Free Radical Biology & Medicine 50 (2011) 988–999

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Free Radical Biology & Medicine

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Original Contribution

Oxidative stress induces protein and DNA radical formation in follicular dendriticcells of the germinal center and modulates its cell death patterns in late sepsis

Saurabh Chatterjee a,⁎, Olivier Lardinois a, Suchandra Bhattacharjee a, Jeff Tucker b, Jean Corbett a,Leesa Deterding c, Marilyn Ehrenshaft a, Marcelo G. Bonini a, Ronald P. Mason a

a Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park,NC 27709, USAb Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USAc Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA

⁎ Corresponding author. Fax: +1 919 541 1043.E-mail address: chatterjees2@niehs.nih.gov (S. Chat

0891-5849/$ – see front matter. Published by Elsevierdoi:10.1016/j.freeradbiomed.2010.12.037

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 July 2010Revised 10 December 2010Accepted 27 December 2010Available online 4 January 2011

Keywords:Oxidative stressFollicular dendritic cellCaspase-3Xanthine oxidaseApoptosomeNecrosisPlasma cellFree radicals

Profound depletion of follicular dendritic cells (FDCs) is a hallmark of sepsis-like syndrome, but the exactcauses of the ensuing cell death are unknown. The cell death-driven depletion contributes toimmunoparalysis and is responsible for most of the morbidity and mortality in sepsis. Here we have utilizedimmuno-spin trapping, a method for detection of free radical formation, to detect oxidative stress-inducedprotein 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 vivoand in HK cells as shown by ELISA and confocal microscopy. The xanthine oxidase inhibitor allopurinol and theiron chelator desferrioxamine significantly decreased the formation of protein radicals, suggesting the role ofxanthine oxidase and Fenton-like chemistry in radical formation. Protein and DNA radical formationcorrelated mostly with apoptotic features at 24 h and necrotic morphology of all the cell types studied at 48 hwith 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, whichmay lead to cell deathof germinal center FDCs.

terjee).

Inc.

Published by Elsevier Inc.

Sepsis-like syndrome is characterized by a severe hyperinflammatorystage followed by a protracted immunosuppressive phase that has beentermed immunoparalysis. At least 25 clinical trials with new agents havefailed, and it is understood that much still has to be learned about thepathophysiological mechanisms that drive sepsis [1]. The loss ofimmunocompetence in the critically ill has been ascribed to the apoptoticdeletion of cells of both the innate and the adaptive immune systems [2].One of the principal components of the innate immunity-adaptiveimmunity cross talk is the antigen-presenting cells of secondary lymphoidorgans such as the spleen. Studies indicate that in septic spleens, there is aprofounddepletion of splenic dendritic cells, including follicular dendriticcells (FDCs), due to apoptosis [3]. This large-scale depletionmay severelycompromiseBandT cell function and impair the ability of thehost to raisean appropriate immune response to secondary bacterial or viralinfections, as is evidenced by the reactivation of the otherwise dormantcytomegalovirus andherpes simplex virus in critically ill individuals [4,5].

Studies in the cecal ligation and puncture model of sepsis haveshown that there is a rapid expansion of FDC networks at 24–36 hfollowed by a rapid depletion, the cause being caspase-3-mediatedapoptosis [3]. That study assumes significance because folliculardendritic cell networks are crucial for the germinal center (GC)reactions in secondary lymphoid organs and present antigens to Bcells in the form of immunocomplexes or iccosomes. In HK cells, anFDC cell line that represents one of the subpopulations of FDCs andthat is characterized by HJ2+GP93+3C8+DRC-1−KIM4−, it wasestablished that GC B cells undergo complex interactions with FDCand T cells in the course of differentiation into memory B and plasmacells [6]. Thus, it is justifiable to speculate that profound depletion ofFDCs in sepsis can have serious consequences on the immune state ofthe critically ill. The scarcity of available evidence, both at clinics andin preclinical studies, about the distinct role of FDC depletion and themechanism behind this event led us to investigate the mode of rapiddepletion of FDCs.

One of the main causes that can lead to such rapid apoptotic celldeath, as proposed by Tinsley and colleagues, could be theinvolvement of reactive oxygen species (ROS) and reactive nitrogenspecies [3]. Involvement of ROS in mechanisms of cell death has been

989S. Chatterjee et al. / Free Radical Biology & Medicine 50 (2011) 988–999

well documented. Free radicals and their associated modifications ofcellular processes may disrupt signal transduction pathways, can beperceived as abnormal, and, under some conditions, may triggerapoptosis [7]. Oxygen metabolites such as superoxide anion and H2O2

in the presence of metal ions such as iron and copper can formhydroxyl radical via Fenton chemistry [8,9] and can have a majorimpact on cellular functions ranging from proliferation and differen-tiation to regulation of cell cycle events, apoptosis, and, underextreme conditions, necrosis [7,10]. Measurements of ROS, especiallyin free radical-mediated processes in cellular and in vivo systems,have been indirect and are generally presumed to cause cellularoxidative damage. Electron spin resonance spectroscopy remains thegold standard for measurement and identification of radical-mediatedreactions. However, with the recent development of immuno-spintrapping technology, it is possible to detect free radical-mediatedreactions onproteins in the formof protein radicalswithin cells [11–14],whichprovides strong evidence in real time of formation of reactive freeradicals on protein amino acid residues and DNA bases.

Using this technique, we investigated the complex interplaybetween the involvement of ROS and profound FDC death associatedwith the acute systemic inflammatory settings in sepsis-like syndrome.In this study we provide evidence for the role of xanthine oxidase-derived superoxide andH2O2 and involvement of Fenton-like chemistryin forming protein-derived radicals in follicular dendritic cells of septicspleen andhuman tonsil-derivedHKcells. The protein radical formationpreceded apoptotic features in both in vitro and in vivo observations at24 h and, interestingly, with a concomitant loss of caspase-3 activity at48 h. Further, we identified nuclear accumulation of DNA-derivedradical adducts thatmay signal late-stagenecrosis [15]. Thismode of celldeath led to a significantdecrease in plasmacell population in the spleenwith no change in germinal center B cell numbers, suggesting a distincteffect of FDC death on the immune system, as it correlateswell with theimmunoparalysis seen in sepsis.

