Complement inhibition alleviates paraquat-induced acute lung injury

Abstract

The widely used herbicide, paraquat (PQ), is highly toxic and claims thousands of lives from both accidental and voluntary ingestion. The pathological mechanisms of PQ poisoning-induced acute lung injury (ALI) are not well understood, and the role of complement in PQ-induced ALI has not been elucidated. We developed and characterized a mouse model of PQ-induced ALI and studied the role of complement in the pathogenesis of PQ poisoning. Intraperitoneal administration of PQ caused dose- and time-dependent lung damage and mortality, with associated inflammatory response. Within 24 hours of PQ-induced ALI, there was significantly increased expression of the complement proteins, C1q and C3, in the lung. Expression of the anaphylatoxin receptors, C3aR and C5aR, was also increased. Compared with wild-type mice, C3-deficient mice survived significantly longer and displayed significantly reduced lung inflammation and pathology after PQ treatment. Similar reductions in PQ-induced inflammation, pathology, and mortality were recorded in mice treated with the C3 inhibitors, CR2-Crry, and alternative pathway specific CR2-fH. A similar therapeutic effect was also observed by treatment with either C3a receptor antagonist or a blocking C5a receptor monoclonal antibody. Together, these studies indicate that PQ-induced ALI is mediated through receptor signaling by the C3a and C5a complement activation products that are generated via the alternative complement pathway, and that complement inhibition may be an effective clinical intervention for postexposure treatment of PQ-induced ALI.

Figure 1.

Dose-dependent effect of paraquat (PQ) on acute lung injury (ALI). Mice were injected intraperitoneally with normal saline (N) or saline-diluted PQ solution at a dose of 10 mg/kg (PQ10), 20 mg/kg (PQ20), or 50 mg/kg (PQ50), and mice were killed for analysis 48 hours later. Weight loss (A), relative lung weight (B), bronchoalveolar lavage fluid (BALF) total protein (C), and myeloperoxidase (MPO) activity (D) were analyzed. *P < 0.05, **P < 0.01 between 0 and 48 hours after PQ injection (A), or between the PQ injection groups and the normal control group (B–D). Data presented are means (±SD); n = 8 per group.

Figure 2.

Time-dependent progression of ALI after PQ poisoning. ALI was assessed at different times after PQ20 treatment by lung index (A), BALF total protein (B), and MPO activity (C). *P < 0.05 or **P < 0.01, relative to the corresponding 0-hour group. Data presented are means (±SD); n = 8 per group.

Figure 3.

Complement activation at early timepoint after PQ injection. (A–L) Immunohistochemical staining for C3, C1q, C5aR and mannan-binding lectin (MBL) in lung and the C3c level in serum after PQ20 poisoning. C3 deposition was strongly elevated at 4 and 24 hours after PQ injection (A–C). C1q depoistion (D–F) was also elevated on interstitial, epithelial base membrane, and lung endothelium at 4 and 24 hours after PQ20 treatment. Expression of C5aR (G–I) was elevated 4 hours after PQ20 treatment, with a further increase in staining seen at 24 hours after PQ20 treatment. No MBL deposition was detected in any group of mice lung (J–L). (M) Changes of C3c levels in serum at indicated times. (N–O) C5aR and C3aR mRNA expression in the lung. Relative C5aR mRNA and C3aR mRNA expression were determined in lung tissue at indicated time points after PQ administration; mRNA expression was assessed by relative real-time quantitative PCR analysis. *P < 0.05 or **P < 0.01, relative to the corresponding 0-hour group. Data presented are means (±SD); n = 7–9 per group. Magnification, ×200.

Figure 4.

Effect of C3 deficiency on lung injury, inflammation, and survival after PQ poisoning. Wild-type (WT) C57 BL/6 mice and C3^−/− mice were treated intraperitoneally with PQ20 or saline. (A–D) Gross appearance of mouse lung sections 30 hours after PQ or saline treatment: (A) WT mice + PQ; (B) WT mice + saline; (C) C3^−/− mice + PQ; (D) C3^−/− mice + saline. (E–H) Hematoxylin and eosin staining of lung sections: (E) WT mice + PQ; (F) WT mice + saline; (G) C3^−/− mice + PQ; (H) C3^−/− mice + saline. Representative images shown, n = 3. (I) MPO activity in lung samples 30 hours after PQ or saline administration in WT or C3 ^−/− mice (n = 3 in each group). (J) Serum concentrations of proinflammatory cytokines 4 hours after PQ poisoning (n = 6 in each group). (K) Survival analysis after PQ injection. (n = 8 per group). *P < 0.05 and **P < 0.01 relative to the corresponding the C3^−/− + PQ group with WT mice + PQ group. Data presented are means (±SD). Magnification, ×200 (E–H).

