The goal of this study was to determine whether Fas deficiency is protective in mechanically ventilated mice exposed to intratracheal LPS. The main finding was that Fas-deficient lpr mice showed decreased neutrophil recruitment in response to combined mechanical ventilation and LPS, even though the cytokine, permeability, and apoptotic responses were similar to those of B6 mice. Interestingly, despite the presence of similar concentrations of KC in the BAL of lpr and B6 mice, only the B6 mice showed extensive KC deposition in the lungs, and this was associated with the presence of anti-KC:KC complexes in the BALF and lung tissue.
In this study, we focused on the initial events of the injury response by studying mice four hours after the initiation of injury, which was induced by combining a very low dose of endotoxin with mechanical ventilation. The main finding was that a functional Fas/FasL system was required for neutrophil migration into the airspaces of the lungs of mechanically ventilated mice exposed to LPS. The changes in BAL neutrophils seen in this study were associated with changes in lung myeloperoxidase (MPO) activity, and because lung MPO activity is a measurement of the total content of neutrophils in the lungs, the data suggests that the Fas/FasL system is required for both neutrophil migration into the airspaces and for neutrophil recruitment into the lungs. This observation confirms other studies that suggest a key role for the Fas/FasL system in neutrophil migration into the airspaces; for example, activation of Fas in the lungs of mice and rabbits results in a neutrophilic alveolitis (1, 2), whereas pharmacologic blockade of the Fas/FasL system attenuates the neutrophilic response to bacteria and bacterial products (3-6).
One potential explanation for the decreased migration of neutrophils into the airspaces of the Fas-deficient mice is that Fas ligation induces release of neutrophilic cytokines such as KC in macrophages and in lung epithelial cells in vitro (7-9). Thus, we had expected to see lower concentrations of KC in the mechanically ventilated lpr mice exposed to LPS, as compared with the B6 animals. However, this was not the case, and the difference in neutrophil migration was not due to differences in soluble KC concentrations.
Another potential mechanism that would explain differences in neutrophil migration with similar concentrations of soluble KC is a difference in neutrophil chemotaxis of lpr and B6 neutrophils. However, our chemotaxis studied showed that, if anything, neutrophils from lpr mice have slightly increased chemotaxis to KC, strongly suggesting that differences in chemotaxis are not the reason for the attenuation in the neutrophilic response seen in the lpr mice.
Neutrophil recruitment and migration appears to be partly dependent on the formation of anti-KC:KC immune complexes, which can bind Fcγ receptors in local leukocytes, enhancing the inflammatory response
. For example, the generation of anti-KC:KC immune complexes in the lungs of mice is followed by acute inflammatory lung injury, and this injury requires the presence of Fcγ receptors
. Furthermore, mice lacking γ chains show attenuated injury in response to LPS, suggesting that this process is relevant for inflammation secondary to bacterial products
. The human equivalent of anti-KC:KC complexes are anti-IL8:IL8 complexes, and these are present in the lungs of patients with ARDS
[25, 26]. In our study, we found that there are less anti-KC:KC complexes in the lungs of the lpr mice, even though the concentrations of KC were similar to those in the B6 mice. Thus, it is possible that the lower numbers of neutrophils seen in the lpr mice were due to decreased formation and deposition of anti-KC:KC complexes in the lungs. Additional studies are necessary to confirm this hypothesis, and it remains possible that differences in other unmeasured chemotactic agents account for the differences in neutrophil recruitment. The mechanism linking the Fas/FasL system with impaired formation and deposition of immune complexes remains unclear. To date, lpr mice, particularly the MRL/lpr strain are known to generate autoantibodies that can result in a lupus-like syndrome
A surprising finding in the present study was that there was no increase in the markers of permeability or apoptosis in the B6 mice exposed to mechanical ventilation and LPS, as compared to the lpr mice; instead, the injury response was limited to neutrophilic inflammation and cytokine release. One explanation is that neutrophil recruitment precedes the development of tissue injury in our model, in which the mice were studied relatively early, four hours after the onset of ventilation. Less clear is the finding that the lpr mice actually had increased caspase-3 activity and TUNEL positive cells compared with the B6 mice. We do not have a clear explanation for this finding, but they seem to be specific to the LPS + MV model, because in another model using mechanical ventilation in which WT and lpr mice were exposed to pneumonia virus of mice (PVM) prior to four hours of MV, we found a decrease in caspase-3 activity in the lpr mice
It is important to emphasize that the decrease in neutrophils seen in the lpr mice does not imply “protection”. It is unclear whether the lung neutrophilic response is directly associated with negative outcomes in ALI/ARDS or not. Most studies of lung injury presume that neutrophilia or increased concentrations of cytokine are deleterious, but all of these studies are limited in that they do not effectively reproduce the series of events seen in the clinical setting, where patients with multiple comorbidities are intubated for prolonged periods of time. Our study should be interpreted as knowledge on the mechanisms of that initiate lung injury, but not extrapolated to the ultimate results of that injury.