Vol.2, No.8, 957-961
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(2010) HEALTH
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Heroin-induced changes of CD34-positive rat thymus
Gentimi Fotini1, Perrea Despina2, Marinos Evangelos3, Konstandi Ourania1,
Katsorchis Theodoros1*
1Department of Cell Biology and Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece;
*Corresponding Author: tkatsor@biol.uoa.gr
2Lab for Experimental Surgery and Surgical Research, Medical School, University of Athens, Athens, Greece
3Lab of Histology and Embryology, Medical School, University of Athens, Athens, Greece
Received 30 March 2010; revised 5 May 2010; accepted 7 May 2010.
Heroin is a well known opioid that causes al-
terations to the immune system of a number of
investigated animals. Only a few studies have
explored the effect of heroin on the lymphocytes
maturation. Thymocyte progenitors originate
from haematopoietic stem cells in the bone mar-
row. The immature T-cells express neither CD4
nor CD8, and are therefore classed as double-
negative (CD4-CD8-) cells. CD34 glycoprotein is
the only defined marker of the immature T-
lymphocytes. In this study we have investigated
the changes induced in CD34+ rat thymocytes
after heroin administration by immunofluores-
cence in frozen rat thymus sections using the
4H11 monoclonal anti-CD34 antibody. There is a
remarkable decrease in the number of CD34+
immature thymocytes when examined 1hour
after last heroin injection and a small recover
when examination took place 20 days after last
injection. The above results suggest a major
effect of heroin administration early in the ma-
turation process of T lymphocytes probably by
increasing the apoptotic cell death and the ne-
gative consequences for the immune system
Keywords: Heroin; Rat Thymus; CD34
Thymocytes are the principal mediators of the sympa-
thetic stimulation that coordinate the organism's acute
responses to a large variety of stressors, including the
administration of narcotic drugs like morphine, an alka-
loid of opium, and its less stable derivative heroin,
which is eventually biotransformed into morphine [1,2].
Heroin binds a) to endorphin receptors throughout the
body causing a feeling of euphoria along with a "relict"
of pain and b) to receptors of neurons that travel from
the spinal cord to the limbic system causing a feeling of
pleasure. Chronic heroin users become physically de-
pendent on the drug; their endogenous endorphin pro-
duction decreases and develops severe tolerance and
withdrawal symptoms. The immunomodulatory proper-
ties of clinically relevant opiates, like morphine, are well
established [3-5]. For example, numerous animal studies
demonstrate that acute or chronic exposure to morphine
produces alterations in many measures of immune status
including in vitro immune responses, like the formation
of plaque-forming cells, lymphocyte proliferation, cyto-
kine production, and natural killer (NK) cell activity
[3-6], and in vivo immune responses, like antibody pro-
duction, graft-versus-host reactions, and contact hyper-
sensitivity responses [7-9]. Previous studies have shown
that some of heroin’s immunomodulatory effects are
similar to those produced by morphine, but some are un-
ique. More specifically, both morphine and heroin pro-
duce a decrease in the proliferative response of splenic T
and B cells, the cytotoxicity of NK cells, and the pro-
duction of IFN-g by stimulated splenocytes.
However, heroin is approximately 10 times more po-
tent than morphine in producing immune alterations in
the spleen. Unlike morphine, the effect of heroin in ex
vivo assays of splenic NK cell activity appears to result
in part from a decrease in effect or NK cells in the spleen.
Another effect unique to heroin is an alteration in the
size of a granulocyte subpopulation in the spleen.
