The Influence of Treatment with Pegylated Interferon-Alfa
and Ribavirin on Neutrophil Function and Death
in Patients with HIV/HCV Coinfection
Elzbieta Jablonowska,1Kamila Wojcik,1and Marek Nocun2
In patients with human immunodeficiency virus (HIV) as well as in patients with hepatitis C virus (HCV)
infection the impairment of neutrophil activity is observed. We decided to analyze how treatment with pegy-
lated interferon-alfa (Peg-IFN-alfa) and ribavirin affects neutrophil function in HIV/HCV coinfected patients.
The study group consisted of 18 patients with HIV/HCV coinfection, on combination antiretroviral treatment
(cART), aged between 27 and 42y (mean 33.1–4.5y). At the beginning of treatment with Peg-IFN-alfa and
ribavirin all patients had an undetectable HIV viral load, and CD4 T-cell counts higher than 350cells/lL. At two
time points, before and after 12wk of treatment with Peg-IFN-alfa and ribavirin, we examined intracellular
levels of reactive oxygen species (ROS), and expression of selected adhesion molecules on whole blood neu-
trophils, along with apoptosis and necrosis of these cells. These analyses were done with flow cytometry. During
anti-HCV therapy undetectable HIV levels were maintained in all patients. Treatment with PEG-IFN-alfa and
ribavirin resulted in increases in the expression of CD11b and CD18, and decreases of CD16 and CD62L.
However, only the change in CD62L expression was statistically significant (p<0.05). Moreover, the treatment
resulted in increased apoptosis of neutrophils, while necrosis remained unchanged. After 12wk of treatment, an
increase in ROS production by neutrophils stimulated with PMA was observed (p<0.01). In HIV/HCV coin-
fected patients on cART, PEG-IFN-alfa and ribavirin treatment caused an activation of neutrophil function, yet it
did not affect the suppression of HIV replication.
infected with hepatitis C virus (HCV) via the same route of
transmission (24,26). It is well known that the prognosis of
individuals with coinfection is worse than in HCV mono-
infected patients. In coinfected patients progression of liver
fibrosis occurs more rapidly, and patients are more likely to
develop liver cirrhosis and liver carcinoma (1,18). Also, the
response to treatment with pegylated-interferon-alfa (Peg-
IFN-alfa) and ribavirin in coinfected patients is not as good as
in individuals infected with HCV alone (16). Antiviral treat-
ment with Peg-IFN-alfa and ribavirin eliminates HCV in less
than half of coinfected patients (22,28).
Interferon-a (IFN-a) displays antiviral, immunoregulatory,
and antiproliferative activity (23,27). This cytokine stimulates
transcriptional activation of hundreds of genes with antiviral
n Europe and the U.S., about one-third of human
immunodeficiency virus (HIV)-positive people are co-
efficacy. IFN-a enhances production of reactive oxygen spe-
cies (ROS), increases expression of adhesive molecules, and
participates in the regulation of apoptosis (2,4,19). Being the
key second messengers, ROS are involved in numerous sig-
naling pathways, and they modulate phagocytosis, secretion,
gene expression, and apoptosis (10). Augmented ROS pro-
duction in patients with chronic hepatitis C can contribute to
the inhibition of HCV replication and thus to the elimination
of HCV (6). However, excessive ROS production can cause
hepatocyte injury (7,9).
In a previous study we observed that as a result of treat-
ment with IFN-a and thymus factor X (TFX), the formation of
free oxygen radicals by resting (unprimed) neutrophils in-
creased both without stimulation and following stimulation
by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Along
with these changes, a compensatory increase in serum anti-
oxidative capacity was observed (14). We decided to ana-
lyze the effect of Peg-IFN-alfa and ribavirin treatment on
1Department of Infectious Diseases and Hepatology, Medical University of Lodz, Lodz, Poland.
2Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Lodz, Poland.
Volume 25, Number 2, 2012
ª Mary Ann Liebert, Inc.
polymorphonuclear leukocytes (PMNs) oxygen metabolism,
the expression of adhesion molecules CD11b, CD16, CD18,
and CD62L, and the death of neutrophils in patients with
Materials and Methods
The study group included 18 patients with HIV/HCV
coinfection, and the control group consisted of 34 patients
with HCV monoinfection. Patients with other systemic or
inflammatory diseases, liver cirrhosis, other causes of liver
disease, previous immunosuppressive or anti-HCV treat-
ment, and pregnant women were excluded from this study.
