Induction of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Granulocyte Colony-Stimulating Factor (G-CSF) Expression in Bone Marrow and Fractionated Marrow Cell Populations by Interleukin 3 (IL-3): IL-3-Mediated Positive Feedback Mechanisms of Granulopoiesis

Abstract

Interleukin 3 (IL-3) induces proliferation and differentiation of mast cell progenitors in vitro, whereas it induces granulocytosis in vivo. In this paper, a positive feedback mechanism of granulopoiesis was studied in order to elucidate the granulocytosis induced by IL-3 in mouse. IL-3 induced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in total bone marrow cells and a marrow adherent cell population. In fractionated marrow cell populations, a different expression pattern of induction by IL-3 stimulation was observed; GM-CSF was expressed in macrophages and fraction 1 (P<1.061) and 2(1.061<P<1.074) of bone marrow cells fractionated by equilibrium density centrifugation, G-CSF was expressed in macrophages and fraction 2 and 3 (1.074<P<1.097), and interleukin 6 (IL-6) in macrophages and fraction 1 to 3. These results indicate a hierarchical regulation of cytokine production and the existence of a positive feedback mechanism in granulopoiesis. IL-6, induced by IL-3, stimulates stem cells into cycle and induces stem cells to respond to IL-3. The stem cells differentiate to granulocyte-macrophage colony-forming cells by the combined effect of IL-3 and IL-6. IL-3 also induces GM- and G-CSF expression which in turn makes granulocyte-macrophage colony-forming cells differentiate to granulocytes. These factors organize a cytokine network in granulopoiesis.     

IL-3 in dendritic cell development and function: a comparison with GM-CSF and IL-4

Dendritic cells (DC) develop in vivo from hematopoietic precursor cells. This process can be mimicked in vitro by growth factor stimulation. Among those factors granulocyte-macrophage colony-stimulating factor (GM-CSF) is the best known and most widely used for generation of rodent and human DC of the myeloid lineage. GM-CSF is often combined with interleukin-4 (IL-4) to suppress macrophage (Mph) outgrowth in cultures of human cells, but this does not apply to the mouse, and detailed analyses on the role of IL-4 are rare. Despite evidence for the importance of GM-CSF for DC development derived from in vitro data, GM-CSF-deficient mice are largely normal with respect to their DC populations. This raised the interest in other growth factors for DC. IL-3 can also support DC growth in vitro, but has been neglected for some years. Now it has been revived by a series of publications. In this review, some new features of myeloid DC regarding their early developmental stages, the GM-CSF/IL-4-interplay, and the role of IL-3 are summarized.     

Effect of Human Recombinant Interleukin 4 on In Vitro Granulopoiesis of Human Bone Marrow Cells

Abstract

Utilizing in vitro colony assay, we investigated the effect of human recombinant interleukin 4 (hIL-4) on granulopoiesis of normal human bone marrow cells. Though hIL-4 itself did not possess any colony-stimulating activity, the number of neutrophil (N) colonies, particularly the number of small colonies, supported by human recombinant G-CSF (hG-CSF) was significantly increased when hIL-4 was used as a costimulant. In contrast, the number of eosinophil (Eo) colonies supported by hlL-5 was decreased when hIL-4 was used as a costimulant. Also, the numbers of N and Eo colonies supported by hIL-3 or hGM-CSF were both significantly decreased when hIL-4 was added. These data suggest that hIL-4 has diverse positive and negative regulatory effects on human neutro- and eosinophilopoiesis as a cofactor of various CSFs.     

