Semisolid Agar Cultures Research Articles

Granulopoiesis in patients with congenital neutropenia.

Granulopoiesis was investigated in five patients with congenital neutropenia (CN) (one Kostmann type, four benign forms). In semisolid agar culture, the marrow cells of all five patients produced normal numbers of CFU-c (colony-forming unit-culture). The size and classification of colonies were normal. In suspension culture in vitro with exogenous colony-stimulating factor (CSF) generated from omental-conditioned medium (OMCM), the myeloid precursors of all patients could proliferate and differentiate into normal polymorphonuclear neutrophils (PMNs). But in the absence of exogenous CSF, myeloid precursors of the patient with Kostmann-type CN did not proliferate or differentiate into PMNs at all. In the four patients with benign neutropenia, however, PMNs were found even without exogenous CSF similar to normal individuals. These results suggest that patients with CN may have normal granulopoietic stem cells with normal proliferative and differentiating capacity in response to exogenous CSF. When a small amount of normal human serum was added to normal marrow cultures stimulated by exogenous CSF, the colony growth increased in a superadditive manner. The enhancing activity of serum from neutropenic patients differed from that of normal serum. Especially, the addition of serum from the patient with Kostmann type CN to normal marrow cultures did not show this enhancement effect. The sera of patients with benign neutropenia had less enhancement effect than did normal control serum. These findings might be interpreted as showing an imbalance between CSF enhancer and inhibitors in the patients' serum.

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation.

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50-100%) and suppressing the formation of macrophage colonies (75-97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12-125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12-250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.

Constitutive Production of Leukemia Differentiation, Colony-Stimulating, Erythroid Burst-Promoting, and Pluripoietic Factors by a Human Hepatoma Cell Line: Characterization of the Leukemia Differentiation Factor

Conditioned medium (CM) obtained from a human hepatoma cell line, SK-HEP-1, contains colony-stimulating factors (CSFs) active on murine and human bone marrow-derived granulocyte and macrophage colony-forming units (CFU-GM) and a factor capable of inducing granulocyte-macrophage differentiation (GM-DF) of murine myelomonocytic leukemic cells WEHI-3B(D+) and human promyelocytic leukemic cells HL-60 when assayed in semisolid agar cultures. The human active granulocyte-macrophage colony-stimulating factor (GM-CSF) for day 7 CFU-GM and the GM-DF for WEHI-3B(D+) and for HL-60 are not separable by acrylamide agarose column chromatography, eluting at an apparent molecular weight between 20,000 and 35,000 daltons, or by isoelectric focusing (isoelectric point, pH 5.4). In addition, SK-HEP-1 CM contains erythroid burst-promoting activity (BPA) and a factor that promotes the growth of human mixed colonies. SK-HEP-1 cells, which grow as an adherent monolayer, appear not to be endothelial or monocytic in origin since by immunofluorescent staining they are negative for Ia (HLA-DR), monocyte antigen 1 and 2, lysozyme, and factor VIII-related antigen. Positive immunofluorescent staining for keratin and fibronectin suggests the possibility that SK-HEP-1 is an epithelial cell line. Constitutive production of GM-DF as well as other hematopoietic activities including GM-CSF, erythroid BPA, and an activity that promotes the growth of human mixed colony progenitors by a human epithelial tumor cell line, SK-HEP-1, suggests that this cell line is a valuable resource for both large-scale production of these factors and the cloning of the gene(s) that code for these regulators.

Constitutive production of leukemia differentiation, colony- stimulating, erythroid burst-promoting, and pluripoietic factors by a human hepatoma cell line: characterization of the leukemia differentiation factor

