Differential Binding and Internalization of Clostridium difficile Toxin A by Human Peripheral Blood Monocytes, Neutrophils and Lymphocytes

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A488) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A488‐associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A488 in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A488‐associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A488‐associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.