Intestinal Mast Cell Activation Research Articles

Spi-B alleviates food allergy by securing mucosal barrier and immune tolerance in the intestine.

Food allergy is a type I allergic reaction induced by mast cells and is mainly activated by allergen-specific immunoglobulin (Ig)E. Spi-B is an E26-transformation-specific (Ets) family transcription factor essential for the differentiation and functional maturation of several immune cell subsets, including mast cells. However, the possible involvement of Spi-B in food allergy remains unclear. In this study, we found that Spi-B-deficient mice were highly susceptible to food allergy to ovalbumin (OVA), as indicated by the exacerbation of diarrhea and elevation of serum IgE levels. These pathological changes were associated with enhanced mast cell infiltration into the intestinal lamina propria. Activation of mast cells in the intestinal mucosa was observed in Spib−/− mice, even under physiological conditions. Accordingly, Spi-B deficiency increased the translocation of fluorescently labeled dextran from the lumen to the serum, suggesting increased intestinal permeability in Spib−/− mice. Moreover, Spib−/− mice showed defects in oral tolerance induction to OVA. These data illustrate that Spi-B suppresses the development of food allergies by controlling the activation of intestinal mast cells and by inducing immune tolerance to food allergens.

Lactobacillus casei ATCC 393 alleviates Enterotoxigenic Escherichia coli K88-induced intestinal barrier dysfunction via TLRs/mast cells pathway

AimsMast cells play a crucial role in gastrointestinal physiology and pathophysiology. This study was conducted to investigate the role of mast cells (MCs) in the protective effect of Lactobacillus casei ATCC 393 (L. casei ATCC 393) on intestinal barrier function. Main methodsThe regulatory effect of L. casei ATCC 393 on intestinal barrier dysfunction and MCs activation induced by Enterotoxigenic Escherichia coli K88 (ETEC K88) were evaluated by porcine mucosal mast cells (PMMCs)-porcine jejunal epithelial cells (IPEC-J2)-L. casei ATCC 393 co-culture experiments in vitro and MCs stabilizer drug experiment in vivo. Key findingsResults showed that L. casei ATCC 393 pretreatment effectively alleviated the reduction of cell viability and increase of permeability in ETEC K88-infected IPEC-J2 cells. L. casei ATCC 393 pretreatment inhibited the increase of proinflammatory cytokines and some other MCs mediators, and decrease of anti-inflammatory cytokines in ETEC K88-infected PMMCs. Cromolyn sodium or L. casei ATCC 393 prevented ETEC K88-induced increase of intestinal epithelial cell permeability in IPEC-J2 cells when co-cultivation with PMMCs. Furthermore, cromolyn sodium or L. casei ATCC 393 pretreatment attenuated ETEC K88-induced increase of MCs mediators, mast cell proteases (MCPs) and carboxypeptidase A3 (CPA3) mRNA levels, and down-regulation of tight junction proteins, Toll-like receptor 2 and 4 (TLR2 and TLR4) expression levels in mice challenged by ETEC K88. SignificanceThese results indicated that intestinal barrier dysfunction caused by ETEC K88 was mediated by intestinal mast cell activation which can be prevented by L. casei ATCC 393 via TLRs signaling pathway.

Moxa-stick moxibustion activates mast cells of small intestine tissue in rats

To explore the effect of moxa-stick moxibustion and joss-stick moxibustion at "Guanyuan" (CV4) on the activity of mast cells in the small intestine tissue in rats. Twelve male SD rats were randomly divided into control, joss-stick moxibustion and moxa-stick moxibustion groups (n=4 rats in each group). Joss-stick or moxa-stick moxibustion was applied to CV4 for 10 min. After moxibustion, the skin temperature of the CV4 region was measured immediately with a thermometer. The mast cells and nerve fibers in the small intestine tissue were displayed by immunofluorescence histochemistry. Compared with the control group, the skin temperature of the CV4 region in both the joss-stick and the moxa-stick groups were significantly increased (P<0.05), while the skin temperature of the moxa-stick group was significantly higher than that in the joss-stick group (P<0.05). There were a large number of tryptase-positive mast cells in the small intestine of rats, some of which were co-expressed with lysosomal-associated membrane protein 1, displaying an activated state. The average numbers of mast cells in the control, joss-stick and moxa-stick groups were 9.2±3.6, 10.8±5.3 and 17.1±6.3, respectively, being significantly higher in the moxa-stick group than in the control and joss-stick groups (P<0.05). In addition, calcitonin gene-related peptide(CGRP) and neuropeptide Y(NPY) positive nerve fibers were found around the mast cells in the small intestinal tissues. Moxa-stick moxibustion gives rise a higher temperature at CV4 to activate mast cells surrounded by CGRP and NPY positive nerve fibers in the small intestine tissue in rats, suggesting an involvement of the sensory and sympathetic nervous system in the activation of intestinal mast cells possibly by way of somatic sympathetic reflex.

