Upregulation of osteoblast apoptosis by malignant plasma cells: a role in myeloma bone disease Franco Silvestris, Paola Cafforio, Marco Tucci, Daniela Grinello and Franco Dammacco Department of Internal Medicine and Oncology (DIMO), University of Bari, Bari, Italy Received 8 November 2002; accepted for publication 10 February 2003 Summary. Typical features of multiple myeloma (MM) are osteolytic lesions and severely affected bone regeneration. This study of 53 MM patients demonstrates an enhance- ment of osteoblast cytotoxicity by malignant myeloma cells via the upregulation of apoptogenic receptors, including Fas ligand (Fas-L) and tumour-necrosis-factor-related apoptosis inducing ligand (TRAIL). Both were significantly increased in the marrow myeloma cells of patients with extensive osteolytic lesions in a fashion similar to the highly malig- nant human myeloma cell line MCC-2. Osteoblasts from these subjects over-expressed Fas and death receptor (DR) 4/5 and underwent dramatic apoptosis when co-cultured with either MCC-2 or autologous myeloma cells. In osteo- blast and myeloma cell co-cultures, monocyte chemoat- tractant protein 1 (MCP-1) mRNA was upregulated in osteoblasts from patients with severe bone disease in parallel with increased CC-chemokine receptor R2 (CCR2) expres- sion, the ligand of MCP-1, in the myeloma cells. This chemokine was shown to activate malignant cell migration in vitro. An upregulation of ICAM-1 expression occurred in osteoblasts from patients with active skeleton disease. This upregulation appeared to be an effect of malignant plasma cell contact, as MCC-2 co-culture greatly enhanced ICAM-1 production by resting osteoblasts from patients without skeleton involvement. Our results suggest that osteoblasts in active myeloma are functionally exhausted and promptly undergo apoptosis in the presence of myeloma cells from patients with severe bone disease. It is suggested that this cytotoxic effect plays a pivotal role in the pathogenesis of defective bone repair. Keywords: apoptosis, Fas-L, multiple myeloma, osteoblasts, TRAIL. Multiple myeloma (MM) is characterized by marrow expan- sion of a malignant plasma cell clone, resulting in the gradual replacement of the haematopoietic matrix and dissemination of osteolytic lesions as nests of tumour cells (Hallek et al, 1998; Callander & Roodman, 2001) 1 . Malig- nant plasma cells are apparently engaged in the deregula- tion of bone resorption by inducing stromal cells to secrete a number of osteoclast activating factors, such as interleukin 1b (IL-1b), IL-6 and tumour necrosis factor (TNF)-b (Michigami et al, 2000). These cytokines stimulate osteo- blasts to enhance the production of trans-membranous RANKL [receptor activator of nuclear factor (NF)-kB ligand] (Lacey et al, 1998; Yasuda et al, 1998), a major osteoclas- togenic factor that may also be directly secreted by myeloma cells (Oyajobi et al, 1998). However, its functional expres- sion by malignant plasma cells remains controversial (Croucher et al, 2001; Giuliani et al, 2001; Pearse et al, 2001; Sezer et al, 2002). Loss of bone in MM is also explained by inadequate regeneration, due to the inhibition of osteoblast function, as there is no evidence of new bone formation within the skeleton lesions or in their vicinity. Osteoblasts are differ- entiated from fibroblasts by the activation of specific genes encoding osteocalcin, a secreted molecule that modulates osteoblast function (Ducy et al, 1996). There is much recent evidence to suggest that these cells are highly sensitive to apoptosis in response to treatment with either growth factors or inflammatory cytokines (Jilka et al, 1998). Upregulated osteoblast apoptosis may thus contribute to both the defective repair of resorption cavities and poor bone remodelling that is detected in a number of chronic disorders. This assumption is supported by evidence that IL-6 and TNFa, secreted by the rheumatoid arthritis synovium, suppress new bone apposition while inducing subchondral bone resorption by RANKL overproduction (Miyasaka et al, 1988; Arend & Dayer, 1990). In addition, TNFa is a direct inducer of apoptosis in murine osteoblasts in vitro (Kitajima et al, 1996). Correspondence: Franco Silvestris, MD, DIMO, University of Bari, Section of Internal Medicine and Clinical Oncology, P.za Giulio Cesare, 11, 70124 Bari, Italy. E-mail: f.silvestris@dimo.uniba.it British Journal of Haematology, 2003, 122, 39–52 Ó 2003 Blackwell Publishing Ltd 39