Apoptosis of Peripheral Blood Lymphocytes is

Induced by Catecholamines
Daniel Petru CIOCA, MD, Noboru WATANABE, MD,
and Mitsuaki ISOBE, MD
SUMMARY
We explored the mechanism through which patients sometimes show immunosuppression after cardiac surgery. To test the hypothesis that commonly used drugs could
cause apoptosis of immune cells, the proapoptotic effects of heparin and catecholamines
(dopamine and dobutamine) on peripheral blood lymphocytes were evaluated. Peripheral
blood lymphocytes were purified from blood samples of normal healthy volunteers.
These cells were cultured in the presence of heparin, dobutamine or dopamine. The apoptosis was quantified by Annexin V fluorescent assay, by DNA content and by morphological assessment. Lymphocytes did not show significant levels of apoptosis induction
after 24 hours of incubation with heparin. Both dopamine and dobutamine demonstrated
a clear apoptosis inducing effect on lymphocytic population after 24 and 48 hours of culture, in concentrations comparable with the clinically used levels. Apoptosis was time
and concentration dependent for both catecholamines. The dopamine and dobutamine
effect on lymphocyte viability was due, at least partially, to lymphocyte receptor
engagement, as proved by blocking the receptor with propranolol. These results suggest
that catecholamines could induce apoptosis of lymphocytes. This finding may be associated with immunosuppression observed in patients undergoing cardiac surgery. (Jpn Heart
J 2000; 41: 385-398)
Key words: Adrenergic, Antagonists, receptor, Cardiovascular surgery, Cell culture,
Necrosis, Second messenger

ACCORDING to many authors, cardiac surgery involves procedures which produce immunological changes in patients undergoing cardiac surgery interventions.1-4) Patients are clinically immunosuppressed after cardiac surgical
operations, with a predisposition to infection. Evidence has been made of antiinflammatory cytokines being released such as IL-10, a true "anticytokine",3,4) and
the cellular immune defense seems to be affected in these circumstances.
The purpose of this research study was to determine if peripheral
blood lymphocytes, as an important immune effector, undergo significant
apoptosis related to some particular conditions commonly present during

First the Department of Internal Medicine, Shinshu University School of Medicine, Nagano, Japan.
Address for correspondence: Mitsuaki Isobe, MD, Department of Cardiovascular Medicine, Tokyo Medical and Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Received for publication December 16, 1999.
385
Revised and accepted January 26, 2000.

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or after cardiac surgery interventions. From the multitude of drugs used

during, pre- or post surgical intervention, two of the most common used
classes of drugs were chosen, namely heparin and catecholamines (dopamine and dobutamine).
Previous studies reported that heparin has a strong antiinflammatory
influence on human polymorphonuclear neutrophils, diminishing the proinflammatory cytokine production, and also a strong proapoptotic effect
on the polymorphonuclear neutrophil population.5,6) In this study we
assessed the effect of heparin on lymphocyte population viability.
The other drug class observed during experiments, catecholamines,
has been previously reported as an apoptosis-inducer in human neuronal
populations and mouse splenocyte population of cells. In this study, we
evaluated the effects of dopamine and dobutamine on lymphocyte apoptosis.
MATERIALS AND METHODS
Peripheral blood samples were obtained from healthy
adult volunteers of both sexes (age 32.1 8.2 years) after informed verbal
consent was obtained. The investigation conformed with the principles
outlined in the Declaration of Helsinki. None of the volunteers had taken
any medications for more than 30 days prior to blood sampling, and none
had taken any - adrenergic antagonist medication in the past. After harvesting, peripheral blood samples were diluted in balanced salt solution
(anhydrous D-glucose 100 mg / l, CaCl2 0.74mg / l, MgCl2 19.92 mg / l,
KCl 40.26 mg / l, Tris 1.7565 g / l, NaCl 7.371 g / l, pH 7.6), and then
carefully layered on top of 3 ml / tube of Ficoll-Paque Research Grade.
Following that, 40 minutes centrifugation at 400g and 18 C resulted in
peripheral blood mononuclear cells (PBMC) isolation. The PBMC layer
was carefully collected and resuspended in 6 ml balanced salt solution,
gently mixed and then recollected by centrifugation at 100 g for 15 minutes at 18 C. The whole washing process was repeated one more time
to eliminate any trace of the Ficoll - Paque.
Cell culture: After the cell isolation procedure was finished, 510 5 cells
were incubated in polystyrene round bottomed tubes for different time
courses, using the specified reagents and concentrations. The culture
medium used was RPMI 1640 supplemented with 10 % fetal calf serum,
2 mM L-glutamine and antibiotics (1 % gentamicin, penicillin 100 U / ml).
When used, - blocker solution was added more than 10 minutes before
the catecholamine solution. Cultures were kept at 37 C in a humidified
Cell preparation:

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atmosphere containing 5 % carbon dioxide.

Antibodies and reagents: Annexin V Fluos was purchased from Boehringer
Mannheim GmbH Germany. Annexin V FITC buffer was prepared according to the manufacturer's instructions (10 mM Hepes / NaOH, pH 7.4,
140 mM NaCl, 5 mM CaCl 2). Propidium iodide was procured from Wako
Pure Chemical Ind. Ltd. (Japan). The nuclear dye bisbenzimide H33342
fluorochrome trihydrochloride (Hoechst 33342) was purchased from Calbiochem-Novabiochem (La Jolla, CA). The CD2 and CD20 monoclonal
antibodies used for lymphocyte population quantification were obtained
from Becton Dickinson, Japan. Catecholamines used in this study were
dopamine from Kyowa Hakko (Japan) and dobutamine from Shionogi Co.,
Ltd. (Japan). The blocker used was propranolol produced by Sumitomo
Pharmaceuticals (Japan).
Assays for apoptosis: The Annexin V fluorescent assay was employed for
determining the apoptotic population of lymphocytes. Briefly, cells
extracted from the culture medium were washed twice in balanced salt
solution and incubated in 100 microliters of Annexin V buffer containing
2 microliters of Annexin V FITC.
Two microliters from the 50
micrograms / ml propidium iodide solution were also added, and after 10
minutes of incubation at room temperature, cells were washed, separated
by centrifugation and subjected to the flow cytometric analysis.
The flow cytometer used was a Becton Dickinson FACScan (Mountain View, CA) equiped with a 488 nm argon blue laser. Debris were
excluded from the analysis by the conventional scatter threshold and the
lymphocyte population was analysed using rigorously defined electronic
gates generated through FSC / SSC criteria. In the region chosen, 98 % of
cells displayed positive fluorescence for the fluorescent markers CD2
(used as a common marker for T and NK lymphocytes) and CD20 (for
the B cells). From the gated population of cells, 20,000 events per sample
were analysed for Annexin V FITC green fluorescence (detected by FL1
at 530 nm) and propidium iodide orange-red florescence (detected by FL2
at 610 nm). Crossover of FITC fluorescence into the PI detection window
was electronically compensated for analogue subtraction at the preamplifier stage.
Fluorescence microscopy was performed using a Nikon Eclipse 600
fluorescence microscope. Cells were stained with Annexin V FITC and
propidium iodide using the same procedure as above and observed under
a 1000x oil immersion objective with a 488 nm blue light excitation and
a 515 nm longpass filter for detection. At the same time, cells were
stained with the nuclear fluorescent dye Hoechst 33342 at 5 micrograms /

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ml and observed under 356 nm excitation radiation. By simply shifting

