By
From the * Division of Gastroenterology and Division of Hematology-Oncology, Department of
Medicine, Veterans Affairs and Duke University Medical Centers, Durham, North Carolina 27705
Although researchers have noted high level activation of rodent mononuclear phagocytes for
nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents
such as interferon (IFN) and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-
serves as
an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear
cells in vitro with IFN-
caused a dose-dependent increase in monocyte NOS2 activity and
NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-
also modulated NOS2, we studied blood cells from
patients with hepatitis C before and after IFN-
therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2
mRNA. However, IFN-
treatment of patients with hepatitis C infection was associated with
a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2
mRNA content. IFN-
-treated patients had significant decreases in levels of serum alanine
aminotransferase and plasma hepatitis C mRNA. The degree of IFN-
-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum
alanine aminotransferase levels. Thus, IFN-
treatment of cells in vitro or administration of
IFN-
to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA
expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been
reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-
in hepatitis C infection.
Nitric oxide (NO)1 is a simple chemical mediator produced endogenously from L-arginine by the action of
NO synthase (NOS), a family of related enzymes encoded
by separate genes (1, 2). The inducible (or immune) form
of the enzyme, NOS2, is found prominently in mononuclear
phagocytes and hepatocytes, and is capable of high level
NO production. Its expression is regulated primarily by transcription, and it is modulated by various cytokines and
microbial products (1). NO has potent antimicrobial activities in vitro and in vivo against a wide array of organisms
including certain viruses (3).
IFN- The purpose of this study was to determine whether
IFN- Subjects.
Control normal subjects were recruited locally. Patients with chronic hepatitis C (diagnosed by second generation
recombinant immunoblot assay and/or hepatitis C virus RNA
measurement and liver biopsy) were recruited consecutively from
the hepatology outpatient clinics at Duke University Medical
Center (DUMC). The study protocol was approved by the
DUMC Institutional Review Board. Informed consent was obtained from each subject before participation. Patients and controls were excluded if they had a coexisting chronic inflammatory
condition, active allergy or infection, malignancy, or if they were
receiving ribavirin, nitroglycerin, or other nitrate-containing medications. Pregnant women were excluded because of reports
of elevated NO production in pregnancy. All subjects and patients were abstinent of alcohol. Patients had no identifiable
cause(s) for chronic hepatitis other than hepatitis C; all had confirmed hepatitis C infection and hepatitis as determined by blood
studies and liver biopsies. Patients received 3-10 million U of recombinant IFN- Blood Mononuclear Cell Preparation.
30 ml of blood was drawn
into lithium heparin. Mononuclear cells were prepared using Ficoll/Hypaque as previously noted (7) and stored at NOS Enzyme Activity and Antigen Analyses.
Cellular extracts
were prepared and analyzed for NOS activity (14C-L-arginine
conversion to 14C-L-citrulline) and antigen content by immunoblot as previously described (7). In some enzyme assays, 2 mM
NG-monomethyl-L-arginine was included to determine if the
conversion to L-citrulline was NOS-mediated. Immunoblots were
done using either a monoclonal anti-NOS2 antibody from Transduction Laboratories (anti-macNOS; Lexington, KY) or from Research and Diagnostic Antibodies (1E8-B8; Richmond, CA). We
used untreated human colon cancer cell line cells (DLD-1; reference 14) as negative controls. DLD-1 cells treated with human
recombinant IFN- Reverse Transcriptase-PCR Analyses.
Total RNA was isolated
by the method of Chomczynski and Sacchi (15), resuspended in
diethyl pyrocarbonate-treated water, and reprecipitated overnight
with cold isopropanol at Measurement of Serum IFN- Statistical Analysis.
Continuous variables were compared using the Student's t test or ANOVA as appropriate. Categorical
variables were compared using the Fisher's exact test. The Bonferroni correction was used to control the type I error rate when
multiple comparisons were performed. Since the NOS activity data
were not normally distributed, we used logarithmic transformation to allow for parametric analysis (ANOVA testing) of the results. The Kruskal-Wallis test was performed on nontransformed
data to confirm these results. To test for an increasing dose-
response relationship between IFN- Freshly isolated monocytes from eight healthy male volunteers showed an increase in NOS activity in response to
IFN-
In an attempt to determine if IFN- Table 1.
