The CD154-CD40-mediated costimulatory pathway is critical for T-B cell cooperation in thymus-dependent (TD) immune response in mammals. However, little is known about its existence and occurrence in lower vertebrates. Here, we report on the identification and functional characterization of CD154 and CD40 homologs from the zebrafish (Danio rerio) model. Zebrafish CD154 is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, whose tertiary structure is a sandwich containing two stacked sheets with “jelly roll” topology, just as the human TNF members do. The zebrafish CD40 is a type I membrane-bound protein with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The consensus TNFR-associated factor (TRAF)2- and TRAF6-binding motifs in mammalian CD40 are found in the cytoplasmic tail of zebrafish CD40, which indicates similar signal transduction mechanisms to higher vertebrates. Zebrafish CD154 and CD40 are widely distributed and can be up-regulated by thymus-dependent Ag. The production of IgM was dramatically decreased by anti-CD154 or soluble CD40, and it was enhanced by soluble CD154 or CD154-encoding plasmid in vivo. Thymus-dependent Ag-induced CD154 expression was inhibited by cyclosporin A, suggesting that CD154 functionally associates with T cells. Double immunofluorescence staining showed that CD40 and membrane IgM colocalized in B cells. CD154-CD40 binding assays showed that CD154 specifically binds to CD40 at homodimeric form. Our results provide the first evidence for the existence of the functional CD154-CD40-mediated costimulatory pathway and helper T cell regulatory mechanism underlying adaptive immunity in a fish species.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN/CD209) has become hot topic in recent studies because of its important roles in immune responses and immune escape. CD209 has been well characterized in humans and several other mammals, but little documentation exists about it in lower vertebrates. This is the first report on the identification and functional characterization of a fish DC-SIGN/CD209 molecule. The zebrafish DC-SIGN/CD209 cDNA translates into 343 aa organized into three domains structurally conserved among vertebrates. An EPN motif essential for interacting with Ca2+ and for recognizing mannose-containing motifs has been identified. Several conserved motifs crucial for internalization and signal transduction are also present within the cytoplasmic tail. Phylogenetic analysis supports the hypothesis that CD209 family members diverged from a common ancestor. The expression of DC-SIGN/CD209 in immune-related tissues can be significantly up-regulated by exogenous Ags and IL-4. This molecule associates with various APCs, including macrophages, B lymphocytes, and a possible dendritic cell-like (CD83+/CD80+CD209+) population. Functionally, T cell activation, Ab (IgM) production, and bacterial vaccination-elicited immunoprotection can be dramatically inhibited by a CD209 blockade after stimulation with keyhole limpet hemocyanin (KLH) in vivo or challenged with Aeromonas hydrophila, suggesting that DC-SIGN/CD209 in zebrafish is crucial for the initiation and development of adaptive immunity. Phagocytosis analysis showed that DC-SIGN/CD209 does not participate in the uptake of KLH Ag, suggesting that other mechanisms might exist that underlie DC-SIGN/CD209 involvement. We hope that the present study will contribute to a better cross-species understanding of the evolutionary history of the DC-SIGN/CD209 family.