perez cruz et all 2003

PHAGOCYTES

Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cellsIsabel Perez-Cruz, Juan M. Carcamo, and David W. Golde

The FAS receptor–FAS ligand system is akey apoptotic pathway for cells of theimmune system. Ligation of the FAS-receptor (CD95) induces apoptosis byactivation of pro–caspase-8 followed bydownstream events, including an in-crease in reactive oxygen species (ROS)and the release of proapoptotic factorsfrom the mitochondria, leading tocaspase-3 activation. We investigated therole of vitamin C in FAS-mediated apopto-sis and found that intracellular accumula-tion of pharmacologic concentrations ofvitamin C inhibited FAS-induced apopto-

sis in the monocytic U937 cell line and infresh human monocytes. Cells wereloaded with vitamin C by exposure todehydroascorbic acid (DHA), thereby cir-cumventing in vitro artifacts associatedwith the poor transport and pro-oxidanteffects of ascorbic acid (AA). Vitamin Cinhibition of FAS-mediated apoptosis wasassociated with reduced activity ofcaspase-3, -8, and -10, as well as dimin-ished levels of ROS and preservation ofmitochondrialmembrane integrity. Mecha-nistic studies indicated that the majoreffect of vitamin C was inhibition of the

activation of caspase-8 with no effect onit enzymatic activity. An independent ac-tion of high intracellular concentrationsof vitamin C on mitochondrial membranestabilization was also detected. Thesestudies illuminate the nature of redox-dependent signaling in FAS-induced apo-ptosis of human monocytes and suggestthat vitamin C can modulate the immunesystem by inhibiting FAS-induced mono-cyte death. (Blood. 2003;102:336-343)

© 2003 by TheAmerican Society of Hematology

IntroductionApoptosis is a suicidal mode of cell death used to eliminate cells inphysiologic and pathologic settings.1,2 While diverse events caninduce apoptosis, such as radiation and lack of growth factors,apoptosis in the immune system is largely regulated by signalingthrough death receptors, including the receptor for tumor necrosisfactor (TNF) and the FAS receptor (FAS-R), also known as CD95.1Apoptosis generally occurs in 2 phases: the commitment to celldeath, including activation of procaspases, followed by an execu-tion phase resulting in cell structure alterations.3Upon FAS-R ligation a set of intracellular signaling proteins is

recruited to the death-inducing signaling complex. The cytosolicpro–caspase-8 is recruited and believed to oligomerize and autoac-tivate.4 Caspase-10 is closely related to caspase-8 and it has beenproposed that both caspases can be recruited to the death-inducingsignaling complex, where they are activated and initiate signalingpathways resulting in apoptosis.5 Active caspase-8 induces translo-cation of the proapoptotic protein BID to the mitochondria, causingan increase in mitochondrial permeability and a loss in mitochon-drial membrane potential (!").6 Oxidative phosphorylation isuncoupled and the mitochondria release reactive oxygen species(ROS), resulting in elevated levels of ROS in cells stimulated viathe FAS pathway.7,8 The increase in cellular ROS is a key event inprogrammed cell death and oxidative molecules themselves caninduce apoptosis, independent of ligation of death receptors.9,10Release of cytochrome C (Cyt C) and other mitochondrial mol-ecules, such as Apaf-1, leads to the downstream activation ofcaspase-9 and the effector caspase-3.11Cellular ROS generation is a natural result of aerobic metabo-

lism and is amplified by cellular activation.12 ROS are widely

involved in receptor-mediated signaling,13-15 and antioxidants caninhibit important signaling pathways in immune cells.16,17 VitaminC is a critical dietary nutrient in humans, since humans cannotsynthesize it as do most nonprimates. Deficiency of vitamin Cseriously impairs immune function, and there is literature pointingto the role of vitamin C in enhanced host defense and themodulation of inflammatory reactions.18-22 Vitamin C is generallythought to enhance immunity and is widely taken as a supplement.Whereas vitamin C is found in the plasma in its reduced form(ascorbic acid [AA]), it is transported by most cells in its oxidizedform, dehydroascorbic acid (DHA), through facilitative glucosetransporters.23 Inside the cell, DHA is reduced to AA.24 Certainspecialized cells transport AA directly through cell-surface Na-ascorbate cotransporters.25 We sought to determine the role ofvitamin C in FAS-induced apoptosis in monocytes and used DHAto load cells with vitamin C, thereby avoiding the pro-oxidanteffects of AA in the presence of free transition metals ubiquitouslyfound in tissue culture.26 We found vitamin C to be a potentinhibitor of FAS-induced apoptosis in fresh monocytes and in U937cells. Our data indicate the primary action of vitamin C to be theinhibition of activation of caspase-8 and a separate effect ondownstream oxidative events.

Materials and methodsCells lines and monocytes

The monocytic cell line U937 was obtained from the American TypeCulture Collection (Manassas, VA) and cultured in complete medium:

From the Program in Molecular Pharmacology and Chemistry and ClinicalChemistry and Medicine, Memorial Sloan-Kettering Cancer Center, NewYork, NY.

Submitted November 25, 2002; accepted February 24, 2003. Prepublished onlineasBlood First Edition Paper, March 6, 2003; DOI 10.1182/blood-2002-11-3559.

Supported by a grant from the National Institutes of Health (CA 30388) and theLebensfeld Foundation.

Reprints: David W. Golde, Memorial Sloan-Kettering Cancer Center, 1275York Ave, New York, NY 10021; e-mail: [email protected].

