Acute lung injury induced by lipopolysaccharide is independent of complement activation

Abstract

Although acute lung injury (ALI) is an important problem in humans, its pathogenesis is poorly understood. Airway instillation of bacterial LPS, a known complement activator, represents a frequently used model of ALI. In the present study, pathways in the immunopathogenesis of ALI were evaluated. ALI was induced in wild-type, C3(-/-), and C5(-/-) mice by airway deposition of LPS. To assess the relevant inflammatory mediators, bronchoalveolar lavage fluids were evaluated by ELISA analyses and various neutralizing Abs and receptor antagonists were administered in vivo. LPS-induced ALI was neutrophil-dependent, but it was not associated with generation of C5a in the lung and was independent of C3, C5, or C5a. Instead, LPS injury was associated with robust generation of macrophage migration inhibitory factor (MIF), leukotriene B(4) (LTB4), and high mobility group box 1 protein (HMGB1) and required engagement of receptors for both MIF and LTB4. Neutralization of MIF or blockade of the MIF receptor and/or LTB4 receptor resulted in protection from LPS-induced ALI. These findings indicate that the MIF and LTB4 mediator pathways are involved in the immunopathogenesis of LPS-induced experimental ALI. Most strikingly, complement activation does not contribute to the development of ALI in the LPS model.

FIGURE 1

Parameters of acute lung injury as reflected by leak of ¹²⁵I-albumin into lung parenchyma (permeability index) as a function of the dose (25–100 μg) of LPS used (A) or as a function of time (0–8 h) after LPS administration (B). C, Effects of systemic neutrophil depletion on LPS-induced ALI. For each bar, n ≥ 5 mice. Histologic features in control lung (D) and lungs injured by deposition of LPS (E). H&E, ×40 (scale bar = 100 μm).

FIGURE 2

A, Effects of anti-C5a (40 μg IgG) administered intratracheally on ALI in the LPS model (50 μg). Lung injury induced by LPS in C5^–/– mice (B) or C3^–/– mice (C). Lung MPO content in WT and C3- and C5-deficient mice (D). E, Number of leukocytes in BAL from C3^–/– and C5^–/– mice in comparison to WT animals with ALI. F, BAL C5a content in LPS-injured lungs as a function of dose of LPS, as measured at 6 h. G, Time-course for BAL C5a content (determined by ELISA) after airway deposition of 50 μg LPS. H, Plasma C5a levels after intraperitoneal vs intratracheal administration of LPS. For each bar, n ≥ 5 mice.

FIGURE 3

A, Intensity of lung injury (measured as permeability index) in the LPS model with isotype-matched IgG1 or with anti-MIF mAb (each at 40 μg mixed with LPS). B, Effects of ISO-1 (the MIF receptor antagonist, 35 mg/kg body weight, administered i.p. 30 min before lung injury induction) or the LTB4 receptor antagonist (1–20 μM) after admixture with the intratracheally administered LPS. Effects of MIF or LTB4 blockade on the buildup of IL-6 (C) and TNF-α (D). For the interventions indicated in C and D, 40 μg neutralizing mAb to mouse MIF or 20 μM of the LTB4 receptor antagonist was mixed with the LPS; when the MIF receptor antagonist (ISO-1) was used, 35 mg/kg body weight was injected i.p. 30 min before intratracheal administration of 50 μg LPS. E, Lung MPO content in LPS-injured lungs of the LPS-treated animals of the ISO-1 (35 mg/kg body weight) or the LTB4 receptor antagonist (20 μM). F, Effects of MIF or LTB4 blockade on the total white cell count in BAL fluids. For each bar, n ≥ 5 mice.

FIGURE 4

Effects of LTB4 blockade by the LTB4 antagonist when administered i.v. in comparison to intratracheal administration on lung MPO content (A) and appearance of leukocytes in BAL fluids (B). Levels of IL-6 (C) and TNF-α (D) in BAL fluids after i.v. application of the LTB4 antagonist. Concentrations of the chemoattractants LIX (E), KC (F), and MIP-2 (G) in BAL fluids after i.v. vs intratracheal administration of the LTB4 antagonist in lung injury. For each bar, n ≥ 5 mice.

FIGURE 5

A, Effects of a dual blockade of MIF (ISO-1; 35 mg/kg body weight) and LTB4 (receptor antagonist; 20 μM) on LPS-induced ALI. Intensity of lung injury as determined by lung permeability. Levels of proinflammatory cytokines IL-6 (B) and TNF-α (C) in the combined presence of ISO-1 and the LTB4 antagonist. D, Buildup of lung MPO after intratracheal LPS administration and ISO-1 + LTB4 antagonist. E, Total leukocyte count in BAL fluids. For each bar, n ≥ 5 mice. Effect of the LTB4 antagonist + ISO-1 on lung histology (G) in comparison to deposition of LPS + DMSO (F). H&E, ×40 (scale bar = 100 μm).