The cytokine receptor gp130 and its soluble form are under hormonal control in human endometrium and decidua

The transmembrane protein gp130 plays a central role in cytokine action as a signal transducing receptor subunit common to all interleukin-6 type cytokines. Endometrial tissue obtained from women with a normal menstrual cycle and decidua obtained from women in the first or second trimester of pregnancy were assessed for gp130 by western blotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis. By immunoblotting, two forms of gp130 were detected: one-the soluble form-of approximately 100 kDa and a larger membrane-bound form of approximately 150 kDa. The latter became clearly visible in the mid to late secretory phase and was more pronounced in decidual tissue of second trimester compared to first trimester. Immunohistochemically, gp130 was located in glandular epithelial cells during the mid to late secretory phase, whereas staining in the proliferative phase was rather weak. In first and second trimester decidua, glandular cells were also positively stained. In addition, the invading trophoblast cells were gp130 positive. Soluble gp130 release was measured in the supernatants from primary endometrial and decidual cell cultures by ELISA and reached maximum values in cell cultures without addition of hormones. In cultured endometrial epithelial cells obtained during the proliferative phase of the cycle, the soluble gp130 release increased significantly under combined estradiol/progesterone supplementation which mimics the secretory phase conditions compared to estradiol supplementation alone. In cultured epithelial cells derived from decidual tissue of first trimester of pregnancy, similar effects of hormonal regulation were observed. Our results suggest that the balance between soluble gp130 and its membrane-bound form may play an important role in regulating cytokine action necessary for blastocyst implantation and for further interaction between the decidualized endometrium and the invading trophoblast.     

Epidermal growth factor in human endometrium: proliferative effects in culture and immunocytochemical localization in normal and endometriotic tissues.

Endometrial proliferation is stimulated by oestradiol. The precise mechanism is poorly understood, but may be mediated by epidermal growth factor (EGF). The aim of the present study was to assess the effects of oestradiol and EGF on glandular and stromal proliferation in human endometrial cell cultures, and to determine the localization of EGF-like immunoreactivity (EGF-IR) using immunocytochemistry in normal and endometriotic tissue. Endometrium was obtained from women undergoing curettage or hysterectomy for benign disease, or laparoscopy for endometriosis. For tissue culture experiments, enriched glandular and stromal cells were prepared by digestion with collagenase and DNAase, and cultured for 4 days with oestradiol or EGF, both alone and in combination. Immunocytochemical studies were performed using sheep primary antibody against EGF, with binding visualized using the unlabelled antibody--enzyme method. In combination, oestradiol and EGF increased mean cell counts from 1.15 +/- 0.06 and 1.36 +/- 0.05 x 10(5)/ml to 1.68 +/- 0.1 (+46%) and 1.94 +/- 0.16 (+43%) x 10(5)/ml, in proliferative and secretory gland preparations respectively (n = 10, P less than 0.01). No effect was seen in stromal cell preparations; however the stimulation in gland preparations was further augmented after the addition of stromal-conditioned medium. EGF-IR was detected in endometrium from normal women, and in normal and ectopic endometrium from women with endometriosis. EGF-IR was seen in glands and stroma and was not related to the phase of the menstrual cycle. EGF may play a role in the oestrogen-stimulated proliferation of normal and endometriotic endometrium.     

Peripheral blood mononuclear cells stimulate progesterone production by luteal cells derived from pregnant and non-pregnant women: possible involvement of interleukin-4 and interleukin-10 in corpus luteum function and differentiation

