Differential modulation of annexin I binding sites on monocytes and neutrophils

Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Effects of beta-carotene, selenium and vitamin A on in vitro polymorphonuclear leukocytic activity in peripartal buffalo (Bubalus bubalus)

The effect of different concentrations of three antioxidans on phagocytic and kill activities of blood polymorphonuclear leukocytes (PMN) isolated from buffaloes during the peripartum period (4 weeks before to 7 weeks after parturition) was investigated in this study. Two concentrations of beta-carotene and vitamin A (10(-6) and 10(-5) M) and one concentration of Se (10(-9) M) were used. Phagocytic activity of PMN treated with beta-carotene (10(-6)M) significantly enhanced (P < 0.05) after parturition (Week 0 until Week 3), whereas the kill activity of the same cells significantly (P < 0.05) increased before and after parturition (at Weeks -4, -3, -2, 0, 1, 2 and 3). The concentration of beta-carotene (10(-5) M) enhanced phagocytosis of PMN only at Weeks 0 and 1 and kill activity at Weeks -4, -3, -2, 0, and 1. Selenium (10(-9)M) significantly (P < 0.05) enhanced phagocytic activity of PMN starting from parturition (Week 0) until Week 3 postpartum. Kill activity increased significantly both before (Weeks -4, -3 and -2) and after (Weeks 0, 1, 2, 3 and 4) parturition. Vitamin A (10(-6) M) significantly enhanced phagocytic activity of PMN at Weeks 0, 1, and 2, whereas, the concentration of beta-carotene (10(-5) M) increased phagocytic activity only at Week 0. Kill activity of PMN increased significantly (P < 0.05) at Weeks -1 and 0 (10(-6)M). These results demonstrate that beta-carotene and selenium significantly enhanced phagocytic and kill activities of PMN isolated from buffaloes around parturition in vitro. Vitamin A enhanced phagocytosis and kill activities but not to the same extent as beta-carotene and selenium. Apparently, the in vitro killing activity of PMN is a distinctive function from phagocytosis and both activities may be enhanced by the use of essential nutrients, especially during the peripartum period. Moreover, beta-carotene is more effective as an antioxidant than vitamin A in enhancing the activities of phagocytic cells.     

The migratory and phagocytic activity of polymorphonuclear leukocytes in rheumatoid arthritis and osteoarthritis patients

Polymorphonuclear neutrophils (PMN) play a central role in the elimination of most extracellular pathogenic microorganisms and any impairment of their functions therefore predisposes to defect immune defence. We investigated the migratory and phagocytic functions of the PMNs isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The results suggest that in RA the number and the migratory but not phagocytic capacity of synovial fluid (SF) neutrophils were enhanced, while in OA they were significantly decreased in synovial fluid cells comparatively with peripheral blood (PB). The migratory function of both PB and SF cells from RA patients was increased comparatively with that of the cells from OA patients. We found the different abnormal functions in synovial fluid neutrophils from RA and OA patients. These results may help to elucidate the underlying mechanism which leads to severe joint destruction and different susceptibility to infectious diseases in patients with rheumatic disorders.     

Effects of storage and incubation conditions on human granulocyte phagocytic, bactericidal, and chemotactic functions

This study was undertaken to determine the effects of different incubation conditions on human granulocyte (PMN) bactericidal, phagocytic, and chemotactic functions. Specifically, (1) how long may a patient's blood be held before assay and maintain original PMN function, and (2) how long may isolated PMNs be incubated for the purpose of exposure to various agents and still maintain original function? PMNs isolated following storage of whole heparinized blood at 4 °C for 24 and 48 hr phagocytized as well as fresh cells and their bactericidal activity was 96 and 85% of control values after 24 and 48 hr, respectively. Chemotaxis decreased to 62% of control after 24 hr. The bactericidal capacity of isolated PMNs stored at 4 °C for 24, 48, and 72 hr decreased to 85, 81, and 78% of controls, respectively. Phagocytosis after 24 hr storage was equal to controls. Chemotaxis was decreased to 59 and 34% of controls after 24 and 48 hr, respectively. Isolated PMNs incubated at 37 °C demonstrated impairment in phagocytic capacity after only 4 hr.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

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.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Abstract Porphyromonas gingivalis culture supernate was found to induce hemotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of aglutination induced by the secretagogues PMA and FMLP. Lipopolysaccaharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors of PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity fot P. gingivalis cells but when PMN were treated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutinatin or enhanced adherence could also lead to decreased phagocytic capacity of the adherant or agglutinated cells.     

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

Summary Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.This work was supported by research grants from the University of North Carolina Research Council and the National Institutes of Health (A1 02430)     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.     

Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.     

