CD40 activation in human pancreatic islets and ductal cells.
OBJECTIVE
CD40 expression on non-haematopoietic cells is linked to inflammation. We previously reported that CD40 is expressed on isolated human and non-human primate islets and its activation results in secretion of IL-8, macrophage inflammatory protein 1-beta (MIP-1beta) and monocyte chemoattractant protein-1 (MCP-1) through nuclear factor-kappaB and extracellularly regulated kinases 1/2 pathways. The objective of this study was to identify the pattern of gene expression, and to study viability and functionality affected by CD40-CD40 ligand (CD40L) interaction in human islets. Furthermore, we have studied the CD40-mediated cytokine/chemokine profile in pancreatic ductal cells, as they are always present in human islet transplant preparations and express CD40 constitutively.


METHODS
CD40-CD40L gene expression modulation was studied by microarray on islet cells depleted of ductal cells. Selected genes were validated by quantitative RT-PCR. The cytokine profile in purified ductal cells was evaluated by Luminex technology, based on the use of fluorescent-coated beads, known as microspheres, and capable of multiplex detection of proteins from a single sample. Glucose-stimulated insulin secretion and islet viability were assessed by perifusion and 7-aminoactinomycin D membrane exclusion, respectively.


RESULTS
Statistical analysis of microarrays identified 30 genes exhibiting at least a 2.5-fold increase across all replicate arrays. The majority of them were related to oxidative stress/inflammation. Prominently upregulated were chemokine C-X-C motif ligand 1 (CXCL1), CXCL2 and CXCL3 belonging to the CXC family of chemokines related to IL-8. CD40-mediated CXCL1 secretion was confirmed by ELISA. The viability or in vitro function was not affected by CD40 activation. In addition to previously reported IL-8, MIP-1beta and MCP-1, CD40 stimulation in ductal cells produced IL-1beta, IFN-gamma, TNF-alpha, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.


CONCLUSIONS
CD40 activation in islets and ductal cells produces cytokines/chemokines with a broad-spectrum range of biological functions.
Klein
D
D
Diabetes Research Institute, Leonard M. Miller School of Medicine, University of Miami, 1450 NW 10th Avenue, Miami, FL 33136, USA.
Timoneri
F
F
Ichii
H
H
Ricordi
C
C
Pastori
R L
RL
D000328:Adult; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002470:Cell Survival; D054360:Chemokine CXCL1 / Q000378:metabolism; D018925:Chemokines / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010183:Pancreatic Ducts / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055815:Young Adult
D019013:CD40 Antigens; D054360:Chemokine CXCL1; D018925:Chemokines; D016207:Cytokines; D023201:CD40 Ligand