Cytometry Part B (Clinical Cytometry) 80B:212–220 (2011)
Original Article
Modulation of the Cellular and Humoral Immune Response to Pediatric Open Heart Surgery by Methylprednisolone ¨ rg Hambsch,1 Jozsef Bocsi,1 Marie-Christin Ha¨nzka,1 Pavel Osmancik,2 Jo 1 3 4 Ingo Da¨hnert, Ulrich Sack, Wilfried Bellinghausen, Peter Schneider,1 Jan Janousˇek,1 Martin Kostelka,5 and Attila Ta´rnok1* 1
2
Department of Pediatric Cardiology, Heart Centre, University of Leipzig, Leipzig, Germany Cardiocenter, Department of Cardiology, 3rd Medical School, Charles University and University Hospital Kralovske Vinohrady, Prague, Czech Republic 3 Institute for Clinical Immunology, IKIT, University Leipzig, Leipzig, Germany 4 Department of Anesthesiology, Heart Centre, University Leipzig, Leipzig, Germany 5 Department of Cardiac Surgery, Heart Centre, University Leipzig, Leipzig, Germany
Background: With the intention to reduce overshooting immune response, glucocorticoids are frequently administered perioperatively in children undergoing open heart surgery. In a retrospective study we investigated extensively the modulation of the humoral and cellular immune response by methylprednisolone (MP). Methods: This study was carried out on blood samples from two groups of children who had undergone surgical correction of atrial or ventricular septal defects, either without (MP2, n 5 10), or with MP administration (MP1, n 5 23, dose median 11 (IQR 10–16) mg kg21 body weight) before cardiopulmonary bypass (CPB, duration median 42 (IQR 36–65) min). EDTA blood was obtained 24 h preoperatively, after anesthesia, at CPB begin and end, 4, 24, and 48 h after surgery, at discharge and at out-patient follow-up (median 8.2 (IQR 3.3–12.2) months after surgery). Complex blood analysis including clinical chemistry and flow cytometry were performed to monitor humoral immune response, differential blood count, lymphocyte subsets, and the degree of activation of various leukocyte subpopulations. Results: The patients’ postoperative courses and follow-up were uneventful. Release of IL-6 and IL8 was reduced and that of the anti-inflammatory cytokine IL-10 upregulated by MP. Significant increase of circulating neutrophils and monocytes as inflammatory reaction to surgery and CPB contact was detected in both groups. However, invasion of monocytes to the periphery was delayed with MP. CD41 and CD81 T-lymphocyte counts were lower with MP treatment. B-lymphocyte count increased significantly after surgery in MP1 but remained constant in MP2 group. Conclusions: MP treatment partially decreased the pro-inflammatory effect of CPB surgery and induced C 2011 International Clinical Cytometry Society anti-inflammatory effect on the cellular and humoral level. V Key terms: pediatric cardiac surgery; congenital heart defects; cardiopulmonary bypass; immune response; methylprednisolone
How to cite this article: Bocsi J, Ha¨nzka M-C, Osmancik P, Hambsch J, Da¨hnert I, Sack U, Bellinghausen W, Schneider P, Janousˇek J, Kostelka M, Ta´rnok A. Modulation of the cellular and humoral immune response to pediatric open heart surgery by methylprednisolone. Cytometry Part B 2011; 80B: 212–220.
Grant sponsor: Deutsche Stiftung fu¨r Herzforschung, Frankfurt, Germany; Grant number: MSM-0021620817; Grant sponsor: Charles University research grant provided by the Ministry of Education, Youth and Physical Education of the Czech Republic; Grant number: MSM 0021620817. *Correspondence to: Attila Ta´rnok, Department of Pediatric Cardiology, Heart Centre, University of Leipzig, Stru¨mpellstr. 39, D-04289 Leipzig, Germany. E-mail:
[email protected].
