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Detection of Hepatitis C Virus RNA in Peripheral Blood Mononuclear Cells
of
Infected Patients byIn Situ Hybridization
By Judit Moldvay, Paul Deny, Stanislas Pol, Christian Brechot, and Eugenia Lamas
W e used in situ hybridization to detect hepatitis C virus
(HCV) infectionofperipheral
blood mononuclearcells
(PBMNC) from 11 patients with chronic active hepatitis.
Using36S-labeled HCV-RNA probe,HCV-RNA-positive
and-negativestrands
were observed in unstimulated
PBMNC from three patients, all of whom were receiving
immunosuppressive drugsafter orthotopic liver transplantation (OLT). HCV-RNA sequences were also identified in
PBMNC from three patients who were not undergoing irn-
munosuppression, after stimulation with either phytohemagglutinin (PHA) or pokeweed mitogen (PWM). In contrast, HCV-RNA was not found in the remaining five patients, who had not undergone OLT and whose cellswere
not stimulated with mitogens. These results show that
mononuclear cells can be infected by HCV and that mitogenic stimulation of
infected cells increasesHCV-RNA replication.
0 1994 by TheAmerican Societyof Hematology.
H
cells were then separated by Ficoll-Hypaque densitygradient centrifugation, washed three times, and resuspended in RPMI.The degree of separation was determined by fluorescence activated cell
sorter analysis (FACSstar, Becton Dickinson) using
monoclonal antibodies (CD3, CD16, CD19; Immunotech SA, France). The cells
(0.5 to 0.7 X 106/mL)were then attached to gelatin-pretreated slides
by cytocentrifugation.After air-drying,the cells(- 10,000per slide)
werefixedwith
4% paraformaldehyde (PFA)for 10 minutes,
washed three times in phosphate-buffered saline (PBS),and dehydrated through a graded seriesofethanols (30%to 100%).The slides
were stored at -20°C.
As patients no. 6 through 8 (Table 1 ) had marked iymphocytopenia, total PBMNC were resuspendedat a density of 0.5 to 0.7 X
106/mLin RPMI for cytocentrifugation and fixation as described
earlier. In cases no. 9 through I 1 (Table 1) total PBMNC were cultured withphytohemagglutinin (PHA; 4 pg/106 cells/mL) and
pokeweed mitogen(PWM; 50 pL/106cells/mL, GIBCO,Grand Island, NY). PHA essentiallystimulates T lymphocytes, while PWM
essentially stimulates B lymphocytes. After48 hours of incubation,
the cells were pelletized, washed,and resuspended at a density 0.5
to 0.7 X 106/mLin RPMI for cytocentrifugationand fixation.
Preparation of probes. We used cDNA correspondingto the 5’
noncoding regionof HCV-RNA(259bp), which is highly conserved
among different viral isolates. This cDNA was subcloned into the
Bluescript SK plasmid(Stratagene,SanDiego,CA).Antisense
RNA was transcribed by T7 RNA polymerase (Pharmacia) after
plasmid linearization with BamHI (Pharmacia). To prepare the
sense probe,we used T, RNA polymerase(Pharmacia) after linearization with EcoRI (Boehringer, Mannheim, Germany). The Bluescript vector without cDNAwas used as a negative control. High35Suridine trispecific activity RNA probes were synthetized using
EPATITIS C VIRUS (HCV) is a small RNA virusresponsible for the vast majority of posttransfusional
and sporadic cases of non-A, non-B hepatitis. Its structure
and pathogenicity are still under investigation.’ Infection
becomes chronic in more than 50%of cases,but the mechanism of viral persistence and the cellular tropism of HCV
are still unclear and there is no cell culture system available.*
There is also little information on the involvement of peripheral blood mononuclear cells (PBMNC) during HCV
infection, but it has recently been shown that HCV-RNA
can be detected by polymerase chain reaction (PCR) in
PBMNC,3v4
although contamination by serum particles during cell isolationcannot be completely ruledout.
