No abstract

Immunoglobulin (Ig) gene promotors are active only in cells of the B‐lymphocyte lineage. Transfection experiments have shown that this is due in part to tissue specific ‘activating’ DNA sequences, so called enhancers. It is not entirely clear whether these sequences are necessary for initial activation or also for maintenance of transcription of a gene. We describe here the isolation and characterisation of a mouse hybridoma cell line that has deleted in vitro the ‘activating’ sequence from the active IgH locus, the only IgH locus it contains. Nevertheless, Ig heavy chain production of the variant cell is not impaired and remains comparable with that of other hybridoma cells. Therefore, a high rate of Ig heavy chain production in antibody‐producing cells is either independent of any sequences enhancing transcription or else these can easily be replaced by other DNA sequences with a similar function that have been moved into the vicinity of the V region.

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

The production of cytokines was analysed in Hodgkin's disease (HD) derived cell lines by enzyme linked immunosorbent tests (ELISA) and Northern blot experiments. Our results demonstrate that HD derived cell lines produce a variety of cytokines, such as IL1 alpha, IL4, IL5, IL6, IL8, TNF alpha, TNF beta and GM-CSF but not IL1 beta, IL2, IL3 and G-CSF. In cell lines with a high expression of CD25 (the light chain of the IL2 receptor), we found soluble IL2 receptors in the supernatants. In addition, receptors for IL6 could be detected in most of the HD derived cell lines. However the growth of HD derived cell lines, which produce IL6 and IL6 receptors could not be inhibited by anti-IL6 antibodies. From our data we conclude, that IL6 and additional cytokines may be involved in the biology of HD.

The comparative analysis of Ig class switch recombination in a priori IgG/IgA-expressing myelomas and hybridomas, in switch variants and in activated normal B cells shows the following characteristics of class switch recombination in activated B cells: It is prevented during most of B cell ontogeny. It happens on both IgH loci of activated and switched B cells. The recombination is programmed in that on both IgH loci of switched cells the same switch regions recombine with Smu. This is true at least for the IgG1 pathway. IgM-expressing cells show no class switch recombination on the inactive IgH locus. Thus, physiological class switch recombination is a programmed rather than a random event and is controlled as such. The initial stages of class switching and the molecules involved in these are largely unclear: What is the nature of the protection of switch regions and how is this protection abrogated? Do specific recombinases exist? What is the role of large transcription units? Is the specificity of class switch recombination a result of specific "opening" of the DNA for transcription? Do all B cells use the same switch mechanism? What is the role of switch factors (such as lymphokines)? These and more questions await answers and although a variety of switch scenarios could be discussed at present a detailed speculation seems premature.