Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques.

We describe a novel method for the isolation and subsequent culture of pulmonary neuroendocrine cells (PNEC) from normal fetal rabbit lung using immunomagnetic techniques with a monoclonal antibody, MOC-1. This surface antigen has originally been identified on small cell carcinoma of the lung. Our immunohistochemical studies have shown that MOC-1 cross-reacts with PNEC of human and rabbit fetal lungs on frozen sections, and in fixed cultures of rabbit fetal lung. Using a combination of mechanical and enzymatic disaggregation, a single-cell suspension of fetal rabbit lung was obtained. These cells were incubated with MOC-1 conjugated to magnetic beads. PNEC were selectively removed from the heterogeneous mixture using a magnet, giving up to 2-fold enrichment compared with our previously reported method. These cells were maintained in culture in a functional state for up to 7 days. The ability to prepare PNEC from rabbit fetal lung offers an opportunity to develop in vitro models to investigate the physiologic and biochemical properties of these cells, and ultimately it may lead to a better understanding of their function in health and disease.     

Anti-bombesin monoclonal antibodies modulate fetal mouse lung growth and maturation in utero and in organ cultures

Fetal pulmonary neuroendocrine cells (PNECs) contain abundant gastrin-releasing peptide (GRP, mammalian bombesin-like peptide [BLP]). Previously, addition of bombesin resulted in increased fetal lung growth and maturation in utero and in organ cultures. A monoclonal antibody (mAb) to bombesin (2A11) blocked baseline automaturation of lung organ cultures in serum-free medium. In the present study, we analyze lung development following daily in utero administration of 2A11 from gestational days 15–18. Fetal lung treated with 2A11 and then harvested on day 18 demonstrated a dose-dependent decrease in surfactant phospholipid synthesis compared to controls treated with MOPC, an unreactive mAb. However, 2A11-treated fetal lung harvested on day 17 showed paradoxical increases in ³H-choline incorporation into saturated phosphatidylcholine, ³H-thymidine incorporation into DNA, and relative numbers of differentiated type II pneumocytes. In serum-containing day 17 lung organ cultures, 2A11 stimulated choline and thymidine incorporation. Since epidermal growth factor (EGF) is the only agent besides bombesin known to stimulate both fetal lung growth and maturation, we added EGF to serum-free cultures and reconstituted the stimulatory effects. A murine EGF receptor mAb (ERA) blocked 2A11-induced lung growth and maturation in serum-containing cultures, and this effect was overcome by adding EGF. In vivo, ERA also blocked stimulatory effects of 2A11 in fetal lung on day 17. These observations suggest that EGF receptor up-regulation may maintain lung growth and maturation if BLP levels are diminished on day 17. Nonetheless, BLPs appear to be involved in lung maturation on day 18, supporting a role for PNECs in normal lung development. © 1993 Wiley-Liss, Inc.     

Radiation-Induced Lung Injury Is Mitigated by Blockade of Gastrin-Releasing Peptide

Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68⁺ macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3⁺ nuclei/cm². GRP blockade with 77427 treatment diminished CD68⁺, GRP⁺, and pSmad2/3⁺ cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally.Radiation fibrosis is a serious complication that affects normal lung following unintentional exposure or due to therapeutic ionizing radiation of thoracic tumors. Despite advances in radiobiology, precise mechanisms by which radiation induces lung injury remain controversial.¹ Classically, radiation-induced lung injury (RiLI) is characterized by a latent period that can last for weeks to months after radiation exposure, followed by 2 stages of overt lung injury that can lead to life-threatening and debilitating pulmonary toxic effects.^2,3 Acute inflammatory lung injury arises 1 to 6 months after radiation exposure, with diffuse alveolar damage, similar to acute respiratory distress syndrome. Later, chronic interstitial and intra-alveolar fibrosis develops, predominantly in irradiated segments, with myofibroblast proliferation and collagen deposition. It is unclear why only approximately 15% of radiation-exposed patients develop RiLI.^1,4 General cytoprotective agents, such as a catalytic antioxidant metalloporphyrin (AEOL10113), can reduce the severity of RiLI by decreasing free radical injury after radiation.⁵Our novel paradigm links gastrin-releasing peptide (GRP) to radiation lung injury. We hypothesized that GRP is a mediator of RiLI: promoting both macrophage accumulation and fibrosis. We propose that ionizing radiation triggers pulmonary neuroendocrine cell (PNEC) hyperplasia, leading to GRP secretion, which then mediates chronic lung injury. GRP receptor (GRPR) gene expression is detected and functional in pulmonary epithelial cells, fibroblasts, endothelial cells, and macrophages.^6–10 GRP is a proinflammatory neuropeptide that functions as an inflammatory cell activator, mitogen, and cell differentiation factor.^8,10,11 GRP is expressed at the highest levels in PNEC in fetal lung,¹² where it can promote lung development.¹³ After birth, GRP production normally decreases, but elevated levels are associated with many inflammatory lung conditions, including chronic lung disease of newborns (bronchopulmonary dysplasia).^14–17 PNEC hyperplasia can be triggered by inflammation or exposure to oxygen or ozone^10,16,18 and can take weeks to reach peak levels.¹⁹The present investigation tests the hypothesis that GRP contributes to radiation-induced pulmonary fibrosis in C57BL/6 mice. One hour post exposure to thoracic radiation (15 Gy), we treated mice i.p. with either PBS or GRP blockade by using small molecule 77427. We have quantified results of immunohistochemistry (IHC) by using image analysis with ImageJ version 1.46e (NIH, Bethesda, MD) to determine whether GRP contributes to radiation-induced inflammatory responses and/or fibrosis, specifically including assessment of active transforming growth factor (TGF)-β signaling.     

Cell biology of pulmonary neuroepithelial bodies—validation of an in vitro model. I. Effects of hypoxia and Ca2+ ionophore on serotonin content and exocytosis of dense core vesicles

Pulmonary neuroendocrine (NE) cells including the innervated clusters of NE cells—neuroepithelial bodies (NEB)—are difficult to study because of their small numbers and diffuse distribution within the airway mucosa of the lung. We have previously reported a method for isolation and culture of NE cells from rabbit fetal lung using a combination of mechanical and enzymatic dissociation followed by gradient centrifugation. This method provides single cell suspension of mixed lung cells enriched in NE cells, particularly those originating from NEB. This study further validates our in vitro model by detailed morphologic characterization of cultured NEB cells using high resolution light microscopy, transmission and scanning electron microscopy, HPLC for detection of serotonin (5-HT), and molecular (Northern blot) analysis of mRNA encoding for 5-HT synthesizing enzymes, tryptophane hydroxylase, and aromatic L-amino acid decarboxylase. In addition to effects of hypoxia on NEB cells in vitro were investigated to define the role of these cells as possible airway chemoreceptors. Exposure of NEB cultures to hypoxia resulted in decreased intracellular content of 5-HT accompanied by increased exocytosis of dense core vesicles (DCV). The amount of 5-HT release correlated with the degree of hypoxia, suggesting modulation by ambient pO₂ levels. The role of Ca²⁺ ions in exocytosis of DCV and 5-HT release from NEB cells was tested in experiments with Ca²⁺ ionophore (A23187). Exposure of cultures to 5 μg/ml of ionophore resulted in up to 40% reduction in 5-HT content of NEB cultures as well as increased exocytosis of DCV. Our overall findings are consistent with a view that NEB cells are chemosensory in nature and that Ca²⁺ signaling pathway is involved in stimulus-secretion coupling. Further refinements in cell separation and culture methodology are required before more detailed investigation of NEB cell membrane properties, signal transduction mechanisms, and intracellular signaling pathways can be carried out. © 1993 Wiley-Liss, Inc.     

人胎肺神经内分泌细胞的发育及其作用

目的：探讨胎肺生长发育中神经内分泌细胞的表达及其功能意义。方法：检测３０例人胎肺组织,采用组织化学Ｇrimelius嗜银反应、免疫组织化学、透射电镜技术及显微图像分析。结果：神经内分泌细胞最早在胚胎第１２周胎肺出现,主要为分泌ＧＲＰ细胞,其分布与细支气管发育分化密切相关;ＣＴ阳性细胞在胚胎１６周胎肺出现;分泌ＣgＡ细胞在２０周 出现。随胎龄增长,神经内泌细胞数量递增,胚胎２４周达高峰。此后细胞数量递减,出生     

