Phagocytosis of Apoptotic Bodies by Hepatic StellateCells Induces NADPH Oxidase and Is Associated With

Liver Fibrosis In VivoShan-Shan Zhan,1 Joy X. Jiang,1 Jian Wu,2 Charles Halsted,3 Scott L. Friedman,4 Mark A. Zern,2 and Natalie J. Torok1

Hepatic stellate cell activation is a main feature of liver fibrogenesis. We have previouslyshown that phagocytosis of apoptotic bodies by stellate cells induces procollagen �1 (I) andtransforming growth factor beta (TGF-�) expression in vitro. Here we have further inves-tigated the downstream effects of phagocytosis by studying NADPH oxidase activation andits link to procollagen �1 (I) and TGF-�1 expression in an immortalized human stellate cellline and in several models of liver fibrosis. Phagocytosis of apoptotic bodies in LX-1 cellssignificantly increased superoxide production both in the extracellular and intracellularmilieus. By confocal microscopy of LX-1 cells, increased intracellular reactive oxygen species(ROS) were detected in the cells with intracellular apoptotic bodies, and immunohistochem-istry documented translocation of the NADPH oxidase p47phox subunit to the membrane.NADPH oxidase activation resulted in upregulation of procollagen �1 (I); in contrast,TGF-�1 expression was independent of NADPH oxidase activation. This was also confirmedby using siRNA to inhibit TGF-�1 production. In addition, with EM studies we showed thatphagocytosis of apoptotic bodies by stellate cells occurs in vivo. In conclusion, these dataprovide a mechanistic link between phagocytosis of apoptotic bodies, production of oxida-tive radicals, and the activation of hepatic stellate cells. Supplementary material for thisarticle can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2006;43:435-443.)

Liver fibrosis is a wound healing process that is elic-ited by various toxic stimuli.1,2 At the center of thefibrogenic process are the hepatic stellate cells

(HSC), which are normally quiescent and produce only

small amounts of extracellular matrix (ECM) compo-nents for the formation of the basement membrane.3 Ex-posure of HSC to soluble factors that include reactiveoxygen species (ROS) or cytokines from damaged hepa-tocytes, activated Kupffer cells, or endothelial cells4 leadsto a morphological and functional transition to myofibro-blast-like cells.5 One of the major profibrogenic mediatorsis transforming growth factor-beta1 (TGF-�1); it is up-regulated in activated HSC, and participates in multiplephases of HSC activation.6,7 The pathways leading toHSC activation are complex and require better elucida-tion.

Apoptosis or programmed cell death is also a commonfeature of chronic liver disease; the end-result of ongoingtoxic stimuli that result in the death of hepatocytes. Apo-ptosis results in the generation of apoptotic bodies (AB),which are subsequently cleared by phagocytosis. After en-gulfing AB, macrophages secrete TGF-�.8 Indeed, wehave previously demonstrated that HSC can phagocytoseAB in vitro, which triggers a profibrogenic response withupregulation of TGF-�1 and procollagen �1 (I) expres-sion.9 This study suggested for the first time that theremay be a direct link between liver injury, apoptosis ofhepatocytes, phagocytosis of AB, and activation of HSC.

Abbreviations: HSC, hepatic stellate cells; ROS, reactive oxygen species; TGF-�1, transforming growth factor beta1; AB, apoptotic bodies; EM, electron micros-copy; BDL, bile duct ligation; FDE, folate deficient/ethanol diet; UV, ultraviolet;SOD, superoxide dismutase; DPI, diphenylene iodonium; TAMRA, carboxytetra-methyl rhodamine succinimidyl ester; PBS, phosphate-buffered saline; RT-PCR,reverse transcription polymerase chain reaction; siRNA, small interfering RNA;DCFDA, 2�,7�-dichlorofluorescein diacetate.

From the Department of Internal Medicine, 1Division of Gastroenterology andHepatology, 2Division of Transplant, 3Division of Endocrinology, Clinical Nutri-tion and Vascular Medicine, UC Davis Medical Center, Sacramento, CA; and the4Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY.

Received April 27, 2005; accepted December 5, 2005.Supported in part by the American Liver Foundation Liver Scholar Award

(NJT), the UC Davis Health System Award (NJT), NIH 1 KO8 DK069765(NJT) and the NIH AA06386 to MAZ.

