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.     

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.     

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.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils

Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils (PMNs). PMNs are recruited into the lungs during inflammation, but as inflammation is resolved, PMNs undergo apoptosis and are phagocytosed by AMs. We determined that SP-A increases AM phagocytosis of apoptotic PMNs 280 +/- 62% above the no protein control value. The increase is dose dependent, and heat-treated SP-A still enhanced uptake, whereas deglycosylated SP-A had significantly diminished ability to enhance phagocytosis. Surfactant protein D also increased phagocytosis of apoptotic PMNs by approximately 125%. However, other proteins that are structurally homologous to SP-A, mannose-binding lectin and complement protein 1q, did not. SP-A enhances phagocytosis via an opsonization-dependent mechanism and binds apoptotic PMNs approximately 4-fold more than viable PMNs. Also, binding of SP-A to apoptotic PMNs does not appear to involve SP-A's lectin domain. These data suggest that the pulmonary collectins SP-A and SP-D facilitate the resolution of inflammation by accelerating apoptotic PMN clearance.     

Concurrent measurement of antigen- and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils

The current study aims to review flow cytometric (FCM) parameters for the quantification of phagocytosis. A limitation of existing methods is their difficulty with accurate quantification of the phagocytic index, i.e., number of beads per phagocyte, in individual cell lines in mixed cell suspensions. We have quantified phagocytosis and the oxidative burst simultaneously using fluorescent beads coated with meningococcal outer membrane vesicles (OMV beads) by the conversion of dihydrorhodamine 123 (DHR-123) to rhodamine 123 (R-123). Both these processes depend on specific serum opsonins. After the incubation, staining with a fluorescent anti-CD14 monoclonal antibody succeeded in discriminating phagocytosing monocytes from neutrophils. The spectral overlaps between OMV beads, R-123, and anti-CD14 could be completely compensated. Percentage of phagocytosis and the phagocytic index were similar in monocytes and neutrophils, but the oxidative burst behaved differently. Two monocyte subpopulations were observed. Both subpopulations spontaneously converted some DHR-123 into R-123, whereas the reaction was triggered by phagocytosis in neutrophils. The total oxidative response increased with increasing phagocytic index in both cell types, but the oxidative burst in monocytes was about twice that of neutrophils. The oxidative ratio (mean R-123 fluorescence value divided by the phagocytic index) declined with time in monocytes, but increased in neutrophils. Our results demonstrate the need for careful attention to technical details. This single-laser, three-color FCM method facilitates the comparative research of phagocytosis and the oxidative burst in monocytes and neutrophils and provides a basis for a number of applications in hematology, infectious medicine, and immunology.     

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.     

Involvement of protein kinase Cdelta in the activation of NADPH oxidase and the phagocytosis of neutrophils

This experiment was performed to clarify the role of protein kinase C (PKC) delta in NADPH oxidase-dependent O(2-) production and actin polymerization followed by phagocytosis in neutrophils. Bovine neutrophils and human neutrophil-like differentiated HL-60 (dHL-60) cells were stimulated with serum-opsonized zymosan (OZ) and fMet-Leu-Phe (fMLP), respectively. Rottlerin, a specific inhibitor of PKCdelta, attenuated the production of O(2-) from NADPH oxidase in both neutrophils and dHL-60 cells. However, it did not inhibit the translocation of p47(phox) from the cytosol to the membrane in either type of cell or the phosphorylation of p47(phox) in dHL-60 cells. GF109203X (GFX), an inhibitor of cPKC, attenuated not only the production of O(2-) but also the translocation of p47(phox) in both cells. Furthermore, rottlerin significantly attenuated the ingestion of opsonized particles and the formation of F-actin in OZ-stimulated neutrophils, whereas, GFX did not affect those phagocytic processes. These results suggest that both PKCdelta and cPKC regulate NADPH oxidase through different pathways, but only PKCdelta regulates the phagocytic function in neutrophils.     

Interaction of two phagocytic host defense systems: Fcγ receptors and complement receptor 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.     

