Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells

Heavy chain ferritin (H-ferritin) Is a component of the Iron-binding protein, ferritin. We have previously shown that H-ferritin Inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to Increased production of Interleukin-10 (IL-10). In the present study we have shown that Induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) In blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes Induced In MoDCs were compared with those Induced by CD40L and their significance tested by Inhibition with monoclonal antibodies. These studies Indicated that H-ferritin Induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), Inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to Induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or Interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin Induces B7-H1 and CD86 (B7-2) on APCs, which In turn Induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may Induce changes In APCs In the vicinity of the tumor and result in suppression of Immune responses by induction of regulatory T cells. (C) 2002 by The American Society of Hematology.

Disturbances in microbiota - cause or effect?

IBD are a group of complex polygenetic diseases also involving environmental factors. Evidence for a role for bacteria in IBD include an increased abundance of mucosa-associated bacteria in IBD (which occurs even where there is no intestinal inflammation), and the positive impact of antibiotics on the progress of both Crohn's disease (CD) and ulcerative colitis (UC) of the pouch - pouchitis. Bacteria are necessary for most animal models of IBD. The increased abundance of mucosal bacteria in IBD is not non-specific because while some mucosal bacteria are more abundant this is not the case for all mucosal bacteria including the very abundant Bacteroides vulgatus. On the other hand, antibiotic treatments are not curative, and the humoral immune Ig response to bacterial antigens which is more evident in CD, appears to be polyclonal. While this argues against a role for specific bacteria causing a classical infection, certain mucosal bacteria may damage the mucosal barrier. This would promote invasion by other commensal mucosal bacteria triggering an immune response. Altered adaptive, and to a lesser extent, innate immunity have been extensively studied, and genetic defects in the CARD15 (or NOD2) gene that encodes a bacterial sensing protein modulating innate and adaptive immunity are strongly associated with ileal CD. However, the penetrance of the homozygous CARD15 frameshift mutation, which is the most strongly CD-associated genotype, is very low with only 4% of humans with this developing CD. Furthermore, mice with the same defects in CARD15 do not develop spontaneous ileitis or colitis. Therefore, there have to be other aetiological factor(s). Altered permeability is a consistent finding in subclinical CD. There are other data to suggest that altered mucin is an early event in UC. We propose that the pathogenesis of IBD is multifactorial involving specific mucosal bacteria, defective barrier function and altered mucosal immunity in an aetiology triangle.

Fate of swallowed interleukin 8 and TNF alpha and effect on the wistar rat gut

To evaluate the passage of cytokines through the gastrointestinal tract, we investigated the digestion of interleukin-8 (IL-8) and tumour necrosis factor α (TNFα), in vitro and in vivo, and their propensity to induce intestinal inflammation. We serially immuno-assayed IL-8 and TNFα solutions co-incubated with each of three pancreatin preparations at pH 4.5 and pH 8. We gavaged IL-8, TNFα and marker into 15 Wistar rats, and measured their faecal cytokine concentrations by ELISA and histologically examined their guts. IL-8 immunoreactivity was extinguished by all pancreatin preparations after 1 h of incubation at 37 °C. TNFα concentration progressively fell from 1 to 4 h with all enzyme preparations. Buffer control samples maintained their cytokine concentrations throughout incubation. No IL-8 or TNFα was detected in any rat faecal pellets. There was no significant proinflammatory effect of the gavaged cytokines on rat intestine. IL-8 and TNFα in aqueous solution could well be fully digested in the CF gut when transit time is normal and exogenous enzymes are provided, although cytokines swallowed in viscous sputum may be protected from such digestion. Copyright © 2011 Elsevier B.V. All rights reserved

Systemic regulation of intestinal iron absorption

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.

