Role of Stromal and Thy 1,2<Superscript>+</Superscript> Cells in the Mechanisms of Action of Immobilized Granulocyte Colony-Stimulating Factor during Cytostatic-Induced Myelosuppression

The effects of immobilized granulocyte colony-stimulating factor (mediated by cells of the hemopoiesis-inducing microenvironment) on hemopoietic precursors of various classes were studied on the model of cytostatic-induced myelosuppression (administration of cyclophosphamide). The action of this preparation was compared with that of the standard preparation of granulocyte colony-stimulating factor. Thy 1,2⁺ cells potentiated the effects of immobilized and standard granulocyte colony-stimulating factors on granulocyte-erythroid-macrophagemegakaryocyte precursors. Stromal cells were shown to potentiate the influence of these agents on granulocyte precursors. Induction of proliferation of precursor cells by the immobilized factor mediated by cells of the hemopoiesis-inducing microenvironment persisted for a longer period compared to that induced by the standard product.     

Effect of cytokines (G-CSF and SCF) on stromal precursor cells

Repeated injections of hemopoietic cytokines (granulocyte colony-stimulating factor and stem-cell factor) to normal mice increase the content of bone marrow stromal precursors, which are capable of transferring hemopoietic microenvironment; cytokines have no effect on osteogenic potencies of stromal precursors. In contrast, injection of granulocyte colonystimulating factor during organization of a hemopoietic microenvironment considerably decreases the number of stromal precursors in the ectopic focus. The role of these stromal effects of cytokines in cytokine-induced mobilization of stem cells from the bone marrow into circulation remains unclear. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 204–206, February, 1998     

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.     

Delayed effects of long-term administration of granulocyte colony-stimulating factor to mice

We studied the effects of chronic administration of granulocyte colony-stimulating factor in nonmobilizing doses to mice. Over 18 months of the study, 55% animals of the treatment group died of unknown cause, blood diseases and tumors were found in 20% mice, and in 5% animals pathological changes were absent. Control mice had no diseases (normal values of total and differential leukocyte count). The diagnoses made over the first 7 months mainly included myeloproliferative diseases. Solid tumors were found at later terms. Suppurative inflammation at the site of injection was observed in all mice after 3-month treatment with granulocyte colony-stimulating factor. Our results indicate that chronic administration of granulocyte colony-stimulating factor in low doses leads to the development of etiologically different tumors and sharply reduced animal life span. The use of granulocyte colony-stimulating factor during allogeneic transplantation of hemopoietic stem cells can be hazardous for donors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 568–573, May, 2008     

Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies

Background  

Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.     

Role of Stem Cells in Adaptation to Hypoxia and Mechanisms of Neuroprotective Effect of Granulocytic Colony-Stimulating Factor

The role of regional cerebral neural precursor cells in restoration of the CNS activity during the development of hypoxic encephalopathy and the participation of the bone marrow mesenchymal stem cells in the formation of blood system reactions in hypoxia are shown. The mechanisms of neuroprotective effect of granulocytic colony-stimulating factor in severe oxygen insufficiency are studied. The specific psychopharmacological effect of granulocytic colony-stimulating factor is due to mobilization and determined homing of the bone marrow mesenchymal stem cells in damaged CNS zones with subsequent differentiation of these cells into specialized elements and due to activation of erythropoiesis. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 4, pp. 203–209, November, 2005     

粒细胞集落刺激因子与血管性痴呆大鼠海马凋亡相关蛋白

背景：研究表明粒细胞集落刺激因子在保护神经元免受各种因素所致的神经元变性和死亡中发挥重要作用。 目的：观察粒细胞集落刺激因子对血管性痴呆大鼠海马组织神经细胞凋亡及Bcl-2、Bax蛋白表达的影响。 方法：采用永久性双侧颈总动脉结扎法建立SD大鼠血管性痴呆模型,以未进行血管结扎的大鼠作为假手术组。造模成功后,治疗组大鼠每日皮下注射粒细胞集落刺激因子50 μg/kg,假手术组和模型组注射等量的生理盐水。分别于造模后7,14,28 d取大鼠海马组织用于检测。 结果与结论：Morris水迷宫结果显示,模型组大鼠逃避潜伏期明显延长(P < 0.01),而治疗组各时间点大鼠逃避潜伏期较模型组缩短(P < 0.01);TUNEL及免疫组织化学结果显示,与模型组比较,治疗组各时间点大鼠海马TUNEL及Bax阳性细胞吸光度值明显减小(P < 0.01),Bcl-2阳性细胞吸光度值明显增加(P < 0.01)。说明粒细胞集落刺激因子可提高血管性痴呆大鼠海马Bcl-2蛋白的表达,抑制Bax蛋白的表达,减少神经细胞凋亡,改善大鼠的学习记忆功能。     

Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice

We produced transgenic mice expressing Tcell receptor-αβ chain genes, derived from the chicken ovalbumin (OVA)-specific I-A^d-restricted CD4⁺CD8^? T helper cell clone 7–3–7. In transgenic mice with H-2^d genetic background (Tg-d mice), delayed-type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg-d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)-2, IL-3, interferon-γ, granulocyte/macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α and MIP-1β, which may play an important role in the attraction of mononuclear cells to an antigen-challenging site.     