C 2011 International Clinical Cytometry Society V
Received 28 July 2010; Revision 14 December 2010; Accepted 3 January 2011 Published online 4 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.20587
IMMUNE EFFECT OF METHYLPREDNISOLONE IN PEDIATRIC HEART SURGERY
Cardiopulmonary bypass (CPB) in children is thought to contribute to several adverse reactions following open heart surgery, including effusions, capillary leak syndrome (CLS), and multiorgan failure (1–4). Escalating pro-inflammatory response is thought to be responsible (2,3,5) and can induce low cardiac output (6–8). According to type of congenital heart disease and surgery, the incidence of post surgical CLS in children with open heart surgery ranges from 4 to 37% (3). To reduce systemic inflammatory reaction induced by CPB and to diminish the peri- and postoperative morbidity several pharmacological and technical therapy interventions were developed (9–12). One of these strategies is the pre- or intraoperative application of glucocorticoids (GC). These steroids have an anti-inflammatory action (13), therefore steroid derivatives such as methylprednisolone (MP) or dexamethasone have been widely used during CPB surgery (14,15). MP also has a protective effect on the vascular endothelium during cardiovascular surgery (16). Available data suggest a clinical benefit of steroid application, but many details are unknown and conditions of steroid administration are highly variable (15). Evidence from adult trials is conflicting regarding the effects of GC on postoperative outcome such as postoperative course, length of hospital stay and survival (14,17). It is also unclear whether results from adults can be directly transferred to children (18). Moreover, little is known how MP modulates the immune response in children in combination with the major stress response to surgery and administration of other drugs. The aim of the study was to analyze the effect of perioperative MP administration on acute cellular and humoral immune reaction in children undergoing simple open heart surgery and comparing it to a similar group of patients without MP administration. Since about 10 years preoperative administration of MP in these children is an internationally accepted standard procedure. Therefore, the present study is very unique and renders important information that will not be repeatable in new prospective studies for ethical reasons. In addition, even in a large cardiac center the number of pediatric patients is relatively low. Thus, it took many years to recruit a homogeneous patient group. We therefore followed the concept of predictive medicine by Cytomics or cell systems biology (19,20) and performed, as comprehensively as possible, a laboratory analysis of the drawn samples. We show that the majority of the analyzed parameters rendered no significant differences between the two groups. We believe that these unique and important studies yield a plethora of information that is of value to a broader community. PATIENTS AND METHODS Selection of Patients Our present study is a retrospective non-randomized analysis of children operated at our Heart Center in the years 1995–2000. Humoral and cellular immune response
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to elective CPB surgery was investigated. All studies were approved by the ethical committee, University of Leipzig. Written informed consent was obtained from the parents of all patients. The majority of the patients enrolled in our earlier studies received MP (MPþ) (21). Only a minority of them did not receive MP (MP). We analyzed the surgical records of all children (n ¼ 198). According to the records, 26 of them did not receive MP before or during surgery. Most of these children underwent correction of an atrial (ASD) or a ventricular septal defect (VSD; total n ¼ 14) while others had diverse congenital heart diseases. To make our patients group as homogenous (and therefore comparable) as possible, we decided to enroll only patients who underwent correction of ASD or VSD. Surgery for more complex congenital heart disease is associated with substantially longer CPB and cross-clamp times carrying prolonged effect of surgical trauma and CPB on the immune system or is known to be associated with latent