A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo

S100A8 (also known as CP10 or MRP8) was the first member of the S100 family of calcium-binding proteins shown to be chemotactic for myeloid cells. The gene is expressed together with its dimerization partner S100A9 during myelopoiesis in the fetal liver and in adult bone marrow as well as in mature granulocytes. In this paper we show that S100A8 mRNA is expressed without S100A9 mRNA between 6.5 and 8.5 days postcoitum within fetal cells infiltrating the deciduum in the vicinity of the ectoplacental cone. Targeted disruption of the S100A8 gene caused rapid and synchronous embryo resorption by day 9.5 of development in 100% of homozygous null embryos. Until this point there was no evidence of developmental delay in S100A8(-/-) embryos and decidualization was normal. The results of PCR genotyping around 7.5-8.5 days postcoitum suggest that the null embryos are infiltrated with maternal cells before overt signs of resorption. This work is the first evidence for nonredundant function of a member of the S100 gene family and implies a role in prevention of maternal rejection of the implanting embryo. The S100A8 null provides a new model for studying fetal-maternal interactions during implantation.

Homocysteine-lowering treatment with folic acid, cobalamin, and pyridoxine does not reduce blood markers of inflammation, endothelial dysfunction, or hypercoagulability in patients with previous transient ischemic attack or stroke - a randomized substudy

Background and Purpose - Epidemiological and laboratory studies suggest that increasing concentrations of plasma homocysteine ( total homocysteine [tHcy]) accelerate cardiovascular disease by promoting vascular inflammation, endothelial dysfunction, and hypercoagulability. Methods - We conducted a randomized controlled trial in 285 patients with recent transient ischemic attack or stroke to examine the effect of lowering tHcy with folic acid 2 mg, vitamin B-12 0.5 mg, and vitamin B-6 25 mg compared with placebo on laboratory markers of vascular inflammation, endothelial dysfunction, and hypercoagulability. Results - At 6 months after randomization, there was no significant difference in blood concentrations of markers of vascular inflammation (high-sensitivity C-reactive protein [P = 0.32]; soluble CD40L [ P = 0.33]; IL-6 [P = 0.77]), endothelial dysfunction ( vascular cell adhesion molecule-1 [P = 0.27]; intercellular adhesion molecule-1 [P = 0.08]; von Willebrand factor [P = 0.92]), and hypercoagulability (P-selectin [P = 0.33]; prothrombin fragment 1 and 2 [P = 0.81]; D-dimer [P = 0.88]) among patients assigned vitamin therapy compared with placebo despite a 3.7-mumol/L (95% CI, 2.7 to 4.7) reduction in total homocysteine (tHcy). Conclusions - Lowering tHcy by 3.7 mumol/L with folic acid-based multivitamin therapy does not significantly reduce blood concentrations of the biomarkers of inflammation, endothelial dysfunction, or hypercoagulability measured in our study. The possible explanations for our findings are: ( 1) these biomarkers are not sensitive to the effects of lowering tHcy (eg, multiple risk factor interventions may be required); ( 2) elevated tHcy causes cardiovascular disease by mechanisms other than the biomarkers measured; or ( 3) elevated tHcy is a noncausal marker of increased vascular risk.

The mechanism of inflammation in RelB deficient mice

RelB, NIK and TRAF6-deficient mice die prematurely with multi-organ inflammatory disease and apparent excessive myelopoiesis. While thymic development of CD4+CD25+ regulatory T cells (Treg) is reduced in TRAF6 deficient mice, the impact of this on inflammation is not known. Here we show that while RelB deficient thymic stroma is unable to sustain the development of Treg, surprisingly, FoxP3hi Treg are increased in the periphery. Peripheral expansion of Treg is driven by GITRligand, expressed by immature monocytes maintained by RelBdeficient stroma. RelB-deficient DC fail to activate Treg suppressor function. The data reveal the dual roles of RelB in both hemopoietic and stromal cells to maintain tolerance and contain inflammation through Treg and DC.

Local inflammation is crucial for T cell mediated rejection of skin graft expressing foreign antigen

Most of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.

