A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokinedeprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.

We have recently found that the inhibitor of plasmacytoma cell growth, restrictin-P, is a stroma derived activin A and that it is an antagonist of interleukin-6 and interleukin-11. The present study was aimed at determining the mode by which this cytokine kills its target cells. On addition of the cytokine there was little or no net increase in cell number, depending on the specific target cells. All plasmacytoma cell lines tested exhibited a similar time dependent inhibition of DNA synthesis and a G0/G1 shift in the cell cycle. Electron microscope examination revealed classical apoptotic features i.e. chromatin condensation and membrane blebbing. DNA fragmentation, measured qualitatively and quantitatively, occurred in all cytokine treated plasmacytoma cell lines. Bovine activin A had an identical capacity to reduce cell viability, to induce G0/G1 shift and to cause DNA fragmentation. X-ray microanalysis of intracellular ions revealed an increase in calcium ions, following exposure of plasmacytoma cells to restrictin-P, accompanied by a decrease in phosphor ions. The cytotoxicity of the inhibitor was augmented in an additive manner by cycloheximide (CHX) indicating that the process did not require de novo protein synthesis. This study thus shows that restrictin-P/stromal activin A kills its target cells by inducing apoptosis. This effect was mediated by subnanogram concentrations and therefore may represent one physiological function of this pleiotropic cytokine.

SUMMARYPeptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines speci®c to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the ab T-cell receptor (TCR) and the CD4 + CD8 ± phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their speci®c peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-speci®c T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-speci®c T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-speci®c T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.

The mesenchymal stroma has been shown to play a crucial role in the development of multiple myeloma, partly by secretion of interleukin (IL)-6, that serves as a growth factor for myeloma cells. However, it is still unclear which other stromal molecules are involved in the pathogenesis of this disease. We chose, as a model system, a mouse plasmacytoma cell line, which does not respond to IL-6. We found that the formation of mouse plasmacytoma tumors, in an in vivo skin transplantation model, is facilitated by co-injection of these tumor cells along with a mesenchymal stromal cell. The tumor promoting effect of the stroma was reproduced in an in vitro model; stromal cells induced the proliferation of plasmacytoma cells under serumfree conditions. This growth promotion could not be mimicked by a series of cytokines including IL-6 and insulin-like growth factor (IGF)-I implying a role for yet unidentified stromal factors. The in vivo formation of plasmacytoma tumors was reduced following administration of activin A, a cytokine member of the transforming growth factor (TGF)␤ superfamily. Furthermore, the in vitro growth promoting effect of the stroma was abrogated by basic fibroblast growth factor (bFGF) which induced a higher stromal expression of activin A. Our results thus show that mesenchymal stroma expresses plasmacytoma growth stimulating activities that overcome the low constitutive level of the plasmacytoma inhibitor, activin A. The expression of activin A is upregulated by bFGF rendering the stroma suppressive for plasmacytoma growth. The balance between the expression of these regulators may contribute to mesenchymal stroma activity and influence the progression of multiple myeloma. Leukemia (2001) 15, 1102-1110.

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