Characteristics of Subjects
, endotoxin, and TNF treatment in vitro can potently increase NOS2 expression and NO production by
rodent macrophages, but it has been difficult to show comparable activation in vitro of normal human mononuclear
phagocytes for high level expression (6). However, cells
from patients with illnesses such as malaria (9), rheumatoid
arthritis (7, 10), and tuberculosis (11) have mononuclear
phagocytes that clearly express NOS2 and produce NO. In
rodent macrophages, exogenous IFN-
cannot activate
macrophages for NO production (12), but macrophage-synthesized IFN-
can augment NO production in an autocrine fashion (13). IFN-
had not previously been investigated thoroughly in human mononuclear phagocytes for
this function.
could activate human monocytes in vitro and in
vivo for NOS2 expression and NO production. We demonstrate here that IFN-
treatment of normal mononuclear
cells in vitro induces increased expression of monocyte
NOS activity, NOS2 antigen, and mRNA content, and production of NO, and that treatment of patients with
chronic hepatitis C virus infection with IFN-
in vivo
causes increased mononuclear cell NOS activity, NOS2
antigen content, and mRNA content.
2b (INTRON®A; 2 × 108 U/mg; Schering-Plough, Kenilworth, NJ) subcutaneously three times per week.
70°C until
use. For some in vitro experiments, monocytes were prepared by
sequential Ficoll/Hypaque-Percoll/adherence technique (6). Freshly
isolated monocytes were cultured at 3 × 105 cells or mononuclear cells 5 × 105 cells/6-mm diameter microtiter plate well in
0.2 ml of DMEM with 10% heated (56°C for 30 min) normal,
pooled human serum.
(100 U/ml), and TNF (100 U/ml), IL-1 (0.5 ng/ml), and IL-6 (200 U/ml) for 3 d were used as positive controls for demonstrating NOS2 antigen. 25-50 µg protein extract
of the cells was used in the individual lanes. A positive immunoblot for NOS2 was one in which a clear band was visible at 130-
131 kD.
20°C. 1 ug of RNA was reverse transcribed with random hexamers and murine leukemia virus reverse
transcriptase (RT; Perkin Elmer, Branchburg, NJ) for 15 min at
42°C. Reactions were stopped by heating for 5 min at 99°C. The
final product was then amplified with 2.5 U of AmpliTaq® DNA
polymerase (Perkin Elmer) and 0.15 µM of sense and antisense primers in PCR buffer containing 100 mM Tris-HCl, 50 mM
KCl, 25 mM MgCl2, and 10 µM deoxyribonucleotide triphosphate. Mixtures were overlaid with mineral oil and amplified for
40 cycles. The primers for NOS2 were 5
-CCT GAG CTC
TTC TTC GAA ATC C-3
(sense) and 5
-AGG ATG TTG
TAG CGC TGG AC-3
(antisense). The expected product is
229 bp. As a control, we used the following primers for the glyceraldehyde-3-phosphate dehydrogenase: 5
-CTA CTG GCG CTG CCA AGG CTG T-3
(sense) and 5
-GCC ATG AGG
TCC ACC ACC CTG T-3
(antisense). The expected product is
390 bp. Final PCR products were separated on a 1% Tris-borate/
EDTA agarose gel and visualized by ethidium bromide staining.
Levels.
Serum IFN-
levels were
done using a previously reported cytokine-specific sandwich
ELISA (Endogen, Cambridge, MA); levels were calculated using
standard curves generated with recombinant IFN-
. The lower
limit of the detection by the assay was 5 pg/ml.
dose in vitro and expression of NOS activity and production of nitrite/nitrate, the Page
test for ordered alternatives was used. We used the Statistical Analysis System (SAS Institute, Inc., Cary, NC). P values are two-sided using
= 0.05 as the reference standard for determining significance.