Conditioned medium (CM) obtained from a human hepatoma cell line, SK- HEP-1, contains colony-stimulating factors (CSFs) active on murine and human bone marrow-derived granulocyte and macrophage colony-forming units (CFU-GM) and a factor capable of inducing granulocyte-macrophage differentiation (GM-DF) of murine myelomonocytic leukemic cells WEHI- 3B(D+) and human promyelocytic leukemic cells HL-60 when assayed in semisolid agar cultures. The human active granulocyte-macrophage colony- stimulating factor (GM-CSF) for day 7 CFU-GM and the GM-DF for WEHI- 3B(D+) and for HL-60 are not separable by acrylamide agarose column chromatography, eluting at an apparent molecular weight between 20,000 and 35,000 daltons, or by isoelectric focusing (isoelectric point, pH 5.4). In addition, SK-HEP-1 CM contains erythroid burst-promoting activity (BPA) and a factor that promotes the growth of human mixed colonies. SK-HEP-1 cells, which grow as an adherent monolayer, appear not to be endothelial or monocytic in origin since by immunofluorescent staining they are negative for Ia (HLA-DR), monocyte antigen 1 and 2, lysozyme, and factor VIII-related antigen. Positive immunofluorescent staining for keratin and fibronectin suggests the possibility that SK- HEP-1 is an epithelial cell line. Constitutive production of GM-DF as well as other hematopoietic activities including GM-CSF, erythroid BPA, and an activity that promotes the growth of human mixed colony progenitors by a human epithelial tumor cell line, SK-HEP-1, suggests that this cell line is a valuable resource for both large-scale production of these factors and the cloning of the gene(s) that code for these regulators.

The in vitro effect of leucocyte alpha-interferon on human myeloma cells in a semisolid agar culture system.

73 bone marrow samples from a total of 50 patients with multiple myeloma (MM) were tested for interferon (IFN) sensitivity in the human tumour colony assay (HTCA). 16 evaluable samples were obtained from untreated patients, 10 from patients during melphalan and prednisone treatment and 6 from patients on IFN treatment or after withdrawal of IFN. The sensitivity to IFN was individually distributed between the bone marrow samples from patients of all 3 groups and a tendency towards IFN resistance could be found in serial assays from patients during MP-treatment and after IFN withdrawal. Stimulation of growth was found in 31% of the cultures, usually at lower doses of IFN (10-100 U/ml) while higher doses (400-4000 U/ml) inhibited growth in 75% of the assays.

Enhancement of colony-stimulating-factor--dependent clonal growth of murine macrophage progenitors and their phagocytic activity by retinoic acid.

The effect of retinoic acid (RA) on the colony-stimulating-factor-dependent clonal growth of myeloid progenitors was assessed in semisolid agar cultures of mouse bone marrow cells using L-cell-conditioned medium that gave rise to macrophage colonies, granulocyte colonies, and mixed macrophage-granulocyte colonies and clusters. RA was found to enhance the overall formation of myeloid colonies (about 50%) and clusters in 7-day cultures. The increase was due to an enhanced formation of macrophage colonies (70-250%) and clusters which reached a maximal value at about 3 microM RA. In 4-day cultures, the effect of RA on macrophage colony formation was biphasic with a maximal enhancement at 10 nM. RA suppressed granulocyte-colony formation in 4-day cultures. RA increased the phagocytic activity of bone-marrow-derived macrophages at all stages of differentiation and/or maturation in culture. The Fc-receptor-mediated erythrophagocytosis as well as the phagocytosis of heat-killed yeast cells (HK-yeast) and starch particles increased by RA treatment in a dose-dependent manner, reaching an increase of 100-200% of the activity expressed in the absence of RA. Peritoneal exudate macrophages likewise exhibited an increased phagocytic response to a variety of particles, at both physiological and pharmacological concentrations of RA. Expression of an RA-mediated increase in phagocytic activity required a prolonged incubation with RA (greater than 19 hr). The data suggest that RA may be of physiological relevance in the regulation of proliferation and function of hemopoietic cells. Therapeutic doses of RA may potentiate macrophage proliferation and function, elements that are crucial at all phases of the various defense mechanisms that the organism possesses.

Indirect serum haemagglutinating antibody response to black pigmented Bacteroides during experimental pure infections in rats.

