In this study, flow cytometry was used to evaluate interleukin-6 (IL-6) production by bone marrow mononuclear cells from 47 patients with multiple myeloma (MM) in different clinical stages and 15 patients with monoclonal gammopathy of undetermined significance. In patients with MM, autocrine IL-6 production paralleled the clinical disease stage. The largest proportion of syndecan-1 ؉ /IL-6 ؉ cells was detected in patients with resistant relapse or primary refractory disease, suggesting that tumor progression involves expansion of myeloma cells producing IL-6. The authors assessed autocrine IL-6 production and in vitro proliferation and apoptosis of myeloma cells in 6 myeloma cell clones (MCCs) and in 2 myeloma cell lines, namely IM-9 and U-266-1970, which showed different sensitivities to the addition of exogenous IL-6. Autocrine IL-6 production was observed in IL-6-independent MCC-2, MCC-3, and MCC-5 cloned from patients with aggressive disease and in the IM-9 cell line. In contrast, IL-6-dependent MCC-1, MCC-4, and MCC-6 were syndecan-1 ؉ and IL-6 ؊ . Blocking experiments with anti-IL-6 monoclonal antibody from clone AH65, which binds IL-6-IL-6R␣ complexes, prevented cell proliferation of IL-6 ؉ MCCs. Flow cytometry evaluations after propidium iodide staining revealed different susceptibilities of MCCs to cell death. IL-6-producing MCCs showed minimal spontaneous and dexamethasone-induced apoptosis, whereas a regular amplitude of apoptosis occurred in the IL-6 ؊ MCCs. These data provide evidence that autocrine IL-6 reflects a highly malignant phenotype of myeloma cells. In fact, autocrine IL-6 production and deregulated apoptosis may induce expansion of selective IL-6 ؉ myeloma cells resistant to spontaneous and drug-induced cell death. IntroductionInterleukin 6 (IL-6), a pleiotropic cytokine produced by a variety of cells, is the most important growth factor for human multiple myeloma (MM). 1-3 Several findings support in vivo and in vitro roles for IL-6 in the disease: specifically, (1) serum IL-6 and IL-6R levels were found to correlate with disease activity 4-6 ; (2) therapy with anti-IL-6 monoclonal antibody (mAb) transiently reversed disease manifestations 7 ; (3) in vitro proliferation of myeloma cells was suppressed by neutralizing mAbs to either IL-6 or its cellular receptors [8][9] ; and (4) inactivation of IL-6 messenger RNA by antisense oligonucleotides inhibited proliferation of plasma cells. 10 Furthermore, other cytokines, such as IL-1, IL-3, and granulocytemacrophage colony-stimulating factor, regulate myeloma cell proliferation in synergy with IL-6 11 or by inducing IL-6 production in myeloma cells or the tumor environment. [12][13] The cellular origin of IL-6 is controversial. Several authors [13][14][15][16] showed that it is produced by the myeloma cells themselves (autocrine hypothesis). Other studies, [17][18][19] however, point to its paracrine production by cells in the bone marrow (BM) and suggest that proliferation of myeloma cells depends on close contact with stromal cells. [20][21...

Bone marrow plasma cells and stromal cells in multiple myeloma (MM) have been shown to be capable of releasing cytokines with angiogenic properties. Plasma cells can also express adhesion molecules controlling their adhesive interactions with endothelial cells. In the present study, we have evaluated by immunohistochemistry the extent of angiogenesis in the bone marrow of: a) 51 patients with active and non-active MM; b) 25 patients with monoclonal gammopathy of undetermined significance (MGUS). Plasma cells were investigated by flow cytometry for the expression of the adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. The results showed that, while angiogenesis was very low or absent in patients with MGUS and non-active MM, it increased markedly in those with active MM. The highest detectability of plasma cell adhesion molecules, except LAM-1, was also found in these patients. The functional significance of these findings is unknown. Their consistent occurrence in the bone marrow of active myeloma patients, however, strongly suggests that more frequent adhesive interactions between plasma cells and their microvasculature underlie tumor dissemination.

IL-6 is a growth factor which interferes in the apoptosis of malignant plasma cells. Here we explore its role in the spontaneous and Fas/FasL-regulated apoptosis of seven myeloma cell clones (MCC). MCC-2 and -7 were constitutively defective in Fas antigen in the presence of large membrane exposure of FasL, and showed a high rate of cell proliferation irrespective of the presence of IL-6. Cytofluorimetric analysis following propidium iodide (PI) staining revealed a minimal extent of spontaneous apoptosis, as in other IL-6-insensitive, though Fas-positive MCC, namely MCC-3 and -5. By contrast, a regular amplitude of apoptosis occurred in the remaining IL-6-dependent clones. Their propensity to cell death, as well as their FasL membrane expression, were promptly down-modulated by the cytokine, whereas no substantial effect was detected in IL-6-independent MCC. Furthermore, we investigated the quantitative secretion of FasL. Both [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) cytotoxicity assay and PI staining of WC8 lymphoblasts from a Fas-transfected mouse lymphoma, incubated with supernatants from MCC, showed a variable cytocidal property, thus confirming the cellular release of FasL. However, a significant elevation of FasL secretion occurred in both Fas- MCC, whereas molecular cloning and sequencing of Fas revealed the presence of a splicing variant, namely Fas Exo4,6Del, in the cDNA from both MCC-3 and -5, which were previously demonstrated to be unresponsive to Fas stimulation. Taken together, these data provide evidence that concurrence of IL-6 insensitivity and deregulation of apoptosis in myeloma cells reflects a high malignancy grade. It is suggested that the secretion of Fas splicing variants in Fas+ plasma cells, as well as the over-production of FasL in Fas- myelomas, are differential mechanisms by which myeloma cells escape host immune surveillance.

Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production. © 1998 by The American Society of Hematology.

SUMMARYTmmunofluorescence (IF) to detect HCV antigens and non-isotopic in situ hybridization (NISH) to detect HCV RNA genome were carried out on bone marrow (BM) and peripheral blood (PB) mononuclear cells (MC) of 11 chronically HCV-infected patients. In four patients (36.4%) HCV antigens were detected in monocytes/macrophages as well as in B lymphocytes in both BMMC and PBMC. Positive T lymphocytes in BMMC were found in three of them, but only one patient showed positive T cells in PBMC. NISH invariably demonstrated minus and plus HCV RNA genomic strands either in monocytes/macrophages or B and T lymphocytes in BMMC and PBMC in the four HCV antigen-positive patients and in two further patients not expressing viral proteins in blood MC. I F signals appeared diffusely distributed within the cytoplasm, or as brilliant granules in distinct submembrane areas or else in cytoplasm membrane. Nuclei never stained. Similarly, NISH displayed HCV RNA accumulation restricted to MC cytoplasm only, nuclei being persistently negative. NISH, however, was unable to detect cell membrane signal. Infection of blood MC is a common event in naturally acquired HCV infection, since none of these patients was conditioned by immunomodulating or immunosuppressive therapies. No difference was found in terms of mean age, length of disease, anti-HCV immune response, type and severity of chronic liver damage between patients with HCV-infected MC and patients without cell infection. These results demonstrate that HCV can infect BMMC and PBMC that represent important extrahepatic sites of virus replication, and may help to explain the immunological abnormalities observed in chronic HCV carriers.Keywords chronic liver disease hepatitis C virus immunofluorescence in situ hybridization mononuclear cells INTRODUCTIONAt least 50% of HCV-infected individuals develop chronic