Human luteal cells have been reported to express human leukocyteantigen-DR and lymphocyte functional antigen-3 on the cell surface,suggesting physiological interaction between luteal cells andT-lymphocytes through the menstrual cycle into early pregnancy.To elucidate the role of peripheral lymphocytes on corpus luteumdifferentiation, the effect of peripheral blood mononuclearcells (PBMC) on steroidogenesis by luteal cells was investigated.The production of Th-2 cytokines such as interleukin (IL)-4and IL-10 by the co-cultured cells was also examined, and theeffects of these cytokines on progesterone production by lutealcells were investigated. Corpora lutea were obtained from eightnon-pregnant women in the luteal phase and five women in earlypregnancy for luteal cell culture. PBMC were isolated from unrelatedwomen in the follicular phase, secretory phase, and early pregnancy.After co-culture with allogenic PBMC for 48 h, progesteroneproduction was significantly enhanced by PBMC from the secretoryphase and early pregnancy in the non-pregnant luteal cell culture.In the pregnant luteal cell culture, a significant increasein progesterone production was also observed by the co-culturewith PBMC from women in early pregnancy, showing that PBMC havea luteotrophic effect. The stimulatory effects of PBMC werealso observed in co-culture conditions which prevented directcell-to-cell interaction with luteal cells, showing the minorinfluence of mixed lymphocyte reaction. By co-culture with PBMC,the production of IL-10, but not IL-4, was significantly augmentedin luteal cell culture derived from non-pregnant women, whereasthe production of both IL-4 and IL-10 was significantly enhancedin the luteal cell culture derived from pregnant women. Moreover,IL-4 and IL-10 promoted progesterone production by culturedluteal cells, especially in the luteal cell culture derivedfrom corpora lutea of early pregnancy. These findings indicatethat PBMC stimulate progesterone production by luteal cellsand suggest the involvement of PBMC in corpus luteum functionand differentiation probably via the Th-2-type lymphocytes.     

X-irradiation reduces the proliferation of astrocytes by cell cycle arrest

Reactive astrogliosis is one of the key components of the cellular response to CNS injury and is considered a major impediment to axonal regeneration. Our previous study demonstrated that cell cycle inhibition treatment can reduce astrocyte activation and proliferation in vivo. In this study, we examined whether reactive astrogliosis can be suppressed by X-irradiation in vitro by modulating cell cycle progression. X-irradiation with low dose (4 Gy) suppressed astrocyte proliferation as demonstrated by immunofluorescence staining with BrdU and Ki67 in monolayer astrocyte cultures and those in scratch-wound model. The proportions of BrdU (+) and Ki67 (+) cells at 12, 24, and 48 h after 4 Gy irradiation were significantly lower than those in control group. FACS analysis of monolayer astrocyte cultures showed that X-irradiation decreased the proportion of astrocytes in S phase at 12 and 24h after irradiation with a dose-dependent manner. Furthermore, after X-irradiation, higher levels of p53 were observed by western blot as compared to control astrocyte cultures. Taken together, these data support that X-irradiation can decrease astrogliosis via arresting the cell cycle progression, which might constitute an effective therapeutic intervention in diseases characterized by excessive proliferation of glial cells.     

Diverse effects of FSH and LH on proliferation of human ovarian surface epithelial cells

The purpose of this study was to evaluate the effects of FSH and LH on growth regulation of normal ovarian surface epithelial (OSE) cells harvested from both premenopausal and postmenopausal women. Ovarian surface epithelial cells were obtained through brushing of the ovarian surface during surgery. FSH and LH were added to the OSE cultures and the proliferative effects were analysed using two different culture models, non-confluent and confluent cells, and two different detection methods, [(3)H]thymidine incorporation and a colorimetric cell number assay. FSH lowered the OSE proliferation under non-confluent conditions (10-27%), and the inhibitory effect was most pronounced among cells from postmenopausal women (P: < 0.01). In the confluent model only cells from postmenopausal women showed significantly (P: < 0.05) decreased proliferation. No effects of LH on OSE cells were detected. The unexpected results of an anti-proliferative effect of FSH on OSE, and the absence of effect by LH, do not support the theory that gonadotrophins are directly involved in ovarian carcinogenesis through an enhanced proliferation of OSE cells.     

Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-gamma ligand, pioglitazone

Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-gamma (PPARgamma) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPARgamma ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids.     

Proliferative stimulus of lung fibroblasts on lung cancer cells is impaired by the receptor for advanced glycation end-products