Materials and methods

Materials

Lipopolysaccharide (LPS; Escherichia coli; strain 55:B5), the ironchelator desferrioxamine mesylate (desferrioxamine), apocynin, thecatalase inhibitor aminotriazole (AT), the cytochrome P450 inhibitor1-aminobenzotriazole (ABT), and allopurinol were obtained fromSigma 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 andwere purchased from Sigma Chemical Co. or Roche MolecularBiochemicals (Mannheim, Germany). All aqueous solutions wereprepared using water passed through a Picopure 2UV Plus system(Hydro Services and Supplies, Research Triangle Park, NC, USA)equipped with a 0.2-μm pore size filter.

Mice

Adult male, pathogen-free, 8- to 10-week-old C57BL6/J mice (TheJackson Laboratory, Bar Harbor, ME, USA) weighing 23–27 g on arrivalwere used in these experiments. The animals were housed for 1 week,one to a cage, before any experimental use. Mice that contained thedisrupted gp91phox (gp91phox−/−; B6.129S6-Cybbtm1Din) (The JacksonLaboratory) or disrupted p47phox (B6.129S2-Ncf1tm1shl N14) (Taconic,Cranbury, NJ, USA) genes were treated identically. In the case of NADPHoxidase, the control animals for knockout experimentswere age-matchedmice of C57BL6/J origin that had normal NADPH oxidase activity. Micehad ad libitum access to food and water and were housed in atemperature-controlled room at 23–24 °C with a 12-h light/darkschedule. All animals were treated in strict accordance with the NIH

Guide for the Humane Care and Use of Laboratory Animals, and theexperiments were approved by the institutional review board.

LPS-induced systemic inflammation model

Systemic inflammationwas induced inmice after LPS administrationas describedpreviously [16,17]. Briefly,mice received a bolus infusion ofLPS (12 mg/kg) (referred to as 0 h). A sham groupwas also included, inwhich normal mice received saline in place of LPS. LPS was dissolved inpyrogen-free saline and administered through the intraperitoneal (ip)route. At+24,+48, and+72 h,mice from the shamgroup and the LPSgroups were sacrificed. For experiments that involved detection ofprotein radical adducts from tissue sections ofmouse spleen, DMPOwasinjected in two divided doses of 1 g/kg. The spleens were collected andsnap-frozen in liquid nitrogen.

Administration of allopurinol, apocynin, and desferrioxamine

Allopurinol, a specific inhibitor of xanthine oxidase, was admin-istered in a single bolus dose of 35 mg/kg through the ip route 30 minbefore LPS treatment. In other studies, desferrioxamine (50 mg/kg)was administered to mice 1 h before LPS injections [18], and apocynin(10 mg/kg) was administered to mice 1 h before LPS injections [19].

Isolation of CD14/CD21-positive follicular dendritic cells from mousespleens

Because there is no specific protocol for isolation of splenic folliculardendritic cells, we chose to follow two distinct methodologies withmodifications [20,21]. Spleens from LPS and LPS+allopurinol- ordesferrioxamine-treated mice were dissected out and placed in35×10-mm petri dishes containing complete Dulbecco's modifiedEagle's medium (DMEM) on ice. The organs were then gently teasedwith a syringe piston and passed through a 75-μm cell strainer. Thespleen cell suspension was then digested using a cocktail consisting of1 mlof 8 mg/ml collagenaseDand1 mlof 10 mg/mlDNase (Sigma)plus1 ml complete DMEMwith 10% fetal bovine serum (FBS). The cellsweredigested for 1 h at 37 °C in a humidified incubator and given a 0.8%NH4Cl treatment to remove the red blood cells. The cells were washedwith DMEM and suspended in azide containing FACS buffer. Cells werestained with anti-CD14–FITC and anti-CD21–PE and sorted on a FACSVantage SE flowcytometer (Becton–Dickinson). The sorted cells were80–90% viable and did not proliferate under culture conditions.However, these cells were used for assays that involved short-termincubation (24–48 h) with the spin trap DMPO. Isolated splenic FDCsstained positive for the mouse FDC marker FDC-M1.

Experiments using a human tonsil-derived follicular dendritic-like cellline, HK

An established FDC-like line (HK cells) was obtained from Dr. Y.S.Choi (Alton Ochsner Medical Foundation, New Orleans, LA, USA) andmaintained as described by Kim et al. [22]. HK cells were maintained inantibiotic-supplemented RPMI 1640 (Invitrogen, Carlsbad, CA, USA)supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin,and 100 μg/ml streptomycin. HK cells after 10–15 passages were usedfor various experiments. To study the effect of acute LPS treatment andformation of protein radicals in follicular dendritic cells, HK cells wereincubatedwith 10 μg/ml LPS, 50 ng/ml TNF-α, and 25 mMDMPO for 24or 48 h. LPS at 10 μg/ml has been found to up-regulate phosphor-IκB-αin FDCs more than lower doses, and thus this dose was selected forreproducing a sepsis-like environment [23]. To see the effects ofxanthine oxidase, NADPH oxidase, catalase, and cytochrome p450enzymes on protein radical formation, HK cells were co-incubated withallopurinol (100 μM), AT (100 μM), ABT (100 μM), apocynin (100 μM),DMPO (100mM), or the iron chelator desferrioxamine (200 μM).

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Immuno-spin trapping of DMPO–protein radicals from cell lysates usingELISA

Cell lysates for use in ELISA for the detection of DMPO–protein nitroneadducts were prepared using a cell sonicator. Because buffers containtraces of metals and other contaminants that could form DMPO–proteinradicals, we compared chelexed phosphate buffer (pH 7.4) containing100 μMdiethylenetriaminepenta acetate (DTPA)withRIPAbuffer (0.05 gof sodium deoxycholate, 100 μl of Triton X-100, and 10 μl of 10% SDS in10 ml of 0.1 M PBS). Results from the pilot experiments found nosignificant difference in the chemiluminescence signal from theseexperiments, and all subsequent lysates were prepared in RIPA buffercontainingprotease inhibitors (Complete,Mini Protease InhibitorCocktailTablets; Roche Applied Science, Indianapolis, IN, USA). After a 30-minincubation of the samples in ice, they were centrifuged at 20,000 g for20 min. The soluble material (supernatant) was stored at 4 °C until use.

DNA extraction from HK cells

Cell pellets were resuspended in digestion buffer and proteinase Ksolution, and DNA extraction was carried out in our laboratory asoutlined in Ramirez et al. [15,24]. The extraction process keeps thenitrone adducts covalently bound to the DNA. DNA concentration and

Fig. 1. Protein radical adducts in FDCs from septic mice and HK cells. (A) CD14/CD21-positiveor LPS-treated mice and cultured for 24 or 48 h using 10% fetal bovine serum-supplemented Rwith 80–90% viability were seeded at a density of 2×105/well with or without DMPO (25 mwas performed using cell supernatants (2 μg protein/well) to detect DMPO–protein radicexperiments. *Pb0.05 compared to sham. #Pb0.05 compared to sham+DMPO-treated micOchsner Medical Foundation (Dr. Y.S. Choi) and cultured according to the protocol providedtreated with either DMPO (25 mM) or LPS+DMPO for 24 or 48 h. TNF-αwas added to the Lharvested and lysed. An anti-DMPO ELISA was performed using cell supernatants to detect Dindependent experiments. *Pb0.05 compared to cells only. #Pb0.05 compared to the DMP

purity were measured from the absorbance at 260 and 280 nm.Purified DNA with low protein content will exhibit an A260/A280 ratiobetween 1.8 and 2.

Confocal laser scanning microscopy (Zeiss LSM 510 UV Meta)

Mice were administered LPS and one of the following: the xanthineoxidase inhibitor allopurinol, the catalase inhibitor 4-aminotriazole, orthe iron chelator desferrioxamine. DMPO was injected in two doses of1 g/kg at 2 and 1 h before sacrifice. Spleens were fixed in 10% neutral-buffered formalin and soaked in 30% sucrose for 24 h. The frozensections (10 μm) were cryocut using a frozen tissue processor (LeicaInstruments, Bannockburn, IL, USA) at the immunohistochemistry corefacility at the NIEHS. Tissue slices were then treated with 0.5% SDS(5 min) for antigen retrieval, permeabilized, and blocked (2% nonfat drymilk; Pierce Biomedical, Rockford, IL, USA). In experiments inwhich HKcells were used, 5×105 cells were plated with 10 μg/ml LPS and 50 ng/ml TNF-α with or without allopurinol or desferrioxamine. Cells wereharvested at 24 or 48 h, fixed in 4% paraformaldehyde, and permeabi-lized with 0.01% Surfact Amps-X100 for 1 h. An antibody specific toDMPO nitrone adducts and Alexafluor 568 goat anti-rabbit antibody(Molecular Probes, Eugene, OR, USA; now Invitrogen) were used asprimary and secondary antibodies, respectively. Experiments that were

cells were flow-sorted from spleen cell suspensions that were from either sham-treatedPMI 1640medium. Each subset of these cells was checked for viability. Cell populationsM). At the end of each time point, cells were harvested and lysed. An anti-DMPO ELISAal adducts. The results are shown as means±SEM and are from three independente. (B) The human tonsil-derived follicular dendritic-like cell line HK was obtained from. Confluent cells between passages 7 and 16 were used for the assays. 5×105 cells werePS-treated cells to mimic a septic environment. At the end of each time point, cells wereMPO–protein radical adducts. The results are shown as means±SEM and are from threeO-only group.

Fig. 2. Localizationof protein radical adducts in FDCs fromsepticmice andHKcells. Spleens fromsham+DMPO- and LPS+DMPO-treatedmicewerefixed in10%neutral-buffered formalin andrehydrated in sucrose solution. Cryocut sections were double stainedwith anti-DMPO antibody and anti-FDC-M1 antibody followed by Alexa 488- and Alexa 567-tagged secondary antibodies.(A) Sham-treated spleen section at 24 h. (B) LPS-treated, DMPO-labeled spleen section. Anti-DMPO staining colocalizeswith FDC-specificmarker FDC-M1. (C) 63×original zoomviewof a TIBMshowing colocalization of protein adducts and FDC-M1-specific cell componentswithin the cell boundary. (D) LPS-only control inwhich noDMPOwas injected into the animal. (E) Spleen slicesshowing rapidly decreasing FDC-M1-specific cells in thegerminal center at 48 and72 hpost-LPS administration. (F)HK cellswere treatedwith eitherDMPOonly or LPS+DMPO for 24and48 h.Adherent cellswere stained for protein radical adducts using anti-DMPOantibody andAlexa 488-tagged secondary antibody and observed under confocalmicroscopy. (G) Thenumber of TIBMsthat are a hallmark of the germinal centerwas counted at 40×magnification at 24, 48, and 72 h. Aminimumof three follicles from three separate experimentswere analyzed per slide. *Pb0.05compared to the 24-h group. (H) Tryptophan oxidation as a result of oxidative stress in FDCs of septicmice. Spleens at 36 h from sham- and LPS-treatedmicewerefixed in 10% neutral-bufferedformalin and rehydrated in sucrose solution. Cryocut sections were double stained with anti-NFK antibody (blue in photomicrograph) and anti-FDC-M1 antibody (red in photomicrograph)followed by Alexa 488- and Alexa 567-tagged secondary antibodies. The sections were analyzed by confocal microscopy at 63× original magnification. Confocal microscopy images arerepresentatives of a pool of images from at least three independent experiments.

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Fig. 3. Nuclear DNA radical formation mediated by xanthine oxidase and Fenton-likechemistry in LPS-treatedHKcells.HK cellswere treatedwith LPS andTNF-α andharvested at48 h.DNAwas isolated fromthenuclear fraction after cell fractionation. ELISA fordetectionofDNA–DMPOnitrone adductswas carried out as per Ramirez et al. [5]. To see the involvementof xanthine oxidase and loosely bound iron, allopurinol (Allo) and desferrioxamine (Des)were co-incubated with LPS. The results are shown as means±SEM and are from threeindependent experiments. *Pb0.05 compared to cells only at 24 h. #Pb0.05 compared tocells only at 48 h. **Pb0.05 compared to the LPS-treated group at 24 h. ##Pb0.05 comparedto the LPS-treated group at 48 h.