The diagnosis of chronic hepatitis was based on the results of
liver biopsy specimen examination. HCV infection was es-
tablished based on the presence of viral genetic material
detected by the RT-PCR method. Combined therapy using
Peg-IFN-alfa 2a (Pegasys; Roche, Basel, Switzerland) and
ribavirin was applied. Pegasys was administered subcuta-
neously once a week in a dose of 180lg. Ribavirin was ad-
ministered orally daily in a dose dependent on the patient’s
weight (those less than 60kg received 1000mg, and those
above 60kg received 1200mg). Upon the introduction of
Peg-IFN-alfa with ribavirin, all patients had received anti-
retroviral treatment and had undetectable HIV viremia (less
than 50 copies/mL). Before and after 12wk of treatment with
Peg-IFN-alfa and ribavirin, intracellular ROS levels, the ex-
pression of adhesion molecules CD11b/MAC-1, CD16,
CD18, and CD62L on neutrophils, as well as apoptosis and
necrosis of these cells were analyzed with the use of flow
cytometry. Out of 18 patients who began treatment with Peg-
IFN-alfa and ribavirin, 15 continued the treatment for at least
12wk, and were subjected to analysis of the studied pa-
rameters at two time points (before treatment and at week 12
of treatment), whereas in three patients these parameters
were assessed solely before treatment.
Flow cytometric analyses were performed on heparinized
whole blood (Li-Heparin, Monovette Blood Collection Sys-
tem; Sarsted, Nu ¨mbrecht, Germany) with the use of a FACS
Canto II Flow Cytometer (Becton Dickinson, San Jose, CA).
Granulocytes were gated using forward and side scatter
(FSC/SSC) dot plots. Ten thousand events within the granu-
locyte gate were counted per sample. The data were analyzed
using FACS Diva software (Becton Dickinson). Prior to each
analysis, the flow cytometer was calibrated by BD Cytometer
Setup & Tracking Beads (BD Biosciences, San Jose, CA). The
percentage of positively labeled cells as well as mean fluo-
rescence intensity (MFI) were used to quantify the effects.
Determination of CD11b/MAC-1, CD16, CD18,
and CD62L expression
A sample of 50lL of heparinized blood was incubated
with monoclonal mouse anti-human antibody against spe-
cific surface antigens: CD11b-PE (clone D12.11), CD62L-PE
(SK11), CD16-FITC (NKP15i), and CD18-FITC (L130) (all
provided by BD Pharmingen, San Jose, CA) for 30min at
room temperature in the dark. Then the erythrocytes were
lysed with BD FACS Lysing Solution (BD Biosciences) for
10min. After lysis the cells were washed once in BD Cell-
WASH solution and resuspended in BD CellFIX solution (BD
Assessment of ROS production
Intracellular ROS levels were measured with the use of a
commercially available assay kit (Phagoburst; Orpegen
Pharma, Heidelberg, Germany) according to the manufac-
turer’s instructions. Briefly, 100lL of blood was transferred
into 5-mL polypropylene FACS tubes and pre-incubated for
10min in an ice cold water bath. The sample of blood was
then mixed with plain buffer (negative control) or one of the
following stimulants: phorbol 12-myristate 13-acetate (PMA,
final concentration: 1.35lM) or a suspension of opsonized
Escherichia coli (0.17–0.33·109bacteria per mL). The tubes
were incubated for 10min at 37?C in a water bath. After
that, the cells were incubated with dihydrorhodamine 123
(DHR 123) for 10min at 37?C. After lysing of erythrocytes
and washing, DNA staining solution was added to exclude
aggregation artifacts of bacteria or cells during the flow
Cell death analysis
Apoptosis and necrosis of granulocytes was measured
using the FITC Annexin V Apoptosis Detection Kit I (BD
Biosciences). According to the manufacturer’s protocol,
blood erythrocytes were lysed by incubation of 200lL of
blood with BD Pharm Lyse Lysing Buffer for 10min at room
was transferred into FACS tubes and incubated for 15min at
room temperature with FITC-conjugated Annexin-V and
propidium iodide (PI). After adding 400lL of Annexin V
Binding Buffer, the samples were measured on a flow cyto-
meter within 30min. Apoptotic cells were defined as cells
demonstrating positive staining for Annexin-V-FITC [An-
were defined as double negative for both stains.
