Targeting ADAM10 in Cancer and Autoimmunity.

Generating inhibitors for A Disintegrin And Metalloproteinase 10 (ADAM10), a zinc-dependent protease, was heavily invested in by the pharmaceutical industry starting over 20 years ago. There has been much enthusiasm in basic research for these inhibitors, with a multitude of studies generating significant data, yet the clinical trials have not replicated the same results. ADAM10 is ubiquitously expressed and cleaves many important substrates such as Notch, PD-L1, EGFR/HER ligands, ICOS-L, TACI, and the "stress related molecules" MIC-A, MIC-B and ULBPs. This review goes through the most recent pre-clinical data with inhibitors as well as clinical data supporting the use of ADAM10 inhibitor use in cancer and autoimmunity. It additionally addresses how ADAM10 inhibitor therapy can be improved and if inhibitor therapy can be paired with other drug treatments to maximize effectiveness in various disease states. Finally, it examines the ADAM10 substrates that are important to each disease state and if any of these substrates or ADAM10 itself is a potential biomarker for disease.

Effect of QiShenYiQi pill on myocardial collagen metabolism in experimental autoimmune myocarditis rats.

OBJECTIVE: To observe the effect of QiShenYiQi pill (QSYQ) on myocardial collagen metabolism in experimental autoimmune myocarditis rats, and to explore its mechanism of action. METHODS: Lewis rats underwent the injection of myocardial myosin mixed with freund's complete adjuvant were randomized into three groups: model, valsartan and QSYQ groups. And we treated rats which were injected phosphate buffered saline (PBS) mixed with freund's complete adjuvant as control group. Rats were intervened and euthanized at 4 and 8 weeks. We use alkaline hydrolysis to detect the content of myocardial hydroxyproline (HYP), and ELISA to detect the level of serum procollagen type I carboxyterminal peptide (PICP), procollagen type III amino-terminal peptide (PIIINP), and collagen C telopeptide type I (CTX-I). Myocardial MMP-1 and TIMP-1 protein expression was detected by immunohistochemistry, and myocardial MMP-1 and TIMP-1 mRNA expression was detected by real-time qPCR. RESULTS: QSYQ reduced the content of myocardial HYP, and this reduction was greater over time. QSYQ also reduced the serum concentration of PICP, PIIINP, CTX-I and the PICP/PIIINP ratio, which further reduced over time, whereas its effect on lowering PICP was significantly greater than that of valsartan at 4 and 8 weeks, and lowering CTX-I was significantly greater than that of valsartan at 8 weeks. In addition, after 4 weeks, QSYQ enhanced the protein and mRNA expression of MMP-1 and TIMP-1, and its effect on highering TIMP-1 was significantly greater than that of valsartan, whereas there was no significant difference in the expression of myocardial MMP-1 or TIMP-1 at 8 weeks. QSYQ reduced the ratio of MMP-1/TIMP-1, which further reduced over time, and the effect of QYSQ was significantly greater than that of valsartan after 4 weeks. CONCLUSION: This study provides evidence that QSYQ can reduce the rate of myocardial collagen synthesis and degradation. It also effectively improved the degree of myocardial fibrosis in experimental autoimmune myocarditis rats and it had a tendency to have a greater effect with longer treatment duration, which is related to the mechanism of regulation of MMP-1 and TIMP-1 expression in the myocardial rat.

Phosphoinositide 3-kinase inhibitor LY294002 ameliorates the severity of myosin-induced myocarditis in mice.

BACKGROUND: Myocarditis, characterized by myocyte necrosis, fibrosis, and degeneration with mononuclear cell infiltration, always causes heart failure in patients. Phosphoinositide 3-kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in cardiac diseases. Although previous research has suggested that PI3K was involved in cardiac diseases such as myocardial infarction, it is still unclear whether the inhibition of PI3K is essential for the treatment of myosin-induced myocarditis. The aim of this study was to explore whether pharmacological blockade of PI3K is able to protect mice against experimental autoimmune myocarditis (EAM). MATERIALS AND METHODS: We used the cardiac myosin-induced murine EAM model to investigate the therapeutic effect of PI3K inhibitor LY294002 on autoimmune myocarditis in mice. RESULTS: LY294002 significantly alleviated EAM injury in mice, as indicated by the reduction of cardiac necrosis, inflammatory infiltrates, and CD3(+) T cells. LY294002 also decreased the expression of p-Akt upon cardiac myosin treatment in the cardiac tissue of the mice. In the present study, LY294002 resulted in a moderate reduction in absolute CD4(+) cell numbers and a significant decrease in the absolute numbers of CD8(+) cells. Consequently, LY294002 increased the CD4(+)/CD8(+) ratio compared with peptide treatment alone. CONCLUSION: This report provides evidence that PI3K inhibitor LY294002 has potent effects against cardiac injury caused by EAM, suggesting that it has therapeutic value for the treatment of myocarditis.

