Giant eosinophil colonies from cultures of bone marrow cells

Summary To date, the small size and slow growth of eosinophil colonies in vitro has hampered study of cloned eosinophils. We found enhanced eosinophil colony size and numbers in methylcellulose cultures of bone marrow cells utilizing defined supplemented bovine calf serum (DSBCS) in combination with EL4 conditioned medium (EL4-CM). At days 9, 16 and 23 significantly more eosinophil colonies and more cells/colony were present in cultures incubated with DSBCS/EL4-CM than in cultures incubated with fetal calf serum/EL4-CM. The ability to generate large numbers of eosinophils in vitro should facilitate study of cloned eosinophils. Supported in part by a grant from the National Institutes of Health, AI 20416, and by the Mayo Foundation. Editor's statement Previous approaches to in vitro culture of eosinophils generally have achieved, at best, mixed cultures of colonies of these cells and granulocyte-macrophage colonies. The improved culture methods described in this report produce more homogeneous eosinophil cultures and larger colonies of these cells. The procedure employs EL4 murine thymoma-conditioned medium, which apparently contains eosinophil colony-stimulating activity in the absence of granulocyte-macrophage colony-stimulating activity. David W. Barnes     

Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.     

Differentiation of a cell line of myeloid leukemia

A cell line was established in vitro from a spontaneous myeloid leukemia of SL strain mice. This cell line was cytologically identified as myeloblast, and its normal differentiation appeared to be completely blocked in mass culture. When the line cells were seeded in soft agar with a conditioned medium from normal cells, either macrophages or neutrophil granulocytes appeared from a single clone. The rate of formation of colonies containing differentiated cells always increased with an increase in the concentration of conditioned medium. The conditioned medium from this line cell was not as effective as was that from normal cells in inducing differentiation.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

Receptors for C3 and IgG on macrophage, neutrophil and eosinophil colony cells grown in vitro.

Macrophage, neutrophil, and eosinophil colony cells from bone marrow culture in semisolid agar medium were studied for membrane C3 and IgG receptors. The capacity of these cells to bind either erythrocytes-19S antibody-complement (EAC) or erythrocyte-7S antibody (EA7S) complexes was measured using the rosette method. Whereas macrophage and neutrophil colony cells showed receptors for both C3 and IgG, eosinophil colony cells appear to bear only IgG receptors. Studies correlating colony age and the presence of receptors showed that 60 to 70% of the cells from 3-day-old macrophage colonies were reactive for EAC and EA7S contrasting with 80 to 90% of the cells from 6- to 12-day-old colonies. Neutrophils behaved somewhat differently: EAC and EA7S reactive cells were seen in colonies after 4 or 5 days in culture and comprised only 50 to 60% of the colony population. Eosinophilic colonies showed 50 to 60% EA7S reactive cells after 6 to 7 days in culture, but no EAC reactive cells were found among these colonies at any time. The characteristics and properties of the receptors detected on colony cells were similar to those on macrophages and neutrophils from normal peritoneal fluid or bone marrow. Most macrophage colony cells were actively phagocytic whereas neutrophils and eosinophilic colony cells failed to show phagocytosis under the same conditions.     

Studies on human cord blood progenitor cells

Analysis of agar cultures throughout a 56-day period determined the concentration and cell cycle status of at least 4 different subclasses of hemopoietic colony forming cells (CFC) in human cord blood (CB). Although the concentration of CFC in CB was not significantly different from bone marrow (BM) in day-12 cultures, neutrophil colonies reached their peak on about day 23 in CB cultures and on day 12 in BM cultures. This suggests that the CFC in CB are more primitive than those in BM. In CB cultures, colonies of small cells contained predominantly neutrophils on day 14 and eosinophils on day 35, while the late developing (day 35) colonies of large cells contained mast-cell-like cells (MCL).     

ULTRASTRUCTURAL ANALYSIS OF HEMATOPOIETIC COLONIES DERIVED FROM HUMAN PERIPHERAL BLOOD : A Newly Developed Method

The ultrastructure of granulocyte colonies derived from normal human peripheral blood leukocytes cultured in semisolid media has been studied by a new method developed for this purpose. Fixation, dehydration, and embedding of the whole content of the Petri dish resulted in a block of Epon containing colonies made up of cells with the spatial orientation of those observed in living cultures. This permitted serial sectioning through entire colonies. Cell maturation in vitro appeared to parallel that of normal marrow. However, even the most mature cells retained cytoplasmic characteristics of more immature cells. This was particularly true for eosinophils which only rarely possessed granules with electron-dense crystalline "cores," a feature typical for mature eosinophils. In addition to the normal-appearing hematopoietic cells found within colonies, very large round or spindle-shaped cells were present between colonies and firmly attached to the bottom of the culture dish. Although the histochemical and functional characterization of these cells awaits further study, it is suggested that they are related to histiocytes or macrophages. The technique described here should prove valuable in studies of the development, differentiation, and interaction of many types of cells.     

Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

REGULATION OF HUMAN MYELOPOIESIS BY PROSTAGLANDIN E AND LACTOFERRIN

Studies on human myeloid stem cell proliferation indicate that progenitor cell populations committed to monocytoid differentiation are preferentially inhibited by prostaglandin E (PGE). the addition of PGE but not PGF to day 7 CFU_GM cultures upon initiation results in the dose-dependent inhibition of total colony and cluster formation. Morpholigical analysis of proliferating clones demonstrates that the effect of PGE on total colony and cluster formation results from the selective inhibition of monocyte-macrophage colony forming cells. Mixed monocytoid/neutrophil colony formation was markedly less sensitive and neutrophil and eosinophil colony formation essentially insensitive to the inhibitory effects of PGE. the inhibition of monocytoid colony formation by PGE₁ extends equally well to day 13 CFU_GM, but not to CFU_GM in suspension culture. the observed effects of PGE₁ on monocytoid committed pre-CFU_GM and day 7 and day 14 CFU_GM indicate that sensitivity to inhibition by PGE increases with progenitor cell maturity. Specificity analysis indicates that prostaglandins of the E series (PGE₁, PGE₂) are by far the most active naturally occurring prostanoate compounds inhibiting CFU_GM proliferation. The addition of iron saturated lactoferrin to liquid cultures of human peripheral blood monocytes, inclusion into mononuclear cell feeder layers or addition to agar cultures proliferating in response to endogenously produced CSFs, results in the equivalent inhibition of CSFs necessary for day 7 and day 14 monocytoid and/or neutrophil and eosinophil colony and cluster formation. These results indicate roles for PGE and lactoferrin in myeloid stem cell regulation in vitro and suggest that they may serve as physiological regulators of granulocyte and monocyte proliferation.     

Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

Eosinophils are produced in the bone marrow from CD34⁺ eosinophil lineage–committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage–committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage–committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage–committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage–committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5.     

B Lymphocyte Colony Formation in vitro: Ultrastructural Development of Individual Colonies

B lymphocyte colony development in agar culture was studied using an electron microscope, and more than 3,000 colony cells were identified and photographed. In early cultures (day 4) lymphoblasts dominated the colonies. From day 5 onwards plasmablasts and small lymphocytes were present in the colonies. From day 6 onward mature plasma cells were observed in increasing numbers. On day 9 of culture the colonies started to degenerate and on day 10 of culture approximately 70% of the colony consisted of pyknotic and degenerating cells. Topographically, the degenerating cells were concentrated in the center of the colony whereas proliferation took place in the periphery. Colony growth occurred in an exponential fashion, the number of viable colony cells being maximal on day 8 of culture (400–600 cells/colony). At this time the frequencies of the four B cell categories were: lymphoblasts 72%, plasmablasts 20%, plasma cells 6%, and small lymphocytes 2%. Recloning experiments showed that dispersed colony cells were capable of forming only small cell clusters. It is concluded that B lymphocyte colony formation reflects a series of B cell developmental stages including the formation of the end cell categories of this lymphocyte lineage.     

Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture

We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.     

Spatial distribution and initial changes of SSEA-1 and other cell adhesion-related molecules on mouse embryonic stem cells before and during differentiation.

We examined the distribution of cell adhesion-related molecules (CAMs) among mouse embryonic stem (ES) cells and the spatial distribution on cell surfaces before and during differentiation. The cell-cell heterogeneity of SSEA-1, PECAM-1, and ICAM-1 among the undifferentiated cells in the ES cell colonies was evident by immunohistochemistry and immuno-SEM, supporting the flow cytometry findings. In contrast, most undifferentiated ES cells strongly expressed CD9. SSEA-1 was located preferentially on the edge of low protuberances and microvilli and formed clusters or linear arrays of 3-20 particles. PECAM-1 and ICAM-1 were randomly localized on the free cell surfaces, whereas CD9 was preferentially localized on the microvilli or protuberances, especially in the cell periphery. Both the SSEA-1(+) fraction and the SSEA-1(-) fraction of magnetic cell sorting (MACS) formed undifferentiated colonies after plating. Flow cytometry showed that these populations reverted separately again to a culture with a mixed phenotype. Differentiation induced by retinoic acid downregulated the expression of all CAMs. Immuno-SEM showed decreases of SSEA-1 in the differentiated ES cells, although some clustering still remained. Our findings help to elucidate the significance of these molecules in ES cell maintenance and differentiation and suggest that cell surface antigens may be useful for defining the phenotype of undifferentiated and differentiated ES cells.     

Eosinophil tissue recruitment to sites of allergic inflammation in the lung is platelet endothelial cell adhesion molecule independent

Platelet endothelial cell adhesion molecule (PECAM or CD31) is a cell adhesion molecule expressed on circulating leukocytes and endothelial cells that plays an important role in mediating neutrophil and monocyte transendothelial migration in vivo. In this study, we investigated whether eosinophils, like neutrophils and monocytes, utilize PECAM for tissue recruitment to sites of allergic inflammation in vivo. Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis. RT-PCR studies demonstrate that eosinophils like neutrophils express the six extracellular domains of PECAM. Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM. The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs did not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, and airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo. Thus, in contrast to studies that have demonstrated that neutrophil and monocyte tissue recruitment is PECAM dependent, these studies demonstrate that eosinophil tissue recruitment in vivo in this model is PECAM independent.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.