in vitro hepatitis bvirus infection of humanbonemarrow...

In Vitro Hepatitis B Virus Infection of Human Bone Marrow CellsJerome B. Zeldis, Hideo Mugishima, Howard N. Steinberg, Emanuel Nir, and Robert Peter GaleHarvard-Thorndike Laboratory, Harvard Digestive Diseases Center, Charles A. Dana Research Institute, Divisions of Gastroenterologyand Hematology/Oncology, Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215;Department of Medicine, Division of Hematology and Oncology, University of California at Los Angeles School of Medicine,Los Angeles, California 90024; Laboratory for Blood Morphology and Cytology, Kaplan Hospital, Rehovot, 76100 Israel

Abstract

Infection of humans with hepatitis B virus (HBV) frequentlyresults in suppression of hematopoiesis; in some cases this maylead to severe bone marrow failure. The mechanism wherebyHBV infection affects hematopoiesis is unknown. In vitro ex-posure of human bone marrow to HBVresults in a dose-depen-dent inhibition of erythroid (erythroid burst forming units,BFU-E; erythroid colony-forming units CFU-E), myeloid (col-ony-forming units-granulocyte macrophage CFU-GM), andlymphoid (CFU-(T-lymphocyficj-TL) hematopoietic stem cells.Inactivation or immunoabsorption of HBVfrom sera resulted inloss of HBV-induced inhibition of hematopoietic stem cells. Denovo gamma interferon was not detectable in the supernatantsof cultures of bone marrow cells with HBV. Antibodies to gammainterferon did not affect the suppression of hematopoietic stemcells by HBV. Hepatitis B surface antigen (HBsAg) was detectedby immune electron microscopy in nuclei of greater than 70% ofimmature hematopoietic cells including myeloblasts, normo-blasts, and lymphoblasts; granulocytes had mostly cytoplasmicHBsAg. Hepatitis B virus core antigen (HBcAg) was also de-tected in about 5% of HBV infected bone marrow cells by im-munoperoxidase staining. These data indicate that HBVcan in-fect hematopoietic cells and their progenitors, thus suggestinga wider range of tropism for HBV than previously reported.These results may provide a basis to study HBV infection invitro, and the effects of HBVon hematopoiesis.

Introduction

Despite the common belief that hepatitis B virus (HBV)' is ex-clusively hepatotrophic, recent evidence suggests that HBVcan

Portions of this study were presented at the 1st Meeting on MolecularBiology of Hepatitis B Virus, Cold Spring Harbor, NY, 1985; the Amer-ican Association for the Study of Liver Diseases, Chicago, 1984; 26thAnnual Meeting of American Society for Hepatology, Miami, FL, 1984,and were published in abstract form in 1984, Blood. 64:1079; and 1984,Hepatology. 4:1047.

Address reprint requests to Dr. Robert Peter Gale, Department ofMedicine/Hematology-Oncology, UCLACenter for Health Sciences, LosAngeles, CA 90024. Dr. Mugishima's current address is Department ofPediatrics, Nihon University School of Medicine, Tokyo, Japan.

Receivedfor publication 14 August 1985 and in revisedform 16 April1986.

1. Abbreviations used in this paper: BFU-E, burst-forming units, ery-throid; CFU-E, colony-forming units, erythroid; CFU-GEMM,CFU-

infect human hematopoietic cells. Romet-Lemonne and col-leagues found that a small percentage of bone marrow cells fromfour HBV-infected patients contained hepatitis B virus surfaceantigen (HBsAg) and hepatitis B core antigen (HBcAg). In ad-dition, they established an HBV-containing lymphoblastoid cellline from the bone marrow of a patient with HBVinfection (1).Others have identified HBVDNAsequences in the peripheralblood cells of patients with HBVinfection (2, 3) and in patientswith acquired immunodeficiency syndrome (AIDS) (4). Infectionof hematopoietic cells by HBVmay explain the moderate bonemarrow failure as well as the rare cases of aplastic anemia ob-served in patients with HBV infection (5). To determine themechanism whereby HBV inhibits normal hematopoiesis, westudied the effect of HBVon the growth of bone marrow pro-genitor cells in vitro. Normal bone marrow cells (MNC) wereexposed to HBV-containing sera and cultured to determine theeffect of HBV infection on five classes of hematopoietic stemcells including granulocyte/macrophage (CFU-GM), erythroid(CFU-E, BFU-E), T lymphocyte (CFU-TL), and pluripotent(CFU-GEMM) progenitor cells. Bone marrow cells exposed toHBVwere also analyzed by immune electron microscopy forHBsAg and by immunoperoxidase staining for HBcAg. Our re-sults suggest a direct effect of HBV on human bone marrowprogenitor cells and may provide a model for studying HBV-induced bone marrow suppression and HBVinfection of humanbone marrow cells.

