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Identification of a novel immunomodulatory gliadin peptide that causes interleukin-8 release in a chemokine receptor CXCR3-dependent manner only in patients with coeliac disease - PubMed

Identification of a novel immunomodulatory gliadin peptide that causes interleukin-8 release in a chemokine receptor CXCR3-dependent manner only in patients with coeliac disease

Karen M Lammers et al. Immunology. 2011 Mar.

Abstract

The autoimmune enteropathy, coeliac disease (CD), is triggered by ingestion of gluten-containing grains. We recently reported that the chemokine receptor CXCR3 serves as a receptor for specific gliadin peptides that cause zonulin release and subsequent increase in intestinal permeability. To explore the role of CXCR3 in the immune response to gliadin, peripheral blood mononuclear cells from both patients with CD and healthy controls were incubated with either pepsin-trypsin-digested gliadin or 11 α-gliadin synthetic peptides in the presence or absence of a blocking anti-CXCR3 monoclonal antibody. Supernatants were analysed for interleukin-6 (IL-6), IL-8, IL-10, IL-13, IP-10 (CXCL10), tumour necrosis factor-α and interferon-γ. Gliadin broadly induced cytokine production irrespective of the clinical condition. However, IL-8 production occurred only in a subgroup of individuals and cells of the phagocytic lineage were the main source. Induction of IL-8 was reproduced by one of a comprehensive panel of synthetic α-gliadin peptides and was abrogated when CXCR3 was blocked before stimulation with either gliadin or this peptide in the CD group but not in the control group, suggesting that gliadin-induced IL-8 production was CXCR3-dependent gliadin induced IL-8 production only in CD.

© 2010 The Authors. Immunology © 2010 Blackwell Publishing Ltd.

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Figures

Figure 1
Figure 1

Pepsin-trypsin-digested (PT-) gliadin is a potent stimulus for cytokine production by peripheral blood monoonuclear cells (PBMC). PBMC from healthy controls (HC) and patients with coeliac disease on a gluten-free diet (CD-GFD) were incubated with medium alone or PT-gliadin (1 mg/ml) for 24 hr. Supernatants were assayed for interleukin-6 (IL-6), IL-8, IL-10, tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and IL-13 production as indicated. Each dot represents a single donor. Horizontal lines are drawn at the median value in each group. *P < 0·05, **P < 0·01, ***P < 0·001, ****P < 0·0001.

Figure 2
Figure 2

Pepsin-trypsin-digested (PT-) gliadin-induced production of interleukin-8 (IL-8) is CXCR3-dependent in peripheral blood mononuclear cells (PBMC) from patients with coeliac disease fed a gluten-free diet (CD-GFD). The PBMC were pre-incubated for 30 min with a blocking anti-human CXCR3 antibody (10 μg/ml), followed by incubation with medium alone or PT-gliadin (1 mg/ml) for 24 hr. Supernatants were assayed for IL-6, IL-8, IL-10, tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-13 concentrations. (a) Blocking of the chemokine receptor, CXCR3, did not change PT-gliadin-induced cytokine production in PBMC from healthy controls (HC), whereas (b) blocking of CXCR3 affected greatly the PT-gliadin-induced IL-8 and, to a much lesser extent, IL-6 production in PBMC from CD-GFD patients, while IL-10, IL-13, TNF-α and IFN-γ production was unchanged. Each dot represents a single donor. Medians and interquartile ranges for each group of donors are also shown. *P < 0·05 relative to isotype control.

Figure 3
Figure 3

Production of interleukin-8 (IL-8) in response to a comprehensive panel of α-gliadin peptides. Eleven peptides from the α-gliadin synthetic peptide library were separately tested as outlined in the Materials and methods section to identify the sequence(s) responsible for IL-8 induction by peripheral blood mononuclear cells from 16 healthy controls (HC) and 17 patients with coeliac disease fed a gluten-free diet (CD GFD). The effect of peptide 4037, corresponding to aa 270–286 of α-gliadin, is shown. Each dot corresponds to a single donor. *P < 0·05 relative to isotype control (iso) from responders (six out of 17 patients). The IL-8 response to the entire set of tested peptides is shown in Fig. S1.

Figure 4
Figure 4

Identification of monocytes and plasmacytoid dendritic cells as cellular sources of gliadin-induced interleukin-8 (IL-8) production. Peripheral blood mononuclear cells (PBMC) from healthy controls (HC) and patients with coeliac disease fed a gluten-free diet (CD-GFD) were subjected to depletion of monocytes (CD14+), B cells (CD19+), myeloid (BDCA1+) or plasmacytoid (BDCA4+) dendritic cells as indicated, or untouched T cells were separated as indicated in the Materials and methods. All fractions were incubated in the presence of medium alone (unstimulated) or pepsin-trypsin-digested (PT-) gliadin (1 mg/ml) for 24 hr. Supernatants were assayed for IL-8. Each dot corresponds to a single donor. Median values are indicated as horizontal lines. Kruskal–Wallis test comparing all suspensions in each group of subjects showed significant differences both in HC (P < 0·01) and CD GFD (P < 0·05). *P < 0·05 at the Dunn's multiple comparison post-test.

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