17beta-estradiol alters the activity of conventional and IFN-producing killer dendritic cells

Estrogens increase aspects of innate immunity and contribute to sex differences in the prevalence of autoimmune diseases and in response to infection. The goal of the present study was to assess whether exposure to 17beta-estradiol (E2) affects the development and function of bone marrow-derived dendritic cells and to determine whether similar changes are observed in CD11c(+) splenocytes exposed to E2 in vivo. E2 facilitated the differentiation of BM precursor cells into functional CD11c(+)CD11b(+)MHC class II(+) dendritic cells (DCs) with increased expression of the costimulatory molecules CD40 and CD86. Exposure of bone marrow-derived dendritic cells to E2 also enhanced production of IL-12 in response to the TLR ligands, CpG and LPS. In contrast, CD11c(+) cells isolated from the spleens of female C57BL/6 mice that were intact, ovariectomized, or ovariectomized with E2 replacement exhibited no differences in the number or activity of CD11c(+)CD11b(+)MHC class II(+) DCs. The presence of E2 in vivo, however, increased the number of CD11c(+)CD49b(+)NK1.1(low) cells and reduced numbers of CD11c(+)CD49b(+)NK1.1(high) cells, a surface phenotype for IFN-producing killer DCs (IKDCs). Ultrastructural analysis demonstrated that CD11c(+)NK1.1(+) populations were comprised of cells that had the appearance of both DCs and IKDCs. CD11c(+) splenocytes isolated from animals with supplemental E2 produced more IFN-gamma in response to IL-12 and IL-18. These data illustrate that E2 has differential effects on the development and function of DCs and IKDCs and provide evidence that E2 may strengthen innate immunity by enhancing IFN-gamma production by CD11c(+) cells.     

Trans-presentation of IL-15 dictates IFN-producing killer dendritic cells effector functions

IFN-producing killer dendritic cells (IKDC) were initially described as B220(+)CD11c(+)CD3(-)NK1.1(+) tumor-infiltrating cells that mediated part of the antitumor effects of the combination therapy with imatinib mesylate and IL-2. In this study, we show their functional dependency on IL-15 during homeostasis and inflammatory processes. Trans-presentation of IL-15 by IL-15Ralpha allows dramatic expansion of IKDC in vitro and in vivo, licenses IKDC for TRAIL-dependent killing and endows IKDC with immunizing potential, all three biological attributes not shared by B220(-)NK cells. However, IL-15 down-regulates the capacity of IKDC to induce MHC class I- or II-restricted T cell activation in vitro. Trans-presentation of IL-15 by IL-15Ralpha allows IKDC to respond to TLR3 and TLR4 ligands for the production of CCL2, a chemokine that is critical for IKDC trafficking into tumor beds (as described recently). We conclude that IKDC represent a unique subset of innate effectors functionally distinguishable from conventional NK cells in their ability to promptly respond to IL-15-driven inflammatory processes.     

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV‐positive tumour cells

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.     

Bone marrow-derived stem cells initiate pancreatic regeneration

We show that transplantation of adult bone marrow-derived cells expressing c-kit reduces hyperglycemia in mice with streptozotocin-induced pancreatic damage. Although quantitative analysis of the pancreas revealed a low frequency of donor insulin-positive cells, these cells were not present at the onset of blood glucose reduction. Instead, the majority of transplanted cells were localized to ductal and islet structures, and their presence was accompanied by a proliferation of recipient pancreatic cells that resulted in insulin production. The capacity of transplanted bone marrow-derived stem cells to initiate endogenous pancreatic tissue regeneration represents a previously unrecognized means by which these cells can contribute to the restoration of organ function.     

Bone marrow-derived dendritic cells generated in the presence of resolvin E1 induce apoptosis of activated CD4+ T cells

In contrast to the role of dendritic cells (DC) in immunity and tolerance, little is known about their possible role in the resolution of inflammatory processes. In addition to the reduction in the number of infiltrating immune cells, the elimination of effector T cells already present at the inflammatory site represents an essential step toward resolution. Recently, lipid mediators such as the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and their metabolites, including resolvin E1 (RvE1), have been shown to accumulate in inflammatory foci during the resolution phase. RvE1 has been reported to reduce immune cell infiltration and proinflammatory cytokine production. In this study we report that DC exposed to RvE1, especially during differentiation, acquire the capacity to induce apoptosis of activated T cells through the induction and activity of indoleamine 2,3-dioxygenase. To our knowledge, this study is the first to report on an omega-3 fatty acid derivative inducing indoleamine 2,3-dioxygenase expression in DC. RvE1-exposed DC maintain an immature chemokine receptor expression pattern even following TLR stimulation, with high CCR5 and no CCR7 expression. This effect implies that DC exposed to RvE1 and pathogens remain at the inflammatory site, instead of migrating to lymph nodes, and induce apoptosis in effector T cells infiltrating the inflammatory site. To our knowledge, the DC described in this study represent a new functional DC subtype, whose essential function resides in the resolution of inflammation.     

