CD40 ligand (CD40L) expressed by activated CD4+ T cells is a family member of membrane bound TNF family ligand and its interaction with CD40 expressed in APC has been shown to contribute in enhancing immune response. Exogenous stimulation through CD40 has been performed using soluble trimeric CD40L, anti-CD40 monoclonal antibody and cells expressing CD40L. Schneider 2 (S2) cells, a cell line derived from Drosophila melanogaster, was transfected with a plasmid vector, pAc5.1/V5-HisA, for the constitutive expression of CD40L (S2-CD40L). Upon incubation of S2-CD40L with B-lymphocytes for 6 days, activated B cells were examined by counting B cell numbers and for activation markers including CD86 and HLA Class II molecules. The activated B cells were tested for its efficient APC function by mixed lymphocyte reactions (MLR) and enzyme-linked Immunospot (ELISPOT) assay. S2-CD40L was cultured for a year and maintained CD40L expression (>90%). S2-CD40L induced B cell activation as demonstrated by increment of total B cells and up-regulation of CD86 and MHC Class II molecules. Activated B cells pulsed with peptide from human cytomegalovirus pp65 antigen efficiently induced both proliferation and IFN-gamma secretion of T cells. Our result suggests that S2-CD40L can efficiently and conveniently generate B cells as a functional APC and represents a potential role for B-cell mediated cancer immunotherapy.
Animals ; Antigen Presentation/*immunology ; Antigens, CD86/metabolism ; B-Lymphocytes/cytology/*immunology/metabolism ; CD4-Positive T-Lymphocytes/immunology ; CD40 Ligand/genetics/*metabolism ; Cell Line ; Cell Proliferation ; Coculture Techniques ; Drosophila melanogaster ; Gene Expression ; Histocompatibility Antigens Class II/metabolism ; Humans ; *Lymphocyte Activation ; Research Support, Non-U.S. Gov't ; Transfection

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.
Animals ; Antigen-Presenting Cells/cytology/*drug effects/immunology/*metabolism ; Antigens, CD86/metabolism ; Carrier Proteins/*pharmacology ; Cell Death/drug effects ; Cell Survival/drug effects ; Cells, Cultured ; Cytokines/metabolism ; Flow Cytometry ; Histocompatibility Antigens Class II/metabolism ; Inflammation Mediators ; Interferon Type II/pharmacology ; Lipopolysaccharides/pharmacology ; Macrophages/cytology/*drug effects/immunology/*metabolism ; Membrane Glycoproteins/*pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Nitric Oxide Synthase Type II/metabolism ; Phagocytosis/drug effects ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/immunology/metabolism ; Up-Regulation/drug effects/genetics

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-alpha, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40-mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPSstimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-alpha and IL-1beta, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.
Antigens, CD40/metabolism/pharmacology ; Antigens, CD80/metabolism ; Antigens, CD86/metabolism ; Blotting, Western ; CD40 Ligand/metabolism/pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Coculture Techniques ; Dendritic Cells/*drug effects/metabolism ; Enzyme-Linked Immunosorbent Assay ; Fluorescein-5-isothiocyanate ; Fluorescent Antibody Technique, Indirect ; Fluorescent Dyes ; Humans ; Interleukin-10/analysis/biosynthesis ; Interleukin-12/analysis/biosynthesis ; Killer Cells, Natural/metabolism ; Leukemia, Myeloid/*pathology ; Lipopolysaccharides/*pharmacology ; Mitogen-Activated Protein Kinase 3/metabolism ; RNA, Messenger/metabolism ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 4/metabolism ; Tumor Necrosis Factor-alpha/pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism