A Dual Role for Diacylglycerol Kinase Generated Phosphatidic Acid in Autoantibody-Induced Neutrophil Exocytosis
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Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.
Human neutrophils contain granules that, when released into the extracellular environment, can promote cellular adhesion, facilitate transmigration into inflamed tissue and provide the cell with essential antimicrobial capability. Because of the high concentration of proteolytic enzymes contained within neutrophil granules, their release from the neutrophil is a tightly controlled and regulated process. For a granule to be successfully released from the cell, key events must occur. First, powered by actin polarization, granules must translocate from the cytosol to the plasma membrane, where they tether and dock at predetermined areas. After this, the granule must fuse and integrate with the plasma membrane to make the granular contents accessible to the outer environment. A number of intracellular signaling molecules are used by the cell to promote each step of exocytosis (1-3). Exacerbation of inflammation is associated with a number of autoimmune diseases where increased release of proteolytic enzymes enhances tissue damage (4,5). Delineating the mechanisms and pathways associated with this aberrant process of exocytosis may reveal a number of targets to control and reduce it.
Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitides such as granulomatosis with polyangiitis (Wegener’s), microscopic polyangiitis and Churg-Strauss syndrome (6). Antibodies with specificities to either proteinase 3 (PR3) or myeloperoxidase (MPO) are believed to contribute to development of acute disease by activating neutrophils within the small vessels of the lung, kidney or other organs. Neutrophil exocytosis of the azurophilic granules is likely to play a role in promoting endothelial cell damage in the blood vessel, with serine proteases and MPO released from activated neutrophils able to induce damage to both endothelial cells and the basement membrane (7, 8, 9).
ANCA IgG promotes distinct signal transduction pathways compared with neutrophil activation by either immune complexes or chemoattractants, and the pathways are independent of phospholipase D involvement (10). During ANCA-induced superoxide production, ANCA IgG F(ab′)2 (fragment antigen-binding) binds to its antigen on the surface of primed cells, resulting in the activation of the heterotrimeric G protein Gi, thereby stimulating phosphatidylinositol 3-kinase (PI3K) type 1γ (PI3Kγ), which activates protein kinase B (10,11). Binding of the Fc fragment of ANCA IgG to either FcγRIIa or FcγRIIIb results in the autophosphorylation of the tyrosine kinases syk and src and the adaptor cbl (12). We have also previously demonstrated the importance of phosphatidic acid (PA) production in the promotion of ANCA-induced neutrophil adhesion in vitro (13). PA production was shown to depend on the activation of the enzyme diacylglycerol kinase (DGK), to phosphorylate the lipid diacylglycerol (DAG). ANCA stimulation results specifically in the phosphorylation of both saturated and monounsaturated forms of DAG (13). These forms of PA are believed to act as signaling secondary messengers compared with polyunsaturated forms of the same molecules that are considered nonsignaling, transient metabolites (14,15).
The azurophilic granules contain the highest concentrations of both MPO and serine proteases compared with other granules and are likely to be released in a highly inflammatory setting (16). We investigated the release of these granules after ANCA IgG activation. Here, we study the signal transduction pathways activated by ANCA IgG that promote the exocytosis of azurophilic granules in vitro, specifically focusing on the activation of DGK through the production of PA. We report that ANCA-induced release of MPO depends on the production of PA, through the activation of DGK. We also demonstrate a dual mechanism for the actions of PA through both the generation of intracellular calcium and the ability to modulate the plasma membrane topography. The work highlights an intriguing pathway that may be activated by ANCAs and that can be further studied and targeted by rational drug design.
Materials and Methods
Total IgG was isolated from anonymized plasmapheresis samples obtained from ANCA-positive patients. All patients fulfilled Chapel Hill definitions and had active renal disease. IgG in serum was isolated on a protein G-Sepharose column (GE Healthcare, Buckinghamshire, UK). Both PR3- and MPO-specific ANCA IgGs were isolated and included in each set of experiments. We found no statistical difference in the ability of either type of ANCA to promote neutrophil activation in any of the assays performed in this study. The protocol was approved by the local ethics committee.