Weaning and postnatal age influence the early time course and nature of intestinal mast cell activation and mucosal inflammation in a porcine model of early life adversity

Abstract Early life adversity (ELA) is a risk factor for later life emergence of functional and inflammatory GI disorders in people and animals. In this study, we compared the early GI immune responses in male piglets exposed to early weaning (EW) at 16–18 d of age, a form of ELA we’ve previously shown result in altered GI developmental and health trajectories similar to ELA in humans, with that of late weaned piglets (LW: 28 d of wean age). RNA transcriptome analysis of ileal mucosa at 24h post-weaning revealed that EW pigs exhibited a greater number of differentially expressed genes (765 vs 110 genes, for EW and LW piglets, respectively), characterized by a large number of upregulated genes associated with immune cell trafficking and inflammation. Mast cells (MC), stress-sensitive innate immune cells which orchestrate the immune response, were acutely activated in EW and LW pigs but in a differential manner. Compared with LW piglets, EW piglets exhibited higher levels of serum histamine which coincided with a downregulation in the gene expression of histamine degrading enzymes, DAO and HNMT in intestinal mucosa. Mast cell tryptase (MCT7) and chymase (CMA1) gene expression were upregulated in ileal and colonic mucosa (within 3h post-weaning) in both EW and LW piglets; however, this response was greater in LW pigs. Together, these data show that weaning stress in piglets induces rapid GI MC activation which precedes immune cell recruitment and intestinal inflammation and that postnatal age has a significant influence on the level and nature of this response. Further investigation of how EW and LW piglets differentially regulate early GI immune responses is expected to provide new insight into the mechanisms of GI and immune disorders associated with ELA.

Nobiletin acts anti-inflammatory on murine IL-10-/- colitis and human intestinal fibroblasts.

Inflammatory bowel disease (IBD) shows increasing prevalence over the last years. We propose that anti-inflammatory plant substances could be used as additional or alternative agents with good compliance in prevention and/or therapy of IBD and its complication intestinal fibrosis. We could recently show that the citrus flavonoid nobiletin acts anti-inflammatory on activation of intestinal mast cells. Here, we analysed the effects of nobiletin on inflammation and fibrosis in IL-10-/- colitis. IL-10-/- and wild-type (WT) mice were orally treated with/without vehicle or nobiletin. Clinical symptoms of colitis and disease activity index (DAI) were assessed, and colon tissue was analysed for tissue damage, cellular infiltration, bowel wall thickness, mast cell number and degranulation, as well as collagen deposition as marker for fibrosis. Human intestinal fibroblasts (hiFB) were treated with nobiletin and the expression of collagen and pro-inflammatory cytokines was measured. Nobiletin treatment of IL-10-/- mice resulted in a reduction of clinical colitis symptoms and a longer survival time. In addition, histological scores of colitis were reduced compared to control groups. Mast cell number and degranulation was lower in nobiletin treated IL-10-/- mice, and correlated positively with DAI. As well, fibrotic marker of collagen deposition was reduced by nobiletin. In hiFB, the expression of collagen as well as of pro-inflammatory cytokines IL-6, TNF and CCL2 was down-regulated by nobiletin treatment. Nobiletin decreases inflammatory symptoms and markers in murine colitis as well as fibrotic collagen deposition and expression. Thus, nobiletin could be a potential new agent in therapy of chronic colitis.

Humoral and cellular responses to casein in patients with food protein–induced enterocolitis to cow's milk

Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy manifesting within 1 to 4hours of food ingestion with repetitive emesis and lethargy. We sought to characterize immune responses to casein in children with FPIES caused by cow's milk (CM). Total IgE and IgM, CM-specific IgG, and casein-specific IgE, IgG, IgG4, and IgM levels, as well as immunoglobulin free light chains, were measured in both patients with active and those with resolved CM-FPIES. Proliferating casein/T-effector cell counts were measured in children with CM-FPIES, children with IgE-mediated CM allergy, and those tolerating CM. Cytokine concentrations in the supernatants were quantified. Serum cytokine and tryptase levels were measured before and after a positive oral food challenge (OFC) result and compared with levels in those with a negative OFC result. We found low levels of CM and casein-specific IgG and casein-specific IgG4 in patients with CM-FPIES versus those tolerating CM (P<.05). Although we found both a high CD4+ Tcell-proliferative response and TH2 cytokines production after casein stimulation in children with CM-FPIES, results were similar to those in control subjects. Significantly lower secretion of IL-10 and higher secretion of IL-9 by casein-stimulated Tcells were found in patients with CM-FPIES versus those with IgE-mediated CM allergy. Lower baseline serum levels of IL-10 and higher tryptase levels were found in active CM-FPIES versus resolved CM-FPIES. We found a significant increase in serum IL-10 and IL-8 levels after a positive OFC result. We confirm the paucity of humoral response in patients with CM-FPIES. IL-10 might play a key role in acquisition of tolerance in patients with CM-FPIES. Increased serum IL-8 levels in patients with active FPIES suggest neutrophil involvement. Elevated baseline serum tryptase levels in patients with active FPIES suggest low-grade intestinal mast cell activation or increased mast cell load.

Curcumin Ingestion Inhibits Mastocytosis and Suppresses Intestinal Anaphylaxis in a Murine Model of Food Allergy.

IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-inflammatory properties, and thus may have the capacity to regulate Th2 cells and mucosal mast cell function during allergic responses. We assessed whether curcumin ingestion during oral allergen exposure can modulate the development of food allergy using a murine model of ovalbumin (OVA)-induced intestinal anaphylaxis. Herein, we demonstrate that frequent ingestion of curcumin during oral OVA exposure inhibits the development of mastocytosis and intestinal anaphylaxis in OVA-challenged allergic mice. Intragastric (i.g.) exposure to OVA in sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1, and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, mast cell activation and expansion, and Th2 responses were also suppressed in mice exposed to curcumin during the OVA-challenge phase alone, despite the presence of elevated levels of OVA-IgE, suggesting that curcumin may have a direct suppressive effect on intestinal mast cell activation and reverse food allergy symptoms in allergen-sensitized individuals. This was confirmed by observations that curcumin attenuated the expansion of both adoptively transferred bone marrow-derived mast cells (BMMCs), and inhibited their survival and activation during cell culture. Finally, the suppression of intestinal anaphylaxis by curcumin was directly linked with the inhibition of NF-κB activation in curcumin-treated allergic mice, and curcumin inhibited the phosphorylation of the p65 subunit of NF-κB in BMMCs. In summary, our data demonstrates a protective role for curcumin during allergic responses to food antigens, suggesting that frequent ingestion of this spice may modulate the outcome of disease in susceptible individuals.

Urinary and faecal N-methylhistamine concentrations do not serve as markers for mast cell activation or clinical disease activity in dogs with chronic enteropathies.

BackgroundThis study sought to correlate faecal and urinary N-methylhistamine (NMH) concentrations with resting versus degranulated duodenal mast cell numbers in dogs with chronic enteropathies (CE), and investigate correlations between intestinal mast cell activation and clinical severity of disease as assessed by canine chronic enteropathy clinical activity index (CCECAI), and between urinary and faecal NMH concentrations, mast cell numbers, and histopathological scores. Twenty-eight dogs with CE were included. Duodenal biopsies were stained with haematoxylin and eosin (H&E), toluidine blue, and by immunohistochemical labelling for tryptase. Duodenal biopsies were assigned a histopathological severity score, and duodenal mast cell numbers were counted in five high-power fields after metachromatic and immunohistochemical staining. Faecal and urinary NMH concentrations were measured by gas chromatography–mass spectrometry.ResultsThere was no correlation between the CCECAI and faecal or urinary NMH concentrations, mast cell numbers, or histopathological score – or between faecal or urinary NMH concentration and mast cell numbers. Post hoc analysis revealed a statistically significant difference in toluidine blue positive mast cells between two treatment groups (exclusion diet with/without metronidazole versus immunosuppression (IS)), with higher numbers among dogs not requiring IS.ConclusionFaecal and urinary NMH concentrations and duodenal mast cell numbers were not useful indicators of severity of disease as assessed by the CCECAI or histological evaluation. The number of duodenal mast cells was higher in dogs that did not need IS, i.e. in dogs responding to an exclusion diet (with/without metronidazole), than in dogs requiring IS. Further studies comparing the role of mast cells in dogs with different forms of CE are needed.

