Disruption of platelet-derived chemokine heteromers prevents neutrophil extravasation in acute lung injury

Abstract

Rationale: Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive.

Objectives: To determine mechanisms of platelet-mediated neutrophil recruitment in ALI.

Methods: Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI.

Measurements and main results: In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to P-selectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution.

Conclusions: Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference.

Figure 1.

Neutrophils and platelets cooperate in the onset of LPS-induced acute lung injury. Mice were challenged with LPS by inhalation and killed 4 hours later. Neutrophils were depleted by injection of an anti-Ly6G antibody (clone 1A8, 50 ng, intraperitoneally), whereas platelets were depleted by application of antiplatelet serum (50 μl, intraperitoneally). (A) Quantification of intravascular (top), interstitial (middle), and alveolar neutrophils (bottom). (B) Protein concentration (top), fluorescein isothiocyanate (FITC)–dextran clearance (middle), and elastase (bottom, uniform bars) and myeloperoxidase (MPO) activity (bottom, hatched bars) in bronchoalveolar lavage fluids. (C) Representative histologic (left) and scanning electron microscopic (right) images of lungs from mice treated as indicated. Scale bar indicates 50 μm for scanning electron microscopy and 250 μm for histology. Quantification of histologic lung sections is shown at bottom. n = 8–10 for each bar. Statistical significance was tested using one-way analysis of variance with Dunnett post hoc test. *Indicates significant difference compared with LPS-treated animals.

Figure 2.

LPS-mediated lung injury is not attenuated by antagonists to P-selectin or by DNase treatment. Mice were treated with antibodies to P-selectin (30 μg) or GPIIb/IIIa (100 μg), or DNase (1 μg) before LPS inhalation, and killed 4 hours later. (A) Quantification of intravascular (top), interstitial (middle), and alveolar neutrophils (bottom). (B) Protein concentration (top), fluorescein isothiocyanate (FITC)–dextran clearance (middle), and elastase (bottom, uniform bars) and myeloperoxidase (MPO) activity (bottom, hatched bars) in bronchoalveolar lavage fluids. n = 8–10 for each bar. Statistical significance was tested using one-way analysis of variance with Dunnett post hoc test. *Indicates significant difference compared with LPS-treated animals.

Figure 3.

Platelet-derived CCL5 and CXCL4 promote neutrophil recruitment in LPS-induced lung injury. (A) Mice challenged with aerosolized LPS or saline (ctrl) were injected with fluorescent beads conjugated with antibodies to CCL5 (top) or CXCL4 (bottom). Bead immobilization in the lung microcirculation was recorded by fluorescence intravital microscopy. Beads per field were manually counted. Scale bar, 50 μm. n = 5 for each bar. Statistical significance was tested using Mann-Whitney tests. *Indicates significant difference between groups. (B and C) Mice were treated with antibodies to CCL5, CXCL4, or a combination of both. The last group was also depleted of platelets. Thereafter mice were exposed to LPS by inhalation and killed 4 hours later. (B) Displayed are intravascular (top), interstitial (middle), and alveolar neutrophil counts (bottom). (C) Protein concentration (top), fluorescein isothiocyanate (FITC)–dextran clearance (middle), and elastase (bottom, uniform bars) and myeloperoxidase (MPO) activity (bottom, hatched bars) in bronchoalveolar lavage fluids. n = 8–10 for each bar. Statistical significance was tested using one-way analysis of variance with Dunnett post hoc test. *Indicates significant difference compared with LPS-treated mice.

Figure 4.

