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.     

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.     

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.     

Single pulses of cytoplasmic calcium associated with phagocytosis of individual zymosan particles by macrophages

We have measured cytosolic free calcium levels ([Ca++]i) in individual macrophages during the phagocytosis of single zymosan particles. We report here that the contact of a macrophage with zymosan results in a rapid transient elevation of [Ca++]i. Each [Ca++]i pulse is symmetrical lasting for up to 30 seconds. In contrast, macrophage spreading is associated with repetitive [Ca++]i spiking occurring in salvos of up to four smaller spikes, each lasting for between 8 and 18 seconds. These qualitative and kinetic differences might suggest that the role of [Ca++]i in phagocytosis is distinct from its role in spreading.     

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.     

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.     

The relationship of detergent-solubilization plasma-membrane components of rabbit alveolar macrophages to an isolated inhibitor of phagocytosis.

1. A plasma-membrane fraction prepared from rabbit alveolar macrophages by hyposmotic borate lysis is described. 2. Rabbit lung lavages, containing a glycoprotein inhibitor of phagocytosis, may be fractionated by preparative isoelectric focusing in the presence of Triton X-100. 3. Chemical analysis indicates that the glycoproteins of the lung lavage contain sialic acid, fucose, mannose, galactose, hexosamine and appreciable quantities of glucose. 4. The relationship of macrophage membrane glycoproteins, solubilized with Triton X-100 in the presence of borate, to the lung lavage glycoproteins is demonstrated immunoelectrophoretically.     

Surface tension influences cell shape and phagocytosis in alveolar macrophages

The effect of surface tension on alveolar macrophage shape and phagocytosis was assessed in vivo and in vitro. Surface tension was regulated in vivo by conditionally expressing surfactant protein (SP)-B in Sftpb-/- mice. Increased surface tension and respiratory distress were produced by depletion of SP-B and were readily reversed by repletion of SP-B in vivo. Electron microscopy was used to demonstrate that alveolar macrophages were usually located beneath the surfactant film on the alveolar surfaces. Reduction of SP-B increased surface tension and resulted in flattening of alveolar macrophages on epithelial surfaces in vivo. Phagocytosis of intratracheally injected fluorescent microbeads by alveolar macrophages was decreased during SP-B deficiency and was restored by repletion of SP-B in vivo. Incubation of MH-S cells, a mouse macrophage cell line, with inactive surfactant caused cell flattening and decreased phagocytosis in vitro, findings that were reversed by the addition of sheep surfactant or phospholipid containing SP-B. SP-B controls surface tension by forming a surfactant phospholipid film that regulates shape and nonspecific phagocytic activity of alveolar macrophages on the alveolar surface.     

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.     

Collagenase from rabbit pulmonary alveolar macrophages.

Rabbit pulmonary alveolar macrophages produce a collagenase which lyses labeled collagen gels, specifically cleaves collagen types I, II and III, is inhibited by ethylenediaminetetraacetate, cysteine, dithiothreitol and serum but is not inhibited by a serine protease inhibitor. Alveolar macrophage collagenase activity can be enhanced by $\text{in vivo}$ BCG activation, $\text{in vitro}$ latex, silica or mycobacterium activation and by $\text{in vitro}$ uncovering of latent enzymatic activity with trypsin treatment. The production of collagenase by unactivated alveolar macrophages and the presence of “latent” collagenase in culture media of alveolar macrophages are examples of significant differences between alveolar and peritoneal macrophages.     

Metabolism of foreign compounds by alveolar macrophages of rabbits

Alveolar macrophcges of rabbits acetylated p-aminobenzoic acid to the same extent as did lung parenchyma and liver. However, microsomes isolated from macrophages lacked detectable activity of aryl hydrocarbon hydroxylase, even in animals treated with 3-methylcholanthrene, an inducer of this enzyme in liver and lung. Similarly, bromobenzene was metabolized by microsomes prepared from liver and lung but not from alveolar macrophages.     

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.     

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).     

Superoxide production by rabbit pulmonary alveolar macrophages.

Nitroblue tetrazolium (NBT) reduction by reduction by alveolar macrophages from normal and BCG-granulomatous rabbit lungs was inhibited by superoxide dismutase (SOD). Superoxide (.O₂^?) might therefore be involved, either direclty or indirectly, in the bactericidal activities of such cells. Cells from BCG-granulomatous rabbits did not, however, reduce significantly more NBT per cell than cells from normal rabbits.     

Identification of mannose 6-phosphate receptors in rabbit alveolar macrophages

Mannose 6-phosphate is an important recognition site involved in transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes. The current study is the first demonstration of functional mannose phosphate receptors in macrophages. The receptor appears to be similar in many respects to that expressed in fibroblasts. Binding at 4 degrees C of a mannose-6-P-containing ligand, alpha-mannosidase from Dictyostelium discoideum, was specific and saturable (KD = 1.6 nM). In the presence of permeabilizing agents (saponin and digitonin), macrophage mannose-6-P receptors gave a distribution of 15-20% on the surface and 80-85% inside. Uptake studies gave a Kuptake value of 4.9 nM. Mannose-6-P, Hansenula holstii phosphomannan, and fructose 1-phosphate were effective inhibitors of alpha-mannosidase uptake. Inhibitors of mannose uptake, such as beta-glucuronidase, mannose-bovine serum albumin, fucose-bovine serum albumin, or mannan had no effect on alpha-mannosidase uptake. Likewise, an inhibitor (fucoidin) of the macrophage receptor which recognizes negatively charged proteins did not inhibit alpha-mannosidase uptake. Uptake was linear over 90 min and inhibited by chloroquine, suggesting that surface receptors recycle. These data demonstrate that macrophages contain receptors which specifically recognize mannose-6-P units and are distinct from the well characterized mannose receptors. The finding that the mannose-6-P receptors play a role at the surface, together with the fact that most of the receptors are intracellular (similar to the mannose receptor) suggests that both carbohydrate receptors play a regulatory role at the surface and intracellularly in transport of lysosomal enzymes.     

The subcellular distribution of iron in rabbit alveolar macrophages.

Rabbit alveolar macrophages were incubated with [⁵⁹Fe], washed, re-incubated with “cold” iron and homogenized. The distribution of radioactivity among the mitochondrial, lysosomal, microsomal and cytosol fractions was determined at short intervals after the onset of incubation. The findings indicate that the mitochondria form a significant iron-binding site during the early stage of iron uptake. A part of the mitochondrial-associated iron is later transferred to the cytosol where it is present in ferritin and in a low molecular weight form. Ferritin is the sole iron-binding protein of the cytosol.     

Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages.

We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average decrease in cell capacitance of 8 pF was seen after exposure of the cells to immune complexes. Cells in which particle uptake was inhibited by cytochalasin B treatment before exposure to immune complexes showed an average increase of 0.5 pF. The decrease in membrane capacitance after exposure of cells to particulate stimuli was absent with the soluble stimulus, platelet-activating factor, further confirming that decreases in membrane capacitance were due to particle uptake. Exposure of cells to sized latex particles resulted in a graded, stepwise decrease in membrane capacitance. The average step size for 0.8-micron particles was 250 fF, and the average step change for the larger 3.2-micron particles was 480 fF, as calculated from Gaussian fits to the step size amplitude histograms. The predicted step size for the individual particles based upon the minimum amount of membrane required to enclose a particle and a specific capacitance of 10 fF/micron2 was 20 and 320 fF, respectively. The step size for the smaller particles deviates significantly from the predicted size distribution, indicating either a possible lower limit to the size of the phagocytic vacuole or multiple particles taken up within a single phagosome. Dynamic interaction between phagocytosis and exocytosis was observed in a number of cells as a biphasic response consisting of an initial rapid increase in capacitance, consistent with cellular exocytosis, followed by stepwise decreases in capacitance.