Monoclonal Antibody Against Bone Marrow Stromal Cells Its Production and Characterization

Bone marrow stromal cells play an essential role in the proliferation and differentiation of hematopoietic stem cells (1,2). As a means of analyzing of the bone marrow microenvironment immunohistochemically, we attempted to produce a rat monoclonal antibody against the murine preadipocyte line HI derived from long-term bone marrow culture (LTBMC) of C57BL/6 mice (3,4). A newly established monoclonal antibody, designated R4-A9, was obtained from a hybridoma prepared by fusion of Y.B2/ 3.0Ag20(YO) rat myeloma cells with spleen cells of LEW rats immunized with HI cells. The immunofluorescence of live HI cells showed that the antigen reacting with this antibody was strongly expressed on the cell surface. The specificity of R4-A9 was assessed immunohistochemically on frozen sections of various tissues from normal adult mice. R4-A9 demonstrated specificity for hematopoietic stroma in bone marrow and spleen. No staining was observed in thymus, lymph nodes or other tissues examined, with the exception of Leydig cells in the testis and the endothelium of small arteries in several organs. Detailed immunohistochemical observations at both the light microscopy and electron microscopy level showed that R4-A9 selectively reacted with the sinusoidal endothelium, peri-sinusoidal adventitial cells (5) (adventitial reticular cells (6)) and intersinusoidal reticular cells (5) and the reticular cells of the splenic red pulp. These findings indicate that reticular cells and the endothelium of the bone marrow possess the common cell surface molecules recognized by R4 A9. SDS-PAGE analysis showed that R4-A9-immuno-precipitated proteins had a molecular mass of 100 kDa under reducing conditions. These data show that R4-A9 is highly specific for reticular cells and the endothelium of murine bone marrow and spleen, providing a new approach for detailed structural analysis of the constituents of bone marrow stroma. Acta Pathol Jpn 41: 499–506, 1991.