Heroin-induced changes of CD34-positive rat thymus cells

doi:10.4236/health.2010.28142

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Vol.2, No.8, 957-961

doi:10.4236/health.2010.28142

(2010) HEALTH

Openly accessible at/HEAL H

Heroin-induced changes of CD34-positive rat thymus

cells

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.

ABSTRACT

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

responses.

Keywords: Heroin; Rat Thymus; CD34

1. INTRODUCTION

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

958

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. MATERIALS AND METHODS

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

Immunofluorescense

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-

ture.

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

3. RESULTS

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

959

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.

4. DISCUSSION

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

960

wn that T-cells－a subset of lymphocytes

rphometrical

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