Original Cardiovascular
Effect of Glyceryl Trinitrate on Staphylococcus aureus Growth and Leukocyte Activation during Simulated Extracorporeal Circulation Vilyam Melki1 Eva Tano2 Lena Douhan Hakansson2 Tadeusz Malinski4 Jan Borowiec1 1 Department of Surgical Sciences, Uppsala University,
Uppsala, Sweden 2 Department of Medical Sciences, Uppsala University, Uppsala, Sweden 3 Department of Clinical Immunology and Transfusion Medicine, Uppsala University, Uppsala, Sweden 4 Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio, United States
Phan-Kiet Tran1
Folke Knutson3
Address for correspondence Vilyam Melki, MD, Department of Cardiothoracic Surgery, Uppsala University Hospital, SE-781 85 Uppsala, Sweden (e-mail: [email protected]).
Thorac Cardiovasc Surg 2014;62:402–408.
Abstract
Keywords
► glyceryl trinitrate ► Staphylococcus aureus ► simulated extracorporeal circulation ► postoperative infection ► cardiac surgery
Background Previously, nitric oxide has been shown to possess antimicrobial effects. In this study, we aim to test the effect of glyceryl trinitrate (GTN) on Staphylococcus aureus growth during simulated extracorporeal circulation (SECC) and also to examine the effect of S. aureus, alone and in combination with GTN, on activation markers of the innate immune system during SECC. Methods In an in vitro system of SECC, we measured GTN-induced changes in markers of leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth. Results GTN had no effect on S. aureus growth after 240 minutes SECC. Staphylococcus aureus reduced the expression of granulocyte Fcγ-receptor CD32 but stimulated the expression of monocyte CD32. Staphylococcus aureus stimulated expression of some leukocyte adhesion key proteins, activation marker CD66b, lipopolysaccharide-receptor CD14, and C3b-receptor CD35. Staphylococcus aureus and GTN addition induced significant increases in monocyte CD63 (lysosomal granule protein) levels. Conclusion GTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation.
Introduction The postoperative wound infections constitute a major health care problem mainly because they are potentially life-threatening complications for patients. Moreover, they enormously increase hospital costs for cardiac procedures. The incidence of surgical-site infections after coronary artery bypass surgery ranges from 0.5 to 14.3%.1 Tegnell et al reported that surgical wound infections occurred in 30% of cardiac surgical
received August 16, 2013 accepted after revision October 27, 2013 published online December 16, 2013
patients.2 The population of cardiac surgical patients has changed in last decades. The ever growing number of older and more fragile patients with severe comorbidities as well as more complex procedures and reoperations possibly contribute to a higher risk of perioperative infections. However, even in the more susceptible population, a fairly low infection incidence of 2% has been reported.3 Staphylococcus aureus (S. aureus) is a very dangerous bacterium and unfortunately rather frequently found in these postoperative infections.4
© 2014 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0033-1363296. ISSN 0171-6425.
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC
Materials and Methods Preparation of Staphylococcus aureus For this study, we used bacterial strain S. aureus, American Type Culture Collection 29213. Preceding each experiment, the strain was grown in 5 mL of Todd-Hewitt broth in plastic tubes at 37°C in air for 5 hours, resulting in approximately 5 108 CFU/mL in an exponential growth phase. Just before the start of each experiment, the bacterial cultures were diluted 1:10 in phosphate-buffered saline (PBS).
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Preparation of Blood and Staphylococcus aureus Infestation For each experiment, blood was freshly drawn from two clinically healthy human volunteers of the same blood type, 450 mL each. The drawn blood was then immediately pooled into a large citrate-phosphate dextrose (CPD) bag and heparinized to 1.4 to 2.8 IU heparin/mL of whole blood (Lövens, Ballerup, Denmark). The CPD bags were then infested with 1 mL of the prepared S. aureus culture, obtaining a final concentration of 1 104 to 1 105 CFU/mL of whole blood. In each experiment, the blood then entered two separate SECC circuits simultaneously by gravity, without foaming; circuits with S. aureus and circuits with S. aureus and GTN. The relationship between magnitude of bacteremia and severity of clinical picture has been extensively investigated. In our model, a final concentration of 1 104 to 1 105 CFU/mL of whole blood would be translated into the clinical range of severe sepsis.10
Simulated Extracorporeal Circulation Assembling of the SECC circuits was made in a standard manner, including a calibrated barely occlusive roller pump Stöckert (Sorin Group GmbH, München, Germany), polyvinylchloride tubing, and hollow fiber membrane oxygenator with hard-shell venous reservoir Jostra Quadrox D Bioline (Maquet, Rastatt, Germany). GTN (1 mg/mL) was delivered to the SECC circuits at 0.5 mL/h by an automated infusion system pump Braun Perfusor (B. Braun, Melsungen, Germany). In a closed circuit, the blood was circulated at 0.5 L/min for 240 minutes. Activated clotting time was measured with Hemotech analyzer (Englewood, Colorado, United States) and kept at more than 270 seconds for the entire duration of the experiments. The oxygenators were ventilated at 0.5 L/min with an oxygen and air mixture of 40 and 60%, respectively, achieving mean levels of PO2 34.3 kPa, PCO2 1.6 kPa, and pH 7.5. Temperature was maintained at 35°C with a thermostatic heat exchanger (Gambro, Lund, Sweden). Measurement of blood gases and pH was done in the AVL Omni Modulator System (AVL Scientific Corp., Roswell, Georgia, United States).
