Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation

Abstract

Acute lung injury (ALI) causes high mortality, but its molecular mechanisms are poorly understood. Acid aspiration is a frequent cause of ALI, leading to neutrophil sequestration, increased permeability, and deterioration of gas exchange. We investigated the role of platelet-neutrophil interactions in a murine model of acid-induced ALI. Acid aspiration induced P-selectin-dependent platelet-neutrophil interactions in blood and in lung capillaries. Reducing circulating platelets or blocking P-selectin halted the development of ALI. Bone marrow chimeras showed that platelet, not endothelial, P-selectin was responsible for the injury. The interaction of platelets with neutrophils and endothelia was associated with TXA(2) formation, with detrimental effects on permeability and tissue function. Activated platelets induced endothelial expression of ICAM-1 and increased neutrophil adhesion. Inhibition of platelet-neutrophil aggregation improved gas exchange, reduced neutrophil recruitment and permeability, and prolonged survival. The key findings were confirmed in a sepsis-induced model of ALI. These findings may translate into improved clinical treatments for ALI.

Figure 1. Platelets control PMN recruitment into the lung in acid-induced ALI.

(A–C) Platelet depletion (by 40%) prior to acid application by busulfan (n = 4–10 mice per group) significantly improved gas exchange (A), reduced intravascular and interstitial PMN accumulation (data not shown), diminished PMNs in the BAL fluid (B), and partially prevented increased vascular permeability (C). Platelet depletion (85%) caused by a polyclonal Ab diminished PMN recruitment into the alveolar space (B) and permeability (C). (D–G) Photomicrographs of lung tissue. H&E-stained paraffin sections from control mice (D) and mice 2 hours after acid administration (E). Acid application induced increased permeability with an influx of protein-rich fluid and cells (E, arrow) into the alveolar space, swelling of the interstitium, and cell accumulation in the interstitial space (E). Glycol pretreatment (F) prior to initiation of ALI induced the same histological changes seen in untreated mice after acid application whereas the pretreatment with busulfan led to reduced morphological changes (G). Original magnification, ×65. Gly, glycol; Bu, busulfan; pre, preimmune serum; Ab, polyclonal anti-platelet Abs. ^#P < 0.05. Scale bars, 50.0 mm.

Figure 2. Acid-induced ALI causes platelet-neutrophil interactions.

(A) Flow cytometry analysis of platelet-neutrophil aggregates after initiation of HCl-induced ALI. CD45, Gr-1, and 7/4 mAbs were used to identify PMNs (data not shown). Neutrophil-platelet aggregates were identified as neutrophils that were also positive for the platelet-specific marker CD41. (B) Thirty minutes after initiation of HCl-induced ALI, platelet-neutrophil interaction in the blood increased significantly. P-selectin Abs almost completely prevented the formation of platelet-neutrophil aggregates (n = 6–9 mice per group). (C) Platelet depletion by Abs prior to initiation of acid-induced ALI reduced the amount of platelet-neutrophil interactions in the blood. (D) Platelet-neutrophil interactions 30 minutes after initiation of acid-induced ALI in pulmonary microvasculature visualized by electron microscopy. Platelets (arrows) attached directly to the endothelium and a neutrophil. Scale bar: 1 μm. Original magnification, ×8000. ^#P < 0.05. P-sel, P-selectin.

Figure 3. Platelet P-selectin plays key role in the development of ALI.

(A–C) Injecting P-selectin Abs 15 minutes after the induction of ALI (n = 4–5 mice per group) improved gas exchange (A) and reduced interstitial (data not shown) and BAL fluid (B) PMNs as well as permeability (C). (D–G) To determine whether hematopoietic (platelet) or nonhematopoietic (endothelial) P-selectin is responsible for neutrophil recruitment in ALI, BM chimeras (WT into WT; WT into Selp^–/–; Selp^–/– into WT; Selp^–/– into Selp^–/–) were tested (n = 4–5 mice per group). Mice lacking platelet P-selectin showed improved gas exchange (D), reduced PMN accumulation in the intravascular (E), interstitial (data not shown), and alveolar compartments (F), and diminished permeability (G) compared with mice expressing hematopoietic P-selectin. ^#P < 0.05; ^ΧP < 0.05 versus platelet Selp^–/–.

Figure 4. Neutrophil-platelet adhesion to untreated HPMECs requires endothelial cell TPs.

(A) Platelet-neutrophil interactions significantly induced PMN adhesion (n = 3 per group). (B) Increased adhesion was partially mediated by endothelial cell TPs. Baseline PMN adhesion is indicated by dotted line. (C) ICAM-1 mRNA in endothelial cells was increased by exposure to activated PMNs or the combination of PMNs and platelets when at least 1 of the cell types was activated (n = 3 per group). (D) The incubation of human platelets with neutrophils induced a 2.5-fold increase in TXB₂ measured in the supernatant, which was further enhanced to 5.5-fold when either PMNs or platelets were activated by TNF-α or thrombin. n = 3 per group. ^&P < 0.05 versus PMNs; ^ΧP < 0.05 versus both and activated PMNs; *P < 0.05 versus vehicle; ^#P < 0.05 versus control; ^†P < 0.05 versus platelets. Act., activated; plt., platelet.

Figure 5. Blocking TXA₂ prevents acid-induced ALI.

(A) ALI induced increased plasma TXB₂ levels at 2 hours. Platelet depletion partially reduced TXB₂ levels (n = 4–5 mice per group). (B–D) Blocking of TPs (n = 4–5 mice per group) significantly improved gas exchange (B), reduced PMN accumulation in the intravascular, interstitial (data not shown), and alveolar compartments (C), and prevented increased vascular permeability (D). ASS showed similar effects regarding PMN recruitment and permeability (C–D), but the effect on gas exchange was not as pronounced as in TP blockade (B). ^#P < 0.05.

Figure 6. Endothelial cell response to TP activation as reflected by F-actin localization and content.

(A) Human pulmonary endothelial cells were treated with 75 or 150 nM of TXA₂ analogue (SQ 29548), and F-actin was localized by phalloidin staining. Images are representative of 3 experiments with similar results. Original magnification, ×175. (B) Activated platelets and activated PMNs induced a significant increase of F-actin formation in endothelial cells (n = 3 per group). The combination of stimulated platelets and PMNs caused a further increase of F-actin polymerization. ^ΧP < 0.05; ^#P < 0.05 versus control. (C) Platelet depletion with busulfan or TP antagonist SQ 29548 prior to induction of ALI led to significant prolongation of survival compared with that of HCl-treated mice (n = 4 per group). All mice treated with a P-selectin Ab survived until termination of the experiment (300 min), as did control mice (data not shown). The HCl group was significantly different from the other groups. P = 0.0002 by log rank test.

Figure 7. Platelets influence sepsis-induced ALI.

(A–D) Four hours after initiation of sepsis-induced ALI by LPS and zymosan, mice displayed reduced gas exchange (A), increased accumulation of neutrophils in the intravascular (B) and alveolar (C) compartments, and permeability (D). Platelet depletion by Ab prior to the induction of the sepsis model caused improved gas exchange, reduced permeability (D), and neutrophil accumulation in the intravascular (B) and alveolar (C) spaces. ^#P < 0.05.