Methods

HBVDNA. pHBV-1, a gift of Dr. Jesse Summers (Fox Chase CancerInstitute), contains the entire genome of an adw hepatitis B virus insertedin the Eco RI restriction site of PBR322. HBVDNAfree of PBR322vector sequences was purified by preparative electrophoresis of Eco RIdigested pHBV-l.

Sera. Serum specimens were obtained from healthy human volunteerswith no history of HBVexposure and who lacked serologic evidence ofprevious or current HBV infection. HBsAg and HBV DNApositiveserum samples were obtained from patients with chronic active hepatitis(3), asymptomatic carrier state (2), and acute hepatitis B (1). HBVDNAnegative samples were obtained from patients with non-A, non-B chronicactive hepatitis (1), acute hepatitis A (1), acute Epstein Barr virus hepatitis(1), hepatocellular carcinoma (1), and HBsAg positive postnecrotic cir-rhosis (1). Sera were filtered with 0.22-tm millipore-GU filter unit (Mil-lipore Corp., Bedford, MA) and stored at -200C.

pluripotent cells; CFU-GM, CFU-granulocyte/macrophage; CFU-TL,CFU-T lymphocytic cells; FACS, fluorescent activated cell sorter; FITC,fluorescein isothiocyanate; HBcAg, hepatitis B virus core antigen; HBsAg,Hepatitis B virus surface antigen; HBV, hepatitis B virus; 2ME, 2-mer-captoethanol; MNC,human bone marrow mononuclear cells; PAP, per-oxidase antiperoxide; PHA-LCM, phytohemagglutinin-stimulated leu-kocyte-conditioned medium; SSC, standard saline citrate.

Hepatitis B Virus Inhibition of Myeloid Differentiation 411

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/86/08/0411/07 $1.00Volume 78, August 1986, 411-417

Bone marrow. Human bone marrow was aspirated from the iliaccrest of normal donors for bone marrow transplantation or from ribsobtained at thoracotomy. Informed consent as approved by an Institu-tional Review Board was obtained from all donors. Bone marrow donorshad no serologic evidence of hepatitis or previous HBVinfection.

Bone marrow inhibition assay. Bone marrow mononuclear cells wereisolated by Ficoll-Hypaque density centrifugation and suspended in RPMI1640 supplemented with 10% heat inactivated (560C for 30 min) fetalcalf serum (FCS). 5 X 106 MNCin 50 MAl were incubated with 150 ,ul ofserial loglo dilutions of human sera at 370C in 5%CO2atmosphere for20 h. The cells were harvested, washed three times, and assayed for CFU-GM, CFU-TL, BFU-E, CFU-E, and CFU-GEMMas follows:

CFU-GM. 2 X 105 MNCwere assayed for CFU-GMin minimalessential medium (MEM)-alpha medium (Irvine Scientific, Irvine, CA),containing 0.3% agar, 20%FCS, and 10% phytohemagglutinin-stimulatedleukocyte conditioned medium (PHA-LCM). CFU-GMwere scored after10 d of culture with colonies consisting of greater than 40 cells.

CFU-TL. 1 X l0 MNCwere plated in eniched MEM-alpha mediumcontaining 20% FCS, 15% bovine serum albumin (BSA), 5 X l0-'2-mercaptoethanol (2ME), and 20% interleukin 2 (6). Colonies werescored after 7 d.

CFU-E, BFU-E, and CFU-GEMM.2 X IO' MNCwere cultured inIscove's modified Dulbecco's medium in the presence of 0.8% methyl-cellulose, 30%FCS; 10%PHA-LCM, 1%deionized BSA(Sigma, St. Louis,MO) 5 X l0-e 2MEand 2 U/ml erythropoietin (Green Cross, Tokyo,Japan) for BFU-E and CFU-GEMMand 0.3 U/ml erythropoietin forCFU-E. BFU-E and CFU-GEMMwere scored after 14 and 16 d ofculture, respectively. CFU-E were scored after 7 d of culture.

