Aberrant Expression of the Insulin-Like Growth Factor-1 Receptor by T Cells from Patients with Graves’ Disease May Carry Functional Consequences for Disease Pathogenesis

Raymond S. Douglas, Andrew G. Gianoukakis, Shweta Kamat and Terry J. Smith
J Immunol March 1, 2007, 178 (5) 3281-3287; DOI: https://doi.org/10.4049/jimmunol.178.5.3281

Abstract

Graves’ disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like growth factor receptor-1 (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD. IGF-1R on fibroblasts, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 ± 4% (mean ± SE; n = 33) in patients with GD compared with 15 ± 3% (n = 21; p < 10−8) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and IGF-1R+CD4+CD3+ cells as those found in the peripheral circulation. GD-derived peripheral T cells express durable, constitutive IGF-1R expression in culture and receptor levels are further up-regulated following CD3 complex activation. IGF-1 enhanced GD-derived T cell incorporation of BrdU (p < 0.02) and inhibited Fas-mediated apoptosis (p < 0.02). These findings suggest a potential role for IGF-1R displayed by lymphocytes in supporting the expansion of memory T cells in GD.

The insulin-like growth factor receptor (IGF-1R)3 pathway plays important and diverse roles in growth and development (1). This tyrosine kinase receptor has been implicated in several metabolic, neoplastic, and immunological diseases (2, 3, 4). IGF-1R and associated IGF-1-binding proteins constitute a cell surface signaling complex (5). An IGF-1 binding domain resides in the extracellular domain of IGF-1Rα, whereas three tyrosine residues represent autophosphorylation sites, namely Tyr1131, Tyr1135, and Tyr1136, within the activation loop of the IGF-1Rβ catalytic domain (6). Phosphorylation at all three is required for optimal receptor activation. This culminates in the recruitment of multiple docking proteins and the generation of intracellular signaling (5).

IGF-1 and IGF-1R play important roles in hemopoietic cell growth and differentiation and normal immune function (7). Peripheral blood T and B cells and monocytes from control human donors express low levels of IGF-1R in vivo (8, 9). Administration of IGF-1 increases the circulating pool of CD4+ T cells and splenic B cells in mice (10, 11), suggesting a role for this growth factor in myelopoietic cell expansion (12). It promotes T cell proliferation during early activation (13) and inhibits apoptosis of both immature and mature T cells through at least three distinct mechanisms (14, 15). IGF-1 stimulates inflammatory cytokine production in T cells and monocytes, including IL-2 (16), TNF-α (17), and IL-8 (18). It can bias lymphocyte development toward a Th2 phenotype by enhancing IL-10 (19), IL-4, and IL-13 synthesis (20) while inhibiting IFN-γ function (21). With regard to B cell differentiation, IGF-1 promotes Ig production in chimeric immunodeficient mice reconstituted with IGF-1R−/− fetal liver cells (22). It also enhances T cell-independent humoral immune responses (22). These findings indicate that functional IGF-1R may be required for T cell-independent B cell responses and support its important role in B cell differentiation and Ab production.

Graves’ disease (GD) is a systemic autoimmune process characterized by several immune system abnormalities, including the production of IgG directed against the thyrotropin receptor, expansion of CD45RO+ T cells (23), and lymphocytic infiltration of the thyroid and connective tissue of the orbit (24). Thyroid-associated ophthalmopathy (TAO) represents the orbital manifestation of GD. Extraocular muscles and fat expand, become inflamed, and are remodeled extensively (24). Cytokines and lipid mediators, synthesized by infiltrating T lymphocytes, monocytes, and mast cells, drive tissue remodeling, including the accumulation of hyaluronan, an abundant nonsulfated glycosaminoglycan (25, 26). The unique phenotype of orbital fibroblasts and their exaggerated responses to cytokines such as IL-1β represent the basis for disease susceptibility of these tissues (25, 27, 28, 29, 30, 31). Why immunocompetent cells are recruited to the orbit in TAO remains uncertain.

