Masayuki Saruta

Expression and functional characterization of FOXP3+CD4+ regulatory T cells in ulcerative colitis†

Background CD4+CD25+ regulatory T cells (TR) can prevent or treat experimental murine colitis but little is known about their potential role in human inflammatory bowel disease (IBD). FOXP3 is a transcription factor that plays a critical role in the development and function of CD4+CD25+ TR. The aim of this study was to examine the presence and functional characteristics of TR cells in colonic lymphoid tissues in patients with ulcerative colitis (UC). Methods FOXP3 expression was assessed by flow cytometry, immunohistochemistry, and reverse‐transcriptase polymerase chain reaction (RT‐PCR). Functional characterization of CD4+CD25+ cells was analyzed by suppression of proliferation and secretion of cytokines by cocultured effector CD4+CD25− T cells. Results FOXP3+CD4+ T cells are increased in the lamina propria (LP) of inflamed and noninflamed areas of UC colon compared to normal colon. CD4+CD25+ T cells in UC mesenteric lymph nodes (MLN) express FOXP3 mRNA and protein and suppress the proliferation of autologous MLN CD4+CD25− T cells. The suppressor activity of MLN CD4+CD25+ T cells is cell contact‐dependent but cytokine‐independent. In addition, CD4+CD25+ T cells potently suppress the production of both Th1 (IFN‐&ggr;, IL‐2) and Th2 (IL‐5, IL‐13) cytokines by cocultured CD4+CD25− T cells. FOXP3+ cells localized in the T‐cell‐rich areas of MLN and occasionally present in the follicles. Conclusions There is an expansion of FOXP3+CD4+ T cells in mucosal lymphoid tissues in UC. CD4+CD25+ isolated from UC MLN express FOXP3 and display features of TR cells in spite of active mucosal inflammation. These data suggest that their suppressor activity may be abrogated in vivo or they are unable to counterbalance the chronic mucosal inflammation in UC. (Inflamm Bowel Dis 2007)

Phenotype and effector function of CC chemokine receptor 9-expressing lymphocytes in small intestinal Crohn's disease

CCL25/CCR9 chemokine ligand/receptor pair has been reported to play an important role in small bowel (SB) immunity and inflammation. We have previously reported an aberrant SB expression of CCL25 in Crohn’s disease (CD) and an increased frequency of CCR9+ T cells in the peripheral blood of patients with SB inflammatory diseases such as CD and celiac disease. In this study, we have characterized the phenotype and effector function of CCR9+ T cells in mucosal lymphoid tissues in CD. We show that CCR9+ T cells isolated from mesenteric lymph nodes (MLN) draining CD SB express a more activated phenotype compared with MLN draining normal SB. Stimulation of CCR9+ T cells isolated from CD SB lamina propria produced more IFN-γ and IL-17 in response to anti-CD3 or IL-12/IL-18 stimulation compared with those isolated from normal SB. The addition of TL1A to the cytokine combination markedly augmented the secretion of IFN-γ, but not IL-17, by CD lamina propria CCR9+ T cells. CCL25 incubation of CD SB lamina propria lymphocytes and MLN lymphocytes increased their adhesion to VCAM-1/Fc in vitro. Finally, the TCRVβ analysis of CCR9+ T cells revealed a diverse TCRVβ repertoire among MLN CCR9+ T cells in patients with SB CD. Our data indicate that CCR9+ T cells in SB CD are proinflammatory and support the rationale for the use of CCR9 antagonists for the treatment of human SB CD.

Urocortin 3/stresscopin in human colon: possible modulators of gastrointestinal function during stressful conditions

