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Apoptosis of Peripheral Blood Lymphocytes is Induced by Catecholamines

Daniel Petru CIOCA, MD, Noboru WATANABE, MD,and Mitsuaki ISOBE, MD

SUMMARYWe explored the mechanism through which patients sometimes show immunosup-

pression after cardiac surgery. To test the hypothesis that commonly used drugs couldcause apoptosis of immune cells, the proapoptotic effects of heparin and catecholamines(dopamine and dobutamine) on peripheral blood lymphocytes were evaluated. Peripheralblood lymphocytes were purified from blood samples of normal healthy volunteers.These cells were cultured in the presence of heparin, dobutamine or dopamine. The apo-ptosis was quantified by Annexin V fluorescent assay, by DNA content and by morpho-logical assessment. Lymphocytes did not show significant levels of apoptosis inductionafter 24 hours of incubation with heparin. Both dopamine and dobutamine demonstrateda clear apoptosis inducing effect on lymphocytic population after 24 and 48 hours of cul-ture, in concentrations comparable with the clinically used levels. Apoptosis was timeand concentration dependent for both catecholamines. The dopamine and dobutamineeffect on lymphocyte viability was due, at least partially, to lymphocyte receptorengagement, as proved by blocking the receptor with propranolol. These results suggestthat catecholamines could induce apoptosis of lymphocytes. This finding may be associ-ated with immunosuppression observed in patients undergoing cardiac surgery. (Jpn HeartJ 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 pro-duce immunological changes in patients undergoing cardiac surgery interven-tions.1-4) Patients are clinically immunosuppressed after cardiac surgicaloperations, with a predisposition to infection. Evidence has been made of antiin-flammatory cytokines being released such as IL-10, a true "anticytokine",3,4) andthe cellular immune defense seems to be affected in these circumstances.

The purpose of this research study was to determine if peripheralblood lymphocytes, as an important immune effector, undergo significantapoptosis 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 Uni-

versity School of Medicine, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.Received for publication December 16, 1999.Revised and accepted January 26, 2000.

386 Jpn Heart JMay 2000CIOCA, ET AL

or after cardiac surgery interventions. From the multitude of drugs usedduring, pre- or post surgical intervention, two of the most common usedclasses of drugs were chosen, namely heparin and catecholamines (dopa-mine and dobutamine).

Previous studies reported that heparin has a strong antiinflammatoryinfluence on human polymorphonuclear neutrophils, diminishing the pro-inflammatory cytokine production, and also a strong proapoptotic effecton the polymorphonuclear neutrophil population.5,6) In this study weassessed 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 neuronalpopulations and mouse splenocyte population of cells. In this study, weevaluated the effects of dopamine and dobutamine on lymphocyte apopto-sis.

MATERIALS AND METHODS

Cell preparation: Peripheral blood samples were obtained from healthyadult volunteers of both sexes (age 32.1 8.2 years) after informed verbalconsent was obtained. The investigation conformed with the principlesoutlined in the Declaration of Helsinki. None of the volunteers had takenany medications for more than 30 days prior to blood sampling, and nonehad taken any - adrenergic antagonist medication in the past. After har-vesting, 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 thencarefully layered on top of 3 ml / tube of Ficoll-Paque Research Grade.Following that, 40 minutes centrifugation at 400g and 18 C resulted inperipheral blood mononuclear cells (PBMC) isolation. The PBMC layerwas carefully collected and resuspended in 6 ml balanced salt solution,gently mixed and then recollected by centrifugation at 100 g for 15 min-utes at 18 C. The whole washing process was repeated one more timeto eliminate any trace of the Ficoll - Paque.Cell culture: After the cell isolation procedure was finished, 5105 cellswere incubated in polystyrene round bottomed tubes for different timecourses, using the specified reagents and concentrations. The culturemedium 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 beforethe catecholamine solution. Cultures were kept at 37 C in a humidified

LYMPHOCYTE APOPTOSIS BY CATECHOLAMINES 387Vol 41No 3

atmosphere containing 5 % carbon dioxide.Antibodies and reagents: Annexin V Fluos was purchased from BoehringerMannheim GmbH Germany. Annexin V FITC buffer was prepared accor-ding to the manufacturer's instructions (10 mM Hepes / NaOH, pH 7.4,140 mM NaCl, 5 mM CaCl2). Propidium iodide was procured from WakoPure Chemical Ind. Ltd. (Japan). The nuclear dye bisbenzimide H33342fluorochrome trihydrochloride (Hoechst 33342) was purchased from Cal-biochem-Novabiochem (La Jolla, CA). The CD2 and CD20 monoclonalantibodies used for lymphocyte population quantification were obtainedfrom Becton Dickinson, Japan. Catecholamines used in this study weredopamine from Kyowa Hakko (Japan) and dobutamine from Shionogi Co.,Ltd. (Japan). The blocker used was propranolol produced by SumitomoPharmaceuticals (Japan).Assays for apoptosis: The Annexin V fluorescent assay was employed fordetermining the apoptotic population of lymphocytes. Briefly, cellsextracted from the culture medium were washed twice in balanced saltsolution and incubated in 100 microliters of Annexin V buffer containing2 microliters of Annexin V FITC. Two microliters from the 50micrograms / ml propidium iodide solution were also added, and after 10minutes of incubation at room temperature, cells were washed, separatedby centrifugation and subjected to the flow cytometric analysis.

