Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.     

The thermal treatment effects on bovine blood neutrophil granulocytes apoptosis and necrosis in vitro

The aim of the study was to evaluate the effect of selected temperatures on viability (apoptosis and necrosis) of bovine blood neutrophil granulocytes (neutrophils) in vitro. The following temperatures were tested: -80, -20, 4, 23, 37 degrees C. Heparinised bovine blood was incubated for 1, 4 and 24 h under respective temperature. Apoptosis and necrosis of neutrophils were detected by light microscopy, transmission electron microscopy (TEM) and flow cytometry (FCM). From selected temperatures, 4 degrees C impaired the neutrophil viability least. The proportion of apoptotic and necrotic neutrophils amounted to (mean +/- SD) 5.25 +/- 3.53% and 0.83 +/- 0.38%; 7.09 +/- 2.07% and 1.64 +/- 0.50%; 35.39 +/- 12.53% and 5.46 +/- 1.46%; after 1, 4 and 24 h incubation, respectively. The temperature (4 degrees C) is the best alternative for short-term storage.     

Ultrastructure of the peripheral blood leucocytes of Oreochromis mossambicus

Peripheral blood leucocytes were examined for their fine structure and their in vivo response to colloidal carbon particles and bacteria, Aeromonas salmonicida . Four main types of leucocyte were identified: lymphocytes, thrombocytes, monocytes and granulocytes. Previously, two types of granulocyte had been identified but at the electron microscope level a third type could be distinguished in the peripheral blood. Monocytes and the type 1 and type 3 granulocytes were found to be phagocytic.     

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)     

The acute response of neutrophil function to a bout of judo training

Intensive exercise training decreases neutrophil functions in athletes. However, no studies to date have investigated the effect of irregular‐interval training, such as is associated with judo training programmes, on neutrophil functions. The purpose of this study was to examine such effects. Thirty‐seven male college judoists participated in this study. Neutrophil oxidative burst activity, phagocytic activity and expression of CD11b and CD16 per cell were measured by ?ow cytometry before and after judo training. Total neutrophil counts increased signi?cantly from 2.98 ± 0.82 to 7.95 ± 1.80 × 10³/µL (p < 0.001). The proportion of neutrophils producing reactive oxygen species (ROS) was increased signi?cantly (p < 0.001). On the other hand, the phagocytic activity decreased after training, as shown by a decrease in the amount of ingested opsonized zymosan per cell (p < 0.001), possibly as a compensatory effect for the increased numbers of ROS‐producing neutrophils. Expression of CD11b and CD16 per cell decreased by 20% and 30%, respectively, after judo training. In conclusion, judo training induced a decrease in phagocytic activity through the lowered expression of CD11b and CD16 on the surface of neutrophils, and increased the oxidative burst activity of neutrophils. Copyright © 2003 John Wiley & Sons, Ltd.     

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.     

Long-term cryopreservation of dog granulocytes

Granulocytes isolated by counterflow centrifugation elutriation (CCE) from leukapheresed dog blood, frozen in liquid nitrogen at ?196 °C, were studied. The effects of long-term cryopreservation on cell recovery and in vitro function were detertmined. In seven separate experiments, an average of 1.7 × 10⁹ granulocytes were obtained. The white cell differential count was 91% granulocytes and 9% mononuclear cells. There was less than 5% red cells presrent and no platelets. Granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5% dimethylsulfoxide (DMS), 6% hydroxyethyl starch (HES), and 4% bovine serum albumin (BSA), pH 7.1. Total volumes of 40 ml were frozen at a cooling rate of 4 °C per minute and stored for periods of 1, 34, 60, 90, and 132 weeks in liquid nitrogen at ?196 °C. Thawing was done at a rate of 190 ° per minute to 10 °C. The recovery of cells was 95%, 105%, 100%, 100%, and 88% respectively. Ethidium bromide exclusion, indicative of viable nuclei, was 91%, 81%, 94%, 89%, and 80% respectively. Virtually all thawed cells ingested opsonized Fluolite particles, but the number ingested was approximately one-half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at ?196 °C with high cellular recovery and at least 50% phagocytic function.     

Host defense function in neutrophils from the American bison (Bison bison)

Selected host defense functions of neutrophils isolated from American bison (Bison bison) were characterized and compared with those of cattle (Bos taurus). Bison neutrophils had a robust chemotactic response to both IL-8 and LTB(4), with maximal responses occurring at 10(-7) M (IL-8) and 10(-8) M (LTB(4)). The magnitude of the chemotactic response to IL-8 was similar in bison and bovine neutrophils (except at 10(-7) M IL-8, where bison had a stronger response). In response to LTB(4), bison neutrophils had a much stronger chemotaxis at both 10(-8) and 10(-7) M than did bovine cells. Production of reactive oxygen species (ROS) in response to phorbol myristate acetate (PMA) and opsonized zymosan (OpZ) was similar between bison and bovine neutrophils. However, the production of ROS in bison neutrophils stimulated with OpZ was primarily intracellular, while extracellular release of ROS was evident in bovine neutrophils stimulated with OpZ. Like bovine neutrophils, bison neutrophils did not generate a respiratory burst in response to fMLF. Granules prepared from bison neutrophils had potent direct killing action on the Gram-negative bacteria Escherichia coli but failed to kill the Gram-positive bacteria Staphylococcus aureus and, at intermediate doses, actually had a permissive effect for this bacteria. Thus, bison neutrophils have potent host defense capabilities similar in quality to those of bovine neutrophils; however, unique differences are present, which may allow bison neutrophils to respond to the distinct immunological challenges that bison encounter.     

