Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex vivo cultured coeliac intestinal mucosa

Abstract

Background: Enteropathy in coeliac disease (CD) is sustained by a gliadin specific Th1 response. Interleukin (IL)-10 can downregulate Th1 immune responses.

Aim: We investigated the ability of recombinant human (rh) IL-10 to suppress gliadin induced Th1 response.

Patients and methods: IL-10 RNA transcripts were analysed by competitive reverse transcription-polymerase chain reaction in duodenal biopsies from untreated and treated CD patients, non-coeliac enteropathies (NCE), and controls. CD biopsies were cultured with a peptic-tryptic digest of gliadin with or without rhIL-10. The proportion of CD80+ and CD25+ cells in the lamina propria, epithelial expression of Fas, intraepithelial infiltration of CD3+ cells, as well as cytokine synthesis (interferon gamma (IFN-gamma) and IL-2) were measured. Short term T cell lines (TCLs) obtained from treated CD biopsies cultured with gliadin with or without rhIL-10 were analysed by ELISPOT for gliadin specific production of IFN-gamma.

Results: In untreated CD and NCE, IL-10 RNA transcripts were significantly upregulated. In ex vivo organ cultures, rhIL-10 downregulated gliadin induced cytokine synthesis, inhibited intraepithelial migration of CD3+ cells, and reduced the proportion of lamina propria CD25+ and CD80+ cells whereas it did not interfere with epithelial Fas expression. In short term TCLs, rhIL-10 abrogated the IFN-gamma response to gliadin.

Conclusions: rhIL-10 suppresses gliadin specific T cell activation. It may interfere with the antigen presenting capacity of lamina propria mononuclear cells as it reduces the expression of CD80. Interestingly, rhIL-10 also induces a long term hyporesponsiveness of gliadin specific mucosal T cells. These results offer new perspectives for therapeutic strategies in coeliac patients based on immune modulation by IL-10.

Figure 1

(A) Interleukin 10 (IL-10) mRNA transcripts in whole duodenal biopsies from patients with coeliac disease (CD), non-coeliac enteropathy (NCE), normal controls, and treated CD patients. The bars represent the median with 95% confidence limits. Mann-Whitney test for unpaired samples: CD versus controls, p<0.002; CD versus treated CD, p<0.02; CD versus NCE, p = 0.7. (B) Interferon γ (IFN-γ) mRNA transcripts in whole duodenal biopsies from patients with CD, NCE, normal controls, and treated CD. Bars represent the median with 95% confidence limits. Mann-Whitney for unpaired samples: CD versus controls, p<0.0001; CD versus treated CD, p<0.0001; CD versus NCE, p<0.0001. (C) Ratio between IL-10 and IFN-γ mRNA transcripts measured in whole duodenal biopsies. Values are mean (SD). IL-10/IFN-γ ratio was markedly reduced (p<0.05) in CD enteropathy in comparison with inflamed non-coeliac, control, and treated CD mucosa.

Figure 2

(A) Southern blot analysis of transcripts for interferon γ (IFN-γ) (upper blot) and β-actin (lower blot) in duodenal mucosa from five untreated coeliac disease (CD) patients cultured in vitro with medium alone (M) and with peptic-tryptic (PT)-gliadin in the absence (PT) or presence of human recombinant interleukin 10 (rhIL-10 50 ng/ml) (PT+IL-10) for 24 hours. Total RNA (700 ng) was used for cDNA preparation. An equivalent amount of cDNA per sample was amplified using specific primers for IFN-γ (26 cycles) and β-actin (18 cycles). Polymerase chain reaction products were then separated on a 1% agarose gel, blotted, and hybridised with oligonucleotide probes specific for IFN-γ and β-actin. In the presence of rhIL-10, gliadin induced IFN-γ mRNA production was reduced in 3/5 patients. (B) Southern blot analysis of transcripts for IFN-γ (upper blot) and β-actin (lower blot) in duodenal mucosa from six untreated CD patients cultured in vitro with medium alone (M) or in the presence of rhIL-10 (50 ng/ml) for 24 hours. Southern blot was performed as in (A). IFN-γ levels were not downregulated in biopsies cultured in the presence of rhIL-10 if compared with biopsies cultured in medium alone; paradoxically, in 4/6 patients there was upregulation of IFN-γ transcripts in the presence of rhIL-10.