Up regulation of nitric oxide synthase-nitric oxide system in the testis of rats undergoing autoimmune orchitis.

BACKGROUND: Male reproductive tract infection and inflammation are important aetiological factors of infertility. Experimental Autoimmune Orchitis (EAO) is a model of chronic inflammation useful to study mechanisms of inflammatory reactions leading to testicular impairment. EAO is characterised by interstitial cell infiltrate of lymphomonocytes, producers of pro-inflammatory cytokines involved in germ cell apoptosis. Nitric oxide (NO), a free radical promoting immune cell activation and apoptosis, is synthesised by conversion of l-arginine to l-citrulline catalysed by NO synthase (NOS). The NOS isoforms are: constitutively endothelial (e) and neuronal (n) NOS and inducible (i) NOS. OBJECTIVES: Although the NO-NOS system was found to be up-regulated by pro-inflammatory mediators in immune and non immune testicular cells, data on its regulation in chronic inflammatory states is lacking. METHODS AND RESULTS: EAO was induced in rats by active immunisation with spermatic antigens and adjuvants; control (C) rats were injected with adjuvants. Untreated normal (N) rats were also studied. We demonstrated that iNOS, eNOS and nNOS was mainly expressed by interstitial cells in N and C rats and that in EAO NOS was up-regulated and also expressed by tubular cells. Constitutive and inducible NOS content (Western blot) as well as NO production and activity increased in the testis of rats with EAO. iNOS content and activity were selectively up-regulated in the testis of rats with orchitis. Flow cytometric analysis of NOS isoforms in testicular macrophages (M) showed that the percentage of ED1(+)ED2(-) and ED1(+)ED2(+) M subsets, expressing constitutive and iNOS isoforms was significantly higher in EAO, but no change in the percentage of ED1(-)ED2(+) resident M was observed compared to C rats. M from EAO rats also released more NO than C and N rats. CONCLUSIONS: In testis of rats with EAO, NO-NOS system was up-regulated and both testicular M and cells from seminiferous tubules contributed to NO increase. NO over production in orchitis was generated mainly by increased iNOS content and activity.

Tyrosine kinase inhibitors as potential drugs for B-cell lymphoid malignancies and autoimmune disorders.

INTRODUCTION: In the last few years, several tyrosine kinase inhibitors (TKIs) have been synthesized and become available for preclinical studies and clinical trials. This article summarizes recent achievements in the mechanism of action, pharmacological properties, and clinical activity and toxicity, as well as the emerging role of TKIs in lymphoid malignancies, allergic diseases, and autoimmune disorders. AREAS COVERED: A literature review was conducted of the MEDLINE database PubMed for articles in English. Publications from 2000 through January 2012 were scrutinized. The search terms used were Bruton's tyrosine kinase (Btk) inhibitors, PCI-32765, GDC-0834, LFM-A13, AVL-101, AVL-292, spleen tyrosine kinase (Syk) inhibitors, R343, R406, R112, R788, fostamatinib, BAY-61-3606, C-61, piceatannol, Lyn, imatinib, nilotinib, bafetinib, dasatinib, GDC-0834, PP2, SU6656 in conjunction with lymphoid malignancy, NHL, CLL, autoimmune disease, allergic disease, asthma, and rheumatoid arthritis. Conference proceedings from the previous 5 years of the American Society of Hematology, European Hematology Association, American Society of Clinical Oncology, and ACR/ARHP Annual Scientific Meetings were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles. EXPERT OPINION: The use of TKIs, especially inhibitors of Btk, Syk, and Lyn, is a promising new strategy for targeted treatment of B-cell lymphoid malignancies, autoimmune disorders and allergic diseases. However, definitive data from ongoing and future clinical trials will aid in better defining the status of TKIs in the treatment of these disorders.

Effects of HMG-CoA reductase inhibitor on experimental autoimmune myocarditis.

