GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

Macrophage colony-stimulating factor drives cord blood monocyte differentiation into IL-10(high)IL-12absent dendritic cells with tolerogenic potential

Immature dendritic cells (DCs) induce tolerance and mature DCs induce inflammatory immune responses. However, the likelihood of maturation of immature DCs in vivo limits its potential application for suppression of unwanted immune reactions in vivo. The aim of this study was to generate DCs with anti-inflammatory properties in both the immature and mature states. GM-CSF combined with IL-4 drives monocyte differentiation into DCs. As M-CSF is a critical cytokine in development of the monocytic lineage and its level is dramatically elevated in immunosuppressive conditions, we investigated whether M-CSF could replace GM-CSF and generate DCs with distinct functions from umbilical cord blood monocytes. Highly purified umbilical cord blood monocytes cultured with M-CSF and IL-4, in a GM-CSF-independent fashion, differentiated into IL-10(high)IL-12absent cells with a DC phenotype (termed M-DC). Single time stimulation with immature DCs (both M-DCs and DCs) derived from cord blood induced hyporesponsive and regulatory CD4+ T cells. In contrast to mature DCs, mature M-DCs induced decreased Th1 differentiation and proliferation of naive CD4+ T cells in both primary and secondary allogeneic MLR and showed tolerogenic potential. These results demonstrate an unrecognized role for M-CSF in alternative differentiation of monocytes into anti-inflammatory M-DCs and suggest that M-CSF-induced DCs may be of use for suppressing unwanted immune responses.     

Inflammasome-derived IL-1β regulates the production of GM-CSF by CD4(+) T cells and γδ T cells

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αβ and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1β upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αβ and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.     

Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.     

Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells

The in vitro production of eosinophils from committed progenitor cells is influenced by interleukin (IL)-5 (eosinophil differentiation factor) and to a lesser extent by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In primary suspension cultures of marrow cells taken from eosinophilic mice, IL-3 induced a modest stimulation of eosinophil production compared to IL-5. In contrast, IL-3 was sevenfold more effective than IL-5 in generating eosinophil progenitors (eosinophil colony-forming units (CFU-eo] from more primitive precursors present in the marrow of normal mice. Pre-incubation of marrow cells in suspension culture with IL-3, but not IL-5, increased the recovery of myeloid precursors responsive to G-CSF, GM-CSF, CSF-1, or IL-3 two- to fourfold while eosinophil progenitor cells responsive to IL-5 were increased by more than 70-fold. Similarly, pre-incubation of bone marrow cells under clonal conditions with IL-3, but not IL-5, resulted in a more than 50 fold increase in CFU-eo responsive to IL-5 over input values. Bone marrow from mice pre-treated with 5-fluorouracil is greatly depleted of progenitor cells directly responsive to IL-3 or IL-5. IL-1 which synergistically interacts with various CSF species to confer a clonogenic response by primitive stem cells present in 5-fluorouracil-treated marrow also failed to stimulate eosinophil production. A marked synergism was observed when IL-1 and IL-3 were combined in the suspension pre-culture phase with a more than sixfold recovery of CFU-eo than induced by either factor alone. Furthermore, pre-culture of 5-fluorouracil-treated marrow cells with a combination of IL-1 and IL-3 resulted in a more than 260-fold increase of CFU-eo over input numbers. These data suggest that the concatenate action of IL-1, IL-3, and IL-5 is an absolute requirement for the in vitro generation of eosinophils from primitive hemopoietic stem cells.     

Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo antigen-presenting capacity.

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

Endogenous granulocyte-macrophage colony-stimulating factor overexpression in vivo results in the long-term recruitment of a distinct dendritic cell population with enhanced immunostimulatory function

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 x 10(8) cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8alpha+CD4- or CD8alpha-CD4- but not CD8alpha+CD4+ or CD8alpha-CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-alpha compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-gamma. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-alpha. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells.     

Butyrate inhibits functional differentiation of human monocyte-derived dendritic cells

Dendritic cells (DCs) play a key role in immune function through antigen presentation by MHC and CD1, as well as cytokine production that shapes the immune response. Here we report that butyrate, a histone deacetylase inhibitor, inhibits the functional differentiation of human monocyte-derived DCs. Mature DCs were generated from monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2 day LPS stimulation. Butyrate treatment throughout the culture period inhibited the expression of CD1 molecules, but not on CD83, CD86, and MHC molecules. The suppression was exerted at protein and mRNA levels. Butyrate-treated immature DCs also showed decreased expression of CD1 molecules. Moreover the butyrate-treated immature DCs showed lower production of IL-12 p40 and IL-6 in response to lipopolysaccharides and induced less Th1 cells in allogenic mixed lymphocyte reactions. Our results imply that histone acetylation is involved in regulating immune responses through regulating functional differentiation of DC. Thus HDAC may be one of the targets for controlling the immune response.     

