Interleukin-4 hyporesponsiveness in inflammatory bowel disease: immune defect or physiological response?
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Humans↗
Biomedical subjects
W F Stenson
Publications and source records attributed to W F Stenson.
Platelet-activating factor acetylhydrolases in Caco-2 cells and epithelium of normal and ulcerative colitis patients.
BACKGROUND & AIMS: Platelet-activating factor (PAF) is a potent inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease and necrotizing enterocolitis. Metabolism by platelet-activating factor acetylhydrolase (PAF-AH) is the major pathway for platelet-activating factor degradation. The aim of this study was to investigate the possible role of intestinal epithelium as a source of PAF-AH. METHODS: Intracellular and secreted PAF-AHs were characterized in human colonic epithelial cells isolated from histologically normal mucosa and inflamed mucosa from patients with ulcerative colitis and in the human intestinal epithelial cell line Caco-2 by measuring the metabolism of [3H]-PAF to [3H]lysoPAF. RESULTS: Human colonic epithelial cells and Caco-2 cells synthesize and secrete PAF-AH as shown by in vitro hydrolysis of [3H]PAF to [3H]-lysoPAF in cell lysates and conditioned media. Both intracellular and secreted PAF-AHs are calcium-independent and substrate-specific for phospholipids similar to PAF. Epithelial cells from involved areas of resections for ulcerative colitis had increased levels of secreted PAF-AH and decreased levels of intracellular PAF-AH compared with epithelial cells from histologically normal areas. CONCLUSIONS: Human colonic epithelial cells and Caco-2 cells produce intracellular and secreted PAF-AHs, which are distinct proteins. This is the first demonstration of PAF-AH production by epithelial cells.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells.
To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.
Animals↗
Mechanisms of transit of lipid mediators of inflammation and bacterial peptides across intestinal epithelia.
The transit of two lipid mediators of inflammation, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), and a formylated peptide produced by intestinal bacteria, N-formylmethionylleucylphenylalanine (FMLP), across Caco-2 cell monolayers was characterized and compared with the transit of mannitol, a hexose known to cross epithelial monolayers by paracellular pathways. The permeability of less mature low-resistance ( < 200 ohm.cm2) monolayers to all four test compounds was similar, but as monolayers matured and the transmonolayer resistance increased, the transit of LTB4, PGE2, FMLP, and mannitol decreased to different degrees, resulting in a selectivity of permeability to the four test compounds in the order LTB4 > PGE2 > mannitol > FMLP. The transit of all four test compounds across Caco-2 cell monolayers was bidirectional, nonsaturable, and energy independent. A small portion of the added LTB4 was incorporated into the cells, whereas the other three compounds were not. Thus the transit of PGE2, mannitol, and FMLP across Caco-2 monolayers appears to be solely by the paracellular pathway, whereas the transit of LTB4 also involves the paracellular pathway but may also involve diffusion through the cell membrane and around tight junctions.
Bacterial Proteins↗
Induction of epithelial arachidonate 12-lipoxygenase at active sites of inflammatory bowel disease.
To determine if epithelial lipoxygenases are regulated by mucosal inflammation, we examined the distribution of arachidonate 12-lipoxygenase in healthy colonic tissue and in involved and uninvolved sections of colon with inflammatory bowel disease. Immunohistochemistry of formaldehyde-fixed, paraffin-embedded intestinal tissue using two anti-12-lipoxygenase antibodies and indirect biotin-avidin-peroxidase detection demonstrated that, in contrast to tissue from normal colon (n = 8), in which 12-lipoxygenase antigen was undetectable in mucosal epithelial cells, the mucosal and glandular epithelium of inflamed colon in ulcerative colitis (n = 4) or Crohn's disease (n = 4) exhibited markedly positive immunostaining with anti-12-lipoxygenase antibodies. Immunoblotting of whole cell extracts with anti-12-lipoxygenase antibodies showed immunoperoxidase staining of a single protein band that comigrated with purified 12-lipoxygenase (in relative molecular weight: M(r) = 72,000) in mucosal samples from inflamed colon from subjects with ulcerative colitis or Crohn's disease but no comparable band in samples from uninvolved sections of the same colons or from colons of subjects without inflammatory bowel disease. Assays of 12-lipoxygenase activity indicated a corresponding increase in enzymatic activity in the same mucosal samples. The increased levels of 12-lipoxygenase antigen in the mucosal and glandular epithelial cells in regions of colon affected by inflammatory bowel disease, the corresponding increases in 12-lipoxygenase activity, and the absence of detectable 12-lipoxygenase antigen or enzymatic activity in the same cell types in noninflamed colonic tissue all suggest that epithelial cell 12-lipoxygenase is induced by local mediators of colonic inflammation.