IL-3 in the development and function of basophils

The β common chain (βc) cytokine family includes granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5, all of which use βc as key signaling receptor subunit. GM-CSF, IL-3 and IL-5 have specific roles as hematopoietic growth factors. IL-3 binds with high affinity to the IL-3 receptor α (IL-3Rα/CD123) and then associates with the βc subunit. IL-3 is mainly synthesized by different subsets of T cells, but is also produced by several other immune [basophils, dendritic cells (DCs), mast cells, etc.] and non-immune cells (microglia and astrocytes). The IL-3Rα is also expressed by immune (basophils, eosinophils, mast cells, DCs, monocytes, and megacaryocytes) and non-immune cells (endothelial cells and neuronal cells). IL-3 is the most important growth and activating factor for human and mouse basophils, primary effector cells of allergic disorders. IL-3-activated basophils and mast cells are also involved in different chronic inflammatory disorders, infections, and several types of cancer. IL-3 induces the release of cytokines (i.e., IL-4, IL-13, CXCL8) from human basophils and preincubation of basophils with IL-3 potentiates the release of proinflammatory mediators and cytokines from IgE- and C5a-activated basophils. IL-3 synergistically potentiates IL-33-induced mediator release from human basophils. IL-3 plays a pathogenic role in several hematologic cancers and may contribute to autoimmune and cardiac disorders. Several IL-3Rα/CD123 targeting molecules have shown some efficacy in the treatment of hematologic malignancies.     

Effects of IL-5, granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-3 on the survival of human blood eosinophils in vitro.

The mechanisms that could affect the lifespan of eosinophils after they have left the bone marrow, and their capacity to respond to activation factors were studied by examining the effects of IL-5, GM-CSF and IL-3 on purified human blood eosinophils in culture. All three cytokines prolonged the lifespan of the majority of blood eosinophils. This effect was dose-dependent: IL-5 greater than GM-CSF greater than IL-3. Light density eosinophils from most patients had a longer lifespan in culture than did normal density eosinophils, with or without the three cytokines. Eosinophil death in the absence of these cytokines occurred by apoptosis. Eosinophils from two patients did not survive when cultured with IL-5, although they survived in the presence of IL-3 or GM-CSF. IL-5, GM-CSF and IL-3 induced the expression of the activation epitope on the eosinophil ribonucleases recognized by monoclonal antibody EG2. We conclude that small amounts of IL-5, GM-CSF and IL-3 prevented programmed cell death in human blood eosinophils and induced the expression of the activation forms of eosinophil ribonucleases. We suggest that differences in the capacity of normal and light density eosinophils to survive in culture, and in the ability of eosinophils from some patients to respond to IL-5 could account for variations in the severity of disease seen in patients with persistent eosinophilia.     

Identification of a fourth ancient member of the IL-3/IL-5/GM-CSF cytokine family,KK34, in many mammals

The related cytokine genes IL-3, IL-5 and GM-CSF map to the (extended) T_H2 cytokine locus of the mammalian genome. For chicken an additional related cytokine gene, KK34, was reported downstream of the IL-3 plus GM-CSF cluster, but hitherto it was believed that mammalian genomes lack this gene. However, the present study identifies an intact orthologue of chicken KK34 gene in many mammals like cattle and pig, while remnants of KK34 can be found in human and mouse. Bovine KK34 was found to be transcribed, and its recombinant protein could induce STAT5 phosphorylation and proliferation of lymphocytes upon incubation with bovine PBMCs. This concludes that KK34 is a fourth functional cytokine of the IL-3/IL-5/GM-CSF/KK34-family (alias IL-5 family) in mammals.While analyzing KK34, the present study also made new identifications of cytokine genes in the extended T_H2 cytokine loci for reptiles, birds and marsupials. This includes a hitherto unknown cytokine gene in birds and reptiles which we designated “IL-5famE”. Other newly identified genes are KK34, GM-CSF(-like), IL-5, and IL-13 in reptiles, and IL-3 in marsupials.     