Shan-Shan Zhan and Joy X. Jiang contributed equally to this study.Address reprint requests to: Natalie Torok, M.D., UC Davis Medical Center,

Patient Support Services Building, 4150 V Street, Suite 3500, Sacramento, CA95817. E-mail: Natalie.Torok@ucdmc.ucdavis.edu; fax: 916-734-7908.

Copyright © 2006 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.21093Potential conflict of interest: Nothing to report.

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Phagocytosis also can activate NADPH oxidase,10 anenzyme primarily found in phagocytic cells, as well asfibroblasts and vascular smooth muscle cells,11,12 where itis termed “nonphagocytic” NADPH oxidase (NOX).NADPH oxidase is a membrane-bound enzyme that cat-alyzes the production of superoxide from oxygen andNADPH. It is dormant in the resting phagocyte but be-comes catalytically active when the cell is activated. Dur-ing activation, the cytosolic subunits, including p47phox,migrate to the cell membrane, where they associate withmembrane-bound subunits to assemble the active en-zyme. The intracellular organelles then fuse with thephagocytic vesicles, resulting in the delivery of superoxideoutward into the extracellular environment and inwardinto the phagocytic vesicle.13 HSC have recently beenshown to express NADPH oxidase,14-16 and thusNADPH oxidase may become activated in HSC duringphagocytosis. Therefore, NADPH oxidase could be a cru-cial component of the signaling cascade linking phagocy-tosis and fibrogenesis.

Here we show that NADPH oxidase activation occursafter phagocytosis of AB by HSC and that this appears tobe an essential linking process between phagocytosis andfibrogenic activation. In addition, electron microscopy(EM) and confocal microscopic studies of livers undergo-ing fibrogenesis from different etiologies indicate thepresence of phagocytosing HSC.

Materials and Methods

Human Biopsy Samples. Human liver biopsy speci-mens from patients with recurrent post-transplantationhepatitis C were used in the EM studies. Informed con-sent was obtained from each patient; specimens were ob-tained according to the guidelines of the approvedinstitutional IRB protocol. The study protocol con-formed to the ethical guidelines of the 1975 Declarationof Helsinki.

Animals. For the bile duct ligation (BDL) experi-ments, male Sprague-Dawley rats were used (CharlesRiver, Portage, MI). BDL was performed with the ratsunder pentobarbital anesthesia (60 mg/kg, intraperitone-ally). The common bile duct was ligated in two locations.Animals were sacrificed 1 week after surgery, and the liverspecimens were fixed and processed for EM studies. Con-trol animals underwent sham operations. Yucatan mi-cropigs (Sinclair Farms, Columbia, MO) on a combinedfolate deficient/ethanol diet with substitution of 40% ofcalories as ethanol17 (FDE) were also used for the EMstudies. After 14 weeks on this diet, the micropigs weresacrificed, and liver samples were obtained and processedfor EM. Stellate cell isolation from rats was performed

according to Geerts et al.18 The animals were housed infacilities approved by the National Institute of Health. Allprocedures were reviewed and approved by the AnimalWelfare Committee of the University of California Davis.

Cell Culture. A human immortalized HSC line LX-1was used.19 For positive control, a mouse macrophage-like cell line RAW 264.7 was used. To generate AB,HepG2 cells were used. LX-1, primary rat stellate cells,and RAW 264.7 were cultured in Dulbecco’s minimumessential medium (Invitrogen, Carlsbad, CA) with 10%fetal bovine saline, HepG2 cells were maintained inMEM, with 10% fetal bovine serum.

Preparation of Apoptotic Bodies. Apoptotic bodies(AB) were generated from HepG2 cells by exposing themto ultraviolet (UV) irradiation (100 mJ/cm2, 142 sec-onds), as described previously.9 AB were collected 48hours after the UV irradiation; at this time 80% of cellswere dead. Of this, 21% were necrotic and 79% apopto-tic.

