ARTICLE
Auteur(s) : Atosa Azarang1, Majid
Mahmoodi1,2, Said Rajabalian1, Majid
Asadi Shekari1, Jafar Nosratabadi1, Nima
Rezaei3
1Neuroscience Research Center, Kerman University of
Medical Sciences, Kerman, Iran
2Cancer Research Center, Medical Sciences/University of
Tehran, Tehran, Iran
3Immunology, Asthma and Allergy Research Institute,
Medical Sciences/University of Tehran, Tehran, Iran
accepté le 26 Octobre 2007
Conflicting results are reported concerning the effect of opioid
drugs on immune function. Several authors have reported that opioid
addicts have an increased susceptibility to various infectious
diseases [1, 2], and morphine or heroin may sensitize users to
virus-bound infection due to the impaired immune function in these
individuals [3, 4]. Similarly, down-regulation of phagocytic cell
function and disturbances in cytokine production have been reported
[1, 5]. It has also been noted that morphine could inhibit
interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α) and
superoxide radical production in vitro by mitogen-stimulated,
peripheral blood mononuclear cells in an opioid-mediated way [6-8].
Morphine inhibits natural killer (NK) cell responses when used
experimentally in animals and in human volunteers [9, 10].
Conversely, some experimental data suggest that the opioid effects
on the immune system are not necessarily deleterious; in fact,
endogenous opioids administered in vitro to human peripheral blood
mononuclear cells augment NK activity in a naloxone-sensitive
manner [11]. Conflicting results also show that results shows that
a low dose of morphine has no effect on the production of
interleukin (IL)-6 and TNF-α by human peripheral mononuclear cells
[6].In cell-mediated immunity, CD4+ T-helper cells are usually
classified into two subsets based on the types of cytokines they
produce. T-helper 1 (Th1) cells produce cytokines such as IFN-γ and
IL-2, are mainly involved in macrophage activation. In contrast,
T-helper 2 (Th2) cells synthesize IL-4 and IL-10, and are mostly
responsible for regulating humoral immune responses and progression
of infection [12]. The delicate balance between Th1 and Th2
cytokines is thought to influence the outcome of disease.
Production of IFN-γ by Th1 cells is usually a predictive index of
good response in cell-mediated immunity, and secretion of IL-10 by
Th2 cells contributes to impaired cellular immunity [12]; therefore
in our study we investigated the in vitro production of IFN-γ and
IL-10 after stimulation of the cells, with phytohemagglutinin (PHA)
or with lipopolysaccharide (LPS), in whole blood from opioid
addicts compared to healthy individuals.
Subjects and methods
Subjects
After ethical approval from the local Ethics Committee, 20 opioid
addicts with no history of alcoholism and/or addiction to other
substances, who were admitted to the Detoxification Department of
Local Health Center (in Kerman, Iran), were enrolled into this
study and provided written consent to participate. The control
group also consented to take part in this study. All subjects were
men, aged 21 to 40 years. None had had any recent infections or a
positive HIV or HBsAg test. They were free of drugs affecting the
immune system and had no history of recent vaccination. Four to
five milliliters of venous blood were drawn from each subject into
a sterile, endotoxin-free, polystyrene tube containing 100 units/mL
sodium heparin. Each blood sample was processed within one hour
after collection. After all the experiments were complete, the
opioid addicts were divided into opium and heroin addiction groups.
An age matched group of 10 healthy individuals, between 20 and 40
years old, served as the control group.
Mitogen and antigen
Culture stimulators were the mitogen, phytohemagglutinin (PHA, 5
μg/mL) (L9017, Sigma, Sweden) and lipopolysaccharide (LPS, 1μg/mL)
(Escherichia coli 0111; B4, Boivin method, Difco Laboratories,
Detroit, MI, USA). Mitogen and LPS were used at optimal stimulatory
concentration as described elsewhere [13, 14].
Stimulation of whole blood
A heparinized, whole blood sample from each subject was diluted to
1:5 with RPMI-1640 (Gibco Life Technologies, Paisly, UK). The
diluted blood was cultured in a one mL volume in endotoxin-free,
polystyrene, round-bottomed sterile tubes with caps and in
duplicate. The cells from the diluted whole blood were either
cultured alone as control or stimulated with LPS (1 μg/mL) or with
PHA (5 μg/mL). The tubes were incubated in a humidified air
atmosphere at 37˚C with 5% CO2 for 72 hours. After
incubation, all antigen-treated, PHA-treated and control tubes were
centrifuged at 300 × g and the supernatants were removed and kept
frozen at -20˚C until assayed for cytokine concentrations. These
fluids were coded and laboratory personnel were blinded throughout
the experimental procedures.
Cytokine measurements
The frozen, culture supernatant fluids were thawed at room
temperature and cytokines, IL-10 and interferon-γ levels were
measured by Enzyme-Linked Immunosorbant Assay (ELISA) using
commercial assay kits supplied by Biosource Europe S.A. (Nivelles,
Belgium) according to the manufacturer’s instructions. The
absorbance of each well was read at 492 nm and cytokine
concentrations in the samples were calculated with a standard curve
generated from recombinant cytokines. Cytokine values were
expressed as picograms/milliliter (pg/mL).
Statistical analysis
The results are presented as the mean ± standard deviation (S.D.).
Student’s t-test was used. Two-group comparison was performed using
the Wilcoxon test (paired) or the Mann–Whitney U-test (unpaired).
Statistical analysis of the data was carried out using SPSS,
version 13. Values of p < 0.05 were considered statistically
significant.
Results
Levels of immune response parameters were evaluated before the
start of detoxification. The base-line characteristics of all
subjects; opium addicts (O; n = 10), heroin addicts (H; n = 10) and
healthy control (N; n = 10) groups for the cytokine production
study are shown in table 1( Table 1 ).
The ages of the subjects ranged from 21 to 58 years. The dosage
range of opium that was used by opium addicts was 500 to 3 000
mg/day. The dosage range of heroin that was used by heroin addicts
was 500 to 2 000 mg/day. The duration of the addiction in the opium
and heroin groups was 8.7 ± 8.03 and 6.7 ± 5.2 years, respectively.
Data for IFN-γ and IL-10 production in cell cultures of whole
blood after stimulation with PHA or with LPS is shown in table 2(
Table 2 ). The mean production of IFN-γ
in response to LPS or to PHA by cells from opium addicts was lower
than corresponding values in the control group (mean ± SD:
67.6 ± 24.5 versus 110.2 ± 60.3 pg/mL in response to LPS). However,
this was not significant (p > 0.05) (table 2, ( figure 1) ). In heroin
addicts, the mean production of IFN-γ in response to LPS or to PHA
was significantly lower than corresponding values in the control
group (35.9 ± 26.3 versus 110.2 ± 60.3 pg/mL in response to LPS,
p < 0.01), (table 2, ( figure 2) ).
With regard to the production of IL-10, it was observed that
much higher levels were produced by the cells in response to LPS or
to PHA in heroin addicts compared with this value in the control
group (71.8 ± 28.4 versus 17.1 ± 13.5 pg/mL in response to PHA,
p < 0.01) (table 2, ( figure 3) ). In opium
addicts, stimulation of the cells with LPS or with PHA resulted in
an elevated level of IL-10 in the culture supernatants compared
with the control group (28.6 ± 14.5 versus 17.1 ± 13.5 pg/mL in
response to PHA, p > 0.05) (table 2).
Table 1 Baseline characteristics (mean ± SD) of the
subjects in the study
|
Opium addicts (n = 10)
|
Heroin addicts (n = 10)
|
Healthy controls (n = 10)
|
|
Age (years)
|
31 ± 10.3
|
28.9 ± 5.7
|
26.7 ± 3.2
|
|
Dosage of opioid addiction (mg/day)
|
500-3 000
|
500-2 000
|
-
|
|
Duration of opioid addiction (years)
|
8.7 ± 8.03
|
6.7 ± 5.2
|
-
|
|
HIV test
|
Negative
|
Negative
|
Negative
|
|
HBs Ag test
|
Negative
|
Negative
|
Negative
|
Table 2 Supernatant cytokine levels, interferon-γ
(IFN-γ) and interleukin-10 (IL-10), of whole blood cells from opium
addicts, heroin addicts and healthy control groups, stimulated in
vitro with lipopolysaccharide (LPS)/phytohemagglutinin (PHA).
Cytokine levels are given in pg/mL. Data are expressed as
mean ± SD
|
Groups
|
Cytokines
|
Cytokine production stimulated by LPS
|
Cytokine production stimulated by PHA
|
|
Opium addicts
|
IFN-γ
|
67.6 ± 24.5
|
162.8 ± 45.6
|
|
IL-10
|
36.1 ± 13.1
|
28.6 ± 14.5
|
|
Heroin addicts
|
IFN-γ
|
35.9 ± 26.3*
|
76.3 ± 40.9*
|
|
IL-10
|
88.3 ± 30.4*
|
71.8 ± 28.4*
|
|
Healthy controls
|
IFN-γ
|
110.2 ± 60.3
|
213.3 ± 61.6
|
|
IL-10
|
26.1 ± 12.8
|
17.1 ± 13.5
|
Discussion
In experimental studies involving the effects of morphine (the most
abundant opioid molecules in opium extracts) on the cells of the
immune system, the literature points to the conclusion that
morphine given in vivo modulates a variety of immune responses
(including T- and B-cell function, macrophages, natural killer cell
and polymorphonuclear leukocyte function) [15, 16]. This assumption
is supported by numerous studies in humans which show that morphine
or heroin (a morphine derivative) inhibits proliferation of T- and
B-cells, suppresses T cell-mediated cytotoxicity and decreases the
activity of NK cells in vitro [10, 17]. Experimental studies have
demonstrated immunosuppressive effects of morphine and heroin.
