Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury (original) (raw)
Activated platelets induce NET formation in human neutrophils. We used neutrophils isolated from normal human volunteers (Figure 1A) to assay for NET formation determined by the colocalization of extracellular DNA, extracellular histone protein, and extracellular myeloperoxidase (MPO). Phorbol 12-myristate 13-acetate–treated (PMA-treated) neutrophils were used as a positive control for NET formation (Figure 1B). When platelets were added to neutrophils, the cells maintained condensed nuclei, without NET formation (Figure 1C). However, when platelets were activated with the PAR-1 agonist, thrombin receptor-activating peptide (TRAP) (20), and then were added to neutrophils, there was robust NET formation (Figure 1D). The addition of TRAP alone to neutrophils also induced rare NET formation (Figure 1E), which was surprising, since human neutrophils do not express PAR-1 (21). It is known, however, that approximately 10%–20% of neutrophils circulate as neutrophil-platelet aggregates (ref. 22 and data not shown). Therefore, NET formation induced by TRAP alone may be explained by preexisting neutrophil-platelet aggregates present in the neutrophil preparation. In separate experiments, we also stimulated platelets with thrombin (0.2 U/ml) or LPS (2 μg/ml), and when added to neutrophils, we observed strong NET induction (data not shown).
Activated platelets induce NET formation in human neutrophils. (A–E) Representative images from direct immunofluorescence staining of DNA (blue), histones (red), and MPO (green) on (A) normal neutrophils incubated in media, (B) showing NET formation in neutrophils treated with PMA (25 nM; as positive control of NETosis) and (D) with platelets activated by TRAP (50 μM). (C) No NET formation was apparent from treatment with platelets alone. (E) Treatment with TRAP alone produced less NET formation than in B or D. n = 6. Scale bar: 10 μm. (F) MPO-DNA ELISA was used to quantify NETs in neutrophil supernatants and is expressed as percentage increase above control (media); mean ± SD (n = 6). *P < 0.05 versus media and platelets groups; †P < 0.05 versus PMA and TRAP-activated platelets groups; ***P < 0.001 versus media and platelets groups.
To further corroborate our fluorescence microscopy experiments and to quantify NET formation, we developed a capture ELISA that detects MPO-associated DNA, similar to an assay previously used in a study of NETs in small-vessel vasculitis (14). To validate this ELISA, we tested the supernatants of neutrophils treated with PMA (NET control) or TNF-α (apoptosis control) (23), neutrophils incubated for 24 hours (apoptosis control) (24), sonicated neutrophils (necrosis control) (25), and unstimulated neutrophils (negative control). The optical density measurements were normalized to those of the negative control and are reported as the percentage of NET formation. With dose dependency (10 nM vs. 25 nM PMA), PMA-treated neutrophils produced a large increase in MPO-DNA complexes, whereas the apoptosis and necrosis controls produced only minimal increases (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI61303DS1). Using this NET-specific assay, we found that neutrophils increased NET production in the presence of PMA- or TRAP-treated platelets but not in the presence of nonactivated platelets (Figure 1F). In parallel to the fluorescence microscopy experiments (Figure 1E), neutrophils treated with TRAP or thrombin (data not shown) showed a small increase in NET production (Figure 1F). The addition of micrococcal nuclease to the coincubation of neutrophils and TRAP-activated platelets more than doubled the soluble NET components detected by the MPO-DNA ELISA (Supplemental Figure 1B).
The canonical Raf/MEK/ERK signaling pathway is essential for PMA-induced NET formation (26). We therefore tested the importance of this pathway in activated platelet-induced NET formation. In the presence of a specific MEK inhibitor (U0126), activated platelet-induced NET formation was substantially diminished (Figure 2, A and C). We have previously reported that thromboxane B2 (TXB2) levels increased in the plasma of mice with TRALI and that blocking TXA2 production with aspirin treatment reduces lung injury and mortality (5). We determined whether thromboxane produced by TRAP-activated platelets is a potential mediator in NET formation. TRAP-activated platelets released TXA2, which we measured by assaying for its degradation product, TXB2 (Supplemental Figure 2). Pretreatment of neutrophils with a selective thromboxane receptor antagonist (SQ29548) prior to the addition of TRAP-activated platelets reduced the production of NETs (Figure 2, A and D).