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performed to examine the xanthine oxidase levels in FDC-M1-positivecells in LPS-treated mouse spleen used anti-mouse xanthine oxidase(Abcam, Cambridge, MA, USA) and Alexa 488-conjugated secondary

Fig. 4. Protein radical adducts in LPS-treated FDCs aremediated by xanthine oxidase and occumice were administered LPS. Wild-type LPS-administered mice received the xanthine oxidasdesferrioxamine (Des), or the cells were incubated with 100 mM DMPO (excess DMPO) as pcell suspensions and cultured for 24 or 48 h using 10% fetal bovine serum-supplemented RPMprotein radical adducts. The results are shown as means±SEM and are from three indepenbetween 7 and 16 passages at a density of 5×105 cells were co-incubated with LPS+DMPO,an excess concentration of DMPO (100 mM) was added to the incubation mixture instead ofharvested and lysed. A direct anti-DMPO ELISA was performed using cell supernatants to detthree independent experiments. *Pb0.05 compared to the LPS-treated group.

antibodies (Invitrogen). N-formylkynurenine, an oxidized product oftryptophan residues, was visualized using rabbit polyclonal antibody[32] in both tissues and HK cells. Confocal images were taken on a ZeissLSM510-UV Meta microscope (Carl Zeiss, Inc., Oberkochen, Germany)using a Plan-NeoFluor 40×/1.3/63× oil DIC objective with differentzoom levels. The 488-nm line from an argon laser was used forproducing polarized light for a DIC image as well as fluorescenceexcitation of the Alexa 488 secondary antibody.

Western blot analysis

Cell lysates from FDCs isolated from mouse spleens were resolved in4–10% Bis–Tris gels using SDS–PAGE and subjected to Western blotanalysis. Antibodies used in these experiments were mouse monoclonalxanthine oxidase (1:1000dilution; Abcam),mousemonoclonal caspase-3(32 kDa), rat monoclonal to LAMP-2 (1:2000; Abcam), and anti-mousegoat polyclonal p47phox (1:2000; Santa Cruz Biotechnology, Santa Cruz,CA, USA). The immunocomplexedmembranes were probed (1 h at roomtemperature) with goat anti-mouse (1:5000; Millipore, Billerica, MA,USA) or anti-goat horseradish peroxidase-conjugated secondary anti-bodies. Immunoreactive proteins were detected using enhanced chemi-luminescence (Immobilon Western chemiluminescence HRP substrate;Millipore). The images were subjected to densitometry analysis usingLabImage 2006 Professional 1D gel analysis software from KapleanBioimaging Solutions (Germany).

r through Fenton-like chemistry. (A)Wild-typemice or p47phox or gp91phox knockoute inhibitor allopurinol (Allo), NADPH oxidase inhibitor apocynin (Apo), or iron chelatorretreatments. CD14/CD21-positive cells from these mice were flow-sorted from spleenI 1640medium. An anti-DMPO ELISA was performed using cell lysates to detect DMPO–dent experiments. *Pb0.05 compared to the LPS-treated group. (B) Confluent HK cellsLPS+Allo, LPS+Des, LPS+Apo for 24 or 48 h. To study the scavenging action of DMPOthe 25 mM that was used for the purpose of spin trapping. At each time point, cells wereect DMPO–protein radical adducts. The results are shown as means±SEM and are from

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Apoptosis detection assay in HK cells

Treated cells were fixed using a 4% formaldehyde solution onMattek uncoated glass-bottomed plates, permeabilized using 50 μl ofcytonin, and incubated for 20 min. The samples were washed twice inDNase-free water, followed by terminal deoxynucleotidyl transferase(TdT) labeling as per the manufacturer's protocol (TACS TdT in situapoptosis detection kit, fluorescein; R&D Systems, Minneapolis, MN,USA). After labeling, the plates were immediately analyzed byfluorescence microscopy.

Caspase-3 activity assay

Caspase-3 activitywas assayed using a fluorimetric detection assaykit (Santa Cruz Biotechnology) following the manufacturer's protocol.Briefly, HK cells at a density of 1×105 were treated with LPS/TNF-αwith or without allopurinol, desferrioxamine, or apocynin in 96-wellplates. For each reaction, 50 μl of the cell lysate was added to eachwellof a 96-well plate. The reaction buffer was diluted and dithiothreitolwas added to a final concentration of 10 mM before use. Two hundredmicroliters of this buffer and 5 μl of DEVD-AFC substrate were addedto each well containing cell lysate. The reaction mixtures wereincubated for 1 h at 37 °C, and the level of free AFC was measuredusing a plate reader with a 400 nm excitation filter and a 505 nmemission filter.

Spleen cell culture from LPS-treated mice

Single-cell suspensions from spleens of LPS-administered mice andmice treated with inhibitors of xanthine oxidase, catalase, and the ironchelator desferrioxamine were cultured after being restimulated with10 ng/ml LPS for 24, 48, or 72 h. Spleen cellswere harvested at these timepoints, stained with CD45R/B220–FITC and CD138–PE, and analyzed in aflow cytometer to examine the differentiation of B cells into plasma cells.Also, to ascertain the number of germinal center B cells, splenocytesweredouble-stained with CD45R/B220–FITC and PNA–PE. The supernatantsfromthese timepointswere analyzed for T helper cell cytokines (TH1andTH2) using a multiple-array ELISA kit (Multi-Analyte ELISArray Kit;SABiosciences, Frederick, MD, USA).

Statistical analyses

All in vivo experiments were repeated three times with three miceper group (N=3; data from each group of three mice were pooled).All in vitro experiments were repeated three times, and the statisticalanalysis was carried out by analysis of variance followed by theKruskal–Wallis nonparametric test for intergroup comparisons.Quantitative data from Western blots as depicted by the relativeintensities of the bands were analyzed by performing a Student t test.Pb0.05 was considered statistically significant.

Results

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

Sepsis-like syndrome and other acute inflammatory insults result incopious generation of ROS, primarily in the form of superoxide radicalsand nonradicals such as H2O2 and peroxynitrite [14,25–28]. Though theinitial generationofROScontributes to thehyperinflammatory state, theresultant cellular stress may alter cell signaling, leading to cell death[29]. FDCs, by functioning as accessory cells in the immune system, playa major role in germinal center reactions of the host. They alsocontribute significantly to B cell differentiation. ROS-related tissuedamage can result in free radical-mediated damage to proteins.