We have reported previously that IGF-1R is expressed by a disproportionately large fraction of fibroblasts from patients with GD (32). When treated with IGF-1 or with IgG derived from these patients (GD-IgG), GD fibroblasts, but not those from control donors, synthesize high levels of IL-16 and RANTES, two powerful T cell chemoattractants (32). In addition, orbital fibroblasts from patients with GD synthesize increased levels of hyaluronan when treated with GD-IgG, an action mediated by IGF-1R (33). Autoantibodies directed against IGF-1R can be detected in almost all patients with GD but in few individuals without the disease (32, 33, 34). We now report increased proportions of IGF-1R+ T cells from patients with GD. IGF-1R expression in T cells is durable in culture and can be further enhanced following TCR activation. An overabundance of IGF-1R+ T cells infiltrate affected orbital tissue in TAO. Unlike CD45RO+ T cells from control donors, a majority of those from patients display IGF-1R. IGF-1 enhances proliferation and inhibits apoptosis of T cells in vitro. These effects are exaggerated in lymphocytes from patients with GD. Thus, the greater abundance of IGF-1R+ T lymphocytes appears to convey functional consequences that might help explain the previously recognized expansion of CD45RO+ memory T cells found in GD.

Materials and Methods

Materials

Ficoll-Hypaque was purchased from Sigma-Aldrich. FacLyse buffer, Cytofix, anti-CD3 CyChrome, anti-IGF-1Rα PE (clone 1H7), anti-CD45RA allophycocyanin, and anti-CD45RO allophycocyanin, and isotype mouse IgG1 FITC, PE, allophycocyanin, and CyChrome were purchased from BD Biosciences. FBS was obtained from Invitrogen Life Technologies. IGF-1 and Des1–3 IGF-1 were supplied by Calbiochem and Gro-Pep, respectively.

Patient samples

Subjects, ages 20–65, were recruited from the patient population of Jules Stein Eye Institute and Harbor-University of California Los Angeles (UCLA) Medical Center. Informed consent was obtained as approved by the Institutional Review Boards of the Center for Health Sciences at UCLA and Harbor-UCLA Medical Center. The study population comprised patients evaluated for GD, all of whom manifested either stable or active TAO. Control subjects were healthy volunteers without known GD or other autoimmune disease who presented for esthetic or functional eyelid surgery. Individuals excluded from the study included those with nonthyroid autoimmune disease, asthma, granulomatous disease, sinusitis, or HIV infection. Patients with GD comprised a clinically heterogeneous group and included those who were hyperthyroid (n = 3) and euthyroid (n = 30). A minority of patients had active inflammatory disease (clinical activity score ≥3; n = 8), whereas most exhibited stable TAO (clinical activity score <3; n = 25). No association between IGF-1R display and disease duration or degree of orbital inflammation could be demonstrated. Bone marrow samples were derived from a patient with stable GD, a healthy volunteer or at necropsy within 24 h of death. Orbital tissue was obtained from surgical waste during orbital decompression surgery in patients with GD or from healthy individuals during cosmetic surgery. The tissue was transported on ice, homogenized, and single-cell suspensions prepared. Tissue was filtered using a 70-μm filter and processed for flow cytometry.

Clinical data including age, sex, medications, smoking history, physical exam, and laboratory values were recorded. Careful examination of the skin failed to detect evidence of thyroid-related dermopathy in any of the study participants.

Flow cytometry

Peripheral blood (∼5 ml) was obtained and stored in tubes containing EDTA. Staining buffer was prepared in PBS containing 4% FBS with 0.1% sodium azide (Sigma Aldrich). Staining for flow cytometry was performed within 24 h of blood collection, according to the manufacturer’s instructions (BD Biosciences). Briefly, 100 μl of whole blood or bone marrow aspirate was placed in 12 × 75-mm polypropylene tubes and fluorochrome-conjugated mAbs were added (1 μg/106 cells). These were then incubated in the dark for 20 min at room temperature. FACSlyse solution (2 ml) was added for 10 min at room temperature to disrupt RBCs. Cells were washed twice with staining buffer, resuspended in Cytofix (BD Biosciences), and kept in the dark at 4°C until cytometric analysis (within 24 h). Analysis was performed on a FACSCalibur flow cytometer (BD Biosciences). Mean fluorescent intensity (MFI) was calculated as a ratio of mean fluorescence sample:isotype fluorescence. Percentage of positive expression was defined as the fraction of cells with increased fluorescent intensity compared with an isotype control.

