The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

Abstract

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Figure 1

LPS degrades the pulmonary ESL via TNF-α. (a) Left, graphical representation (not to scale) of the in vivo endothelial glycocalyx, forming a substantial ESL that excludes large molecules (for example, dextrans) from the vessel surface. Right, representative images of mouse subpleural microvessels (MV) using simultaneous differential interference contrast (DIC) and FITC-dextran (FITC) microscopy. Differences in DIC and FITC vascular widths (inclusive and exclusive of the ESL, respectively) reflect ESL thickness (brackets). Scale bars, 10 μm. A, alveolus. (b) Assessment of pulmonary ESL thickness within subpleural microvessels (mean DIC diameter 18.11 ± 1.01 μm, mean FITC diameter 14.17 ± 0.96 μm) of wild-type mice injected with intravenous saline, LPS (20 μg per g body weight) or TNF-α (200 ng) at t = 0 min and imaged at 0, 30, 60 and 90 min. n = 5 mice per group; *P < 0.05 in comparison to other groups. (c) Assessment of pulmonary ESL thickness of TNFR1-deficient Tnfrsf1a^tm1Imx mice treated at t = 0 min with intravenous saline or LPS (20 μg per g body weight); n = 3 or 4 mice per group. Data are represented as means ± s.e.m.

Figure 2

Heparanase mediates LPS-induced ESL degradation. (a) Assessment of pulmonary ESL thickness within subpleural microvessels of wild-type mice treated with heparinase-III or heat-inactivated heparinase-III (1 U) at t = 0. n = 4–6 mice per group; *P < 0.05. (b,c) Expression of active (50 kDa) and inactive (65 kDa) endothelial heparanase (b, representative of three independent experiments) and heparan sulfate degradation activity (c, n = 4 per group) in cultured mouse lung microvascular endothelial cells treated with TNF-α (5 and 50 ng ml⁻¹) or saline control for 30 min; *P < 0.05. (d) Assessment of pulmonary ESL thickness within subpleural microvessels of wild-type mice injected with intravenous vehicle or LPS (20 μg per g body weight) in addition to the heparanase inhibitor heparin (5 U administered intravenously at t = 0); n = 4–6 mice per group. (e) Assessment of pulmonary ESL thickness within subpleural microvessels of wild-type mice injected with intravenous vehicle or LPS (20 μg per g body weight) in addition to NAH (150 μg administered intravenously at t = 0); n = 4 or 5 mice per group. (f) Assessment of pulmonary microvascular ESL thickness in Hpse^−/− mice injected with intravenous saline or LPS (20 μg per g body weight); n = 3 mice per group. Data are represented as means ± s.e.m.

Figure 3

LPS-induced neutrophil adherence is dependent upon ESL degradation. (a) Adherence of adoptively transferred GFP⁺ neutrophils within subpleural microvessels before and 30–45 min after intravenous saline, LPS (20 μg per g body weight) or LPS (20 μg per g body weight) with heparin (5 U) in wild-type mice or 30–45 min after intravenous LPS (20 μg per g body weight) in Hpse^−/− mice. Representative images reflect changes occurring within a single, serially imaged, low-powered field. Scale bars, 40 μm. n = 3 mice per group; *P < 0.05 compared to saline. PMN, polymorphonuclear leukocytes. (b) Visualization of anti-ICAM-1–coated fluorescent microspheres within wild-type mouse subpleural microvessels, simultaneously imaged by DIC and fluorescence microscopy. Images obtained 45 min after intravenous saline, LPS (20 μg per g body weight), LPS (20 μg per g body weight) with heparin (5 U) or heparinase-III (1 U). Isotype-matched antibody-coated microspheres serve as controls for nonspecific adhesion. Scale bars, 20 μm. n = 3 or 4 mice per group; *P < 0.05 compared to saline. Data are represented as means ± s.e.m.

