NKT cells belong to a distinct subset of T cells that recognize hydrophobic antigens presented by major histocompatibility complex class I-like molecules, such as CD1d. Because NKT cells stimulated by antigens can activate or suppress other immunocompetent cells through an immediate production of a large amount of cytokines, they are regarded as immunological modulators. CD1d-restricted NKT cells are classified into two subsets, namely, type I and type II. CD1d-restricted type I NKT cells express invariant T cell receptors (TCRs) and react with lipid antigens, including the marine sponge-derived glycolipid α-galactosylceramide. On the contrary, CD1d-restricted type II NKT cells recognize a wide variety of antigens, including glycolipids, phospholipids, and hydrophobic peptides, by their diverse TCRs. In this review, we focus particularly on CD1d-restricted type II NKT cells that recognize endogenous hydrophobic peptides presented by CD1d. Previous studies have demonstrated that CD1d-restricted type I NKT cells usually act as pro-inflammatory cells but sometimes behave as anti-inflammatory cells. It has been also demonstrated that CD1d-restricted type II NKT cells play opposite roles to CD1d-restricted type I NKT cells; thus, they function as anti-inflammatory or pro-inflammatory cells depending on the situation. In line with this, CD1d-restricted type II NKT cells that recognize type II collagen peptide have been demonstrated to act as anti-inflammatory cells in diverse inflammation-induction models in mice, whereas pro-inflammatory CD1d-restricted type II NKT cells reactive with sterol carrier protein 2 peptide have been demonstrated to be involved in the development of small vessel vasculitis in rats.

We previously generated a rat model that developed systemic connective tissue diseases, including synovitis, myositis, and small-vessel vasculitis (SVV), and established a vascular endothelial cell-reactive T-cell clone, VASC-1, from the model. VASC-1 was determined to be a type II natural killer T-cell clone. In this study, we attempted to identify the antigen recognized by VASC-1. The monkey-derived cell line COS-7 was used because VASC-1 does not bind naturally to COS-7, although the amino acid sequences are well conserved between monkey CD1d and rat CD1d. We generated 98 COS-7 clones transfected with miscellaneous rat cDNA and screened them for VASC-1 binding. Consequently, we found one clone, 4D2, which could bind to VASC-1. Sequencing identified the rat cDNA introduced into 4D2 as sterol carrier protein 2 (SCP2). When VASC-1 was co-cultured with SCP2 knockdown rat vascular endothelial cells, VASC-1 binding was reduced significantly. Moreover, we designed a series of rat SCP2 peptides and introduced them into COS-7 cells. On the basis of VASC-1 binding and proliferation, we revealed that the peptide rSCP2 included the epitope recognized by VASC-1. Furthermore, immunization with rSCP2 accelerated the development of SVV in the rat model. The collective findings suggest that type II natural killer T cells reactive with autologous SCP2 are implicated in vascular inflammation in the rat model.

NKT cells are defined as T cells that recognize hydrophobic antigens presented by class I MHC-like molecules, including CD1d. Among CD1d-restricted NKT cells, type I and type II subsets have been noted. CD1d-restricted type I NKT cells are regarded as proinflammatory cells in general. On the contrary, accumulated evidence has demonstrated an anti-inflammatory property of CD1d-restricted type II NKT cells. In our earlier study using a rat model with vasculitis, we demonstrated the pro-inflammatory function of CD1d-restricted type II NKT cells and identified that one such cell recognized P 518-532 of rat sterol carrier protein 2 (rSCP2 518-532 ), which appeared on vascular endothelial cells presented by CD1d. Based on this evidence, we attempted to detect human CD1d-restricted type II NKT cells in peripheral blood using hSCP2 518-532 , the human counterpart of rSCP2 518-532, together with a CD1d tetramer in flow cytometry. First, we determined the binding of hSCP2 518-532 to CD1d. Next, we detected CD3-positive hSCP2 518-532 -loaded CD1d (hSCP2 518-532 /CD1d) tetramer-binding cells in peripheral blood of healthy donors. The abundance of TGF-β-producing cells rather than TNF-α-producing cells in CD3-positive hSCP2 518-532 /CD1d tetramer-binding cells suggests the anti-inflammatory property of SCP2-loaded CD1d (SCP2/CD1d) tetramer-binding type II NKT cells in healthy individuals. Furthermore, we compared cytokine profile between healthy individuals and patients with vasculitis in a pilot study. Interestingly, the percentage of TGF-β-producing cells in SCP2/CD1d tetramerbinding type II NKT cells in vasculitic patients was significantly lower than that in healthy controls despite the greater number of these cells. Although further studies to clarify the mechanism and significance of this phenomenon are needed, SCP2/CD1d tetramer-binding type II NKT cells in peripheral blood should be examined in more detail to understand the pathophysiology of vasculitides in humans.

Objective. Recent studies have suggested that aPS-PT antibody is one of the most relevant autoantibodies to APS. This study aimed to demonstrate the pathogenicity of aPS-PT antibody in vivo. Methods. At first, cultured rat vascular endothelial cells (RECs) were exposed to calf thymus-derived histones. Two hours later, lactate dehydrogenase release from the RECs and expression of PS on the cell surface were assessed. Next, we administered an i.v. injection of calf thymus-derived histones into Wistar rats (12.5 µg/g weight of 8-week-old female rats), and 2 h later they were given an i.v. injection of aPS-PT mAb (1.25 mg/g weight, n = 6) or an equal dose of rat IgM as controls (n = 5). Three days later, histological examination was conducted. Results. Calf thymus-derived histones (>12.5 µg/ml) could injure RECs in vitro. Simultaneously, annexin V could bind to the RECs; thereby, this result indicated that cell-free histone exposure of vascular endothelial cells induced cell surface expression of PS, which is naturally present inside the plasma membrane. Thrombosis developed with higher frequency in the rats given an i.v. injection of aPS-PT mAb than in controls. Conclusion. We established a rat model of thrombosis induced by i.v. injection of aPS-PT mAb.

Brain-derived neurotrophic factor (BDNF) is a well-known humoral protein that induces growth of neurons. Recent studies have suggested that BDNF could act as an angiogenesis inducer similar to vascular endothelial growth factor (VEGF). Angiogenin is a strong mediator of angiogenesis. It has particular characteristics both as a secreted protein and a transcription factor. After being incorporated into the cytoplasm, angiogenin is immediately transferred to the nucleus and then mediates the angiogenic effects of angiogenesis inducers, including VEGF. The aim of this study is to determine the association between BDNF and angiogenin. At first, we determined the secretion of angiogenin from human umbilical vein endothelial cells (HUVEC) induced by BDNF with enzyme-linked immunosorbent assay. Next, we determined BDNF-induced nuclear translocation of angiogenin by immunofluorescent staining. In addition, we examined the mRNA expression of angiogenin in HUVEC before and after BDNF stimulation by quantitative reverse transcriptase-polymerase chain reaction. As a result, we noted that BDNF induced angiogenin secretion and nuclear translocation without an increase in the mRNA expression in HUVEC. Furthermore, we demonstrated that BDNF-induced HUVEC proliferation was significantly suppressed when neomycin, a specific inhibitor of nuclear translocation of angiogenin, was administered. These findings indicate that nuclear translocation of angiogenin is critically involved in BDNF-induced proliferation of HUVEC. In conclusion, angiogenin contributes to angiogenesis induced by BDNF.

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