Flow cytometric evaluation of leukocyte function in rat whole blood

The aim of this study was to establish a standard flow cytometric method to measure the phagocytic function of and intracellular hydrogen peroxide (H2O2) production by rat leukocytes. Thirty-six adult, male Sprague-Dawley rats were included in this study. Whole-blood specimens from the inferior vena cava were collected in a heparinized tube and ethylenediaminetetraacetic acid (EDTA) anticoagulated tube. The phagocytic function of and intracellular H2O2 generation by leukocytes were measured with FACS Vantage trade mark flow cytometer (Becton Dickinson, San Jose, CA), using fluorescent microspheres and dihydrorhodamine-123 as probes, respectively. Several conditions were optimized in this study, including anticoagulants (heparin and EDTA), fluorescent probes (0.75- and 1.72-microm-diameter microspheres), incubation time, and concentration of the chemicals used in the experiment. Neutrophils, monocytes, and lymphocytes could be clearly defined and separated in whole blood by flow cytometry and tested for phagocytosis and intracellular H2O2 generation without the need for further purification and handling of the cells. Intracellular H2O2 production by and phagocytic function of neutrophils and monocytes were inhibited in EDTA-anticoagulated blood compared with heparin- anticoagulated blood (P < 0.01). Neutrophils showed similar phagocytic function to 0.75- and 1.72-microm microspheres, but monocytes showed weak phagocytic activity to 1.72-microm beads compared with 0.75-microm beads (P < 0.01). In conclusion, a flow cytometric method to measure the phagocytic function of and intracellular H2O2 production by rat leukocytes has been developed. Quantitative flow cytometric analysis of rat leukocyte function is convenient and feasible and provides a reliable and rapid assay to assess phagocytosis and intracellular H2O2 production by rat neutrophils and monocytes.     

Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.     

Poly(I):poly(C)-enhanced alveolar and peritoneal macrophage phagocytosis: quantification by a new method utilizing fluorescent beads

Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess the baseline levels of phagocytosis for rat alveolar and peritoneal macrophages from the same animal and the kinetics and level of enhanced phagocytosis for alveolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.     

Flow-cytometric Studies of the Phagocytic Capacities of Equine Neutrophils

Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and un-quenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.     

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.     

Neutrophil dysfunction induced by hyperglycemia: modulation of myeloperoxidase activity

Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the May-Grunwald-Giemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils. Copyright ? 2012 John Wiley & Sons, Ltd.     

Signaling pathways in phagocytosis

Phagocytosis is an uptake of large particles governed by the actin-based cytoskeleton. Binding of particles to specific cell surface receptors is the first step of phagocytosis. In higher Eucaryota, the receptors able to mediate phagocytosis are expressed almost exclusively in macrophages, neutrophils, and monocytes, conferring immunodefence properties to these cells. Receptor clustering is thought to occur upon particle binding, that in turn generates a phagocytic signal. Several pathways of phagocytic signal transduction have been identified, including the activation of tyrosine kinases and (or) serine/threonine kinase C in pivotal roles. Kinase activation leads to phosphorylation of the receptors and other proteins, recruited at the sites of phagocytosis. Monomeric GTPases of the Rho and ARF families are likely to be engaged downstream of activated receptors. The GTPases, in cooperation with phosphatidylinositol 4-phosphate 5-kinase and phosphatidylinositol 3-kinase lipid modifying enzymes, can modulate locally the assembly of the submembranous actin filament system leading to particle internalization. BioEssays 21:422–431, 1999. © 1999 John Wiley & Sons, Inc.     

Altered phagocytosis by monocytes from HLA-DR2 and DR3-positive healthy adults is Fc gamma receptor specific