The Role of Hemopoietic Growth Factors in Self-Renewal and Differentiation of IL-3-Dependent Multipotential Stem Cells

Abstract

A multipotent hemopoietic cell line has been employed to assess the influence of the hemopoietic growth factors, IL-3, GM-CSF, G-CSF, and CSF-1 on the processes of self-renewal, the generation of lineage restricted progenitor cells and the production of mature neutrophils and macrophages. At a high concentration of IL-3, the cells undergo self-renewal and demonstrate little or no ability to undergo differentiation in the presence of the other growth factors. In the absence of IL-3, the cells show minimal (GM-CSF) or no (G-CSF or CSF-1) ability to respond to these other growth factors. When combined with a low concentration of IL-3, the ability of the cells to respond to GM-CSF, G-CSF, and CSF-1 is enhanced and a selective preference for the neutrophil or macrophage lineage is seen depending on the combination used, i.e., the presence of CSF-1 preferentially promotes macrophage development and G-CSF preferentially promotes neutrophil development. Conditions optimal for neutrophil development were seen using a combination of low IL-3 concentrations plus GM-CSF plus G-CSF. In such conditions, the cells undergo extensive proliferation and progressively lose their clonogenic potential (i.e., differentiation self-renewal) and acquire the biochemical markers characteristic of fully mature phagocytes.     

人酸性成纤维细胞生长因子单克隆抗体的制备

以纯化的重组人酸性成纤维细胞生长因子（ｈａＦＧＦ）为抗原，免疫ＢＡＬＢ／Ｃ小鼠，取脾细胞与ＳＰ２／０细胞融合，获得一株稳定分泌抗ｈａＦＧＦ的单克隆抗体的杂交瘤细胞株，基ＤＮＡ含量为脾细胞与ＳＰ２／０细胞ＤＮＡ含量之和，所分泌抗体为小鼠ＩｇＧ１亚类，免疫印迹显示：此单抗只与大肠杆菌中的ｈａＦＧＦ有结合，与牛ａＦＧＦ则没有结合反应。     

Production of Non-pathogenic Human Monoclonal Antibodies to Desmoglein 3 from Pemphigus Vulgaris Patient

Pemphigus vulgaris is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3, a 130 kDa epidermal protein. To further characterize the epitope(s) of pemphigus vulgaris antigen we established two human-human hybridoma by fusion of the peripheral blood mononuclear cells with a human and mouse heterohybridoma. These hybridomas designated as MAb Dsg-3: 06 and MAb Dsg-3: 10 and stable in culture and demonstrated yield of monoclonal antibodies specific for pemphigus vulgaris. Immunofluorescence, immunoblot, ELISA assays demonstrated that both the monoclonal antibodies bind to the intercellular cement substance and to 130 kDa protein present in the skin and specifically binds to recombinant desmoglein 3 protein, but not to desmoglein 1 protein. The IgG subclass distribution study demonstrated that both the antibodies are of IgG1 subclass in nature. Both the antibodies were non-pathogenic as demonstrated in vitro by their inability to produce acantholysis in normal human skin in organ culture or in vivo by the induction of disease in neonatal BALB/c mice. The relevance and value of these monoclonal antibodies in the pathogenesis of pemphigus vulgaris is discussed.     

The host environment promotes the development of primary and metastatic squamous cell carcinomas that constitutively express proinflammatory cytokines IL-1a, IL-6, GM-CSF, and KC

Human and murine squamous cell carcinomas (SCC) have been reported to produce proinflammatory cytokines IL-1a, IL-6, GM-CSF, and IL-8 or KC. Production of individual members of the proinflammatory cytokine family has been associated with increased tumor growth or metastasis in a variety of neoplasms. In this study, we determined whether the expression of these cytokines occurs as a result of the events of cel-lular transformation or culture, or is promoted by interaction of neoplastic cells with factors or cells in the host environment. We compared the expression of proinflammatory cytokines following the spontaneous transformation of murine keratinocytes in vitro, and following the formation of tumors and metastases from these transformed keratinocytes in syngeneic recipients in vivo. Using sensitive ELISA assays, we found that cultures of the in vitro transformed Balb/c SCC line Pam 212 do not produce elevated levels of proinflam-matory cytokines IL-1a, IL-6, GM-CSF and KC, indicating that transformation or culture alone is insufficient to account for the level of cytokine expression detected in patient and experimental tumors. In contrast, Pam reisolates from primary and metastatic tumors were obtained which constitutively produce markedly elevated levels of cytokines IL-1a, IL-6, KC and GM-CSF. The increase in the expression of these cytokines by SCC in vivo occurred independent of T and B lymphocyte-mediated immunity, since increases in expression of the cytokines was observed in lines reisolated from immunodeficient athymic nude and SCID Balb/c congenic mice. The increased expression of cytokines appeared to result from additional events in vivo, rather than due to selection of a pre-existing cytokine-producing subpopulation, since clones of the parental cell line expressed lower cytokine levels than cloned reisolates, and clones of the non-secreting parental cell line that formed tumors in vivo secreted elevated levels of cytokines following reisolation. We conclude that the development of SCC that express proinflammatory cytokines is promoted by tumor-host interaction(s) that are independent of specific T and B cell immunity©Lippincott Williams & Wilkins     