Each assay consisted of I ml of the respective mixture plated induplicate in 35-mm LUX tissue culture dishes (Miles Laboratories, Na-perville, IL) and incubated at 370C in 5%CO2and 95% humidity. Thenumber of colonies formed from bone marrow incubated with HBVwere compared to that incubated with normal AB+ control serum andthe data expressed as percent inhibition. Each assay was run in triplicateand the results are reported as the mean number of colonies for a par-ticular dilution of serum.

Inactivation of HBV+SeraHeat inactivation. I ml of sera was incubated in a 1000C water bath inan Eppendorf centrifuge tube for 1 min and then allowed to cool toroom temperature.

Urea dialysis. Sera were placed in a dialysis membrane and dialyzedagainst 4 Murea or phosphate-buffered saline (PBS) for 48 h with twochanges of dialysate, and then against three changes of PBS.

Immunoabsorption of HBsAg and HBVDNA. The HBsAg+ HBVDNA+serum was repeatedly immunoabsorbed to a mouse monoclonalanti-HBs Sepharose column until >95% of the HBsAg was removedfrom the eluate as judged by endpoint titration of serial dilutions in animmunoassay for HBsAg. The serum eluate was concentrated to theoriginal volume and sterilized by 0.22 Mmfiltration. The column waswashed with 10 vol of PBSand eluted with a pH 2.4 glycine-HCl buffer.The eluate was neutralized, dialyzed against PBS, and concentrated tothe original volume. The immunoabsorbed serum was demonstrated tohave no HBVDNAby dot-blot analysis and <95% of initial HBsAgconcentration. In contrast, the eluate had both HBsAg and HBVDNA.The mouse monoclonal anti-HBs Sepharose column was the gift of Dr.Jack Wands.

Absorption of sera with bone marrow cells. 5 X l05 MNCin RPMI1640 were incubated with an equal volume of sera for 2 h at 4VC. Thecells were pelleted at 400 g and the supernatant removed.

Monoclonal anti-gamma interferon antibodies. Monoclonal anti-gammainterferon antibodies with a neutralizing effect of 1,600 nU/unitgammainterferon were obtained from Dr. Jan T. Vilcek (7). Bone marrowcells were incubated with HBV+serum and a volume of antibody capableof neutralizing 40 U/ml of gammainterferon. After 20 h of incubation,the MNCwere washed twice and assayed for CFUJ-E and CFU-GMasdescribed.

HBVDNAassayThe sensitivity of this assay is 2.8 X 104 HBVDNAmolecules per 50 Mlserum. The preparation of the serum samples was performed as described(8), Biodyne Nylon Membrane (Pall-Ultra Fine Corp, Glen Cove, NY)was used instead of nitrocellulose. The membranes were prehybridizedand hybridized to 32P-labeled HBVDNAby standard techniques (9).The filters were washed at room temperature with 2 X standard salinecitrate (SSC) (1 X SSC= 0.15 MNaCl, 0.0 1 5 Msodium citrate pH 7.0)with 0.5% SDS for 5 min, then 2 X SSC with 0.1% sodium dodecylsulfate (SDS) for 15 min, then twice in 0.1 X SSC with 0.5% SDS at680C for 1 h each. The filters were air-dried and autoradiographed over-night and for 7 d at -70'C using Kodak X-Omat XARfilm (EastmanKodak Co., Rochester, NY) and a Cronex Lightning-Plus enhancingscreen (DuPont, Wilmington, DE). 32P-labeled HBVwere produced bynick translation using standard techniques (9). The 32P-labeled HBVDNAprobe (sp act 1-4 X 108 cpm/Mg DNA) did not cross hybridizewith pBR322 DNA.

Fluorescent activated cell sorter (FACS) analysis of humanbone marrow cellsAfter 20 h incubation with HBVor control serum, 1 X 106 MNCwerewashed twice with 0.1 %sodium azide (NaN2); then incubated with 200Ml of murine monoclonal anti-HBs antibody (1:100) or an identical classof murine hybridoma protein (5 Mg) without anti-HBs activity for 45min at 240C; then washed with PBS with NaN2. 200 Ml of fluoresceneisothiocyanate (FITC)-conjugated goat antimouse IgG antibody (Tago,Burlingame, CA) was added and the cells incubated at 4°C for 45 min.Cells were washed and resuspended in 1 ml of PBSwith NaN2. Sampleswere examined with FACS (Cytofluorograf 504, Ortho, Raritan, NJ).The murine monoclonal antibodies were the gift of Dr. Jack Wands.