Blood Analysis At five different time points, blood samples were taken; immediately after heparinization and infestation with S. aureus, then at 30, 60, 180, and 240 minutes of SECC. The first blood sample was taken directly from the CPD bag and the subsequent samples from the SECC circuits. At each time point, 4 mL was collected in a tube containing 7.2 mg ethylenediaminetetraacetic acid (EDTA) for blood cells count and leukocyte differential. Samples for myeloperoxidase (MPO) analysis were collected in serum separator tubes, and samples for activated complement factor 3 (C3a) and terminal complement complex (TCC) were collected in EDTA tubes, then centrifuged at 2,000 rpm for 10 minutes, and the plasma immediately frozen and stored at – 70°C for later quantitative analysis. MPO in plasma was detected with radioimmunoassay kit according to the manufacturer’s recommendation (Pharmacia Diagnostics AB, Uppsala, Sweden) and quantified in Wallac Thoracic and Cardiovascular Surgeon
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We recently published observations on how nitric oxide (NO) enhances the growth of S. aureus under conditions with increased oxidative stress, for example, simulated extracorporeal circulation (SECC).5 NO is a small short living signaling molecule. It is a free radical endogenously synthesized in the human body from L-arginine and oxygen by three types of NO synthase (NOS) enzymes; neuronal NOS found mostly in neuronal cells, endothelial NOS produced in a high degree by endothelial cells, and inducible NOS (iNOS) commonly found in phagocytes such as neutrophils, monocytes, and macrophages. NO, generated by iNOS in phagocytes, can be involved in the immune response to infection. NO may exert either harmful or beneficial effects, which elucidates its role in many fundamental physiological and pathological processes of the body.6 In clinical practice, glyceryl trinitrate (GTN) is one of the most important NO donors used for blood pressure control. GTN has been shown to exert a potent inhibitory effect on bacterial growth, including S. aureus, in sodium chloride 0.9%, glucose 5%, ethanol 10%, and Mueller-Hinton broth.7 Peroxynitrite is a NO-derived oxidant, formed during the reaction between NO and superoxide radicals, acting as a key component of nitroxidative stress associated with pathophysiological conditions such as inflammatory or cardiovascular disorders. Peroxynitrite demonstrates very strong bactericidal potential, which magnitude depends on many complex factors of kinetics and cellular radical milieu.8 Effect of GTN on bacterial growth has neither been studied in whole blood nor during extracorporeal circulation (ECC). A wide variety of proteins, functioning either as receptors or as ligands, are expressed on the cell surfaces of leukocytes. Leukocyte regulation, activation, function, and cell–cell communication are directed by the interactions of these numerous cell surface proteins. The interactions can be long-lasting stable or transient, as seen in the immune system, where the cells bind, communicate, and then separate.9 In this study, we tested the effect of GTN, an important NO donor widely used in clinical practice, on S. aureus growth during SECC. We also examined the effect of S. aureus alone and in combination with GTN on activation markers of the innate immune system during SECC. We chose for this study a set of the Fcγ receptors known to be involved in the inflammatory processes including CD11b, CD 14, CD 16, CD 32, CD35, CD63, CD64, CD65, and CD66b.
Melki et al.
Glyceryl Trinitrate Effect on S. aureus Growth during SECC 1260 Multigamma Counter (Wallac Oy, Turku, Finland). Using an automatic cell counter (Medonic CA 620 Loke 16-parameter system, Boule Diagnostics AB, Stockholm, Sweden), hemoglobin concentration, platelet, leukocyte, and leukocyte subset counts were determined. Quantitative analysis of plasma C3a and TCC was performed with a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden).