Effects of (-)-RO363 at human atrial beta-adrenoceptor subtypes, the human cloned beta(3)-adrenoceptor and rodent intestinal beta(3)-adrenoceptors

1 Chronic treatment of patients with beta-blockers causes atrial inotropic hyperresponsiveness through beta(2)-adrenoceptors, 5-HT4 receptors and H-2-receptors but apparently not through beta(1)-adrenoceptors despite data claiming an increased beta(1)-adrenoceptor density from homogenate binding studies. We have addressed the question of beta(1)-adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the beta(1)-adrenoceptor selective partial agonist (-)-RO363 and by carrying out both homogenate binding and quantitative beta-adrenoceptor autoradiography in atria obtained from patients treated or not treated with beta-blockers. In the course of the experiments it became apparent that (-)-RO363 also may cause agonistic effects through the third atrial beta-adrenoceptor. To assess whether (-)-RO363 also caused agonistic effects through beta(3)-adrenoceptors we studied its relaxant effects in rat colon and guinea-pig ileum, as well as receptor binding and adenylyl cyclase stimulation of chinese hamster ovary (CHO) cells expressing human beta(3)-adrenoceptors. 2 beta-Adrenoceptors were labelled with (-)-[I-125]-cyanopindolol. The density of both beta(1)- and beta(2)-adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (-)-RO363 for beta(1)-adrenoceptors (pK(i) = 8.0-7.7) and beta(2)-adrenoceptors (pK(i) = 6.1-5.8) were not significantly different in the two groups. 3 (-)-RO363 increased atrial force with a pEC(50) of 8.2 (beta-blocker treated) and 8.0 (non-beta-blocker treated) and intrinsic activity with respect to (-)-isoprenaline of 0.80 (beta-blocker treated) and 0.54 (non-beta-blocker treated) (P<0.001) and with respect to Ca2+ (7 mM) of 0.65 (beta-blocker treated) and 0.45 (non-beta-blocker treated) (P<0.01). The effects of (-)-RO363 were resistant to antagonism by the beta(2)-adrenoceptor antagonist, ICI 118,551 (50 nM). The effects of 0.3-10 nM (-)-RO363 were antagonized by 3-10 nM of the beta(1)-adrenoceptor selective antagonist CGP 20712A. The effects of 20-1000 nM (-)-RO363 were partially resistant to antagonism by 30-300 nM CGP 20712A. 4 (-)-RO363 relaxed the rat colon, partially precontracted by 30 mM KCl, with an intrinsic activity of 0.97 compared to (-)-isoprenaline. The concentration-effect curve to (-)-RO363 revealed 2 components, one antagonized by (-)-propranolol (200 nM) with pEC(50)=8.5 and fraction 0.66, the other resistant to (-)-propranolol (200 nM) with pEC(50)=5.6 and fraction 0.34 of maximal relaxation. 5 (-)-RO363 relaxed the longitudinal muscle of guinea-pig ileum, precontracted by 0.5 mu M histamine, with intrinsic activity of 1.0 compared to (-)-isoprenaline and through 2 components, one antagonized by (-)-propranolol (200 nM) with pEC(50)=8.7 and fraction 0.67, the other resistant to (-)-propranolol with pEC(50)=4.9 and fraction 0.33 of maximal relaxation. 6 (-)-RO363 stimulated the adenylyl cyclase of CHO cells expressing human beta(3)-adrenoceptors with pEC(50)=5.5 and intrinsic activity 0.74 with respect to (-)-isoprenaline (pEC(50)=5.9). (-)-RO363 competed for binding with [I-125]cyanopindolol at human beta(3)-adrenoceptors transfected into CHO cells with pK(i)=4.5. (-)-Isoprenaline (pk(i)=5.2) and (-)-CGP 12177A (pK(i)=6.1) also competed for binding at human beta(2)-adrenoceptors. 7 We conclude that under conditions used in this study, (-)-RO363 is a potent partial agonist for human beta(1)- and beta(3)-adrenoceptors and appears also to activate the third human atrial beta-adrenoceptor. (-)-RO363 relaxes mammalian gut through both beta(1)- and beta(3)-adrenoceptors. (-)-RO363, used as a beta(1)-adrenoceptor selective tool, confirms previous findings with (-)-noradrenaline that beta(1)-adrenoceptor mediated atrial effects are only slightly enhanced by chronic treatment of patients with beta-blockers. Chronic treatment with beta(1)-adrenoceptor-selective blockers does not significantly increase the density of human atrial beta(1)- and beta(2)-adrenoceptors.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mu mol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mu mol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mu mol/l; P<0.05), PACAP also stimulated FSH secretion in cells from some animals although this effect was small, The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist; but not by (N-Ac-Tyr(1), D-Arg(2))-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mu mol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mu mol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mu mol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mu mol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.