Expression of proinflammatory and Th1 but not Th2 cytokines is enhanced in gastric mucosa of Helicobacter pylori infected children.

BACKGROUND: Helicobacter pylori-induced gastric inflammation is thought to be largely regulated by cytokines. PATIENTS AND METHODS: The expression of interferon-gamma, interleukin-12, interleukin-4, interleukin-10, interleukin-8, and interleukin-17 mRNA was examined on gastric mucosal samples from 24 children by semiquantitative reverse transcription polymerase chain reaction and southern blotting. Biopsy-based tests, serology, and urea 13C breath test were used to assess Helicobacter pylori status. Gastric biopsies were also evaluated for bacterial density, chronic inflammation, and acute inflammatory activity. RESULTS: Interferon-gamma, interleukin-12, interleukin-8 and interleukin-17 expression was higher in Helicobacter pylori-infected (n=13) than uninfected (n=11) children. Conversely, interleukin-4 and interleukin-10 expression did not differ between Helicobacter pylori-infected and uninfected children. In Helicobacter pylori-infected children, interferon-gamma, interleukin-12, interleukin-8 and interleukin-17 expression correlated with bacterial density, and Interferon-gamma and interleukin-12 expression with chronic inflammation score. CONCLUSIONS: The findings of this study indicate that, in children, Helicobacter pylori-induced inflammatory response would favour production of proinflammatory cytokines and development of cell-mediated immunity, namely Th1 response.

Up-regulation of the IL-12 receptor beta 2 chain in Crohn's disease.

Crohn' s disease (CD) is a chronic intestinal inflammatory disorder characterized by aberrant mucosal Th1 cell activation and production of IL-12, the major Th1-driving factor. The T cell response to IL-12 is dependent on the expression of a specific receptor composed of two subunits, termed IL-12Rbeta1 and IL-12Rbeta2. The content of IL-12Rbeta2, as measured at the mRNA level, is crucial in regulating Th1 differentiation. In this study we therefore investigated IL-12Rbeta2 RNA transcripts in CD. IL-12Rbeta2 expression was increased in active CD as well as Helicobacter pylori (HP)-associated gastritis and Salmonella colitis compared with that in inactive CD, ulcerative colitis, noninflammatory controls, and celiac disease. In contrast, IL-12Rbeta1 transcripts were expressed at comparable levels in all samples. In CD, IL-12Rbeta2 expression strictly correlated with tyrosine phosphorylation of STAT4, a key component of the IL-12-dependent Th1 polarization. This was associated with a pronounced expression of IFN-gamma. Transcripts for IL-12/p40 were detected in CD, HP-positive, and Salmonella colitis patients, but not in celiac disease, indicating that IL-12Rbeta2 up-regulation occurs only in IL-12-associated Th1 gastrointestinal diseases. Finally, we showed that stimulation of lamina propria mononuclear cells with IL-12 enhanced IL-12Rbeta2, suggesting that IL-12 regulates IL-12Rbeta2 expression in human gastrointestinal mucosa. The data show that the signaling pathway used by IL-12 to induce Th1 differentiation is increased at the site of disease in CD, further supporting the view that IL-12/IL-12R signals contribute to the inflammatory response in this condition.

Up-regulation of IL-17 is associated with bioactive IL-8 expression in Helicobacter pylori-infected human gastric mucosa.

Helicobacter pylori (HP)-associated gastritis is characterized by an increased number of acute and chronic inflammatory cells secreting cytokines that contribute to maintain and expand the local inflammation. Locally induced IL-8 is believed to play a major role in the HP-associated acute inflammatory response. Factors/mechanisms that regulate IL-8 induction are, however, not fully understood. In the present study we investigated whether HP infection is associated with an increased production of IL-17, a T cell-derived cytokine capable of modulating IL-8 gene expression. We showed that both IL-17 RNA transcripts and protein were expressed at a higher level in the whole gastric mucosal and lamina propria mononuclear cell samples from HP-infected patients than in those from uninfected subjects. HP: eradication was associated with a marked down-regulation of IL-17 expression. The addition of a neutralizing anti-IL-17 Ab to the gastric lamina propria mononuclear cell cultures resulted in a significant inhibition of IL-8 secretion, indicating that IL-17 contributes to enhance IL-8 in the HP-colonized gastric mucosa. Consistently, stimulation of MKN 28 cells, a gastric epithelial cell line, with IL-17 increased IL-8 secretion. Finally, conditioned medium from the IL-17-stimulated MKN 28 cell cultures promoted the in vitro polymorphonuclear leukocyte migration. This effect was inhibitable by a neutralizing IL-8 but not IL-17 Ab. Together, these data indicate that biologically active IL-17 production is increased during HP: infection, suggesting the possibility that this cytokine may play an important role in the inflammatory response to the HP colonization.