PURPOSE: Myocarditis is an acute inflammatory disease of the heart and is often a precursor of dilated cardiomyopathy. Experimental autoimmune myocarditis (EAM) has been used as a model for human myocarditis. The purpose of this study was to investigate the therapeutic role of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, rosuvastatin, on the development of EAM. METHODS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with murine cardiac α-myosin heavy chain (MyHc-α(614-629) [Ac-SLKLMATLFSTYASAD-OH]). High-dose (10 mg/kg/day) or low-dose (1 mg/kg/day) rosuvastatin or vehicle was administered orally by gastric gavage to mice with EAM from day 0 to day 21 after immunization. On day 21 after immunization, echocardiography was carried out and the severity of myocarditis was detected by histopathological evaluation. Levels of serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 were measured by ELISA. Histopathology was performed using haematoxylin and eosin. With apoptosis examined by Tunel, the expression of active caspase-3 in myocardium was investigated by immunohistochemistry. RESULTS: Rosuvastatin attenuated the histopathological severity of myocarditis. Cardiac function was improved in the two rosuvastatin-treated groups compared to the non-treated EAM group (LVFS: high-dose rosuvastatin group [group H], 0.38 ± 0.10%; low-dose rosuvastatin group [group L], 0.34 ± 0.06%; non-treated EAM group [group N], 0.29 ± 0.07%. LVEF: group H, 0.80 ± 0.09%; group L, 0.71 ± 0.07%; group N, 0.68 ± 0.07%). Furthermore, treatment with rosuvastatin decreased the expression levels of TNF-α (group H, 65.19 ± 7.06 pg/ml; group L, 108.20 ± 5.28 pg/ml; group N, 239.34 ± 11.65 pg/ml) and IL-6 (group H, 14.33 ± 2.15 pg/ml; group L, 19.67 ± 3.04 pg/ml; group N, 40.39 ± 7.17 pg/ml). The rates of expression of active Caspase-3 and myocardial apoptosis were positively correlated with the scores for myocardial pathology. CONCLUSIONS: These results demonstrate that administration of rosuvastatin can ameliorate EAM progression, inhibit apoptosis of cardiomyocytes, and preserve cardiac output, and they also suggest rosuvastatin may be a promising novel therapeutic strategy for the clinical treatment of myocarditis.

The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy.

Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway.

Selective inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050.

Inhibiting signal transduction induced by inflammatory cytokines offers a new approach for the treatment of autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown promising oral disease-modifying antirheumatic drug potential with efficacy similar to anti-TNF biologics. Direct and indirect inhibition of the JAKs, with small molecule inhibitors like CP-690,550 and INCB018424 or neutralizing Abs, such as the anti-IL6 receptor Ab tocilizumab, have demonstrated rapid and sustained improvement in clinical measures of disease, consistent with their respective preclinical experiments. Therefore, it is of interest to identify optimized JAK inhibitors with unique profiles to maximize therapeutic opportunities. INCB028050 is a selective orally bioavailable JAK1/JAK2 inhibitor with nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple proinflammatory cytokines including IL-6 and IL-23 at concentrations <50 nM. Significant efficacy, as assessed by improvements in clinical, histologic and radiographic signs of disease, was achieved in the rat adjuvant arthritis model with doses of INCB028050 providing partial and/or periodic inhibition of JAK1/JAK2 and no inhibition of JAK3. Diminution of inflammatory Th1 and Th17 associated cytokine mRNA levels was observed in the draining lymph nodes of treated rats. INCB028050 was also effective in multiple murine models of arthritis, with no evidence of suppression of humoral immunity or adverse hematologic effects. These data suggest that fractional inhibition of JAK1 and JAK2 is sufficient for significant activity in autoimmune disease models. Clinical evaluation of INCB028050 in RA is ongoing.

FLT3 inhibitors for the treatment of autoimmune disease.

BACKGROUND: Autoimmune diseases encompass a broad range of illnesses with a variety of underlying causes, some of which are known and some of which remain elusive. OBJECTIVE: The focus of this review will be on describing the development of a new type of therapy that could potentially treat T cell-mediated autoimmune diseases. Unlike traditional therapies, which have primarily focused on suppressing T cells directly, targeting the step of antigen presentation may allow a less toxic therapy in which autoimmunity is lessened without compromising the entire immune system. This review will outline the science behind the development of the therapy, the roles of dendritic cells in generating autoimmune disease, and the function of the FLT3 receptor in this process.

Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category.