Cell proliferation assay

BrdU incorporation was assessed according to the manufacturer’s instructions (Calbiochem). Cells (1 × 106 cells/ml) were cultured for 24–72 h in 96-well plates in RPMI 1640 medium containing nothing, IGF-1 (10 nM), Des 1–3 IGF-1 (10 nM), plate-bound anti-TCR (10 μg/ml; BD Biosciences), or PHA (2 μg/ml; Invitrogen Life Technologies). This PHA concentration yielded submaximal proliferation in preliminary experiments. During the last 6–12 h of culture, BrdU (20 μl, 0.05 M solution) was added and the cells were centrifuged at 500 × g. Once the supernatant was decanted, fixative/denaturing solution was added, incubated for 30 min, and decanted. Samples were incubated with anti-BrdU Ab followed by peroxidase-labeled goat anti-mouse IgG according to the manufacturer’s instructions. The fluorogenic substrate was added, and emission was determined using a Wallac Victor 1420 fluorometer (PerkinElmer) at 320 nm excitation and 460 nm emission wavelengths.

PBMC and T lymphocyte preparation

PBMCs were prepared using a technique described previously (35). Briefly, whole blood was diluted 1/2 in PBS and layered over Ficoll-Hypaque, centrifuged at 500 × g for 25 min, and washed three times in PBS. A total of 3 × 106 cells/ml was cultured in RPMI 1640 medium supplemented with PHA (2 μg/ml) or in plates coated with anti-CD3 (BD Biosciences) or anti-TCR (BD Biosciences) Abs (10 μg/ml). Purified CD3+ T cells were negatively selected using magnetic cell sorting (BD IMag) according to the manufacturer’s instructions (BD Biosciences). For T cell preparation, PBMCs were incubated in a mixture of biotinylated Abs not binding T lymphocytes (BD Biosciences). Streptavidin particles were added to the cell suspension and T cells collected following magnetic depletion. They were enriched to >97% purity without activation by analysis of CD69 and CD25 before and after enrichment.

Apoptosis assay

Cells were stained with annexin V-FITC and 7-aminoactinomycin D (7-AAD) (BD Pharmingen) according to the manufacturer’s directions and analyzed by flow cytometry. Cell debris was excluded from analysis by adjusting the forward light scatter threshold. Cells staining positively with annexin V-FITC, but excluding 7-AAD, were regarded as those undergoing apoptosis.

Statistics

Values are reported as the mean ± SE. Statistical analysis was performed using a two-tailed Student’s t test with a confidence level >95%.

Results

IGF-1R is expressed by a greater proportion of T cells derived from patients with GD compared with those from normal donors

A disproportionate fraction of fibroblasts from patients with GD express IGF-1R (32). We therefore investigated the cell surface display of IGF-1R by PBMCs. As with their fibroblasts, donors with GD exhibit a larger fraction of peripheral blood T cells (CD3+, IGF-1R+) expressing IGF-1R compared with controls. Fig. 1⇓ contains a representative histogram of IGF-1R expression by T cells from patients and controls, and demonstrates that a substantially greater fraction of T cells from the former express IGF-1R. Cumulative data in Fig. 1⇓ demonstrate that 48 ± 5% (mean ± SE) of T cells from patients (n = 33) express IGF-1R, whereas the receptor was detected in 15 ± 3% cells from control donors (n = 21; p < 10−8; GD vs controls). The range of IGF-1R expression was considerably greater among patient-derived T cells (20–95% CD3+ IGF-1R+) compared with controls (1–29%). The MFI of the CD3+IGF-1R+ population was similar for GD and control T cells, suggesting similar levels of receptor density. The abundance of CD4+ and CD8+ T cells was similar in controls and patients with GD, as anticipated; however, that of IGF-1R+CD4+ and IGF-1R+CD8+ lymphocytes was substantially greater in GD (n = 6) than that in controls (n = 6) (CD4+ T cells, p < 0.01; CD8+ T cells, p < 0.01). The fraction of IGF-1R+ T cells appears durable because serial examination of five patients revealed similar relative abundance of CD3+IGF-1R+ cells over a 1-year period (data not shown).

FIGURE 1.
FIGURE 1.

