Prolactin is an autocrine growth factor for the Jurkat human T-leukemic cell line

Despite convincing evidence of cooperation between IL-2 and endogenous prolactin (PRL) during T cell activation, the individual role of PRL as a T-cell lineage cytokine remains to be defined. We have examined the production and function of PRL on the Jurkat human T-leukemic cell line, which does not constitutively produce IL-2. The majority of Jurkat cells expressed PRL receptor (R) under standard culture conditions, whereas appearance of the α chain of the IL-2-R required PHA–PMA stimulation, as did IL-2 synthesis. Western blotting revealed a predominant band at 23.5 kDa and a weaker band at 25.5 kDa in both Jurkat cell lysates and human (h) pituitary PRL. Metabolic labeling of the cell lysates with -methionine and immunoprecipitation with an antiserum against hPRL showed that both forms of PRL are actively synthesized by the Jurkat cell line. PRL released in the medium was biologically active in the rat Nb2 lymphoma mitogenic assay. Depletion of medium PRL with two polyclonal anti-hPRL antisera inhibited the growth of Jurkat cells in a dose-dependent manner, as evaluated by cell number and -TdR uptake. Purified pituitary or recombinant hPRL at a wide range of concentrations had no significant effect on their growth, but reversed the blocking activity of the anti-hPRL antibody. Recombinant IL-2 had no effect on the antibody-induced growth inhibition. Taken as a whole, these results demonstrate that PRL can act as an autocrine T cell growth factor independently of IL-2 and are the first evidence of its involvement in human leukemic growth and possibly in leukemic transformation.     

白黎芦醇对GH3细胞增殖和PRL合成的影响

目的 探讨白黎芦醇对垂体腺瘤GH3细胞增殖和泌乳素合成的影响，及其对雌激素的拮抗作用。方法 在无血清无酚红的培养条件下，白黎芦醇单独或与雌二醇联合作用于GH3细胞，用MTT法测定细胞增殖，用免疫荧光法、RT-PCR和Western印迹法测定泌乳素的表达情况。结果 白黎芦醇对GH3细胞增殖具有刺激和抑制双相作用，呈时间一剂量依赖性。并且白黎芦醇使GH3细胞中PRL阳性细胞比例下降。同时，白黎芦醇抑制泌乳素的合成。白黎芦醇对雌二醇诱发的细胞增殖和泌乳素合成均有拮抗作用．但对泌乳素合成的拮抗作用较强，而雌二醇刺激细胞增殖作用较其诱发泌乳素合成作用强。结论 白黎芦醇对GH3细胞增殖和泌乳素合成均有抑制作用，从两方面发挥着抗肿瘤作用。     

Tumor associated release of interleukin-10 alters the prolactin receptor and down-regulates prolactin responsiveness of immature cortical thymocytes

The soluble factors produced either by Ehrlich's ascites carcinoma (EAC) or thymic adherent cells (TAC) of tumor-bearing mice comprising of CD11b(+) and CD11c(+) antigen-presenting cells caused a sharp decrease in prolactin (PRL)-induced ConA-mediated effect on survival of PNA(+) thymocytes. Similar suppression of PRL-induced effect was observed when the cells were cocultured with TAC of EAC-bearing mice. Anti-IL-10 antibody could reverse the PRL inability to induce ConA-mediated effect on PNA(+) thymocyte survival, indicating the presence of IL-10 in EAC culture supernatant (EAC sup) and thymic microenvironment. IL-10 could block PRL-induced proliferation of PNA(+) thymocytes without affecting spontaneous apoptosis. IL-10 altered the expression of the long-form (LF) of PRL-R and reduced the PRL binding of the cells, suggesting down-regulation of the PRL effect on PNA(+) thymocyte by the cytokine. Induction of tumor, which was found to increase the IL-10 secretion by TAC, also modified the PRL-R (LF) to PRL-R (SF). Since PRL plays a role in survival, proliferation and differentiation of lymphoid progenitor cells, the tuning of PRL action by IL-10 may be a possible mechanism of depletion of immature cortical thymocytes and thymic atrophy in tumor-bearing mice.     

