Modulation of dendritic cell activation chemokines and cellular injury.

Dendritic cells (DC) are professional antigen presenting cells expressing MHC class II, derived from a common marrow precursor. They are motile, diffused and have a spidery shape with many long cytoplasmic processes. The aim of this project was to test the hypothesis that cellular injury induces the activation and functional maturation of DC. To test the effects of injury on DC activation, immature DCs were used as substrate for DC activation assays. They were obtained from their precursor in peripheral blood mononuclear cells (PBMNCs) by culturing them GM-CSF and IL-4. Expression of surface B7 was measured by immunofluorescence and flowcytometry. beta- chemokines were used as potential injury mediators, including: RANTES, MIP-1alpha, MIP-1beta, MCP-1, -2, -3 and -4, as well as other inflammatory cytokines such as TNF-alpha and IL-1. They were screened on immature DCs to examine whether or not they modulate B7-1 and B7-2. A model of cellular injury was established to investigate whether the injured parenchymal cells deliver signals to initiate DC activation or upregulation of B7-1/B7-2 by release of soluble mediators. H2O2 was used as an injury mediator to injure renal tubular epithelial cells (RTECs). RANTES, MIP-1alpha and MIP-1beta upregulated B7-1. MCP-1, -2, -3 and -4 downregulated the expression of HLA-DR greatly. Furthermore, MCP-1, -2, -3 and -4 upregulated B7.2, while and -4 and MCP2 upregulated B7.1. We observed that immature DCs could not be readily stimulated with chemokines and pro-inflammatory cytokines IL-1 and TNF-alpha unless GM-CSF and IL4 were used continuously. The supernatant of injured renal epithelial cells had an effect on DC activation. These findings may explain the role of DCs as a link between the innate and the adaptive immune response, as well as being an active participant in determining the outcome of an antigen encounter.

Apoptosis role of FAS/FAS ligand system in the regulation of myelopoiesis.

OBJECTIVES: CD34+ cells and colony forming unit-granulocyte and macrophage (CFU-GM) from human bone marrow were used to investigate the role of Fas/FasL system in the regulation of myelopoiesis. METHODS: Fas and FasL expression in CD34+ cells and in day 14 CFU-GM were measured by RT-PCR and immunofluorescence respectively. The functional assays for the CFU-GM were measured by a standard colony assay and the proliferative capacity of CFU-GM was measured by replating the primary colony and observing the secondary colony formation. Human marrow cells were treated with IETD (caspase-8 inhibitor) or anti-Fas CH-11 Mab. RESULTS: Treatment with the CFU-GM with IETD significantly increased, the proliferative capacity, while anti-Fas CH-11 Mab markedly reduced it. Fas and FasL expression were demonstrated using RT-PCR and immunofluorescence respectively. CONCLUSION: Fas, FasL, and caspase activation are likely to play an important role in the regulation of myelopoiesis.