Immune electromicroscopy of MNC1 X 1O0 bone marrow cells were incubated for 20 h with sera as describedabove. Samples were washed twice with RPMI 1640 medium and in-cubated with murine monoclonal anti-HBs (diluted 1:100) for 45 min.Cells were washed and fixed for 10 min with Karnovsky fixative (10)and analyzed. For surface labeling, cells were washed and incubated for1 h with 150 Mul of goat antimouse immunoglobulin linked to colloidalgold particles (size±20 nm) (GAMG20 Janssen Pharmaceutia N.V.B-2340, Beerse-Belgium, diluted 1:1), washed and refixed for 24 h or moreprior to myeloperoxidase staining (11). For intracellular labeling, cellswere treated with Karnovsky fixative for 24 h and embedded. Ultra-thinsections were labeled with GAMG-20for 15 min and stained with uranylacetate.

Preparations were examined with an electron microscope. Black grainsappeared in cells containing HBsAg.

Immunoperoxidase detection of HBcAg1 X 106 MNCwere incubated with sera as described above and thentransferred to a microscope slide using a cytospin (Shandon-Southern,England). The cells were then stained for HBcAg by the protocol andreagents of the DAKOperoxidase antiperoxide (PAP) kit for HBcAg(DAKO Corp., Santa Barbara, CA) (12). Samples were air-dried, incu-bated with 3% H202 in methanol for 5 min to inactivate endogenousperoxidase. Slides were rinsed with 0.05 MTris buffer (pH 7.6) andincubated with rabbit anti-HBc or nonimmune rabbit serum for 20 min.Cells were washed again with Tris buffer and incubated with a swineantirabbit immunoglobulin for 20 min, rinsed, and incubated with solublehorseradish peroxidase rabbit anti-horseradish peroxidase complex for20 min. Samples were incubated with the peroxidase substrate containing3-amino-9-ethylcarbazole, 0.1 Macetate buffer and 0.3% H202 beforecounterstaining with hematoxylin to demonstrate nuclei.

Results

Co-culture of human bone marrow mononuclear cells (MNC)with HBV containing sera resulted in a dose-dependent inhi-

412 Zeldis, Mugishima, Steinberg, Nir, and Gale

bition of the five types of stem cells studied. For example, ex-posure of bone marrow cells to an undiluted HBVDNApositiveserum resulted in suppression of CFU-GM, BFU-E, and CFU-TL colony formation by 65±3, 46+6, and 49±3%, respectively,compared with controls (Fig. 1). Although the absolute numberof colonies in each assay varied for different bone marrow spec-imens, percent inhibition was internally consistent for each ex-periment. Incubation of bone marrow cells with serial logl0 di-lutions of HBVcontaining sera resulted in the gradual loss ofthe HBV-mediated suppression. Inhibition of CFU-GMandCFU-TL was still observed using sera diluted 1:100. Other serathat had higher concentrations of HBV DNA inhibited thenumber of CFU-GMand BFU-E by as much as 100% comparedto control (data not shown). CFU-E was also inhibited by HBVDNA+sera. Undiluted HBVDNA+sera from three patientsinhibited CFU-E by 38±7, 72±3, and 100±0%. In a preliminarystudy, CFU-GEMMwas also found to be inhibited by 75% ofcontrol by undiluted HBVserum. (Exposure to control serumgave 8±2 colonies per 2 X I05 MNC,while undiluted HBVseraproduced 2±1 CFU-GEMMper 2 X 105 MNC.)

The effect of preincubation time on subsequent BFU-E col-ony formation by two different HBVDNAcontaining sera isillustrated in Fig. 2. Although no inhibition of BFU-E was ob-served after 3 h of preincubation with undiluted serum 1, com-plete suppression was seen after 20 h preincubation of undilutedserum but not serum diluted 1:100. Serum 2, which containeda tenfold higher concentration of HBVDNA, completely inhib-ited BFU-E after 3 h incubation when undiluted, but not at a1:1,000 dilution. Similar results were obtained with CFU-GMand CFU-E.

Nine HBVDNAnegative sera from patients with chronicnon-A, non-B hepatitis, hepatitis A, Epstein-Barr virus-relatedhepatitis, hepatoma and healthy normal volunteers had no sig-nificant effect on hematopoietic stem cells (<20% inhibition).In these experiments, significant inhibition of CFU-GMassayby >30% correlated with the presence in serum of HBVDNAsequences. CFU-GMwere inhibited by all HBVDNApositivesera whether or not circulating mononuclear cells from the pa-tient contained HBVDNA(Table I).