Interaction between oligodendroglia and immune cells: mitogenic effect of an oligodendrocyte precursor cell line on syngeneic T lymphocytes.

We analyzed cellular interactions between T lymphocytes and a recently established immortal glial line, L3 that retains several properties of immature oligodendrocytes (Aloisi et al., J Neurosci Res 27:16-24, 1990). L3 oligodendrocytes (L3-OL) cannot be induced to express class II antigens, nor do they specifically present antigen to syngeneic specific T lymphocyte. However, L3-OL strongly enhance the proliferation of freshly activated, interleukin-2(IL-2)-dependent T-line lymphocytes and concanavalin A (ConA)-activated lymphoblasts, irrespective of their antigen specificity or surface phenotype (CD4+ or CD8+). Resting and some activated T cells were susceptible to the mitogenic effect of L3-OL only in the presence of exogenous IL-2, not of other cytokines. The mitogenic effect of L3-OL did not depend on cell viability. It was observed in paraformaldehyde-fixed L3-OL cells and in membrane preparations, but not in culture supernatant. Neither intact L3-OL cells nor membrane preparations had direct IL-2 activity. The conclusion that the mitogenic effect of L3-OL cells is exerted by membrane structures acting as a costimulatory factor(s) of IL-2 is supported by the finding that it is largely blocked by a monoclonal anti-IL-2 receptor antibody. The effect is distinct from membrane-bound IL-1, membrane-bound tumor necrosis factor-alpha (TNF-alpha), IL-3, or IL-6 and cannot be reconstituted by these cytokines.     

Interleukin regulation of prolactin and corticotropin from pituitary adenoma

Recent findings indicate that lymphokines, leukocyte-derived hormones, interact with the hypothalamic-pituitary axis. We examined the role of neurotrophic lymphokines in the neuroendocrine system. Specifically, the action of Interleukin (IL)-1b, IL-2 and IL-6 upon the anterior pituitary hormones, growth hormone (GH), prolactin (PRL) and adrenocoticotropic hormone (ACTH) were studied in rodent pituitary adenoma cell lines. Hormone release by GH and PRL-producing rat adenoma cells (GH3) and ACTH-producing mouse adenoma cells (AtT-20) was analyzed by radioimmunoassay (RIA). Recombinant (r) IL-1beta decreased PRL release from GH3 in a dose-dependent fashion. IL-1 inhibition of PRL production occurred in parallel with IL-1 inhibition of DNA synthesis in GH3 as measured by [(3)H] thymidine incorporation. This result strongly indicates that IL-1 alters PRL production by adenoma cells at the translational level. Low dose IL-2 (10 U/ml) enhanced ACTH production from AtT-20, but higher concentrations of IL-2 failed to affect the release of ACTH. IL-2 did not change the incorporation of [(3)H] thymidine in AtT-20. Previous studies showed that IL-1 and IL-6 induce a significant secretion of ACTH via the hypothalamic-pituitary axis. However, IL-1 and IL-6 failed to affect ACTH secretion by AtT-20. Blood-derived cytokines have direct effects on hormone secretion by pituitary adenoma cells in vitro.     

Expression of SOCS genes in normal and leukemic human leukocytes stimulated by prolactin, growth hormone and cytokines

