NEDD4-2 associates with γc and regulates its degradation rate

Interleukin-2 (IL-2) is a cytokine that regulates proliferation, differentiation and survival of various lymphoid cell subsets. Its actions are mediated through its binding to the IL-2 receptor which is composed of three subunits (IL-2Rα, IL-2Rβ and γ_c). Only β and γ_c have been shown to transduce intra cellular signals. The γ_c chain is shared by the interleukin-2, 4, 7, 9, 15 and 21 receptors, and is essential for lymphocyte functions. The regulation of γ_c expression level is therefore critical for the ability of cells to respond to these cytokines. In the present work, we show that the IL-2R constitutively associates with the ubiquitin ligase NEDD4-2, and to a lesser extent NEDD4-1. We identified the specific binding site on γ_c. And we show that the loss of NEDD4 association on γ_c is accompanied by a dramatic increase of the half-life of the receptor subunit.     

Molecular cloning and genomic structure of an interleukin-8 receptor-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Chemokines are small proteins (70-100 amino acids) which play an important role in recruitment and activation of leucocytes to migrate to the site of inflammation. Based on the position of the first two conserved cysteines, chemokines are classified into four subfamilies: C, CC, CXC and CX3C. To date, many members of CC and CXC have been found and studied extensively [1]. Chemokines exert effects on their target cell via chemokine receptors, which are G-protein coupled receptors containing seven transmembrane domains with an extracellular N-terminus and an intracellular C-terminus [2]. Interleukin 8 (IL-8) belongs to the CXC chemokine subfamily. It can activate and attract migratory neutrophils to an inflammation site. Two IL-8 receptors, CXCR1 and CXCR2, have been identified in mammals [3-6]; both of these receptors have high affinity for IL-8 and are expressed on the neutrophil. CXCR1 just binds IL-8; however, CXCR2 binds IL-8 and other structurally related chemokines such as growth-related oncogene (GRO) a, GRObeta, GROgamma, neutrophil-activating peptide-2 (NAP-2) and epithelial cell-derived neutrophil activating peptide-78 (ENA-78) [7, 8]. Several studies on fish chemokine receptors have been reported [9-11]. Thus far, however, IL-8 and CXCR1 and CXCR2 proteins from rainbow trout have not been reported: however, the sequence of a rainbow trout IL-8 has been noted (GenBank Accession No. AJ279069 [12]). Cloning of the IL-8 receptor is important to study the function of IL-8/CXCR1 and (CXCR2) in inflammation and signal transduction in fish. This paper reports the molecular cloning and genomic structure of an IL-8 receptor-like gene from four homozygous clones of rainbow trout: Oregon State University (OSU), Hot Creek (HC), Arlee (AR) and Swanson (SW).     

Cloning of two rainbow trout nucleotide-binding oligomerization domain containing 2 (NOD2) splice variants and functional characterization of the NOD2 effector domains

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic pattern recognition receptor (PRR), which is involved in innate antibacterial and antiviral responses. Here, two NOD2 splice variants, trNOD2a and trNOD2b, are reported in rainbow trout Oncorhynchus mykiss, that share 63% and 61% similarity with human NOD2, respectively. These two trout NOD2 splice variants were shown to be constitutively expressed in thymus, gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and brain, with the expression of trout NOD2 (trNOD2) mainly contributed by trNOD2a in all the examined tissues. PolyI:C transfection up-regulated the expression of trNOD2a and trNOD2b in RTG-2 cells. The expression of trNOD2a/b was modulated by the inflammatory stimulant interferon-γ (IFN-γ) or interleukin-1β (IL-1β). Overexpression of trout NOD2 effector domains resulted in induced expression of proinflammatory cytokines including IL-1β, tumor necrosis factor-α (TNF-α), IL-6 and IL-8, the antibacterial peptide cathelicidin-2, a variety of caspases including caspase-2, -6, -7, -8, -9, and type I and type II IFN. These results suggest that fish NOD2 functions in inflammatory events, possibly via NF-κB activation, regulation of apoptosis, and triggering of antibacterial and antiviral defences.     