Influence of the iron saturation of bacteria on the phagocytic capacity of neutrophils

The comparative study of the adsorptive capacity of human blood neutrophils with respect to "iron saturated" cells of bacteria belonging to different systematic groups was carried out. A decrease in the phagocytic number by 22-76% of the control level was shown. In experiments with iron-saturated Gram positive bacteria the phagocytic number was noted to be less than the physiological norm.     

Chemiluminescence properties of human,canine and rat polymorphonuclear cells

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct different densities were used. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/I), phorbol myristate acetate (PMA, 2.8 × 10^?6 mol/I), calcium ionophore A 23187 (10^?5 mol/l) and N-formylmethionyl-leucyl-phenylalanine (FMLP, 3.5 × 10^?6 mol/l). The isolated cells of the three species responded to all of the various stimuli. Species-related sensitivity could be ordered: human > canine > rat. Response to the various agents in the human cells can be ranked: PMA ? A 23187 > zymosan > FMLP; for the dog: A 23187 > PMA > zymosan > FMLP; and for the rat: zymosan ? PMA > FMLP ? A 23187. Time course and peak maximum response were different upon stimulation in the absence and presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.     

Phagocytic capacity of cytokineplasts from human blood polymorphonuclear leukocytes

Cytokineplasts (CKP) are membrane-bounded anucleate cytoplasmic fragments, induced from polymorphonuclear leukocytes (PMN) by the brief application of heat; derived from the cortical cytoplasm that gathers at the leading front of migrating PMN; and endowed with many of the motile properties of the parent cell. In this study we examine their phagocytic capacity by quantitative methods. CKP ingest Staphylococcus aureus and Serratia marcescens somewhat less avidly than do the corresponding intact PMN, yet rather impressively when one considers how restricted a portion of the parent cell they represent. Under the conditions employed, CKP killed about half as many of the bacteria presented to them as did their parent PMN. Thus, despite a heat-associated loss of demonstrable respiratory burst oxidase activity and a paucity of cytoplasmic granules, the organelle-depleted CKP deals with bacteria in a way that mimics its parent PMN.     

Early degranulation of human neutrophils: immunocytochemical studies of surface and intracellular phagocytic events.

Degranulation of azurophil and specific granules after phagocytic challenge with E. coli for 5 sec to 10 min was investigated in the human polymorphonuclear neutrophil (PMN). PMN were stained simultaneously with fluorescein and rhodamine-labeled monospecific antisera to myeloperoxidase (MPO) and lactoferrin (LF) to identify azurophil and specific granules, respectively, within single cells. Fixation was designed to preserve or disrupt differential permeability of cell membrane to fluorescent conjugates in order to study granule translocation. Within 5 sec after phagocytic challenge, MPO and LF appeared on the cell surface coating the bacteria as granule contents leaked from the incompletely formed phagolysosomes. The phagocytic cup, shown by scanning electron microscopy as large and circular, appeared by immunofluorescent markers to be outlined by curvilinear staining for both granule markers, and was always coincident with bacterial localization. MPO and LF appeared singly or simultaneously on the cell surface, suggesting that degranulation to the surface was random. Sequential phagocytic events were demonstrated by comparing staining intensities for each granule marker on the surface and intracellularly within single cells. LF sometimes appeared on the cell surface independent of the nascent phagosome, suggesting that perturbation of the cell membrane by bacteria may cause some specific granule extrusion not limited to the phagosome. These results imply that bacteria make contact with granule-associated anti-microbial substances within 5 sec after phagocytosis is initiated and that free communication of granule constituents occurs between the newly forming phagolysosome and the extracellular space.     

Production of H2O2 by bovine blood and milk polymorphonuclear leucocytes

The ability of bovine polymorphonuclear leucocytes (PMN) to release H2O2 was investigated. Resting PMN suspended in buffer released only small amounts of H2O2 which was appreciably increased during phagocytosis of heat killed coliforms. However, in the presence of bovine serum (BS), foetal calf serum (FCS) and milk whey (MW) no increase of H2O2 could be detected unless sodium azide (NaN2) was added. It appears that the enzyme content of these fluids (catalase and lactoperoxidase) consumed the released H2O2 and NaN2, which inactivates these enzymes, abolished this interference. Live organisms required BS or MW both for phagocytosis and for H2O2 production. Bovine IgG2 and, to a lesser extent, IgG1 but not SIgA or IgM stimulated the release of H2O2 independently of phagocytosis; this indicates the presence of receptors specific for IgG2 and IgG1 on the cell surface. Ingestion of casein micelles triggered the greatest burst of H2O2 production by cells suspended in buffer. In general, PMN isolated from blood were more active than cells isolated from milk. Since the extracellular release of H2O2 reflects the intracellular level of H2O2, the lower metabolic activity of milk PMN may contribute to the lesser intracellular bactericidal activity of milk leucocytes. The possibility that the release of H2O2 may activate extracellularly the lactoperoxidase system, known to be bactericidal in milk, is discussed.     

