Norbert Lügering

Mesenteric lymph nodes are critical for the induction of high-dose oral tolerance in the absence of Peyer's patches.

We have previously demonstrated the loss of oral tolerance (OT) in lymphotoxinα − / − (LTα − / −) and TNFα / lymphotoxinα deficient (TNFα / LTα − / −) mice which have defective Peyers patches (PP) and lymph node (LN) development. We have now studied OT in BALB / c mice with differential defects of the gut‐associated lymphoid tissue (GALT) caused by inhibition of LTβR signaling during fetal development. Treatment of pregnant mice with LTβR‐IgG (LTβRIgG) and TNFR I(55)‐IgG (TNFR55IgG) abrogates the formation of PP (LTβRIgG) or of PP and mesenteric LN (MLN) (LTβRIgG / TNFRIgG) without genetically deleting the respective cytokine pathways. OT was readily induced in mice without PP but retaining MLN (PP null / LN +). In contrast, OTcould not be induced in mice lacking both MLN and PP (PP null / MLN null) as shown by the inability of these mice to suppress IFN‐γ secretion or DTH reactions. We next assessed OT in 129 × B6 LTα − / − mice with and without MLN. Timed treatment of pregnant LTα − / − mice with an agonist anti‐LTβR mAb induces formation of MLN but not of PP in LTα − / − mice. LN + LTα −/ − mice developed OT while LN LTα − / − mice were resistant to OT induction. Taken collectively, the data show that in the presence of MLN PP are not required for OT induction and that the presence of MLN is sufficient for OT induction in the LTα − / − model.

Circulating antiinflammatory cytokine IL-10 in patients with inflammatory bowel disease (IBD).

IBD is characterized by increased serum concentrations of different cytokines. IL‐10 inhibits the production of proinflammatory cytokines such as IL‐1, tumour necrosis factor‐alpha (TNF‐a), interferon‐gamma (IFN‐γ) and IL‐6 through inhibitory action on Th1 cells and macrophages, and it is thought to be a suppressor type cytokine. In the present study we determined serum concentrations of IL‐10 in patients with ulcerative colitis (UC) and Crohns disease (CD). We measured human IL‐10 by our own newly established ELISA system using PharMingen antibodies. Serum antibodies were assessed in 44 patients with UC, 40 patients with CD, and in 30 healthy controls. Human IL‐10 serum levels were significantly increased in patients with active UC (144 ± 34 pg/ml (mean ± s.e.m.), P <0.001) and in active CD (132 ± 32 pg/ml, P <0.001) compared with healthy controls (44.9.5pg/ml). Only patients with active CD and active UC presented with significantly increased IL‐10 serum levels, while patients with inactive disease did not show any significant increase. There was no statistically significant difference between IL‐10 serum levels in patients with CD or UC. Compared with clinical disease activity indices there was a significant correlation between IL‐10 serum concentration and CDAI in patients with CD (r= 0.45, P <0.01) and CAI in VC patients (r= 0.39, P <0.05). Comparing IL‐10 serum levels with serum concentrations of other proinflammatory cytokines there was a significant correlation to scrum levels of sIL‐2R (r= 0.417, P <0.05) and IL‐6 (r= 0.387, P <0.05) in patients with CD. Serum cytokine levels in patients with UC did not show any significant correlation to IL‐10 serum concentration. IL‐10 is elevated in serum of patients with active CD and UC. suggesting that IL‐10 acts as a naturally occurring damper in the acute inflammatory process of IBD.

Immunohistochemical Distribution and Serum Levels of the Ca2+ -Binding Proteins MRP8, MRP14 and Their Heterodimeric Form MRP8/14 in Crohn’s Disease

In previous histochemical studies the distribution of the two Ca(2+)-binding proteins MRP8 and MRP14 as well as their heterocomplex MRP8/14 has been demonstrated in different inflammatory diseases. Monoclonal antibodies against MRP8 and MRP14 and their heterodimer MRP8/14 (27E10 epitope) were used to investigate immunohistochemically the distribution of these proteins in routinely processed small and large bowel tissues from patients with Crohns disease (CD). Furthermore, we used a sandwich immunoassay to measure serum concentrations of MRPs in 62 patients were simultaneously assessed by the Crohns disease activity index (CDAI) and the severity activity index of Goebell (SAI). In our immunohistochemical study, MRP8, MRP14 and heterocomplex MRP8/14 were demonstrated in the majority of granulocytes and macrophages in active CD. Additionally, a strong complex MRP8/14 immunoreactivity was present in epithelial cells adjacent to ulcerative and fissuring lesions in the bowel. Serum MRP8/14 concentrations were significantly (p < 0.0001) increased in patients with active CD (CDAI > 150, SAI > 120). No correlations were found for level of MRP14 and MRP8 alone, respectively. The follow-up of individual patients with initially active CD showed a further increase in MRP8/14 levels during acute attacks of the inflammatory process. We suggest that our assay for MRP8/14 discriminates well between active and inactive CD and may have considerable potential in the analysis of clinical disease activity in CD patients. Our morphological results confirm the finding of increased MRP8/14 serum levels in patients with active CD.

