Efficient Ex Vivo Generation of Human Dendritic Cells from Mobilized CD34p+ Peripheral Blood Progenitors

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.     

The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells

Notch-mediated cellular interactions are known to regulate cell fate decisions in various developmental systems. A previous report indicated that monocytes express relatively high amounts of Notch-1 and Notch-2 and that the immobilized extracellular domain of the Notch ligand, Delta-1 (Delta(ext-myc)), induces apoptosis in peripheral blood monocytes cultured with macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage CSF (GM-CSF). The present study determined the effect of Notch signaling on monocyte differentiation into macrophages and dendritic cells. Results showed that immobilized Delta(ext-myc) inhibited differentiation of monocytes into mature macrophages (CD1a+/-CD14+/- CD64+) with GM-CSF. However, Delta(ext-myc) permitted differentiation into immature dendritic cells (CD1a+CD14-CD64-) with GM-CSF and interleukin 4 (IL-4), and further differentiation into mature dendritic cells (CD1a+CD83+) with GM-CSF, IL-4, and tumor necrosis factor-alpha (TNF-alpha). Notch signaling affected the differentiation of CD1a-CD14+ macrophage/dendritic cell precursors derived in vitro from CD34+ cells. With GM-CSF and TNF-alpha, exposure to Delta(ext-myc) increased the proportion of precursors that differentiated into CD1a+CD14- dendritic cells (51% in the presence of Delta(ext-myc) versus 10% in control cultures), whereas a decreased proportion differentiated into CD1a-CD14+ macrophages (6% versus 65%). These data indicate a role for Notch signaling in regulating cell fate decisions by bipotent macrophage/dendritic precursors.     

Acceleration of endothelial-like cell differentiation from CD14+ monocytes in vitro

OBJECTIVE: In vitro differentiation of endothelial cells has potential applications in vascular tissue engineering and cell-based therapy for many diseases. The objective of this study was to develop a new strategy that utilizes cytokines and lipopolysaccharide (LPS) to accelerate endothelial-like cell differentiation from peripheral blood CD14(+) monocytes. METHODS: Peripheral blood CD14(+) monocytes were purified with immunobeads and cultured with an angiogenic growth factor-rich growth medium (EGM-2) with or without initial treatment of LPS in combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 4 days (the day 4 cultures). The cells were then continuously cultured in EGM-2 medium for an additional 4 or 10 days (the day 8 or day 14 cultures). Cell markers were determined by flow cytometry analysis and immunofluorescence staining. Cytokine/chemokine profile was studied by Bio-Plex immunoassay. RESULTS: In the group of initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2, the majority of suspended CD14(+) monocytes were attached and changed their morphology to endothelial-like cells, which expressed high levels of endothelial cell markers CD31, von Willebrand factor, and vascular endothelial growth factor receptor-1 as well as two major endothelial tight junction proteins zonula occludens -1 and occludin in the day 8 cultures. Endothelial nitric oxide synthase expression was substantially increased. Endothelial-like cells were also able to uptake acetylated low-density lipoprotein and bind to Ulex europeus lectin. In addition, endothelial-like cells showed a unique cytokine/chemokine profile with substantial increases of macrophage inflammatory protein-1beta, IL-6, granulocyte colony-stimulating factor, and IL-8. CONCLUSION: Initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2 is an effective strategy for acceleration of endothelial-like cell differentiation from peripheral blood CD14(+) monocytes in vitro.     

Activities of granulocyte-macrophage colony-stimulating factor and interleukin-3 on monocytes