The humoral immune response during experimental infection with black pigmented bacteroides was studied by the indirect haemagglutination test. Both Bacteroides melanimogenicus (ATCC 25845) and B. intermedius (ATCC 25611), grown in semi-solid agar culture, produced pure subcutaneous, intra-abdominal and lung infections. In each of these infection models, B. intermedius was found to be more pathogenic than B. melaninogenicus on the basis of gross pathology of the lesion, bacteriological and histopathological findings, and the capacity to produce persistent infection. Cytoplasmic extracts of these strains were used as an antigen for the indirect haemagglutination test. In all the infections, B. intermedius provoked a better and higher antibody response than did B. melaninogenicus, suggesting a potent immunogenic property of the former strain. Peak antibody titres in both groups during all the above infections were seen between the 10th and 15th days post infection (p.i.), which was precisely 3-5 days after peak lesion was achieved. A significant IHA antibody titre persisted up to days 30-37 p.i. These findings suggest that the antibodies to the black pigmented Bacteroides are not protective, but may play a role in the pathogenesis of the diseases.

Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice.

Abstract BXSB mice, a recently developed autoimmune strain, develop a human lupus-like disease with B cell hyperplasia in peripheral lymphoid organs. Unlike other experimental models of autoimmunity and human lupus, BXSB male mice manifest accelerated autoimmune phenomena through the influence of a Y chromosome-linked enhancing factor. The present studies were performed to investigate the features of B lymphopoiesis in BXSB mice and to determine whether differences exist between BXSB males and females in this respect. B lineage cell populations in the marrow of BXSB mice were identified phenotypically by studying the cytoplasmic mu-heavy chains of IgM (c mu), and functionally by their ability to acquire clonability and sIg in short-term liquid cultures. Male BXSB mice became deficient in both the precursors of functional B cells and c mu + pre-B cells by the age of 8 to 12 wk. This followed a transient increase in this population, which peaked when the mice were 2 to 4 wk old. In females, substantial numbers of functional B cell precursors and c mu + cells were maintained until more than 4 mo of age. Cells lacking Ig but bearing a B lineage cell antigen (14.8) were elevated in numbers in both BXSB males and females until 16 wk of age when compared to normal strains of mice. At the time pre-B cells and functional B precursors were elevated in numbers, some sIg- cells were shown to form colonies in mitogen-stimulated semisolid agar cultures without a period of preculture. Most of these sIg- cells seemed to bear the B lineage cell antigen (14.8). They were independent of both G-10 adherent regulatory cells and Thy-1+ cells for their colony formation. These results indicate that B lymphocyte formation may be maintained in a hyperactive state in BXSB females, whereas males become deficient in B cell precursors very early in life. This early decline might be related to the accelerated development of autoimmune disease in BXSB mice. Bone marrow transplantation studies showed that these unusual characteristics of B lymphopoiesis were reciprocally transferable with unseparated bone marrow cells between BXSB males and females. This finding indicates that sex hormones are not a critical variable in abnormal B lymphocyte formation in this strain, and that the premature deficiency of immediate B precursors in males may be regulated by a genetic factor(s) located on the Y chromosome.

Differentiation of human acute myeloid leukaemia cells in response to exogenous and endogenous stimuli: Different patterns of response suggested by a bioassay system

The object of this study was to devise quantitative bioassay systems suitable for the analysis of differentiation in acute myeloid leukaemia (AML) in response to both endogenous (bone marrow) and exogenous stimuli. Dose response analyses of the relationship between exogenous differentiation stimulus (supplied by peripheral blood mononuclear leucocytes) and clone cell maturity in double layer semi-solid agar cultures, were used to identify a measure of response with a linear relationship to differentiation stimulus on which bioassay systems could be based. All 7 AML samples tested at presentation and 1 sample from a patient during the regenerative phase after therapy, exhibited some response to exogenous differentiation stimulus. Only in the latter was there no significant difference in the response of test and reference cells. The other 7 showed marked variation in the pattern of the response in vitro. There was no close correlation between differentiation capacity in vitro and extent of differentiation observed in vivo. This discrepancy might be related to varying availability of endogenous differentiation stimulus in different patients. The technique for assaying differentiation response was adapted to demonstrate and quantify the endogenous differentiation stimulus (the stimulus provided by the leukaemic patient's marrow cells). The results suggest that in some patients the level of this stimulus may be a major determinant in the level of differentiation achieved in vivo.

Precocious and enhanced functional maturation of B lineage cells in New Zealand Black mice during embryonic development.