NET formation is dependent on thromboxane production and MEK signaling and increases endothelial permeability. (A) Quantification of NET release in cell supernatant (MPO-DNA ELISA). Neutrophils were pretreated with the MEK inhibitor (U0126, 10 μM) or a thromboxane receptor antagonist (SQ29548, 10 μM) for 10 minutes before the addition of platelets and TRAP. Pretreatment of neutrophils with U0126 or SQ29548 inhibited NET formation compared with that in neutrophils treated with TRAP-activated platelets. Mean ± SD (n = 6); *P < 0.05, ***P_ < 0.001 versus media and platelets; †_P_ < 0.05 versus TRAP-activated platelets. (**B**–**D**) Representative images from direct immunofluorescence staining of DNA (blue), histone (red), and MPO (green), showing NET formation in neutrophils treated with (**B**) TRAP-activated platelets and less NET formation in neutrophils pretreated with (**C**) U0126 or (**D**) SQ29548. _n_ > 6; Scale bar: 10 μm. (E) Permeability of endothelial cell monolayers (HUVEC) measured in a Transwell system. In selected experiments, HUVECs were primed with LPS (2 μg/ml) for 24 hours prior to the experiment. Permeability was measured by 125I-albumin flux across endothelium over 1 hour and was increased in cells treated with cytomix (0.5 ng/ml) or in LPS-primed endothelium with TRAP-activated platelets or PMA. Mean ± SD (n = 12); *P < 0.05, **P < 0.01, ***P < 0.001 versus HUVECs without treatment. †_P < 0.05, †††P < 0.001 versus HUVECs treated LPS and with neutrophils and TRAP-activated platelets.
NETs increase the permeability of primed endothelial cells. A hallmark of ALI is increased lung endothelial protein permeability. Therefore, we next explored the relationship between NET formation and endothelial cell permeability in a transwell model. HUVECs were grown to confluency on transwell inserts suspended in cell culture media, and the permeability across this endothelial monolayer was measured. Cytomix (TNF-α, IL-1β, and IFN-γ) induced a 2- to 3-fold increase in endothelial permeability, but the addition of neutrophils, platelets, or neutrophils activated to produce NETs failed to induce permeability changes (Figure 2E). However, when the endothelial cells were primed with LPS for 24 hours, NETs induced by PMA or by activated platelets produced an increase in permeability similar to that produced by cytomix (Figure 2E). Additionally, the permeability changes induced by NETs were attenuated by the MEK inhibitor (U0126) and the thromboxane receptor antagonist (SQ29548) (Figure 2E). Similar results were also obtained using human lung microvascular endothelial cells in which NETs (treated with a combination of LPS, neutrophils, platelets, and TRAP) induced protein permeability changes that were attenuated with MEK inhibition (Supplemental Figure 3). We conclude from these experiments that NETs increase permeability in LPS-primed endothelial cells.
NETs are produced in TRALI. Using the mouse model of TRALI, we previously demonstrated that neutrophils and platelets are required for injury development (5, 6). Since both neutrophils and platelets become sequestered in the lung microcirculation during TRALI (5, 6), we reasoned that intimate platelet-neutrophil interactions are produced that lead to NET formation. We prepared single-cell suspensions of lung tissue from mice in which TRALI was induced and used flow cytometry to detect neutrophil-platelet aggregates. In lungs removed only 2 minutes after injection of the H2Kd mAb, there was a greater than 2-fold increase in the staining intensity for the platelet-specific marker, CD41, on the surface of CD11b+Ly6G+ cells (Supplemental Figure 4A). Using transmission electron microscopy, we detected neutrophils that were simultaneously adherent to lung endothelial cells, platelets, and red blood cells, providing further evidence for the spatial interactions among these cells in TRALI (Supplemental Figure 4B).
Previously, we demonstrated that aspirin treatment decreases platelet sequestration in the lung, decreases lung vascular permeability and edema, and increases survival in experimental TRALI (5). We therefore hypothesized that aspirin treatment would decrease NET formation by decreasing platelet activation. We found that NETs were present in abundance in the lung microcirculation of mice with TRALI (compare Figure 3A with Figure 3B), and soluble NET components were higher in the peripheral blood (Figure 3E). Treatment of mice with aspirin prior to the induction of TRALI decreased NET formation (Figure 3, C and E). In addition, platelet sequestration increased in areas of the lung in which NETs were present (Figure 3B), and aspirin treatment decreased NET-associated platelets (Figure 3C). The speed by which NETs formed after H2Kd mAb injection was remarkable. With higher doses of mAb (4.5 mg/kg), we detected NET formation and associated platelet trapping only 5 minutes after mAb injection (Figure 3D).
NETs are present in TRALI mouse lungs, and aspirin decreases NET formation. (A–D) Representative images of NET formation detected by immunofluorescence in the lung microcirculation of (A) control mice (LPS plus isotype control mAb) and (B) mice with TRALI (LPS plus H2Kd mAb, 1.0 mg/kg), (C) with or without aspirin treatment at 2 hours after mAb injection. Platelet (CD41) staining was increased in TRALI mice and localized to areas of NET formation. Aspirin treatment decreased platelet staining to levels observed in control mice. (D) Mice treated with a higher dose of H2Kd mAb (4.5 mg/kg) had NET formation at 5 minutes after mAb injection. Scale bar: 20 μm. (E) NET formation was quantified (MPO-DNA ELISA) in mouse plasma and plotted as mean ± SD (n > 4). **P < 0.01, ***P < 0.001 versus group receiving no treatment; ††P < 0.01 versus TRALI plus DMSO group.
NETs are therapeutic targets in TRALI. We next took aim at NET components as a therapeutic target in TRALI. Extracellular histones are directly toxic to primary human endothelial cells (HUVECs), and, in a mouse model of endotoxemia, the neutralization of extracellular histones with a blocking mAb decreases mortality (27). We hypothesized that extracellular histones may mediate the increased endothelial permeability observed in our HUVEC (Figure 2E) and human lung microvascular endothelial cell (Supplemental Figure 3) experiments and in the TRALI mouse model. Accordingly, we administered a histone-blocking mAb (BWA3) that recognizes histone 2A (H2A) and histone 4 (H4) (28) to mice with TRALI (immediately prior to H2Kd mAb injection). We observed a decrease in lung edema, lung vascular permeability to protein, and mortality (Figure 4A), compared with that after a control antibody injection. This treatment strategy also reduced NET formation detected in plasma samples (Figure 4C), which implies that extracellular histones may propagate NET formation.
Platelets and extracellular histones are therapeutic targets in TRALI. (A) Mice with TRALI (LPS plus H2Kd mAb) administered BWA3 mAb (10 mg/kg, i.v.) or IgG control (10 mg/kg) given immediately prior to H2Kd mAb. BWA3 mAb decreased extravascular lung water and lung vascular permeability to 125I-labeled albumin and decreased mortality compared with that in the IgG control group. Mean ± SD (n = 9). *P < 0.05 versus IgG control group. **P < 0.01. (B) Mice with TRALI (LPS plus H2Kd mAb) administered tirofiban (0.5 μg/g, i.v.) or PBS given immediately prior to H2Kd mAb. Tirofiban decreased extravascular lung water and lung vascular permeability to 125I-labeled albumin and decreased mortality compared with that in the PBS group. Mean ± SD (n = 7–9). **P < 0.01 versus PBS group. (C and D) MPO-DNA ELISA was used to quantify NET formation in the plasma of mice treated with (C) BWA3 mAb or (D) tirofiban compared with that in treatment controls and normal mouse plasma. Mean ± SD (n = 6). ***P < 0.001 versus control group; ††P < 0.01 versus IgG control and PBS groups. (E and F) Lung sections stained for NET formation (DNA, histone, and MPO) and for platelets (CD41). Representative images of mice with (E) TRALI given diluent (PBS) compared with those with (F) TRALI administered tirofiban. Mice treated with tirofiban have decreased NET formation and associated platelet sequestration compared with the PBS group. (n = 6). Scale bar: 20 μm.
Since platelets bind to extracellular histones (29), we reasoned that the increase in platelet sequestration in areas of NETosis could reflect this binding. Moreover, the binding of platelets to histones may lead to platelet activation and propagation of platelet-platelet binding around NETs, much like the platelet aggregation that occurs with thrombosis. Therefore, we tested the effect of tirofiban, an inhibitor of the platelet glycoprotein IIb/IIIa receptor that mediates platelet-platelet binding through fibrinogen (30). Mice that were injected with tirofiban at the time of H2Kd mAb challenge were protected from lung edema and lung vascular permeability to protein (Figure 4B), and were also completely protected from mortality (Figure 4B). Tirofiban treatment also decreased soluble NET components (Figure 4D), NETs detected in lung tissue (Figure 4, E and F), and platelet sequestration in areas of NET formation (Figure 4, E and F). These results indicate that NET-induced platelet aggregation is an important feature of TRALI.
