Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

Characteristics of the functional activity of human blood neutrophils using the nitroblue tetrazolium reduction test]

A study was made of the test of restoration of the nitroblue tetrazolium (NBT) by the intact blood neutrophils and the neutrophils stimulated with various endotoxin doses of Gram-negative bacteria in 70 healthy persons. A high sensitivity of the test with the NBT for the quantitative assessment of the neutrophil response under conditions stimulating the phagocytic activity was demonstrated. A functional nonhomogeneity of the neutrophil population, the most distinct with the minimal stimulating action, was noted.     

Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.     

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.     

Phagocytosis of Candida albicans by rabbit alveolar macrophages and guinea pig neutrophils.

Studies of host-parasite relationships at the cellular level, using Candida albicans and rabbit alveolar macrophages or guinea pig neutrophils are presented. Guinea pig neutrophils killed the intracellular candida cells presumed by myeloperoxidase-halide-hydrogen peroxide system. In contrast, rabbit alveolar macrophages did not kill the intracellular candida cells although their phagocytic rate was almost comparable to that of neutrophils. Phagocytizing macrophages were eventually destroyed by the intracellular proliferation of candida cells and formation of germ tubes and pseudomycelia. No significant improvement of candidacidal activity was observed with macrophages from normal and immunized rabbits in immune serum. The mode of phagocytosis by macrophages and neutrophils were also studied under the scanning electron microscope.     

Protective effect of acidic mannan fraction of bakers' yeast on experimental candidiasis in mice

An acidic fraction of bakers' yeast mannan, WAM025, showed a significant protective effect against Candida albicans infection in mice, but a neutral fraction of the same bakers' yeast mannan, WNM, did not exhibit this effect. Moreover, pretreatment with WAM025 resulted in a marked reduction of proliferation of C. albicans cells in the organs of the infected mice. We investigated the stimulative effect of these mannan fractions on the function of mouse peritoneal phagocytes, and found that mice administered WAM025 showed a greater increase in the number of peritoneal exudate cells, macrophages and polymorphonuclear leucocytes (PMN), than the mice treated with WNM, especially in the proportion of PMN. Peritoneal phagocytes, PMN and macrophages obtained from WAM025-treated mice showed marked candidacidal activity. Of the phagocytes, PMN were responsible for the larger part of the candidacidal activity. The myeloperoxidase activities of PMN and macrophages in WAM025-treated PEC were greater than in untreated macrophages. The myeloperoxidase activity of WAM025-treated PMN was significantly greater than that of WAM025-treated macrophages. This activity paralleled the active oxygen-releasing activity of the phagocytes. On the other hand, the phagocytic activity of phagocytes from mice administered WNM or WAM025 for C. albicans cells was identical to that of untreated phagocytes. WAM025 seems to cause enhance elimination of the pathogen from mice, by increasing the number and candidacidal activity of phagocytic cells.     

Rac GTPase isoform-specific regulation of NADPH oxidase and chemotaxis in murine neutrophils in vivo. Role of the C-terminal polybasic domain

The Rho family GTPase Rac acts as a molecular switch for signal transduction to regulate various cellular functions. Mice deficient in the hematopoietic-specific Rac2 isoform exhibit agonist-specific defects in neutrophil chemotaxis and superoxide production, despite expression of the highly homologous Rac1 isoform. To examine whether functional defects in rac2(-/-) neutrophils reflect effects of an overall decrease in total cellular Rac or an isoform-specific role for Rac2, retroviral vectors were used to express exogenous Rac1 or Rac2 at levels similar to endogenous. In rac2(-/-) neutrophils differentiated from transduced myeloid progenitors in vitro, increasing cellular Rac levels by expression of either exogenous Rac1 or Rac2 increased formylmethionylleucylphenylalanine- or phorbol ester-stimulated NADPH oxidase activity. Of note, placement of an epitope tag on the N terminus of Rac1 or Rac2 blunted reconstitution of responses in rac2(-/-) neutrophils. In rac2(-/-) neutrophils isolated from mice transplanted with Rac-transduced bone marrow cells, superoxide production and chemotaxis were fully reconstituted by expression of exogenous Rac2, but not Rac1. A chimeric Rac1 protein in which the Rac1 C-terminal polybasic domain, which contains six lysines or arginines, was replaced with that of the human Rac2 polybasic domain containing only three basic residues, also reconstituted superoxide production and chemotaxis, whereas expression of a Rac2 derivative in which the polybasic domain was replaced with that of Rac1 did not and resulted in disoriented cell motility. Thus, the composition of the polybasic domain is sufficient for determining Rac isoform specificity in the production of superoxide and chemotaxis in murine neutrophils in vivo.     

Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles

In this study we tested the hypothesis that after administration of a single intraperitoneal dose of concanavalin A (Con-A) to mice, the proportion of neutrophils and macrophages in the peritoneal exudate and their phagocytic and candidacidal activities should change with time. The number of neutrophils in the peritoneal exudate was greatly increased 6 h after administration of Con-A, and those cells were able to kill both intracellular and extracellular yeast and germ tube forms of Candida albicans. Addition of catalase to the culture medium reduced the killing of C. albicans, suggesting that the candidacidal activity depended on the myeloperoxidase system. The survival of mice pretreated with Con-A and submitted to an inoculum of C. albicans 6 h afterwards was twice higher than that of controls, which suggests that neutrophils were able to clear the experimental infection. One day after the treatment, the population of neutrophils in the exudate was about 45%, but after 2 days it was reduced to only 5% and the candidacidal activity was also reduced. After 4 days the exudate contained over 95% of macrophages, the candidacidal activity reached a maximum, and the phagocytosis mediated by both complement receptors and mannose receptors was increased. Uptake of FITC-mannose-BSA by macrophages was maximal on about the 4th day and was inhibited by mannan, suggesting that treatment with Con-A increased the activity of mannose receptors. These results support the hypothesis that activation of cellular immunity by Con-A occurred in two phases, one dominated by neutrophils, and the other by macrophages expressing increased activity of mannose receptors.     

Improved functional recovery of human granulocytes after cryopreservation

Human peripheral blood phagocytes (90% neutrophils) were cryopreserved with either 5 or 10% dimethyl sulfoxide (DMSO) and stored in the liquid phase of liquid nitrogen. Modifications to the freezing method included the elimination of dextran from the freezing medium, addition of the bulk of the DMSO at −5 °C, elimination of heparin and centrifugation from all postreconstitution procedures, and the use of deoxyribonuclease to minimize post-thaw granulocyte agglutination.Substantial numbers of the cryopreserved phagocytes, as assayed by nitroblue tetrazolium and chemotactic activity, showed comparable functional activity to fresh cells. Post-thaw cell dialysis further improved functional capacity although probably not as a consequence of DMSO removal.     

Functional and metabolic activity of peripheral blood neutrophils in acute viral hepatitis B and C

The functional metabolic activity of peripheral blood neutrophils in acute virus hepatitis B (VHB) and/or virus hepatitis C (VHC) was evaluated. 48 patients were examined; of these, VHB was diagnosed in 28 patients and VHC was diagnosed in 9 patients and the mixed form of virus hepatitis (VHB + VHC), in 11 patients. Determination of adhesive capacity of neutrophils, production of superoxidase anion in the nitro blue tetrazolium (NBT) test, activity of myeloperoxidase (MPO) and acidic phosphatase (AP), the amount of cation proteins (CP) was made. Most pronounced functional dysbalance of neutrophil leukocytes and considerable changes in biochemical characteristics of the activity of the infectious process in patients with the mixed form of virus hepatitis were established. These data demonstrated that in acute virus hepatitis B and C at the peak of the disease such characteristics of the functional activity of neutrophils as results of the NBT test, the activity of MPO and AP, as well as the amount of CP, were highly informative.     

Reconstitution of the diiron sites in hemerythrin and myohemerythrin

The first reconstitutions of functional diiron sites in the nonheme O2-carrying proteins hemerythrin (Hr) and myohemerythrin (myoHr) have been achieved. Both proteins are reconstituted under anaerobic conditions, and the procedure consists of (i) denaturation of the native met form with 6 M guanidinium chloride in the presence of sodium dithionite and 2,2'-dipyridyl, (ii) separation of the apoprotein from the other reagents and products, (iii) addition of an iron(II) stock solution to the apoprotein in the presence of 2-mercaptoethanol, and (iv) several cycles of slow dilution and reconcentration by ultrafiltration to remove excess reagents. Iron analyses indicate that the apoproteins have been essentially completely freed of iron and that reconstituted Hr contains its full complement of iron, i.e., approximately 2 Fe/subunit. Ferrous rather than ferric iron appears to be necessary for recovery of the native structures for both myoHr and Hr. In the case of Hr, reconstitution was successful only when iron(II) was added to apoHr prior to removal of denaturant. ApoHr is essentially insoluble at pH 7 in the absence of denaturants but remains soluble when denaturant is removed in the presence of ferrous iron, which leads to recovery of the octameric structure containing all of its diiron sites. Iron(II) apparently stabilizes the native or a nearly native structure during reconstitution. OxymyoHr and oxyHr are the major initial products of reconstitution. The yield of oxymyoHr from apomyoHr was approximately 87%. In contrast to reconstituted oxymyoHr, where essentially all of the iron appears to be functional, approximately 30% of the diiron sites in the reconstituted oxyHr are unable to bind O2 at ambient p(O2).(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid centrifugation method for the isolation of polymorphonuclear leucocytes from human blood

