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. 2010 Sep 7;107(36):15850-5.
doi: 10.1073/pnas.1000494107. Epub 2010 Aug 23.

Identification of dendritic antigen-presenting cells in the zebrafish

Geanncarlo Lugo-Villarino  1 , Keir M Balla, David L Stachura, Karina Bañuelos, Miriam B F Werneck, David Traver
Affiliations

Identification of dendritic antigen-presenting cells in the zebrafish

Geanncarlo Lugo-Villarino et al. Proc Natl Acad Sci U S A. .

Abstract

In mammals, dendritic cells (DCs) form the key link between the innate and adaptive immune systems. DCs act as immune sentries in various tissues and, upon encountering pathogen, engulf and traffic foreign antigen to secondary lymphoid tissues, stimulating antigen-specific T lymphocytes. Although DCs are of fundamental importance in orchestrating the mammalian immune response, their presence and function in nonmammalian vertebrates is largely unknown. Because teleosts possess one of the earliest recognizable adaptive immune systems, we sought to identify antigen-presenting cells (APCs) in the zebrafish to better understand the potential origins of DCs and their evolutionary relationship to lymphocytes. Here we present the identification and characterization of a zebrafish APC subset strongly resembling mammalian DCs. Rare DCs are present in various adult tissues, and can be enriched by their affinity for the lectin peanut agglutinin (PNA). We show that PNA(hi) myeloid cells possess the classical morphological features of mammalian DCs as revealed by histochemical and ultrastructural analyses, phagocytose-labeled bacterial preparations in vivo, and exhibit expression of genes associated with DC function and antigen presentation, including il12, MHC class II invariant chain iclp1, and csf1r. Importantly, we show that PNA(hi) cells can activate T lymphocytes in an antigen-dependent manner. Together, these studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from teleosts to mammals, and indicate that the zebrafish may serve as a unique model to study the origin of APC subsets and their evolutionary role as the link between the innate and adaptive immune systems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of DCs in zebrafish. (A) PBS (Left) or S. aureus Alexa 488 (Center) was injected i.p., and IPEX cells were collected after 16 h. Phagocytes (black gate, Center) were detected by Alexa 488 fluorescence (5.06 ± 1.84%, n = 11) and separated according to their light-scatter characteristics (Right) as erythrocytes (red gate), lymphocytes (blue gate), myelomonocytes (green gate), or eosinophils (orange gate). (B) IPEX phagocytes within the myelomonocyte gate (green) were isolated (1.1 × 104 ± 962 phagocytes/fish) and subjected to cytospin analysis followed by WG staining. (Upper) Phagocytic myelomonocytes, including neutrophils (i, 40 ± 15%), monocytes, and Mφs (ii and iii, 52 ± 12%). (Lower) DCs (iv–vi, 7 ± 2.5%); n = 6, ± indicates SD. (Scale bar: 5 μm.)
Fig. 2.
Fig. 2.
Enrichment of zebrafish DCs by flow cytometry and lectin-binding affinity. Single-cell suspensions were prepared from WKM and labeled with PNA. (A) Contour plot (Left) demonstrates the distribution of cells in WKM by light scatter: erythrocytes (red), lymphocytes (blue), precursors (purple), myelomonocytes (green), and eosinophils (orange). Histogram (Right) shows PNA binding (black unshaded histogram) and negative control (shaded gray histogram) within the myelomonocyte gate. (B) Cytospin analysis of PNAhi myelomonocytes purified from WKM and stained with WG: neutrophils (i), monocytes (ii), Mφs (iii), and DCs (iv). (C) DC-like cells isolated from other organs and peripheral sites based on PNA binding: skin (i), gut (ii), IPEX (iii), and gills (iv). (Scale bar: 5 μm.)
Fig. 3.
Fig. 3.
Morphological characterization of zebrafish DCs. PNAhi myelomonocytes were isolated from WKM (A and B) or skin (C) by FACS. (A) Cells were stained for WG, H&E, AP (magenta precipitate), NAE (black precipitate), MPX (brown precipitate), PAS (red precipitate), and TB (purple precipitate). (Upper) Putative DCs for each stain. (Lower) Positive staining controls: Mφs (i–iii), monocyte (iv), neutrophil (v), eosinophil (vi), and mast cell (vii). (Scale bar: 5 μm.) (B) TEM was performed to examine the ultrastructure of PNAhi myelomonocytes. (Upper) DCs. (Lower) Other leukocytes: Mφ (i), neutrophil (ii), and eosinophil (iii). Denoted features include nucleus (N), apoptotic corpse (AC), cigar-shaped granules (red arrowheads), round, electron-dense granules (blue arrowheads), and mitochondria (green arrowheads). (Scale bar: 1 μm.) (C) TEM analysis of Birbeck-like granules from PNAhi DCs in skin. Magnified granule regions: vacuole (red arrowheads) and rod structure (blue arrowheads). (Scale bar: 200 nm.)
Fig. 4.
Fig. 4.
Further enrichment of zebrafish DCs for functional analyses. (A) Single-cell suspensions were prepared from mpx:eGFP transgenic WKM and labeled with PNA. Myelomonocytes (green gate) were divided into mpx− (black box) and mpx+ (blue box). These populations were further subdivided into mpx− PNA− (gray box), mpx− PNA+ (red box), and mpx+ PNA+ (blue box) fractions. (B) After 4 h of culture, mpx− PNA− (gray), mpx− PNA+ (red), and mpx+ PNA+ (blue) fractions were collected and stained with MGG to assess cell morphology. Differentials are presented as mean ± SD, n = 6. (C) mpx− PNA+ (red bar), mpx+ PNA+ (blue bar), and mpx− PNA− (black bar) myelomonocyte fractions were cultured for 16 h with and without LPS. The abundance of il-12p40, csf1r, and iclp1 transcripts were measured by qPCR. Expression is presented as fold induction over nonstimulated controls. Asterisks denote populations with no detectable expression of the transcript. Bars represent mean, n = 3. Error bars represent SD.
Fig. 5.
Fig. 5.
Functional characterization of Zebrafish DC-enriched populations. (A Upper) In vivo phagocytosis by PNAhi myelomonocytes. S. aureus Alexa 488 was injected i.p., and 16 h later cells were collected from IPEX, stained with PNA, and analyzed by FACS. Cells were gated on the myelomonocyte gate and divided into the following populations: PNA− (red gate and bar), PNAhi (blue gate and bar), and PNAhi phagocytes (green gate and bar). (Lower) qPCR analysis of il-12p40 transcript in these populations relative to that in WKM (black bar, arbitrary unit set at 1). Data are shown as average of fold change over WKM ± SD (n = 4). Asterisk indicates transcript not detected. (B) Illustration of the antigen-presentation assay using KLH to prime a polyclonal T-cell response, with the lck:eGFP zebrafish transgenic line as the source for naive (Tnaive) or KLH-primed (TKLH) T lymphocytes. (C) Proliferation responses as measured by dilution of PKH26 of lck+-TKLH cells stimulated with either PNAhi (Upper) or PNA− (Lower) myelomonocytes loaded with KLH protein. (D) lck+-TKLH cells stimulated with PNAhi myelomonocytes loaded (Upper) or not loaded (Lower) with KLH. (E) lck+-TKLH (Upper) or lck+-Tnaive (Lower) cells stimulated with PNAhi myelomonocytes loaded with KLH. T-cell proliferation results (C–E) are representative of three independent experiments. For all proliferation assays, the APC/T-cell ratio = 5:1. Histograms show T-cell proliferation as modeled by the FlowJo proliferation tool. %DIV = percent of T cells that divided.
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