IL-10 signaling in dendritic cells controls IL-1ß-mediated IFNγ secretion by human CD4+ T cells: relevance to inflammatory bowel disease.

Uncontrolled interferon γ (IFNγ)-mediated T-cell responses to commensal microbiota are a driver of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is crucial for controlling these T-cell responses, but the precise mechanism of inhibition remains unclear. A better understanding of how IL-10 exerts its suppressive function may allow identification of individuals with suboptimal IL-10 function among the heterogeneous population of IBD patients. Using cells from patients with an IL10RA deficiency or STAT3 mutations, we demonstrate that IL-10 signaling in monocyte-derived dendritic cells (moDCs), but not T cells, is essential for controlling IFNγ-secreting CD4+ T cells. Deficiency in IL-10 signaling dramatically increased IL-1ß release by moDCs. IL-1ß boosted IFNγ secretion by CD4+ T cells either directly or indirectly by stimulating moDCs to secrete IL-12. As predicted a signature of IL-10 dysfunction was observed in a subgroup of pediatric IBD patients having higher IL-1ß expression in activated immune cells and macroscopically affected intestinal tissue. In agreement, reduced IL10RA expression was detected in peripheral blood mononuclear cells and a subgroup of pediatric IBD patients exhibited diminished IL-10 responsiveness. Our data unveil an important mechanism by which IL-10 controls IFNγ-secreting CD4+ T cells in humans and identifies IL-1ß as a potential classifier for a subgroup of IBD patients.

IL-10 signaling prevents gluten-dependent intraepithelial CD4+ cytotoxic T lymphocyte infiltration and epithelial damage in the small intestine.

Breach of tolerance to gluten leads to the chronic small intestinal enteropathy celiac disease. A key event in celiac disease development is gluten-dependent infiltration of activated cytotoxic intraepithelial lymphocytes (IELs), which cytolyze epithelial cells causing crypt hyperplasia and villous atrophy. The mechanisms leading to gluten-dependent small intestinal IEL infiltration and activation remain elusive. We have demonstrated that under homeostatic conditions in mice, gluten drives the differentiation of anti-inflammatory T cells producing large amounts of the immunosuppressive cytokine interleukin-10 (IL-10). Here we addressed whether this dominant IL-10 axis prevents gluten-dependent infiltration of activated cytotoxic IEL and subsequent small intestinal enteropathy. We demonstrate that IL-10 regulation prevents gluten-induced cytotoxic inflammatory IEL infiltration. In particular, IL-10 suppresses gluten-induced accumulation of a specialized population of cytotoxic CD4+CD8αα+ IEL (CD4+ CTL) expressing Tbx21, Ifng, and Il21, and a disparate non-cytolytic CD4+CD8α- IEL population expressing Il17a, Il21, and Il10. Concomitantly, IL-10 suppresses gluten-dependent small intestinal epithelial hyperproliferation and upregulation of stress-induced molecules on epithelial cells. Remarkably, frequencies of granzyme B+CD4+CD8α+ IEL are increased in pediatric celiac disease patient biopsies. These findings demonstrate that IL-10 is pivotal to prevent gluten-induced small intestinal inflammation and epithelial damage, and imply that CD4+ CTL are potential new players into these processes.

Macrophage-mediated gliadin degradation and concomitant IL-27 production drive IL-10- and IFN-γ-secreting Tr1-like-cell differentiation in a murine model for gluten tolerance.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

Increased production of interleukin-21, but not interleukin-17A, in the small intestine characterizes pediatric celiac disease.

Celiac disease (CD) is caused by inflammatory CD4(+) T-cell responses to dietary gluten. It is unclear whether interleukin (IL)-21 and IL-17A contribute to CD onset and lesion severity; therefore, we investigated IL-21 and IL-17A expression in biopsies from pediatric CD patients with different histopathological scores. High numbers of IL-21-producing cells were observed in pediatric CD lesions, even Marsh 1-2 lesions, whereas increased numbers of IL-17 secreting cells were not observed. Intraepithelial lymphocytes, CD4(+) T cells and also neutrophils secreted IL-21. Flow cytometry of lamina propria cells revealed a large population of IL-21- and interferon-γ (IFN-γ)-secreting CD3(+) T cells that did not secrete IL-17A. Adult CD patient biopsies also contained high numbers of IL-21-positive cells; however, enhanced numbers of IL-17-positive cells were observed in a small subgroup of patients with severe lesions. As duodenal tissue damage increases contact with microbe-associated molecular patterns, we hypothesized that microbial sensing by Toll-like receptors (TLRs) modulates T cell-derived cytokine secretion. Costimulation with TLR3 ligands during polyclonal T-cell activation significantly increased IL-21 secretion, whereas TLR2 ligands selectively enhanced IL-17A. These results demonstrate that an IL-17A-independent increase in IL-21 production by CD4(+) T cells is characteristic of pediatric CD. We hypothesize that incidental IL-17 secretion is caused by tissue damage rather than gluten-specific responses.

