A role for matrix metalloproteinases in granulocyte infiltration and testicular remodelation in a seasonal breeding teleost

The testicular cystic structure and the abrupt morphological changes that the fish testis undergoes during the reproductive cycle (RC) make it an interesting model for studying the regulation of spermatogenesis, in particular the role of matrix metalloproteinases (Mmps). The gilthead seabream is a seasonal breeding teleost whose testis undergoes drastic remodeling events, especially during the post-spawning stage when a massive infiltration of a immune cell type, the acidophilic granulocytes, occurred. Bearing this in mind, we studied the gilthead seabream testis gelatinolytic activities involved in migration and tissue remodeling and its regulation by endocrine, immune and tissue stimuli. Thus, we demonstrated that the germinal epithelium of the testis showed gelatinolytic activity during spermatogenesis and post-spawning but not during resting, when only scarce interstitial cells were stained. Moreover, the precursor and mature forms of two gelatinases, Mmp2- and Mmp9-like, were active in the gonad, whose activities were up-regulated by 17β-estradiol (E₂) but not by lipopolysaccharide (LPS)/bacterial DNA (VaDNA) in testicular cell suspensions. E₂ and LPS/VaDNA also up-regulated a variety of cytokines and chemokines. We also cloned mmp9, mmp13, tissue inhibitors of Mmps (timp)-2a and timp2b genes and found that all of them were expressed in the gonad in a RC stage-dependent manner. Interestingly, mmps and timps were highly expressed by the testicular acidophilic granulocytes. Moreover, in these cells, the gelatinolytic activity seemed to correspond to the precursor and mature forms of putative Mmp2 and Mmp9 gelatinases, while the main gelatinolytic activity seemed to correspond to the mature form of Mmp2 in head–kidney acidophilic granulocytes. Finally, although none of the stimuli used were able to induce the gelatinolytic activity of Mmp9-like in head–kidney acidophilic granulocytes, the expression of mmp9, timp2a and timp2b were all up-regulated by LPS/VaDNA in these cells, while only mmp9 and timp2a expression increased upon stimulation with gelatin.     

The type II interleukin-1 receptor (IL-1RII) of the bony fish gilthead seabream Sparus aurata is strongly induced after infection and tightly regulated at transcriptional and post-transcriptional levels

Interleukin-1beta (IL-1beta) is the prototypic pro-inflammatory cytokine. All the biological effects of IL-1beta are mediated through interaction with type 1 IL-1 receptor (IL-1RI), whereas another receptor, called type 2 IL-1R (IL-1RII), lacks an intracellular signalling domain and acts as a decoy receptor that down-regulates responses to IL-1beta. Although both receptors are present in bony fish, their expression and biological role in the regulation of IL-1beta activity in non-mammalian vertebrates remain to be established. In this study, a homologue of mammalian IL-1RII was isolated and characterized in the gilthead seabream (Sparus aurata). The seabream IL-1RII harboured two Ig-like domains in its extracellular region and a short cytoplasmic tail lacking a signalling domain. The seabream IL-1RII cDNA showed an unexpectedly long 3'UTR compared with that from other species and contained three ATTTA instability motifs, which seem to be responsible for its relatively short half-life (less than 2h). The expression of seabream IL-1RII was dramatically up-regulated after infection with Vibrio anguillarum in all the immune tissues examined and was even more strongly induced than the IL-1beta gene in the head kidney, spleen and liver. Strikingly, the mRNA levels of IL-1RII were 15-fold higher than those of IL-1beta in the liver, suggesting a role for this organ in the neutralization of IL-1beta leaking into the systemic circulation from the sites of inflammation. In vitro, bacterial DNA and flagellin increased the mRNA levels of IL-1RII in macrophages, while only flagellin was able to weakly induce its expression in acidophilic granulocytes. Finally, the seabream IL-1RII was localized in the plasma membrane when expressed in HEK293 cells and was able to bind IL-1beta.     