2',5'-Oligoadenylate trimer core and the cordycepin analog augment the tumoricidal activity of human natural killer cells

Interferon (IFN) augments the lytic activity of natural killer (NK) cells, inhibits the transformation of human peripheral blood lymphocytes (PBL) by Epstein Barr virus (EBV), and induces a 2',5'-oligoadenylate (2',5'-An) synthetase. Exogenous 2',5'-An by itself can inhibit the transformation of human PBL by EBV. The present studies report that 2',5'-An and its cordycepin analog also augmented the tumoricidal activity of human NK cells. Incubation of nylon wool-passed PBL for 1 to 2 hr with the 5'-dephosphorylated core trimer of 2',5'-An boosted natural killing of tumor target cells modestly, but consistently. The cordycepin analog (3'-deoxyadenylate) also augmented NK activity. The optimal concentration both of 2',5'-A3 core and of 2',5'-3'dA3 core was 50 microM, and the optimal time for this effect was 2 hr of treatment. Kinetic analysis revealed that 2',5'-A3 core increased the lytic rate of NK cells by about one-third. This increase was due to an even greater increase (about 50%) in the lytic activity of individual NK cells, coupled with a slight decrease in the number of actual NK effector cells. In contrast, 3',5'-A3 core did not increase NK activity even at 300 microM, at which point it was toxic. In addition, to rule out a pro-drug effect as the basis for the boosting of NK activity by 2',5'-A3 core and by 2',5'-3'dA3 core, the effect of adenosine and cordycepin monomers on NK activity was tested. Neither adenosine nor cordycepin, tested at 150 microM (three times the optimal concentration of the trimer cores), boosted NK activity. The addition of 2'-deoxycoformycin (2 microM) had no effect on the actions of adenosine and cordycepin monomers. The data presented here demonstrate that 2',5'-A3 core and its analog 2',5'-3'dA3 core have another IFN-like action, augmentation of NK activity, in addition to inhibiting EBV-induced transformation.     

Bone marrow-derived cells: A potential approach for the treatment of xerostomia

Transplantations of bone marrow-derived cells (BMDCs) are traditionally used for hematologic diseases, but there are increasing numbers of clinical trials using BMDC treatments for non-hematologic disorders, including autoimmune diseases. BMDCs are recently reported to improve organ functions. This paper will review available reports supporting the role of BMDCs in reducing xerostomia (i.e. re-establishing salivary gland functions) due to head and neck irradiation for cancer therapies and in Sj?gren's syndrome. There are reports that BMDCs provide a beneficial effect on the saliva production. BMDCs positively affect blood vessels stability and regeneration in irradiated salivary glands. Also, BMDCs provide an immunomodulatory activity in mice with Sj?gren's-like disease. While the exact mechanisms by which BMDCs improve organ functions remain controversial, there is preliminary evidence that a combination of them (such as cell transdifferentiation, vasculogenesis, and paracrine effect) occur in salivary glands.     

Nitric oxide-mediated tumoricidal activity of murine microglial cells

Experimental metastases in the brain of mice are infiltrated by microglia, and parabiosis experiments of green fluorescent protein (GFP(+)) and GFP(-) mice revealed that these microglia are derived from circulating monocytes (GFP(+), F4/80(+), and CD68(+)). These findings raised the question as to whether microglia (specialized macrophages) possess tumoricidal activity. C8-B4 murine microglia cells were incubated in vitro in medium (control) or in medium containing both lipopolysaccharide and interferon-γ. Control microglia were not tumoricidal against a number of murine and human tumor cells, whereas lipopolysaccharide/interferon-γ-activated microglia lysed murine and human tumor cells by release of nitric oxide. Parallel experiments with murine peritoneal macrophages produced identical results. Neither activated microglia nor activated macrophages lysed nontumorigenic murine or human cells. Collectively, these data demonstrate that brain metastasis-associated microglia are derived from circulating mononuclear cells and exhibit selective and specific tumoricidal activity.     