While previous investigations have explored the ef-
fects of heroin in the immune system responses only a
few of them have studied its effects on lymphocytes
maturation and cell death. It is well known that the thy-
mus represents the major site of T lymphocyte matura-
tion [l]. Different steps of thymocyte differentiation have
been identified on the basis of both phenotypic and func-
G. Fotini et al. / HEALTH 2 (2010) 957-961
Copyright © 2010 SciRes. http://www.scirp.org/journalT /Openly accessible at/HEALH
tional criteria [l,10]. The study of human thymic T cell
progenitors has been hampered by the lack of markers as
well as differentiation assays that unequivocally define
these cells, although there is consensus that the most
immature human thymocytes lack CD3, CD4, and CD8
[8,9,11,12]. The only well-defined marker expressed by
a fraction of immature thymocytes is represented by the
non lineage-specific CD34 [7] a 120-kD cell surface
antigen that is expressed on pluripotent hematopoietic
stem [13-15] cells and on precursors that are committed
to several hematopoietic lineages, including the B [16],
myeloid, and erythroid lineages [13,14]. Studies have
shown that thymic CD34+ cells have a very limited
myeloid differentiation capacity and differentiate in vitro
mostly into CD1a+-derived but not CD14+-derived den-
dritic cells (DC).
In this study we have investigated the effects of heroin
administration in CD34+ immature rat T-cell lympocytes
using the 4H11 monoclonal anti-CD34 antibody in order
to find some evidence on how–at very early stages- the
lymphocytes maturation process is affected and what the
consequences are for the rest of the immune system re-
sponse. The results presented are in agreement with pre-
vious studies and extend our knowledge in relation to the
apoptotic mechanisms in thymus after heroin admini-
stration. For the first time an immunohistochemical &
morphometrical analysis of thymus is made in order to
quantitate cell apoptosis successfully, using CD34 as a
specific marker.
2.1. Experimental Animals
Thirty 2 weeks old male Wistar rats, weighing 50gr each,
were housed in cages in a ventilated room with alternat-
ing light cycle, 12h dark/12h light, at about 25°C and fed
a standard chow diet and water ad libitum. They were
divided in experimental groups A, B and C containing
ten animals per group.
2.2. Protocol of Heroin Administration
The experimental animals in groups B and C (ten ani-
mals each) received subcutaneous heroin injections daily,
of increasing dosage for eighteen consecutive days, as
follows: Starting dose was 1 mg heroin/kg animal body
weight for days 1-3. The dose was doubled to 2, 4 and 8
mg heroin/kg per animal body weight, during days 4-6,
7-9 and 10-12, respectively. The dose was next increased
to 12 mg heroin/kg animal body weight for days 13-15
and 16 mg/kg of animal body weight for days 16-18.
The dose was next increased to 12 mg heroin/kg animal
body weight for days 13-I5and 16 mg/kg of animal body
weight for days 16-18 [5]. By analogy to the corre-
sponding heroin volumes, the ten control rats (group A)
received equivalent daily injections of isotonic saline for
eighteen days. All heroin treated group B and all control
rats (group A) were sacrificed 1 h after the last injection
while group C (ten animals) rats where sacrificed 20
days after the last injection.
2.3. Tissue Preparation and
Immediately after dissection, the thymus tissues were
immersed in 0.1 M cacodylate buffer pH 7.4 containing
4% paraformaldehyde and 5% glutaraldehyde for 10 min.
They were washed in the same buffer supplemented with
25% sucrose for 5 to 10 min, transferred to the same
buffer supplemented with 2% glyoxylic acid and 25%
sucrose for 15 min and quick-frozen in liquid nitrogen.
Cryosectioning was performed at –30°C by adjusting the
microtome at 2 to 4 μm. Six and ten thymus sections per
experimental and control animal group A and B respec-
tively, were picked up on slides, mounted and fixed for
20 min in 3.5% formaldehyde sodium in PBS (pH 7.2)
and rinsed in PBS.
Tissue sections were exposed to CD34 antibodies (di-
luted 1:100) for 60 min, rinsed in PBS and incubated
with anti-mouse conjugated to fluorescein (1:300) for 30
min. All incubations were carried out at room tempera-
2.4. Visualization
The microscopic observation was carried out under a
ZEISS AXIOPHOT microscope with excitation filter 13
(max. 400 nm) and barrier filter 3.