Assay for Superoxide Production as the Measure ofNADPH Oxidase Activity. To evaluate superoxide re-lease, a superoxide dismutase (SOD)-inhibitable cyto-chrome c reduction assay was performed.20,21 LX-1 cellswere plated at a density of 106/mL and incubated with theAB in the presence or absence of diphenylene iodonium(DPI, 10 �mol/L, for 30 minutes) (Sigma-Aldrich, St.Louis, MO), an NADPH oxidase inhibitor.12 The super-natant was collected at 1 hour, 6 hours, and 24 hours afterincubation with the AB. Alternatively, to study intracel-lular release of superoxide, cell lysates were obtained ac-cording to standard methods using RIPA lysis buffer (atthe above time points). Cytochrome c (160 �mol/L)(Sigma-Aldrich) was added in the presence (100 U/mL)or absence of SOD (Sigma-Aldrich) and incubated for 20minutes at 37°C. The values of superoxide-dependentreduction of cytochrome c were obtained by subtractingthe absorbance readings at 550 nm of the samples withoutSOD from those with SOD. The amount of reducedcytochrome c was calculated,21 and the final result wasreported as fold changes above the control value. To vi-sualize superoxide production, LX-1 cells were incubatedwith a redox-sensitive dye 2�,7�-dichlorofluorescein diac-etate (DCFDA) (20 �mol/L; Molecular Probes Inc., Eu-gene, OR), for 30 minutes at 37°C after phagocytosis ofcarboxytetramethyl rhodamine succinimidyl ester(TAMRA)-labeled apoptotic bodies. To inhibit p47phoxmigration, the cells were preincubated with the inhibitorML-7 (75 �mol/L, for 30 minutes) (Calbiochem, SanDiego, CA). Image analysis and co-localization of signalswas performed by confocal microscopy (Zeiss, Thorn-wood, NY). The excitation and emission wavelengths for

436 ZHAN ET AL. HEPATOLOGY, March 2006

the DCFDA were 488 nm and 505 nm, and for TAMRA543 nm and 580 nm, respectively.

Immunohistochemistry. LX-1 cells were incubatedwith TAMRA-labeled AB for 6 hours, and then were fixedwith 4% paraformaldehyde for 20 minutes. Cells werepermeabilized by 0.1% Triton-X 100 [in phosphate-buff-ered saline (PBS) with 1% bovine serum albumin] for 30minutes followed by washing and then incubated with ananti-p47phox rabbit antibody (1:2000) (Upstate, NY) at4°C, overnight. After washing with PBS, a goat anti-rab-bit secondary antibody conjugated with FITC (1:300)was applied for 1 hour at room temperature. For tissuestaining, 4 �m micropig liver sections were boiled in ci-trate buffer for 5 minutes. Slides were then incubated in50 mmol/L NH4Cl/PBS at room temperature for 30minutes. Blocking was done with 2% bovine serum albu-min for 30 minutes, then incubation with first antibodies[E-cadherin 1:1,000, BD Transduction Laboratories (SanJose, CA)., anti-SMA 1:200, Epitomics Inc., Burlingame,CA)]. The secondary antibodies were Cy3-conjugated Af-finipure Goat Anti-Mouse, 1:1000, (Jackson ImmunoRe-search Lab. Inc., West Grove, PA), and Alexa Fluor 488anti-rabbit IgG 1:1000, (Molecular Probes, Eugene, OR;Invitrogen, Carlsbad, CA). The images were analyzed byconfocal microscopy.

Real-Time Polymerase Chain Reaction to StudyActivation of HSC. Real-time quantitative reverse tran-scription polymerase chain reaction (RT-PCR) was per-formed to evaluate the effects of superoxide productionon the expression of TGF-�1 and procollagen �(1) I.After incubation with AB for 48 hours, LX-1 cells wereharvested, total RNA extracted, using the TRIzol reagent(Invitrogen). One microgram of RNA was reverse-tran-scribed using dT15-oligonucleotide and Moloney murineleukemia virus reverse transcriptase. To inhibit NADPHoxidase activity, cells were preincubated with DPI. Real-time quantitative RT-PCR was done using a TagManSystem (Applied Biosystems, Foster City, CA), using thefollowing primers: TGF-�1; forward 5�-TCTCTC-CGACCTGCCACAGA-3�, reverse 5�-GATCGCGC-CCATCTAGGTT-3�, procollagen �(1) I; forward 5�-AAAGCAGAAACATCGGATTTGG-3�, reverse 5�-CGTGTCATCCCTTGTGCCGCA-3�. The resultswere expressed as a ratio of product copies/mL to cop-ies/mL of the housekeeping gene (GAPDH) from thesame RNA sample and PCR run.