To evaluate the possible role of the recently described family of suppressors of cytokine signaling (SOCS) factors in the human lympho-hemopoietic system, we have monitored SOCS factor expression, both constitutive and induced by either cytokines, prolactin (PRL) or growth hormone (GH), using polymerase chain reaction in normal and leukemic cells. CIS (cytokine-inducible SH2-containing protein), SOCS-2 and SOCS-3 were constitutively expressed in peripheral blood mononuclear leukocytes. SOCS-3 expression was enhanced by PRL or by IFN-gamma. In bone marrow cells and granulocytes, CIS expression was induced and SOCS-2 enhanced by IFN-gamma and by PRL. In tonsillar cells, CIS expression was increased and SOCS-2 was induced by IL-1beta, IL-6, PRL and GH. SOCS-3 expression was enhanced by IL-1beta. The expression of SOCS-7 was increased by IL-6, PRL and GH. In Raji B-lymphoma cells, the expression of SOCS-2 and SOCS-7 was enhanced by IL-1beta. In THP-1 myeloid leukemia cells pretreated with TPA (to induce receptors for IFN-gamma), IFN-gamma induced SOCS-2. Jurkat cells expressed more SOCS-2 when exposed to PRL. Original observations in this work include the first report on SOCS-7 induction by cytokines. Also our data shed new light on the possible involvement of PRL and GH in the cytokine network. These hormones could modulate the transduction of signals originating from receptors for various cytokines.     

Effects of an estrogen receptor antagonist on proliferation, prolactin secretion and growth factor expression in the MMQ pituitary prolactinoma cell line

The aim of this study was to investigate the functional role of estrogen receptor α (ERα) in MMQ pituitary prolactinoma cells in the absence of estrogen with respect to proliferation, prolactin (PRL) secretion, and expression of growth factors. MMQ cells were treated with the ERα antagonist fulvestrant, then proliferation and PRL secretion were measured using MTS and enzyme-linked immunosorbent assays. Levels of ERα, vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9) and B cell leukemia/lymphoma-2 (BCL-2) were measured using quantitative polymerase chain reaction and western blot analysis. Fulvestrant acted as a potent inhibitor of ERα expression, and significantly inhibited cell proliferation (by up to 57.6±2.2%) and PRL secretion (by up to 81.0%). Fulvestrant also significantly altered the expression levels of VEGF, MMP-9 and BCL-2. We conclude that ERα plays an important functional role in pituitary prolactinomas and is also involved in the expression of particular growth factors, even in the absence of estrogen. Fulvestrant treatment may be an effective therapy for such tumors.     

Myeloid leukemic cells express and secrete bioactive pituitary-sized 23 kDa prolactin

Prolactin (PRL) is a 23 kDa polypeptide hormone of pituitary origin which is of major importance for reproduction. In addition, PRL has immunomodulatory effects and can be produced in small quantities in nonpituitary tissues. To address possible autocrine or paracrine functions of PRL in leukemia, we characterized immunoreactive PRL from the culture medium of leukemic cells. The myeloid cell line Eol-1 expresses the long extrapituitary type mRNA for PRL and synthesizes immunoreactive PRL with a molecular weight of 23 kDa. The biological activity in Eol-1 culture medium was determined using the Nb2 bioassay. This activity co-eluted with recombinant human (rh) PRL on an S-200 Sephacryl gel filtration column and could be blocked by anti-PRL antiserum. Western blot analysis and Nb2 bioassays also suggest that acute myelogenous leukemic blasts secrete bioactive 23 kDa PRL in one out of three tested patients.     

Astrocyte-induced regulatory T cells mitigate CNS autoimmunity

Although astrocytes presumably participate in maintaining the immune privilege of the central nervous system (CNS), the mechanisms behind their immunoregulatory properties are still largely undefined. In this study, we describe the development of regulatory T cells upon contact with astrocytes. Rat T cells pre-incubated with astrocytes completely lost the ability to proliferate in response to mitogenic stimuli. The cells were blocked in G0/G1 phase of the cell cycle, expressed less IL-2R, and produced significantly lower amounts of interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2), IL-10, or tumor necrosis factor (TNF). These anergic cells completely prevented mitogen-induced growth of normal T lymphocytes, as well as CNS antigen-driven proliferation of autoreactive T cells. The suppressive activity resided in both CD4+ and CD8+ T-cell compartments. Heat-sensitive soluble T-cell factors, not including transforming growth factor-beta (TGF-beta) or IL-10, were solely responsible for the observed suppression, as well as for the transfer of suppressive activity to normal T cells. The administration of astrocyte-induced regulatory T cells markedly alleviated CNS inflammation and clinical symptoms of CNS autoimmunity in rats with experimental allergic encephalomyelitis. Finally, the cells with suppressive properties were readily generated from human lymphocytes after contact with astrocytes. Taken together, these data indicate that astrocyte-induced regulatory T cells might represent an important mechanism for self-limitation of excessive inflammation in the brain.     