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2003 by The American Society of Hematology

336 BLOOD, 1 JULY 2003 ! VOLUME 102, NUMBER 1

RPMI containing 100 U penicillin, 100 U streptomycin (Gemini Bioprod-ucts, Woodland, CA), L-glutamate, and 10% fetal calf serum (OmegaScientific, CA). Human monocytes were isolated from peripheral bloodobtained from the New York Blood Center (NY). The following day,peripheral blood mononuclear cells were separated by Ficoll gradient(Accu-Prep;Accurate Chemicals and Scientific,Westbury, NY). Monocyteswere immediately purified by negative selection using the magnetic-activated cell separation monocyte isolation kit (Miltenyi Biotec, Auburn,CA) following the manufacturer’s instructions. Monocytes were incubatedin RPMI containing 100 U penicillin, 100 U streptomycin, L-glutamate, and20% human AB serum (Irvine Scientific, Santa Ana, CA). Purity wasassessed by staining with anti-CD3 (RPE-Cy5; DAKO, Glostrup, Den-mark), anti-CD56 (phycoerythrin [PE]; Becton Dickinson, San Diego, CA),anti-CD20 (peridinin chlorophyll protein [PerCP]; Becton Dickinson), andanti-CD14 (fluorescein isothiocyanate [FITC]; Becton Dickinson) fluores-cent antibodies. The cells were suspended in staining buffer consisting of1% heat-inactivated fetal calf serum and 0.1% (wt/vol) sodium azide(Sigma, St Louis, MO) in phosphate-buffered saline (PBS), pH 7.4,containing the fluorescent antibody (Ab) for 30 minutes at 4°C. Afterstaining, the cells were washed with buffer, fixed in 1% paraformaldehydeand analyzed by flow cytometry (FACScalibur; Becton Dickinson) within24 hours of staining. The results were analyzed using the CELLQuestsoftware (Becton Dickinson). Monocyte preparations were always higherthan 95% purity.

Detection of CD95

The antihuman CD95-RPE Ab (DAKO) was used to detect the CD95antigen in cell lines and monocytes by flow cytometry. Cell-surface stainingand analysis were performed as described in the previous section.

Induction of apoptosis

An anti-FAS Ab and soluble FAS ligand (FASL) were used to induceapoptosis. For the induction of apoptosis in monocytes, an anti-FAS Ab(immunoglobulin G1 [IgG1], clone DX2; R and D Systems, Minneapolis,MN) or an isotype controlAb (R and D Systems) were plate-immobilized ina 12–flat well plate overnight. The plate was washed twice in serum-freeRPMI and blocked with RPMI containing 10% human type AB serum for30 minutes at 37°C. The plate was then washed and the cells were addedimmediately. For induction of apoptosis in U937 cells, 10 #L protein G (2.5mg/mL suspension in agarose beads; Boehringer Mannheim, Mannheim,Germany) per #g anti-FAS Ab or control Ab was mixed in cold serum-freeRPMI for 5 minutes at room temperature (RT) before addition to the cellsuspension. To induce apoptosis with soluble FASL, Super FASL (AlexisBiochemicals, Lausen, Switzerland) was added to U937 cells or to freshlyisolated monocytes. Cells treated with anti-FAS Ab, FASL, or control cellswere incubated at 37°C in an environment of 5% CO2 in air.

Detection of apoptosis

The annexin V–FITC/propidium iodine (PI) apoptosis detection kit (Pharm-ingen, San Diego, CA) was used to determine the frequency of apoptosis inU937 cells, following the manufacturer’s instructions. Alternatively, PIstaining of DNAwas used to analyze the sub-G1 region by flow cytometry.For PI staining, cells were washed twice with PBS and fixed with 70%ethanol for at least 30 minutes at 4°C. Cells were then washed twice withPBS and resuspended in 50 #L of a 100 U/mL RNAse-A solution(Boehringer Mannheim) and 20 #L PI (0.1 mg/mL; Sigma) per 1 $ 105cells for 30 minutes at RT in the dark and kept at 4°C until flow cytometryacquisition. Apoptosis in monocytes was determined by analysis of thesub-G1 region by DNA staining and flow cytometry. For PI staining, themonocytes were incubated with trypsin for 5 minutes at 37°C, washed twicewith PBS, and then stained as described in the previous section.

Vitamin C uptake

Vitamin C uptake was measured as intracellular accumulation afterincubation of cells with DHA or AA at 37°C, as previously described.23,24To determine the accumulation of vitamin C supplied as DHA, triplicate