To determine if the HBVvirion is responsible for inhibitionof CFU-GM, HBVDNApositive sera were treated to inactivate

80

z T

0

00

1:1 1:10 1:102 1 :103DILUTION

Figure 1. Effect on hematopoietic colony formation of incubatingMNCwith serum containing HBVDNA. The data charted are themean inhibitions and SEMof colony formation produced by exposureto a HBVDNA+serum compared to the number of colonies of anegative control serum (see Methods). The serial log10 dilutions of serawere made in RPMI 1640 media. The number of colonies for the con-trol serum for CFU-GMwas 224±8 (95% CI) per I05 cells; for BFU-E, 230±4; and for CFU-TL, 1,228±41.

A. 3 HourPreincubotion

w

LimLL0z0co

za:.

B. 24 Hour Preincubation

[H RH

[Ii 1H: 1:102 1:1 1:102 1:IO3

#1 #*2

Figure 2. The effect of two HBVDNA+sera on BFU-E after a 3- and24-h preincubation. MNCwere incubated with AB+ control serum orHBVDNA+sera for 3 or 24 h; washed four times and plated into theBFU-E assay as described in the methods. Plotted are the mean valuesof three simultaneous determinations and the SEM. The sera used inthe 3 h incubation were diluted 1:1. The mean colony number and the95% CI for the 3-h control was 13.9±3.2 per 105 cells and for the 24-hcontrol, 58.6±13.4.

or remove infectious HBVby three techniques: (a) heat (1 minat 980C); (b) dialysis against 4 Murea; and (c) removal of HBVusing a mouse monoclonal anti-HBs affinity column. The effectof these treatments on HBVmediated suppression of CFU-GMare shown in Fig. 3. Both heat inactivation and urea dialysis,which eliminate infectivity of HBV, removed the inhibitory effect

Table I. Inhibition of CFU-GMby Sera

HBVDNAin

Mononuclear Inhibition ofPatient Diagnosis Serum* cells+ CFU-GMt

I HBVCAH + + 37±62 HBVCAH + + 37±63 HBVCAH + - 71±34 HBVCarrier + - 37±105 HBVCarrier + - 40±66 HBVCarrier + - 30±57 HCC/HBsAg- - - 17±78 NANBCAH - - 7±69 HAVHepatitis - - 2±7

10 EBV Hepatitis - - 10±61 1 Cirrhosis/HBsAg+ - ND 15±7

* HBVDNAdetermined from 50 ,dl of sera by a dot blot techniquedescribed in Methods. +, HBVDNApositive; -, no detectable HBVDNAafter 7 d autography. +HBVDNAwas assayed by extractingDNAfrom mononuclear fraction of a Ficoll-Hypaque fraction of pe-ripheral blood; shearing the DNA, electrophoresing the DNAin 0.7%agarose, Southern blotting to nitrocellulose (NC) and exposing the NCfilter to radioactive HBVDNAprobes (2).f Inhibition of <20% was considered not statistically significant. Eachvalue is the mean of three measurements with the same undilutedserum. Listed are the percentage inhibition and the standard error ofthe mean. The control serum produced 157±5.6 colonies per 2 X 105cells.


100 - Figure 3. Percent inhibition of CFU-GMby treating a

serum with heat, urea dialysis, and immunoabsorp-80 - tion. This serum was treated by several techniques de-

0 scribed in Methods. After each treatment, the serumu. 60 - T was assayed for its effect on the CFU-GM. "HBVz F [17] DNA+serum" refers to an untreated HBVDNA+°40 - serum obtained from a patient with chronic active

II IIhepatitis. This serum was heat inactivated, dialyzed

Z 20- against 4 Murea (urea dialysis) or PBS (PBS dialysis);

0 Li-. Ur~eeLi Li T Liimmunoabsorbed (HBV DNA+depleted), immuno-

HBV DNA+ Heat Urea PBS HBV DNA+ HBV DNA+ MNC purified (HBV DNA+eluted), and preabsorbedSerum Inactivation Inactivation Dialysis Eluate Depleted Absorption against MNC(MNCabsorption). The results shown

are the percent inhibition ±SEMof CFU-GMproduced by 1:1 dilution of the samples. The control serum produced 205±14 (95% CI) coloniesper 2 X l0 MNC.

of HBVDNApositive sera on CFU-GM. In contrast, dialysisof HBVDNA+ sera against PBS did not suppress CFU-GM.Serum depleted of HBVDNA following passage through ananti-HBs immunoabsorption column no longer inhibited CFU-GM. The HBVDNAcontaining eluate recovered from the col-umn markedly suppressed CFU-GM. The inhibitory activity ofHBVcontaining sera thus coincided with HBV infectivity andthe presence of HBsAg immunoreactivity and of HBVDNA.

