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The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Cultured mast cells (CMC) derived from the bone marrow of mice express the receptor encoded by the W (c-kit) locus (W receptor), and the WCB6F1(+/+)-3T3 fibroblasts express the ligand encoded by the Sl locus (stem cell factor [SCF]). CMC attach to the fibroblasts through the W receptors and cell-bound SCF. We investigated the effect of phorbol 12-myristate 13-acetate (PMA) and recombinant murine SCF (rmSCF) on the attachment. PMA induced both the internalization and shedding of W receptors, whereas rmSCF induced only the internalization. Moreover, both PMA and rmSCF reduced the expression of c-kit mRNA levels in CMC. Addition of either PMA or rmSCF to the coculture of CMC and fibroblasts resulted in the inhibition of attachment. Since the magnitude of the attachment between CMC and fibroblasts may be manipulated by changing the doses of either PMA or rmSCF, the present experimental system may be useful as a model for the attachment between blood cells and stromal cells.

The effects of culture matrix (agar, collagen, and methylcellulose) on differentiation of mast cells were investigated. Because berberine sulfate-positive colonies in agar were composed of macrophages but not of mast cells, the naphthol AS-D chloroacetate esterase reaction was used to identify mast-cell colonies. When bone marrow cells of WBB6F1 mice were cultured with conditioned media containing interleukin 3 (IL-3), numbers of mast-cell colonies were greater in collagen cultures than in agar and methylcellulose cultures. Electron microscopic examination revealed that mast cells developing in collagen and agar cultures were more mature than those developing in methylcellulose cultures. However, when bone marrow-derived cultured mast cells or purified peritoneal mast cells were plated, the efficiency of colony formation and size of colonies were comparable among agar, collagen, and methylcellulose cultures. Therefore, all three matrices tested had similar effects on the proliferation of mast cells. Collagen appeared to be suitable for differentiation of bone-marrow precursors and their maturation. Agar appeared to be suitable only for maturation.

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.

Although precursors of mast cells are derived from the bone marrow, phenotypes of mast cells are influenced by the tissues in which final differentiation occurs. Connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC) are different in morphological, biochemical, immunological, and functional criteria. The purpose of the present study was to obtain information about the differentiation process of MMC. First, we compared changes in irradiation susceptibility in mice during the differentiation process of CTMC and MMC. The decrease in irradiation susceptibility was remarkable in the CTMC differentiation process, but it was moderate in that of MMC. Some morphologically identifiable CTMC in the peritoneal cavity had proliferative potential and were highly radioresistant, whereas such a radioresistant population of MMC was not detectable in the gastric mucosa. Second, we estimated the turnover of CTMC and MMC by determining the proportion of mast cells that were labeled with continuously administered bromodeoxyuridine. The turnover of MMC was significantly faster than that of CTMC. The absence of the radioresistant mast cell population in the gastric mucosa appeared to be related to the short life span of MMC.

Bone marrow-derived mast cell precursors form large mast cell colonies in methylcellulose and are designated as L-CFU-Mast. The effect of differentiated mast cells on recruitment and differentiation of L-CFU-Mast was investigated by using genetically mast cell-deficient WBB6F1-W/Wv mice. Giant granules of C57BL/6-bgJ/bgJ (Chediak-Higashi syndrome) mice were used as a marker to identify the origin of L-CFU-Mast and differentiated mast cells. Practically no L-CFU-Mast are present in the peritoneal cavity of WBB6F1-W/Wv mice. When bone marrow cells of WBB6F1(-)+/+ mice were i.v. injected, the concentration of +/+(-)type L-CFU-Mast increased in the peritoneal cavity of WBB6F1-W/Wv mice and became several times greater than that of nontreated WBB6F1(-)+/+ mice. This increase of L-CFU-Mast was suppressed by a prior i.p. injection of bgJ/bgJ-type cultured mast cells. The differentiation of the +/+(-)type L-CFU-Mast to morphologically identifiable mast cells was also suppressed by the i.p. injection of bgJ/bgJ-type cultured mast cells. The present results suggest that the suppression of recruitment and differentiation of L-CFU-Mast is a physiological function of differentiated mast cells.