The role of B-cells in immunity against adult Strongyloides venezuelensis

BackgroundStrongyloides venezuelensis has been used as a tool and model for strongyloidiasis research. Elimination of S. venezuelensis adult worms from mice has been particularly associated with proliferation and activation of intestinal mast cells and eosinophils. To date, the role of B-cells in the protective mechanism against adult Strongyloides infection in experimental animals has not been reported in the literature. Therefore, the present study was carried to investigate the role of B-lymphocytes in immunity against adult S. venezuelensis infection using mice with a targeted deletion of the JH locus.MethodsJHD knockout mice with its wild-type Balb/c mice were infected by intra-duodenal implantation of adult S. venezuelensis. Fecal egg count, intestinal worm recovery, mucosal mast cells and eosinophils were counted.ResultsAt day 11 post infection, parasites in wild-type mice stopped egg laying, while in JHD knockout mice parasites continued to excrete eggs until the end of the observation period, day 107. The higher number of parasite eggs expelled in the feces of JHD knockout infected mice was a consequence of higher worm burdens, which established in the small intestine of these animals. On the other hand worm fecundity was comparable in both groups of mice. Both B-cell-deficient mice and wild-type mice, showed an influx of mucosal mast cells and eosinophils. The absolute numbers in JHD knockout mice were lower than those seen in wild-type mice at day 11, but not to a level of significance. JHD knockout mice could not recover from infection despite the recruitment of both types of cells.ConclusionOur findings highlight a role of B cells in mucosal immunity against invasion of adult S. venezuelensis and in its expulsion. Therefore, we conclude that B-cells together with mucosal mast cells and eosinophils, contribute to immunity against adult S. venezuelensis by mechanism(s) to be investigated.

Su1777 GIP Activation of Signaling Pathways Traffick PepT1 Proteins to the Apical Membrane of Intestinal Epithelial Cells

Background: In our previous study, using lymph fistula rat model, we have shown that fat ingestion stimulates release of mast cell mediators such as histamine, rat mucosal mast cell protease II (RMCP II), and prostaglandin D2 (PGD2) into intestinal lymph; however, it is unclear whether these responses are different depending on the type of fatty acid ingested. Aim: The purpose of this study was to determine the effects of dietary long-chain fatty acids with varying degrees of saturation on intestinal mast cell activation. Methods: We investigated lymphatic mast cell mediator concentrations after administration of three types of triacylglycerols, trilinolein (C18:2 n-6), trilinolenin (C18:3 n-3) or triolein (C18:1 n-9) by using the lymph fistula rat model. After cannulation of the major mesenteric lymph duct and duodenum of male Sprague-Dawley rat, a 3 ml bolus mixture of phosphate-buffered, taurocholatestabilized emulsion containing 120μmol of trilinolein or trilinolenin or triolein was provided to each animal through the intraduodenal feeding tube. The emulsion medium without triacylglycerol was used as control. Lymph was continuously collected for 6 hours and analyzed for RMCPII, histamine, PGD2, triglyceride and protein content. Results: Infusion of trilinolein and trilinolenin significantly increased the lymphatic release of mast cell mediators and influenced lymphatic protein concentration. In contrast, triolein did not induce the release of mast cell mediators. The peak RMCPII concentration after infusion of trilinolein (432.97 ± 77.53 ng/ml) and trilinolenin (317.50 ± 47.59 ng/ml) were significantly greater than triolein (141.46 ± 23.46 ng/ml) ( P,0.01, P,0.05, respectively). Similar to the RMCPII response, the peak values of histamine were significantly greater than triolein (12.97 ± 0.62ng/ml) for trilinolein (34.2 ± 1.7 ng/ml, P,0.01, n=3) and trilinolenin (19.85 ± 1.06ng/ ml, P,0.01, n=3). Infusion of trilinolein induced a significant increase of lymphatic PGD2 concentration peaked at 2 hour compared with the control (1461.23 ± 133.12 vs. 842.30 ± 129.02 pg/ml, P,0.05), but triolein did not induce (978.43 ± 153.74 pg/ml). Conclusion: These observations identify that the intestinal mast cell activation induced by dietary fat is dependent on the degree of unsaturation in fatty acid.