To study the ROS-induced deactivation of FDCs in sepsis-likesyndrome, we used immuno-spin trapping [11,14] to quantify thegeneration of protein-derived radicals at 24 and 48 h post-LPS adminis-tration in FDCs isolated from LPS-treated mice and in HK cells. Weobserved a significant increase in protein radical adducts at 24 h in bothFDCs isolated from LPS-treated septic spleens and HK cells treated withLPS and TNF-α, as detected by anti-DMPO ELISA (Pb0.05; Figs. 1A and B).In CD14/CD21-positive cells, a subset of FDCs isolated from septic spleens,there was a sevenfold increase in the protein radical adducts in FDCsisolated from the LPS-treated group co-incubated with the spin trapDMPO at 24 h (Pb0.05). Experiments with HK cells costimulated with10 μg of LPS and 50 ng/ml TNF-α showed a significant increase in proteinradical adducts at 24 and 48 h (Fig. 1B).

To localize the site of radical formation in spleen tissues of LPS-treatedmice, spleen sections were stained with the mouse FDC marker FDC-M1and anti-DMPO antibody and visualized using confocal microscopy.Results indicated that protein radical adducts colocalized in FDC-M1-positive cells in the germinal center of the spleen and at 24 h weresignificantly higher than in sham-treated spleens in which no germinalcenter formation was seen (Figs. 2A and B). As expected, we could notlocate germinal center formation in sham-treated spleensbyPNA-positivestaining (data not shown).We could localize and identify several tingiblebody macrophages (TIBMs), a hallmark of germinal centers in the LPS-treated spleen(datanot shown).A63×magnified imageof a tingiblebodymacrophagewith endocytosed FDC debris is shown in Fig. 2C. A control inwhich no DMPOwas administered to mice given LPS is shown in Fig. 2D.There was a gradual decline in the FDC-M1-positive cells at 48 and 72 hcompared to 24 h, in agreementwith thefindings of Tinsley et al. [3], whoshowed depletion of FDCs in the septic spleen (Fig. 2E). Interestingly, weidentifieddistinct colocalizationofbothFDC-M1-positive andanti-DMPO-positive cellular debris within the TIBMs, suggesting engulfment of FDCsby thesemacrophages as early as 24 h (Fig. 2C). This colocalizationmaybeof significance because the germinal center cells that undergo apoptosisare rapidly engulfed by TIBMs to prevent autoimmune reactions andpossible inflammatory spillages. It may also explain the apoptotic modeof cell death among FDCs in early sepsis. The numbers of TIBMs in agerminal center may reflect the efficacy of removal of the cell debrisarising from apoptosis. TIBM numbers were significantly reduced at48 h in the septic spleen, indicating a change in the inflammatorymicroenvironment in the spleen (Fig. 2G).

To reproduce the inflammatory microenvironment of FDCsobserved in the germinal centers of treated spleens of septic mice,we examined HK cells treated with LPS and TNF-α co-incubated with25 mM DMPO. Confocal imaging showed two distinct and time-dependent localization patterns of DMPO nitrone adducts in HK cells(Fig. 2F). At 24 h incubation, protein radical adducts primarilycolocalized in the cytosol, whereas at 48 h intense and punctatenuclear staining was observed (Fig. 2F).

N-formylkynurenine (NFK) and kynurenine are formed from theoxidation of tryptophan and tryptophan residues through a numberof reactions [30,31]. We used a recently developed antiserumagainst NFK to detect NFK-containing proteins in splenic FDCs [32].FDCs that stained positive with FDC-M1, a marker for mousegerminal center FDCs, had a significant increase in NFK immuno-reactivity in LPS-treated spleens compared to sham-treated spleensat 36 h but not at 24 h as shown by confocal microscopy (Fig. 2H). Adetailed time-response experiment for the generation of NFKadducts after LPS administration using a low-magnification confocalmicroscopy image of the germinal center was also conducted(Supplementary Fig. 1).

The distinct punctate staining with anti-DMPO antibody withinthe nucleus of HK cells at 48 h prompted us to analyze the formationof DNA-derived radical adducts [15]. The nuclear fraction wasisolated by cell fractionation using a manufacturer's protocol. TheDNA was isolated and analyzed for DNA–nitrone adducts by ELISA.Results indicated that there was a very significant increase in the

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Fig. 6. Protein radicals in FDCs modulate procaspase-3/caspase-3 activity. (A) CD14/CD21-positive cells from sham-treated, LPS-treated, LPS+allopurinol (Allo)-treated,LPS+desferrioxamine (Des)-treated, and LPS+apocynin (Apo)-treated mice wereflow-sorted from spleen cell suspensions and cultured for 24 or 48 h using RPMI 1640medium supplemented with 10% fetal bovine serum. For experiments to study the roleof excess DMPO in scavenging protein radicals, sorted cells were incubated with100 mM DMPO. Cell lysates collected at these time points were used to measurecaspase-3 activity using a caspase-3 activity kit following the manufacturer's protocol.The results are shown as means±SEM and are from three independent experiments.*Pb0.05 compared to the sham-treated group at 24 h. **Pb0.05 compared to the LPS-treated group at 24 h. #Pb0.05 compared to the sham-treated group at 48 h. (B) HKcells were untreated or co-incubated with LPS, LPS+Allo, LPS+Apo, or LPS+Des for24 and 48 h. The cell lysates were used for assaying caspase-3 activity. The results areshown as means±SEM and are from three independent experiments. $Pb0.05compared to the cells-only group at 48 h. $$Pb0.05 compared to the LPS-treated cellsgroup at 48 h.

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formation of DNA-derived radicals in the nucleus of HK cells at 48 h(Fig. 3).

To assess the role of the lysosome as a possible organelle where theprotein radicals and their stable oxidation products can be generatedand/or mobilized, we used confocal laser scanning microscopy andfluorescence imaging to detect the DMPO–nitrone adducts. Resultsindicated that there were very few areas where punctate staining ofboth lysosomal marker LAMP-2 and DMPO–nitrone adducts could becolocalized (Supplementary Fig. 2).

