Instant Blood-mediated Inflammatory Reaction During Islet Transplantation: The Role of Toll-like Receptors Signaling Pathways

Abstract

The instant blood-mediated inflammatory reaction (IBMIR) leads to massive destruction of transplanted islets. Islet isolation and time of culture may elicit the release of potent activators of Toll-like receptors (TLRs) signaling pathways during IBMIR. This work sought to evaluate the role of TLR signaling pathways to mediate inflammatory reactions. Isolated rat pancreatic islets were cultured for 12, 24, or 48 hours. Their viability was assessed by fluorescein diacetate/propidium iodide and their functionality, by glucose stimulation tests. Endotoxin levels were quantified using the Limulus Amebocyte Lysate assays. After RNA extraction and reverse transcription, we performed polymerase chain reaction (PCR) arrays. Samples obtained immediately after isolation were defined as controls. Eighty-four genes belonging to the TLR signaling pathways, were compared with control samples. After culture, islets were viable and functional with low endotoxin levels (< 0.1 endotoxin units/mL) showed TLR activation not due to exogenous contamination. Analysis of PCR arrays highlighted significant up-regulation of TLR-2. After 24 hours of culture, TLR-2 was up-regulated to 6.8 ± 0.6–fold ( P < .001) compared with controls but decreased to 4.3 ± 1.4–fold after 48 hours. In the same way, expression of myeloid differentiation primary response gene 88 (Myd88) was significantly up-regulated (3.2 ± 0.4–fold [ P < .001]) compared with controls. After 12 hours of culture, interleukin-10 gene expression was significantly up-regulated at 11.6 ± 3.7– fold ( P < .05), reaching 17.5 ± 8.3 after 24 hours. Finally, the cyclo-oxygenase-2 gene expression was up-regulated to 509 ± 67.1–fold ( P < .05) after 12 hours of culture. These data confirmed the implication of TLR signaling pathways in early inflammatory events.