cell samples were incubated with several concentrations of DHA from asolution containing 0.5 #Ci (0.0185 MBq) L-14C-AA (specific activity 8.0mCi/mmol (296 MBq/mmol); Perkin-Elmer Life Sciences, MA), 1.0 to 4.0mM AA (Sigma), and 86 U/mL ascorbate oxidase (Sigma) in incubationbuffer (15.0 mM HEPES [N-2-hydroxyethylpiperazine-N%-2-ethanesul-fonic acid], 135.0 mM NaCl, 5.0 mM KCl, 1.8 mM CaCl2, and 0.8 mMMgCl2 [pH 7.4]). Cells were then washed twice with cold PBS and lysedwith 0.5 mL sodium dodecyl sulfate (SDS)–lysis solution (10.0 mM Tris[tris(hydroxymethyl)aminomethane]–HCl [pH 8.0] and 0.2% SDS). Cell-associated radioactivity was quantified by scintillation spectrometry. Todetermine the accumulation of vitamin C supplied as AA, cells wereincubated with varying concentrations of AA from a solution prepared with0.5 #Ci (0.0185 MBq) L-14C-AA, 1.0 to 4.0 mM L-AA, and 0.1 mM1,4-dithiothreitol in incubation buffer and cell-associated radioactivitymeasured by scintillation spectrometry. Additionally, accumulation ofvitamin C supplied as DHA was determined by high-performance liquidchromatography (HPLC) electrochemical detection (ESA, Chelmsford,MA).27 Cells incubated with DHAwere washed twice in cold PBS and thenlysed in 60% methanol. Lysates were centrifuged, the supernatants filteredthrough 0.2-#m nylon membrane filters (Nalgene, Rochester, NY) andequal amounts of each sample injected onto a modified C18 column (no.70-4160; ESA). The column was equilibrated at 30°C with a mobile phasebuffer consisting of 50 #M sodium phosphate, 50 #M sodium acetate, 189#M dodecyltrimethylammonium chloride, and 3.66 #M tetraoctylammo-nium bromide in 30:70 methanol-water (vol/vol, HPLC grade; J. T. Baker,Phillipsburg, NJ), pH 4.8. The flow rate was 0.3 mL/min. Under theseconditions, the retention time of AA was 4.9 minutes. Peak areas for AAwere determined using a dominant potential for AAof 100 mV, andAAwasquantified from a calibration curve.

Cell volume determination

Estimation of cell volume was performed as previously described.28 Briefly,5 $ 106 cells were incubated for 60 minutes at RT in 200 #L incubationbuffer containing 1.0 mM 3-oxy-methyl-D-glucose and 5 #Ci (0.185 MBq)3H-3-oxy-methyl-D-glucose. Uptake was stopped by adding 2 #L of 2.0mM cytochalasin B (Sigma), which blocks glucose transport and preventsthe efflux of trapped glucose. The cells were then washed twice in ice-coldPBS containing 20 #M cytochalasin B and cell-associated radioactivitydetermined by scintillation spectrometry. Glucose reached equilibrium after60 minutes of incubation, therefore the amount of radioactivity accumu-lated inside the cells is in direct proportion to the intracellular volume. Thecell volume estimated for monocytes was 0.15 #L per 106 cells and 1.0 #Lper 106 U937 cells.

Vitamin C loading

Cells were washed twice in incubation buffer at pH 7.4, and DHA (Aldrich,Milwaukee, WI) was added. After incubation with DHA for up to 60minutes at 37°C, the cells were washed twice in serum-free RPMI.

Detection of ROS

Intracellular ROS in U937 were estimated by 2%7% dichlorofluoresceindiacetate (DCFH-DA) fluorescence.8 Cells were washed twice in Krebs-Ringer buffer (20.0 mM HEPES, 10.0 mM dextrose, 127.0 mM NaCl, 5.5mM KCl, 1.0 mM CaCl2, and 2.0 mM MgSO4 [pH 7.4]) and stained with0.1 #g/mLDCFH-DA (Molecular Probes, Eugene, OR) and 1.0 mMmaleicacid diethyl ester (Sigma) in Krebs-Ringer buffer. Fluorescence wasdetermined by flow cytometry after incubation at 37°C, and the data wereanalyzed using CELLQuest software.

Assessment of mitochondrial membrane potential (!")

U937 cells were washed twice with PBS, stained with 40 nM 3,3%-dihexyloxacarbocyanine iodide (DiOC(6)(3); Molecular Probes) with orwithout 100 #M carbonyl cyanide m-chlorophenylhydrazone (Sigma) as acontrol for membrane potential disruption, and incubated at 37°C in thedark for 15 minutes before determination of fluorescence by flow cytom-etry. These experiments and the ROS quenching studies were not performed

VITAMIN C INHIBITS FAS-INDUCEDAPOPTOSIS 337BLOOD, 1 JULY 2003 ! VOLUME 102, NUMBER 1

in monocytes because cell handling and the required trypsinization led toerratic results in these assays.

Cytochrome C release

Release of CytC into the cytosol was detected with a CytC enzyme-linkedimmunosorbent assay (ELISA) kit (MLB, Nagoya, Japan), following themanufacturer’s instructions. To obtain cytosolic fractions, cells werewashed twice with ice-cold PBS, resuspended in cold buffer (1.28 M NaCl,50.0 mM KCl, 50.0 mM MgSO4, 13.0 mM CaCl2, 0.5 M HEPES, 1.0 mMphenylmethylsulfonyl fluoride, 10% vol 2.5 M sucrose, 10.0 mM 1,4-dithiothreitol, 1.0 mM reduced glutathione, and 1% glycerol) and homoge-nized mechanically. This total cellular extract was centrifuged at 2000 rpmfor 3 minutes at 4°C and the cytosolic supernatant recovered and analyzedfor the presence of cytochrome C.

Caspase assays

Caspase activity was measured using the caspase-3 (Sigma), caspase-8, orcaspase-10 (R and D Systems) colorimetric assay kits, following themanufacturer’s instructions. Briefly, 1$ 107 cells were lysed and equalquantities of protein loaded in MaxiSorp 96-well plates (Nunc, Roskilde,Denmark). The caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Sigma)or caspase-8 inhibitor benzylloxycarbonyl-Ileu-Glu-Thr-Asp-fluoromethylketone (Z-IEDT-FMK; BioVision Research Products, CA) was added as acontrol at 20-#M final concentration. The plate was incubated at 37°Cand color development quantified by reading with an ELISA plate readerat 405 nm.