Xanthine oxidase and Fenton-like chemistry are involved in proteinradical formation in germinal center FDCs and HK cells

To begin to identify the source of radicals and H2O2 in LPS-treatedmice and HK cells, we used the xanthine oxidase inhibitor allopurinoland the iron chelator desferrioxamine in experiments analyzing both

Fig. 5. Oxidative stress in sepsis generates cytosolic and nuclear radical adducts in a time-dependLPS+allopurinol (Allo)+DMPO-treated, and LPS+desferrioxamine (Des)+DMPO-treatedmice488- and Alexa 567-tagged secondary antibodies. Sections were visualized under confocal microsindependent experiments. (B) LPS+DMPO, LPS+Allo+DMPO, or LPS+Des+DMPO for 24 andAlexa 488-tagged secondary antibody and observed under confocal microscopy. (C) Spleen sectioxidase localization using confocal microscopy. (D) Western blot analysis of xanthine oxidase levNADPH oxidase knockout mice were analyzed for protein radical adducts using confocal microsco

DNA and protein radical formation. ELISA showed that co-incubationwith either allopurinol or desferrioxamine significantly decreasedDNA–DMPO adduct formation in HK cells (Fig. 3). Similarly, analysisof protein radical accumulation in HK cells and septic mouse spleencells also implicated both xanthine oxidase and Fenton chemistry inradical formation. Protein–DMPO adduct accumulation in HK cellswas significantly reduced by both allopurinol and desferrioxamine atboth 24 and 48 h. Pretreatment of mice with desferrioxaminesignificantly reduced the accumulation of protein–DMPO nitroneadducts in spleens of LPS-induced septic mice at both 24 and 48 h.Although allopurinol also attenuated the production of proteinradicals at 24 h, this effect was lost by 48 h.

Confocal analysis was then used to confirm and expand theinhibitor experiments. DMPO adduct accumulation is reduced by bothallopurinol and desferrioxamine in both spleen (Fig. 5A) and HK(Fig. 5B) cells, paralleling the ELISA results (Figs. 4A and B). Confocalanalysis also allowed the detection of an increase in xanthine oxidaseprotein in mouse spleen due to LPS treatment as well as accumulationof xanthine oxidase in HK cells (Fig. 5C). Western analysis of thesecells corroborated the confocal images, showing higher amounts ofxanthine oxidase in LPS-treated spleen and HK cells. The role ofNADPH oxidase was ascertained by using gp91phox knockout andp47phox knockout mice and injecting apocynin into LPS-treated miceand co-incubating with HK cells. Apocynin injection or co-incubationdid not result in a significant decrease in DMPO–nitrone adductformation in either FDCs derived from the septic mice or HK cells.Similarly, both p47phox and gp91phox knockout mice showed nosignificant reduction in DMPO–nitrone adduct formation in their FDCsduring sepsis (Figs. 4A and B). Further Western analysis ofimmunoblotted proteins for the p47phox subunit of NADPH oxidaserevealed no immunoreactivity for this protein. In addition, confocalanalysis showed that spleens from LPS-treated NADPH oxidaseknockout mice form amounts of DMPO adducts equal to those ofthe wild type. This experiment, however, also showed that theseknockout spleens had low recruitment of FDCs to the germinalcenters. Cells incubated with 100 mM DMPO showed a significantdecrease in DMPO–nitrone adducts (Figs. 4A and B).

Caspase-3 activity and mode of cell death

The only study [3] that links FDC cell death to sepsis stronglysuggests that caspase-mediated apoptosis leads to FDC depletion,resulting in immunosuppression. Alternatively, studies with HK cellsindicate a clear role for TGF-β in opposing TNF-α-mediated apoptosis[33]. To study the involvement of caspase-3-mediated apoptosis, wemeasured caspase-3 activity in FDCs isolated from the septic spleen andin HK cells. In the CD14/CD21-positive cells from LPS-treated mice, thecaspase 3 activity showed an increase at 24 h, which was restored tocontrol levels by allopurinol, excessDMPO, or desferrioxamine (Fig. 6A).At 48 h, caspase-3 activity had decreased in cells from the LPS-treatedmice, whereas the activity in cells from mice coadministered LPS andallopurinol, excess DMPO, or desferrioxamine had control levels ofactivity.

The caspase-3 activity in HK cells (Fig. 6B) responded somewhatdifferently compared to the CD14/CD21-positive spleen cells. At 24 hall treatments had equal activity, whereas at 48 h the LPS-treated cellsshowed a significant reduction in activity that was restored to controllevels by desferrioxamine and excess DMPO, but not by allopurinol.Apocynin had no effect in cells derived from septic mice or in HK cells

ant manner. (A) Cryocut spleen sections from sham+DMPO-treated, LPS+DMPO-treated,were double stainedwith anti-DMPO antibody and anti-FDC-M1 antibody followed by Alexacopy. Confocal microscopy images are representatives of a pool of images from at least three48 h. Adherent cells were stained for protein radical adducts using anti-DMPO antibody andons from sham- and LPS-treated mice and LPS-treated HK cells were analyzed for xanthineels in CD14/CD21-positive and HK cells. (E) Spleen sections from LPS-treated wild-type andpy.

Fig. 7. Cell death patterns in FDCs in early and late sepsis. (A) CD14/CD21-positive cells from sham-treated (represented as “cells”) and LPS-treated mice were flow-sorted fromspleen cell suspensions and cultured for 24 or 48 h using RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were labeled with annexin–Alexa 488 conjugate andFDC-M1 antibody (Alexa 567-conjugated secondary antibody) at the desired time points and analyzed by fluorescence microscopy. A DIC image for each group was analyzed for themorphological features. (B) HK cells were treated with LPS (10 μg/ml), LPS+allopurinol (Allo), and LPS+desferrioxamine (Des) for 24, 48, or 72 h. At the desired time points, cellswere fixed, permeabilized with cytonin, and analyzed for TdT–fluorescein labeling using a fluorescence microscope. TdT-positive cells were counted in a 40× magnification field ofview. Results are represented for 24-h cell counts. The results are shown as means±SEM and are from three independent experiments.*Pb0.05 compared to the cells-only group.#Pb0.05 compared to the LPS-treated group. (C) Photomicrographs of TdT–fluorescein labeling of HK cells at 72 h showing extracellular and disintegrated chromatin structures(a phenomenon likely to be seen in necrosis) compared to untreated cells. (D) Trypan blue dye exclusion test was carried out with HK cells at 24, 48, or 72 h for assessing the viabilityof cells in culture after treatment with LPS (10 μg/ml), LPS+Allo, and LPS+Des. The results are shown as means±SEM and are from three independent experiments.*Pb0.05compared to the cells-only group. #Pb0.05 compared to the LPS-treated group.