Figure 3

mRNA transcripts for interferon γ (IFN-γ) (A) and interleukin 2 (IL-2) (B) were analysed by quantitative reverse transcription-polymerase chain reaction in treated coeliac disease and control duodenal biopsies after challenge for eight hours in medium alone or with peptic-tryptic (PT)-gliadin in the presence or absence of human recombinant IL-10 (rhIL-10 50 ng/ml). Values are mean (SEM) of the number of transcripts/μg total RNA. A significant increase in IFN-γ and IL-2 was observed in CD mucosa but not in controls in the presence of PT-gliadin (p<0.03). Addition of rhIL-10 downregulated Th1 cytokine synthesis in CD mucosa (p<0.03).

Figure 4

(A) Immunohistochemical staining for CD25+ cells in treated coeliac disease (CD) biopsies challenged with peptic-tryptic (PT)-gliadin in vitro in the presence or absence of human recombinant interleukin 10 (rhIL-10). A significant increase in the number of CD25+ cells/mm² of lamina propria was observed in CD biopsies cultured in the presence of PT-gliadin in comparison with medium alone (p<0.003). Addition of rhIL-10 together with PT-gliadin resulted in marked inhibition of CD25 expression (p<0.008). Values are mean (SEM). (B) Immunohistochemical staining for CD80+ cells in treated CD biopsies challenged with PT-gliadin in vitro in the presence or absence of rhIL-10. A significant increase in the number of CD80+ cells/mm² of lamina propria was observed in CD biopsies cultured in the presence of PT-gliadin in comparison with medium alone (p<0.003). Addition of rhIL-10 together with PT-gliadin inhibited CD80 expression (p<0.007). Values are mean (SEM). (C) Immunohistochemical staining for CD3+ intraepithelial lymphocytes in treated CD biopsies challenged with PT-gliadin in vitro in the presence or absence of rhIL-10. Migration of CD3+ T cells into the epithelial compartment was observed on gliadin challenge (p<0.05). Addition of rhIL-10 significantly reduced the number of CD3+ T cells in the epithelial compartment (p<0.01). Values are mean (SEM).

Figure 5

Immunofluorescence staining on duodenal mucosa from a coeliac patient cultured in vitro with peptic-tryptic (PT)-gliadin (A), with PT-gliadin and human recombinant interleukin 10 (rhIL-10) (B), or with medium only (C). In comparison with (C), in (A) there is an increase in CD3+ intraepithelial lymphocytes (in red) and of CD25+ cells (in green), particularly in the subepithelial region. There is also a significant increase in CD3+CD25+ activated T cells (in yellow). In (B), addition of rhIL-10 inhibited T cell activation and intraepithelial lymphocyte infiltration. Original magnification 40×.

Figure 6

Fas expression in the epithelium of jejunal mucosa from a coeliac patient cultured in vitro with medium only (A) or with peptic-tryptic (PT)-gliadin (B). In (B), intense staining for Fas is detected in almost all epithelial cells. Expression is particularly evident in the basolateral membrane of the enterocytes. A similar pattern was observed culturing the mucosa with PT-gliadin in combination with human recombinant interleukin10 (data not shown). Original magnification 100×.

Figure 7

Short term T cell lines (TCLs) were generated from treated CD mucosa challenged for 24 hours with gliadin (gliadin) or gliadin plus human recombinant interleukin 10 (gliadin+IL-10). TCLs were expanded for three weeks by repeated stimulation with APCs, gliadin, and IL-15 and subsequently tested for gliadin specificity by interferon γ (IFN-γ) and IL-10 ELISPOT and for proliferation to exogenously added IL-2. Representative results of TCLs obtained from one of the two CD patients analysed are shown. (A) TCLs from IL-10 treated explants did not produce IFN-γ in response to gliadin. Autologous peripheral blood mononuclear cells pulsed overnight with medium or gliadin (100 μg/ml) were used as APC. (B) IL-10 treated TCLs retained their capacity to proliferate in response to cytokines. No significant differences in proliferative responses to exogenously added IL-2 (100 U/ml) were observed in both control TCLs (gliadin) and IL-10 treated TCLs. (C) Increased frequency of IL-10 producing T cells in response to gliadin stimulation in TCLs obtained from IL-10 challenged biopsies