RNA Interference. The target sequence to ratTGF-�1 small interfering RNA (siRNA) was AA-CAACGCAATCTATGACAAA (Qiagen, Valencia,CA), which corresponds to nucleotides 761-780 of ratTGF-�1 mRNA (NM #021578). For negative control,labeled (Alexa Fluor 488) or unlabeled siRNA was used

(Qiagen, Valencia, CA). Primary rat stellate cells werecultured and when they reached log phase growth approx-imately 60% confluence, were treated with 50 nmol/LsiRNA in RiboJuice (Novagen, Madison, WI), accordingto the manufacturer’s instructions. Random siRNA in thesame concentration was used as control.

Transmission Electron Microscopy. Specimens (hu-man liver biopsies, BDL rat liver, and micropig liver) werepre-fixed in Karnovsky’s fixative (2.5% glutaraldehydeand 2.0% paraformaldehyde) in 0.08 mol/L phosphatebuffer (pH 7.3).22 The tissues were secondarily fixed in1% freshly prepared osmium tetroxide and 1.5% potas-sium ferrocyanide in double distilled water for 1 hour.After being washed with cold double distilled water 3times, the tissue blocks were dehydrated through an as-cending concentration of ethanol (30%, 50%, 70%,95%, and 100%), and three changes of 100% ethanol,embedded in a 100% Epon-araldite resin, and polymer-ized at 70°C. The embedded blocks were sectioned usinga diamond knife on a Leica, Ultracut UCT (Leica Micro-systems, Deerfield, IL). Sections were placed on a coatedgrid, and stained with uranyl acetate and lead citrate be-fore being examined in the Philips 120 BioTwin ElectronMicroscope at 80KV (Fei Company, Hillsboro, OR)equipped with a Gatan/MegaScan, model 794/20 digitalcamera (Gatan Inc., Pleasanton, CA).

Statistical Analysis. All data represent at least threeexperiments and are expressed as the mean � SEM. Dif-ferences between groups were compared using ANOVAfor repeated measures and the post-hoc Bonferroni test tocorrect for multiple comparisons. A P value of less than.05 was considered statistically significant.

Results

Phagocytosis of Apoptotic Bodies Induces NADPHOxidase and Release of Intra- and Extracellular Su-peroxide Anions. To assess the impact of phagocytosis ofAB on superoxide production, AB were generated by ex-posing HepG2 cells to UV radiation (100 mJ/cm2), andincubated with LX-1 cells, an immortalized human HSCline, either in the presence or absence of diphenylene io-donium (DPI, 10 �mol/L for 30 minutes). DPI is a fla-voprotein inhibitor that attacks one of the components ofthe NADPH oxidase complex, and inhibits flavocyto-chrome b558, and thus inhibits NADPH oxidase activity.Based on the cytochrome c reduction assay, phagocytosisof AB significantly increased superoxide anion produc-tion and release in both the extracellular and intracellularmilieus (Fig. 1). The extracellular release of superoxideanion (Fig. 1A) increased by 17.4-fold (1 hour), 22.2-fold(6 hours), and 15.1-fold (24 hours). Preincubation with

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DPI inhibited the release of superoxide. Macrophageswere used as a positive control for phagocytosis-inducedsuperoxide production, whereas LX-1 cells without ABexposure served as a negative control to establish the levelof baseline superoxide anion production in LX-1 cells. Inparallel, intracellular production of superoxide was alsoincreased significantly after engulfment of AB, by 11.4-fold (1 hour), 27.4-fold (6 hours), and 22.6-fold (24hours) compared with the negative control. Again, DPIprevented superoxide production, the cells producingonly a baseline amount of superoxide. These biochemicalstudies verified the hypothesis that engulfment of AB byHSC indeed induces a significant production of superox-ide anion, with release into both the intracellular andextracellular milieus.

