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 (R) 488 (toxin A(488)) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 degrees C/ice. The ability of trypan blue to quench cell surface-associated (but not cytoplasmic) fluorescence was also investigated. At 37 degrees C, toxin A(488)-associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A(488) in neutrophils was greater on ice than at 37 degrees C. Studies using trypan blue suggested that over 3 h at 37 degrees C, most of the toxin A(488)-associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 degrees C and ice/4 degrees C), there was minimal toxin A(488)-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.