transforming-growth-factor-beta and Mast-Cell-Sarcoma

In 58 patients with mast cell disease (MCD) and three with basophilic leukemia, bone marrow (54 cases) or skin tissue (four cases) was studied immunohistochemically for expression of Kit (c-kit protein), the different isomers of transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), and their respective receptors. Kit was expressed in all cases of MCD but in none of basophilic leukemia. Expression pattern of cytokines and their receptors was variable in systemic MCD with (SMCD-HD) or without (SMCD) associated hematologic disorder. However, type I TGF-beta receptor (TGFbeta1R) was not expressed in 30% of SMCD-HD patients or in patients with mast cell leukemia, but the remaining cases of MCD showed near uniform expression. The associated hematologic disorders in TGFbeta1R-negative cases of SMCD-HD were prognostically less favorable than those associated with TGFbeta1R-positive cases of SMCD-HD. The results confirm the diagnostic value of Kit immunohistochemistry in MCD and suggest a biologically relevant heterogeneity in TGFbeta1R expression among patients with SMCD-HD.

Topics: Bone Marrow; Cytokines; Fibroblast Growth Factor 2; Humans; Immunoenzyme Techniques; Leukemia, Mast-Cell; Mast-Cell Sarcoma; Mastocytosis; Proto-Oncogene Proteins c-kit; Retrospective Studies; Serine Endopeptidases; Transforming Growth Factor beta; Tryptases

Our prior work shows that cultured BR cells derived from dog mastocytomas secrete the 92-kDa proenzyme form of gelatinase B. We provided a possible link between mast cell activation and metalloproteinase-mediated matrix degradation by demonstrating that alpha-chymase, a serine protease released from secretory granules by degranulating mast cells, converts progelatinase B to an enzymatically active form. The current work shows that these cells also secrete gelatinase A. Furthermore, gelatinases A and B both colocalize to alpha-chymase-expressing cells of canine airway, suggesting that normal mast cells are a source of gelatinases in the lung. In BR cells, gelatinase B and alpha-chymase expression are regulated, whereas gelatinase A expression is constitutive. Progelatinase B mRNA and enzyme expression are strongly induced by the critical mast cell growth factor, kit ligand, which is produced by fibroblasts and other stromal cells. Induction of progelatinase B is blocked by U-73122, Ro31-8220, and thapsigargin, implicating phospholipase C, protein kinase C, and Ca2+, respectively, in the kit ligand effect. The profibrotic cytokine TGF-beta virtually abolishes the gelatinase B mRNA signal and also attenuates kit ligand-mediated induction of gelatinase B expression, suggesting that an excess of TGF-beta in inflamed or injured tissues may alter mast cell expression of gelatinase B, which is implicated in extracellular matrix degradation, angiogenesis, and apoptosis. In summary, these data provide the first evidence that normal mast cells express gelatinases A and B and suggest pathways by which their regulated expression by mast cells can influence matrix remodeling and fibrosis.

Topics: Animals; Chymases; Collagenases; Dogs; Drug Synergism; Enzyme Induction; Gelatinases; Lung; Mast Cells; Mast-Cell Sarcoma; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Organ Specificity; Serine Endopeptidases; Signal Transduction; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

We examined the role of the CD28/B7 interaction in regulation of NK cell activity. Cells transfected with B7 enhanced IL-12-induced production of IFN-gamma by IL-2-activated, CD28+ NK cells, but not by resting CD28- NK cells. The ability of B7 transfectants to enhance NK cell production of IFN-gamma was dependent on the intracellular adhesion molecule-1/LFA-1 interaction and could be inhibited by TGF-beta, but not IL-10. Since IL-12-induced production of IFN-gamma by NK cells is associated with resistance to certain infections, we examined whether the CD28/B7 interaction regulated NK cell responses during infection. Infection of SCID mice with Toxoplasma gondii resulted in the appearance of a population of CD28+ NK cells, NK cell production of IFN-gamma, and increased NK cell cytolytic activity. Administration of CTLA4-Ig to SCID mice infected with T. gondii inhibited these latter two effects and resulted in a significant increase in parasite burden. The stimulus for CD28 expression by NK cells in SCID mice infected with T. gondii appeared to be independent of IL-2. However, mRNA for IL-15, a cytokine with properties similar to those of IL-2, was detected in tissues of SCID mice infected with T. gondii. In vitro experiments demonstrated that IL-15 could stimulate resting NK cells to express functionally active CD28 as well as enhance the production of IFN-gamma by SCID splenocytes stimulated with T. gondii. Together our data demonstrate that the interaction of CD28+ NK cells with B7 regulates NK cell production of IFN-gamma associated with resistance to infection and that IL-15 may be involved in these events.