Confocal Microscopy for Detection of OxidativeRadicals in HSC Phagocytosing AB. We next used amorphological approach to co-localize labeled AB andoxidative radicals within LX-1 cells. After cells engulfedTAMRA-labeled AB, they were incubated with the redox-

sensitive dye, DCFDA, and the images were analyzed byconfocal microscopy (Fig. 2). An increase in DCF signalwas noted 6 hours after exposure to AB. Fig 2A depicts theco-localization of the DCF signal (green) and intracellularAB (red). Preincubation with DPI reduced the produc-tion of oxidative radicals, corresponding to our biochem-ical data (Fig. 2B). LX-1 cells without exposure to ABexhibited only low levels of DCF signal (Fig.2C). Macro-phages were used as a positive control, and after exposureto AB, intense fluorescent signal was noted in these cells(Fig. 2D). These data therefore corroborate our previousbiochemical results, showing that engulfment of AB inHSC induces production of oxidative radicals. DCFDA,however, is not specific for superoxide anion detectionand NADPH oxidase activation. Therefore, we comple-mented these findings with a morphological methoddemonstrating that phagocytosis of AB induces NADPHoxidase.

p47phox Translocation in HSC Phagocytosing AB.To specifically document activation of NADPH oxidase,TAMRA-labeled AB were added to HSC as described,then immunohistochemistry was performed to detect theintracellular location of the p47phox subunit of NADPHoxidase. On activation of the enzyme, the p47phox sub-unit migrates to the cell membrane (Supplementary Fig.Fig. 1. Phagocytosis of AB induces significant increase in superoxide

production and release into the (A) extra, and (B) intracellular milieu.This is measured at 1 hour, 6 hours, and 24 hours after the addition ofAB. Preincubation of the cells with the NADPH oxidase inhibitor DPI (10�mol/L for 30 minutes) prevents production of superoxide. Macrophageswere used as a positive control. LX-1 cells without AB were the negativecontrol. Four different experiments *P � .05. AB, apoptotic bodies; DPI,diphenylene iodonium.

Fig. 2. Phagocytosis of AB by HSC increases production of oxidativeradicals. Six hours after exposure to AB, an increase in DCFDA signalnoted in HSC as assessed by confocal microscopy. (A) Represents theDCFDA signal (green) and intracellular AB (red). (B) Preincubation with10 �mol/L DPI for 30 minutes decreased the production of oxidativeradicals. (C) Control HSC (without exposure to AB) with baseline DCFDAsignal. (D) Macrophages used as positive control. Bar � 10 �m. AB,apoptotic bodies; HSC, hepatic stellate cells; DCFDA, 2�,7�-dichlorofluo-rescein diacetate; DPI, diphenylene iodonium.

438 ZHAN ET AL. HEPATOLOGY, March 2006

1a; Supplementary material can be found on the HEPA-TOLOGY website: http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). Next, HSC werepreincubated with ML-7, an inhibitor of the myosin lightchain kinase, known to prevent migration of the cytosolicsubunits to the membrane, and therefore NADPH oxi-dase activation. Indeed, this inhibited translocation of thep47phox to the membrane, and resulted in only a cytoso-lic signal, similar to the control cells (Supplementary Fig.1b). In control HSC, without exposure to AB, p47phox iscytosolic (Supplementary Fig. 1c). These experimentstherefore confirmed that phagocytosis of AB by HSC re-sults in NADPH oxidase activation.

Phagocytosis of AB Induces Procollagen �1 (I) andTGF-�1 Expression. Expression of procollagen �1 (I)and TGF-�1 mRNAs was quantified after 48 hours incu-bation with AB. To inhibit NADPH oxidase, cells werepreincubated with DPI before addition of the AB. Expres-sion of procollagen �1 (I) increased significantly in cellsphagocytosing AB; this was decreased by inhibitingNADPH oxidase, suggesting that upregulation of theprocollagen �1 (I) in this case was NADPH-oxidase andsuperoxide-dependent (Fig. 3A). Conversely, althoughphagocytosis of AB induced upregulation of TGF-�1,this was not prevented by inhibiting NADPH oxidase,indicating that TGF-�1 signals by a different mechanismafter phagocytosis, which does not involve NADPH oxi-dase activation (Fig. 3B).

