Activation of trout macrophages and production of CRP after immunization with Vibrio anguillarum

To examine the mechanism of the protection of rainbow trout (Salmo gairdneri) against Vibrio anguillarum in the early stage of immunization, the activation of macrophages and production of C-reactive protein (CRP) were investigated. Fish immunized with formalin-killed bacteria emulsified in Freund's complete adjuvant (FCA) resisted intraperitoneal challenge with living bacteria seven and ten days after immunization. The activation of macrophages was demonstrated by a significant increase of the chemiluminescent (CL) response and phagocytic activity. These fish also showed a significant increase of the CRP level in sera. Fish immunized with V. anguillarum alone or injected with FCA, however, did not resist the challenge. Though FCA itself increased CRP level and the sera enhanced phagocytic activity, increase of CL activity was weak. These results indicated that the increase of CL activity and opsonising effect of CRP on the phagocytosis of specifically activated macrophages concern to host defense in the early stage of infection.     

Molecular characterization of interleukin-6 in the gilthead seabream (Sparus aurata)

A cDNA clone, designated sbIL-6 (seabream interleukin-6), was obtained from a cDNA library of enriched immune-stimulated sequences from gilthead seabream. The deduced sbIL-6 protein corresponds to a 225-amino acid protein with a putative 24-amino acid signal peptide, four conserved alpha helices and one N-linked glycosylation site. At the amino acid level sbIL-6 shares 23-26% identity with mammalian IL-6 sequences and 30-51% identity with other fish IL-6 sequences. The structure of the sbIL-6 gene consisted of 5 exons and 4 introns, spanning 2.4 kb. Healthy fish expressed sbIL-6 in white muscle, skin, spleen, anterior intestine and stomach, while no expression was detected in brain, gill, head kidney, posterior intestine and adipose tissue. A significant up-regulation of sbIL-6 expression was observed after lipopolysaccharide (LPS), Vibrio anguillarum DNA (VaDNA) and peptidoglycan treatment in cultured seabream head kidney leukocytes. Using purified immune cells, sbIL-6 expression was induced similarly in macrophages and acidophilic granulocytes by VaDNA but LPS was more effective in inducing sbIL-6 expression in acidophilic granulocytes than in macrophages. Furthermore, in vivo infection of seabream with live V. anguillarum caused significant increases in sbIL-6 mRNA expression in the thymus, peritoneal exudate, head kidney and gills. In summary, our study provides further evidence for the existence of distinct IL-6 genes in lower vertebrates and for the strong induction of their expression by immune stimuli, supporting the notion of a potentially important role for this cytokine in fish.     

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.     

Neutrophil NADPH oxidase is reduced at the Anaplasma phagocytophilum phagosome

The intracellular organism Anaplasma phagocytophilum causes human granulocytic ehrlichiosis and specifically infects and multiplies in neutrophilic granulocytes. Previous reports have suggested that, for its survival, this bacterium suppresses the neutrophil respiratory burst. To investigate the mechanism of survival, we first assessed the kinetics of A. phagocytophilum entry into neutrophils by using double-labeling confocal microscopy. At 30, 60, 120, and 240 min of incubation, 25, 50, 55, and 70% of neutrophils contained bacteria, respectively. The neutrophil respiratory burst in the presence of A. phagocytophilum was assessed by a kinetic cytochrome c assay and by measurement of oxygen consumption. Neutrophils in the presence of A. phagocytophilum did not produce a significant respiratory burst, but A. phagocytophilum did not inhibit the neutrophil respiratory burst when phorbol myristate acetate was added. Immunoelectron microscopy of neutrophils infected with A. phagocytophilum or Escherichia coli revealed that NADPH oxidase subunits gp91(phox) and p22(phox) were significantly reduced at the A. phagocytophilum phagosome after 1 and 4 h of incubation. In neutrophils incubated simultaneously with A. phagocytophilum and E. coli for 30, 60, and 90 min, gp91(phox) was present on 20, 14, and 10% of the A. phagocytophilum phagosomes, whereas p22(phox) was present in 11, 5, and 4% of the phagosomes, respectively. Similarly, on E. coli phagosomes, gp91(phox) was present in 62, 64, and 65%, whereas p22(phox) was detected in 54, 48, and 48%. We conclude that A. phagocytophilum does not suppress a global respiratory burst and that, under identical conditions in the same cells, A. phagocytophilum, but not E. coli, significantly reduces gp91(phox) and p22(phox) from its phagosome membrane.     

Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in pneumococcal meningitis in the rabbit

The inflammatory response in bacterial meningitis is mediated by cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1), which are produced in the subarachnoid space by different cells, e.g., leukocytes, astrocytes, and microglia. The recruitment of leukocytes into the cerebrospinal fluid (CSF) has been shown to contribute to the neurological damage in this disease, a process which could be enhanced by treatment with antibiotics. In this study, we have used a rabbit meningitis model for two sets of experiments with intracisternal (i.c.) injections of Streptococcus pneumoniae. First, pneumococcal cell wall (PCW) components were injected i.c., inducing an inflammatory response with pleocytosis and increased levels of CSF TNF-alpha) and IL-1 at 6 and 12 h after PCW injection. Treatment with fucoidin, known to inhibit leukocyte rolling, abolished pleocytosis and inhibited the release of TNF-alpha and IL-1. In the second experiment, live pneumococcal bacteria were injected i.c. and treatment with one dose of ampicillin (40 mg/kg of body weight intravenously) was given 16 h after induction of meningitis, causing a sevenfold increase in CSF leukocytes over a 4-h period. CSF IL-1 levels at 16 h were high but did not increase further at 20 h. Also, CSF TNF-alpha levels were high at 16 h and tended to increase at 20 h. Fucoidin treatment prevented the antibiotic-induced increase of CSF leukocytes but had no effect on the TNF-alpha and IL-1 levels. Taken together, fucoidin reduced CSF TNF-alpha and IL-1 levels in acute bacterial meningitis induced by PCW fragments but had no effect later in the course of the disease, when live bacteria were used and an inflammatory increase was caused by a dose of antibiotics.     

Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection

To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate approximately 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1beta and TNFalpha2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.     

Activity of polymorphonuclear leukocytes in the presence of sulfide.

Polymorphonuclear leukocytes (PMN) isolated from human blood were exposed to various levels of hydrogen sulfide. The effect on respiratory burst, myeloperoxidase activity, and capacity to phagocytose and kill bacteria were studied. A 1-h exposure of the PMN to 1 mM sulfide did not decrease their myeloperoxidase activity or their capacity to initiate a respiratory burst. Actually the products of the respiratory burst rapidly oxidized sulfide. The phagocytosis and killing of bacteria in the presence of 1 mM sulfide was only decreased to a minor extent. Myeloperoxidase in cell extract was, however, almost completely inhibited by 1 microM sulfide. These results indicate that hydrogen sulfide does not easily permeate PMN. PMN may be able to function in infected sites with high sulfide levels such as in the gingival pockets of periodontal disease. In the oxygenated areas of these sites the PMN may actually help in the detoxification of sulfide.     

Experimental transmission of<Emphasis Type="Italic"> Cryptosporidium molnari</Emphasis> (Apicomplexa: Coccidia) to gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) and European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> L.)

Cryptosporidium molnari was experimentally transmitted to gilthead sea bream (Sparus aurata) and European sea bass (Dicentrachus labrax) by oral infection with infected stomach scrapings. The infection was also cross-transmitted from infected gilthead sea bream to sea bass by cohabitation. The course of the infection was assessed after necropsy by three microscopic diagnostic methods and their sensitivity was compared. At the end of all the experiments the prevalence of infection reached 100%. In the oral experiments, both fish hosts appeared infected as early as 7 days post exposure (p.e.), but gilthead sea bream exhibited a higher intensity of infection and infection proceeded at a faster rate than in sea bass. The cellular host reaction was stronger in sea bass than in sea bream, whereas the histopathological effect was lower in the former. Transmission could be favoured by cannibalism among cohabiting fish. This is the first report on piscine Cryptosporidium transmission. The implications for the aquaculture industry are discussed.     

Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro

Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P <0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P <0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.     

Induction of CD 14 antigen on the surface of U937 cells by an interleukin-6 autocrine mechanism after culture with formalin-killed Gram-negative bacteria

ABSTRACT: In order to better understand the regulation of CD 14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD 14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram-negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116–54. Various cytokines such as interleukin-1β, interleukin-2, interleukin-6, interferon-γ and tumor-necrosis factor-α were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116–54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116–54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116–54 also induced strong expression of CD 14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD 14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6.     

Hydrocortisone suppresses the chemiluminescent response of striped bass phagocytes

Phagocytic cells obtained from the pronephros of striped bass (Morone saxatilis) were exposed in vitro to various levels of hydrocortisone acetate. This treatment reduced the normal ability of the cells to generate a chemiluminescent response when exposed to bacteria or phorbol myristate acetate. Although the levels of hydrocortisone were higher than is physiological for fish, suppression was dose dependent and was not attributable to a reduction in cell viability. Whereas phagocytic chemiluminescence has been linked to the respiratory burst and bactericidal activity, the possibility exists that stress-induced elevations in serum corticosteroids lead to increased susceptibility to infection.     

Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape

Francisella tularensis is a Gram-negative bacterium and the causative agent of tularemia. Recent data indicate that F. tularensis replicates inside macrophages, but its fate in other cell types, including human neutrophils, is unclear. We now show that F. tularensis live vaccine strain (LVS), opsonized with normal human serum, was rapidly ingested by neutrophils but was not eliminated. Moreover, evasion of intracellular killing can be explained, in part, by disruption of the respiratory burst. As judged by luminol-enhanced chemiluminescence and nitroblue tetrazolium staining, neutrophils infected with live F. tularensis did not generate reactive oxygen species. Confocal microscopy demonstrated that NADPH oxidase assembly was disrupted, and LVS phagosomes did not acquire gp91/p22(phox) or p47/p67(phox). At the same time, F. tularensis also impaired neutrophil activation by heterologous stimuli such as phorbol esters and opsonized zymosan particles. Later in infection, LVS escaped the phagosome, and live organisms persisted in the neutrophil cytosol for at least 12 h. To our knowledge, our data are the first demonstration of a facultative intracellular pathogen, which disrupts the oxidative burst and escapes the phagosome to evade elimination inside neutrophils, and as such, our data define a novel mechanism of virulence.     

Interactions of Mycobacterium lepraemurium with resident peritoneal macrophages; phagocytosis and stimulation of the oxidative burst

Live Mycobacterium lepraemurium or 60Co-gamma-irradiated organisms stimulated a very weak oxidative burst compared with similar numbers of heat-killed organisms or with live M. microti. This was found to reflect the poor uptake of the living organisms rather than an absolute failure to stimulate an oxidative burst. It is possible, however, that phagocytosis of fewer than 3-4 bacteria may not trigger the respiratory burst. Pre-incubating live M. lepraemurium with sera from infected mice, but not with fresh normal mouse sera, resulted in enhanced phagocytosis with a concomitant increase in the oxidative burst. The level of opsonic activity correlated with the M. lepraemurium specific antibody titres. The opsonic activity appeared to be mediated by antigen-antibody activation of the classical complement pathway as heat-inactivation destroyed the activity.     

Induction of CD14 antigen on the surface of U937 cells by an interleukin-6 autocrine mechanism after culture with formalin-killed gram-negative bacteria.

In order to better understand the regulation of CD14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram-negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116-54. Various cytokines such as interleukin-1 beta, interleukin-2, interleukin-6, interferon-gamma and tumor-necrosis factor-alpha were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116-54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116-54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116-54 also induced strong expression of CD14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6.     

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.