The flow cytometer used was a Becton Dickinson FACScan (Moun-tain View, CA) equiped with a 488 nm argon blue laser. Debris wereexcluded from the analysis by the conventional scatter threshold and thelymphocyte population was analysed using rigorously defined electronicgates generated through FSC / SSC criteria. In the region chosen, 98 % ofcells displayed positive fluorescence for the fluorescent markers CD2(used as a common marker for T and NK lymphocytes) and CD20 (forthe B cells). From the gated population of cells, 20,000 events per samplewere analysed for Annexin V FITC green fluorescence (detected by FL1at 530 nm) and propidium iodide orange-red florescence (detected by FL2at 610 nm). Crossover of FITC fluorescence into the PI detection windowwas electronically compensated for analogue subtraction at the preampli-fier stage.

Fluorescence microscopy was performed using a Nikon Eclipse 600fluorescence microscope. Cells were stained with Annexin V FITC andpropidium iodide using the same procedure as above and observed undera 1000x oil immersion objective with a 488 nm blue light excitation anda 515 nm longpass filter for detection. At the same time, cells werestained with the nuclear fluorescent dye Hoechst 33342 at 5 micrograms /


ml and observed under 356 nm excitation radiation. By simply shiftingthe filters, cells could be observed with both fluorescent systems used. Amanual cell count was performed on an improved Neubauer hemocytom-eter, 1000 cells being counted each time, respecting the standard countingconvention on each square of the hemocytometer. The cell shrinkage withnuclear fragmentation, or marked condensation of the chromatin withreduction of nuclear size, or both, plus specific shifting of nuclear fluo-rescence intensity, were considered typical features of apoptotic cells.

Apoptotic cell death of lymphocytes stained with propidium iodidewas measured by flow cytometry according to the method described byNicoletti, et al.7) Briefly, cells were resuspended in 1 ml of hypotonic pro-pidium iodide solution (0.1 % sodium citrate, 50 micrograms / ml propid-ium iodide, 0.1 % Triton X - 100) and stored in the dark at 4 C until theywere analysed. Cellular debris were excluded from analysis by raising theforward scatter threshold, and the DNA content of intact nuclei wasrecorded on a logarithmic scale. The percentage of apoptotic cells wasdetermined by measuring the hypodiploid fraction, results being concor-dant with those from the other methods used.Statistical analysis: Each set of determinations was performed at least onfive peripheral blood samples from different donors. Data obtained byflow cytometry were collected and stored on hard support and later ana-lysed using specific software (Lysis II and Cell Quest, Becton Dickinson,Mountain View, CA). Results were expressed as percentage of apoptoticcells and statistical analysis was performed using ANOVA. A value of pless than 0.05 was considered significant. Three methods were used foranalysing the significance of results, namely Fisher's Protected Least Sig-nificant Difference, Scheffe's F and Bonferronni-Dunn's Procedure as amultiple comparison procedure.

RESULTS

The results of the determination of the apoptotic elements followingdifferent incubation times and different concentrations of the studied phar-macological agents are shown in Figures 1-6.Heparin does not influence peripheral blood lymphocyte apoptosis: Using thedescribed methods, lymphocyte behavior was tested in the presence ofheparin, at concentrations ranging from 1U / ml to 80U / ml. Even atheparin concentrations higher than the clinically achieved levels, the lym-phocyte population did not exhibit a significant increase in the percentageof apoptotic elements, compared with the control (untreated) samples (Fig-


ure 1).Catecholamines induce peripheral blood lymphocyte apoptosis: After 24hour incubation of lymphocytes with catecholamines, there was a signifi-cant apoptosis inducing effect in a concentration-dependent manner. Atypical dot plot obtained by flow cytometric analysis of the lymphocytesstained with Annexin V FITC and propidium iodide is shown in Figure2. In the case of dopamine, the effect became significant at concentrationsbetween 510-7 M to 10-6 M, and for dobutamine the apoptosis inductionwas significant at even lower concentrations (below 510-7 M). Both cat-echolamines had a similar and comparable effect (Figure 3A).

