Surface immunoglobulin-mediated signal transduction involves rapid phosphorylation and activation of the protooncogene product Raf-1 in human B-cells.

The protooncogene product, Raf-1, is a serine/threonine kinase and has been implicated as an intermediate in signal transduction mechanisms. We examined neoplastic and normal B cells for phosphorylation and activation of Raf-1 protein in response to anti-immunoglobulin antibody (anti-Ig). Anti-Ig induced rapid phosphorylation of Raf-1 protein in both neoplastic B-cells of hairy cell leukemia and normal tonsillar B-cells which proliferated well in response to anti-Ig. The increase in phosphorylation was due primarily to an increase in phosphoserine. The immune complex kinase assay using Histone V-S as an exogenous substrate also showed an increase in Raf-1-associated kinase activity. An inhibitor of protein kinase C, H7, inhibited the proliferation as well as the Raf-1 phosphorylation in response to the proliferative signal of anti-Ig. Further, downregulation of protein kinase C by the treatment with 12-phorbol 13-myristic acid significantly abrogated the induction of Raf-1 phosphorylation. These results suggest that, in human B-cells, Raf-1 protein may be involved in the signal transduction pathway mediated by surface immunoglobulin, and that it may be, at least partially, phosphorylated by activated PKC.

Proliferation of human myeloid leukemia cell line associated with the tyrosine-phosphorylation and activation of the proto-oncogene c-kit product.

We investigated the expression, degree of phosphorylation, and activation of the proto-oncogene c-kit product before and after stimulation with the c-kit ligand in a human factor-dependent myeloid leukemia cell line, MO7E. The culture supernatant of the BALB/3T3 fibroblast cell line, which contains the ligand for the murine c-kit product, was found to stimulate proliferation of the MO7E cell line in a dose-dependent manner. The proliferation was significantly inhibited by a tyrosine kinase inhibitor, genistein. An immunoblot technique with a monoclonal antibody specific for phosphotyrosine, showed that there was rapid, dose-dependent tyrosine-phosphorylation of the c-kit product in response to murine c-kit ligand. Furthermore, the murine c-kit ligand increased autokinase activity of the c-kit product in vitro. Similar results were obtained with human stem cell factor (SCF), a recombinant human ligand for the c-kit product. These results suggest that the phosphorylation and activation of the c-kit product are involved in proliferative signals of some human leukemia cells, as well as of normal hematopoietic cells.

Expression and functional role of the proto-oncogene c-kit in acute myeloblastic leukemia cells.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is thought to play an important role in hematopoiesis. In a series of human acute myeloblastic leukemia (AML), the expression of the c-kit proto-oncogene and its product was studied by means of Northern blot and immunoblot analyses. The c-kit mRNA was expressed in 20 of 25 cases of AML, and in those cases the product of the c-kit proto-oncogene was detected by immunoblotting with anti-c-kit antibody. The expression of c-kit transcripts and protein was barely detectable in normal bone marrow cells as a control. The expression of c-kit transcript did not correlate with the French-American-British classification nor clinical manifestations. In 6 of 11 cases that expressed c-kit product, AML cells were found to proliferate in response to recombinant human stem cell factor (rhSCF), the ligand for c-kit, and the synergistic stimulation of AML cells was observed by rhSCF and granulocyte-macrophage colony-stimulating factor. Immunoblotting with anti-phosphotyrosine antibody showed that the c-kit receptor protein was detectably phosphorylated in 7 of 12 cases tested before the stimulation with rhSCF, while the rhSCF treatment resulted in an increased tyrosine phosphorylation of c-kit in AML cells. These results indicate that c-kit proto-oncogene is expressed in most cases of AML and is functional in terms of supporting proliferation.

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts.

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

Proliferative potential of murine peritoneal mast cells after degranulation induced by compound 48/80, substance P, tetradecanoylphorbol acetate, or calcium ionophore A23187.

Proliferative potential of degranulated mast cells was investigated. Mast cells were collected from the peritoneal cavity of mice, and degranulation was induced by compound 48/80, substance P, 12-O-tetradecanoylphorbol 13-acetate (TPA), or calcium ionophore A23187. The potentiality of colony formation in methylcellulose was not reduced by treatment of various concentrations of compound 48/80, substance P and TPA. When degranulation was induced by compound 48/80, substance P or TPA, proportion of highly degranulated mast cells containing less than five granules was rather small. In contrast, considerable proportion of highly degranulated mast cells was obtained after the treatment with the low concentration (0.1 microgram/ml) of A23187. These highly degranulated mast cells, which were individually picked up by the micromanipulator, proliferated not only in methylcellulose but also in the skin of mast cell-deficient WBB6F1-W/Wv mice. Inasmuch as we have already shown the proliferation of IgE-sensitized and Ag-stimulated mast cells, degranulated mast cells appear to retain the proliferative potential in general.

Analyses of thrombocytopenia in idiopathic thrombocytopenic purpura-prone mice by platelet transfer experiments between (NZW x BXSB)F1 and normal mice.

Male (NZW x BXSB) F1 (W/B F1) mice, which develop lupus nephritis, myocardial infarction, and thrombocytopenia, showed reduced platelet lifespan (PLS) and increased platelet-associated antibody (PAA) values. There were statistically significant correlations between the increase in PAA values and either the reduction in PLS or the decrease in platelet counts. This and the results of platelet transfer experiments between old male W/B F1 mice and either female W/B F1 or normal BALB/c mice indicate that PAAs on the platelet surface play a crucial role in the destruction of platelets in W/B F1 mice. The mechanism of thrombocytopenia observed here appears similar to that of human idiopathic thrombocytopenic purpura (ITP). Therefore, we think that W/B F1 mice are a potentially useful animal model for investigating the effectiveness and mode of action of therapeutic agents in human ITP, and that they may provide additional information on the basic mechanisms of this autoimmune phenomenon.

[Regulatory mechanisms of mast cell differentiation].

Mast cells are a progeny of the multipotential hematopoietic stem cell. Most of progenies of the stem cell complete their differentiation within the bone marrow, but precursors of mast cells leave the bone marrow, migrate in blood, and invade into tissues. After the invasion, precursors proliferate and differentiate into mast cells. An appreciable proportion of mast cells retain proliferative potential after differentiation, and even after degranulation, some mast cells can proliferate and recover the original morphology. Proliferation of mast cells are regulated by both T cell-derived factors (i.e., IL-3 and IL-4) and fibroblast-derived factor(s). Mice of either W/Wv or Sl/Sld genotype lack mast cells, but mast cells do develop when bone marrow cells of W/Wv or Sl/Sld mice were cultured in the presence of T cell-derived factors. Mast cells derived from W/Wv mice cannot respond fibroblast-derived factor(s) and fibroblasts derived from Sl/Sld mice cannot support mast cells of normal mouse origin. Phenotypes of mast cells are determined by the environment in which the mast cells differentiated. However, when mast cells are transplanted into a new environment which is different from the original one, the mast cells acquire the phenotype which are dependent on the second environment.

Proliferative potential of degranulated murine peritoneal mast cells.

The fate of mast cells after degranulation was investigated. Purified peritoneal mast cells of WBB6F1-+/+ mice were sensitized with monoclonal anti-dinitrophenol (DNP) IgE antibodies and stimulated with DNP conjugated with human serum albumin. Mast cells were vitally stained with neutral red, and highly degranulated mast cells were identified under a phase-contrast microscope and individually picked up with the micromanipulator. When these highly degranulated mast cells were individually plated in methylcellulose, their potential to produce a cluster or a colony was comparable to that of morphologically intact mast cells. Moreover, when highly degranulated mast cells were injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, the proportion of injection sites at which mast cell clusters appeared was comparable to the value observed when morphologically intact mast cells were injected. The present result indicates that proliferative potential of mast cells is not reduced by their degranulation.