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(Figs. 6A and B). 4-Aminotriazole had no effect on either CD14/CD21-positive or HK cells at 24 or 48 h (data not shown).

To study the pattern of cell death, we used confocal microscopy tovisualize binding of annexin V to the phosphatidyl serine moieties on

the membrane. Results indicated that FDCs isolated from septicspleens (CD14/CD21-positive cells) had marked annexin V binding onthe cell membrane at 24 h, but the staining was diffuse in cells thatwere observed at 48 h (Fig. 7A). The DIC images at 24 h showed

Fig. 8. Oxidative stress in late sepsis modulates B cell differentiation after death ofFDCs in septic mice. (A) B cell differentiation into plasma cells is affected by oxidativestress-induced FDC death. Spleen cells from sham-, LPS-, LPS+allopurinol (Allo)-, andLPS+desferrioxamine (Des)-treated mice were stimulated with 10 ng/ml LPS andincubated for 24, 48, or 72 h. At the end of each time point, cells were harvested andlabeled with FITC-conjugated CD45R and PE-conjugated CD138 and analyzed by flowcytometry. Flow plots for the results at 72 h are shown. (B) Column graph of double-positive cells at 72 h from sham, LPS, LPS+Allo, and LPS+Des groups. The results areshown as means±SEM and are from three independent experiments.*Pb0.05compared to the sham-treated group.

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typical apoptotic characteristics such as cell shrinkage and conden-sation of the nucleus, but were in sharp contrast to the 48-h view,which showed disintegration of the cell boundary, a characteristic ofnecrotic morphology.

Because the cells isolated from LPS-treated spleens were flow-sorted, which can cause stress-induced damage and perturb the resultsof the annexin V analysis, we carried out TdT-labeling experimentswithHK cells (Fig. 7B). This labeling showed that LPS-treated cells had asignificant increase in apoptotic nuclei at 24 h, which was substantiallyreturned to control levels by the xanthine oxidase inhibitor allopurinol(Pb0.05). Neither desferrioxamine nor 4-aminotriazole (data notshown), however, had a significant effect on apoptosis in these cells(Fig. 7B). Confocal analysis of HK cells at 72 h revealed unstained nucleiwith theTdT–fluorescein label predominantly locatedoutside the nuclei(Fig. 7C). Most of the cells observed had disintegrated cell membraneswith the fluorescein localized in structures that did not resemblehealthy cellular components, suggesting cellular necrosis. The cells thatscreened positive for TdT–fluorescein stain in the nucleus weresignificantly reduced at 48 and 72 h compared to 24 h in both celltypes studied (data not shown).

Trypan blue dye exclusion tests for assessing cell viability showeda time-dependent increase in nonviable cells in LPS-treated HK cells.Groups co-incubated with allopurinol or desferrioxamine hadsignificantly fewer nonviable cells compared to the LPS-treatedgroup at the 72-h time point, suggesting that at 72 h superoxide,H2O2 (from the xanthine/xanthine oxidase system), and looselybound iron in proteins or free iron had a significant role in the celldeath process (Pb0.05; Fig. 7D).

Protein radical formation and posttranslational protein oxidation areassociated with FDC death and defects in B cell differentiation in thegerminal center

The germinal center constitutes the dynamic microenvironmentwhere antigen-activated B cells rapidly expand and differentiate,generating plasma cells and memory B cells [34]. Therefore, depletionof FDCs in the germinal center could derail the process of B celldifferentiation, causing large-scale apoptosis of B cells and ultimatelyamplifying the risk of secondary infections due to a diminishedimmune response.

Activation of B cells leads to their differentiation into IgG-secretingplasma cells [35]. To investigate whether FDC death affects B celldifferentiation in the germinal center, we used flow cytometry to analyzesplenic cell populations from sham- and LPS-treated mice at 24, 48, and72 h. CD138, a cell-surface marker for plasma cells, was used along withthe B cell marker CD45R [43]. When spleen cells from sham- and LPS-treated septic mice were isolated and restimulated, there was asignificantly smaller number of CD45R/CD138-positive cells at 72 hcompared to the sham-treated group (Pb0.05; Figs. 8A and B). Therewas,however, no significant difference at the 24- and 48-h time points (datanot shown). These results suggest that the defect in differentiation ofgerminal center B cellsmight be due to the subtle depletion of FDCs in latesepsis (i.e., at time points N48 h) and arewell correlatedwith the necroticphase of cell death. Desferrioxamine administration to septicmice had nosignificant effect on CD45R/CD138-positive cell number compared to thesham-treated group, indicating that chelation of iron might have led toreduced toxicity at 72 h, causing less FDC depletion and less cell death.

Discussion

Mortality andmorbidity in sepsis are now known to be the results ofsevere immunosuppression, termed immunoparalysis. This condition iscaused primarily by the profound depletion of lymphocytes, dendriticcells, interdigitating cells, and follicular dendritic cells; enhanceddendritic cell survival attenuates LPS-induced immunosuppression[2,3,36–38]. The rapid depletionof the immuneeffector cells is primarily

due to apoptotic-like processes, and the molecular mechanismsinvolved are ascribed to both intrinsic and extrinsic pathways [2]. Ourstudies for the first time identify the involvement of novel free radical-mediated, posttranslational oxidation of proteins and DNA in cell death

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of FDCs both in septicmice and in an FDC-like cell line, HK. Injury toDNAcanhave severepathophysiological consequences such as cell death andcarcinogenic transformation [39].

In our previous studies, under conditions where severe inflam-mation and reactive oxygen species generation can influencemortality and morbidity in early sepsis, we identified the role ofxanthine oxidase in radical formation and nitration of tyrosine residueson carboxypeptidase B1, resulting in an amplification of the inflamma-tion cascade in spleen cells [14,17]. In this study, we focused on FDCs inthe spleen germinal center and onhuman tonsil-derivedHKcells, whichare a subtype of germinal center FDCs. We detected protein radicalformation and tryptophan oxidation in FDCs in the septic spleen at 24 h(Figs. 1A and 2). LPS also stimulated the generation of protein and DNAradicals in HK cells (Figs. 1B, 2F, and 3).

