Dendritic Cells Abstracts

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-1-.

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-γ.

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-γ 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, Th1-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.

Human Monocytes differentiate into Dendritic Cells (DCs) or Macrophages according to the nature of environmental signals.

Monocytes stimulated with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) plus InterLeukin-4 (IL-4) yield DCs.

We tested here whether Interferon-gamma (IFN-γ), a potent activator of Macrophages, may modulate Monocyte differentiation.

Addition of IFN-γ to IL-4 plus GM-CSF-stimulated Monocytes switches their differentiation from DCs to CD14^- CD64⁺ Macrophages.

IFN-γ increases Macrophage Colony-Stimulating Factor (M-CSF) and IL-6 production by IL-4 plus GM-CSF-stimulated Monocytes.

By acting at the transcriptional level and acts together with IL-4 to up-regulate M-CSF but not IL-6 production. IFN-γ also increases M-CSF Receptor internalization.

Results from neutralizing experiments show that both M-CSF and IL-6 are involved in the ability of IFN-γ to skew Monocyte differentiation from DCs to Macrophages. Finally, this effect of IFN-γ is limited to early stages of differentiation.

When added to immature DCs, IFN-γ up-regulates IL-6 but not M-CSF production and does not convert them to Macrophages, even in the presence of exogenous M-CSF.

In conclusion, IFN-γ shifts Monocyte differentiation to Macrophages rather than DCs through Autocrine M-CSF and IL-6 production.

These data show that IFN-γ controls the differentiation of Antigen-Presenting Cells and thereby reveals a new mechanism by which IFN-γ orchestrates the outcome of specific Immune Responses.

That Monocytes can differentiate into Macrophages or Dendritic Cells (DCs) makes them an essential link between Innate and Adaptive Immunity.

However, little is known about how interactions with pathogens or T-Cells influence Monocyte engagement toward DCs.

We approached this point in cultures where Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and InterLeukin-4 (IL-4) induced Monocytes to differentiate into immature DCs.

Activating Monocytes with soluble CD40⁺ Ligand (CD40⁺L) led to accelerated differentiation toward mature CD83⁺ DCs with up-regulated human Leukocyte Antigen-DR, CoStimulatory molecules and CD116⁺ (GM-CSF receptor), and down-regulation of molecules involved in Antigen capture.

Monocytes primed by Phagocytosis of AntiBody-opsonized, killed Escherichia Coli differentiated into DCs with an immature phenotype, whereas Zymosan priming yielded active DCs with an intermediate phenotype.

Accordingly, DCs obtained from cultures with CD40⁺L or after Zymosan priming had a decreased capacity to Endocytose Dextran, but only DCs cultured with CD40⁺L had increased capacity to stimulate Allogeneic T-Cells.

DCs obtained after E. Coli or Zymosan priming of Monocytes produced high levels of proinflammatory Tumor Necrosis Factor- and IL-6 as well as of regulatory IL-10, but they produced IL-12p70 only after secondary CD40⁺ ligation.

Thus, CD40⁺ ligation on Monocytes accelerates the maturation of DCs in the presence of GM-CSF/IL-4.

Whereas Phagocytosis of different microorganisms does not alter and even facilitates their potential to differentiate into immature or active DCs, the maturation of which can be completed upon CD40⁺ ligation.

In vivo, such differences may correspond to DCs with different trafficking and T-Helper Cell-stimulating capacities that could differently affect induction of Adaptive Immune Responses to infections.

It was observed that Interferon-beta (IFN-ß) prevents the down-regulation of the InterLeukin-3 Receptor alpha chain (IL-3R-), which spontaneously occurs during culture of human Monocytes.

The functionality of IL-3R was demonstrated by the fact that IL-3 rescued IFN-ß-treated Monocytes from Apoptosis.

Monocytes cultured in the presence of IFN-ß and IL-3 acquire a Dendritic morphology and express high levels of HLA Antigen Class I and Class II and CoStimulatory molecules.

When stimulated by either LipoPolySaccharide or Fibroblasts expressing CD40⁺ Ligand (CD40⁺L) transfectants, Dendritic Cells (DCs) generated in IFN-ß and IL-3 secreted high levels of IL-6, IL-8.

And Tumor Necrosis Factor- but low levels of IL-12 in comparison with DCs generated in IL-4 and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).

In mixed Leukocyte culture, IL-3-IFN-ß DCs induced a vigorous proliferative response of Allogeneic Cord blood T-Cells and elicited the production of high levels of IFN-γ and IL-5 by naive adult CD4⁺ T-Cells.

Finally, IL-3-IFN-ß DCs were found to produce much higher levels of IFN- than IL-4-GM-CSF DCs in response to Poly (I:C) but not to Influenza Virus.

