Spleen Cell Conditioned Medium Research Articles

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.

Stimulation of mast cell chemotaxis by interleukin 3.

PWM-activated spleen cell-conditioned medium (SCCM) and a variety of purified hematopoietic growth factors were tested for their ability to stimulate chemotaxis of mouse connective tissue mast cells (CTMC). Of the agents tested, only IL-3 and SCCM promoted mast cell chemotaxis. Neither IL-2, IL-4, GM-CSF, nor endotoxin had any significant mast cell chemotactic activity. Neutralizing antibodies to mouse IL-3 blocked greater than 90% of the chemotactic activity of SCCM, suggesting that IL-3 is the predominant mast cell chemotactic factor produced by activated spleen cells. Our results demonstrate that mature connective tissue type mast cells are capable of moving toward a gradient of spleen cell-derived IL-3 and suggest that movement of mature mast cells toward lymphokines may influence the accumulation of mast cells at sites of inflammatory or immune reactions.

Thiol-sensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4.

A series of permanent IL-3-dependent cell lines have been established from normal BALB/c or C3H bone marrow using alpha-thioglycerol-supplemented culture medium and PWM-stimulated spleen cell-conditioned medium as a source of IL-3. The cell lines and derivatives cloned in agar resembled "mucosal type" mast cells with respect to phenotypic and functional properties. In this report we demonstrate that in vitro growth of these mast cell lines was not only dependent on IL-3 and synergistically enhanced by IL-4, but in addition regulated by alpha-thioglycerol which could be replaced by 2-ME or cysteamine. We show that these thiol-sensitive mast cell lines respond to a mast cell growth enhancing activity (MEA) present in spleen cell-conditioned medium and acting in concert with IL-3. Partially purified MEA was not able to stimulate the growth of IL-3-dependent 32Dcl.23 cells, IL-2-dependent CTLL-2 cells or the mouse T cell line F4/4K.6 (L3T4+) adapted to grow in purified IL-4. Moreover, 11B11 hybridoma-derived anti-IL-4 mAb specifically neutralizing mouse Il-4 were unable to abolish the bioactivity of MEA. PWM, CSF-1, GM-CSF, IL-1, IL-2, IL-5, IL-6, IL-7, IFN-gamma, TGF-alpha, TNF-alpha, NGF, or EPO did not substitute for MEA in our standard proliferation assay.

Partial purification of a mast cell growth-enhancing activity and its separation from IL-3 and IL-4.

A growth factor acting synergistically with IL-3 on thiol-sensitive "mucosal type" bone marrow-derived mast cell lines, and therefore termed mast cell growth enhancing activity, is present in PWM stimulated spleen cell conditioned medium. Mast cell growth enhancing activity can be partially purified and completely separated from IL-3, IL-4, and IL-5, and for the most part from IL-6 and GM-CSF using strong cation exchange and Procion red affinity chromatography. Mast cell growth enhancing activity binds to Con A-Sepharose and can be digested with trypsin and chymotrypsin. It shows a Mr ranging from 37 to 43 kDa under nonreducing SDS-PAGE and a main isoelectric point ranging from 6.2 to 7.3.

Recombinant human erythropoietin has little influence on megakaryocytopoiesis in mice

The availability of a preparation of recombinant human erythropoietin (rEp) prompted us to investigate the role of Ep in the regulation of megakaryocytopoiesis in mice, using experimental procedures by which the effects on the mitotic and post-mitotic compartment of megakaryocytes could be evaluated separately. In agar cultures of murine bone marrow cells, either serum-depleted or serum-supplemented, rEp (0.1-2 U/ml) did not stimulate megakaryocyte colony formation and when it was added to suboptimal amount of spleen cell-conditioned medium (SCM), it failed to show a significant synergistic activity. On the contrary, rEp increased the number of megakaryocytic colonies developed from splenic precursors in the presence of suboptimal amounts of SCM, although it was unable per se to stimulate colony formation. The effects of rEps on megakaryocyte maturation and platelet production were studied in vivo evaluating the incorporation of 75Se-selenomethionine into platelets, the platelet count and platelet size, and the number of megakaryocyte precursors (small acetylcholinesterase positive cells, sAchE) in the bone marrow of mice injected with 1-8 U of rEp. No modification of these parameters was found in comparison with control mice. On the other hand, rEp increased the number of recognizable splenic megakaryocytes in a dose-dependent fashion. These data suggest that rEp has little influence on megakaryocytopoiesis, at least at the doses we used and which are known to elicit a maximal response of erythropoiesis. However, a subset of megakaryocytes with particular kinetic properties, such as those in the spleen of mice, may be responsive to relatively high doses of rEp. The significance of this observation in the overall regulation of megakaryocytopoiesis remains to be determined.

Analysis of murine megakaryocyte colony size and ploidy: effects of interleukin-3.

Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in-vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin-3 (IL-3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM, a crude source of megakaryocyte colony-stimulating activity) or IL-3, the modal ploidy of day-5 colony megakaryocytes was 16N (range 2N-128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM-SCM and at all concentrations of IL-3 tested. Increasing concentrations of IL-3 in cultures of either unfractionated marrow cells or nonadherent T-lymphocyte-depleted (NATD) marrow cells stimulated similar dose-dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL-3-stimulated cultures of NATD marrow cells was significantly less (P less than 0.001) than the mean ploidy of megakaryocytes in IL-3-stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM-SCM-stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day-5 murine megakaryocyte colonies are structured as continua and that IL-3 stimulates an increase in mean colony size and numbers without affecting ploidization. T-lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM-SCM can substitute for this activity.

Granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (CSF-1) synergize to stimulate progenitor cells with high proliferative potential.

The ability to expand a population of bone marrow progenitors capable of forming macrophage colonies of high proliferative potential (HPP-CFC) was achieved using a culture system and signal requirements not previously shown to support the growth of HPP-CFC. Using bone marrow cells from untreated animals (not 5-FU-treated), culture conditions designed primarily for the detection of GM-CFC or M-CFC progenitors, and a more stringent criteria for HPP-CFC colony size (greater than or equal to 2 mm diameter), HPP-CFC progenitor expansion was demonstrated following simultaneous addition of rGM-CSF and CSF-1 to bone marrow cultures. Examination of the sequence and temporal requirements for rGM-CSF and CSF-1 addition necessary for the development of HPP-CFC-like colonies revealed that addition of the second factor could be delayed for up to 5 days and still result in the development of significant numbers of HPP-CFC colonies. Based on a comparison with human spleen cell conditioned medium (HSCM) and interleukin 1 (rIL 1), as sources of "synergistic activity" (SA) for the development of HPP-CFC-like colonies in combination with CSF-1, the combination of GM-CSF and CSF-1 appears to represent a novel pathway for stimulating the expansion of HPP-CFC progenitors with high proliferative potential.

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.

Establishment of megakaryoblastic cell lines by coinfection of Abelson murine leukemia virus and recombinant SV40-retrovirus.

Murine embryonic cells including yolk sac prepared from 8‐day embryos were co‐infected with Abelson murine leukemia virus (A‐MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M‐SV40). By coinfection with A‐MuLV and M‐SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A‐MuLV or M‐SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single‐cell transfer, 6 acetyl‐cholinesterase (AchE)‐positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins lIb and IlIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen‐stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.

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.

Megakaryocyte colony-stimulating factor and burst-promoting activity in LPS-treated mouse spleen cell-conditioned medium.

In order to clarify the mechanism(s) of increased splenic hematopoiesis noted in lipopolysaccharide (LPS)-injected mice, the effects of spleen cell-conditioned medium (SPCM) on megakaryocyte colony (CFU-meg) formation and early erythroid (BFU-e) differentiation were investigated. After spleen cells from LPS-injected mice were incubated for 3 days, the SPCM was assayed for megakaryocyte colony-stimulating factor (Meg-CSF) in CFU-meg assay and for burst-promoting activity (BPA) and erythropoietin (Epo) in erythroid colony assays (i.e., CFU-e, BFU-e). Colony formation of CFU-meg and BFU-e peaked with the addition of 30 and 10-15% SPCM, respectively. Spleen cells from LPS-injected mice produced Meg-CSF and BPA when compared with controls. However, conditioned medium from spleen cells depleted of phagocytic cells had low Meg-CSF and BPA. SPCM did not contain detectable quantities of Epo. It appears likely that local splenic production of Meg-CSF and BPA may affect proliferation of CFU-meg and erythroid progenitor cells in the spleen.

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

Bovine brain gangliosides were applied to primary cultures of murine bone marrow cells to examine the role of gangliosides in development of megakaryocytes. Megakaryocytes in the cultures were detected by staining for a cytoplasmic enzyme, acetylcholinesterase, and divided into two types, 1) immature megakaryocytes which were stained less intensely, and 2) mature ones which were stained intensely. A medium containing total ganglioside fraction from bovine brain increased the number of both immature and mature megakaryocytes in the presence of pokeweed mitogen-stimulated murine spleen cell conditioned medium. Between the two cell types, the number of the mature cells was more significantly increased than the immature cells. The ganglioside GD1a could substitute for the total ganglioside mixture. The results suggested that bovine brain gangliosides potentiated both megakaryocytic proliferation and maturation in vitro.

Mouse peritoneal macrophages produce CFU-gm and CFU-meg growth-promoting factors.

The effect of peritoneal macrophage-conditioned medium (macrophage CM) from lipopolysaccharide-injected mice on granulocyte-macrophage colony (CFU-gm) and megakaryocyte colony (CFU-meg) formation was examined using a plasma clot culture system. Macrophage CM stimulated mouse bone marrow cells to form CFU-gm and CFU-meg in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium, but it had no effect on colony formation in the absence of exogenous colony-stimulating factors (CSF). CFU-gm and CFU-meg colony formation was enhanced by low concentrations of exogenous GM-CSF or Meg-CSF alone. These data demonstrate that macrophage CM contains an activity that sensitized CFU-gm and CFU-meg to exogenous CSF.

