M. G. Gregoretti

Cytokines involved in the progression of multiple myeloma.

We have investigated which of the cytokines that are relevant in the in vitro growth of multiple myeloma (MM) malignant plasma cells are actually produced in vivo by MM patients. To this end, we have measured the levels of IL‐1β, IL‐3, IL‐4, IL‐6, IL‐7, IL‐8 and tumour necrosis factor‐alpha (TNF‐α) both in sera and in the supernatant of bone marrow (BM) stromal cell cultures from patients with MM and monoclonal gammopathy of undetermined significance (MGUS). The significance of our findings is three‐fold. First, IL‐6 and IL‐8 are produced by MM BM stromal cells, while IL‐1β, TNF‐α, IL‐4 and IL‐7 are not. Second, IL‐3 is the only cytokine consistently raised in serum samples; we have also detected low levels of serum IL‐6 in a minority of cases, usually in advanced stage of the disease. Third, MM BM stromal cells are active IL‐6 and IL‐8 producers, while both normal and MGUS BM stromal cells are low producers, thus suggesting that in the BM of MM a number of environmental cells, that would normally be quiescent, are instead activated and that, in MM, activated BM stromal cells play an active role in supporting the progressive expansion of the B cell clone.

Characterization of bone marrow stromal cells from multiple myeloma

We have cultured multiple myeloma (MM) bone marrow (BM) stromal cells that are able to sustain the in vitro growth of monoclonal B-cells. Our aim was to evaluate which adhesion molecules are expressed and which extracellular matrix proteins are produced by these cells and whether they differ from the stromal cells that can be grown under the same experimental conditions from the BM of monoclonal gammopathies of undetermined significance (MGUS) and of normal donors. MM BM stromal cells that support malignant B-cell development have a striking proliferative ability that is absent in MGUS and normal donors of the same age group and are formed by four major different cell populations. Two kinds of HLA-DR+, CD10+ fibroblast-like cells can be recognized through the expression (or the lack) of alpha-smooth muscle actin isoform; further, macrophages and osteoclasts can be identified. Fibroblast-like cells that express alpha-smooth muscle actin isoform, often organized along stress fibers in a periodic fashion, may be considered as myofibroblasts. Fibroblast-like cells react strongly with antibodies to CD54 (ICAM-1), integrin beta 1, beta 3, beta 5 and some of associated alpha chains. Integrin beta 1 is diffusely exposed on the surface while beta 3 is clustered in focal contacts in association with vinculin. A still undetermined subpopulation of fibroblasts is highly positive for alpha v beta 5 that is clustered at focal contacts as shown by association with stress fiber termini and by interference reflection microscopy. A major difference between MM and normal donor BM stromal cells involves lower deposition and simpler organization of the extracellular matrix proteins (fibronectin, laminin, collagen type IV) deposited by MM fibroblast-like cells. CD14+ macrophages from MM, MGUS and normal donor BM are CD11a+ (alpha L), CD11b+ (alpha M), CD11c+ (alpha X), CD54+ (ICAM-1), CD56+ (N-CAM), beta 1 and beta 2 (CD18) integrin positive. The integrin beta 1 is diffusely expressed on the surface, while beta 2 is concentrated in podosomes. MM osteoclasts show a weak diffuse staining with CD54 and CD56 MoAbs; beta 1 integrin has a diffuse surface expression, while beta 3 integrin is concentrated in the podosomes. Normal donor osteoclasts are CD54- and the staining with CD56 is barely visible. These findings lead us to suggest that the microenvironment provided by MM BM may be significantly different from that of normal BM indicating its potential role in controlling the local proliferation and differentiation of malignant B-lineage cells.

Bone Marrow Microenvironment and the Progression of Multiple Myeloma

The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1 beta and M-CSF activating BM stromal cells to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-1 beta, TNF-beta, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population.

BCL-2 in B-chronic lymphocytic leukemia.

B-chronic lymphocytic leukemia (B-CLL) is a human B-cell malignancy characterized by the relentless accumulation of long-lived mature B cells that have two distinctive features. First, more than 99% of the circulating malignant lymphocytes are in the Go phase of the cell cycle (Andreeff et al. 1980; Carlsson et al. 1988). Second, B-CLL cells express the CDS surface molecule (Caligaris-Cappio and Janossy 1985). Crucial to our understanding of the development and natural history of the disease is to define which is the cellular origin of B-CLL and which mechanisms favour the progressive accumulation of malignant resting CD5+ B cells. The phenotype of B-CLL cells (Caligaris-Cappio and Janossy 1985, Freedman and Nadler 1990; Schena et al. 1992) suggests a similarity with mature B lymphocytes that are found in the mantle zone of secondary follicles and lends credit to the hypothesis that the normal counterpart of B-CLL may be a long-lived, recirculating subpopulation of mantle zone B cells (Galton and MacLennan 1982). As, in adult lymphoid tissues, CD5+ B lymphocytes are located within the mantle zone of secondary follicles (Kipps et al. 1991) it is not unreasonable, though still unproven, to suggest that the CD5+ B cell population might be the actual normal counterpart of B-CLL.