Recombinant caspase-8 in vitro assay

Recombinant caspase-8 (Alexis Biochemicals) was incubated with differentconcentrations ofAAand activity measured using the caspase-8 assay kit (Rand D Systems). Background was determined from triplicates of eachconcentration of vitamin C without the enzyme and subtracted from theexperimental results. The results were expressed as the percentage ofcaspase activity in the absence of vitamin C.

Caspase-8 Western blot

Cells were lysed in a buffer containing 30 mM Tris-HCl (pH 7.5), 150mM NaCl, 10% glycerol, and 1% triton and protease inhibitors. Celllysates were separated by SDS–polyacrylamide gel electrophoresis onprecast Tris-HEPES-SDS-polyacrylamide gradient minigels (4%-20%;Gradipore, Frenchs Forest, Australia) according to the manufacturer’sprotocols. The gels were then transferred onto a nitrocellulose mem-brane (Bio-rad Laboratories, Hercules, CA) in a buffer containing 25mM Tris (pH 8.3), 192 mM glycine, and 20% methanol in a semidrysystem (Bio-rad) for 15 minutes (15 V) at 25°C. Residual binding siteson the membrane were blocked by incubating the membrane inTris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.4] and 0.15 M NaCl),containing 5% nonfat dry milk and 0.1% Tween-20, for 2 hours at 25°C(blocking buffer). Membranes were incubated with monoclonal anti–caspase-8 (12F5; Alexis Biochemicals) or anti-&tubulin (H-235; SantaCruz Biotechnology, Santa Cruz, CA) Abs (1:1000 dilution) for 16 hoursat 4°C. The primary Ab was removed, and the blots were washed 3 timesin TBS. To detect Ab reaction, the blots were incubated for one hourwith anti–mouse or anti–rabbit IgG horseradish peroxidase (HRP)–conjugated Abs (Bio-rad), diluted 1:3000 in blocking buffer, washedwith TBS, and the protein bands were revealed using the enhancedchemiluminescence (ECL'plus) Western blotting detection system(Amersham Pharmacia Biotech, Piscataway, NJ) before exposure toBioMax film (Kodak, Rochester, NY).

Statistical analysis

The paired 1-tail distribution Student t test was applied to compare resultswith a criterion for statistical significance of a P value of .05 or less.

ResultsVitamin C inhibits FAS-induced apoptosis

FAS-R (CD95) was expressed in U937 cells and monocytes, andboth anti-FAS Ab and FASL induced apoptosis in these cells in adose-dependent manner (Figure 1). Incubation of U937 cells for 3hours with FASL induced up to a 3.5-fold increase in apoptosis,whereas incubation with anti-FAS Ab caused a 2-fold increase(Figure 1B-C). In monocytes, 3-hour treatment with FASL resultedin up to a 7-fold increase in apoptosis, whereas incubation withanti-FASAb resulted in a 4-fold increase (Figure 1E-F). An isotypecontrol Ab did not cause apoptosis in either cell type (data notshown). FASL was more efficient than anti-FAS Ab in inducingapoptosis in both U937 cells and monocytes (Figure 1).We investigated the effect of loading cells with vitamin C on

FAS-R–dependent apoptosis. U937 cells and monocytes transportvitamin C in the form of DHA through facilitative glucosetransporters.29,30 As measured by 14C-substrate uptake, there waslittle evidence for direct transport ofAA, whereas DHAwas readilytaken up by the cells (Figure 2A-D). We estimated the internalvolume of U937 cells at 1.0 #L/106 cells and monocytes at0.15 #L/106 cells from tritiated methylglucose equilibrium stud-ies.23,28 Based on this internal volume and cell-associated radioac-tivity, the intracellular accumulation of AA in U937 cells andmonocytes was estimated. The intracellular AA concentrationcalculated using this method was higher than values determined byHPLC electrochemical detection (Figure 2A,D). The small accumu-lation of vitamin C in cells incubated with AAwas probably due tooxidation of AA to DHA in air.The frequency of apoptosis induced by anti-FASAb or FASL in

U937 cells and monocytes was prominently reduced by cellularloading with vitamin C (Figure 2). Data regarding the intracellularAA concentrations reported in these experiments were based onHPLC electrochemical detection. The frequency of apoptosis

Figure 1. FAS-R ligation induces apoptosis in U937 cells and in monocytes. (A)U937 cells express the FAS-R (CD95) as determined by flow cytometry. (B-C) FASLand anti-FAS Ab induce apoptosis in U937 cells in a dose-dependent manner. (D)Fresh humanmonocytes express CD95 as determined by flow cytometry. (E-F) FASLand anti-FAS Ab induce apoptosis in monocytes in a dose-dependent manner. Thefrequency of apoptosis for U937 cells was determined by annexin V'/PI( staining;apoptosis in monocytes was determined by the frequency of events in the sub-G1region. The results are presented as the fold increase in the frequency of apoptosisover control cells left untreated for 3 hours and are representative of at least 5independent experiments for each cell type.