Fc gamma receptor-dependent mononuclear phagocyte system (MPS) clearance of opsonized erythrocytes is prolonged in healthy adults with the class II alloantigens HLA-DR2, DR3, or DQw1, despite normal receptor-specific Fc ligand binding by monocytes in these groups. To investigate the basis for the MPS dysfunction, we determined the phagocytic capacity of blood monocytes from 66 disease-free adults and analyzed the data according to the HLA type of the subjects. The data demonstrate decreased phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes from HLA-DR2, DR3, or DQw1-positive subjects compared with normals without these B cell alloantigens (2.87 +/- 0.83 erythrocytes/monocyte vs 3.87 +/- 1.05, p less than 0.004; 3.01 +/- 0.94 vs 3.87 +/- 1.05, p less than 0.02; 3.18 +/- 0.89 vs 3.87 +/- 1.05, p less than 0.02, respectively). Because HLA-DR2 and DQw1 are in linkage disequilibrium, we compared EA phagocytosis in subjects with DQw1 alone to normals without HLA-DR2, DR3, or DQw1. Among subjects positive only for DQw1, no significant decrease in phagocytosis could be seen (3.46 +/- 0.95 vs 3.87 +/- 1.05, p = NS). To determine whether these differences represented an Fc receptor-specific dysfunction or a more generalized decrease in phagocytic activity, we compared the quantitative phagocytosis of EA with that of neuraminidase-treated erythrocytes (EN), which is Fc receptor independent and beta-glucan receptor mediated. No segregation of phagocytic capacity for EN by HLA class II phenotypes could be demonstrated (DR2, 2.68 +/- 1.30 erythrocytes/monocyte; DR3, 2.95 +/- 1.30; DQw1, 2.84 +/- 1.15; others, 3.06 +/- 1.14). Our data indicate that the decrease in phagocytosis by blood monocytes from normal individuals with HLA-DR2 or DR3, the class II alloantigens associated with systemic lupus erythematosus and other autoimmune diseases, is specific for the Fc receptor-mediated process. This alteration of Fc receptor function among immunogenetically defined individuals may contribute to their predisposition to autoimmune disease. These differences may also apply to other Fc receptor-initiated cellular functions.     

Interaction of non-adherent suspended neutrophils to complement opsonized pathogens： a new assay using optical traps

Phagocytosis of opsonized pathogens by circulating non-adherent neutrophils is an essential step in host defense, which when overwhelmed contributes to sepsis. To investigate the role played by ligation of complement receptors CR3 and CR4 in non-adherent neutrophils, we designed a novel assay system utilizing dual optical traps, respectively, holding a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface. We chose anti-CD 18 as an example ligand, mimicking the bacterial opsonizing complement fragment iC3b. Presentation of anti-CD 18-coated beads elicited both pseudopodial protrusion and subsequent phagocytosis. This is in sharp contrast to previously reported responses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We used this same new assay to probe actomyosin pathways in the neutrophil＇s pseudopodial and phagocytic response. Disruption of actin or inhibition of myosin light-chain kinase dose-dependently reduced pseudopod formation and phagocytosis rates. In summary, i） the new dual trap assay can be used to study the responses of suspended neutrophils to a variety of ligands, and ii） in a first application of this technique, we found that local ligation of CR3/4 in unactivated neutrophils in suspension induces pseudopod formation and phagocytosis at that site, and that these events occur via an actomyosindependent pathway.     

Transmembrane signaling: an ion-flux-independent model for signal transduction by complexed Fc receptors

Fluxes of Na+/K+ that precede effector functions in stimulated phagocytes are thought to play a role in signal transduction. To examine this hypothesis, phagocytosis, phagosomal acidification, and superoxide anion generation (O2-) were stimulated in media in which the Na+ was replaced with K+ or choline+. Counts of particles internalized and assessment of acidification of the phagosomes by acridine orange staining indicated that Na+/K+ fluxes were not necessary for phagocytosis or phagosomal acidification in J774.2 macrophages. Phagocytosis mediated by the ionophoretic Fc receptor gamma 2b/gamma 1 of J774.2 macrophages was equally independent of a Na+ gradient. Na+/K+ fluxes did not dictate the rate of O2- generation in human monocytes. Therefore, in at least these three effector functions, Na+/K+ fluxes stimulated by Fc- and non-specific receptor binding play neither a signaling nor an enhancing role. An ion-flux-independent model for transmembrane signaling by the Fc receptor is proposed. Others have shown that there is an apparent dependence on the external Na+ concentration for O2- generation and lysosomal secretion by neutrophils. These neutrophils had been pre-treated with NH4+ during a routine purification step. O2- generation stimulated by opsonized zymosan or phorbol myristate acetate, by monocytes or monocyte-derived macrophages, and phagocytosis of opsonized zymosan by J774.2 macrophages, showed dependence on external Na+ only if these cells had been pre-treated with NH4+. Brief NH4+ pre-treatment would be expected to acidify the cytoplasm of the cells. The reversal of this acidification is known to require Na+ for H+ extrusion through the Na+/H+ antiport, thus explaining the apparent Na+ dependence.