Head kidney neutrophils of carp (Cyprinus carpio L.) are functionally modulated by the haemoflagellateTrypanoplasma borreli

In the present work responses of carp (Cyprinus carpio) head kidney-derived neutrophils to the blood parasite T. borreli, and the consequences of these responses for parasite survival and other host response mechanisms, were studied. In co-cultures of head kidney leucocytes (HKL) with viable and lysed T. borreli a prominent shape change of neutrophilic granulocytes towards increased size and complexity was observed. In addition, the longevity of neutrophils in vitro was prolonged in the presence of T. borreli antigens. In these cultures, neutrophils also exhibited an increased phagocytosis activity. An up regulation of the production of nitric oxide (NO) and reactive oxygen species (ROS) was observed in T. borreli- and mitogen-stimulated HKL cultures. However, addition of live, fluorescence-labelledT. borreli to previously stimulated HKL cultures, revealed neither killing nor phagocytosis of the parasite by activated neutrophils. Moreover, viable T. borreli, when added to HKL cultures of infected carp, reduced their phagocytosis activity and NO production. Supernatants of co-cultures between T. borreli and HKL also contained mediators, which suppressed a mitogen-induced proliferative response of peripheral blood leucocytes (PBL) in vitro. Thus, while T. borreli itself appeared not to be sensitive to responses of activated neutrophils, the flagellates interferes with the production of immunomodulatory signals of these cells, probably resulting in a partial immunosuppression, which may favour the parasite development in vivo.     

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide,which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were tested in vitro. Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli-stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600 micromol l(-1) NO(-)(2)) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.     

Minor effect of depletion of resident macrophages from peritoneal cavity on resistance of common carp Cyprinus carpio to blood flagellates

Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection.     

Modulation of carp (Cyprinus carpio) neutrophil functions during an infection with the haemoparasite Trypanoplasma borreli

Trypanoplasma borreli is an extracellular blood parasite of common carp (Cyprinus carpio) transmitted by fish-biting leeches. The infestation with this parasite in juvenile carp may range between 75% and 100%, especially in fish recovering from the first hibernation period. T. borreli is perfectly adapted to its prolonged survival in a cyprinid host. Elevated numbers of activated neutrophils in peripheral blood and tissues are reported during T. borreli infection, but in context of the disease, the direct reason for elevated neutrophil numbers and their role during the infection remain unclear. In this study, a quantitative transmigration system, permitting the harvest of highly pure (> or = 97%) neutrophil populations was applied to investigate the modulation of carp neutrophil functions during T. borreli infection. We demonstrate time-dependent kinetics of a serum-induced down-regulation of neutrophil chemotaxis and an up-regulation of ROS production during the course of infection. With highly pure neutrophil populations, we could show that this divergent alteration of neutrophil functions was neither caused by T. borreli metabolites nor by the parasite itself. Moreover, when added to highly purified neutrophils, parasite metabolites did not alter the leukotriene B4-induced neutrophil chemotaxis nor the Staphylococcus aureus-induced ROS production. We conclude that the haemoparasite T. borreli does not interact with neutrophils directly, but indirectly modulates their functions via serum factors induced by parasite interaction with other components of the immune system.     

Flow cytometric analysis of proliferative responses of carp Cyprinus carpio peripheral blood leukocytes to mitogens and to the hemoflagellate Trypanoplasma borreli

The activation of carp peripheral blood leukocytes (PBL) was analysed radiometrically and by means of flow cytometry (FCM) in order to compare the results obtained with both methods. The qualitative and quantitative FCM analyses of cellular morphology and viability resulted in a further characterisation of proliferative responses of carp PBL to Trypanoplasma borreli in vivo and in vitro. The lymphocyte population of PBL from T. borreli-infected carp exhibited a marked shift in forward scattered light (FSC; cell size). When PBL from healthy carp were stimulated with mitogens in vitro, a lymphoid population with increased FSC profiles was also observed. The number of these cells coincided to ratios of 3H-thymidine incorporation, recorded from corresponding cultures. Thus, it was concluded that the increase in size of stimulated lymphocytes could be due to blastogenic transformation. The advantage of the FCM procedure is that activation and proliferation of carp lymphocytes can be monitored without labelling the cells. Cocultures of mitogen-stimulated carp PBL and T. borreli revealed the ability of the parasite to suppress lymphocyte proliferation in vitro.     

Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection

Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of l-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate l-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor Aminoguanidine reverts the attenuation in arginase-blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of l-arginine. The whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.     