Sources of cellular superoxide and hydrogen peroxide can often betraced to either NADPH oxidase or xanthine oxidase [14,40], and wetherefore assessed the roles of these enzymes on radical formation.Spleens fromLPS-treatedNADPHoxidase knockoutmice (bothgp91phoxand p47phox) accumulated DMPO radicals equal to those found in wild-type animals. Further administration of apocynin, an inhibitor of NADPHoxidase, did not affect the DMPO–nitrone adduct formation, suggestingthat NADPH oxidase did not contribute to radical formation in these cells(Figs. 4 and 5E). Xanthine oxidase, however, does play a role. Not only areamounts of xanthine oxidase increased (Figs. 5C andD), but cotreatmentsof LPS-treated mice and HK cells with the inhibitor allopurinol reducedaccumulation of both DNA and protein radical adducts (Figs. 3–5).

Transition metals are also recognized as important in thegeneration of H2O2 and as catalysts for free radical reactions [41,42].To study the role of iron-mediated production of hydroxyl radicals, weused the iron chelator desferrioxamine. In general, desferrioxaminereduction of radical adduct accumulation was similar to that ofallopurinol inhibition. ELISA and confocal microscopy showed areduction of both DNA and protein radical formation in HK cells andprotein radical formation in FDCs from LPS-treated mice pretreatedwith desferrioxamine (Figs. 3, 4B, and 5B), signifying a contributingrole for iron-mediated radical production in our sepsis model.

Reactive oxygen species are known inducers of both apoptosis andnecrosis under inflammatory conditions [29]. Many agents that triggerapoptotic modes of cell death are stimulators of cellular oxidativemetabolism; and thus ROS have been proposed as commonmediators

Fig. 9. Scheme showing the proposed mechanism of oxidative stress-induced modulation ofin late sepsis.

of cell death [7]. Earlier studies have indicated that there is a significantincrease in immunoreactivity of active caspase-3 in the germinalcenter FDCs, suggesting apoptosis as amajor cause of their depletion insepsis [3]. Interestingly, our results for caspase-3 activity were time-dependent (Fig. 6), showing an increase at 24 h and amarked decreaseat 48 h in both cell types. Caspase-3 activity was restored to untreatedlevels by allopurinol and desferrioxamine in cells derived from theseptic spleen and by desferrioxamine in HK cells (Fig. 6). The failure ofallopurinol to restore caspase-3 activity in HK cells at 48 h mightindicate an as yet unknownmechanism of ROS generation that is iron-dependent in a transformed cell line such as HK.

The observed caspase-3 activity was correlated with apoptoticfeatures as seen by annexin V staining in FDCs from septic spleens andby numbers of TdT–fluorescein-positive nuclei of HK cells at 24 h(Fig. 7), suggesting a time- andROS-dependentmodulation of cell deathin septic FDCs. Various modes of caspase-3-independent cell death areknown [43]. It has been proposed that the necrosis-inducing effects aredue to the sensitivity of the caspases to oxidative inactivation [44]. Insome cell types, TNF-α, a pleiotropic cytokine, elicits necrotic cell deatheither spontaneously orwhen caspases are blockedby inhibitors. Recentevidence of secondary necrosis of immune cells during inflammationhas been ascribed to a failure of phagocytosis [45].

Interestingly, our results show high numbers of TIBMs in thegerminal center of septic mice at 24 h, which decreased significantlyover time (Fig. 2G). A highermagnification confocal image of one suchTIBM showed that cell debris from FDCs had internal protein radicaladducts at 24 h. This confirms that initially, after LPS treatment, therewas apoptosis of FDCs. The result assumes significance because this isthe first evidence of protein free radical adducts derived from anendocytosed FDC in a TIBM (Fig. 2C). These events also confirm therole of TIBMs as major scavengers of FDCs in the germinal center. Thedecrease in the number of TIBMs at subsequent times also correlateswell with a possible failure of the efficient removal of apoptotic cells,leading to alternate cell death. Our results thus suggest that sepsisalong with a concomitant release of TNF-α and ROS might regulatethe process of cell death from an initial apoptotic mode to a secondarynecrotic mode at later time points. Thus, future studies are needed toestablish amore direct role of caspase-3 inactivation by ROS generationand subsequent protein and DNA radical formations, in modulating celldeath patterns in the germinal center microenvironment.

FDC immunodepletion and altered B cell differentiation in the germinal center of spleen

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Our studies in both FDCs isolated from the septic spleen andHK cellsprovided clear evidence of apoptotic mechanisms at 24 h and necrosispost-LPS administration. It seems reasonable, therefore, to suggest thatincreased ROS generation coupledwith protein and DNA radical adductformation leads to rapid depletion of FDCs from the septic spleen. Insummary, we report a novel mechanism of modulation of cell deathpatterns of FDCs in sepsis. Based on our evidence from this study, wepropose amechanism (Fig. 9) whereby association of xanthine oxidase-derived superoxide anion radicals and hydrogen peroxide leads to theformation of protein andDNA radicals, causingmodulation of caspase-3activity, alterations in cell death patterns, and decreased plasma cellnumbers. The decreased plasma cell numbers might indicate that thereis decreased differentiation of B cells into CD138-positive plasma cells(Fig. 8). Thismight be one of the principal factors that contributes to theimmunoparalysis seen in sepsis.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.freeradbiomed.2010.12.037.

Acknowledgments

The authors sincerely acknowledge Tiwanda Marsh, JeoffreyHurlburt, and Holly Rutledge for excellent technical assistance. Wealso thankDr. Carl Bortner for help in analyzingflowcytometry data andDr. Shyamal Peddada of the Statistics Branch, NIEHS, for generous helpin statistical analysis. We also sincerely thank Dr. AnnMotten andMaryMason for help in the careful editing of the manuscript. This work wassupported by the IntramuralResearchProgramof theNational Institutesof Health and the National Institute of Environmental Health Sciences(Z01 ES050139-13).

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