Melki et al. locyte and monocyte populations were gated and the FITCfluorescence measured. The fluorescence intensity above background (isotype control) of granulocytes and monocytes was determined and expressed as mean fluorescence intensity. In case of CD35 and CD64, expression was also given as relative number of positive cells (%), defined as the relative number of cells that expressed CD35, respectively, CD64 to a higher extent than background (isotype control).
Preparation of Leukocytes Leukocytes were prepared as previously described.11,12 Briefly, 1 mL heparinized blood was fixated with 0.4% paraformaldehyde and the erythrocytes were lysed by incubation with 0.85% (w/v) NH4Cl in Tris-HCl buffer [Tris(hydroxymethyl)aminomethane 0.01 mol/L, pH 7.4]. Finally, the cells were washed with PBS containing human serum albumin (HSA) (0.1%, w/v) and diluted to the concentration of 2.5 106/mL.
Labeling of Leukocytes with Antibodies to Cell Surface Antigens A total of 50 μL samples of the leukocyte suspension were mixed with optimally titrated fluorescein isothiocyanate (FITC)-, phycoerythrine-, or unlabeled mouse monoclonal antibodies (mab) against CD11b, CD35, CD63, CD64, CD65, CD66b (Beckman Coulter, Fullerton, California, United States), CD16, CD14, (Dako, Glostrup, Denmark), and CD32 (BectonDickinson, San Diego, California, United States) and incubated for 30 minutes at 4°C. After incubation, the cells were washed with PBS and thereafter diluted in 300 μL PBS with HSA. Leukocytes were also labeled by an identical procedure with negative isotype controls for mouse immunoglobulin G1 (IgG1) and IgG2 (Dako). After labeling, the cells were kept on ice until analysis.
Bacteriological Analysis Blood samples (5 mL) from the infected GTN and control circuits were collected at 0, 30, 60, 180, and 240 minutes. According to a standardized protocol, the samples were then diluted empirically, spread on agar plates, and incubated for 2 days at 37°C. The quantification of bacterial growth was determined by manual count of the number of colony forming unit.
Statistical Analysis Data are presented as median and range from five independent experiments. Statistical analysis was performed using nonparametric ANOVA (Kruskal–Wallis), and p values 0.05 were considered significant. All the statistical analyses were
Flow Cytometry Flow cytometric analysis was performed on an EPIC MCL-XL flow cytometer (Beckman Coulter). Identification of granulocytes was based on their forward scatter/side scatter (FSC/ SSC) dot-plot profile and of monocytes on the FSC/SSC dotplot profile and positive staining with anti-CD14. The granu-
Fig. 1 Growth of S. aureus during SECC, without ( ) and with GTN (•). The data are presented as the median of the number of S. aureus CFU/mL whole blood with range (n ¼ 5). Thoracic and Cardiovascular Surgeon
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Fig. 2 Changes in levels of leukocyte Fcγ receptors during SECC, S. aureus infested without ( ) and with GTN (•). Expression of (A) granulocyte CD32 and (B) monocyte CD32. Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5).
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC
Results GTN Has No Effect on Staphylococcus aureus Growth during SECC
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of leukocyte CD16 and CD64. Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD32.
Staphylococcus aureus affects the Expression of Key Proteins Involved in Leukocyte Adhesion
Staphylococcus aureus were introduced into parallel SECC circuits as described in methods. GTN was added to one of these parallel circuits. We performed five independent parallel SECC runs, each lasted 240 minutes. In both groups, bacterial counts were significantly reduced (p 0.05) over time. Addition of GTN did not generate any intergroup differences (►Fig. 1). To explore possible immunological mechanisms involved in the potential reductive effect of GTN on S. aureus growth, we analyzed a panel of markers of inflammatory response.
Expression of key molecules involved in cell adhesion was determined by measuring the level of CD11b (adhesion and C3bi receptor), CD65 (E-selectin ligand), and CD162 (Pselectin ligand). Addition of S. aureus induced significant increases in expression of granulocyte and monocyte CD11b, monocyte CD65, and monocyte CD162 over time (p 0.01) (►Fig. 3A–D), indicating an activation of these cells. Granulocyte expression of CD162 was though reduced over time. Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD11b, CD65, and CD162.