The disabled dendritic cell.

Dendritic cells are important antigen-presenting cells of the immune system that induce and modulate immune responses. They interact with T and B lymphocytes as well as with natural killer cells to promote activation and differentiation of these cells. Dendritic cells generated in vitro from monocytes by use of the cytokines GM-CSF and IL-4 are increasingly used clinically to enhance antitumor immunity in cancer patients. However, recent studies revealed that the functional repertoire of monocyte-derived dendritic cells may be incomplete. Important functions of monocyte-derived dendritic cells such as migration or the ability to induce natural killer cell activation or type 2 T helper cell differentiation appear to be impaired. We propose that all these deficiencies relate to a single biochemical deficiency of monocyte-derived dendritic cells. IL-4, which is used to generate monocyte-derived dendritic cells, suppresses phospholipase A2, the enzyme that liberates arachidonic acid from membrane phospholipids and contributes to the synthesis of platelet-activating factor. Monocyte-derived dendritic cells must therefore fail to generate platelet-activating factor as well as arachidonic acid derivatives such as prostaglandins, leukotrienes, and lipoxins, collectively referred to as eicosanoids. Since eicosanoids and platelet-activating factor are known to play an important role in processes such as leukocyte migration, natural killer cell activation, and type 2 T helper cell differentiation, the deficiency in eicosanoid and platelet-activating factor biosynthesis may be responsible for the observed handicaps of monocyte-derived dendritic cells.     

Cytokines in the generation and maturation of dendritic cells: recent advances

Dendritic cells (DCs) are extremely efficient antigen presenting cells (APCs) that are potent stimulators of both T and B cell-mediated immune responses. Although DCs are normally present in very small numbers in the peripheral blood (PB), recent advances have made it possible to generate relatively large numbers of cells in culture. DCs can be differentiated in vitro from various cellular sources, including bone marrow (BM), cord blood (CB) and PB mononuclear cells (PBMCs). Although a wide variety of conditions have been reported to be able to support DC generation, the majority of research and clinical protocols to date differentiate DCs from precursors using granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with either tumor necrosis factor-(TNF-)alpha or interleukin (IL)-4. However, a diverse array of cytokines has been shown to be able to induce DC differentiation under a variety of conditions. According to recent reports, cytokines such as IL-2, IL-6, IL-7, IL-13, IL-15 and hepatocyte growth factor (HGF), in combination or even, in some cases, alone, can contribute to the generation of DCs from either monocytes or CD34+ cells. Although the majority of cytokine combinations include GM-CSF, some do not. For example, Flt3 ligand (FL), in conjuction with IL-6 (in the absence of GM-CSF), has been reported to be able to induce DC differentiation from BM cells in a murine system. Other agents can play a dual role in DC activity. CD40 ligand (CD40L), as a single agent, has been shown to be able to generate DCs from PB monocytes, while numerous other reports have also demonstrated its role as a potent maturation factor. In contrast, for other cytokines such as IL-16 or IL-17, although there is no data for a role in DC generation, they have been reported to be involved in promoting DC maturation in vitro as defined by upregulation of costimulatory molecules, major histocompatibility complex (MHC) antigens and antigen presenting/T lymphocyte stimulatory capacity. Furthermore, cytokines such as stem cell factor (SCF) and FL have been shown to dramatically enhance in vivo DC recovery. The wide variety of cytokines and conditions that have been shown to be able to influence DC differentiation and activity to amply demonstrate the extreme heterogeneity found in the DC population, something that is reflected in the diverse phenotypes, functions and ontogeny displayed by DCs. This diversity may account for the large number of roles that have been attributed to DCs in the development and function of the immune system and, in turn, emphasizes the potential as well as the challenges of modifying specific aspects of the immune response system by manipulating specific DC subpopulations.     

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes

 We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon γ (IFNγ) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFNγ and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFNγ secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to up-regulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor. Received: 30 January 1996 / Accepted: 22 March 1996     

The presence of GM-CSF and IL-4 interferes with effect of TGF-beta1 on antigen presenting cells in patients with multiple sclerosis and in rats with experimental autoimmune encephalomyelitis

The possibility to generate and expand tolerogenic dendritic cells (DC) with TGF-β1 in vitro opens new therapeutic perspectives for the treatment of autoimmune diseases. In the present study, GM-CSF+IL-4 induced the differentiation of DC from adherent peripheral blood mononuclear cells, which had a higher expression of HLA-DR, CD86 and CD1a and the capacity to stimulate T cells. TGF-β1 alone slightly promoted the generation of antigen presenting cells (APC) with higher expression of CD14, but did not differentiate them into E-cadherin + Langerhans cell (LC)-like DC. TGF-β1-driven APC exhibited the morphology, phenotypes and functions of tolerogenic immature DC, and had lower capacity to stimulate T cells. In vivo experiment demonstrates that TGF-β1-treated APC exhibited the therapeutic potential in Lewis rats with experimental autoimmune encephalomyelitis (EAE), followed by increase of IL-10 production in lymph nodes and decrease of inflammatory cells in spinal cords. Most importantly, GM-CSF/IL-4 used in DC preparation abolished the effect of TGF-β1 to induce tolerogenic APC in vitro and in vivo. The results reveal that the usage of GM-CSF for the generation of tolerogenic DC should not be copied from DC preparation for anti-tumor therapy.     

