100-soluble fractions, and the Triton X-100-insoluble pellet following apical infection. Apical infection with C. jejuni also caused rapid activation of NF-kappaB and AP-1, phosphorylation of extracellular signal-regulated kinase, Jun N-terminal protein kinase, and p38 mitogen-activated protein kinases, and basolateral secretion of the CXC chemokine interleukin-8 (IL-8). Basolateral infection with C. jejuni caused a more rapid decrease in TER, comparable redistribution of tight-junction proteins, and secretion of more IL-8 than that seen with apical infection. These results suggest that compromised barrier function and increased chemokine expression contribute to the pathogenesis of C. jejuni-induced enterocolitis.

Campylobacter jejuni↗

PubMed1986

Effect of dietary vitamin D3 and cadmium on the lipid composition of rat intestinal brush border membranes.

Vitamin D deficiency in cadmium-exposed rats was observed along with enhanced tissue cadmium accumulation. In relation to the barrier function, the changes in the lipid composition have been studied in the intestinal brush border membranes prepared from rats raised on diets differing in vitamin D status in the absence or presence of cadmium. In an analysis of lipid composition, vitamin D3 treatment resulted in an increase of phospholipid content, and cadmium ingestion resulted in a decrease of cholesterol and glycolipid contents in the duodenal brush border membranes. On the other hand, vitamin D3 and cadmium showed no significant effect on the lipid composition of the jejunal brush border membranes. Further analysis of the fatty acid composition in duodenal brush border membrane lipids showed that vitamin D3 treatment led to an increase of the proportion of fatty acids (18:1 and 18:2 in the total and phospholipid fraction) and also shorter chain fatty acids in neutral lipid fractions in the absence of cadmium. However, vitamin D3 treatment in the presence of cadmium led to a decrease of the proportion of fatty acid (18:2 in the total and phospholipid fraction) and also shorter chain fatty acids in neutral lipid fractions. Vitamin D-dependent alterations of the membranes might act as a barrier in cadmium-exposed rats.

Animals↗

PubMed2002

Altered intestinal morphology and immunity in patients with acute necrotizing pancreatitis.

PURPOSE/BACKGROUND: Impairment of gut barrier function has been demonstrated in patients with severe acute pancreatitis and may contribute to the development of local and systemic septic complications. The underlying mechanisms, however, remain unclear. Against this background, our aims were to investigate the small intestinal epithelial morphology and mucosal immunity in patients with severe acute pancreatitis. METHODS: Segments of terminal ileum from three patients with severe necrotizing acute pancreatitis who underwent pancreatic debridement and ileocolic resection for doubtful or evident segmental colonic viability were available for the study. Control specimens were available from seven patients who underwent gastric bypass and distal ileal resection for morbid obesity. Sections were cut and stained with hematoxylin and eosin for the measurement of villous height and crypt depth, and with toluidine blue for the determination of mucosal mast cell counts. Only adequately oriented specimens were deemed suitable for computer-aided image analysis. Results were expressed as the villous height/crypt depth ratio (VH/CD) and mucosal mast cell index (ratio of mast cell count/length of muscularis mucosa). RESULTS: Microscopy of the small intestine from controls was normal. The villous height and VH/CD were significantly reduced in patients with acute pancreatitis compared with controls (median, 0.47 mm vs 0.68 mm, and 1.9 vs 2.8, respectively; P < 0.00001). The mast cell index was significantly reduced in patients with acute pancreatitis compared with controls (median, 5.88 cells/mm vs 8.58 cells/mm; P= 0.001). A positive correlation was observed between the mast cell index and the height of the villi ( r= 0.23; P= 0.027). CONCLUSIONS: Patients with necrotizing acute pancreatitis have an altered intestinal morphology and depleted mucosal mast cells. These factors may contribute to the impairment of gut barrier function in patients with severe acute pancreatitis.

Aged↗

PubMed2026

Potential contribution of the microbiota-gut-brain axis to doxorubicin-associated cognitive impairment: Mechanisms, evidence, and therapeutic opportunities.

