Jeroen P.J Saeij

Molecular and functional characterization of a fish inducible-type nitric oxide synthase

Abstract Using an oligonucleotide primer based on a partial goldfish inducible nitric oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated from an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1127-amino acid protein with 57% sequence identity to human iNOS. Like mammalian NOSs, carp iNOS protein contains putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADPH. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Inducibility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or the protozoan blood flagellate Trypanoplasma borreli. These stimulators produced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transciption factor NF-κB was shown to play a role in the induction of iNOS transcription.

The immune response of carp to Trypanoplasma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation

Although Trypanoplasma borreli induces the production of non-specific antibodies, survival of infection is associated with the production of T. borreli specific antibodies, able to lyse this parasite in the presence of complement. During the lag phase of this acquired immune response, innate immune mechanisms must limit multiplication of T. borreli. A heat-labile fraction of T. borreli together with CpG motifs in the DNA of this parasite are responsible for the induction of nitric oxide (NO) and probably also for the induction of expression of the inflammatory cytokines tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta by carp phagocytes in vitro. In the signal transduction pathway leading to activation of phagocytes, protein tyrosine kinase and protein kinase C are involved and probably collaborate in activation of the transcription factor nuclear factor (NF)-kappaB. In vivo, carp intraperitoneally injected with T. borreli up-regulate expression of TNFalpha, IL-1beta and mRNAs for acute phase response proteins (complement factor 3, serum amyloid A and alpha-2-macroglobulin).

Daily handling stress reduces resistance of carp to Trypanoplasma borreli: in vitro modulatory effects of cortisol on leukocyte function and apoptosis.

Carp subjected to daily handling stress were much more susceptible to Trypanoplasma borreli infection than control fish. In a search for the cellular mechanisms involved, it was observed that cortisol suppressed T. borreli-induced expression of interleukin-1beta, tumor necrosis factor-alpha, serum amyloid A and inducible nitric oxide synthase. An NF-kappaB-inhibitor could replicate cortisol-induced apoptosis of activated peripheral blood leukocytes. In contrast, although this NF-kappaB-inhibitor induced apoptosis of neutrophilic granulocytes, cortisol prevented apoptosis of these cells, suggesting the latter process to be NF-kappaB-independent. Carp leukocytes, upon induction of apoptosis, exhibit a number of sequential metabolic alterations. First, the mitochondrial transmembrane potential (DeltaPsi(m)) is disrupted and glutathione levels are depleted, followed by exposure of phosphatidylserine on the outer cell membrane. In vitro, cortisol could inhibit NO production induced by low concentrations of lipopolysaccharide (LPS), but remarkably, enhanced NO production induced by high concentrations of LPS. However, no differences in NO production were observed in stressed versus non-stressed infected carp.

Differential expression and haplotypic variation of two interleukin-1β genes in the common carp (Cyprinus carpio L.)

Interleukin-1beta (Il-1beta) is a central component in innate immunity and the inflammatory response of mammals. Only recently, the first non-mammalian IL-1beta sequences were published. In this study, we describe a second IL-1beta sequence (IL-1beta2) in carp with 74% amino acid identity to the carp IL-1beta1 sequence. The existence of two IL-1beta copies in the carp genome probably originates from the tetraploid nature of the species. In contrast to the first carp Il-1beta sequence, IL-1beta2 is represented by multiple genes with 95-99% identity. Detection of several IL-1beta2 sequences within individual homozygous fish suggests the presence of multiple copies of the Il-1beta2 gene in the carp genome, possibly as a result of subsequent gene duplication of IL-1beta2. In vivo, constitutive mRNA expression of both IL-1beta genes was found in healthy carp. IL-1beta2 mRNA expression could be up-regulated in head kidney cells similar to carp IL-1beta1, in vivo by infection with Trypanoplasma borreli and in vitro by stimulation with lipopolysaccharide (LPS). Cortisol, the major glucocorticoid in fish, is an endocrine-derived factor mediating Il-1beta expression. Although constitutive IL-1beta expression was inhibited by a physiological dose of cortisol, cortisol synergistically enhanced LPS-induced IL-1beta expression in carp. Involvement of the transcription factor nuclear factor (NF)-kappaB in expression of IL-1beta1 and IL-1beta2 was demonstrated. Ratio of IL-1beta expression was determined and this showed IL-1beta1 mRNA expression to be at least tenfold higher compared with IL-1beta2. The possibilities of IL-1beta2 being a functional gene or approaching pseudogene status are discussed

