Intratracheal IL-6 protects against lung inflammation in direct, but not indirect, causes of acute lung injury in mice

Abstract

Introduction: Serum and bronchoalveolar fluid IL-6 are increased in patients with acute respiratory distress syndrome (ARDS) and predict prolonged mechanical ventilation and poor outcomes, although the role of intra-alveolar IL-6 in indirect lung injury is unknown. We investigated the role of endogenous and exogenous intra-alveolar IL-6 in AKI-mediated lung injury (indirect lung injury), intraperitoneal (IP) endotoxin administration (indirect lung injury) and, for comparison, intratracheal (IT) endotoxin administration (direct lung injury) with the hypothesis that IL-6 would exert a pro-inflammatory effect in these causes of acute lung inflammation.

Methods: Bronchoalveolar cytokines (IL-6, CXCL1, TNF-α, IL-1β, and IL-10), BAL fluid neutrophils, lung inflammation (lung cytokines, MPO activity [a biochemical marker of neutrophil infiltration]), and serum cytokines were determined in adult male C57Bl/6 mice with no intervention or 4 hours after ischemic AKI (22 minutes of renal pedicle clamping), IP endotoxin (10 µg), or IT endotoxin (80 µg) with and without intratracheal (IT) IL-6 (25 ng or 200 ng) treatment.

Results: Lung inflammation was similar after AKI, IP endotoxin, and IT endotoxin. BAL fluid IL-6 was markedly increased after IT endotoxin, and not increased after AKI or IP endotoxin. Unexpectedly, IT IL-6 exerted an anti-inflammatory effect in healthy mice characterized by reduced BAL fluid cytokines. IT IL-6 also exerted an anti-inflammatory effect in IT endotoxin characterized by reduced BAL fluid cytokines and lung inflammation; IT IL-6 had no effect on lung inflammation in AKI or IP endotoxin.

Conclusion: IL-6 exerts an anti-inflammatory effect in direct lung injury from IT endotoxin, yet has no role in the pathogenesis or treatment of indirect lung injury from AKI or IP endotoxin. Since intra-alveolar inflammation is important in the pathogenesis of direct, but not indirect, causes of lung inflammation, IT anti-inflammatory treatments may have a role in direct, but not indirect, causes of ARDS.

Figure 1. Bronchoalveolar (BAL) fluid proinflammatory cytokines, neutrophils, and IL-10 after acute kidney injury (AKI), intraperitoneal (IP) endotoxin, or intratracheal (IT) endotoxin.

BAL fluid proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, (D) IL-1β; and (E) neutrophils; and (F) IL-10 (an anti-inflammatory cytokine) were determined 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), or IT endotoxin (direct lung injury) (n = 3–7). *P<0.01 versus IP vehicle and P = NS versus Sham; **P<0.01 versus IP vehicle and P<0.01 versus Sham.

Figure 2. Lung inflammation after acute kidney injury (AKI), intraperitoneal (IP) endotoxin, or intratracheal (IT) endotoxin.

Lung (A) CXCL1 protein, and lung (B) MPO activity (a biochemical indicator of lung neutrophils) were measured 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), or IT endotoxin (direct lung injury) (n = 5–7).

Figure 3. Serum proinflammatory cytokines, renal function, and serum IL-10 after acute kidney injury (AKI), intra-peritoneal (IP) endotoxin, or intratracheal (IT) endotoxin.

Serum proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, (D) IL-1β; and renal function by (E) serum creatinine and (F) BUN; and serum IL-10 (an anti-inflammatory cytokine) were measured 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), and IT endotoxin (direct lung injury)(n = 5–7).

Figure 4. Bronchoalveolar lavage (BAL) fluid proinflammatory cytokines, volume, and IL-10 after intratracheal (IT) IL-6 to healthy mice.