Chemotherapy-induced cognitive impairment (CICI), often termed chemobrain, is a clinically important complication of cancer treatment that can affect memory, attention, executive function, and processing speed during and after therapy. Doxorubicin is of particular mechanistic interest because brain parenchymal exposure is limited, yet preclinical studies consistently identify neuroinflammatory, oxidative, vascular, and synaptic abnormalities after treatment. This critical narrative review evaluates whether intestinal injury and disruption of the microbiota-gut-brain axis may contribute to these central effects. Preclinical evidence indicates that doxorubicin can alter microbial community structure, injure the intestinal barrier, modify SCFA-associated taxa or predicted functions, alter selected metabolite profiles, and promote systemic inflammatory and metabolic signaling. These peripheral changes could interact with brain endothelial cells, glia, mitochondria, hippocampal neurogenesis, and synaptic-plasticity pathways. However, the proposed doxorubicin-gut-brain pathway remains a predominantly preclinical and incompletely tested framework. No longitudinal human study has yet established, within the same patients, the temporal sequence linking doxorubicin exposure, microbiome or metabolome changes, systemic inflammation, and objective cognitive outcomes. Existing animal studies also vary in dose, regimen, tumor context, sampling time, microbiome methodology, and control of behavioral or microbiological confounders, while causal rescue experiments remain limited. Key priorities are therefore longitudinal human cohorts with pretreatment baselines and repeated multi-omics and cognitive assessments; animal studies that test temporal precedence and causal rescue or pathway blockade in the same model; mediation analyses that determine whether microbial or metabolic changes lie between treatment and cognitive dysfunction; and mechanism-informed clinical trials that demonstrate target engagement, cognitive benefit, oncology safety, and preservation of antitumor efficacy. Microbiome-directed interventions are promising but remain investigational for doxorubicin-associated CICI.

blood–brain barrier↗

PubMed2003

No effect of MDR1 C3435T variant on loperamide disposition and central nervous system effects.

BACKGROUND: The MDR1 gene encodes the efflux transporter P-glycoprotein, which is highly expressed in the small intestine and in the blood-brain barrier. A major function of P-glycoprotein is to limit the absorption and central nervous system exposure of numerous xenobiotics. A genetic polymorphism in the MDR1 gene (C3435T) has been associated with changes in the intestinal expression level and function of P-glycoprotein. The aim of this study was to investigate the effect of this polymorphism on disposition and brain entry of the P-glycoprotein substrate loperamide. METHODS: Healthy white volunteers were genotyped for the MDR1 C3435T polymorphism, and a 16-mg oral dose of loperamide was administered to 8 subjects with the 3435TT genotype and 8 subjects with the 3435CC genotype. Plasma levels of loperamide were determined by liquid chromatography-tandem mass spectrometry. Loperamide-induced respiratory depression was detected as the ventilatory response to carbon dioxide and was used as a measure of central nervous system side effects. RESULTS: We found no significant difference in loperamide pharmacokinetics between individuals homozygous for the C and the T alleles in position 3435 of MDR1, as follows: peak plasma drug concentration, 3164 +/- 1053 pg/mL and 3021 +/- 984 pg/mL; area under the concentration-time curve from 0 to 8 hours, 14414 +/- 4756 pg. h/mL and 14923 +/- 6466 pg. h/mL; and time to peak plasma drug concentration, 3.9 +/- 1.4 hours and 3.9 +/- 2.6 hours for the MDR1 3435CC and 3435TT genotypes, respectively (P >.05, for all parameters). Hypercapnic ventilatory response changed only minimally after ingestion of loperamide (the coefficient of variation during the 0- to 8-hour period was 21% +/- 14% for the sample population), and there was no MDR1 3435 genotype-related effect on respiratory response. Carriers of the 2 major MDR1 haplotypes, MDR1*1 and MDR1*13, did not differ in their response to loperamide. CONCLUSION: There was no association between the MDR1 C3435T variation and plasma levels or central nervous system effects of the P-glycoprotein substrate loperamide in a white study population. The MDR1 haplotype structure was quite variable and supports the use of haplotypes instead of single nucleotide polymorphisms in determining clinical consequences of genetic variation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Alteration in epithelial permeability and ion transport in a mouse model of total parenteral nutrition.