Figure 4

Heparanase contributes to septic acute lung injury. (a) Assessment of pulmonary endothelial permeability (filtration coefficient, K_f) in wild-type or Hpse^−/− mice 6 h after intraperitoneal LPS (40 μg per g body weight in 500 μl saline) or saline. Wild-type mice were pretreated with subcutaneous saline (200 μl), heparin (5 U in 200 μl saline) or NAH (150 μg in 200 μl saline) 3 h before LPS. Hpse^−/− mice received no pharmacologic pretreatment. n = 5–7 mice per group; *P < 0.05 compared to all other groups. (b,c) Myeloperoxidase (MPO) activity within lung homogenates of wild-type (b) or wild-type and Hpse^−/− (c) mice treated as described in a, normalized to saline/saline (b) or saline/wild type (c) control. n = 4–8 mice per group; *P < 0.05 compared to saline control. (d) Representative z-stacked imaging (1-μm increments) of heparanase (red) and the endothelial cell marker thrombomodulin (green) in LPS- or saline-treated wild-type mouse lungs, as described in a. Bottom left, area of positive heparanase immunofluorescence in ten random low-power lung fields, normalized to saline control. Bottom right, percentage of area positive for thrombomodulin immunofluorence that is additionally positive for heparanase, as quantified in ten random low-power fields. Scale bars, 50 μm. n = 5 mice per group; *P < 0.05. Data are represented as means ± s.e.m.

Figure 5

Heparanase is apparent in human sepsis and lung injury. (a) Heparan sulfate degradation activity measured in plasma collected from healthy donors and three groups of mechanically ventilated individuals: those with altered mental status-induced respiratory failure, pneumonia- or aspiration-induced respiratory failure or respiratory failure associated with antecedent nonpulmonary sepsis. *P < 0.05 compared to healthy donors. (b) Heparanase immunofluorescence in normal human lung tissue and in lung biopsies with diffuse alveolar damage. *P < 0.05 compared to normal lung tissue. (c) Top, confocal fluorescent images of normal human lung showing minimal heparanase expression. Representative fluorescent images of a lung from a patient with diffuse alveolar damage, with high heparanase expression (red) within capillaries (arrow) and conduit vessels (arrowhead), ascertained by the endothelial marker CD31 (green). Nuclei stained with DAPI. Bottom, H&E staining, with capillaries (arrow) and conduit vessels (arrowhead) noted. Scale bars, 50 μm. Data are represented as means ± s.e.m.

Figure 6

Heparin is a lung-protective treatment in established sepsis. (a) Assessment of pulmonary endothelial permeability (K_f) in wild-type mice 6 h after intraperitoneal LPS administration (40 μg per g body weight in 500 μl saline). Mice received saline (200 μl) or heparin (5 U in 200 μl saline) subcutaneously 3 h after LPS administration (3 h before measurement of K_f). n = 3 or 4 per group. *P < 0.05. (b) Pulmonary heparanase expression (red) after CLP in wild-type mice. Images are representative of two or three mice per group. Scale bars, 100 μm. (c) Mouse pulmonary K_f measured 48 h after CLP surgery. Wild-type mice received saline (200 μl) or heparin (5 U in 200 μl saline) subcutaneously 24 h after CLP (24 h before measurement of K_f). n = 3 per group. *P < 0.05. (d) H&E staining of representative (n > 4 per group) lungs of wild-type or Hpse^−/− mice 48 h after CLP or sham surgery with ensuing exposure to 60% oxygen (FiO₂). Scale bars, 500 μm in low-powered images. High-powered imaging (bottom left; scale bar, 50 μm) details an area of neutrophilic alveolitis. (e) Pulmonary neutrophil infiltration 48 h after CLP and hyperoxia with or without delayed heparin treatment as assessed by histology (top) and myeloperoxidase activity (bottom). Mice were treated with subcutaneous saline (200 μl) or heparin (5 U in 200 μl saline) 24 h after CLP. Scale bars, 100 μm. n = 4 per group; *P < 0.05. (f) Survival of wild-type (n = 14) and Hpse^−/− (n = 6) mice exposed to CLP and 60% hyperoxia. Survival of wild-type mice after sham surgery and 60% hyperoxia was 100% (n = 8). Data are represented as means ± s.e.m.