Phagocytosis of staphylococci by human polymorphonuclear leukocytes is enhanced in the presence of endothelial cells.

The role of various surfaces in the phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) was studied. Uptake of both opsonized and unopsonized staphylococci on the surface of a monolayer of human venous endothelial cells was compared with uptake on an inert plastic surface, with an assay that uses radiolabeled bacteria. Uptake of unopsonized S. aureus was threefold higher on the endothelial cell surface than on the plastic surface and was followed by efficient killing of the phagocytosed staphylococci. Uptake of unopsonized S. aureus on endothelial cells was not inhibited by treatment of the PMN with pronase or 2-deoxy-D-glucose and was only partially inhibited by cytochalasin B treatment of the PMN. The supporting effect of endothelial cells on the phagocytosis of unopsonized S. aureus was not due to opsonization of the bacteria by immunoglobulin or complement from the endothelial cell surface, nor to coating with fibronectin.     

Interaction between human polymorphonuclear leucocytes and Staphylococcus aureus in the presence and absence of opsonins.

Phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN) in the presence and absence of opsonins was studied with an assay which allows interaction between PMN and bacteria on a surface. The kinetics of uptake, the activity of the metabolic burst, and the degranulation during phagocytosis of opsonized and unopsonized bacteria were compared. Uptake of unopsonized S. aureus proceeded at a slower rate, but unopsonized staphylococci induced metabolic activity and degranulation in the PMN to the same extent as opsonized bacteria. Treatment of PMN with a metabolic inhibitor (2-deoxy-D-glucose) or with an inhibitor of microfilament function (cytochalasin B) totally inhibited the capacity of PMN to ingest unopsonized S. aureus, whereas uptake of opsonized bacteria was much less affected. Treatment of the PMN with pronase prevented uptake of unopsonized bacteria, but had no effect on the uptake of opsonized bacteria. Uptake was not inhibited by mannose. Recognition of S. aureus by the PMN was not dependent on the presence of the cell wall components protein A or teichoic acid. The presence of a capsule inhibited uptake.     

Phagocytosis by polymorphonuclear leukocytes of Staphylococcus aureus and Pseudomonas aeruginosa adherent to plastic,agar, or glass

Phagocytic cells may encounter bacteria in vivo that are stationary or adherent to a surface. In this study, recently developed in vitro techniques were adapted to evaluate the interaction of polymorphonuclear leukocytes (PMN) with adherent Staphylococcus aureus and Pseudomonas aeruginosa. By measuring the uptake of radiolabeled bacteria, we found that normal human PMN readily phagocytize these organisms when they are attached to plastic or when they are grown on the surface of nutrient agar. Bacteria adherent to glass elicited a chemiluminescent response, and such organisms were phagocytized and killed by PMN. Opsonization of S. aureus and P. aeruginosa was not required for surface phagocytosis, chemiluminescence, or killing. These new methods should allow evaluation of certain biological and clinical aspects of surface phagocytosis in host defense.     

Opsonization of Staphylococcus aureus protects endothelial cells from damage by phagocytosing polymorphonuclear leukocytes.

When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.     

Activation of human polymorphonuclear leucocytes by particulate zymosan is related to both its major carbohydrate components: glucan and mannan.

Unopsonized particulate zymosan and its major carbohydrate component glucan were phagocytosed under serum-free conditions by adherent polymorphonuclear leucocytes (PMN) in a dose- and time-dependent manner. Preincubation of PMN monolayers with mannan did not cause a reduction in the phagocytosis of either particle. The phagocytic response was inhibited by preincubation of the cells with trypsin at a concentration that did not inhibit the phagocytosis of sheep erythrocytes coated with IgG or of latex particles. Homology of the recognition mechanisms for glucan and zymosan was confirmed when cells cultured on fixed glucan or on fixed zymosan failed to ingest either particle to more than 40% of control phagocytosis. Similarly, zymosan and glucan activated PMN in suspension, in a dose- and time-dependent manner, to generate reactive oxygen species which were measured as luminol-dependent chemiluminescence (CL). There was, however, a four-fold greater CL response to zymosan. Preincubation of PMN with mannan resulted in a significantly decreased CL response to zymosan, while the response to glucan was unaffected. The CL response was also sensitive to a range of concentrations of trypsin. In contrast, two other complex polysaccharide particles (barley-derived beta-glucan and algae-derived laminarin) were not phagocytosed by PMN, nor did they cause the generation of CL, despite the fact that they possessed the capacity, in common with zymosan and glucan, to activate the alternative pathway of complement. The identification of a trypsin-sensitive recognition mechanism on the surface of human PMN for unopsonized zymosan and glucan represents a response not hitherto characterized. Furthermore, our data indicate that the phagocytosis of unopsonized zymosan by human PMN is dependent primarily on its glucan content, but that its capacity to activate the respiratory burst may involve mannan and the recruitment of a second cell surface recognition mechanism.     

