Michael J. Coenen

A Small Molecule Antagonist Inhibits Thyrotropin Receptor Antibody-Induced Orbital Fibroblast Functions Involved in the Pathogenesis of Graves Ophthalmopathy

CONTEXT Graves ophthalmopathy (GO) is an autoimmune disorder characterized by increased adipogenesis and hyaluronan (HA) production by orbital fibroblasts. Circulating autoantibodies (thyroid-stimulating antibodies [TSAbs]) directed at the thyrotropin receptor (TSHR) on these cells stimulate or augment these cellular processes. A recently developed drug-like small molecule inverse agonist of TSHR, NCGC00229600, termed 1, binds to TSHR and blocks basal and stimulated signal transduction. OBJECTIVE The purpose of this article was to determine whether 1 might inhibit HA production and relevant signaling pathways in orbital fibroblasts cultured in the presence of monoclonal TSAbs or bovine TSH (bTSH). DESIGN Primary cultures of undifferentiated GO orbital fibroblasts (n = 13) were untreated or treated with a TSAb (M22 or MS-1) or bTSH in serum-free medium, with or without 1 or a TSHR neutral antagonist, NCGC00242595, termed 2, which does not inhibit basal signaling but does inhibit stimulated signaling. MAIN OUTCOME MEASURES cAMP production, Akt phosphorylation (Ser473pAkt in media and immunoblotting for pAkt/total Akt), and HA production were analyzed. RESULTS Compound 1 inhibited basal cAMP, pAkt, and HA production and that stimulated by M22 in undifferentiated orbital fibroblasts. Inhibition of HA production was dose-dependent, with a half-maximal inhibitory dose of 830 nM. This compound also inhibited MS-1- and bTSH-stimulated cAMP, pAkt, and HA production. Compound 2 did not inhibit basal HA production but did inhibit M22-stimulated HA production. CONCLUSIONS Because cAMP, pAkt, and HA production are fibroblast functions that are activated via TSHR signaling and are important in the pathogenesis of GO, small molecule TSHR antagonists may prove to be effective in the treatment or prevention of the disease in the future.

Short‐term treatment with interferon‐α/β promotes remyelination, whereas long‐term treatment aggravates demyelination in a murine model of multiple sclerosis

The mechanisms by which type I interferons (IFN) reduce the rate and severity of exacerbations in multiple sclerosis are unknown. We utilized a model of multiple sclerosis to determine the extent of demyelination and remyelination in Theilers murine encephalomyelitis virus (TMEV)‐infected SJL/J mice treated with mouse IFN‐α/β for a short (5 weeks) or a long (16 weeks) period. All mice were chronically infected with TMEV to simulate the clinical situation in multiple sclerosis. Short‐term IFN‐α/β treatment increased the percent of remyelinated spinal cord white matter by threefold when compared with phosphate‐buffered saline (PBS) treatment (P < 0.02), but it did not affect the extent of demyelination. In contrast, long‐term IFN‐α/β treatment increased the extent of demyelination by twofold (P < 0.03). Long‐term treatment increased the absolute area of remyelination, but the percent remyelination as a function of area of demyelination was not changed because of increased demyelination. An immunomodulatory mechanism may have contributed to the effect of IFN‐α/β on white matter pathology because treated mice had higher anti‐TMEV IgGs in serum and demonstrated decreased numbers of B and T lymphocytes infiltrating the central nervous system (CNS). There was no correlation between the level of anti‐ IFN‐α/β antibodies and the extent of demyelination or remyelination. These results indicate that the length of type I IFN treatment may have paradoxical effects on demyelination and remyelination. J. Neurosci. Res. 59:661–670, 2000

Expression of the human histocompatibility leukocyte antigen DR3 transgene reduces the severity of demyelination in a murine model of multiple sclerosis.

The role of various MHC genes in determining the progression of multiple sclerosis (MS) remains controversial. The HLA-DR3 gene has been associated with benign relapsing MS in some genetic epidemiologic studies, but with disease progression in others. We induced demyelination in highly susceptible B10.M and B10.Q mice expressing the DR3 (HLA-DRB1*0301) transgene to determine directly the effects of a human transgene by infecting them with Theilers murine encephalomyelitis virus (TMEV). DR3+ mice experienced a dramatic reduction in the extent and severity of demyelination compared with DR3- littermate controls, whereas anti-TMEV antibody titers, delayed-type hypersensitivity responses, and levels of infectious virus, virus antigen, and virus RNA were similar in both groups. To address a possible mechanism of how the human transgene is reducing virus-induced demyelination, we analyzed cytokine expression in the lesions and also determined whether B10.M mice can respond to peptides derived from the DR3 molecule. Intense staining for IFN-gamma and IL-4, T helper (TH) 1 and TH2 cytokines, respectively, was found in the lesions of TMEV-infected DR3- mice but not in the DR3+ transgenic mice at day 21 after infection. DR3 peptides elicited strong proliferative responses in B10.M mice but not in B10.M (DR3+) mice. These experiments are the first to demonstrate that a human class II DR gene can alter the severity of demyelination in an animal model of MS without influencing viral load. These experiments are consistent with a mechanism by which DR3 reduces demyelination by altering the cytokine expression in the lesions, possibly by deleting T cells involved in virus-induced pathology.

Eosinophil ribonucleases and their cutaneous lesion-forming activity.

Eosinophil granule proteins are deposited in cutaneous lesions in many human diseases, but how these proteins contribute to pathophysiology is obscure. We injected eosinophil cationic protein (ECP or RNase 3), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 (MBP1) intradermally into guinea pig and rabbit skin. ECP and EDN each induced distinct skin lesions at ≥2.5 μM that began at 2 days, peaking at ∼7 days and persisting up to 6 wk. These lesions were ulcerated (ECP) or crusted (EDN) with marked cellular infiltration. EPO and MBP1 (10 μM) each produced perceptible induration and erythema with moderate cellular infiltration resolving within 2 wk. ECP and EDN localized to dermal cells within 2 days, whereas EPO and MBP1 remained extracellular. Overall, cellular localization and RNase activity of ECP and EDN were critical for lesion formation; differential glycosylation, net cationic charge, or RNase activity alone did not account for lesion formation. Ulcerated lesions from patients with the hypereosinophilic syndrome showed ECP and EDN deposition comparable to that in guinea pig skin. In conclusion, ECP and EDN disrupt skin integrity and cause inflammation. Their presence in ulcerative skin lesions may explain certain findings in human eosinophil-associated diseases.