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.     

BALB/3T3 fibroblast-conditioned medium attracts cultured mast cells derived from W/W but not from mi/mi mutant mice, both of which are deficient in mast cells.

Proliferation of murine mast cells is induced by both T-cell-derived and fibroblast-derived growth factors. Because the most potent T-cell-derived mast cell growth factor, interleukin-3, promotes the migration of mast cells, we investigated whether fibroblast-derived growth factors had the chemoattractive activity as well. Conditioned medium (CM) of BALB/3T3 fibroblasts induced the migration of cultured mast cells (CMC) derived from normal (+/+) mice. BALB/3T3-CM contained the mast cell growth factor (MGF)/stem cell factor (SCF)/kit ligand (KL), which is the ligand for the receptor encoded by the W (c-kit) gene. CMC derived from the spleen of W/W mice lack the extracellular domain of the W (c-kit) receptor, and W/W CMC did not proliferate in response to BALB/3T3-CM. However, W/W CMC did migrate normally toward BALB/3T3-CM and, moreover, the antibody to the extracellular domain of the W (c-kit) receptor did not inhibit the chemoattractive activity of +/+ CMC toward BALB/3T3-CM. These results indicated that MGF/SCF/KL itself did not represent the major chemoattractive activity. On the other hand, BALB/3T3-CM induced neither proliferation nor migration of CMC derived from mi/mi mice. Both W/W and mi/mi mice are deficient in mast cells, but the present results suggest that the mechanism of the abnormality is different between W/W and mi/mi mice.     

Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts.

The receptor encoded by the W (c-kit) locus (W receptor) is expressed on the surface of cultured mast cells (CMC) derived from normal (+/+) mice, whereas its ligand encoded by the Sl locus (Sl ligand) is expressed on the surface of fibroblast cell lines derived from murine embryos. Involvement of W receptors and Sl ligands in attachment of CMC to fibroblasts was investigated. CMC were cocultured with fibroblasts; nonattaching CMC were removed and the remaining CMC were counted. CMC derived from mice of the W/W genotype did not express the extracellular domain of W receptors, and attachment of W/W CMC to +/+ fibroblasts was significantly impaired. Fibroblasts derived from embryos of the Sl/Sl genotype did not express Sl ligands, and the attachment of +/+ CMC to Sl/Sl fibroblasts was also impaired. The Wv and W42 alleles are point mutations at the intracellular tyrosine kinase domain. Attachment of either Wv/Wv, W/Wv, or W/W42 CMC to +/+ fibroblasts was comparable with that of +/+ CMC. Moreover, the addition of monoclonal antibody against the extracellular domain of W receptors inhibited the attachment of +/+ CMC to +/+ fibroblasts. Thus, the extracellular domain of W receptors appeared to be necessary for attachment of CMC to fibroblasts.     

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.     

Characterization of cultured mast cells derived from Ws/Ws mast cell- deficient rats with a small deletion at tyrosine kinase domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.     

Suppressive effect of Sl/Sld mouse embryo-derived fibroblast cell lines on diffusible factor-dependent proliferation of mast cells

Two modes of mast cell growth are present, one dependent on diffusible growth factors (interleukins [IL] 3 and 4) and another dependent on contact with fibroblasts. The 3T3 fibroblast cell lines derived from WCB6F1-+/+ mouse embryos supported the proliferation of cultured mast cells (CMC), whereas the 3T3 fibroblast cell lines from WCB6F1-Sl/Sld mouse embryos did not. To investigate the relationship between growth factor-dependent and fibroblast-dependent growths of mast cells, we cocultured CMC and 3T3 fibroblasts in the presence of diffusible growth factors. WCB6F1-+/+ mouse embryo-derived 3T3 cells did not affect the growth factor-dependent proliferation of CMC, but WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells significantly suppressed the proliferation. Close cell-to-cell contact was necessary for the suppression. The NWS1 fibroblast cell line was established from the spleen cells of an adult WBB6F1-+/+ mouse. Although the NWS1 cell line had no supporting effect on the proliferation of CMC in the absence of diffusible growth factors, it did not suppress the proliferation of CMC induced by the growth factors. The present result suggests that a product of mutant Sl genes may be involved in the suppressive activity of WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells.     

Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of [35S] sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate [35S] proteoglycans. When "MMC-like" cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1- W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these "second generation PMC" formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.     

In vitro duplication and in vivo cure of mast-cell deficiency of Sl/Sld mutant mice by cloned 3T3 fibroblasts.