胎儿小肠生长抑素、胃泌素及血管活性肠肽免疫反应细胞的个体发生

目的：探讨生长抑素（SS）、胃泌素（Gas）、血管活性肠肽（VIP）免疫反应（IR）细胞在胎儿小肠中的个体发生及其分布。方法：免疫组织化学方法和图像分析技术。结果：SS-IR细胞和Gas-IR细胞于11周即可见于小肠三段绒毛上皮和尚未分化完全的肠腺细胞间。随着胎龄增长,SS-IR细胞数量和细胞内SS由少至多,其中以十二指肠细胞最多,回肠最少。Gas-IR细胞和细胞内的Gas在十二指肠中随胎龄增长逐渐增多;21周后空肠中则有所减少,回肠中则消失。VIP-IR细胞数量少,整个胎期细胞数量、形态及免疫染色强度均未见明显变化。结论：SS、Gas和VIP在胎儿小肠的内分泌细胞中表达,提示内分泌激素在胎儿小肠的发育过程中起调节作用。     

对胎儿胃粘膜三种内分泌细胞的观察

目的探讨胎儿胃粘膜三种内分泌细胞的发生过程。为胚胎学发展提供资料。方法收集2-10个月胎儿53例,新生儿3例,利用免疫组织化学方法,检测胃窦粘膜中胃泌素细胞（G）,生长抑素细胞（SS）和5-羟色胺细胞（5-HT）,计数进行组间比较。结果胎龄5-9个月胎儿胃窦粘膜中G、SS、5-HT细胞迅速增多,以G细胞数目最多,足月胎儿及新生儿G细胞减少。结论胃粘膜三种内分泌细胞随胎龄增多,在胎儿生长发育旺盛期6-8月数目最多。     

胎儿子宫内膜内分泌细胞的免疫组化观察

目的：观察胎儿子宫内膜内分泌细胞的发育规律、形态特征及含物，探讨其生物学意义。方法：12～39w胎儿子宫内膜20例，采用嗜铬素A免疫组化染色标记内分泌细胞；血清素，生长抑素及胃泌素释放肽的免疫组化染色，观察胎儿子宫内膜内分泌细胞的内含物。结果：最早在16w子宫膜内膜上皮出现CgA阳性细胞，分布在子宫上皮及腺上皮，以靠近基底处较多。各胎龄组子宫内膜均未见血清素、生长抑素及胃泌素释放肽阳性细胞。结论：胎儿子宫内膜与成人子宫内膜的内分泌细胞在分布、数量和类型上存在差异。内分泌细胞在胚胎早期出现，提示内分泌细胞对子宫内膜的发育具有特殊意义。     

胚胎及新生大鼠胃肠道5-羟色胺及胃泌素免疫反应细胞的形态学研究

本研究用免疫组织化学ＰＡＰ法，较系统地观察了５－羟色胺、胃泌素免疫反应细胞在胚胎及新生大鼠胃肠道的个体发生及分布。结果表明：５－羟色胺免疫反应细胞最早见于第１９ｄ胚胎的小肠各段。胚胎第２１ｄ及新生期分布于胃肠道各段。５－羟色胺免疫反应细胞形态多样。胚胎期胃肠道中胃泌素免疫反应细胞也始见于第１９ｄ胚胎鼠的胃和小肠。胚胎第２１ｄ和新生期，胃泌素免疫反应细胞渐多，但也仅见于胃和小肠各段，结肠和直肠中未见胃泌素免疫反应细胞。本文对大鼠胃肠道两种免疫反应细胞在胚胎发育中的可能功能进行了讨论。     

Postnatal development of intestinal endocrine cell populations in the water buffalo

The frequency and distribution of 11 endocrine cell populations were studied in the intestine of differently aged buffalo, grouped on the basis of diet: 2-d-olds (suckling), 5-mo-olds (weaning) and 5-y-olds (ruminant adult diet). The endocrine cell populations were identified immunocytochemically using antisera against 5-hydroxytryptamine (5-HT), somatostatin, gastrin, cholecystokinin (CCK), COOH-terminal octapeptide of gastrin/CCK, neurotensin, motilin, gastric inhibitory polypeptide (GIP), secretin, glucagon/glicentin (GLU/GLI) and polypeptide YY (PYY). In adult buffalos the regional distribution of endocrine cells is similar to that of other adult ruminants. During postnatal development, these cell types showed the following changes in their frequency and distribution: (1) 5-HT, neurotensin and gastrin/CCK immunoreactive cells (i.c.) showed a decrease in frequency with age; (2) somatostatin i.c. frequency remained stable with age; (3) motilin, GIP, secretin and CCK i.c. showed a slight increase in frequency with age; (4) GLU/GLI and PYY i.c. decreased in frequency with age in the small intestine, caecum and proximal colon and an increase in frequency in the rectum. It was hypothesised that the endocrine cell types, whose presence and localisation is substantially stable in all examined ages, probably contain substances that are strictly necessary for intestinal function. In contrast the hormones contained in the cell populations that decreased with age, are probably involved in physiological needs during the milk and weaning diet or play a role in intestinal growth.     

