Proinsulin is encoded by an RNA splice variant in human blood myeloid cells

Abstract

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of "immunological self" and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

Fig. 1.

A subpopulation of blood mononuclear cells expresses epitopes for endocrine self-antigens. PBMC purified from healthy individuals were labeled with FITC-conjugated mAbs to proinsulin (GS9A8), GAD65 (GAD6), 21-OH 1, glucagon, or somatostatin and with isotype control antibodies (thin lines in the histogram plots) and analyzed by flow cytometry. Dead cells were identified by propidium iodide nuclear staining and excluded from the analysis. The Lower Right plot shows labeling of the same cell population by biotin (streptavidin-PE)-conjugated antibody to proinsulin and FITC-conjugated antibody to GAD65. The data shown are representative of 15 individuals studied.

Fig. 2.

Myeloid lineage cells express an epitope for proinsulin. (a) PBMC purified from healthy individuals were labeled with PE-conjugated mAbs to T cell (CD3), B cell (CD19), NK cell (CD56), dendritic cell (CD11c, CD1a), and monocyte/macrophage cell (CD11c, CD14) markers and with FITC-conjugated mAb (GS9A8) to proinsulin and analyzed by flow cytometry. Binding of isotype control antibodies is represented by the shaded histograms. (b) Dendritic cells were purified from buffy coat-derived PBMC as described in Materials and Methods, labeled with markers of myeloid (HLA-DR, CD1c) or plasmacytoid (HLA-DR, CD123) dendritic cell subsets and with FITC-conjugated antibody (GS9A8) to proinsulin, and analyzed by flow cytometry. (c) FACS-sorted CD11c⁺ and CD11c⁻ PBMC (each 5 × 10⁶ per milliliter) were solubilized in 8% SDS and spotted onto nitrocellulose membrane, in parallel with serial dilutions of recombinant human proinsulin, and immunoblotted with the antiproinsulin mAb KL1.

Fig. 3.

Blood cells transcribe self-antigen genes, including a proinsulin RNA splice variant. (a) RNA was freshly isolated from PBMC, reverse-transcribed into cDNA with random hexamer oligonucleotides, and amplified by PCR using nested sets of sequence-specific primers that spanned introns to avoid amplifying genomic DNA. GAPDH was used as a housekeeping gene. Electrophoresis in 1% agarose and staining with ethidium bromide demonstrated cDNAs for proinsulin, GAD65, and 21-OH in all five individuals examined (one example shown). Product identities were confirmed by DNA extraction and sequencing. (b) Native (N) and splice variant (SV) proinsulin RNAs from the pancreas and flow-sorted PBMC were quantified by real-time PCR (Upper), and the PCR products were identified by staining with ethidium bromide after electrophoresis in 1% agarose (Lower).

Fig. 4.

Introduction of proinsulin siRNA reduces expression of immunoreactive proinsulin. As described in Materials and Methods, PBMC (5 × 10⁶ per milliliter) in RPMI medium 1640 containing 10% FCS were incubated in uncoated plastic flasks in 5% CO₂/air at 37°C for 3 h. Adherent cells (5 × 10⁶) were resuspended in transfection medium for human monocytes with or without (“mock transfection”) 500 nM siRNA and transfected. Cells, including some not transfected, were then incubated in transfection medium for human monocytes in 5% CO₂/air at 37°C for 48 h. After washing and the addition of FcR blocking reagent, cells were stained with KL1-FITC (proinsulin) (Upper) or biotinylated-GAD6-streptavidin-PE (GAD65) (Lower) and HLA-DR-APC mAbs or isotype control mAbs and analyzed by flow cytometry. siRNA1–siRNA3 are to the second exon of insulin; the −ve siRNA, used as a negative control, is a scrambled version of siRNA1.