338 PEREZ-CRUZ et al BLOOD, 1 JULY 2003 ! VOLUME 102, NUMBER 1

induced by FASL (200 ng/mL) in U937 cells was 12% anddecreased to 7.8% in cells loaded with approximately 13 mMvitamin C (P ) .01) (Figure 2B). U937 cells incubated 3 hourswith 1 #g/mL anti-FAS Ab had a frequency of apoptosis of 5.8%,which decreased to 4.0% when the cells were loaded withapproximately 13 mM vitamin C (P ) .03) (Figure 2C). Thepercentage of reduction in apoptosis for U937 cells loaded with 13mM vitamin C compared with control ranged from 22% to 27%(mean, 25%) when the cells were challenged with anti-Fas Ab andfrom 26% to 50% (mean 35%) when the cells were treated withFASL. Vitamin C loading did not affect the expression of CD95 inthese cells (data not shown). Monocytes loaded with vitamin C alsohad a reduced frequency of apoptosis after treatment with anti-FASAb or FASL. FASL (100 ng/mL) induced 37.1% apoptosis inmonocytes, and when these cells were loaded with approximately14 mM vitamin C the frequency was reduced to 20.0% (P ) .04)(Figure 2E). Monocytes treated with 0.1 #g/mL anti-FAS Ab for 3hours showed 7.2% apoptosis, which was reduced to 5.1% in cellsloaded with approximately 14 mM vitamin C (P ) .047) (Figure2F). This represents a mean reduction in apoptosis of 31% to44%. The lowest intracellular concentration of AA used in theseexperiments was 4 mM, which approximates the concentrationof AA reported in monocytes obtained from blood of healthydonors (3 mM).31,32 AA (4 mM) conferred significant protectionfrom FAS-induced apoptosis in monocytes (P ) .02)(Figure 2E-F).

Vitamin C quenches ROS produced by FAS-R ligation

ROS levels are elevated in cells stimulated via the FAS pathway,and it has been suggested that ROS are an important component ofFAS signaling.8,33,34 Since vitamin C is a potent antioxidant andquenches ROS in cells under oxidative stress,35 we examined theeffect of vitamin C on ROS accumulation after FAS ligation.Incubation of U937 cells with anti-FASAb for 3.5 hours resulted inapproximately a 2.5-fold increase in cellular ROS, whereas loadingthe cells with vitamin C reduced ROS accumulation in the cells(Figure 3A). FASL incubation for 3 hours induced a 2-fold increase

in the levels of ROS, which were quenched by loading cells withvitamin C (Figure 3B). The production of ROS correlated with thefrequency of apoptosis in cells incubated with FASL in theseexperiments (data not shown).

Vitamin C reduces mitochondrial damage induced byFAS-R ligation

In apoptotic cells, the decrease in mitochondrial membrane differ-ential potential (!") occurs before chromatin condensation andDNA fragmentation,36 and precedes the enhanced release of ROSby the mitochondria.7 Since vitamin C prevented the increase incellular ROS levels induced by FAS ligation, we studied the effectof vitamin C on mitochondrial apoptosis in U937 cells culturedwith FASL by using the dye DiOC(6)(3). Cells with healthymitochondria have a high uptake of DiOC(6)(3) and therefore a highfluorescence signal, whereas cells with damaged mitochondriashow reduced fluorescence. Only 1.1% of control U937 cells haddull DiOC(6)(3) fluorescence (Figure 4A, upper left panel), whereas

Figure 3. Vitamin C inhibits ROS generated by FAS-R ligation. (A) U937 cellswere loaded with approximately 13 mM vitamin C prior to treatment with 1 #g/mLanti-FASAb for 3.5 hours and then stained with DCFH-DA to detect intracellular ROSby detection of dye fluorescence within 90 minutes by flow cytometry. (B) U937 cellswere loaded with vitamin C, treated with 200 ng/mL FASL for 3 hours, and stainedwith DCFH-DA for 30 minutes for fluorescence measurement. The results arepresented as the mean fluorescence intensity of DCFH-DA in arbitrary fluorescenceunits and are representative of 5 independent experiments.

Figure 2. Vitamin C decreases FAS-induced apopto-sis in U937 cells and monocytes. (A) Intracellularaccumulation of vitamin C in U937 cells after exposure to1.0 mM DHA or AA. Results are presented as theintracellular concentration of accumulated AA, deter-mined by cell-associated radioactivity. The standard de-viation is smaller than the symbols in the graphs, and wasalways less than 10% of the values for each point. (B-C)U937 cells loaded with vitamin C were treated for 3 hourswith 200 ng/mL FASL or 1 #g/mL anti-FAS Ab, andapoptosis was measured by annexin V'/PI( staining orby analysis of the sub-G1 region. The intracellular accu-mulation of AA was estimated by HPLC. (D) Accumula-tion of AA in monocytes after exposure to 0.1 mM DHAorAA, calculated by cell-associated radioactivity. (E-F)Monocytes were loaded with different amounts of vitaminC estimated by HPLC analysis before treatment with 100ng/mL FASL or 0.1 #g/mL anti-FAS Ab for 3 hours tomeasure apoptosis by analysis of the sub-G1 region.Asterisks indicate statistically significant differences(P ! .05) using the Student t test between control andcells loaded with vitamin C before challenge with FASL.These experiments were repeated 6 times for each celltype with similar results. A representative experimentis shown. Error bars represent the SD of triplicate values.