From each experimental animal biopsy, thirty photo-
micrographs were taken in total. Photographs 1-3 are
representative. These were analyzed by a computer run-
ning the program Image-Pro plus (Media Cybernetics)
Animal care and use was approved by the University
ethics committee
Baseline distribution of transmembrane glycoprotein
mucosaline (CD34) containing particles in thymocytes
frontal sections from placebo and heroin treated rats,
sacrificed 1 hour (group A and B) after the last injection
with normal saline and heroin respectively as well as
twenty [16] days (group C) after induce of heroin, was
analyzed by histofluorescence using the anti-CD34 an-
tibody detecting significant differences between the
three groups. Fully T-cell comitted CD34+ immature
thymocytes are normaly detected in untreated animals.
Figure 1(a) shows a typical pattern of fluorescent gly-
coptotein signals classified as distinct grains and aggre-
G. Fotini et al. / HEALTH 2 (2010) 957-961
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Openly accessible at
gates, under baseline conditions. Figures 1(b) and 1(c)
show that there is a significant decrease in the number of
the CD34+ glycoprotein signals 1 hour after heroin ad-
ministration and a remarkable recovery 20 days after the
last treatment. Despite the detected recovery, there is still
a significant difference between the fluorescent signals
corresponding to the CD34+ thymocytes detected in the
untreated and the heroin administed animals.
Using the same program both the microphotographs
and the pictures of the control sections and the ones after
heroin administration, were analysed. The coverage of
each field of vision of the aggregates and grains of
Group A is 43% of their respective Group B is 29% and
Group C is 37%.
The histogram in Figure 2 resulted from the computer
analysis of photographs of thymocytes stained with an-
ti-CD34 antibody in sections, from heroin injected and
naïve animals, summarizing the mean number of fluo-
rescent particles, including grain (diameter less than 0.8
μm) and aggregate (diameter greater than 1 μm) per op-
tical field.
The fundamental role of the thymus in the develop-
ment of an effective immune system is well established
as well as the negative effects that the opioids such as
heroin cause to the immune responses [12,17-19]. The
results in the present study clearly demonstrate that her-
oin administration decreases the cellularity of rat thymus.
This is in accordance with results from similar investiga-
tions in rat spleen and mice thymus. As deduced from
(a) (b) (c)
Figure 1. Fluorescent photomicrographs of rat thymus sections.
The animals were sacrificed, one hour posi repetitive placebo or
heroin administration. (a) Saline treated control group; (b) Her-
oin treated group sacrificed 1 hour after last drug injection.
Fluorescent grains are indicated by arrowhead, aggregates by
arrow; (c) Heroin treated group sacrificed twenty days after last
drug injection. All magnifications were (× 800). Each photo-
graph represented approximately one optical field from each
group of animals. Straight arrows represent aggregates while
dotted arrows represent grains.
Figure. 2. The histogram resulted from the image analysis of the
photomicrographs. X = Medium term of the number of aggre-
gates and grains from 350 fields of observation. Z = 1-3 the me-
dium term of aggregates of the control group A (1), the group B,
animals sacrificed one our after heroin administration (2) and the
group C, animals sacrificed 20 days after heroin administration
(3). Z = 4-6 the medium term of the grains of the control group A
(4), the group B, animals sacrificed one our after heroin admini-
stration (5) and the group C, animals sacrificed 20 days after
heroin administration (6) P > 0.05.
