Mercury in blood cells—Altered elemental profiles

The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure.     

Galvanotaxis of human granulocytes

The galvanotactic response of human granulocytes was investigated theoretically and experimentally. The basic results are: (i) The granulocytes move towards the anode. (ii) The directed movement has been quantified by two different polar order parameters-the McCutcheon index and the average of cos . (iii) The polar order parameters are a function of the applied electric field (= dose-response curve). (iv) The inverse of the galvanotactic constant of migrating cells (analogous to the Michaelis-Menten constant) has a value of-0.2±0.03 V/mm. (v) The galvanotactic response of granulocytes is a non-cooperative process with a cooperativity coefficient of 1±0.2. (vi) The galvanotactic constant is a function of pH. (vii) The protein essential for the galvanotactic response is very likely a G-protein.     

A sensitive procedure for quantifying the phagocytic competence of blood granulocytes

The phagocytic activity of blood granulocytes can be quantitatively assayed by ingestion of opsonised paraffin oil droplets containing the dye Oil Red-O (Stossel, T. P., Mason, R. J., Hartwig, J., and Vaughan, M. (1972)J. Clin. Invest. 51:615–624). We have modified this assay by incorporating [³H]glycerol into the oil droplets which allows a more sensitive and reproducible measurement of the phagocytic competence of blood granulocytes even at very low cell counts. Comparative studies after one day storage of the blood at 4°C is feasible since they retain 84 % of the phagocytic capacity measured when isolated from fresh blood.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

Mechanisms towards compensation of long-term haemopoietic injury in mice after 5 Gy irradiation:In vivo andin vitro enhancement of superoxide anion production by granulocytes

This paper analyzes the long-term (6 and 12 months) function of mouse granulocytes after total body irradiation with a single dose (5 Gy) of X-rays. Superoxide anion production has been investigated in granulocytes from peripheral blood, and also in those harvested from long term bone marrow cultures, with the aim of correlating the environmental damage induced by radiation with the functional properties of granulocytes. Anin vivo andin vitro enhancement of superoxide anion production and protein levels in granulocytes from irradiated mice is described. The presence of some colony stimulating factor in the supernatant of cultures from irradiated mice could play an important role in the priming of granulocytes.     

The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.     

Individual variation of nucleoside triphosphate pyrophosphohydrolase activity in human erythrocytes,granulocytes, lymphocytes,and platelets

Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5–125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/10⁷ cells, granulocytes have 17–201 units/10⁷ cells, lymphocytes have 91–462 units/10⁷ cells, and platelets have 1.1–7.1 units/10⁷ platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.This work was supported by funds allocated by the Agricultural Experiment Station, Michigan State University. Michigan Agricultural Experiment Station Journal No. 8727.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Acidophilic granulocytes of the marine fish gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) produce interleukin-1β following infection with<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis>

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1 (IL-1) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1 intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1 following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1 were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed. This work was supported by the Spanish Ministry of Science and Technology (grants BIO2001-2324-C02-02 and AGL2002-03529, and fellowship to J. García-Castillo), Spanish Ministry of Education, Culture and Sport (fellowship to E. Chaves-Pozo) and Fundación Séneca, Coordination Centre for Research (grant PI-51/00782/FS/01 and fellowship to P. Pelegrín).E. Chaves-Pozo and P. Pelegrín contributed equally to this work.     

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.