In vitro studies have shown that the early responses of these cells to toxin A are characterized by the loss of barrier function and the secretion of cytokines [18–20], which include the potent neutrophil chemoattractant interleukin (IL)-8 [21–24]. The cells
subsequently undergo programmed cell death [24–26]. Following the loss of epithelial cells, lamina propria macrophages/monocytes, neutrophils and lymphocytes (many of these cells recruited from the systemic circulation) will be exposed Z-VAD-FMK manufacturer to C. difficile toxins. We have previously reported that human monocytes/macrophages are more sensitive to C. difficile toxin A–induced cell death than lymphocytes [27, 28]. These studies lead us to postulate that the greater sensitivity of monocytes to C. difficile toxin A–induced cell death is because of their ability to internalize more of the toxin than lymphocytes. Binding and internalization of
toxin A by human neutrophils also remain to be characterized. In this study, we have investigated cell surface binding and internalization of fluorescently labelled toxin A to human peripheral blood neutrophils, lymphocytes and monocytes. Purification of toxin A. Toxin A was purified selleck chemicals from a toxigenic strain of C. difficile, VPI 10463, as previously described . Following culture (at 37 °C for 48 h) in brain heart infusion broth, the crude Casein kinase 1 culture filtrate was applied to a bovine thyroglobulin affinity chromatography column, exploiting the ability of toxin A to bind the thyroglobulin at 4 °C, but not 37 °C [29, 30]. Thus, C. difficile culture filtrate was applied to the column at 4 °C, and elution of bound toxin A was undertaken using prewarmed (to 37 °C) buffer. Following two sequential anion-exchange chromatography steps on Q-Sepharose-FF (GE Healthcare, Little Chalfont, UK) and Mono Q columns (GE Healthcare), purified fractions of toxin A were assessed
for cytotoxicity using the Vero cell assay . Aliquots of purified toxin were stored at −80 °C prior to use. Labelling and characterization of toxin A. Purified toxin A was labelled with Alexa Fluor® 488 by the protocol outlined in the manufacturers’ guidelines (Protein Labelling kit; Invitrogen Ltd., Paisley, UK). In brief, purified toxin was incubated with the reactive dye for 1 h at room temperature, before unincorporated dye was separated from the labelled toxin protein by size exclusion gel filtration through Bio-Rad BioGel P-30 (Bio-Rad, Hemel Hempstead, UK) fine resin (molecular weight cut-off, MWCO, >40 kDa). Fractions containing labelled protein were pooled and concentrated by passing through a 100-kDa MWCO concentrator column (Centricon; Millipore, Billerica, MA, USA). Elution buffer was also exchanged with phosphate-buffered saline (PBS, pH 7.4) as the toxin A488 carrier buffer.