Su1776 Activation of Rat Intestinal Mast Cells Depends on Types of Unsaturated Long-Chain Fatty Acids

Background: In our previous study, using lymph fistula rat model, we have shown that fat ingestion stimulates release of mast cell mediators such as histamine, rat mucosal mast cell protease II (RMCP II), and prostaglandin D2 (PGD2) into intestinal lymph; however, it is unclear whether these responses are different depending on the type of fatty acid ingested. Aim: The purpose of this study was to determine the effects of dietary long-chain fatty acids with varying degrees of saturation on intestinal mast cell activation. Methods: We investigated lymphatic mast cell mediator concentrations after administration of three types of triacylglycerols, trilinolein (C18:2 n-6), trilinolenin (C18:3 n-3) or triolein (C18:1 n-9) by using the lymph fistula rat model. After cannulation of the major mesenteric lymph duct and duodenum of male Sprague-Dawley rat, a 3 ml bolus mixture of phosphate-buffered, taurocholatestabilized emulsion containing 120μmol of trilinolein or trilinolenin or triolein was provided to each animal through the intraduodenal feeding tube. The emulsion medium without triacylglycerol was used as control. Lymph was continuously collected for 6 hours and analyzed for RMCPII, histamine, PGD2, triglyceride and protein content. Results: Infusion of trilinolein and trilinolenin significantly increased the lymphatic release of mast cell mediators and influenced lymphatic protein concentration. In contrast, triolein did not induce the release of mast cell mediators. The peak RMCPII concentration after infusion of trilinolein (432.97 ± 77.53 ng/ml) and trilinolenin (317.50 ± 47.59 ng/ml) were significantly greater than triolein (141.46 ± 23.46 ng/ml) ( P,0.01, P,0.05, respectively). Similar to the RMCPII response, the peak values of histamine were significantly greater than triolein (12.97 ± 0.62ng/ml) for trilinolein (34.2 ± 1.7 ng/ml, P,0.01, n=3) and trilinolenin (19.85 ± 1.06ng/ ml, P,0.01, n=3). Infusion of trilinolein induced a significant increase of lymphatic PGD2 concentration peaked at 2 hour compared with the control (1461.23 ± 133.12 vs. 842.30 ± 129.02 pg/ml, P,0.05), but triolein did not induce (978.43 ± 153.74 pg/ml). Conclusion: These observations identify that the intestinal mast cell activation induced by dietary fat is dependent on the degree of unsaturation in fatty acid.

Mast Cell Corticotropin Releasing Factor Receptor Subtypes Play Opposing Roles in Mediating Stress‐Induced Colonic Barrier Dysfunction

It has long been known that gastrointestinal disorders are profoundly influenced by life stress; however the mechanisms are poorly understood. Previous research showed that psychological stress causes disturbances in intestinal epithelial barrier function via corticotropin releasing factor (CRF)‐dependent activation of intestinal mast cells (MC). The precise pathway of MC activation by CRF in the stress response remains unclear. Here we definitively explore the role of MC CRF receptors 1 and 2 (CRF1 and CRF2) in stress‐induced intestinal barrier dysfunction. MC‐deficient mice (W‐sh/W‐sh) were repleted with bone marrow‐derived mast cells (BMMCs) from wild type (WT), CRF1KO, or CRF2 KO mice. Following the successful repletion of BMMCs, mice were subjected to 3 hours of restraint stress (RS). Transepithelial electrical resistance (TER) and paracellular flux of FITC‐dextran (FD4) were measured in Ussing chambers as indices of barrier function. There was a significant increase (p=0.003) in the rate of FD flux and reduction in TER in the colon of WT mice subjected to RS but not in W‐sh/W‐sh mice. Repletion of W‐sh/W‐sh mice with WT BMMCs restored stress‐induced changes in FD4 flux and TER (p=0.01). Repletion of W‐sh/W‐sh mice with CRF1 KO BMMCs ameliorated stress‐induced increases in FD4 flux. In contrast, repletion of W‐sh/W‐sh mice with CRF2 KO BMMCS exacerbated stress‐induced barrier dysfunction compared with WT BMMC controls (p&lt;0.05). These data definitively show that MC CRF1 mediates stress‐induced intestinal barrier dysfunction while CRF2 may play a critical barrier protective role. These opposing roles of CRF receptors may help in identifying novel preventative and therapeutic targets to mitigate stress‐related intestinal disorders.

