Bone marrow-derived IL-13Rα1-positive thymic progenitors are restricted to the myeloid lineage

The earliest thymic progenitors (ETPs) were recently shown to give rise to both lymphoid and myeloid cells. Whereas the majority of ETPs are derived from IL-7Rα-positive cells and give rise exclusively to T cells, the origin of the myeloid cells remains undefined. In this study, we show both in vitro and in vivo that IL-13Rα1(+) ETPs yield myeloid cells with no potential for maturation into T cells, whereas IL-13Rα1(-) ETPs lack myeloid potential. Moreover, transfer of lineage-negative IL-13Rα1(+) bone marrow stem cells into IL-13Rα1-deficient mice reconstituted thymic IL-13Rα1(+) myeloid ETPs. Myeloid cells or macrophages in the thymus are regarded as phagocytic cells whose function is to clear apoptotic debris generated during T cell development. However, the myeloid cells derived from IL-13Rα1(+) ETPs were found to perform Ag-presenting functions. Thus, IL-13Rα1 defines a new class of myeloid restricted ETPs yielding APCs that could contribute to development of T cells and the control of immunity and autoimmunity.     

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.     

Stromal cell regulation of lymphoid and myeloid differentiation

In vitro microenvironmental influences seem to be critical for both B lymphocyte and myeloid differentiation. Studies on murine Dexter cultures and Whitlock-Witte lymphocyte cultures suggest the presence of two critical stromal regulatory cells: an alkaline-phosphatase-positive epithelioid cell and a macrophage. Further data suggest that these cells are capable of producing colony stimulating factor-1, granulocyte-macrophage CSF, a myeloid synergizing activity, and probably separate B cell growth factors. Isolation of a cell line from Dexter stroma was accomplished and this line produced CSF-1, GM-CSF, a pre-B cell and myeloid synergizing activity, and an activity acting on differentiated B cells. We speculate that the Dexter and Whitlock-Witte in vitro culture systems are regulated by factors produced by the two adherent cell types. A lineage nonspecific factor capable of inducing cells into the B lineage or synergizing with interleukin-3, GM-CSF, and CSF-1 is produced, which presumably acts on early stem cells. In addition, the cell line produces GM-CSF, CSF-1, and a factor acting on differentiated B cells. We speculate that in these culture systems, these "terminal differentiating hormones" regulate the final pathway of differentiation, whereas the pre-B-synergizing activity supports early stem cells that can then respond to the other differentiating hormones.     

Hyperactivation of STAT3 is involved in abnormal differentiation of dendritic cells in cancer

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However, the molecular mechanisms of this process remain elusive. In this study, we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3, which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells, which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation, and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus, this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3.     

Maintenance of granulocyte-monocyte progenitor cells in liquid cultures of human foetal liver

These studies describe a liquid suspension culture system for normal myeloid cells derived from human foetal liver. A simple one-step fractionation procedure was employed to obtain a cell population capable of expanding into all stages of myeloid differentiation, including committed myeloid progenitor cells (GM-CFC). Cell proliferation in these cultures resulted in the maintenance of early myeloid populations for up to a month. In order to extend myeloid cell maintenance, a specific factor in the form of media conditioned by human endothelial cells (endo C.M.) was used. Addition of endo C.M. to foetal liver cultures resulted in increased myeloid proliferation coupled to extensive myeloid differentiation. Clonally derived foetal liver culture cells proliferated for up to 2 months in the presence of endo C.M. before maturing into macrophages. These results show that endo C.M. exert an extensive proliferative effect on early myeloid cells as well as inducing their differentiation. The large quantity of cells in early stages of myeloid differentiation provided by foetal liver cultures may be useful for biochemical and molecular biology studies of myelopoiesis. In addition, these cultures are a potential source from which to derive normal myeloid lines. The separation of the potent proliferative activity present in endo C.M. may yield an effector which maintains human myeloid cell proliferation.     

Tumor necrosis factor mediates autocrine growth inhibition in a chronic leukemia

Autocrine production of growth factors may contribute to the rapid and fatal proliferation of acute hematologic malignancies. We have investigated whether the more controlled growth of less aggressive malignancies such as chronic myeloid leukemia (CML) may be associated with autocrine production of growth inhibitory factors. TNF inhibits the growth of both normal and leukemic hemopoietic progenitor cells. We find that exogenous TNF reduces the viability and DNA synthesis of purified myeloid cells from patients with CML and inhibits myeloid colony formation by patient progenitor cells. However, unlike progenitor cells from normal donors, patient myeloid progenitor cells also constitutively express mRNA for TNF and secrete functional TNF protein in culture. This endogenous TNF impedes the growth of CML cells because anti-TNF mAb shown to neutralize bioactive human TNF increases CML cell DNA synthesis whereas non-neutralizing anti-TNF mAb has no effect. Production of TNF by CML cells is not associated with production of lymphotoxin (TNF-beta), IL-1 or IL-6. TNF-mediated autocrine growth inhibition may contribute to the maintenance of the stable, chronic phase of this disease and similar mechanisms may operate in other malignancies to limit tumor proliferation. Competition between autocrine growth promoting and inhibiting factors may underlie the observed differences in biologic behavior between acute and chronic malignancies.     

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.     

Retroviral transfer of antisense sequences results in reduction of c-Abl and induction of apoptosis in hemopoietic cells

The c-abl proto-oncogene is ubiquitously expressed during mammalian development. Activated forms of c-Abl proteins are oncogenic and have been shown to suppress apoptosis. The biological role of normal c-Abl protein is unknown. In this study, we have introduced c-abl antisense sequences into various hemopoietic cells by retroviral gene transfer. Introduction and expression of the antisense sequence effectively reduced the amount of c-Abl protein in a number of transduced hemopoietic cells, that consequently underwent apoptosis. When factor-dependent cell lines were examined, we observed that the addition of sufficient amounts of growth factors could suppress apoptosis in myeloid but not in lymphoid lines. The ability of myeloid cells to be rescued by growth factors correlated with upregulation of mRNA level of IL-3 receptor subunits. Our data suggest that c-Abl provides an anti-apoptotic signal during mammalian cell growth, and that myeloid and lymphoid cells are different in their resistance to apoptosis.     

The role of myofibroblasts in upregulation of S100A8 and S100A9 and the differentiation of myeloid cells in the colorectal cancer microenvironment

Background/aimS100A8/A9 and myeloid cells in the tumor microenvironment play an important role in cancer invasion and progression, and the effect of tumor-infiltrated myofibroblasts on myeloid cells in the tumor microenvironment is relatively unknown. Accordingly, we investigated the role of myofibroblasts in the upregulation of S100A8/A9 as well as in the differentiation of myeloid cells in the colorectal cancer (CRC) microenvironment.Materials and methodsTo investigate the interactions among cancer cells, myofibroblasts, and inflammatory cells in the microenvironment of CRC, we used 10 CRC cell lines, 18CO cells and THP-1 cells, which were co-cultured with each other or cultured in conditioned media (CM) of other cells. Expression of S100A8/A9 was evaluated via Western blot, immunohistochemical staining and immunofluorescence. The secreted factors from the cell lines were analyzed using cytokine antibody array. Flow cytometry analysis was performed to analyze the differentiation markers of myeloid cells.Results18CO CM induced increased expression of S100A8/A9 in THP-1 cells. Increased expression of S100A8/A9 was noted in inflammatory cells of the peri- and intra-tumoral areas, along with myofibroblasts in colon cancer tissue. S100A8/A9-expressing inflammatory cells also exhibited CD68 expression in colon cancer tissue, and 18CO CM induced differentiation of THP-1 cells into myeloid-derived suppressor cells (MDSCs) or M2 macrophages expressing S100A8/A9. Significant amounts of IL-6 and IL-8 were detected in 18CO CM, compared to those in both controls and THP-1 CM, and tumor-infiltrated myofibroblasts expressed IL-8 in colon cancer tissue. Finally, neutralizing antibodies to IL-6 and IL-8 attenuated 18CO CM-induced increased expression of S100A8/A9.ConclusionsThe upregulation of S100A8/A9 in tumor-infiltrated myeloid cells could be triggered by IL-6 and IL-8 released from myofibroblasts, and myofibroblasts might induce the differentiation of myeloid cells into S100A8/9-expressing MDSCs or M2 macrophages in the CRC microenvironment.     

