Reuse water: Exposure duration, seasonality and treatment affect tissue responses in a model fish.

Partially remediated gray (reuse) water will likely find increasing use in a variety of applications owing to the increasing scarcity of freshwater. We aimed to determine if a model fish, the goldfish, could sense reuse water using olfaction (smell), and if 30min or 7d (acute) and 60d (sub-chronic) exposures would affect their olfactory responses to natural odorants. We examined olfaction as previous studies have found that numerous chemicals can impair the olfactory sense, which is critical to carrying out numerous life-sustaining behaviors from feeding to mating. We also examined if fish olfactory and liver tissues would mount a response in terms of biotransformation enzyme gene expression, and whether treatment of reuse water with UV/H2O2 ameliorated adverse effects following reuse water exposure. We found that fish olfactory tissue responded to reuse water as it would to a natural odorant and that UV/H2O2 treatment had no influence on this. With acute exposures, olfactory impairment was apparent regardless of water type (e.g. responses of 23-55% of control), but in sub-chronic exposures, only the untreated reuse water caused olfactory impairment. The exposure of fish to reuse water increased the expression of one enzyme (CYP1A; >2.5-6.5 fold change) and reuse water treatment with UV/H2O2 reversed the effect. There was a seasonal effect that was likely due to changes in water quality (60d summer exposure impaired olfaction whereas spring and fall exposures did not). Overall, the data suggest that reuse water may be detected by olfaction, impair olfactory responses in fish receiving unavoidable exposures, and that exposure duration and season are important factors to consider regarding adverse effects.

Effects of polymer-coated metal oxide nanoparticles on goldfish (Carassius auratus L.) neutrophil viability and function.

Exposure effects from polyacrylic acid (PAA) metal-oxide nanoparticles (TiO2, CeO2, Fe2O3, ZnO) on fish neutrophil viability and effector functions (degranulation, respiratory burst, inflammatory gene expression) were investigated using primary kidney goldfish (Carassius auratus L.) neutrophils as a model. Several studies have reported cytotoxic effects of NPs but there are limited reports on their potential to perturb the innate immune system of aquatic organisms. PAA-TiO2 significantly decreased neutrophil viability in a time and dose-dependent manner at all measured time points (0-48 h) and concentrations (0-200 µg/mL). Maximum viability decreased by (mean ± SEM): 67.1 ± 3.3%, 78.4 ± 4.2% and 74.9 ± 5.0% when exposed to 50, 100 and 200 µg/mL for 48 h, respectively. PAA-ZnO also significantly decreased neutrophil viability but only at 48 h exposures at higher concentrations. Neutrophil degranulation increased by approximately 3% after 30 min and by 8% after 4 h when exposed to sublethal doses (10 µg/mL) of PAA-CeO2 or PAA-Fe2O3. All PAA-NPs induced an increase in neutrophil respiratory burst when exposed to 10 µg/mL for 30 and 60 min, however, PAA-Fe2O3 was the only NP where the response was significant. Lastly, NPs altered the expression of a number of pro-inflammatory and immune genes, where PAA-TiO2 most significantly increased the mRNA levels of pro-inflammatory genes (il-1b, ifng) in neutrophils by 3 and 2.5 times, respectively. Together, these data demonstrate that goldfish neutrophils can be negatively affected from exposures to PAA-coated NPs and are functionally responsive to specific core-material properties at sublethal doses. These changes could perturb the innate response and affect the ability of fish to respond to pathogens.

Dietary lipids containing gangliosides reduce Giardia muris infection in vivo and survival of Giardia lamblia trophozoites in vitro.

We examined whether a ganglioside supplemented diet affected the course of Giardia muris infection in mice and survival of Giardia lamblia trophozoites in vitro. Female CD-1 mice were fed 1 of 5 experimental diets: standard lab chow as a control diet; semi-synthetic diets containing 20% (w/w) triglyceride based on the fat composition of a conventional infant formula; triglyceride diet; triglyceride diet containing a low level of ganglioside (0.1% w/w); and triglyceride diet containing a high level of ganglioside (1.0% w/w of diet). After 2 weeks of feeding, mice were inoculated with G. muris by gastric intubation and fed the experimental diets during the course of the infection. Cysts released in the faeces and trophozoites present in the small intestine were enumerated at various times post-infection. The average cyst output and the number of trophozoites during the course of the infection in mice fed ganglioside-containing diet were found to be significantly lower (3-log10 reduction) compared to animals fed control diets. The results of in vitro growth studies indicated that gangliosides may be directly toxic to the parasites. Thus, gangliosides have a protective effect against G. muris infection in vivo and affect the survival of G. lamblia trophozoites in vitro.