Generation of F(ab′)2 Fragments
Both ANCA and normal IgG preparations were pepsin digested to generate F(ab′)2 fragments. Preparations of antibody (0.5 mg) were dialyzed overnight in acetate buffer (0.1 mol/L sodium acetate, 0.1 mol/L acetic acid, pH 4) and then incubated with pepsin (20 µg/mL) in acetate buffer for 24 h. The preparation was then dialyzed overnight in 2× phosphate-buffered saline. Fc fragments were removed through incubation with immobilized protein G beads (Thermo Scientific). Concentration of F(ab′)2 was determined by spectrophotometry and generation of F(ab′)2 fragments confirmed on an sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel.
Blood from healthy donors was obtained by venipuncture into acid citrate dextrose, and neutrophils were isolated by centrifugation over a Percoll discontinuous gradient (GE Healthcare) as described previously (17). Informed consent was received from all donors, and the local ethics committee approved the protocol.
Isolated neutrophils were diluted to 2.5 × 106/mL in Hanks buffered salt solution with 10 mmol/L 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) (pH 7.4) and primed for 15 min with tumor necrosis factor (TNF)-α (2 ng/mL) (NIBSC, Potters Bar, UK) in the presence of cytochalasin B (10 µmol/L) (Sigma) at 37°C. Neutrophils (2.5 × 105) were added to wells of a round-bottom 96-well plate and stimulated with 200 µg/mL ANCA IgG, normal IgG or f-Met-Leu-Phe (fMLF) (1 µmol/L) (Sigma) as a positive control for 15 min at 37°C. After stimulation, cells were spun at 400g for 5 min and supernatants were removed. The activity of MPO within each sample was investigated using the o-phenylenediamine dihydrochloride (OPD) substrate (Sigma) (according to the manufacturer’s instructions). The reaction was stopped with 100% glacial acetic acid and read at 450 nm (Multiskan Thermo-Fisher, Waltham, MA, USA). Optical density readings from each sample were normalized as a percentage of the unstimulated control. Anti-MPO and anti-PR3 IgG had no effect on the enzymatic activity of MPO. Exocytosis was also measured after neutrophil adhesion. Neutrophils (2.5 × 106/mL) were primed with TNF-α (2 ng/mL) in the absence of cytochalasin B for 15 min and stimulated in 96-well flat-bottom plates previously coated with fibrinogen (25 µg/mL) as described previously (13). After 2 h of treatment, supernatants were removed and spun down to remove any unbound neutrophils and MPO release was measured as above. After the assay, neutrophils were found to be over 90% viable by trypan blue (Sigma) staining.
Inhibition of DGK and Phospholipase D Activity
Neutrophils, being terminally differentiated cells, are not amenable to many molecular and genetic techniques that may be used in other cell types to investigate signal transduction pathways. Therefore, we used specific chemical inhibitors to study the role of secondary messengers in ANCA-induced activation. All treatments had no significant effect on neutrophil viability analyzed by trypan blue exclusion and forward/side scatter properties using flow cytometry. For the study of DGK, we used the specific inhibitor R59022, which directly targets Ca2+-sensitive isoforms of DGK selectively (3,18,19). For the study of phospholipase D (PLD), we used the inhibitor VU0155069, which at concentrations >1 µmol/L, inhibits the two mammalian isoforms of PLD (PLD1 and PLD2). Concentrations of inhibitors used were selected by known selectivity of isoforms in cell systems and had no effect on neutrophil viability in vitro assays and no effect on TNF-α priming. As a result, neutrophils were incubated with the DGK-selective inhibitor R59022 (18 µmol/L) (Calbiochem, La Jolla, CA, USA) or the PLD-selective inhibitor VU0155069 (1 µmol/L) for 10 min (Cayman Chemicals, Ann Arbor, MI, USA) before priming. All incubations were carried out before neutrophil priming at 37°C. DMSO (Sigma) used as a vehicle control, at the highest concentration, did not have a significant effect on neutrophil activation.
Neutrophils were analyzed for the surface expression of CD63 using a monoclonal fluorescein isothiocyanate (FITC)-conjugated antibody at 2 µg/mL (AbD Serotec, Oxford, UK) and an IgG isotype control (BD Pharmingen, Oxford, UK). Cells were incubated with antibody for 15 min at room temperature before being washed and fixed with 2% paraformaldehyde. Samples were acquired on a FACScalibur flow cytometer using Cell Quest software (Becton Dickinson, Oxford, UK) with 1 × 104 cells acquired per tube. List mode data were analyzed using WinDMI (2.9) software.