A rapid one-step gradient centrifugation method to prepare PMN leucocytes has been worked out by which a 98--99% pure, washed granulocyte suspension of 98% viability can be obtained in 30--40 minutes. The cells prepared by this method displayed higher NBT reduction upon ingesting the same soluble DNA-anti DNA complexes than those prepared by the dextran sedimentation method.     

Indicators of spontaneous and stimulated nitroblue tetrazole test of polymorphous-nuclear leucocytes in acute dysentery patients

140 healthy individuals and 93 sick with acute dysentery were subjected to an examination by spontaneous and by bacterial preparations stimulated reaction with nitroblue tetrazole (NBT test). Indicators in healthy persons were normal in the spontaneous, and increased in the NBT test, stimulated by bacterial preparations. Indicators of the spontaneous NBT test in patients with acute dysentery were raised with a maximum in the period of early convalescence. Stimulation by a live shigella culture--the dysentery vaccine--revealed by means of Sonne diagnostic high, and when endotoxin from Serratia marcescens and dysenterin was used as an inductor, mild indicators of NBT test activity. When a polyvalent agglutinating dysentery serum was used as a stimulator, the activity increased considerably, and a simultaneous use of serum and vaccine had an inhibiting effect on the indicators of the stimulated NBT test. The obtained results testify the sufficient high reserve possibilities of leucocytes towards complete phagocytosis and the efficiency of the NBT test, stimulated by bacterial preparations for the study of functional and metabolic activity of leucocytes in the process of acute bacterial dysentery.     

Neutrophil activation in vitro

Kinetics of spontaneous and induced reactions of Nitroblue tetrazolium (NBT) restoration by neutrophils was investigated in vitro. The index of activated neutrophils (IAN), commonly characterizing cell activation degree depending on the reaction time (t), was described by the equation: IAN = [NBT0](1-exp[-k1t]) + k0t; where (NBT0) is intracellular concentration of NBT, which reflects membrane permeability, k1 and k0--speed constants of the first and zero order reactions, characterizing, respectively, stimulation and spontaneous activities of enzymatic systems involved in NBT restoration. We detected the ability of heparin (2.0-7.5 mkg/ml) or chondrotin sulphate (0.25-2.5 mkg/ml) to activate neutrophils and presumably to maintain in vivo their spontaneous activity. The values of kinetics equation parameters enable us to speculate about relative hyper-, hypo- and normal function of neutrophils, related to the functional state of the whole organism. The value of [NBT0] changes from 8 to 103 conventional units, and a high correlation exists between this index and the reaction speed constants, differing in stimulated and spontaneously activated cells. The latter are represented by neutrophils only, which, as shown before, produce active forms of oxygen, used, probably, not for phagocytosis needs. The share of such cells makes approximately 30-40% of the neutrophil recirculating pool, and positively correlates with [NBT0], (r = 0.58; P < 0.05). This is determined by a very high membrane permeability of this neutrophil subpopulation. The detected different spontaneous and stimulated ability, various membrane permeability and enzyme system activity confirm the neutrophil functional unequality, which defines their activation peculiarity.     

