Chemotactic selection with insulin, di-iodotyrosine and histamine alters the phagocytotic responsiveness of Tetrahymena

Chemotactic selection is a method by which populations of cells exposed to ligands can be isolated and subsequently cultivated. We used Tetrahymena pyriformis GL cultures selected by chemotactic selection to insulin (10 nM), histamine (0.1 nM) and di-iodotyrosine (T2, 10 nM) to study the phagocytotic capacity under the induction of selector hormones. Our results show a long-lasting link between chemotactically selected cultures and phagocytotic activity. Cells selected to histamine produced the highest phagocytotic activity upon a second exposure to the selector hormone. T2 selection was also strongly effective, however, the phagocytosis stimulation was not specific to the hormone given later. Insulin selected sub-populations had different phagocytotic responses to the control substance itself, whereas histamine selected sub-populations seem to be heterogeneous in the phagocytotic response to histamine. For insulin, the increased endocytotic or metabolic activity was demonstrated by the lack of non-phagocytotic cells. These experiments call attention to the evolutionary role of selection in the later developing receptor-hormone relationship.     

Presence and localization of histidine decarboxylase enzyme (HDC) and histamine in Tetrahymena pyriformis.

Histidine decarboxylase (HDC) enzyme and its function under hormonal influences were studied in a low level of phylogeny. HDC protein is present in the unicellular ciliate Tetrahymena and its expression was not altered by insulin or histamine treatment. Starvation for 24 h enormously decreased the quantity of histamine in the cells. However, insulin influenced the activity of the HDC enzyme, demonstrated by the seven-fold quantity of histamine in the starved cells after insulin treatment. Insulin also increased the uptake of histamine from the tryptone-yeast extract medium. HDC was found in different parts of the cytoplasm, mainly in the periphery (epiplasm) of the cells. The experiments demonstrated the uptake and synthesis of histamine by Tetrahymena as well as the possibility of hormonal regulation of HDC activity.     

Studies into secretions of Tetrahymena: enzymes secreted into inorganic medium.

The peptides secreted by Tetrahymena cells into inorganic medium were chromatographed. Six fractions showing a marked enzyme-like activity were examined for influence on certain physiological parameters of Tetrahymena. The enzymatically active fractions increased the phagocytic activity of Tetrahymena and decreased its binding capacity for lectins and hormone (insulin), but enhanced insulin imprinting at primary interaction. It remains to be clarified whether these effects were due to the enzymatic or other components of the fractions investigated, or to lack of the compensatory influence of the fractions not studied.     

Immunological and cytotoxicological responses of the Asian clam, Corbicula fluminea (M.), experimentally exposed to cadmium

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^-1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^-1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^-1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

In vitro effect of biogenic amines on the hormone content of immune cells of the peritoneal fluid and thymus. Is there a hormonal network inside the immune system?

Immune cells contain different hormones and hormone-like molecules, such as insulin, endorphin, triiodothyronine (T3) histamine, serotonin. In earlier in vitro experiments insulin down-regulated histamine, serotonin and T3 content of thymus cells. Now we studied the effect of biogenic amines on the endorphin, T3, serotonin and histamine content of rat peritoneal and thymic cells. Cells were obtained from male rats of 100g body weight. 100 ng/ml serotonin or 300 ng/ml histamine was added for 30 min. After that the cells were prepared for flow cytometric analysis with antibodies to endorphin, T3, histamine and serotonin as primary antibodies and anti-rabbit IgG as secondary antibody. Finishing the measurements the cells were also studied by confocal microscopy. T3 concentration (binding of anti-T3 antibody) increased in peritoneal mast cells after serotonin treatment and in the monocyte-macrophage-granulocyte group after histamine treatment. Thymocytes' T3 content radically decreased after both treatments. Serotonin and histamine treatment also radically reduced the amine content of each other. Endorphin level was resistant to hormonal treatments. The results call attention to a possible hormonal network inside the immune system in which hormones produced by the immune cells themselves can influence each other.     

Effect of insulin and isoproterenol on macrophage phagocytic activity

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.     

Immunological and cytotoxicological responses of the Asian clam,Corbicula fluminea (M.), experimentally exposed to cadmium

Abstract

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^?1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^?1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^?1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

Changes in some endocrinological and non-specific immunological parameters during seawater exposure in the brown trout

The influence of autumnal progressive and direct seawater transfers on ionic parameters, plasma growth hormone (GH) and thyroid hormones (TH) and also on the non–specific immune traits phagocytic activity, lysozyme and non–specific cytotoxicity were examined in 45–55 g brown trout ( Salmo trutta ). In both experiments, the seawater transfer induced the same pattern of endogeneous modifications but they were more pronounced and more lasting after the direct seawater transfer than after the progressive one. In seawater–transferred trout, there was a significant transitory increase of the plasma osmolarity, chloride concentration, GH levels and a transient decrease of the TH. The phagocytic activity of the pronephric leucocytes and the lysozyme concentrations were significantly higher in seawater–transferred trout than in controls. Nevertheless, the non–specific cytotoxicity should not be modified after the seawater exposure. Moreover significant positive correlations were observed between plasma GH and chemiluminescence or lysozyme increases. These data support the hypothesis that GH is involved in the salmonids' non–specific immune potential, especially by stimulating the macrophage functions.     

Phagocytosis as a Biomarker of Immunotoxicity in Wildlife Species Exposed to Environmental Xenobiotics

In the present paper, we are reviewing experimental evidencedemonstrating that phagocytic cells, such as macrophages, maybe used as a biomarker of immunotoxicity in wildlife studies.We will first present data obtained after exposure in vitrowith selected chemicals showing the comparative sensitivityof phagocytic cells from different species. These results demonstratethat, at least for metals, each species produce a similar shapeddose-response curve, although considerate interspecies sensitivityis evident. These results also demonstrate the sensitivity ofthe phagocytic activity, suggesting indeed that this functioncould be used to monitor exposure to chemicals. The similarshaped dose-response curves imply that mechanisms of actionmay also be similar. Furthermore, based on the relative speicessensitivity, sentinel species could be selected for field monitoring.Such an approach may also be useful to establish correctionfactors required to extrapolate results between species. Thissensitivity of the phagocytic activity of macrophages will befurther under controlled conditions in laboratory animal models.Finally, the reliability of this approach will be demonstratedusing case studies with wildlife species.     

Suppression of nonspecific resistance of the body under the effect of extremely high frequency electromagnetic radiation of low intensity

The dynamics of leukocyte number and functional activity of peripheral blood neutrophils under whole-body exposure of healthy mice to low-intensity extremely-high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.15 mW/cm2, 20 min daily) was studied. It was shown that the phagocytic activity of peripheral blood neutrophils was suppressed by about 50% (p < 0.01 as compared with the sham-exposed control) in 2-3 h after the single exposure to EHF EMR. The effect persisted for 1 day after the exposure, and then the phagocytic activity of neutrophils returned to the norm within 3 days. A significant modification of the leukocyte blood profile in mice exposed to EHF EMR for 5 days was observed after the cessation of exposures: the number of leukocytes increased by 44% (p < 0.05 as compared with sham-exposed animals), mostly due to an increase in the lymphocyte content. The supposition was made that EHF EMR effects can be mediated via the metabolic systems of arachidonic acid and the stimulation of adenylate cyclase activity, with subsequent increase in the intracellular cAMP level. The results indicated that the whole-body exposure of healthy mice to low-intensity EHF EMR has a profound effect on the indices of nonspecific immunity.     

A functional study of concanavalin A-histamine binding site overlap in Tetrahymena phagocytosis test

Treatment with histamine stimulated the phagocytotic activity of the Tetrahymena to a measurable degree, which was still demonstrable after a week (about 40 generations). Concanavalin A, which binds to the same membrane binding site as histamine, inhibited the stimulatory action of subsequently added histamine, but did not in itself influence phagocytotic activity in any way. The inhibitory effect of Con A on the histamine binding site proved to be dose-dependent. These observations stress the importance of investigating the functional context--as sole realistic measure--of receptor--ligand bindings.     

Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena

FITC-insulin binding to previously hormone-treated Tetrahymena was studied by flow cytometry and confocal microscopy. Hormones produced by Tetrahymena were chosen for study and the hormone concentrations were administered between 10(-6) and 10(-21)M for 30 min. Endorphin, serotonin and insulin significantly reduced the hormone binding however histamine did not influence it at all. Endorphin, serotonin and insulin were significantly effective down to 10(-18)M and the effect of insulin and endorphin suggest a similar mechanism. The results call attention to the efficacy of very low hormone concentrations, which can influence the hormone content (earlier experiments) and receptor binding capacity (present study) of a unicellular organism. This seems to be very important, as in wild (natural) conditions the dilution of signaling materials secreted by a water-living protozoan is very high. In addition, the results point to the selectivity of response, as not all of the hormones that deeply influence other physiological indices (e.g. histamine) have an effect on insulin content or insulin receptors.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress

In this present study, Oreochromis mossambicus tilapia were transferred to cold water at 12°C for various time intervals (1, 4, 8, 24, and 48 hr) and its innate immune response was analyzed by studying cellular and humoral parameters. In vivo, alternative complement pathway activity in blood plasma was rapidly increased at 1 hr of cold water (12°C) exposure. Lysozyme activity and cortisol levels of plasma were increased at 4 and 1 hr, respectively. Surprisingly, only plasma cortisol levels remained unchanged through 24 hr of cold water transfer. Phagocytic ability, phagocytic capacity, and respiratory burst (RB) activity of head kidney (HK) leukocytes and splenocytes showed no any significant changes. In peripheral blood leukocytes, phagocytic capacity, and RB activity were increased at 24 hr of cold water exposure. The expressions of genes involved innate immunity in splenocytes and HK leukocytes of tilapia cold water exposure were analyzed, messenger RNA (mRNA) expressions of HSP70, HSP90, and immunoglobulin M failed to change upon exposure to cold stress. Major histocompatibility complex-I and II mRNAs were significantly increased in tilapia splenocytes at 1 hr of cold water transferred. Whereas myxovirus (Mx) expression was increased in splenocytes and HK leukocytes of tilapia after 1 hr of cold water exposed. Our result reveals that the exposure of tilapia to acute cold stress condition significantly enhances plasma acid phosphatase activity and both phagocytic capacity and RB activity. Furthermore, cold stress significantly stimulates Mx gene expression in splenocytes and HK leukocytes.     

Effects of exogenous growth hormone on insulin action and glucose metabolism in the dwarf 'little' mouse

The in vivo actions of growth hormone (GH) on insulin activity and glucose homeostasis were examined in the GH-deficient Little mouse. The insulin-like action of GH was revealed during glucose tolerance tests on the animals after acute treatment with the hormone and the insulin-antagonistic action was demonstrated in both glucose tolerance tests and insulin tolerance tests on the mice after chronic GH infusion. The primary mechanism of the GH actions is to influence the responses of the target tissues to circulating insulin in vivo. The pancreatic function seems to be of little importance in the alteration of glucose metabolism after acute exposure to GH as no significant change of the levels of plasma insulin was detected. It is concluded that the GH-deficient Little mouse is an ideal laboratory model for the elucidation of the molecular mechanism of the interaction between insulin and GH in the regulation of carbohydrate metabolism.     

Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.     

Alteration in cellular functions in mouse macrophages after exposure to 50 Hz magnetic fields

The aim of the present study is to investigate whether extremely low frequency electromagnetic fields (ELF-EMF) affect certain cellular functions and immunologic parameters of mouse macrophages. In this study, the influence of 50 Hz magnetic fields (MF) at 1.0 mT was investigated on the phagocytic activity and on the interleukin-1beta (IL-1beta) production in differentiated macrophages. MF-exposure led to an increased phagocytic activity after 45 min, shown as a 1.6-fold increased uptake of latex beads in MF-exposed cells compared to controls. We also demonstrate an increased IL-1beta release in macrophages after 24 h exposure (1.0 mT MF). Time-dependent IL-1beta formation was significantly increased already after 4 h and reached a maximum of 12.3-fold increase after 24 h compared to controls. Another aspect of this study was to examine the genotoxic capacity of 1.0 mT MF by analyzing the micronucleus (MN) formation in long-term (12, 24, and 48 h) exposed macrophages. Our data show no significant differences in MN formation or irregular mitotic activities in exposed cells. Furthermore, the effects of different flux densities (ranging from 0.05 up to 1.0 mT for 45 min) of 50 Hz MF was tested on free radical formation as an endpoint of cell activation in mouse macrophage precursor cells. All tested flux densities significantly stimulated the formation of free radicals. Here, we demonstrate the capacity of ELF-EMF to stimulate physiological cell functions in mouse macrophages shown by the significantly elevated phagocytic activity, free radical release, and IL-1beta production suggesting the cell activation capacity of ELF-EMF in the absence of any genotoxic effects.     

Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC(50-96 h)) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms.     

Effects of hydroxyurea on the phagocytosis of microspheres in V79 cells

Phagocytosis of fluorescent microspheres in V79 cells arrested in the S phase by hydroxyurea (HU) was investigated by flow cytometry. The phagocytic activity increased with the exposure time of HU. No dependency on the HU concentration was observed in the concentration used. Cell size (projection area) and membrane fluidity, two factors effected by HU treatment, were examined in relation to phagocytic activity, and were found to be significantly increased. The elevation in phagocytic activity could be interpreted in terms of the alteration in the two physiological factors.     

The effect of insulin on the growth of transplanted tumors in mice

The growth of four murine transplantable tumours, Ehrlich ascites or solid tumour, an aplastic carcinoma, B-16 melanoma, and a thymoma, were suppressed in mice treated daily with insulin (2 IU). Since insulin increased the number of plaque-forming cells and the phagocytic activity of the liver and spleen cells, the retardation of the tumour growth was ascribed to immunological mechanisms.