42% of U937 cells cultured with FASL for 3 hours incorporatedlittle DiOC(6)(3) (Figure 4A, upper right panel). We found thatloading the cells with vitamin C conferred protection fromFASL-induced mitochondrial damage, and these cells maintainedhigh DiOC(6)(3) fluorescence (Figure 4A, lower left panel). Controlcells treated with cyanide m-chlorophenylhydrazone showed amarked decrease in mitochondrial !" (data not shown). Caspase-8activation precedes the reduction in mitochondrial !" and thechange in mitochondrial membrane permeability.37 Thus, themitochondrial damage and the mitochondrial collapse should beprevented by incubation with the specific caspase-8 inhibitorZ-IETD-FMK. The addition of 0.2 #M Z-IETD-FMK inhibitedapproximately 60% of FASL-induced mitochondrial !" losscompared with nearly 100% inhibited with vitamin C (Figure 4A,lower panels). We therefore reasoned that vitamin C was inhibitingevents at both the mitochondrial level and upstream sites in theapoptosis cascade. The caspase-8 inhibitor Z-IETD-FMK and

vitamin C, however, prevented FASL-induced apoptosis in similarproportions (Figure 4B).Apoptotic cells release cytochrome C (Cyt C) from themitochon-

dria into the cytosol,38 and we sought to determine the effect ofvitamin C on this process. The amount of Cyt C found in thecytosolic fractions of cells incubated with FASL was nearly twicethat found in control cells (Figure 4C). In cells loaded with vitaminC, there was a marked reduction of Cyt C in the cytoplasmicfractions, even below the control levels. These results indicate thatvitamin C loading results in inhibition of the release of Cyt C frommitochondria after FAS-R ligation.

Vitamin C reduces FAS-induced caspase-3 andcaspase-10 activation

A consequence of mitochondrial damage during apoptosis isactivation of caspase-3,39 and we studied the effect of vitamin C onthe activation of this caspase. Activation of caspase-3 was inducedby FASL incubation in U937 cells and in monocytes (Figure5A-B). The activity of caspase-3 induced by FASL in U937 cellsloaded with vitamin C was modestly reduced (30%), although thedifference in caspase activity of cells with or without vitamin C wasstatistically significant (Figure 5A,P ) .039). In contrast, caspase-3activity induced by FASL in monocytes loaded with vitamin C wasmarkedly reduced (Figure 5B, P ) .02).The results suggested that caspase-3 in U937 could be activated

by pathways independent of mitochondrial signaling, and wetherefore analyzed the effect of vitamin C on alternative mecha-nisms for caspase-3 activation in U937 cells. Caspase-10 ishomologous to caspase-8 and is activated upstream in the apoptosiscascade and can activate caspase-3.40 We explored the effect ofvitamin C on caspase-10 activity in U937 cells treated with FASL.The activity of caspase-10 increased 36% after incubation withFASL and was reduced by about 50% when the cells were loadedwith vitamin C (Figure 6). We did not detect caspase-10 activity inmonocytes after treatment with FASL (data not shown), and to ourknowledge caspase-10 activity in monocytes has not been reported.

Vitamin C inhibits caspase-8 activation induced via FASL

ROS production, mitochondrial damage, and Cyt C release aredownstream consequences of initiator caspases such as caspase-8.37 We therefore addressed the hypothesis that vitamin C inhibitedFAS-dependent caspase-8 activation. In U937 cells incubated with

Figure 4. Vitamin C prevents mitochondrial damage induced by FAS-R ligation.(A) U937 cells were loaded with 13 mM vitamin C prior to 3 hours of treatment withFASL (200 ng/mL) and were stained with 40 nM DiOC(6)(3) for 15 minutes at 37°C.Control cells were treated with 0.2 #M Z-IEDT-FMK. DiOC(6)(3) fluorescence wasanalyzed by flow cytometry. The intensity fluorescence of DiOC(6)(3) is shown on thex-axis, and the forward scatter is represented on the y-axis. The numbers in the figureindicate the frequency of the population with low intensity of DiOC(6)(3) fluorescencefor each treatment and are representative of 7 independent experiments (except forthe experiment with Z-IEDT-FMK, which was performed twice). (B) Cells culturedunder the conditions described in panel A were stained with annexin-PI to assessfrequency of apoptosis. These results are the mean of 3 independent experiments,performed in duplicates, and are presented as the fold increase in apoptosis overuntreated control cells. (C) U937 cells were loaded with approximately 13 mM vitaminC prior to treatment with 200 ng/mL FASL. Cytosolic cell extracts were obtained andanalyzed by ELISA for the presence of Cyt C. The results show the normalized Cyt Ccontent for each experimental condition in arbitrary units based on the Cyt C cytosoliccontent of control cells and represent 2 independent experiments. Asterisk indicatesstatistically significant differences (P ) .039) using the Student t test between controland cells loaded with vitamin C before challenge with FASL. Error bars represent theSD of triplicate values.

Figure 5. Effect of vitamin C on caspase-3 activity. (A) U937 cells loaded withapproximately 13 mM vitamin C prior to treatment with FASL (200 ng/mL) were lysed,and the activity of caspase-3 in the cell lysates was determined as described in“Materials and methods.” (B) Monocytes loaded with approximately 14 mM vitamin Cprior to treatment with FASL (100 ng/mL) were lysed, and the activity of caspase-3 inthe cell lysates analyzed. The results are presented as the percentage increase incaspase activity compared with cells without treatment and are the mean of triplicatesamples. These results are representative of 3 independent experiments for each celltype. Asterisks indicate statistically significant differences (in A, P ) .03 and in B,P ) .02) in caspase activity between cells loaded with vitamin C and control.


