The lectin-like domain of tumor necrosis factor improves lung function after rat lung transplantation--potential role for a reduction in reactive oxygen species generation

Abstract

Objective: To test the hypothesis that the lectin-like domain of tumor necrosis factor, mimicked by the TIP peptide, can improve lung function after unilateral orthotopic lung isotransplantation. Because of a lack of a specific treatment for ischemia reperfusion-mediated lung injury, accompanied by a disrupted barrier integrity and a dysfunctional alveolar liquid clearance, alternative therapies restoring these parameters after lung transplantation are required.

Design: Prospective, randomized laboratory investigation.

Setting: University-affiliated laboratory.

Subjects: Adult female rats.

Interventions: Tuberoinfundibular peptide, mimicking the lectin-like domain of tumor necrosis factor, mutant TIP peptide, N,N'-diacetylchitobiose/TIP peptide, and amiloride/TIP peptide were instilled intratracheally in the left lung immediately before the isotransplantation was performed. An additional group received an intravenous TIP peptide treatment, 1.5 mins before transplantation. Studies using isolated rat type II alveolar epithelial cell monolayers and ovine pulmonary endothelial cells were also performed.

Measurements and main results: Intratracheal pretreatment of the transplantable left lung with the TIP peptide, but not with an inactive mutant TIP peptide, resulted in significantly improved oxygenation 24 hrs after transplantation. This treatment led to a significantly reduced neutrophil content in the lavage fluid. Both the effects on oxygenation and neutrophil infiltration were inhibited by the epithelial sodium channel blocker amiloride. The TIP peptide blunted reactive oxygen species production in pulmonary artery endothelial cells under hypoxia and reoxygenation and reduced reactive oxygen species content in the transplanted rat lungs in vivo. Ussing chamber experiments using monolayers of primary type II rat pneumocytes indicated that the primary site of action of the peptide was on the apical side of these cells.

Conclusions: These data demonstrate that the TIP peptide significantly improves lung function after lung transplantation in the rat, in part, by reducing neutrophil content and reactive oxygen species generation. These studies suggest that the TIP peptide is a potential therapeutic agent against the ischemia reperfusion injury associated with lung transplantation.

Figure 1

Oxygenation at 24 hrs after transplantation. At kill, 24 hrs after reperfusion of the left-sided lung transplant, the Pao₂/Fio₂ ratio was measured after excluding the native right-sided lung by clipping the right-sided stem bronchus and right-sided pulmonary artery. The animals were tracheotomized and ventilated with an Fio₂ of 1.0. The TIP peptide significantly increased gas exchange compared with all other study groups. *p < .003 vs. NaCl. Data are mean ± sem. i.t., intratracheally.

Figure 2

Bronchoalveolar lavage (BAL) polymorphonuclear neutrophilic leukocytes (PMN) count in the transplanted rats. The TIP peptide intratracheal group had a significantly reduced BAL PMN content compared with all other study groups. *p < .032 vs. NaCl. The absolute PMN count (×10⁴ cells/mL) is given as mean ± sem. i.t., intratracheally.

Figure 3

A, Electron paramagnetic resonance waveform spectra of pulmonary artery endothelial cells treated for 120 mins with either saline, TIP peptide (50 μg/mL) or mutant TIP (mTIP) (50 μg/mL) and kept either in normoxia (120 mins) or in hypoxia (90 mins)/reoxygenation (30 mins) conditions; B, intratracheally (i.t.) relative superoxide generation in all groups, as compared with normoxia of control cells. *p < .05 vs. normoxia. Data represent mean ± sem; n = 6.

Figure 4

Reactive oxygen species (ROS) generation (assessed by electron paramagnetic resonance measurements in lung homogenates), expressed as μM ROS/mg of protein in transplanted left and corresponding nontransplanted right lungs in control rats treated with 500 μL NaCl i.t. intratracheally (i.t.) (n = 5) vs. TIP peptide-treated rats (n = 6). Lungs were isolated and snap-frozen 24 hrs after transplantation. Data represent mean ± sem.

Figure 5

Transepithelial current (Isc) (μA/cm²) in confluent monolayers of primary rat alveolar epithelial type II cells treated for 1 hr with the TIP peptide (10 μg/mL) or the mutant TIP. The TIP or its mutant form was added in either (top) the apical or (bottom) the basolateral side of the cell monolayers. In some experiments, the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor 172 (10 μM) or the epithelial sodium channel complex inhibitor amiloride (100 μM) was added together with the TIP. *p < .05 vs. control; **p < .005 vs. control. Data are mean ± sem; n = 3.