The results are presented as arithmetic mean–standard
deviation (mean–SD) for the normally distributed parame-
ters, or as a median and interquartile range [median (Q1–
Q3)] for data showing a departure from normality (the
assumption of normality was assessed with the Shapiro-Wilk
test). An independent samples t-test was conducted to
compare the normally distributed unpaired data sets. Sta-
tistical comparisons of non-normally distributed variables
were performed either with the Mann-Whitney U test or the
Wilcoxon signed-rank test (for independent and paired data,
respectively). A chi-square test was used for testing an as-
sociation between categorical variables.
Profile of the patients
There were no statistically significant differences in age,
gender, ALT and GTP activity, HCV viral load, or
NEUTROPHILS AND PEG-IFN-ALFA AND RIBAVIRIN TREATMENT 167
histopathological changes between the group of HIV/HCV
coinfected patients and the control group (patients with HCV
monoinfection) (Table 1). Before antiviral treatment, expres-
sion of CD11b/MAC-1, CD16, CD18, and CD62L antigens,
and ROS production, as well as the percentage of apoptotic
and necrotic whole blood neutrophils of HIV/HCV coin-
fected patients did not differ significantly from the group of
patients with HCV monoinfection (Table 2). At the 12th week
of antiviral therapy, the HCV viral load in HIV/HCV coin-
fected patients decreased significantly, from 2580 (549–6415)
IU/mL (·103) to 0.002 (0.002–374) IU/mL (·103; p<0.0001).
During treatment with Peg-IFN-alfa and ribavirin all HIV/
HCV patients were on combination antiretroviral therapy
(cART) and had undetectable HIV viremia (less than 50
Influence of antiviral therapy on CD11b/MAC-1, CD16,
CD18, and CD62L expression
Treatment with Peg-IFN-alfa and ribavirin resulted in an
increase in the expression of CD11b/MAC-1 and CD18, and
a decrease in CD16 and CD62L; however, only the change in
CD62L expression was statistically significant (Fig. 1. and
Influence of antiviral therapy on ROS production
After 12wk of treatment intracellular ROS production of
unstimulated neutrophils remained unchanged, but after
stimulation with PMA, ROS levels increased statistically
significantly. An increase in ROS production was also trig-
gered by the suspension of opsonized Escherichia coli; how-
ever, these changes were not of statistical significance (Fig. 2
and Table 3).
Influence of antiviral therapy on granulocyte death
Antiviral therapy in patients with HIV/HCV coinfection
significantly increased the percentage of apoptotic whole
blood neutrophils, whereas the percentage of necrotic cells
was not affected (Fig. 3 and Table 3).
Impairment of neutrophil function has been demonstrated
in both symptomatic and asymptomatic patients with HIV
infection (20,21). Neutrophils isolated from HIV-infected
patients displayed an impairment of chemotaxis and
phagocytosis and dysregulated production of ROS (5,29).
Schwartz et al. demonstrated that neutrophils extracted from
asymptomatic HIV-infected patients had an exaggerated re-
sponse to LPS and a diminished inhibitory response to
S100A8/A9. The authors concluded that this dysregulated
response would contribute to the onset of bacterial or fungal
infections and increased transactivation of HIV (25). Elbim
et al. showed that unstimulated PMNs and monocytes
from HIV-infected patients are activated even in the early
asymptomatic stage of the disease. Unstimulated phagocytes
from these patients expressed higher H2O2 production
and CD11b adhesion molecule expression, and decreased L-
selectin expression (8).
In our study we compared PMN oxygen metabolism, the
expression of adhesion molecules CD11b/MAC-1, CD16,
Table 1. Characteristics of the Patients
HCV viremia (IU/mL·103)
Histopathology staging (S)a
Histopathology grading (G)a
aEstimation according Batt and Ludwig’s scale.
GPT, glutamic-pyruvic transaminase; HCV, hepatitis C virus;
ALT, alanine aminotransferase; HIV, human immunodeficiency
Table 2. Studied Parameters Before Peg-IFN-alfa and Ribavirin Treatment
HCV patients (n=34)HCV/HIV patients (n=18)
Adhesion molecules CD16
PI(+) Annexin V(–)
HCV, hepatitis C virus; HIV, human immunodeficiency virus; PI,- propidium iodide; PMA, phorbol 12-myristate 13-acetate; ROS, reactive
oxygen species; MFI, mean fluorescence intensity.
