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The Journal of Immunology, 1999, 162: 2266-2274.
Copyright © 1999 by The American Association of Immunologists

Fc{gamma} Receptor-Mediated Activation of Phospholipase D Regulates Macrophage Phagocytosis of IgG-Opsonized Particles1

David J. Kusner2,*,{dagger},{ddagger}, Clifton F. Hall3,* and Stephen Jackson{dagger},{ddagger}

* Department of Medicine, the {dagger} Inflammation Program, and the {ddagger} Graduate Program in Immunology at the University of Iowa and Veterans Affairs Medical Center, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Receptors for the Fc portion of IgG (Fc{gamma}Rs) integrate the innate and acquired components of immunity by coupling the specific recognition of IgG Abs to the activation of phagocytic leukocytes. Knowledge of the molecular mechanisms that regulate phagocyte stimulation by Fc{gamma}Rs may permit therapeutic modulation to augment immunoprotective aspects and minimize damage to host tissues in diverse inflammatory diseases. Since phospholipase D (PLD) has been linked to the stimulation of cytotoxic leukocyte responses, we characterized Fc{gamma}R-dependent activation of PLD in human macrophages. IgG-coated SRBCs (EIgG) stimulated a 9.4-fold increase in PLD activity compared with SRBCs treated with control Ab (p < 0.001), determined by formation of the PLD-specific product phosphatidylethanol in the presence of 0.5% ethanol. Levels of phosphatidic acid, the physiologic product of PLD-mediated catalyzis, were significantly increased in the absence of ethanol (6.4-fold, p < 0.001). PLD activity was also stimulated by immune complex-coated latex beads or cross-linking of Abs specific for Fc{gamma}RI, Fc{gamma}RII, or Fc{gamma}RIII. Phagocytosis of EIgG was reduced by two inhibitors of PLD-mediated signaling, 2,3-diphosphoglycerate or 1-butanol. Addition of purified PLD restored control levels of phagocytosis in cells in which endogenous PLD was inhibited. The tyrosine kinase inhibitors genistein and herbimycin A caused concordant reductions in Fc{gamma}R-stimulated PLD activity and phagocytosis. These studies demonstrate that Fc{gamma}R-mediated phagocytosis is accompanied by tyrosine kinase-dependent activation of PLD and support the hypothesis that stimulation of PLD functions to regulate the ingestion of IgG-opsonized particles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The inflammatory response consists of soluble and cellular components that function in a highly integrated manner to promote host defense, but inflammation also contributes to the pathophysiology of a wide range of infectious and noninfectious diseases, including sepsis, acute respiratory distress syndrome, myocardial infarction, and rheumatoid arthritis 1, 2, 3, 4 . Cell-surface receptors for the Fc portion of IgG (Fc{gamma}Rs) are critical regulators of inflammation, which integrate the humoral and cellular arms of the specific inflammatory response and link these to innate immunity provided by phagocytes and complement 3, 5, 6 . The binding of the Fc portion of IgG to Fc{gamma}Rs on the surface of monocytes, macrophages (MPs)4, and neutrophils activates a broad spectrum of antimicrobial responses, including phagocytosis, secretion, cytokine synthesis, Ab-dependent cellular cytotoxicity, and generation of reactive oxidant species 3, 4, 5, 6 . Thus, an understanding of the regulation of Fc{gamma}R-dependent responses may provide a locus for therapeutic modulation of the inflammatory response.

There are three classes of Fc{gamma}Rs, Fc{gamma}RI, RII, and RIII, and isoform diversity is present within each class 3, 6 . MPs contain all three Fc{gamma}R subtypes, whereas more restricted expression occurs in monocytes and neutrophils. Several components of the signal transduction cascades coupled to Fc{gamma}Rs have been described in recent years, including protein tyrosine kinases (PTKs) of the Src and Syk families, protein kinase C (PKC), Ca2+, phosphatidylinositol 3-kinase, and the Ras/Raf-1/MAP kinase pathway 7, 8, 9, 10, 11, 12, 13, 14 . A major challenge of current research is to characterize the mechanisms that link these signaling components to each other and to the primary functional responses of phagocytic leukocytes.

Phospholipase D (PLD) is a major signaling component in myeloid leukocytes, linking the stimulation of receptors for chemoattractants, chemokines, and cytokines to generation of reactive oxidant species and granule secretion 15, 16 . Of particular relevance to Fc{gamma}R-dependent leukocyte functions is the recent demonstration that phagocytosis via the functionally analogous CRs is closely linked to activation of PLD 17, 18, 19, 20 . Two recent reports have demonstrated that stimulation of Fc{gamma}Rs on neutrophils 21 and monocytic leukemia cells 22 results in activation of PLD. Gewirtz and Simons 21 stimulated human neutrophils with high valency immune complexes and demonstrated that the resultant activation of PLD was tightly coupled to secretion of azurophilic granules. Utilizing Ab-mediated cross-linking of Fc{gamma}RI on {gamma}-IFN-differentiated U937 cells, Melendez et al. 22 showed that activation of PLD was required for elevation of intracellular Ca2+ via this receptor and for the vesicular trafficking of internalized immune complexes for lysosomal degradation. Several important questions regarding Fc{gamma}R-dependent activation of PLD remain unresolved, including: 1) Is PLD activated during phagocytosis of IgG-opsonized particles?; 2) If so, is Fc{gamma}R-dependent stimulation of PLD required for particle ingestion?; and 3) Is each class of Fc{gamma}Rs (Fc{gamma}RI, RII, and RIII) coupled to stimulation of PLD activity? The objective of this study was to evaluate these questions utilizing monocyte-derived MPs and both particulate and soluble agonists for Fc{gamma}Rs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

HEPES, trypan blue, leupeptin, PMSF, aprotinin, purified PLD from Streptomyces chromofuscus, peanut, cabbage, phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) from Bacillus cereus, phospatidylethanol (PEt), and phosphatidic acid (PA) were obtained from Sigma (St. Louis, MO). RPMI 1640 and PBS were from Life Technologies Laboratories (Grand Island, NY). Teflon wells were obtained from Savillex (Minnetonka, MN). SRBCs were from BioWhittaker (Walkersville, MD). All organic solvents (HPLC grade) were obtained from Fisher (Fairlawn, NJ). 1-O-[3H]lyso-platelet-activating factor (lysoPAF) and the reagents for Western blot detection by ECL were from Amersham (Arlington Hts., IL). HSA and genistein were purchased from Calbiochem (San Diego, CA). Herbimycin A was from Biomol (Plymouth Meeting, PA). Polystyrene microspheres were purchased from Polysciences (Warrington, PA).