OBJECTIVE: To investigate the effects of total parenteral nutrition administration on intestinal ion transport and intestinal epithelial permeability. Additionally, to assess the role of interferon-gamma on the total parenteral nutrition-induced loss of epithelial barrier function. DESIGN: Randomized, controlled study. SETTING: Experimental laboratory, University of Michigan Medical School, Ann Arbor. SUBJECTS: Adult wild-type and interferon-gamma knockout mice. INTERVENTIONS: Wild-type mice received total parenteral nutrition or enteral diet (control group) for 7 days. Segments of small bowel from the mice were mounted in Ussing chambers. Short circuit current, as an indictor of active ion transport, was constantly monitored. Epithelial barrier function was assessed by measuring transepithelial resistance and transmural passage of 51Cr-EDTA and 3H-mannitol. Intestinal intraepithelial lymphocyte-derived interferon-gamma protein expression was detected with enzyme-linked immunosorbent assay and confirmed by using intracellular staining and flow cytometry. To investigate the effect of total parenteral nutrition on intestinal ion transport, we used a secretory agonist, carbachol, and an absorptive agent, glucose. MEASUREMENTS AND MAIN RESULTS: Total parenteral nutrition significantly increased small-bowel permeability. Ion transport in the total parenteral nutrition group was significantly increased. To stimulate ion transport, we found that increases in short circuit current induced by carbachol and glucose were higher in the total parenteral nutrition group compared with the control group. Intestinal intraepithelial lymphocyte interferon-gamma protein expression significantly increased with the administration of total parenteral nutrition. Intestinal permeability in interferon-gamma knockout total parenteral nutrition mice was significantly lower than in wild-type mice receiving total parenteral nutrition. CONCLUSION: Total parenteral nutrition has significant effects on intestinal epithelial physiology, stimulating ion secretion and reducing epithelial barrier function. Interferon-gamma appears to play an important role in the loss of the epithelial barrier function that is associated with total parenteral nutrition.

Animals↗

PubMed1999

Impairment of HIV polymorphonuclear leukocyte transmigration across T84 cell monolayers: an alternative mechanisms for increased intestinal bacterial infections in AIDS?

Our objective was to study the influence of HIV infection of polymorphonuclear leukocytes (PMN) on transepithelial migration. To date, reports of functional PMN chemotaxis in AIDS are contradictory. This is the first attempt to assess this function via an in vitro model allowing transmigration of neutrophils through an intestinal epithelial barrier. PMN were isolated from 45 HIV-infected patients and 45 healthy volunteers. PMN transmigration across T84 epithelial cells was initiated by applying either various concentrations of formyl-met-leu-phe peptide (f-MLP) or interleukin-8 and assayed by quantification of myeloperoxidase activity. CD11b, CD18, and CD47 expression on PMN was compared before and after transepithelial migration by flow cytometry analysis. CD11b expression was studied by electron microscopy. Apoptosis of transmigrated HIV PMN and control PMN was investigated by morphology and DNA fragmentation characterization. Compared to control PMN, HIV PMN exhibited a decrease in transepithelial migration that directly correlated with CD4+ counts. Basal and transepithelial migration-mediated expression of CD11b, CD18, and CD47 were unmodified in HIV PMN compared to control PMN. Electron microscopy labeling confirmed no difference in CD11b expression on HIV and control PMN. The index of apoptosis in transmigrated HIV PMN and control PMN was identical. These data provide evidence of a defect in the f-MLP-induced chemotaxis of PMN from HIV-infected patients across an intestinal epithelial barrier. This defective migration is not due to a quantitative modification of CD11b, CD18 and CD47 on HIV PMN suggesting a more subtle alteration. The impairment in the transmigration function may contribute in vivo to an increased susceptibility to intestinal bacterial infection in HIV-infected patients.

Adult↗

PubMed2000

[Effect of the long-term enteral administration of sorbents on the structural organization of microvillus mucosa in the small intestine].

Long term use of diatomite and ceolite, SUMS-1 sorbents in ration exerts irreversible effect on structural organization of microarea of mucosa small intestinal villus causing changes that indicate activation of absorption, barrier function of interstitium, stimulation of the ansendothelial fluid perfusion into blood and lymphatic capillaries. SUMS-1 and ceolite showed the highest stimulative effect.

Adsorption↗

PubMed1997

Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases.

The inflammatory bowel diseases (IBDs) are characterized by intestinal inflammation of unknown etiology. Two distinct disorders, Crohn's disease and ulcerative colitis, have been identified. Three theories of IBD etiology are currently under consideration: 1) reaction to a persistent intestinal infection, 2) existence of a defective mucosal barrier to luminal antigens, and 3) a dysregulated host immune response to ubiquitous antigens. In each of these theories, either pathogenic or resident luminal bacteria constantly stimulate the mucosal and systemic immune systems to perpetuate the inflammatory cascade. Chronicity of inflammation results from an interaction of the persistent stimulus of microbial antigens with genetically determined host susceptibility factors that determine the individual's immune response or mucosal barrier function. The pathogenesis of IBD involves a series of steps, beginning with the breach of the intestinal mucosal barrier by infectious agents or toxins. The defective barrier exposes lamina propria immune cells to the continual presence of resident luminal bacteria, bacterial products, or dietary antigens, which perpetuates the inflammatory cascade. Many immunoregulatory abnormalities are noted in IBD, including the ratio of proinflammatory to immunosuppressive cytokines, selective activation of T(H) lymphocyte subsets, and abnormalities in epithelial antigen presentation. When activated during the initial inflammatory process, macrophages and T lymphocytes secrete a host of cytokines, which recruit other inflammatory cell types, thereby continuing the process. Tissue injury is the net result of the soluble products of the activated inflammatory cells. Knowledge of the pathogenesis in IBD suggests that the ultimate goals of therapy should be to block the proinflammatory mediators toward the proximal, rather than the distal, end of the cascade, to decrease the constant antigenic drive of luminal bacteria, and to correct the dysregulated immune response.