综述:髓系衍生的抑制性细胞与肿瘤免疫耐受关系的研究进展

髓系衍生的抑制性细胞(myeloid-derived suppressor cells,MDSCs),是在肿瘤等病理因素的作用下髓系细胞发生分化障碍所产生的不同阶段髓系祖细胞的集合,具有广谱而强大的免疫抑制功能,是免疫系统的重要负性调节组件之一.研究表明:肿瘤微环境中的多种细胞因子或生长因子可通过激活相应的信号通路促进MDSCs扩增及活化,MDSCs进而通过多种机制抑制包括T细胞在内的多种免疫细胞的功能而促进肿瘤个体免疫耐受的发生.临床研究表明:肿瘤患者体内MDSCs的水平与肿瘤临床病程进展密切相关,基于MDSCs的免疫治疗也有望成为肿瘤免疫治疗的新策略.本文主要介绍了肿瘤中MDSCs的表型鉴定、扩增及活化机制、发挥免疫抑制作用的途径及机制、肿瘤中MDSCs的临床意义以及本领域需要解决的问题,以期对MDSCs在肿瘤免疫耐受中的作用进展提供参考.     

Isolation and characterization of My23, a myeloid cell-derived antigen reactive with the monoclonal antibody AML-2-23

In this study, we describe the isolation and characterization of My23, a human myeloid antigen defined by the monoclonal antibody (MoAb) AML-2-23. Cells of the HL-60 human promyelocytic cell line, when cultured in the presence of 1,25-dihydroxyvitamin D3 (calcitriol), express a surface protein of approximately 50 to 55 kilodaltons (Kd) which was immunoprecipitated with the AML-2-23 MoAb. Furthermore, after 2 days of exposure to calcitriol, HL-60 cells began to release My23 into culture medium, as determined by the ability of culture supernatant from these cells to block the binding of AML-2-23 to myeloid cells. My23 release was almost totally inhibited by incubation of cells at 4 degrees C, and was partially blocked by treatment of cells with cycloheximide or tunicamycin. The culture supernatant blocking factor, soluble My23, was identified as a 45 to 50 Kd protein by Western blot/immune overlay, using AML-2-23 and an 125I-labeled second antibody. My23, which was affinity-purified from culture supernatant, retained the ability to block AML-2-23 binding to myeloid cells. The affinity-purified antigen migrated on SDS-PAGE as a diffuse band in the m.w. range of 44 to 52 Kd. On treatment with endoglycosidase, the apparent m.w. of My23 decreased to approximately 40,000, indicating the presence of carbohydrate residues on My23. Serum from mice immunized with the purified antigen reacted with the same spectrum of myeloid cells as AML-2-23 MoAb, reacted with the My23 soluble protein in immunoblots, and competed with AML-2-23 for binding to myeloid cells. Binding of this antiserum to myeloid cells was blocked by cell supernatant from both monocytes and calcitriol-treated HL-60 cells, suggesting, along with results from m.w. determinations of the two preparations, that the soluble and cell surface forms of My23 are similar. Moreover, based on our finding that human plasma specifically inhibits the binding of AML-2-23 to myeloid cells, My23 may also be released in vivo. The enhanced expression of My23 on activated and more mature myeloid cells and its shedding or secretion by these cells is consistent with a functional role for My23.     

Acellular bone marrow extracts significantly enhance engraftment levels of human hematopoietic stem cells in mouse xeno-transplantation models