Antibodies

Rabbit anti-SRBC Ab was from Sigma. 4G10 anti-P-Tyr mAb was from Upstate Biotechnology (Lake Placid, NY). Anti-CD64 mAb (8H9), originally prepared by Dr. James E.K. Hildreth (Johns Hopkins University, Baltimore, MD), was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). mAbs to CD32 (IV.3) and CD16 (3G8) were from Medarex (Annandale, NJ). Rabbit anti-mouse IgG (R{alpha}M) F(ab')2 fragments were purchased from Cappel (Durham, NC). Polyclonal Ab to myeloperoxidase was kindly provided by Dr. William M. Nauseef (University of Iowa).

Preparation of MPs and test particles

PBMCs were isolated from healthy adult volunteers by density-gradient centrifugation on Ficoll-Hypaque and cultured in RPMI 1640, 20 mM HEPES (pH 7.4) and 20% autologous serum for 5 day in Teflon wells, as previously described 19 . Following purification of MP by adherence to tissue culture plastic for 2 h at 37°C, monolayers were washed and incubated in RPMI-HEPES with 2.5% serum for use in experiments. MDM viability was assessed by exclusion of trypan blue, and monolayer density by nuclei counting with napthol blue-black stain 19, 23 . EIgG were prepared as previously described 24, 25 by incubation of washed erythrocytes with rabbit polyclonal anti-SRBC Ab in PBS, followed by repeated washings and resuspension in RPMI 1640. Control particles consisted of SRBCs incubated with PBS alone or with rabbit polyclonal irrelevant Ab (anti-myeloperoxidase) and were otherwise processed identically to EIgG.

To confirm that EIgG-stimulated PLD activity was mediated via MP Fc{gamma}Rs, select experiments were conducted with a second IgG-opsonized particle, immune complex-coated (IC) microspheres 26 . Polystyrene beads (3.0-µm diameter) were washed twice in PBS, incubated in 8% glutaraldehyde for 16 h followed by 0.1% BSA for 5 h, both at 25°C. Beads were washed in PBS, incubated for 16 h in 0.5 M ethanolamine, and then resuspended in rabbit polyclonal anti-BSA Ab (0.2 mg/ml) followed by incubation for 4 h at 25°C 26 . Beads coated with either BSA alone or BSA and irrelevant Ab served as controls. All incubations were performed on a sample rotator. Opsonized microspheres were washed twice with PBS (4°C), resuspended in RPMI 1640, and counted in a hemacytometer. IC beads were kept on ice before use in experiments.

Determination of phagocytosis of IgG-opsonized particles

Phagocytosis of EIgG and IC beads were performed by phase contrast microscopy, as previously described 24, 25, 26 . MPs (~2 x 105) were adhered for 2 h at 37°C in RPMI 1640 and 10% autologous serum to chromic acid-cleaned glass coverslips placed in 24-well tissue culture plates. Nonadherent cells were removed by repeated washing of the monolayer, followed by addition of RPMI 1640 and 2.5% heat-inactivated autologous serum for assays. Phagocytic particles were incubated with MPs at a particle:MP ratio of 10:1 for time periods ranging from 30 to 120 min. Noningested SRBCs were hypotonically lysed by incubation with 0.0375 M NaCl for 5 s, followed by restoration of isotonicity with 3 M NaCl 25 . The number of ingested particles per 100 MPs was determined for triplicate samples for each condition and expressed as the phagocytic index. The total number of cell-associated EIgG (adherent + ingested) was determined in parallel samples not treated with hypotonic saline, and the adherence index (number of adherent particles per 100 MPs) was calculated by subtraction of the number of ingested particles from the total number of cell-associated particles. Phagocytosis of IC beads was determined by the change in opacity that occurs following ingestion and was confirmed in select experiments by fluorescence quenching of extracellular FITC-labeled microspheres with trypan blue 26, 27 .

Measurement of PLD activity

MPs were cultured in 6-well tissue culture plates at ~2.0 x 106 MPs/well and radioisotopically labeled with [3H] lyso-PAF; 5 uCi/well, for 90 min at 37°C in RPMI 1640/20 mM HEPES with 2.5% serum 19 . Following repeated washings to remove unincorporated radioactivity, MPs were incubated with EIgG or control SRBCs at a particle:MDM ratio of 30:1. In certain assays, 0.5% ethanol was added 5 min before the particles to permit detection of the specific transphosphatidylation product, [3H]PEt, as a metabolically stable index of PLD activity 15 . Following a 30 min incubation, reactions were terminated with 1.67 volumes of ice-cold methanol, MPs were scraped and transferred to polypropylene tubes, and 3.3 volumes of CHCl3 were added for extraction 19 . Following phase separation, the CHCl3 layer was dried, and [3H]PA and [3H]PEt were isolated by TLC in an ethyl acetate/iso-octane/acetic acid (9:5:2) solvent system 28 by comigration with pure phospholipid standards. Quantitation of [3H]-cpm in PA and PEt was performed by liquid scintillation spectrophotometry, and counts were normalized for total cpm in phospholipid to correct for potential differences in labeling between experiments.