A homogeneous population of mast cells was obtained by culturing bone marrow cells of WBB6F1(-)+/+ mice. The proliferation of the cultured mast cells in diffusion chambers was investigated to examine whether the diffusion chamber technique was applicable for study of the regulation of mast cell proliferation. WBB6F1-W/Wv mice are genetically deficient in mast cells. When cultured mast cells of WBB6F1(-)+/+ mouse origin were directly injected into the peritoneal cavity of WBB6F1-W/Wv mice, the mast cells survived. In contrast, WBB6F1(-)+/+ mouse-derived cultured mast cells did not survive in diffusion chambers implanted in the peritoneal cavity of either WBB6F1-W/Wv or WBB6F1(-)+/+ mice. Because the coinoculation of NIH/3T3 cells supported the proliferation of mast cells in diffusion chambers, a certain type of cells in the peritoneal cavity appeared to have the same mast cell-supporting activity as NIH/3T3 cells. The magnitude of either interleukin 3-dependent or NIH/3T3 cell-dependent proliferation of mast cells in diffusion chambers was not significantly influenced by the genotype of chambers recipients (i.e., WBB6F1(-)+/+ or WBB6F1-W/Wv mice), suggesting that the previously reported inhibitory effect of mast cells on differentiation of mast cells may be mediated by direct contact between mast cells.

Although mice possessing two mutant genes at the W locus have a defect in multipotential hematopoietic stem cells that form macroscopic colonies in the spleen of irradiated mice, the number of neutrophils in the blood of these mutant mice is normal or nearly normal. We investigated neutrophil production using the NFSA fibrosarcoma of C3H mouse origin, which induces neutrophilia accompanied by production of a neutrophil-macrophage colony-stimulating factor by the tumor. When the NFSA tumor was transplanted to (C57BL/6 X C3H/He)F1-Wv/Wx or to congenic +/+ mice, neutrophilia developed in mice of both genotypes. However, there was a significant difference between the degree of neutrophilia that developed in them; there was a 107-fold increase in the +/+ mice, but only a 28-fold increase in the Wv/Wx mice four weeks after tumor transplantation. This result is consistent with the concept that doubly heterozygous W mice have multipotential stem cells with diminished ability to respond to stimulation. The unperturbed condition may not provide a sufficient stimulus to demonstrate the defect in neutrophil production in doubly heterozygous W mutant mice.

When spleen cells (1.5 x 10(8)) from beige C57BL/6 (bgJ/bgJ, Chediak-Higashi syndrome) mice were injected into nonirradiated (C57BL/6 x CBA)F1 hybrid mice, the hematopoietic stem cells of the hybrid host were eradicated by graft-versus-host (GVH) reaction, and peripheral neutrophils of the host changed from normal type to beige type with giant sudanophilic granules in 20 days after the cell injection. Effect of splenectomy on the establishment of such hematopoietic chimerism was investigated. The splenectomy carried out either before or after the cell injection reduced the proportion of mice in which more than 90% of peripheral neutrophils became of beige type. Since the mortality of mice which received splenectomy at various days prior to or after the cell injection paralleled the proportion of establishment of chimerism, the splenectomy does not seem to affect directly the establishment of chimerism but indirectly through the modification of the intensity of GVH reaction.

We investigated the fate of donor stem cells that were injected into hosts with a normal concentration of spleen colony-forming unit (CFU-S). Radiation chimeras were used as hosts. When CFU-S concentration in the marrow and spleen recovered to preirradiation levels after the initial bone marrow transplantation, the subsequent transplantation was done without reirradiation. Giant granules of beige C57B1/6 (bg) mice were used as a marker and proliferation and differentiation of the stem cells of the subsequent donor origin were evaluated by measuring the proportion of neutrophils with giant granules. No beige-type neutrophils were detectable at week 24 after transplantation of 5 X 10(7) marrow cells from bg mice to intact (WB X C57B1/6)F1 (F1) mice, which were used as control recipients. In contrast, transplantation of 5 X 10(7) marrow cells to radiation chimeras resulted in the appearance of neutrophils of second-donor origin. The proportion of beige-type neutrophils was 12% at week 24 after transplantation of bg marrow cells to F1-to-F1 (syngenic) or C57B1/6-+/+-to-bg (B6-to-bg) (congenic) chimeras; the proportion of beige-type neutrophils was 43% when bg marrow cells were transplanted to B6-to-F1 semiallogenic chimeras; the proportion of normal-type neutrophils was 82% when F1 marrow cells were injected to bg-to-F1 semiallogenic chimeras. Thus, the interaction of the host hematopoietic microenvironment with the stem cells of the initial donor as well as with the stem cells of the second donor seems to influence the proliferation and differentiation of the latter stem cells.