Human umbilical cord blood-derived stromal cells are superior to human umbilical cord blood-derived mesenchymal stem cells in inducing myeloid lineage differentiation in vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.     

High-dose intravenous melphalan in a patient with multiple myeloma and oliguric renal failure

A light-chain myeloma was diagnosed as the underlying disease in a 52-year-old woman with acute oliguric renal failure. The patient was erroneously treated with high-dose intravenous melphalan (60 mg/m²). Because of this overdose treatment with granulocyte colony-stimulating factor was initiated, but pronounced absolute leukopenia (white blood cell count < 0.5 × 10⁹/l) developed and lasted for 13 days. Following melphalan treatment a continuous increase in urine volume was accompanied by a decrease of serum creatinine and blood urea nitrogen. Within 10 days after the administration of melphalan the patient no longer required hemodialysis. We conclude that high-dose chemotherapy in combination with hematopoietic growth factors should be considered in individual cases with newly diagnosed light-chain nephropathy.Abbreviations BUN blood urea nitrogen - G-CSF granulocyte colony-stimulating factor - WBC white blood cell count Correspondence to: M. Pecherstorfer     

Interleukin-12 activates human γδ T cells: synergistic effect of tumor necrosis factor-α

γδ T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium-stimulated monocytes activate cord blood γδ T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for γδ T cell activation in vitro: interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-α and granulocyte/macrophage colony-stimulating factor were examined. Recombinant human IL-12 stimulated γδ T cells, but not αβ T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL-12-primed γδ T cell numbers increased to a greater extent in the culture to which exogenous IL-2 (5 U/ml) was added. Anti-TNF-α monoclonal antibody inhibited IL-12-induced up-regulation of CD25 on γδ T cells, suggesting that endogenous TNF-α may play a role in IL-12-induced activation of γδ T cells. Recombinant TNF-α synergistically augmented IL-12-induced activation of γδ T cells. Furthermore, IL-12 up-regulated TNF receptors on γδ T cells in vitro: TNF-α binding to its receptor induced CD25 expression on the γδ T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL-12 and TNF-α were produced by mycobacterial lysate-stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, γδ T cells can be quickly and antigen-nonspecifically activated by soluble factors including IL-12 and TNF-α, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.     

The CD2 and CD28 adhesion molecules induce long-term autocrine proliferation of CD4+ T cells

In vitro human T lymphocyte activation requires two-signal triggering delivered by lectins, phorbol esters or antibodies directed against surface molecules. Stimulation of adhesion molecules by CD2 and/or CD28 antibodies defines alternative activation pathways. Activation by CD2 + CD28 monoclonal antibodies induces high-level, long-lasting and monocyte-independent proliferation of highly purified T cells. Limiting dilution cultures showed that CD28 in association with CD2 or CD3, without addition of exogenous cytokines, induced single-cell proliferation. CD2 + CD28 stimulation induced long-term interleukin (IL)-2-dependent autocrine proliferation of CD4⁺ T cell clones. We tried to elucidate this long-term proliferation by evaluating cytokine secretion and cytokine dependency. CD28 associated to CD3 or CD2 induced high levels of IL-2, tumor necrosis factor (TNF) and IL-4 secretion for 10 days, in contrast to CD3 alone which induced only TNF secretion. Cytokines of the monocytic lineage were also secreted, such as colony-stimulating factor-1, granulocyte macrophage colony-stimulating factor or IL-1, the latter being more specific of CD2 + CD28 activation. Blocking antibodies confirmed the crucial role of IL-2 in CD2 + CD28 activation. Anti-IL-4, anti-IL-7 receptor or anti-TNF antibodies had no effect on proliferation. Stimulation with CD2 + CD28 induced long-term autocrine (at least for IL-2) proliferation for CD4⁺ T cells, with no evidence for the implication of another cytokine among those tested other than IL-2. This represents a model for long-term autocrine growth for non-leukemic cells.     

