Histamine induces NF-κB controlled cytokine secretion by orbital fibroblasts via histamine receptor type-1.

Mast cells and their products are likely to be involved in regulating orbital fibroblast activity in Graves' Ophthalmopathy (GO). Histamine is abundantly present in granules of mast cells and is released upon mast cell activation. However, the effect of histamine on orbital fibroblasts has not been examined so far. Orbital tissues from GO patients and controls were analyzed for the presence of mast cells using toluidine blue staining and immunohistochemical detection of CD117 (stem cell factor receptor). Orbital fibroblasts were cultured from GO patients and healthy controls, stimulated with histamine and cytokines (IL-6, IL-8, CCL2, CCL5, CCL7, CXCL10 and CXCL11) were measured in culture supernatants. Also hyaluronan levels were measured in culture supernatants and hyaluronan synthase (HAS) and hyaluronidase (HYAL) gene expression levels were determined. In addition, histamine receptor subtype gene expression levels were examined as well as the effect of the histamine receptor-1 (HRH1) antagonist loratadine and NF-κB inhibitor SC-514 on histamine-induced cytokine production. Mast cell numbers were increased in GO orbital tissues. Histamine stimulated the production of IL-6, IL-8 and CCL2 by orbital fibroblasts, while it had no effect on the production of CCL5, CCL7, CXCL10, CXCL11 and hyaluronan. Orbital fibroblasts expressed HRH1 and loratadine and SC-514 both blocked histamine-induced IL-6, IL-8 and CCL2 production by orbital fibroblasts. In conclusion, this study demonstrates that histamine can induce the production of NF-κB controlled-cytokines by orbital fibroblasts, which supports a role for mast cells in GO.

Current perspectives on the role of orbital fibroblasts in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease (GD; Graves' hyperthyroidism) characterized by orbital tissue inflammation, expansion, remodeling and fibrosis. Although the initiating trigger of GO is still indistinct, excessive orbital fibroblast activity is at the heart of its pathogenesis. Orbital fibroblasts are activated by cellular interactions with immune cells and the soluble factors they secrete. Orbital fibroblasts, especially from GO patients, express the thyrotropin receptor (TSH-receptor; TSHR), and activation of the orbital fibroblast population by stimulatory autoantibodies directed against the TSHR may provide an important link between GD and GO. Furthermore, stimulatory autoantibodies directed against the insulin-like growth factor-1 receptor have been proposed to contribute to orbital fibroblast activation in GO. Activated orbital fibroblasts produce inflammatory mediators thereby contributing to the orbital inflammatory process in GO. Moreover, orbital fibroblasts exhibit robust proliferative activity and extracellular matrix (especially hyaluronan) synthesizing capacity and can differentiate into adipocytes and myofibroblasts with disease progression, thereby contributing to tissue expansion/remodeling and fibrosis in GO. Orbital fibroblasts, especially those from GO patients, exhibit a hyper-responsive phenotype when compared to fibroblasts from other anatomical regions, which may further contribute to GO pathogenesis. Fibrocytes have been identified as additional source of orbital fibroblasts in GO, where they may contribute to orbital tissue inflammation, adipogenesis and remodeling/fibrosis. This review addresses our current view on the role that orbital fibroblasts fulfill in GO pathogenesis and both established as well as less established not fully crystallized concepts that need future studies will be discussed.

Platelet-derived growth factor: a key factor in the pathogenesis of graves' ophthalmopathy and potential target for treatment.

Activation of orbital fibroblasts resulting in excessive proliferation, cytokine and hyaluronan production and differentiation into adipocytes, is a main determinant of orbital tissue inflammation and tissue expansion in Graves' ophthalmopathy (GO). During the last years we have shown that the platelet-derived growth factor (PDGF) isoforms PDGF-AA, PDGF-AB and PDGF-BB are increased in orbital tissue from GO patients with active and inactive disease. These PDGF isoforms exhibit the capacity to stimulate proliferation, hyaluronan and cytokine/chemokine production by orbital fibroblasts. Moreover, PDGF-AB and PDGF-BB increase thyroid stimulating hormone receptor (TSHR) expression by orbital fibroblasts, which enhances the orbital fibroblast activating capacity of the THSR stimulatory autoantibodies present in Graves' disease (GD) patients. Of these PDGF isoforms PDGF-BB exhibits the strongest orbital fibroblast activating effects, which is likely related to its ability to bind both the PDGF-receptor (PDGF-R)α and PDGF-Rß chains. Thus the PDGF-system fulfills important roles in orbital fibroblast activation in both active and inactive GO, which supports a therapeutic rationale for blocking PDGF signaling in GO. Tyrosine kinase inhibitors (TKIs) may be candidates to target PDGF signaling. Of several TKIs tested dasatinib exhibited the highest potency to block PDGF-R signaling in orbital fibroblasts and may represent a promising compound for the treatment of GO as it was effective at low dosage and is associated with less side effects compared to imatinib mesylate and nilotinib. In this review the contribution of PDGF to the pathophysiology of GO as well as therapeutic approaches to target this PDGF-system will be addressed.

Whole orbital tissue culture identifies imatinib mesylate and adalimumab as potential therapeutics for Graves' ophthalmopathy.