Increased fraction of peripheral blood T cells from patients with GD display IGF-1R compared with those from control donors. PBMCs were stained with anti-CD3 and IGF-1R Abs, as described in Materials and Methods, and subjected to multiparameter flow cytometry. A and B, The open histograms represent staining with isotype control Abs. Data derived from single, representative samples from each source. C, Fraction of IGF-1R+CD3+ T cells from individual patients with GD and control donors. D, Analysis of IGF-1R display in T cells from the aggregate of multiple patients with GD and control donors; 48 ± 4% GD T cells (n = 33) display IGF-1R compared with 15 ± 3% control T cells (n = 21; p < 1 × 10−8). Data are expressed as mean ± SE.

An increased fraction of IGF-1R+ T cells are found in orbital tissue derived from patients with TAO

The phenotypic profile of T cells derived from the orbital tissue of patients with TAO mirrors that in the peripheral circulation and exhibits a disproportionately large fraction of IGF-1R+ T cells. Orbital and peripheral blood lymphocytes were isolated from the same donors. A relatively small percentage of both CD4+ T cells and total T cells are IGF-1R+ in the peripheral blood of control donors (29% and 28%, respectively). A similarly small fraction of T cells express IGF-1R in orbital tissue from these same donors (CD4+ 23%, total CD3+ 26%) (Fig. 2⇓). In contrast, orbital T cells from a patient with TAO are overrepresented by the IGF-1R+ phenotype (CD4+, 51%; CD3+, 56%) and thus resemble those in the peripheral blood (CD4+, 64%; CD3+, 61%).

FIGURE 2.
FIGURE 2.

Disproportionate IGF-1R+ peripheral blood CD4+CD3+ and CD3+ T cells from patients with GD reflect the T cell population infiltrating orbital connective tissue in TAO. Peripheral blood (PB) and orbital connective tissue T cells from a single patient with GD and those from a single control donor were examined for IGF-1R display by flow cytometry. Data are representative of three experiments. Bottom panels, Similar, nearly uniform IGF-1R display was found in bone marrow-derived (BM) CD3+ T cells from a patient with GD and those from a donor without autoimmune disease. PB (inset) and BM T cells were stained with anti-CD3 Abs and with anti-IGF-1R Abs (solid) or isotype control Abs (open) and subjected to flow cytometry.

We next investigated whether the increased fraction of peripheral and orbital IGF-1R+ T cells of patients with GD mirrored those in their bone marrow. As demonstrated in Fig. 2⇑, marrow-derived T cells (CD3+CD19−CD45+ CD15−CD14−) from two control donors and a patient with GD uniformly express IGF-1R. The expected increased fraction in peripheral blood IGF-1R+ T cells is found in GD (Fig. 2⇑, inset). Thus, the relative abundance of IGF-1R+ bone marrow T cells appears substantially greater than that found in the peripheral circulation of both control donors and those with GD.

CD45RO+IGF-1R+ phenotype accounts for the increased fraction of IGF-1R+ T cells in patients with GD

The fraction of CD45RA+IGF-1R+ T lymphocytes from patients and control donors appears similar (Fig. 3⇓). In contrast, CD3+CD45RO+IGF-1R+ cells account for a small fraction of T cells from control donors (8 ± 4%; n = 5) but a substantially greater proportion of those from patients (57 ± 12%; n = 5; p < 0.01 vs control). This is true for both CD4+ and CD8+CD45RO+ T cells. Thus, both CD4+ and CD8+ memory T lymphocytes account for the increased fraction of IGF-1R+ T cells in patients with GD.

FIGURE 3.
FIGURE 3.

Disproportionate IGF-1R+CD45RO+ memory T cells from patients with GD. The fraction of CD3+, CD4+, and CD8+ T lymphocytes expressing IGF-1R was determined using multiparameter flow cytometry by gating on populations of CD3+, CD4+, or CD8+, CD45RA+ or CD45RO+ T cells and is represented as a histogram (solid) compared with isotype controls (open). A, Naive CD45RA+ lymphocytes from a patient with GD and a control donor demonstrate a similar, frequent display of IGF-1R. B, The fraction of memory CD45RO+ lymphocytes expressing IGF-1R is dramatically greater in lymphocytes from a patient with GD compared with a control. GD CD8+CD45RO+ T lymphocytes uniformly express IGF-1R, compared with infrequent control CD8+CD45RO+ cells. T cell expression of IGF-1R was representative of our aggregate observations.