The same aspect remained after 48 hours incubation with the samecatecholamine concentrations. At 48 hours the percentage of apoptoticcells was higher, both in control (catecholamine negative) and catechola-mine-positive samples, but the significant difference remained, with do-butamine showing a slightly stronger effect (Figure 3B).

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


Significant induction of apoptosis by catecholamines could bedetected 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 knownagents, at this higher concentrations the main effect was necrosis, whichprevails over the apoptosis. At these high catecholamine concentrations,there was a marked loss of viability among of catecholamine-treated lym-phocytes, compared with the control (untreated) samples, for which apo-ptosis was at least partially responsible (Figure 5). Propranolol blocks apoptosis induction by catecholamines: Propranolol was

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 thepercentage of early apoptotic cells detected by Annexin V FITC plus propidium iodide co-staining method.


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


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

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


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 with10-4M propranolol. A significant difference appears between the percentage of apoptotic cells treated only withdobutamine, in which apoptosis is induced by the catecholamine, and the - blocker preincubated cells, wherethe proapoptotic effect of dobutamine is totally inhibited (A). The proapoptotic effect of dopamine is partially,but not totally inhibited by the - blocker pretreatment (B).


used to block the - receptors for 10 minutes before catecholamines wereadded to the culture. The quantity of - blocker used was ten timeshigher (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 timecourse of spontaneous apoptosis. The samples treated with a 10-4 M con-centration of propranolol did not differ from control samples (untreated),regarding their apoptotic lymphocyte populations.

As shown in Figure 6A, the apoptosis induced by dobutamine wastotally inhibited by the - blocker pretreatment. No significant differencewas observed between control samples (untreated) and catecholaminetreated samples preincubated with 10-4 M propranolol. However, the apo-ptosis-inducing effect of dopamine was only partially blocked by the lym-phocyte pretreatment with propranolol. The concentration-response curveis shown in Figure 6B. Percentages of apoptotic lymphocytes are stillslightly increased by dopamine treatment, but the effect is significantlyreduced compared with the samples without - blocker preincubation.

DISCUSSION

Factors involved in the immunosuppression following cardiac surgery: It is clearthat surgical interventions, especially cardiac surgical procedures, producemajor alterations in immune system functions. The immune disturbancewas proved both by the clinical follow-up of patients subjected to cardiacsurgical interventions and by many in vitro studies.1-4) Many factors havebeen implicated: major surgical trauma combined with hypothermia andespecially cardiopulmonary bypass,8) blood trans-fusions,9,10) and anaesthe-sia techniques using opioids.11)

The complex network of cytokines is deeply disturbed following car-diac surgical intervention, and there are many studies describing suchchanges. IL-1, IL-6, IL-8 and TNF- were reported to increase shortlyafter the beginning of surgical intervention.12,13,15) Other cytokines likeIFN- and IL-2 were found to be significantly reduced,16) and suppressivecytokines such as IL-10, IL-4 and interleukin-1 receptor antagonist (IL-1ra) have been found to increase, thus influencing the immune balance.13-15) Open-heart surgery was also associated with profound suppression incellular parameters of immunity, with a decrease in the percentage of totallymphocytes, CD3+ lymphocytes (T cells) and especially CD3+ CD4+

(helper) T lymphocytes.2,16,17)

Lymphocytes and neutrophils behave differently after cardiac surgery: Besidesits anticoagulant activity, heparin is known to have anti-hypertensive, anti-


inflammatory and anti-proliferative effects. Neutrophil function is inhi-bited in vitro by heparan sulfate.18) It was also demonstrated that heparinoligosaccharides bind L- and P- selectin and inhibit acute inflammation.19)

In 1996, Manaster, et al. demonstrated that heparin induces apoptosis inhuman peripheral blood neutrophils.5)

Therefore, the effect of heparin administered to cardiac surgerypatients was tested in this study on the peripheral blood lymphocyte pop-ulation. As shown in Figure 1, even at high concentrations, lymphocytepopulation's apoptotic percentage was not affected by heparin, thus indi-cating that there are significant differences between neutrophils and lym-phocytes regarding the proapoptotic effect of this drug. In fact, it seemsthat neutrophils and lymphocytes behave differently or even oppositelyafter cardiac or other major elective surgery intervention. Neutrophils tendto reduce their apoptotic percentage, and consequently increase their num-ber after surgical intervention or during the systemic inflammatoryresponse syndrome20-23). Lymphocytes seem to behave differently regardingtheir number and apoptotic induction.Catecholamines induce apoptosis via - receptor stimulation: As for catechola-mines, it was demonstrated by Bergquist, et al. that catecholamines exerta powerful impact on the immune system by downregulation of prolifer-ation and differentiation of lymphocytes.23,24) In their experiments, expo-sure of lymphocytes to catecholamines at concentrations as low as 10-8M decreased proliferation and differentiation. Our observations are inagreement with these data. At higher concentrations (510-7 M - 10-5 M)apoptosis is induced in peripheral blood lymphocytes. Catecholaminesappear to act by suppressing the activity of immunocompetent cells, apo-ptosis being a final outcome of increased levels of catecholamines.