BACKGROUND AND AIMS: Biologicals and small inhibitory molecules are used to treat inflammatory diseases, but their efficacy varies upon clinical application. Using a whole orbital tissue culture system, we tested the potential efficacy of imatinib mesylate (a tyrosine kinase inhibitor that blocks platelet-derived growth factor (PDGF)-receptor, c-Abl and c-Kit activity) and adalimumab (an anti-TNF-α antibody) for the treatment of Graves' ophthalmopathy (GO). METHODS: Orbital fat tissue from GO patients (n=10) was cultured with or without imatinib mesylate or adalimumab. PDGF-B and tumour necrosis factor (TNF)-α mRNA expression levels were determined in the primary orbital tissue, and interleukin (IL)-6 and hyaluronan were measured in tissue-culture supernatants. RESULTS: Imatinib mesylate significantly (p=0.005) reduced IL-6 and hyaluronan production. The inhibition of hyaluronan production correlated positively and significantly (p<0.05) with the PDGF-B mRNA level in the primary tissue. Adalimumab also significantly (p=0.005) reduced IL-6 production. The amount of IL-6 inhibition correlated positively with the TNF-α mRNA level in the primary tissue, but this was not significant. CONCLUSIONS: Imatinib mesylate can be expected to reduce inflammation and tissue remodelling in GO, while adalimumab can be mainly expected to reduce inflammation. This in vitro tissue-culture model may, in future, prove valuable to test novel therapeutics for their presumed effect in GO as well as in other inflammatory diseases.

The orbital fibroblast: a key player and target for therapy in graves' ophthalmopathy.

Orbital fibroblasts play a key role in the pathogenesis of Graves' ophthalmopathy (GO). We discuss some major aspects by which orbital fibroblasts contribute to GO and the important role of PDGF-BB herein. Finally, we propose the orbital fibroblast, and especially the PDGF system acting on orbital fibroblasts, as an important therapeutic target in GO.

Platelet-derived growth factor-BB: a stimulus for cytokine production by orbital fibroblasts in Graves' ophthalmopathy.

PURPOSE: Graves' ophthalmopathy (GO) is characterized by the infiltration of immune cells into the orbit, a process in which cytokines play a central role. Orbital fibroblasts are potent producers of cytokines on different stimuli. Recently, the authors showed increased expression of the PDGF-B chain in GO orbital tissue. The dimeric PDGF-BB molecule has been described to activate the NF-kappaB pathway, which is well recognized for its role in regulating cytokine production. This study was conducted to determine the role of PDGF-BB in the production of proinflammatory cytokines by orbital fibroblasts in GO. METHODS: Orbital, lung, and skin fibroblasts were stimulated with PDGF-BB, and cytokine (IL-1beta, IL-6, IL-8, IL-16, CCL2, CCL5, CCL7, TNF-alpha) production was measured by ELISA. Involvement of NF-kappaB activation through PDGF signaling was investigated by electrophoretic mobility shift assay, specific NF-kappaB inhibitors, and the PDGF-receptor kinase inhibitor imatinib mesylate. RESULTS: IL-6, IL-8, CCL2, CCL5, and CCL7 production by orbital fibroblasts was increased by PDGF-BB stimulation, whereas IL-16, IL-1beta, and TNF-alpha production was not affected. PDGF-BB induced NF-kappaB activity in orbital fibroblasts, and both NF-kappaB inhibitors and imatinib mesylate reduced PDGF-BB-induced cytokine production. Similar, but less vigorous, effects of PDGF-BB on cytokine production were observed in lung and skin fibroblasts. CONCLUSIONS: PDGF-BB is a potent inducer of proinflammatory cytokines via the NF-kappaB pathway in orbital fibroblasts, whereas cytokine production by fibroblasts from other anatomic locations showed a moderate response. These data suggest a possible role for PDGF-BB in regulating orbital inflammation in GO and identify the PDGF signaling cascade as a therapeutic target in GO.

Imatinib mesylate and AMN107 inhibit PDGF-signaling in orbital fibroblasts: a potential treatment for Graves' ophthalmopathy.

PURPOSE: Excessive orbital fibroblast proliferation and hyaluronan production are characteristic of Graves' ophthalmopathy (GO) and are driven by local mediators. Imatinib mesylate and AMN107 are tyrosine kinase inhibitors that inhibit fibroblast proliferation and collagen production in lungs and skin. This study was conducted to determine whether imatinib mesylate and AMN107 inhibit orbital fibroblast proliferation and hyaluronan production induced by PDGF-BB and TGF-beta(1) and whether expression of the genes PDGF-B and TGF-B(1) (growth factors suggested to play a role in GO) are increased in GO orbital tissues. METHODS: PDGF-B and TGF-B(1) mRNA levels were determined in orbital tissues of 13 patients with GO and 5 control patients. Orbital fibroblasts were cultured from eight patients with GO and three control patients and the effect of imatinib mesylate and AMN107 on PDGF-BB and TGF-beta(1)-induced orbital fibroblast proliferation, signaling cascades, hyaluronan synthase (HAS) gene expression and hyaluronan production were determined. RESULTS: PDGF-B and TGF-B(1) mRNA levels were significantly increased in GO orbital tissues. Imatinib mesylate and AMN107 inhibited PDGF-BB-induced orbital fibroblast proliferation, HAS induction and hyaluronan production by blocking PDGF-receptor phosphorylation. TGF-beta(1) induced HAS expression and hyaluronan production. This induction was not inhibited by imatinib mesylate or AMN107, due to the inability of TGF-beta(1) to activate c-Abl kinase activity in orbital fibroblasts. CONCLUSIONS: Imatinib mesylate and AMN107 inhibit orbital fibroblast proliferation and hyaluronan production induced by PDGF-BB; a factor highly expressed in orbital tissue from patients with GO. The drugs, however, had no effect on TGF-beta(1)-induced HAS expression and hyaluronan production. Nevertheless, imatinib mesylate and AMN107 should be considered as treatment candidates for GO.