Chronic Disease↗

PubMed2004

Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells.

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.

Cell Line↗

PubMedDate not supplied

Influence of nutrient delivery on gut structure and function.

Food is an important stimulus for the growth of gastrointestinal mucosa. Gut structure is influenced by the route of nutrient administration, dietary composition and the availability of specific nutrients. The alterations in intestinal structure and function that occur when enteral nutrition is withheld suggests that the ingestion of food results in physiologic responses that are responsible for the maintenance of gut mass during the fed state. The mechanism of mucosal suppression that occurs during starvation, stress, and total parenteral nutrition is not completely understood but may involve the absence of luminal substrates, decreased pancreaticobiliary secretions and alterations in the endocrine or paracrine events that normally accompany eating, digestion, and absorption. Enterocytes prefer glutamine and ketone bodies as oxidative fuels, whereas colonocytes utilize short chain fatty acids. Although enteral delivery of nutrients is the preferred route for maintenance of intestinal mass, provision of specific nutrients and hormonal stimulation during parenteral alimentation has been shown to be important in maintaining mucosal structure and function. If not adequately maintained, the intestine becomes susceptible to a variety of injuries which may result in impaired ability to digest and absorb nutrients and loss of mucosal barrier function.

Animals↗

PubMed1981

[Microcirculatory bed of the rat small intestine after nerve reflex isolation].

Intravital biomicroscopy of small intestinal wall vessels was used in experiments on rats undergoing total neuroreflex isolation of the small intestine to study the time course of changes in the diameter of intramural arterioles and venules of the first, second, third and fourth grades. It was demonstrated that neuroreflex isolation of the small intestine is accompanied by long-term deranged coordination of the vascular bed. These shifts appeared to be the earliest and the most significant in the major part of resistive vessels, determining the hydrostatic pressure in the capillaries and shifts in histohematic barriers, as well as in specific functions of the small intestine.

Animals↗

PubMed2002

The ability of endotoxin-stimulated enterocytes to produce bactericidal factors.

OBJECTIVE: Bactericidal peptides, specifically defensins, are produced by polymorphonuclear cells. Intestinal epithelial cells also produce bactericidal peptides, perhaps as part of their barrier function, to the greatest load of endogenous bacteria present in the body. We sought to determine whether and under what conditions intestinal cell lines could produce bactericidal compounds. DESIGN: Laboratory investigation. SETTING: Children's burn hospital. SUBJECTS: Caco-2, IEC-6, and HT-29 cell lines. INTERVENTIONS: Three different enterocyte lines were cultured for 1 day +/- lipopolysaccharide (1 or 10 microg/mL), and their supernatants were tested for bactericidal activity. Also, reverse transcription-polymerase chain reaction of Caco-2 cells was performed to assess the expression of defensin-6 mRNA. MEASUREMENTS AND MAIN RESULTS: After culture, enterocytes all were found to release one or more soluble factors with bactericidal activity (as measured fluorometrically by using a metabolizable dye) when stimulated by lipopolysaccharide (1 microg/mL). The bactericidal activity of these culture supernatants was saturated by increased bacterial load, additive to the effects of normal human peripheral blood polymorphonuclear cells, and was reduced by serial supernatant dilution. Enterocyte stimulation with larger amounts of lipopolysaccharide (10 microg/mL) resulted in greater bactericidal activity. After supernatant fractionation based on molecular weight, the bactericidal effect was best retained in the <10-kDa fraction. In addition, the expression of mRNA for defensin-6, a bactericidal peptide produced by neutrophils, was seen in Caco-2 cells. CONCLUSION: Enterocytes are shown to produce a soluble, low molecular weight, bactericidal compound in response to endotoxin stimulation. The expression of defensin-6 mRNA in Caco-2 cells suggests that intestinal cells may release defensins as bactericidal peptides. This experimental system provides an in vitro model to study the activity and production of bactericidal factors by enterocytes.

Analysis of Variance↗