The receptor for advanced glycation end-products (RAGE) is highly expressed in lung tissue, especially at the site of the alveolar epithelium, but its expression is reduced in lung carcinomas. Because epithelial-mesenchymal interactions are suggested to contribute to cancer progression, we investigated the RAGE-dependent impact of fibroblasts on tumor cell growth. Cocultivation of human lung cancer cells (H358) with lung fibroblasts (WI-38) improved their proliferation in monolayer and spheroid culture models, the number of H358 cells in the S/G2 cell cycle phase increased, and there was less spontaneous cell death. Overexpression of full-length human RAGE reduced the proliferative stimulus of fibroblasts as seen in monolayers (cell number, cell cycle), spheroid cultures (spheroid size), and in a colony-forming assay compared with mock-transfected cells. Comparable results were observed by culturing H358 cells with and without RAGE overexpression in the presence of conditioned medium taken from WI-38 cells, or in response to selected growth factors, such as basic fibroblast growth factor. Moreover, we clearly showed that the fibroblast-induced proliferation correlates with activation of the p42/44 mitogen-activated protein kinase, but not with Akt kinase activation. On the basis of lung cancer as an age-related disease, we additionally proved the impact of senescent WI-38 fibroblasts. Here, we show that senescent fibroblasts improve H358 cell proliferation to the same extent as do presenescent fibroblasts. From our data, we conclude that re-expression of RAGE in lung cancer cells impairs the proliferative stimulus mediated by fibroblasts. Therefore, lung cancer progression may be enhanced by the RAGE downregulation in human lung carcinomas.     

Peripheral blood mononuclear cells in early pregnancy promote invasion of human choriocarcinoma cell line, BeWo cells

BACKGROUND: During the establishment of the maternal blood circulation around the implanting human embryo, maternal peripheral blood mononuclear cells (PBMC) directly contact trophoblasts. To determine the physiological significance of this interaction, the effects of PBMC obtained from pregnant women on the proliferative and invasive properties of a human choriocarcinoma cell line, BeWo cells, were examined. METHODS AND RESULTS: PBMC were obtained from women in early pregnancy and from women in the secretory phase of the menstrual cycle. PBMC from pregnant women significantly increased the number of invading BeWo cells in an invasion assay without affecting the proliferation of BeWo cells (P +/- 0.05). No significant changes were observed in the co-cultures with PBMC from non-pregnant women. The addition of conditioned medium, which was prepared by 2 days of incubation with PBMC from pregnant women, also enhanced BeWo cell invasion in a dose-dependent manner. Moreover, when PBMC obtained from non-pregnant women were incubated with recombinant HCG (0-10 IU/ml) for 2 days, significant augmentation of the effect on BeWo cell invasion was observed in the conditioned medium from HCG-treated PBMC (P +/- 0.05). CONCLUSION: This study indicated that soluble factor(s) secreted from PBMC promote BeWo cell invasion. It also showed the possible involvement of HCG in the regulation of BeWo cell invasion by PBMC. These findings suggest crosstalk between maternal PBMC and trophoblasts via soluble factor(s), which may play an important role in early embryo implantation.     

Uterus and endometrium: Effects of daily low dose mifepristone on endometrial maturation and proliferation

Following an ovulatory control cycle, six women took 2 mg ofmifepristone daily for 30 days. Endometrial biopsies were collectedin the control cycle between 7 and 11 days after the plasmaluteinizing hormone (LH) surge and on the corresponding dayof the treatment cycle (days 19–28). In order to investigatethe effects of unopposed oestrogen on the endometrium, persistentproliferative endometrium was obtained from six women with anovulatoryinfertility due to polycystic ovarian syndrome (PCOS) on a similarcycle day (days 21–23) following a progestogen-inducedwithdrawal bleed. Endometrium was evaluated for histology andimmuno-localization of oestrogen receptors (ER), progesteronereceptors (PR) and the cell proliferation markers [proliferatingcell nuclear antigen (PCNA) and Ki67]. Treatment with mifepristoneinhibited ovulation in four of the six subjects. In the twosubjects in whom ovulation did occur, secretory transformationwas delayed, suggesting that successful implantation of a blastocystwould be unlikely. In subjects who remained anovulatory duringtreatment, the histology and pattern of steroid receptor expressionwas similar to proliferative phase endometrium. In women withPCOS, mitoses and intense immunostaining for ER, PR and cellproliferation markers were observed in both glands and stroma.Although PCNA and Ki67 immunostaining were also present in mifepristone-treatedendometrium from subjects who did not ovulate, there were nomitoses and significantly less ER immunostaining in spite ofexposure to unopposed oestrogen for a similar duration. SincePCNA and Ki67 detect cells throughout all stages of the cellcycle this would suggest that mifepristone might affect theentry of cells into the mitotic phase of the cell cycle and,therefore, might prevent endometrial hyperplasia. These findingsadd further evidence to support the contraceptive potentialand antiproUferative activity of daily low dose mifepristone.     

Angiogenesis in the human corpus luteum: simulated early pregnancy by HCG treatment is associated with both angiogenesis and vessel stabilization.