In vitro studies have demonstrated that NETs may be dismantled using DNase1, which helps to untangle the web of extracellular chromatin (31). Extracellular DNA of NETs could damage the lung microcirculation by impeding blood flow and potentially creating zones of ischemia. To test this hypothesis, we treated mice undergoing TRALI with DNase1, using a dose similar to that used in experimental studies of autoimmune kidney disease (32). When we initiated the treatment simultaneously with H2Kd mAb injection, we found that the pretreatment strategy with DNase1 protected mice from lung edema and lung vascular permeability (Figure 5A) and reduced NET formation and also platelet sequestration in the lung (Figure 5, C and D). DNase1 actually increased soluble NET components detected in the peripheral blood compared with the diluent control (Figure 5B), indicating that DNase1 cleaves extracellular DNA in the lung microcirculation that then enters the systemic circulation. Injection of DNase1 5 minutes after mAb injection, when NETs were already forming (Figure 3D), also reduced lung edema and protected mice from mortality (Figure 5, E and F).
DNase1 treatment decreases TRALI and NETs. (A) Mice treated with DNase1 (10 mg/kg, i.v.) or diluent immediately prior to H2Kd mAb injection are protected from TRALI with decreased extravascular lung water and decreased lung vascular permeability to 125I-labeled albumin (EVPE). Mean ± SD (n = 7). **P < 0.01 versus diluent group. (B) MPO-DNA ELISA was used to quantify NET formation in the plasma of mice pretreated with DNase1 or diluent compared with that in normal mouse plasma. Mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. (C and D) Lung sections stained for NET formation (DNA, histone, and MPO) and for platelets (CD41). Representative images of mice with TRALI given (C) diluent or (D) DNase1. Mice treated with DNase1 have decreased NET formation and associated platelet sequestration compared with the diluent-treated group. (n = 3). Scale bar: 20 μm. (E and F) Mice treated with DNase1 (10 mg/kg, i.v.) or diluent 5 minutes after H2Kd mAb injection are protected from TRALI with (E) decreased extravascular lung water and (F) decreased mortality compared with that in the diluent group. Mean ± SD (n = 8). *P < 0.05, **P < 0.01 versus diluent group.
NETs in human TRALI and ALI. To establish the clinical significance of our findings, we sought evidence of NET formation in patients with TRALI and ALI. TRALI autopsy reports are rare, as are available tissue samples, but we were able to test lung tissue from a fatal case of HLA class I antibody–associated TRALI (33). For a control, we used a case of fatal transfusion-associated circulatory overload (TACO), which is characterized by hydrostatic rather than permeability pulmonary edema. In the TRALI case, we detected areas in lung vessels in which many neutrophils probably were undergoing NETosis, with large, decondensed nuclei and abundant extranuclear histones and extracellular MPO (Figure 6B). In the TACO control case, we found intra-alveolar neutrophils, but we detected no NET formation (Figure 6A).
NETs are present in human TRALI lungs and plasma. (A and B) Human lung paraffin sections were stained for histone (red), MPO (green), and DNA (blue) and analyzed by confocal microscopy. (B) In the TRALI fatality case, we observed clumps of NET-forming neutrophils in the intravascular compartment. (A) In the TACO fatality case, neutrophils were found in mainly intra-alveolar locations, but no NET formation was detected. Scale bar: 10 μm. (C) MPO-DNA ELISA was used to quantify NET components in the plasma of patients, and the mean optical density of plasma obtained from normal, human blood donors (n = 6) was used as the control. Plasma from individuals with cardiac disease (n = 6), individuals before TRALI and after TRALI (paired samples, n = 14), and individuals before ALI and after ALI (paired samples, n = 9) are compared. Horizontal bars represent the mean; symbols represent individual samples. *P < 0.05 versus donor blood group; ***P < 0.001 versus donor blood, cardiac disease, and before TRALI groups; †P < 0.05 versus donor blood, cardiac disease, and before ALI groups.
Finally, we used the MPO-DNA ELISA to test plasma samples for NET formation in patients with TRALI and other causes of ALI. Plasma samples were obtained from a case-control study of TRALI and ALI in which blood was collected before and after the development of lung injury (34). There was a striking increase in NETs in the plasma samples after development of TRALI and, to a lesser degree, in samples after development of ALI, compared with that in normal human plasma and with patients with acute cardiac syndromes (Figure 6C).