R. Stoll

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.

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.

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.

Immunoregulatory properties of IL-13 in patients with inflammatory bowel disease; comparison with IL-4 and IL-10

Activated monocytes with increased expression of proinflammatory cytokines play a major role in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL‐4 and IL‐10 can effectively suppress the proinflammatory response of activated monocytes. IL‐13 is a recently described antiinflammatory agent in vitro. The aim of our study was to determine the in vitro immunosuppressive capacity of IL‐13, IL‐4 and IL‐10 in patients with IBD. Peripheral blood monocytes were isolated from 27 patients with ulcerative colitis (UC), 27 patients with Crohns disease (CD) and 16 healthy controls. Cells were stimulated with pokeweed mitogen (PWM) after treatment with IL‐13, IL‐4 and IL‐10, and secretion of IL‐1β, tumour necrosis factor‐alpha (TNF‐α) and IL‐6 was assessed using sandwich ELISA systems. Peripheral blood monocytes secreted significantly increased amounts of TNF‐α and IL‐6 under stimulation with PWM in patients with CD, while UC patients showed significantly elevated levels of IL‐1β. The antiinflammatory cytokines IL‐13, IL‐4 and IL‐10 were all capable of inhibiting monocyte secretion of IL‐1β in a dose‐dependent manner. With regard to IL‐13 and IL‐4, there was no significant suppression of TNF‐α and IL‐6 in patients with active IBD. By contrast, IL‐10 was able to down‐regulate all proinflammatory cytokines in active IBD as well as in controls. Proinflammatory cytokines from patients with inactive IBD could be significantly down‐regulated by all three immunoregulatory cytokines. The inhibitory effect of IL‐13 on TNF‐α and IL‐6 production in differentiated macrophages was diminished in IBD patients, as well as in controls. In disease controls we also observed a reduced inhibition of TNF‐α and IL‐6 after treatment with IL‐13. In conclusion, the antiinflammatory activity of IL‐13 is partially reduced in patients with active IBD. The hyporesponsiveness of activated and differentiated monocytes to IL‐13 and IL‐4 does not seem to be a disease‐specific phenomenon.

Mucosal adaptation to aspirin induced gastric damage in humans. Studies on blood flow, gastric mucosal growth, and neutrophil activation.

The gastropathy associated with the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin is a common side effect of this class of drugs, but the precise mechanisms by which they cause mucosal damage have not been fully explained. During continued use of an injurious substance, such as aspirin, the extent of gastric mucosal damage decreases and this phenomenon is named gastric adaptation. To assess the extent of mucosal damage by aspirin and subsequent adaptation the effects of 14 days of continuous, oral administration of aspirin (2 g per day) to eight healthy male volunteers was studied. To estimate the rate of mucosal damage, gastroscopy was performed before (day 0) and at days 3, 7, 14 of aspirin treatment. Gastric microbleeding and gastric mucosal blood flow were measured using laser Doppler flowmeter and mucosal biopsy specimens were taken for the estimation of tissue DNA synthesis and RNA and DNA concentration. In addition, the activation of neutrophils in peripheral blood was assessed by measuring their ability to associate with platelets. Aspirin induced acute damage mainly in gastric corpus, reaching at day 3 about 3.5 on the endoscopic Lanza score but lessened to about 1.5 at day 14 pointing to the occurrence of gastric adaptation. Mucosal blood flow increased at day 3 by about 50% in the gastric corpus and by 88% in the antrum. The in vitro DNA synthesis and RNA concentration, an index of mucosal growth, were reduced at day 3 but then increased to reach about 150% of initial value at the end of aspirin treatment. It is concluded that the treatment with aspirin in humans induces gastric adaptation to this agent, which entails the increase in mucosal blood flow, the rise in neutrophil activation, and the enhancement in mucosal growth.

Synergistic Effect of Immunoregulatory Cytokines on Peripheral Blood Monocytes from Patients with Inflammatory Bowel Disease