Enzyme treatment of Trypanosoma danilewskyi (Laveran and Mesnil) increases its susceptibility to lysis by the alternative complement pathway of goldfish, Carassius auratus (L.).

This study examined whether in vitro-cultured Trypanosoma danilewskyi were susceptible to lysis in the presence or absence of anti-parasite antibodies and complement. Cultured trypanosomes were resistant to lysis by either immune or non-immune goldfish serum. However, trypanosomes treated with the proteolytic enzyme trypsin, which destroys surface proteins of the parasites, became susceptible to lysis when exposed to either immune or non-immune goldfish serum. The lysis by goldfish serum was dependent on the presence of heat-labile factors and occurred at 4 and 20 degrees C. The lysis was also dependent on the presence of Mg(2+) ions but not Ca(2+) ions. Furthermore, treatment of the parasites with different sialidases did not enhance their susceptibility to lysis by goldfish serum. Trypsinized parasites regained resistance to lysis after at least 6-h cultivation in the absence of trypsin and the restoration of full resistance was observed after 24-h cultivation. The resistance to lysis was abrogated when the protein synthesis inhibitor, puromycin, was added to the cultures. These results suggest that trypsinized trypanosomes were susceptible to lysis by goldfish complement (alternative pathway) and that protective surface proteins of the parasite were required for the resistance of normal trypanosomes to lysis.

Immunization of goldfish with excretory/secretory molecules of Trypanosoma danilewskyi confers protection against infection.

Trypanosoma danilewskyi is a protozoan that lives in blood and other tissues of fish. In the aquaculture industry, economic losses may be substantial, since the prevalence of infection may approach 100% and the parasite may cause significant mortality in farmed carp. Most of the surviving fish acquire resistance after elimination of the primary infection. In this study, we examined whether protection against infection could be induced in naïve goldfish immunized with excretory-secretory (ES) products of the parasite. The ES extracts were administered in conjunction with Freund's complete or incomplete adjuvant (FCA and FIA, respectively). Parameters used to assess the efficacy of immunization after challenge infection, included prevalence of infection, abundance of parasites, and presence of parasite-specific antibodies. Intraperitoneal inoculation of ES products in FCA conferred protection against T. danilewskyi infection (P<0.05). Administration of ES products (with or without FIA) conferred insignificant levels of protection. In an attempt to identify the immunogenic ES molecules, we assessed whether anti-parasite antibodies present in the serum collected from experimentally infected fish or rabbits immunized with ES recognized parasite ES antigens. An immunoblot using rabbit anti-parasite antibody revealed a recognition profile of molecules (two antigens of approximately 102-104 and 70-72kDa) similar to that of immune goldfish serum. While the antigens that confer protection need further molecular characterization, our results suggest that the administration of ES products may allow for the design of control strategies for T. danilewskyi.

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation.

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.

Transcriptional regulation of hemopoiesis.

The regulation of blood cell formation, or hemopoiesis, is central to the replenishment of mature effector cells of innate and acquired immune responses. These cells fulfil specific roles in the host defense against invading pathogens, and in the maintenance of homeostasis. The development of hemopoietic cells is under stringent control from extracellular and intracellular stimuli that result in the activation of specific downstream signaling cascades. Ultimately, all signal transduction pathways converge at the level of gene expression where positive and negative modulators of transcription interact to delineate the pattern of gene expression and the overall cellular hemopoietic response. Transcription factors, therefore, represent a nodal point of hemopoietic control through the integration of the various signaling pathways and subsequent modulation of the transcriptional machinery. Transcription factors can act both positively and negatively to regulate the expression of a wide range of hemopoiesis-relevant genes including growth factors and their receptors, other transcription factors, as well as various molecules important for the function of developing cells. The expression of these genes is dependent on the complex interactions between transcription factors, co-regulatory molecules, and specific binding sequences on the DNA. Recent advances in various vertebrate and invertebrate systems emphasize the importance of transcription factors for hemopoiesis control and the evolutionary conservation of several of such mechanisms. In this review we outline some of the key issues frequently identified in studies of the transcriptional regulation of hemopoietic gene expression. In teleosts, we expect that the characterization of several of these transcription factors and their regulatory mechanisms will complement recent advances in a number of fish systems where identification of cytokine and other hemopoiesis-relevant factors are currently under investigation.