Stimulation of MPs with anti-Fc{gamma}R Abs

Cross-linking of surface-bound mAbs to Fc{gamma}RI, RII, and RIII was performed as described by Park et al. 11 with minor modifications. F(ab')2 fragments of anti-Fc{gamma}R Abs were prepared by digestion with pepsin, as previously described 29 , and partially purified by chromatography on a protein A-Sepharose column, followed by molecular sizing. F(ab')2 fragments of anti-Fc{gamma}R Abs (0.2–1.0 µg/ml) were added to MP monolayers (2 x 106 cells/sample) and incubated at 4°C for 15 min. Unbound primary Ab was removed by washing and stimulation of MPs was initiated by addition of 37°C medium containing 10 µg/ml of R{alpha}M (F(ab')2 fragment) Ab for different times. Incubations were terminated with ice-cold methanol, and PLD activity was determined as noted above.

Determination of the effects of inhibitors of signal transduction on PLD activity and phagocytosis

To inhibit PLD-dependent generation of PA, MPs were incubated with 2,3-diphosphoglycerate (2,3-DPG; 0.3–10 mM) for 15 min or 1-butanol (0.1–1.0%) for 5 min at 37°C before stimulation of Fc{gamma}Rs. Levels of [3H]PEt or PA were determined as a measure of PLD activity, and phagocytosis of EIgG was performed as outlined above. To analyze the role of tyrosine kinases in Fc{gamma}R-dependent activation of PLD and phagocytosis, the specific PTK inhibitors, genistein and herbimycin A, were utilized. The pretreatment interval for genistein (1–100 µM) was 15 min, whereas herbimycin A was added to MP monolayers 18 h before addition of IgG-opsonized particles. The effects of each inhibitor on MP viability and monolayer density were determined in parallel experiments. The additions of 2,3-DPG, ethanol, genistein, or herbimycin A did not affect the viability of MPs at the concentrations noted above 19 . Only herbimycin A caused a decrease in monolayer density, which was dose-dependent (maximal reduction of 18 ± 5% at 0.6 µg/ml). For the PLD assay, [3H]PEt cpm were normalized to total [3H]-cpm in phospholipid to correct for the herbimycin A-induced reduction in monolayer density.

Analysis of protein tyrosine phosphorylation

MPs were adhered to 12-well tissue culture plates (~1.0 x 106 cells/well) and cultured in RPMI 1640 (pH 7.4), 20 mM HEPES and 2.5% serum. Following addition of Fc{gamma}R-dependent stimuli, control particles, Abs, or buffer alone, MPs were incubated for 30 min at 37°C in 5% CO2. At the end of the incubation, monolayers were washed once with ice-cold PBS, 1 mM vanadate, and then lysed in 250 µl of 1.0% Triton X-100, 20 mM Tris (pH 8.0), 140 mM NaCl, 1 mM Na3VO4, 2 mM EDTA, 10% glycerol, 1 mM PMSF, 20 µM leupeptin, and 0.15 U/ml of aprotinin 19, 28 . Following incubation on ice for 1 h, lysates were centrifuged at 14,000 x g for 15 min at 4°C, and supernatants were transferred to separate tubes. The detergent-insoluble pellet was washed in lysis buffer and then solubilized in 100 µl of 2x SDS-sample buffer, whereas supernatants were boiled in an equal volume of electrophoresis sample buffer. SDS-PAGE, transfer to PVDF membranes, and Western blotting with 4G10 anti-P-Tyr mAb with detection by enhanced chemiluminescence were performed as previously described 19, 28 . To correct for the effects of herbimycin A on monolayer density, protein concentrations of Triton X-100 extracts were determined by the bicinchoninic acid method 19 , and equal amounts of protein were loaded in each lane.

Data analysis

Data from each experimental group were subjected to an analysis of normality and variance. Differences between experimental groups composed of normally distributed data were analyzed for statistical significance using Student’s t test. Nonparametric evaluation of other data sets was performed with the Mann-Whitney Rank Sum test. Analysis of correlation was performed with the Spearman Rank Order test 30 .


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Macrophage phagocytosis of IgG-opsonized particles is associated with marked increases in PLD activity

We studied the activity of PLD in resting and Fc{gamma}R-stimulated human monocyte-derived MPs labeled with [3H] lyso-PAF. Resting MPs exhibited low basal PLD activity, determined by formation of the specific transphosphatidylation product [3H]PEt in the presence of 0.5% ethanol. Incubation of MPs with the well-characterized Fc{gamma}R-dependent stimulus, IgG-coated SRBCs (EIgG), resulted in significant activation of PLD (9.4-fold, p < 0.001, n = 8) compared with control samples incubated with either SRBC treated with an irrelevant Ab or SRBCs alone (Fig. 1GoA). In the absence of ethanol, EIgG stimulated the production of PA, the physiologic product of PLD-mediated catalysis (6.4-fold increase over control, p < 0.001, n = 5; Fig. 1GoB). To further confirm a PLD-type enzyme activity in EIgG-treated MPs, incubations were performed in the presence of various concentrations of ethanol (0–0.75%, v/v). The generation of PEt in EIgG-treated MPs was directly proportional to the concentration of ethanol, whereas PA production declined with increasing concentrations of ethanol (Fig. 2Go). This inverse relationship between PEt and PA generation, dependent on ethanol concentration, is due to the unique transphosphatidylation capacity of PLD and constitutes definitive evidence for stimulation of this class of phospholipases 15 .



