| Objectives. Dendritic cells (DCs) are very important in integrating innate immunity with acquired immunity. Although DCs are studied very widely, little is known about the functional and structural differences among various DCs, and also about immature and mature DCs. The broad objective of this study was to identify some unique molecules involved in DC development from bone marrow and determine the onset, tissue-specificity and biological role of such markers in the context of APC. Methods. We used published protocols to generate and characterize DCs from BALB/c mouse bone marrow after cultivation in IMDM plus GM-CSF. Cultivated immature DC lysates were used to raise polyclonal antibodies in rabbits, and antisera tested for DC-specificity by using immunodiffusion, and Western blot. We generated specific antibody reagents (Reagent A and Reagent B) by repeated adsorption of polyclonal antisera on normal cells. Results. The Reagent A was shown to recognize a novel GPI-linked cell-surface lactoferrin protein, we termed CSP82, on BM cells including hematopoietic stem cells (HSCs). Cross linking of this protein by Reagent A caused BM cells differentiation into prodendritic cells (BM4 cells) which were shown to express a novel inducible cytosolic phosphoprotein termed DP58 recognized by Reagent B. DP58 was found also in neuronal nuclei as a constitutive protein. Once the sequences of both CSP82 and DP58 were determined, we developed specific anti-CSP82 and anti-DP58 peptide antibodies, which were subsequently used in all studies reported here.; Conclusion. We have identified and partially characterized two proteins, a novel, DP58, a cytosolic phosphoprotein, a biomarker of pro-DCs and a constitutive nuclear protein in neuronal nuclei. Unlike antibacterial and usually secreted iron-binding lactoferrin, CSP82 occurs as the glycosylphosphatidylinositol (GPI)-linked mouse lactoferrin precursor glycoprotein on undifferentiated BM cells. Cross-linking of CSP82 with anti-CSP82 peptide antibody induced DP58 in the absence of GM-CSF and initiated DC differentiation. |