Addition of Short-Chain Phosphatidate and Lysophosphatidylcholine
Short-chain lipid phosphatidate (Di8:0) (PA; Sigma), 1,2-dioctanoyl-sn-glycerol (Di8:0) (DAG, Cayman Chemicals, Tallinn, Estonia) and lysophosphatidylcholine (LPC) (Sigma) was reconstituted in chloroform:methanol (2:1), vacuumdried, resuspended in phosphate-buffered saline and sonicated. PA, DAG (final concentration 12.5 µmol/L) and LPC (250 nmol/L) were added to cells after priming and before neutrophil stimulation at 37°C. The working concentration of both PA and LPC was added at the lowest concentration that did not cause significant exocytosis in unstimulated cells during titration experiments. The concentration of PA is within the range of what is found physiologically in stimulated neutrophils (20). All treatments had no significant effect on neutrophil viability analyzed by trypan blue exclusion and forward/side scatter properties using flow cytometry.
Neutrophils were treated as above, and the cells were then fixed with 2% paraformaldehyde before the addition of wheat-germ agglutinin conjugated to alexa fluor 594 (5 µg/mL) to label plasma membranes (LIVE plasma membrane stain; Invitrogen, Paisley, UK). Neutrophils were then resuspended in permeabilization buffer (e-Bioscience, San Diego, CA, USA) and labeled with anti-CD63 monoclonal FITC-conjugated antibody L (AbD Serotec) or IgG isotype (BD Pharmingen) both at 2 µg/mL or incubated with phalloidin-FITC (10 µmol/L) (Sigma). Staining was carried out for 20 min at room temperature. The stained cells were spun onto glass slides, mounted in SlowFade® Gold antifade buffer (Invitrogen) and viewed using a Zeiss LSM 510 confocal scanning microscope equipped with a c-apochromat 100×/1.2 oil immersion objective (Carl Zeiss, Jena, Germany). All images were processed and analyzed using LSM 510 software (version 2.3; Carl Zeiss). Isotype- and concentration-matched control antibodies yielded consistently negative results.
[Ca2+] Measurement by Spectrofluorimetry
Neutrophils were resuspended at 2 × 106 cells/mL in 1.4 mmol/L Ca2+ Hanks balanced salt solution + 20 mmol/L HEPES buffer + 7.5% NaHCO3 and loaded with Indo-1 AM ester (Invitrogen) at 1 µmol/L (21). To confirm that the cells were resting and produced a stable baseline, the ratio of the fluorescent intensity at the two emission wavelengths was observed for 100 s, and ANCAs (200 µg/mL) were added through a light occlusive stopper in the top of the fluorimeter. After addition of the stimulus, the trace was observed until a stable plateau response was achieved. A calibration file was generated to allow calculation of [Ca2+] from the ratio of fluorescence intensity, as described previously (22). To assess the relative contributions of intracellularderived Ca2+ and the influx of Ca2+ from the extracellular space, investigations were performed under Ca2+ free conditions. Neutrophils were transferred into Ca2+ free Hanks balanced salt solution containing HEPES buffer (HBH). Any remaining Ca2+ was removed by the addition of ethyleneglycoltetraacetic acid (EGTA) (0.4 mmol/L) immediately before stimulation, thus minimizing the time that the cells spent in Ca2+ free medium. ANCAs (200 µg/mL) were then added, and the trace was observed until a stable plateau response was observed. A source of Ca2+ (CaCl2) (1.4 mmol/L) was then added to the cell suspension to determine the influence of system operating channels and enable the rate and magnitude of Ca2+ influx to be assessed.
Where required, data were analyzed by one-way repeated-measures analysis of variance (ANOVA) with a Bonferroni posttest or paired Student t test using Graphpad Prism software (Graphpad, La Jolla, CA, USA).
Neutrophils Release MPO to Intact ANCA IgG, Dependent on Actin Cytoskeleton Modulation
ANCA-Induced Azurophilic Granule Exocytosis Is Driven by DGK Activation and Phosphatidic Acid Production
DGK Does Not Affect Azurophil Granule Movement
Membrane Fusion Modulation Cannot Restore Exocytosis in DGK-Inhibited Cells
DGK, through the Production of PA, Promotes the Release of Intracellular Calcium by ANCAs
Actions of LPC and Ionomycin Combine to Restore Exocytosis in R59022-Inhibited Cells
Pathophysiological mediators of neutrophil activation, including the vasculitis-associated autoantibody ANCA, can use distinct signaling pathways to induce exocytosis of the cell’s azurophilic granules. We previously reported that ANCA IgG induces neutrophil adhesion through a pathway involving DGK-catalyzed monounsaturated or saturated PA formation, although this pathway is not used for ANCA-mediated superoxide release (13). In this study, we now report that ANCA-induced DGK-mediated formation of PA promotes exocytosis of the azurophilic granules by dual effects on calcium and membrane fusion.
Although the complete signaling pathways promoting ANCA-induced neutrophil exocytosis are yet to be fully defined, it is clear from this report that ligation of both the Fc and F(ab′)2 portions of ANCA IgG are required to promote the release of azurophilic granules. The work also confirms earlier studies examining superoxide release (32) where both fragments, through ligation, were shown to promote distinct signaling pathways. It is possible that ligation of receptors by the intact IgG molecule brings key receptors and signaling molecules together in the plasma membrane to promote adequate signal transduction. This result is feasible considering that the ANCA antigens have been reported to sit in distinct lipid rafts near both the β2 integrin complex and the low-affinity IgG receptor FcγRIIIb (33).
A degree of actin disruption induced by cytochalasin B has been shown, in a number of previous studies, to be required for chemoattractant-induced azurophilic granule release (3,25), bringing into question the ability of neutrophils to release their azurophilic granules physiologically. In this study, we were able to demonstrate that primed neutrophils stimulated with either autoantibody or fMLF, and left to adhere onto fibrinogen, released significant levels of MPO, suggesting that actin cytoskeleton modulation induced by adhesion and transmigration is sufficient to promote the release of the azurophilic granules. The mechanisms of neutrophil adhesion and exocytosis may well be entwined, since CD63 localizes with the β2 integrin CD11/CD18 in distinct lipid rafts of the plasma membrane and signal transduction downstream of CD63 promotes the distinct activation of the CD11/CD18 to promote increased neutrophil adhesion (34,35).
Exocytosis of granules is a complex and ordered process, beginning with the movement of granules from the cytosol to the plasma membrane, recognition and docking of the granule and complete fusion between vehicle and target membrane resulting in the release of granule constituents into the extracellular environment. Using confocal microscopy, we observed that DGK activation plays little or no role in the movement of granules from the cytosol to the membrane, pointing to more defined roles at the plasma membrane concerning granule fusion. Fusion of the target and vehicle membrane is achieved through the actions of SNARE (soluble N proteins ethylmalimide-sensitive factor accessory protein [SNAP] receptor) family proteins. SNARE family members are found on both the target membrane and the vehicle membrane and, according to the SNARE hypothesis, opposite SNARE complexes form a transient bridge promoting a trans-SNARE complex that promotes the fusion of two opposing biological membranes (36).
With PA likely to exert its effects at the plasma membrane, we aimed to investigate the role of the phospholipid in its ability to modulate membrane fusion and promote the release of intracellular Ca2+. R59022-inhibited neutrophils exhibited diminished calcium generation upon ANCA treatment that was restored in the presence of exogenous PA. However, adding exogenous calcium into the cell with the ionophore ionomycin did little to restore exocytosis in R59022-inhibited cells, indicating that exocytosis did not solely depend on PA-induced calcium release. The ability of PA to modulate membrane fusion was assessed by adding LPC into R59022-inhibited cells; here, also, the addition of LPC had no restorative effect on exocytosis. The observation that exocytosis was only restored when R59022-inhibited cells were treated with ANCA in the presence of both ionomycin and LPC indicates dual or multiple roles for PA that are generated through neutrophil activation by ANCA.