the filters, cells could be observed with both fluorescent systems used. A
manual cell count was performed on an improved Neubauer hemocytometer, 1000 cells being counted each time, respecting the standard counting
convention on each square of the hemocytometer. The cell shrinkage with
nuclear fragmentation, or marked condensation of the chromatin with
reduction of nuclear size, or both, plus specific shifting of nuclear fluorescence intensity, were considered typical features of apoptotic cells.
Apoptotic cell death of lymphocytes stained with propidium iodide
was measured by flow cytometry according to the method described by
Nicoletti, et al. 7) Briefly, cells were resuspended in 1 ml of hypotonic propidium iodide solution (0.1 % sodium citrate, 50 micrograms / ml propidium iodide, 0.1 % Triton X - 100) and stored in the dark at 4 C until they
were analysed. Cellular debris were excluded from analysis by raising the
forward scatter threshold, and the DNA content of intact nuclei was
recorded on a logarithmic scale. The percentage of apoptotic cells was
determined by measuring the hypodiploid fraction, results being concordant with those from the other methods used.
Statistical analysis: Each set of determinations was performed at least on
five peripheral blood samples from different donors. Data obtained by
flow cytometry were collected and stored on hard support and later analysed using specific software (Lysis II and Cell Quest, Becton Dickinson,
Mountain View, CA). Results were expressed as percentage of apoptotic
cells and statistical analysis was performed using ANOVA. A value of p
less than 0.05 was considered significant. Three methods were used for
analysing the significance of results, namely Fisher's Protected Least Significant Difference, Scheffe's F and Bonferronni-Dunn's Procedure as a
multiple comparison procedure.
RESULTS
The results of the determination of the apoptotic elements following
different incubation times and different concentrations of the studied pharmacological agents are shown in Figures 1-6.
Heparin does not influence peripheral blood lymphocyte apoptosis: Using the
described methods, lymphocyte behavior was tested in the presence of
heparin, at concentrations ranging from 1U / ml to 80U / ml. Even at
heparin concentrations higher than the clinically achieved levels, the lymphocyte population did not exhibit a significant increase in the percentage
of apoptotic elements, compared with the control (untreated) samples (Fig-

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Figure 1. Apoptosis of peripheral blood lymphocytes cultured for 24 hours in the absence (control samples) or
presence of different concentrations of heparin. Results are expressed as percentage of Annexin V FITC positive cells from the propidium iodide negative population.

ure 1).
After 24
hour incubation of lymphocytes with catecholamines, there was a significant apoptosis inducing effect in a concentration-dependent manner. A
typical dot plot obtained by flow cytometric analysis of the lymphocytes
stained with Annexin V FITC and propidium iodide is shown in Figure
2. In the case of dopamine, the effect became significant at concentrations
between 510 -7 M to 10-6 M, and for dobutamine the apoptosis induction
was significant at even lower concentrations (below 510-7 M). Both catecholamines had a similar and comparable effect (Figure 3A).
The same aspect remained after 48 hours incubation with the same
catecholamine concentrations. At 48 hours the percentage of apoptotic
cells was higher, both in control (catecholamine negative) and catecholamine-positive samples, but the significant difference remained, with dobutamine showing a slightly stronger effect (Figure 3B).
Catecholamines induce peripheral blood lymphocyte apoptosis:

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Figure 2. Representative analysis dot plots for the effect of dobutamine on peripheral blood lymphocytes.
Cells were cultured for 24 hours in the absence (control) or presence of different concentrations of dobutamine
(dot plots for 10-6M, 5x10-6M and 10-5M are shown). The numbers in the right lower quadrant represent the
percentage of early apoptotic cells detected by Annexin V FITC plus propidium iodide co-staining method.

Significant induction of apoptosis by catecholamines could be

detected as early as 4 hours after incubation with higher concentrations
(10-5 M, 10 -4 M, 10-3 M) (Figure 4). However, similar to other known
agents, at this higher concentrations the main effect was necrosis, which
prevails over the apoptosis. At these high catecholamine concentrations,
there was a marked loss of viability among of catecholamine-treated lymphocytes, compared with the control (untreated) samples, for which apoptosis was at least partially responsible (Figure 5).
Propranolol blocks apoptosis induction by catecholamines: Propranolol was

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Figure 3. Apoptosis of peripheral blood lymphocytes cultured for 24 hours (A) and 48 hours(B) in the absence
or presence of dopamine or dobutamine in concentrations ranging from 510-7M to 10-5M. A significant difference exists between spontaneous apoptotic percentage in control samples and apoptotic percentage in the catecholamine treated samples.

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Figure 4. Apoptosis of peripheral blood lymphocytes cultured for 4 hours in the absence or presence of dopamine in higher concentrations (10-5M, 10-4M, 10-3M). After only four hours of incubation, a significant difference
appears between spontaneous apoptotic percentage of cells (in control samples) and apoptotic percentage of
cells in the dopamine treated samples.

