The effects of aging on pulmonary oxidative damage, protein nitration, and extracellular superoxide dismutase down-regulation during systemic inflammation

Abstract

Systemic inflammatory response syndrome (SIRS), a serious clinical condition characterized by whole-body inflammation, is particularly threatening for elderly patients, who suffer much higher mortality rates than the young. A major pathological consequence of SIRS is acute lung injury caused by neutrophil-mediated oxidative damage. Previously, we reported an increase in protein tyrosine nitration (a marker of oxidative/nitrosative damage) and a decrease in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in the lungs of young mice during endotoxemia-induced SIRS. Here we demonstrate that during endotoxemia, down-regulation of EC-SOD is significantly more profound and prolonged, whereas up-regulation of iNOS is augmented, in aged compared to young mice. Aged mice also showed 2.5-fold higher protein nitration levels, compared to young mice, with particularly strong nitration in the pulmonary vascular endothelium during SIRS. Additionally, by two-dimensional gel electrophoresis, Western blotting, and mass spectrometry, we identified proteins that show increased tyrosine nitration in age- and SIRS-dependent manners; these proteins (profilin-1, transgelin-2, LASP 1, tropomyosin, and myosin) include components of the actin cytoskeleton responsible for maintaining pulmonary vascular permeability. Reduced EC-SOD in combination with increased oxidative/nitrosative damage and altered cytoskeletal protein function due to tyrosine nitration may contribute to augmented lung injury in the aged with SIRS.

Figure 1. Histological analysis demonstrating age-associated severity of acute lung injury during systemic inflammation

Mice at 6 and 26 months of age were injected with LPS (2.5 mg/kg, ip) and sacrificed 24 h later. Non-injected mice were used as controls. Sections from formalin-fixed, paraffin embedded lung samples were stained with H&E. Arrow heads indicate granular leukocytes, arrows indicate edematous thickened alveolar walls, and stars indicate deposit of proteinaceous debris. Inflammation and edema are distinct in 26-month-old mice with LPS.

Figure 2. Western blot analysis demonstrating that LPS-mediated decrease in pulmonary EC-SOD is more profound in aged mice than young mice

(A) Time course of lung EC-SOD levels in young (4 months) and aged (24 months) mice after LPS injection (2.5 mg/kg, ip). Each lane represents a pooled protein sample derived equally from 5 mice. (B) Densitometric analysis of A. (C) Lung EC-SOD levels in young and aged mice 12 h after LPS injection. Each lane represents a protein sample from an individual mouse (n=5 per age group). (D) Densitometric analysis of C. (E). Lung EC-SOD levels in young (4 months), middle aged (14 months), and aged (24 months) mice 12 h after LPS injection (2.5mg/kg). Each lane represents a pooled protein sample derived equally from 3 mice. (F) Northern hybridization analysis demonstrating that down-regulation of lung EC-SOD mRNA expression after LPS injection was more profound in aged mice than in young mice. Young (4 months) and aged (24 months) mice were injected with LPS (2.5 mg/kg, ip) and total lung RNA was isolated and used for Northern blot analysis. Each lane represents a pooled protein sample derived equally from 5 mice.

Figure 3. Age-associated increase in protein tyrosine nitration in the lungs during systemic inflammation

Systemic inflammation was induced in young (4 months) and aged (24 months) mice by injection with LPS (2.5 mg/kg, ip); mice were sacrificed 6 and 12 h later and the lung tissues harvested for analysis. Age and sex-matched non-injected mice were used as controls. (A) Immunohistochemical localization of nitrotyrosine to the vasculature of aged mice after LPS injection. Arrows indicate strongly stained vascular endothelium. (B) Western blot analyses for nitrotyrosine on whole-lung proteins and densitometric analysis of each lane. Total intensity throughout each lane was normalized by β-actin. (C) Northern hybridization analysis demonstrating that induction of lung iNOS mRNA after LPS injection was augmented in aged mice compared to young mice. Young (4 months) and aged (24 months) mice were injected with LPS (2.5 mg/kg, ip) and total lung RNA was isolated and used for Northern blot analysis. Each lane represents a pooled protein sample derived equally from 5 mice.

Figure 4. 2D western blot analyses and mass spectrometry identification of pulmonary proteins that exhibit an age-associated increase in tyrosine nitration during systemic inflammation

Systemic inflammation was induced in young and aged mice by injection with LPS (2.5 mg/kg, ip); mice were sacrificed 12 h later and the lung tissues harvested for analysis. Age and sex-matched non-injected mice were used as controls. (A) Western blot analysis of 2 dimensional gels detecting tyrosine nitration of lung proteins from young control mice (top left panel); young LPS injected mice (bottom left panel); aged control mice (top right panel); and aged LPS injected mice (bottom right panel). Each gel contained total lung protein derived equally from 4 mice. Numbers indicate proteins identified by mass spectrometry in Table 1. (B) Enlarged view of individual nitrotyrosine positive protein spots. Numbers on bottom correspond to spots in (A).