重组IL-12对人乳腺癌细胞自噬的研究

目的 探讨重组人IL-12（rIL-12）对乳腺癌细胞自噬的影响。方法 两株乳腺癌细胞（MDA-MB-231和MCF7）分别用rIL-12处理,经免疫蛋白印迹技术和细胞免疫荧光技术检测其自噬微管相关蛋白轻链3（LC3）的变化,以观察自噬情况;另外,用透射电镜观察rIL-12处理乳腺癌细胞前后自噬小体的变化。分别用胰岛素样生长因子1（IGF-1）激活磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）通路,再加入rIL-12处理后,蛋白免疫印迹法检测相关通路蛋白PI3K/Akt, 哺乳动物雷帕霉素靶点（TOR）磷酸化变化及LC3的表达。结果 与空白组相比,rIL-12组细胞的自噬标志蛋白LC3表达增高,差异有统计学意义（P＜0.05）,且增加程度呈时间和浓度依赖性;荧光显微镜观察自噬体形成的结果显示,与空白组相比,rIL-12处理组的细胞核周点状聚集的绿色荧光增多;使用电镜观察到rIL-12处理组明显有自噬小体形成。与rIL-12组相比,IGF-1+rIL-12组的LC3Ⅱ表达减少,差异有统计学意义（P＜0.05）。结论 rIL-12可以激活乳腺癌细胞自噬,并通过抑制PI3K/Akt信号通路的机制,促进自噬标志蛋白LC3,特别是LC3Ⅱ的表达。     

Murine Macrophages Cultured with IL-4 Acquire a Phenotype Similar to That of Epithelioid Cells from Granulomatous Inflammation

Epithelioid cells (ECs) found in granulomas are thought to derive from mononuclear phagocytes. Although GM-CSF and/or IL-4 are known to promote cell differentiation their role in the development of ECs has never been demonstrated. Here we showed that mouse macrophages treated exclusively with recombinant IL-4 (rIL-4) differentiate into epithelioid-like cells. Macrophages cultivated with rIL-4 presented a fried-egg shape, and ultrastructural studies revealed membrane interdigitations, cytoplasmic vesicles, prominent Golgi complex, and rough endoplasmic reticulum. Compared with controls, rIL-4 treated cells displayed increased expression of MHC class II molecules and of Migration Inhibitory Factor-Related Protein-14. Whereas mannose receptor-mediated phagocytosis was increased, Fc-receptor mediated phagocytosis and the production of nitric oxide were decreased in treated cultures. All these features overlap those reported for ECs from granulomatous lesions. In conclusion, treatment of mouse peritoneal macrophages with rIL-4 drives their in vitro differentiation to an epithelioid phenotype and provides a tool to investigate the biology of ECs.     