Lactoferrin: affinity purification from human milk and polymorphonuclear neutrophils using monoclonal antibody (II 2C) to human lactoferrin, development of an immunoradiometric assay using II 2C, and myelopoietic regulation and receptor-binding characteristics

Several investigators have now confirmed our original report demonstrating the myelopoietic suppressive activity of lactoferrin (LF) in vitro. In order to further clarify this activity, we used the recently produced and purified neutralizing antibody (II 2C) to LF to set up an immunoradiometric assay specific for LF and to affinity purify LF from lysates of peripheral blood polymorphonuclear neutrophils (PMN) obtained from healthy donors. Iron-saturated purified PMN LF was as active as iron-saturated affinity purified milk LF as a suppressor of the release of granulocyte-macrophage colony stimulating factors (GM-CSF) from mononuclear human peripheral blood leukocytes. The activities of both the PMN LF and milk LF were inactivated by preincubation with monoclonal anti-LF antibody (II 2C). In order to evaluate the methods of iron saturation of LF in vitro as measures of their functional activities, milk LF was iron saturated by four different methods, including ferric citrate, ferric ammonium sulphate, ferric chloride with nitriloacetate, and ferric chloride alone. The functional characteristics of all four preparations of LF saturated with iron in vitro were relatively equal and were more active than native LF. Resident mouse peritoneal macrophages separated into subpopulations of GM-CSF-producing cells by velocity sedimentation were evaluated for their LF-receptor binding capacity and for sensitivity to the suppression of GM-CSF release by LF. Iron saturated LF suppressed release of GM-CSF from only those fractions containing LF-receptor bearing cells, although not all fractions containing cells bearing receptors for LF responded to the suppressive activity of LF. These studies provide further evidence for the myelopoietic regulatory activity in vitro of PMN-derived LF, which is mediated through populations of mononuclear phagocytes having receptors for LF.     

Human adipose tissue–derived mesenchymal stromal cells and their phagocytic capacity

Mesenchymal stromal cells (MSCs) have evidenced considerable therapeutic potential in numerous clinical fields, especially in tissue regeneration. The immunological characteristics of this cell population include the expression of Toll‐like receptors and mannose receptors, among others. The study objective was to determine whether MSCs have phagocytic capacity against different target particles. We isolated and characterized three human adipose tissue MSC (HAT‐MSC) lines from three patients and analysed their phagocytic capacity by flow cytometry, using fluorescent latex beads, and by transmission electron microscopy, using Escherichia coli, Staphylococcus aureus and Candida albicans as biological materials and latex beads as non‐biological material. The results demonstrate that HAT‐MSCs can phagocyte particles of different nature and size. The percentage of phagocytic cells ranged between 33.8% and 56.2% (mean of 44.37% ± 11.253) according to the cell line, and a high phagocytic index was observed. The high phagocytic capacity observed in MSCs, which have known regenerative potential, may offer an advance in the approach to certain local and systemic infections.     

Lochial secretions of Escherichia coli- or Arcanobacterium pyogenes-infected bovine uteri modulate the phenotype and the functional capacity of neutrophilic granulocytes

It has been suggested that in cases of puerperal endometritis of cattle infected with Escherichia coli and Arcanobacterium pyogenes, the neutrophils are compromised in their defense capacity or downregulated functionally. In addition to direct bacterial effects, contents of lochial secretions and secreted products of locally activated polymorphonuclear neutrophilic granulocytes (PMNs) may also account for changes in function of freshly immigrating neutrophils. In this study, lochial secretions were obtained from healthy cows and from cows infected by E. coli or A. pyogenes. Separated uterine PMN of infected cows displayed an altered phenotype and function which correlated with the degree of bacterial contamination. Concurrently tested circulating PMN showed no such changes. Infected lochial secretions sterilized by filtration also changed the phenotype of blood PMN. Lochial secretions of healthy cows displayed only minor effects. The effects on PMN function in infected cows varied: ingestion was less affected, whereas generation of reactive oxygen species (ROS) was severely depressed. Concurrently tested purified bacterial products (solubles and fragments) of E. coli and A. pyogenes did not induce the phenotypical and functional changes observed in blood PMN. Since infected lochia also contained high numbers of immigrated and probably activated PMN, the influence of supernatants from phorbol myristate acetate-activated PMN were tested on freshly isolated blood PMN. Such supernatants also increased the expression of certain surface molecules and inhibited the ROS generation. Thus, reduced function and altered phenotypes of PMN which immigrate into the uteri of cows with bacterial endometritis is due not only to interactions with bacteria or bacterial products, but is also to the uterine milieu.