To investigate whether antibodies to HLA might accountfor inhibition of CFU-GM, HBVDNA+serum was absorbedagainst bone marrow cells from the same donor used in the stemcell assays. Preabsorption did not abrogate the inhibitory effectof HBVDNA+serum on CR1-GM. Additional evidence againsta role of anti-HLA antibodies is the finding that the same seruminhibited progenitors from individuals of different HLA typesand that different HBVDNA+sera inhibited bone marrow froma single donor. As indicated, HBVDNA- sera from individualshad no effect on CFU-GM.

Gammainterferon can inhibit CFU-GMin vitro (13) andmay play a role in the suppression of hematopoiesis in somecases of aplastic anemia (14, 15). The HBV DNA+ sera westudied typically contained 5-15 U/ml of gammainterferon vs.5 U/ml in normal sera (as determined by the VSV/WISH assay[16]). It was therefore necessary to determine whether the ob-served inhibition of bone marrow progenitor cells with HBVDNA+sera was mediated by gammainterferon. Incubation ofHBVDNA+serum with a monoclonal anti-gamma interferonantibody prior to addition of bone marrow cells did not affectthe serum's ability to inhibit CFU-GMor CFU-E colony for-mation (Fig. 4). Furthermore, preincubation of bone marrowcells with 10 to 1,000 U/ml gammainterferon for 20 h did notalter the number of CFU-GM(data not shown). Finally, theconcentration of gammainterferon recovered from these culturesdecreased with incubation time consistent with the conclusionthat gammainterferon was not induced during culture of bonemarrow cells with HBV.

Having established that HBVcan inhibit hematopoiesis invitro, we next determined if HBV-associated antigens could bedetected in bone marrow cells exposed to HBVcontaining sera.Bone marrow was incubated with HBVDNA+ sera, washed,incubated with murine anti-HBs antibodies or hyperimmuneanti-HBs sera, and examined for indirect immunofluorescenceby fluorescence microscopy or using a FACS. 1-2% of MNCco-cultured with HBVcontaining sera exhibited surface fluo-rescence for HBsAg by indirect immunofluorescence; cells eitherincubated with normal control sera or stored with sera without

anti-HBs activity were negative. FACS analysis indicated thatthe HBV-infected bone marrow cells incubated with anti-HBsmurine monoclonal antibodies contained a substantial popu-lation (5%) of brightly fluorescent positive cells. Only MNCin-cubated with HBV-containing sera and stained with anti-HBsmonoclonal antibody exhibited fluorescence in a size populationof cells consistent with hematopoietic cells. No fluorescence wasobserved either in MNCincubated with HBVDNA- ABserumand stained with anti-HBs or in MNCincubated with HBVDNA+sera and stained with control monoclonal antibody withno anti-HBs activity.

To better define the population of MNCcontaining viralantigens, immunoelectron microscopic analyses of bone marrowcells cultured with and without HBVDNA+serum were per-formed. Surface labeling with murine monoclonal anti-HBsantibodies was only observed in <10% of the myeloperoxidase-positive myeloblasts and myelocytes. Lymphocytes and eryth-rocyte precursors did not have HBsAg on their surface. In con-trast, intracellular labeling with monoclonal anti-HBs antibodieswas detected in nuclei and cytoplasm of all nucleated cells ex-amined. The labeling pattern of the nucleus and cytoplasm wasdifferent among the various hematopoietic cell types (Table II).Greater than 70% of nuclei of myeloblasts, normoblasts, andlymphocytes were HBsAg+; granulocytes contained predomi-

r (,0

z0U 40Li.0z0

mn 20Tz

a0

HBVDNA+ HBV DNA* ControlSerum Serum Serum

+ +Anti-IFNy Anti -IFNr

Figure 4. Monoclonal anti-gamma interferon antibodies do not inhibitthe HBVDNA+serum's suppression of CFU-E and CFLJ-M. Anexcess of monoclonal anti-gamma interferon antibody (Anti-INFy)was incubated with MNCand HBVas described in Methods. Themean number of colonies of the control were 456± (95% CI) for CFU-E and 47±3 (95% CI) for CFU-GM.