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  		FIGURE 1. Macrophage phagocytosis of EIgG is associated with activation of PLD. Human MPs were radiolabeled with [3H]-lyso-PAF for 90 min, washed, and then incubated under the indicated conditions in the presence (A) or absence (B) of 0.5% ethanol. At 30 min, lipids were extracted and PLD activity assayed as accumulation of [3H]PEt (A) or [3H]PA (B) by TLC/liquid scintillation spectrophotometry. "Basal" represents PLD activity at 30 min in the absence of stimulation, whereas "E" denotes that following addition of untreated SRBC. "E-Ab" indicates SRBC incubated with control irrelevant Ab, and "EIgG" denotes opsonization of SRBC with specific Ab. All SRBC preparations were processed and washed in an identical manner and utilized at a particle:MP ratio of 10:1. [3H]-cpm in PEt (A) and PA (B) were normalized for total cpm in phospholipid to correct for potential differences in substrate labeling between experiments. The increase in PEt and PA in the presence of EIgG was statistically significant compared with Basal, E, or E-Ab (p < 0.001 for each). Data represent mean ± SEM of duplicate determinations from 8 identical experiments and were analyzed for statistical significance using the Mann-Whitney Rank Sum test.

 


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  		FIGURE 2. EIgG-stimulated production of PA and PEt is consistent with a PLD-type transphosphatidylation reaction. MPs were incubated with the specified concentrations of ethanol for 5 min before exposure to EIgG. Thirty minutes later, PEt (open squares) and PA (filled squares) were quantitated as described in Materials and Methods. Open and filled circles on the ordinate denote levels of PEt and PA, respectively, in the absence of EIgG and ethanol. Results represent mean ± SD of duplicate samples from one of three identical experiments.

 
The extent of Fc{gamma}R-dependent phagocytosis was determined in parallel samples by phase-contrast microscopy, utilizing brief hypotonic lysis of extracellular erythrocytes to distinguish between adherent and ingested particles 25 . At an EIgG:MP ratio of 10:1, the level of phagocytosis at 30 min, expressed as the phagocytic index, was 475 ± 33 EIgG ingested/100 MPs. Analysis of the time-dependence of EIgG-stimulated PLD activity demonstrated significantly increased levels of PEt within 1 min of particle addition and continued generation of this metabolically stable index of PLD activity throughout the 60-min assay (Fig. 3Go). The onset of the maximal rate of PEt production preceded the period of maximal phagocytosis (data not shown), supporting the hypothesis that PLD activity may be causally related to ingestion of IgG-opsonized particles.



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  		FIGURE 3. Kinetics of PLD activation during MP phagocytosis of EIgG. MPs were incubated with EIgG at a particle:MP ratio of 10:1. At the indicated times, chloroform/methanol extracts were prepared and [3H]PEt measured as an index of PLD activity (filled squares). Open squares represent PEt accumulation in control samples, incubated with SRBC treated with irrelevant control polyclonal Ab. Results are mean ± SD of duplicate determinations from a single experiment representative of three.

 
To confirm and extend the analysis of PLD activation by IgG-opsonized particles, additional studies were conducted with IC microspheres. Latex (polystyrene) beads (3.0-µm diameter) were coated with BSA via condensation with surface-bound glutaraldehyde. Following elimination of excess aldehyde sites with 0.5 M ethanolamine, beads were incubated with rabbit polyclonal anti-BSA Ab to form specific immune complexes (IC beads) that result in phagocytosis of the microspheres via Fc{gamma}Rs 26 . In addition to confirming the data shown above with a second IgG-opsonized particle, IC beads were chosen because: 1) their size is close to that of pathogenic bacteria, and 2) IC beads do not possess ligands for any other class of phagocytic receptor 26 . At a particle:MP ratio of 10:1, the phagocytic index of IC beads was 614 ± 28 (n = 4). In contrast, control beads coated with BSA alone, or BSA and Ab, were not phagocytosed to a significant extent (23 ± 5 and 38 ± 3 beads/100 MP). Incubation of IC beads with [3H]-lyso-PC-labeled MPs, in the presence of 0.5% ethanol, resulted in significant stimulation of PLD activity, measured as a 7.2-fold increase in generation of PEt, compared with that induced by BSA-coated beads or microspheres incubated with irrelevant Ab (p < 0.001 for each comparison, n = 7; Fig. 4GoA). In the absence of the transphosphatidylation substrate, ethanol, IC beads stimulated the accumulation of [3H]PA (6.7-fold increase compared with control beads, p < 0.001, n = 4; Fig. 4GoB). As expected, incubation of MPs in various concentrations of ethanol (0–0.75%, v/v) resulted in inverse production of PEt and PA, confirming the activation of PLD activity. The kinetics of macrophage PLD activation by IC beads were similar to that induced by EIgG, in that levels of PEt were significantly increased by 1 min and continued to accumulate throughout the 60-min assay period (data not shown). Therefore, the experiments with IC beads confirm that MP phagocytosis of IgG-opsonized particles is associated with stimulation of significant levels of PLD activity.



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  		FIGURE 4. IC polystyrene beads stimulate MP PLD activity. Polystyrene microspheres (3.0-µm diameter) were coated with BSA via glutaraldehyde condensation, as described in Materials and Methods. Following washing in PBS, microspheres were either utilized directly (BSA beads), incubated with polyclonal anti-BSA Ab (IC beads), or with irrelevant Ab control (Ab beads). Microspheres were incubated with [3H]-lyso-PAF-labeled MPs at a bead:MP ratio of 10:1 for 30 min at 37°C. "Basal" denotes MP monolayers incubated in the absence of microspheres. PLD activity was determined by accumulation of [3H]PEt (A) in the presence of 0.5% ethanol or [3H]PA (B) in the absence of ethanol. Results represent mean ± SEM of seven experiments performed in triplicate for PEt and four experiments for PA.

 
Each class of MP Fc{gamma}Rs is specifically coupled to activation of PLD

To determine whether stimulation of each class of MP Fc{gamma}R is coupled to activation of PLD, cross-linking of surface-bound mAbs specific to Fc{gamma}RI (8H9), Fc{gamma}RII (IV.3), or Fc{gamma}RIII (3G8) were utilized. To minimize potential interference due to binding of the Fc region of these IgGs to Fc{gamma}Rs, F(ab')2 fragments of each mAb were generated via pepsin cleavage of intact IgG 29 , as outlined in Materials and Methods. The F(ab')2 fragment of the secondary Ab (R{alpha}M IgG) was used for cross-linking. Table IGo demonstrates that Ab-mediated stimulation of each class of macrophage Fc{gamma}R (Fc{gamma}RI, RII, or RIIIA) resulted in stimulation of significant levels of PLD activity. The rank order of potency for stimulation of PLD activity was: anti-Fc{gamma}RII > anti-Fc{gamma}RI > anti-Fc{gamma}RIII. Incubation of MPs with anti-Fc{gamma}R Abs in the absence of the secondary R{alpha}M Ab, with the secondary Ab alone, or with isotype-matched irrelevant mAbs did not result in increased PLD activity. Previous work has indicated that each class of MP Fc{gamma}Rs is capable of mediating the phagocytosis of IgG-opsonized particles 31, 32, 33 . The demonstration that Fc{gamma}RI, RII, and RIIIA are each coupled to activation of PLD supports the hypothesis that the resultant stimulation of PLD is tightly associated with phagocytosis via Fc{gamma}Rs.


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  		Table I. Stimulation of MP PLD activity by anti-Fc{gamma}R Abs1

 
Inhibition of Fc{gamma}R-dependent PLD activity is associated with reduction of phagocytosis

To evaluate the hypothesis that Fc{gamma}R-dependent activation of PLD is required for phagocytosis of IgG-opsonized particles, we utilized two complementary inhibitors of PLD-mediated signal transduction, 2,3-DPG and 1-butanol, and determined their effects on the ingestion of EIgG. Although no specific inhibitor of PLD is currently available, 2,3-DPG possesses several advantages as an inhibitor of PLD, including: 1) a defined mechanism of action as a competitive inhibitor, 2) low toxicity to intact cells, 3) inhibition of PLD-dependent phagocyte responses, including FMLP-stimulated superoxide generation and CR-dependent phagocytosis, and 4 lack of inhibition of PI-PLC (the other major source of diglycerides in activated phagocytes) 19, 34 . Preincubation of MP monolayers with 2,3-DPG for 15 min before addition of EIgG resulted in concentration-dependent reductions in Fc{gamma}R-stimulated PLD activity (Fig. 5GoA). The maximal concentration of 2,3-DPG utilized (10 mM) produced a 76 ± 5% reduction in PLD activity (p < 0.001, n = 4) without affecting MP viability (determined via exclusion of trypan blue) or monolayer density (assessed by nuclei counting with napthol blue-black stain) 19, 23 . 2,3-DPG-mediated inhibition of PLD activity was paralleled by dose-dependent reductions in phagocytosis of EIgG (Fig. 5GoA) with an inhibition of 82 ± 6% at 10 mM 2,3-DPG.



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  		FIGURE 5. Inhibition of EIgG-stimulated PLD activity is associated with reductions in phagocytosis. A, MPs labeled with [3H]-lyso-PAF were incubated with the indicated concentrations of 2,3-DPG for 15 min at 37°C before the addition of EIgG. Thirty minutes after stimulation with EIgG, incubations were terminated, and [3H]PEt levels were determined as a measure of PLD activity (solid bars). Phagocytosis of EIgG was assayed in parallel experiments by phase contrast microscopy (striped bars). MPs were preincubated with either 1-butanol (B) or 2-butanol (C) for 5 min at 37°C, followed by addition of EIgG for 30 min. In B and C, [3H]PA was determined as a measure of PLD activity. Data are presented as the percentage of PLD activity (solid bars) and phagocytic index (PI, striped bars) of control cells, and represent the mean ± range of four identical experiments, conducted in triplicate, for each condition.

 
Short-chain primary alcohols, such as 1-butanol and ethanol, inhibit PLD-mediated generation of PA by substituting for water as the nucleophilic acceptor of the phosphatidyl moiety 15 . In this way, such compounds inhibit PLD-dependent responses without blocking enzyme turnover, thus providing a complementary method of PLD inhibition, mechanistically distinct from that of 2,3-DPG. Preincubation of MPs with 1-butanol (0.1–0.75%, v/v) for 5 min at 37°C resulted in concentration-dependent inhibition of Fc{gamma}R-dependent PLD activity, quantitated as reductions in EIgG-stimulated accumulation of PA. Similar to the results with 2,3-DPG, inhibition of PLD activity by 1-butanol was accompanied by dose-dependent reductions in the extent of phagocytosis (Fig. 5GoB). The maximal concentration of 1-butanol used in these experiments (0.75%) produced a 82 ± 7% reduction in EIgG-dependent PLD activity and a 87 ± 5% inhibition of phagocytosis. Neither the viability of MPs nor the density of the monolayer were altered by incubation with 1-butanol (data not shown). Also, maximal concentrations of 1-butanol and 2,3-DPG did not alter the stability of the EIgG preparations. As a control for the effects of 1-butanol on PLD activity and phagocytosis, we utilized 2-butanol, a structurally related aliphatic alcohol that cannot function as a nucleophilic acceptor in the transphosphatidylation reaction catalyzed by PLD 22 . Preincubation of MPs with 2-butanol, at the same concentrations used with 1- butanol, did not affect Fc{gamma}R-mediated activation of PLD or the extent of EIgG ingestion (Fig. 5GoC). Ethanol, an alternative transphosphatidylation substrate for PLD, produced dose-dependent (0.1–1.0%) inhibition of EIgG-stimulated PLD activity and phagocytosis, which closely paralleled the effects of 1-butanol (data not shown).