2b treatment in vitro. The increase peaked at 500 U/ml (Fig. 1 A). Similarly, production of NO (measured as
nitrate and nitrite, the stable catabolites of NO) increased
with IFN-
2b treatment (Fig. 1 B). Increasing doses of IFN-
2b were associated with significantly increased NOS activity and nitrite/nitrate production (P <0.001). Studies also
demonstrated that treatment of monocytes in vitro with
IFN-
2a (Roferon®; Roche Laboratories, Nutley, NJ) augmented NOS activity and NO production (data not shown).
In immunoblot studies, normal subject monocytes from four
out of six treated in vitro with IFN-
2b had increased expression of NOS2 antigen (Fig. 2 A). We did RT-PCR analysis of RNA from blood mononuclear cells to determine if IFN-
induced increased levels of NOS2 mRNA.
Mononuclear cells from normal subjects were cultured for
3 d without or with 500 U/ml IFN-
. Untreated cells from
zero out of six normal individuals had NOS2 mRNA expression, whereas all those treated with IFN-
in vitro expressed NOS2 mRNA (six out of six) (Fig. 2 B).
Fig. 1.
(A) Enhancement of normal blood monocyte NOS enzyme activity by treatment with IFN- in vitro.
Purified blood monocytes from eight
separate normal individuals were cultured for 3 d with the indicated
amounts of IFN-
, and analyzed for
NOS activity. The points show the
mean ± one SEM. (B) Enhancement of
normal blood monocyte NO production by treatment with IFN-
in vitro.
Purified blood monocytes from eight
separate normal individuals were cultured as noted in Fig. 3 A, and then supernatant media samples were assessed
for content of nitrite/nitrate. The points
show the mean ± one SEM.
[View Larger Versions of these Images (15 + 13K GIF file)]
Fig. 2.
(A) Immunoblot
analyses of extracts of mononuclear cells treated in vitro with
IFN-. Cells were cultured with
(+) or without (
) 500 U/ml of
IFN-
for 3 d. Extracts were
then made as described in the
Methods section, and equivalent
amounts of cellular protein were
analyzed. Antibody anti-MacNOS was used. Cells from two
separate, normal individuals were
analyzed. Analysis of the first is
shown in lanes 1 and 2, and that
of the second is in lanes 3 and 4. DLD-1 + signifies extracts from
the human colon cancer cell line DLD-1 treated with IFN-
, IL-1, and
TNF in vitro. Results demonstrate that IFN-
treatment induces NOS2
antigen expression. (B) RT-PCR analysis of mRNA of mononuclear
cells treated in vitro with IFN-
. Cells from two separate, normal individuals were cultured with (+) or without (
) 500 U/ml of IFN-
for
12 h. RNA was extracted and analyzed as described in the Methods section for NOS2 and glyceraldehyde-3-phosphate dehydrogenase mRNA.
M, molecular weight markers; D, cells of the human colon cancer cell
line DLD-1 treated with IFN-
, IL-1, and TNF; and W, distilled water.
[View Larger Versions of these Images (22 + 37K GIF file)]
Fig. 3.
(A-C) NOS enzyme activity in extracts of blood
mononuclear cells (A), serum
ALT (B), and plasma hepatitis C
RNA levels (C). The bars show
the mean + one SEM for samples taken from the different subject groups. Control, normal control subjects; Hep C, patients with hepatitis C not on therapy;
Hep C + IFN, patients with hepatitis C on IFN- therapy. For
NOS measurements, n = 9 for
Control, 18 for Hep C, and 15 for Hep C + IFN; for serum
ALT, n = 15; and for plasma
hepatitis C RNA, n = 4. (D)
NOS enzyme activity in extracts
of blood mononuclear cells from
six individual patients before and
after IFN-
treatment. The lines
connect the values from an individual patient's samples before
and after IFN-
therapy. The solid
squares show the means ± one
SEM of the groups.
