Grant-in-Aid for Scientific Research (C)
|Research Institution||The University of Tokyo|
TSUNO Nelson The University of Tokyo, Dept.Transfusion Medicine, Professor, Assistant, 医学部・附属病院, 助手 (50282637)
OSADA Takuya The University of Tokyo, Dept.Transfusion Medicine, Professor, Assistant, 医学部・附属病院, 助手 (90272559)
SHIBATA Yoichi The University of Tokyo, Dept.Transfusion Medicine, Professor, Chairman, 医学部・附属病院, 教授 (30010474)
|Project Fiscal Year
1997 – 1998
Completed(Fiscal Year 1998)
|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥2,000,000 (Direct Cost : ¥2,000,000)
|Keywords||Endothelial cells / Autoantibodies / Autommune diseases / Mixed-passive hemagglutination (MPHA) assay / Human platelet antigens (HPAs) / Flow-cytometry / Western blotting / 内皮細胞 / 自己抗体 / 自己免疫疾患 / MPHA / 血小板特異抗原(HPA) / フローサイトメトリー / Western Blotting / Western Biotting / 抗内皮細胞抗体 / MPHA法 / Human Platelet Antigen (HPA) / 臓器移植|
Autoantibodies to many cell surface, intracytoplasmic or even nuclear antigens have been implicated in the development of autoimmune diseases. Recently, anti-neutrophil cytoplasmic antibody has been associated with the development of autoimmune diseases such as rapidly progressive nephropathy.
Endothelial cells are a monolayer of flat cells covering the internal surface of all blood cells, and consequently are systemically distributed and are abundantly present in many organs. Anti-endothelial cells have also been reported in many autoimmune diseases such as connective tissue disease and vasculitis, but there is no consensus yet on its clinical significance. There is consensus on the fact that there is a need for standardization for determining the precise pathophysiologic and immunologic role of anti-endothelial cell antibodies. Another important point is the need of a stable methodology for their detection.
In this project, we aimed to develop a stable and sensitive system for detectio
n of anti-endothelial antibodies and investigate the clinical significance of these antibodies in human diseases.
(1) Development of an anti-endothelial cell antibody detection system.
Firstly endothelial cells were isolated from human umbilical veins (HUVEC) and transferred to the culture system. Freshly isolated HUVEC were cryopreserved until use. Using the PCR technique, the HUVEC were typed for ABO blood group arid Human Platelet Anti germ (HPA). Group O HUVEC were seeded on U-bottomed 96-well microtiter plates in MCDB-151 medium and the cells were allowed to adhere and spred. Cells were then fixed with gluraldehyde and proceeded for MPHA.
(2) Expression of adhesion molecules on endothelial cells : comparison with flow-cytometry
Expression of adhesion molecules on HUVEC was investigated by MPHA and compared with flow-cytometry. Exactly the same pattern of expression was found.
(3) Screening of Patient sera for anti-endothelial cell antibody
Patient sera was investigated for the presence of anti-endothelial cell antibody using the MPHA.Reactivity with endothelial cells was found in approximately 3-5% of the patient sera.
(4) Characterization of the antigenic determinant of endothelial cell reactive sera
Endothelial cells were homogenized amid the protein extracts were used for western blotting. Endothelial cell reactive sera were used for the immnunoblotting. Almost all sera found to be reactive with endothelial cells recognized a 40-45 kDa protein, suggestive to be the HLA antigen. Also, most of these sera reacted with human platelets and granulocytes, and were found to be anti-HLA antibodies by LCT.
As our objective is to find an autoantibody or alloantibody that specifically reacts with endothelial cell antigens, more stud y is needed.
(5) Detect ion of human platelet antigen (HPA) on endothelial cells
As endothelial cells are similar to human platelets regarding the membrane protein expression, the expression of HPAs on endothelial cells was investigated. Endothelial cells were found to express the HPA-1, HPA-4 and HPA-5, but not HPA-2 and -3. These results are in accordance with the findings that GPIIb and GPIa are not expressed on HUVEC.An interesting finding was that HPA-4b is expressed on HUVEC but HPA-4a is riot. Considering that HPA-4 is found on the glycoprotein GPIIIa, and this glycoprotein exists in two forms, i.e., complexed with GPIIb or alphaV, we can speculate that the GPIIbIIIa complex is necessary for the reactivity of anti-HPA-4a antibodies. More studies are needed to clarify this phenomenom. Less