The mechanisms underlying increases in the susceptibility of the elderly to the adverse effects of inhaled air pollutants are unknown. Innate immune system functioning declines with advancing age and this may contribute to xenobiotic-induced hyperresponsiveness. Altered oxidant/antioxidant balance may also be a key factor. To evaluate these possibilities, we compared inflammatory mediator and antioxidant expression in lungs of younger and older mice following exposure to inhaled DE.
Exposure of older but not younger mice to DE was associated with rapid and progressive morphological and structural alterations in the lung, which included neutrophil and macrophage accumulation in the tissue, focal cellular infiltrates of plasma cells and macrophages in the interstitum and patchy thickening of alveolar septa. These findings are consistent with previous reports of morphological and structural changes in lungs of older rodents and humans exposed to PM (Tankersley et al., 2003
; Sunil et al., 2007a
; Tsagareli et al., 2008
) and confirm that aging is associated with increased sensitivity to PM-induced lung injury.
An accumulation of protein and inflammatory cells in BAL fluid and an increase in LDH activity have been used as markers of injury to the lower lung (Bhalla, 1999
). Despite pronounced structural and histologic evidence of lung pathology in older mice after exposure to DE, no significant changes were noted in BAL protein or LDH activity. This suggests that these measures are not sensitive indicators of lung injury in the elderly. Alternatively, DE-induced injury may not be prominent in the lower lung of older mice. Interestingly, in older mice, BAL cell number was reduced 24 h following single or repeated exposure to DE. This is most likely due to increased adherence of inflammatory cells to alveolar epithelium, which is consistent with our histologic findings. In younger animals, increases in BAL cell number and protein were detected after single or repeated exposure to DE, whereas BAL LDH activity increased only after repeated exposure. However, no major structural alterations were noted in the lung of younger mice. These findings further indicate the limitations in the use of BAL protein and cell number as general markers of acute lung injury.
Nitric oxide possesses complex redox chemistry and is capable of forming a variety of cytotoxic reactive nitrogen species which are thought to contribute to lung pathology and disease (Dweik et al., 2001
; Ricciardolo et al., 2006
). At higher DE concentrations, biological effects may also be induced by the interaction between particles and co-pollutant gases, which are present at higher levels. The present studies demonstrate significantly greater levels of NOx in BAL from older animals following exposure to DE (single or repeated) relative to younger mice, providing additional evidence that elderly mice are more susceptible to adverse pulmonary effect of inhaled DE. NOx has been implicated in the pathogenesis of acute respiratory distress syndrome and bronchopulmonary dysplasia in animal models of lung injury (Linke et al., 2001
; Haczku et al., 2002
), and they may play a similar role in the increased responsiveness of elderly mice to inhaled DE.
Lipocalin 24p3 (Lcn2, NGAL) was originally identified as a liver-derived acute phase protein produced in response to hepatic injury (Liu and Nilsen-Hamilton, 1995
). More recent studies have shown that 24p3 is upregulated in a number of tissues including the lung and kidney following injury, as well as in diseases such as colorectal and pancreatic cancer and inflammatory bowel disease (Nielsen et al., 1996
; Missiaglia et al., 2004
; Mishra et al., 2006
; Roudkenar et al., 2007
; Sunil et al., 2007b
). It has also been suggested that 24p3 is important in the response of cells and tissues to oxidative stress (Roudkenar et al., 2007
). We found that exposure of older, but not younger mice to DE, was associated with a rapid induction in 24p3 mRNA expression in the lung. cDNA microarray studies revealed a similar upregulation of 24p3 after exposure of mice to the combination of DE and lipopolysaccharide (Yanagisawa et al., 2004
). Our findings that in older mice 24p3 is upregulated after exposure to DE alone suggest that this may be a highly sensitive biomarker of early oxidative stress in the elderly.