Mouse strain susceptibility to trypanosome infection: an arginase-dependent effect

We previously reported that macrophage arginase inhibits NO-dependent trypanosome killing in vitro and in vivo. BALB/c and C57BL/6 mice are known to be susceptible and resistant to trypanosome infection, respectively. Hence, we assessed the expression and the role of inducible NO synthase (iNOS) and arginase in these two mouse strains infected with Trypanosoma brucei brucei. Arginase I and arginase II mRNA expression was higher in macrophages from infected BALB/c compared with those from C57BL/6 mice, whereas iNOS mRNA was up-regulated at the same level in both phenotypes. Similarly, arginase activity was more important in macrophages from infected BALB/c vs infected C57BL/6 mice. Moreover, increase of arginase I and arginase II mRNA levels and of macrophage arginase activity was directly induced by trypanosomes, with a higher level in BALB/c compared with C57BL/6 mice. Neither iNOS expression nor NO production was stimulated by trypanosomes in vitro. The high level of arginase activity in T. brucei brucei-infected BALB/c macrophages strongly inhibited macrophage NO production, which in turn resulted in less trypanosome killing compared with C57BL/6 macrophages. NO generation and parasite killing were restored to the same level in BALB/c and C57BL/6 macrophages when arginase was specifically inhibited with N(omega)-hydroxy-nor-L-arginine. In conclusion, host arginase represents a marker of resistance/susceptibility to trypanosome infections.     

Some immune parameters in carp cyprinus Carpio susceptible and resistant to the haemoflagellate Trypanoplasma borreli

The present study addresses aspects of the (specific) immune response of carp to the haemoflagellate Trypanoplasma borreli. Sera of resistant carp contained antibodies, which agglutinated the flagellates in vitro. When flagellates were incubated in fish sera from resistant carp, binding of antibodies to flagellates could be demonstrated by flow cytometry, and T. borreli were effectively killed. Heat-treatment of the sera prevented killing, indicating that complement activation is important for the control of a T. borreli infection. Sera of carp that were highly susceptible to infection with T. borreli contained no antibodies capable of binding to or killing the parasite. Furthermore, specific antibodies were not generated after experimental infection. This lack of antibody production in susceptible carp is not due to a general unresponsiveness of lymphoid cells, since peripheral blood leukocytes (PBL) from susceptible and resistant carp responded to mitogenic stimuli in vitro with lymphocyte proliferation in a similar manner. However, viable flagellates were significantly less able to stimulate proliferation of PBL from susceptible carp. In vitro-produced culture supernatants of freshly isolated PBL from both carp lines (but not those of head kidney cells) differentially modulated the mitogen-induced proliferation of PBL from susceptible and resistant carp. The supernatants enhanced the proliferation of leukocytes obtained from individuals from the same carp line, but suppressed the mitogen-induced proliferation of PBL from the other line tested. This indicates that lymphoid cells from susceptible and resistant carp differ in their spectrum of spontaneously produced immunomodulatory mediators. Whether this is decisive for a T. borreli-specific and successful immune response is discussed.     

Enhancer-mediated control of macrophage-specific arginase I expression

Arginase I expression in the liver must remain constant throughout life to eliminate excess nitrogen via the urea cycle. In contrast, arginase I expression in macrophages is silent until signals from Th2 cytokines such as IL-4 and IL-13 are received and the mRNA is then induced four to five orders of magnitude. Arginase I is hypothesized to play a regulatory and potentially pathogenic role in diseases such as asthma, parasitic, bacterial, and worm infections by modulating NO levels and promoting fibrosis. We show that Th2-inducible arginase I expression in mouse macrophages is controlled by an enhancer that lies -3 kb from the basal promoter. PU.1, IL-4-induced STAT6, and C/EBPbeta assemble at the enhancer and await the effect of another STAT6-regulated protein(s) that must be synthesized de novo. Identification of a powerful extrahepatic regulatory enhancer for arginase I provides potential to manipulate arginase I activity in immune cells while sparing liver urea cycle function.     

The induction of nitric oxide response of carp macrophages by transferrin is influenced by the allelic diversity of the molecule

The central role of transferrin (Tf) as an iron transporting protein has been extended by observations that modified versions of Tf also participate in the regulation of innate immunity. We report on the isolation of two carp Tf proteins (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography, and describe the activation of head kidney-derived carp macrophages by cleaved Tf. We demonstrate the superiority of the D-type over the G-type Tf in inducing nitric oxide (NO) and confirm previous observations that full-length Tf cannot induce NO in fish macrophages. We believe that cleaved Tf fragments should be considered to be "alarmins". We discuss the possibility that parasites such as Trypanoplasma borreli cleave Tf and use Tf fragments to their advantage by modulating the NO induction in carp macrophages.     

Macrophage cytotoxicity against Entamoeba histolytica trophozoites is mediated by nitric oxide from L-arginine.

