Anti-KCA-3, a monoclonal antibody reactive with a rat complement C3 receptor, distinguishes Kupffer cells from other macrophages.
A new mAb, designated anti-KCA-3, was developed against rat Kupffer cells. The reactivity of anti-KCA-3 was restricted to macrophages with preferential binding to Kupffer cells; only a few macrophages in the spleen, lymph nodes, lungs, and intestine stained with the antibody. A very small number of peritoneal resident and exudate macrophages reacted with the antibody and no reactivity was seen within the thymus, skin, heart, kidneys, brain, peripheral blood, and bone marrow. KCA-3 was expressed predominantly by the Kupffer cells in the periportal region rather than in the centrilobular region of the hepatic lobules. The cells in the portal tract did not stain with the antibody. The staining of the cytosmears and FACS analysis of the Kupffer cell fraction isolated from hepatic sinusoidal cells by centrifugal elutriation revealed that as many as 62% and 49% of the cells were stained with anti-KCA-3, respectively. Immunoelectron microscopic study of the liver indicated that expression of KCA-3 on Kupffer cells was limited to the plasma membrane facing the sinusoid rather than the space of Disse. Immunoprecipitation and SDS-PAGE analysis demonstrated KCA-3 to have a m.w. of approximately 50 kDa under both reducing and nonreducing conditions. After treatment of KCA-3 with N-glycanase, there was no significant change in the m.w., indicating KCA-3 was not highly glycosylated. C3b- and iC3b-mediated rosette formation between Kupffer cells and sensitized SRBC was inhibited by the antibody, implying that KCA-3 functioned as a complement C3 receptor or complement receptor-associated molecule. Furthermore, KCA-3 was eluted from C3b-Sepharose but not HSA-Sepharose after incubation with Kupffer cell lysate, indicating that KCA-3 directly binds C3b. The cell distribution, ligand-binding specificity, and biochemical properties of the protein were found to be different from the complement C3 receptors previously described. Because OX42 (antibody reactive with the rat CR3 receptor) inhibited complement C3-mediated rosette formation with peritoneal resident macrophages but not with Kupffer cells, the findings suggest that C3-mediated binding to Kupffer cells and to peritoneal macrophages is mediated by two different receptors. We conclude that anti-KCA-3 recognizes a novel type of complement C3 receptor preferentially expressed on Kupffer cells.
Maruiwa
M
M
Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Mizoguchi
A
A
Russell
G J
GJ
Narula
N
N
Stronska
M
M
Mizoguchi
E
E
Rabb
H
H
Arnaout
M A
MA
Bhan
A K
AK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D003179:Complement C3b / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D007728:Kupffer Cells / Q000276:immunology*; D016177:Macrophage-1 Antigen / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D011233:Precipitin Tests; D051381:Rats; D012397:Rosette Formation
D000911:Antibodies, Monoclonal; D016177:Macrophage-1 Antigen; D003179:Complement C3b