Tilo Andus

Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes

The three monokines interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL‐6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C‐reactive protein, serum amyloid A, fibrinogen, α1‐antitrypsin, α1‐antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL‐1β as well as TNFα, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C‐reactive protein. These data suggest that IL‐6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.

Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa

BACKGROUND & AIMS Transcription factors of the nuclear factor kappaB (NF-kappaB) family play an important role in the regulation of genes involved in inflammation. In inflammatory bowel diseases, proinflammatory cytokines known to be regulated by NF-kappaB are involved. The aim of this study was to investigate the role of NF-kappaB activation during mucosal inflammation in situ. METHODS A monoclonal antibody, alpha-p65mAb, was applied for immunofluorescence and immunohistochemical analysis that recognizes activated NF-kappaB. Electrophoretic mobility shift assay was used to directly demonstrate the presence of active DNA-binding NF-kappaB. RESULTS Using the alpha-p65mAb antibody, activated NF-kappaB could be found in biopsy specimens from inflamed mucosa but was almost absent in uninflamed mucosa. The number of cells showing NF-kappaB activation correlated with the degree of mucosal inflammation but was not significantly different between inflamed mucosa from patients with Crohns disease, ulcerative colitis, and nonspecific colitis or diverticulitis. NF-kappaB activation was localized in macrophages and in epithelial cells as identified by double-labeling techniques. Electrophoretic mobility shift assay with isolated lamina propria mononuclear cells and epithelial cells confirmed these results. CONCLUSIONS This study shows for the first time the activation of NF-kappaB during human mucosal inflammation in situ. In addition to macrophages, epithelial cells contained activated NF-kappaB, indicating an involvement in the inflammatory process.

Recombinant human B cell stimulatory factor 2 (BSF-2/IFN-β2) regulates β-fibrinogen and albumin mRNA levels in Fao-9 cells

Conditioned medium from human monocytes contains a partially characterized hepatocyte‐stimulating factor that simultaneously elevates the mRNA levels of the acute‐phase protein β‐fibrinogen and decreases albumin mRNA in rat hepatoma cells. We demonstrate that recombinant human B‐cell stimulatory factor 2, which is identical to interferon‐β2/26 kDa protein and interleukin‐HP1, exhibits the same activity as hepatocyte‐stimulating factor. Furthermore, a specific antibody against B‐cell stimulatory factor 2 was able to inhibit hepatocyte‐stimulating factor in conditioned medium from human monocytes. Our data show that hepatocyte‐stimulating factor and B‐cell stimulatory factor 2 are functionally and immunologically related proteins.

Evidence for continuous stimulation of interleukin-6 production in Crohn's disease.

Increased serum concentrations of acute-phase proteins can be found in active inflammatory bowel disease. Because interleukin 6 (IL-6) is one of the main mediators of acute-phase protein synthesis by the liver, the serum concentrations of IL-6 and the acute-phase proteins C-reactive protein, alpha 1-antitrypsin, and alpha 1-acid glycoprotein were determined in 70 patients with Crohns disease (CD) and 23 patients with ulcerative colitis (UC). Disease activities were determined by established clinical activity indices. Serum IL-6 concentrations were significantly (P less than 0.005) increased in patients with CD (mean +/- SEM, 6.8 +/- 0.9 U/mL) compared with patients with UC (mean, less than 4 U/mL) and healthy controls (mean, less than 4 U/mL). Of patients with CD, 68.5% had serum IL-6 concentrations of greater than or equal to 4 U/mL, compared with 21.7% of patients with UC and 0% of healthy controls. There was a tendency toward higher serum IL-6 concentrations in patients with active CD than in patients with inactive disease. However, these differences were not statistically significant. There was no correlation between IL-6 serum concentrations and clinical activity indices, possibly because of the short circulatory lifetime and rapid hepatic clearance of IL-6 from the portal venous blood. In contrast to serum IL-6, acute-phase proteins, which have a longer circulatory lifetime, were significantly correlated with clinical activity indices. Only the follow-up of individual patients with initially highly active disease showed a further increase in IL-6 levels during acute exacerbations of the inflammatory process. The results show that most patients with even moderately active CD have significantly increased serum concentrations of IL-6, most probably reflecting a continuous stimulation of IL-6-producing cells.