Adult↗
Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C.
Caco-2 cells are an enterocyte-like cell line derived from a human colonic adenocarcinoma. Paracellular permeability was assessed in monolayers of these cells by transmonolayer resistance and by the permeation of [3H]mannitol across the monolayer. Paracellular permeability was increased by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (50 nM), carbachol (500 microM), and the combination of carbachol (50 microM) and monolein (100 microM), an inhibitor of diacylglycerol kinase, as manifested by a decrease in transmonolayer resistance and an increase in mannitol permeation. The effects of all of these stimuli on transmonolayer resistance were inhibited by staurosporine (3 nM), an inhibitor of PKC. The effects of carbachol plus monolein were also inhibited by atropine (0.1 microM), a muscarinic antagonist. Treatment of the monolayers with each of the stimuli was associated with translocation of PKC activity from cytosol to a membrane-associated state. Stimulation of Caco-2 cell monolayers with phorbol myristate acetate or with the combination of carbachol and monolein was also associated with phosphorylation of the MARCKS protein, an endogenous substrate of PKC. These data support the hypothesis that intestinal paracellular permeability is regulated by the activity of enterocyte PKC and demonstrate that the increase in paracellular permeability induced by binding of carbachol to the muscarinic receptor is mediated by activation of PKC.
Adenocarcinoma↗
Dietary supplementation with fish oil in ulcerative colitis.
OBJECTIVE: To determine the efficacy of fish oil supplementation in patients with active ulcerative colitis. DESIGN: Multicenter, randomized, double-blind, placebo-controlled, crossover trail with 4-month treatment periods (fish oil and placebo) separated by a 1-month washout. SETTING: Four gastroenterology divisions. PATIENTS: Twenty-four patients with active ulcerative colitis entered the study. Five dropped out, and one was noncompliant. Eighteen patients completed the study. All patients had active disease as manifested by diarrhea and rectal inflammation. INTERVENTIONS: Treatment with prednisone and sulfasalazine was continued. Fish oil supplementation consisted of 18 Max-EPA (eicosapentaenoic acid) capsules daily (eicosapentaenoic acid, 3.24 g; and docosahexaenoic acid, 2.16 g). Placebo supplementation consisted of 18 identical capsules containing isocaloric amounts of vegetable oil. MEASUREMENTS: Patients were evaluated at study entry and after each diet period. Evaluations included a review of symptoms, flexible sigmoidoscopy, rectal biopsy, and rectal dialysis to measure prostaglandin E2 and leukotriene B4 levels. RESULTS: Fish oil supplementation resulted in a significant decrease in rectal dialysate levels of leukotriene B4 from 71.0 to 27.7 pg/mL (average change, -43.3 pg/mL; 95% CI, -83 to -3.6). Significant improvements were seen in acute histology index (average change, -8.5 units from a baseline of 10.5 units; CI, -12.9 to -4.2) and total histology index (average change, -8.5 units from a baseline of 14.80; CI, -13.2 to -3.8) as well as significant weight gain (average weight gain, 1.74 kg, CI, 0.94 to 2.54). No significant changes occurred in any variable during the placebo period. Seven patients received concurrent treatment with prednisone. During the fish oil supplementation period, the mean prednisone dose decreased from 12.9 mg/d to 6.1 mg/d and rose from 10.4 mg/d to 12.9 mg/d during the placebo diet period (P greater than 0.20). CONCLUSIONS: Four months of diet supplementation with fish oil in patients with inflammatory bowel disease resulted in reductions in rectal dialysate leukotriene B4 levels, improvements in histologic findings, and weight gain.
