Quantitative studies of phagocytosis by alveolar macrophages

The initial rate of phagocytosis by rabbit alveolar macrophages of paraffin oil emulsions stabilized with albumin was markedly increased by prior incubation of the emulsion with serum. The active component(s) of serum was non-dialyzable and heat-labile and was absent from zymosan-treated serum. Magnesium and calcium were both required for the maximal rate of phagocytosis. At 4 °C or in the presence of 1 mM N-ethylmaleimide, the rate of phagocytosis was less than 2% of the control (37° C) rate. The initial rates of phagocytosis of this emulsion by alveolar macrophages from rabbits injected with Freund's adjuvant were not demonstrably different from those observed with macrophages from normal rabbits. Per of mg of cell protein, polymorphonuclear leukocytes ingested serum-treated emulsion more rapidly than did macrophages, but per cell the rates were not different.     

Transmembrane potential changes during phagocytosis in rat alveolar macrophages

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol-12-myristate-13-acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mM K+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.     

Disorder of the phagocytosis process in alveolar macrophages in thermal injury

Bacteriological assay, cytochemical studies of succinate and malate dehydrogenases, acid phosphatase, glycogen and lipids, as well as electron microscopy were used in experiments on 75 rabbits to examine over time phagocytosis of alveolar macrophages and some mechanisms of its disturbance after burn trauma. It was established that the phagocytic function of alveolar macrophages gets disturbed shortly after trauma, remaining depressed up to the time of convalescence. It was demonstrated that the mechanism by which phagocytic function gets disturbed differs with time following trauma. Primary depression of phagocytosis occurs immediately after burn. At the height of burn disease the cells develop an energy deficient state, whereas the time of convalescence is marked by the emergence of poorly differentiated forms of macrophages having the reduced phagocyte capacity.     

Experimental and calculated parameters on particle phagocytosis by alveolar macrophages.

Phagocytosis of three types of fluorescein-labeled test particles by rat alveolar macrophages (AM) were studied: spherical silica (3.2 microm), heat-killed Candida albicans (3.8 microm), and heat-killed Cryptococcus neoformans (6.1 microm) opsonized with specific IgG. These particles should attach to scavenger, mannose, and Fc receptors, respectively. Both control AM and AM pretreated for 20 h with interferon-gamma (12.5 or 50 U/ml) were studied. The sum of the number of attached and ingested particles per AM (accumulated attachment) was used as a measure of the attachment process, and the number of ingested particles per AM divided by the accumulated attachment (ingested fraction) was used as a measure of the ingestion process. The average ingestion time (IT), which is also a measure of the ingestion process, was calculated from the experimental data. The ingestion process was independent of the attachment process. IT increased with the time of observation. This is explained by the fact that IT determined from observation times shorter than the whole distribution of IT for a certain particle results in a shorter IT than the real average IT. C. albicans (mannose receptor) had the fastest ingestion process, C. neoformans opsonized with specific IgG (Fc receptor) had ingestion that was nearly as fast, and the silica particles (scavenger receptors) had the slowest ingestion process. Treatment with interferon-gamma markedly impaired the attachment process for all three types of particles (and three types of receptors) but clearly impaired the ingestion process only for silica particles (scavenger receptors).     

Chemiluminescence associated with phagocytosis of foreign particles in rabbit alveolar macrophages

Rabbit alveolar macrophages exhibit a chemiluminescent response which is associated with phagocytosis of zymosan and polystyrene-butadiene particles. The chemiluminescence reaches a peak in 15 to 25 minutes and then gradually diminishes over the next 1 to 3 hours. During the time of maximal light emission there appears to be no actual uptake of particles, but the response is dependent upon the particle concentration. The metabolic inhibitor, DNP (2,4-dinitrophenol), causes a rapid inhibition of the chemiluminescent response. The addition of ATP to the medium prior to exposure of the cells to particles causes the chemiluminescent response to be greatly diminished, i.e., 0.3mM ATP virtually abolishes the response. These experiments suggest that some metabolic response of the cell to phagocytosis is responsible for the chemiluminescence.     