BAL fluid proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, (D) IL-1β; and (E) volume; and (F) IL-10 (an anti-inflammatory cytokine) were determined in normal mice (no IT treatment of any kind), and two hours after IT vehicle (0.1% BSA), low dose IT IL-6 (25 ng), and high dose IT IL-6 (200 ng). (n = 3–5 for normal mice; n = 10–20 for vehicle, IL-6 low dose, and IL-6 high dose). * P<0.05 vs. Normal, ** P<0.01 vs. Normal, #P<0.05 vs. Vehicle.

Figure 5. Lung proinflammatory cytokines, lung MPO activity, and lung IL-10 after intratracheal (IT) IL-6 to healthy mice.

The proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, (D) IL-1β; and (E) MPO activity; and (F) IL-10 (an anti-inflammatory cytokine) were determined in the lung in normal mice (no IT treatment of any kind), and two hours after IT vehicle (0.1% BSA), low dose IT IL-6 (25 ng), and high dose IT IL-6 (200 ng). (n = 3–5 for normal mice; n = 10–20 for vehicle, IL-6 low dose, and IL-6 high dose). * P<0.05 vs. Normal, ** P<0.01 vs. Normal, #P<0.05 vs. Vehicle.

Figure 6. Serum proinflammatory cytokines and serum IL-10 after intratracheal (IT) IL-6 to healthy mice.

Serum proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, and (D) IL-1β; and (F) IL-10 (an anti-inflammatory cytokine) were determined in normal mice (no IT treatment of any kind), and two hours after IT vehicle (0.1% BSA), low dose IT IL-6 (25 ng), and high dose IT IL-6 (200 ng). (n = 3–5 for Normal mice; n = 10–20 for Vehicle, IL-6 Low Dose, and IL-6 High Dose). * P<0.05 vs. Normal, ** P<0.01 vs. Normal, #P<0.05 vs. Vehicle.

Figure 7. Bronchoalveolar (BAL) fluid proinflammatory cytokines after intratracheal (IT) IL-6 treatment in acute kidney injury (AKI), intraperitoneal (IP) endotoxin, and IT endotoxin.

The proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, and (D) IL-1β were measured in BAL fluid 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), or IT endotoxin (direct lung injury) in mice treated with 200 ng of IT IL-6 or IT vehicle (veh) (0.1% BSA) 30 minutes prior to injury induction (n = 5–7). *P<0.01 versus IT vehicle plus AKI).

Figure 8. Lung inflammation after intratracheal (IT) IL-6 treatment in acute kidney injury (AKI), intraperitoneal (IP) endotoxin, and IT endotoxin.

(A) Lung CXCL1 (a neutrophil chemokine), and (B) lung MPO activity (a biochemical indicator of lung neutrophils) were measured 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), and IT endotoxin (direct lung injury) in mice treated with 200 ng of IT IL-6 or IT vehicle (veh) (0.1% BSA) 30 minutes prior to injury (n = 5–7).

Figure 9. Serum proinflammatory cytokines after intratracheal (IT) IL-6 treatment in acute kidney injury (AKI), intraperitoneal (IP) endotoxin, and IT endotoxin.

The proinflammatory cytokines (A) IL-6, (B) CXCL1, (C) TNF-α, and (D) IL-1β were measured in the serum 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), and IT endotoxin (direct lung injury) in mice treated with 200 ng of IT IL-6 or IT vehicle (veh) (0.1% BSA) 30 minutes prior to injury (n = 5–7).

Figure 10. Bronchoalveolar (BAL) fluid, lung, and serum IL-10 after intratracheal (IT) IL-6 treatment in acute kidney injury (AKI), intraperitoneal (IP) endotoxin, and IT endotoxin.

The anti-inflammatory cytokine IL-10 was determined in the (A) BAL fluid, (B) lung, and (C) serum 4 hours after AKI (indirect lung injury), IP endotoxin (indirect lung injury), and IT endotoxin (direct lung injury) in mice treated with 200 ng of IT IL-6 or IT vehicle (veh) (0.1% BSA) 30 minutes prior to injury (n = 5–7).