Topics: Abatacept; Animals; Antigens, CD; Antigens, Differentiation; B7-1 Antigen; CD28 Antigens; CTLA-4 Antigen; Female; Immunoconjugates; Injections, Intraperitoneal; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-15; Interleukin-2; Killer Cells, Natural; Lymphocyte Activation; Lymphocyte Function-Associated Antigen-1; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, SCID; Toxoplasma; Toxoplasmosis, Animal; Transfection; Transforming Growth Factor beta

Spontaneous and induced fetal resorptions have been associated with the infiltration and activation of GM1-positive natural killer (NK)-like cells. Predominance of these cells in the decidua and their reduced lytic activity suggest that regulation of their killing activity could be important for the survival of the fetus. It has therefore been hypothesized that the embryo was regulating NK lytic activity. To test this hypothesis, human and rabbit lymphocytes were cultured with various concentrations of interleukin-2. Their ability to kill 51Cr-labelled NK and lymphokine-activated killer (LAK)-sensitive targets was assessed in the presence of rabbit blastocoelic fluid taken at day-12 of pregnancy (BF D-12). BF D-12 dramatically suppressed the killing activity of NK and LAK cells. This effect was observed on K562 (NK-sensitive targets), P815 cells (LAK-sensitive targets), and freshly isolated cells in rabbit trophoblastic cell preparation. Elimination of prostaglandin E2 (PGE2), but not transforming growth factor beta 2 (TGF beta 2) or 6 keto prostaglandin F1 alpha (6KPGF 1 alpha), by affinity chromatography, completely abolished BF biological activity. These findings clearly suggest that PGE2 in BF regulates the killing activity of NK and LAK cells, and that the semiallograft embryo plays an active role in its own protection. To our knowledge, it is the first demonstration that PGE2 from the embryo inhibits NK and LAK cell lytic activity.

Topics: Animals; Blastocyst; Body Fluids; Cytotoxicity, Immunologic; Dinoprostone; Female; Humans; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Mast-Cell Sarcoma; Mice; Pregnancy; Rabbits; Transforming Growth Factor beta; Tumor Cells, Cultured

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.

Topics: Animals; Calcium; Cell Adhesion; Cyclic AMP; Cycloheximide; Dose-Response Relationship, Drug; Endothelium, Vascular; Ethers, Cyclic; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Protein Kinases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor beta (TGF-beta) is a potent immunosuppressive cytokine that is produced by neoplastic and normal cells. It has not been demonstrated directly, however, that TGF-beta can inhibit antigen-specific T-cell responses to tumor cells in vitro. We show here that generation of antitumor cytotoxic T-lymphocyte (CTL) activity in mixed-lymphocyte tumor cultures of splenocytes from DBA/2 mice immunized with the syngeneic P815 mastocytoma + Corynebacterium parvum was consistently and profoundly inhibited when 0.675 to 10 ng/ml of TGF-beta were added on Day 0 of culture. TGF-beta added on Day 1 or later had little or no effect. In contrast to the results with P815 immune mice, mixed-lymphocyte tumor cultures established with splenocytes from P815 tumor-bearing hosts showed variable degrees of inhibition by TGF-beta, depending on the stage of the ongoing in vivo immune response. Addition of recombinant murine tumor necrosis factor alpha (1,000 or 10,000 units/ml) partially reversed inhibition of CTL responses by TGF-beta, while recombinant interleukin 2 nearly completely reversed the suppression. These data indicate that one level at which TGF-beta may act to inhibit mixed-lymphocyte tumor cultures is that of cytokine production. To determine whether TGF-beta also has any direct effect on CTL, P815-specific CTL clones derived from tumor-bearing host mice were utilized. We found that proliferation of rested CTL clones in response to tumor cells + interleukin 2 was inhibited by 5 ng/ml of TGF-beta, while the interleukin 2-dependent reactivation of cytolytic activity was not affected by TGF-beta. In contrast to rested CTL, when TGF-beta was added to cultures of previously activated CTL, proliferation was not inhibited. These data demonstrate that TGF-beta has profound inhibitory effects on the in vitro generation of effector CTL from tumor-specific murine splenocytes, and this inhibition may be an indirect result of suppressed cytokine production as well as a direct antiproliferative effect on CTL.