However, controversial results have been reported in clinical
studies. We thus decided to evaluate the in vitro cytokine
production of cells in whole blood from a group of opium addicts
and a group of heroin addicts.
In our study, the group of opium addicts showed higher
production of IL-10 and lower production of IFN-γ compared to the
healthy control group (table 2, ( figure 1) ), but this
difference was not significant (p > 0.05). Similar results have
been reported by Peterson et al., who found intact production of
IFN-γ by concavanalin A- (con-A) stimulated peripheral blood from
patients maintained on methadone [18]. However, we found comparable
production of IL-10 by cells of whole blood from opium addicts. In
addition, a previous study by Brugo et al., showed no evidence of
in vitro suppression after con-A stimulation of peripheral blood
from heroin addicts or patients maintained on methadone [19].
Another study by Shine et al. indicated a normal pattern of T-cell
subsets in a group of healthy, intravenous drug abusers and
patients maintained on methadone [20]. Similarly, a normal T-cell
response to both con-A and tetanus toxoid antigen in another group
of healthy addicts has been reported [21].
We found that immune cells from heroin addicts presented a
significantly enhanced production of IL-10 and lower production of
IFN-γ (p < 0.01) after PHA or LPS stimulation in comparison with
immune cells from a healthy control group (table 2, ( figure 2) ). This finding
is supported by the result from Govitrapong et al. who demonstrated
a decrease in the response of T-lymphocytes to a wide range of
concentrations of PHA in heroin addicts [22]. This result is also
supported by experimental studies by Fecho et al. who showed that
administration of heroin in rats produced a dose-dependent
decreases of B- and T-cell proliferations and interferon-gamma
production in the spleen [23]. This has also been demonstrated by
Roy and his colleagues, who investigated the effect of morphine
treatment on Th1/Th2 cytokine production in an experimental study
of an LPS-mediated sepsis model, which in animals treated with both
LPS and morphine showed a significant shift towards a Th2 cytokine
profile [24].
The nature of the immunomodulatory activity of the opioids has
been the subject of a great deal of research over the last ten
years. There is increasing evidence that effects of opioids on the
immune response are mediated at several levels. Fecho and his
colleagues have demonstrated that sympathoadrenal activity is
involved in the suppressive effects of acute morphine treatment on
the proliferative response of splenic T and B cells to Con A, LPS
or ionomycin/phorbol myristate acetate [25]. Wang et al. who
investigated the role of the mu-opioid receptor in chronic,
morphine-mediated immunosuppression in mice, showed that
morphine-induced immunosuppression may be mediated directly by the
mu-opioid receptor and they provide evidence that the inhibition of
IFN- γ synthesis and activation of macrophage-cytokine synthesis is
corticosterone-independent, and only partially dependent on
sympathetic activation [26]. McCarthy and colleagues made a review
on the impact of opioid treatment on antibody responses, and
suggest that the molecular basis for this effect is likely due to
the modulation of both cytokine and cytokine receptor expression
[27]. Other authors have reported that opioid peptides, bound to
various types of opioid receptors, detected on various cell types
including blood mononuclear elements, which differentiate as
macrophages in tissues, elicited a stimulatory effect [28]. Vallejo
et al. believe that opiates behave like cytokines, modulating the
immune response by interaction with their receptors in the central
nervous system and in the periphery. So peripheral
immunosuppression is mediated, at least in part, by opioid
receptors located in the central nervous system, and
intrathecally-administered opioids do not exert the same
immunosuppressive effects. This may have important clinical
implications for those patients receiving long-term opioid therapy
for malignant and nonmalignant pain [29].
In conclusion, the results from our study indicate that opioid
addiction could cause an in vitro shift in the Th1/Th2 cytokine
balance of peripheral CD4+ cells towards a Th2 response, and that
opioid addicts demonstrate reduced mitogenic responsiveness of
lymphocytes compared to healthy individuals.
Acknowledgement
This work was supported by a grant from Neuroscience Research
Center, Kerman University of Medical Science, Kerman, Iran.
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