Independent Phagocytosis Research Articles

Longitudinal Characterization of Phagocytic and Neutralization Functions of Anti-Spike Antibodies in Plasma of Patients after Severe Acute Respiratory Syndrome Coronavirus 2 Infection

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

Exploiting Endocytosis for Non-Spherical Nanoparticle Cellular Uptake

Several challenges exist for successful nanoparticle cellular uptake—they must be able to cross many physical barriers to reach their target and overcome the cell membrane. A strategy to overcome this challenge is to exploit natural uptake mechanisms namely passive and endocytic (i.e., clathrin- and caveolin-dependent/-independent endocytosis, macropinocytosis and phagocytosis). The influence of nanoparticle material and size is well documented and understood compared to the influence of nanomaterial shape. Generally, nanoparticle shape is referred to as being either spherical or non-spherical and is known to be an important factor in many processes. Nanoparticle shape-dependent effects in areas such as immune response, cancer drug delivery, theranostics and overall implications for nanomedicines are of great interest. Studies have looked at the cellular uptake of spherical NPs, however, fewer in comparison have investigated the cellular uptake of non-spherical NPs. This review explores the exploitation of endocytic pathways for mainly inorganic non-spherical (shapes of focus include rod, triangular, star-shaped and nanospiked) nanoparticles cellular uptake. The role of mathematical modelling as predictive tools for non-spherical nanoparticle cellular uptake is also reviewed. Both quantitative structure-activity relationship (QSAR) and continuum membrane modelling have been used to gain greater insight into the cellular uptake of complex non-spherical NPs at a greater depth difficult to achieve using experimental methods.

Phagocytosis of microparticles increases responsiveness of macrophage-like cell lines U937 and THP-1 to bacterial lipopolysaccharide and lipopeptide

Following bacterial infection, macrophages produce pro-inflammatory cytokines in response to bacterial cell components, including lipopolysaccharide (LPS) and lipopeptide, and simultaneously phagocytize and digest the invading bacteria. To study the effects of phagocytosis on pro-inflammatory responses, we determined if phagocytosis of polystyrene latex beads with ~ 1 µm diameter increases pro-inflammatory cytokine expression by human macrophage-like U937 and THP-1 cells stimulated with LPS. Treating macrophage-like cells with beads coated with IgG to facilitate Fcγ receptor-mediated phagocytosis increased LPS-induced expression of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. Treatment with beads coated with poly-l-lysine to facilitate Fcγ receptor–independent phagocytosis also increased LPS-induced cytokine expression. Our results indicate that LPS-induced pro-inflammatory responses are enhanced by bead phagocytosis regardless of the uptake mechanism. Additionally, phagocytosis enhanced LPS-induced NF-κB activation, suggesting that Toll-like receptor (TLR) 4 signaling is enhanced by phagocytosis. Furthermore, bead phagocytosis enhanced pro-inflammatory responses in U937 cells stimulated with lipopeptide, a ligand for the TLR2/TLR6 heterodimeric receptor. In conclusion, microparticle phagocytosis by macrophage-like U937 and THP-1 cells enhances the innate immune response induced by bacterial components.

Inorganic arsenic can be potent granulotoxin in mammalian neutrophils in vitro

An important outcome arising out of occupational/environmental exposure to arsenic (As) is immunotoxicity. To determine the impact of inorganic As on innate immune cells, effects of a low dose of NaAsO2 (i.e. 20 ng As/ml) on select parameters associated with human and bovine neutrophils (PMN) were evaluated in vitro. PMN isolated from the blood of healthy individuals and cows (n = 8/treatment) were pre-incubated with NaAsO2 for 12 h before effects on PMN phagocytosis, transcription of TLR2, TLR4 and CD64 in human PMN – as well as on phagocytosis-dependent/-independent cell chemiluminescence (CL), phagocytosis and killing of Staphylococcus aureus and Escherichia coli, PMN H2O2 production and necrosis and TLR4 transcription in bovine PMN – were assessed. Relative to control (no As) PMN, treatment with As significantly decreased phagocytic capacity and CD64 mRNA, but increased TLR2 and TLR4 mRNA, in human PMN. In bovine PMN, while As also led to increased TLR4 mRNA abundance, it resulted in decreases in phagocytosis-dependent and -independent CL, PMN H2O2 production, PMN phagocytosis and killing of both E. coli and S. aureus by PMN. Considering the broad roles of PMN in immunology, the results of these studies increase our understanding of functional consequences of As exposure in inducing immunotoxicity and increasing susceptibility to (infectious) diseases in mammals.

Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells

Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.

S46 Phagocytosis By Blood Neutrophils Is Not Attenuated In Patients With Chronic Obstructive Pulmonary Disease

Rationale All COPD phenotypes have airway neutrophilia but,despite this, bacteria associated infections are common, relate to decline and a significant proportion of patients have persistent airway colonisation. This is suggestive of innate immune dysfunction. In vitro studies have shown reduced neutrophil migratory accuracy in COPD (Sapey, Stockley et al . 2011) however, the ability of the neutrophil to contain bacterial infection upon arrival at a site of infection is poorly understood. Literature regarding the phagocytic ability of neutrophils from patients with COPD is conflicting and inconclusive. It is unclear whether responses change depending on the bacterial species present. We hypothesised that neutrophil phagocytosis during COPD is impaired, predisposing patients to increased inflammation and reduced bacterial clearance. Methods Blood neutrophils were isolated from stable-state COPD patients and healthy age-matched controls (HC). Phagocytosis of both opsonised (with 10% pooled COPD serum) and unopsonised pHrodo™-conjugated Staphylococcus aureus bioparticles (SA, n = 20), or E scherichia coli bioparticles (EC, n = 10) and fluorescently labelled disease-relevant bacteria, Haemophilus influenzae (HI, n = 10) and Streptococcus pneumoniae (SP, n = 10) was assessed, at regular intervals over 60 min, using flow-cytometry. Results were confirmed using time-lapse video microscopy. Results Peak phagocytosis was achieved at 60 min for unopsonised bacteria and 30 min for opsonised bacteria. There were no differences in time to peak phagocytosis between bacterial species. Blood neutrophils from patients with COPD and HC displayed similar phagocytic ability, in both percentage of neutrophils with phagocytic activity and the amount of SA, EC, HI or SP ingested (as indicated by MFI) (COPD vs. HC, p > 0.05 for all). This was ubiquitous to both opsonin independent and opsonin-dependant phagocytosis, and was consistent across all time points measured. A typical comparison is shown in figure one, with unopsonised SA data. Conclusions Phagocytic ability of blood neutrophils from patients with COPD to ingest Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae and Haemophilus influenzae is not altered compared to age-matched healthy controls. This should be replicated in lung neutrophils to assess whether transmigration to the tissues affects function.

Functional Selection of Phagocytosis-Promoting Genes: Cell Sorting–Based Selection

Phagocytosis is a critical host defense mechanism that clears invading pathogens, apoptotic cells, and cell debris; it is an essential process for normal development, tissue remodeling, immune response, and inflammation. Here, a functional selection strategy was used to isolate novel phagocytosis-promoting genes. After the retroviral transfer of mouse brain cDNA library into NIH3T3 mouse fibroblast cells, cell sorting was used to select the cells that phagocytosed fluorescent zymosan particles. The cDNAs were retrieved from the selected cells and identified by DNA sequencing as eIF5A, Meg3, Tubb5, Sparcl-1, Uchl-1, Bsg (CD147), Ube2v1, and Pamr1. The phagocytosis-promoting activity for some of these cDNAs was confirmed by transient transfection in the independent phagocytosis assays. Thus, the unbiased selection procedure successfully identified multiple phagocytosis-promoting genes. The selection method can be applied to other cell-based assays where cells with a desired phenotype can be physically separated. Moreover, the new gene targets uncovered in this study could be relevant to biomolecule screening in search of phagocytosis-regulating agents. In a small-scale screen, a series of imidazopyridine compounds was tested to identify the small molecules that modulate eIF5A-mediated phagocytic activity. Several compounds that influenced the phagocytic activity can be further used as chemical-genetic tools to delineate the mechanisms of eIF5A action and be potential drug candidates that are capable of therapeutically modulating phagocytic activity.