Intestinal CRF Receptor Subtypes Mediate Distinct Intestinal Barrier Function Responses Through Intestinal Mast Cell Activation

Psychological stress is a major contributing factor in the onset and exacerbation of gastrointestinal disease through poorly understood mechanisms. Our previous studies in a porcine model showed that stress-induced disturbances in mucosal barrier function are regulated by peripheral activation of CRFr subtypes CRFr1 and CRFr2. Furthermore, results from these studies suggested that CRFr1 mediates deleterious mucosal barrier responses to stress while CRFr2 may play a protective role. The objective of this study was to further elucidate the role of CRFr subtypes in stress intestinal barrier dysfunction. Porcine ileum was mounted on Ussing Chambers and exposed to CRFr1 and CRFr2 agonist treatments: 1) Control 2) CRF;(1μM;CRFr1/r2 agonist) 3) UCN2;(0.1μM CRFr2 agonist) 4) Stressin 1 (2nM;CRFr1 agonist). Intestinal barrier function was measured in terms of mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) over a 180-minute period on Ussing chambers. CRF treatment increased (p < 0.005) paracellular flux of FD4 compared with controls. Ileal mucosa treated with the CRFr1 agonist Stressin 1 exhibited the greatest FD4 flux compared with all other treatments (p< 0.001) whereas treatment with the CRFr2 agonist UCN2 had no effect on baseline ileal barrier function. However, when added in combination with Stressin 1, UCN2 inhibited (p<0.05) Stressin 1-induced increases in FD4 flux. To determine if the effects of CRFr activation on barrier function were mediated via intestinal mast cell activation, ileal tissue was pre-treated with the mast cell stabilizer cromolyn (10 -4M) and then CRFr agonists were applied to tissues. Blockade of mast cell activation prevented increases in paracellular flux induced by CRF and Stressin 1 (p <0.05). Overall, these data demonstrate CRFr1 and CRFr2 play distinct roles in regulation of stress-induce mucosal barrier dysfunction with CRFr1 mediating intestinal barrier dysfunction and CRFr2 mediating protection, potentially through negative regulation of CRFr1 pathways. In addition, these data show that CRFr1mediated intestinal barrier dysfunction is mediated through activation of intestinal MCs.

Deficiency of BACH1 Ameliorates TNBS-Induced Colitis in Mice Thorough the Anti-Inflammatory Function of HO-1 Highly Expressed Macrophages