The killing of Entamoeba histolytica trophozoites by phagocytes involves oxidative and nonoxidative mediators. In this study, we determine whether L-arginine-derived nitric oxide (NO) is involved in the killing of E. histolytica trophozoites by activated murine macrophages in vitro. Elicited peritoneal and bone marrow-derived macrophages activated with IFN-gamma alone or with IFN-gamma and LPS killed 62 to 73% of amebae, concomitant with increased levels of nitrate (NO2). Depletion of L-arginine by addition of arginase to culture medium abrogated macrophage amebicidal activity. NG-monomethyl L-arginine, an L-arginine analog, competitively inhibited NO2 release and amebicidal activity in a dose-dependent fashion, without affecting H2O2 production; however, the addition of excess L-arginine competitively restored macrophage amebicidal effects. In culture, sodium nitrite and sodium nitroprusside were cytotoxic to E. histolytica and this was reversed by the addition of myoglobin. Exogenously added FeSO4 prevented macrophage cytotoxicity. Addition of superoxide dismutase, a scavenger of O2-, partially inhibited amebicidal activity, without influencing NO2 production. Untreated and LPS-exposed macrophages produced high levels of H2O2 independent from NO2 production and amebicidal effects. However, the addition of catalase, a scavenger of H2O2, inhibited both amebicidal activity and NO2 production by activated macrophages. Our results demonstrate that NO is the major cytotoxic molecule released by activated macrophages for the in vitro cytotoxicity of E. histolytica and that O2- and H2O2 may be cofactors for the NO effector molecule.     

The parasitemia of cloned Trypanoplasma borreli Laveran and Mesnil, 1901, in laboratory-infected common carp (Cyprinus carpio L.)

The course of parasitemia of cloned Trypanoplasma borreli in laboratory-infected common carp was investigated. In 25-42-g carp kept at 20 C, the prepatent period was 8 days; after a phase of exponential growth, the parasitemia peaked at day 39 postinjection (PI) at a level of about 10(3) T. borreli/microliters blood. This maximum was followed by a chronic phase of about 6 wk with large numbers of T. borreli. At 20 wk PI, T. borreli was absent in infected carp. In 2.2-g carp kept at 20 C, the prepatent period was 4 days only, and the parasitemia peaked at day 23 PI. At 30 C, T. borreli was present in the blood only for 12 wk, and the number of T. borreli did not exceed 162 trypanoplasms/microliters blood. Carp kept at 8 and 15 C showed retarded development of parasitemia. The prepatent period lasted longer and the generation time was increased, but the level of parasitemia was not affected. Carp, inoculated at 8 C and then warmed to 20 C on days 27 and 55 PI, developed a parasitemia of 10(4) flagellates/microliters blood and showed high mortalities. During the prepatent period, T. borreli was found in the muscle tissue of the inoculation area but in no other tissue. In the kidney, T. borreli was found 27 hr PI, whereas in the circulating blood it was manifest at day 3 PI. At the same time it was manifest in the liver and spleen.     

Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism

Macrophages infected with amastigotes of Leishmania major and treated with IFN-gamma in vitro develop potent antimicrobial activities that eliminate the intracellular parasite. This antileishmanial activity was suppressed in a dose dependent fashion by NG-monomethyl-L-arginine (NGMMLA), a competitive inhibitor of nitrite, nitrate, nitric oxide and L-citrulline synthesis from L-arginine. Excess L-arginine added to infected macrophage cultures reversed the inhibitory effects of NGMMLA. Addition of arginase to culture media inhibited intracellular killing by IFN-gamma-treated cells. Similar effects were seen with macrophages obtained from BCG-infected C3H/HeN mice. Increased levels of nitrite, an oxidative product of the L-arginine-dependent effector mechanism, was measured in cultures of infected IFN gamma-treated macrophages as well as infected BCG-activated macrophages. Nitrite production correlated with development of antileishmanial activity. Nitrite production and microbicidal activity both decreased when in vivo or in vitro-activated macrophages were cultured in the presence of either arginase or NGMMLA. Nitric oxide synthesized from a terminal guanidino nitrogen atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe-dependent enzymatic pathways vital to the survival of amastigotes within macrophages.     

Inverse relation in the de novo arginase synthesis and nitric oxide production in murine and rat peritoneal macrophages in long-term cultures in vitro.

1. The de novo synthesis of arginase was much higher in murine than in rat peritoneal macrophages. This process was inhibited irreversibly by protein synthesis inhibitors and reversibly by glycolysis blockers. 2. Rat macrophages produce more nitric oxide (NO) than murine cells. NO production was inhibited by the inhibitors of protein synthesis or glycolysis. 3. The loading of macrophages by exogenous arginine for 24 hr in vitro resulted in the increase of arginase and nitrite in macrophages to different extents. 4. No great differences in lysozyme production was observed. 5. The proportion of arginine taken up and incorporated is contrasted in murine and rat macrophages.     

