Briefly, fibrocytes were incubated with teprotumumab at graded concentrations (0, 5, 50, and 500 g/mL) at 4C for 2 hours
Briefly, fibrocytes were incubated with teprotumumab at graded concentrations (0, 5, 50, and 500 g/mL) at 4C for 2 hours. (S473) levels were analyzed by Western blot. TSHR and IGF-1R display was assessed by circulation cytometry. Results: Fibrocyte display of IGF-1R and TSHR was reduced with teprotumumab, as were IGF-1- and TSH-dependent phosphorylated Akt levels. TSH induction of IL-6 and IL-8 mRNA and protein was also reduced by the monoclonal antibody. Conclusions: Teprotumumab attenuates the actions of both IGF-1 and TSH in fibrocytes. Specifically, it blocks the induction of proinflammatory cytokines by TSH. These results provide, at least in part, the molecular rationale for interrogating the therapeutic efficacy of this antibody in TAO. Thyroid-associated ophthalmopathy (TAO) is the inflammatory orbital manifestation of Graves’ disease (GD) (1). The molecular mechanisms underlying TAO remain obscure. At the heart of GD is the generation of activating antibodies directed against the TSH receptor (TSHR) (2). In addition, antibodies that activate the IGF-1 receptor (IGF-1R) have also been detected (3,C6). TSHR and IGF-1R have been shown to form a physical and functional complex in orbital fibroblasts and thyroid epithelial cells (7). Moreover, blocking IGF-1R appears to attenuate TSH-dependent signaling (7). These findings have been confirmed recently by another laboratory group (8). They suggest that blocking IGF-1R using the monoclonal antibody (mAb) antagonist LGD-6972 might reduce both TSHR- and IGF-1-dependent signaling and therefore interrupt pathological activities initiated through both receptors. Monoclonal antibodies directed LGD-6972 against IGF-1R have been developed and assessed as a therapeutic strategy for several types of solid tumors and lymphomas (9,C11). Teprotumumab (RV 001, R1507) is usually a fully human mAb that binds to the ligand binding extracellular -subunit domain name of IGF-1R (12). This molecule is currently under phase 2 clinical investigation in patients with moderate to severe, active TAO. Its potential for attenuating the actions of TSH has not been investigated previously. In TAO, the orbit appears to be infiltrated by fibrocytes, bone marrow-derived progenitor cells of the monocyte lineage (13). These cells express leukocyte and fibroblast surface antigens and amazingly high levels of functional TSHR (14, 15). Fibrocytes participate in wound healing and tissue remodeling and appear to be involved Rabbit Polyclonal to DPYSL4 LGD-6972 in the pathogenesis of pulmonary fibrosis and rheumatic arthritis (16, 17). Fibrocytes activated by TSH and IgGs from patients with GD express several proinflammatory cytokines, including IL-1, IL-1 receptor antagonist, IL-6, IL-8, and TNF (13, 15, 18). Besides the orbit, fibrocytes also infiltrate the thyroid gland in GD, providing a potential mechanistic link between affected tissues (19). In this study, we statement for the first time that teprotumumab decreases TSHR and IGF-1R display by fibrocytes and attenuates TSH-dependent IL-6 and IL-8 expression and Akt phosphorylation. These findings further help elucidate the interplay between TSHR and IGF-1R that may be crucial to the pathogenesis of TAO and support the therapeutic rationale for blocking IGF-1R in this disease. Patients and Methods Patient samples Patients with GD (n = 6) were recruited from the patient population of the Kellogg Vision Center at the University or college of Michigan. Informed consent was obtained in compliance with policies of the Institutional Review Table of the University or college of Michigan Health System. Research methods followed the tenets of the Declaration of Helsinki. Fibrocyte cultures Fibrocytes were generated from LGD-6972 peripheral blood mononuclear cells isolated from leukocyte reduction filters provided by the American Red Cross or from blood of patients with GD and were cultured as explained previously (13, 20). Briefly, peripheral blood mononuclear cells were isolated by centrifugation over Ficoll-PaquePlus (catalog no. 17C1440-03; GE Healthcare Bio-Science). After washing, cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin combination (catalog no. LGD-6972 15140C122; Life Technologies). Each culture well of a six-well plate was inoculated with 107 cells and incubated at 37C in a 5% CO2 atmosphere. After 7 days, cultures were rinsed, and nonadherent cells were removed by gentle aspiration. Medium was changed every 3 days. After 10 to 14 days of cultivation, culture purity was verified to be >90% fibrocytes.