Hematopoietic stem cells (HSC) derived from cord blood (CB), bone marrow (BM), or mobilized peripheral blood (PBSC) can differentiate into multiple lineages such as lymphoid, myeloid, erythroid cells and platelets. The local microenvironment is critical to the differentiation of HSCs and to the preservation of their phenotype in vivo. This microenvironment comprises a physical support supplied by the organ matrix as well as tissue specific cytokines, chemokines and growth factors. We investigated the effects of acellular bovine bone marrow extracts (BME) on HSC in vitro and in vivo. We observed a significant increase in the number of myeloid and erythroid colonies in CB mononuclear cells (MNC) or CB CD34+ cells cultured in methylcellulose media supplemented with BME. Similarly, in xeno-transplantation experiments, pretreatment with BME during ex-vivo culture of HSCs induced a significant increase in HSC engraftment in vivo. Indeed, we observed both an increase in the number of differentiated myeloid, lymphoid and erythroid cells and an acceleration of engraftment. These results were obtained using CB MNCs, BM MNCs or CD34(+) cells, transplanted in immuno-compromised mice (NOD/SCID or NSG). These findings establish the basis for exploring the use of BME in the expansion of CB HSC prior to HSC Transplantation. This study stresses the importance of the mechanical structure and soluble mediators present in the surrounding niche for the proper activity and differentiation of stem cells.     

Contribution of myeloid-derived suppressor cells to tumor-induced immune suppression, angiogenesis, invasion and metastasis

Growing evidence suggests that myeloid-derived suppressor cells (MDSCs), which have been named "immature myeloid cells" or "myeloid suppressor cells" (MSCs), play a critical role during the progression of cancer in tumor-bearing mice and cancer patients. As their name implies, these cells are derived from bone marrow and have a tremendous potential to suppress immune responses. Recent studies indicated that these cells also have a crucial role in tumor progression. MDSCs can directly incorporate into tumor endothelium.They secret many pro-angiogenic factors as well. In addition, they play an essential role in cancer invasion and metastasis through inducing the production of matrix metalloproteinases (MMPs), chemoattractants and creating a pre-metastatic environment. Increasing evidence supports the idea that cancer stem cells (CSCs) are responsible for tumorigenesis, resistance to therapies, invasion and metastasis.Here, we hypothesize that CSCs may "hijack" MDSCs for use as alternative niche cells, leading to the maintenance of stemness and enhanced chemo- and radio-therapy resistance. The countermeasure that directly targets to MDSCs may be useful for against angiogenesis and preventing cancer from invasion and metastasis. Therefore, the study of MDSCs is important to understand tumor progression and to enhance the therapeutic efficacy against cancer.     

Up-regulation of alpha2,6 sialylation during myeloid maturation: a potential role in myeloid cell release from the bone marrow

The mechanisms by which mature myeloid cells are released from the bone marrow into the peripheral blood are not clearly understood. Glycosylation is likely to play an important role, as has been shown in the homing of lymphocytes to lymph nodes and of neutrophils to inflamed endothelia. Cell surface sialylation is an important component of many cellular adhesive interactions, both as ligand-promoting interactions, as occurs in selectin and sialoadhesin-mediated adhesion, and for reducing cell adhesion as in some cancer cells. We have studied the expression of cell surface alpha2,6-linked sialic acid in the maturation of normal bone marrow myeloid cells, the expression of alpha2,6-sialyltransferase mRNA, and the role of sialylation in the adherence of myeloid cells to bone marrow stroma. Our data show that there is a dramatic increase in cell surface alpha2,6-sialylation during the late stage of maturation. This up-regulation is restricted to specific glycoproteins including CD11b and CD18. It is associated with a relative increase in the level of alpha2,6-sialyltransferase mRNA compared with alpha2,3-sialyltransferase mRNA. The changes in mature bone marrow myeloid cells are associated with reduced cell binding to fibronectin and cultured bone marrow stroma. Our data strongly suggest that alpha2,6-sialylation may be important in the interaction between maturing myeloid cells and bone marrow stroma and may govern the release of cells from the bone marrow into the peripheral blood.     

24p3 in differentiation of myeloid cells

24p3 is a secreted lipocalin that has been variously related to apoptosis, proliferation, and the neutrophil lineage of blood cells. We have investigated the expression of 24p3 mRNA and protein in myeloid cell lines induced to differentiate by insulin-like growth factor 1 (IGF-1) and the granulocytic-colony simulating factor (G-CSF). Both these growth factors, which cause myeloid cells to differentiate into granulocytes, induced a marked increase in the expression of both 24p3 protein and mRNA. The mRNA especially appeared early after the cells were induced with either IGF-1 or G-CSF, at a time when the cells were still proliferating and are morphologically undifferentiated. 24p3 can be considered an early marker of granulocytic differentiation.