Altered IgG(4) renal clearance in patients with inflammatory bowel diseases. Evidence for a subclinical impairment of protein charge renal selectivity.

BACKGROUND: A loss of intestinal glycosaminoglycans (GAGs) has been shown in inflammatory bowel diseases (IBD). Since GAGs are involved in the regulation of renal protein filtration and GAGs disruption is associated with anionic proteinuria, we examined whether changes in the selectivity of renal protein filtration occur in IBD. METHODS: From 46 patients with IBD (17 with Crohn's disease (CD), and 29 with ulcerative colitis (UC)) and 21 healthy subjects, urine and serum samples were obtained. Albumin, total IgG and IgG(4) clearances were measured using sensitive methods. Serum p-ANCA and TNF-alpha were tested. RESULTS: Median IgG(4) clearance was 0.041 ml/ min/10(-3) in patients with UC and 0.10 ml/ min/10(-3) in CD patients, both significantly higher than in controls (0.03 ml/min/10(-3)) (P<0.03). IgG(4) clearance was above the upper normal limit in 9/17 CD (53%) and in 10/29 UC (34.5%). Eighteen of 19 patients showing abnormal IgG(4) clearance were taking mesalazine. In patients on maintenance oral mesalazine, IgG(4) clearance was higher than that in patients off treatment (0.12 vs 0.03 ml/min/10(-3), P=0.04). No clinical/laboratory sign of renal dysfunction was documented in patients with altered IgG(4) clearance and maintained on mesalazine treatment. CONCLUSION: Renal protein charge permselectivity is impaired in 40% of patients with IBD with no overt proteinuria. Our data suggest that altered IgG(4) clearance may represent a subclinical marker of renal involvement in IBD.

Interferon-gamma (IFN-gamma) and prostaglandin E2 (PGE2) regulate differently IL-12 production in human intestinal lamina propria mononuclear cells (LPMC).

IL-12 modulates Th1 immune response during chronic colitis. Mechanisms regulating IL-12 synthesis in human intestine are poorly understood. The aim of this study was to investigate the effect of IFN-gamma and PGE2 on lipopolysaccharide (LPS)-stimulated LPMC IL-12 production. Normal LPMC cultures were run in the presence or absence of IFN-gamma and/or PGE2 before LPS stimulation. To examine the role of endogenous PGE2 on LPS-stimulated IL-12 release, LPMC cultures were added of indomethacin before LPS stimulation. IL-12, IL-10 and IL-8 were measured by ELISA. No IL-12 was detected in either unstimulated or LPS-stimulated LPMC cultures. In contrast, LPMC released IL-8 (650 +/- 125 pg/ml) and IL-10 (75 +/- 25 pg/ml) in response to LPS. Treatment of LPMC with IFN-gamma facilitated LPS-stimulated IL-12, whereas it completely abrogated IL-10 production. IL-12 release by LPMC stimulated with IFN-gamma and LPS was significantly inhibited by exogenous IL-10. The addition of PGE2 to IFN-gamma-treated LPMC cultures inhibited in a dose-dependent manner LPS-induced IL-12 secretion. Furthermore, IL-12 was detectable (85 +/- 25 pg/ml) in the supernatants of LPMC cultures treated with indomethacin and LPS. In contrast to the effect on IL-12, PGE2 significantly augmented LPS-stimulated LPMC IL-10 production. However, the inhibition of IL-12 by PGE2 was only partially reversed by anti-IL-10. In a simplified model of LPS tolerance, we finally showed that monocyte-derived macrophages exhibited reduced IL-12 production after repeat LPS stimulation. In these cell cultures, indomethacin abrogated the induction of LPS desensitization. IFN-gamma and PGE2 modulate differently the LPMC responsiveness to LPS in terms of IL-12 synthesis.

Changes in the mucosal expression of interleukin 15 in Helicobacter pylori-associated gastritis.

Transcripts for interleukin (IL) 15 were detected in the gastric mucosal samples of 5/5 (100%) patients with no evidence of Helicobacter pylori infection and in 4/14 (28%) H. pylori-infected patients (P< 0.05). Both IL-15 mRNA and IL-15 protein were detected in 1/6 (17%) patients who successfully underwent H. pylori eradication therapy, before treatment and in 5/6 (83%) cases after eradication. Even though a parallel significant (P < 0.03) improvement of gastritis score occurred after eradication, the severity of gastritis did not differ according to the mucosal IL-15 expression among H. pylori-infected patients, irrespective of the CagA serology. This study demonstrates, for the first time, that transcripts for IL-15 are expressed in the human gastric mucosa. Changes occurring during H. pylori colonisation and after eradication raise the hypothesis that H. pylori may down-regulate IL-15 expression in the gastric mucosa.

