Human fibrocytes express multiple antigens associated with autoimmune endocrine diseases

Abstract

Context: Factors common to multiple autoimmune diseases have been sought vigorously. Graves' disease (GD) and type 1 diabetes mellitus (T1DM) involve end-organ remodeling. Fibrocytes participate in inflammatory diseases and were recently shown to express thyroid-specific proteins such as the thyrotropin receptor and thyroglobulin.

Objective: The objective of the study was to determine whether a broader repertoire of autoantigen expression, such as proteins associated with T1DM, can be ascribed to fibrocytes.

Design, setting, and participants: Fibrocytes and fibroblasts were collected and analyzed from healthy individuals and those with autoimmune diseases in an academic clinical practice.

Main outcome measures: Real-time PCR, Western blot analysis, gene promoter analysis, cell transfections, and flow cytometric cell sorting were performed.

Results: Islet cell antigen ICA512 (IA-2) and islet cell autoantigen of 69 kDa (ICA69), two islet-specific proteins implicated in T1DM, are expressed by fibrocytes from healthy donors and those with T1DM, GD, and multiple sclerosis. Both transcripts are detected by PCR, the proteins are resolved on Western blots, and both gene promoters are active in fibrocytes. Levels of ICA69 are substantially higher than those of IA-2 in fibrocytes. ICA69 localizes to CD34(+) GD orbital fibroblasts putatively derived from fibrocytes, whereas higher levels of IA-2 are found in CD34(-) fibroblasts.

Conclusions: In addition to autoantigens implicated in thyroid autoimmunity, fibrocytes and derivative fibroblasts express multiple autoantigens associated with T1DM. This expression results from active gene promoters and abundant steady-state mRNA encoding ICA69 and IA-2. These latest findings demonstrate that fibrocytes express antigens relevant to multiple forms of endocrine autoimmunity. They suggest the potential for these cells playing a direct role in immune reactivity directed at the thyroid and pancreatic islets.

Figure 1.

Fibrocytes express ICA69 and IA-2 protein. Cultured fibrocytes were lysed and proteins separated on 10% SDS-PAGE, transferred onto nitrocellulose filters, and detected using the primary and secondary antibodies described in Materials and Methods. Bands were detected by chemiluminescence with the SuperSignal West Pico chemiluminescent substrate (Pierce/Fisher). Mouse recombinant ICA69 and human full-length IA-2 were used as standards.

Figure 2.

ICA69 mRNA levels (A) and IA2 mRNA levels (B) in fibrocytes from individuals with type 1 diabetes (T1D Fibrocyte), with T1D and Graves' disease (T1D+GD), Graves' disease (GD Fibrocyte), MS (MS Fibrocyte), and healthy individuals (Healthy Fibrocyte). Also quantified were levels in pancreas, orbital fibroblasts from individuals with GD (GD-OF), and skin fibroblasts (Dermal). ICA69 mRNA and IA-2 mRNA were quantified by reversed transcribing RNA. cDNA was subjected to quantitative real time-PCR. Values were normalized to their respective GAPDH signals. Data are expressed as the mean ± SD of triplicate independent determinations.

Figure 3.

Tg mRNA (A) and TSHR mRNA (B) levels in fibrocytes and fibroblasts from healthy individuals and those with a variety of autoimmune diseases, as defined in the legend to Figure 2. The respective mRNAs were quantified by reversed transcribing RNA. cDNA was subjected to quantitative real-time-PCR. Values were normalized to their respective GAPDH signals. Data are expressed as the mean ± SD of triplicate independent determinations.

Figure 4.

ICA69 and IA-2 gene promoter activities in NMB7 cells and fibrocytes. A, Sequence diagram of a fragment of human ICA69 gene promoter extending from −1012 to +19 nt. Mutations of the CREB (−174 to −171 nt) and Sp1/GC elements (−196 nt to −193 nt) are shown with the wt sequences below their emboldened, mutated counterparts. Activities of wild-type (WT) and mutated (Mut) ICA69 gene promoter/reporter constructs transfected into NMB7 cells (B) and fibrocytes (C) are shown. D, Activity of the IA-2 gene promoter/reporter construct transfected into NMB7 cells and fibrocytes. Fibrocyte cultures were transfected with plasmid containing empty reporter or one fused with the respective constructs, as described in Materials and Methods. Data are expressed as the mean ± SD. *, P < .05 vs wild type.

Figure 5.

A, Left panel, Distribution of CD34⁺ and CD34⁻ fibroblasts in a mixed (parental) orbital fibroblast culture from a patient with severe TAO, as determined by flow cytometry, demonstrates two distinct populations. Right panel, Cytometric sorting results in two pure populations. ICA69 (B) and IA-2 (C) mRNA levels in pure CD34⁺ and CD34⁻ fibroblast subsets from the patient shown in panel A. Mixed (parental) strain of GD-OF containing CD34⁻ and CD34⁺ cells was sorted into pure subsets by fluorescence-activated cell sorting. Cells were then cultured for 48 hours and RNA extracted and subjected to real-time PCR for the respective mRNA levels. Values were normalized to their respective GAPDH signals and expressed as the mean ± SD of three independent determinations. P < .05, CD34⁺ vs CD34⁻ strain. Results shown are representative of four separate experiments.