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Expanding immunological
‘self’: expression of self-antigens, including a proinsulin
mRNA splice variant, by human blood myeloid cells
P. Narendran,
A.M. Neale, K.P. Jensen, R.J. Steptoe, L.C. Harrison, in collaboration
with G. Morahan, Genetics and Bioinformatics Division; Martin Lackmann,
Department of Biochemistry & Molecular Biology, Monash University;
O. Madsen, Hagedorn Research Institute, Copenhagen
Self-antigens
are expressed by medullary epithelial cells in the mouse thymus, where
they contribute to immune tolerance through the deletion of self antigen-reactive
T cells. In the mouse and human thymus, self-antigens have also been
shown to be expressed by cells displaying phenotypic markers of dendritic
and monocytic antigen-presenting cells. Despite the emphasis on central
tolerance, the deletion of self antigen-reactive T cells in the thymus
is incomplete and autoreactive T cells can be detected in peripheral
blood, where distinct regulatory mechanisms operate to avert autoimmune
disease. These mechanisms, including T-cell deletion, T-cell anergy
and induction of regulatory T cells, depend classically on the uptake
and presentation of self-antigens by specialised antigen-presenting
cells. However, the possibility must be considered that, as in the thymus,
self-antigens could be expressed ectopically by circulating antigen-presenting
cells and contribute to peripheral immune tolerance.
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Figure
1: Sub-populations of (CD11c+ and CD14+) cells in human blood
label express epitopes for endocrine self-antigens. Blood cells
purified from healthy donors were labelled with FITC-conjugated
monoclonal antibodies to proinsulin (GS9A8), GAD65 (GAD6), 21-hydroxylase
(21-OH 1), glucagon and somatostatin, and with isotype control
antibodies (thin lines in the histogram plots), and analysed by
flow cytometry. Dead cells, identified by propidium iodide nuclear
staining, were excluded from the analysis. The bottom right-hand
panel shows labelling of the same cell population by biotin (streptavidin-PE)-conjugated
proinsulin antibody (GS9A8) and FITC-conjugated GAD65 antibody.
The data are representative of 15 individuals studied.
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Figure
2: Human blood myeloid cells that express a proinsulin B24-C36 epitope
preferentially transcribe a proinsulin splice variant mRNA. Blood
cells were labelled with a monoclonal antibody (GS9A8) that recognises
the B-C chain junction of human proinsulin and flow-sorted into
GS9A8+ and GS9A8- cells for RNA extraction. Native and splice variant
proinsulin mRNAs from sorted cells and from the pancreas were quantified
by real-time PCR and the products identified by staining with ethidium
bromide after electrophoresis in 1% agarose. |
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We show that
myeloid lineage cells in human peripheral blood express surface epitopes
for self-antigens and transcribe low levels of self-antigen genes. In
the case of proinsulin, these cells express a known epitope for B- and
T-cells located across the junction of the insulin B chain and the connecting
(C) peptide (Martinez et al, J Clin Invest 2003 [download
PDF]). Furthermore, they preferentially transcribe a proinsulin
mRNA splice variant whose expression level correlates with allelism
at the insulin gene locus, which modifies susceptibility to type 1 diabetes.
The synthesis and expression of self-antigens by peripheral blood myeloid
cells extends the network of ‘self’ and, by analogy with the
thymus, may be involved in regulating immune tolerance.
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