Inhibition of TGF-�1 Expression Does Not InhibitSuperoxide Production After Phagocytosis. RNA in-terference using siRNA was used to inhibit expression ofTGF-�1 in primary rat stellate cells. Incubation with 50nmol/L siRNA decreased TGF-�1 expression by 62% incells exposed to apoptotic bodies (Fig. 4A). Procollagen�1 (I) expression decreased by 30% after incubation withTGF-�1 siRNA (Fig. 4B). Superoxide production in-duced by phagocytosis decreased only by 13% after incu-bation with the TGF-�1 siRNA (Fig. 4C). The data fromthese experiments support our previous findings and aresuggestive of two different signaling pathways induced byphagocytosis of apoptotic bodies: an NADPH oxidase-dependent pathway, and an NADPH-oxidase indepen-dent pathway involving TGF-�1 activation.

Phagocytosis of AB in Different Liver FibrosisModels. To ascertain whether phagocytosis of AB byHSC occurs in vivo as well, we studied EM sections oflivers of rats 7 days after BDL, micropigs fed with FDEdiet for 14 weeks23 and human liver biopsy samples withrecurrent post-transplantation hepatitis C. The biopsysamples were from three different patients with stage 1fibrosis. Mechanistically distinct models (cholestatic, al-cohol-induced, and viral) were employed in different spe-

cies to establish that these findings were generic to allforms of fibrotic stage liver disease. Activated HSC losetheir fat droplets and have well-developed rough-surfacedendoplasmic reticulum and large Golgi apparatus. An in-creased amount of microfilaments is seen underneath theplasma membrane, a feature of myofibroblasts or acti-vated HSC.2,24-26 Activated HSC localize in close proxim-ity to collagen fibers. In vivo, engulfed AB becomephagosomes, and at a later stage, phagolysosomes that arereadily identified as double membrane-bound intracyto-plasmic organelles containing remnants of cell organelles,and chromatin derived from apoptotic cells. Phagocy-tosed necrotic debris morphologically would appear dif-ferent because the membrane integrity is lost duringnecrosis: therefore, the uptake does not occur as discreteparticles, and a double membrane would not be foundaround them.27 In all three models many HSC were seenwith phagosomes or phagolysosomes, and these cells dis-played condensed bundles of microfilaments, a typicalattribute of active HSC (Figs. 5-7). Moreover, the cells

Fig. 3. Real-time PCR of procollagen �1(I) (A) and TGF-�1 (B)expression in LX-1 cells. (A) Phagocytosis of AB induces upregulation ofprocollagen �1(I). This is decreased by the preincubation of LX-1 cellswith DPI (10 �mol/L for 30 minutes). (B) Phagocytosis of AB inducesupregulation of TGF-�1. This is not inhibited by the preincubation of LX-1cells with DPI, suggesting that upregulation of TGF-�1 occurs by anNADPH oxidase–independent pathway. The expression of procollagen �1(I) and TGF-�1 is normalized to the expression of GAPDH. LX-1 cellswithout exposure to AB used as controls. Three different experiments;*P � .05. PCR, polymerase chain reaction; TGF-�1, transforming growthfactor beta1; DPI, diphenylene iodonium; AB, apoptotic bodies.

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did not have fat droplets, but rather had well-developedrough-surfaced endoplasmic reticulum and large Golgiapparatus, and they also appeared to be adjacent to colla-gen bundles. Specimens from three rats after BDL, twomicropigs, and from the three patients with post-trans-plantation hepatitis C were studied. Reviewing three dif-ferent sections (each four grids) for every specimen,33.1% of HSC in human livers with HCV, 30.7% ofHSC in BDL rats, and 25.8% of HSC in micropigs fedwith FDE diet contained intracellular AB. In contrast,

HSC from control micropig liver exhibited the character-istic resting phenotype with lipid droplets, no increasedintracellular microfilaments, and intracellular AB rarelywere seen (data not shown).

Co-localization of Apoptotic Bodies and ActivatedStellate Cells in the Micropig Liver. To addresswhether the cells with intracellular apoptotic bodies arereally stellate cells, we performed double labeling experi-ments. Micropig liver sections were stained for alpha-smooth muscle actin and E-cadherin. E-cadherin hasrecently been recognized as a marker for intracellular ap-optotic bodies.28 E-cadherin is a membrane protein onepithelial cells, and it is retained on apoptotic bodies de-rived from these cells.28 Figure 8 shows several stellatecells (arrows) in the micropig liver with intracellular, E-cadherin–positive apoptotic bodies.