内源性骨髓间充质干细胞与骨折微环境中的相关趋化因子

背景：研究表明,骨髓间充质干细胞定向迁移依赖于损伤局部表达趋化因子与细胞表面相应受体的相互作用。然而,哪些趋化因子在介导骨髓间充质干细胞向骨折部位定向迁移过程中起关键作用,目前尚不清楚。 目的：对内源性骨髓间充质干细胞进行示踪,观察其在骨修复中的作用;检测并筛选出骨折微环境中表达量高且与骨髓间充质干细胞趋化规律相关的因子。 方法：建立C57BL/6小鼠荧光/普通骨髓嵌合体,利用嵌合体动物模型制造胫骨骨折模型,在不同时相点检测骨折部位组织绿色荧光蛋白阳性细胞占所有细胞的比例、来源于骨髓间充质干细胞的成骨细胞占全部成骨细胞的比例,判断来源于骨髓的骨髓间充质干细胞在骨折修复中的作用;利用免疫组化等方法检测骨折部位不同时相点趋化因子的蛋白表达水平。 结果与结论：骨折局部绿色荧光蛋白阳性细胞占所有细胞比例在术后1,5,14 d分别为(3.011±0.911)%,(9.031±0.145)%,(12.064±0.145)%;来源于骨髓间充质干细胞的成骨细胞占全部成骨细胞的50%以上;骨折后各时相点微环境中基质细胞衍生因子1、集落刺激因子、肝细胞生长因子、单核细胞趋化蛋白1、间质金属蛋白酶9有不同程度的表达,粒细胞集落刺激因子无明显表达。基质细胞衍生因子1的表达量相对最高。经相关分析认为：基质细胞衍生因子1在骨折微环境中的表达与骨髓间充质干细胞趋化规律具有相关关系。结果表明骨髓内的骨髓间充质干细胞参与骨折修复并在其中起到了重要作用;基质细胞衍生因子1促进骨髓间充质干细胞的定向趋化并参与骨组织的修复。     

微环境对心肌干细胞动员和分化的影响

在成体心脏和骨髓中存在多能性心肌干细胞(cardiac stem cell,CSC)。当心肌发生缺血坏死时,心肌局部微环境发生改变,在粒细胞集落刺激因子、基质细胞衍生因子、骨形态发生蛋白-2和胰岛素样生长因子-Ⅰ等各类因子的作用下,CSC可被动员至心肌缺血区,向心肌细胞、内皮细胞等定向分化,从而修复缺血坏死的心肌。因此,尝试改善心肌局部微环境对于CSC动员或移植治疗缺血性心脏病将具有重要意义。     

Fatal hematophagic histiocytosis after granulocyte-macrophage colony-stimulating factor and chemotherapy for high-grade malignant lymphoma

Fatal hematophagic histiocytosis occurred in two patients after they had received granulocyte-macrophage colony-stimulating factor (GM-CSF) in addition to chemotherapy for malignant non-Hodgkin's lymphoma. In one patient GM-CSF promoted the activity of subclinical hematophagic histiocytosis, resulting in severe pancytopenia and multiorgan failure. In the other patient the syndrome that caused persistent bone marrow failure began after the institution of GM-CSF therapy. Exogenous GM-CSF appears to upregulate preexisting hematophagic histiocytosis and may even contribute to its de novo initiation. It is therefore conceivable that endogenous GM-CSF also plays an essential role in the pathogenesis of this syndrome.Abbreviations CMV cytomegalovirus - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor Correspondence to: A. Schaffner     

脊髓损伤后运动功能恢复中粒细胞集落刺激因子的作用

背景：近期有文献报道了粒细胞集落刺激因子在脑梗死模型及急性脊髓损伤模型中的神经保护作用,但应用的动物模型均非击打模型,与人体脊髓损伤的病理生理过程存在一定差距。 目的：观察粒细胞集落刺激因子对Allen’s脊髓损伤大鼠模型运动功能恢复的影响。 方法：使用改良Allen’s法制作T10节段Wistar大鼠脊髓损伤撞击模型,随机分为2组,粒细胞集落刺激因子组应用粒细胞集落刺激因子治疗,vehicle组注射等剂量PBS。于造模后第1,7,14,21,28,35天分别应用BBB运动功能评分法和Rivlin斜板实验评估大鼠运动功能,造模后第7,14,21,28,35天使用网格步行实验评估大鼠四肢肌力。 结果与结论：所有大鼠造模后均出现后肢瘫痪症状。造模后第7,14,21,28,35天粒细胞集落刺激因子组BBB运动功能评分及Rivlin斜板实验评分高于vehicle组(P < 0.05-0.01),造模后第14,21,28,35天粒细胞集落刺激因子组网格行走实验错误数低于vehicle组(P < 0.05-0.01),结果显示,粒细胞集落刺激因子治疗后大鼠运动功能及四肢肌力恢复情况均优于vehicle组。提示粒细胞集落刺激因子疗法对脊髓损伤起到了积极的治疗效果。     

Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.     

Dependence of neovascularization mechanisms on the molecular microenvironment

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57Bl/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.