Antimicrobial mechanisms of fish phagocytes and their role in host defense.

Phagocytosis is a primitive defense mechanism in all multicellular animals. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens. These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, macrophages and neutrophils can be activated to produce a number of highly toxic molecules. Production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens. Studies in fish suggest that the biological basis of these inducible killing mechanisms is similar to those described in mammals. More recent work suggest novel roles for regulating these killing responses in fish. In this review, we describe the biological basis of these killing mechanisms and how they are regulated in fish.

Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes.

Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the goldfish. Flow cytometric analysis of the initial T-PKM cultures, identified three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. >8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, goldfish IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and flow cytometry, we identified two ROI-producing sub-populations in goldfish IVDKM but only a single ROI-producing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the flow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and goldfish is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a final differentiation step in vitro.

Characterisation of growth enhancing factor production in different phases of in vitro fish macrophage development.

We previously described the release of macrophage growth factor(s) (MGF) into culture supernatants (CCM) by a goldfish macrophage cell line (GMCL) and in vitro derived kidney macrophages (IVDKM). In this study, we report that IVDKM growth can be subdivided into three developmental phases, defined using both morphological and flow cytometric characteristics: a lag phase, a proliferative phase, and a senescence phase. Analysis of the growth inducing capabilities of CCM indicated that maximum activity was consistently found in supernatants isolated from IVDKM cultures during the proliferative phase of development. In contrast, CCM from the senescence phase proved to be poor inducers of macrophage growth. Overall, we identify a link between the seeding-CCM composition, the extent of IVDKM growth and the rate of entrance into a senescent state characterised by IVDKM apoptotic cell death. Use of IVDKM CCM obtained at the peak of macrophage growth maximised macrophage growth factor (MGF) activity, and prevented the introduction of negative regulators of IVDKM proliferation, which will contribute significantly to our MGF purification efforts. Furthermore, the collection of IVDKM, prior to their commitment into apoptotic pathways, will prove to be essential in the selection of specific cell subsets for studies of antimicrobial mechanisms of macrophages.

Inactivation of Cryptosporidium parvum oocysts using medium- and low-pressure ultraviolet radiation.

The effect of ultraviolet radiation from low- and medium-pressure mercury arc lamps on Cryptosporidium parvum oocysts was studied using a collimated beam apparatus. Experiments were conducted using parasites suspended in both filtered surface water and phosphate buffered laboratory water. Inactivation of oocysts was measured as reduction in infectivity using a CD-1 neonatal mouse model and was found to be a non-linear function of UV dose over the range of germicidal doses tested (0.8-119 mJ/cm2). Oocyst inactivation increased rapidly with UV dose at doses less than 25 mJ/cm2 with two and three log-units inactivation at approximately 10 and 25 mJ/cm2, respectively. The cause of significant leveling-off and tailing in the UV inactivation curve at higher doses was not determined. Maximum measured oocyst inactivation ranged from 3.4 to greater than 4.9 log-units and was dependent on different batches of parasites. Water type and temperature, the concentration of oocysts in the suspension, and the UV irradiance did not have significant impacts on oocyst inactivation. When compared on the basis of germicidal UV dose, the oocysts were equally sensitive to low- and medium-pressure UV radiation. With respect to Cryptosporidium, both low- and medium-pressure ultraviolet radiation are attractive alternatives to conventional chemical disinfection methods in drinking water treatment.

Sequential inactivation of Cryptosporidium parvum using ozone and chlorine.