Polymorphonuclear neutrophil dysfunctions in streptozotocin-induced type 1 diabetic rats

Since conflicting results have been reported on non-specific immune response in type 1 diabetes, this study evaluates polymorphonuclear neutrophil (PMN) functions in the infection free Long Evan diabetic rats (type 1) by using tests that include: polarization assay, phagocytosis of baker\'s yeasts (Saccharomyces cerevisiae) and nitroblue tetrazolium (NBT) dye reduction. Polarization assay showed that neutrophils from diabetic rats were significantly activated at the basal level compared to those from the controls (p < 0.001). After PMN activation with N-formylmethionyl-leucyl-phenylalanine (FMLP), control neutrophils were found to be more polarized than those of the diabetic neutrophils and the highest proportions of polarization were found to be 67 % and 57 % at 10(-7) M FMLP, respectively. In the resting state, neutrophils from the diabetic rats reduced significantly more NBT dye than that of the controls (p < 0.001). The percentages of phagocytosis of opsonized yeast cells by the neutrophils from control and diabetic rats were 87 % and 61 %, respectively and the difference was statistically significant (p < 0.001). Evaluation of the phagocytic efficiency of PMNs revealed that control neutrophils could phagocytose 381 +/- 17 whereas those from the diabetic rats phagocytosed 282 +/- 16 yeast cells, and the efficiency of phagocytosis varied significantly (p < 0.001). Further, both the percentages of phagocytosis and the efficiency of phagocytosis by the diabetic neutrophils were inversely related with the levels of their corresponding plasma glucose (p = 0.02; r = -0.498 and p < 0.05; r = -0.43, respectively), which indicated that increased plasma glucose reduced the phagocytic ability of neutrophils. Such relationship was not observed with the control neutrophils. These data clearly indicate that PMN functions are altered in the streptozotocin (STZ)-induced diabetic rats, and hyperglycemia may be the cause for the impairment of their functions leading to many infectious episodes.     

Immunotoxicity of carbaryl in chicken

Effect of methyl carbonate pesticide, carbonyl, was studied on macrophage functions, lymphocyte proliferation and delayed type hypersensitivity response. Sixteen adult chicken, vaccinated against Newcaslte disease, were procured and randomly divided in two experimental groups. Chicken of group I served as control, while group II birds were given carbaryl at 20 ppm (No observable effect level, NOEL) in feed for 3 months. To measure the functional activity of phagocytic cells, nitroblue tetrazolium (NBT) reduction test was performed on peripheral blood leucocytes. Concanvalin A (Con-A) and lipopolysaccharide stimulated proliferation of T and B lymphocytes was assessed using MTT dye method. At the end of experiment, the phagocytic capacities of macrophages were significantly reduced in carbaryl treated group. Lymphocyte proliferation responses to Con-A and LPS were (23 and 28%, respectively lower) in chicken fed with carbaryl. Delayed hypersensitivity reaction to tuberculin was reduced to 77% of control values indicating inhibition of cell mediated immune response. The present study suggested of immunosuppressive effect of (NOEL dose carbaryl) in chicken.     

The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterisation of neutrophils and macrophages in the normal and inflamed peritoneal cavity

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.     

Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils.

A cytochrome b with a midpoint oxidation-reduction potential of -245mV (cytochrome b-245) that is a major component of the microbicidal oxidase system of human neutrophil leucocytes has been identified in human eosinophils, monocytes and macrophages at concentrations similar to that found in human neutrophils. It was absent from a variety of other cells. This cytochrome is present in phagocytic leucocytes and probably plays an important part in the specialized activities of these cells.     

Effect of indole-3-acetic acid administration by gavage and by subcutaneous injection on rat leukocytes

This study was done to investigate the effect of indole-3-acetic acid (IAA) administered subcutaneously and by gavage on neutrophil function and cytotoxicity in neutrophils and lymphocytes. A gavage administration resulted in an increase in phagocytic capacity in neutrophils in a dose depended manner for 1 mg, 2 mg, 18 mg, and 40 mg of IAA per kg of body mass, respectively, compared with the control. Similarly, subcutaneous administration of IAA at 2, 18, and 40 mg per kg of body mass promoted a significant rise in phagocytosis by neutrophils. H2O2 production in neutrophils from treated rats by gavage was similar to those receiving subcutaneous IAA treatment, and did not show a significant difference between treatments and control. IAA treatment, whether by gavage or subcutaneous, did not produce an alteration in antioxidant enzyme activities or in glucose-6-phosphate dehydrogenase activity of either neutrophils or lymph nodes. Subcutaneous IAA administration did not alter the neutrophil and lymphocyte death as deduced by unaltered membrane integrity, DNA fragmentation and mitochondrial transmembrane potential, compared with controls. In conclusion, IAA administration either subcutaneously or by gavage could increase the phagocytic capacity by neutrophils and this acid administration did not have prooxidant effects or cytotoxic effects on neutrophils and lymphocytes.