BACKGROUND: This study examined changes in the luteal vasculature throughout the menstrual cycle and during simulated pregnancy with human chorionic gonadotrophin (HCG) in the human. METHODS: Endothelial cell and pericyte area were assessed by quantitative immunocytochemistry for CD34 and alpha-smooth muscle actin respectively, taking into consideration the dynamics of lutein cell hypertrophy and atrophy throughout the cycle and after HCG treatment. Endothelial cell proliferation was detected by Ki-67/CD34 dual staining and a proliferation index was obtained. The molecular regulation of angiogenesis was studied by examining changes in vascular endothelial growth factor (VEGF) immunostaining. RESULTS: The early luteal phase is associated with intense angiogenesis, as indicated by high endothelial cell proliferation, and by the mid-luteal phase a mature vasculature was apparent, as shown by maximal endothelial cell and pericyte areas. During the late luteal phase, decreased endothelial proliferation, endothelial cell and pericyte area indicated vascular regression. HCG treatment induced a second burst of total and endothelial cell proliferation and a concomitant increase in endothelial cell and pericyte areas. VEGF protein was expressed throughout the luteal phase and a significant increase was found after HCG treatment. CONCLUSION: Luteal rescue with HCG is associated with a second wave of angiogenesis and vascular stabilization.     

Cell proliferation and vascular morphology in the marmoset corpus luteum

Luteal formation is associated with angiogenesis and low progesterone production. Maximal mid-luteal phase progesterone production is concurrent with extensive vascularization, and luteolysis occurs when steroidogenesis decreases. Angiogenic cell proliferation and vascular changes have not been examined in the marmoset. The aim of this study was to examine vascular morphology throughout the luteal phase by identifying: (i) von Willebrand factor VIII antigen (vW)-immunopositive endothelial cells; (ii) Ki67-positive proliferating cells; and (iii) bromodeoxyuridine-positive proliferating cells. Marmoset corpora lutea were examined throughout the cycle, and natural regression was compared with induced luteolysis after administration of a prostaglandin F(2alpha) analogue or gonadotrophin-releasing hormone (GnRH) antagonist. Steroidogenic and endothelial cells were positive for proliferation markers. Endothelial cell proliferation was highest during luteal formation, then decreased and remained low during the luteal phase and functional regression, however endothelial cell proliferation increased during structural regression. Endothelial cell proliferation was unchanged by induced regression. The area of vW immunostaining was highest during luteal formation, decreased thereafter and remained constant during the luteal phase and regression. Distribution of immunostaining indicated the presence of an extensive capillary network, but during structural regression the numbers of capillaries decreased and numbers of microvessels increased. These results suggest that vascular changes are concurrent with changes in the functional status of the marmoset corpus luteum.     

敲降ZEB1基因表达对人脂肪间充质干细胞增殖、周期与凋亡的影响

目的探讨敲降ZEB1基因表达对人脂肪间充质干细胞(h ADSCs)增殖、周期和凋亡的影响。方法胶原酶消化法提取人脂肪组织中原代间充质干细胞,对其进行免疫学表型、成骨和成脂分化能力鉴定后用于实验。脂质体转染法将siRNA转入h ADSCs,实时定量PCR和Western blot检测ZEB1、细胞增殖、周期和凋亡相关基因mRNA和蛋白的表达。MTS法检测细胞增殖。流式细胞术检测细胞周期和细胞凋亡率。结果与si-NC转染组相比,si-ZEB1转染组可使ZEB1 mRNA和蛋白表达水平降低(P0.01),细胞增殖能力明显下降(P0.05),增殖相关基因CCND1、MKI67、MYC和PCNA表达显著下调(P0.05或P0.01);G1期细胞比例从50.71%增加到58.94%(P0.01),S期和G2期细胞比例分别减少了6.16%和2.07%;细胞凋亡率上升了10.2%,促凋亡相关基因TP53和BAX表达上调,抗凋亡基因MCL1和BCL2表达下调(P0.05或P0.01)。结论 ZEB1具有促进h ADSCs增殖和周期进展,抑制细胞凋亡的作用。     

Lymphocyte dysfunction in cartilage hair hypoplasia. II. Evidence for a cell cycle specific defect in T cell growth