Association of humoral markers of inflammation and dehydroepiandrosterone sulfate or cortisol serum levels in patients with chronic inflammatory bowel disease

Objectives:Dehydroepiandrosterone sulfate (DHEAS) and cortisol are multifunctional adrenal hormones with immunomodulating properties. DHEAS levels were found to be very low in chronic inflammatory diseases. This study aimed to shed more light on the interrelation between DHEAS and cortisol (and humoral markers of inflammation) in chronic inflammatory bowel disease.Methods:DHEAS and cortisol serum levels were measured by ELISA in the serum of 66 normal subjects, 115 patients with Crohns disease (CD) and 64 patients with ulcerative colitis (UC). Humoral markers of inflammation and disease activity scores were assessed by standard techniques.Results:DHEAS was lower in patients with CD (p < 0.005) and UC (p < 0.005) than in controls, which was, in part, dependent on previous corticosteroid treatment (p < 0.01). In CD patients, z-normalized DHEAS was inversely correlated with blood sedimentation rate (p= 0.017). Z-normalized DHEAS was negatively correlated with interleukin-6 (IL-6) in the form of a trend (p= 0.068), and z-normalized DHEAS was significantly positively correlated with hemoglobin (p= 0.001) but not with the Crohns disease activity index. Cortisol, however, was positively correlated with blood sedimentation rate (p= 0.034) and C-reactive protein (p= 0.006). In contrast, in UC patients no such correlation of z-normalized DHEAS or cortisol and parameters of humoral inflammatory activity or Rachmilewitz index exist.Conclusions:DHEAS as a marker of inflammation was low in CD and UC. In CD patients, low DHEAS and high cortisol serum levels were associated with higher humoral inflammatory activity. With respect to humoral inflammatory activity in CD patients, DHEAS and cortisol seem to be inversely regulated, which may have an impact on several immune functions, such as IL-6 secretion.

Uncoupling of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis in inflammatory bowel disease?

In inflammatory diseases such as Crohns disease (CD) and ulcerative colitis (UC), one would expect that TNF or IL-6 stimulates the hypothalamus, which activates the hypothalamus-autonomic nervous system (HANS) axis and the hypothalamic-pituitary-adrenal (HPA) axis in a parallel fashion. The study was initiated in order to investigate the parallelism of the HANS and HPA axes. We measured a typical marker of the HANS axis (neuropeptide Y, NPY) and of the HPA axis (serum cortisol). Plasma NPY was positively correlated with serum cortisol in control subjects (R(Rank)=0.259, p=0.026), which is a sign for the parallel activation of the two axes in healthy subjects. However, serum cortisol was not correlated with plasma NPY in CD or UC patients. In the active CD or UC, inclusion of patients with and without prior prednisolone therapy revealed a negative correlation between the serum cortisol and plasma NPY (CD: R(Rank)=-0.285, p<0.05; UC: R(Rank)=-0.510, p<0.01). This study demonstrates that the two stress axes seem to act in a parallel fashion in control subjects but are uncoupled in IBD patients. Uncoupling of these two axes may be partly due to prior corticosteroid therapy, whereas inverse coupling is a result of simultaneous corticosteroid therapy. It is discussed how the uncoupling of the two anti-inflammatory stress axes can appear.

Alterations of the phenotype of colonic macrophages in inflammatory bowel disease

Background: Intestinal macrophages play an important role in mucosal inflammation. In normal colonic mucosa we recently demonstrated a unique macrophage phenotype with attenuated immune functions. Here we present an analysis of the alterations of the phenotype of colonic macrophages in inflammatory bowel disease (IBD). Methods: Intestinal macrophages were isolated from biopsies of patients with IBD (n = 20). Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, HLA‐DR, CD44, CD11b, CD11c and CD3/CD19 expression. Results: In IBD there was an increase in expression not only of CD14 compared to control mucosa (36.0%±13.2% vs. 10.5%±3.8%, P<0.0001) but also of CD16 (28.6%±10.3% vs. 10.1%±3.9%, P<0.0001), HLA‐DR (53.1%±15.9% vs. 27.3%± 9.2%, P<0.0005), CD11b (42.8%±14.2% vs. 17.4%±6.8%, P<0.0001) and CD11c (35.1% ± 15.9% vs. 17.8%±10.4%, P<0.005.). Furthermore, a hitherto undescribed new population of macrophages could be detected by flow cytometry only in patients with ulcerative colitis (CD16++, CD11b++, CD14low, CD33low, CD11c‐) accounting for 5.8% of all cells isolated. Conclusion: In contrast to colonic macrophages from normal mucosa, there is a significantly higher expression of CD14, CD16, HLA‐DR, CD11b and CD11c in IBD, indicating additional macrophage populations in the inflamed mucosa. This may reflect either a recruitment of new cells from the circulation or a change in phenotype of resident cells.