The activation of gilthead seabream professional phagocytes by different PAMPs underlines the behavioural diversity of the main innate immune cells of bony fish

Phagocytic cells form the cellular arm of the innate immune system. A primary role of these cells is an ability to discriminate large number of potential pathogens from self, using a restricted number of receptors. In the gilthead seabream, acidophilic granulocytes and macrophages have been described as the professional phagocytes of this species. However, no direct functional comparisons between these two phagocytic lineages exist for the seabream or for other teleost species. Therefore, purified fractions of acidophilic granulocytes and macrophages were used to characterize the ability of these cells to recognize and respond to different pathogen-associated molecular patterns (PAMPs) and the data obtained were then correlated with the expression of several pattern-recognition receptors (PRRs). The time course of the respiratory burst of acidophilic granulocytes stimulated with different PAMPs showed that muramyldipeptide (MDP), the ligand for NOD2 in mammals, induced maximal activation earlier than several ligands for toll-like receptors (TLRs), including bacterial DNA, flagellin and lipopolysaccharide (LPS). In addition, all these PAMPs strongly increased the phagocytic and bactericidal activities of acidophilic granulocytes, while other PAMPs, including poly I:C, Pam3CSK(4) and zymosan, failed to do so. The stimulation of acidophilic granulocytes and macrophages by PAMPs also resulted in the up-regulation of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), cyclooxygenase-2 (COX-2) and TLRs, although the kinetics and expression profiles observed for each cell type differed. These results suggest different roles for professional phagocytes of fish in the recognition and elimination of pathogens and in the regulation of adaptive immune responses.     

Distribution of the professional phagocytic granulocytes of the bony fish gilthead seabream (Sparus aurata L.) during the ontogeny of lymphomyeloid organs and pathogen entry sites

Although it is believed that fish fry depend fundamentally on their innate defence mechanisms, the ontogeny of fish innate immune cells is poorly understood. In the present study, we have used a specific monoclonal antibody against acidophilic granulocytes (AGs), the main professional phagocytic cell type of the bony fish gilthead seabream, to study their localization during the development of the main lymphomyeloid organs, namely the head kidney, spleen and thymus, and of the two major portals for pathogen entry, namely the gills and intestine. AGs were observed in the posterior intestine and in the blood earlier than in the haematopoietic kidney (21 vs. 27 days post-hatching, dph). AGs were observed scattered between other cells of the haematopoietic lineage in the head kidney of larvae, but were grouped around the blood vessels of this organ in juveniles and adults, where they were also much more numerous. In the spleen and in the thymus, AGs were observed much later (62 dph) and appeared scattered. AGs were also observed in the gill lamella and the posterior intestine near the anus throughout development.     

Production and mechanism of secretion of interleukin-1beta from the marine fish gilthead seabream

Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.     

Molecular characterization, 3D modelling and expression analysis of sea bass (Dicentrarchus labrax L.) interleukin-10

Interleukin-10 (IL-10) is a pleiotropic cytokine generally known for its relevance in the resolution of inflammation, but that also has immunostimulatory properties. Here is described the isolation and characterization of the sea bass IL-10 (sbIL-10) cDNA and gene. The sbIL-10 gene encodes a 187 amino acid protein and comprises a five exon-four intron structure as other known IL-10 genes. Important structural residues are maintained in the sbIL-10 protein, including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10. The 3D structure of sbIL-10 was predicted. This first homology model of a fish IL-10 reveals a high degree of compatibility between the dimeric quaternary architectures of sbIL-10 and its mammalian counterparts. The phylogenetic analysis clusters sbIL-10 with other IL-10s, apart from IL-10-related molecules. The involvement of IL-10 in sea bass immune responses was demonstrated by investigating the expression profiles of IL-1beta and IL-10 in the head-kidney and spleen following intraperitoneal injection of UV-killed Photobacterium damselae ssp. piscicida. Furthermore, involvement of IL-10 in the resolution of inflammation is for the first time suggested in fish, due to the delayed maximal mRNA levels of sbIL-10 compared to those of the pro-inflammatory IL-1beta.     

TLR agonists extend the functional lifespan of professional phagocytic granulocytes in the bony fish gilthead seabream and direct precursor differentiation towards the production of granulocytes

Neutrophils are major cells participants in innate host responses. They are short-lived leukocytes, although microbial products activate intracellular signaling cascades that prolong their survival by inhibiting constitutive apoptosis. To gain insight into the phylogeny of this important cell type, we examined the ability of toll-like receptor agonists to extend the lifespan of gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils. The results obtained demonstrated that apoptosis was also the default state of seabream acidophilic granulocytes and that toll-like receptor agonists were able to dramatically extend their functional lifespan (up to 10 days) by inhibiting apoptosis and inducing a long lasting activation of phagocytic and respiratory burst activities, together with the expression of genes coding for several proinflammatory molecules. This process was independent on contaminating cells and interleukin-1β production. In addition, the results showed that p38 mitogen-activated protein kinase, but not nuclear factor κB, c-Jun terminal kinase or phosphatidylinositol 3-kinase, was involved in the inhibition of acidophilic granulocyte apoptosis following toll-like receptor engagement. Finally, stimulation of head kidney hematopoietic precursor cells with toll-like receptor agonists promoted their terminal differentiation to acidophilic granulocytes. These results demonstrated that the extension of neutrophil lifespan by microbial products is conserved in lower vertebrates although the magnitude of the response is much higher in fish.     