Figure 5.

CR2-Crry and CR2-factorH (fH) alleviate lung injury with less C3 deposition and prolong survival of mice with PQ-induced ALI, respectively. (A–D) Gross appearance of mouse lung 30 hours after PQ injection(n = 3 in each group): (A) normal mouse group; (B) PQ + PBS group; (C) PQ + CR2-Crry group; (D) PQ + CR2-fH group. (E–H) Histological examinations of lung tissues of mice 30 hours after PQ injection: (E) normal mouse group; (F) PQ + PBS group; (G) PQ + CR2-Crry group, (H) PQ + CR2-fH group. (I–L) C3 deposition at lung tissues of mice 30 hours after PQ injection (n = 3 in each group): (I) normal mouse group; (J) PQ + PBS group; (K) PQ + CR2-Crry group; (L) PQ + CR2-fH group. (M) Survival analysis in ALI model with CR2-Crry or CR2-fH intravenous administration 0 hours after PQ poisoning. **P < 0.01 and *** P < 0.001 relative to the corresponding PQ + CR2-Crry group or PQ + CR2-fH group with PQ + PBS group. Data presented are means (±SD); n = 5 per group. Magnification, ×200 (E–L).

Figure 6.

Localization of CR2-Crry in tissues of PQ-poisoned mice treated with CR2-Crry immediately after PQ administration. (A) Biodistribution of CR2-Crry in main tissues of mice by radiolabeled ¹²⁵I-CR2-Crry (2 μg) intravenous injection into control mice and groups of mice with 20 mg/kg PQ administration at 0 hours (n = 4–6 per group). Groups of mice were killed at 4 and 24 hours after ¹²⁵I-CR2-Crry injection, and serum and other main tissues were collected for analysis. (B–D) Localization of CR2-Crry in lung tissues of mice 4 hours after PQ injection by indirect immunofluorescent staining with 7G6 antibody in PQ + CR2-Crry group (B), PQ + PBS group (C), and normal mouse group (D). CR2-Crry deposition is denoted by green fluorescence on interstitial tissue and vascular endothelium; n = 4–6 in each group. Data presented are means (±SD) (A). Magnification, ×200 (B–D).

Figure 7.

Blockade of the binding of anaphylatoxin with their receptors alleviates lung injury and prolongs survival of mice with PQ-induced ALI. Four groups of mice were designed, with 11–13 mice in each group (saline, PQ + PBS, PQ + C3aR antagonist, and PQ + C5aR monoclonal antibody [mAb] groups). Mice received C5aR mAb 20/70 (600 μg/kg) or C3aR antagonist (1 mg/kg) by intravenous injection 50 minutes before and 2 hours after intraperitoneal injection of 20 mg/kg PQ. (A–D) Gross appearance of mouse lung 30 hours after PQ injection (n = 3). (A) PBS group, (B) PQ + PBS group, (C) PQ + C3aR antagonist group, and (D) PQ + C5aR antibody group. (E–H) Histological examinations of lung tissues of mice 30 hours after PQ injection in each group (n = 3). (E) PBS group, (F) PQ + PBS group, (G) PQ + C3aR antagonist group, and (H) PQ + C5aR antibody group. (I) Comparison of proinflammatory cytokine concentration in serum of PQ + PBS and PQ + C3aR antagonist groups 4 hours after PQ poisoning (n = 6). (J) Survival analysis of mice in each group after PQ administration (n = 8–10). *P < 0.05 and **P < 0.01 relative to the corresponding PQ + C3aR antagonist group or PQ + C5aR antibody group with PQ + PBS group. Data presented are means (±SD). Magnification, ×200 (E–H).