BackgroundTumour necrosis factor (TNF) is upregulated in the alveolar space early in the course of ventilator-induced lung injury (VILI). Studies in genetically modified mice indicate that the two TNF receptors play opposing roles during injurious high-stretch mechanical ventilation, with p55 promoting but p75 preventing pulmonary oedema.AimTo investigate the effects of selective inhibition of intra-alveolar p55 TNF receptor on pulmonary oedema and inflammation during ventilator-induced lung injury using a newly developed domain antibody.MethodsAnaesthetised mice were ventilated with high tidal volume and given an intratracheal bolus of p55-specific domain antibody or anti-TNF monoclonal antibody (‘pure’ VILI model). As a model of enhanced inflammation, a subclinical dose of lipopolysaccharide (LPS) was included in the intratracheal antibody bolus (LPS+VILI model). Development of lung injury was assessed by respiratory mechanics and blood gases and protein levels in lavage fluid. Flow cytometry was used to determine leucocyte recruitment and alveolar macrophage activation, while lavage fluid cytokines were assessed by ELISA.ResultsThe ventilation protocol produced deteriorations in respiratory mechanics and gas exchange with increased lavage fluid protein levels in the two models. The p55-specific domain antibody substantially attenuated all of these changes in the ‘pure’ VILI model, while anti-TNF antibody was ineffective. In the LPS+VILI model, p55 blockade prevented deteriorations in respiratory mechanics and oxygenation and significantly decreased neutrophil recruitment, expression of intercellular adhesion molecule 1 on alveolar macrophages, and interleukin 6 and monocyte chemotactic protein 1 levels in lavage fluid.ConclusionsSelective inhibition of intra-alveolar p55 TNF receptor signalling by domain antibodies may open new therapeutic approaches for ventilated patients with acute lung injury.

BackgroundTumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator-induced lung injury. In the current study, we explored the efficacy of this antibody in mouse models of acid-induced lung injury to investigate the longer consequences of treatment.MethodsWe employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting “dummy” dAb, 1 or 4 h before acid instillation.ResultsAcid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation, and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase.ConclusionThese results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 h. Together with our previous data, the current study lends support toward the clinical targeting of p55 for patients with, or at risk of ARDS.

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.