Staphylococcus aureus Reduces the Expression of Granulocyte Fcγ-Receptor CD32 but Stimulates the Expression of Monocyte CD32
Staphylococcus aureus Stimulates the Levels of Lipopolysaccharide Receptor, C3b Receptors, and Leukocyte Activation Marker
Staphylococcus aureus significantly reduced the expression of granulocyte CD32 and stimulated the expression of monocyte CD32 (p 0.05) over time (►Fig. 2A, B). Theoretically, the reduced expression of granulocyte CD32 may be due to an internalization of the receptor. There were no significant changes, with or without addition of GTN, for the expression
To screen for other potential effects of GTN on other components of the innate immune system, we also analyzed the levels of CD14 (lipopolysaccharide receptor), CD35 (C3b receptor), and CD66b (a marker of granulocyte activation). Addition of S. aureus induced significant increases in monocyte CD14, leukocyte CD35, and granulocyte CD66b levels
Fig. 3 Changes in expression of leukocyte key proteins, involved in cell adhesion, during SECC, S. aureus infested without () and with GTN (•). Expression of (A) granulocyte CD11b, (B) monocyte CD11b, (C) monocyte CD65, and (D) monocyte CD162. Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5). Thoracic and Cardiovascular Surgeon
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performed with the Statistica 10.0 software for Windows (StatSoft, Tulsa, Oklahoma, United States).
Melki et al.
Glyceryl Trinitrate Effect on S. aureus Growth during SECC (p 0.01) over time (►Fig. 4A–D). Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD14, CD35, and CD66b.
Staphylococcus aureus Stimulates Expression of Lysosomal Granule Membrane Protein The levels of granulocyte CD63 (lysosomal granule protein) were analyzed. Addition of S. aureus resulted in a significant increase in monocyte CD63 levels (p 0.01) over time. Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD63 (►Fig. 5).
Staphylococcus aureus Increases the Levels of TCC, C3a, and MPO Staphylococcus aureus induced a significant increase of TCC, C3a, and MPO levels (p 0.01) over time. Addition of GTN to the circuits did not generate any significant intergroup changes for TCC, C3a, and MPO.
Discussion Intravenous preparation of GTN has previously been shown to effectively reduce the growth of various bacterial strains, including S. aureus. GTN in different physiological saline solutions was bactericidal.7 In this study, though, for the first
Melki et al. time, we have shown that GTN does not affect S. aureus growth in whole blood during SECC. During our experiment, we also screened several important proteins and markers involved in the recruitment and activation of leukocytes in response to S. aureus infection, which has allowed us to address the question whether or not GTN addition may affect the activity of the innate immune system. Our study confirms that SECC with whole blood infested with S. aureus, induces an activation of both granulocytes and monocytes, as reflected by increases in the majority of measured parameters at most time points. Addition of GTN did not significantly alter expression of the investigated cell surface receptors. A weakness of this study is the low number of experiments performed, and also that the experiments were performed at only one GTN dose. The GTN dose delivered in this study was correlated to clinically established levels. We intend though to perform additional studies including longer SECC times and GTN dose response correlation. On the contrary, the hypothesis is new and, since GTN is an important NO donor widely used in clinical practice for patients with angina pectoris, pulmonary hypertension, and systemic blood pressure control, the results could be of major clinical importance, and further investigations are therefore desirable. NO has been shown to facilitate the killing of the intracellular pathogen
Fig. 4 Changes in levels of components of the innate immune system during SECC, S. aureus infested without ( ) and with GTN (•). Expression of (A) monocyte CD14, (B) granulocyte CD35, (C) monocyte CD35, and (D) granulocyte CD66b. Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5). Thoracic and Cardiovascular Surgeon
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Fig. 5 Changes in leukocyte expression of lysosomal granule membrane protein CD63 during SECC, S. aureus infested without () and with GTN (•). Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5).