Assessment by a fluorochrome microassay of phagocytic killing of group B streptococci adherent to glass

An in vitro adherence assay was developed to study the interaction between bovine polymorphonuclear cells (PMN) and group B streptococci (GBS) on glass. The kinetics of uptake and killing of GBS in the presence and absence of opsonins were compared by staining bacteria with a fluorochrome (acridine orange). Opsonization was not required for the PMN to ingest and kill glass-adherent GBS. Nevertheless, the presence of serum during the phagocytosis incubation, or preopsonization of bacteria, both increased the rate of ingestion and the number of bacteria per PMN at the end of the reaction (90 min). Precolostral calf serum (PCS), almost devoid of antibodies, was as effective as normal bovine serum in this respect. By contrast, PCS was not an effective opsonin source when phagocytosis took place in suspension, demonstrating further that different underlying mechanisms operate depending on the physical presentation of bacteria to phagocytes. It was noted that PMN monolayers were able to ingest unopsonized bacteria that merely settled on the glass surface during phagocytosis incubation. The method should permit investigations on the mechanisms involved in surface phagocytosis, a defense mechanism of potential importance in local infections.     

Opsonin-dependent and independent surface phagocytosis of S. aureus proceeds independently of complement and complement receptors.

We examined the mechanism of surface phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN). Surface phagocytosis of unopsonized bacteria occurred, but was significantly enhanced by the presence of serum. The serum requirement was low, and a maximal effect occurred with serum concentrations of 0.25-0.5%. The opsonic effect of serum was not removed by heat inactivation of complement but was adsorbed, at low serum concentrations, by protein A, indicating that opsonin-dependent surface phagocytosis requires IgG but not C3. The requirement of opsonin-dependent surface phagocytosis for IgG was demonstrated further with purified IgG preparations as the sole opsonin. Activation of PMN by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA) increased opsonin-independent surface phagocytosis by 47% and 66%, respectively, but had no effect on opsonin-dependent surface phagocytosis. Blockade of the PMN iC3b receptor (CR3), which has lectin-like properties, by a panel of monoclonal antibodies against the alpha- and beta-chains of CR3 did not inhibit the surface phagocytosis of opsonized or unopsonized S. aureus, and one antibody (NIMP-R10) enhanced opsonin-independent surface phagocytosis. These results indicate that the mechanism of surface phagocytosis is quite different to that observed in suspension assays. Opsonin-independent surface phagocytosis occurs and is enhanced by PMN activation, opsonin-dependent surface phagocytosis is dependent on IgG and not complement, and neither opsonin-independent nor -dependent surface phagocytosis proceeds through CR3.     

Polyamino acid enhancement of bacterial phagocytosis by human polymorphonuclear leukocytes and peritoneal macrophages.

Cationic polyamino acids are known to enhance a variety of cell-cell interactions by virtue of their ability to alter electrostatic forces of cell surfaces. In this study, the effect of polyamino acids on phagocytosis of 3H-labeled bacteria by human polymorphonuclear leukocytes (PMNs) and peritoneal macrophages was investigated. Negatively charged and neutral polyamino acids did not influence phagocytosis of unopsonized Staphylococcus epidermidis, whereas protamine, poly-L-arginine, and poly-L-lysine stimulated phagocytosis in a dose-dependent manner. At 50 micrograms/ml, greater than 30% uptake by PMNs was seen with each of these cationic polyamino acids. Although cationic polyamino acids promoted PMN and peritoneal macrophage phagocytosis of unopsonized S. epidermidis, Staphylococcus aureus M (encapsulated) and M variant (unencapsulated), and Escherichia coli J5, little effect was seen with the parent E. coli O111:B4 or a serotype O222:H16 strain. Pretreatment of bacteria and phagocytes separately demonstrated that the phagocytosis-promoting property of polyamino acids is manifest predominantly on the bacteria. Bacteria pretreated with cationic polyamino acids also elicited a PMN chemiluminescent response, and PMN-associated bacteria were killed, as determined by a fluorochrome microassay. Thus, cationic polyamino acids promote the phagocytosis and killing of many but not all bacterial strains, and in this respect polyamino acids function as opsonins.     

