CD40-CD40 ligand interaction activates proinflammatory pathways in pancreatic islets.

Pancreatic islet transplantation is becoming an alternative to insulin therapy in patients suffering from brittle type 1 diabetes. A major obstacle to the procedure is the early graft loss caused by nonspecific inflammation at the site of implantation. We recently discovered that CD40, a member of tumor necrosis factor (TNF) receptor family, is expressed in pancreatic beta-cells. CD40 expression in nonhematopoietic cells is generally associated with inflammation. Therefore, we investigated the potential proinflammatory role of CD40 in human and nonhuman primate islets. Islet beta-cells responded to CD40L interaction by secreting interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein (MIP)-1beta, the latter a chemokine first reported to be produced by islets. Induction of IL-8 and MIP-1beta was confirmed at the transcriptional level by quantitative RT-PCR. MIP-1beta expression in beta-cells was verified by double-immunofluorescence staining. CD40-CD40L interaction activates extracellular signal-regulated kinase 1/2 and nuclear factor-kappaB pathways in insulinoma NIT-1 cells, and inhibitors of either pathway suppress cytokine/chemokine production in islets. Moreover, ligation of CD40 receptor upregulates intercellular adhesion molecule-1, associated with inflammation, at both transcriptional and translational levels. Our results in vitro indicate that the CD40 receptor expressed by beta-cells could be activated in vivo, inducing proinflammatory responses contributing to early islet graft loss after transplantation.

Caspase-9 and effector caspases have sequential and distinct effects on mitochondria.

Proapoptotic Bcl-2 family members alter mitochondrial permeability resulting in the release of apoptogenic factors that initiate a caspase cascade. These changes are well described; however, the effects of caspases on mitochondrial function are less well characterized. Here we describe the consequence of caspase-9 and effector caspase inhibition on mitochondrial physiology during intrinsic cell death. Caspase inhibition prevents the complete loss of mitochondrial membrane potential without affecting cytochrome c release. When effector caspases are inhibited, mitochondria become uncoupled and produce reactive oxygen species. Interestingly, the effector caspase-mediated depolarization of the mitochondria occurs independent of the activity of complexes I-IV of the electron transport chain. In contrast, caspase-9 inhibition prevents mitochondrial uncoupling and ROS production and allows for continued electron transport despite the release of cytochrome c. Taken together, these data suggest that activated caspase-9 prevents the accessibility of cytochrome c to complex III, resulting in the production of reactive oxygen species, and that effector caspases may depolarize mitochondria to terminate ROS production and preserve an apoptotic phenotype.

Immune cell populations in nonhuman primate islets.

Islet transplantation is a promising cellular therapy for the treatment of type 1 diabetes (T1D). The immunogenicity of isolated islets has been of interest to the transplant community for many years, as upon transplantation, islets are damaged or destroyed through specific and nonspecific inflammatory and immune events. Antigen presenting cells (APC) are crucial intermediates in the generation of both innate and specific immune responses, and it has long been understood that some APC are resident in islets in situ, as well as after isolation. Our aim was to identify and characterize intraislet resident populations of APC and other immune cells in islets from nonhuman primates (Macaca fascicularis) in situ (pancreas biopsies obtained prerecovery) and after isolation using immunohistochemistry, confocal microscopy, and flow cytometry. The numbers of cells obtained in situ are similar to those in islets postisolation. Each isolated islet equivalent contains an average of 21.8 immune cells, 14.7 (67%) of which are APC. Many of these APC are dentritic cells and, surprisingly, 50% are B lymphocytes. The number of islet-resident immune cells increases with islet size, with greater numbers in large versus small islets (p < 0.001). The APC were localized around the exterior or spread evenly throughout the islets, with no definitive orientation identified. This knowledge will be useful to develop tailored modulation strategies to decrease immunogenicity, enhance engraftment, and ultimately prevent islet rejection.