Urocortin 3 (Ucn 3) or stresscopin (SCP) is a new member of the corticotropin-releasing factor (CRF) neuropeptide family and is a specific ligand for CRF type 2 receptor (CRF2). CRF receptors are known to be expressed in the gastrointestinal tract and are considered to play pathophysiological roles, for example, in gastrointestinal motility under stress. We, therefore, examined Ucn 3 expression in the normal human large intestine obtained from surgery and autopsy in order to clarify this local response to stress in human intestine. Both immunohistochemistry and mRNA in situ hybridization demonstrated Ucn 3 expression in myenteric and submucosal nervous plexus, in vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) of blood vessels in subserosa, in smooth muscle layers of the large intestine, and in enterochromaffin cells. In contrast to Urocortin 1 (Ucn 1), Ucn 3 was hardly detected in lamina propria (LP) inflammatory cells in colonic mucosa. In addition, immunohistochemistry demonstrated CRF2 expression in myenteric and submucosal nervous plexus, in smooth muscle layers, in VECs, in VSMCs and in lamina propria inflammatory cells. Immunoreactive Ucn 3 was also detected in the large intestine by RIA, with high concentrations detected in the rectum (15.4+/-9.5 pmol/g wet weight, mean+/-SEM, n=3) and sigmoid colon (6.5+/-3.5 pmol/g wet weight, n=5). Reverse-phase HPLC of the human large intestine disclosed peaks eluting in the position of synthetic Ucn 3 or SCP. These findings all suggest that Ucn 3 plays some physiological or pathological roles in the modulation of gastrointestinal functions during stressful conditions in different manners from Ucn 1.

Visilizumab induces apoptosis of mucosal T lymphocytes in ulcerative colitis through activation of caspase 3 and 8 dependent pathways

Visilizumab, a humanized low-Fc receptor binding anti-CD3 antibody, induces rapid clinical response in patients with steroid-refractory ulcerative colitis (UC). Several effective treatments in IBD have been linked to the induction of mucosal T cell apoptosis. The aim of the present study was to evaluate the effect of visilizumab on the apoptosis of lamina propria (LP) and peripheral blood (PB) lymphocytes isolated from patients with UC. Visilizumab induced dose- and time-dependent apoptosis of LP T cells isolated from non-IBD individuals, UC or CD patients. Maximal effect was seen at a concentration of 100 ng/ml and it was 33% for normal, 34% for UC and 23% for CD LP T cells following 24 h stimulation. Visilizumab induced apoptosis predominantly of CD4(+) LP T cells, whereas CD8(+) LP T cells were relatively resistant to apoptosis. Visilizumab did not induce apoptosis of PB T cells from UC patients. Visilizumab-induced apoptosis of LP T cells was dependent on caspase 3 and 8, but not caspase 9 activation and did not involve the Fas/FasL pathway. Low-Fc receptor binding Abs such as visilizumab may be highly effective for the treatment of UC through induction of apoptosis of LP T cells and rapid elimination of lesional pathogenic T cells in the gut mucosa.

High-frequency haplotypes in the X chromosome locus TLR8 are associated with both CD and UC in females.

Background: TNF‐&agr; and IL‐1 have been associated with mucosal inflammation in both Crohns disease (CD) and ulcerative colitis (UC). Innate immune defects have been associated with CD, specifically CARD15/NOD2. Recently, Toll‐like receptor 8 (TLR8) signaling has been shown to enhance generation of both cytokines. Interestingly, TLR8 is located on the X chromosome and inflammatory bowel disease (IBD) has been associated with abnormalities of the X chromosome. The aim was to test whether TLR8 haplotypes are associated with IBD. Methods: Subjects (735 CD, 343 UC, 245 controls) were genotyped. Single nucleotide polymorphisms (SNPs) were chosen to tag common Caucasian haplotypes. Results: Both “risk (H4)” and “protective (H1)” TLR8 haplotypes were observed associated with CD in females. Eighteen percent of CD females had H4 compared with 9% of controls (P = 0.02). Fifty‐nine percent of CD females had H1 compared with 72% of controls (P = 0.01). H1 was also negatively associated with UC in females (59% of UC, 72% of controls P = 0.03). Diplotype analysis of CD, UC, and all IBD in females revealed that 2 protective haplotypes (H1/H1) had a markedly diminished odds ratio, 0.4–0.5. The presence of a risk haplotype (H4 / not H1) had a significantly increased odds ratio, 2.0–2.2. Thus, the risk for IBD was 4–5 times higher in females with 1 risk haplotype than with the protective/protective diplotype. Conclusions: TLR8 is an X‐linked IBD susceptibility gene with both common predisposing and protecting haplotypes. These associations further emphasize the importance of genetic variation in innate immunity as determinants, not only of CD, but of UC as well.