Table II. Distribution of HBsAg in Cytoplasm and Nucleus ofDifferent Hemopoietic Cells as Determined by a Gold Label*

Cell Cytoplasm labeling Nuclear labeling

Myeloblasts 20 80Promyelocytes and myelocytes 50 50Metamyelocytes 55 45Granulocytes 76 24Normoblasts 30 70Lymphocytes 15 85Red blood cells 0 0

* MNCincubated with control sera instead of HBVDNA+sera didnot have two or more gold particle clusters. Data are expressed as thepercentage of 50 cells of each type of bone marrow cell that containedclusters of gold particles.

nantly cytoplasmic HBsAg. Mature erythrocytes which lack nu-clei, contained no HBsAg. Fig. 5 illustrates cytoplasmic and nu-clear HBsAg detected by immunoelectron microscopy. Althoughthe density of HBsAg detected in the MNCwas low, results werereproducible. Furthermore, MNCpreincubated with sera fromhealthy controls or MNCincubated with a control murine my-eloma protein were negative. Intracellular inclusions resemblingvirions or core particles were not seen on electronmicroscopyof MNCincubated for 24 h with HBV.

Bone marrow cells incubated with HBVDNA+sera were

assayed for HBcAg by the PAPtechnique. HBcAg was detectedin MNCincubated with HBV+sera but not in controls. About5%of cells stained positive for HBcAg.

Discussion

HBVinfections are associated with mild suppression of hema-topoiesis and rarely with cases of severe aplastic anemia. Ourinvestigations of the in vitro effects of HBVon colony formationmay provide a model to investigate these phenomena. Our stud-ies indicate that suppression of hematopoiesis in vitro is directlyrelated to an effect of HBVon hematopoietic progenitor cells.Exposure of bone marrow cells to HBVDNA+sera results inthe in vitro suppression of erythroid (CFU-E; BFU-E), myeloid(CFU-GM), and lymphoid (CFU-TL). Preliminary data alsosuggest an effect on pluripotential (CFU-GEMM) stem cellgrowth. Viral inactivation studies (Fig. 3) and serial dilution ofHBVDNA+sera suggest that the inhibitory effect of HBV ismediated by infectious virions ("Dane particles"). Sera thatlacked HBVDNAbut contained HBsAg, corresponding to thenoninfectious "22-nm particles" and "pencil shapes," had noeffect on bone marrow progenitors. Inactivation of HBVDNA+sera by heat or 4 Murea treatment that eliminate the infectivitybut not the immunogenicity of HBV, resulted in the loss ofCFU-GMinhibition. Removal of viral particles by immunoab-sorption using an anti-HBs column similarly eliminated CFU-GMinhibition. Exposure of HBVDNA+serum to anti-gammainterferon antibodies did not alter inhibition of CFU-E and CFU-

Figure 5. Immune electron microscopy of bonemarrow mononuclear cells incubated withHBVDNA+serum. (A) Myeloperoxidase posi-tive myelocyte with gold particles on the sur-face (X 4,500); (B) Myeloblast with nuclear la-beling (X 14,000); (C) Neutrophil with both cy-toplasmic and nuclear labeling (X 4,500); and(D) normoblast with nuclear labeling(X 20,000). Only MNCincubated with HBVand stained with murine monoclonal anti-HBsantibodies had clusters of two or more goldparticles. MNCincubated with HBVandstained with nonspecific monoclonal antibodyor MNCincubated with control sera andstained with monoclonal anti-HBs antibodiesdid not have clusters of gold particles.


GM, suggesting that gammainterferon does not play a role inthis process. These results are consistent with the hypothesis ofa direct effect of HBVon hematopoietic stem cells.

Demonstration of both HBsAgand HBcAg in the cytoplasmand nucleus of mature and immature bone marrow mononuclearcells is consistent with the hypothesis that the virus is able toinfect hematopoietic cells. FACS analysis demonstrated that-5% of MNChave HBsAg on their surface. This may be due

to HBVbinding to receptors on the hematopoietic cells. Whetherthere is viral replication or active transcription in these cells isunknown but is presently under study. The absence of intra-cellular virus and core particles on electron microscopy makesit unlikely that active viral production was completed duringthe 20-h incubation of bone marrow cells with HBV. Recentwork with the duck hepatitis virus, another hepadna virus, in-dicated that at least 6 d are needed before viral assembly occurs.These studies and other recently reported investigations extendthe range of tropism of HBV in man (1-3, 17, 18). Recently,Laure et al. (4) demonstrated that T cell clones derived fromAIDS patients contain HBVDNA.