Impaired survival of T cell receptor Vγ3+ cells in interleukin-4 transgenic mice

The mouse epidermis contains a network of Thy-1⁺ dendritic T cells. Most of these cells express a homogeneous T cell receptor (TCR) configuration (Vγ3/ Vδ1) with only negligible junctional diversity. Because fetal thymocytes are precursors of these dendritic epidermal T cells (DETC) and the addition of interleukin (IL)-4 to fetal thymic organ cultures causes an early arrest in thymopoiesis, we examined DETC development in transgenic (tg) mice expressing IL-4 under the control of major histocompatibility complex class I regulatory sequences. Immunohistologic examination of epidermal sheets and polymerase chain reaction analysis of total skin RNA from IL-4 tg mice failed to reveal TCR Vγ3⁺ DETC and Vγ3 mRNA, respectively. In contrast, the sizes of TCR γδ subpopulations in lymphoid organs were unchanged in these mice. Although the numbers and staining intensities of TCR Vγ3⁺ thymocytes in early fetal (days 14–17) IL-4 tg mice were similar to those of littermate controls, we observed a preferential death of these cells in thymic organ cultures from IL-4 tg mice. We observed further that epidermal sheets prepared from 9-day-old mice whose mothers had been treated with an IL-4-neutralizing antibody from day 12 to day 18 of pregnancy contained DETC numbers similar to those of controls. However, upon termination of the anti-IL-4 treatment, DETC ceased to expand. We conclude that IL-4 impairs the survival of TCR Vγ3⁺ cells.     

Effects of catecholamines on fetal rat cardiocytes in vitro

Norepinephrine stimulates the growth in size of non-dividing, neonatal cardiac muscle cells, and it can stimulate the growth in numbers of dividing hepatocytes and endothelial cells in culture. The objective of this study was to test the hypothesis that in dividing fetal cardiocytes, norepinephrine would stimulate growth in cell number rather than in cell size. Fourteen-day fetal heart cells were placed in serum-free or serum-supplemented cultures in the presence or absence of norepinephrine (NE), NE plus propranolol, or isoproterenol for 4 days. Almost 90% of the cardiocytes in serum-supplemented medium were in the cell cycle as determined by proliferating cell nuclear antigen (PCNA) antibody staining during this period. In addition, between days 2 and 4 of culture, 35% and 40% of these cardiocytes were labeled with ³H-thymidine. After 4 days the cardiocytes increased in cell number in the serum-supplemented NE cultures as compared to serum-free cultures. In contrast, there was no significant change in cardiocyte volume between any of the groups examined. It was concluded that in dividing muscle cell populations the effect of norepinephrine was to enhance cell proliferation rather than to stimulate cell growth in size.     

Gastrin releasing peptide and neuropeptide Y exert opposing actions on circadian phase

Microinjection of gastrin releasing peptide (GRP) into the third ventricle or the suprachiasmatic nucleus (SCN) induces circadian phase shifts similar to those produced by light. Administration of GRP during the day does not alter circadian phase. In contrast, neuropeptide Y (NPY) induces phase shifts of circadian rhythms during the day but has little effect when administered at night, similar to the effects of most non-photic stimuli. NPY inhibits the phase shifting effects of light, and GRP is thought to be part of the photic signaling system within the SCN. This experiment was designed to test whether GRP and NPY inhibit each other's effects on circadian phase. Adult male Syrian hamsters equipped with guide cannulas aimed at the SCN were housed in constant darkness until stable free-running rhythms of wheel running activity were apparent. Microinjection of GRP during the early subjective night induced phase delays that were blocked by simultaneous administration of NPY. During the middle of the subjective day, microinjection of NPY caused phase advances that were blocked by simultaneous administration of GRP. These data suggest that GRP and NPY oppose each other's effects on the circadian clock, and that the actions of NPY on the photic phase shifting mechanism in the SCN occur at least in part downstream from retinorecipient cells.     

IgE receptors on human lymphocytes III. Expression of IgE receptors on mitogen-stimulated human mononuclear cells

Human peripheral blood mononuclear cells (PBMC) were tested for the expression of Fee-receptor (FcεR) after stimulation with various mitogens in the absence of IgE. FcεR were found on virtually all the cells from 19 Epstein-Barr virus-transformed B cell lines including those derived from cord blood, from one agamma-globulinemic patient and VDS-0 pre-B cells. Hence, the data clearly indicate that FcεR may be expressed on very immature B cells. PBMC cultures stimulated with either pokeweed mitogen (PWM), phytohemagglutinin (PHA) or concanavalin A displayed an early increase of their content in FcεR-bearing cells followed by a decrease to levels below those of the control cultures. After fractionation of the PWM-stimulated cultures into T and B cell-enriched preparations, most of the FcεR⁺ cells were in the B cell fractions and the same low levels of FceR⁺ cells were found in the T cell fractions isolated from the PWM-stimulated and from the control cultures. Double-labeling experiments, employing biotinylated F(ab')₂ monoclonal antibody to FcR and either fluorescein isothiocyanate-conjugated B1 or Mo2 monoclonal antibodies, indicated that PWM mainly exerted its effect on B cells and on monocytes. This effect was T cell dependent and it was mediated by soluble factors of T cell origin. At the peak of the PHA or concanavalin A response, most of the FceR-bearing cells were found in the B cell fraction but the T cells isolated from mitogen-stimulated cultures contained significantly more FcεR⁺ cells than those from the control cultures, suggesting that T cell mitogens had increased the expression of FcεR on some T cells. This view was supported by the finding of a higher proportion of FceR⁺ cells in PHA-stimulated than in control cultures of highly purified T cells with a maximum response at the end of the culture period. Double-labeling experiments at the peak (day 2) of the peripheral blood mononuclear cell response indicated that the expression of FcεR was increased on B cells (Bl⁺) and on monocytes (Mo2⁺). By using the same approach at the peak of the T cell response (day 7), it was found that T cells isolated from PHA-stimulated cultures expressed more FcεR than those isolated from control cultures. Moreover, in unstimulated cultures, FcεR was mainly expressed on T helper cells (Leu 3⁺) whereas in PHA-stimulated cultures FcεR was expressed on both T helper and T suppressor/cytotoxic cells (Leu 2⁺).     

Persistent and selective effects of inflammation on smooth muscle cell contractility in rat colitis

Intestinal inflammation affects smooth muscle contractility contributing to altered motility, but changes to the individual smooth muscle cells are not well described. We used video microscopy to study the contractility of circular smooth muscle cells (CSMC) isolated from the rat mid-descending colon throughout the course of TNBS-induced colitis, measuring their shortening response to carbachol (CCh), 5-HT, histamine or high K⁺. In control CSMC, CCh caused a maximal shortening response of 28 (2%), similar to that for 5-HT of 27 (1%), but by day 4 of colitis, these responses were decreased by 35% and 37%, respectively. By day 36, all aspects of cholinergic contraction returned to control levels, while 5-HT-induced contraction remained significantly attenuated. In contrast, the contractile responses to histamine remained similar at all time points. K⁺-induced contraction was impaired only on day 4, and the maximal response remained substantially greater than CCh or 5-HT. Colitis caused a 121% increase in CSMC length by day 2 that persisted through day 36, independent evidence for phenotypic change. We conclude that impaired CSMC contractility at both the receptor and non-receptor levels contribute to altered smooth muscle function during colitis. Persistent changes in contractile response remained detectable after resolution of inflammation, and similar events may occur in post-enteritis syndromes seen in humans.     

Growth of Fetal Thymocytes in Organ Culture: Effect of Recombinant Lymphokines on Thymocyte Maturation

Abstract

Interleukin 2 (IL2) has a dose-dependent inhibitory effect on the growth and phenotypic maturation of thymocyte populations grown in fetal thymus organ culture. Addition of IL2 (100 U/ml) to 14-day fetal thymus organ cultures induces the appearance of a population of lymphokine-activated killer (LAK) cells which lyse allogeneic, syngeneic, and syngeneic tumor cell targets. The addition of the monoclonal antibody, PC-61, blocks the IL2-dependent growth and activation of LAK cells but does not influence the maturation of CD4⁺ CD8⁺ fetal thymocytes. These data imply that IL2 is not a major regulator of normal fetal thymocyte maturation. The effects of a range of recombinant lymphokines (IL1α, IL1β, IL3, IL4, GM-CSF, G-CSF, M-CSF) on the proliferation and phenotypic maturation of fetal 14-day thymocytes in organ culture has been analysed. Two significant changes were seen. First, IL1α and IL1β inhibited growth and the expression of the CD4 and CD8 antigens in organ culture, and second, GM-CSF increased the expression of Mac-1 cells. IL4, which has known T cell growth-promoting activity, IL3, G-CSF, and M-CSF did not alter either normal growth or surface antigen expression in fetal thymocytes. While some of these lymphokines may function as accessory molecules in fetal thymocyte development, our experiments suggest that they do not have a significant influence on thymocyte maturation when used alone.     