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. We examined the role of the interleukin (IL)-23-IL-17 and IL-12-T helper cell (Th)1 pathways in the pathogenesis of EAU. IL-23 but not IL-12 was necessary to elicit disease by immunization with the retinal antigen (Ag) interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. IL-17 played a dominant role in this model; its neutralization prevented or reversed disease, and Th17 effector cells induced EAU in the absence of interferon (IFN)-gamma. In a transfer model, however, a polarized Th1 line could induce severe EAU independently of host IL-17. Furthermore, induction of EAU with IRBP-pulsed mature dendritic cells required generation of an IFN-gamma-producing effector response, and an IL-17 response by itself was insufficient to elicit pathology. Finally, genetic deficiency of IL-17 did not abrogate EAU susceptibility. Thus, autoimmune pathology can develop in the context of either a Th17 or a Th1 effector response depending on the model. The data suggest that the dominant effector phenotype may be determined at least in part by conditions present during initial exposure to Ag, including the quality/quantity of Toll-like receptor stimulation and/or type of Ag-presenting cells. These data also raise the possibility that the nonredundant requirement for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and help provide a balance in the current Th1 versus Th17 paradigm.

Differential regulation of human NK cell-mediated cytotoxicity by the tyrosine kinase Itk.

NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses where NK cells play key regulatory roles.

[The prevalence of thyro-peroxidase antibodies and thyroid function in Turner's syndrome]. / Wystepowanie przeciwcial przeciw peroksydazie tarczycowej a funkcja tarczycy w zespole Turnera.

INTRODUCTION: Higher frequency of autoimmune diseases in patients with Turner's syndrome (TS) compared with the general population has been described. 5 to 10% of cases occur before adolescence. The goal of the study was to determine the prevalence of thyro-peroxidase antibodies (TPO-Ab) in correlation with karyotype, clinical symptoms and hormonal thyroid function in TS patients. MATERIAL AND METHODS: 96 girls with TS, aged 0.5-19.8 years (mean age 12.3+/-5.0) and 58 girls matched for age and BMI (control group) were analysed. The diagnosis of TS was established basing on clinical features and karyotype analysis. 54 had X monosomy, 7--isochromosome, 1--other X chromosome aberration, 11--mosaicism 45,X/46,XX, 3--45,X/47,XXX, 1--45,X/46,XX/47,XXX, 19--mosaicism with structural aberration: 12--45,X/46,X,i(Xq), 2--others, 5--with Y chromosome. In all children TSH, FT(4), FT(3), TPO-Ab, cholesterol, triglyceride levels, physical and ultrasonographic examination were performed. RESULTS: 25% of TS patients were positive for TPO-Ab. This frequency was significantly higher (p=0.0017) than that seen in the control group (5.2%). Positive titers of TPO-Ab were found: in 42% of girls with isochromosome (46,X,i(Xq) and 45,X/46,X,i(Xq)), 22.2% with X monosomy, and 17.4% with other karyotypes. The percentage of positive TPO-Ab titres increased with cumulative age of TS patients. It was 6.7% at the age of 10 years and almost doubled (12.1%) one year later. The next strong increase was observed at the age of 16 (up to 19.1%) and gradually rose to 20 years of age. Mean age of seronegative patients was significantly lower than that of seropositive patients (p=0.018). Only 2 patients manifested symptoms of hyperthyroidism requiring short period of antithyroid treatment. Others did not reveal any clinical features of thyroid dysfunction, although developed thyroid abnormalities such as elevated TSH (11.4%) or goiter (28%). Lack of correlation between TPO-Ab, thyroid hormones and lipid levels was associated with L-thyroxine supplementation, in patients with mildly elevated TSH, prior to the study. CONCLUSIONS: Patients with TS, especially with isochromosome, have antithyroid antibodies more frequently than their co-evals. Therefore, it is important to monitor TPO-Ab from about the age of 10 years even in asymptomatic patients. However, in routine clinical practice, both the thyroid examination and TSH level (even in asymptomatic patients) should be screened yearly for early detection of subclinical hypothyroidism and risk of more severe growth retardation in girls with TS.

IL-converting enzyme/caspase-1 inhibitor VX-765 blocks the hypersensitive response to an inflammatory stimulus in monocytes from familial cold autoinflammatory syndrome patients.