Bioactive IL-18 expression is up-regulated in Crohn's disease.

An imbalance of immunoregulatory factors is believed to contribute to uncontrolled mucosal Th1 cell activation in Crohn's disease (CD). IL-18, a macrophage-like cell-derived cytokine, is involved in Th1 clone development, and IFN-gamma production. Therefore, IL-18 expression was investigated in CD. Whole mucosal intestinal tissue and lamina propria mononuclear cells (LPMC) of 12 CD and 9 ulcerative colitis (UC) patients and 15 non-inflammatory bowel disease (IBD) controls were tested for IL-18 by semiquantitative RT-PCR and Western blot analysis. Transcripts for IL-18 were found in all samples tested. However, increased IL-18 mRNA accumulation was detected in both mucosal and LPMC samples from CD in comparison to UC and controls. In CD, transcripts for IL-18 were more abundant in the mucosal samples taken from involved areas. An 18-kDa band consistent with mature IL-18 was predominantly found in CD mucosal samples. In mucosal samples from non-IBD controls, IL-18 was present as a 24-kDa polypeptide. Consistently, active IL-1beta-converting enzyme (ICE) subunit (p20) was expressed in samples from either CD or UC, whereas, in colonic mucosa from non-IBD controls, ICE was synthesized as precursor (p45) only. To confirm that IL-18 produced in CD tissue was functionally active, CD LPMC were treated with a specific IL-18 antisense oligonucleotide. In these cultures, IL-18 down-regulation was accompanied by a decrease in IFN-gamma expression. In aggregate, our data indicate that IL-18 up-regulation is a feature of CD and suggest that IL-18 may contribute to the local immunoinflammatory response in CD.

Response of human intestinal lamina propria T lymphocytes to interleukin 12: additive effects of interleukin 15 and 7.

BACKGROUND/AIM: Interleukin (IL) 12 is involved in the mucosal response during intestinal inflammation but its role is not fully understood. The response of human lamina propria T lymphocytes (T-LPL) to IL-12 in terms of interferon gamma (IFN-gamma) release and proliferation was investigated, exploring whether IL-15 and IL-7 cooperate with IL-12. The role of accessory molecules (CD2 and CD28) was also investigated. METHODS: Unstimulated and phytohaemagglutinin preactivated T-LPL cultures were incubated with or without the initial addition of cytokines, anti-CD2 or anti-CD28 antibodies. IFN-gamma mRNA was detected by reverse transcriptase polymerase chain reaction, and protein secretion was measured by enzyme linked immunosorbent assay (ELISA). RESULTS: IFN-gamma mRNA was induced in T-LPLs by IL-12 and IL-15 but not IL-7, whereas IFN-gamma was measured only in IL-12 stimulated T-LPL cultures. IL-12 induced IFN-gamma release was not abrogated by neutralising anti-IL-2 antibody or by cyclosporin A. IL-12 synergised with either anti-CD2 or anti-CD28 antibodies in inducing IFN-gamma synthesis. In preactivated T-LPLs, IL-7 enhanced IFN-gamma release induced by both IL-12 and anti-CD2, whereas IL-15 potentiated only IL-12 induced IFN-gamma synthesis. IL-12 did not induce proliferation of either unstimulated or preactivated T-LPLs and it did not enhance the CD2/CD28 stimulated T-LPL proliferative response. No transcript for IL-12 receptor beta1 subunit was detected in freshly isolated and activated T-LPLs whereas the beta2 subunit mRNA was consistently found in T-LPL samples. CONCLUSIONS: IL-12 induces human T-LPLs to produce and release IFN-gamma, and IL-15 and IL-7 cooperate with IL-12 in expanding the IFN-gamma mucosal response.