Psychological stress is a major contributing factor in the onset and exacerbation of gastrointestinal disease through poorly understood mechanisms. Our previous studies in a porcine model showed that stress-induced disturbances in mucosal barrier function are regulated by peripheral activation of CRFr subtypes CRFr1 and CRFr2. Furthermore, results from these studies suggested that CRFr1 mediates deleterious mucosal barrier responses to stress while CRFr2 may play a protective role. The objective of this study was to further elucidate the role of CRFr subtypes in stress intestinal barrier dysfunction. Porcine ileum was mounted on Ussing Chambers and exposed to CRFr1 and CRFr2 agonist treatments: 1) Control 2) CRF;(1μM;CRFr1/r2 agonist) 3) UCN2;(0.1μM CRFr2 agonist) 4) Stressin 1 (2nM;CRFr1 agonist). Intestinal barrier function was measured in terms of mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) over a 180-minute period on Ussing chambers. CRF treatment increased (p < 0.005) paracellular flux of FD4 compared with controls. Ileal mucosa treated with the CRFr1 agonist Stressin 1 exhibited the greatest FD4 flux compared with all other treatments (p< 0.001) whereas treatment with the CRFr2 agonist UCN2 had no effect on baseline ileal barrier function. However, when added in combination with Stressin 1, UCN2 inhibited (p<0.05) Stressin 1-induced increases in FD4 flux. To determine if the effects of CRFr activation on barrier function were mediated via intestinal mast cell activation, ileal tissue was pre-treated with the mast cell stabilizer cromolyn (10 -4M) and then CRFr agonists were applied to tissues. Blockade of mast cell activation prevented increases in paracellular flux induced by CRF and Stressin 1 (p <0.05). Overall, these data demonstrate CRFr1 and CRFr2 play distinct roles in regulation of stress-induce mucosal barrier dysfunction with CRFr1 mediating intestinal barrier dysfunction and CRFr2 mediating protection, potentially through negative regulation of CRFr1 pathways. In addition, these data show that CRFr1mediated intestinal barrier dysfunction is mediated through activation of intestinal MCs.

Gastrointestinal dysfunction induced by early weaning is attenuated by delayed weaning and mast cell blockade in pigs

Our previous work has demonstrated that weaning at 19 days of age has deleterious effects on mucosal barrier function in piglet intestine that are mediated through peripheral CRF receptor signaling pathways. The objectives of the present study were to assess the impact of piglet age on weaning-associated intestinal dysfunction and to determine the role that mast cells play in weaning-induced breakdown of mucosal barrier function. Nursing Yorkshire-cross piglets were either weaned at 19 days of age (early-weaned, n = 8) or 28 days of age (late-weaned, n = 8) and housed in nursery pens. Twenty-four hours postweaning, segments of midjejunum and ascending colon from piglets within each weaning age group were harvested and mounted on Ussing chambers for measurements of transepithelial electrical resistance and serosal-to-mucosal [(3)H]mannitol fluxes. Early weaning resulted in reductions in transepithelial electrical resistance and increases in mucosal permeability to [(3)H]mannitol in the jejunum and colon (P < 0.01). In contrast, postweaning reductions in intestinal barrier function were not observed in piglets weaned at 28 days of age. Early-weaned piglet intestinal mucosa had increased expression of CRF receptor 1 protein, increased mucosal mast cell tryptase levels, and evidence of enhanced mast cell degranulation compared with late-weaned intestinal mucosa. Pretreatment of piglets with the mast cell stabilizer drug cromolyn, injected intraperitoneally 30 min prior to weaning, abolished the early-weaning-induced intestinal barrier disturbances. Our results indicate that early-weaning stress induces mucosal dysfunction mediated by intestinal mast cell activation and can be prevented by delaying weaning.

Concurrent exposure to thermal stress and oral Ag induces intestinal sensitization in the mouse by a mechanism of regulation of IL‐12 expression

The mechanism of food allergy remains unclear. The absorption of intact protein Ag into the intestinal tissue is a prerequisite in the development of intestinal sensitization. Previous studies indicate that thermal stress compromises the intestinal barrier function. Mice were concurrently exposed to thermal stress and oral Ag. Intestinal sensitivity, levels of serum-specific IgE, IL-4 and INF-gamma were assessed. Intestinal dendritic cell, Th1 and Th2 functions were determined. The mice that were treated with thermal stress and oral Ag showed high levels of serum Ag-specific IgE, intestinal mast cell activation in response to oral Ag challenge, suppression of IL-12 expression in the intestinal dendritic cells, inhibition of T-bet expression and Th1 function and marked increases in (GATA)3 expression and Th2 function. Mice exposed to thermal stress alone or oral Ag alone did not show any signs of the intestinal sensitization. Pretreatment with IL-12 inhibited the intestinal sensitization induced by the concurrent exposure to thermal stress and Ag gavage. We conclude that although Ag absorption is essential, Ag absorption alone is insufficient; other accessory factors that can disturb the local immune homeostasis are also required for the induction of intestinal sensitization. The present study illustrates that concurrent exposure to thermal stress and oral Ag can prove to be a factor in the induction of intestinal sensitization by a mechanism of regulating IL-12 expression.