Cytotoxicity of lymphokine activated peritoneal macrophages against Trichomonas vaginalis]

Trichomonas vaginalis is a parasitic flagellate in the urogenital tract of human. Innate cytotoxicity of macrophages against T. vaginalis has been recognized, but any report on the cytotoxicity of lymphokine-activated macrophages to T. vaginalis is not yet available. The present study aimed to elucidate the lymphokine-activated cell mediated cytotoxic effect against T. vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured by counting the release of 3H-thymidine from prelabeled protozoa, and tested in U-bottom microtiter plates. Nitrite concentration in culture supernatants was measured by standard Griess reaction. The results obtained are as follows: 1. The cytotoxicity of macrophages was increased by addition of rIL-2 or rIFN-gamma. 2. Cytotoxicity of macrophages was reduced by addition of rIL-4 to rGM-CSF, rIL-2 or rIFN-gamma. 3. Crude lymphokine mixed with anti-IL-2 decreased the cytotoxicity of macrophages. 4. In case of macrophages cultured with rIFN-gamma or rIL-4, the concentration of nitrite was related with cytotoxicity of macrophages against T. vaginalis, but the cytotoxicity of macrophages cultured with rIL-2 and rIFN-gamma was decreased in spite of its high production of nitrite. From the results obtained, it is assumed that rIL-2 and rIFN-gamma enhance the cytotoxicity of macrophages while rIL-4 inhibits the cytotoxicity against T. vaginalis, and that the production of nitrite does not relate with the cytotoxicity of macrophages, but nitric oxide may play a role as an inhibitory factor on the proliferation of T. vaginalis.     

TGF-β1 exerts opposing effects on grass carp leukocytes: implication in teleost immunity, receptor signaling and potential self-regulatory mechanisms

In fish immunity, the regulatory role of transforming growth factor-β1 (TGF-β1) has not been fully characterized. Here we examined the immunoregulatory effects of TGF-β1 in grass carp peripheral blood leukocytes (PBL) and head kidney leukocytes (HKL). It is interesting that TGF-β1 consistently stimulated the cell viability and the mRNA levels of pro-inflammatory cytokines (Tnfα and Ifnγ) and T/B cell markers [Cd4-like (Cd4l), Cd8α, Cd8β and Igμ] in PBL, which contrasted with its inhibitory tone in HKL. Further studies showed that grass carp TGF-β1 type I receptor, activin receptor-like kinase 5 (ALK5), was indispensable for the immunoregulatory effects of TGF-β1 in PBL and HKL. Notably, TGF-β1 persistently attenuated ALK5 expression, whereas immunoneutralization of endogenous grass carp TGF-β1 could increase ALK5 mRNA and protein levels. It is consistent with the observation that TGF-β1 decreased the number of ALK5(+) leukocytes in PBL and HKL, revealing a negative regulation of TGF-β1 signaling at the receptor level. Moreover, transient treatment with TGF-β1 for 24 h was sufficient to induce similar cellular responses compared with the continuous treatment. This indicated a possible mechanism by which TGF-β1 triggered the down-regulation of ALK5 mRNA and protein, leading to the desensitization of grass carp leukocytes toward TGF-β1. Accordingly, our data revealed a dual role of TGF-β1 in teleost immunity in which it can serve as a positive or negative control device and provided additional mechanistic insights as to how TGF-β1 controls its signaling in vertebrate leukocytes.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

Modulation of fish phagocytic cells by N-terminal peptides of proopiomelanocortin (NPP)

N-terminal peptide of proopiomelanocortin (NPP, or pro-gamma-MSH) has shown to exhibit biological activity such as stimulation of adrenal mitogenesis and prolactin release-inhibiting factor activity. Structurally, studies reveal a significant difference between fish NPP from that of tetrapods, as NPPs from carp and salmonid lack gamma-MSH. Thus, fish NPP may exhibit functions different from that of mammals. The activation of phagocytic cells by NPP was analysed using rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio. Rainbow trout and carp macrophages incubated with chum salmon NPP significantly enhanced the production of superoxide anion in comparison with control macrophages (without hormones). Both rainbow trout and carp macrophages had shown increased phagocytosis when stimulated administered with NPP. The above results were complemented by in vivo studies where NPP was administered to rainbow trout and carp. NPP significantly increased superoxide anion production as well as phagocytosis in macrophages. These results show that NPP in lower vertebrates activates the function of the phagocytic cells.