Download Withdrawal-induced Inhibition of Phagocytosis in Morphine Tolerant

Withdrawal-induced
Inhibition of Phagocytosis
in Morphine Tolerant
Macrophages may be Due
to an Increase in cAMP
Cristhian J. Ildefonso¹, Fernando L. Renaud¹ and Ana M. Lugo-Chinchilla²
¹Biology Department, University of Puerto Rico, Rio Piedras Campus, and
²Biology Department, Interamerican University, Bayamón Campus.
Abstract
We have previously demonstrated that acute exposure to 100nM morphine
inhibits phagocytosis by murine peritoneal macrophages, whereas chronic treatment
induces a state of putative tolerance. The tolerant state is also accompanied by
putative dependence, since withdrawal from tolerant cells inhibits phagocytosis.
Tolerant and dependence to morphine in the nervous system are based at least in
part on an upregulation of the cAMP pathway. In the present study the role of this
pathway in tolerant and dependent macrophages was investigated. We found that
morphine does not change basal levels of cAMP during acute and chronic treatment.
On the other hand, drug withdrawal from tolerant cells results in a transient increase
in cAMP levels, which suggests that this cyclic nucleotide could be involved in the
inhibition of phagocytosis. This was confirmed by the fact that artificially increasing
levels of cAMP in tolerant cells with dibutyryl cAMP also resulted in inhibition.
Furthermore, addition of an inhibitor of protein kinase A (PKA), Rp-Adenosine 3’,5’cyclic monophosphorothioate triethylammonium salt, prior to drug withdrawal from
tolerant cells, abrogates withdrawal-induced inhibition of phagocytosis, thus
suggesting that activation of PKA by the increase in cAMP is necessary for inhibition
to take place. We conclude that, as in the nervous system, morphine exerts its
effects in macrophages via the cAMP pathway. ( Sponsored by NIH Grant
SO6GM08102, the FIPI Program of the University of Puerto Rico, and by NIH-MARC
Honors Program, Grant number 078210)
Macrophages at work
Macrophages are cells of the immune system that have
the capability to phagocyte any foreign body. They are a
differentiated form of blood monocytes that reside in a
specific region of the body of an organism.
Effects of 100 nM Morphine on
Phagocytosis by Murine Peritoneal
Macrophages
% of Phagocytosis
105
100
95
90
*
85
80
75
0
0.5
24
Time of exposure (hrs)
“*” Significant difference (P<0.05) between this value and that of
the control group.
Specific Research Aims
• Study the pathway utilized by morphine to
exerts its effects on macrophages.
• Study the cellular basis by which morphine
withdrawal from tolerant macrophages
inhibits phagocytosis.
% of cAMP
Effects of 100nM Morphine on the
cAMP levels of Macrophages
160
140
120
100
80
60
40
20
0
0.0 min
30 min
25min +
5min PGE1
8hr
Time of exposure to 100nM Morphine
Cells were cultured with 100nM Morphine for different times and one
group was exposed also to Prostaglandin 1. This was followed by a
radioimmunoassay to measure the cAMP levels. There were no significant
difference (P<0.05) between any group value and that of the control
group.
Effect of 100nM Morphine
Withdrawal on cAMP levels
% of cAMP
400
350
*
300
250
200
150
100
*
*
10
30
50
0
0
5
Tim e of w ithdraw al (m in)
Macrophages were cultured with 100nM Morphine (M) for 8 hr before drug
withdrawal (Wd). cAMP levels were determined at different times after M
Wd by a radioimmunoassay (RIA). “*”Significant difference (P<0.05)
between value and that of the control cells.
Effect of Dibutyryl cAMP on Phagocytosis by
Opiate-naïve Cells
% of Phagocytosis
100
90
*
80
* *
*
*
70
60
50
0
10
20
30
40
50
60
70
80
90
100
Dibutyryl cAMP (nM)
Macrophages were cultured in RPMI and exposed to different
concentrations of dibutyryl cAMP, followed by a phagocytosis assay.
“*”Significant difference (P<0.05) between the value and that of the control
group.
Effect of Dibutyryl cAMP on Phagocytosis by
Morphine Tolerant Macrophages
% of Phagocytosis
100
90
*
80
*
*
*
70
60
50
0
2
4
6
8
10
Dibutyryl cAMP (nM)
Macrophages were cultured with 100nM Morphine for 8hrs and
then exposed to different concentrations of dibutyryl cAMP,
followed by a phagocytosis assay. “*” Significant difference
(P<0.05) between the value and that of the control group.
Summary of Effects of Dibutyryl cAMP
and of Drug withdrawal
on Phagocytosis by Macrophages
70
% of Phagocytosis
65
60
55
50
*
45
*
40
35
30
M 24hrs
Withdrawal
5nM Db
Treatment
Withdrawal and dibutyril cAMP (Db) groups were cultivated with
100nM Morphine and then exposed for 30mins. to Db or drug
withdrawal with RPMI. “*” Significant difference (P<0.05) between
the value and that of the control group.
Effect of 20nM Butyrate on
Phagocytosis of Tolerant
Macrophages
% of Phagocytosis
120
100
80
*
*
60
40
20
0
RPMI
M 0.5hr
M 8.0hr
M 8.0hr +
Wd 1hr
M 7.5hr +
Butyrate
0.5hr
Treatment
“*” Significant difference (P<0.05) between the value and that of
control group.
Effects of Rp-Adenosine 3’,5’-cyclic
monophosphorothioate on
Morphine Withdrawal
105
% of Phagocytosis
100
95
90
85
*
*
80
75
1
2
3
4
5
Treatment
1= Control cells; 2= Morphine (M) 0.5 hr; 3= M 24hrs; 4= M 24hr
+Withdrawal (Wd) 1hr; 5= M 24hr+ Rp-Adenosine 2hr + Wd 1hr.
“*”Significant difference (P<0.05) between this value and that of
the control group.
Conclusions
The present work has let us to conclude that
chronic morphine, as in the nervous system, exerts
its effects on macrophage phagocytosis through
the cAMP pathway. This information should be
taken into account when using opioids as
analgesics, or in the therapy of drug addiction.
Pathway utilized by Morphine in
Macrophages
Acknowledgments
This project was sponsored by NIH Grant
SO6GM08102, the FIPI Program of the
University of Puerto Rico, and by NIHMARC U*STAR Grant Number
5T34GM07821-23.