Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Abstract

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.

Long‐term bone marrow cultured stromal cells regulate myeloma tumour growth in vitro: studies with primary tumour cells and LTBMC‐dependent cell lines

“…Recently we and others showed that IL-6 production is induced in LTBMC or osteoblastic cells after cellular interaction of myeloma cells (Uchiyama et al, 1993;Lokhorst et al, 1994;Barillé et al, 1995). Using in situ hybridization we showed that IL-6 was produced by the LTBMC (Lokhorst et al, 1994). These results indicated that growth of the myeloma tumour might be regulated by direct contact with the BM stromal environment.…”

“…LTBMC. LTBMC were grown as described (Lokhorst et al, 1994). Briefly BM mononuclear cells (BMMC) were seeded in 24-well culture plates.…”

“…Both cell lines were plasma cell lines as examined in cytocentrifuge preparations stained with May-Grü nwald-Giemsa, FACS analysis and immunofluorescence microscopy. Protocols for analysis of surface and cytoplasmic antigen expression by flow cytometry (FACScan (Becton Dickinson, Erembodegem-Aalst, Belgium)) or immunofluorescence microscopy on cytospin preparations have been described previously (Lokhorst et al, 1987(Lokhorst et al, , 1994Bloem et al, 1988;Ahsmann et al, 1992). Fluorescent stained preparations were viewed with a Leitz Orthoplan phase-contrast immunofluorescence microscope equipped with K2 and N2 filter systems for FITC and TRITC examination, respectively, and with 4 × eyepieces and 63 × and 95 × objectives.…”

“…Purification of myeloma cells from BM aspirates. Myeloma plasma cells from BM aspirates were purified by highresolution cell sorting on the basis of CD38 expression and forward and orthogonal light scatter properties on a FACStar Plus (Becton Dickinson) as described before (Lokhorst et al, 1994). In short, in the two-parameter dot-plot projection of forward light scatter and orthogonal light scatter an electronic plasma cell gate was created via a back-gating procedure of the bright CD38 positive cells.…”

“…the stage of disease (Merico et al, 1993;Lokhorst et al, 1994). Recently we and others showed that IL-6 production is induced in LTBMC or osteoblastic cells after cellular interaction of myeloma cells (Uchiyama et al, 1993;Lokhorst et al, 1994;Barillé et al, 1995).…”