Appendectomy is an independent protective factor for ulcerative colitis: results of a multicentre case control study. The Italian Group for the Study of the Colon and Rectum (GISC)

BACKGROUND: Different exogenous factors are believed to play a role in the pathogenesis of ulcerative colitis. Smoking habits and other risk factors have received much attention. It has recently been reported that appendectomy decreases the risk of ulcerative colitis. AIM: Aim of the study was to further examine the role of appendectomy in ulcerative colitis. METHODS: A large multicentre case control study was performed. Cases were all patients with a recent new diagnosis of ulcerative colitis (from 1990 to 1994) at participating centres. One or two controls attending the orthopaedic and surgical units were considered and matched to cases for age (+/- 5 years), sex and year of diagnosis. A total of 536 cases and 755 controls were enrolled. Mean age of cases was 37.9 years (range 2-92). Assessment of exposure was done by examining the clinical records and by interview, if necessary. Smoking habits, alcohol consumption, use of oral contraceptives, type of occupation and area of residence were also recorded. Odds ratio and 95% confidence intervals were calculated by conditional logistic regression analysis. RESULTS: Forty-one out of the 536 cases (7.6%) and 150 out of the 755 controls (19.9%) had been submitted to appendectomy. A total of 110 out of 536 cases (20.5%) and 135 out of 753 (17.9%) controls had had tonsillectomy. Seven out of 41 cases and 15 out of 755 controls underwent appendectomy for recurrent pain. In all ulcerative colitis patients, appendectomy had been performed before the onset of disease. When data were adjusted for the confounding variables, ulcerative colitis patients were less likely to have had appendectomy compared with controls (odds ratio = 0.3, confidence interval = 0.19-0.48). There was no significant association of ulcerative colitis with tonsillectomy (odds ratio = 1.09, confidence interval = 0.76-1.58). The well recognized inverse association of ulcerative colitis with cigarette smoking was also shown in this study. CONCLUSIONS: The present data emerging from a large multicentre study, confirm that appendectomy has a protective role for the development of ulcerative colitis.

Antineutrophil cytoplasmic antibodies are present in long standing type 1 diabetics but do not correlate with selective proteinuria.

Antineutrophil cytoplasmic antibodies (ANCA) are present in systemic vasculitis with or without renal involvement and in inflammatory bowel diseases, conditions which share damage in proteoglycan content of basal membrane. In diabetes, there is a reduction in proteoglycans in the kidney basal membrane, responsible for the decrease in fixed anionic charges and, consequently, for the prevalent anionic proteinuria (albumin, IgG4) even in the early preclinical stage of nephropathy. The aims of this study were to search for the presence of ANCA in long-standing type 1 diabetic patients and to evaluate possible correlations with size- and/or charge-selective proteinuria. Twenty-two type 1 diabetic patients (duration of diabetes 24 years, range 9-30) selected and grouped according to albumin excretion rate values, were studied together with 21 age and sex comparable normal subjects. ANCA, albumin excretion rate, and the clearances of albumin, of prevalently cationic total IgG (IgG) and of anionic IgG4 were evaluated. ANCA were measured using ELISA and indirect immunofluorescence methods; albumin, IgG and IgG4 were tested with RIA or ELISA methods developed in our laboratory. ANCA were found in five patients, three of whom showed proteinuria. 33.3% and 18.2% of patients with normal IgG and albumin clearances respectively had elevated IgG4 clearance. This study shows for the first time the presence of ANCA in long-standing type 1 diabetic patients and confirms a prevalent anionic protein excretion in these patients, but does not show a correlation between the presence of ANCA and proteinuria, even if the presence of ANCA in diseases sharing alterations in proteoglycan content of vascular basal membrane is noteworthy.

Low Prevalence of p-ANCA in Unaffected Relatives of Patients with Ulcerative Colitis from a Mediterranean Area.

: Antineutrophil-cytoplasmic-autoantibodies (p-ANCA) are strongly associated with ulcerative colitis (UC) and may represent an indicator of genetic susceptibility to UC. To further examine whether p-ANCA may serve as a genetic marker of UC we evaluated the frequency of p-ANCA in unaffected family members of UC from a defined geographic area. A total of 110 patients with UC and 90 unaffected family members (first- or second-degree relatives) were tested. Controls included: 58 Crohn's disease (CD) patients with 25 unaffected relatives and 52 irritable bowel syndrome (IBS) patients with 20 healthy family members. p-ANCA were detected by enzyme-linked immunoassay followed by immunofluorescence. p-ANCA were detected in 57 UC patients (51.8%). Six of 90 (6.6%) unaffected relatives were positive for p-ANCA and 4 of these were from two families. In 3 of 35 families the proband and at least one unaffected relative were p-ANCA-positive. In five families with more than one member affected by UC, p-ANCA were detected in 2 of 19 (10.5%) unaffected relatives. Six CD patients (10.3%) and one (1.9%) in the IBS group were positive for p-ANCA. One family member was positive in the CD family group and 1 was positive in the control family group. In the group of families recruited for this study, p-ANCA were not more frequent in unaffected relatives of UC patients than in controls, suggesting that at least in the geographic area considered for this study p-ANCA may not represent a definite subclinical marker of susceptibility for UC.