Familial cold autoinflammatory syndrome (FCAS) and the related autoinflammatory disorders, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, are characterized by mutations in the CIAS1 gene that encodes cryopyrin, an adaptor protein involved in activation of IL-converting enzyme/caspase-1. Mutations in cryopyrin are hypothesized to result in abnormal secretion of caspase-1-dependent proinflammatory cytokines, IL-1beta and IL-18. In this study, we examined cytokine secretion in PBMCs from FCAS patients and found a marked hyperresponsiveness of both IL-1beta and IL-18 secretion to LPS stimulation, but no evidence of increased basal secretion of these cytokines, or alterations in basal or stimulated pro-IL-1beta levels. VX-765, an orally active IL-converting enzyme/caspase-1 inhibitor, blocked IL-1beta secretion with equal potency in LPS-stimulated cells from FCAS and control subjects. These results further link mutations in cryopyrin with abnormal caspase-1 activation, and support the clinical testing of caspase-1 inhibitors such as VX-765 in autoinflammatory disorders.

Effect of dietary n-3 and n-6 oils with and without food restriction on activity of antioxidant enzymes and lipid peroxidation in livers of cyclophosphamide treated autoimmune-prone NZB/W female mice.

OBJECTIVE: Cyclophosphamide (CTX), an alkylating agent, is extensively used in the treatment of lupus nephritis, but its administration has been associated with free radical mediated oxidative stress. The present study was designed to investigate the effect of dietary corn oil (CO), fish oil (FO) and food restriction (FR) on the activities of hepatic antioxidant enzymes, fatty acid composition and lipid peroxidation following CTX administration in autoimmune-prone NZB/W female mice. METHODS: Autoimmune-prone NZB/W female mice were fed either ad libitum (AL) or food restricted (60% of AL intake), semipurified diets containing 5% CO or 5% FO supplemented with equal levels of antioxidants and injected with either phosphate buffered saline (PBS), or CTX (50 mg/kg body weight) every 10 days. Proteinuria was measured biweekly. The treatment was stopped at 10 months and diets were continued until the mice were killed at 12 months. Fatty acid composition, activity of antioxidant enzymes and lipid peroxidation were analyzed in liver homogenates, and anti-DNA antibodies were analyzed in the serum. RESULTS: Mice in the FO/AL dietary group exhibited significantly higher liver catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities compared to the CO/AL dietary group. CTX significantly decreased SOD and GSH-Px activity in the FO/AL group and CAT and GSH-Px in the CO/AL group. In AL fed mice given CTX, activities of CAT, GSH-Px and GST were significantly higher in mice fed FO diets than in mice fed CO diets. FR increased the activity of enzymes in both the CO and FO diet groups. In FR mice, CTX decreased CAT and GSH-Px activity in both the CO and FO dietary groups, but glutathione S-transferase (GST) only in the CO group. The decrease in SOD activity was not significant in either of the restricted groups. CTX significantly increased generation of thiobarbituric acid reactive substances (TBARS) in both AL groups. FR significantly decreased lipid peroxidation in both the CO and FO groups, with or without CTX. CTX decreased serum anti-DNA antibody levels in both the CO and FO dietary groups. FR also decreased antibody titer in both the CO and FO dietary groups, and it was decreased further with CTX treatment. FO fed animals had higher levels of n-3 fatty acids, whereas CO fed animals had high levels of n-6 fatty acids. CTX significantly increased 20:4 and decreased 18:1 in CO/AL fed animals, whereas it increased 18:1 and decreased 22:6 in FO/AL fed animals. CONCLUSIONS: Results obtained in the present study suggests that FO and, more significantly, FO combined with FR can have a beneficial effect in hepatic tissues subjected to CTX induced oxidative stress by regulating the activity of antioxidant enzymes. In addition, the study also indicates that n-3 and n-6 dietary lipids are susceptible to lipid peroxidation, particularly in the presence of a prooxidant like CTX, and that FR is beneficial in decreasing lipid peroxidation. The study also suggests that FO and CTX can have additive effects in preventing kidney disease in NZB/W mice.

Lethal autoimmune myocarditis in interferon-gamma receptor-deficient mice: enhanced disease severity by impaired inducible nitric oxide synthase induction.

BACKGROUND: Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease. METHODS AND RESULTS: We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis. CONCLUSIONS: Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis.

Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.

An inhibitor of inducible nitric oxide synthase ameliorates experimental autoimmune myocarditis in Lewis rats.

We studied the effect of nitric oxide (NO) on experimental autoimmune myocarditis (EAC) in rats. We examined the role of inducible nitric oxide synthase (iNOS), an enzyme that produces NO, on hearts affected with EAC, by testing the effects of aminoguanidine (AG), a selective iNOS inhibitor, on the course of EAC. Western blotting detected iNOS in the affected cardiac tissues, but not in CFA immunized cases. Immunohistochemically, the majority of ED1+ macrophages in the EAC lesions were positive for iNOS and nitrotyrosine. A high dose of AG (200 mg/kg/day) significantly reduced the incidence of EAC (p < 0.05) and ameliorated the histological score for the cardiac inflammation (p < 0.01) compared with the low dose AG (100 mg/kg/day) and vehicle treated groups. The immunoblot analysis showed that a high dose of AG effectively suppressed iNOS in hearts affected with EAC. An iNOS band was barely detected in the high dose AG (200 mg/kg) treated group, while it was distinctively visualized in the vehicle and low dose AG (100 mg/kg) treated groups. These results suggest that iNOS is upregulated in EAC lesions and increased NO production plays an important role in the development of EAC. In addition, selective iNOS inhibitors may have a therapeutic role in treating certain autoimmune diseases including EAC.

Inhibition of inducible nitric oxide synthase intensifies injury and functional deterioration in autoimmune interstitial nephritis.

T lymphocytes are exquisitely sensitive to the antiproliferative effects of nitric oxide. We examined the effects of oral administration of two nitric oxide synthase inhibitors, Nw-nitro-L-arginine methyl ester (L-NAME) and L-N6-(1-iminoethyl)lysine (L-NIL), on the course of T cell-dependent autoimmune interstitial nephritis in Brown Norway rats. Kidneys from rats immunized to produce interstitial nephritis display a net generation of nitric oxide end products. By immunohistochemical staining, the cytokine-inducible nitric oxide synthase (iNOS) is expressed in cortical tubular epithelial cells. Treatment with either inhibitor results in markedly more severe disease following immunization. Animals receiving L-NAME were hypertensive, while those treated with L-NIL, a highly selective inhibitor of iNOS, were not. Evaluation of the expression of IFN-gamma, IL-2, and IL-4 in diseased kidneys by quantitative reverse transcriptase-PCR demonstrated that L-NAME-treated animals displayed significantly augmented levels of IFN-gamma and IL-2 with preserved ratios of IFN-gamma/IL-4 and IL-2/IL-4, while L-NIL-treated animals had augmented levels of IL-2 and IFN-gamma with augmented IFN-gamma/IL-4 and IL-2/IL-4 ratios. Animals treated with L-NAME or L-NIL both had augmented Ag-specific IgG responses. The L-NAME group demonstrated increases in both the IgG2a and IgG1 subtypes, with a constant IgG2a/IgG1 ratio, while the L-NIL group demonstrated an increase in the ratio of the IgG2a/IgG1 response. These Ab and cytokine data suggest that the L-NIL-treated animals had a skewing of their immune response toward a Th1-like response. We conclude that in autoimmune interstitial nephritis, generation of nitric oxide through the iNOS pathway has host-protective effects, and suggest that this may be broadly applicable to T cell-mediated pathologies.

Modulation of antioxidant enzymes and programmed cell death by n-3 fatty acids.

Studies from our laboratory indicate that n-3 (fish oil, FO) lipids at 10% (w/w) in a nutritionally adequate, semipurified diet, and supplemented with equal levels of antioxidants, extended the life span of lupus-prone (NZB/NZW)F1 (B/W) female mice as compared to n-6 (corn oil, CO) lipids. The early rise of autoimmune disease in CO-fed mice was closely linked to the loss of T-cell function. Both IL-2 production and IL-2 receptor expression were reduced due to the loss of naive T-cells and a rise in memory T-cells. Proliferative response to both mitogens and superantigens (staphylococcal enterotoxins A and B) was higher in FO-fed 6.5-mon-old mice. These changes paralleled decreased PGE2 production by splenic cells from FO-fed mice. Analysis of mRNA expression in different organs revealed differential effects of dietary lipids. In FO-fed mice, transforming growth factor beta 1 (TGF beta 1) expression was decreased in kidneys, but splenic tissues had higher expression of TGF beta mRNA. As TGF beta promotes programmed cell death (PCD), we studied the effects of CO and FO on PCD rates in lymphocytes. Both propidium iodide staining and DNA fragmentation were elevated in lymphocytes of FO-fed mice when compared to CO-fed mice of similar age. Also, increased PCD correlated closely with increased Fas gene expression. Thus, in addition to various other antiinflammatory effects, dietary FO appears to increase PCD and prevent accumulation of self-reactive immune cells in lymphoid organs. Further studies are required to dissect the pro- and antiinflammatory mechanisms associated with dietary n-3 and n-6 lipids in modulating autoimmune disorders or malignancy during aging.