[View Larger Versions of these Images (8 + 7 + 9 + 14K GIF file)]
treatment in vivo
augmented NOS2 expression, we studied patients with
hepatitis C before and after IFN-
therapy. Table 1 displays characteristics of the subjects and details of their treatments. As expected, hepatitis C patients receiving IFN-
treatment had higher levels of IFN-
than did normal subjects or hepatitis C patients not receiving IFN-
. There
was an overall difference in the blood mononuclear cell
NOS activity levels among the three groups (P <0.004;
Fig. 3 A). Untreated patients with chronic hepatitis C and
healthy controls had comparable NOS activity (ability to
convert L-arginine to L-citrulline) in freshly isolated blood
mononuclear cells. The activity was inhibited by >80% by
inclusion of N-monomethyl-L-arginine in the assaying
(data not shown), indicating that the activity was mediated
by NOS. Patients receiving IFN-
2b therapy had significantly higher NOS activity levels than did healthy controls
(adjusted P <0.05) and hepatitis C patients not receiving
IFN-
2b therapy (adjusted P <0.05). Although IFN-
2b
treatment caused increases in NOS2 levels, levels of serum
alanine aminotransferase (ALT; an indicator of active hepatitis) and plasma hepatitis C RNA decreased with IFN-
2b
therapy (Fig. 3, B and C; P = 0.002 and 0.02, respectively, by paired Students t test). When we analyzed samples from
individuals both before and after receiving IFN-
2b, there
was a significant increase in NOS activity after the IFN-
2b treatment in six patients in whom paired samples were
available at baseline and during IFN-
2b therapy (2-8-wk
time interval) (P <0.02, paired t test; Fig. 3 D).
Normal subjects
n = 9 (5 M, 4 F)
Age 39.4 ± 5.8 yr*
Total number of hepatitis C patients
n = 26 (17 M, 9 F)
Chronic hepatitis (18)
Chronic hepatitis with cirrhosis (8)
Hepatitis C patients not on IFN-
treatment (never treated or
blood sampled before started on IFN-
treatment)
n = 18 (14 M, 4 F)
Age 43.8 ± 10.5 yr
Hepatitis C patients on IFN-
treatment
n = 15 (10 M, 5 F)
Age 40.7 ± 10.5 yr
3 to 10 million units IFN-
three times/wk
1-40 wk of treatment
26.4 ± 12.0 h from last injection
until blood draw
Serum IFN-
levels
Normal subjects:
13.8 ± 0.2 pg/ml
Hepatitis C:
14.5 ± 0.8 pg/ml
Hepatitis C on IFN-
:
34.0 ± 13.9 pg/ml
*
Mean ± SD.
P <0.04.
M, male; F, female.
To determine if the increase in NOS activity after IFN-
treatment was accompanied by an increase in NOS2 protein, we analyzed cells for NOS2 antigen content. NOS2
antigen in extracts of blood mononuclear cells was detectable by immunoblot analysis in zero out of seven of the untreated hepatitis C patients and in zero out of five of the
healthy control subjects tested. However, eight out of eight
samples from hepatitis C patients treated with IFN-
2b had cells with detectable NOS2 antigen. These differences
in NOS2 antigen expression among the groups were significant (P <0.00001, Fisher's exact test). Similarly, analysis of
matched sets of cells from hepatitis C patients before and
after IFN-
2b treatment revealed zero out of four with detectable NOS2 before treatment and four out of four with
detectable NOS2 after treatment (Fig. 4 A). Using RT-PCR analyses with cells isolated from six normal individuals and examined without any in vitro culture, we could
find no NOS2 mRNA (zero out of six) (Fig. 4 B). In two
out of two patients with hepatitis C not on IFN-
treatment, we noted relatively low level expression of NOS2
mRNA, while three out of three hepatitis C patients on
IFN-
treatment had relatively higher levels of NOS2
mRNA expression. Fig. 4 B shows representative results.
There was a statistically significant correlation between
the fold increase in NOS2 antigen immunoblot band density and the degree of reduction of the serum ALT level
(fraction of pretreatment ALT level after IFN- therapy);
with increasing NOS2 antigen expression, there was a
greater treatment-associated decrease in serum ALT (Fig.