The formation of the trans-SNARE complex does not happen readily, since the outer membrane leaflet needs to bend in a negative curvature to preserve membrane integrity, while the inner leaflet requires opposite positive curvature (37). PA is able to promote a negative curvature in the membrane, since it is principally a cone-shaped molecule with relatively small phospholipid head groups (38). It has been reported that inhibiting PA production in the plasma membrane, but not in the donor granule membrane, inhibits granule fusion and release (39). PA may also promote the formation of the SNARE fusion complex directly by interacting with syntaxin, acting as structural support and reducing the energetic barrier required to fuse two membranes (40). In many cell types, including neutrophils, PA was demonstrated to modulate levels of intracellular Ca2+ (41), which in turn tightly regulates SNARE fusion (42,43). The SNARE complex alone has no Ca2+ sensory capacity yet uses an additional factor (synaptotagmin-1) for this role (44). The complete role of synaptotagmin-1 is yet to be fully elucidated, but reports suggest that the molecule is able to promote positive curvature of the vesicle membrane as well as binding distinct anionic phospholipids (such as PA) and syntaxin in the plasma target membrane (45,46).
Phosphatidic acid also plays a role in promoting neutrophil activation in response to fMLF treatment (47). However, in our experiments, fMLF-induced MPO release did not depend on PA generated by the actions of DGK. A number of previous reports suggested that PA generated as a result of fMLF treatment of neutrophils is likely to be the product of a PLD-dominant and not a DGK-driven pathway (48,49). We can also confirm that fMLF promotes neutrophil activation through a PLD pathway, since VU0155069 was able to significantly inhibit fMLF-induced MPO release in vitro. Although both ANCA IgG and fMLF are able to induce PA generation, it is likely that these are indicative of two separate pools with distinct properties. For example, PA generated through the actions of PLD may have roles in chemotaxis and modulation of the actin cytoskeleton (50,51), whereas it is evident from work here that the actions of DGK activated by ANCA IgG ligation do not modulate the actin cytoskeleton. We have previously documented the importance of the PI3K pathway in promoting ANCA-induced neutrophil activation (11), and this is likely to be central in promoting changes to the actin cytoskeleton (52).
It remains unclear which upstream pathways lead to DGK activation and the production of PA in ANCA-stimulated neutrophils. However, it is evident from this study that ligation of both the F(ab′)2 and Fc portions are required for ANCA-induced MPO release, and signaling pathways downstream of this ligation may promote the activation of DGK and the production of PA. We previously demonstrated that PI3K inhibition abrogates ANCA-induced neutrophil adhesion in vitro, which can be subsequently reversed by adding PA (13). Indeed, downstream products of PI3K including phosphatidylinositol3,4,5-trisphosphate (PIP3) are known to be activators of DGK (53). Downstream pathways of FcγR ligation may also promote DGK activation, for example, src was previously identified as an activating tyrosine kinase of DGKs as well as inducing Ca2+ generation in ANCA-stimulated neutrophils (12,54). We also cannot rule out the possibility that these distinct mechanisms of PA activity are provided by different pools of PA. Both the location and timing of PA generation may have different effects on ongoing signal transduction pathways (55). The generation of PA may directly modulate both membrane fusion and calcium generation; however, it is also feasible that PA promotes the activation of downstream signaling pathways, which in turn may promote both the membrane modulation, calcium generation and other processes involved in neutrophil exocytosis. It has been reported that PA may directly promote the activation of a number of tyrosine and serine-threonine kinases (41,56,57), which may have similar effects to what we have reported here. For example, PA is known to promote activation of sphingosine kinase (58), which also acts as a modulator of intracellular calcium release in neutrophils (59,60).
In conclusion, we have demonstrated that the release of azurophilic granules from ANCA-stimulated neutrophils depends on the production of PA through the activation of DGK. We propose that PA promotes azurophil granule membrane fusion through its dual ability to modulate membrane fusion and promote increases in intracellular calcium. This pathway may be key, not only to the neutrophils’ ability to release harmful proteins into their microenvironment, but also for the neutrophils’ ability to adhere. Involvement of these molecular mechanisms in ANCA-induced exocytosis suggests that they are worthy of consideration as novel targets for treating vasculitic glomerulonephritis.
C Savage is currently employed by GlaxoSmithKline PLC. The authors declare that they have no other competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
The work was funded by a Stuart Strange Vasculitis Trust postdoctoral fellowship.
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