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 responsible for the failure of a large number of islet transplants. Evidence is accumulating to single out endotoxin contamination of the various reagents needed 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 were compared. Graft function was assessed using a syngeneic murine model of marginal islet mass transplantation. Pro-inflammatory cytokine production by islets was measured by ELISA in culture supernatants, and quantitative 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 Liberase solution similarly delayed graft function. After 18 hr in culture, collagenase-isolated islets released higher amounts of proinflammatory cytokines compared with Liberase-isolated islets (interleukin-6: 2,185+/-1,174 pg/ml vs. 520+/-201 pg/ml; tumor necrosis factor-alpha: 304+/-298 pg/ml vs. 0; IL-1beta: 12.5 pg/ml+/-12.5 vs. 0). This observation correlated with higher cytokine mRNA expression in collagenase-isolated islets. The percentage of apoptotic islet cells immediately after isolation was 17.2%+/-9.4 in collagenase-isolated islets and 7.1%+/-2.1 in Liberase-isolated islets.


CONCLUSIONS
We propose that endotoxin contamination is the primum movens of a chain of events that involves intra-islet cytokine production and release and islet cell apoptosis, and endotoxin contamination can ultimately lead to primary nonfunction in vivo. This emphasizes the fact that using endotoxin-free reagents during isolation is a key factor for successful islet transplantation.
Berney
T
T
Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.
Molano
R D
RD
Cattan
P
P
Pileggi
A
A
Vizzardelli
C
C
Oliver
R
R
Ricordi
C
C
Inverardi
L
L
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D017364:Collagenases / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis*; D004731:Endotoxins / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D013820:Thermolysin / Q000494:pharmacology
D016207:Cytokines; D004731:Endotoxins; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin; D020780:Matrix Metalloproteinase 9