Defects of in vitro B and T lymphocyte function and impaired delayed type hypersen-sitivity reactions, as well as an increased risk of lethal viral infections have been reported in cartilage hair hypoplasia (CHH), an autosomal recessive form of short limbed dwarfism. We have previously found an intrinsic proliferative defect that affected several cell types from CHH individuals. In order to further evaluate it we developed continuous T cell lines (CTCL) from CHH and normal individuals. The T cells from cultures of CHH and normal individuals were indistinguishable with respect to cell surface antigens characteristic of fully differentiated T cells, as defined by monoclonal antibody analysis. However, CHH T cells produced significantly less interleukin 2 (IL2) than normal T cells and the growth of CHH CTCL in response to exogenously supplied IL2 was markedly diminished (cell cycle 120-165 hr) compared to normal CTCL (cell cycle 48-60 hr). Furthermore, the exogenous IL2 was not absorbed from growth medium by CHH CTCL at the same rate as normal CTCL. Both production and utilization of IL2 are cell cycle specific events that occur during G₁ phase before the onset of DNA synthesis (S phase). Thus, CHH T lymphocytes appear to have a defect related to G₁ phase that results in a longer cell cycle for individual cells, and leads to decreased proliferation of the population. We postulate that this G₁ phase defect is present in multiple cell types in CHH and that analysis of continuous T cell lines from CHH individuals may permit the identification of this defect.     

低营养及低氧状态下NIH3T3细胞增殖及细胞周期的变化及对bFGF的影响

目的： 体外模拟慢性创面缺氧、低营养环境，观察成纤维细胞在该状态下增殖及细胞周期的变化及对外源性生长因子（bFGF）的反应,探讨低氧、低营养条件下成纤维细胞的病理生理变化。方法: 单纯缺氧环境采用厌氧培养箱，通入混合气，氧分压（PO₂）分为27 mmHg和44 mmHg 2个水平；低营养环境则控制培养液新生牛血清（NCS）浓度。用MTT法检测细胞活性以及其对外源性生长因子的反应，用流式细胞仪检测细胞周期。结果: PO₂ 44 mmHg时细胞增殖速度较同期对照组无明显差异；PO₂ 27 mmHg时，细胞增殖速度较同期对照组明显减慢（P<0.01），细胞被阻滞于G₀期，S期细胞比例明显减少，bFGF未显示促增殖作用。NCS浓度为0.5%的低营养状态下细胞增殖速度较同期对照组明显减慢（P<0.01），细胞被阻滞于G₀-G₁期（P<0.01）；bFGF能明显改善低营养状态下的增殖减慢（P<0.01），使G₂-M期细胞比例增加（P<0.05）。结论: 27 mmHg PO₂或NCS浓度为0.5%的低营养环境使细胞阻滞于G₀-G₁期，影响成纤维细胞增殖；bFGF可以改善低营养条件下细胞增殖减慢的状态，但对极度缺氧条件下的成纤维细胞增殖障碍无明显作用。     

Ro/SSA inhibits the autologous mixed lymphocyte reaction.

To test the hypothesis that the Ro/SSA autoantigen can be recognized as antigenic by the human immune system, lymphocytes obtained from normal volunteers were used in in vitro assays evaluating the ability of Ro/SSA (mol. wt 60 kD) to induce B and/or T cell responses. Bovine Ro/SSA strongly inhibited the autologous mixed lymphocyte reaction in a dose-dependent manner without similar effects on concurrently performed allogeneic mixed lymphocyte reactions or T cell proliferation induced by phytohaemagglutinin. Using three colour FACS analysis, Ro/SSA was found to decrease the percentage of CD4+CD45+RA+ T cells in the proliferative, S+(G2+M), phase of the cell cycle. Associated with the decrease in the percentage of suppressor-inducer cells, was the finding that Ro/SSA was able to augment RF production in pokeweed mitogen stimulated cultures of peripheral blood lymphocytes.     

Uterus and endometrium: Inhibition of human endometrial stromal cell proliferation by interleukin 6

We investigated the ability of interleukin 6 (IL-6) to modulatehuman endometrial stromal cell growth in vitro Stromal cellproliferation in response to treatment with varying concentrationsof IL-6 was determined. Endometrial tissue was obtained from10 normally cycling women during the secretory phase of theirmenstrual cycle. Treatment with IL-6 resulted in a dose- andcell-density-dependent inhibition of endometrial stromal cellproliferation in vitro. The maximal inhibition was observedwith 200 pg/ml of IL-6 and at a concentration of 10⁵ cells/well.During in-vitro culture, stromal cells produced low amountsof IL-6 and demonstrated the presence of IL-6 receptor. Thesedata demonstrate that IL-6 acts as a growth-regulatory signalfor human endometrial stromal cells. We postulate that IL-6may contribute to the maintenance of homeostasis in normal endometriumand that perturbation of IL-6 mediated responses may play arole in disorders of the endometrium such as endometrial cancerand endometriosis.     

