Decreased arthritis severity in cathepsin L-deficient mice is attributed to an impaired T helper cell compartment.

OBJECTIVE: Cathepsin L (CL) is potentially involved in joint destruction and in antigen presentation in rheumatoid arthritis. In order to define the roles of this protease in arthritis development we analysed the antigen-induced arthritis (AIA) in CL-deficient (CL(-/-)) mice. METHODS: Antigen-induced arthritis was induced in CL(-/-) and wild-type mice. Complete CL deficiency resulted in an impaired positive selection of conventional CD4(+) T helper (Th) cells and finally in a reduced number of Th cells. Thus, we addressed the effect of this phenotype by rescuing CD4(+) Th cell numbers by transgenic expression of the human CL-like protease cathepsin V (hCV) in thymic epithelium of CL(-/-) mice [Tg(K14-hCV);CL(-/-)]. The arthritis development was monitored by measuring joint swelling. Joint inflammation and destruction were assessed histopathologically. RESULTS: The severity of AIA was decreased in CL(-/-) mice characterized by reduced swelling, decreased inflammation and destruction, and diminished cellular and humoral immune responsiveness. AIA in Tg(K14-hCV);CL(-/-) mice was associated with a reconstitution of all parameters by normalization of the ratio of regulatory to conventional T cells. CONCLUSIONS: Cathepsin L has a significant impact on AIA severity by influencing the selection of Th cell populations in the thymus, but seems not play any significant role in the direct joint destruction.

TGF-beta and fibrosis in different organs - molecular pathway imprints.

The action of transforming-growth-factor (TGF)-beta following inflammatory responses is characterized by increased production of extracellular matrix (ECM) components, as well as mesenchymal cell proliferation, migration, and accumulation. Thus, TGF-beta is important for the induction of fibrosis often associated with chronic phases of inflammatory diseases. This common feature of TGF-related pathologies is observed in many different organs. Therefore, in addition to the description of the common TGF-beta-pathway, this review focuses on TGF-beta-related pathogenetic effects in different pathologies/organs, i. e., arthritis, diabetic nephropathy, colitis/Crohn's disease, radiation-induced fibrosis, and myocarditis (including their similarities and dissimilarities). However, TGF-beta exhibits both exacerbating and ameliorating features, depending on the phase of disease and the site of action. Due to its central role in severe fibrotic diseases, TGF-beta nevertheless remains an attractive therapeutic target, if targeted locally and during the fibrotic phase of disease.

Imatinib mesylate attenuates fibrosis in coxsackievirus b3-induced chronic myocarditis.

AIMS: Coxsackievirus B3 (CVB3)-induced chronic myocarditis in mice is accompanied by severe fibrosis and by sustained elevation of platelet-derived growth factor (PDGF)-A, -B, and -C levels in the cardiac tissue. To test if PDGF stimulation of resident fibroblasts causally contributes to fibrosis, we employed inhibition of PDGF receptor signalling with the orally available kinase inhibitor Imatinib. METHODS AND RESULTS: Chronic myocarditis was induced by CVB3 infection of major histocompatibility complex (MHC) class II knockout (B6Aa(0)/Aa(0)) mice. The mice were treated with 100 mg/kg Imatinib or vehicle, respectively, twice daily for 34 days. Expression of PDGF-C and of inflammatory cytokines were analysed by semi-quantitative RT-PCR. PDGFalpha receptor phosphorylation was detected by immunoblotting of cardiac tissue extracts and in situ by immunohistochemistry. Fibrosis formation was analysed by Sirius-Red staining and hydroxyproline (HP) determination. Fibronectin, and tenascin expression was analysed by RT-PCR and immunohistochemistry. Matrix metalloproteinase (MMP) activity was assessed with collagen, synthetic peptides, and gelatine as substrates. Imatinib significantly inhibited the myocarditis-related PDGFalpha receptor activation in the heart tissue. The virus titres in the hearts, inflammatory infiltrations, and elevated PDGF levels were unaffected by the Imatinib treatment. A significant attenuation of fibrosis occurred in Imatinib-treated animals. The Sirius Red-stained fibrotic area was reduced from 5.30 +/- 0.50 to 3.21 +/- 0.35%, and the HP content was reduced from 362 +/- 43 to 238 +/- 32 microMol/10 mg dry weight vs. 190 +/- 27 in uninfected controls. The expression of fibronectin, EIIIA+ fibronectin, and tenascin C were likewise reduced. The diminished matrix protein deposition was not caused by elevated MMP activity, since MMP activity was not changed or even reduced under Imatinib. CONCLUSION: The data suggest a causal role for elevated PDGF expression and PDGF receptor activity in the pathogenesis of cardiac fibrosis.

TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-theta signaling and promote autoimmune myocarditis.

The serine/threonine kinase, protein kinase C-theta (PKC-theta), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-theta may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-theta in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with alpha-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-theta-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-theta was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-theta in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-theta-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-x(L), but exogenous IL-6 and TGF-beta was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-theta signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.

Elevated expression of PDGF-C in coxsackievirus B3-induced chronic myocarditis.

AIMS: Coxsackievirus B3 (CVB3) is a frequent cause of human chronic myocarditis and subsequent fibrosis, leading to dilated cardiomyopathy. The molecular processes underlying the development of fibrosis are poorly understood. Enhanced levels of platelet-derived growth factors (PDGFs), especially PDGF-C, have recently been linked with the development of different forms of fibrosis. Therefore, the expression of PDGF was analysed in hearts of CVB3-infected major histocompatability complex class II knockout mice. The latter were recently established as mouse model mimicking the chronic inflammation and fibrosis characteristic for this disease. METHODS AND RESULTS: Expression of PDGF was analysed by reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry. Hearts of C57BL/6 mice served as controls because infection of these animals leads to acute cardiac inflammation, but the hearts heal without signs of chronic inflammation. In uninfected hearts, basal expression of PDGF, notably PDGF-C, was detectable throughout the heart. The chronic inflammatory process was associated with elevated and sustained expression of all tested PDGF isoforms. Immunostaining and in situ hybridization analysis localized enhanced PDGF levels to areas with highest virus load and inflammatory infiltrations, adjacent to fibrotic areas. CONCLUSION: PDGF may participate in fibrosis development in CVB3-induced myocarditis. Therefore, PDGF signalling may be considered a target for therapeutic interference.

Coxsackievirus B3-induced myocarditis: differences in the immune response of C57BL/6 and Balb/c mice.

Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.