In addition to HBV, other types of acute viral infections,including non-A, non-B hepatitis, hepatitis A, and Epstein-Barrvirus hepatitis are associated with bone marrow suppression.However, sera from patients with these infections have no effecton bone marrow progenitor cells. Their inability to suppressbone marrow progenitor cells may be related to the presence oflow concentrations of virus. Indeed, it has been shown that duringthe clinical phase of acute hepatitis A infection, the serum con-centration of virus is very low. The lack of an inhibitory effectby sera of patients with other infectious agents may be analogousto the gradual loss of inhibition observed with the serial dilutionof HBVDNA+sera.

In their study of a parvovirus-like agent and its effect onhematopoiesis, Young et al. reported that acute phase sera frompatients with HBV-associated hepatitis failed to inhibit bonemarrow progenitor cells in vitro (19). This lack of effect may beattributed to the exposure of bone marrow cells only to a 1:100dilution of sera. As demonstrated in Figs. 1 and 2, the inhibitoryeffect of HBVon hematopoiesis may be readily diluted out ata 1:100 concentration of sera containing only a moderate amountof HBVDNA. Sera that contain the parvovirus-like agent de-scribed by Young et al. (19) inhibit CFU-E at greater dilutionsthan sera that contain HBV. The greater potency of the par-vovirus-like agent containing sera may be a reflection of a higherconcentration of active virus in serum as compared to thatof HBV.

Gammainterferon has been demonstrated to be producedby the peripheral blood mononuclear cells of patients who haveaplastic anemia. Some patients with aplastic anemia also havevery high concentrations of gamma interferon in their serum.This has led to the hypothesis that gammainterferon may be amediator of the hematopoietic suppression that occurs in aplasticanemia (14, 15). However, our results suggest that HBVsuppression of colony formation is not mediated by gammain-terferon. The addition of monoclonal anti-gamma interferonantibody did not affect HBVsuppression of CFU-E and CFU-GM. It should be emphasized furthermore, that bone marrowcells were preincubated with HBVsera prior to cell culture, andthus only exposed to low levels of gammainterferon (10-20 U/ml) for 18 to 24 h. Wehave demonstrated that preincubationof bone marrow cells with gammainterferon up to 1,000 U/ml

did not affect colony formation. These results are similar to thosereported by Toretsky et al. (20) who showed that transient ex-posure to bone marrow cells to gamma interferon did not sig-nificantly suppress colony formation. A dose-dependentsuppression of colony formation was only observed when gammainterferon was added in continuous culture to bone marrowcells (unpublished observations, 13, 15, 20).

Though both alpha and gamma interferon are induced byacute viral infections, a putative role for alpha interferon in HBVsuppression of hemopoiesis was not studied. It should be notedthat the dose-response suppression of colony formation by alphainterferon is similar to gammainterferon for CFU-GEMM,BFU-E, and day 7 CFU-GM. Day 14 CFU-GM, which were assayedin the present study, were significantly less sensitive to alphainterferon (13). Thus, while alpha interferon's role in thesuppression of hematopoiesis observed by HBVin vitro cannotbe excluded, it is unlikely.

Since hematopoietic cells are easily obtained, and becausethere are extensive in vivo and in vitro data regarding theirgrowth, progenitor-progeny relationships, and growth-factor re-quirements, the effects of HBVon bone marrow progenitor cellscan be readily studied in vitro. To date, there is no in vitromodel to examine HBV infection of human hepatocytes andhepatoma cells. The infection of human bone marrow cells withHBVthus may provide a model to study the cellular and mo-lecular events associated with HBV infection.

Acknowledgments

Dr. Susan Wormsley (Cytokinetics, La Jolla, CA), performed the cyto-fluorographic analyses; Dr. Annette Maluish (National Cancer Institute/FCRC, Frederick, MD), the analysis of gamma-interferon; Dr. JackWands (Massachusetts General Hospital, Boston, MA) donated the mu-rine monoclonal antibodies and the murine monoclonal anti-HBs Se-pharose column; and Mr. T. Shimron assisted with the electron micros-copy. Dr. Robertson Parkman provided the gold-linked antibody. Neu-tralizing monoclonal antibody to gamma-interferon was kindly providedby Dr. Jan T. Vilcek (New York University Medical Center).

Supported in part by grant CA23175 and CA33012 from the NationalCancer Institute. Dr. J. Zeldis is a Pfizer postdoctoral fellow. Dr. H.Steinberg is the recipient of a Leukemia Society of America Special Fel-lowship Award.

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