Adult↗
Caco-2 cell transfection by rat intestinal alkaline phosphatase cDNA increases surfactant-like particles.
The rat enterocyte produces a particle with surfactant-like properties (including a whorled appearance, enrichment for dipalmitoyl phosphatidylcholine, and ability to lower surface tension) that also is enriched for intestinal alkaline phosphatase. Human Caco-2 cells grown on polycarbonate filters were utilized to study the secretion of these particles and exhibited whorls and strands of unilamellar membranes, particularly concentrated at the apical pole or near junctional complexes. Concentrated culture medium from these cells separated on continuous NaBr gradients revealed a fraction at density = 1.07 g/l enriched for phosphatidylcholine and intestinal alkaline phosphatase. This fraction contained membranous sheets containing alkaline phosphatase, detected by immunolocalization. Phosphatidylcholine comprised 54% of phospholipid in this fraction, compared with 20% in brush borders. When Caco-2 cells were transfected with cDNA encoding rat intestinal alkaline phosphatase, cellular phosphatase activity increased twofold, but activity in the medium increased 14-fold to > 200 (average 32)-fold. Ultrastructurally, compared with mock-transfected cells or cells transfected with human placental alkaline phosphatase, transfection with rat intestinal alkaline phosphatase cDNA led to intracellular and extracellular accumulation of surfactant-like particles. We conclude that surfactant-like particles are produced by Caco-2 cells, and their production can be enhanced by transfection with a cDNA encoding a protein known to be associated with such particles.
Alkaline Phosphatase↗
Metabolism of oxygenated derivatives of arachidonic acid by Caco-2 cells.
Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated separately with 3H8-labeled preparations of three different lipid mediators of inflammation: 5-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4. Both [3H8]5-hydroxyeicosatetraenoic and [3H8]12-hydroxyeicosatetraenoic acids were taken up and metabolized by Caco-2 cells, but [3H]leukotriene B4 remained unmetabolized in the incubation medium. [3H]5-hydroxyeicosatetraenoic acid was esterified into cellular phospholipids (15%) and triglycerides (4%) but did not undergo beta-oxidation. When [3H]12-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, 14% underwent two cycles of beta-oxidation to form [3H]8-hydroxyhexadecatrienoic acid, and 3% underwent three cycles of beta-oxidation to form [3H]6-hydroxytetradecadienoic acid, both of which were released into the media. [3H]12-Hydroxyeicosatetraenoic acid was also esterified into cellular phospholipids (13%), but none was esterified into cellular triglycerides.
Adenocarcinoma↗
The role of the mucosal immune system in inflammatory bowel disease.
Continued delineation of the major factors that lead to intestinal inflammation will provide critical insights into many of the pathophysiologic events leading to tissue destruction in IBD. The exploration of exciting and important new areas, such as the role of adhesion molecules, proinflammatory cytokines, and the activation of lymphocytes and phagocytes, will contribute significantly to a better understanding of the mechanisms that sustain the intestinal inflammatory process. Determining the mechanisms of amplification and perpetuation of intestinal inflammation as well as learning more about the natural suppression of intestinal inflammation by the normal cellular and cytokine networks of the mucosal immune system will open exciting new therapeutic approaches. It is encouraging to see realistic and testable working models emerge from the combined efforts of many committed investigators who have been engaged in studying the role of the mucosal immune system in the pathophysiology of IBD. A great deal more remains to be learned in this rapidly advancing area, and we can look forward with confidence to continued advances in the study of IBD.