Immune modulation by fish kinetoplastid parasites: A role for nitric oxide

Trypanoplasma borreli and Trypanosoma carassii are kinetoplastid parasites infecting cyprinid fish. We investigated the role of nitric oxide (NO) in immune modulation during T. borreli and T. carassii infection of carp. Phagocytic cells from different organs produced NO and serum nitrate levels increased, demonstrating that T. borreli activates NO production in vivo. In contrast, T. carassii did not induce NO production in vivo and inhibited LPS-induced NO production in vitro. Production of NO was detrimental to the host as T. borreli-infected carp treated with the inducible NO synthase inhibitor aminoguanidine had a higher survival than infected control carp. This detrimental effect can be explained (in part) by the toxicity of NO to cells in vitro as NO inhibited the proliferative response of blood and spleen leukocytes. Head-kidney phagocytes were resistant to the immunosuppressive effects of NO in vitro. The NO-inducing activity of T. borreli may be an adaptation developed to ensure survival and immune evasion in the fish host. Apparently, T. carassii has adopted another strategy by deactivating specific functions of phagocytes. Both strategies may ensure long-term survival of the parasite.

Major histocompatibility genes in cyprinid fishes : theory and practice

Summary: The first teleostean MHC sequences were described for carp. Subsequent studies in a number of cyprinid fishes showed that the class I sequences of these fishes are of particular interest. Two distinct lineages (Cyca‐Z and Cyca‐U) are found in the common and ginbuna crucian carp, but only the U lineage is present in zebrafish and other non‐cyprinid species. The presence of the Z lineage is hypothesised to be the result of an allotetraploidisation event. Both phylogenetic analyses and amino acid sequence comparisons suggest that Cyca‐Z sequences are non‐classical class I sequences, probably similar to CD I. The comprehensive phylogenetic analyses of these sequences revealed different phylogenetic histories of the exons encoding the extracellular domains. The MHC genes were studied in laboratory and natural models. The natural model addressed the evolution of MHC genes in a Barbus species flock. Sequence analysis of class I and class II supported the species designation of the morphotypes present in the lake, and as a consequence the trans‐species hypothesis of MHC polymorphism. The laboratory model involves the generation of gynogenetic clones, which can be divergently selected for traits such as high and low antibody response. The role of MHC molecules can be investigated further by producing a panel of isogenic lines.

Identification and characterization of a fish natural resistance- associated macrophage protein (NRAMP) cDNA

Abstract The mouse Lsh/Ity/Bcg locus regulates natural resistance to intracellular pathogens, and the Nramp1 gene was isolated as its candidate. Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. In the present study, a full-length cDNA for carp NRAMP has been isolated and characterized. Nucleotide and predicted amino acid sequence analysis indicate that the carp NRAMP encodes a 548 amino acid membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionarily conserved consensus transport motif. The peptide sequence identity among carp and human NRAMP2 is 78%, and 65% with human NRAMP1. Reverse transcription-polymerase chain reaction revealed that carp NRAMP is ubiquitously expressed. Phylogenetic analysis, using neigbor-joining, showed that the carp NRAMP protein clustered together with mammalian NRAMP2 proteins.

Different capacities of carp leukocytes to encounter nitric oxide-mediated stress: a role for the intracellular reduced glutathione pool

Carp head kidney (HK) phagocytes can be stimulated by lipopolysaccharide (LPS) to produce nitric oxide (NO). High production of NO can suppress the carp immune system. Carp peripheral blood leukocytes (PBL) are highly susceptible but HK phagocytes are relatively resistant to the immunosuppressive effects of NO. This study demonstrates that the antioxidant glutathione plays an important role in the protection against nitrosative stress. Carp HK phagocytes, especially the neutrophilic granulocytes, contain higher levels of glutathione than PBL. Moreover, freshly isolated carp neutrophilic granulocytes have higher mRNA levels than PBL of glucose-6-phosphate dehydrogenase (G6PD), manganese superoxide dismutase (MnSOD) and gamma-glutamylcysteine synthetase (gamma-GCS). Since these molecules are part of the glutathione redox cycle, neutrophilic granulocytes have a higher capacity than PBL to maintain glutathione in a reduced state following nitrosative stress. When stimulated with LPS, neutrophilic granulocytes upregulate the expression of G6PD, MnSOD and gamma-GCS.