The existence of - adrenergic receptors on human lymphocytes iswell documented.25) The primary intracellular mediator (second messen-ger) for the - adrenergic complex is cyclic adenosine monophosphate(cAMP). Dopamine, in concentrations ranging from 10-6 M to 10-4 M, wasreported to elevate cAMP levels, and this dopamine-induced cAMPincrease in mouse lymphocytes was mediated via - adrenoceptors.26)Kouassi, et al. also demonstrated that this cAMP increase can be com-pletely inhibited by propranolol.26) These findings correlate very well withour results. Other studies demonstrated that theophylline, which inhibitsintracellular cAMP degradation by phosphodiesterases, is able to induceapoptosis in B chronic lymphatic leukemia lymphocytes.27) CAMP wasalso found to have an important role in leukocyte functions by regulatingchemotactic responsiveness and spontaneous motility.28) In lymphocytes, at


concentrations between 10-7 M-10-5 M, isoproterenol increased cAMP atlevels between 200 % and 400 %,29) which roughly corresponds with ourresults regarding the increase in the percentage of apoptotic lymphocytes.

These findings indirectly suggest a link between catecholamine cAMPincrease via - receptor stimulation and apoptosis induced in the samecells at similar concentrations. Indeed, in our study, blocking - receptorsby propranolol leads to a complete inhibition of the proapoptotic effectfor dobutamine and a partial inhibition for dopamine. We did not succeedin completely blocking dopamine with a nonselective - adrenergic antag-onist (propranolol) possibly because lymphocytes also express specificdopa-mine receptors on their surface, which determine an increase incAMP via the stimulation of adenylyl cyclase.26)

Another possible explanation for the difference between dopamineand dobutamine is that dopamine acts also through an oxidative mecha-nism. In neuronal populations apoptosis is strongly induced by culture inthe presence of micromolar concentrations of dopamine,30,31) and the mech-anism seems to be through an activation of the c - jun N - terminal kinase(JNK) / stress-activated protein kinase (SAPK) pathway.32,33) This pro-grammed cell death through oxidative stress triggered by dopamine canbe effectively blocked by scavengers of reactive oxygen species like N-acetylcysteine, reduced glutathione and dithiothreitol.34) There is no med-ical literature data to suggest that the same oxidative mechanism could beinvolved in the case of dobutamine.

Dobutamine hydrochloride is a racemic mixture. The (+) - enantiomerpreferentially stimulates - receptors, whereas the () - enantiomer pref-erentially stimulates 1-receptors, which activates hydrolysis of phosphati-dylinositol-4,5 biphosphate to inositol-1,4,5-triphosphate (IP3) anddiacylgly-cerol. IP3 releases intracellular Ca2+, which could favour apop-tosis induction, thus explaining the difference between the dopamine anddobutamine proapoptotic potency. The mechanism involved in the proap-optotic effect of dopamine and dobutamine on peripheral blood lympho-cytes is a subject for further research.

Our observations suggest that catecholamine administration topatients subjected to cardiac surgical interventions is one of the factorsinvolved in the immunological changes which may occur in the case ofthese patients. Identification of the molecular mechanisms controlling thisprocess will be of significant value in understanding the regulation of theimmune response, and the impact of this in vitro study to the clinical man-agement of the surgical patients should be the subject of future studies.


ACKNOWLEDGEMENTS

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

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Figure 1. Apoptosis of peripheral blood lymphocytes cultured for 24 hours in the absence (control...Figure 2. Representative analysis dot plots for the effect of dobutamine on peripheral blood lymp...Figure 6. Apoptosis of the peripheral blood lymphocytes incubated in the absence or presence of d...Figure 3. Apoptosis of peripheral blood lymphocytes cultured for 24 hours (A) and 48 hours(B) in ...Figure 4. Apoptosis of peripheral blood lymphocytes cultured for 4 hours in the absence or presen...Apoptosis of Peripheral Blood Lymphocytes is Induced by CatecholaminesDaniel Petru Cioca, MD, Noboru Watanabe, MD, and Mitsuaki Isobe, MDMaterials and MethodsCell preparation:Cell culture:Antibodies and reagents:Assays for apoptosis:Statistical analysis:

ResultsHeparin does not influence peripheral blood lymphocyte apoptosis:

DiscussionFactors involved in the immunosuppression following cardiac surgery:Lymphocytes and neutrophils behave differently after cardiac surgery:Catecholamines induce apoptosis via b-receptor stimulation:

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