TNFα is a macrophage-derived cytokine implicated in the pathogenesis of lung injury induced by a number of air pollutants (Kelley, 1990
; Schins and Borm, 1999
; Fakhrzadeh et al., 2004
; Gowdy et al., 2008
). Recent studies suggest that TNFα plays a dual role in the pathogenic response, initially promoting inflammation and cytotoxicity and subsequently initiating tissue repair (Lehmann et al., 2005
; Gosselin and Rivest, 2007
). This latter activity is thought to be due to TNFα-induced upregulation of antioxidants, stimulation of epithelial cell proliferation and extracellular matrix turnover (Sasaki et al., 2000
; Ryter et al., 2002
; Chiu et al., 2003
). In accord with previous studies (Saber et al., 2006
; Gowdy et al., 2008
), we observed a significant increase in expression of TNFα in the lung 24 h after a single exposure of younger mice to DE. Interestingly, this response was most notable at the lower DE dose. It may be that higher doses of DE cause suppression of inflammatory responses in the lung of younger mice (Yin et al., 2002
). Although TNFα expression was also increased in lungs of older animals 24 h following a single exposure to DE, this response was attenuated relative to younger mice. A similar attenuation of TNFα production in lung or BAL fluid has previously been described in older animals after induction of endotoxin shock and after exposure to silica or terpene oxidation products (Corsini et al., 2004
; Ito et al., 2007
; Sunil et al., 2007a
). Age-related decreases in production of TNFα have also been described in isolated monocytes and macrophages (Lloberas and Celada, 2002
; Boehmer et al., 2004
). After a single DE exposure, serum TNFα levels were increased significantly, but only in older mice. The origin of TNFα in serum is unknown and may reflect its rapid release in the lung following DE exposure. Alternatively, TNFα may be released by extra-pulmonary tissues responding to DE-induced oxidative stress and this remains to be determined.
IL-6 and IL-8 are known to be important in regulating inflammatory cell trafficking into injured tissues (Murtaugh et al., 1996
; McClintock et al., 2008
). Previous studies have reported DE-induced increases in IL-6 and IL-8 in cultured bronchial epithelial cells (Steerenberg et al., 1998
). Similarly, we noted significant increases in IL-6 and IL-8 mRNA expression in lungs of younger and older animals after exposure to DE (single or repeated). Whereas the kinetics of IL-6 mRNA expression appeared to be independent of age, IL-8 expression increased in older animals immediately after DE exposure. These findings are consistent with reports of increased production of IL-8 and IL-8-like cytokines (e.g., MIP-2) with advancing age in rodents and humans (Himi et al., 1997
; Pulsatelli et al., 2000
). IL-8 is a potent chemoattractant for neutrophils (Matsuzaki et al., 2006
). Our findings of increased expression of IL-8 in older mice following exposure to DE are consistent with our histologic data of increased accumulation of neutrophils in the lungs and with increased levels of NOx in BAL of these animals.
Antioxidants play a critical role in host defense by scavenging and detoxifying oxidants (Heffner and Repine, 1991
; Lang et al., 2002
). With increasing age, constitutive levels of tissue antioxidants and the capacity to respond to oxidative stress decline (Lykkesfeldt and Ames, 1999
; Squier, 2001
; Servais et al., 2005
). Thus, while expression of antioxidant enzymes, such as SOD, rapidly decreases in younger animals as it is utilized following exposure to PM, in older mice this response is reduced or absent (Sagai et al., 1993
; Sunil et al., 2007a
). We found that MnSOD was constitutively expressed in lungs of younger animals, predominantly in alveolar macrophages. Moreover, exposure of younger animals to DE resulted in a rapid reduction in MnSOD expression. In contrast, constitutive expression of MnSOD was not evident in lungs of older mice and DE exposure did not alter its expression. It has been suggested that oxidative stress is a key mechanism underlying the adverse health effects of ambient PM (Li et al., 2004
). A lack of constitutive MnSOD in the lung of older mice may contribute to their increased susceptibility to lung injury induced by inhaled DE.
In summary, the present studies demonstrate that single or repeated inhalation of DE results in significant structural and inflammatory changes in the lungs of older but not younger mice. However, no consistent differences were noted between the two exposure protocols, suggesting that the impact of diesel is rapid. Alternatively, more prolonged exposures may be required for differences in the effects of single and repeated DE exposures to be observed. It should be noted that the exposure doses used in our studies are relevant to human exposure in occupational and environmental settings (USEPA, 2002
). Occupational exposures to DE can exceed 1000 μg/m3
, and levels of PM in the world's largest cities have been shown to exceed 300 μg/m3
; USEPA, 2002
). The data presented in our studies showing altered production of inflammatory mediators and reduced MnSOD in lungs of older mice suggest a potential mechanism for the increased susceptibility of the elderly to inhaled PM. However, at present we cannot exclude the possibility that some of these biological effects are due to different levels of NOx and COx in the DE. Identification of key inflammatory mediators involved in the response of older mice to DE may help in the design of new and effective approaches to mitigating pulmonary pathology in the geriatric population.