FASL there was a 65% increase in caspase-8 activity, while vitaminC loading resulted in activity reduced to control levels (Figure 7A).A caspase-8 Western blot analysis indicated that cellular loadingwith vitamin C did not modify the expression of pro–caspase-8 inU937 cells (Figure 7B). In monocytes, FASL induced caspase-8activity by 130%, whereas vitamin C–loaded cells showed littleactivation (Figure 7C).The inhibitory effect of vitamin C on the activity of caspase-8 in

cells cultured with FASL could be due to a direct effect of vitaminC on the enzyme or to inhibition of enzyme activation. It has beenreported that vitamin C can directly inhibit certain enzymes.41,42 Asvitamin C is accumulated intracellulary in its reduced form,AA, weinvestigated if AA had a direct inhibitory effect on caspase-8activity. The activity of recombinant caspase-8 was assayed in thepresence of AA (1.0-20.0 mM). To inhibit the conversion of AA toDHA, 1,4-dithiothreitol was added to the reaction mixture. Therewas no significant effect of AA on the observed activity ofrecombinant caspase-8 (Figure 7D), while 20 #M Z-IEDT-FMKcompletely inhibited the activity of caspase-8 (not shown). There-fore, the data indicate that vitamin C is a potent inhibitor of theactivation of caspase-8 without a direct effect on enzymatic activity.These results indicate that vitamin C inhibits apoptosis induced

by FAS-R in monocytes and U937 cells largely by preventing the

activation of caspase-8. The molecular mechanisms of inhibition ofthe activation of caspase-8 are not known. We postulate that theactivity of caspase-8 is dependent on production of ROS at thelevel of the FAS-R and that the antioxidant properties of vitamin Ccould inhibit this early step of FAS-initiated apoptosis signaling.There is support for this notion in the recent finding that preloadingHL-60 cells with vitamin C inhibits the processing of pro–caspase-8 induced by hydrogen peroxide.43 Vitamin C also main-tains the integrity of the mitochondrial membrane, which isdisrupted during FAS signaling, and inhibits further downstreameffects. Thus, the results suggest that vitamin C inhibitsFAS-R–induced apoptosis in mononuclear phagocytes because ofits antioxidant capabilities.

DiscussionDietary vitamin C is critical for normal host defense, and vitamin Cis widely believed to enhance immune function when usedpharmacologically.18-20 Vitamin C may also have anti-inflamma-tory properties.21,44-46 There is now a substantial body of evidencedefining a central role for oxidative reactions in cell signal-ing12-15,47,48 and showing that antioxidants can inhibit importantsignaling pathways in immune cells.16,17 For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleu-kin-3 (IL-3) signaling involves ROS,17 and our evidence suggeststhat vitamin C inhibition of GM-CSF signaling occurs at the levelof JAK-2 kinase activation.46 In this study, we undertook tounderstand the role of vitamin C in FAS-mediated signaling forapoptosis in human monocytes and the monocytic cell line U937.Vitamin C circulates in the plasma as AA; however, it is

generally transported into cells as DHA through the facilitativeglucose transporters.23,24 Inside the cell, DHA is rapidly reduced toAA.24,28 In this way cells can accumulate high intracellularconcentrations of vitamin C. For example, the intracellular concen-tration of AA in mononuclear cells is reported to be about 3 mM,with circulating concentrations of AA in the range of 50 #M.31,32Specialized cells can transport AA directly through sodium/ascorbate cotransporters.25AA is often used in in vitro experiments,leading to confounding results due to the lack of direct transport ofascorbate and because AA acts as a pro-oxidant in the presence ofthe free transition metals ubiquitously found in tissue culture.26 Wetherefore used DHA to load cells with vitamin C24 and analyzed itseffect on the intracellular events mediating FAS-induced apoptosisin monocytic cells.

Figure 6. Effect of vitamin C on caspase-10 activity. U937 cells were loaded with13 mM vitamin C prior to FASL treatment (200 ng/mL) and lysed to determine activityof caspase-10. The results are presented as the percentage increase in caspaseactivity compared with cells without treatment and are the mean of triplicate samples.These results are representative of 2 independent experiments. Asterisk indicates astatistically significant difference (P ) .033) in caspase-10 activity of cells loaded withvitamin C compared with control.

Figure 7. Effect of vitamin C on caspase-8 activity. (A) U937 cells were loaded with 13 mM vitamin C prior to FASL treatment. The cell lysates were analyzed for caspase-8activity. Error bars represent the SD of triplicate values. (B) AWestern blot was performed to analyze the expression of pro–caspase-8 in U937 cells loaded with 13 mM vitaminC (arrow, upper panel). Detection of &-tubulin was used as control for protein loading (arrow, lower panel). (C) Monocytes were loaded with 14 mM vitamin C prior to FASLtreatment and the cell lysates were analyzed for caspase-8 activity. The results are the mean of triplicate samples expressed as the percentage increase in caspase activitycompared with untreated cells and represent 3 (U937) or 2 (monocyte) experiments. (D) The activity of recombinant caspase-8 (1.25 U/well) with and without vitamin C wasdetermined as detailed in “Materials and methods.” These results are expressed as the percentage of activity in relation to control wells (without vitamin C) and arerepresentative of 3 independent experiments. Asterisks indicate statistically significant differences (P ) .0005) in caspase activity of cells loaded with vitamin C beforechallenge with FASL.