RAGE signaling by alveolar macrophages influences tobacco smoke-induced inflammation

Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.     

Pulmonary surfactant obtained from starved rats enhances phagocytosis of alveolar macrophages

Phagocytic activity of alveolar macrophages (AM) was enhanced by pulmonary surfactant obtained from bronchoalveolar lavage fluid of rats starved for 2 days, as compared to fed. The enhanced activity of phagocytosis was dependent on the dose of surfactant. The prepared surfactant showed a different protein to phospholipid ratio of 0.108 in fed and 0.234 in 2 days starved, because of an increased ratio of protein in surfactant from 2 days starved rats. F(ab')2 anti-surfactant protein inhibited the enhanced AM phagocytosis by surfactant. These results suggested that the enhancement of AM phagocytosis in 2 days starved rats was on account of an increase of protein in their surfactant compared to fed.     

Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages

Chronic inhalation of crystalline silica is an occupational hazard that results in silicosis due to the toxicity of silica particles to lung cells. Alveolar macrophages play an important role in clearance of these particles, and exposure of macrophages to silica particles causes cell death and induction of markers of apoptosis. Using time-lapse imaging of MH-S alveolar macrophages, a temporal sequence was established for key molecular events mediating cell death. The results demonstrate that 80 % of macrophages die by apoptosis and 20 % by necrosis by clearly distinguishable pathways. The earliest detectable cellular event is phago-lysosomal leakage, which occurs between 30 and 120 min after particle uptake in both modes of death. Between 3 and 6 h later, cells undergoing apoptosis showed a dramatic increase in mitochondrial transmembrane potential, closely correlated with activation of both caspase-3 and 9 and cell blebbing. Externalization of phosphatidyl serine and nuclear condensation occurred 30 min–2 h after the initiation of cell blebbing. Cells undergoing necrosis demonstrated mitochondrial membrane depolarization but not hyperpolarization and no caspase activation. Cell swelling followed the decrease in mitochondrial membrane potential, distinguishing necrosis from apoptosis. All cells undergoing apoptosis followed the same temporal sequence, but the time lag between phago-lysosomal leakage and the other events was highly variable from cell to cell. These results demonstrate that crystalline silica exposure can result in either apoptosis or necrosis and each occurs in a well-defined but temporally variable order. The long time gap between phago-lysosomal leakage and hyperpolarization is not consistent with a simple scenario of phago-lysosomal leakage leading directly to cell death. The results highlight the importance of using a cell by cell time-lapse analysis to investigate a complex pathway such as silica induced cell death.     

Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages

Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMphi) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMphi and peritoneal macrophages (PMphi) from normal C57BL/6 mice. AMphi were deficient relative to PMphi both in percentage containing apoptotic thymocytes (19.1 +/- 1% vs 96 +/- 2.6% positive) and in phagocytic index (0.23 +/- 0.02 vs 4.2 +/- 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mphi types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage. To confirm these studies, apoptotic thymocytes were given intratracheally or i.p. to normal mice, and then AMphi or PMphi were recovered 30-240 min later. Ingestion of apoptotic thymocytes by AMphi in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMphi capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages

There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca²⁺]_i. However, the mechanism of silica- induced cytotoxicity relative to [Ca²⁺]_i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca²⁺]_i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca²⁺]_i in a dose-dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca²⁺]_o markedly inhibited the [Ca²⁺]_i rise (≈90%), suggesting that Ca²⁺ influx from extracellular source is a major mechanism for silica-induced [Ca²⁺]_i rise. When used at low doses but sufficient to cause a transient [Ca²⁺]_i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca²⁺]_o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca²⁺ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca²⁺ influx is inhibited by the Ca²⁺ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca²⁺ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca²⁺]_i rise was not due to the impairment of Ca²⁺-pumps, which restricts Ca²⁺ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca²⁺]_i rise. Taken together, these results suggest that the elevation of [Ca²⁺]_i caused by silica is due mainly to Ca²⁺ influx through plasma membrane Ca²⁺ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca²⁺ leakage during phagocytosis was attributed to the silica-induced [Ca²⁺]_i rise. © 1993 Wiley-Liss, Inc.     

Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages

Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.     

The protective action of disodium cromoglycate on alveolar macrophages in phagocytosis of fibrogenic silica.

Treatment of fibrogenic silica (DQ-12) with Disodium Cromoglycate (DSCG) prior to its in-vitro and in-vivo phagocytosis by alveolar macrophages prevents the destruction of the cells and the fibrosis of lung tissue which are a consequence of phagocytosis. However, the treatment of alveolar macrophages with DSCG before phagocytosis of the silica had no, or a negligible, protective effect on the cells. Acid phosphatase activity which was significantly enhanced above the control in cells phagocytosing the silica was returned to the range found in phagocytosis of inert dust when silica treated with DSCG was phagocytized. The inhibitor of DNA- dependent RNA synthesis actinomycin D caused an increase of acid phosphatase activity. DSCG did not depress the phagocytic ability of alveolar macrophages. It appears that the catabolic enzyme process predominant in cells phagocytosing DQ-12 was under control in cells phagocytosing DQ-12 treated with DSCG and that DSCG probably acted as a regulator of the factors permitting catabolism. From the results it is suggested that the equilibrium of the enzyme reactions which accompany phagocytosis was such that the integrity of the phagocytes was preserved.     

Radiation effects on expression of FC-receptor of alveolar macrophages of rat and their specific phagocytosis

BCG-activated alveolar macrophages (AM) of Wistar rats were irradiated with different doses of gamma-ray in vitro. The effects of radiation on the expression of their Fc-receptor and specific phagocytic activity were observed. AM, after irradiation with doses of 0, 100, 300 and 500 Gy, showed decreasing phagocytic activity to chicken red blood cells (CRBC) opsonized with anti-CRBC antibody with no change in phagocytic indices. The expression of Fc-receptor of AM was, however, increased.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.     

Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages

BackgroundIn the lung, macrophages attempt to engulf inhaled high aspect ratio pathogenic materials, secreting inflammatory molecules in the process. The inability of macrophages to remove these materials leads to chronic inflammation and disease. How the biophysical and biochemical mechanisms of these effects are influenced by fiber length remains undetermined. This study evaluates the role of fiber length on phagocytosis and molecular inflammatory responses to non-cytotoxic fibers, enabling development of quantitative length-based models.MethodsMurine alveolar macrophages were exposed to short and long populations of JM-100 glass fibers, produced by successive sedimentation and repeated crushing, respectively. Interactions between fibers and macrophages were observed using time-lapse video microscopy, and quantified by flow cytometry. Inflammatory biomolecules (TNF-α, IL-1α, COX-2, PGE₂) were measured.ResultsUptake of short fibers occurred more readily than for long, but long fibers were more potent stimulators of inflammatory molecules. Stimulation resulted in dose-dependent secretion of inflammatory biomolecules but no cytotoxicity or strong ROS production. Linear cytokine dose-response curves evaluated with length-dependent potency models, using measured fiber length distributions, resulted in identification of critical fiber lengths that cause frustrated phagocytosis and increased inflammatory biomolecule production.ConclusionShort fibers played a minor role in the inflammatory response compared to long fibers. The critical lengths at which frustrated phagocytosis occurs can be quantified by fitting dose-response curves to fiber distribution data.General significanceThe single physical parameter of length can be used to directly assess the contributions of length against other physicochemical fiber properties to disease endpoints.     

Effect of phagocytosis by human polymorphonuclear leukocytes and rabbit alveolar macrophages on 2-deoxyglucose transport.

2-Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was 0.66 x 10(-3) micromoles/45 sec/10(6) PMN and 5.09 x 10(-4) micromoles/45 sec/10(6) AM. The rate of 2-deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2-deoxyglucose transport and intracellular fate of 2-deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites.