Hitoshi Akedo

Distribution of C3-Step Regulatory Proteins of the Complement System, CD35 (CRl), CD46 (MCP), and CD55 (DAF), in Hematological Malignancies

The distribution and levels of three membrane proteins, CD35, CD46, and CD55, which serve as complement regulators, were examined in normal peripheral blood and hematologically malignant cells. CD35 was negative in most leukemia cells regardless of the type of leukemia, although granulocytes, monocytes, and some populations of lymphocytes were CD35+. CD46 was present in all blood cells except erythrocytes, and levels were 2-8 times higher in most leukemia cells than in their mature counterparts, particularly in CML and CLL cells, except for those of B cell lineage. CD55, a widely-distributed phosphatidyl inositol-anchored protein, was more frequently lost in NHL cells than in other types of hematological malignancies. In this review, we discuss the roles, mechanisms, and clinical applications of cell-associated complement regulatory proteins in hematological malignancies.

A monoclonal antibody against human decay-accelerating factor (DAF, CD55), D17, which lacks reactivity with semen-DAF☆

Human decay-accelerating factor (DAF, CD55) is a phosphatidyl inositol-anchored glycoprotein consisting, from the N-terminus, of 4 short consensus repeats (SCR), a Ser/Thr (ST)-rich region providing O-glycosylation sites, and the membrane-anchoring unit. A mAb, named D17, was raised against purified erythrocyte-DAF. This mAb recognized DAF on blood cells and most cell lines as determined by flow cytometry and immunoblotting. Its reactivity was similar to but weaker than that of two other well-characterized mAbs to DAF, IA10 (seeing an epitope within SCR1) and 1C6 (seeing an epitope within SCR3). The reactivity of D17 with erythrocyte DAF became increased by treatment with sialidase/O-glycanase, suggesting that its epitope is located close to the O-glycosylation sites, probably within the ST-rich region or SCR4. D17 barely blocked the decay-accelerating activity of DAF. Using the three mAbs, tissue-associated and soluble forms of DAF were identified by SDS-PAGE/immunoblotting and immunohistochemical staining. IA10 and 1C6 recognized a 50 kDa protein in spermatozoa lysate and two proteins of Mr 70 and 55 kDa, respectively, in seminal fluid. These represented membrane-associated and soluble forms of DAF, which were neither recognized by mAb against membrane cofactor protein (MCP, CD46) and C3b/C4b receptor (CR1, CD35) nor by non-immune IgG. In contrast to IA10 and 1C6, D17 did not recognize either spermatozoa-DAF or seminal plasma-DAF, or the deglycosylated or untreated forms of them. Immunohistochemical analysis showed that testis was stained with IA10 but not with D17.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of protein A-rosette assay for screening of monoclonal antibodies to human complement regulatory proteins.

Mice were immunized with purified membrane cofactor protein (MCP) and its monoclonal antibodies were screened by protein A(PA)-rosette assay. In this assay, the culture supernatants of hybridoma cells were layered over fixed MCP-bearing cells, and after washing, PA-coated sheep erythrocytes were applied as an indicator to these MCP-bearing cells. No purified antigen was therefore required throughout the screening. More than 300 of the supernatants harvested were successfully examined within 6 h. Each resultant antibody consisted of a single subclass of IgG, and reacted only with MCP in both transblotted and surface-labeled materials. The sensitivity of this assay was then assessed with these purified antibodies. As little as 0.5 micrograms of IgG1 or 0.01 micrograms of IgG2a was found to be detectable with more than 30% rosette formation. There were variations among cell lines in the sensitivity to the PA-rosette assay and the sensitivity did not correlate with the quantity of MCP surface expression in any of the cell lines. K562 gave the lowest background (nonspecific rosette formation) and the best specificity for anti-MCP of the 20 MCP-positive cell lines tested. Cell lines suitable for the detection of monoclonal anti-decay-accelerating factor and anti-C3b/C4b receptor were also examined and CCRF-SB and HSB2, and peripheral blood granulocytes, were found to be proper cell lines for screening the decay-accelerating factor and C3b/C4b receptor, respectively. Clones for anti C3b/C4b receptor were successfully obtained using granulocytes by the PA-rosette assay. This method needs no purified antigen and facilitates the rapid screening and purification of positive clones against cell-surface complement regulatory proteins.