IGF-1R expression in T cells is induced by activation through the CD3 complex

We next investigated whether IGF-1R display on T cells from patients with GD is durable in culture and can be up-regulated by CD3 activation (Fig. 4⇓). Peripheral blood T cells from those patients and control donors were isolated and placed in culture without or with anti-CD3 Ab (10 μg/ml). As expected, an increased fraction of GD-derived T cells expressed IGF-1R (68%) compared with controls (25%) immediately following isolation. These levels remained constant for 72 h in culture without stimulation. Addition of anti-CD3 Ab expanded IGF-1R+ T cells after 72 h in cultures from both sources. In each experiment (n = 5), fewer IGF-1R+ T cells (unstimulated and anti-CD3-stimulated) were found in control donor cultures than those from patients with GD (CD3-stimulated GD T cells, 84 ± 6%; control T cells, 57 ± 5%; p < 0.05). The MFI of CD3+IGF-1R+ T cells derived from GD and control donors was similar.

FIGURE 4.
FIGURE 4.

Durable, high-level IGF-1R display by peripheral blood T cells from patients with GD and controls can be enhanced by CD3 activation in vitro. PBMCs were isolated from a patient and a control donor and cultured for up to 72 h without or with immobilized anti-CD3. IGF-1R display was assessed using multiparameter flow cytometry. Horizontal line was determined from isotype control. Data are representative of four experiments.

IGF-1 and GD-IgG selectively stimulate proliferation and inhibit apoptosis of GD T cells

IGF-1R activation has been shown previously to enhance normal T cell proliferation and confer resistance to apoptosis (36, 37). We next investigated whether the increased fraction of circulating IGF-1R+ T cells found in GD conveys a functionally different phenotype. PBMCs were stimulated with plate-immobilized anti-CD3 (10 μg/ml) in the absence or presence of IGF-1 (10 nM). IGF-1 significantly enhances T cell proliferation compared with cells receiving anti-CD3 Ab alone for 48 h (Fig. 5⇓A). Moreover, Des 1–3 IGF-1 (10 nM), an analog that binds and selectively activates IGF-1R (5), also enhances the mitogenic effects of anti-CD3 Ab. In contrast, PBMCs from control donors failed to respond to IGF-1 or Des 1–3 IGF-1, as measured by BrdU incorporation (p < 0.02). As expected, IGF-1 failed to influence cell proliferation in the absence of anti-CD3 Ab (data not shown). GD-IgG also binds IGF-1R and initiates signaling in fibroblasts from donors with GD (38). In this study, it promotes proliferation of GD-derived T cells but fails to influence control cells (Fig. 5⇓B). As data in that figure demonstrate, purified T cells (>97%) stimulated with either PHA (Fig. 5⇓C) or immobilized TCR (data not shown) responded to IGF-1 similarly.

FIGURE 5.
FIGURE 5.

IGF-1 and GD-IgG potentiate proliferation and IGF-1 promotes resistance to apoptosis in T cells from a patient with GD. A, PBMCs were isolated and cultured with immobilized anti-TCR Ab without or with IGF-1 (10 nM) or Des 1–3 IGF-1 (10 nM). Cells were pulse-labeled with BrdU and assayed after 48 h, as described in Materials and Methods (*, p < 0.02 vs control). B, Increased proliferation of GD T cells following stimulation with GD-IgG in vitro. BrdU incorporation after incubation without or with GD-IgG or control-IgG (*, p < 0.05 vs control IgG). C, IGF-1 potentiates PHA-induced proliferation of GD T cells. T cells (>97% pure) from a control donor or a patient with GD were stimulated with PHA (2 μg/ml) without or with IGF-1 (10 nM). BrdU incorporation was measured after 48 h (*, p < 0.05 vs control). D, IGF-1 inhibits apoptosis in T cells from patients with GD, as assessed using flow cytometry for detection of early apoptotic cells that express annexin-v but not 7-AAD. E, IGF-1 inhibits apoptosis of T cells from patients with GD (solid) but not those from control donors (cross-hatched) when stimulated by anti-CD3 (*, p < 0.05) and anti-Fas (*, p < 0.05). Data are expressed as a mean ± SE of the fraction of apoptotic cells not receiving IGF-1 and are representative of five separate experiments.

