The mechanism of macrophage-mediated antiapoptosis in myeloma cells. (A) Western blot analysis showing the protein expression of cleaved PARP (cPARP), cleaved caspase-3 (cCas-3), Bcl-xL, Bcl-2, Bad, and Bax in ARP-1 myeloma cells cultured alone or cocultured with tMφs in the presence of melphalan (5 μM). The level of β-actin served as loading control. Results from 1 representative experiment of 3 performed with ARP-1 are shown. Similar results were obtained with other myeloma cell lines. (B) Levels of IL-6 in normal medium, TCCM, and in the supernatants of nMφs and tMφ, measured by enzyme-linked immunosorbent assay. (C) Dot plots showing apoptotic myeloma cells in culture medium (Med), in cocultures with tMφ, and in coculture with tMφs and IL-6–neutralizing antibody (αIL-6) in the presence or absence of melphalan (Mel). Numbers inside dot plots indicate the percentages of live cells. (D) Percentages of melphalan (Mel)–induced, annexin V–positive apoptotic myeloma (ARP-1) cells in culture medium only (Med) or in cocultures with untreated tMφs or with cytochalasin D (CD)–pretreated tMφs (CD-tMφ). Infiltration of Mφs in the bone marrow of myeloma patients. (E) Immunochemistry staining by CD68 antibody to identify Mφs in bone marrow biopsies from a control patient without malignancy and from a randomly selected myeloma patient. Representative results from experiments with bone marrow biopsies from 1 of 4 myeloma and 4 control patients examined are shown. (F) Percentages of infiltrated Mφs in bone marrow biopsies of MM and control patients. Data were derived from the numbers (mean ± SD) of CD68⁺ Mφs in a total of 1000 cells counted in bone marrow biopsies of 4 patients with MM and 4 controls. **P < .01.