Leishmania donovani, the causative agent of visceral leishmaniasis, by stimulating phagosomal maturation.13 In a wider perspective, our results may promote further studies in patients with leishmaniasis and/or other bacterial infections. One may question whether our observed changes are not only due to the time effect of SECC. To clarify this, we have performed further experiments with similar settings. Preliminary results from these unpublished experiments indicate that SECC by itself induces increased expression of leukocyte activation markers CD11b, CD14, CD35, and CD162, but seem to have no effect on CD16, CD32, CD65, CD63, and CD66b. An interesting issue is whether GTN had any effect at all on the innate immune system. Cartwright et al stated that physiologically relevant NO levels did not affect lymphocyte adhesion to endothelium and expression of proteins, such as cell adhesion molecule-1 (C-CAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin.14 ECC always leads to activation of the innate immune system as a part of systemic inflammatory response in which the excessive oxidative stress plays an important role.15 The interpretation of our results may be complicated by the fact that the ECC-induced activation of different cascade systems alters the pro- and anti-inflammatory balance of the body many times during postoperative period. In this process, NO is deeply involved, although its role is still not fully understood. NO may temporarily move this inflammatory balance in beneficial or harmful direction depending on actual status in biochemical and/ or redox milieu.16 Worth considering is also that the ability of S. aureus to develop resistance to innate immunity by S. aureus adaptive response to nitrosative stress.17 Also, other recent studies of host–pathogen interaction give us reasons to revise our understanding of the nature of S. aureus infection. The bacteria produce several factors to evade neutrophil response. These include chemotaxis inhibitory protein of staphylococci, staphylococcal complement inhibitor, clump-
Melki et al.
ing factor A, and extracellular adherence protein.18,19 For survival, S. aureus can in fact hide inside neutrophils.20 The very presence of granulocytes may therefore generate better S. aureus survival. In vitro studies have shown that GTN have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. GTN induces an antimitogenic effect and inhibits cytokine production and expression, cell-mediated cytotoxicity, and antibody production.21 Since our observed changes were mostly detected at the late stage of SECC and, due to the short viability of neutrophils, therefore make them difficult to relate to settings of more time prolonged ECC such as extracorporeal membrane oxygenation or dialysis, where also there is a continuous release of new neutrophils from the bone marrow. In our model, we observed a steadily decreasing number of neutrophils, probably due to a combination of adhesion to the foreign surfaces, mechanical destruction, and apoptosis. What role apoptosis of immune cells play in our settings is unclear. Therefore, it would be of interest to perform further studies to find out mechanisms behind it and elucidate potential clinical impact.
Acknowledgments This study was supported by Swedish Heart-Lung Foundation (V.M.), and Sellanders’ Family Fund and the Heart Lung Fund, Uppsala University Hospital (J.B.).
Note This study was approved by the appropriate ethics committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Conflict of Interest None.
References 1 Harrington G, Russo P, Spelman D, et al. Surgical-site infection
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rates and risk factor analysis in coronary artery bypass graft surgery. Infect Control Hosp Epidemiol 2004;25(6): 472–476 Tegnell A, Arén C, Ohman L. Coagulase-negative staphylococci and sternal infections after cardiac operation. Ann Thorac Surg 2000; 69(4):1104–1109 Collart F, Feier H, Kerbaul F, et al. Primary valvular surgery in octogenarians: perioperative outcome. J Heart Valve Dis 2005; 14(2):238–242, discussion 242 Saadatian-Elahi M, Teyssou R, Vanhems P. Staphylococcus aureus, the major pathogen in orthopaedic and cardiac surgical site infections: a literature review. Int J Surg 2008;6(3): 238–245 Melki V, Tran PK, Tano E, Knutson F, Borowiec JW. Enhanced growth of Staphylococcus aureus after nitric oxide supplementation during simulated extracorporeal circulation. Thorac Cardiovasc Surg 2010; 58(2):81–85
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC
Glyceryl Trinitrate Effect on S. aureus Growth during SECC
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6 Kobzik L. Translating NO biology into clinical advances: still
14 Cartwright JE, Whitley GS, Johnstone AP. Endothelial cell adhesion
searching for the right dictionary? Am J Respir Cell Mol Biol 2009;41(1):9–13 Bátai I, Kerényi M, Tekeres M. The growth of bacteria in intravenous glyceryl trinitrate and in sodium nitroprusside. Anesth Analg 1999;89(6):1570–1572 Ferrer-Sueta G, Radi R. Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. ACS Chem Biol 2009;4(3):161–177 Russwurm S, Vickers J, Meier-Hellmann A, et al. Platelet and leukocyte activation correlate with the severity of septic organ dysfunction. Shock 2002;17(4):263–268 Yagupsky P, Nolte FS. Quantitative aspects of septicemia. Clin Microbiol Rev 1990;3(3):269–279 Hamblin A, Taylor M, Bernhagen J, et al. A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins. J Immunol Methods 1992;146(2):219–228 Torsteinsdóttir I, Arvidson NG, Hällgren R, Håkansson L. Monocyte activation in rheumatoid arthritis (RA): increased integrin, Fc gamma and complement receptor expression and the effect of glucocorticoids. Clin Exp Immunol 1999;115(3):554–560 Winberg ME, Rasmusson B, Sundqvist T. Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages. Exp Parasitol 2007;117(2):165–170