Antibody production in vitro: The effects of anti-allotypic serum on the secondary response of rabbit spleen cell suspensions to sheep erythrocytes SRC

Rabbit anti-allotypic sera directed against rabbit immunoglobulins stimulated an increase in the number of direct PFCs in suspensions of cells from the spleens of rabbits killed after a boost of SRC. This effect occurred within the first 24 hours of incubation of these cells with antiserum in the absence of antigen in the culture medium. High levels of these antisera were much less stimulatory than low during the early period of culture, but more prolonged incubation with antiserum in high concentrations resulted in increased numbers of PFC in these cultures. Amounts of antisera which were stimulatory in the absence of SRC in the culture medium were found to inhibit the stimulatory effect of antigen in PFC during the first 1–2 days of culture. Like high concentrations of anti-allotypic sera alone, this early inhibition was followed by a stimulatory increase of PFC on more prolonged culture in the presence of both SRC and anti-allotypic serum.

Anti-allotypic sera affected the levels of PFC in these studies even in the absence of any change in the rate of DNA synthesis, as measured by the incorporation of [³H]thymidine. Further to this, it was found that concentrations of antisera which stimulated increased uptake of [³H]thymidine during 24–48 hours of culture, actually depressed the levels of PFC in these cultures. It was, therefore, concluded that the action of anti-allotypic sera on PFC in these cultures was by means of a direct effect on the process of production of antibodies by these cells and was not dependent on the stimulation of cell division in the antibody-producing population. This stimulatory effect of anti-allotypic sera was prevented by Actinomycin-D and the inhibitory effects of complement on PFC in cultures incubated with anti-allotypic sera indicated that antibodies had combined with receptor molecules on these cells. The effects of the combination of SRC and anti-allotypic sera on the levels of PFC would suggest that the same cell population in these cultures was being affected by both these agents and that the receptor molecule for them was specific antibody.

The effect of anti-allotypic sera of the PFC in these rabbit spleen cell suspensions was shown to have some degree of non-specificity. Since normal rabbit serum as well as rabbit antiserum against a Proteus vulgaris OX19 did not have any effect it was considered possible that this non-specific stimulation of PFCs was also due to antibodies directed against antigenic determinants in rabbit immunoglobulins.

     

Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7

B cell development is influenced by interactions between B cell progenitors and stromal cells. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. Flt3 ligand (FL) is a growth factor which stimulates the proliferation of stem cells and early progenitors. Mice deficient for the FLT3 receptor exhibit severe reductions in early B lymphoid progenitors. We have previously described a clonal assay in vitro which allows us to follow the entire B cell differentiation pathway from uncommitted progenitors to mature, immunoglobulin-secreting plasma cells. The growth factor combination of interleukin (IL)-11, mast cell growth factor (MGF) and IL-7 was shown to maintain the differentiation of these hematopoietic precursors into B cell progenitors capable of giving rise to functionally mature B cells in secondary cultures. Here, we show that FL in combination with IL-11 and IL-7 is sufficient to support the differentiation of uncommitted progenitors from day 10 yolk sac (AA4.1⁺) or day 12 fetal liver (AA4.1⁺ B220^? Mac-1^? Sca-1⁺) into the B lineage. The frequency of B cell progenitors obtained in these conditions was similar, if not better, than the frequency of B cell precursors that arose when cultured in IL-11+MGF+IL-7. Furthermore, the growth factor combination of IL-11+FL+IL-7 was able to maintain the potential of bipotent precursors giving rise to both the B and myeloid lineages in secondary cultures. We also show that FL synergizes with IL-7 in the proliferation of committed B220⁺ pro-B cells and may contribute to the maintenance of an earlier pro-B cell population. Together, these results show that FL is important in supporting the differentiation and proliferation of early B cell progenitors in vitro.