immunological abnormalities, e.g., Glenn-Fontan surgery (22–24). Inclusion criteria. ASD or VSD repair, open heart surgery with CPB, age range 3–16 years. All patients included in the study had normal heart functions. Exclusions criteria. Left ventricular ejection fraction < 50%, known chromosomal abnormalities (e.g., trisomy 21) or other genetic defects, minimal-invasive surgery of the congenital heart disease, infections before or during surgery. This resulted in 10 children not receiving MP (MP) and 23 who received MP (MPþ) being enrolled in this study. The MPþ patients received, between anesthesia onset and begin of CPB, 250 mg MP either in a single dose or in two doses of 125 mg each. This resulted in a median dose of 11 mg kg1 body weight (IQR: 10–16 mg kg1). Anesthesia and Antibiotic Regime General anesthesia and myorelaxation were performed with midazolam, fentanyl, etomidate, propofol, and pancuronium. CPB was performed with a Stockert roller pump (Stockert Instrumente GmbH, Munich, Germany) and hollow-fiber oxygenator (DIDECO, Mirandola, Italy). Priming solution consisted of a crystalloid solution, mannitol (4–6 ml kg1 body weight), human albumin, and compatible fresh blood. During bypass the hematocrit level was kept at 22–30% and the flow-rate was maintained at 2.7–3.5 l m2 BSA per min. Hypothermia (28– 35 C) was induced by cooling the priming solution in the extracorporeal circuit and circulating blood with heat exchanger. During the cooling period all patients received sodium nitroprussid (0.3–2.0 lg/kg/min) for vasodilatation. Bretschneider’s or St. Thomas cardioplegic solutions were used for myocardial perfusion. Antibiotic regime consisted of Cephazolin (50 mg kg1 body weight) in three separate doses. During rewarming, also heated humidified inspired gases and intravenous administration of sodium nitroprussid were used. At end of the bypass, patients underwent
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FIG. 1. Time course of serum components induced by cardiovascular surgery in the MPþ (closed symbols) and the MP group (open symbols). Data display median values of 23 children with MP and 10 without MP treatment. Error bars indicate IQR. Data at sampling time CPBI and CPBII were corrected for hemodilution as shown in Patients and Methods. A. Cortisol (MP) and methylprednisolon with cortisol (MPþ); B. IL-6; C. IL-8; D. sEselectin; E. IL-10; F. C1-inhibitor. The symbols in the graphs indicate statistically significant differences: * ¼ MP vs. MPþ by Wilcoxon test; þ ¼ time point compared to 1d- (pre-operative value), for MPþ patients by Mann-Whitney U-Test; ^ ¼ time point compared to 1d- for MP patients by Mann-Whitney U-Test.
ultrafiltration. Then, normal flow was re-established and the patient was rewarmed and heparin was neutralized by protamine sulphate. If necessary, catecholamine (dopamine, dobutamine, or epinephrine) was infused before the patient was weaned off the bypass.
Samples for serum and plasma were sedimented at 2,800g, and supernatants were frozen at 80 C in aliquots within 1 h after sampling. From the same blood samples, hematocrit values were determined.
Postoperative Care
Serum Concentration of Cortisol, MP, Complement, Cytokines, and Soluble Adhesion Molecules
After surgery all patients were mechanically ventilated. To optimize blood pressure and diuresis catecholamines and diuretics were infused. Laboratory assays including blood count, electrolytes, renal and liver tests, and coagulation analysis were performed immediately after the surgery and 16–24 h later. Echocardiography, chest X-ray, and sonography were performed, whenever necessary. Blood Samples Blood samples (5 ml patient1) were drawn 1 day before surgery before any medication (1d-), after onset of anesthesia (anaesth), shortly after CPB onset (mean SD, 14 6 min; range, 10–30 min) (CPB1), before CPB termination during reperfusion and rewarming (CPB2), 4–6 h after the end of surgery (4 hþ), 1 (1 d) and 2 days (2 d) after surgery and at discharge (>3 d; 9.8 3.6 days postoperatively), and at out-patient follow-up (3 m; time after surgery: 9.1 7.0 months (range: 2–30 months); obtained from 25 patients only (16 MPþ/9 MP). EDTA anticoagulated blood and native blood were collected for flow cytometry and serology, respectively.