Cell cycle differences in vitro between primitive hematopoietic cell populations from adult and umbilical cord blood

Lineage-negative (Lin(-)) cell populations, obtained by negative selection from umbilical cord blood (UCB) and adult mobilized peripheral blood (aMPB), were cultured in serum-free liquid cultures supplemented with a mixture of seven stimulatory cytokines. On specific days, proliferation potential was assessed and cell cycle status was determined by DNA content. Expression of the cell cycle regulators cyclin D3 (cD3), cyclin-dependent kinase 4 (cdk4), p21(cip1/waf1) (p21), and p27(kip1) (p27) was also determined. As expected, UCB cells showed significantly higher proliferation potentials than aMPB cells, particularly during the first 7 days of culture. During this period of time, higher numbers of cell cycles were observed in UCB cells (7-9 cycles), as compared to aMPB cells (5-6 cycles). Higher levels of cD3, cdk4, and p27 were also detected in UCB cells. Our results confirm that UCB cells possess an intrinsically higher proliferation potential, as compared to aMPB cells, and suggest that such a biological difference is due, at least in part, to differences in cell cycle status. This, in turn, seems to result from the differential expression of cell cycle regulatory molecules.     

RACK1高表达促进人结肠癌细胞的生长和增殖

目的：研究RACK1高表达对人结肠癌细胞增殖能力的影响。方法：采用脂质体转染术分别建立 RACK1表达下调的SW620细胞系、RACK1表达上调的SW480细胞系以及对照细胞系;采用CCK-8细胞增殖测定、软琼脂集落形成实验、EdU掺入实验以及流式细胞术检测RACK1表达改变对 SW620和SW480细胞增殖的影响;采用Western blot分析RACK1表达改变对G1/S期限制点调控蛋白Cyclin D1和p27蛋白表达的影响。结果：下调RACK1的表达抑制SW620细胞的生长、软琼脂集落形成能力,降低EdU标记的S期细胞数目,阻滞细胞周期于G1/S期;而上调RACK1的表达增强 SW480细胞的生长、软琼脂集落形成能力,增加EdU标记的S期细胞数目,促进细胞周期G1/S期进程。结论：RACK1高表达促进人结肠癌细胞的生长和增殖。     

Differential activity of recombinant lymphokines on mouse B cell proliferation and cell cycle progression are revealed by 5-bromo-2'-deoxyuridine/Hoechst 33258 dye flow cytometry

Activation of resting mouse B cells with anti-mu chain antibodies (anti-mu) leads to cell proliferation. We have investigated the effect of recombinant T cell interleukins (IL 2 to IL 6) on such anti-mu-induced proliferation. No proliferative response was detected when IL 2, IL 3 and IL 6, either alone or in combination with anti-mu, were studied. Furthermore, neither IL 4 nor IL 5 could induce proliferation when added alone to B cell cultures. However, when combined with anti-mu, IL 4 as well as IL 5 stimulated cell growth. Analysis by 5-bromo-2'-deoxyuridine/Hoechst 33258 flow cytometry revealed distinct effects of IL 4 and IL 5 on B cell growth. In the presence of anti-mu, both IL 4 and IL 5 co-stimulated unfractionated splenic B cells. However, when B cells were separated into subpopulations by density, IL 4 proved to be a cell cycle progression factor, stimulating the majority of resting B cells to enter the cell cycle. In contrast, IL 5 had little effect on the resting fraction of B cells. Rather, IL 5 acted as a co-competence factor, stimulating predominantly low-density B cells. Following exposure of anti-mu alone, most B cells accumulated in the G1 of the second cycle. Upon addition of IL 4, the cells acquired the ability to progress into the next S phase compartment. Contrary to what is seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti-mu plus IL 4 stimulation. This phenomenon was not due to a differential cell cycle progression rate. Our findings provide an analytical basis for fractionating cell-cycle-compartment-specific B cells for their molecular study.