The promotion of plasmacytoma tumor growth by mesenchymal stroma is antagonized by basic fibroblast growth factor induced activin A.

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 serum-free 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)beta 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.

A peptide based on the sequence of the CDR3 of a murine anti-DNA mAb is a better modulator of experimental SLE than its single amino acid-substituted analogs.

A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA 16/6 Id(+) monoclonal antibody was previously shown to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated proliferative responses. Single amino acid-substituted analogs of pCDR3 were designed and analyzed for their ability to stimulate or inhibit the proliferation of a pCDR3-specific T-cell line. Alterations in positions 9 and 10 neutralized the proliferative potential of pCDR3, whereas alterations in positions 6-8 and 11-15 retained the proliferative potential of the peptides. Similar to pCDR3, its analogs Ala11 and Nle13 inhibited efficiently the in vivo priming of lymph node cells either to pCDR3 or to the human monoclonal anti-DNA 16/6 Id(+) antibody. Substituting both positions 11 (Tyr --> Ala) and 13 (Met --> Nle) reduced this inhibitory capacity compared to the single substituted analogs. Also, truncation of pCDR3 at the C- and/or N-terminus obliterated the inhibitory activities of the peptide. Analogs Ala11 and Nle13 immunomodulated serological and clinical smanifestations of experimental SLE. Nevertheless, the original pCDR3 was a more efficient modulator of the disease.

Immune response of SLE patients to peptides based on the complementarity determining regions of a pathogenic anti-DNA monoclonal antibody.

We have examined the humoral and cellular responses of SLE patients to peptides based on the complementarity-determining regions (CDR) of a monoclonal anti-DNA antibody with a major idiotype- 16/6 Id, in comparison to their responses to the whole 16/6 Id-bearing antibody. Sera of 63% of the SLE patients had antibodies that bound the 16/6 Id, 80% had antibodies to one of the CDR-based peptides, and 40% of the patients reacted with both CDRs. Sera of only a few controls reacted with either the 16/6 Id (6%) or the CDR based peptides (4%) (P < 0.01). Peripheral blood lymphocytes (PBL) of 39% of the patients proliferated in response to the 16/6 Id or to one of the CDR-based peptides (37%), while in the control group the proliferation rates were 66% to the 16/6 Id and 59% to one of the CDR-based peptides (P < 0.05). The correlation between (both) the humoral and cellular immune responses to the CDR-based peptides and to the 16/6 Id suggests the relevance of these peptides to the 16/6 Id and provides additional information on the pathogenic moiety of the latter antibody.

A peptide based on the CDR3 of an anti-DNA antibody of experimental SLE origin is also a dominant T-cell epitope in (NZBXNZW)F1 lupus-prone mice.

A molecular homology has been demonstrated between sequences of the heavy chain variable regions of the anti-DNA, anti-cardiolipin monoclonal antibody, 2C4C2, isolated from C3H.SW mice with induced systemic lupus erythematosus, and sequences of the anti-DNA monoclonal antibody BW16 originating in the lupus-prone (NZBXNZW)F1 mice. It was of interest to determine whether these homologous sequences function also as immunodominant T-cell epitopes, in order to establish a connection between spontaneous and induced experimental models. Therefore, three peptides were designed and synthesized based on the complementarity determining region (CDR)1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 2C4C2. In the present study, we compare these peptides with the CDR1- and CDR3-based peptides of another murine anti-DNA antibody; namely, 5G12. The comparison was carried out by analyzing the ability of the peptides to induce T-cell activation in (NZBXNZW)F1 lupus-prone mice and in mouse strains susceptible to induction of experimental systemic lupus erythematosus. Immunization of (NZBXNZW)F1 mice with the 2C4C2 mAb or with its CDR-based peptides, as well as immunization with the 5G12-based CDR peptides, induced significant lymph node proliferation to the pCDR3 of the 5G12 mAb. Naive (NZBXNZW)F1 splenocytes exhibited activation to the same peptide. It is also shown that MHC class II molecules of (NZBXNZW)F1 macrophages bind preferentially the 5G12-based pCDR3. It is proposed that the CDR3-based peptide of 5G12 mAb of experimental lupus is also a dominant and relevant epitope in the (NZBXNZW)F1 lupus-prone mice.

Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody.

Peptides 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 specific 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 alphabeta T-cell receptor (TCR) and the CD4+ CD8- phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific 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-specific 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-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.

The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. Identification as a stroma-derived activin A.

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 cytokine-deprived 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.

Restrictin-P/stromal activin A, kills its target cells via an apoptotic mechanism.

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.

Dynamic changes in cytokine secretion by stromal cells during prolonged maintenance under protein-free conditions.

Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities. Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells. We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%). The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable. Stromal cells of the MBA-2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of non-woven fabric matrix. After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in protein-free medium. Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF) and restrictin-P, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells. In addition, interleukin-6 (IL-6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml. Concomitantly, the production of restrictin-P diminished and reached its lowest levels at the end of 10 months. The results may imply a possible causal relationship between the expression of IL-6 and restrictin-P, since no similarly significant changes were observed in the titers of M-CSF and TGF-beta. This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serum-free conditions.

Fertility impairment and improved fetal survival induced by a tumor cell line in mice.

The BL6-T2 tumor, a regressor melanoma line in C57BL/6J mice shown by us previously to be abortifacient, has been cloned in vitro. Clones obtained have been tested in vivo for tumorigenicity and effect on gestations, in order to correlate the immunogenicity of the tumor and its effect on fetal survival. Results demonstrate that high immunogenicity and impairment of fertility do not occur in the same tumor clones. We also show that this tumor produces and secretes GM-CSF and CSF-1 and that trophoblast cells express in situ mRNA encoding for the CSF-1 receptor. We consequently hypothesize that this tumor exerts its abortifacient effect not via its strong immunogenicity but via cytokines it secretes.

Colony-stimulating factor-1 blocks early pregnancy in mice.

To elucidate the mechanisms underlying the suspected immune-related pregnancy failures in humans, we established experimental systems to induce pregnancy blocking and abortion in mice. One system, based on the preimmunization of C57BL/6J females with a syngeneic regressor tumor, is described. Such females fail to develop normal gestations when mated to C57BL/6J x DBA/2 F-1 (B6D2F-1) males or DBA/2 males but sustain normal pregnancies when impregnated by CBA/J or C57BL/6 males. An investigation into the cause of these male-specific pregnancy failures led us to identify colony-stimulating factor-1 (CSF-1) as responsible for both pregnancy-block and resorption of embryos. Indeed, injection of very small amounts of CSF-1 into plugged females, for the first 5 days of pregnancy, was sufficient to block B6D2F-1-induced gestations but had no effect on CBA/J-mated females. It also induced a high rate of fetal resorptions in the sensitive mating. These results suggest a novel mechanism underlying pregnancy failures: a mechanism based on cytokines and their effect on early embryonic development in certain mating combinations.

Restrictin-P: the first member of a putative family of novel inhibitors.

MBA-2.1 cells produce an activity, designated restrictin-P, which is specifically inhibitory to the growth of plasmacytomas and mature B cell lymphomas. We examined whether the activity of this stromally derived glycoprotein could be attributed to a well-characterized growth factor. Restrictin-P-producing cells were therefore screened for the expression of transcripts of a variety of growth suppressors. With the exception of TGF-beta 1, none was produced in detectable amounts by these cells. Furthermore, recombinant forms of the inhibitory molecules tested did not exert a biological effect similar to that of restrictin-P. Restrictin-P was shown to elicit a G0/G1 arrest in the cell cycle of its target cells, as soon as 24 h after their exposure to the inhibitor. This effect could not be mimicked by TGF-beta 1. We suggest that restrictin-P is part of a novel family of inhibitors which are required for the maintenance of cell-type specificities in the hematopoietic microenvironment.