The role of the IGF axis in IGFBP-1 and IGF-I induced renal enlargement in Snell dwarf mice.

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is generally believed to inhibit IGF action in the circulation. In contrast, IGFBP-1 has been reported to interact with cell surfaces and enhance IGF-I action locally in some tissues. Renal IGFBP-1 levels are found elevated in various conditions characterized by renal growth (e.g. diabetes mellitus, hypokalemia). To test whether IGFBP-1 is a renotropic factor, IGFBP-1 was administered alone or in combination with IGF-I to Snell dwarf mice, an in vivo model without compensatory feedback effects on growth hormone (GH) secretion. In three control groups of Snell dwarf mice, placebo, GH or IGF-I was administered. Compared with placebo, kidney weight increased in all treated groups, however, with different effects on kidney morphology. Administration of IGF-I, alone or in combination with IGFBP-1, tended to increase glomerular volume, while no changes were seen in the other groups. Administration of IGFBP-1 or IGFBP-1+IGF-I both caused dilatation of the thin limbs of Henle's loop, while GH or IGF-I administration had no visible effect. Furthermore, IGF-I administration resulted in an increased mean number of nuclei per cortical area and renal weight, whereas GH, IGF-I+IGFBP-1 or IGFBP-1 caused a decreased renal nuclei number. In situ hybridization and immunohistochemistry showed specific changes of the renal IGF system expression patterns in the different groups. Particularly, IGFBP-1 administration resulted in extensive changes in the mRNA expression of the renal IGF system, whereas the other administration regimen resulted in less prominent modifications. In contrast, administration of IGFBP-1 and IGFBP-1+IGF-I resulted in identical changes in the protein expression of the renal IGF system. Our results indicate that IGFBP-1, alone or in combination with IGF-I, demonstrated effects on the renal tubular system that differ from the effects of IGF-I.

Human insulin-like growth factor (IGF) binding protein-1 inhibits IGF-I-stimulated body growth but stimulates growth of the kidney in snell dwarf mice.

The actions of insulin-like growth factor-I (IGF-I) are modulated by IGF binding proteins (IGFBPs). The effects of IGFBP-1 in vivo are insufficiently known, with respect to inhibitory or stimulatory actions on IGF-induced growth of specific organs. Therefore, we studied the effects of IGFBP-1 on IGF-I-induced somatic and organ growth in pituitary-deficient Snell dwarf mice. Human GH, IGF-I, IGFBP-1, and a preequilibrated combination of equimolar amounts of IGF-I and IGFBP-1 were administered sc during 4 weeks. Treatment with IGF-I alone induced a significant increase in body length (108% of control) and weight (112%) as well as an increase in weight of the submandibular salivary glands (135%), kidneys (124%), femoral muscles (111%), testes (129%), and spleen (126%) compared with saline-treated controls. IGFBP-1 alone induced a significant increase in weight of the kidneys (152% of control). Coadministration of IGF-I with IGFBP-1 neutralized the stimulating effects of IGF-I on body length and weight as well as on the femoral muscles and testes. In contrast, the weights of the submandibular salivary glands (143%) were not significantly different from those of IGF-I-treated animals, whereas the weights of the kidneys (171%) and spleen (156%) were significantly increased compared with IGF-I-treated mice. The effect of IGFBP-1 plus IGF-I on kidney weight was not significantly greater than the effect of IGFBP-1 alone. Western ligand blotting showed induction of the IGFBP-3 doublet as well as IGFBPs with molecular masses of 24 kDa, most probably IGFBP-4, by human GH, IGF-I alone, and IGF-I in combination with IGFBP-1. Our data show that coadministration of IGFBP-1 inhibits IGF-I-induced body growth of GH-deficient mice but significantly stimulates the growth promoting effects of IGF-I on the kidneys and the spleen. These data warrant further investigation because differences in concentrations of IGFBP-1 occurring in vivo may influence IGF-I-induced anabolic processes.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo.

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.

The IGF system during fetal-placental development of the mouse.

Insulin-like growth factors (IGF-I and -II) promote cellular mitosis and differentiation and have been implicated in fetal and placental growth. Together with the IGF receptors and IGF binding proteins (IGFBPs) they form a complex network, with tissue specific activity. This review will discuss the data generated to elucidate the functions of the IGF system during mouse development.

mRNA expression patterns of the IGF system during mouse limb bud development, determined by whole mount in situ hybridization.

During limb development the primary limb bud requires various signals to differentiate. Insulin-like growth factor (IGF)-I and IGF-II serve as ubiquitous cellular growth promoters and are modulated by their binding proteins (IGFBPs), which inhibit or augment IGF bioavailability. This is the first study to give a complete overview of the mRNA expression patterns of Igf-1, Igf-2, type 1 Igf receptor (Igf1r) and six Igf binding proteins (IGFBP-1-6) in embryonic mouse limbs, at various stages of development, by whole mount in situ hybridization (ISH). Our results show that all the members of the Igf system, except Igfbp-1 and -6, have specific spatio-temporal mRNA expression patterns. IGFBP-2 and -5 are found in the apical ectodermal ridge (AER), and IGF-I and IGFBP-4 in the region of the zone of polarizing activity (ZPA). IGF-II and IGF1R are found in regions of pre-cartilage formation. At 13.5 days post coitus (dpc) the IGF system colocalizes with apoptosis areas; IGFBP-2, -4 and -5 are found in the interdigital zone, while IGFBP-3 and IGF-I border this region. Furthermore, IGFBP-3, -4 and -5 are found in the phalangeal joint areas, at an early stage of joint formation. This supports the hypothesis that the IGF system may be involved in chondrogenic differentiation of mesenchyme and the regulation of apoptosis in the developing limb.

Gene expression of the insulin-like growth factor system during mouse kidney development.

Expression of the insulin-like growth factor (IGF) system was investigated in mouse renal development and physiology, using non radioactive in situ hybridization and quantitative RT-PCR. IGF-I mRNA levels increased after birth and were confined to distal tubules and peritubular capillaries in the outer medulla. IGF-II mRNA levels were high in developing kidneys and peaked after birth. The type I receptor mRNA expression pattern mostly parallelled those of IGF-I and IGF-II. The IGF binding proteins (IGFBP's) showed weak mRNA expression for IGFBP-1 and -6. High fetal mRNA levels were measured for IGFBP-2, showing a similar profile in time as observed for IGF-II. Low fetal IGFBP-3 and -5 mRNA levels increased after birth. IGFBP-2, -4 and -5 mRNA expression was localized to differentiating cells. In the mature kidney predominant expression was confined to proximal tubules (IGFBP-4), thin limbs of Henle's Loop (IGFBP-2), glomerular mesangial cells (IGFBP-5) and peritubular capillaries of the medulla (IGFBP-5). IGFBP-3 mRNA was exclusively expressed in endothelial cells of the renal capillary system. Distinct mRNA expression for each member of the IGF system may point to specific roles in development and physiology of the mouse kidney.

Insulin-like growth factor binding protein-2, 28 kDa and 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles, obtained from normal and polycystic ovaries.

In order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with regular menstrual cycles and of subjects with cycle abnormalities and polycystic ovaries (diagnosed as polycystic ovary syndrome (PCOS)) and analyzed by Western ligand blotting. IGFBPs of 43 kDa, 37 kDa, 31 kDa, a doublet around 28 kDa and a minor band of 24 kDa were detected in follicle fluid of normal non-dominant (size < 10 mm) and atretic (androstenedione/estradiol ratio > 4) follicles of both regularly menstruating women and PCOS patients. The 43 and 37 kDa IGFBPs could be identified as IGFBP-3 and the 31 kDa IGFBP as IGFBP-2, whereas the 28 kDa IGFBP could not be identified as IGFBP-1, all by immunoblotting techniques. A dramatic decrease in IGFBP-2, the 28 kDa and 24 kDa IGFBPs was observed in follicular fluid of dominant follicles (size > 10 mm) of both regular menstruating individuals and one PCOS patient as compared with follicular fluid of normal non-dominant or atretic follicles. These observations indicate that the PCOS follicle may not be different from normal with respect to IGFBP profiles. Furthermore, these results suggest that at least one of these IGFBPs might be involved in human folliculogenesis.