Interleukin-10 enhances the CD14-dependent phagocytosis of bacteria and apoptotic cells by human monocytes

Monocytes are centrally involved in both specific and nonspecific immunity by secretion of regulatory immune mediators, phagocytosis, and presentation of antigens. Recent work has shown that monocytes can phagocytose bacteria independently from Fc gamma, complement, and scavenger receptors via a CD14-mediated process. Furthermore, incorporation of cells undergoing apoptosis is also mediated by CD14. In this study we investigated the regulation of monocytic CD14-dependent phagocytosis by the immunoregulatory cytokines interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1). In this study an in vitro human whole-blood assay was used to test regulation of CD14-dependent phagocytosis of fluorescence-labeled E. coli by IL-10, IFN-gamma, and TGF-beta1 in monocytes from healthy donors. Phagocytosis by monocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH) and its regulation by IL-10 was also investigated. Finally, regulation of monocytic incorporation of apoptotic Jurkat cells by IL-10 was analyzed. For the CD14 blockade, murine anti-CD14 IgG2a antibody RMO52 was used. We observed that IL-10, suggested to be a monocyte-deactivating cytokine, strongly increased the monocytic CD14-dependent phagocytosis of E. coli. In contrast, IFN-gamma and TGF-beta1 depressed monocytic CD14 incorporation of E. coli. Compatible with this, IL-10 upregulated CD14 expression on monocytes, whereas IFN-gamma and TGF-beta1 downregulated its expression. IL-10 also increased the monocytic CD14-dependent and -independent phagocytosis of apoptotic cells. As expected, IL-10 strongly increased the CD14-independent phagocytosis but had no influence on the CD14-dependent phagocytosis of monocytes from a PNH patient. In conclusion, our data support a general role of IL-10 for activating monocytic scavenger functions, which are at least partly mediated by CD14. This is in line with the fact that IL-10 promotes the development of monocytes to macrophages. The contrasting effects of IL-10 and IFN-gamma on monocytic CD14-dependent phagocytosis may reflect a further mechanism counterbalancing antigen-presentation and nonimmunogenic scavenging of bacterial and cellular debris. TGF-beta, however, may be an inhibitor of both systems.

Leucocyte mobilization and function in vitro of blood and exudative leucocytes after inguinal herniotomy.

The effect of surgical trauma on leucocyte mobilization in vivo and the function of blood and exudative leucocytes was studied in 15 patients after inguinal herniotomy. Mobilization of leucocytes to skin windows with chambers was reduced 48 h after anaesthesia (P less than 0.01) compared with 21 healthy volunteers. Blood leucocyte chemotaxis was reduced during the period of anaesthesia (P less than 0.05). Serum independent phagocytosis was increased from 24 h after anaesthesia (P less than 0.01) but without a corresponding increase in the serum dependent phagocytosis. The oxidative metabolic activity of resting leucocytes in terms of Nitro Blue Tetrazolium (NBT) reduction decreased 24-48 h after anaesthesia without any change in NBT reduction during phagocytosis. In contrast, exudative leucocytes exhibited reduced phagocytosis independent of serum factors (P less than 0.05) and increased NBT reduction by resting leucocytes. It is concluded that a diminished accumulation of leucocytes as inflammatory lesions occurs postoperatively and that changes in the function of circulating leucocytes do not always reflect the changes in function of the exudative leucocyte.

Positive Regulation of Phagocytosis by SIRPβ and Its Signaling Mechanism in Macrophages

SIRPbeta (signal-regulatory protein beta) is a transmembrane protein that is expressed in hematopoietic cells but whose functions are unknown. We have now cloned mouse SIRPbeta cDNA and have shown that the gene is expressed in various tissues in addition to cells of the macrophage lineage. Engagement of SIRPbeta by specific monoclonal antibodies promoted Fcgamma receptor-dependent or -independent phagocytosis in mouse peritoneal macrophages. It also induced marked activation of MAPK and the upstream kinase MEK but weak activation of Akt. MEK inhibitors markedly blocked the promotion of phagocytosis by SIRPbeta, whereas an inhibitor of phosphoinositide 3-kinase partly blocked such response. In addition, inhibitors of myosin light chain kinase or of myosin ATPase blocked the promotion of phagocytosis by SIRPbeta. Furthermore, SIRPbeta induced the formation of filopodia and lamellipodia in macrophages as well as the translocation of activated MAPK to these structures. It also elicited tyrosine phosphorylation of DAP12, Syk, and SLP-76, and a Syk inhibitor blocked the promotion of phagocytosis and activation of MAPK by SIRPbeta. Our results suggest that engagement of SIRPbeta promotes phagocytosis in macrophages by inducing the tyrosine phosphorylation of DAP12, Syk, and SLP-76 and the subsequent activation of a MEK-MAPK-myosin light chain kinase cascade.

