Isolation of genes selectively expressed in mouse bone marrow derived dendritic cells

Professional antigen presenting cells such as dendritic cells (DCs) and macrophages share similar characteristics; however, they differ in ability to initiate an immune response. DCs are much more potent in stimulating naïve T cells. To further define the molecular determinants of DC differentiation and function, cDNA subtraction was performed using sorted MHC class II hi, B7hi bone marrow derived DCs as tester and interferon-γ/LPS treated bone marrow derived macrophages as driver. Analysis of 117 resulting clones revealed a diverse pattern of DC selective gene expression including known genes whose expression in DCs had not been previously demonstrated as well as multiple novel genes. These genes included membrane receptors (B7-1, the CCR7 chemokine receptor, synaptogyrin-2, a novel c-type lectin, and a novel B7 family member), chemokines (such as ABCD-1/TARC), nuclear proteins, and signal transduction molecules. In addition to providing new DC markers, identification of these genes will help to elucidate the molecular basis for important DC function.; A novel member of the c-type lectin supergene family was identified in a screen for genes selectively expressed in mouse bone marrow derived DC. As this gene was shown to be most highly expressed in DC it was named dendritic cell lectin-1 (dcl-1). Quantitation by real time PCR demonstrated that dcl-1 mRNA is increased 7 fold in DCs relative to activated macrophages. The dcl-1 genomic locus (CLECSF4) was shown to be composed of six exons residing on distal mouse chromosome 6 within the NK gene complex. Transient expression of tagged dcl-1 indicates that DCL-1 is likely a cell surface receptor with type II membrane topology. Additionally, DCL-1 was shown to be a 35 kilodalton (kd) glycoprotein that can form a 70kd disulfide linked dimer.; Expression of Mitogen Inducible Nuclear Orphan Receptor (MINOR) was identified in mature mouse bone marrow derived DCs. Quantitation by real time PCR demonstrated that MINOR mRNA is increased 100 fold in mature DC relative to activated macrophage. As MINOR is closely related to the Nur77 steroid hormone receptor family of transcription factors, which regulate apoptosis in T cells, an analogous role for MINOR in DC apoptosis was investigated. Expression of MINOR could induce apoptosis in a bone marrow derived dendritic cell line, DC2.4, but not in 293T or NIH3T3 cells.; A novel member of the FYVE zinc finger family was identified in a screen for genes selectively expressed in mouse bone marrow derived DCs. In addition, this gene was found to share homology with dual specific phosphatases of the myotubularin gene family. As the 5.7kb cDNA was isolated from mouse bone marrow derived DCs this gene has been termed dc fyve dsp. Assays for transient expression of DC FYVE DSP indicate early endosomal localization. Treatment of transfectants with the PI3-kinase inhibitor wortmannin mislocalized DC FYVE DSP to cytosol in a majority of cells. Additionally, a truncation removing the FYVE domain consensus sequence resulted in the appearance of large perinuclear vesicles. Specific detection of endogenous DC FYVE DSP was achieved by confocal microscopy and confirms localization on dendritic cell endosomes. Double staining experiments for colocalization indicate that DC FYVE DSP resides on intracellular compartments that are RAB5 positive but Mannose-6-Phosphate Receptor and MHC class II negative. Our data strongly suggest that DC FYVE DSP is a regulator of vesicular trafficking in dendritic cells.