Localization of the epitope of a monoclonal antibody against human insulin-like growth factor binding protein-1, functionally interfering with insulin-like growth factor binding.

In order to identify regions in insulin-like growth factor binding protein-1 involved in the binding of IGFs, we tested three monoclonal antibodies, designated MAb A, B, and C on their interference with IGF-binding. Monoclonal A interfered with the binding of IGF to IGFBP-1 as determined by immunoprecipitation whereas monoclonal B and C did not. Furthermore MAb A was found to abolish IGFBP-1 inhibition of IGF stimulation in an in vitro proliferation assay. The epitopes of all three monoclonal antibodies were found to be located within the C-terminal part of IGFBP-1. The regions surrounding residue 188-196 and 222-227 are especially important for antibody recognition. These results indicate that MAb A functionally interferes with the binding of IGF to IGFBP-1. Furthermore, we suggest that part of the epitope of MAb A is located at or sterically near the IGF binding domain of IGFBP-1.

Gene expression of the IGF binding proteins during post-implantation embryogenesis of the mouse; comparison with the expression of IGF-I and -II and their receptors in rodent and human.

The IGF binding proteins (IGFBPs) comprise at least six distinct species which may modulate the action of IGFs. IGFs are important regulators of fetal growth and differentiation. We have studied the mRNA expression of the six IGFBPs during post-implantation embryogenesis (day 11-18) by in situ hybridization techniques. Expression of IGFBP-1 was detected in mouse conceptuses after day 12 of gestation and seemed restricted to the liver. Transcripts for IGFBP-2, -4 and -5 were detected in various tissues and were found in all stages tested. In contrast, expression of IGFBP-3 and -6 could be detected only weakly in late gestational embryos. Comparison of the expression pattern of IGFBP-2, -4 and -5, which were found widely distributed in mouse conceptuses, revealed that IGFBP-2 was expressed mainly in the ectodermal layer and also in the mesoderm derived part of the tongue (day 13.5). Transcripts for IGFBP-4 however, only were detected in the mesoderm derived tissues, whereas expression of IGFBP-5 was restricted to the ectodermal layer. A similar distribution pattern was observed in the lung. In general, expression of IGFBP-2 and -5 was detected in the same cells, whereas IGFBP-4 and -5 were expressed mainly in different cell types. In rodents as in the human there is widespread expression of the genes coding IGFs, the IGFBPs and the receptors during pre- and postimplantation embryogenesis. These data support the assumption that the IGFs play an important role during embryogenesis.

Structural aspects of the IGFBP family.

To date six IGF binding proteins (IGFBP) have been characterized. Analysis of the amino acid sequence reveals that the IGFBPs are clearly distinct but are sharing regions with strong homology. Specifically the hydrophobic cysteine rich N-terminal region and to a lesser extend the C-terminal part are preserved. The alignment of the cysteine molecules is strongly conserved across the 6 IGFBPs. The middle one-third region, where no cysteines are present (except for IGFBP-4) is most divergent. IGFBP-3 and -4 are glycosylated, whereas IGFBP-1 and -2 contain an Arg-Gly-Asp sequence near the carboxyl terminus. Determination of the number of free-SH groups of IGFBP-1 and -3 has revealed that most likely all cysteine residues are involved in disulfide bond formation. All members of the IGFBP family bind IGF-I and IGF-II with about equal affinity. Studies involving deletion mutation and site-directed mutagenesis of IGFBP-1 and -3 have suggested that the three-dimensional structure of the protein plays an important role in IGF binding. However at present it is unclear whether the IGFBPs share one or more specific IGF binding domain. The predominant function of the IGFBPs is to allocate IGF in the various body fluid compartments and tissues and to modulate IGF binding to receptors. For this purpose there exists a very sophisticated control of the routing of circulating IGF both from and to the cell. There is mounting evidence that the structure of the IGFBP proteins plays a key role in the regulation of IGF bioavailability, by modulating its molecular size, capillary membrane permeability, target tissue specificity, cell membrane adherence and IGF affinity.