molecule expression and lymphocyte adhesion to endothelial cells: effect of nitric oxide. Exp Cell Res 1997;235(2):431–434 Christen S, Finckh B, Lykkesfeldt J, et al. Oxidative stress precedes peak systemic inflammatory response in pediatric patients undergoing cardiopulmonary bypass operation. Free Radic Biol Med 2005;38(10):1323–1332 Dyson A, Singer M. Animal models of sepsis: why does preclinical efficacy fail to translate to the clinical setting? Crit Care Med 2009; 37(1, Suppl):S30–S37 Richardson AR, Dunman PM, Fang FC. The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity. Mol Microbiol 2006;61(4):927–939 Foster TJ. Immune evasion by staphylococci. Nat Rev Microbiol 2005;3(12):948–958 Rooijakkers SH, van Kessel KP, van Strijp JA. Staphylococcal innate immune evasion. Trends Microbiol 2005;13(12):596–601 Anwar S, Prince LR, Foster SJ, Whyte MK, Sabroe I. The rise and rise of Staphylococcus aureus: laughing in the face of granulocytes. Clin Exp Immunol 2009;157(2):216–224 Shoker AS, Yang H, Murabit MA, Jamil H, al-Ghoul A, Okasha K. Analysis of the in vitro effect of exogenous nitric oxide on human lymphocytes. Mol Cell Biochem 1997;171(1-2):75–83
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Effect of Glyceryl Trinitrate on Staphylococcus aureus Growth and Leukocyte Activation during Simulated Extracorporeal Circulation Vilyam Melki1 Eva Tano2 Lena Douhan Hakansson2 Tadeusz Malinski4 Jan Borowiec1 1 Department of Surgical Sciences, Uppsala University,
Uppsala, Sweden 2 Department of Medical Sciences, Uppsala University, Uppsala, Sweden 3 Department of Clinical Immunology and Transfusion Medicine, Uppsala University, Uppsala, Sweden 4 Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio, United States
Phan-Kiet Tran1
Folke Knutson3
Address for correspondence Vilyam Melki, MD, Department of Cardiothoracic Surgery, Uppsala University Hospital, SE-781 85 Uppsala, Sweden (e-mail: [email protected]).
Thorac Cardiovasc Surg 2014;62:402–408.
Abstract
Keywords
► glyceryl trinitrate ► Staphylococcus aureus ► simulated extracorporeal circulation ► postoperative infection ► cardiac surgery
Background Previously, nitric oxide has been shown to possess antimicrobial effects. In this study, we aim to test the effect of glyceryl trinitrate (GTN) on Staphylococcus aureus growth during simulated extracorporeal circulation (SECC) and also to examine the effect of S. aureus, alone and in combination with GTN, on activation markers of the innate immune system during SECC. Methods In an in vitro system of SECC, we measured GTN-induced changes in markers of leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth. Results GTN had no effect on S. aureus growth after 240 minutes SECC. Staphylococcus aureus reduced the expression of granulocyte Fcγ-receptor CD32 but stimulated the expression of monocyte CD32. Staphylococcus aureus stimulated expression of some leukocyte adhesion key proteins, activation marker CD66b, lipopolysaccharide-receptor CD14, and C3b-receptor CD35. Staphylococcus aureus and GTN addition induced significant increases in monocyte CD63 (lysosomal granule protein) levels. Conclusion GTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation.
Introduction The postoperative wound infections constitute a major health care problem mainly because they are potentially life-threatening complications for patients. Moreover, they enormously increase hospital costs for cardiac procedures. The incidence of surgical-site infections after coronary artery bypass surgery ranges from 0.5 to 14.3%.1 Tegnell et al reported that surgical wound infections occurred in 30% of cardiac surgical
received August 16, 2013 accepted after revision October 27, 2013 published online December 16, 2013
patients.2 The population of cardiac surgical patients has changed in last decades. The ever growing number of older and more fragile patients with severe comorbidities as well as more complex procedures and reoperations possibly contribute to a higher risk of perioperative infections. However, even in the more susceptible population, a fairly low infection incidence of 2% has been reported.3 Staphylococcus aureus (S. aureus) is a very dangerous bacterium and unfortunately rather frequently found in these postoperative infections.4
© 2014 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0033-1363296. ISSN 0171-6425.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
402
Glyceryl Trinitrate Effect on S. aureus Growth during SECC
Materials and Methods Preparation of Staphylococcus aureus For this study, we used bacterial strain S. aureus, American Type Culture Collection 29213. Preceding each experiment, the strain was grown in 5 mL of Todd-Hewitt broth in plastic tubes at 37°C in air for 5 hours, resulting in approximately 5 108 CFU/mL in an exponential growth phase. Just before the start of each experiment, the bacterial cultures were diluted 1:10 in phosphate-buffered saline (PBS).