Studies by our group and others5”clearly indicate, that in
the infected liver tissue, HCV-RNA-positive
and -negative
strands can be detected by in situ hybridization in scattered
hepatocytes. We have alsodetected HCV-RNA in cells present in inflammatory infiltrates, strongly suggesting the involvement of mononuclear c e k 5
To assess definitely whether HCV indeed infects
PBMNC, weused in situ hybridization with35S-labeled
HCV-RNA probes to test blood samples from l 1 patients
with chronic active hepatitis C.
MATERIALS ANDMETHODS
Patients. Bloodsampleswere obtained from 1 1 patients (six
women and five men; mean age, 46 years) with histologicallyconfirmed chronic active hepatitis (Table I). All patients were antiHCV-positive (recombinant immunoblot assay
2;
Ortho
Diagnostic
Systems,
California), hepatitis B surface antigen
(HBsAg)-and human immunodeficiency virus (HIV)-negative,
and
had elevated ALT values(1.5 to five times upper limit of normal).
Three patients (no. 6,7, and 8) had undergone liver transplantation
(2 1, 15, and 17 months previously).
Controls. PBMNC
from
four healthy,
anti-HCV-negative
blood donors served as negativecontrols. To check the specificity of
the HCV probes, we used HCV-PCR-positive and -negative liver
tissue sections sections placed
on the same slides.
Preparation of cells. PBMNC were prepared from heparinized
venous blood by Ficoll-Hypaque density gradient centrifugation
(Ficoll-Hypaque;Pharmacia, Uppsala, Sweden).The PBMNC pellet was washed
three times by centrifugationin RPMI 1640medium
(Seromed, Berlin,Germany) and the cells were counted in a hematocytometer.
In cases no. 1 through 5 (Table l), we separated T lymphocytes
from other white blood cells(B lymphocytes, monocytes,and polymorphonuclear cells) byrosette formation. PBMNC at a density of
10 X 106/mLwere incubated overnight withneuraminidase-treated
sheep red blood cells and complement-free fetal calf serum. The
Bfood,Vol83, No 1 (January l), 1994: pp 269-273
From INSERM U-370, CHU Necker, Paris: the Laboratoire de
Bacteriologie Virologie, Hopital Avicenne, Bobigny, France; the
Unith d’Hhpatologie, Hopital Laennec. Paris: and Hybridotest, Institut Pasteur, Paris, France.
Submitted March 16, 1993; accepted September l , 1993.
Supported by grants from Community Economic European, the
Association pour la Recherchecontre le Cancer,Ligue Nationalcontre le Cancer, Institut National de la Santi et de la RechercheM6dicale, and Diagnostic Pasteur-Sanoji.
J.M. is the recipient of an ARC
fellowship
Address reprint request to Eugenia Lamas, PhD, INSERM U370, CHUNecker, 156, ruedeVaugirard, 75742, Paris CEDEXIS,
France.
The publicationcosts of this article were defrayed in part by page
charge payment.This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
01994 by The American Society
of Hematology.
VVV6-497I/94/83Vl-VV28$3.OV/V
269
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270
MOLDVAY ET AL
in three samples (Table 2 ) , corresponding to the OLT recipients. An antisense probe that detects positive HCV-RNA
strands gave signals in all three of these cases (Fig 1A and
Patient No.
Age/Sex
Dlagnosls
Anti-HCV
B), while an HCV-RNA sense probe that detects negative
1
55/F
CAH
POS
strands
gave signals in two cases (Fig IC and D). PBMNC
2
35/F
CAH
POS
from three of the negative patients were also examined after
46/M
3
CAH
POS
stimulation with PHA and PWM; positive HCV-RNAs
4
50/F
CAH
POS
5
strands were found in all three, and negative HCV-RNA
CAH
POS
36/M
6'
CAH
5 a / ~
POS
strands in two, regardless of the mitogen used. No signals
7'
CAH
POS
43/M
were detected in PBMNC from the remaining five patients
CAH
a*
48/F
POS
(Table 2, patientsno. 1 through 5), who had not undergone
9t
CAH
35/M
POS
liver transplantation and whose cells were not exposed to
CAH
1O t
61/F
POS
mitogen.