TLR8-mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4+ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohns disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4+ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4+ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.

Fecal calprotectin is a clinically relevant biomarker of mucosal healing in patients with quiescent ulcerative colitis.

Calprotectin is an abundant protein in neutrophils, which infiltrate the mucosa during inflammation. Fecal calprotectin (FC) level has shown correlation with disease activity in ulcerative colitis (UC) patients. Additionally, FC level is expected to indicate mucosal healing (MH). This study was to see the significance of FC for predicting MH in patients with quiescent UC.

Expression of peptide YY in human brain and pituitary tissues

Expression of peptide YY (PYY) in the human brain and pituitary tissues was studied by radioimmunoassay, immunocytochemistry, and reverse transcription polymerase chain reaction. The polyclonal antibody raised against human PYY(1-36) in a rabbit was used in the assay, which showed 100% cross-reactivity with PYY(3-36) and no significant cross-reactivity with other peptides including neuropeptide Y and pancreatic polypeptide. The highest concentration of immunoreactive PYY was found in the hypothalamus (0.44+/-0.060 pmol/g of wet weight, mean +/- SEM, n=8), followed by the pituitary (0.41+/-0.16 pmol/g of wet weight, n=3). Reverse-phase high performance liquid chromatography of tissue extracts of human rectum and cortical brain showed a peak eluted in the position of authentic PYY(1-36) and PYY(3-36). Immunocytochemistry showed positive immunostaining for PYY in neurons of the paraventricular, arcuate, and supraoptic nuclei of the human hypothalamus. Moreover, reverse transcription polymerase chain reaction analysis showed expression of mRNA for PYY in human brain and pituitary tissues. The present study has shown for the first time expression of PYY in the human brain and pituitary tissues, suggesting a role for PYY as a neurotransmitter, in the neuroendocrine physiology, such as regulation of appetite and energy expenditure and modulation of pituitary hormone secretion.

Expression of urocortin 3/stresscopin in human adrenal glands and adrenal tumors

Urocortin 3 (Ucn 3)/stresscopin (SCP) is a novel peptide of the corticotropin-releasing factor (CRF) family and is a specific ligand for the CRF type 2 receptor. In the present study, we studied expression of Ucn3/SCP in the normal adrenal and adrenal tumors by radioimmunoassay and reverse transcriptase-polymerase chain reaction (RT-PCR). High concentrations of immunoreactive (IR)-Ucn3 were present in the normal portions of adrenal glands (4.2+/-0.51 pmol/g wet weight, mean+/-S.E.M., n = 14), and the levels were higher than those in the brain. IR-Ucn3 was also detected in the tumor tissues of aldosterone-secreting adenomas (6.2+/-0.6 pmol/g wet weight, n = 10), cortisol-secreting adenomas (5.0+/-1.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.9+/-0.4 pmol/g wet weight, n = 7). Reverse phase high performance liquid chromatography showed that IR-Ucn3 in normal portions of adrenal glands and aldosterone-secreting adenomas was eluted mainly in the positions of Ucn3 and SCP with several minor peaks eluting earlier. The RT-PCR showed expression of Ucn3 mRNA in normal portions of adrenal gland (positive ratio; 4/4), aldosterone-secreting adenomas (3/4), cortisol-secreting adenomas (1/3) and pheochromocytomas (6/7). These findings indicate that Ucn3 is produced in normal adrenal and adrenal tumors (both adrenocortical tumors and pheochromocytomas), and suggest that Ucn3 acts as an autocrine or paracrine regulator in normal adrenal and adrenal tumors.

Lymphocyte Homing Antagonists in the Treatment of Inflammatory Bowel Diseases

Lymphocyte homing antagonists represent promising therapeutic agents for the treatment of idiopathic inflammatory bowel disease (IBD). Several critical molecules involved in the recruitment of inflammatory cells in the intestine, including integrins and chemokine receptors, have been successfully targeted for the treatment of IBD. These agents have shown great promise for the induction and maintenance of remission for both Crohn disease and ulcerative colitis. This article discusses currently approved prototypic agents for the treatment of IBD (natalizumab, anti-α4 integrin; vedolizumab, anti-α4β7 integrin), and several other agents in the same class currently under development.