Antibody Formation↗
Trafficking of exogenous fatty acids within Caco-2 cells.
Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes.
Adenocarcinoma↗
Aggregated bovine IgG inhibits mannose receptor expression of murine bone marrow-derived macrophages via activation.
We previously described the presence of an inhibitory protein contained in the 20 to 40% (NH4)2SO4 precipitable fraction of FCS that down-regulates expression of mannose receptors on bone marrow-derived macrophages. We now identify aggregated bovine IgG as the main inhibitory component. Heat-aggregated bovine IgG was capable of down-regulating expression of the macrophage mannose receptor in a dose-dependent manner without inducing changes in ligand affinity whereas neither F(ab')2 fragments nor nonaggregated IgG displayed any inhibitory effect. Depleting of IgG from heat inactivated FCS by protein G affinity chromatography completely removes the inhibitory activity. Moreover, readdition of the Ig eluate from the protein G chromatography column restored inhibition in a dose-dependent manner. Macrophages were able to clear exogenously added aggregated bovine IgG, thus leading to loss of inhibitory activity in macrophage-conditioned media as compared to sham-conditioned media containing aggregated IgG. These results indicate that aggregated IgG down-regulates mannose receptor expression by macrophage activation via interaction with Fc-gamma R.
Animals↗
Monomeric IgG2a promotes maturation of bone-marrow macrophages and expression of the mannose receptor.
The macrophage mannose receptor, a 172-kDa lineage-specific glycoprotein, partakes in nonopsonin-mediated phagocytosis by recognition of terminal mannose residues on targeted particles. Because appearance of the receptor progresses with monocyte/macrophage differentiation, its expression is indicative of the maturational state of the cell. Monomeric IgG2a and IgG2b up-regulate mannose-receptor surface expression and biosynthesis by murine bone-marrow macrophage precursors as much as 7- to 12-fold in a dose-dependent manner. IgG2a accelerates macrophage mannose-receptor expression by several days during in vitro bone-marrow differentiation; however, treated and control cells ultimately express equivalent levels of receptor. Moreover, the effect is independent of cell cycle or ambient levels of colony-stimulating factor 1. The coinduction of another maturation-dependent lineage-specific antigen, F4/80, and the fact that macrophage precursors respond to IgG2a only within the first day of culture, indicate that the targeted cell is an early myelomonocytic precursor, responsive only during a short, early developmental window. The effect is specific for immunoglobulin molecules of the IgG2a and IgG2b subclasses and probably involves an Fc gamma-receptor signal-transduction pathway but not macrophage priming or activation. Most importantly, a paracrine mechanism of immunoglobulin-mediated bone-marrow macrophage differentiation is suggested by experiments in which basal levels of mannose-receptor expression are reduced by continual removal of B-cell-generated IgG from marrow cultures. Thus, IgG2a and IgG2b prompt mannose-receptor synthesis and bone-marrow macrophage differentiation and may, therefore, play a role in the regulation of macrophage differentiation in host defense.
Animals↗
Thickening of muscularis mucosae in Crohn's disease.
Crohn's disease (CD) of the bowel showed a statistically significant thickening of the muscularis mucosae when compared with disease controls. In areas of gross stricture in CD, the muscularis mucosae comprised almost 10% of total wall thickness. Similar findings were also present in a previously characterized experimental model of CD (trinitrobenzene sulfonic acid-induced colitis in rats), particularly in what appeared to be grossly strictured areas. Taken together, these findings suggest that increased mass of muscularis mucosae smooth muscle may be responsible in part for the commonly observed stricture formation in CD. As extreme muscularis mucosae hyperplasia appears to be peculiar to CD, it may serve as an additional marker differentiating CD from other diseases.
Adolescent↗
The synthesis and molecular dynamics of phospholipids having hydroxylated fatty acids at the sn-2 position.