Cortisol modulates the induction of inflammatory gene expression in a rainbow trout macrophage cell line

Glucocorticoid actions on the immune system are diverse and cell type dependent, and little is known about cell type-specific interactions and cross-talk between hormones and cytokines. In this study we have analyzed the gene expression patterns of the rainbow trout macrophage cell line RTS-11 by quantitative PCR, after exposure to combinations of cortisol plus a pro-inflammatory cytokine (e.g. recombinant trout IL-1β, IFN-γ), type I IFN or a PAMP (LPS or poly I:C). Several key genes of the inflammatory process were targetted to assess whether any modulation of their expression occurred due to the addition of cortisol to this cell line. Incubation of macrophages for 3 or 6 h with a physiological concentration of cortisol caused a decrease in expression of IL-6 and IL-8, but no significant changes were observed for the other genes examined. Co-stimulation of cortisol with the inflammatory agents resulted in a general suppression of genes related to the inflammatory response. Cortisol inhibited the up-regulation of IL-8 by all the stimulants after 3 h of co-incubation. Suppression of the up-regulation of IL-6 by rIL-1β, rIFN-γ and poly I:C, of γIP by rIFN-γ or poly I:C, and of Cox-2 by rIL-1β was seen after 6 h. In contrast, cortisol in combination with the pro-inflammatory agents has a synergistic effect on IL-10 expression, an anti-inflammatory molecule, suggesting that the activation of certain macrophage functions that lead to the resolution of inflammation occurs in fish macrophages in response to cortisol treatment.     

Crohn's disease alters MHC-rafts in CD4+ T-cells

Clusters of MHCI, ICAM-1, CD44, CD59, IL-2R, and IL-15R molecules have been studied on the surface of CD4(+) T-cells from peripheral blood and lymph nodes of patients in Crohn's disease and healthy individuals as controls by using a dual-laser flow cytometric fluorescence resonance energy transfer (FRET) technique and fluorescently stained Fabs. When cells from patients in Crohn's disease are compared to those of controls, the surface expression level for the MHCI reduced by ～45%, for CD44 enhanced by ～100%, and for IL-2Rα, IL-15Rα, and common γ(c) enhanced by ～50%, ～70%, and ～130%, respectively. Efficiencies of FRET monitoring homoassociation for the MHCI and CD44 reduced, that for IL-2Rα enhanced. While efficiencies of FRET monitoring the association of γ(c) and ICAM-1 with the MHCI reduced, those monitoring association of IL-2/15Rα, CD44, and CD59 with MHCI enhanced. Efficiencies of FRET measured between the MHCI and IL-2Rα, IL-15Rα differently enhanced to the advantage of IL-15Rα, the one measured between γ(c) and IL-2Rα reduced, suggesting modulations in the strength of interaction of MHCI with IL-2R, IL-15R, and γ(c). The increases in density of surface bound cTx and in the associations of the receptors with the G(M1)-ganglioside lipid molecules suggest stronger lipid raft interactions of the receptors. The observed alterations of MHC-rafts in Crohn's disease--summarized in models of receptor patterns of diseased and control cells--may have functional consequences regarding signaling by the raft components.     

Two copies of the genes encoding the subunits of putative interleukin (IL)-4/IL-13 receptors,IL-4Rα, IL-13Rα1 and IL-13Rα2, have been identified in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) and have complex patterns of expression and modulation