Myeloid-derived Suppressor Cells Adhere to Physiologic STAT3- vs STAT5-dependent Hematopoietic Programming,Establishing Diverse Tumor-Mediated Mechanisms of Immunologic Escape

The receptor tyrosine kinase inhibitor, sunitinib, is astonishingly effective in its capacity to reduce MDSCs in peripheral tissues such as blood (human) and spleen (mouse), restoring responsiveness of bystander T lymphocytes to TcR stimulation. Sunitinib blocks proliferation of undifferentiated MDSCs and decreases survival of more differentiated neutrophilic MDSC (n-MDSC) progeny. Ironically, sunitinib’s profound effects are observed even in a total absence of detectable anti-tumor therapeutic response. This is best explained by the presence of disparate MDSC-conditioning stimuli within individual body compartments, allowing sensitivity and resistance to sunitinib to coexist within the same mouse or patient. The presence or absence of GM-CSF is likely the major determinant in each compartment, given that GM-CSF’s capacity to preempt STAT3-dependent with dominant STAT5-dependent hematopoietic programming confers sunitinib resistance and redirects differentiation from the n-MDSC lineage to the more versatile monocytoid (m-MDSC) lineage. The clinical sunitinib experience underscores that strategies for MDSC and Treg depletions must be mindful of disparities among body compartments to avoid sanctuary effects. Ironically, m-MDSCs manifesting resistance to sunitinib also have the greatest potential to differentiate into tumoricidal accessory cells, by virtue of their capacity to respond to T cell-secreted IFN-γ or to TLR agonists with nitric oxide and peroxynitrate production.     

Detection of IL-5 and IL-1 receptor antagonist in bronchoalveolar lavage fluid in acute eosinophilic pneumonia

BACKGROUND: Acute eosinophilic pneumonia is an idiopathic cause of respiratory failure, characterized by very high numbers of alveolar eosinophils without significant blood eosinophilia. OBJECTIVE: The purpose of this study was to determine which cytokines are associated with acute eosinophilic pneumonia. METHODS: Soluble IL-1 type II receptor and the cytokines IL-1β, IL-1ra, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-α were measured in serum and in bronchoalveolar lavage fluid from two patients with acute eosinophilic pneumonia during both acute and convalescent phases. RESULTS: Compared with patients with adult respiratory distress syndrome, the patients with acute eosinophilic pneumonia had high bronchoalveolar lavage fluid levels of IL-5, IL-1ra, and soluble type II IL-1 receptor but not IL-1β, tumor necrosis factor-α>, IL-3, or granulocyte-macrophage colony-stimulating factor. Bronchoalveolar lavage fluid levels of IL-5 and IL-1ra fell after resolution of symptoms. In the serum of patients with acute eosinophilic pneumonia, IL-5 was not detectable, and IL-1ra was initially high but fell after corticosteroid treatment. CONCLUSION: Acute eosinophilic pneumonia is characterized by locally high levels of IL-5, IL-1ra, and soluble type II IL-1 receptor in the alveolar space. (J ALLERGY CLIN IMMUNOL 1996;97:1366-74.)     

IL-1 up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1

A factor-dependent human hemopoietic cell line, TF-1, requiresinterleukln 3 (IL-3) or granulocyte/acrophage colony-stimulatingfactor (GM-CSF) for its long-term growth. We have found thatIL-4, IL-5, and IL-6 also support the growth of TF-1 and thatIL-1 enhances the proliferative effect of these cytoklnes. Augmentationby IL-1 is associated with up-regulatlon of the receptors forIL-3, IL-5, GM-CSF, and erythropoietln (Epo). IL-1 increasedthe number of binding sites forIL-3 and Epo without changingtheir affinities. In contrast, IL-1 Increased the number ofhigh affinity binding sites forGM-CSF and IL-5, whereas thetotal number of binding sites was unchanged. Chemical crossllnkingexperiments Indicated that the receptors for IL-3, IL-5, andGM-CSF were composed of two components and that the molecularmasses of the larger components of these cytokine receptorswere quite similar (120 kd). The enhanced expression of thelarger components of theIL-3, IL-5, and GM-CSF receptors byIL-1 may be responsible for IL-1-induced up-regulation of thesereceptors. These observations are consistent with the modelthat the receptors for IL-3 and GM-CSF share a common component.