ceramide-3 and Burkitt-Lymphoma

Fresh biopsy cells from cases of Burkitt's lymphoma (BL) display a homogeneous cell surface phenotype. The cells were found to be reactive with the pan B cell marker B1, and consistently co-expressed the BL-associated glycolipid antigen, BLA, and the common acute lymphoblastic leukemia antigen, CALLA, but lacked the B cell "activation" antigens characteristically expressed on EB virus-transformed normal B cells. Microscopic and cell sorter analysis of cells isolated from a series of fresh normal tonsils have identified a subpopulation of normal B cells carrying the same cell surface markers. That BLA and CALLA could be co-expressed on individual B cells was demonstrated by two-color immunofluorescence (IF) of tonsils in suspension, and immunoperoxidase (IP) staining of serial tonsil sections. These BLA+, CALLA+, "activation" antigen- cells were further characterized as B1+, sIgM+, sIgD-, C3d/EB virus receptor+ and were susceptible to virus-induced transformation in vitro. IF studies on Percoll-fractionated tonsillar cell populations and direct examination of IP-stained tonsil semi-thin sections indicated that the BLA+, CALLA+ cells were localized in germinal centers. Their morphological characteristics matched those of BL cells, and their location within germinal centers was consistent both with the known phenotype of germinal center tonsillar B cells and with the description of BL as a proliferation of centroblasts. We suggest that this population of tonsillar germinal center B cells provides the normal counterpart of BL tumor cells.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; B-Lymphocytes; Burkitt Lymphoma; Cell Separation; Glycosphingolipids; Humans; Neprilysin; Palatine Tonsil; Phenotype

A monoclonal immunoglobulin M (IgM) antibody (38-13) which recognizes Burkitt's lymphoma (BL) cells, by reacting with the neutral glycolipid Gal alpha 1 leads to 4-Gal beta 1 leads to 4-Glc beta 1 leads to 1-ceramide, was recently characterized. This monoclonal IgM was coupled to either ricin A chain or gelonin. The two different immunotoxins obtained retained the apparent immunological specificity of 38-13 IgM, as shown by flow cytofluorometry analysis and complement-dependent cytotoxicity test. The BL Ramos cells and the apparently irrelevant Epstein-Barr virus-containing lymphoblastoid Priess cells were used as targets in in vitro assays of the cytotoxic properties of the two immunotoxins by measuring the inhibition of protein synthesis. Isolated ricin A chain, gelonin, and 38-13 IgM exhibited very low intrinsic cytotoxicity on both target cells. 38-13 ricin A chain and 38-13 gelonin conjugates exerted toxic effects on both target cells which were about 6000-fold and 3000-fold higher than uncoupled ricin A chain and gelonin, respectively. The toxicity of these conjugates almost reached that of intact ricin. On Ramos BL cells, the kinetics of action of the 38-13 ricin A chain conjugate was almost as fast as that of intact ricin, because 50% protein synthesis inhibition was reached after 3 hr. In contrast, the kinetics of action in the non-BL Priess was much slower (50% protein synthesis inhibition after 10 hr). An obviously irrelevant immunotoxin (anti-trinitrophenol IgM-ricin A chain) had no significant cytotoxic effect on BL Ramos and non-BL Priess cells. An excess of D-galactose was shown previously to inhibit the 38-13 IgM from binding to the reactive glycolipid antigen bearing a terminal galactose. An excess of D-galactose (0.1 M) inhibited the cytotoxic effect of the two 38-13 immunotoxins, whereas it did not prevent the cytotoxic effect of the anti-trinitrophenol immunotoxin on the same trinitrophenol labeled target cells. These data suggest that the cytotoxic effect observed with 38-13 immunotoxins on non-BL Priess cells was mediated through their binding to a very low number of antigenic sites undetectable by conventional immunological methods. The main characteristics of 38-13 immunotoxins appear to be their fast kinetics of action and the very low number of antigenic sites required for the expression of their toxic effects. These properties could be related to the glycolipid nature of the reacting antigen. Such glycolipid antigens would repre

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Burkitt Lymphoma; Cell Line; Flow Cytometry; Glycosphingolipids; Humans; Immunoglobulin M; Kinetics; Plant Proteins; Protein Biosynthesis; Ribosome Inactivating Proteins, Type 1; Ricin