Mammalian interleukin-4 (IL-4) and IL-13 are T helper type 2 (Th2) cytokines with pleiotropic functions in immunity. They signal through receptors containing IL-4Rα and IL-2Rγ or IL-13Rα1. In addition, a decoy receptor, IL-13Rα2, is known to exist and modulates the function of IL-13. The existence of fish orthologues to mammalian IL-4 and IL-13 is still under debate. However, the receptor chains have been predicted in zebrafish, and we have previously cloned IL-2Rγ and IL-13Rα2 in rainbow trout. In this study, we have cloned a further five novel trout IL-4/13 receptors. Thus, each of the IL-4Rα, IL-13Rα1 and IL-13Rα2 chains has two copies. The identities of the receptors is supported by homology analysis, characteristic domain structure, phylogenetic tree analysis and synteny analysis in zebrafish. However, the characteristic WSXWS motif of structural importance in mammalian type I cytokine receptors is missing in all fish IL-4Rα and IL-13Rα1 molecules. All the receptors have a characteristic domain structure that is similar to their mammalian counterparts except for IL-13Rα1b that has the N-terminal Ig domain missing. Since this Ig domain is a specific and critical binding unit for IL-13 but not for IL-4 signalling, its absence potentially converts the IL-13Rα1b into a receptor that can only signal via IL-4 ligation. The existence of duplicated receptor genes perhaps suggests that more ligands still remain to be discovered that will bind these receptors. The duplicated receptors are differentially expressed in most tissues and cell lines examined, and their expression can be modulated by LPS, polyIC and IFN-γ in cell lines. In contrast, the T-cell stimulant phytohaemagglutinin increased the expression of IL-4Rα1 and IL-4Rα2, but not IL-13Rα1/2, suggesting a role of an IL-4-like molecule in T-cell growth/activation in fish.     

Thermal Tolerance of Diploid Versus Triploid Rainbow Trout and Brook Trout Assessed by Time to Chronic Lethal Maximum

Synopsis An effect of ploidy on thermal tolerance in juvenile trout was assessed in a series of tests comparing time to chronic lethal maximum (CLMax). Diploid and triploid fish were produced from a common spawn for three different groups each of brook trout Salvelinus fontinalis and of rainbow trout Oncorhynchus mykiss. One or two CLMax tests were performed per group, on between 15 and 50 individuals per ploidy within groups. The tests involved exposure of fish to a progressive 2°C day⁻¹ water temperature increase and recording of the time at which each individual fish reached loss of equilibrium (LE). The time to LE data were rank transformed and analyzed as a randomized complete block design. Although relative performance varied among trials, the analysis indicated overall differences due to ploidy were small and nonsignificant among both brook trout and rainbow trout. Size proved to be significantly correlated with time to LE in the brook trout trials, but not in the rainbow trout trials. Two of the six groups included a large proportion of fish which had received a heat shock following fertilization, but were not successfully triploidized. In both cases, thermal tolerance of the heat-shocked diploids was similar to that of the non-heat shocked control diploids, indicating no persistent effect of the heat shock on thermal tolerance.     

γ-Tocotrienol inhibits lipopolysaccharide-induced interlukin-6 and granulocyte colony-stimulating factor by suppressing C/EBPβ and NF-κB in macrophages

Cytokines generated from macrophages contribute to pathogenesis of inflammation-associated diseases. Here we show that γ-tocotrienol (γ-TE), a natural vitamin E form, inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 production without affecting tumor necrosis factor α (TNF-α), IL-10 or cyclooxygenase-2 (COX-2) up-regulation in murine RAW264.7 macrophages. Mechanistic studies indicate that nuclear factor κB (NF-κB), but not c-Jun NH(2)-terminal protein kinase, p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs), is important to IL-6 production and that γ-TE treatment blocks NF-κB activation. In contrast, COX-2 appears to be regulated by p38 MAPK in RAW cells, but γ-TE has no effect on LPS-stimulated p38 phosphorylation. Despite necessary for IL-6, NF-κB activation by TNF-α or other cytokines is not sufficient for IL-6 induction with exception of LPS. CCAAT/enhancer-binding protein (C/EBP) β appears to be involved in IL-6 formation because LPS induces C/EBPβ up-regulation, which parallels IL-6 production, and knockdown of C/EBPβ with small interfering RNA results in diminished IL-6. LPS but not individual cytokines is capable of stimulating C/EBPβ and IL-6 in macrophages. Consistent with its dampening effect on IL-6, γ-TE blunts LPS-induced up-regulation of C/EBPβ without affecting C/EBPδ. γ-TE also decreases LPS-stimulated granulocyte colony-stimulating factor (G-CSF), a C/EBPβ target gene. Compared with RAW264.7 cells, γ-TE shows similar or stronger inhibitory effects on LPS-triggered activation of NF-κB, C/EPBβ and C/EBPδ and more potently suppresses IL-6 and G-CSF in bone marrow-derived macrophages. Our study demonstrates that γ-TE has antiinflammatory activities by inhibition of NF-κB and C/EBPs activation in macrophages.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Lipopolysaccharide from Rhodobacter capsulatus counteracts the effects of toxic lipopolysaccharides and inhibits the release of TNF-α, IL-6, and IL-1β in human whole blood

