SummaryThe effect of nerve growth factor (NGF) on proliferation/differentiation of mast cells was investigated in vitro. Although NGF alone neither supported colony formation of bone marrow-derived cultured mast cells (BMCMC) nor induced development of mast cell colonies from nonadherent bone marrow cells (NBMC), addition of NGF to the suboptimal dose of interleukin 3 (IL3) significantly increased the numbers of mast cell colonies produced by BMCMC or NBMC in methylcellulose. When stimulated by IL3 alone, cells in mast cell colonies were not stained by berberine sulfate, a fluorescent dye. In contrast, mast cells developing in methylcellulose cultures obtaining both 11,3 and NGF were stained by berberine sulfate. The fluorescence was abolished by the treatment of heparinase but not of chondroitinase ABC, suggesting that mast cells stimulated by 11,,3 and NGF produced and stored heparin proteoglycan . The histamine content of BMCMC maintained by IL-3 was also increased by addition of NGF. Since BMCMC showed mucosal mast cell-like phenotype, NGF appeared to induce the phenotypic change to connective tissue-type mast cells (CTMC) . In the culture containing BMCMC, 3T3 fibroblasts, and IL-3, the phenotypic change of BMCMC to CTMC was observed as well . Since NGF was detected in this coculture and since addition of anti-NGF monoclonal antibody suppressed the phenotypic change, NGF produced by fibroblasts appeared to induce the phenotypic change. Neither BMCMC alone nor IL3 alone increased the concentration of NGF. Therefore, there is a possibility that BMCMC stimulated by IL3 may induce the production and/or release of NGF by fibroblasts .

We investigated the inhibitory activity of nerve growth factor (NGF) on apoptosis of rat peritoneal mast cells (PMCs) and compared it with that of recombinant stem cell factor (rSCF), which is a mast cell growth factor. When PMCs were incubated up to 72 hours in the presence of control medium, internucleosomal fragmentation of DNA indicating apoptosis was detected by agarose gel electrophoresis and flow cytometry. The aged PMCs showed morphological changes typical for apoptosis, such as chromatin condensation and loss of microvilli of the cell membrane. Addition of NGF or rSCF prevented development of the characteristic DNA fragmentation and decreased the proportion of apoptotic cells with low DNA content values in a dose-dependent manner. Polyclonal antibody to NGF completely abolished the inhibitory activity of NGF but not of rSCF. NGF-induced PMCs were in the G0/G1 phase of the cell cycle, but rSCF transited them from the G0/G1 phase to the S/G2M phase, suggesting that NGF, unlike rSCF, may have no proliferation activity to PMCs. By flow cytometric analysis with antibodies to NGF receptors p75LNGFR and p140trk, we defined that PMCs expressed p140trk but not p75LNGFR. Addition of herbimycin A or K-252a, tyrosine kinase inhibitors, to NGF resulted in blockage of the NGF-induced p140trk phosphorylation and restriction of the inhibitory activity of NGF on apoptosis of PMCs. These results indicated that NGF suppressed apoptosis of rat PMCs through the p140trk tyrosine phosphorylation and possessed no proliferative activity. Thus, NGF may act as a key factor to promote survival of connective tissue-type mast cells.

A cell line (KMY-J) was established from a transplantable tumor (MM-KMY) derived from a spontaneous malignant meningioma arising in an aged F344 rat, and three cloned cell lines (KMY-1, KMY-2 and KMY-3) were induced from the parent KMY-J. Morphologically, KMY-J and tumors induced in syngeneic rats by KMY-J showed cell pleomorphism. All neoplastic cells in KMY-J and its tumors were immunoreactive to vimentin; occasional cells reacted to ED1 (rat macrophage/histiocyte-specific antibody) and alpha-smooth muscle actin (alpha-SMA), indicating expression of histiocytic or myofibroblastic immunophenotypes of meningioma cells. In contrast, KMY-1, KMY-2 and KMY-3 consisted of a uniform cell population differing from each other. KMY-1-induced tumors were similar histologically to meningeal fibrosarcomas. Dendritic cells seen in KMY-2 cultures gave an appearance of arachnoid trabecular cells. In KMY-3 and its tumors, large round cells and multinucleated giant cells were predominant. Cells of these cloned cell lines also reacted to vimentin, but were negative for ED1 and alpha-SMA. By the bioassay using PC12 cells and reverse transcription-polymerase chain reaction for nerve growth factor (NGF) mRNA, production of NGF was demonstrated in the parent and cloned cell lines. The present cell lines may prove useful for studying the histological features of meningeal tumors and the bioactive factors produced by meningeal cells.

No abstract

The effect of 2.5S nerve growth factor (NGF) on survival, phagocytosis, and superoxide production of murine neutrophils was examined and compared with the effects of interleukin-3 (IL-3) and recombinant GM colony-stimulating factor (rGM-CSF). NGF enhanced the viability of neutrophils isolated from peripheral blood and peritoneal cavity in a dose-dependent way. IL-3 50 U/mL had no effect. rGM-CSF 50 U/mL had an effect similar to that of 50 ng/mL NGF. NGF also enhanced the phagocytosis of hydrophilic microspheres by peritoneal neutrophils, and the activity of NGF was greater than that of IL-3 or rGM-CSF. NGF enhanced the superoxide production induced by phorbol 12-myristate 13- acetate (PMA), which acts at postreceptor sites, and that induced by opsonized zymosan, a receptor-mediated ligand. The stimulating activity of NGF was largely comparable to that of rGM-CSF. The present data show that NGF bound to neutrophils enhances their survival, phagocytosis, and superoxide production. Thus, we postulate that NGF plays an important role in the inflammatory processes.