It was concluded that Monocytes cultured in the presence of IL-3 and IFN-ß differentiate into DCs with potent helper T-Cell stimulatory capacity despite their low secretion of IL-12.

Langerhans Cells (LCs) represent a subset of immature Dendritic Cells (DCs) specifically localized in the Epidermis and other Mucosal Epithelia.

As surrounding Keratinocytes can produce InterLeukin-15 (IL-15), a Cytokine that utilizes IL-2Rγ chain, we analyzed whether IL-15 could skew Monocyte differentiation into LCs.

Monocytes cultured for 6 d with Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) and IL-15 differentiate into CD1a⁺ HLA-DR⁺ CD14^-DCs (IL-15-DCs).

Agents such as LipoPolySaccharide (LPS), Tumor Necrosis Factor-alpha (TNF-), and CD40L induce maturation of IL-15-DCs to CD83⁺, DC-LAMP⁺ Cells.

IL-15-DCs are potent Antigen-Presenting Cells able to induce the primary (Mixed Lymphocyte Reaction [MLR]) and secondary (recall responses to flu-matrix peptide) Immune Responses.

As opposed to cultures made with GM-CSF/IL-4 (IL-4-DCs), a proportion of IL-15-DCs expresses LC markers: E-Cadherin, Langerin, and CC Chemokine Receptor-6 (CCR-6).

Accordingly, IL-15-DCs, but not IL4-DCs, migrate in response to Macrophage Inflammatory Protein (MIP)-3/CCL20.

However, IL-15-DCs cannot be qualified as "genuine" Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules.

Thus, our results demonstrate a novel pathway in Monocyte differentiation into Dendritic Cells.

The local Cytokine environment and the presence of stimulatory signals determine whether circulating Monocytes will finally acquire characteristics of Dendritic Cells (DCs) or Macrophages.

Because FcepsilonRI expressed on professional APCs, e.g., Monocytes and DCs, has been suggested to play a key role in the pathophysiology of Atopic Diseases, we evaluated the effect of receptor ligation on the generation of Monocyte-derived DCs (MoDCs).

Aggregation of FcepsilonRI at the initiation of the IL-4-GM-CSF-driven differentiation resulted in the emergence of Macrophage-like cells with a strong expression of the Mannose Receptor and a low level of CD1a and the DC-specific markers CD83 and the Actin-bundling protein (p55).

These cells sustained the ability to take up FITC-labeled Escherichia coli by Phagocytosis and were significantly less efficient in stimulating purified Allogeneic T-Cells.

In addition, receptor ligation of FcepsilonRI at the beginning of the culture prevented the generation of MoDCs, mainly due to a dramatic increase in the IL-10 production.

These results suggest that FcepsilonRI aggregation prevents the generation of CD1a⁺ MoDCs and imply a novel pivotal function of this receptor in modulating the differentiation of Monocytes.

Dendritic Cells (DCs) play a critical obligate role in presenting Antigens to T-Cells for activation. In the process, upon Antigen capture, DCs undergo maturation and become more stimulatory.

Human Myeloid DCs can be generated from various sources, including blood, bone marrow, and CD34⁺ Stem Cells.

As such, plastic-adherent Monocytes from circulation have served as a ready source for generating Myeloid DCs in culture in Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and InterLeukin-4 (IL-4).

For translational research in active specific ImmunoTherapy, especially in cancer, with the belief that they are essentially stimulatory or "Immunogenic."

Here we show that in vitro cultures of plastic-adherent circulating Monocytes in GM-CSF and IL-4 followed by further maturation in Interferon-gamma plus bacterial superantigens.

(DC maturing agents) can give rise to two diametrically opposite types of DCs-one stimulatory and another inhibitory.

The stimulatory DCs express higher amounts of CoStimulatory molecules, synthesize IL-12, and efficiently stimulate naive Allogeneic T-Cells in Mixed Lymphocyte Reaction (MLR).

The inhibitory DCs, in contrast, express lower concentrations of the critical CoStimulatory molecules, synthesize large amounts of IL-10, and are nonstimulatory in Allogeneic primary MLR.

Moreover, while the stimulatory DCs further amplify proliferation of T-Cells in Lectin-driven proliferation assays, the inhibitory DCs totally block T-Cell proliferation in similar assays, in vitro.

Most interestingly, neutralization of the endogenously derived IL-10 with anti-IL-10 AntiBody in DC cultures repolarizes the inhibitory DCs toward stimulatory phenotype. Accordingly, these observations have important implications in translational research involving Myeloid DCs.

Copyright 2000 Academic Press.