Inactivation of bovine-derived C. parvum oocysts was studied at bench-scale in oxidant demand free 0.05 M phosphate buffer using free chlorine alone or ozone followed by free chlorine at temperatures of 1 degrees C, 10 degrees C and 22 degrees C at pH 6. Animal infectivity using neonatal CD-1 mice was used for evaluation of oocyst viability after treatment. Kinetic models based on the linear Chick-Watson model were developed for free chlorine inactivation and ozone/free chlorine sequential inactivation for 0.4 or 1.6 log-units of ozone primary kill. At 22 degrees C. ozone pre-treatment increased the efficacy of free chlorine for about 4-6 times depending on the level of ozone primary kills. Gross kills of the ozone/free chlorine sequential inactivation were a function of ozone primary kills and increased linearly with the free chlorine C(avg)t (arithmetic average of the initial and final residual x contact time) product. Temperature was critical for both single and sequential inactivation, and the efficacy of free chlorine after 1.6 log-units of ozone primary inactivation decreased by a factor of 1.8 for every 10 degrees C temperature decrease. Given an ozone primary kill of 1.6 log-units, the free chlorine C(avg)t products required for a gross kill of 3.0 log-units were 1000, 2000 and 3,300 mgmin/L for 22 degrees C. 10 degrees C and 1degrees C, respectively.

Products of proteolytic cleavage of transferrin induce nitric oxide response of goldfish macrophages.

Enzymatic cleavage product of transferrin induced the production of nitric oxide (NO) by LPS-stimulated goldfish macrophages. A NO-inducing factor was purified from the supernatants of mitogen-stimulated goldfish kidney leukocytes using fast performance liquid chromatography (FPLC) and the purified proteins analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry. The proteins were identified as truncated forms of transferrin, having approximate molecular weights (MW) of 33, 35, and 37kDa (kilodaltons). The precursor form (i.e. full-length) of transferrin did not enhance NO production by LPS-stimulated goldfish macrophages, but enzymatic cleavage of this precursor form correlated with enhanced production of NO by goldfish macrophages. Enzymatic cleavage of transferrin was dependent on the presence of stimulated kidney leukocytes and was shown to occur in response to both mixed lymphocyte reactions (MLR) and the mitogenic stimulation of goldfish kidney leukocytes. Time course analysis revealed that 24h after kidney leukocyte MLR or mitogen stimulation, cleaved transferrin products appeared in the supernatants of cultured cells, which was related to the on-set of NO-inducing activity of these preparations. To confirm these findings, bovine transferrin was digested in vitro using protease XXVII. The resulting cleavage products had approximate MW of 33, 35, and 37kDa. When these peptides were subjected to the purification protocols used to purify a NO-inducing factor from goldfish leukocyte supernatants, they were shown to elute to identical fractions. To examine the potential role of fish transferrin in mediating goldfish NO production, carp transferrin was purified from serum and following protease-digestion and purification by FPLC, the truncated proteins were found to elute to similar fractions as bovine transferrin. Furthermore, mitogen-stimulated leukocyte supernatants prepared in the absence of bovine serum (carp serum only) retained NO-inducing activity, indicating that this response was not an artifact of bovine serum components (i.e. bovine transferrin). Anti-bovine and anti-carp transferrin polyclonal antibodies identified the presence of truncated forms of transferrin in the active fractions of FPLC-separated mitogen-stimulated leukocyte supernatants prepared in the presence of bovine or carp serum, respectively. Thus, our results suggest a novel role for fish transferrin as one of the factors that mediates teleost macrophage antimicrobial functions.

Generation and functional analysis of distinct macrophage sub-populations from goldfish (Carassius auratus L.) kidney leukocyte cultures.

Three distinct sub-populations of macrophages derived from goldfish kidney leukocyte cultures were generated and characterised. The sub-populations designated as R1, R2 and R3-type macrophages had distinct morphological, cytochemical and flow cytometric profiles, and also differed in their anti-microbial functions after activation with macrophage activation factors (MAF) and bacterial lipopolysaccharide (LPS). The R1-type macrophages were small cells that contained acid phosphatase, but lacked myeloperoxidase and non-specific esterase. The R2-type macrophages were morphologically similar to mature tissue macrophages of mammals, and were positive for acid phosphatase, myeloperoxidase and non-specific esterase. The R3-type macrophages were round cells with eccentrically placed nuclei and resembled mammalian monocytes. This sub-population stained for acid phosphatase, myeloperoxidase and non-specific esterase. The R2 and R3-type macrophages exhibited distinct functional responses after activation with MAF and/or LPS. R2-type macrophages were potent producers of nitric oxide, while R3-type macrophages produced little or no nitric oxide after activation with MAF and LPS. The R2 and R3-type macrophages also exhibited unique respiratory burst responses (ROI) after treatment with MAF and/or LPS. After treatment with MAF and LPS, activated R2 macrophages were primed for ROI after only 6 h of stimulation with the activating agents, and continued to exhibit a strong ROI response for an extended cultivation period (48 h). In contrast, activated R3-type macrophages showed an early ROI response (6 h after treatment with MAF and LPS), which decreased significantly by 48 h after treatment with the activating agents. Our results suggest that the analysis of the mechanisms of induction of fish anti-microbial responses may be dependent upon the concerted actions of functionally distinct macrophage sub-populations.