We have devised a general method for the synthesis of phospholipids containing hydroxylated fatty acids and have utilized this methodology to synthesize two naturally occurring hydroxylated lecithins (i.e. 1-palmitoyl-2-[15(S)-hydroxy-5E, 8E,11E,13Z-eicosatetraenoyl]-sn-glycero-3-phosph ocholine (1-palm-2-15HETE PC) and 1-palmitoyl-2-[5-hydroxy-6Z,8E,11E, 14E-eicosatetraenoyl]-sn-glycero-3-phosphocholine (1-palm-2-5HETE PC]. After protection of the hydroxylated fatty acid as its t-butyldimethylsilyl derivative, the anhydride of the protected fatty acid was formed utilizing dicyclohexylcarbodiimide and subsequently was condensed with a regiospecific lysolecithin utilizing pyrrolidinopyridine as catalyst to form the protected hydroxylated lecithin. Finally, hydroxylated lecithins were formed after removal of the t-butyldimethylsilyl group with acetic acid. Electron spin resonance spectroscopy was utilized to interrogate the molecular dynamics of lipid bilayers comprised of mixtures of these hydroxylated lecithins and naturally occurring lecithins. Remarkably, the molecular dynamics of spin-labeled phospholipids in liposomes comprised of cholesterol and phosphatidylcholine were altered substantially after addition of only 3.5 mol% 1-palm-2-15HETE PC as assessed by the order parameter of 16-doxylstearoyl phosphatidylcholine.
Chemical Phenomena↗
Prostaglandin E specifically upregulates the expression of the mannose-receptor on mouse bone marrow-derived macrophages.
The macrophage mannose receptor (MMR) facilitates the binding and internalization of microorganisms and glycoproteins with terminal mannose residues. The receptor is progressively upregulated as bone marrow precursor cells mature into macrophages and thus may serve as a marker of differentiation. Prostaglandins of the E series (PGE) are known inhibitors of monocyte and macrophage precursor proliferation, an effect often associated with cellular maturation. MMR expression was therefore assessed after exposure of bone marrow macrophage precursor (BMMP) cells to these prostanoids. Receptor expression was determined by ligand binding and via immunoprecipitation of newly synthesized receptor molecules. PGE1 and PGE2 at 10(-9)-10(-6) M upregulated MMR surface expression and biosynthesis four- to sixfold in a dose-dependent manner. BMMPs responsive to prostaglandins were characterized by plastic adherence, F4/80 antigen expression, and nonspecific esterase activity. Prostaglandins accelerated the expression of the MMR in cells by 48-72h, with maximal levels of receptor expression being identical in control or treated cells. Thus, prostaglandins enhanced mannose receptor expression in adherent but not fully differentiated macrophage precursors. This effect is specific for PGE and is mimicked by dibutyrl cyclic AMP. These results indicate that prostaglandins accelerate MMR expression and hence the differentiation of macrophage precursor cells. Cells resident in the bone marrow secrete abundant prostaglandins, suggesting that a paracrine mechanism may exist to regulate MMR expression and function.
Adenylyl Cyclases↗
Role of eicosanoids as mediators of inflammation in inflammatory bowel disease.
An unknown initiating event in inflammatory bowel disease (IBD) activates the immune system, which is followed by infiltration of the intestinal mucosa with inflammatory cells and the production of soluble mediators of inflammation. These mediators of inflammation include the metabolites of arachidonic acid. The results of research on arachidonic acid metabolites are reviewed, and it is concluded that the major arachidonic metabolites in human IBD mucosa are the lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid. These metabolites are found in much higher concentrations in mucosa from patients with IBD than from healthy controls. Significantly more chemotactic activity is found in IBD mucosa than in healthy mucosa, and most of this activity is attributable to LTB4. Enhanced synthesis of LTB4 could account for much of the inflammatory response in IBD. Inhibition of the lipoxygenase pathway could be the mechanism that accounts for the therapeutic efficacy of mesalazine.
Arachidonic Acids↗
Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells.
Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).
Arachidonic Acid↗