IGF-1 inhibits T cell apoptosis provoked by either anti-Fas or anti-CD3 Abs (Fig. 5⇑, D and E). Apoptosis was assessed by annexin V cell surface exposure and exclusion of the vital dye, 7-AAD. The fraction of early apoptotic T cells following addition of either Ab was similar in cultures from GD and control donors (17.2% and 16.2%, respectively; Fig. 5⇑D). Addition of IGF-1 reduced the T cell apoptosis to 6.8% in cultures from patients compared with 15.5% in controls. Similarly, addition of IGF-1 significantly inhibited Fas-induced apoptosis in T cells from GD patients but not in controls (Fig. 5⇑E). Thus, it would appear that IGF-1 exerts both mitogenic and antiapoptotic actions in peripheral circulating T cells derived from patients with GD.

Discussion

We have previously found an increased fraction of IGF-1R+ fibroblasts cultured from the orbit, skin, and thyroid of patients with GD (32). Treatment of these IGF-1R+ fibroblasts with either IGF-1 or GD-IgG results in the synthesis of two powerful T cell chemoattractants, IL-16 and RANTES (38, 39) as well as the generation of hyaluronan (33). In this study, we report a similarly expanded population of IGF-1R+ T cells. This increase is evident both in peripheral blood and orbital connective tissue infiltrates. As with their fibroblasts, overrepresentation of IGF-1R+ T cells in patients with GD conveys functional consequences because IGF-1 selectively increased cell proliferation while attenuating Fas-mediated apoptosis. In aggregate, our results support a potential role for IGF-1R in the pathogenesis of GD as a determinant of immune responses through fibroblast and lymphocyte activation and expansion.

Up-regulation of the IGF-1/IGF-1R pathway has been implicated previously in the pathogenesis of human disease. With regard to malignancies, neoplastic transformation can result in elevated IGF-1R levels, the consequence of altered tumor suppressor gene function and constitutive Akt activity (40). In hemopoietic cell lines, p53 mutations are linked to increased IGF-1R gene expression and exaggerated proliferative responses to IGF-1 (41). Expression of wild type p53 diminishes IGF-1R levels and the magnitude of these cellular responses (42). Constitutively active Akt and Src-activated Akt up-regulate IGF-1R levels in pancreatic cancer cells, promoting cell survival (40). Akt activity also mediates the antiapoptotic effects of IGF-1 in human T cells (43).

With regard to autoimmune diseases, IGF-1R+ cells pervade affected intestinal tissues in Crohn’s disease, focused primarily in the mucosa and submucosa (44). Expression of IGF-1R by submucosal fibroblasts and adipocytes is confined to areas of fibrosis and inflammation. This is also true in chronic inflammatory lung diseases where increased numbers of IGF-1R+ and IGF-1+ fibroblasts and infiltrating macrophages are found in pulmonary fibrosis and systemic sclerosis (45, 46). IGF-1 generated by infiltrating cells promotes lung fibroblast proliferation (47). A similar pattern has been described in GD where IGF-1 and IGF-1R are also expressed in thyroid tissue (48, 49). Analogous to orbital fibroblasts, cultured thyrocytes express IGF-1R whereas IGF-1 and GD-IgG induce IL-16 and RANTES in a time-dependent manner (50). These findings suggest that the actions of IGF-1 and GD-IgG may participate directly in recruitment of inflammatory cells to targeted tissues in GD.

Our current studies disclose nearly uniform IGF-1R display by early myeloid cells in control bone marrow and in that from a patient with GD (Fig. 2⇑). This expression is retained by peripheral erythrocytes, monocytes, and NK cells (7). IGF-1R is also strongly expressed on T cells within the marrow but not by the majority of control mature T cells (Fig. 2⇑) (8, 9). A greater proportion of circulating mature lymphocytes retain the IGF-1R+ phenotype in GD, implying that the gradient between receptor display before and following release from the marrow is diminished in the disease. The mechanism underlying this phenomenon is currently undefined.