Acceleration of a murine T-cell leukemia associated with loss of interleukin-3 producing activity.

L-8313 is a murine T-cell leukemia cell line the cells of which constitutively produce interleukin-3 (IL-3). S2-8313 is a unique subline of L-8313 which has lost IL-3 producing activity. Although the growth of S2-8313 cells in the bone marrow is comparable to that of L-8313 cells, the survival time of C3H mice grafted with S2-8313 cells is significantly shorter than that of C3H mice grafted with L-8313 cells. The accelerated death observed in C3H mice bearing S2-8313 cells is attributable to early development of granulocytopenia and thrombocytopenia, which resulted from the loss of the IL-3 producing activity.

Failure of W/Wv mouse-derived cultured mast cells to enter S phase upon contact with NIH/3T3 fibroblasts.

Although W/Wv mutant mice are profoundly deficient in tissue mast cells, these mice do have cells with similar features of mast cells that develop from their bone marrow cells as efficiently as those from congenic +/+ mice in pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). With cultured mast cells (CMCs), we analyzed the mechanism of mast-cell deficiency in tissues of W/Wv mice. CMCs were established from bone marrow cells of W/Wv and congenic +/+ mice with PWM-SCM, and then co-cultured with various mouse fibroblast cell lines without PWM-SCM. All the examined mouse embryo-derived fibroblast cell lines maintained CMCs derived from +/+ mice, but not CMCs from W/Wv mice, for greater than 2 weeks. Mast cells in S phase were observed only in CMCs derived from +/+ mice under these conditions. The poor survival of W/Wv CMCs as compared with +/+ CMCs was not owing to a differential death rate but to the inability of W/Wv CMCs to continue active proliferation on fibroblasts without PWM-SCM. By synchronizing CMCs at the G1 phase of the cell cycle, the defect in W/Wv CMCs was further characterized as a failure to transit G1 and enter the S phase upon contact with fibroblasts. This finding indicates the indispensable function of the W gene product(s) for this response.

Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors.

Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. "Large" mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas "medium" and "small" mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chédiak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast.

Formation of mast-cell colonies in methylcellulose by mouse skin cells and development of mucosal-like mast cells from the cloned cells in the gastric mucosa of W/Wv mice.

Connective tissue-type mast cells, other than those located in the serosal cavity, are fixed in the tissues. For study of the differentiation processes of mast cells in connective tissue, an in vitro method for producing mast-cell colonies is required. The authors enzymatically dispersed the cells from the skin of either neonatal or adult mice and plated them in methylcellulose containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). More than 97% of the colonies that developed consisted of mast cells alone. The clonal nature of the mast-cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when a mixture of skin cells from C57BL/6-bgJ/bgJ and C57BL/6-+/+ mice was plated, most of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. In spite of depletion of T-cell-derived factors, concentrations of mast cells and mast cell colony-forming units (CFU-Mast) in the skin of nude athymic mice are normal. However, PWM-SCM was necessary for in vitro development of mast-cell colonies from the skin of nude mice. The concentration of CFU-Mast in the skin of genetically mast-cell-deficient WBB6F1-W/Wv mice was negligible when compared with the value observed in the skin of control WBB6F1-+/+ mice. Individual mast-cell colonies derived from the skin of neonatal WBB6F1-+/+ mice were lifted from the methylcellulose, and cells from each colony were injected into the wall of the glandular stomach and the skin of WBB6F1-W/Wv mice. Most of the mast cell that appeared at the skin injection sites of the WBB6F1-W/Wv mice stained with berberine sulfate, indicating that they contained heparin. In contrast, the mast cells that appeared in the stomach mucosa of the recipient WBB6F1-W/Wv mice did not stain. This suggests that CFU-Mast located in the skin have not been committed to the connective tissue type. The present method may be useful for investigation of the mechanisms of mast-cell differentiation in connective tissue other than the serosal cavity.