Recovery,structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide

The recovery, structure and function of dog granulocytes were determined before and after freeze-preservation. Leucocytes were isolated from defibrinated or anti-coagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6%)-isopaque (33.9%). Granulocytes isolated by these procedures were examined for changes in O₂ consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7.5%) and autologous serum or HBSS-minus and 20% autologous serum at the rate of ?1 °C/min to ?80 °C and stored in liquid N₂ vapor.After freeze-preservation, O₂ consumption associated with phagocytosis was decreased by 54 and 64% for granulocytes isolated from defibrinated or from ACD-anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze-preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD-anticoagulated blood. Chemotactic response after freeze-preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods. However, nuclear, cytoplasmic, and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze-preservation in DMSO to a degree not previously reported.It is concluded that dog granulocytes freeze-preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes.     

A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway

The ligand specificity of the human monocyte receptor that mediates phagocytosis of particulate activators of the human alternative complement pathway was defined by inhibiting the phagocytic response with glycans known to be present in zymosan. When monocytes in monolayers were preincubated with 100 micrograms/ml of beta-glucan and then incubated with 1.25 to 2.5 X 10(6) zymosan particles, the percentage of cells that exhibited phagocytosis was inhibited in a time-dependent manner; maximal inhibition occurred within 20 min of preincubation. beta-Glucan inhibited monocyte phagocytosis of zymosan and rabbit erythrocytes (Er) in a similar dose-dependent fashion and at 100 micrograms/ml reduced monocyte ingestion of 5 X 10(6)/ml zymosan and 2 X 10(8)/ml Er by 63 +/- 8% and 68 +/- 16% (mean +/- SD, n = 3), respectively. The other glycan constituent of zymosan, mannan, was less than 1% as active, and 10 mg/ml of mannan reduced the number of monocytes ingesting zymosan and Er by 56 +/- 12% and 26 +/- 11%, respectively. At concentrations as high as 500 micrograms/ml, beta-glucan had no effect on monocyte Fc, C3b, or fibronectin receptor-mediated functions. Enzymatic hydrolysis of beta-glucan and alpha-mannan with beta-glucosidase or beta-glucanase before their incubation with monocytes abrogated their inhibitory capacity, whereas hydrolysis with alpha-mannosidase or alpha-glucosidase did not. Neither of the two alpha-glucans tested (dextran T-70 and nigeran) affected monocyte ingestion of zymosan particles or sheep erythrocytes (Es) sensitized with rabbit 7S anti-Es (EsIgG) at concentrations as high as 2 mg/ml. In contrast, a number of beta-glucans were active against zymosan but not EsIgG ingestion with a 75% reduction in the number of monocytes ingesting zymosan occurring with 100 micrograms/ml laminarin, 500 micrograms/ml soluble pachyman, and 900 micrograms/ml of soluble pustulan. The galactan, agarose, either in suspensions at 2 mg/ml or in a soluble portion at 600 micrograms/ml failed to affect monocyte ingestion of zymosan particles or Er. Thus, the monocyte receptor for particulate activators that is specifically inhibited by beta-glucan at a rate compatible with a phagocytic process and that recognizes beta-glucans but not alpha-glucans, mannan, or galactan is a beta-glucan receptor.     

Luminol dependent chemiluminescence of quartz and zymosan stimulated neutrophils

The effect of the opsonization by zymosan and quartz particles on the chemiluminescence was investigated on human neutrophil granulocytes. Opsonization of zymosan enhanced the chemiluminescence response, while opsonized quartz inhibited the chemiluminescence reaction. Calcium ionophore A 23187 treatment did not influence the chemiluminescence of quartz but the light signal in the presence of quartz decreased rapidly. In parallel experiments the protein pattern of zymosan treated neutrophils was investigated by high resolution two-dimensional polyacrylamide gel electrophoresis.     

Influence of HIV-infection on the phagocytic activity of monocytes/macrophages and granulocytes

Because of the great importance of phagocytosis as a key process in host defence, the influence of HIV-infection on the phagocytic activity of monocytes/macrophages (M0/MAC) and granulocytes was investigated. Therefore, blood samples from the peripheral blood of 70 HIV-infected individuals were incubated with fluorescein isothiocyanate (FITC) labeled Escherichia coli. The uptake of the bacteria was monitored by flow cytometer analysis. A strong and significant increase in the relative number of phagocytic granulocytes was observed ranging from 12.8% in an uninfected control collective to over 30% in AIDS patients. This effect was obtained for all patients and independent of the stage of disease. For monocytes, only marginal changes were found in their phagocytic function. These data suggest that the high susceptibility of HIV patients for secondary infections is not linked to a loss of phagocytic ability of monocytes/macrophages and/or granulocytes.     

Plunder of Human Blood Leukocytes Containing Ingested Material,by Other Leukocytes: Where Is the Fusagen That Allows Preservation of Membrane Integrity and Motile Function?

In studying phagocytosis of zymosan particles by human blood monocytes in phase-contrast videomicroscopy, we found that monocytes loaded with zymosan particles became chemotactic for polymorphonuclear leukocytes (PMN) which closed on them and purloined their particle content. This despoliation usually occurred in monocytes that had begun to swell—prefiguring their death. The violent seizure of their contents by the aggressing PMN often tore the monocytes apart. However, some apparently healthy monocyte survived the removal of zymosan content by PMN or, more commonly, its removal by another monocyte. PMN—a much hardier cell in slide preparations—that were similarly loaded with zymosan particles, also attracted PMN. The latter could remove zymosan from the target cell without killing it. Thus, leukocytes were sacrificing significant portions of themselves without losing residual membrane integrity and motile function. Their behavior with respect to other particles (e.g., bacteria) will be of interest. We suggest that the membrane fusagen resides in the inner membrane leaflets when they are brought together in an extreme hourglass configuration. This event may be similar to the fragmentation of erythrocytes into intact pieces, the formation of cytokineplasts, the rear extrusion of content by migrating cells on surfaces, and the phagocytic process itself.     

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.     

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.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Effect of the EM Bokashi® Multimicrobial Probiotic Preparation on the Non-specific Immune Response in Pigs

The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I – control and II – experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.

     

Phagocytic cells in blood from rainbow trout, Salmo gairdneri (Richardson), characterized by flow cytometry and electron microscopy

An in vitro assay for estimating the proportion of phagocytic cells among peripheral leucocytes from rainbow trout by flow cytometry (FCM) and fluorescence microscopy was evaluated. Data from FCM were compared with fluorescence microscopic observations and good correlation ( r = 0.87) was found. The influence of various culture conditions, such as serum type, duration of incubation and temperature, on the in vitro phagocytic assay was investigated. Cultures supplemented with brown trout serum and incubated for 18 h at 19° C were considered to give optimal conditions for phagocytosis. The proportion of phagocytic cells detected in the peripheral blood leucocyte preparation was 3.3 ± 1.5% with FCM and 5.5 ± 2.4% with fluorescence microscopy. The applicability of the method was demonstrated in a preliminary study with arsenic. In a concentration of 1 μg ml⁻¹, arsenic increased the proportion of actively phagocytic cells, but, at a high concentration, 100 μg ml⁻¹, it decreased the phagocytic activity. Electron microscopy was used for morphological classification of the peripheral leucocytes throughout the study.     

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

POTASSIUM AND AMINO ACID TRANSPORT IN HUMAN LEUKOCYTES EXPOSED TO PHAGOCYTIC STIMULI

Influxes of potassium and amino acids were measured in suspensions of human polymorphonuclear leukocytes (PMNs) under resting conditions and after various phagocytic stimuli. Both ouabain-sensitive (or pump) and ouabain-insensitive (or leak) influxes of K were determined. In 5 mM external K, mean total K influx was 0.69 nmol/10⁶ cells x min, of which 52% was ouabain-sensitive. Ouabain binding was irreversible, and, as in erythrocytes, was inhibited by K. At external concentrations of 0.1 mM, influxes of lysine and leucine were entirely carrier-mediated, with means of 0.021 nmol/10⁶ cells x min, and 0.019 nmol/10⁶ cells x min, respectively. After incubation of PMNs with zymosan or latex particles, the K pump was reduced more than 60%, whereas amino acid influxes were inhibited only by 30%. PMNs were also exposed to cytochalasin B before challenge by particles: the drug prevented phagocytosis but not surface binding of zymosan, nor did it influence transport of K or amino acids. After pretreatment of PMNs with cytochalasin B, interaction of zymosan with their surface resulted in the same degree of inhibition of influxes of K and amino acids as when the cells were permitted to phagocytose the particles. In contrast, exposure of PMN to latex particles, which do not bind to cytochalasin B-treated cells, after pretreatment of cells with cytochalasin B did not result in inhibition of influxes. Treatment of cells with colchicine had no effect on either membrane transport or its inhibition after exposure to various phagocytic stimuli. These results indicate that the surface membranes of PMNs are functionally heterogeneous with respect to the association of transport sites for the different solutes. Moreover, loss of specific membrane functions from phagocytosing cells may result from the surface-at-tachment phase of particle-cell interactions, since the interactions of zymosan particles with PMNs in the absence of phagocytosis also inhibited transport of solutes.