Theiler's murine encephalomyelitis virus-induced cardiac and skeletal muscle disease.

The DA strain of Theiler's murine encephalomyelitis virus, a member of the cardiovirus genus of picornaviruses, induces a restricted and persistent infection associated with a demyelinating process following intracerebral inoculation of mice; both virus infection and the immune response are believed to contribute to the late white matter disease. We now report that intraperitoneal inoculation with DA produces an acute myositis that progresses to a chronic inflammatory muscle disease in CD-1 mice as well as several inbred mouse strains. Some mouse strains also develop central nervous system white matter disease and a focal myocarditis. Infectious virus in skeletal muscle falls to undetectable levels 3 weeks postinoculation (p.i.), although viral genome persists for at least 12 weeks p.i., the longest period of observation. Severe combined immunodeficient animals have evidence of muscle pathology as long as 5 weeks p.i., suggesting that DA virus is capable of inducing chronic muscle disease in the absence of an immune response. The presence in immunocompetent mice, however, of prominent muscle inflammation in the absence of infectious virus suggests that the immune system also contributes to the pathology. T lymphocytes are the predominant cell type infiltrating the skeletal muscle during the chronic disease. This murine model may further our understanding of virus-induced chronic myositis and help to clarify the pathogenesis of human inflammatory myopathies.     

Expression and potential role of inducible nitric oxide synthase in the central nervous system of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. We examined the pathogenic roles of nitric oxide (NO) and inducible NO synthase (iNOS) in TMEV-induced demyelinating disease (TMEV-IDD). The presence of iNOS was confirmed in the spinal cords of TMEV-infected mice using immunohistochemical staining with anti-iNOS antibody on day 0 (control) and days 15, 30, 60, and 120. Aminoguanidine (AG), a specific inhibitor of iNOS, was injected intraperitoneally (ip) on 1, 3, 5, 8, 10, and 12 days post-TMEV inoculation as induction phase or 15, 17, 19, 22, 24, and 26 days as effector phase. Control animals in each experiment received phosphate-buffered saline (PBS) ip at similar time intervals. Few iNOS-positive cells were observed in the spinal cords of naive SJL/J mice. In the early phase (day 15) of TMEV-IDD, an increase of iNOS-positive cells was detected in the leptomeninges and perivascular space of the spinal cords. The number of iNOS-positive cells was increased and reached its peak on day 60, when histology of the animals showed peak infiltration with inflammatory cells. The clinical course of TMEV-IDD on each day postintracerebral infection was significantly reduced in mice treated with AG in the effector phase, and there was no significant difference between mice treated with AG in induction phase versus those administered PBS. Thus, NO production via iNOS appears to be a pathogenic factor in the effector phase of TMEV-IDD.     

Sex-specific quantitative trait loci govern susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination

Susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination (TMEVD), a mouse model for multiple sclerosis (MS), is genetically controlled. Through a mouse-human comparative mapping approach, identification of candidate susceptibility loci for MS based on the location of TMEVD susceptibility loci may be possible. Composite interval mapping (CIM) identified quantitative trait loci (QTL) controlling TMEVD severity in male and female backcross populations derived from susceptible DBA/2J and resistant BALBc/ByJ mice. We report QTL on chromosomes 1, 5, 15, and 16 affecting male mice. In addition, we identified two QTL in female mice located on chromosome 1. Our results support the existence of three linked sex-specific QTL on chromosome 1 with opposing effects on the severity of the clinical signs of TMEV-induced disease in male and female mice.     

Effect of lactoferrin on the production of tumor necrosis factor-alpha and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor-alpha and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor-alpha production or nitric oxide production, but lipopolysaccharide-stimulated tumor necrosis factor-alpha production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin.     

Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.     

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.     

Over-expression of GTP-binding proteins and GTPase activity in mouse astrocyte membranes in response to Theiler's murine encephalomyelitis virus infection

Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease that resembles human multiple sclerosis. In order to delineate the early events in this virus-induced neuroinflammatory disease, we have analyzed global GTPases gene activation following TMEV infection of murine brain astrocytes. DNA hybridization microchip analysis demonstrated that 10 sequences described as GTPbinding proteins and GTPases in different protein databases were over-expressed, in response to this infectious agent in astroglial cells. We have first characterized both the GTP-binding and GTPase activities in uninfected astrocyte membranes from a biochemical point of view. The increase in such activities was further validated in TMEV-infected astrocytes, peaking 2-4 h after infection. Over-expression is also induced by the inflammation-related chemokines interleukin-6 and interferon-gamma but not by interleukin-1alpha or tumor necrosis factor-alpha. From the many GTPases that could be over-expressed we have studied two, because of its biological significance; Ras p21 and the subunit alphai2 of G proteins. Western blots revealed increases in both proteins after infection with TMEV, in accordance with the previous enzymologic results. An increase in the active form of Ras (the GTP bound form) in cell lysates was also confirmed by affinity binding to a glutathione-S-transferase-fusion protein, following TMEV infection. A final demonstration of physiological up-regulation is provided by UV cross-linking of membrane proteins with the hydrolysis-resistant GTP agonist GTP [gamma-(35)S]. This technique allow us to detect, after SDS-PAGE, the increase of two further majoritary GTPbinding proteins with MW of 62 and 49 KDa. A quantitative analysis of four selected genes coding for p21 ras, Galphai2 subunit of protein G, Munc-18 and protein interacting with C kinase 1, was performed by real-time RT-PCR to verify the microarray results. The study of GTPase activity and of the above genes by RT-PCR in brains of sick mice, demonstrated a significative increase in mRNA coding for p21ras and protein interacting with C kinase 1 in vivo. Here we demonstrate that one of the mechanisms triggered by TMEV infection of astrocytes is the up-regulation of proteins related to GTP metabolism, one important signal transduction system in mammalian cells.     

The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and chronic, inflammatory demyelinating disease in susceptible mice. TMEV antigen(s) and RNA genome have been detected in astrocytes, oligodendrocytes, and macrophages during persistence. Whether there is a predominant cell type in which TMEV persists has not been resolved. Since TMEV-induced demyelinating lesions are infiltrated with macrophages and a number of other persistent viruses show near-exclusive tropism for these phagocytic cells, we used two-color immunofluorescent staining with conventional and confocal microscopy to colocalize TMEV to cells that stain with monoclonal antibodies (MOMA-2) [unknown antigen], Mac-1 [CD11b], FA-11 [CD66], and 2F8 [scavenger receptor]) to macrophages in BeAn-infected SJL mice. A predominant virus antigen burden within macrophages infiltrating demyelinating lesions was seen. A dichotomy of cells staining for virus antigen(s) was found with infected cells containing either a large or small virus antigen load. Ninety percent of cells with a large virus antigen load were large phagocytes (20 to 50 microns) that were readily detected at low power (5x objective). Cells with smaller amounts of virus antigen(s) turned out to be either these same large phagocytic cells or much smaller cells, approximately equal to 10 microns in diameter. Forty percent of cells with a small virus antigen load were macrophages. The unidentified approximately equal to 10-microns cells that are virus antigen positive and macrophage negative in this study could still be macrophages, or they may be oligodendrocytes. The fact that virus was detected in the cytoplasm and not phagolysosomes of macrophages and the sheer mass of fluorescently stained virus proteins in some macrophages suggest that TMEV persists in these phagocytic cells by active virus replication.     