llt
47/M
CAH
POS
The proportion of positively labeled cells approximated
Abbreviations: CAH, chronic active hepatitis; POS, positive
0.01%to 0.039'0, although it reached 0.3% in one case with
* Orthoptopic liver transplant(OLT) recipients.
the antisense probe after stimulation with PHA (Table 2,
t PBMNC were also examined after mitogen stimulation.
patient no. 1 1; Fig 2A through E). There was also a slightly
but not significantly higher proportion of labeled cells after
mitogenic stimulation with PHA than with PWM. As laphosphate ( U T P specific activity, > 1,000 Ci/mmol; Amersham,
beled cells were usually entirely covered by silver grains, it
UK) according to the supplier's recommendations. Probe length
was impossible to distinguish between nuclear and/or cytowas reduced to a mean size of approximately 50 nucleotides by alplasmic labeling (Fig 2E), or to compare the
labeling intenkali hydrolysis, and unincorporated nucleotides were removed by
sity with the antisense and sense probes; however, there was
gel filtration on G75 sephadex. After precipitation, the probe was
an apparent difference in favor of the antisense probe.
redissolved in a hybridization buffer (50% deionized formamide,
To check the specificity of the signals obtained in our ex0.3 mol/L NaCI, 20 mmol/L Tris-HCI, pH 7.4,5 mmol/L EDTA,
perimental conditions, several controls were used. PBMNC
10 mmol/L NaPO,, pH 8. 10%dextran sulfate, 1 X Denhardt's sofrom four healthy anti-HCV-negative blood donors showed
lution, and 50 pg/mL total yeast RNA) at a final concentration of
no labeling when hybridized with the HCV-RNA antisense
500,000 cpm/20 pL and stored at -2o'C.
In situ hybridization. The slides were rinsed in a 0.85% saline
or sense probes. Two of them were also studied after stimusolution for 5 minutes and postfixed in freshly prepared 4% PFA for lation with PHA and with PWM. These two samples showed
20 minutes. They were then rinsed twice inPBS for 5 minutes, and
no labeling with HCV-RNA antisense or sense probes (Fig
the cells digested withproteinase K (Merck, France; 10 pg/mL for 7
2F). Similarly, a cDNA-free 35S-labeledBluescript SK plasminutes). The slides were rinsed
in PBS for 5 minutes, refixed in 4% mid probe, also used as a negative control, showed no hyPFA for 5 minutes, dipped in distilled water, and acetylated in a
bridization with any of the 1 1 PBMNC samples (Fig 1 E and
0.09-mol/L solution of triethanolamine with acetic anhydride ( l /
F). Liver tissue sections from HCV-positive patients con400 vol/vol, 10minutes). They werethen rinsed in PBS and 0.85%
taining HCV-RNAs detected by PCR were used as positive
saline for 5 minutes each, dehydrated through a series of ethanol
controls. Using both HCV antisense and sense probes, spe(30%to loo%), and allowed to dry for at least 2 hours before hyTable 1. Clinical and Histopathological Characteristics
of the Patients
bridization.
The probewas denatured for 2 minutes at 8WC, then applied to
the slides, which were covered with
a siliconized coverslip. Hybridization was performed at 52°C for 16 hours in a humid chamber.
The coverslips were gently floated
off ina 5X SSC buffer (1X SSC
= 0.15 mol/L NaCI, 0.0 15 mol/L sodium citrate) and 10 mmol/L
dithiotreitol at 42°C followed by a stringent wash at 60'C in 50%
deionized formamide, 2X SSC, and 0.1 mol/L dithiotreitol. The
slides werethen rinsed ina washing buffer,and unhybridized probe
was digested with RNase A (20 pg/mL) and T, (2 pg/mL; Boehringer) in washing buffer for 30 minutes at 37°C. After washes at
42°C in 2X SSC, 0% SSC, and 0.1 X SSC (15 minutes each), the
slides were dehydratedin a series of ethanols containing 0.3 mol/L
of ammonium acetate. The slides were exposedfor 10 to 15 days at
4°C using Kodak NTB2 emulsion (Eastman Kodak, Rochester,
NY). The slides werecounterstained with Harris hematoxylin.