The Cytoplasmic Domain of Human FcγRIa Alters the Functional Properties of the FcγRI·γ-Chain Receptor Complex

The gamma/zeta-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding alpha-chain alters the biological properties of the receptor complex. Using macrophage FcgammaRIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human FcgammaRIa. Both receptors functionally associate with the endogenous murine gamma-chain. However, we have established that the CY of FcgammaRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcgammaRIa CY leads to slower kinetics of receptor-specific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent FcgammaRIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates FcgammaRIa-specific secretion of interleukin-6 but does not affect production of interleukin-1beta. These results demonstrate a functional role for the CY of FcgammaRIa and provide a general model for understanding how multiple receptors that utilize the gamma-chain can generate diversity in function.

Purification and Characterization of a Phagocytosis Inducing Factor: Role in Cell-Substratum Adherence

The presence of two different signaling molecules is essential to induce opsonin- independent phagocytosis of particulate activators of the human alternative complement pathway by human monocytes. In addition to the involvement of a low Mrpeptide cytokine or phagocytosis inducing factor (PIF) , we have now established that the participation of bacterial lipopolysaccharide is also required. PIF has been demonstrated to be present in human cell lines of different origins, e.g., WISH cells, Raji cells, U937 cells, HL-60 cells and M21 cells. PIF has been purified to apparent homogeneity from the U937 cell line by anion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sephadex G-50. On the basis of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass has been estimated to be approximately 1,600 Da. PIF also plays an important role in the regulation of cell-substratum adherence.

Enhancement of phagocytosis by corticostatin I (CSI) in cultured mouse peritoneal macrophages

Corticostatin I (CSI) is one of the corticostatic peptides which inhibit ACTH-stimulated steroidogenesis. To clarify the function of CSI on the immune system, the effect of CSI on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. In the presence of Ca2+ and Mg 2+, CSI enhanced phagocytosis of latex beads in a dose-dependent manner. Unstimulated phagocytosis in physiological solution consisted of Ca2+ and Mg2+-dependent and -independent phagocytosis. Divalent cations-independent phagocytosis was sensitive to CSI. Present results suggest that the enhancement of phagocytosis by CSI may be one of the mechanisms modulating the immune response regarding infection and inflammation. Present study also showed that one of defensin HNP-1 enhanced phagocytosis.

Beta-Endorphin enhances phagocytosis of latex particles in mouse peritoneal macrophages.

The effects of beta-endorphin (beta End) on phagocytosis in peritoneal macrophages were examined by using flow cytometry (FCM). Beta End enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), alpha-endorphin (alpha End), gamma-endorphin (gamma End), alpha End (18-31) and beta End (28-31) had no such activity. Beta End (1-27) and beta End (6-31) enhanced phagocytosis less effectively than beta End did. Naloxone did not inhibit the enhancement of phagocytosis induced by beta End. Unstimulated control phagocytosis was partially suppressed in Ca2(+)-free EGTA-containing solution and even in this solution beta End enhanced phagocytosis. However, the enhancement was suppressed in the solution containing BAPTA-AM. The present study showed that beta End enhanced extracellular Ca2+ ([Ca2+]o)-dependent and -independent phagocytosis and that the enhancement is largely dependent on intracellular Ca2+ ([Ca2+]i). These results support the contention that beta End is one of the mediators that modulates the immune system.

Inhibitory effect of vasoactive intestinal peptide (VIP) on phagocytosis in mouse peritoneal macrophages

The effect of VIP on phagocytosis in peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that VIP suppressed phagocytosis in a dose-dependent manner. VIP(1–12) did not suppress phagocytosis. VIP(10–28) was more suppressive than VIP(1–28). A known VIP-antagonist (N-Ac-Tyr 1, d-Phe 2)-growth hormone-releasing factor (GRF) (1–29)-NH 2 suppressed phagocytosis less than VIP. Control phagocytosis was partially suppressed in Ca 2+-free solution. Phagocytosis was suppressed by VIP further in Ca 2+-free solution than in the normal solution. Phagocytosis was suppressed in a known phosphodiesterase inhibitor IBMX-containing solution. The degree of suppression by VIP was the same in the presence or the absence of IBMX. These results suggest that VIP suppresses extracellular Ca 2+-dependent and -independent phagocytosis, that the C-terminal fragment of VIP is essential for VIP action, that the suppression is mediated by cAMP and that the inhibition of macrophage phagocytosis by VIP is one of the mechanisms which modulates immune responses by the nervous system.