Bone marrow-derived dendritic cells reverse the anergic state of CD4+CD25+ T cells without reversing their suppressive function

Dendritic cells (DC) are potent inducers of immunity to foreign Ags, but also contribute to self-tolerance by induction of regulatory T cells or deletion/anergy of self-reactive T cells. In this study, we have studied the capacity of DC to activate naturally occurring CD4+CD25+ regulatory T cells as well as the ability of CD4+CD25+ T cells to suppress the DC-mediated activation of CD4+CD25- T cells. Mature bone marrow-derived dendritic cells, but not splenic DC, were able to induce the proliferation of CD4+CD25+ T cells in the presence of a polyclonal stimulus and in the absence of exogenous IL-2. The DC-induced proliferative response of the CD4+CD25+ T cells was partially dependent on IL-2 produced by a small number of contaminating CD25+ effector cells. Because bone marrow-derived dendritic cells induce proliferation of both CD4+CD25+ and CD4+CD25- T cells in vitro, it was impossible to assay the suppressive function of the CD4+CD25+ T cells using [3H]TdR uptake or CFSE dilution. We therefore measured IL-2 production in cocultures of CD4+CD25+ and CD4+CD25- T cells using the IL-2 secretion assay. Surprisingly, CD4+CD25+ T cells markedly suppressed IL-2 secretion by the CD4+CD25- T cells without inhibiting their proliferation. Collectively, these results suggest that Ag presentation by DC can induce the expansion of CD4+CD25+ T cells while simultaneously activating their ability to suppress cytokine secretion by effector T cells.     

Prostaglandin E2 promotes the survival of bone marrow-derived dendritic cells

Since dendritic cells (DC) participate in both innate and adaptive immunity, their survival and expansion is tightly controlled. Little is known about the mechanisms of DC apoptosis. PGE(2), an arachidonic acid metabolite, plays an essential role in DC migration. We propose a novel function for PGE(2) as a DC survival factor. Our studies demonstrate that PGE(2) protects DC in vitro against apoptosis induced by withdrawal of growth factors or ceramide. DC matured in conditions that inhibit endogenous PGE(2) release are highly susceptible to apoptosis and exogenous PGE(2) re-establishes the more resistant phenotype. The antiapoptotic effect is mediated through EP-2/EP-4 receptors and involves the PI3K --> Akt pathway. PGE(2) leads to increased phosphorylation of Akt, protection against mitochondrial membrane compromise, and decreased caspase 3 activity. Macroarray data indicate that PGE(2) leads to the down-regulation of a number of proapoptotic molecules, i.e., BAD, several caspases, and granzyme B. In vivo, higher numbers of immature and Ag-loaded CFSE-labeled DC are present in the draining lymph nodes of mice inoculated with PGE(2) receptor agonists, compared with animals treated with ibuprofen or controls injected with PBS. This suggests that PGE(2) acts as an endogenous antiapoptotic factor for DC and raises the possibility of using PGE(2) agonists to increase the survival of Ag-loaded DC following in vivo administration.     

TRANCE counteracts FasL-mediated apoptosis of murine bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most potent APCs known to date. Despite their potency, DCs are short-lived. During the course of an immune response, DCs interact with cognate T cells, which upon activation express both DC survival and pro-apoptotic factors. This raises the question how DC longevity is regulated by these signals. In this study, we have assessed the roles of FasL (CD95L) and tumor necrosis factor-related activation-induced cytokine (TRANCE) in regulating the survival of murine bone marrow-derived DCs (BMDCs). We have shown for the first time that TRANCE protects DCs from FasL-mediated apoptosis, and that the quantitative balance between TRANCE and FasL can modulate BMDC survival in vitro. In addition, by quantifying adoptively transferred BMDCs in draining lymph nodes (LNs), we have shown that treating DCs with FasL prior to the transfer decreases the quantity of donor DCs capable of migrating to the LN, presumably due to FasL-mediated apoptosis of donor DCs in vivo. Furthermore, we have shown that TRANCE can counteract FasL and reverse such decrease. Taken together, these results suggest that the interplay between FasL and TRANCE play a role in regulating the survival of DCs.     

Cryptococcus neoformans activates bone marrow-derived conventional dendritic cells rather than plasmacytoid dendritic cells and down-regulates macrophages

Induction of IL-12 and IL-23 is essential for protective immunity against Cryptococcusneoformans. The contribution of dendritic cells vs. macrophages to IL-12/23 production in response to C. neoformans infection is unclear. Activation of conventional bone marrow-derived dendritic cells (BMDC), plasmacytoid BMDC, and bone marrow-derived macrophages (BMMPhi) was assessed by analyzing cytokine responses and the expression of MHC-II, CD86, and CD80 in each cell type. Cryptococcus neoformans induced the release of IL-12/23p40 by BMDC, but not by BMMPhi, in a TLR2- and TLR4-independent but MyD88-dependent manner. Conventional BMDC rather than plasmacytoid BMDC up-regulated MHC-II and CD86, while BMMPhi down-regulated MHC-II and CD86 in response to C. neoformans. The up-regulation of MHC-II and CD86 on BMDC required MyD88. Our data point to conventional DC as critical IL-12/23-producing antigen-presenting cells during cryptococcosis.     

Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells

A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as “non-dendritic cells” or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these “junk cells” are actually qualified dendritic cells.     

Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells

Surfactant protein A (SP-A) is an innate immune molecule that regulates pathogen clearance and lung inflammation. SP-A modulates innate immune functions such as phagocytosis, cytokine production, and chemotaxis; however, little is known about regulation of adaptive immunity by SP-A. Dendritic cells (DCs) are the most potent antigen-presenting cell with the unique capacity to activate naive T cells and initiate adaptive immunity. The goal of this study was to test the hypothesis that SP-A regulates the differentiation of immature DCs into potent T cell stimulators. The data show that incubation of immature DCs for 24 h with SP-A inhibits basal- and LPS-mediated expression of major histocompatibility complex class II and CD86. Stimulation of immature DCs by SP-A also inhibits the allostimulation of T cells, enhances dextran endocytosis, and alters DC chemotaxis toward RANTES and secondary lymphoid tissue chemokine. The effects on DC phenotype and function are similar for the structurally homologous C1q, but not for SP-D. These studies demonstrate that SP-A participates in the adaptive immune response by modulating important immune functions of DCs.     

Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

By inducing and amplifying dendritic cells (DCs) derived from the bone marrow of asthma murine in vitro, cytokines mRNA were expressed, and the functions of DCs were investigated. Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4, and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates. Large numbers of DCs have been obtained from bone marrow, and they expressed interleukin-13 (IL-13), interleukin-9 (IL-9), and interleukin-3 (IL-3) mRNA. Moreover, the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups (P<0.05). But, the level of IL-3 mRNA showed no discrepancy between the two groups (P>0.05). DCs are very important in the forming and developing of asthma, which implies that the therapy targeted at DCs will possibly become a new goal. __________ Translated from Journal of Nanjing Normal University (Natural Science), 2005, 28(2): 96–100 [译自: 南京师范大学学报 (自然科学版), 2005, 28(2): 96–100]     

Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

By inducing and amplifying dendritic cells(DCs)derived from the bone marrow of asthma murine in vitro,cytokines mRNA were expressed,and the functions of DCS were investigated.Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4,and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates.Large numbers of DCS have been obtained from bone marrow,and they expressed interleukin-13(IL-13),interleukin-9(IL-9),and interleukin-3(IL-3)mRNA.Moreover.the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups(P＜0.05).But,the level of IL-3 mRNA showed no discrepancy between the two groups(P＞0.05).Des are very important in the forming and developing of asthma,which implies that the therapy targeted at DCs will possibly become a new goal.     

Bone marrow-derived cells as progenitors of lung alveolar epithelium.

We assessed the capacity of plastic-adherent cultured bone marrow cells to serve as precursors of differentiated parenchymal cells of the lung. By intravenously delivering lacZ-labeled cells into wild-type recipient mice after bleomycin-induced lung injury, we detected marrow-derived cells engrafted in recipient lung parenchyma as cells with the morphological and molecular phenotype of type I pneumocytes of the alveolar epithelium. At no time after marrow cell injection, did we detect any engraftment as type II pneumocytes. In addition, we found that cultured and fresh aspirates of bone marrow cells can express the type I pneumocyte markers, T1alpha and aquaporin-5. These observations challenge the current belief that adult alveolar type I epithelial cells invariably arise from local precursor cells and raise the possibility of using injected marrow-derived cells for therapy of lung diseases characterized by extensive alveolar damage.     

小鼠骨髓源树突状细胞体外诱导培养及初步鉴定

用重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组小鼠白细胞介素4（rmIL-4）体外诱导小鼠骨髓细胞分化为树突状细胞,进行形态学变化观察,分析细胞表面分子,刺激T细胞增殖,探讨小鼠骨髓源树突状细胞（BMDC）体外诱导培养并进行初步鉴定。体外培养9d后BMDC可达80%以上,光镜下可见典型的树突状细胞形态。清楚表达成熟期主要表面标志物,可显著刺激同种异体混合淋巴细胞增殖。获得了较高纯度的BMDC,避免了使用传统磁珠分离方法所带来的成本高,操作复杂,产出率低的弊端,为研究BMDC功能以及运用开展下游实验提供材料。