Oxidative response of human neutrophils, monocytes, and alveolar macrophages induced by unopsonized surface-adherent Staphylococcus aureus.

In contrast to results with bacterial suspensions, phagocytosis of unopsonized bacteria readily occurs when bacteria are adhered to glass or plastic surfaces. However, in contrast to neutrophils, alveolar macrophages produced much less DNA denaturation as measured by acridine orange metachromasia of phagocytized Staphylococcus aureus. We have studied the phagocytosis of unopsonized surface-adherent S. aureus and the subsequent production of reactive oxygen species by peripheral blood neutrophils, monocytes, and alveolar macrophages. Phagocyte-free systems were then used to show the relationship of the reactive oxygen species produced by neutrophils and alveolar macrophages and the denaturation of unopsonized S. aureus DNA with acridine orange. Peripheral blood neutrophils, monocytes, and alveolar macrophages from normal human volunteers were added to vials with adherent S. aureus without opsonin. Bacterial uptake and luminol- and lucigenin-dependent chemiluminescence were measured. Neutrophils developed much greater luminol-dependent chemiluminescence than monocytes or alveolar macrophages. Compared with neutrophils and monocytes, alveolar macrophages developed significantly greater concentrations of superoxide, as measured by lucigenin-dependent chemiluminescence and ferricytochrome c reduction. These findings suggested that products of the myeloperoxidase-hydrogen peroxide-halide pathway were generated when peripheral blood neutrophils were stimulated and that alveolar macrophages primarily produced superoxide. When these reactive oxygen species were generated in phagocyte-free systems containing S. aureus, products of the myeloperoxidase-hydrogen peroxide-halide pathway produced denaturation of S. aureus DNA, whereas superoxide did not. Thus, differences in reactive oxygen species produced during phagocytosis may be related to the different capacities of neutrophils and alveolar macrophages to denature unopsonized adherent S. aureus DNA.     

Chemiluminescent response of polymorphonuclear leukocytes to Streptococcus pneumoniae and Haemophilus influenzae in suspension and adhered to glass.

We measured the luminol- and lucigenin-enhanced chemiluminescent response of human polymorphonuclear leukocytes (PMN) stimulated by various strains of Streptococcus pneumoniae and Haemophilus influenzae. In the absence of opsonin, phagocytosis of either bacterial species elicited good PMN response when the bacteria were adhered to a surface but minimal PMN response when they were in suspension. When 10% pooled human serum was used as a source of opsonin, a moderate to excellent chemiluminescent PMN response was elicited during phagocytosis of opsonized bacteria both in suspension and adhered to surface. We conclude that opsonin significantly enhances PMN chemiluminescence when a suspension-type assay is used and that opsonin-independent mechanisms play a significant role in the chemiluminescent response of PMN during phagocytosis of adherent bacteria.     

Regulation of human neutrophil type 3 complement receptor (iC3b receptor) expression during phagocytosis of Staphylococcus aureus and Escherichia coli.

Human neutrophils (PMN) express a receptor for iC3b, a cleavage product of C3b. CR3 is an important receptor for phagocytosis of opsonized bacteria and its expression is enhanced by cell activation. We examined PMN CR3 expression during phagocytosis using flow cytometry and a CR3-specific monoclonal antibody. After 30 min phagocytosis of opsonized S. aureus and E. coli, CR3 expression increased to 151% and 221% of controls, respectively. Unopsonized S. aureus had no effect on CR3; however, unopsonized E. coli enhanced CR3 expression despite not being phagocytosed. Time-kinetic studies indicated a rapid initial fall in CR3 after addition of bacteria to PMN, followed by enhanced expression within 5-10 min. The initial fall in CR3 probably represented CR3 internalization rather than receptor destruction, as superoxide dismutase, catalase and protease inhibitors had no effect on this. Correlation of CR3 expression with the PMN oxidative response, measured with the intracellular fluorescent probe DCF-DA, demonstrated a dichotomy. Opsonized S. aureus and E. coli caused an oxidative response from PMN but unopsonized E. coli, which caused significant CR3 up-regulation, did not. CR3 up-regulation with unopsonized and opsonized E. coli was markedly inhibited by Polymyxin B, suggesting a role for endotoxin. These experiments indicate that CR3 expression can be regulated during phagocytosis, and the mechanisms responsible are distinct from those involved in the oxidative burst. CR3 up-regulation following exposure to bacteria in vivo may enhance neutrophil function at sites of infection.     

