Pulmonary transmigration reduction of human neutrophils by the Clostridium botulinum C2 toxin subunit CIIa in an in vitro extravasation model

Abstract

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury. ARDS is characterized by increased pulmonary vascular permeability leading to edema, severe hypoxemia, accumulation of activated immune cells, diffuse bilateral pulmonary infiltrates, right-to-left-shunt, increased dead space, and decreased lung compliance. The diffuse alveolar damage leads to deteriorated gas exchange up to respiratory failure, associated with a high mortality. No effective pharmacotherapy is available. Various harmful factors and diseases – both pulmonary and extrapulmonary – can cause ARDS. An exaggerated immune response with an excessive extravasation of neutrophils into the lung and subsequent immune-mediated tissue damage is a key factor in this detrimental process. A selective inhibition of neutrophil transmigration into lung tissue may represent an effective approach for the amelioration or prevention of ARDS in critical ill patients. Within this thesis, I tested whether the pore-forming, non-catalytic subunit C2IIa of the binary Clostridium (C.) botulinum C2 toxin enables selective down-modulation of excessive neutrophil transmigration. I therefore developed a model that mimics (pulmonary) extravasation of neutrophils in a sensitive, but cost-effective and non-labor intensive and non-time-consuming manner. The model allows pharmacodynamic analyses, including mass transmigration and migration kinetics. Endothelial barrier integrity can be validated in parallel. The setup – based on a Boyden chamber assay – consisted of an apical and basolateral compartment, separated by a microporous membrane representing a physical barrier that cells can overcome only by active migration. Human umbilical vein endothelial cells (HUVEC) were cultivated on the apical side of the porous membrane. Primary human neutrophils were isolated, fluorescently labeled and subsequently seeded into the upper chamber. Chemotactic agents in the lower chamber initiated neutrophil transmigration from apical to basolateral. The transmigration of neutrophils was continuously monitored from the top by recording the fluorescence signal in a plate reader. The isolation of primary human neutrophils and experimental conditions were optimized. (Co-)Culture-conditions suitable for the model were characterized. N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) ± tumor necrosis factor-alpha (TNF-α) was established as transmigration control, while cytochalasin B was implemented as transmigration-inhibitory control. A facile method for evaluating the assay was developed. Subsequently, the effect of C2IIa alone on mass transmigration and apparent transmigration rate was analyzed. Moreover, impact of C2IIa on barrier integrity was examined. Treatment of neutrophils with C2IIa reduced chemotactic mass transmigration and apparent transmigration rate of translocating neutrophils. There seems to be a correlation between neutrophil adherence and the effect of C2IIa, since already adherent neutrophils were less inhibited in their transmigration by C2IIa. There are initial evidences that pore formation by C2IIa with subsequent calcium (Ca2+) influx is the mode of action. The results in this work revealed that HUVEC treated with C2IIa maintained intact barrier integrity, supporting the hypothesis that C2IIa selectively targets neutrophils and does not exhibit cytotoxic effects on HUVEC. In conclusion, the potentially selective neutrophil transmigration down-modulating effect may extend our current understanding of the mechanism of intoxication by the complete binary C2 toxin. Moreover, these novel findings might represent an effective approach for the amelioration or prevention of ARDS in critical ill patients, and could thus provide a pharmacological strategy for the reduction of mortality from severe injuries or diseases.