The Roles Of Calcium Ionophore In Generating Of Dendritic Cells And It's Signal Mechanisms

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that prime naive T lymphocytes and activate antigen specific cytotoxic T lymphocytes. The superior ability of DCs to present antigens to T cells has led to the development of DC-based strategies for the purpose of enhancing the immune response against tumors and severe viral infections. It is well known that normal monocytes (Mos), as well as immature CD34~+ haemopoietic progenitor cells (HPCs) and certain myelogenous leukaemia cell lines, can be differentiated in vitro with certain factors, and induced to acquire functional and phenotypic DC characteristics. Most frequently used in these studies were the cytokine combinations of granulocyte-macrophage colony- stimulating factor(GM-CSF), tumor necrosis factor- α (TNF- α ), interleukin (IL)-4 or/and stem cell factor(SCF). In the studies presented here, we first showed the immunophenotype and functions of DC induced by conventional cytokine combinations, and then investigated the roles of calcium ionophore (CI) in generating of dendritic cells from Mos or HPCs or acute myloid leukemia (AML) cells and it's signal mechanisms.Mature DCs are typically characterized by expression of MHC class I, MHC class II, the co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) and the DC lineage marker CD83. We found that treatment with GM-CSF and IL-4 caused Mos to develop the morphologic properties of DCs within 4 days, but co-stimulatory molecule up-regulation and CD 14 down-regulation were heterogeneous, and CD83expression was infrequent after 10 days of culture continuously by GM-CSF, IL-4 and TNF- α . When the two-step method, consisting of 1 week of the first step culture for the expansion and proliferation of CD34~+ HPCs in the presence of GM-CSF, TNF- α and SCF and another 1 week of the second step culture for the induction of DCs in the presence of GM-CSF, IL-4 and TNF- α , we found that the total nucleated cells increased 113±29-fold (n=5) compared with the number of CD34~+ cells at the time of starting culture. Many of the nucleated cells acquired DC characteristic appearance and phenotypes, which were marked expressions of HLA-DR, CD40, CD86 and adhesion molecules such as CD54, but the expressions of B7-1/CD80 and the activation marker CD83 were not sufficient. In addition to low expressions of costimulatory molecules, adhesion molecules and the activation molecule CD83 of DCs, there are some problems induced by cytokine combinations, such as long time culture (at least 2 weeks), high payment and low output, in the conventional culture system involving in cytokine combinations. On the other hand, these culture systems contain animal or human serum that is not safe and consistent in experimental researches and clinical applications. For all these reasons, it is essentially to find the new methods and serum-free culture systems to drive DC differentiation more rapidly and more efficiently.Recent laboratory observations indicated that pharmacologic agents that mobilize intracellular calcium can be used to enhance APC functions in human Mo, apparently bypassing initial receptor/ligand requirements involving cytokines or ligands previously shown to influence such development. So we tried to use calcium ionophore (CI) as the main agent to generate DCs more effectively from human Mo, CD34+ HPC and AML cells, and to investigate it's signal transduction mechanisms. We found that each of these myeloid subpopulations responded to CI treatment by developing the morphologic characteristics and functions of highly activated, mature DCs. When Mos were treated with CI for 20 hours, full morphologic differentiation occurred, and many immunophenotypic alterations occurred promptly within 40 hours of culture, including decreased expression of the monocyte/macrophage-associated molecule CD 14 (LPS receptor), increased expressions of major histocompatibility complex (MHC) molecules, upregulation of B7.2/CD86, CD40, and CD54 expression, and de novo expression of both B7.1/CD80and the DC-associated activation marker CD83. In addition, enhanced T-cell sensitizing efficiency was evident within the first 40 hours of treatment. When CD34+ HPC were treated with two-step cultured cells with CI, the expression of co-stimulatory molecules such as CD86 and CD80 was up-regulated. The expression of mature DC marker CD83 was remarkably induced by treatment with CI for a short duration (20 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with CI also showed a stronger allo-T cell stimulatory capacity compared with the cells without CI treatment. Therefore, Mos and CD34+ HPCs all can be induced rapidly by CI to manifest appearance and functions of mature DCs.It is well known that cytokine (CKs) induce DC differentiation through the pathways of receptor/ligand and activate nuclear transcription factor-xB (NF-kB) . In order to analyze the intracellular signaling pathways by which CI promotes the acquisition of DC-associated properties from human Mo, we employed 3 antagonists (W-7, CsA, KT5926) that block Ca2-mediated signaling pathways. W-7 is the antagonist of calmodulin (CaM). Cl-induced rapid acquisition of morphological, immunophenotypicl and functional DC-characteristics from Mo was broadly attenuated by CaM antagonists W-7. However, antagonists of signaling pathways downstream to CaM displayed more selective inhibitory effects. Calcineurin (CaN) antagonist cyclosporin A (CsA) diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in Cl-treated Mos, and strongly attenuated the capacity to stimulate the proliferation of allogenic T cells of Cl-treated Mos, but displayed minimal effect on Cl-induced CD14 down-regulation on Mos. In contrast, the calmodulin-dependent protein kinase (CaMK) antagonist KT5926, while displaying only modest effects on Cl-induced costimulatory molecule and CD83 expressions, strongly blocked CD 14 down-regulation. These antagonists had little or no effect on CK-mediated signaling pathways. These results were consistent with Ca2+-dependent mechanisms for CI- induced differentiation of Mos, and indicated that multiple discrete signaling pathways downstream to calcium mobilization and CaM activation may be essential in regulating this process. It also indicated that calcium-mobilizing agents have the advantage of bypassing initial surface receptor/ligand signal transduction mediated by CKs. CI, which raisesintracellular Ca2+ levels by permeabilization of cell membranes to Ca2+ by using a pivotal calmodulin/calcineurin/calmodulin-dependent kinaser axis for transmission of its activation signal through the cytoplasm, can induce DC differentiation from Mos more rapidly than cytokine combinations. However, cytokine signal transduction displays synergism to calcium-dependent signaling pathways during DC differentiation.