Glucose Stimulates Phagocytosis of Unopsonized Pseudomonas aeruginosa by Cultivated Human Alveolar Macrophages

Glucose has previously been shown to increase the in vitro phagocytosis of unopsonized Pseudomonas aeruginosa by freshly explanted murine peritoneal macrophages (PM) and cultivated alveolar macrophages (AM). This study examined the effect of glucose on the same phagocytosis process in human AM in order to determine whether this phenomenon is conserved among species. Freshly explanted human AM phagocytosed unopsonized P. aeruginosa at a low level (2 bacteria/macrophage/30 min), whereas mouse AM ingested a negligible number of P. aeruginosa (0.01 bacterium/macrophage/30 min). Glucose had no effect on this or other phagocytic processes in freshly explanted mouse or human AM. However, following in vitro cultivation for 72 h, human AM phagocytosed three to four times more unopsonized P. aeruginosa than did freshly explanted cells, but only in the presence of glucose. This glucose-inducible phagocytic response had also been observed in cultivated murine AM. Although similar increases were also detected for the phagocytosis of latex particles and complement-coated sheep erythrocytes by cultivated human AM, these processes were not glucose dependent. The lack of response to glucose in freshly explanted mouse AM was attributed to insufficient glucose transport; however, freshly explanted human AM exhibited significant facilitative glucose transport activity that was inhibitable by cytochalasin B and phloretin. Taken together, these results suggest that the process of glucose-inducible phagocytosis of unopsonized P. aeruginosa is conserved among macrophages from different species, including humans, and that AM, but not PM, required cultivation for this glucose effect to occur. Glucose transport by AM appears to be necessary but not sufficient for phagocytosis of unopsonized P. aeruginosa.     

Potential of human polymorphonuclear leukocytes to synthesize and secrete sulfated proteoglycans

To analyze the function of proteoglycans (PG) in different types of leukocytes, both the relative amounts and specific types of proteoglycans produced by cultured human peripheral blood polymorphonuclear leukocytes (PMN) were were determined and compared to mononuclear leukocytes (PBMC). Media from 3-day cultured PMN contained significantly less (less than 10%) 35SO2-4-labeled PG than media from PBMC cultures. Incorporation of 35SO2-4 into cell-associated material was comparable for both types of white blood cells. In contrast to PBMC, PMN could not increase their synthesis or secretion of PG after exposure to concanavalin A or phorbol-12-myristate-13-acetate. Various inducers of leukocyte chemotaxis also failed to enhance PG production by PMNs. Release of prelabeled PG from PMNs could be induced by exposure to either opsonized or unopsonized zymosan (yeast) as well as the bacteria S. aureus, suggesting that particle ingestion may be accompanied by PG exocytosis. Both chondroitinase ABC and AC digested greater than 90% of PMN 35S-labeled material in media and 75% in cell lysates; HNO3 treatment removed less than 5% of N-linked 35SO4 from radiolabeled media and 25% from cells. Treatment with 0.5 N NaOH released shortened glycosaminoglycan chains from 35S-labeled PMN cell lysates. beta-D-xylosides did not stimulate an increase in polysaccharide chain production by cultured PMNs. These data suggest that PMNs can produce chondroitin 4-sulfate PG whose synthesis is not affected by treatments that alter PMN functions; in contrast to PBMCs, PMNs will actively release these molecules when exposed to micro-organisms that stimulate phagocytosis.     

Leukocyte spreading behavior on vascular biomaterial surfaces: consequences of chemoattractant stimulation

Chemoattractant-induced phenomena of polarity and migration of polymorphonuclear leukocytes (PMN) are believed to play a key physiological role in controlling bacterial infections on implantable vascular biomaterials. Our study targeted the spreading behavior of human PMN adherent to expanded polytetrafluoroethylene (ePTFE), pretreated with various plasma proteins, in response to the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMLP). To this end, a novel imaging configuration was developed to allow in situ reconstructive analysis of PMN 3-D morphology on opaque ePTFE surfaces, using optical sectioning confocal microscopy. Following fMLP stimulation, PMN morphological polarity was enhanced on all substrates studied except fibrinogen treated ePTFE. 3-D PMN morphometry revealed that in the absence of fMLP, overall cell spreading was minimized on albumin-treated ePTFE and maximized on fibrinogen and immunoglobulin-G-treated ePTFE. Following fMLP stimulation, overall PMN spreading increased markedly on untreated and albumin-coated ePTFE, while it stayed invariant on IgG and plasma treated ePTFE, and decreased on fibrinogen-treated ePTFE. Spatial analysis of PMN spreading following fMLP stimulation revealed enhanced PMN attachment on untreated and albumin treated ePTFE and diminished attachment on fibrinogen and plasma treated ePTFE. Thus, chemoattractant stimulation altered a wide range of PMN spreading attributes on ePTFE, including morphological polarity, substrate attachment, and 3-D membrane spreading, in a substrate dependent manner. These chemoattractant-induced spreading responses may also have important consequences for PMN phagocytosis. We report that fMLP stimulation led to enhanced unopsonized particulate phagocytosis on untreated and albumin treated ePTFE, but caused no discernible change in phagocytosis on other protein substrates. Thus, chemoattractant modulation of PMN spreading on ePTFE is highly substrate-regulated, and manifests in concerted effects on PMN phagocytosis.     

Direct activation of human polymorphonuclear leukocytes by Pseudomonas aeruginosa

Brief exposure of polymorphonuclear leukocytes (PMNs) to unopsonized Pseudomonas aeruginosa resulted in significant enhancement of PMN adherence and 3H-deoxyglucose uptake, two important indicators of PMN activation. Dose-response and time-course experiments show that maximal stimulation of PMNs occurred at a PMN: bacteria ratio of 10:1 and exposure time of 10 min. The PMN-stimulatory capacity of P. aeruginosa was completely or partially abolished by treatment with formalin, heat, ultrasonication and ultraviolet (UV) irradiation, all of which affect the integrity of ligands on the bacteria surface. The putative glycoprotein ligand(s) contains galactose rather than glucose as shown by the ability of galactose in the medium to abrogate PMN-stimulatory activity. This PMN-stimulatory activity was not mediated by soluble factor(s) released from either P. aeruginosa or PMNs as shown by experiments with appropriate supernatants. Finally, PMN-stimulating activity was also observed with P. maltophilia and P. pseudomallei, but not by the other Pseudomonas spp. such as P. cepacia and P. fluorescens. These results suggest that P. aeruginosa and other Pseudomonas spp. may have little capacity to circumvent PMN host defences. This may account in part for their limited pathogenic role as opportunistic micro-organisms, for the severe inflammatory lesions seen in Pseudomonas infections when they do occur, and is consistent with the fact that infection with these organisms is commonly associated with impaired quantity or function of PMNs.     

Interference with granulocyte function by Staphylococcus epidermidis slime.

The interaction of Staphylococcus epidermidis slime with human neutrophils (PMN) was examined by using isolated slime and allowing bacteria to elaborate slime and other extracellular products in situ. S. epidermidis slime was found to contain a chemoattractant. Incubation of PMN with 50 micrograms or more of slime per ml inhibited subsequent chemotaxis of the PMN to n-formyl-methionyl-leucyl-phenylalanine by 27% and to zymosan-activated serum by 44 to 67% with increasing slime concentrations. S. epidermidis slime stimulated little degranulation of untreated PMN. After pretreatment of PMN with 5 micrograms of cytochalasin b per ml, slime predominantly induced release of specific granule contents (33.8% lactoferrin release by 250 micrograms of slime per ml versus 10% myeloperoxidase release by 250 micrograms of slime per ml). By a surface phagocytosis assay, PMN uptake of radiolabeled S. epidermidis which were incubated for 18 h on a plastic surface for slime expression was less than that for S. epidermidis adhered to the plastic for 2 h or grown in unsupplemented nutrient broth. These results suggest that S. epidermidis slime interaction with PMN may be potentially detrimental to host defense and may contribute to the ability of this organism to persist on surfaces of foreign bodies in the vascular or central nervous system.     