Suppression of phagocytosis by adrenocorticotropic hormone in murine peritoneal macrophages

Phagocytosis of latex beads by peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that adrenocorticotropic hormone (ACTH) suppressed phagocytosis in a dose-dependent manner. ACTH (1–24) was more suppressive than ACTH (1–39). Control phagocytosis was partially suppressed in Ca 2+-free solution. Phagocytosis was suppressed by ACTH in this solution to the same degree as in the normal solution. Suppression by ACTH was reduced in phosphodiesterase inhibitor-containing solution. These results suggest that (1) ACTH suppresses extracellular Ca 2+-dependent and -independent phagocytosis, (2) the suppression is not mediated by cAMP and (3) the inhibition of macrophage phagocytosis by ACTH is one of the mechanisms that modulate immune responses in stressful situations.

Canine distemper virus increases procoagulant activity of macrophages

Inflammatory demyelination in canine distemper has been proposed to be due to a “bystander” mechanism, in which macrophages play an important role. In the present work we studied whether infection of macrophages by canine distemper virus (CDV) results in changes of macrophage functions, including Fc receptor-dependent and -independent phagocytosis, release of reactive oxygen species (ROS), and procoagulant activity (PCA). As a source of macrophages, dog bone marrow cells were seeded in teflon bags and grown for 1–2 weeks, at which time a marked enrichment of macrophages was noted. These cells were infected with the A75/17 strain of CDV. We could not detect any significant difference between uninfected and CDV-infected macrophages with respect to Fc receptor-dependent or -independent phagocytosis or with respect to the release of ROS. However, from Day 4 p.i. to the end of our observation period (10 days p.i.), PCA was up to 10-fold higher in CDV-infected unstimulated macrophage cultures than in uninfected unstimulated cultures of the same age. Increase in PCA was not due to the inoculation procedure by itself nor to components of the inoculum other than CDV; in particular, PCA was not due to contaminating endotoxin. Thus, several important macrophage functions do not appear to be impaired by CDV infection. The marked increase of macrophage PCA expression suggests that certain macrophage functions may even be enhanced as a result of infection. Such macrophage activation might contribute to the pathogenesis of the disease.

Kinetics of Phagocytosis and Phagosome-Lysosome Fusion in Hamster Lung and Peritoneal Macrophages

The time course of phagocytosis and phagosome-lysosome fusion (PLF) in lung and peritoneal macrophages (LMs and PMs) was measured. Lysosomes in unelicited hamster LMs and PMs were labeled with lucifer yellow. Macrophages then phagocytized heat-killed Saccharomyces cerevisiae (yeast) and were evaluated at several time points for the degree to which yeast particles were adherent vs. internalized and for the presence or absence of PLF as based on the presence or absence of lucifer yellow in yeast-containing phagosomes. A three-compartment model (adherent, ingested, fused) of independent phagocytosis and PLF was developed; the number of yeast particles in each compartment was counted, and rate constants for ingestion and fusion were determined. Comparison of rate constants showed that ingestion was significantly faster in PMs (0.047 +/- 0.005 min-1) than in LMs (0.016 +/- 0.005 min-1) (mean +/- pooled SEM; P less than 0.001). Similarly, PLF was significantly faster in PMs (0.109 +/- 0.013 min-1) than in LMs (0.046 +/- 0.013 min-1) (P less than 0.003).

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis.

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.

Alginase treatment of mucoid Pseudomonas aeruginosa enhances phagocytosis by human monocyte-derived macrophages.

Mucoid Pseudomonas aeruginosa colonizes and infects the respiratory tract of most older patients with cystic fibrosis. These bacteria resist both opsonin-dependent and -independent phagocytosis by human polymorphonuclear leukocytes and monocyte-derived macrophages. Resistance to phagocytosis is thought to be mediated in part by the mucoid exopolysaccharide associated with the bacterial surface. The purpose of this study was to determine whether degradation of the mucoid exopolysaccharide by alginase enhances bacterial susceptibility to nonopsonic phagocytosis by macrophages. Eight phagocytosis-resistant mucoid P. aeruginosa isolates from patients with cystic fibrosis were studied. The bacteria were treated with a preparation of alginase from Bacillus circulans, and phagocytosis by macrophages was measured by a visual inspection assay. Alginase degradation of mucoid exopolysaccharide was measured by the periodate-thiobarbituric acid assay and by indirect immunofluorescence with a mouse monoclonal antibody to the mucoid exopolysaccharide. Alginase degraded the mucoid exopolysaccharide of all eight mucoid strains tested. Phagocytosis was enhanced in five of the eight strains. Alginase-enhanced phagocytosis was magnesium dependent and heat labile. Alginase may be a useful tool for studying the biological properties of P. aeruginosa mucoid exopolysaccharide.