Reduction of macrophage activation after antioxidant enzymes gene transfer to rat insulinoma INS-1 cells.
BACKGROUND
After transplantation, islet damage occurs through oxidative stress and host immune rejection mediated in part by macrophage activation. We investigated the influence of the overexpression of catalase (CAT) and Cu/Zn superoxide dismutase (Cu/Zn SOD) by rat insulinoma INS-1 beta cells exposed to oxidative stress on their viability and murine macrophage activation.


METHODS
INS-1 cells were infected with adenoviral vectors containing CAT (AdCAT) or Cu/Zn SOD (AdSOD) genes. After 72 hours, noninfected and infected INS-1 cells were exposed to oxidative stress and their viability was assessed using a colorimetric assay. Murine peritoneal exudate macrophages (mPEM) incubated with the supernatant of infected and stressed INS-1 cells were tested for chemotaxis and cytokine release (TNF-alpha, IL-alpha and IFN-gamma).


RESULTS
After infection, AdCAT and AdSOD gene transfer protected INS-1 cells from the toxicity of different oxidative reagents. The exposure of non-infected INS-1 cells to oxidative stress stimulated mPEM chemotaxis. INS-1 cells infection with AdCAT or AdSOD reduced significantly mPEM chemotaxis from 2.41 +/- 0.31 to 1.61 +/- 0.17 and from 2.53 +/- 0.24 to 1.27 +/- 0.14 respectively (n = 5; p < 0.05). Cytokine release by mPEM was stimulated after exposure to stressed noninfected INS-1 cell supernatant. CAT and Cu/Zn SOD overexpression by infected INS-1 cells decreased significantly the release of TNF-alpha from 268.18 +/- 30.18 to 81.40 +/- 23.58 pg/ml and from 446.96 +/- 75.47 to 20.37 +/- 2.38 pg/ml respectively (n = 6; p < 0.001). The overexpression of these enzymes also reduced significantly the release of IL-1beta and IFN-gamma.


CONCLUSIONS
CAT or Cu/Zn SOD gene transfer to INS-1 cells preserved them from oxidative damage and reduced the macrophage activation induced by these pancreatic cells. Therefore, protection of pancreatic beta cells against oxidative injury by antioxidant enzymes gene transfer is an effective approach to overcome the deleterious actions of macrophages in pancreatic islet transplantation.
Centre européen d'etude du Diabète , Faculté de Médecine, Strasbourg, France.
Véronique
V
Karsten
Séverine
S
Sigrist
Christine
C
Moriscot
Pierre-Yves
PY
Benhamou
Patricia
P
Lemarchand
Alain
A
Belcourt
Philippe
P
Poindron
Michel
M
Pinget
Laurence
L
Kessler
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002374:Catalase / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007340:Insulinoma / Q000201:enzymology* / Q000235:genetics; D008262:Macrophage Activation / Q000187:drug effects / Q000502:physiology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000502:physiology*; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000235:genetics; D051381:Rats; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics*
D000975:Antioxidants; D017077:Culture Media, Conditioned; D016207:Cytokines; D016877:Oxidants; C002385:linsidomine; D006861:Hydrogen Peroxide; D008981:Molsidomine; D002374:Catalase; D013482:Superoxide Dismutase