IGF-1 plays a critical role in early development and expansion of hemopoietic cells in bone marrow and thymus (12). It induces granulopoiesis and the formation of granulocyte-monocyte colonies in bone marrow cultures (51). IGF-1 also enhances the proliferation of promyelocytic, erythroid, and lymphocytic cell lines and bone marrow blast colony formation in vivo in patients with myeloid leukemia (52, 53). Expansion of CD4+ T cells and splenic B cells occurs in mice following systemic administration of IGF-1 (11, 12). IGF-1R can be detected in thymic tissue, and administration of IGF-1 in vivo promotes T cell and stromal repopulation of atrophic thymus in diabetic rats (54) and following cyclosporine A treatment (55). These effects are mediated through coordinate promotion of proliferation and inhibition of apoptosis. IGF-1R appears to mediate proliferative effects of IGF-1 in T and B lymphoblasts, several T cell lines including Jurkat cells, and T cells infected with human T cell leukemia virus-1 or -2 (56, 57, 58). Jurkat cells and those infected with human T cell leukemia virus-1 constitutively express IGF-1R and respond to IGF-1. These effects are inhibited by receptor-blocking mAbs. Although IGF-1 does not typically enhance the proliferation of mature inactive T cells, activation through TCR induces IGF-1R expression in vitro and facilitates IGF-1-dependent proliferation (59). In agreement with previous studies, our results demonstrate that anti-CD3 Ab, PHA, and anti-TCR Ab expand IGF-1R+ cells (Fig. 4⇑) (60, 61, 62). T cells from patients with GD stably express IGF-1R and faithfully maintain their exaggerated proliferative response to IGF-1 in culture (Fig. 5⇑). In addition, disease-derived T cells treated with IGF-1 exhibit greater resistance to apoptosis. Thus, increased IGF-1R display by T cells in GD may underlie lymphocyte expansion through at least two distinct mechanisms.

IGF-1 has been shown previously to inhibit the apoptosis of immature CD45RA+ and mature CD45RO+ T cells by attenuating PHA-induced Fas expression and by inhibiting apoptosis resulting from growth factor withdrawal (36). This latter action appears independent of Fas/Fas ligand (36). IGF-1 also promotes the transition of cord blood CD45RA+ T cells to a CD45RO+ phenotype by increasing the RA+ to RO+ conversion following Ag stimulation (36). This progression results from the survival of Ag-specific (CD4+) T cells and Ag-driven expansion of effector (CD8+) T cells. Overrepresentation in peripheral blood of CD4+CD45RO+ and CD8+CD45RO+ T cells in GD has been demonstrated previously (23, 63). In this study, we report that most naive CD4+ and CD8+CD45RA+ T cells, both from patients with GD and from control donors, display IGF-1R. In agreement with previous reports, few IGF-1R+CD45RO+ T cells were found in control donors (9). In contrast, the vast majority of CD45RO+ T cells from patients with GD, particularly those with the CD8+ phenotype, display IGF-1R. IGF-1 and GD-IgG may directly promote the survival or expansion of Ag-specific T cells in GD through their interaction with IGF-1R. These observations should thus be viewed in the context of exaggerated IGF-1R expression by fibroblasts in this disease and the functional consequences of the interactions with anti-IGF-1R-activating GD-IgG by those cells (32). Generation of chemoattractants IL-16 and RANTES could constitute the basis for T cell infiltration in affected tissues (38). Our new findings imply that the disproportionate number of IGF-1R+CD45RO+ T cells found in GD may underlie memory T cell expansion in this disease, and thus further implicate IGF-1R in its pathogenesis.

Acknowledgments

We are grateful to Debbie Hanaya for her expert assistance in the preparation of this manuscript and to Dr. Robert Goldberg for patient recruitment. Furthermore, we acknowledge the invaluable assistance of the Harbor-UCLA General Clinical Research Center staff in their efforts to facilitate these studies.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was funded in part by Grants EY016339 (to R.S.D.), RR017304 (to A.G.G.), EY008976, EY011708, and DK063121 (to T.J.S.), and RR00425 from the National Institutes of Health. We gratefully acknowledge generous support from Steve and Kathleen Flynn and the Bell Charitable Foundation.

  • ↵2 Address correspondence and reprint requests to Dr. Terry J. Smith, Division of Molecular Medicine, Harbor-University of California Los Angeles Medical Center, Building C-2, 1124 West Carson Street, Torrance, CA 90502. E-mail address: tjsmith{at}ucla.edu

  • ↵3 Abbreviations used in this paper: IGF-1R, insulin-like growth factor receptor-1; GD, Graves’ disease; TAO, thyroid-associated ophthalmopathy; MFI, mean fluorescent intensity; 7-AAD, 7-aminoactinomycin D.

  • Received October 23, 2006.
  • Accepted December 8, 2006.

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