Previous reports suggest that large numbers of immunoglobulin-secreting cells appear in tissues of NZB strain mice from the time of birth. In this study, we investigated the development of B lineage cells during embryonic life and found that they were present 2-3 d earlier and in higher numbers in NZB embryos than several other strains of mice. That is, liver cell suspensions from NZB embryos contained larger numbers of surface Ig (sIg)- cells that could form B cell colonies in mitogen-dependent semisolid agar culture. Sephadex G-10-adherent cell depletion diminished numbers of colonies and this was partially restored by addition of humoral factors. The latter were partially purified from serum of very young NZB mice. These findings document that abnormal changes take place in B lineage cells and possibly also in cells that regulate their maturation in NZB strain mice at a very early stage of development.

Survival and repopulation of irradiated 'pre-CFU-c' in mice.

Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c, burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). The CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely 'pre-CFU-c', in the maturation sequence of granulopoiesis, and this maturation of the 'pre-CFU-c' to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.

Retinoic acid enhances the growth of only one subpopulation of granulomonocyte precursors.

The effect of various concentrations of retinoic acid on the growth of committed granulomonopoietic precursors (CFU-GM) was studied on semisolid agar cultures. Retinoic acid increased the growth of day 14 CFU-GM but had no significant effect on day 7 CFU-GM. Since the latter are believed to represent the progeny of day 14 CFU-GM it is suggested that the effect of retinoic acid is confined to the early stages of granulomonocytic differentiation.

Analysis of the effects of erythrocytes on mitogen-dependent clonal proliferation of murine B lymphocytes

Addition of intact erythrocytes to semisolid agar cultures of murine B cells dramatically improves cloning efficiency and affects colony morphology. In this study, we investigated possible mechanisms through which this might occur. Specific modification of sheep erythrocyte (SRBC) membranes by treatment with trypsin but not other enzymes improved colony potentiation and erythrocytes from rats, mice, and humans were also effective after trypsin treatment. In addition, autoantibody-coated murine erythrocytes were superior to normal cells in this regard. These observations suggest that erythrocytes enhance lymphocyte survival and/or proliferation by means of particular membrane-mediated processes. The possible importance of erythrocytes as scavengers of toxic hydroxyl radicals was also investigated. Deliberately generated radicals formed by addition of dihydroxyfumaric acid and iron were effectively countered by addition of SRBC. More detailed analyses revealed that of several endogenously produced toxic species, hydrogen peroxide may be the most important under ordinary culture conditions. That is, addition of catalase but not Superoxide dismutase or mannitol improved cloning efficiency in cultures lacking SRBC. These studies suggest that erythrocytes have a beneficial effect on lymphocyte survival and function in culture through at least two mechanisms.

Stimulation of human and murine bone marrow cell colony formation by colony-stimulating factors obtained from the urine of mice bearing leukocytosis-inducing fibrosarcoma.

Dialyzed urine of mice bearing leukocytosis-inducing fibrosarcoma stimulated granulocyte colony formation in semisolid agar culture of human bone marrow cells. Removal of phagocytic cells prior to stimulation did not interfere with the formation of these colonies in the culture. On the other hand, macrophage colonies were predominantly produced when murine bone marrow cells were stimulated by the dialyzed mouse urine. The activity of colony-stimulating factor (CSF) in the urine of normal mice was less than 1/100 of that in the urine of tumor-bearing mice. DEAE-cellulose column chromatography separated the activity stimulating human granulocyte colony formation from that stimulating murine macrophage colony formation. Further purification showed that a sialoglycoprotein with an apparent molecular weight of 80,000 corresponded to the macrophage CSF, which was devoid of activity toward human cells. The molecular properties of the human-active granulocyte CSF could not be studied further, because it was quite unstable.

B lymphocyte lineage cells in newborn and very young NZB mice: evidence for regulatory disorders affecting B cell formation.

