|DEL CACHO, EMILIO|
Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2008
Publication Date: 7/22/2008
Citation: Lillehoj, H.S., Del Cacho, E., Gallego, M., Lopez-Bernard, F., Sanchez-Acedo, C. 2008. Isolation of chicken follicular dendritic cells. Journal of Immunological Methods. 334:59-69.
Interpretive Summary: Fundamental knowledge on poultry immune system and the role played by each component of poultry immune system is ciritcal to develop novel strategies against many poultry diseases of economic importance. As in mammals, avian follicular dendritic cells (FDC) are central players in humoral immunity. In recent years, considerable progress has been made in understanding the basis of the FDC activity by the way of studies performed in vitro using purified populations of mammalian FDC. Despite their important role in driving high-affinity antibody responses, it has not been possible to study chicken dendritic cells due to a lack of isolation protocols and absence of FDC-specific reagents. In this paper, scientists at the University of Zaragoza, Spain and an ARS scientist describe a new method for the isolation of avian FDC which are CD45- and express IgG on their surface. The FDC identity was confirmed by morphological, phenotypical and functional characteristics. In conclusion, the protocol described here demonstrates for the first time a novel method for isolating pure avian FDC that will aid in the functional analysis of dendritic cells in avian species and this method should facilitate future studies on the cellular function of avian FDC in normal and disease states for poultry scientists.
Technical Abstract: The aim of the present study was to isolate chicken follicular dendritic cells (FDC). A combination of methods involving panning, iodixanol density gradient centrifugation, and magnetic cell separation technology made it possible to obtain functional FDC from the cecal tonsils from chickens, which had been infected with Eimeria tenella. CD45- dendritic cells were selected using the specific monoclonal antibody against chicken CD45, which is a marker for chicken leukocytes, whereas it is not expressed on chicken FDC. Isolated FDC were characterized morphologically, phenotypically and functionally. The phenotype of selected cells was consistent with FDC in that they expressed IgG, IgM, complement factors C3 and B, ICAM-1, and VCAM-1, but lacked cell surface markers characteristic of macrophages, T-, and B-cells. Furthermore, transmission electron microscopy confirmed their characteristic dendritic morphology. In addition, FDC identity was further confirmed by their ability to trap chicken immune complexes (ICs) on their surface, whereas they did not trap naive antigen (ovalbumin) or ICs generated with mammalian immunoglobulins. Moreover, co-culturing FDC with B-cells in the presence of allogeneic or autologous cells resulted in enhanced B-cell proliferation and immunoglobulin production. The lack of MHC restriction, a functional characteristic feature of FDC, further reinforces the identity of the isolated cells as chicken FDC.