Although the strong correlation between inhibition of PLD activity and phagocytosis by these two, mechanistically distinct, inhibitors lessens the possibility that nonspecific effects contributed to the reductions in particle ingestion, the lack of a highly specific PLD inhibitor limits the strength of the conclusions drawn from these results. Therefore, to further evaluate whether the effects of 2,3-DPG and 1-butanol on EIgG phagocytosis were due to mechanisms other than inhibition of PLD activity, purified PLD from S. chromofuscus was utilized to reconstitute PLD-dependent signaling in MPs in which endogenous PLD activity had been inhibited. Although exogenous PLD does not precisely mimic the spatial and temporal production of PA resulting from agonist stimulation of endogenous phospholipase activity, addition of purified PLD preparations has provided valuable information on PLD-dependent signal transduction in many cell types, including MPs, fibroblasts, and smooth muscle cells 19, 35, 36, 37 .

To determine the approximate levels of S. chromofuscus PLD required to reconstitute PLD activity in 2,3-DPG-treated MPs to that of control EIgG-stimulated cells, levels of PEt accumulation were first determined at various concentrations of exogenous lipase. Addition of 40 U/ml of S. chromofuscus PLD resulted in levels of PEt production that closely approximated those produced in control cells stimulated by EIgG (Fig. 6GoA). Increases in PEt accumulation were associated with parallel augmentation of phagocytosis (Fig. 6GoB). Most importantly, restoration of phagocytosis to near normal levels occurred in samples in which PLD activity was essentially fully reconstituted by addition of 40 U/ml of S. chromofuscus PLD. Heat-inactivation of S. chromofuscus PLD (80°C, 30 min) eliminated its ability to reconstitute control levels of PA accumulation and EIgG ingestion (data not shown), indicating a requirement for catalytic activity in the restoration of Fc{gamma}R-dependent phagocytosis. The phospholipase specificity required for reconstitution of control levels of phagocytosis in MPs whose endogenous PLD activity was inhibited by 2,3-DPG is demonstrated in Table IIGo. Purified preparations of PLD from cabbage and peanut, like S. chromofuscus PLD, were able to restore near-normal levels of phagocytosis in 2,3-DPG-treated MPs. In contrast, purified PI-PLC or PC-PLC did not augment the ingestion of EIgG by 2,3-DPG-treated cells (Table IIGo) at the concentration effective with the PLD enzymes (40 U/ml).



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  		FIGURE 6. Exogenous PLD reconstitutes the accumulation of PEt in 2,3-DPG-treated MPs and restores phagocytosis of EIgG. Parallel determinations of PLD activity and phagocytosis of EIgG were determined under the following conditions: Basal, incubation of MPs with buffer alone; EIgG, addition of SRBCs opsonized with specific Ab at a particle:MP ratio of 10:1; + DPG, preincubation with 10 mM 2,3-DPG for 15 min at 37°C, followed by EIgG; + PLD, preincubation with 2,3-DPG before the concurrent addition of EIgG and the indicated concentrations of S. chromofuscus PLD. Levels of [3H]PEt (A) and phagocytosis (B) were determined at 30 min, as described in Materials and Methods. Ingestion of EIgG is expressed as the phagocytic index, i.e., the number of ingested particles per 100 MPs. Results are mean ± SEM of four experiments, each conducted in triplicate.

 

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  		Table II. Exogenous PLD, but not PI-PLC or PC-PLC, restores control levels of phagocytosis in 2,3-DPG-treated MPs1

 
Tyrosine kinase inhibitors produce concordant reductions in Fc{gamma}R-stimulated PLD activity and phagocytosis

Another approach to test the hypothesis that activation of PLD is functionally coupled to phagocytosis via Fc{gamma}Rs is to inhibit signal transduction events that lead to activation of PLD and determine their effects on particle ingestion. We chose to utilize tyrosine kinase inhibitors, since activation of PTKs is known to be a proximal event following the stimulation of all three classes of Fc{gamma}Rs 6, 38 . Genistein and herbymicin A were selected because of their high specificity, complementary mechanisms of action, and relatively low toxicities. Genistein (3–30 µM), which is a competitive inhibitor of PTKs with respect to ATP 39 , significantly reduced EIgG-stimulated PLD activity and phagocytosis (Figs. 7Go, A and C). Preincubation of MPs with 30 µM genistein for 15 min at 37°C inhibited the accumulation of [3H]PEt by 71 ± 7% and reduced phagocytosis by 67 ± 4%. Herbimycin A (0.1–0.6 µg/ml), which results in irreversible inhibition of PTKs by promoting their oxidation and subsequent proteosome-dependent degradation 40 , also resulted in parallel, concentration-dependent reductions in Fc{gamma}R-mediated PLD activity and phagocytosis (Figs. 7Go, B and D). Consistent with previous studies by other investigators 41, 42 , genistein and herbimycin A also produced dose-dependent reductions in EIgG-stimulated tyrosine phosphorylation of MP proteins (data not shown). Neither genistein nor herbimycin A altered MP viability or stability of EIgG (data not shown).



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  		FIGURE 7. Tyrosine kinase inhibitors reduce EIgG-stimulated PLD activity and phagocytosis. MPs were preincubated with the indicated concentrations of genistein for 15 min at 37°C (A and C) or herbimycin A for 18 h (B and D) before addition of EIgG for an additional 30 min. PLD activity was determined in [3H]-lyso-PAF-labeled MPs by accumulation of [3H]PEt (A and B). MPs incubated with 0.1% DMSO, followed by SRBC treated with irrelevant Ab, served as the control (Ctr) for the PLD assay. Parallel determinations of phagocytosis were conducted with unlabeled MPs (C and D). Addition of 0.1% DMSO alone did not alter PLD activity or phagocytosis. Results are mean ± SEM of three experiments conducted in triplicate.