Sl/Sld mutant mice are profoundly deficient in tissue mast cells as a result of a defect in the microenvironment promoting the development of these cells. To facilitate the analysis of the Sl mutation, we attempted to establish an in vitro system in which the in vivo defect of Sl/Sld mice could be reproduced. 3T3 cell lines were established from 17-day-old embryos of Sl/Sld and congenic +/+ genotypes and were cocultured with mast cells obtained in vitro from the bone marrow of +/+ mice. All eight 3T3 cell lines derived from +/+ of T-cell-derived growth factors. By contrast, none of eight 3T3 cell lines from Sl/Sld embryos supported mast cells under similar conditions. The defect in Sl/Sld 3T3 cells was further characterized as a failure to induce the G1-to-S transition in synchronized mast cells upon contact, suggesting that the Sl gene product is indispensable for this activity. When 3T3 cells of +/+ genotype, grown on pieces of cellulose acetate membrane, were transplanted into the peritoneal cavity of Sl/Sld mice, mast cells appeared locally in the transplanted 3T3 cell layers. These results suggested an essential role of fibroblasts in vivo as the tissue microenvironment promoting the development of mast cells and that they are defective in Sl/Sld mice. The present coculture system duplicated mast-cell deficiency of Sl/Sld mice in vitro and should prove useful for analysis of the Sl gene product.     

The c-fms gene complements the mitogenic defect in mast cells derived from mutant W mice but not mi (microphthalmia) mice.

Mutations at three loci in the mouse--W, Steel Sl), and microphthalmia (mi)--can lead to a deficiency in melanocytes and mast cells. As well, W and Sl mutants can be anemic and sterile, whereas mi mice are osteopetrotic due to a monocyte/macrophage defect. Recent data have shown that the c-kit receptor tyrosine kinase is the gene product of the W locus, whereas Sl encodes the ligand for this growth factor receptor. We show here that ectopic expression of c-fms, a gene that encodes a macrophage growth factor receptor that is closely related to the c-kit receptor, complements mutations at the W locus in an in vitro mast cell/fibroblast coculture system but is unable to reverse the inability of mi/mi mast cells to survive under these conditions. Furthermore, mast cells expressing the c-fms receptor survive on a monolayer of fibroblasts homozygous for the Sl mutation. These results suggest that ligand binding to the c-kit or c-fms receptor activates identical or overlapping signal transduction pathways. Furthermore, they suggest that mi encodes a protein necessary for transducing signals mediated by way of either the c-kit or c-fms receptor.     

Anemia and mast cell depletion in mutant rats that are homozygous at "white spotting (Ws)" locus

Mice possessing two mutant alleles at the W or Sl locus are anemic and deficient in mast cells. These mouse mutants have black eyes and white hair. Because homozygous mutant rats at the newly found white spotting (Ws) locus were also black-eyed whites, the numbers of erythrocytes and mast cells were examined. Suckling Ws/Ws rats showed a severe macrocytic anemia and were deficient in mast cells. When bone marrow cells of normal (+/+) control or Ws/Ws rats were injected into C3H/He mice that had received cyclophosphamide injection and whole-body irradiation, remarkable erythropoiesis occurred in the spleen of +/+ marrow recipients but not in the spleen of Ws/Ws marrow recipients. When skin pieces of Ws/Ws embryos were grafted under the kidney capsule of nude athymic rats, mast cells did develop in the grafted skin tissues. Therefore, the anemia and mast cell deficiency of Ws/Ws rats were attributed to a defect of precursors of erythrocytes and mast cells. Because the magnitude of the anemia decreased and that of the mast cell deficiency increased in adult Ws/Ws rats, this mutant is potentially useful for investigations about differentiation and function of mast cells.     

Hybrid resistance to parental bone marrow-derived cultured mast cells in the skin but not in the peritoneal cavity of (WB X C57BL/6)F1-W/Wv mice

When cultured mast cells of (WB X C57BL/6)F1-+/+(WBB6F1-+/+) and WB-+/+(WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. Although in vitro colony-forming ability is comparable between cultured mast cells of WB mice and those of WBB6F1-+/+ mice, the number of WB mast cells necessary for the appearance of mast cell clusters in the skin of WBB6F1-W/Wv mice was significantly larger than the number of WBB6F1-+/+ mast cells. In spite of the presence of such an apparent hybrid resistance in the skin of WBB6F1-W/Wv mice to mast cells of the WB parent, both WB and WBB6F1-+/+ mast cells grow in the peritoneal cavity of WBB6F1-W/Wv mice with comparable efficiency. This is a demonstration of the tissue-related (nonrecirculating) expression of hybrid resistance against nonmalignant hematopoietic cells.     

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.     

C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression

The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c- kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein- containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5- day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.