Study of visceral hypersensitivity in irritable bowel syndrome

Visceral hypersensitivity is highly prevalent in most functional bowel disorders, such as irritable bowel syndrome (IBS), and activation of intestinal mast cells (MC) may play a role because they have been found in close proximity to gastrointestinal mucosal sensory nerve terminals containing neuropeptides and a bi-directional pathway connecting the central nervous system, gut, and MC has been demonstrated. The current study appraised the status of rectal visceral perception, as well as the changes in the MC and substance P (SP) in the intestinal mucosa of patients with IBS. The study group comprised 42 patients with IBS and 19 healthy subjects who underwent anorectal manometry and rectal perception thresholds to balloon distension. The MC and the SP-ergic terminals in the mucosa were stained for respective histochemical and immunohistochemical investigations. The results were presented both qualitatively and quantitatively by color image analyzer, based on analysis of the intensity and area of stained fibrils. The structural relationship between the MC and nerve terminals was studied by electron microscopy, using an in situ embedding technique. The anorectal resting pressure, squeezing pressure and relaxation pressure were normal in both groups. The sensation threshold, defecation threshold and pain threshold in diarrhea-predominant IBS and the pain thresholds in constipation-predominant IBS were much lower than in the controls. Rectal compliance decreased in IBS. The number of MC in the terminal ileum, the ileocecal junction and the ascending colon was significantly elevated in IBS (P < 0.01), and the MC showed great variation. A significantly increased concentration of SP was found in the colon of the IBS patients compared with the controls. There was a positive correlation between the profiles of mucosal MC and the SP-ergic terminals, and MC were closely adjacent to SP-ergic terminals in the lamina propria. As altered rectal perception is present in almost all patients with IBS, it might be a reliable biological characteristic of the disease. Alterations in the MC and SP of the intestinal mucosa may be important factors in visceral hypersensitivity.

Intestinal mast cell activation and degranulation following physiological stress in IBD: electron microscopy study

Intestinal mast cell activation and degranulation following physiological stress in IBD: electron microscopy study

Mast cells: a possible link between psychological stress, enteric infection, food allergy and gut hypersensitivity in the irritable bowel syndrome.

Intestinal mast cell activation (degranulation), which results from previous enteric infection and/or intestinal allergy, may play a central role in the gut hypersensitivity in both motor response and visceral perception in the Irritable Bowel syndrome. This occurs through various mediators acting on enteric neurons and smooth muscle cells. Psychological stress may trigger this sensitive alarm system via the brain-gut axis.

IgE and mast cell responses on intestinal allergen exposure: A murine model to study the onset of food allergy

Objective: Allergic reactions to food are characterized by enhanced allergen-specific IgE serum levels and the activation of intestinal mast cells. Here we describe a murine model for the onset of food allergy and the role of cytokines in the regulation of food-induced IgE responses. Methods: Mice were primed systemically with low doses of alum-precipitated ovalbumin. Subsequent intragastric challenge led to enhanced sensitization. Results: Compared with baseline ovalbumin-specific IgE levels before challenge (0.23 ± 0.06 optical density [OD] units), ovalbumin-challenged mice showed significantly elevated IgE levels (0.86 ± 0.23 OD units) after intragastric challenge, which were not observed in control animals (0.29 ± 0.06 OD units). IgE levels mirrored intestinal mast cell activation, measured by decreased histamine levels in duodenal specimens, in ovalbumin-challenged mice (92.6 ± 7.9 ng/0.1 gm tissue weight) but not in saline-challenged mice (135.4 ± 18.3 ng/0.1 gm tissue weight), compared with baseline levels (141.1 ± 4.1 ng/0.1 gm tissue weight). Changes in IgE and histamine levels after intragastric challenge could be blocked by treating the animals with neutralizing antibodies against IL-4 or IL-10. Although it is generally accepted that ingestion of food allergens leads to a state of immunologic unresponsiveness (i.e., oral tolerance), it is shown here that low-dose systemic priming followed by intragastric challenge leads to sensitization instead of unresponsiveness. Conclusions: Our murine model shows an important correlation between T h2 cytokines, IgE production, and histamine release. Hence, this in vivo model provides a useful tool with which the complex mechanism underlying sensitization to food allergens can be studied. (J Allergy Clin Immunol 1997;99:94-9.)