Molecular characterization of the nonspecific cytotoxic cell receptor (NCCRP-1) demonstrates gilthead seabream NCC heterogeneity

Teleost fish NCCs (nonspecific cytotoxic cells) are thought to be the evolutionary precursors of the mammalian NK cells. A novel mechanism mediating the NCC-mediated cytotoxicity has been described in teleosts. Now, this NCC receptor protein-1 (NCCRP-1) was characterized in gilthead seabream. The NCCRP-1 full-length sequence contains 1036 bp with an open reading frame of 702 bp. A comparison of the predicted 233-amino acid protein with several fish orthologues indicates a highly conserved sequence containing the F-box associated (FBA) domain and proline-rich motifs (PRM) characteristics of this family. The phylogenetical tree shows that seabream NCCRP-1 belongs to the NCCRP subfamily within the FBA family of proteins. This is a single copy gene with a constitutive and ubiquitous expression as determined by RT-PCR and flow cytometry. The results show that lymphocytes, monocyte/macrophages and acidophilic granulocytes from lymphoid tissues express the receptor, both at gene and protein level. Immunofluorescence microscopic observations confirm the previous results. The implications of this receptor on seabream NCC activity are discussed.     

Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor

Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr(+)) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr(+) cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.     

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 10⁹ heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.     

Characterization of twin toll-like receptors from rainbow trout (Oncorhynchus mykiss): evolutionary relationship and induced expression by Aeromonas salmonicida salmonicida

Structure and function of factors contributing to the innate immune system of lower vertebrates, including fish are only sparsely characterized. We retrieved with RT-PCR cDNA copies of two closely related Toll-like receptors (TLR) from liver RNA of the rainbow trout (Oncorhynchus mykiss). The cDNA sequences are homologous to 95.6%. The phylogenetic analysis of their deduced amino acid sequences places these twin factors closely to other known TLRs from fish. The twin factors are equally expressed in all tissues analysed, most abundantly in spleen and head kidney and lowest in adipose tissue. Formalin-inactivated Aeromonas salmonicida pathogens induce their expression up to eight-fold in vitro in peripheral blood lymphocytes and in tissues from spleen and head kidney. Our sequence information will be useful to establish expression constructs for these factors necessary to analyse the pathogen specific signal transduction activating the innate immune defence in fish.     

Natural and synthetic estrogens modulate the inflammatory response in the gilthead seabream (Sparus aurata L.) through the activation of endothelial cells

Sex steroids are known to deeply alter processes other than fish reproduction, including fish growth, intermediary metabolism, osmoregulation and immunity. We have previously reported that 17β-estradiol (E(2)), the main fish estrogen, promotes the mobilization of acidophilic granulocytes from the head kidney, the bone marrow equivalent in fish, to the gonad in the bony fish gilthead seabream (Sparus aurata L.). The aim of this study was to investigate the effects of E(2) and 17α-ethinylestradiol (EE(2)), an endocrine disruptor with strong estrogenic effects commonly found in the aquatic environment, on the ability of gilthead seabream endothelial cells (ECs) to promote leukocyte infiltration. E(2) and EE(2) were seen to affect ECs in different ways. Thus, E(2) was able to increase the production of nitric oxide (NO) and up-regulate the expression of the key activation markers, interleukin-1β, CC chemokine ligand 4, interleukin-8, E-selectin and matrix metalloproteinase 9, when used alone or combined with bacterial DNA. In contrast, EE(2) failed to affect NO release and reduced the up-regulation of the above genes promoted by bacterial DNA. Moreover, we found that leukocyte adhesion to ECs was enhanced by E(2) treatment. Collectively, these results suggest that estrogens modulate fish leukocyte trafficking during an inflammatory process by activating ECs.     