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes.

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.

Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages.

We recently demonstrated that a goldfish macrophage cell line (GMCL) and primary in vitro-derived kidney macrophage (IVDKM) cultures contain three distinct macrophage subpopulations. Morphological, cytochemical, functional, and flow cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at distinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of mature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fluorescent dye and analysis of the data using the MODFIT software. Our results showed that IVDKM cultures proliferated non-synchronously, suggesting the presence of a temporal control mechanism regulating the number of cells entering the paths towards maturation. Such control is most evident during early progenitor proliferation and differentiation events. Our results showed that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observation of the mature macrophage-like subpopulation indicated that: 1) they appear to develop from both, the differentiation of monocyte-like cells, and direct differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage-like cells appeared to be capable of self-proliferation. Our results suggest the presence of alternate pathways of fish macrophage development other than the classical hematopoietic pathway.

Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice.

In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.

Comparison of animal infectivity and nucleic acid staining for assessment of Cryptosporidium parvum viability in water.

Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.

Inhibition of macrophage activity by mitogen-induced goldfish leukocyte deactivating factor.

Macrophage activating and deactivating cytokines have been characterized in mammalian systems but little is known about these immunoregulatory molecules in fish. Using gel permeation and chromatofocusing fast performance liquid chromatography (GP-FPLC and C-FPLC) we partially purified a macrophage deactivating factor (MDF) from mitogen-induced goldfish kidney leukocytes. Inhibition of the macrophage-derived nitric oxide (NO) response induced by this MDF was time-, dose- and temperature-dependent. Macrophages pre-treated for 6 or 24 h with MDF before activation with macrophage activating factors (MAF) and/or bacterial lipopolysaccharide (LPS) exhibited a down-regulation in their NO response, while those treated with MDF 24 h after activation with MAF and LPS did not. MDF treatment also impaired the NO response of goldfish macrophages infected with the mammalian protozoan parasite Leishmania major. These results suggest that MDF exhibits its inhibitory effect downstream of the converging intracellular pathways induced by LPS and/or L. major. The novel teleost MDF has an approximate Mr of 15 kD and a pI < 4, and is the first endogenous molecule of teleosts known to down regulate macrophage antimicrobial responses.

Treatment of the macrophage-like P388D.1 cells with bacterial lipopolysaccharide and interferon-gamma causes long-term alterations in calcium metabolism.

The intracellular calcium concentrations ([Ca2+]i) of P338D.1 macrophage-like cells, activated with interferon-gamma (IFN-gamma) and/or bacterial lipopolysaccharide (LPS) were determined using fura-2/AM and ratiometric imaging techniques. Treatment of macrophages with IFN-gamma and LPS resulted in significant downward shift in [Ca2+]i, 8, 16 and 24 h but not at 1 and 4 h after treatment. The decrease in [Ca2+]i also occurred when macrophages were treated with LPS only, but not after exposure of the cells to recombinant IFN-gamma, indicating that LPS was an essential signal in the observed changes in [Ca2+]i of activated macrophages. The IFN-gamma and/or LPS alteration in the [Ca2+]i, paralleled the in vitro nitric oxide production of the activated macrophages, 8, 16 and 24 h after treatment. The decrease in the [Ca2+]i may be caused by vigorous buffering and storing of Ca2+ by macrophages to below the normal resting quantities, following the reported transient increase in Ca2+ during the priming stage of macrophage activation. Thus, the downward shift in [Ca2+]I may play a physiological role in the activation processes of macrophages for antimicrobial responses.