Topics: Animals; Immunologic Memory; Interleukin-2; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; T-Lymphocytes; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF beta) promotes deposition of extracellular matrix and is associated with fibrotic conditions both in experimental animals and in humans. Although a role for mast cells has been suspected in the pathogenesis of fibrosis, no potent mediator capable of stimulating fibroblast growth or extracellular matrix deposition has been identified in mast cell supernatants. We report here the constitutive production of TGF beta 1 by four dog mastocytoma cell lines. TGF beta 1 was identified by characteristic biologic activity, blockade of biologic effect by specific neutralizing antibody, and by recognition of a band with the appropriate migration by western blot. TGF beta 1 mRNA, but not TGF beta 2 or TGF beta 3 mRNA, was also produced constitutively by all four cell lines. Quantitation by bioassay revealed baseline TGF beta secretion of approximately 1 ng/10(6) cells over 48 h. Stimulation of mastocytoma cells with phorbol ester increased the rate of release of TGF beta 1, most markedly in the first 30 min after stimulation, without increasing TGF beta 1 mRNA. Dog mastocytoma cells produced TGF beta 1 primarily in a latent form, inactive until treated with acid. Both pure TGF beta 1 and TGF beta-containing mastocytoma cell-conditioned media inhibited mitogenesis and proliferation in dog mastocytoma cell lines, suggesting that mast cell tumor lines would not grow preferentially based on their ability to produce TGF beta. These studies may make possible further investigation of the mechanism by which mast cells contribute to the induction of fibrosis.

Topics: Animals; Cell Division; Dogs; Mast-Cell Sarcoma; RNA, Messenger; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured

Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Understanding the mechanisms which prevent maternal immunological rejection of the fetus remains a fundamental unsolved problem in immunology. We have previously reported that macrophages are inhibited by maternal decidual stromal cells residing at the maternal-fetal interface. In view of the central role of macrophages in cell-mediated immunity, this inhibition may contribute to preventing maternal antifetal responses. We now report that it was the solid phase signals embedded in the extracellular matrix (ECM) made by decidual cells which are responsible for inhibiting macrophage-mediated lysis of TNF-alpha-resistant P815 mastocytoma cells. The latter macrophage function is acquired after stimulation by interferon gamma and endotoxin. All these macrophage functions were also inhibited by ECM isolated from the Engelberth-Holm-Swarme (EHS) tumor. This tumor ECM has a similar biochemical composition to decidual ECM. This ECM inhibited the effector, as opposed to the stimulator, phase of macrophage-mediated tumor lysis. Laminin, type IV collagen, and heparan sulfate proteoglycans, the major known components of decidual and EHS ECMs, did not inhibit the above macrophage functions. Altogether these data indicate that macrophages were inhibited by solid phase signals embedded in decidual and EHS ECMs. Whether the solid phase signals in these two ECMs are biochemically identical remains to be determined. To our knowledge, such signals are a novel pathway of inhibiting macrophage functions which may be important in understanding the maternal-fetal immunologic relationship, and the pathogenesis of perinatal infections. Furthermore, the ability of EHS tumor ECM to inhibit macrophage functions may indicate that some tumors may defend themselves against host macrophage responses using solid phase signals. This may be important in understanding some host-tumor relationships.

Topics: Animals; Chondroitinases and Chondroitin Lyases; Collagen; Cytotoxicity, Immunologic; Decidua; Extracellular Matrix; Female; Heparin Lyase; Hyaluronoglucosaminidase; Immunity, Cellular; Laminin; Listeria monocytogenes; Macrophages; Male; Mast-Cell Sarcoma; Mice; Mice, Mutant Strains; Neoplasms, Experimental; Polysaccharide-Lyases; Rats; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Cell Division; Cell Line; Cytoplasmic Granules; Dogs; Mast Cells; Mast-Cell Sarcoma; Transforming Growth Factor beta