Two neolignans from Perilla frutescens and their inhibition of nitric oxide synthase and tumor necrosis factor-alpha expression in murine macrophage cell line RAW 264.7

Two neolignans were isolated from leaves of Perilla frutescens (Labiatae) as inhibitors of nitric oxide syntheses (IC50 5.9 microM and 53.5 microM, respectively) and tumor necrosis factor-alpha in lipopolysaccharide-activated RAW 264.7 cells. Their structures were identified as 1beta,2alpha,3beta,4alpha-1,2-dimethyl-3,4-bis-(2,4,5-trimethoxyphenyl)-cyclobutane (magnosalin), and 1alpha,2beta,3beta,4alpha-1,2-dimethyl-3,4-bis-(2,4,5-trimethoxyphenyl)-cyclobutane (andamanicin), and their activities were confirmed as resulted from the suppressed expression of inducible nitric oxide synthase enzyme and from the secretion of tumor necrosis factor-alpha from activated macrophages.     

Extracellular inosine participates in tumor necrosis factor-alpha induced nitric oxide production in cultured Sertoli cells

Recent reports have described purinergic modulation of tumor necrosis factor-alpha (TNF-α) signaling in neutrophils and astrocytes. In Sertoli cells, both TNF-R1 and TNF-R2 TNF-α receptors are present and this cytokine modulates many functions of these cells related to the maintenance of spermatogenesis. Sertoli cells express distinct purinoreceptors and previous work has shown that these cells secrete extracellular nucleotides and their metabolites. In this work, we studied the possible role of extracellular purines in TNF-α signaling in cultured Sertoli cells. This cytokine increased inosine concentration from 30 min to 6 h, with no effect at 24 h. Both TNF-α and inosine increased nitrite accumulation and nitric oxide synthase activity. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor, abolished the TNF-α induced inosine increase, nitrite accumulation and nitric oxide synthase activity. These results suggest that extracellular inosine acts as intermediary in TNF-α stimulated nitric oxide production in cultured Sertoli cells.     

Effect of anti-B7-1 and anti-B7-2 mAb on Theiler's murine encephalomyelitis virus-induced demyelinating disease

We examined the role of B7-1 and B7-2, costimulatory molecules critical to full activation of T cells, in the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to B7-1 resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of TNF-alpha and IFN-gamma in the spleen cells was decreased. The delayed-type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. In contrast, treatment with Abs to B7-2, resulted in no effect on TMEV-IDD. These data suggest that B7-1 is critically involved in the pathogenesis of TMEV-IDD and that Abs to B7-1 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.     

Production of nitric oxide and tumor necrosis factor-alpha by Smilacis rhizoma in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which, Smilacis rhizoma (SR) regulates nitric oxide (NO) production. When SR was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, SR had no effect on NO production by itself. The increased production of NO from rIFN-gamma plus SR-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus SR caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of SR on TNF-alpha production significantly. These findings demonstrate that SR increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of SR.     

Alpha-MSH peptides inhibit production of nitric oxide and tumor necrosis factor-alpha by microglial cells activated with beta-amyloid and interferon gamma.

Alpha-melanocyte stimulating hormone (alpha-MSH) is an ancient tridecapeptide with potent inhibitory activity in all major forms of inflammation. The anti-inflammatory message sequence of alpha-MSH resides in the COOH-terminal tripeptide alpha-MSH[11-13]. We tested the influence of alpha-MSH[1-13] and of alpha-MSH[11-13] in a cultured murine microglia cell line known to produce nitric oxide (NO(-)(2)) and tumor necrosis factor (TNFalpha) when stimulated with beta-amyloid protein (Abeta). Melanocortin peptides significantly inhibited release of both NO(-)(2) and TNFalpha into cell-free supernatants from microglia stimulated with Abeta[1-42] or Abeta[25-35] peptides and interferon gamma (IFNgamma). Northern blot analysis demonstrated that alpha-MSH[1-13] and alpha-MSH[11-13] inhibited accumulation of inducible nitric oxide synthase (iNOS) and TNFalpha mRNA was triggered by Abeta stimulation. Abeta/microglial interaction is believed to promote the progression of inflammatory and neurodegenerative changes in senile plaques in Alzheimer's disease. Our data indicate that alpha-MSH peptides might be used to modulate the local response of the brain to Abeta deposition in this neurodegenerative disease.