Figure 5. After 24 hours of incubation with higher dopamine concentrations (10-5M, 10-4M, 10-3M), peripheral
blood lymphocytes display a loss of viability compared with control (catecholamine untreated) samples.

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Figure 6. Apoptosis of the peripheral blood lymphocytes incubated in the absence or presence of dobutamine
(a) and dopamine (b) in concentrations ranging from 510-7M to 10-5M and the influence of preincubation with
10-4M propranolol. A significant difference appears between the percentage of apoptotic cells treated only with
dobutamine, in which apoptosis is induced by the catecholamine, and the - blocker preincubated cells, where
the proapoptotic effect of dobutamine is totally inhibited (A). The proapoptotic effect of dopamine is partially,
but not totally inhibited by the - blocker pretreatment (B).

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used to block the - receptors for 10 minutes before catecholamines were

added to the culture. The quantity of - blocker used was ten times
higher (10 -4 M) than the maximal catecholamine concentration used (10-5 M).
Both at 24 and 48 hours, propranolol by itself did not alter the time
course of spontaneous apoptosis. The samples treated with a 10 -4 M concentration of propranolol did not differ from control samples (untreated),
regarding their apoptotic lymphocyte populations.
As shown in Figure 6A, the apoptosis induced by dobutamine was
totally inhibited by the - blocker pretreatment. No significant difference
was observed between control samples (untreated) and catecholamine
treated samples preincubated with 10 -4 M propranolol. However, the apoptosis-inducing effect of dopamine was only partially blocked by the lymphocyte pretreatment with propranolol. The concentration-response curve
is shown in Figure 6B. Percentages of apoptotic lymphocytes are still
slightly increased by dopamine treatment, but the effect is significantly
reduced compared with the samples without - blocker preincubation.
DISCUSSION
Factors involved in the immunosuppression following cardiac surgery: It is clear

that surgical interventions, especially cardiac surgical procedures, produce

major alterations in immune system functions. The immune disturbance
was proved both by the clinical follow-up of patients subjected to cardiac
surgical interventions and by many in vitro studies.1-4) Many factors have
been implicated: major surgical trauma combined with hypothermia and
especially cardiopulmonary bypass,8) blood trans-fusions,9,10) and anaesthesia techniques using opioids. 11)
The complex network of cytokines is deeply disturbed following cardiac surgical intervention, and there are many studies describing such
changes. IL-1 , IL-6, IL-8 and TNF- were reported to increase shortly
after the beginning of surgical intervention.12,13,15) Other cytokines like
IFN- and IL-2 were found to be significantly reduced,16) and suppressive
cytokines such as IL-10, IL-4 and interleukin-1 receptor antagonist (IL1ra) have been found to increase, thus influencing the immune balance. 1315)
Open-heart surgery was also associated with profound suppression in
cellular parameters of immunity, with a decrease in the percentage of total
lymphocytes, CD3 + lymphocytes (T cells) and especially CD3 + CD4+
(helper) T lymphocytes. 2,16,17)
Lymphocytes and neutrophils behave differently after cardiac surgery:
Besides
its anticoagulant activity, heparin is known to have anti-hypertensive, anti-