We examined the actions of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on human monocytes, using a serum-free culture system. GM-CSF and IL-3 did not promote the differentiation of monocytes into macrophages but rather into cells with a phenotype compatible with that of immature dendritic cells (DCs). The addition of fetal bovine serum to serum-free cultures with GM-CSF or IL-3 restored the differentiation of monocytes into macrophages. Cells generated with GM-CSF or IL-3 elicited phagocytic activity. Cells generated in the presence of GM-CSF or IL-3, followed by the addition of tumor necrosis factor-alpha, displayed a phenotype of mature DCs, and primed and stimulated immunogenic peptide-specific T lymphocytes. Surprisingly, GM-CSF and IL-3 inhibited macrophage colony-stimulating factor (M-CSF)-dependent differentiation of monocytes into macrophages and induced differentiation into immature DCs. We asked if the inhibition of M-CSF-dependent differentiation into macrophages by GM-CSF or IL-3 was associated with the expression of M-CSF receptors (M-CSFR). GM-CSF or IL-3 down-regulated the expression of M-CSFR. These data demonstrate that GM-CSF and IL-3 primarily support the differentiation of monocytes into DCs and inhibit M-CSF-dependent differentiation into macrophages by suppressing the expression of M-CSFR, thereby promoting differentiation into DCs.     

Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes

We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN- gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.     

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.     

Calcium signaling inhibits interleukin-12 production and activates CD83(+) dendritic cells that induce Th2 cell development

Mature dendritic cells (DCs), in addition to providing costimulation, can define the Th1, in contrast to the Th2, nature of a T-cell response through the production of cytokines and chemokines. Because calcium signaling alone causes rapid DC maturation of both normal and transformed myeloid cells, it was evaluated whether calcium-mobilized DCs polarize T cells toward a Th1 or a Th2 phenotype. After human monocytes were cultured for 24 hours in serum-free medium and granulocyte-macrophage colony-stimulating factor to produce immature DCs, additional overnight culture with either calcium ionophore (CI) or interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and soluble CD40L resulted in phenotypically mature DCs that produced interleukin-8 (IL-8) and displayed marked expression of CD80, CD86, CD40, CD54, CD83, DC-LAMP, and RelB. DCs matured by IFN-gamma, TNF-alpha, and soluble CD40L were additionally distinguished by undetectable CD4 expression, marked secretion of IL-12, IL-6, and MIP-1beta, and preferential ability to promote Th1/Tc1 characteristics during T-cell sensitization. In contrast, DCs matured by CI treatment were distinguished by CD4 expression, modest or absent levels of IL-12, IL-6, and MIP-1beta, and preferential ability to promote Th2/Tc2 characteristics. Calcium signaling selectively antagonized IL-12 production by mature DCs activated with IFN-gamma, TNF-alpha, and soluble CD40L. Although the activation of DCs by calcium signals is largely mediated through calcineurin phosphatase, the inhibition of IL-12 production by calcium signaling was independent of this enzyme. Naturally occurring calcium fluxes in immature DCs, therefore, negatively regulate Dc1 differentiation while promoting Dc2 characteristics and Th2/Tc2 polarization. Calcium-mobilized DCs may have clinical usefulness in treating disease states with excessive Th1/Tc1 activity, such as graft-versus-host disease or autoimmunity.     

The differential effect of pentoxifylline on cytokine production by alveolar macrophages and its clinical implications.

The aim of this study was to investigate the effects of pentoxifylline (PTX) on the production of TNF-alpha, IL-1 beta, IL-6 and GM-CSF by lipopolysaccharide (LPS)-stimulated alveolar macrophages (AM). AM and peripheral blood monocytes (PBM) from 10 patients were cultured for 24 h in the presence of LPS (10 micrograms ml-1) and PTX at concentrations of 2.0 mM, 1.0 mM, 0.5 mM, 0.1 mM and 0.01 mM. TNF-alpha and GM-CSF were measured from the culture supernatants of both the AM and PBM from all 10 patients and IL-1 beta and IL-6 from the culture supernatants of the AM from five patients. The TNF-alpha production by AM was significantly suppressed in the presence of PTX at concentrations of 2.0 and 1.0 mM, while production of IL-1 beta, IL-6 and GM-CSF remained unaffected. In PBM cultures, PTX significantly suppressed the production of TNF-alpha and GM-CSF, at all tested concentrations. The present study provides evidence that PTX selectively suppresses the production of TNF-alpha by LPS-stimulated AM and may have a role in the treatment of lung diseases where TNF-alpha is involved. The mode of administration of PTX should take into account the suppressive effect of this drug on GM-CSF production by PBM.