Interleukin-1 activity in lesioned peripheral nerve.

The cytokine interleukin-1 (IL-1) is involved in a wide range of inflammatory and immune responses. As such, IL-1 could play a role in peripheral nerve repair mechanisms. Specifically, by its already established properties as a regulator of nerve growth factor (NGF) synthesis, and as a chemotactant to macrophages. We examined, therefore, IL-1 production in injured mouse peripheral nerve. Injured nerve segments were incubated in serum free medium to produce conditioned medium (CM) that was then tested for IL-1 activity in a thymocyte proliferation assay. CM induced thymocyte proliferation in a dose-dependent manner. Proliferation was inhibited by the M20 IL-1 inhibitor, the IL-1 receptor antagonist, and antisera raised against recombinant mouse IL-1 alpha. Inhibitions produced by these three specific inhibitors of IL-1-induced thymocyte proliferation strongly suggest that proliferation induced by CM was mediated largely by IL-1 secreted by non-neuronal cells residing in the damaged nerve. IL-1 activity was detected within hours after lesion, and 1 week thereafter. The rapid and prolonged production of IL-1 indicates that IL-1-dependent mechanisms can play roles in the response of the peripheral nerve to injury: degeneration and regeneration. The regulation of NGF synthesis, and the recruitment of white blood cells, macrophages in particular, from blood into the damaged nerve tissue, are two such mechanisms.     

催乳素对神经母细胞瘤细胞系Neuro2a增殖的调节作用

目的:探讨催乳素(Prolactin,PRL)对小鼠神经母细胞瘤细胞系Neuro2a细胞增殖的影响。方法:用不同浓度PRL作用于无血清培养的各Neuro2a细胞实验组,通过细胞计数,观察PRL对细胞增殖的影响。采用RT-PCR方法检测PRL受体基因在Neuro2a细胞的表达。结果:与对照组比较,10-7mol/LPRL对Neuro2a细胞的增殖具有明确的时间依赖性调节作用。应用PRL第一天可见Neuro2a细胞数明显增加(P<0.01),第三天时Neuro2a细胞增殖达最大峰值,较对照组增高228%(P<0.001)。第四天时实验组与对照组细胞数无明显差异。PRL受体基因在Neuro2a细胞中有明确的表达。结论:PRL能够促进Neuro2a细胞的增殖。提示PRL可能在神经母细胞瘤的发生和发展中发挥了一定作用。     

4-羟基他莫昔芬对泌乳素腺瘤GH3细胞增殖和分泌的影响

目的探讨雌激素受体拮抗剂4-羟基他莫昔芬（OHTam）对泌乳素腺瘤GH3细胞增殖和分泌泌乳素的影响。方法用逆转录多聚酶链反应（RT—PCR）的方法测定泌乳素腺瘤GH3细胞中雌激素受体-mRNA（ER—mRNA）的表达,并观察在去激素培养条件下以及不同浓度的OHTam和雌二醇（E2）对GH3细胞生长速度、生长抑制率、雌激素受体-mRNA（ER—mRNA）及泌乳素分泌水平的影响。结果在E2（10^-8mol／L）组细胞吸光度为0．561±0．018,抑制率为-0．06,细胞计数、生长抑制率、ER—mRNA及泌乳素分泌水平与去激素培养组差异显著（P〈0．05）;E2（10^-6mol／L）＋OHTam（10^-6mol／L）组吸光度为0．504±0．014,抑制率为0．08,细胞计数、ER—mRNA及泌乳素分泌水平与E2（10^-8mol／L）组差异显著（P〈0．05）。结论泌乳素腺瘤GH3细胞有雌激素受体表达,E2可以促进GH3细胞的增殖和泌乳素的分泌,而雌激素受体拮抗剂OHTam能够有效抑制雌激素的作用。     