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inflammatory and anti-proliferative effects. Neutrophil function is inhibited in vitro by heparan sulfate.18) It was also demonstrated that heparin
oligosaccharides bind L- and P- selectin and inhibit acute inflammation. 19)
In 1996, Manaster, et al. demonstrated that heparin induces apoptosis in
human peripheral blood neutrophils. 5)
Therefore, the effect of heparin administered to cardiac surgery
patients was tested in this study on the peripheral blood lymphocyte population. As shown in Figure 1, even at high concentrations, lymphocyte
population's apoptotic percentage was not affected by heparin, thus indicating that there are significant differences between neutrophils and lymphocytes regarding the proapoptotic effect of this drug. In fact, it seems
that neutrophils and lymphocytes behave differently or even oppositely
after cardiac or other major elective surgery intervention. Neutrophils tend
to reduce their apoptotic percentage, and consequently increase their number after surgical intervention or during the systemic inflammatory
response syndrome20-23). Lymphocytes seem to behave differently regarding
their number and apoptotic induction.
Catecholamines induce apoptosis via - receptor stimulation: As for catecholamines, it was demonstrated by Bergquist, et al. that catecholamines exert
a powerful impact on the immune system by downregulation of proliferation and differentiation of lymphocytes. 23,24) In their experiments, exposure of lymphocytes to catecholamines at concentrations as low as 10 -8
M decreased proliferation and differentiation. Our observations are in
agreement with these data. At higher concentrations (510-7 M - 10-5 M)
apoptosis is induced in peripheral blood lymphocytes. Catecholamines
appear to act by suppressing the activity of immunocompetent cells, apoptosis being a final outcome of increased levels of catecholamines.
The existence of - adrenergic receptors on human lymphocytes is
well documented. 25) The primary intracellular mediator (second messenger) for the - adrenergic complex is cyclic adenosine monophosphate
(cAMP). Dopamine, in concentrations ranging from 10 -6 M to 10-4 M, was
reported to elevate cAMP levels, and this dopamine-induced cAMP
increase in mouse lymphocytes was mediated via - adrenoceptors. 26)
Kouassi, et al. also demonstrated that this cAMP increase can be completely inhibited by propranolol. 26) These findings correlate very well with
our results. Other studies demonstrated that theophylline, which inhibits
intracellular cAMP degradation by phosphodiesterases, is able to induce
apoptosis in B chronic lymphatic leukemia lymphocytes. 27) CAMP was
also found to have an important role in leukocyte functions by regulating
chemotactic responsiveness and spontaneous motility.28) In lymphocytes, at

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concentrations between 10-7 M-10-5 M, isoproterenol increased cAMP at

levels between 200 % and 400 %,29) which roughly corresponds with our
results regarding the increase in the percentage of apoptotic lymphocytes.
These findings indirectly suggest a link between catecholamine cAMP
increase via - receptor stimulation and apoptosis induced in the same
cells at similar concentrations. Indeed, in our study, blocking - receptors
by propranolol leads to a complete inhibition of the proapoptotic effect
for dobutamine and a partial inhibition for dopamine. We did not succeed
in completely blocking dopamine with a nonselective - adrenergic antagonist (propranolol) possibly because lymphocytes also express specific
dopa-mine receptors on their surface, which determine an increase in
cAMP via the stimulation of adenylyl cyclase.26)
Another possible explanation for the difference between dopamine
and dobutamine is that dopamine acts also through an oxidative mechanism. In neuronal populations apoptosis is strongly induced by culture in
the presence of micromolar concentrations of dopamine,30,31) and the mechanism seems to be through an activation of the c - jun N - terminal kinase
(JNK) / stress-activated protein kinase (SAPK) pathway.32,33) This programmed cell death through oxidative stress triggered by dopamine can
be effectively blocked by scavengers of reactive oxygen species like Nacetylcysteine, reduced glutathione and dithiothreitol.34) There is no medical literature data to suggest that the same oxidative mechanism could be
involved in the case of dobutamine.
Dobutamine hydrochloride is a racemic mixture. The (+) - enantiomer
preferentially stimulates - receptors, whereas the () - enantiomer preferentially stimulates 1-receptors, which activates hydrolysis of phosphatidylinositol-4,5 biphosphate to inositol-1,4,5-triphosphate (IP3) and
diacylgly-cerol. IP3 releases intracellular Ca2+, which could favour apoptosis induction, thus explaining the difference between the dopamine and
dobutamine proapoptotic potency. The mechanism involved in the proapoptotic effect of dopamine and dobutamine on peripheral blood lymphocytes is a subject for further research.
Our observations suggest that catecholamine administration to
patients subjected to cardiac surgical interventions is one of the factors
involved in the immunological changes which may occur in the case of
these patients. Identification of the molecular mechanisms controlling this
process will be of significant value in understanding the regulation of the
immune response, and the impact of this in vitro study to the clinical management of the surgical patients should be the subject of future studies.

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ACKNOWLEDGEMENTS
We would like to express many special thanks to Mr. Susumu Ito from the
Shinshu University Hospital Blood Transfusion Service for his excellent technical
advice and skillful technical assistance.

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