Disruption of CCL5–CXCL4 heteromer formation abrogates neutrophil recruitment in LPS-induced acute lung injury. (A) Correlation of leukocyte counts and CCL5–CXCL4 heteromers in bronchoalveolar lavage (BAL) fluid from patients with acute lung injury and adult respiratory distress syndrome. (B) Quantification of CCL5–CXCL4 heteromers in supernatants of homogenates of lungs from mice exposed to LPS and having received antiplatelet serum (50 μl) or MKEY (50 μg). n = 3 for each bar. Statistical significance was tested using Kruskal-Wallis test with Dunn post hoc test. *Indicates significant difference to all other groups. (C) Isolated neutrophils were perfused over human umbilical vein endothelial cells (HUVEC) treated with tumor necrosis factor (TNF) (50 ng/ml, 12 h). In addition, recombinant CCL5 and CXCL4 were complexed and immobilized on HUVEC. Thereafter, neutrophils were perfused in presence or absence of MKEY. n = 8 for each bar. Statistical significance was tested using one-way analysis of variance (ANOVA) with Dunnett post hoc test. *Indicates significant difference compared with HUVEC treated with CCL5–CXCL4 in absence of MKEY. (D–F) Mice were treated with MKEY (50 μg) 1 hour before or after LPS inhalation, scrambled MKEY (sMKEY, 50 μg), antibodies to CCL5 and CXCL4, or platelet-depleting serum. Four hours after LPS inhalation, mice were killed. (D) Displayed are intravascular (top), interstitial (middle), and alveolar neutrophil counts (bottom). (E) Protein concentration (top), fluorescein isothiocyanate (FITC)–dextran clearance (middle), and elastase (bottom, uniform bars) and myeloperoxidase (MPO) activity (bottom, hatched bars) in BAL fluids. n = 8–10 for each bar. Statistical significance was tested using one-way ANOVA with Dunnett post hoc test. *Indicates significant difference compared with mice receiving LPS. (F) Representative photographs of histologic (left) and scanning electron analyses (right) of mice receiving phosphate-buffered saline (ctrl) or LPS in presence or absence of MKEY. Scale bars indicate 50 μm for scanning electron microscopy and 250 μm for histology. Quantification of histologic lung sections (bottom). n = 8–10 for each bar. Statistical significance was tested using one-way ANOVA with Dunnett post hoc test. *Indicates significant difference compared with LPS-treated animals.

Figure 5.

Acid- or sepsis-induced acute lung injury (ALI) is abrogated by disrupting CCL5–CXCL4 heteromers. ALI was induced by intratracheal acid application (Acid) or by cecal ligation and puncture (CLP). MKEY (50 μg) was injected 1 hour before or 1 hour after ALI induction. Mice were killed 4 (Acid) or 24 hours (CLP) after ALI induction. (A) Displayed are intravascular (top), interstitial (middle), and alveolar neutrophil counts (bottom). (B) Protein concentration (top), fluorescein isothiocyanate (FITC)–dextran clearance (middle), and elastase and myeloperoxidase (MPO) activity (bottom) in bronchoalveolar lavage fluids. n = 7 for each bar. Statistical significance was tested using Kruskal-Wallis test with Dunn post hoc test. *Indicates significant difference compared with ALI control mice.

Figure 6.

MKEY does not affect neutrophil degranulation and adhesion. Neutrophils were pretreated with MKEY (1 or 3 h, 1 or 10 μM) and then activated with N-formyl-methionine-leucine-phenylalanine (fMLP). (A and B) MFI of surface expression of β₂ (A, top) or β₁ integrin (B, top) as measured by fluorescence-activated cell sorter analysis after staining with directly conjugated antibodies. Adhesion of neutrophils perfused over immobilized intercellular adhesion molecule 1 (A, bottom) and fibronectin (B, bottom) at 1 dyne/cm². Number of adherent neutrophils per field is displayed. n = 3–6 for fluorescence-activated cell sorter experiments, and 8–10 for flow chamber experiments. Statistical significance was tested using Kruskal-Wallis test with Dunn post hoc test. *Indicates significant difference compared with fMLP treatment. MFI = mean fluorescence intensity.

Figure 7.

MKEY does not affect neutrophil antimicrobial activity. (A) Neutrophils pretreated with N-formyl-methionine-leucine-phenylalanine (fMLP) (10 μM, 15 min) and MKEY (3 h at indicated doses) were incubated with IgG- or complement-opsonized fluorescent Escherichia coli. Uptake was recorded by flow cytometry. n = 4 for each bar. (B) Neutrophils were labeled with the reactive oxygen species (ROS)-sensitive dye H₂DCFDA and ROS formation was recorded by flow cytometry after fMLP-stimulation in presence or absence of MKEY. Data indicate fluorescence intensity 30 minutes after fMLP exposure. n = 6 for each bar. Statistical significance was tested using Kruskal-Wallis test with Dunn post hoc test. *Indicates significant difference compared with fMLP treatment. (C) Formation of colony-forming units (CFU) after hypotonic lysis of neutrophils that had phagocytozed E. coli (D21) for 20 minutes. n = 5 for each bar. (D) Bacterial clearance in mice that underwent CLP. Mice received vehicle control or MKEY (50 μg) and cultures of lung homogenate or plasma were made on Luria Bertani agar overnight and the colonies were enumerated. n = 7 for each bar. MFI = mean fluorescence intensity.