Expression of chicken DEC205 reflects the unique structure and function of the avian immune system

Abstract

The generation of appropriate adaptive immune responses relies critically on dendritic cells, about which relatively little is known in chickens, a vital livestock species, in comparison with man and mouse. We cloned and sequenced chicken DEC205 cDNA and used this knowledge to produce quantitative PCR assays and monoclonal antibodies to study expression of DEC205 as well as CD83. The gene structure of DEC205 was identical to those of other species. Transcripts of both genes were found at higher levels in lymphoid tissues and the expression of DEC205 in normal birds had a characteristic distribution in the primary lymphoid organs. In spleen, DEC205 was seen on cells ideally located to trap antigen. In thymus it was found on cells thought to participate in the education of T cells, and in the bursa on cells that may be involved in presentation of antigen to B cells and regulation of B cell migration. The expression of DEC205 on cells other than antigen presenting cells (APC) is also described. Isolated splenocytes strongly expressing DEC205 but not the KUL01 antigen have morphology similar to mammalian dendritic cells and the distinct expression of DEC205 within the avian-specific Bursa of Fabricius alludes to a unique function in this organ of B cell diversification.

Figure 1. Structure of the chicken DEC205 gene.

At the top, those parts of the 35 exons containing coding sequence are drawn to scale and labelled with their lengths in nucleotide residues. Introns are not to scale although their lengths relative to each other are approximately as drawn. The protein domain structure is shown below using shaded rectangles drawn to scale, with annotations: S, signal sequence; FNII, fibronectin type II; CTLD, C-type lectin like domain; TM, transmembrane region; Cyto, cytoplasmic region. Dotted lines connect the ends of domains to the encoding positions in exons. The dashed box indicates the three domains which were used to make the recombinant fusion protein used to raise monoclonal antibodies.

Figure 2. Transcript levels of DEC205 and CD83 genes in tissues.

The vertical scale represents transcript levels relative to the reference 28S RNA on a logarithmic (base 2) scale. Bars indicate the levels in each of the tissues indicated below the horizontal axis. Error bars indicate the standard error of three assays carried out on RNA preparations from tissue samples from different birds.

Figure 3. Localisation of cells expressing DEC205 in normal chicken spleen.

The four panels show a set of serial sections of the same area from the normal spleen of a juvenile bird, stained with antibodies recognising the indicated molecules. Bu-1 reveals B cells and CD4 a subset of T cells. KUL01 is a marker for monocyte/macrophage lineage cells. DEC205 is detected using the antibody FG9. The dotted curves indicate the approximate outlines of the same peri-ellipsoid lymphocyte sheaths (pe) in each section. Other labelled features are ellipsoid (e), aterioles (a) and peri-arteriolar lymphocyte sheath (pa) .

Figure 4. DEC205 and CD83 expressing cells in the spleen of a MDV infected bird.

The four panels show a set of serial sections of the same area from the spleen of a juvenile bird five days after infection with Marek's disease virus. The sections are stained with antibodies recognising the indicated molecules. Bu-1 revealing B cells and CD4 & CD8 revealing T cells. DEC205 is detected using the antibody FG9 and CD83 using GE8. Labelled features are artery (a) and germinal centre (gc).

Figure 5. DEC205 and CD83 expressing cells in normal thymus.

The four panels show a set of serial sections of the same area from the thymus of a juvenile bird. The sections are stained with antibodies recognising the indicated molecules. Bu-1 revealing B cells and CD4 & CD8 revealing T cells. DEC205 is detected using the antibody FG9 and CD83 using GE8. Cortical (c) and medullary (m) areas of a thymic lobe are labelled.

Figure 6. DEC205 expression in the juvenile bursa.

The left panel shows a lower magnification of a section of bursal tissue stained with the DEC205 antibody FG9. The right panel shows a higher power view of the region of the same section indicated by the dashed lines. Two blood vessels in the interfolicular space are labelled (v). The cortex (c) and medulla (m) of the bursal follicle are also indicated. The arrowhead marks the layer of stained cells at the cortico-medullary boundary.

Figure 7. Distinct populations of adherent splenocytes expressing DEC205.

Adherent spleen cells were stimulated with LPS for three hours, then permeabilised and stained with FG9 anti DEC205 antibody (green, Alexa Fluor 488) and KUL01 monocyte/macrophage marker (red, Alexa Fluor 568). Nuclei were stained with DAPI (blue). The left panel shows the pseudocolour image from the confocal microscope, and the right panel shows a differential interference contrast image of the same cells. KUL01 positive cells with rounded morphology are indicated with large arrowheads and KUL01 negative cells with flattened and extended morphology are indicated with double arrows.

Figure 8. Increased expression of DEC205 on LPS stimulation of adherent spleen cells.

The upper two panels show images from two chambers of the same slide, one having been incubated with LPS for one hour (A) and one without (B) The cells were stained with FG9 anti DEC205 antibody (green) and GE8 anti CD83 antibody (red). Nuclei were stained with DAPI (blue). Staining, confocal camera settings and image processing were identical for both images. Surface staining of ungated (C) or cells gated for CD11c expression (D) following stimulation with LPS or CD40 ligand fusion protein are also shown.

Figure 9. Surface mobilisation of MHC class II upon LPS activation of DEC205-expressing adherent cells.

Adherent spleen cells were stimulated with LPS for three hours, permeabilised, and stained with AD6 anti-DEC205 antibody (green) and 2G11 anti MHC class II antibody (red). Nuclei were revealed with DAPI (blue). The inset shows an image of an unstimulated cell stained in the same way.

Figure 10. Adherent cells expressing DEC205 are non-pahgocytic.

The top pair of panels shows confocal images of fresh adherent cells stained with both DEC205 and KUL01 antibodies. The left hand image reveals low level expression of DEC205 on the KUL01 positive cells. The same cell preparation was incubated with fluorescent microbeads for three hours and then fixed and permeablised before staining with either DEC205 antibody FG9 (middle) or KUL01 (bottom), and examination by confocal microscopy. The pseudocoloured fluorescence images are on the left with beads in yellow and antibody staining in red. At the right are the corresponding differential interference contrast images. Red arrows point to cells that are strongly stained with DEC205 antibody but have no internalised fluorescent beads. Yellow arrows point to cells that have internalised fluorescent beads. The white arrow points to a cell that is not stained with KUL01.

Figure 11. DEC205 expression on leucocyte subsets.

Splenocytes, bursal cells and thymocytes recovered by enzymatic digestion were stained with DEC205 antibody and antibodies recognising each of the indicated antigens. The figure shows histograms of the DEC205 antibody fluorescence for cells gated for positive expression of the markers indicated at the left. Controls are the staining of appropriate subclass control antibodies on cells gated for the same markers.