literature, CD34 use as a specific marker was enough to
reveal the effects of heroin. Although there is supporting
evidence that adaptive mechanisms exist leading to tol-
erance prior to the development of abstinence to the drug
since a small recovery take place after the discontinu-
ance of heroin administration. These results are in ac-
cordance with previous studies performed in our labora-
tory investigating the baseline and heroin-suspended
levels of the CD34 glycoprotein-containing particles
including grain (diameter less than 0.8 μm and aggregate
(diameter greater than 1 μm forms, in rat cell thymus
immature cells [18]. Results from both studies show that
heroin administration to heroin-tolerant or not rats causes
formation of unusually large intracellular glycoprotein
aggregates (diameter greater than 1 μm) in thymus cells
and support a direct role for these formations in the mod-
ulation of biogenic glycoprotein bio-availability. The ra-
tional for our selection of the thymus in this study, was
based on reports with tolerant nits indicating that opiates
induce minimal changes in the glycoprotein pool of this
thymus structure [6]. In previous studies, the parallel
changes detected by electrophysiological immunocy-
lochemical and autoradiographic techniques in thymus
cells following opiate administration, suggest that bio-
genic glycoprotein interact directly with heroin and oil-
ier narcotic analgesic drugs [20,21]. According to our
findings it is proposed that the adaptation to drug expo-
sure involves multiple homeostatic interactions, with
sympathetic activation at the level of proteins reorgani-
zation and redistribution playing major roles in rat im-
G. Fotini et al. / HEALTH 2 (2010) 957-961
Copyright © 2010 SciRes. http://www.scirp.org/journalT /
wn that T-cellsa subset of lymphocytes
E., Osipenko, O.N., Haskó, G. and
J.M. and Bei-
yamin, R.
t, D. and Rather, M. (1979) Catecholamine biosyn-
quin, F., Setterblad,
Lysle, D.T. (1999) Enhance-
hymus functions. Per-
Openly accessible at/HEALH
mune system.
It is well kno
ay a major role in the immune response effectiveness.
Their maturation process takes place in the thymus [22].
Different steps of thymocyte differentiation have been
identified on the basis of both phenotypic and functional
criteria [22,23]. Immature thymocytes have been nega-
tively defined on the basis of the lack of surface expres-
sion of markers present at later stages of differentiation
including CD3, TCR, CD4 and CD8 [24-28,29]. The
only well-defined marker expressed by a fraction of im-
mature thymocytes is represented by the non lineage-
specific CD34 [28,30]. In spite of its expression on he-
matopoietic progenitors, the function of CD34 remains
unresolved [31,32]. The 4H11 monoclonal antibody used
in our studies reacts with human CD34, also known as
mucosialin. According to our findings there is remark-
able decrease of the CD34+ immature T lymphocytes
after heroin administration suggesting that heroin can
affect thymocytes survival or proliferation. These results
in relation with recent investigations [33,34] or in studies
performed in our laboratory, strongly suggest an apoptotic
role of heroin on immature T-cells. The growth- inhibitory
or apoptosis-inducing effects of morphine in neurons
and immunocytes are directly associated with morphine
tolerance [33,35] or receptor desensitization as assessed
by a lack of morphine-stimulated GTPase activity at
concentrations that inhibit tumor growth [16]. Drugs that
prevented the development of morphine tolerance in rats
also prevented cell death [33,34] and vice versa [33].
Recent investigations concerning the cellularity of an-
other organ implicated in the immune process have
shown similar results on the effects of heroin in splenic
leucocytes [36]. Even a single injection of heroin pro-
duces a dose-dependent, naltrexone-reversible decrease
in the total number of leukocytes in the rat spleen. The
heroin-induced decrease in splenic leukocytes is not ac-
companied by a heroin induced increase in circulating
leukocytes. Heroin does not increase the number of ne-
crotic leukocytes in the spleen, but does increase the
number of apoptotic leukocytes in the spleen. The her-
oin-induced increase in leukocyte apoptosis is clearly
evident when splenocytes are isolated 1 or 3 h after in-
jection, and the effect of heroin is maintained after 24 h
of culture [36]. Similar findings have been shown in
monocytes by inhibiting their response to activating
stimuli after heroin administration [30,37].
In conclusion, in the present work a mo
udy is made for the first time in order to show immu-
nohistochemically that heroin administration to rats causes
formation of unusually large intracellular aggregates with
glycoprotein thymus cells, a finding supporting a direct
role for these formations in the modulation of biogenic
glycoprotein bio-availability and provide evidence of the
apoptotic effects of heroin in immature thymocytes af-
fecting their maturation and differentiation process.
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