Characterisation of fractalkineCX3CL1 and fractalkine receptor CX3CR1 expression in abdominal aortic

1
Characterisation of fractalkine/CX3CL1 and
fractalkine receptor (CX3CR1) expression in
abdominal aortic aneurysm disease

• A Patel1, VP Jagadesham1,2,
• KE Porter3, DJA Scott2, SR Carding1

1.Research Institute of Molecular Cellular
Biology. University of Leeds 2. Leeds Vascular
Institute. The General Infirmary at Leeds 3.
Institute for Cardiovascular Research. University
of Leeds
2
Overview

• Background
• Hypothesis and aims
• Experimental approach
• Results
• Proposed model

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Background

• Abdominal aortic aneurysm (AAA)
• Prevalence 1.3-12.7
• Inflammatory infiltrate
• T cells 58.1
• B cells 41.1
• NK cells 7.3
• Macrophages 2
• Autoimmune disease

Wilmink 1998, Forester 2005, Lindholt 2006
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Background

• CX3CR1
• - Expressed on NK cells, monocytes and T
  lymphocytes

• Fractalkine
• Unique chemokine
• Expressed on vEC and vSMC

• Increased chemoattraction and adhesion
• Increased NK cell cytotoxicity

• Associated with autoimmune conditions

MacSweeny 1994, Bazan 1997, Yoneda 2000
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Hypothesis and aims

• Hypothesis
• The fractalkine-CX3CR1 interaction contributes to
  the accumulation of leucocytes in AAA tissue
• Aims
• CX3CR1 in AAA tissue
• CX3CR1 in peripheral blood of AAA and control
  patients

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CX3CR1 expression in AAA tissue
Patient Demographics

• Method
• Immunohistochemistry
• Microwave antigen retrieval
• Horse-radish peroxidase based
• Antibody to CX3CR1
• Microscope evaluation of staining
• Five fields of view

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Immunohistochemistry images of CX3CR1 cells
A
B
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Quantification of CX3CR1 cells in AAA tissue by
IHC
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CX3CR1 expression in peripheral blood

• Method
• Flow cytometry (FC)
• Cell isolation
• Fluorochrome-conjugated antibodies
• Emit light at different wavelengths
• Records the emission
• Quantitative analysis
• Peripheral blood mononuclear cells (PBMCs)

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CX3CR1 expression in peripheral blood
Patient demographics
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Phenotyping of CX3CR1 cells using a FACS plot
CX3CR1
Control
CD45
CD3
CD56
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Quantification of CX3CR1 PBMCs of AAA and
control patients
Percentage of CX3CR1 PBMCs
Haematopoetic cells
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Quantification of CX3CR1 PBMCs of AAA and
control patients
Percentage of CX3CR1 PBMCs
T cells
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Quantification of CX3CR1 PBMCs of AAA and
control patients
Percentage of CX3CR1 PBMCs
NK cells
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Quantification of Fractalkine and CX3CR1 AAA
tissue MNCs

• Method Flow cytometry
• Results

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Fractalkine stromal cells

• Method Flow cytometry
• Results

Ctrl. Ab Media TNFa
vSMC
53.4
vEC
66.7
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Conclusions

• Fractalkine and CX3CR1 in AAA patients
• CX3CR1 cells in AAA tissue
• Predominantly in adventitia
• CX3CR1 cells in peripheral blood
• Expression on NK cells and T cells
• Fractalkine cells in AAA tissue

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Proposed model

• The Fractalkine-CX3CR1 interaction may contribute
  to the extravasation of leucocytes seen in AAA
  tissue

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Acknowledgements

• Professor Simon Carding
• Institute of Molecular and Cellular Biology
• University of Leeds
• Mr Vamshi Jagadesham
• Leeds Vascular Institute
• The General Infirmary at Leeds
• Dr Karen Porter
• Institute for Cardiovascular Research
• University of Leeds
• Professor Julian Scott
• Leeds Vascular Institute
• The General Infirmary at Leeds

University of Leeds