Myeloid dendritic cells from B6.NZM Sle1/Sle2/Sle3 lupus-prone mice express an IFN signature that precedes disease onset

Patients with systemic lupus erythematosus show an overexpression of type I IFN-responsive genes that is referred to as "IFN signature." We found that B6.NZMSle1/Sle2/Sle3 (Sle1,2,3) lupus-prone mice also express an IFN signature compared with non-autoimmune C57BL/6 mice. In vitro, myeloid dendritic cells (mDCs) (GM-CSF bone marrow-derived dendritic cells; BMDCs) from Sle1,2,3 mice constitutively overexpressed IFN-responsive genes such as IFN-β, Oas-3, Mx-1, ISG-15, and CXCL10 and members of the IFN signaling pathway STAT1, STAT2, and IRF7. The IFN signature was similar in Sle1,2,3 BMDCs from young, pre-autoimmune mice and from mice with high titers of autoantibodies, suggesting that the IFN signature in mDCs precedes disease onset and is independent from the autoantibodies. Sle1,2,3 BMDCs hyperresponded to stimulation with IFN-α and the TLR7 and TLR9 agonists R848 and CpGs. We propose that this hyperresponse is induced by the IFN signature and only partially contributes to the signature, as oligonucleotides inhibitory for TLR7 and TLR9 only partially suppressed the constitutive IFN signature, and pre-exposure to IFN-α induced the same hyperresponse in wild-type BMDCs as in Sle1,2,3 BMDCs. In vivo, mDCs and to a lesser extent T and B cells from young prediseased Sle1,2,3 mice also expressed the IFN signature, although they lacked the strength that BMDCs showed in vitro. Sle1,2,3 plasmacytoid DCs expressed the IFN signature in vitro but not in vivo, suggesting that mDCs may be more relevant before disease onset. We propose that Sle1,2,3 mice are useful tools to study the role of the IFN signature in lupus pathogenesis.     

The BCL1 B lymphoma responds to IL-4, IL-5, and GM-CSF

Proliferation in vitro of the in vivo passaged murine B cell tumor line BCL1 has been used as a standard assay for mouse interleukin-5 (IL-5) for a number of years. We demonstrate that this line will also respond to human IL-5. The response to murine IL-5 is abrogated by transforming growth factor-beta and to a lesser extent by interferon-gamma. This suggests a possible regulatory role for these lymphokines in the proliferation of B cells induced by IL-5. Other purified recombinant lymphokines were also tested for their ability to induce BCL1 proliferation. The lymphokines IL-1, IL-2, IL-3, and IL-6 had no effect on the growth of BCL1. In contrast, IL-4 and more surprisingly granulocyte-macrophage colony-stimulating factor (GM-CSF) also induced proliferation of this cell. These effects could be inhibited by specific antibodies directed against the respective lymphokines. These data suggest that GM-CSF, as well as IL-4 and IL-5, may be yet another regulator of neoplastic and possibly even normal B-cell growth and differentiation.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Induction of dendritic cell maturation by IL-18

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.     

GM-CSF signalling boosts dramatically IL-1 production

GM-CSF is mostly known for its capacity to promote bone marrow progenitor differentiation, to mobilize and mature myeloid cells as well as to enhance host immune responses. However the molecular actions of GM-CSF are still poorly characterized. Here we describe a new surprising facet of this "old" growth factor as a key regulator involved in IL-1β secretion. We found that IL-1β release, a pivotal component of the triggered innate system, is heavily dependent on the signaling induced by GM-CSF in such an extent that in its absence IL-1β is only weakly secreted. GM-CSF synergizes with LPS for IL-1β secretion mainly at the level of pro-IL-1β production via strengthening the NF-κB signaling. In addition, we show that expression of Rab39a, a GTPase required for caspase-1 dependent IL-1β secretion is greatly augmented by LPS and GM-CSF co-stimulation suggesting a potential GM-CSF contribution in enhancing IL-1β exocytosis. The role of GM-CSF in regulating IL-1β secretion is extended also in vivo, since GM-CSF R-/- mice are more resistant to LPS-mediated septic shock. These results identify GM-CSF as a key regulator of IL-1β production and indicate GM-CSF as a previously underestimated target for therapeutic intervention.