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.

The colony-forming ability of hematopoietic cells derived from 18 species of rodents was examined on the mouse macrophage layer which formed on a cellulose acetate membrane inserted into the peritoneal cavity of mice. Bone marrow cells of 12 xenogeneic species made a considerable number of colonies on the mouse macrophage layer. However, there was no parallelism between the colony-forming ability of the cells and donor's taxonomic relationship to the mouse.

The cause of the severe anemia in Sl/Sld mice is attributed to (1) hypoproduction of erythrocytes due to a defect in the erythropoietic microenvironment and (2) bleeding from stomach ulcers. Sl/Slt mice also showed a moderate anemia, but bleeding from stomach ulcers was excluded as a cause of the anemia, because no significant amount of radioactivity was excreted in feces after the injection of 59Fe-labeled erythrocytes. The activity of erythropoiesis in the bone marrow and spleen was compared between Sl/Slt and congenic +/+ mice using three different criteria: the number of erythroblasts, 59Fe incorporation, and the number of erythropoietic precursor cells. All three parameters in the femur were lower, and those in the spleen were higher in Sl/Slt mice than in +/+ mice, suggesting that the low erythropoietic potential in the bone marrow of Sl/Slt mice is partially compensated by the spleen. In fact, splenectomy aggravated the anemia of Sl/Slt mice. The enhanced erythropoiesis in Sl/Slt spleens may explain our previous finding that numbers and sizes of spleen colonies were normal when bone marrow cells were injected into irradiated Sl/Slt mice. Sl/Slt mice may be a useful model for studying biological characteristics of the hematopoietic microenvironment.

Irradiated mice infused with bone marrow cells developed colonies both in the spleen and on the macrophage layer of the cellulose acetate membrane which had been inserted into their peritoneal cavity. Factors influencing the ratio of spleen to macrophage-layer colonies were examined. The ratio of the colonies depended on the condition of the recipients rather than the condition of the injected cells provided there was no marked histoincompatibility between donors and recipients.

Concentration of mast-cell precursors in the mesenteric lymph node of (WB X C57BL/6)F1 hybrid mice (WBB6F1) were evaluated by a limiting dilution method. Cells from WBB6F1-+/+ mice were injected directly into the skin of WBB6F1-W/Wv mice which genetically lack tissue mast cells. Concentrations of mast-cell precursors were calculated from the proportion of injection sites at which mast cells appeared. Although immunization with horse serum significantly increased the concentration of mast-cell precursors in the mesenteric lymph node, the concentration in the lymph node remained about 10% that observed in the peripheral blood mononuclear cells. Since the bone marrow origin of mast-cell precursors in the mesenteric lymph node was demonstrated by using giant granules of beige (C57BL/6-bgj/bgj) mice as a marker, the immunization seemed to increase the migration of bone-marrow-derived mast-cell precursors from the peripheral blood to lymph node.

Mutant mice of Sl/Sld genotype are depleted of tissue mast cells due to a defect of stromal cells which may support the differentiation of mast cells. Number of mast cells did not increase in the skin of Sl/Sld mice grafted on the back of the congenic +/+ mice even 50 weeks after the transplantation. When the wound was produced in the central part of the Sl/Sld skin graft, a considerable number of mast cells appeared within the scar resulting from the wound. The appearance of mast cells in the scar was shown to be due to the differentiation of circulating precursors into mast cells rather than the simple migration of mature mast cells by using giant granules of bgJ/bgJ mice as a marker for newly-formed mast cells. Therefore, the present results seem to suggest that normal stromal cells of the +/+ host origin migrate into the wound produced in the grafted skin of Sl/Sld mice and support the differentiation of circulating precursors into mast cells.