Dendritic Cells (DCs) may arise from multiple lineages and progress through a series of intermediate stages until fully mature, at which time they are capable of optimal Antigen presentation and T-Cell activation.

High cell surface expression of CD83⁺ is presumed to correlate with full maturation of DCs, and a number of agents have been shown to increase CD83⁺ expression on DCs.

We hypothesized that InterLeukin-12 (IL-12) expression would be a more accurate marker of functionally mature DCs capable of activating Antigen-specific T-Cells.

We used combinations of signaling through CD40⁺, using CD40⁺ Ligand trimer (CD40⁺L), and Interferon-gamma to demonstrate that CD83⁺ expression is necessary but not sufficient for optimal production of IL-12 by DCs.

Phenotypically mature DCs could be induced to produce high levels of IL-12 p70 only when provided 2 simultaneous stimulatory signals.

By IntraCellular Cytokine detection, we determined that only a subset of cells that express high levels of CD80⁺ and CD83⁺ generate large amounts of IL-12.

DCs matured with both signals are superior to DCs stimulated with the individual agents in activating antigen-specific T-Cell in vitro.

These findings have important implications regarding the identification, characterization, and clinical application of functionally mature DCs.

Peripheral blood Monocytes are common precursor cells of Dendritic Cells (DCs) and Macrophages. We have searched for factors with the potential to regulate the differentiation of Monocytes to DCs and Macrophages.

When CD14⁺ Monocytes are cultured with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and InterLeukin-4 (IL-4), the CD14⁺ CD1a^- population, which consists of Macrophages, was found in the serum-containing cultures but not in the Serum-free cultures.

Addition of IL-6 receptor-neutralizing MonoClonal AntiBody (mAb) or gp130-neutralizing mAb to the Serum-containing cultures resulted in a decreased population of CD14⁺ CD1a^- Cells.

An increase in the CD14⁺ CD1a^- population with reduction in CD14^- CD1a⁺ DCs was observed with the addition of IL-6 to cultures.

Whereas IL-11, Leukaemia Inhibitory Factor, Oncostatin-M or Macrophage Colony-Stimulating Factor did not affect the differentiation of Monocytes in the presence of GM-CSF plus IL-4.

This effect of IL-6 was blocked by Tumour Necrosis Factor-alpha (TNF-), LipoPolySaccharide (LPS), IL-1beta, CD40⁺ ligand (CD40⁺L) and Transforming Growth Factor beta-1 (TGF-ß-1). Among these factors, TNF- was most potent in interfering with the action of IL-6.

These results suggest that IL-6 inhibits the differentiation of Monocytes to DCs by promoting their differentiation toward Macrophages, which is modulated by factors such as TNF-, LPS, IL-1-ß, CD40⁺L and TGF-ß1.

Objective
Because of its potent ImmunoSuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-DihydroxyVitamin (D3) (Calcitriol) on differentiation, maturation, and function of Dendritic Cells (DC).

Materials And Methods
Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNF-. DCs were derived from CD34⁺ progenitors using SCF plus GM-CSF plus TNF-

For differentiation studies, cells were exposed to Calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function.

Results
When added at day 0, Calcitriol blocked DC differentiation from Monocytes and inhibited the generation of CD1a⁺ Cells from progenitor cells while increasing CD14⁺ Cells.

Exposure of immature DCs to Calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the CoStimulatory molecules CD40 and CD80, and MHC Class II expression, whereas the Monocyte/Macrophage marker CD14 was clearly reinduced.

In addition, Calcitriol hindered TNF--induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of CoStimulatory molecules.

In contrast, the mature CD83⁺ DCs remained CD1a⁺ CD14^- when exposed to Calcitriol. The capacity of Cytokine-treated cells to stimulate Allogeneic and Autologous T-Cells and to take up soluble Antigen was inhibited by Calcitriol.

Conclusion
The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by Calcitriol, may explain some of its ImmunoSuppressive properties.

Monocytes can give rise to either Antigen Presenting Dendritic Cells (DCs) or scavenging Macrophages. This differentiation is initiated when Monocytes cross the Endothelium. But the regulation of DC and Macrophage differentiation in tissues remains elusive.

When stimulated with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and InterLeukin-4 (IL-4), Monocytes yield DCs. However, we show here that the addition of Fibroblasts switches differentiation to Macrophages.

On contact with Monocytes, Fibroblasts release IL-6, which up-regulates the expression of functional M-CSF receptors on Monocytes. This allows the Monocytes to consume their Autocrine M-CSF.

Thus, the interplay between IL-6 and M-CSF switches Monocyte differentiation to Macrophages rather than DCs, and IL-6 is an essential factor in the molecular control of Antigen Presenting Cell development.