Proinflammatory cytokines induce NF-kappaB-dependent/NO-independent chemokine gene expression in MIN6 beta cells.
BACKGROUND
Interactions between chemokines IP-10, MCP-1, and RANTES and their receptors may mediate graft rejection following islet transplantation. The mechanisms regulating chemokine gene expression in pancreatic islet cells have not been well characterized. We examined the cytokine-induced gene expression profiles for several chemokines in a transformed pancreatic beta-cell line (MIN6) cotreated with an inhibitor of nitric oxide synthase and in a mutated clone of MIN6 made to overexpress a dominant negative inhibitor of NF-kappaB (IkappaBalphaM).


METHODS
MIN6 and MIN6-IkappaBalphaM (Bm) cells were cultured in mixtures of IL-1beta and TNF-alpha or IL-1beta, TNF-alpha, and IFN-gamma plus/minus the iNOS inhibitor L-NMMA. RT-PCR and RNase Protection Assay were used to measure mRNA expression for the following chemokines: IP-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES. Enzyme linked immunosorbant assay was used to measure IP-10 and MCP-1 protein release.


RESULTS
Cytokine-treated MIN6 and Bm demonstrated increased expression of genes for IP-10 and MCP-1. Expression in MIN6 was first detected at 2 h of incubation and peaked at 6 h. MIN6 demonstrated a more marked increase in chemokine gene expression for both IP-10 and MCP-1 and a more marked increase in IP-10 protein release than did Bm. There was no detectable gene expression for MIP-1alpha, MIP-1beta, or RANTES from MIN6 or Bm. L-NMMA completely blocked NO production from MIN6 and Bm but had no effect on chemokine gene expression in either MIN6 or Bm.


CONCLUSIONS
These results suggest that beta cells produce a complement of rejection-relevant chemokines in response to a proinflammatory stimulus and that pathways governing cytokine-induced chemokine gene expression in MIN6 are dependent on NF-kappaB but independent of NO.
Baker
Marshall S
MS
Department of Surgery, Division of Transplantation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Chen
Xiaojuan
X
Rotramel
Alizah
A
Nelson
Jeffrey
J
Kaufman
Dixon B
DB
D000818:Animals; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D054357:Chemokine CXCL10; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology*; D004338:Drug Combinations; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D018836:Inflammation Mediators / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism* / Q000503:physiopathology; D019402:Macrophage Inflammatory Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D054357:Chemokine CXCL10; D018925:Chemokines; D019743:Chemokines, CXC; D016207:Cytokines; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D007375:Interleukin-1; D019402:Macrophage Inflammatory Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse