Acute lung injury by gastric fluid instillation: activation of myofibroblast apoptosis during injury resolution

Background: Gastric contents aspiration in humans has variable consequences depending on the volume of aspirate, ranging from subclinical pneumonitis to respiratory failure with up to 70% mortality. Several experimental approaches have been used to study this condition. In a model of single orotracheal instillation of gastric fluid we have shown that severe acute lung injury evolves from a pattern of diffuse alveolar damage to one of organizing pneumonia (OP), that later resolves leaving normal lung architecture. Little is known about mechanisms of injury resolution after a single aspiration that could be dysregulated with repetitive aspirations. We hypothesized that, in a similar way to cutaneous wound healing, apoptosis may participate in lung injury resolution by reducing the number of myofibroblasts and by affecting the balance between proteases and antiproteases. Our aim was to study activation of apoptosis as well as MMP-2/TIMP-2 balance in the sub-acute phase (4-14 days) of gastric fluid-induced lung injury. Methods: Anesthesized Sprague-Dawley rats received a single orotracheal instillation of gastric fluid and were euthanized 4, 7 and 14 days later (n = 6/group). In lung tissue we studied caspase-3 activation and its location by double immunofluorescence for cleaved caspase-3 or TUNEL and alpha-SMA. MMP-2/TIMP-2 balance was studied by zymography and Western blot. BALF levels of TGF-beta 1 were measured by ELISA. Results: An OP pattern with Masson bodies and granulomas was seen at days 4 and 7 that was no longer present at day 14. Cleaved caspase-3 increased at day 7 and was detected by immunofluorescence in Masson body-alpha-SMA-positive and -negative cells. TUNEL-positive cells at days 4 and 7 were located mainly in Masson bodies. Distribution of cleaved caspase-3 and TUNEL-positive cells at day 14 was similar to that in controls. At the peak of apoptosis (day 7), an imbalance between MMP-2 activity and TIMP-2 expression was produced by reduction in TIMP-2 expression. Conclusions: Apoptosis is activated in Masson body-alpha-SMA-positive and -negative cells during the sub-acute phase of gastric fluid-induced lung injury. This mechanism likely contributes to OP resolution, by reducing myofibroblast number and new collagen production. In addition, pre-formed collagen degradation is favored by an associated MMP-2/TIMP-2 imbalance.