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Preparation of Blood and Staphylococcus aureus Infestation For each experiment, blood was freshly drawn from two clinically healthy human volunteers of the same blood type, 450 mL each. The drawn blood was then immediately pooled into a large citrate-phosphate dextrose (CPD) bag and heparinized to 1.4 to 2.8 IU heparin/mL of whole blood (Lövens, Ballerup, Denmark). The CPD bags were then infested with 1 mL of the prepared S. aureus culture, obtaining a final concentration of 1 104 to 1 105 CFU/mL of whole blood. In each experiment, the blood then entered two separate SECC circuits simultaneously by gravity, without foaming; circuits with S. aureus and circuits with S. aureus and GTN. The relationship between magnitude of bacteremia and severity of clinical picture has been extensively investigated. In our model, a final concentration of 1 104 to 1 105 CFU/mL of whole blood would be translated into the clinical range of severe sepsis.10
Simulated Extracorporeal Circulation Assembling of the SECC circuits was made in a standard manner, including a calibrated barely occlusive roller pump Stöckert (Sorin Group GmbH, München, Germany), polyvinylchloride tubing, and hollow fiber membrane oxygenator with hard-shell venous reservoir Jostra Quadrox D Bioline (Maquet, Rastatt, Germany). GTN (1 mg/mL) was delivered to the SECC circuits at 0.5 mL/h by an automated infusion system pump Braun Perfusor (B. Braun, Melsungen, Germany). In a closed circuit, the blood was circulated at 0.5 L/min for 240 minutes. Activated clotting time was measured with Hemotech analyzer (Englewood, Colorado, United States) and kept at more than 270 seconds for the entire duration of the experiments. The oxygenators were ventilated at 0.5 L/min with an oxygen and air mixture of 40 and 60%, respectively, achieving mean levels of PO2 34.3 kPa, PCO2 1.6 kPa, and pH 7.5. Temperature was maintained at 35°C with a thermostatic heat exchanger (Gambro, Lund, Sweden). Measurement of blood gases and pH was done in the AVL Omni Modulator System (AVL Scientific Corp., Roswell, Georgia, United States).
Blood Analysis At five different time points, blood samples were taken; immediately after heparinization and infestation with S. aureus, then at 30, 60, 180, and 240 minutes of SECC. The first blood sample was taken directly from the CPD bag and the subsequent samples from the SECC circuits. At each time point, 4 mL was collected in a tube containing 7.2 mg ethylenediaminetetraacetic acid (EDTA) for blood cells count and leukocyte differential. Samples for myeloperoxidase (MPO) analysis were collected in serum separator tubes, and samples for activated complement factor 3 (C3a) and terminal complement complex (TCC) were collected in EDTA tubes, then centrifuged at 2,000 rpm for 10 minutes, and the plasma immediately frozen and stored at – 70°C for later quantitative analysis. MPO in plasma was detected with radioimmunoassay kit according to the manufacturer’s recommendation (Pharmacia Diagnostics AB, Uppsala, Sweden) and quantified in Wallac Thoracic and Cardiovascular Surgeon
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We recently published observations on how nitric oxide (NO) enhances the growth of S. aureus under conditions with increased oxidative stress, for example, simulated extracorporeal circulation (SECC).5 NO is a small short living signaling molecule. It is a free radical endogenously synthesized in the human body from L-arginine and oxygen by three types of NO synthase (NOS) enzymes; neuronal NOS found mostly in neuronal cells, endothelial NOS produced in a high degree by endothelial cells, and inducible NOS (iNOS) commonly found in phagocytes such as neutrophils, monocytes, and macrophages. NO, generated by iNOS in phagocytes, can be involved in the immune response to infection. NO may exert either harmful or beneficial effects, which elucidates its role in many fundamental physiological and pathological processes of the body.6 In clinical practice, glyceryl trinitrate (GTN) is one of the most important NO donors used for blood pressure control. GTN has been shown to exert a potent inhibitory effect on bacterial growth, including S. aureus, in sodium chloride 0.9%, glucose 5%, ethanol 10%, and Mueller-Hinton broth.7 Peroxynitrite is a NO-derived oxidant, formed during the reaction between NO and superoxide