5). The correlation was statistically significant (P <0.001 by
ANOVA, and <0.05 by Spearman testing). Because of a
lack of certain simultaneous determinations, we could analyze data from only five of the eight subjects before and after treatment. While the NOS2 antigen immunoblot band
density and the degree of reduction of the serum ALT level
correlated, the correlation between NOS activity (pico
moles citrulline/mg protein) and ALT reduction was not
significant. Nevertheless, these data suggest that the augmentation in NOS2 levels may be causally related to the
observed decreases in serum ALT levels and decrease in
liver inflammation associated with IFN-
therapy.
IFNs are a family of proteins with established antiviral
and immunomodulatory properties (16). While much is
known regarding the possible molecular and cellular modes
of action of IFNs, the precise mechanism(s) by which IFN-
mediates its anti-hepatitis C virus effect in vivo is not known.
Our study shows that IFN-
2b treatment of patients with
hepatitis C increases their blood mononuclear cell NOS
enzyme activity and their NOS2 antigen and mRNA expression. In addition, we demonstrate that treatment of purified monocytes or mononuclear cells from healthy donors
with IFN-
2b or IFN-
2a in vitro enhances their NO
production, and NOS enzyme activity, and their NOS2
antigen and mRNA expression. Among isolated mononuclear cells, monocytes are those most likely to express
NOS2 and produce NO (6, 7). Our work provides good
evidence that IFN-
serves to activate human mononuclear cells (most likely mononuclear phagocytes) for NOS2
expression and NO production both in vitro and in vivo. More than 90% of patients with chronic hepatitis C have
circulating immune complexes (17), and immune complexes
may enhance NO formation by rodent mononuclear phagocytes (18, 19); thus, immune complexes and IFN-
could
have cooperated in vivo to enhance NOS2 expression.
NO has potent antimicrobial activities against a wide array of organisms. These include protozoa, fungi, and bacteria (including Leishmania major, Mycobacterium leprae, Mycobacterium tuberculosis, Toxoplasma gondii, Cryptococcus neoformans, and Schistosoma mansoni; references 3, 4). Also, NO is a critical effector for macrophage-mediated tumor cell cytotoxicity (20). In rodent systems, pharmacological inhibition of NOS or genetic disruption of NOS2 reduces host resistance to infection (3, 4, 21, 22). NO can inhibit infection with DNA viruses (e.g., ectromelia, Herpes simplex virus, vaccinia, and Epstein-Barr virus) and RNA viruses (e.g., vesicular stomatitis virus and Coxsackie virus; references 5, 23).
Based on our results, we think that IFN--induced
NOS2 expression and NO production may be responsible
(at least in part) for the anti-hepatitis C effects of IFN-
in
vivo. There was a statistically significant correlation between the degree of increase in NOS2 antigen in blood
mononuclear cells and the degree of improvement in hepatitis as reflected by a decrease in serum ALT. We do not know the precise mechanism of this possible NO-mediated
inhibition; NO likely affects both cellular and viral factors
that modify the infectivity. With vaccinia virus, NO-mediated interference with ribonucleotide reductase function
appears to be important (30, 31). NO may also damage nucleic acids, alter cellular growth and differentiation, and
modify a variety of transcription factors (2, 5, 27, 32, 33);
all of these effects might alter viral infectivity. There have
been no demonstrations of NO antiviral activity for hepatitis C. There is currently no efficient and reliable cell culture system for growth of hepatitis C virus in vitro, and
chimpanzees are the only suitable nonhuman hosts for hepatitis C virus growth in vivo.