Association of autonomic nervous hyperreflexia and systemic inflammation in patients with Crohn's disease and ulcerative colitis

The autonomic nervous system modulates gastrointestinal motility, secretion and mucosal immunity. Its dysfunction may be of pathogenetic importance in inflammatory bowel disease (IBD). This study aimed at investigating the autonomic nervous function in patients with IBD. Forty-seven patients with IBD, 28 with Crohns disease (CD) and 19 with ulcerative colitis (UC), were investigated by means of 5 cardiovascular and 2 pupillary standardized autonomic nervous function tests. In CD and UC, cardiovascular autonomic neuropathy was very rare (0%, 5%), whereas pupillary autonomic neuropathy was more prevalent (21%, 21%). In contrast to autonomic neuropathy, overall cardiovascular (CD: 29%, UC: 26%) and pupillary autonomic hyperreflexia (46%, 37%) were found more often. Patients with CD and UC demonstrated elevated percentiles in the respiratory sinus arrhythmia test as compared to controls (RSA: 82.3 +/- 3.9%, 80.0 +/- 5.9%, controls: 50.0% +/- 1.5%, p < 0.0001). CD patients with, as compared to patients without, RSA hyperreflexia had significantly higher CDAIs (p < 0.001), increased erythrocyte sedimentation rates (p < 0.005) and more often extraintestinal disease manifestations (p < 0.001). UC patients with, as compared to patients without, pupillary latency time hyperreflexia had lower hemoglobin (p < 0.05), lower albumin (p < 0.01) and increased erythrocyte sedimentation rates (p < 0.05). Autonomic hyperreflexia was significantly associated with more severe inflammation and systemic disease in IBD. Hyperreflexia may be a response to inflammation or a pathogenetic element that drives mucosal inflammation.

O- and N-glycosylation lead to different molecular mass forms of human monocyte interleukin-6

The biosynthesis and secretion of human interleukin‐6 (IL‐6) was studied in monocyte cultures stimulated with endotoxin. After labeling with [35S]methionine and immunoprecipitation with a specific antiserum one major (24 kDa) and four minor (27.5, 23.3, 22.5 and 21.8 kDa) molecular mass forms of IL‐6 could be found in the cells and media. Incubation of monocyte media with sialidase and subsequently with endo‐α‐N‐acetylgalactosaminidase, which cleaves Ga1(β1‐3)Gal‐NAc from serine or threonine, led to the formation of only two forms of IL‐6 with apparent molecular masses of 25 and 21.8 kDa. The latter had an electrophoretic mobility indistinguishable from that of 125I‐labeled recombinant human IL‐6. The results suggest that human monocyte IL‐6 carries O‐glycosidically bound carbohydrates with a Ga1(β1‐3)Gal‐NAc core to which only sialic acid is bound. Differences in O‐glycosylation are the major cause for the molecular heterogeneity of IL‐6. A small part of IL‐6 (27.5 kDa form) is in addition N‐glycosylated. Incubation of monocytes with tunicamycin and 1‐deoxymynnojirimycin and treatment of IL‐6 with endoglucosaminidase H suggested that the 27.5 kDa form of IL‐6 carries at least one N‐linked complex‐type oligosaccharide chain.

Prior bowel resections, perianal disease, and a high initial Crohn's Disease Activity Index are associated with corticosteroid resistance in active Crohn's disease

OBJECTIVES:Some patients with Crohns disease (CD) do not respond to corticosteroid therapy. Furthermore, corticosteroids frequently cause side effects. Thus, predictive parameters for treatment refractoriness would be helpful for treatment decisions.METHODS:A total of 300 patients with active CD (i.e., with a Crohns Disease Activity Index [CDAI] >200) entered the study. Treatment started with 60–100 mg/day prednisolone equivalent, which was then tapered to 10–15 mg/day within 6 wk and maintained at that dose for another 4 wk. After 10 wk of treatment, response to steroids was defined by a CDAI <150, steroid resistance by a CDAI always ≥150 and steroid dependency by a relapse after dose reduction. Of 239 eligible patients, 196 were responders, 26 were steroid resistant, and 17 were steroid dependent.RESULTS:Prior bowel resections, a high initial CDAI, and perianal disease were associated with steroid resistance. Of the steroid resistant patients 53.9% were bowel-resected compared to 20.4% of the responders (relative risk = 3.63; 95% CI = 1.79–7.36). Perianal disease was observed in 42.3% of steroid resistant patients versus 21.9% of responders (relative risk = 2.28; 95% CI = 1.12–4.66) and initial CDAI was 347 ± 91 in resistant patients versus 301 ± 81 in responders (p < 0.05). Parameters for steroid dependent patients were not significantly different from those of responders.CONCLUSIONS:In this study (thus far the largest study for the evaluation of predictive factors for treatment refractoriness to corticosteroids in CD), only prior bowel resection, perianal disease, and a high initial CDAI were found to be predictive of resistance to steroid treatment.