Plasma concentration of MP and cortisol were measured by a Cortisol ELISA (IBL, Hamburg, Germany, Fig. 1A). This ELISA had a cross reactivity with MP of 5%. From CPB2 and 4 hþ values the half life of MP was estimated by:
t1=2 ¼ t ln 2=ðln C0 ln Ct Þ where t1/2 is the half life, t the time between two concentration measurements, C0 the initial concentration (t ¼ 0, here CPB2), and Ct the concentration after time t (here 4 hþ) (25,26). The serum concentrations of the complement components C3, C4, C5, C1-inhibitor, and C3d were determined by radial immunodiffusion (The Binding Site, Heidelberg, Germany) with serum or EDTA-plasma (C3d). Total hemolytic complement CH100 was determined by lysis of antibody coated sheep erythrocytes (The Binding Site). Interleukin (IL) 8, IL-6, soluble-(s)Eselectin, and sL-selectin (all from R&D Systems, Oxon, UK), sICAM-1 (Bender Med Systems), C5a (Enzygnost, Behringwerke AG, Marburg, Germany) were quantified
Cytometry Part B: Clinical Cytometry
IMMUNE EFFECT OF METHYLPREDNISOLONE IN PEDIATRIC HEART SURGERY
in serum by enzyme linked immune assay (ELISA). The lower limits of detection were 1.1 mg l1 (C3d), 0.1 lg l1 (C5a), and 4 pg ml1 (IL-6 and IL-8) 142 U ml1 (CH 100), 155 mg l1 (C3), 58 mg l1 (C4), 20 mg l1 (C5), 45 mg l1 (C1-Inhibitor), 68.75 ng ml1 (sICAM-1), 8.19 ng ml1 (sE-selectin) and 62.73 ng ml1 (sL-selectin). The measured serum concentration of a biologically active compound is relevant for the response of the patient. However, in order to compare patients with slightly varying hemodilution serum component concentration and absolute cell count values were corrected for hemodilution according to Hambsch et al. (21):
½Ckorr ¼ ½Csample HK1d =HKsample where [C]korr is the corrected concentration, [C]sample that in the sample, HKsample the hematocrit value in the sample and HK1d- the preoperative HK value. Correction for hemodilution was done for all samples except the out-patient follow-up sample. Cell Phenotyping and Antigen Expression Phenotyping of leukocytes was done as detailed elsewhere by the whole blood technique (24,27). About 40 ll EDTA blood was mixed with a cocktail of directly fluorescence dye-conjugated monoclonal antibodies. Cells were stained for 15 min at room temperature in the dark, after adding 1 ml of a lysing solution (BD Biosciences, San Jose, CA) the samples were mixed and incubated for 10 min at room temperature in the dark. The cells were spun down at 300g, and the supernatant discarded. The cells were washed twice in 1 ml phosphate buffered saline pH ¼ 7.4 (PBS, Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) and finally resuspended in 200 ll 0.5% paraformaldehyde (Sigma) in PBS. Antibodies for lineage and activation markers were obtained from following providers: BD Biosciences (CD3—T cells, CD4—T-helper/inducer cells, CD8—T-cytotoxic/suppressor cells, CD14—LPS receptor on monocytes, CD45—pan leukocyte antigen, HLA-DR—MHC II, CD11a—LFA-1, CD11b—Mac-1, CD69—early activation antigen), Caltag (Hamburg, Germany), (CD16—Fc-c Receptor III on neutrophils and NK-subset, CD19—B cells, CD25—IL2-R, CD56—N-CAM on NK subset), BeckmanCoulter Corp. (Hialeah, FL) (CD54—ICAM-1), and DAKO (Glostrup, Denmark) (CD18—b2-integrin). Background fluorescence was quantified after staining with appropriate control antibodies (BD Biosciences). Antibodies were labeled with the fluorescent dyes fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCPTM), or allophycocyanin (APC) in cocktails of three- or four-color combinations. Cells were measured on a dual laser flow cytometer (FACSCalibur; BD Biosciences) calibrated with calibration microbeads (Spherotech, Libertyville, IL) for standardization (28,29). Flow cytometric data were analyzed with the CellQuest software package (BD Biosciences). The percent-
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age of leukocyte subsets was quantified, and their cell counts per blood volume were calculated based on the differential blood count. For each cell subset the mean fluorescence intensity (MFI) as a measure of antigen expression and activation degree was determined after subtraction of the fluorescence intensity of control antibody-stained cells (28,29). Statistical Analysis Data distribution was tested for normality (Kolmogorov-Smirnov test). Because the majority of the analyzed laboratory parameters were not normally distributed data are presented as median and inter quartile range (IQR) and only nonparametric tests were performed (exception: multivariate ANOVA,). Changes with time