Characterization of a human basophil-like cell promoting activity.

Biologic and biochemical properties of a human basophil-like cell promoting activity (BaPA), which induces growth of metachromatically staining cells from normal bone marrow cells in a liquid culture system have been examined. In order to study this T cell factor, an assay was developed based on the intracellular histamine content of the cultured human bone marrow cells. Many lymphokines, including granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin 1 alpha and 1 beta, interleukin 2, and interferon-alpha and gamma, did not exhibit any significant activity in the assay. By employing this assay, BaPA was purified approximately 500-fold from lectin-stimulated spleen cell-conditioned medium. BaPA has a molecular weight of 23,000 on high performance liquid chromatography gel filtration and displays isoelectric points between 5.8 and 7.3. It is heat stable up to 80 degrees C for 30 min and resistant to 6 M guanidine hydrochloride, whereas it is rather sensitive to sulfhydryl reagents. BaPA has no stimulating activity on mouse bone marrow cells.

Establishment and in vitro differentiation of a chicken monocytic leukemia cell line.

A chicken monocytic leukemia Cell line was established from a spontaneous myelocytic leukemia. The cell line consisted of adherent round and polygonal cells. About 65% of the cells formed rosettes with chicken antibody-coated sheep erythrocytes (ChA-SEs) and only 18% of the cells phagocytized them. Nonspecific esterase activity was present in 60% of the cells. The cells treated with phytohaemagglutinin-stimulated chicken spleen cell-conditioned medium (PHA-SCM) exhibited an increase in Fc receptor expression, nonspecific esterase activity and Fc receptor-mediated phagocytosis associated with development of petal-like ruffles on the cell surface. In addition, the cells could form rosettes with rabbit antibody-coated erythrocytes (RaA-SEs). In contrast, the cells treated with lipopolysaccharide (LPS) showed disappearance of ruffle structurcs. The cells exhibited expression of Fc receptor, but no Fc receptor-mediated phagocytosis. Nonspecific esterase activity showed focal staining pattern. The chicken monocytic leukemia cell line was induced to differentiate into individual distinct macrophage-like cells by PHA-SCM and LPS.

Fibroblast-dependent growth of mouse mast cells in vitro: duplication of mast cell depletion in mutant mice of W/Wv genotype.

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.

Lymphokine mediated production of an epidermal cell proliferation factor by cultured murine bone marrow cells.

The effect of cultured bone marrow cell supernatant (BMS) was studied on the proliferative response of cells of the transformed murine epidermal cell line Pam 212. Elevated DNA synthesis was found in Pam 212 cells cultured with BMS from bone marrow cells grown in spleen cell conditioned medium with concanavalin A (ConA). Pam cell proliferative activity was related to the histamine content in the culture supernatant. Neither spleen cell conditioned medium with IL1, IL2, or IL3 activity, nor ConA alone, showed any effect on Pam cell growth. A soluble mediator from the cultured bone marrow cells with mast cell characteristics was thought to be responsible for the stimulation of Pam cell growth, and Con A appeared to be a prerequisite for generation of this factor by the bone marrow cells.

The limitation of growth of Trypanosoma musculi in vitro

Trypanosoma musculi grow readily in vitro provided their growth is supported by mammalian cells. In the presence of murine spleen cells, or spleen cell-conditioned medium, the parasites increase by 100-fold, or more, in a period of 5–6 days. Growth ceases abruptly and death of the parasites soon follows. The reason for the termination of growth has been obscure and is the subject of this report. Termination of growth is not due to an immunological process; not even of ablastin affecting epimastigote reproduction. Instead it appears that other growth inhibitory substances are responsible. Culture medium, collected from spent cultures on day 8 after initiation, inhibits T. musculi growth in fresh medium in dose-dependent fashion. No inhibitory substances were present in medium collected earlier, during the phase of rapid parasite growth. These inhibitory substances appeared to be derived from the parasites rather than the cocultivated spleen cells.

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)

Normalization of Anti-Tick Response of Mast Cell-Deficient W/W v Mice by Intracutaneous Injection of Cultured Mast Cells

Genetically mast cell-deficient (WB X C57BL/6)F1-W/Wv mice show a defect in manifestation of resistance against larval Haemaphysalis longicornis ticks. In order to obtain direct evidence for anti-tick roles of mast cells, we examined whether intracutaneous injection of cultured mast cells normalized the defect of W/Wv mice. Bone marrow cells of (WB X C57BL/6)F1-+/+ mice were cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium. More than 95% of the cultured cells were identified as immature mast cells 4 wk after the initiation of the culture; the cells were harvested and directly injected into the skin of W/Wv mice. Mast cells appeared at the injection sites, where the resistance against the ticks was observed. Thus, mast cells developing at the injection sites seem to play an essential role for manifestation of resistance.