Technical Reference #3279

3279.

Oxidative stress induces protein and DNA radical formation in follicular dendritic cells of the germinal center and modulates its cell death patterns in late sepsis Saurabh Chatterjee; Olivier Lardinois; Suchandra Bhattacharjee; Jeff Tucker; Jean Corbett; Leesa Deterding; Marilyn Ehrenshaft; Marcelo G. Bonini ; Ronald P. Mason, National Institute of Environmental Health Sciences; National Institutes of Health, Free Radical Biology & Medicine, 50(3279), (2011)
Link To Paper

Abstract:
Profound depletion of follicular dendritic cells (FDCs) is a hallmark of sepsis-like syndrome; but the exact causes of the ensuing cell death are unknown. The cell death-driven depletion contributes to immunoparalysis and is responsible for most of the morbidity and mortality in sepsis. Here we have utilized immuno-spin trapping; a method for detection of free radical formation; to detect oxidative stress-induced protein and DNA radical adducts in FDCs isolated from the spleens of septic mice and from human tonsilderived HK cells; a subtype of germinal center FDCs; to study their role in FDC depletion. At 24 h postlipopolysaccharide administration; protein radical formation and oxidation were significantly elevated in vivo and in HK cells as shown by ELISA and confocal microscopy. The xanthine oxidase inhibitor allopurinol and the iron chelator desferrioxamine significantly decreased the formation of protein radicals; suggesting the role of xanthine oxidase and Fenton-like chemistry in radical formation. Protein and DNA radical formation correlated mostly with apoptotic features at 24 h and necrotic morphology of all the cell types studied at 48 h with concomitant inhibition of caspase-3. The cytotoxicity of FDCs resulted in decreased CD45R/CD138- positive plasma cell numbers; indicating a possible defect in B cell differentiation. In one such mechanism; radical formation initiated by xanthine oxidase formed protein and DNA radicals; which may lead to cell death of germinal center FDCs.

Keywords:
<div><font face=AdvTT5235d5a9 size=1 color=black>Oxidative stress; Follicular dendritic cell; Caspase-3</font></div> <div><font face=AdvTT5235d5a9 size=1 color=black>Xanthine oxidase; Apoptosome; Necrosis; Plasma cell; Free radicals</font></div>

Materials & Methods:
Apoptosis detection assay in HK cells Treated cells were fixed using a 4% formaldehyde solution on Mattek uncoated glass-bottomed plates; permeabilized using 50 μl of cytonin; and incubated for 20 min. The samples were washed twice in DNase-free water; followed by terminal deoxynucleotidyl transferase (TdT) labeling as per the manufacturer's protocol (TACS TdT in situ apoptosis detection kit; fluorescein; R&D Systems; Minneapolis; MN; USA). After labeling; the plates were immediately analyzed by fluorescence microscopy.

Microscopic Technique
fluorescence microscopy

Cell Type(s)
HK cells