Effect of transforming growth factor-β1 and -β2 on Schwann cell proliferation on neurites

Mechanisms regulating Schwann cell proliferation during development are unclear. Schwann cell division is known to be driven by an unidentified mitogen present on the surface of axons, but it is not known whether other molecules play a role in regulating this proliferation. Transforming growth factor-beta (TGF-β) which is found in the developing peripheral nervous system (PNS) and is mitogenic for neuron-free Schwann cells in vitro could be involved. We have investigated the effects of TGF-β 1, TGF-β 2 and antibodies to TGF-β and TGF-β 2 on axon driven Schwann cell proliferation. Rat embryonic dorsal root ganglion neurons (DRG) neurons and Schwann cells from the sciatic nerve were isolated, purified and recombined in vitro. Confirming earlier reports by others, we observed that TGF-β 1 and TGF-β 2 added to the culture medium stimulated the proliferation of Schwann cells in the absence of neurons. However, when added to neuron-Schwann cell co-cultures, TGFβ caused a variable response ranging from no effect to moderate inhibition of Schwann cell proliferation in different experiments. A stimulation of Schwann cell proliferation by TGFβ was never observed in neuron-Schwann cell co-cultures. Antibodies to TGF-β and TGF-β 2 did not influence axon driven Schwann cell proliferation. To further determine the role of TGF-β in Schwann cell proliferation and myelination, we studied Schwann cell proliferation in cultures from mice in which the TGF-β 1 gene was delected by homologous recombination. Neuron-Schwann cell cultures from wild-type, heterozygous and homozygous mice were used. No differences were observed in either Schwann cell proliferation or myelination between cultures obtained from homozygous mutants and their heterozygous and wild-type controls. These findings suggest that TGF-β does not function as a part of the mitogenic mechanism presented by neurons to Schwann cells, but that the presence of active TGFβ in the cellular environment might regulate the degree of proliferation induced by neuronal contact. Copy 1995 Wiley-Liss, Inc.     

Interleukin-6 upregulates expression of KDR and stimulates proliferation of human cerebrovascular smooth muscle cells.

Interleukin-6 (IL-6) may play multiple roles in angiogenesis and vascular remodeling. Our previous study showed that a promoter polymorphism (174G>C) in IL-6 is associated with brain arteriovenous malformation hemorrhage; tissue expression is related to genotype. In this study, we investigated the effects of IL-6 on human cerebral smooth muscle cells (HCSMCs) and smooth muscle cells isolated from brain arteriovenous malformation surgical specimens (AVM SMCs) and surgical controls (control HCSMCs--from structurally normal temporal lobe taken during surgical treatment of epilepsy patients). We found that IL-6 (1.1+/-0.27 versus 0.37+/-0.04 pg/mL, n=5, P<0.05) and endogenous vascular endothelial growth factor (VEGF) receptor II (kinase domain-containing receptor (KDR), 15+/-3 versus 1.5+/-3 pg/mL, n=5, P<0.05) were increased in brain AVM SMCs compared with control HCSMCs. Further research revealed that IL-6 could stimulate SMC proliferation, VEGF release, and KDR activation in control HCSMCs. It could also stimulate KDR phosphorylation in control HCSMCs, further confirming a unique role of IL-6 in the triggering of KDR. Interleukin-6 could increase matrix metalloproteinase-9 (MMP-9) secretion through activating KDR in control HCSMCs (P<0.05 versus control). Inhibiting IL-6-induced KDR could reduce MMP-9 activity at least 50% compared with the control group (P<0.05). Increased MMP-9 activity was accompanied by increased control HCSMC proliferation, and blocking MMP-9 activity significantly reduced IL-6-induced control HCSMC proliferation (P<0.05). Collectively, our results show that IL-6 could activate, amplify, and maintain the angiogenic cascade in HCSMCs. A novel role of IL-6 during HCSMC proliferation is upregulating KDR expression and phosphorylation. The results may contribute to the angiogenic phenotype of human brain vascular diseases, such as brain AVM.     

Infusion of anti-nerve growth factor into the cisternum magnum of chick embryo leads to decrease cell production in the cerebral cortical germinal epithelium

There has been considerable recent progress in understanding the processes involved in cerebral cortical development. Several mitogenic and trophic factors have been implicated in the processes of cortical cell proliferation and differentiation. Anti-nerve growth factor (NGF) antibody was administered to 15 days chick foetuses through the cisternum magnum. Control group received phosphate buffered saline (PBS). To identify cells born in the cerebral cortex at the time of antibody or PBS injection, 5'-bromo-2'- deoxyuridine was administered to the foetuses by intravenous injection into an outlying vein using micromanipulation. After injection, the foetuses were re-incubated for another 3 days. All the foetuses were collected on day 18, the brains fixed in paraformaldehyde, cut with a microtome and stained with methyl green pyronin and anti-NGF antibody. Quantitative measurements showed that the thickness of the germinal epithelium (GE) and cerebral cortex in the anti-NGF antibody injected foetuses was decreased when compared with normal control embryos. The number of cells produced in the GE of antibody injected foetuses was decreased when compared with normal control embryos. The results from this study using neutralizing antibody suggests that NGF is an important factor in cerebral cortical development, stimulating neuronal precursor proliferation.     

Modulatory effect of prolactin on the resting and mitogen-induced activity of T, B, and NK lymphocytes

Prolactin (PRL) has been shown to contribute to the development of lymphoid tissues and maintenance of physiological immune function. Here we show that the role of the hormone extends to the control of the effector phase of the immune response. In addition to triggering resting lymphocytes to cell division, the hormone can also control the magnitude of their response to polyclonal stimuli. Concentrations of PRL in the physiological range increased the [3H]thymidine, [3H]uridine, and [3H]leucine incorporation of unstimulated NK cells cultured in serum-free conditions. The same concentrations of the hormone increased the response of NK, T, and B cells to the mitogenic stimuli interleukin 2 (IL2), phytohemagglutinin (PHA), and staphylococcus aureus cowan, respectively, the effect being maximally evident in the presence of suboptimal concentrations of the mitogens. By contrast concentrations of PRL five- to tenfold the physiological levels inhibited the mitogenic response to IL2 and PHA. These data indicate a double-faceted regulatory role of this hormone in vivo.     

Requirement of PI3-kinase activity for the nuclear transport of prolactin in cloned murine T lymphocytes.

The murine T-cell clone, L2, requires both IL2 and PRL to proliferate. Proliferation and selected IL2-driven gene expression are blocked by treatment with rapamycin. Since prolactin translocation to the nucleus is IL2 dependent and required for proliferation, experiments were performed to identify activation pathways that might be involved in nuclear transport and proliferation. L2 cells were stimulated with IL2 in the presence and absence of the mTOR inhibitor rapamycin, PI3-kinase inhibitors (wortmannin, LY294002), the p38 MAP kinase inhibitor SB203580 and the vitamin D analog calcipotriol. The immunosuppressant rapamycin markedly inhibited IL2-induced proliferation and prolactin translocation to the nucleus. Similarly, wortmannin and LY294002 inhibited IL2-induced proliferation and markedly decreased the amount of prolactin transported to the nucleus. SB203580 and calcipotriol partially inhibited IL2-induced proliferation but had no effect on prolactin translocation. None of the inhibitors affected Lucifer Yellow uptake indicating that rapamycin, wortmannin and LY294002 did not inhibit early endosomal formation but rather worked to inhibit prolactin translocation at a later point in the retrograde transport pathway.