Molecular polymorphism and expression analysis of MHC class II B gene from red sea bream (Chrysophrys major)

MHC class II (major histocompatibility complex class II) plays an important role in the immune response of vertebrates. Its function is to present antigenic peptides to the T-cell receptor. In order to study the function and molecular polymorphism of class II B gene in fish, we have isolated cDNAs encoding class II B from spleen cDNA library of red sea bream (Chrysophrys major) by using EST sequencing, and examined genomic organization, molecular polymorphism and expression of red sea bream class II B gene. As in other vertebrates, five exons and four introns were identified in red sea bream class II B gene. Seven class II B alleles were identified from seven individuals of red sea bream. The deduced amino acid sequence of red sea bream MHC class II B 1(Chma-DAB*0101) had 87.1, 85.1, 87.1, 90.4, 87.1, 90.8% identity with those of red sea bream class II B 2, 3, 4, 5, 6, 7(Chma-DAB*0201-Chma-DAB*0701), respectively, and had 75.2, 74.5, 55.9, 55.1, 34.3 and 30.4% identity with those of striped sea bass, cichlid, rainbow trout, Atlantic salmon, mouse and human, respectively. Four different class II B alleles were observed in a single individual and two different 3' untranslated region (3' UTR) sequences from this individual may infer the existence of two loci at least. Semi-quantitative RT-PCR demonstrated that high expression was detected in liver, head kidney, kidney, intestine, gill, stomach, hear and spleen, low expression in muscle and blood. Challenge of red sea bream with the pathogenic bacteria, Vibrio anguillarum, resulted in a significant decrease in the expression of MHC class II B mRNA from 5 to 72 h after infection in liver, spleen, head kidney and intestine, followed by a recovery to normal level after 96 h.     

Natural and synthetic estrogens modulate the inflammatory response in the gilthead seabream (Sparus aurata L.) through the activation of endothelial cells

Sex steroids are known to deeply alter processes other than fish reproduction, including fish growth, intermediary metabolism, osmoregulation and immunity. We have previously reported that 17β-estradiol (E₂), the main fish estrogen, promotes the mobilization of acidophilic granulocytes from the head kidney, the bone marrow equivalent in fish, to the gonad in the bony fish gilthead seabream (Sparus aurata L.). The aim of this study was to investigate the effects of E₂ and 17α-ethinylestradiol (EE₂), an endocrine disruptor with strong estrogenic effects commonly found in the aquatic environment, on the ability of gilthead seabream endothelial cells (ECs) to promote leukocyte infiltration. E₂ and EE₂ were seen to affect ECs in different ways. Thus, E₂ was able to increase the production of nitric oxide (NO) and up-regulate the expression of the key activation markers, interleukin-1β, CC chemokine ligand 4, interleukin-8, E-selectin and matrix metalloproteinase 9, when used alone or combined with bacterial DNA. In contrast, EE₂ failed to affect NO release and reduced the up-regulation of the above genes promoted by bacterial DNA. Moreover, we found that leukocyte adhesion to ECs was enhanced by E₂ treatment. Collectively, these results suggest that estrogens modulate fish leukocyte trafficking during an inflammatory process by activating ECs.     

Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: the first evidence of Th2-type cytokine in fish

Interleukin-4 (IL-4) is one of the key cytokines in Th2 mediated immune responses, which has been shown to regulate the responses of many immune cytokines, such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1) and TNF-alpha. Much work on IL-4 has been done in human and several mammal species while little in fish. In this study, we have cloned and characterized the full-length cDNA of IL-4 in Tetraodon. The Tetraodon IL-4 cDNA is 834bp in length and contains a short 5'UTR of 39bp, a 3'UTR of 375bp and an open reading frame of 420bp translating into a protein of 139aa with a predicted molecular mass of 16.131kDa. The Tetraodon IL-4-encoding gene with the same organization as the mammalians and birds consists of four exons and three introns. The encoded protein shows 11-16% identities to other homologues. RT-PCR was optimized to estimate the expression level of IL-4 in Tetraodon. The results showed that IL-4 is constitutively expressed in all selected tissues, including head kidney, spleen, liver, brain, gill, muscle and heart, although low levels were observed in head kidney, spleen, and liver. The ubiquitous expression of IL-4 is consistent with a postulated role in immune cytokines regulation. Stimulating the fish with a mixed stimulant that contained 2 microg ConA, 2 microg PHA, and 2 microg PMA, significantly up-regulated the expression of IL-4 in most tissues examined, which potentially indicated that IL-4 was involved in the immune inflammatory responses triggered by mitogens. This is the first report of cloning and characterization of IL-4 cDNA and gene in fish.     

Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells

The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream (Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5'-UTR region of 84 bp, an ORF of 729 bp and a 3'-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, alpha1, alpha2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the alpha1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA+LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.