Previous studies have shown that B6.TgL mice display abnormal T-cell differentiation and functions, and spontaneous inflammation in the colon and lung. The colonic phenotype in B6.TgL mice was histopathologically defined as an inflammatory bowel disease resembling ulcerative colitis in humans. In the present study, the undetermined lung disease in B6.TgL mice was characterized as a PAH-like disease. PAH is a clinical category of PH that comprises many different disease entities. Pathological manifestations of the PAH-like disease in B6.TgL mice are not completely parallel to those of idiopathic PAH. The differences between idiopathic PAH and the present animal model include the caliber of the affected arteries, the primarily affected layer of vascular wall, and the participation of massive lymphocytic perivascular infiltration. Further investigations are needed to define the present lung pathology as any type of PAH. An increasing body of evidence implicates the role of immune-mediated mechanisms in the pathogenesis of PAH. A type of PAH occurs secondarily to collagen vascular disorders, such as systemic sclerosis and mixed connective tissue disease (MCTD). Interestingly, PAH with MCTD presents with a prominent characteristic of endothelial degeneration and proliferation, probably due to the pathogenic contribution of autoantibodies to endothelial cells [28, 29]. This characteristic may be a pathological consequence of immune-mediated mechanisms shared with the present model. The findings in the B6.TgL mice provide a possible insight into an implication of an OX40L-derived signal in the immune-mediated mechanism of endothelial pathology in PAH.
PAH is associated with endothelial cell dysfunction and vasoconstriction. There is no direct evidence for a link between these pulmonary vascular manifestations and abnormality in situ of OX40L-derived signal. However, it has been shown that OX40L-derived signals have a pathologic impact on the endothelial cell functions of systemic arteries. Recent studies have demonstrated an association of OX40L gene polymorphism with the susceptibility to atherosclerosis in humans , and the critical contribution of OX40-OX40L interactions to atherogenesis in low-density lipoprotein receptor-deficient mice . The endothelial cells of the systemic arteries and those of the pulmonary arteries are exposed to different conditions, i.e., blood pressure and oxygen tension. It is interesting to know whether the OX40-OX40L interactions yield a different response on pulmonary endothelial cells than on systemic endothelial cells, and whether the OX40L gene polymorphism is associated with any type of PAH in humans.
Our present immunological studies performed using TgL and non-TgL strains of mice with different genetic backgrounds--B6 and BALB--revealed the respective effects of TgL and strain-dependent genetic background on immune phenotypes in the lung. Previous studies have demonstrated that OX40L-derived signals promote the expansion of effector/memory CD4+ T cells [17, 19] and naturally arising Treg cells , and enhance the production of IL-13 and IL-17 by CD4+ T cells [33–35]. To examine which TgL-dependent immune aberrations are correlated with the onset of the PAH-like disease, we examined TgL-dependent immune phenotypes in the lungs of 2 different strains at a pre-disease stage. The findings indicate that B6-specific genetic factors influence the expansion of effector/memory CD4+ T cells and Treg cells in advance of the onset of lung disease. A possible role of Treg cells has been documented in the development of PAH in humans . Furthermore, it is clearly shown that B6-specific genetic factors increase the number of IL-17-producing CD4+ T cells as well as secretion of IL-13 and IFNγ (data not shown) by lung tissue resident CD4+ T cells. IL-17 producing CD4 T cells, namely Th17 cells are well known that participates in the pathogenesis of various organ-specific autoimmune diseases, such as inflammatory bowel disease and rheumatoid arthritis [36, 37]. Although the role of Th17 cells in PAH in humans has not been determined, our present findings suggest that they indeed play a role in PAH. IL-13 serves as an important mediator in pulmonary inflammation [5, 38, 39], suggesting a causal contribution of IL-13 to the pathogenesis of the present model. The presence of immunological findings provides an insight into PAH-prone immune condition in the lung: the increase of proinflammatory effectors, IL-13 and Th17, and the decrease of an anti-inflammatory effector, Treg.
The present genome-wide genetic approach demonstrated a new susceptibility locus controlling the onset of a PAH-like disease in our model. Previous genetic studies performed on familial PAH have shown mutations in 2 genes responsible for susceptibility to PAH: bone morphogenetic protein receptor 2 gene (BMPR2)  and activin-like kinase type-1 gene (ALK-1) . Our identified locus includes neither of these genes, nor, to the best of our knowledge, any gene involved in their signal transduction pathways. However, Nos3 and Hgf genes were particularly noted within this locus. Nitric oxide (NO) is known as a potent endothelial cell-derived vasodilator and an inhibitor of smooth muscle proliferation. Endothelial NO production largely depends on NOS3/eNOS (encoded by Nos3). NOS3-deficient mice showed reduced pulmonary vascular proliferation and remodeling to chronic hypoxia [42, 43]. Several studies have reported the preventive role of NO in the development of PH in mice and humans. The polymorphism of human Nos3 gene is associated with high-altitude pulmonary edema and PH in patients with chronic obstructive pulmonary disease . On the other hand, Hgf, which encodes hepatocyte growth factor (HGF), suppresses vascular medial hyperplasia and matrix accumulation in advanced PH in rats . These findings have underscored the role of NOS3/eNOS or HGF as a pathogenic modifier in the present PH model.
A T-cell subset, type II helper T cell (Th2), plays an important role in the pathogenesis of PAH in mice . In this regard, it is noteworthy that the 2 loci (the transgene locus and the susceptibility locus) have a strong impact on Th1/Th2 balance. The OX40 signal promotes a Th2-prone condition in mice . On the other hand, NO and HGF serve as inducible factors for type I helper T cells (Th1) [46, 47]. Therefore, in the TgL strains of mice, the 2 loci are mutually counterbalanced, and the net Th1/Th2 proportion depends mainly on the polymorphic effect of the susceptibility locus. In a B6 genetic background, an effect of the susceptibility locus may suppress Th1 responses and maximize Th2 augmentation in the lung conferred by the OX40L transgene, resulting in the B6-specific onset of PH.