Pokeweed Mitogen-stimulated Spleen Cell Research Articles

PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors.

PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. We demonstrate that mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU. 1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways.

A study of mast cells in autoimmune mice: the proliferative response of autoimmune mast cells to cytokines.

To investigate the mechanisms underlying the increased number of mast cells in autoimmune mice, the proliferative response of autoimmune mast cells to cytokines was examined. Bone marrow cells from autoimmune NZB mice produced scarcely any bone marrow derived mast cells (BMMCs) in the presence of interleukin 3 (IL-3), but were able to generate BMMCs when cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). In contrast, NZB BMMCs showed very little proliferation in the presence of PWM-SCM, but proliferated strongly when cultured with stem cell factor (SCF). Non-autoimmune NZW BMMCs showed a strong proliferative response to both IL-3 and PWM-SCM, but proliferated weakly in culture with SCF. Autoimmune NZB x NZW F1 (B/W) BMMCs shared the proliferative activities of both NZB and NZW BMMCs, showing strong proliferation in response to IL-3, PWM-SCM and SCF. All strains (including other non-autoimmune strains) except for NZW demonstrated synergism between PWM-SCM and SCF. This study suggests that the strong proliferative response of autoimmune mast cells to SCF plays a major role in, and that other cytokines are partially responsible for, increasing the number of mast cells in autoimmune mice. These mechanisms are discussed in relation to both constitutive and inducible hematopoiesis.

Sex Differences and the Effects of Alcohol on Immune Response in Male and Female Rats

Although it is clear that both alcohol and sex hormones impact immune function, very little information is available on the effects of alcohol on immune response in males versus females. We decided to determine if the alterations in immune response resulting from alcohol feeding might be expressed differently in males and females. To accomplish this we utilized pair-fed male and female Sprague-Dawley rats. Animals were fed a liquid diet for 60 days containing 30% of their calories as ethanol, and after 1 week this concentration was increased to 45% ethanol. Controls received liquid control diet of the same caloric and nutritional composition, and immune status was monitored with in vivo and in vitro techniques. Ethanol feeding significantly reduced the phytohemagglutinin skin response in males (p = 0.020) and females (p = 0.012). The concanavalin A blastogenic response of spleen cells prepared from female rats fed ethanol was significantly depressed with respect to spleen cells prepared from female rats fed the control diet (p = 0.0071). Alcohol also appeared to depress spleen cell blastogenic response in males, but this trend did not quite reach significance (p = 0.071). Spleen cells from groups of ethanol and control male and female rats were labeled with fluorescent monoclonal antibodies and run on a Fluorescent-Activated Cell Sorter. Ethanol significantly increased the percentage population of CD4 (T-helper cell) in males (p = 0.017), but not in females, and promoted an apparent, although nonsignificant, increase in the CD4/CD8 ratio in both sexes. An ELISA was used to measure IgM and IgG antibody elaborated by pokeweed mitogen-stimulated spleen cells in cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Clonal proliferation of murine lymphohemopoietic progenitors in culture.

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.

Aspergillus antigen-induced eosinophil differentiation in a murine model.

Eosinophilia is a prominent feature of the cellular response in allergic and parasitic diseases. Allergic bronchopulmonary aspergillosis due to colonization of the lungs of some asthmatics with Aspergillus fumigatus is characterized by high levels of serum immunoglobulin E and peripheral blood (PB) and lung eosinophilia. This study investigates the role of eosinophils in the pathogenesis of allergic bronchopulmonary aspergillosis by using a mouse model. BALB/c mice were immunized intranasally and intraperitoneally with A. fumigatus antigens (Ag), and the eosinophils in PB and bone marrow (BM) were enumerated. Eosinophilopoiesis in BM cultures was studied in the presence of murine recombinant interleukin-5 (mrIL-5) and supernatants from pokeweed mitogen-stimulated spleen cells as the source of eosinophil differentiation factors. Eosinophils were quantitated by direct counting and by estimating eosinophil peroxidase activity. The results indicate that the percentage of eosinophils in the PB (5.77 +/- 1.17) and the BM (11.19 +/- 4.31) of mice exposed to A. fumigatus Ag was higher than in controls (PB, 2.42 +/- 0.76; BM, 5.12 +/- 2.79; P less than 0.01 for both). Similarly, a significant increase in eosinophils was observed in the BM population from mice exposed to A. fumigatus Ag compared with that in controls when cultured with murine recombinant interleukin-5 (23.13 +/- 7.14 versus 13.77 +/- 5.79, P less than 0.01), indicating that the mice exposed to A. fumigatus Ag had significantly greater numbers of eosinophil precursors in their BM. This study demonstrates that A. fumigatus Ag may be involved in the in vivo commitment of stem cells in the eosinophil differentiation pathway.

Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.

Mechanism of Mast Cell Deficiency in Mutant Mice of mi/mi Genotype: An Analysis by Co-Culture of Mast Cells and Fibroblasts

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.

The mast cell-committed progenitor. II. W/Wv mice do not make mast cell-committed progenitors and S1/S1d fibroblasts do not support development of normal mast cell-committed progenitors.

We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.

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.

Recombinant retrovirus-mediated gene transfer to normal haemopoietic cells.

Several of parameters seeking to increase the infection frequency of normal haemopoietic cells by recombinant retroviruses containing a drug selectable marker and/or a haemopoietic growth factor gene have been compared. By using bone marrow cells pretreated with 5-fluorouracil, cocultivation of cells for at least 4 days with virus producing fibroblasts, and addition of pokeweed mitogen stimulated spleen cell conditioned medium, from 30 to 100% of haemopoietic spleen colony forming cells (CFU-S) could be infected. Because drug (G-418) selection was found to select subsets of CFU-S, a retrovirus lacking the drug selectable marker Neor, but containing a GM-CSF gene, was used to study the effects of endogenous GM-CSF production on CFU-S differentiation. Infected CFU-S produced equivalent numbers of erythroid and neutrophil-macrophage committed progenitor cells to control uninfected CFU-S. However, the progeny of infected CFU-S produced GM-CSF, some cells grew autonomously and neutrophil numbers were greatly increased at the expense of erythroblasts. These results suggest that unregulated GM-CSF production, although not altering commitment, alters the amplication phase following commitment.

Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor.

Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with 125I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with 125I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor with an approximate Ka of 10(8)-10(9) M-1. CFU-GM expressed an average of 3783 +/- 4 receptors per cell; normal bone marrow cells, 1518 +/- 242 receptors per cell; and peritoneal exudate cells, 2025 +/- 216 receptors per cell. Affinity crosslinking studies demonstrated that 125I-labeled GM-CSF bound specifically to two species of Mr 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The Mr 70,000 species is thought to be a proteolytic fragment of the intact Mr 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

Effects of hypoxia on megakaryocytopoiesis and granulopoiesis

Previous studies have reported that exposure of mice to hypoxic conditions results in a decrease in blood platelets. To further explore the effect of hypoxia on megakaryocytopoiesis, megakaryocytes and their progenitor cells (CFU-M) were studied in hypoxic mice. Mice were exposed to hypoxia by enclosure in cages covered with dimethyl-silicone rubber membranes for up to 10 d. At various times during the hypoxic and ex-hypoxic periods the total megakaryocytes and CFU-M were determined in the humerus and spleen. CFU-M were assayed in the soft gel colony forming assay using pokeweed mitogen-stimulated spleen cell conditioned medium (PWCM) as a source of colony stimulating activity. After 10 d of hypoxia, packed red cell volume (PRCV) increased to 148% of control levels and blood platelets decreased to 40% of controls. Total megakaryocytes and CFU-M per humerus decreased to 18% and 50% of controls respectively. 4 d into the ex-hypoxic phase, PRCV was still increased at 128% of controls while marrow megakaryocytes and CFU-M increased to normal levels. Platelet recovery was somewhat slower, returning to normal by d 6. In contrast to the findings in the marrow, total spleen megakaryocytes and CFU-M increased to about 3- and 5-fold of control levels respectively by 6 d of hypoxia. During the exhypoxic phase, CFU-M decreased to normal on d 4, followed by a rebound of 3-fold control values on d 8. Spleen megakaryocytes decreased more slowly, returning to normal by d 10. A marked granulocytosis was observed during the hypoxic phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic action of recombinant-derived murine interleukin-1 on the augmentation of colony stimulating activity on murine granulocyte-macrophage hematopoietic stem cells in vitro.

This communication reports studies that were designed to investigate the role of recombinant-derived murine interleukin-1 on granulocyte macrophage (CFU-GM) progenitor stem cells in vitro. Interleukin-1 (IL-1, 5-25 units/ml culture) in a dose-dependent fashion, stimulated CFU-GM with a maximum effect at 25 units (147.3 +/- 6.4 colonies/10(5) nonadherent-derived marrow cells). At a higher concentration (50 units/ml) this stimulation was significantly reduced. IL-1, in combination with various types of conditioned media: pokeweed mitogen stimulated spleen cell, mouse lung and WEHI-3 cell, as a source of colony-stimulating activity (CSA), produced more CFU-GM when compared to controls (conditioned media alone). Furthermore, when assayed for their esterase activity, IL-1 increased both types of nonspecific and specific esterase content in CFU-GM colonies. Also, the actual ratio between neutrophilic and monocyte/macrophage colonies was reduced when compared to cultures stimulated in the presence of CSA, indicating that IL-1 increased myeloid differentiation. In the presence of indomethacin, an effective inhibitor of prostaglandin synthesis (1 microgram/ml), greater numbers of CFU-GM were present with IL-1 and/or CSA than without indomethacin. Cultures with anti-CSA demonstrated a reduced number of CFU-GM when plated in the presence of CSA but not with IL-1, demonstrating the specificity of IL-1 to stimulate CFU-GM in the presence of anti-CSA antibody. These results indicate a role for IL-1 in modulating granulopoiesis in vitro.

Effect of insulin on murine megakaryocytopoiesis in a liquid culture system.

To examine the influence of insulin on megakaryocytopoiesis, we tested its effect on murine bone marrow cultures in a liquid culture system. In the presence of pokeweed mitogen-stimulated spleen cell conditioned medium in culture, insulin markedly enhanced megakaryocyte colony formation and increased the number and size of free megakaryocytes seen after 7 days. Many of the cells in cultures with insulin, however, were classified as immature, since they had a basophilic cytoplasm, a low cytoplasmic/nuclear ratio and low acetylcholinesterase activity. It is suggested that insulin potentiates murine marrow megakaryocytopoiesis in vitro, but that this is not accompanied by differentiation of the cells from the immature to mature state.

Reproducible generation of autonomous malignant sublines from non-tumorogenic murine interleukin 3-dependent mast cell lines.

Murine interleukin 3 (IL-3)-dependent permanent mast cell lines derived from normal mouse bone marrow were established using pokeweed mitogen-stimulated spleen cell conditioned medium (SCM) as a source of IL-3. When propagated continuously in media containing a high concentration of IL-3 (20% SCM or 20 U/ml murine recombinant IL-3 (rIL-3], all the cell lines remained strictly factor-dependent in vitro and non-tumorogenic in vivo. However, we were able to reproducibly generate autonomous sublines from cultures supplemented with low amounts of IL-3 (1% SCM or 2 U/ml rIL-3). Abrogation of exogeneous growth factor dependency was always associated with neoplastic transformation. In newly generated autonomous sublines an autocrine mechanism of growth regulation was evident in vitro.

Production of Granulocyte-Macrophage Colony-Stimulating Factor by Abelson Virus-Induced Tumorigenic Mast Cell Lines

We have recently described a system that supports the development of continuously growing and tumorigenic cell lines after infection of individual multilineage hematopoietic colonies with Abelson murine leukemia virus (A-MuLV). We now provide definitive evidence that these transformed lines express features characteristic of mast cells. Although these lines have been maintained in some cases for more than a year in the absence of exogenous growth factors other than those present in fetal calf serum, colony formation could consistently after 2 months, and variably after 5 months, be shown to be increased several fold when pokeweed mitogen-stimulated spleen cell conditioned medium (CM) was added to the cultures. CM from the A-MuLV-transformed lines was then tested for its ability to stimulate hematopoietic colony formation by cells from both fetal and adult tissues. Four of four randomly selected cell lines produced factors that were active on erythropoietic, granulopoietic, and in some cases pluripotent progenitors. Removal of viral particles from the CM from one of the lines (27d1) by either heat inactivation or high-speed centrifugation did not alter the colony-stimulating activity detected. When CM from 27d1 cells was tested for its ability to stimulate the proliferation of interleukin 3 (IL3) granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent FDC-P1 cells, a positive result was obtained. This stimulatory activity was not reduced in the presence of neutralizing anti-IL 3 immunoglobulin (Ig), suggesting that the activity detected was GM-CSF and not IL 3. This was confirmed by the lack of expression of the IL 3 gene in 27d1 cells as determined by Northern analysis of 27d1 cell RNA. Furthermore, S1 analysis of mRNA from 27d1 cells as well as two other lines indicated that the GM-CSF gene in all three was transcriptionally active. Taken together, these data suggest that A-MuLV transformation of normal mast cells or their precursors under certain conditions commonly activates the production of GM-CSF.

Production of granulocyte-macrophage colony-stimulating factor by Abelson virus-induced tumorigenic mast cell lines

We have recently described a system that supports the development of continuously growing and tumorigenic cell lines after infection of individual multilineage hematopoietic colonies with Abelson murine leukemia virus (A-MuLV). We now provide definitive evidence that these transformed lines express features characteristic of mast cells. Although these lines have been maintained in some cases for more than a year in the absence of exogenous growth factors other than those present in fetal calf serum, colony formation could consistently after 2 months, and variably after 5 months, be shown to be increased several fold when pokeweed mitogen-stimulated spleen cell conditioned medium (CM) was added to the cultures. CM from the A-MuLV-transformed lines was then tested for its ability to stimulate hematopoietic colony formation by cells from both fetal and adult tissues. Four of four randomly selected cell lines produced factors that were active on erythropoietic, granulopoietic, and in some cases pluripotent progenitors. Removal of viral particles from the CM from one of the lines (27d1) by either heat inactivation or high-speed centrifugation did not alter the colony-stimulating activity detected. When CM from 27d1 cells was tested for its ability to stimulate the proliferation of interleukin 3 (IL3) granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent FDC-P1 cells, a positive result was obtained. This stimulatory activity was not reduced in the presence of neutralizing anti-IL 3 immunoglobulin (Ig), suggesting that the activity detected was GM-CSF and not IL 3. This was confirmed by the lack of expression of the IL 3 gene in 27d1 cells as determined by Northern analysis of 27d1 cell RNA. Furthermore, S1 analysis of mRNA from 27d1 cells as well as two other lines indicated that the GM-CSF gene in all three was transcriptionally active. Taken together, these data suggest that A- MuLV transformation of normal mast cells or their precursors under certain conditions commonly activates the production of GM-CSF.

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells.

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. 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 mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.

A self-renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow.

We have established permanent lines of nonadherent cells from fresh normal mouse bone marrow in media containing pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). These lines continuously produced erythropoietic progenitor cells (detected by their ability to form erythroid bursts in semi-solid medium containing erythropoietin) together with cells having characteristics of the mast cell lineage (as demonstrated by metachromatic staining with toluidine blue, histamine content and membrane receptors for IgE). Sixteen such cell lines have been established in sixteen attempts. Cloning experiments were carried out to determine the nature of the progenitor cell(s) responsible for the permanence of these cultures. When cells were cultured in methylcellulose medium containing PWSCM, colonies were observed which reached macroscopic size after 4 weeks of incubation. Replating of individual primary colonies resulted in secondary colony formation, indicating the presence of progenitor cells with self-renewal potential. Forty-seven primary colonies were picked and their cells were suspended in liquid culture medium containing PWSCM. Of these, twenty-one could be expanded to establish permanently growing sublines. Sixteen of these sublines were found to be composed of both erythroid progenitors and mast cells. In five sublines only mast cells could be seen; none of the sublines appeared to be purely erythroid. Karyotypic analysis of mast cells and of erythroid cells of seven sublines derived from individual colonies which arose in cocultures of male and female cells revealed that the mast cells and erythroid cells were both of the same sex in each of the seven sublines; this demonstrates the single cell origin of each colony and of the two lineages derived from it. We conclude that these nonadherent, factor-dependent cell lines are maintained by self-renewal and differentiation of bipotential progenitor cells apparently restricted to the erythroid and mast cell lineages.

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase.

A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum +/- pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE-positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.