RESULTS
We have examined PBMNC of 1 1 patients with chronic
active hepatitis C (Table 1). For three patients, the samples
were obtained during the follow-up after orthotopic liver
transplantation (OLT).
All I 1 PBMNC samples were first examined without mitogenic cell stimulation. HCV-RNA molecules were found
Table 2. HCV-RNA Detection in PBMNC
PBMNC
No
Stirnulatlon
Patient No.
HCV-RNAs
A
S
1
2
3
No
No
No
-
-
-
-
4
No
-
-
5
6'
7"
8'
9
10
No
Yes
-
-
11
Yes
Yes
Yes
Yes
Yes
-
+
+
+
Mitogen
Stimulation
nt
nt
nt
nt
nt
+
nt
nt
nt
-
+
-
-
-
-
-
-
S
A
+
+
+
+
+++t
-
Abbreviations: A, HCV-RNA
antisense
probe (detects positive
strands); S, HCV-RNA sense probe (detects negative strands): nt. not
tested.
* OLT recipients.
t Semiquantitativeestimation.
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IN
HEPATITIS
271
VIRUS RNA
Fig 1. In situ hybridization
on unstimulated PBMNC from
HCV-infected patients after
OLT. (A) An antisense HCVRNA probe (500,000 cpm/
slide) labeled a single cell. (B)
Dark-field view of the same cell.
(C) A sense HCV-RNA probe
(500,000 cpmlslide) also labeled a single cell. (D) Dark-field
view of the same cell. (E) No signal was detected with an unrelated Bluescript-RNA probe
(500,000 cpm/slide). (F) Darkfield view of the same zone.
(A-F: hematoxylin and eosin
[H&E]; original magnification
X 500.)
cific signals were observed in scattered hepatocytes and in
cells of the inflammatory infiltrates, whereas negative control tissue was unlabeled’ (data not shown).
DISCUSSION
This study clearly demonstrates that HCV-RNA sequences can be detected in PBMNC from patients on immunosuppressive therapy (ie, after OLT). and after in vitro
mitogenic stimulation. Based on PCR results, it has been
previously suggested that HCV infects PBMNC.3 but in
these experiments, contamination by infected serum particles during cell preparation could not be ruled out completely. Although one samplecontained0.3%oflabeled cells
after PHA stimulation, with the HCV antisense probe, labeled cells were generally present at verylow frequency
(0.01%to 0.03%). This range of values is in keeping with
that reported in PBMNC from HIV-infected patients using
HIV-RNA probe (0.001%to O.OI%).x Similarly, less than
0.03% of PBMNC from calves infected with bovine immunodeficiency-like virus contain the viral RNA.9 In contrast,
using in situ hybridization. 1% to 10%of mononuclear cells
from hepatitis B virus (HBV)-infected patients were found
to contain HBV-DNA sequences.’”
