Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis

Background. Primary nonfunction resulting in immediate graft loss is respon sible for the failure of a large number of islet transplants. Evidence is a ccumulating to single out endotoxin contamination of the various reagents n eeded for islet isolation as a major cause of early graft loss. Methods. Islets isolated with endotoxin-containing (400 endotoxin units/ml) collagenase type V and "endotoxin-free" (3.1 endotoxin units/ml) Liberase( TM) were compared. Graft function was assessed using a syngeneic murine mod el of marginal islet mass transplantation. Pro-inflammatory cytokine produc tion by islets was measured by ELISA in culture supernatants, and quantitat ive reverse transcriptase-PCR. Islet cell apoptosis was measured using the annexin assay. Results. Graft function was significantly delayed when islets were isolated with endotoxin-containing collagenase. Addition of endotoxin to the Libera se(TM) solution similarly delayed graft function. After 18 hr in culture, c ollagenase-isolated islets released higher amounts of proinflammatory cytok ines compared with Liberase(TM)-isolated islets (interleukin-6: 2185+/-1174 pg/ml vs. 520+/-201 pg/ml; tumor necrosis factor-alpha: 304+/-298 pg/ml vs . 0; IL-1 beta: 12.5 pg/ml+/-12.5 vs. 0). This observation correlated with higher cytokine mRNA expression in collagenase-isolated islets, The percent age of apoptotic islet cells immediately after isolation was 17.2%+/-9.4 in collagenase-isolated islets and 7.1%+/-2.1 in Liberase(TM)-isolated islets . Conclusions. We propose that endotoxin contamination is the primum movens o f a chain of events that involves intra-islet cytokine production and relea se and islet cell apoptosis, and endotoxin contamination can ultimately lea d to primary nonfunction in vivo. This emphasizes the fact that using endot oxin-free reagents during isolation is a key factor for successful islet tr ansplantation.