The central observation of this work was a significant inhibitionof FAS-induced apoptosis by vitamin C loading in monocytes andU937 cells. We used pharmacologic concentrations of vitamin C toascertain the effect of vitamin C loading on the apoptotic process.The lowest intracellular concentration of AA used in these experi-ments was higher than the physiologic intracellularAA reported fornormal human monocytes (3 mM).31,32 Loading monocytes with 4mM AA conferred significant protection from FAS-R–inducedapoptosis. Viability of U937 cells and monocytes was not impairedby pharmacologic concentrations of vitamin C. The general cellularlevel of ROS increased after FAS-R ligation, and that increase wasabrogated by loading cells with vitamin C. The liberation of ROSduring the apoptotic process is largely due to uncoupling ofoxidative metabolism in the mitochondria.7 Others have noted thatN-acetyl-L-cysteine reduces ROS in monocytes after FAS liga-tion,8 possibly through an increase in glutathione. The release ofROS in apoptosis is preceded by a decrease in mitochondrialmembrane potential (!"),7 and we found that the mitochondrial !"was maintained in U937 cells loaded with vitamin C and stimulatedwith FAS, suggesting that vitamin C inhibited events occurringbefore injury to the mitochondria.FASL-induced aggregation of FAS-R is believed to cause

caspase-8 autoactivation, presumably related to proximity. VitaminC loading, however, prominently inhibited FASL-mediated activa-tion of caspase-8 in U937 cells and in monocytes. In a cell-freesystem AA did not inhibit the enzymatic activity of recombinantcaspase-8, and therefore these results suggest that vitamin Cinhibits the activation of caspase-8, rather than its enzymaticactivity.We postulate that local or “micro-oxidation” is required forcaspase-8 activation and that vitamin C inhibits that process byquenching ROS. This thesis implies that FAS ligation leads to thelocal generation of ROS, which is required for caspase-8 activa-tion. Indeed, a rapid and transient synthesis of oxygen radicalsfollowing FAS ligation has been reported that is independent ofcaspase activation,49,50 indicating that the initiation of FAS-mediated signaling is temporally associated with local generationof ROS. ROS production after receptor ligation has been noted forthe IL-1R, TNF*-R, and other cytokine receptor systems,12 andthere is abundant evidence for ROS-mediated signaling for growthfactor receptors.13-15,17,51Both vitamin C and the caspase-8 inhibitor Z-IETD-FMK

inhibited apoptosis to a similar degree. The loss of mitochondrial!" as a consequence of FAS-R ligation was inhibited by bothvitamin C and Z-IETD-FMK, although to a different extent, withalmost complete protection provided by vitamin C. The greaterstabilization of the mitochondrial membrane resulting from highconcentrations of vitamin C in cells challenged with FAS may bedue to vitamin C’s capability to quench ROS generated at thereceptor level and elsewhere in the cell. Nevertheless, this effect

did not result in more protection from apoptosis than the selectiveinhibition of caspase-8. This result is consistent with the notion thatthe major mechanism by which vitamin C prevents apoptosis isthrough the inactivation of caspase-8; however, the data also pointto an independent effect of vitamin C on mitochondrial membranestabilization.We studied the downstream consequences of inhibition of

caspase-8 activation by vitamin C by measuring the liberation ofCyt C into the cytosol and the subsequent activation of caspase-3.Cyt C was not detected in the cytosol of FAS-stimulated U937 cellsloaded with vitamin C, confirming that mitochondrial integrity waspreserved in cells protected with vitamin C. Also, a FAS-inducedincrease in caspase-3 activity in monocytes was not detected incells loaded with vitamin C. Vitamin C reduced basal levels ofcaspase-3 activity in these cells likely because there is some degreeof oxidatively-induced apoptosis occurring in vitro.52 In U937cells, however, vitamin C did not greatly inhibit caspase-3,maintaining 70% of FAS-induced activity. This phenomenon maybe due to the activity of caspase-10, which can activate caspase-353and which was active in U937 cells incubated with FAS (38%above basal).Beneficial effects of vitamin C on immune function are

frequently cited, and it is widely assumed that vitamin C enhancesimmunity via its antioxidant function. Blood monocytes migrate totissues where some mature into macrophages, which participateimportantly in the modulation of the adaptive immune system asphagocytes and antigen-presenting cells and by producing cyto-kines. Upon maturation into macrophages, monocytes down-regulate the expression of caspase-8 and up-regulate the expressionof FAS-associated death domain–like IL-1 beta-converting enzyme-inhibitory protein, a natural caspase-8 inhibitor. Macrophagestherefore are resistant to FAS-induced apoptosis.54,55 Before matu-ration, however, monocytes are highly susceptible to FAS-inducedapoptosis while resting and during bacterial phagocytosis.52,56 Herewe present evidence that the intracellular accumulation of vitaminC in monocytes causes resistance to FAS-induced apoptosis byinhibiting caspase-8 activation. A strong antioxidant such asvitamin C could prolong the active life of monocytes before theyreach a FAS-insensitive stage. Maintenance of monocyte viabilityby vitamin C may be a physiologic mechanism of vitamin C in hostdefense. Further, the pharmacologic use of vitamin C administeredas DHA57 could be a means to favorably affect host defense inpathologic states.

AcknowledgmentsWe appreciate the technical help of O. Borquez, C. Tat, andA. Pedraza.

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