We only detected positive signals with unstimulated
PBMNC from the three OLT recipients (Table 2). probably
because of the drug-induced immunosuppression in these
patients. Interestingly. HCV-RNAs were previously found
either in PBMNC by means of PCR. or in inflammatory
infiltrates ofthe liver by in situ hybridization. only in HCVpositive patients with immunodeficiency caused by HIV
coinfection, a clinical situation with increased HTV viremia 3.5
These results are in parallel to those obtained with woodchuck hepatitis virus (WHV): an increase in WHV-DNA
replication was found in PBMNC of infected animals after
lipopolysaccharide stimulation.’’ It has also recently been
reported that viral replication can be increased by PHA
stimulation in HBV-infected PBMNC.” The detection of
infected cells with the HCV sense probe that recognizes
HCV-RNA-negative strands confirms our previous findings in liver tissue.’ Negative RNA molecules are probably
intermediates in the replication ofthe HCV genome. In the
Flmiviruse.~,negative RNA strands indeed serve as templates during replication. and as HCV is related to Fhvivirtlscs. it can be supposed that HCV follows the same replicative pattern. Using PCR, it has been shown in the liver
and in the serum that the number
of negative strands is usu-
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272
MOLDVAY ET AL
4
Fig 2. In situ hybridization
on PBMNC from an HCV-infected patient (A-E) and from a
healthy subject (F) after PHA
stimulation in vitro. (A) Before
stimulation, an antisense HCVRNA probe (500,000 cpm/
slide), gave no signal. (B) Same
picture on dark field. (C) After
PHA stimulation, an antisense
HCV-RNA
probe (500,000
cpm/slide) labeled several cells.
(D) Dark-field view of the same
cells. (E) Oneof
the labeled
cells at a higher magnification; the silvergrains entirely
cover the nucleus andthe cytoplasm. (F) After PHA stimulation,an antisense HCV-RNA
probe (500,000 cpm/slide)
gave no signal.(A, B: H&E; original magnification X 312.5. C,
D, F: H&E; original magnification X 500. E: H&E; original
magnification X 1,250.)
ally I O to 100 times lower than the number of positive
strand^.^ In the present study, we found only a slight difference in the density of silver grains with the sense and antisense probes. but itwas in favor of the antisense probe
(which detects positively stranded HCV-RNA molecules).
We also found aslightly higher proportion of labeled cells
after stimulation with PHA than with PWM (0.03%and
0.01%.respectively), but were unable to identify clearly
which subpopulations of PBMNC were infected by HCV.
This was due to the
low number of cells disposed to perform
theexperiments. Thisquestion could be investigated further
by means of in situ PCR combined with FACS selection after antibody labeling.'3 However, the fact that cells were labeled after stimulationwith PHA and PWM suggests that T
and B lymphocytes are both infected. This can be correlated
with data presented recently by Zignego et al, who found
that HCV-RNAs were detected only in T and B lymphocytes in some cases by means of PCR. but not in the monocyte macrophage fraction.' Our results are also consistent
with the recent demonstration that the humanlymphocytic
cell line MOLT4 can be infected by HCV.I4 However, it is
noteworthy that a recent report suggested that theHCV capsid antigen might be detected in some HCV-positive patients in the circulating monocytes."
In conclusion, we provide morphological evidence of infection by HCV of circulating mononuclear cells. Similar
observations have been made with related viruses such as
(Thiel Heinz-Jurgen,
the F/uvirirtr.sc>.sand thePe.s/ivirtrse.vs'6
personal communication, 1993). HCV infection of
PBMNC might lead to the selection of variants, as is the
case with lymphocytic choriomeningitis virus." Moreover,
HCV-infected cells may become functionally defective and
thus lead to viral persistence and chronicinfection.2 Finally,
infected PBMNC may serve as an HCV reservoir, causing
reinfection of liver graft after OLT."
ACKNOWLEDGMENT
Wethank Lucienne Chatenoud, Patricia Moreau, and Nicole
Chanteloup for their help. and David Young for editing the manuscript.