As adults, NZB mice have a severe deficiency of identifiable precursors of B lymphocytes, but this is preceded by a time when B cells and their immediate precursors are present in bone marrow in elevated numbers. Our present studies indicate that the final stages of B cell formation may be proceeding at a greater than normal rate at 4 wk of age. At this time, large numbers of sIg- B cell precursors can be identified in NZB but not in normal CBA/H or DBA/2 marrow that can respond to mitogens in semisolid agar cultures without preculture. Colony formation by these precursors was dependent on the presence of Sephadex G-10-adherent cells in the suspensions. In this respect, 4-wk-old NZB marrow was similar to spleen and liver of normal CBA/H newborn mice. Mixing experiments suggested that hyperactive regulatory cells present in young NZB marrow may promote functional maturation of normal pre-B cells from CBA/H or DBA/2 mice. In addition, potent substances present in the serum of young but not older NZB mice produced the same effect. Factor-mediated enhancement of clonal proliferation by sIg- precursors was not dependent on adherent cells and required more than brief exposure of the cells to young NZB serum. These are all indications that abnormalities of B lineage differentiation in young NZB marrow may be attributable to microenvironmental elements. Congeneic, B cell-deficient NZB.xid mice, however, lacked hyperactive regulatory cell function and serum factors that characterize NZB mice of this age. A study was also made of B cell precursors in newborn spleen and liver of NZB mice, and abnormal shifts of populations of B lineage cells were already present by that time. These observations support previous contentions that humoral immunity is precocious and abnormally regulated in NZB mice.

TS/A: a new metastasizing cell line from a BALB/c spontaneous mammary adenocarcinoma.

A metastasizing mouse cell line (TS/A), originated from a mammary adenocarcinoma which arose spontaneously in a BALB/c female retired breeder, has been established in vitro. It displayed a remarkable morphologic heterogeneity, which is evident in plastic adherent cultures (cell types ranging from epithelial-like to fibroblast-like) as well as in semi-solid agar cultures. The TS/A line exhibited the presence of specific cytoplasmic estradiol receptor, with a binding activity of 16 fmoles/mg cytosol protein. The in vivo growth pattern was as follows: a s.c. inoculum of 105 cells caused a 100 per cent tumor take and kill in syngeneic animals; mean survival time was 54 +/- 1 days; it did not show significant transplant immunogenicity in syngeneic animals; it was able to give rise to both spontaneous lung metastases and artificial lung colonies; it had a high capacity to grow in H-2 matched, minor histocompatibility antigen incompatible hosts (10(6) cells killed 100 per cent DBA/2 mice in 58 +/- 2 days). This line of spontaneous mammary tumor cells is proposed as a useful model for studies on the heterogeneity of the neoplastic population in relation to metastatic spread, on tumor immunogenicity, and on therapy of mammary neoplasia.

Effect of mouse interferon on growth and differentiation of mouse bone marrow cells stimulated by two different types of colony-stimulating factor

The effects of mouse L-cell interferon (IFN) on growth of mouse bone marrow cells and their differentiation into macrophages and granulocytes were investigated in a liquid suspension culture system with two different types of colony-stimulating factor (CSF). Within 7 days, most bone marrow cells differentiated into macrophages in the presence of macrophage colony-stimulating factor (M-CSF) derived from mouse fibroblast L929 cells, but into both granulocytes (40%) and macrophages (23%) in the presence of a granulocyte-macrophage colony- stimulating factor (GM-CSF) from mouse lung tissue. IFN inhibited growth of bone marrow cells with both M-CSF and GM-CSF, but had 20 times more effect on bone marrow cells stimulated with M-CSF than on those stimulated with GM-CSF. A low concentration of IFN (50 IU/ml) stimulated production of macrophages by GM-CSF in liquid culture medium, whereas it selectively inhibited colony formation of macrophages in semisolid agar culture. IFN caused no detectable block of late stages of differentiation; mature macrophages and granulocytes were produced even when cell proliferation was inhibited by IFN. These results indicate that IFN preferentially affects growth and differentiation of the cell lineage of macrophages among mouse bone marrow cells.

