Interleukin 18 and associated markers of T helper cell type 1 activity in coeliac disease - PubMed (original) (raw)
Interleukin 18 and associated markers of T helper cell type 1 activity in coeliac disease
V M Salvati et al. Gut. 2002 Feb.
Abstract
Background: Coeliac disease (CD) is caused by a T helper cell type 1 (Th1) response in the small intestinal mucosa to dietary gluten. Paradoxically, interleukin (IL)-12, the major Th1 inducing factor, is undetectable in the mucosa of active CD. IL-18 is a recently described cytokine capable of promoting T cell interferon (IFN)-gamma production and facilitating Th1 cell polarisation.
Aim: To examine expression of IL-18 and IL-18-associated Th1 proteins in CD.
Methods: IL-18 and IFN-gamma RNA transcripts were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). IL-18 and caspase-1 protein expression were assessed by western blotting. Caspase-1 activity was determined using a commercially available assay. RNA transcripts for the IL-18 receptor subunits, IL-1 receptor related protein (IL-1 Rrp) and accessory protein-like subunit (AcPL), and IL-18 induced Th1 specific T box transcription factor (T-bet) were measured by RT-PCR and Southern blotting.
Results: IL-18 RNA transcripts were found in all mucosal samples analysed, with no difference between CD patients and controls. By western blot analysis, a large protein of approximately 24 kDa, corresponding to the immature IL-18, was detected in all mucosal samples from CD patients and controls. In contrast, mature IL-18 was only seen in CD patients. Immunoreactivity corresponding to both immature and mature caspase-1 was present in both CD and control samples. Tissue homogenates from CD patients and controls expressed similar levels of caspase-1 activity. IL-1Rrp and AcPL were seen in all samples but were expressed at greater levels in the mucosa of CD patients. T-bet was also upregulated in CD.
Conclusions: Active IL-18 is expressed in CD as well as other markers of Th1 polarisation.
Figures
Figure 1
Interferon γ (IFN-γ) mRNA transcripts in duodenal biopsies from eight patients with coeliac disease and 11 normal controls. Each point indicates the number of transcripts/μg total RNA in the mucosal sample taken from a single patient or control. Bars represent the median. In coeliac patients, IFN-γ mRNA transcripts were significantly increased compared with controls (p<0.0002).
Figure 2
(A) Interleukin (IL)-18 mRNA transcripts in duodenal biopsies from 16 patients with coeliac disease and 14 normal controls. Each point indicates the number of transcripts/μg total RNA in the mucosal sample taken from a single patient or control. Bars represent the median. No significant difference was observed. (B) Representative western blot of IL-18 in duodenal mucosal tissue from four patients with coeliac disease and three normal controls. The sample shown in lane 4 was taken from a patient with coeliac disease on a gluten free diet. Anti-IL-18 antibody detected a protein corresponding to the size of human recombinant IL-18 in all coeliac disease samples but not in controls. A 24 kDa IL-18 was also detected in all samples from coeliac disease patients and controls. Sizes of protein standards are given. One representative experiment of two independent experiments is shown.
Figure 3
(A) Representative western blot of caspase-1 in duodenal mucosal tissue from patients with coeliac disease and normal controls. Samples were the same as those analysed for IL-18 in fig 2B ▶. Caspase-1 antibody detected a protein of 20 kDa corresponding to the mature form in both coeliac disease and control samples. One representative experiment of two independent experiments is shown. (B) Levels of caspase-1 activity in mucosal samples from four coeliac disease patients and three controls. Functional assay was performed as indicated in materials and methods. Optical density was read at 450 nm and the results expressed as arbitrary units. Values are mean (SEM). Caspase-1 activity was not significantly different in coeliac disease and control samples.
Figure 4
Southern blot analysis of transcripts for accessory protein-like subunit (AcPL) (top blot), interleukin 1 receptor related protein (IL-1Rrp) (middle blot), and β-actin (bottom blot) in duodenal mucosa from five patients with coeliac disease and five normal controls. Total RNA (1 μg), extracted as indicated in materials and methods, was used for cDNA preparation. cDNA (2 μl) for AcPL and IL-1Rrp, and β-actin (1 μl) were amplified for 28, 30, and 23 cycles, respectively. Polymerase chain reaction products were then run on an agarose gel, blotted, and hybridised with oligonucleotide probes specific for AcPL, IL-1Rrp, and β-actin. One representative experiment is presented out of the 10 coeliac disease and 10 controls studied in total.
Figure 5
Southern blot analysis of transcripts for T box transcription factor (T-bet) and β-actin in duodenal mucosa from five patients with coeliac disease and five normal controls (A) and in colonic mucosal samples from three patients with Crohn's disease and three normal controls (B). Total RNA (1 μg), extracted as indicated in materials and methods, was used for cDNA preparation. T-bet (2 μl) and β-actin (1 μl) were amplified for 33 and 23 cycles, respectively. Polymerase chain reaction products were then run on an agarose gel, blotted, and hybridised with probes specific for T-bet and β-actin. For the duodenal specimens, one representative experiment is presented out of the 10 coeliac disease and 10 controls studied in total, while for the colonic samples 3/4 patients with Crohn's disease and 3/4 controls are shown. The other patients showed a similar pattern of response. Consistent upregulation of T-bet mRNA expression was detected in coeliac disease and Crohn's mucosa.
References
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