REFERENCES
I . Houghton MA. Weiner AH. Han J. Kuo G . Choo Q-L Molecular biology of the hepatitis C viruses: Implication for diagnosis,
development and control of viral disease. Hepatology 2:38 I , 1991
2. Ahmed R. Stevens JG: Viral persistence, in Fields BN, Knipe
DM (eds):Virology. New York. NY. Raven. 1990. p 242
3. Zignego AL. Macchia D. Monti M, Thiers V. Mazetti M.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
HEPATITISC VIRUS RNA
IN
PBMNC
Foschi M,Magi E, Romagnani S, Gentilini P, Brkhot C: Infection
of peripheral mononuclear blood cellsby hepatitis C virus. J Hepato1 15:382, 1992
4. Fong TL, Shindo M, Feinstone SM, HoofnagleJH, Di Bisceglie AM: Detection of replicative intermediates of hepatitis C viral
RNA in liver and serum of patients with chronic hepatitis C. J Clin
Invest 88:1058, 1991
5 . Lamas E, Baccarini P, Housset C, Kremsdorf D, Brkhot C:
Detection of hepatitis C virus (HCV) RNA sequences
in liver tissue
by in situ hybridization.J Hepatol 16:219,1992
6. Yamada S, Koji T, Nozawa M, KiyosawaK, Nakane P K Detection of hepatitis C virus (HCV) RNA in paraffin
embedded tissue
sections ofhuman liver of non-A, non-B
hepatitis patients by in situ
hybridization. J Clin Invest6:40, 1992
7. Blight K, Trowbridge R, Rowland R, Gowans E: Detection of
hepatitis C virus RNAby in situ hybridization. Liver 12:286, 1992
8. Harper ME, Marselle LM, Gallo RC, Wong-Staal F Detection of lymphocytes expressinghuman T-lymphotropic virus type
111 in lymph nodes and peripheral blood from infectedindividuals
by in situ hybridization.Proc Natl Acad Sci USA83:772, 1986
9. Carpenter S, Miller LD, Alexandersen S, Whetstone CA, Van
Der Maaten MJ, Viuff B, Wannemuehler Y , Miller JM, Roth J
Characterization of early pathogenic effectsafter experimental infection of calves with bovine immunodeficiency-like virus.
J Virol
66: 1074, 1992
10. HadchouelM,Pasquinelli
C, Fournier JG, Hugon RN,
Scotto J, Bernard 0, Brichot C: Detection of mononuclear cells
expressing hepatitis B virus in peripheral blood fromHBsAg positive and negative patients by in situ hybridization.J Med Virol24:
27, 1988
273
1 1. Korba BE, Cote PJ, Gerin JL: Mitogen-induced replication
of woodchuckhepatitis virus in cultured peripheral bloodlymphocytes. Science 24
1 :I2 13, 1988
12. Bouffard P, LamelinJ-P, Zoulim F, Lepot D, Trepo C: Phytohemagglutinin and concanavalin A activate hepatitis B virus in
peripheral bloodmononuclear cells ofpatients with chronic hepatitis B virus infection.J Med Virol37:255, 1992
13. Omar B, Hauptman SP, Lischner HW, SachsM, Pomerantz
1 provirus in
RJ: Detection of human immunodeficiency virus type
mononuclear cells by in situ polymerase chain reaction. N Engl J
Med 326: 1385, 1992
14. Shimizu YK, Iwamoto A, Hijikata M, Purcell RH, Yoshikuba H: Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc Natl Acad Sci USA 89:5477,
1993
15. Bouffard P, Hayashi R, AcevedoR, Levy N, Zeldis JB:Hepatitis C virus is detectedin a monocyte/macrophagesubpopulation
of peripheral blood mononuclear cells of infected patients.
J Infect
Dis 166:1276, 1992
16. Monath T P Flaviviruses, in Fields
BN, Knipe DM (eds): Virology. NewYork, NY, Raven, 1990,p 763
17. King C-C, de Fries
R, Kolhekar SR, Ahmed R In vivo selection of lymphocyte-tropicand macrophage tropic variants of lymphocytic choriomeningitisvirus during persistent infection. J Virol
64561 1, 1990
18. Firay C, Samuel D, Thiers V, Gigou M, Pichon F, Bismuth
A, Reynes M, Maisonneuve P, BismuthH, Brichot C: Reinfection
of liver graftby hepatitis C virus (HCV) after liver transplantation.
J Clin Invest 89:136 1, 1992
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1994 83: 269-273
Detection of hepatitis C virus RNA in peripheral blood mononuclear
cells of infected patients by in situ hybridization
J Moldvay, P Deny, S Pol, C Brechot and E Lamas
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