Transcript DC-SIGNEMIG.ppt

DC-SIGN/HLA-DR+ cells increased in coeliac duodenum
Montalvillo E.1, García-Vallejo J.J.2, Martínez-Abad B.1, Vallejo-Diez S.1,EscuderoHernández C.1, Bernardo D3. Garrote J.A.1,4, Arranz E.1
1. Immunology Lab, Dept of Paediatrics & Immunology, University of Valladolid, IBGM-CSIC, Spain; 2. Department of Molecular Cell Biology and Immunology, VU
University Medical Centre, Amsterdam, the Netherlands; 3. Imperial College London, St. Mark’s and Northwick Park Hospital, United Kingdom; 4.
Molecular Genetics, Hospital Universitario Rio Hortega, Valladolid, Spain.
E-mail: [email protected]
INTRODUCTION:
• Coeliac Disease (CD) is an intestinal inflammatory disorder induced by the ingestion of dietary gluten which occurs in genetically predisposed individuals. The only treatment is a strict
gluten-free diet (GFD), after which a complete remission of the clinical expression is found.
• DC-SIGN is a C-type lectin and has a high affinity for the ICAM3 molecule. It binds various microorganisms by recognizing high-mannose-containing glycoproteins on their envelopes.
Besides functioning as an adhesion molecule, recent study has shown that DC-SIGN can initiate innate immunity by modulating toll-like receptors. DC-SIGN together with other C-type
lectins is involved in recognition of tumors by dendritic cells and DC-SIGN is also a potential engineering target for dendritic cell based vaccine.
OBJECTIVE:
• We aimed to determine the DC-SIGN expression by immunofluorescence in duodenal biopsies from coeliac patients and controls.
PACIENTS & METHODS:
• We obtained endoscopic duodenal biopsies from 3 aCD, 3 GFD and 3 HC patients. Tissue was washed three times in PBS before to be embedded in OCT
compound. Then, the samples were frozen in liquid nitrogen and stored at -80C for long-term use. After cutting 8μm tissue sections, slides were air dried overnight.
We used acetone solution to fix the tissue sections. To rehydrate and to wash them we used PBS. Blocking was performed with 3% BSA in PBS before antibody
staining.
• The staining was done adding rabbit DC-Sign anti-human ([1:1000], labelled with α-rabbit AlexaFluor 488 [1:500]) and mouse HLA-DR anti-human or mouse CD4
anti-human (both [1:500], labelled with α-mouse AlexaFluor 647[1:500]) for 1h at room temperature. We used the corresponding isotypes as controls.
• Slides were mounted using Vinol mounting medium with DAPI [1:10000] and viewed in a Leica DM6000 microscope. For the analysis, we used Leica Application
Suite for Advanced Fluorescence software (Leica Microsystems, 2.3.5 version)
Healthy Control
RESULTS
In active Coeliac Disease CD4+ T cell infiltration
occurs in both the lamina propria and the
epithelium. As shown in Figure 1, the T cells
infiltration is accompanied with an increase in both
the number of DC-SIGN+ cells and the expression
level of DC-SIGN on HLA-DR+ cells in the lamina
propria. This appears to be specific of active CD, as
healthy controls do not show CD4+ T cell infiltration
and/or increased presence of DC-SIGN+. Diettreated CD patients also show a lower CD4+ T cell
infiltrate and DC-SIGN expression. This decreased
depends on the time elapsed since the start of the
gluten-free diet (fig. 1).
Gluten Free Diet
Celiac Disease
40x
Figure 1. Immunofluorescence analysis of DC-Sign (green) and CD4 (red)
c
in duodenal biopsies from aCD, GFD and healthy control patients, with a
DAPI (blue) nuclear staining
Active Celiac Disease
20x
2a
2c
2c
Active Celiac Disease
20x
Figure 2. Immunofluorescence analysis of DC-Sign (green) and HLA-DR
(red) in duodenal biopsies from aCD, GFD and healthy control patients,
with a DAPI (blue) nuclear staining.
Healthy Control
Gluten Free Diet
Celiac Disease
By Immunofluorescence, DC-SIGN was found to
be most highly expressed in the lamina propria
cells of aCD patients (Fig. 2). In these patients, we
have observed that all the DC-SIGN+ cells are
HLA-DR+ (Fig. 2a). On the contrary, both healthy
controls and diet-treated celiac patients show
less DC-SIGN/HLA-DR+ cells (2b). Also these
samples show some HLA-DR+ cells that do not
express DC-SIGN (2c).
2b
2b
40x
CONCLUSSIONS:
• In aCD CD4+ T cell infiltration occurs in both the lamina propria and the epithelium.
• The T cells infiltration is accompanied with an increase in both the number of DC-SIGN+ cells and the expression level of DC-SIGN on HLA-DR+ cells in the
lamina propria. This appears to be specific of aCD, as HC do not show CD4+ T cell infiltration and/or increased presence of DC-SIGN+.
• GFD patients also show a lower CD4+ T cell infiltrate and DC-SIGN expression. This decreased depends on the time elapsed since the start of the glutenfree diet.
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS: This project has been partially funded by University of Valladolid (VA016A10-2), Health Institute Carlos III
(PI10/01647) , Junta de Castilla y Leon (FPI, Montalvillo E.) and University of Valladolid (FPI-UVa; Escudero-Hernández C., Martínez-Abad B.)