DiscussionOur data strongly support a model in which hepato-

cyte AB are engulfed by HSC, resulting in TGF-�1 andprocollagen �1 (I) upregulation and consequently fibro-genesis. Furthermore, NADPH oxidase activation and re-sulting superoxide production seem to be importantcomponents linking phagocytosis to procollagen �1 (I)upregulation. Indeed, p47phox�/� mice do not developalcoholic liver damage29 and do not develop significantfibrosis after bile duct ligation.30

We found that NADPH oxidase plays a critical role insignaling between phagocytosis and fibrogenesis in the liver.NADPH oxidase activation appears to induce both intracel-lular (into the phagolysosomes) and extracellular superoxiderelease. Release of ROS may affect neighboring cells in sev-eral ways; for example, it can induce neutrophil extravasationand activation, thereby further exacerbating the inflamma-tory response.31 ROS can directly damage cells by causingmembrane damage through lipid peroxidation32 and mito-chondrial dysfunction,33 as well as DNA damage either bydirect mutation by DNA strand breaks34 or by impairing thefunction of DNA repair enzymes.35,36 These effects may leadto further cell damage, culminating in apoptosis of neighbor-ing hepatocytes,37 thereby reinforcing the cycle of apoptosis,phagocytosis, and fibrogenesis. Our results also indicate thatthe generation of superoxide induced by the phagocytic pro-cess has a direct effect on fibrogenesis. Previously, it wasdemonstrated that superoxide directly affects procollagen�1(I) expression by a C/EBPB-dependent pathway.38 Also,superoxide production induced by the CYP2EI system wasshown to result in collagen 1 upregulation at the transcrip-tional level.39 Taken together, our data provide a novelmechanism connecting NADPH oxidase activation, pro-duction of superoxide, and upregulation of procollagen �1(I) expression.

Fig. 4. Inhibition of TGF-�1 expression in primary rat HSC by siRNA.Primary rat stellate cells were incubated with 50 nmol/L siRNA to TGF-�1and then exposed to AB. As control, transfection with random siRNA wasperformed. (A) Expression of TGF-�1 was inhibited by 62%. (B) Expres-sion of procollagen � 1(I) was inhibited by 30% by incubation withTGF-�1 siRNA. (C) Production of superoxide showed no significantchange after incubation with TGF-�1 siRNA. *P � .05, average of fourexperiments for (A), three experiments for (B), and four experiments for(C). TGF-�1, transforming growth factor beta1; HSC, hepatic stellatecells; siRNA, small interfering RNA.

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The clearance of AB is thought to be an evolutionarily-conserved process to limit inflammatory responses sec-ondary to tissue injury.8,40 One of the main mediators ofthe anti-inflammatory effect is TGF-�1, produced byphagocytic cells after ingestion of AB in several systems8,41

as well as in ours. It is also a potent profibrogenic cytokinein the liver.42 Therefore, excessive apoptosis during ongo-ing tissue injury of various causes could trigger the fibro-genic response through enhanced TGF-�1 production.This hypothesis may explain recent observations that thedetection of caspase activation in sera of patients withhepatitis C as a marker of increased apoptotic activityappears to be associated with a more aggressive fibrogenicprocess.43 We also observed TGF-�1 upregulation in ourculture model of phagocytosis. In contrast to collagen,however, this upregulation does not appear to be depen-

dent on NADPH oxidase activation and superoxide pro-duction; yet it appears to be a direct consequence of thephagocytic process itself. Phagocytosis-induced TGF-�1upregulation may reflect a different signaling pathway.This was confirmed by using siRNA to TGF-�1; inhibit-ing its expression did not affect phagocytosis-induced su-peroxide production (Fig. 4). This is in accordance withthe recent observations of Svegliati-Baroni et al.,44 show-ing neutralizing antibodies to TGF-�1 failed to inhibitacetaldehyde-dependent collagen gene transcription.44

Our data show a major effect on procollagen �1 (I) ex-pression after phagocytosis by both NADPH oxidase–dependent and independent pathways.