 
The extent of phagocytosis of EIgG correlates strongly with the level of Fc{gamma}R-stimulated PLD activity

The concordance between Fc{gamma}R-dependent PLD activity and MP phagocytosis of EIgG, in terms of kinetics, response to inhibitors of PLD or PTKs, and effects of exogenous PLD support the hypothesis that the ingestion of IgG-opsonized particles is regulated by PLD. To obtain a quantitative assessment of the strength of the relation between the level of Fc{gamma}R-dependent PLD activity and phagocytosis of EIgG, we calculated the correlation coefficient between these two variables for all experiments in which both parameters were measured. Although our hypothesis is that the extent of phagocytosis is dependent on the level of PLD activity, no assumption was made in this analysis regarding the symmetry of the relation between these variables. Therefore, the strength of the association was determined by an analysis of correlation, rather than regression, since, in the latter case, variables are designated as "independent" or "dependent" before analysis 30 . Fig. 8Go demonstrates the scatter plot of the data, which are expressed as the percentage of control values (PLD activity and phagocytosis) for each experiment to minimize confounding due to additional variables that may differ between experiments, such as the efficiency of radiolabeling, the intrinsic phagocytic capacity of cells from different donors, etc. The correlation coefficient for the association between PLD activity and phagocytosis of EIgG was 0.956, which was highly significant (p < 0.001, n = 15). A similar analysis was performed utilizing the data from all experiments in which PLD activity was modulated and the effects on ingestion of IC beads determined. The correlation coefficient for the association between PLD activity and phagocytosis of IC beads was 0.944, which was also highly significant (p < 0.001, n = 12). The marked degree of correlation between PLD activity and the ingestion of EIgG and IC beads provides strong support for the hypothesis that stimulation of PLD functions to regulate Fc{gamma}R-mediated phagocytosis.



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  		FIGURE 8. Scatter diagram of the correlation between macrophage PLD activity and phagocytosis of EIgG. Data were derived from each set of experiments in which EIgG-associated PLD activity was modulated and the effect on phagocytosis determined. Values for PLD activity and the phagocytic index were expressed as the percentage of control values for untreated MPs. Each data point represents the mean ± range of at least 3 experiments, performed in duplicate or triplicate, for each condition, of a total of 15 different conditions. The association between PLD activity and phagocytosis was analyzed by the Spearman Rank Order Correlation test, which yielded a correlation coefficient, r = 0.956, which was highly significant (p < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fc{gamma}Rs play a central role in both physiologic immune responses and pathologic inflammatory states, by integrating the functions of lymphocytes, phagocytes, NK cells, and the complement system. Definition of the molecular mechanisms that regulate Fc{gamma}R-dependent functions of myeloid leukocytes will be essential to the development of therapeutic strategies to modulate the specificity and magnitude of the immune response. Phagocytosis is central to Fc{gamma}R-mediated functions since it serves to activate the primary antimicrobial mechanisms of myeloid leukocytes and also amplifies specific immunity by providing access to the Ags of the ingested particle. In this study, we have provided evidence to support the conclusions that: 1) phagocytosis of IgG-opsonized particles by human MPs is associated with activation of a major signal transduction pathway of myeloid cells, PLD, 2) each class of macrophage Fc{gamma}Rs, Fc{gamma}RI, RII, and RIIIA, is coupled to stimulation of PLD, and 3 PLD functions in the regulation of phagocytosis of IgG-opsonized particles.

Our first conclusion, that Fc{gamma}R-mediated MP phagocytosis is associated with activation of PLD, is supported by the use of two well-defined IgG-opsonized particles, EIgG and IC latex beads 24, 26, 42, 43 . The fact that cross-linking surface-bound F(ab')2 fragments of mAbs to Fc{gamma}RI, RII, or RIII also stimulated significant levels of PLD activity provides further support for our first conclusion and defines the second. This latter data is in agreement with the recent report by Melendez et al. 22 in which Ab-mediated cross-linking of Fc{gamma}RI on {gamma}-IFN-differentiated U937 cells stimulated PLD activity. In addition to stimulation via cross-linking Abs or phagocytic particles, Gewirtz and Simons 21 demonstrated that high valency immune complexes activate PLD inneutrophils.

The commonality in stimulation of PLD via diverse Fc{gamma}R agonists is particularly interesting in light of data by Davis et al. 31 that demonstrate differences in the regulation of Fc{gamma}RI-mediated endocytosis of immune complexes vs phagocytosis of IgG-opsonized particles, with regards to their dependence on tyrosine kinase activity and transmembrane and cytosolic domains of Fc{gamma}RI. Therefore, stimulation of Fc{gamma}Rs by diverse soluble and particulate agonists results in uniform activation of certain signal transduction pathways, whereas other signaling components are activated in a particle-specific manner. Rossi and colleagues 44 demonstrated that zymosan, opsonized with sera from rabbits immunized with Saccharomyces cerevisiae, stimulated neutrophil PLD activity. Although, the receptor that mediated this response was not defined, it is likely that Fc{gamma}Rs played a significant role. Previous studies from multiple investigators have linked activation of PLD in myeloid leukocytes to the regulation of two major Fc{gamma}R-dependent responses, superoxide generation and granule secretion (reviewed in Refs. 15 and 16). Along with the recent work of Melendez et al. 22 , indicating an essential role for PLD in vesicular trafficking of endocytosed immune complexes to lysosomes, and the present study, it is clear that stimulation of PLD is a key step in the activation of phagocytic leukocytes by Fc{gamma}Rs.

Our third conclusion, that Fc{gamma}R-dependent activation of PLD functions to regulate phagocytosis, is supported by several complementary experimental approaches. Two inhibitors of PLD-dependent signal transduction, 2,3-DPG and 1-butanol, which utilize different mechanisms of inhibition, produced concentration-dependent reductions in EIgG-stimulated PLD activity, which strongly correlated with inhibition of phagocytosis. Addition of purified PLD to 2,3-DPG-treated MPs at a concentration that reconstituted EIgG-associated generation of PEt restored phagocytosis to the level of control cells. Purified PLD preparations from phylogenetically diverse sources (Streptomyces, peanut, cabbage) shared this reconstituting activity, whereas purified PI-PLC and PC-PLC did not. Although we acknowledge that exogenous PLD does not precisely mimic activation of the endogenous isoform(s), this approach has yielded valuable insights regarding the physiologic functions of several mammalian phospholipases, including PLD 19, 35, 36, 37 . Since PA, the primary signaling-competent product of PLD catalyzis, remains membrane-bound following its generation, exogenous PLD may exert its primary effects on the plasma membrane, though to our knowledge, this has not been experimentally verified. In fact, some of the uncertainties regarding the mechanisms of action of exogenously supplied phospholipases are shared for their endogenous counterparts. For example, the mechanism by which intracellular PLD gains access to its substrate, phosphatidylcholine, which is enriched on the outer leaflet of the plasma membrane, is unknown 15, 45 . However, despite these mechanistic uncertainties, considerable evidence indicates that exogenous phospholipases can utilize plasma membrane lipids as substrates to catalyze the formation of bioactive lipid products and mimic the function of their endogenous homologues 19, 35, 36, 37 .

Further evidence to support a regulatory role for PLD in phagocytosis of IgG-opsonized particles was obtained with the use of two complementary PTK inhibitors, genistein and herbimycin A. Each of these produced dose-dependent reductions in Fc{gamma}R-dependent PLD activity and phagocytosis of EIgG. A potential limitation of the PTK inhibitors is that since tyrosine kinases probably promote phagocytosis via regulation of multiple downstream effectors (e.g., PLD, phosphoinositide 3-kinase, actin polymerization, etc.), reduction in the activity of any single proposed effector by PTK inhibitors does not constitute specific evidence for the involvement of that effector in phagocytosis. However, part of the value of the experiments with PTK inhibitors is that if inhibition of phagocytosis occurred without reductions in Fc{gamma}R-mediated PLD activity, this would strongly argue against our third conclusion. Overall, the marked correlation between PLD activity and phagocytosis of EIgG or IC latex particles, strongly supports a fundamental role for this phospholipase in the regulation of particle ingestion. However, we recognize that even this high degree of association does not prove a causal effect of PLD on the regulation of phagocytosis. Additional evaluation of the proposed regulatory role for PLD in Fc{gamma}R-mediated phagocytosis will include more specific genetic manipulation of PLD activity and modulation of PA levels by complementary metabolic pathways.

We have previously demonstrated that phagocytosis of complement-opsonized particles by human MPs is also characterized by a strong correlation with PLD activity 19 . Multiple studies have detailed the similarities and differences in phagocytosis mediated by Fc{gamma}Rs vs CRs, including biochemical requirements, mechanisms of particle ingestion, and consequences for subsequent phagocyte activation and microbicidal activity 1, 46, 47, 48, 49 . A common role for PLD in phagocytosis via Fc{gamma}Rs and CRs may be due to the fact that the PLD/phosphatidate phosphohydrolase pathway is the major route of diglyceride production in stimulated phagocytes 15, 16 with the resultant activation of phagocytosis-promoting signaling components, such as PKC 1, 38, 46 . McPhail and colleagues 51 have recently described a PA-stimulated protein kinase that functions in the activation of superoxide generation, and the role of PLD in promoting Fc{gamma}R-mediated phagocytosis may involve stimulation of this, or a related, PA-activated protein kinase.

The biophysical consequences of the focal generation of PA may also contribute to the involvement of PLD in phagocytosis, particularly since this amphipathic phospholipid promotes fusion of purified lipid bilayers and functions in vesicle trafficking and fusion in the Golgi complex 15, 45 . The second messenger functions of PA may bridge these biochemical and biophysical considerations via stimulation of several lipid-modifying pathways, including phospholipases A2 and C, and phosphoinositide 4- and 5- kinases, which result in production of lipids with both signaling and membrane-perturbing properties 15, 45, 50 . Future studies will address the proposed regulatory role for PLD in Fc{gamma}R- and CR-mediated phagocytosis, characterize the biochemical requirements for specific lipid second messengers in particle ingestion, and define the consequences of these proximal signaling events for the subsequent phagocyte responses which contribute to both the antimicrobial and tissue-damaging effects of the immune response.


   Acknowledgments
 
We thank Dr. William M. Nauseef for his support and generous gift of anti-myeloperoxidase Ab and gratefully acknowledge the support provided by the Graduate College, University of Iowa (to J.A., via the Summer Research Opportunities Program).


   Footnotes
 
1 These studies were supported by a Merit Review grant and a Career Development Award (to D.J.K.) from the Department of Veterans Affairs. Back

2 Address correspondence and reprint requests to Dr. David J. Kusner, Division of Infectious Diseases, Department of Internal Medicine, University of Iowa, 200 Hawkins Drive SW 34-I, GH, Iowa City, IA 52242. E-mail address: Back

3 Current address: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870. Back

4 Abbreviations used in this paper: MP, macrophage, PLD, phospholipase D; PA, phosphatidic acid; PEt, phosphatidylethanol; 2,3-DPG, 2,3-diphosphoglycerate; PI-PLC, phosphatidylinositol-specific phospholipase C; PC-PLC, phosphatidylcholine-specific phospholipase C; CR, complement receptor; lyso-PAF; PTK, protein tyrosine kinase, PKC, protein kinase C; IC, immune complex-coated; R{alpha}M, rabbit anti-mouse. Back

Received for publication September 16, 1998. Accepted for publication November 9, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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