Cyclooxygenase-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigen.

Intestinal inflammatory diseases are mediated by dysregulated immune responses to undefined luminal antigens. Feeding hen egg-white lysozyme to mice expressing a transgenic T-cell receptor that recognizes hen egg-white lysozyme peptide 46-61 resulted in no intestinal pathology; however, simultaneous administration of cyclooxygenase-2 inhibitors and dietary hen egg-white lysozyme resulted in increased proliferation of lamina propria mononuclear cells and crypt epithelial cells, crypt expansion and villus blunting. Lamina propria mononuclear cells produce high levels of cyclooxygenase-2-dependent arachidonic acid metabolites, which act as immunomodulators in the immune response to dietary antigen. These findings establish that cyclooxygenase-2-dependent arachidonic acid metabolites are essential in the development and maintenance of intestinal immune homeostasis.

Genetic risk factors for perception of symptoms in GERD: an observational cohort study.

BACKGROUND: Genetic polymorphisms in G-protein beta-3 subunit (GNß3) and beta-2 adrenergic receptor (ADRB2) are associated with pain and gut hypersensitivity, which can overlap with gastroesophageal reflux disease (GERD). AIM: To evaluate relationships between single nucleotide polymorphisms (SNPs) within GNß3 and ADRB2 systems, and reflux symptom burden, GERD phenotypes from ambulatory reflux monitoring, and quality of life. METHODS: Symptomatic adults undergoing ambulatory reflux testing were recruited and phenotyped based on acid burden and symptom reflux association; major oesophageal motor disorders and prior foregut surgery were exclusions. A comparison asymptomatic control cohort was also identified. Subjects and controls completed questionnaires assessing symptom burden on visual analog scales, short-form health survey-36 (SF-36), and Beck Anxiety and Depression Inventories (BAI and BDI). Genotyping was performed from saliva samples; 6 SNPs selected from each of the two genes of interest were compared. RESULTS: Saliva from 151 study subjects (55.3 ± 1.2 years, 63.6% F) and 60 control subjects (50.9 ± 2.2 years, 66.7%) had sufficient genetic material for genotyping. Study subjects had higher symptom burden, worse total and physical health, and higher anxiety scores compared to controls (P ≤ .002). Tested SNPs within ADRB2 were similar between study subjects and controls (P > .09). Study subjects with recessive alleles in 3 GNß3 SNPs (Rs2301339, Rs5443, and Rs5446) had worse symptom severity (P = .011), worse mental health (P = .03), and higher depression scores (P = .005) despite no associations with GERD phenotypes or reflux metrics. CONCLUSIONS: Genetic variation within GNß3 predicts oesophageal symptom burden and affect, but not oesophageal acid burden or symptom association with reflux episodes.

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.

Microbial sensing by goblet cells controls immune surveillance of luminal antigens in the colon.

The delivery of luminal substances across the intestinal epithelium to the immune system is a critical event in immune surveillance, resulting in tolerance to dietary antigens and immunity to pathogens. How this process is regulated is largely unknown. Recently goblet cell-associated antigen passages (GAPs) were identified as a pathway delivering luminal antigens to underlying lamina propria (LP) dendritic cells in the steady state. Here, we demonstrate that goblet cells (GCs) form GAPs in response to acetylcholine (ACh) acting on muscarinic ACh receptor 4. GAP formation in the small intestine was regulated at the level of ACh production, as GCs rapidly formed GAPs in response to ACh analogs. In contrast, colonic GAP formation was regulated at the level of GC responsiveness to ACh. Myd88-dependent microbial sensing by colonic GCs inhibited the ability of colonic GCs to respond to Ach to form GAPs and deliver luminal antigens to colonic LP-antigen-presenting cells (APCs). Disruption of GC microbial sensing in the setting of an intact gut microbiota opened colonic GAPs, and resulted in recruitment of neutrophils and APCs and production of inflammatory cytokines. Thus GC intrinsic sensing of the microbiota has a critical role regulating the exposure of the colonic immune system to luminal substances.

Genetic variation in the beta-2 adrenergic receptor (ADRB2) predicts functional gastrointestinal diagnoses and poorer health-related quality of life.

BACKGROUND: The beta-2 adrenergic receptor (ADRB2) is an important target for epinephrine, a neurotransmitter in pain signalling. ADRB2 haplotypes affect receptor expression and ligand response, and have been linked to painful non-GI disorders. AIMS: To assess whether ADRB2 polymorphisms (rs1042713, rs1042714) are risk alleles for functional GI (FGID) and extraintestinal functional (EIFD) diagnoses, and whether ADRB2 predicts GI symptoms and health-related quality of life (HRQOL). METHODS: Of 398 subjects (49.6 ± 2.9 years, 68.0% female), 170 (42.5%) met Rome III criteria for ≥1 FGID [IBS (n = 139, 34.9%); functional dyspepsia (FD, n = 136, 34.1%), functional chest pain (FCP, n = 25, 6.2%)], while 228 were healthy controls. FGID subjects reported on bowel symptom severity and burden (10-cm VAS), frequency (days/last 2 weeks), EIFD, psychiatric diagnoses and HRQOL (SF 36). Multivariable models determined the contribution of ADRB2 polymorphisms to HRQOL, and mediational analyses assessed functional diagnoses as potential intermediates. RESULTS: rs1042714 minor G alleles were associated with FGID diagnoses (OR 1.8; 95% CI 1.2-2.7; P = 0.009), particularly FD (OR 2.1, 95% CI 1.3-3.3), with trends towards IBS (P = 0.19) and FCP (P = 0.06) diagnoses. Within IBS, G allele carriers had more severe bowel symptoms (P = 0.025), and symptomatic days (P = 0.009). G allele carriers had greater numbers of EIFD (1.0 ± 0.1 vs. 0.4 ± 0.07, P < 0.001) and poorer HRQOL. The effect of ADRB2 on HRQOL was partially mediated by FGID, EIFD and psychiatric diagnoses. CONCLUSIONS: ADRB2 minor alleles at rs1042714 predict FGID and EIFD, and may influence bowel symptom severity and HRQOL. These findings provide indirect evidence of sympathetic nervous system role in FGID pathophysiology.

Spontaneous and continuous cyclooxygenase-2-dependent prostaglandin E2 production by stromal cells in the murine small intestine lamina propria: directing the tone of the intestinal immune response.

The mechanisms allowing the gastrointestinal immune system to avoid an inappropriate inflammatory response to nonpathogenic luminal Ags are poorly understood. We have previously described a role for cyclooxygenase (COX)-2-dependent arachidonic acid metabolites produced by the murine small intestine lamina propria in controlling the immune response to a dietary Ag. To better understand the role of COX-2-dependent arachidonic acid metabolites produced by the lamina propria, we examined the pattern of expression and the cellular source of COX-2 and COX-2-dependent PGE(2). We now demonstrate that non-bone marrow-derived lamina propria stromal cells have basal COX-2 expression and that COX-2-dependent PGE(2) production by these cells is spontaneous and continuous. The other mucosal and nonmucosal lymphoid compartments examined do not share this phenotype. In contrast to the majority of descriptions of COX-2 expression, COX-2 expression by lamina propria stromal cells is not dependent upon exogenous stimuli, including adhesion, LPS signaling via Toll-like receptor 4, or the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-1 beta. These findings, in conjunction with the known immunomodulatory capacities of PGs, suggest that COX-2 expression by the small intestine lamina propria is a basal state contributing to the hyporesponsiveness of the intestinal immune response.

Microbiological assay of blood serum for the vitamin B12 status of dairy cows.

Four vitamin B12 assays were compared using blood sera from Friesian cows on winter diets or grazing. In herd 1, ten animals were blood-sampled three times at monthly intervals and the vitamin B12 concentration of the sera determined by the Poteriochromonas malhamensis and Lactobacillus delbrueckii assays. At all three sampling dates the results produced by the P. malhamensis assay were significantly greater than those produced by the L. delbrueckii assay. Cows in herd 2 were divided into two groups, each of 30 animals. One group was given a soluble glass bolus releasing cobalt and the other was unsupplemented. Milk yields were recorded throughout the experimental period and herbage samples were taken at intervals for cobalt determinations. Blood samples were taken at intervals from January to September 1983 and the vitamin B12 concentration of the sera determined by four different assay methods: the P. malhamensis and L. delbrueckii microbiological assays and the Becton Dickinson and RIA Products 'No-boil' radioassays. The last of these failed to detect vitamin B12 in any sample. There was a significant difference between the results obtained by the three other assays, with the Becton Dickinson radioassay consistently producing the lowest result and the P. malhamensis assay the highest result of the three. There was no significant effect of cobalt supplementation on milk yield.

Positive and negative selection of an antigen receptor on T cells in transgenic mice.

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.

Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice.

The major problem in the study of T-cell development is that of tracking thymocytes of a given specificity. Recent studies have exploited natural correlations between the expression of a particular V beta gene segment and T-cell receptor (TCR) specificity. We and others (refs 5, 6 and M. Davis, personal communication) have taken an alternative approach. We have generated transgenic mice expressing the alpha beta antigen receptor from the cytotoxic T-lymphocyte clone 2C (ref. 7). In transgenic mice of the same haplotype as the 2C clone, the 2C TCR was expressed on 20-95% of peripheral T cells. Very few of these T cells carried the CD4 antigen; the vast majority were CD4-CD8+ and were able to lyse targets with the same specificity as the original 2C clone. These results indicate that the alpha beta heterodimer transfers specificity to recipient cells as expected from earlier studies, and that receptor specificity in T-cell repertoire selection is determined by both alpha beta heterodimer and CD4 or CD8 accessory molecules.

Positive selection of transgenic receptor-bearing thymocytes by Kb antigen is altered by Kb mutations that involve peptide binding.

A specific interaction between the class I major histocompatibility complex molecule Kb and thymocytes expressing the antigen receptor from the cytolytic T lymphocyte 2C enhances maturation of T cells of the CD8 lineage in transgenic mice. By analyzing transgenic mice backcrossed to Kbm mutant strains of mice, we have identified five bm mutations of the Kb antigen-encoding gene that alter the positive selection of thymocytes induced by Kb antigen. Compared with Kb, Kbm10 and Kbm1 did not induce significant maturation of 2C T-cell receptor-bearing thymocytes, and Kbm8 antigen positively selected for transgenic thymocytes only weakly. Altering residue 77 of Kb molecule from aspartic acid to serine made Kbm3 and Kbm11 allogeneic targets for the 2C antigen receptor and caused deletion of transgenic thymocytes. This deletion spared T cells that expressed low levels of CD8, a result differing from the total deletion of CD8-bearing T cells seen in mice that expressed the original target alloantigen Ld. This evidence indicates that (i) self-peptides bound to thymic major histocompatibility complex molecules can influence the positive selection of thymocytes and (ii) thymocytes with apparently weak interaction with self-major histocompatibility complex antigens can escape clonal deletion.