Effect of Mouse Interferon on Growth and Differentiation of Mouse Bone Marrow Cells Stimulated by Two Different Types of Colony-Stimulating Factor

The effects of mouse L-cell interferon (IFN) on growth of mouse bone marrow cells and their differentiation into macrophages and granulocytes were investigated in a liquid suspension culture system with two different types of colony-stimulating factor (CSF). Within 7 days, most bone marrow cells differentiated into macrophages in the presence of macrophage colony-stimulating factor (M-CSF) derived from mouse fibroblast L929 cells, but into both granulocytes (40%) and macrophages (23%) in the presence of a granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse lung tissue. IFN inhibited growth of bone marrow cells with both M-CSF and GM-CSF, but had 20 times more effect on bone marrow cells stimulated with M-CSF than on those stimulated with GM-CSF. A low concentration of IFN (50 IU/ml) stimulated production of macrophages by GM-CSF in liquid culture medium, whereas it selectively inhibited colony formation of macrophages in semisolid agar culture. IFN caused no detectable block of late stages of differentiation; mature macrophages and granulocytes were produced even when cell proliferation was inhibited by IFN. These results indicate that IFN preferentially affects growth and differentiation of the cell lineage of macrophages among mouse bone marrow cells.

Changes in B lineage cell population in liver and spleen of normal neonatal mice.

The frequency and characteristics of B lymphocyte lineage cells in neonatal murine liver and spleen were studied during the first 10 days after birth. These were distinguished as B cells with surface IgM (slgM), immediate precursors of B cells (pre-B cells) lacking slgM but containing micron-heavy chains of IgM, and earlier precursors that did not synthesize immunoglobulin but could be detected with monoclonal 14.8 antibody. Experiments were also done to relate these to cells capable of clonal proliferation in mitogen-containing semisolid agar cultures and cells that acquire this function only after preculture in liquid medium. Newborn liver contained large numbers of early precursors as well as pre-B cells, and culture studies revealed that a majority of the colony-forming B cells present at that time were slg-. Adherent accessory cells in newborn liver suspensions facilitated the maturation of these into functional B cells in vitro. At most ages, however, numbers of slg+ B cells detected in that tissue were surprisingly low. Possible explanations for this include a rapid exit of newly formed B cells and their immediate precursors from liver and/or a high rate of abortive lg gene rearrangements during the neonatal period. In contrast, whereas the spleen contained early precursors and pre-B cells at birth, these cells steadily declined in number with age as the numbers of slgM+ B cells increased. Adherent cells in liver but not spleen of immunodeficient CBA/N mice suppressed B lymphocyte formation in semisolid or liquid cultures. These observations document population dynamics in B lineage cells during a critical period of development.

Effects of 12-0-Tetradecanoylphorbol-13-Acetate (TPA) on the Proliferation of Granulocyte-Macrophage Colony-Forming Cells

Abstract It has been suggested that 12–0-tetradecanoylphorbol-13-acetate (TPA) may stimulate the proliferation of granulocyte-macrophage (GM) colony- forming cells (CFC) via the GM colony-stimulating factor (CSF) receptor. GM-CFC in unfractionated mouse bone marrow and light density fetal liver (LDFL) cells were induced by TPA to form colonies in the absence of exogenously added GM-CSF. The colonies induced by TPA (10(- 8)M) were smaller than normally seen with maximal concentrations of GM- CSF, and less than 30% of the GM-CFC formed colonies in the presence of TPA. The number of colonies stimulated by TPA in the absence of GM-CSF was dependent on the number of cells plated. When fewer than 10,000 bone marrow cells or 3000 LDFL cells were plated in the 1-ml semisolid agar cultures, no colonies were stimulated by the TPA. Similarly, GM- CFC purified from the LDFL cells stimulated with TPA did not form colonies. However, when the fetal liver accessory cells (macrophages) were recombined with cell-sorter-purified GM-CFC, colony formation was again observed in the presence of TPA (10(-7)-10(-8) M). The number of colonies formed from the CFC was dependent on the number of accessory cells present, suggesting that the macrophages were induced by TPA to produce CSF. Although the purified GM-CFC required CSF for proliferation, TPA (10(-8) M) increased (5–10-fold) the sensitivity of the GM-CFC to GM-CSF. These observations indicate that TPA does not stimulate GM-CFC proliferation directly, but rather by inducing GM-CSF production by accessory cells and by increasing the responsiveness of GM-CFC to GM-CSF.