Interferon-β attenuates lung inflammation following experimental subarachnoid hemorrhage

Aneurysmal subarachnoid hemorrhage (SAH) affects patients at a relatively young age and has an incidence of 5 to 7 per 100,000 person-years. SAH carries a bad prognosis with a case fatality rate of 35% [1]. Systemic complications including cardiac failure, pneumonia, and pulmonary edema, occur in more than half the patients with SAH, and are an important contributor to poor outcome [2].

Acute lung injury (ALI) is commonly associated with brain injury and has an incidence between 5 and 30% in SAH patients [2‐5]. The development of ALI after SAH is an independent predictor of increased mortality and poor neurological outcome in patients suffering from SAH. One of the major components of ALI is the formation of pulmonary edema. Neurogenic pulmonary edema (NPE) is a life-threatening complication in patients suffering from SAH and is associated with a three-fold increase in poor outcome [6].

The proposed etiology of NPE is a massive sympathetic discharge following severe brain injury leading to intra-alveolar accumulation of protein-rich edema fluid and hemorrhage. The pathogenesis of NPE is still incompletely understood. A well-established theory on the pathophysiology of NPE is the blast-theory, which proposes combined hydrostatic and high permeability mechanisms for the formation of NPE due to the sympathetic storm caused by an acute increase in intracranial pressure [7]. Recently, it has been suggested that a systemic inflammatory response may also play a pivotal role in the development of pulmonary dysfunction after traumatic brain injury [8, 9]. The effects, however, of SAH on inflammatory mediators in secondary organs including the lung are still unknown.

Current therapy is mainly supportive since no effective drugs are available to treat NPE. Mechanical ventilation (MV) is necessary in the majority of patients to assure adequate oxygenation. However, MV can also contribute to lung injury, called ventilation-induced lung injury (VILI) [10]. A staggering 40% of SAH patients on MV develop VILI [11].

Interferon-β (IFN-β) is a small protein with immunomodulatory properties that has been approved for treatment of multiple sclerosis. It has been shown that IFN-β decreases pro-inflammatory cytokines and inhibits the migration of lymphocytes across the blood-brain barrier by decreasing the expression of chemokines and adhesion molecules on endothelial cells [12].

The aim of the present study was to investigate whether experimental SAH contributes to lung inflammation. Moreover, we examined the effects of IFN-β treatment on the expression of inflammatory mediators in the lung associated with brain injury in a rat model of SAH.

We report here that SAH induces the influx of neutrophils into the lung and expression of pulmonary adhesion molecules, chemokines, and TNF-α. More importantly, we are the first to show that IFN-β effectively abolishes the SAH-induced expression of all pro-inflammatory mediators in the lung.

SAH rats showed a case fatality rate of 50% after seven days. The mortality observed by us is common in experimental SAH-models and resembles other findings [15‐17]. Treatment with IFN-β resulted in a case fatality rate of 63%, although this increase did not statistically differ from the placebo group.

Neutrophils play an essential role in the development of lung inflammation. We observed a four-fold increase in MPO, indicating that neutrophils are recruited to the lung following SAH. The influx of neutrophils involves a complex cascade of events. The early response cytokine, TNF-β, most probably initiates the inflammatory response by activating the endothelial cells resulting in increased chemokine expression and upregulation of adhesion molecules [18]. In support of this concept we showed increased expression of pulmonary TNF-α following SAH. In addition, SAH induced enhanced pulmonary expression of the endothelial adhesion molecules E-selectin, ICAM-1, and VCAM-1, which play a pivotal role in the rolling across the endothelium and firm adhesion of neutrophils to the endothelium, respectively [18]. Furthermore, we observed marked SAH-induced upregulation of the chemokines MIP-1α, MIP-2, and CINC-1, which are responsible for the chemotactic activity of neutrophils. Therefore, we conclude that SAH induces a pro-inflammatory environment in the lung which may represent an important risk factor for the development of NPE.

Although several groups reported the occurrence of lung injury after brain injury, the underlying mechanisms are largely unknown. It has been suggested that increased intracranial production of pro-inflammatory cytokines results in the release of systemic pro-inflammatory mediators, thereby promoting secondary organ injury [8]. Although this is a tempting hypothesis, we show here de novo synthesis of pro-inflammatory mediators, suggesting that spill-over of cytokines is not the primary cause of lung inflammation following SAH. A second proposed mechanism underlying secondary organ injury is increased capillary permeability elicited by catecholamines due to sympathetic nerve stimulation after brain injury [7]. Catecholamines can activate the transcription factor NFκB in macrophages thereby promoting the production of TNF-α chemokines, and adhesion molecules [19, 20]. Therefore, we propose that sympathetic activation of the lung could have led to the local release of cytokines and chemokines in our model.

The major finding of our study was that IFN-β treatment strongly attenuates the SAH mediated pulmonary inflammation. The decreased influx of neutrophils in response to IFN-β administration was accompanied by decreased expression of TNF-α, chemokines, and adhesion molecules in the lung. Although this is an interesting finding, one should keep in mind that IFN-β therapy may also worsen bacterial pneumonia [21], although until now only one case-report has been published. However, the increase in incidence of bacterial pneumonia was only described for a situation in which long-term treatment with IFN-β was applied [22]. IFN-β is a potent immunomodulator with diverse effects. Several studies have shown that IFN-β reduces the migration of inflammatory cells across the blood-brain barrier [12]. This is likely accomplished by reducing the expression of endothelial adhesion molecules, ICAM-1 and VCAM-1, and by downregulating the production of chemokines [23, 24]. Although the immunomodulatory effects of IFN-β have been described in brain-related diseases including multiple sclerosis and focal cerebral ischemia, we are the first to describe the modulatory effects of IFN-β in pulmonary inflammation. The exact mechanisms by which IFN-β attenuates SAH-induced lung inflammation need to be further clarified. In our study, IFN-β was administered systemically; therefore it may be possible that IFN-β had a direct effect on pulmonary cells. For example, Kiss et al. showed that IFN-β treatment ameliorated vascular leakage in ALI via upregulation of 5'-nucleotidase (CD73) on pulmonary endothelial cells [25]. Although we did not look at CD73, it could be a possible explanation for our findings. IFN-β could also have an indirect effect by inhibiting either the activation of sympathetic nervous system or reducing the systemic pro-inflammatory environment and subsequently preventing the upregulation of pro-inflammatory mediators in the lung. Although we did not measure catecholamines, we could not see an effect of IFN-β treatment on TNF-α levels in the blood (data not shown). Finally, IFN-β could have a direct effect on cerebral inflammatory responses after SAH, thereby indirectly regulating the lung inflammation. Our preliminary data do not confirm this hypothesis since IFN-β did not have any effect on the SAH-induced cerebral inflammation (manuscript in preparation, Tiebosch et al.)

In conclusion, SAH induces a pro-inflammatory environment in the lung, which can be efficiently blocked by IFN-β. Therefore, our data strongly suggest that IFN-β may be an attractive clinical candidate to prevent SAH mediated lung inflammation.

We show here that subarachnoid hemorrhage (SAH) results in the upregulation of pro-inflammatory mediators in the lung as well as recruitment of neutrophils into the lung. In addition, we report that treatment with IFN-β completely abolishes the SAH-induced pulmonary inflammation. Our data imply that SAH is associated with pulmonary inflammation and that IFN-β may be an attractive therapeutic candidate to prevent SAH-mediated lung inflammation.