Essential Role of IL-4 and IL-4Rα Interaction in Adaptive Immunity of Zebrafish: Insight into the Origin of Th2-like Regulatory Mechanism in Ancient Vertebrates

Abstract

The roles of IL-4 and IL-4Rα in Th2-mediated immunity have been well characterized in humans and other mammals. In contrast, few reports have been documented in ancient vertebrates. Several putative IL-4– and IL-4Rα–like molecules were identified recently from a few fish species, providing preliminary insight into the occurrence of Th2-type immunity in teleosts. However, functional determination still is required to address this hypothesis. To this end, these two molecules were characterized functionally in zebrafish (Danio rerio). Besides the identification of a full-length IL-4Rα molecule and an isoform lacking most of the cytoplasmic region as predicted previously, two novel alternatively spliced soluble variants with the extracellular domain only also were identified. Zebrafish IL-4Rα (DrIL-4Rα) shared overall conserved structural features of the IL-4Rα family. Immunofluorescence staining showed that DrIL-4Rα distributed on B cells. In vitro binding assays demonstrated that zebrafish IL-4 (DrIL-4) can bind specifically to DrIL-4Rα. In vivo administration of DrIL-4 significantly upregulated B cell proliferation and Ab production. These DrIL-4–elicited immune responses were downregulated by the administration of zebrafish soluble IL-4Rα or by DrIL-4Rα blockade using anti–DrIL-4Rα Abs. In addition, Th2-related cytokines or transcription factors were upregulated by DrIL-4. The DrIL-4–DrIL-4Rα interaction promoted CD40 expression on B cells and enhanced the CD154–CD40 costimulatory response, both of which are crucial for the initiation of Th2-type immunity. To our knowledge, this is the first report showing that a possible Th2-mediated regulatory mechanism may have appeared before the divergence of teleosts and mammals. These results add greater insight into the evolutionary history of adaptive immunity.

Conserved IL-2Rγc Signaling Mediates Lymphopoiesis in Zebrafish

“…Motifs important for downstream pathways are also conserved on several receptor subunits, including the conserved STAT5 docking sites (YXXV/L/M) on Il-2rb, Il-4ra, and Il-7ra (87)(88)(89), and a docking site for SHC, which contributes to activation of the PI3K and ERK pathways, on Il-2rb (64) (data not shown). Ligands of the IL-2R family identified in zebrafish include Il-4, Il-15, Il-15-like, and Il-21 (29,90), with potential Il-2 (91-93) and Il-7 ligands (94) characterized in other teleosts, but not Il-9 (95). Therefore, the phenotypes observed following knockdown of il-2rgc.a and jak3 could potentially be due to defective signaling via several cytokines.…”

“…Therefore, the phenotypes observed following knockdown of il-2rgc.a and jak3 could potentially be due to defective signaling via several cytokines. Zebrafish Il-4 has been shown to stimulate B cell proliferation and Ab production in adult fish (29), but its role in T cell development has not been determined.…”

“…Like mammals, zebrafish undergo distinct phases of hematopoiesis, with lymphoid progenitors seeding the thymus early in development following the establishment of definitive hematopoiesis (24). Zebrafish also shows broad conservation of cytokine receptors and downstream signaling pathway components (25,26), particularly those known to facilitate the development of blood and immune cells (27)(28)(29)(30)(31).…”

Comparison of Two Chemically-Induced Colitis-Models in Adult Zebrafish, Using Optical Projection Tomography and Novel Transcriptional Markers

“…The key Th-1 cytokines IFN-γ and IL-12 were not significantly regulated, whereas IL-4/13A and IL-4/13b-hallmark cytokines of the Th2 pathway-were significantly upor down regulated, respectively, after 72 hpi in both treated groups. At the moment it is not clear if the two paralogous IL4-/13-genes in zebrafish are functionally comparable to their mammalian counterparts, but is has been suggested that IL-4/13Acorrespond to IL-4 in zebrafish [55]. In two recent studies, IL-4/13b gene expression was elevated in zebrafish and carp kidney cell lines exposed to antigens, whereas IL-4/13a expression was unaffected [54] [56].…”

Production of Carp Immunoglobulin M Exposed with Whole Protein from Myxobolus koi Spore through Feed as an Immunostimulant

“…T lymphocytes in non-specific response would produce interferon that can activate performance macrophages through phagocytosis mechanism in the face of parasites, bacteria, viruses and foreign particles that are considered as an antigen [10]. Whereas, in the specific immune response, cytokines, including IL-4, IL-5 and IL-13 with the Th cells, will activate B lymphocytes and T lymphocytes [11].…”