Original Contribution
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

https://doi.org/10.1016/j.freeradbiomed.2010.12.037Get rights and content

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 tonsil-derived HK cells, a subtype of germinal center FDCs, to study their role in FDC depletion. At 24 h post-lipopolysaccharide 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.

Section snippets

Materials

Lipopolysaccharide (LPS; Escherichia coli; strain 55:B5), the iron chelator desferrioxamine mesylate (desferrioxamine), apocynin, the catalase inhibitor aminotriazole (AT), the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT), and allopurinol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Alexis Biochemicals (San Diego, CA, USA). All other chemicals were of analytical grade and were purchased from Sigma

LPS-induced sepsis-like syndrome forms cytosolic and nuclear protein radicals in germinal center FDCs and HK cells

Sepsis-like syndrome and other acute inflammatory insults result in copious generation of ROS, primarily in the form of superoxide radicals and nonradicals such as H2O2 and peroxynitrite [14], [25], [26], [27], [28]. Though the initial generation of ROS contributes to the hyperinflammatory state, the resultant cellular stress may alter cell signaling, leading to cell death [29]. FDCs, by functioning as accessory cells in the immune system, play a major role in germinal center reactions of the

Discussion

Mortality and morbidity in sepsis are now known to be the results of severe immunosuppression, termed immunoparalysis. This condition is caused primarily by the profound depletion of lymphocytes, dendritic cells, interdigitating cells, and follicular dendritic cells; enhanced dendritic cell survival attenuates LPS-induced immunosuppression [2], [3], [36], [37], [38]. The rapid depletion of the immune effector cells is primarily due to apoptotic-like processes, and the molecular mechanisms

Acknowledgments

The authors sincerely acknowledge Tiwanda Marsh, Jeoffrey Hurlburt, and Holly Rutledge for excellent technical assistance. We also thank Dr. Carl Bortner for help in analyzing flow cytometry data and Dr. Shyamal Peddada of the Statistics Branch, NIEHS, for generous help in statistical analysis. We also sincerely thank Dr. Ann Motten and Mary Mason for help in the careful editing of the manuscript. This work was supported by the Intramural Research Program of the National Institutes of Health

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