within individual groups (MPþ or MP) were analyzed by the Friedman test. The time courses of MPþ and MP groups were compared using multivariate ANOVA (MANOVA). Single time-points within one group were compared to the 1d- values by paired Wilcoxon test, identical time-points between MPþ and MP by nonparametric Mann-Whitney U-test. All statistical analyses were performed using the SPSS program package (SPSS V.8; Knowledge Dynamics, Canyon Lake, TX). Values were regarded significantly different if P < 0.05. In multiple tests the significance levels were corrected according to Bonferroni: P ¼ 0.05/n where n ¼ number of time-points analyzed for a single analyte tested. If not stated otherwise, in the following only data rendering statistically significant differences or changes are discussed. RESULTS Clinical Characteristics Clinical data of the patients are shown in Table 1. Both groups did not differ with respect to age, gender, body weight, and duration of surgery. Intubation time, ICU days and time until discharge from the clinic were identical. Pleural and pericardial effusions were detected in both groups mostly without hemodynamic significance and had disappeared until discharge. The drainage volume did not differ on the day of surgery and the following days between MPþ and MP group. All children were discharged well. Altogether, the postoperative courses could be regarded as normal and uneventful. Post-operatively none of the patients developed symptoms of cardiac insufficiency. Laboratory Results From all analyzed immunological parameters only 11 showed significantly different values either according to MANOVA test and/or at identical time-points according to Wilcoxon test. Table 2 summarizes the results for MANOVA and Friedmann test for these parameters. Serum MP and Cortisol In the MP group the postoperative cortisol level increased from base line level of 100 ng ml1 up to 250
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Table 1 Patient Characteristics and Perioperative Data
Age (years) Gender (m/f) Weight (kg) Height (cm) Duration of surgery (min) CPB time (min) Cross-clamping time (min) Hypothermia ( C) Duration of Intensive Care (h) Total Drainage Volume (ml kg1 body weight) Methylprednisolone (mg) Methylprednisolone (mg kg1 body weight) NS ¼ not significant (Mann Whitney U-test,
MPþ-group (n ¼ 23) Median (IQR)
MP-group (n ¼ 10) Median (IQR)
Significance MPþ vs. MP
6.5 (5.2 ; 7.4) 0 10/13 21 (15 ; 26) 120 (106 ; 128) 120 (103 ; 134) 42 (36 ; 65) 21 (14 ; 35) 32 (30 ; 32) 43 (29 ; 48) 6.7 (4.34 ; 9.21) 250 (250 ; 250) 11 (10 ; 16)
7.9 (6.5 ; 9.7) 0 4/6 26 (22 ; 27) 128 (121 ; 132) 110 (106 ; 118) 51 (42 ; 59) 27 (14 ; 32) 32 (30 ; 32) 43 (29 ; 48) 5.67 (3.75 ; 7.52) 0 0
NS NSa NS NS NS NS NS NS NS NS
a
Except gender: X2-test).
ng ml1 at 4hþ (Fig. 1A). All patients from the MPþ group had during and up to 4 h after surgery about 100 higher cortisol ELISA values than the MP patients. Maximal values were obtained at CPB2 (median: 11,940 lg l1) followed by the 4hþ concentration (median 3,200 lg l1). At 1dþ serum levels did not significantly differ from those of MP patients. From these values half life of MP in the blood was calculated as 2.62 h (median, IQR: 2.53–2.93 h).
< 0.001, Friedman) and the time courses between the MPþ and MP group were different (IL-6: P < 0.001, IL8: P ¼ 0.014; MANOVA). The concentrations were higher without MP administration for both cytokines (significant at 4hþ). The concentration of sE-selectin (Fig. 1D) was in both groups also significantly altered by surgery (P < 0.001) and the time courses differed significantly between MPþ and MP (P ¼ 0.001; MANOVA). sE-selectin serum level was under MP administration perioperatively reduced.
Humoral Parameters Serum concentration of the following compounds did not show significant MP related changes: IL2 receptor, TNF-a, Histamine, IL-1b, IL-12p70, sL-selectin, sP-selectin, sICAM-1, CH-100, C3, C3d, C3d/C3-ratio, C4, C5, C5a, C5a/C5 ratio, hematocrit, erythrocyte, and platelet counts. Their time courses were found to be similar with and without MP (MANOVA) and there were no significant differences at identical time-points between MPþ and MP. This is in agreement with our earlier reports (21,30,31). Proinflammatory Cytokines and Endothelium Activation IL-6 and IL-8 concentrations (Figs. 1B and 1C) were significantly affected by surgery in both groups (both P
MANOVA
IL6 IL8 sE-Selectin IL10 C1-inhibitor Neutrophil cell count Monocyte cell count NK cell count CD4 cell count CD8 cell count B-cell count