Although there are no firm data in this area, it is possiblethat integrins may be involved in the signal transductionduring phagocytosis leading to TGF-�1 upregulation. In

Fig. 5. HSC in rat liver after BDL. (A) Shows HSC with phagocytosed AB (box). (B) Enlarged image of the AB. Prominent rough ER is seen throughoutthe cell, characteristic of HSC. (C) Further enlargement of the same HSC shows microfilament bundles (arrow). HSC, hepatic stellate cells; BDL, bileduct ligation; AB, apoptotic bodies; ER, endoplasmic reticulum.

Fig. 6. HSC in pig liver specimen fed with folate-deficient/ethanol diet. (A) Several HSC, collagen bundles (arrowheads), one HSC withphagocytosed AB (arrow). (B) Enlarged image of the AB. (C) Further enlargement of the same HSC reveals prominent microfilament bundles (arrow).HSC, hepatic stellate cells; AB, apoptotic bodies.

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macrophages after phagocytosis, TGF-�1 induces cross-talkbetween ERK and p38 MAPK and this mediates selectivesuppression of pro-inflammatory cytokines.45 Therefore, viathese signaling pathways, TGF-�1 may also mediate a fibro-genic stimulus, independent of NADPH oxidase. This isconsistent with the findings that inhibition of the p38MAPK activity reduced type I collagen mRNA levels in pri-mary rodent HSC.46,47

Our findings demonstrate the presence of AB withinHSC in vivo in three different models of fibrosis, confirm-ing that phagocytic activity is indeed a physiologicallyrelevant characteristic of these cells. The phagosomes in-deed represent AB, because they are discrete particlessurrounded by a double membrane. By immunohisto-chemistry these intracellular particles are E-cadherin pos-itive, further supporting our data that these are AB.

Phagocytosis of necrotic particles could also occur in theliver; however, it is a different phenomenon. Their recog-nition, tethering, and phagocytosis are described as dis-tinctively different processes compared with thephagocytic process of apoptotic bodies,27,37 and necroticparticles are not surrounded by a double membrane. HSCare known to transdifferentiate from a resting fat-storingcell into a migratory, contractile myofibroblast on activa-tion. Phagocytic activity is yet another function that thesecells can perform, proving their impressive plasticity.

Howimportant is thephagocytosisbyHSCintheprocessofliver fibrogenesis? HSC are located in close proximity to hepa-tocytes; therefore, they may be more apt to phagocytose ABfrom hepatocytes than the more distant Kupffer cells. Further-more, phagocytosis by HSC seems to directly upregulate profi-brogenicgenes.Our in vivodata,detectingphagocytosingHSCin livers, provide evidence about the relevance of this finding inthe disease process. Thus, phagocytosis by HSC is likely to be acommon, universal pathway leading to fibrosis independent ofthe etiology of the liver disease.

Therefore, our studies indicate that phagocytosis of ABby HSC is an important process in fibrogenesis, linkingchronic liver injury and apoptosis of hepatocytes to fibro-genesis. We must develop a better understanding of howthe signaling pathways induced by phagocytosis result infibrogenesis. Preventing phagocytosis-induced signalingevents could be a promising approach to develop success-ful therapy for liver fibrosis.

Acknowledgment: The authors thank Grete Adamsonfor providing assistance with the EM studies and Dr. An-dreea Catana for expert assistance.

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Fig. 7. HSC in a liver biopsy specimen from a patient with post-transplantation hepatitis C. (A) HSC contains several intracellular AB (box), as wellas prominent microfilaments (arrow). (B) Enlarged image of the AB. It is surrounded by double-layered membrane (arrow) (C) Enlarged image of themicrofilaments. HSC, hepatic stellate cells; AB, apoptotic bodies.

Fig. 8. Co-localization of AB and HSC in micropigs fed with FDE diet.E-cadherin–positive AB (red, arrows) in HSC (ASMA, green) in themicropig liver. (A) phase image, bar � 10 �m. (B) Higher magnificationof the same field; bar � 20 �m. (C) and (D) Higher-magnificationimages of different fields (bars � 20 �m and 10 �m, respectively).HSC, hepatic stellate cells; AB, apoptotic bodies.

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