Active inflammatory bowel disease (IBD) ischaracterized by increased monocyte secretion ofproinflammatory cytokines. Immunoregulatory cytokinessuch as Interleukin (IL)-4, IL-10, and IL-13 are capable of inhibiting the proinflammatory cytokineresponse of activated monocytes. The aim of our studywas to determine the effect of differentantiinflammatory cytokines under various cultureconditions and to evaluate combinations of antiinflammatorycytokines in down-regulating monocyte response in IBD.Peripheral monocytes from patients with active IBD wereisolated and stimulated with pokeweed mitogen (PWM). IL-4, IL-10, IL-13 and a combination ofIL-4/IL-10 and IL-10/IL-13 were added at differentconcentrations and different times. Secretion ofIL-1beta and TNF-α was assessed using sandwichELISA systems. There was a diminished down-regulationof TNF-α by IL-4 and IL-13 in IBD when thecytokines were added at the time of stimulation, whilethere was a significantly higher down-regulation when monocytes were primed with these Th-2 cytokines24 hr before activation. IL-10 plus IL-4 and IL-10 plusIL-13, respectively, inhibited the proinflammatorycytokine response of monocytes as well as matured macrophages much more than IL-4, IL-10, orIL-13 alone. Even at suboptimal concentrations for eachcytokine alone, a combination of cytokines showedsynergistic inhibitory effects. In summary, acombination of antiinflammatory cytokines is more effectivein down-regulating the response of activated monocytesthan using the cytokines alone and thus may have apotential therapeutic benefit for patients with IBD.

Colon carcinoma cell lines stimulate monocytes and lamina propria mononuclear cells to produce IL-10

Cytokines released from tumour cells may have function as signals to neighbouring immune and inflammatory cells. Several studies have shown that the immunoregulatory cytokines IL‐10 and transforming growth factor‐beta 1 (TGF‐β1) as well as prostaglandin‐E2 (PGE2) play an important role in tumour‐induced immunosuppression. The aim of the study was to investigate the effect of colon carcinoma cell lines on IL‐10 production in peripheral monocytes (PBMC) and lamina propria mononuclear cells (LPMC). We examined four colon carcinoma cell lines (HT‐29, Caco‐2, Colo‐320 and HCT‐116) and determined their production of TGF‐β1, IL‐10 and PGE2. Peripheral monocytes were isolated by density gradient centrifugation and LPMC were isolated from surgical specimens using a collagenase digestion method. Monocytes and LPMC were cultured with colon carcinoma cell conditioned medium or in co‐culture with colon carcinoma cells. Supernatants were then determined for the production of IL‐10 by ELISA assays. All colon carcinoma cell lines stimulated peripheral monocytes as well as LPMC to produce markedly increased levels of IL‐10. Colon cancer cells secreted negligible levels of IL‐10, but high amounts of TGF‐β1 and PGE2. Neutralization of TGF‐β1 by administration of anti‐TGF‐β as well as neutralization of PGE2 with anti‐PGE2 antisera reduced the IL‐10 production of monocytes markedly, indicating that tumour cell‐derived TGF‐β1 and PGE2 are major factors for IL‐10 stimulation. In vitro stimulation of monocytes with TGF‐β1 and PGE2 could confirm that TGF‐β1 as well as PGF2 at picogram concentrations were able to prime monocytes for enhanced IL‐10 production. Our results demonstrate that colon carcinoma cell lines enhance the ability of monocytes and intestinal macrophages to produce IL‐10. The stimulation of monocyte IL‐10 by colon cancer cell‐derived TGF‐β1 and PGE2 may act as a tumour‐protecting mechanism by impairing the activation of anti‐tumour cytokines.

IL-10 Synergizes with IL-4 and IL-13 in Inhibiting Lysosomal Enzyme Secretion by Human Monocytes and Lamina Propria Mononuclear Cells from Patients with Inflammatory Bowel Disease

Tissue injury and inflammation in inflammatorybowel disease (IBD) are associated with enhancedmonocytic lysosomal enzyme release. In this study,peripheral monocytes and lamina propria mononuclearcells (LPMNC) were isolated from IBD patients andnormal controls. Cells were stimulated withlipopolysaccharide after treatment with IL-13, IL-4, andIL-10, and enzyme secretion was assessed by using thecorresponding p-nitrophenyl glycosides as substrates.Molecular forms of cathepsin D were examined to describethe mode of enzyme release. IL-10 and IL-4 stronglydown-regulate enzyme secretion in IBD monocytes. IBD monocytes showed a diminished responsiveness tothe inhibitory effect of IL-13. Impaired monocyteresponse was not found with combinations of IL-13 andIL-10 or IL-4 and IL-10. LPMNC from involved IBD mucosa showed significantly higher enzyme secretioncompared with LPMNC from noninvolved IBD mucosa butresponded inefficiently to either IL-4, IL-13, or IL-10alone. However, combined treatment with IL-10 and IL-4 or IL-10 and IL-13 strongly suppressedenzyme release by these cells. Both the precursor andmature forms of cathepsin D were elevated in IBDpatients. While IL-13 reduced mainly the precursor form, the effect of IL-4 and IL-10 concerns both theprecursor and mature form of cathepsin D. Our resultsfavor the potent clinical utility of combined treatment,thus improving chances of developing effective treatments for human IBD.