168 JABLONOWSKA ET AL.
CD18, and CD62L, and apoptosis/necrosis of neutrophils in
two groups of patients: those infected with HCV alone and
those with HIV/HCV coinfection. Moreover, we analyzed
the effect of Peg-IFN-alfa and ribavirin treatment on these
parameters in HIV/HCV coinfected patients. In our previous
study, similarly to the findings of other authors (7), we ob-
served increased ROS production in patients with HCV
monoinfection compared to healthy individuals, whereas in
our present study we did not find any differences in ROS
production in HCV monoinfected and HIV/HCV coinfected
patients. This may be related to the fact that patients with
HIV/HCV conifection were on cART with undetectable HIV
whole blood neutrophils were identified and gated based on forward and side scatter (FSC/SSC) dot plot (A). Histograms
show CD16 (B), CD18 (C), CD11b/MAC-1 (D), and CD62L (E) expression (expressed as mean fluorescence intensity) on
neutrophils before (shaded areas) and after (open areas) 12wk of combined therapy with Peg-IFN-alfa and ribavirin. Gates
(dashed line) were set up according to isotype control data.
Flow cytometric analysis of expression of specific surface markers on whole blood granulocytes. Human peripheral
NEUTROPHILS AND PEG-IFN-ALFA AND RIBAVIRIN TREATMENT169
Table 3. Studied Parameters in HIV/HCV Coinfected Patients Before and at Week 12 of Treatment
with Peg-IFN-alfa and Ribavirin
Before treatment (n=15)At 12 weeks of treatment (n=15)
PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; MFI, mean fluorescence intensity; HCV, hepatitis C virus; HIV, human
trophils were identified and gated based on DNA staining (A) and forward and side scatter (FSC/SSC) dot plot (B).
Histograms show mean fluorescence intensity of dihydrorhodamine 123 (DHR) staining of unstimulated cells (C), or cells
stimulated either with phorbol 12-myristate 13-acetate (PMA; D), or suspension of opsonized Escherichia coli (E), before
(shaded areas) and after (open areas) 12wk of combined therapy with Peg-IFN-alfa and ribavirin. Gates (dashed line) were
set up according to unstimulated cells.
Flow cytometric analysis of ROS production by whole blood granulocytes. Human peripheral whole blood neu-
170 JABLONOWSKA ET AL.
viral loads, and CD4 T-cell counts were higher than 350
During treatment with Peg-IFN-alfa and ribavirin we
observed an increase in ROS production in HIV/HCV
coinfected patients. The same effect of IFN treatment was
observed by us and by other authors in HCV-monoinfected
patients (6,11,14,15,17). Our present study confirms the results
of our previous research conducted in HCV-monoinfected
patients (15), which demonstrated that treatment with Peg-
IFN-alfa and ribivirin causes activation of PMNs.
Co-occurrence of an increase in ROS production and a
decrease in HCV viremia seen after 12wk of treatment with
Peg-IFN-alfa and ribavirin may indicate that neutrophils
play an important role in the elimination of HCV infection in
HIV/HCV coinfected patients. Yet the increased ROS pro-
duction seen during treatment with Peg-IFN-alfa and riba-
virin may result in transient transactivation of HIV. Our
observations are in accord with the results of other authors,
who showed that ROS activate NF-jB transactivation fac-
tor and induce HIV-LTR transactivation in monocytic cell
lines (13), and potentiate monocyte production of proin-
flammatory cytokines (3,12). However, in patients on cART
during treatment with Peg-IFN-alfa and ribavirin, we
did not observe a transient increase of HIV viremia, which
we believe indicates that cART is a safe therapy for use with
Peg-IFN-alfa and ribavirin in HIV/HCV coinfected patients.
Here we conclude that in HIV/HCV coinfected patients
on cART, Peg-IFN-alfa and ribavirin treatment causes acti-
vation of neutrophil functions, yet it does not affect the
suppression of HIV replication.
The study was supported by an internal grant from the
Medical University of Lodz (502-11-724), and the Nofer In-
stitute of Occupational Medicine (IMP 11.3). The skillful
technical assistance of Jolanta Wojno-Kulinska and Barbara
Kur is gratefully acknowledged.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Elzbieta Jablonowska
Department of Infectious Diseases and Hepatology
Medical University of Lodz
Received October 23, 2011; accepted December 13, 2011.
172JABLONOWSKA ET AL.