Human neutrophil swelling induced by immune complexes and aggregated IgG

Using a Coulter counter method, the effects of various types of IgG-dependent phagocytic stimuli on human neutrophil (PMN) swelling were determined. Human heat aggregated IgG, ovalbumin-antiovalbumin (OV-anti-OV) immune complexes, and opsonized latex particles all induced PMN swelling. The OV-anti-OV immune, complexes were effective, whether prepared at antigen-antibody equivalence (insoluble) or at 4 or 9 times antigen excess (soluble). Swelling of PMN occurred at 37°C, but not at 4°C. Complement was not present in any of the experiments. In contrast to the above results, native IgG, OV-anti-OV F(ab)₂ immune complexes and unopsonized latex particles did not induce PMN swelling. These results suggest that the PMN swelling observed in this study is due to Fc-dependent, complement-independent membrane stimulation and/or phagocytosis.Supported by: U.S. Public Health Service Program/Project Grant AM09989.     

Phagocytosis and killing of Actinobacillus pleuropneumoniae by alveolar macrophages and polymorphonuclear leukocytes isolated from pigs.

To study the cellular response of phagocytic cells to Actinobacillus pleuropneumoniae, we investigated whether porcine alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) are able to phagocytize and intracellularly kill A. pleuropneumoniae in vitro. Bacterial cultivation methods of A. pleuropneumoniae were used to assess in vitro phagocytosis and the ability to kill. A specific-pathogen-free pig was killed, blood was collected, and PMN were isolated and counted. The AM were isolated by lung lavage of the same animal and counted. In addition, convalescent-stage serum was collected from a specific-pathogen-free pig that was infected with A. pleuropneumoniae. Both porcine AM and porcine PMN effectively phagocytized A. pleuropneumoniae in the presence of convalescent-stage pig serum. PMN killed 90 to 99% of the bacteria intracellularly, whereas AM did not. Because A. pleuropneumoniae produces exotoxins that kill porcine AM and porcine PMN, we incubated equal amounts of bacteria and phagocytic cells and tested the viability of the cells 120 min later. In the presence of convalescent-stage pig serum, A. pleuropneumoniae was toxic to AM but not to PMN. Probably in porcine AM, intracellular released toxins of A. pleuropneumoniae lessen the ability of the cell to kill the bacterium. Consecutive lysis of AM and release of viable A. pleuropneumoniae may initiate the characteristic porcine pleuropneumonia.     

Preservation of Fc gamma receptor-mediated particle phagocytosis and binding with rat alveolar macrophages adhered to a plastic substrate in a serum free system

Functional differences between adherent macrophages and macrophages in suspension have been repeatedly reported. During the course of developing serum-free phagocytic assays to evaluate Fc gamma receptor (Fc gamma R)-mediated phagocytosis by rat alveolar macrophages (AM) we found Fc gamma R-mediated phagocytosis by Rat AM in suspension was markedly greater than when these cells were bound to plastic substrate in protein-free medium. This dissimilarity in performance was related, in part, to a plastic substrate-associated loss in Fc gamma R-mediated phagocytosis in that this cell function was preserved when monolayers of AM were formed in culture medium containing a low concentration of bovine serum albumin (BSA). The results of particle binding studies suggest the diminished phagocytic activities of AM in monolayers formed in the absence of BSA was due to a loss in the functional expression of Fc gamma R.     

Preservation of Fcγ Receptor-Mediated Particle Phagocytosis and Binding with rat Alveolar Macrophages Adhered to a Plastic Substrate in a Serum Free System

Functional differences between adherent macrophages and macrophages in suspension have been repeatedly reported. During the course of developing serum-free phagocytic assays to evaluate Fcγ receptor (FcγR)-mediated phagocytosis by rat alveolar macrophages (AM) we found FcγR-mediated phagocytosis by Rat AM in suspension was markedly greater than when these cells were bound to plastic substrate in protein-free medium. This dissimilarity in performance was related, in part, to a plastic substrate-associated loss in FcγR-mediated phagocytosis in that this cell function was preserved when monolayers of AM were formed in culture medium containing a low concentration of bovine serum albumin (BSA). The results of particle binding studies suggest the diminished phagocytic activities of AM in monolayers formed in the absence of BSA was due to a loss in the functional expression of FcγR.