The outcome of pathological process during sepsis caused by Gram-negative bacteria depends on the reaction of human blood cells to bacterial structural components, lipopolysaccharides (LPS). A general inflammatory response develops due to the increased production of proinflammatory cytokines. One of the current methods of prevention of inflammatory response is the inhibition of LPS binding to cellular receptors. We have studied the efficacy of antagonistic properties of LPS from Rhodobacter capsulatus on the production of TNF-α, IL-6, and IL-1β cytokines induced by toxic LPS from Salmonella typhimurium and Escherichia coli in human whole blood. LPS from R. capsulatus in concentrations of 0.1 and 1 μg/mL did not induce synthesis of TNF-α, IL-6, or IL-1β. Measurements of cytokine levels showed that LPS from R. capsulatus exerted a clear protective effect against toxic LPS. In particular, LPS from R. capsulatus fullly inhibited the production of TNF-α and IL-1β and significantly decreased the IL-6 production induced by LPS from S. typhimurium. Additionally, LPS from R. capsulatus antagonized the effects of LPS from E. coli, fully inhibiting the TNF-α production and decreasing the IL-6 and IL-1β levels by 60% and 70%, respectively. Thus, LPS from R. capsulatus acts as a potent antagonist of cell activation induced by toxic LPS.     

Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin-1β and transforming growth factor-β

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1β and TGF-β increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1β; 1 ng/ml TGF-β) and time-dependent (IL-1β 12-hr peak; TGF-β 24-hr peak). Effect of IL-1β and TGF-β on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with ¹²⁵l-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1β (4.7 × 10⁶/cell) and TGF-β (3.9 × 10⁶/cell)-treated cells, compared to control (3.0 × 10⁶/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1β and TGF-β have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q. © 1993 Wiley-Liss, Inc.     

Long‐term impacts of invasive species on a native top predator in a large lake system

1. Declining abundances of forage fish and the introduction and establishment of non‐indigenous species have the potential to substantially alter resource and habitat exploitation by top predators in large lakes. 2. We measured stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) in field‐collected and archived samples of Lake Ontario lake trout (Salvelinus namaycush) and five species of prey fish and compared current trophic relationships of this top predator with historical samples. 3. Relationships between δ¹⁵N and lake trout age were temporally consistent throughout Lake Ontario and confirmed the role of lake trout as a top predator in this food web. However, δ¹³C values for age classes of lake trout collected in 2008 ranged from 1.0 to 3.9‰ higher than those reported for the population sampled in 1992. 4. Isotope mixing models predicted that these changes in resource assimilation were owing to the replacement of rainbow smelt (Osmerus mordax) by round goby (Neogobius melanostomus) in lake trout diet and increased reliance on carbon resources derived from nearshore production. This contrasts with the historical situation in Lake Ontario where δ¹³C values of the lake trout population were dominated by a reliance on offshore carbon production. 5. These results indicate a reduced capacity of the Lake Ontario offshore food web to support the energetic requirements of lake trout and that this top predator has become increasingly reliant on prey resources that are derived from nearshore carbon pathways.