Long-Term Effectiveness of Azathioprine in IBD Beyond 4 Years: A European Multicenter Study in 1176 Patients

In Crohn’s disease the optimal duration of azathioprine treatment is still controversial and for ulcerative colitis only limited data are available to support its efficacy. Charts of 1176 patients with IBD from 16 European centers were analyzed. Flare incidences and steroid dosages were assessed for the time before and during treatment and after discontinuation. Within the first 4 years, azathioprine suppressed flare incidence and steroid consumption in both diseases (P < 0.001). While in CD discontinuation after 3–4 years did not lead to reactivation, this was the case in UC. However, continuation beyond 4 years further improved clinical activity in CD and steroid requirement in both diseases (P < 0.001). Discontinuation of azathioprine may thus be considered after 3–4 years in CD patients in complete remission without steroid requirement. In all other CD patients and for UC patients in general, continuation seems beneficial. These results support a novel differential algorithm for long-term azathioprine therapy in IBD.

Role of M Cells in Intestinal Barrier Function

Abstract: M cells are known as specialized epithelial cells of the follicle‐associated epithelium of the gastrointestinal tract. As M cells have a high capacity for transcytosis of a wide range of microorganisms and macromolecules, they are believed to act as an antigen sampling system. The primary physiological role of M cells seems to be the rapid uptake and presentation of particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response. In contrast to absorptive enterocytes, M cells do not exert direct defense mechanisms to antigens and pathogens in the gut lumen. Therefore, they provide functional openings of the epithelial barrier. Although M cells represent a weak point of the epithelial barrier, even under noninflamed conditions, there seems to be a balance between antigen uptake and immunological response. The low number of M cells in the gastrointestinal tract and the direct contact to immune cells in the lamina propria usually prevent the occurrence of mucosal inflammation. During chronic intestinal inflammation we observe an increase of M cell number and apoptosis selectively in M cells. M cell damage seems to be responsible for the increase of the uptake of microorganisms that is observed during intestinal inflammation. Under inflammatory conditions in the intestine, the maintenance of the epithelial barrier is broken and M cells seem to play a major role during this process.

Human Intestinal Microvascular Endothelial Cells Express Toll-Like Receptor 5: A Binding Partner for Bacterial Flagellin

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

The myeloic related protein MRP8/14 (27E10 antigen)— usefulness as a potential marker for disease activity in ulcerative colitis and putative biological function

Abstract. MRP8, MRP14 and their heterodimer MRP8/14 (27E10 antigen) are myeloic related proteins which have been shown to have a major role in inflammatory and immunological responses. In the present study monospecific antibodies against MRPs were used to investigate immunohistochemically the distribution of these proteins in routinely processed bowel tissues from 23 patients with ulcerative colitis (UC). MRP8, MRP14 and their heterocomplex MRP8/14 were demonstrated in the majority of granulocytes and macrophages in tissues of patients with active UC. Furthermore by employing the ELISA technique we measured MRP8/14 serum levels in 62 patients with UC and the results were compared with those for healthy controls. Disease activities were determined by established clinical activity indices. Serum MRP8/14 concentrations were significantly (P<0.0001) increased in patients with active ulcerative colitis. No enhancement of serum levels were found for MRP 14 and MRP8 alone, respectively. The follow‐up of individual patients with initially active disease showed a decrease of MRP8/14 serum levels in parallel with clinical improvement following the start of therapy. It is thus concluded that MRP8/14 accurately reflects the degree of disease activity in UC. Further, possible biological function of MRPs seems to be associated with the heterodimeric form (27E10 antigen) rather than with individual proteins. Our morphological results confirm the finding of enhanced MRP8/14 serum levels in patients with active UC.

IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells

Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and chemokines. MCP‐1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL‐4, IL‐10 and IL‐13 have been described to exert anti‐inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP‐1 by activated intestinal epithelial cells. We examined Caco‐2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP‐1 was determined under stimulated and non‐stimulated conditions. IL‐4, IL‐10 and IL‐13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL‐1β or tumour necrosis factor‐alpha (TNF‐α), we observed markedly increased concentrations of MCP‐1 in supernatants of Caco‐2 cells and intestinal epithelial cells. IL‐4, IL‐10 and IL‐13 all had the capacity to down‐regulate the production of MCP‐1 in Caco‐2 cells as well as in freshly isolated epithelial cells. Caco‐2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL‐1β or TNF‐α for MCP‐1 production. As MCP‐1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP‐1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis.

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.

IL‐10 induces apoptosis in human monocytes involving the CD95 receptor/ligand pathway

The cytokine IL‐10 exerts potent immunosuppressive and anti‐inflammatory effects, although the mechanisms of this action remain largely unknown. In the present study, we investigated the effects of IL‐10 in human peripheral blood monocytes. We were able to demonstrate that IL‐10 dose‐ and time‐dependently triggers apoptosis in these cells as detected by annexin‐V staining, the nick end labeling (TUNEL) procedure, electron microscopy and analysis of DNA laddering. IL‐10‐induced apoptosis required the activation of proteases of the caspase family, since a peptide caspase inhibitor attenuated cell death and, in addition, the proteolytic activation of caspase‐8 was observed. Since caspase‐8 has been implicated as a regulator of apoptosis mediated by death receptors, we investigated a potential involvement of the CD95 receptor/ligand system. Indeed, treatment of monocytes with IL‐10 induced a dose‐dependent up‐regulation of CD95 receptor and ligand expression on the monocyte surface. Furthermore, a CD95 ligand‐neutralizing antibody significantly inhibited IL‐10‐induced apoptosis. In summary, our data show that IL‐10 triggers monocyte apoptosis involving the CD95 system via an autocrine or paracrine process. Therefore, at least part of the anti‐inflammatory properties of IL‐10 may involve induction of apoptosis in monocytes.