In a subset of patients with hepatitis C virus infection,
treatment with IFN effectively inhibits viral replication and
reduces liver injury, but the effect is usually brief, with an
overall response rate of 10-25%. Predictors of a poor response to IFN include viral factors such as high serum hepatitis C virus RNA levels, viral genotype 1, and the absence
of sequence mutations in the NS5 region of the viral genome; host-specific factors such as age, weight, duration of
infection, and immune status also are apparently important
factors (34). The responses of patients to IFN- treatment
correlate inversely with plasma iron saturation and liver
iron content (the response is worse with more iron). In
some cases, depletion of iron by repeated phlebotomy is associated with reduction in serum ALT abnormalities, reduction in plasma hepatitis C virus RNA levels, and improved responsiveness to IFN-
treatment (35). We
speculate that iron in these patients may blunt IFN-
-
induced NOS2 expression (38), or quench NO antiviral effects (26). NO targets iron- and thiol-containing proteins such as hemoglobin, guanylate cyclase, ribonucleotide reductase, aconitase, and mitochondrial electron transport
enzymes, and glyceraldehyde phosphate dehydrogenase (2,
20, 31, 39). Excess iron decreases NOS2 mRNA transcription, and reduction of iron (e.g., by treatment with the
iron chelator deferoxamine) can increase NOS2 mRNA
levels in vitro (38).
Patients receiving IFN- for a variety of indications
(e.g., hepatitis B or C and malignancies) may develop "autoimmune" illnesses with inflammation similar to rheumatoid arthritis (RA) and SLE (40). Our work and that by
others has indicated that NO may be a mediator of inflammation in human autoimmune diseases such as RA and
SLE. NO is increased in synovial fluid and serum of patients with RA (44). Synovial tissues from patients with
RA contain increased amounts of NOS2 and overproduce
NO (10, 45), and RA patients overproduce NO systemically and have blood mononuclear cells with increased
NOS2 expression and NO production (7, 46). It is possible
that the IFN-
treatment-related inflammatory illnesses are
due (at least in part) to an IFN-
-mediated increase in NO
production.
This study provides the first evidence that IFN- can
augment NOS2 expression and NO production by human
blood mononuclear cells in vitro and in vivo. It is not
known whether the magnitude of NOS expression will be
predictive of response to IFN-
treatment in patients with
hepatitis C virus infection; future studies may determine
this. Amaro et al. recently showed that patients with hepatitis C had reduced levels of serum nitrite as compared to control subjects. However, the nitrite levels were higher in
hepatitis C patients who responded to IFN-
therapy (47).
In their study, serum nitrate levels were not measured, dietary intake of nitrite and nitrate was not controlled, and
levels of NOS activity and NOS2 antigen were not measured (47).
Although in this study we focus on monocytes as the
producers of NO, hepatocytes can also express NOS2 and
produce large amounts of NO after activation (48). We did
not test hepatocytes for the ability to produce NO in response to IFN-. Kane et al. recently reported that tissue in
60% of liver biopsies from hepatitis C patients (but none of
"normal" subjects) expressed NOS2 mRNA by RT-PCR
analysis (49). The authors did not report whether the patients were receiving IFN-
treatment. IFN-
-induced
NOS2 expression and NO production by hepatocytes
would provide an efficient manner of delivering an antiviral effector molecule to the offending pathogen in its primary cellular target. In addition to indirectly stimulating
endogenous NO production by treatment with agents such
as IFN-
, delivery of NO per se, perhaps selectively to the liver (50), might be effective in inhibiting hepatitis C virus or other viruses in vivo.
Address correspondence to Dr. J. Brice Weinberg, VA and Duke University Medical Centers, 508 Fulton Street, Durham, NC 27705-3875. Phone: 919-286-6833; FAX: 919-286-6891; E-mail: brice{at}acpub.duke.edu
Received for publication 12 June 1997 and in revised form 2 September 1997.
1 Abbreviations used in this paper: ALT, alanine aminotransferase; DLD-1, human colon cancer cell line; NO, nitric oxide; RA, rheumatoid arthritis; RT, reverse transcriptase; S, synthase.We thank Dr. Robert Webber for the anti-NOS2 antibody 1E8-B8.
This study was supported in part by a Smith-Kline Beecham Research Clinical Award from the American Digestive Health Foundation (A.I. Sharara), Schering-Plough Pharmaceuticals (A.I. Sharara), the Veterans Affairs Research Service (J.B. Weinberg), the James R. Swiger Hematology Research Fund (J.B. Weinberg), National Institutes of Health grant AR-39162 (J.B. Weinberg), and National Institutes of Health training grant AI-07217 (D.J. Perkins).
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