radicals, acting as a key component of nitroxidative stress associated with pathophysiological conditions such as inflammatory or cardiovascular disorders. Peroxynitrite demonstrates very strong bactericidal potential, which magnitude depends on many complex factors of kinetics and cellular radical milieu.8 Effect of GTN on bacterial growth has neither been studied in whole blood nor during extracorporeal circulation (ECC). A wide variety of proteins, functioning either as receptors or as ligands, are expressed on the cell surfaces of leukocytes. Leukocyte regulation, activation, function, and cell–cell communication are directed by the interactions of these numerous cell surface proteins. The interactions can be long-lasting stable or transient, as seen in the immune system, where the cells bind, communicate, and then separate.9 In this study, we tested the effect of GTN, an important NO donor widely used in clinical practice, on S. aureus growth during SECC. We also examined the effect of S. aureus alone and in combination with GTN on activation markers of the innate immune system during SECC. We chose for this study a set of the Fcγ receptors known to be involved in the inflammatory processes including CD11b, CD 14, CD 16, CD 32, CD35, CD63, CD64, CD65, and CD66b.
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC 1260 Multigamma Counter (Wallac Oy, Turku, Finland). Using an automatic cell counter (Medonic CA 620 Loke 16-parameter system, Boule Diagnostics AB, Stockholm, Sweden), hemoglobin concentration, platelet, leukocyte, and leukocyte subset counts were determined. Quantitative analysis of plasma C3a and TCC was performed with a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden).
Melki et al. locyte and monocyte populations were gated and the FITCfluorescence measured. The fluorescence intensity above background (isotype control) of granulocytes and monocytes was determined and expressed as mean fluorescence intensity. In case of CD35 and CD64, expression was also given as relative number of positive cells (%), defined as the relative number of cells that expressed CD35, respectively, CD64 to a higher extent than background (isotype control).
Preparation of Leukocytes Leukocytes were prepared as previously described.11,12 Briefly, 1 mL heparinized blood was fixated with 0.4% paraformaldehyde and the erythrocytes were lysed by incubation with 0.85% (w/v) NH4Cl in Tris-HCl buffer [Tris(hydroxymethyl)aminomethane 0.01 mol/L, pH 7.4]. Finally, the cells were washed with PBS containing human serum albumin (HSA) (0.1%, w/v) and diluted to the concentration of 2.5 106/mL.
Labeling of Leukocytes with Antibodies to Cell Surface Antigens A total of 50 μL samples of the leukocyte suspension were mixed with optimally titrated fluorescein isothiocyanate (FITC)-, phycoerythrine-, or unlabeled mouse monoclonal antibodies (mab) against CD11b, CD35, CD63, CD64, CD65, CD66b (Beckman Coulter, Fullerton, California, United States), CD16, CD14, (Dako, Glostrup, Denmark), and CD32 (BectonDickinson, San Diego, California, United States) and incubated for 30 minutes at 4°C. After incubation, the cells were washed with PBS and thereafter diluted in 300 μL PBS with HSA. Leukocytes were also labeled by an identical procedure with negative isotype controls for mouse immunoglobulin G1 (IgG1) and IgG2 (Dako). After labeling, the cells were kept on ice until analysis.
Bacteriological Analysis Blood samples (5 mL) from the infected GTN and control circuits were collected at 0, 30, 60, 180, and 240 minutes. According to a standardized protocol, the samples were then diluted empirically, spread on agar plates, and incubated for 2 days at 37°C. The quantification of bacterial growth was determined by manual count of the number of colony forming unit.
Statistical Analysis Data are presented as median and range from five independent experiments. Statistical analysis was performed using nonparametric ANOVA (Kruskal–Wallis), and p values 0.05 were considered significant. All the statistical analyses were
Flow Cytometry Flow cytometric analysis was performed on an EPIC MCL-XL flow cytometer (Beckman Coulter). Identification of granulocytes was based on their forward scatter/side scatter (FSC/ SSC) dot-plot profile and of monocytes on the FSC/SSC dotplot profile and positive staining with anti-CD14. The granu-
Fig. 1 Growth of S. aureus during SECC, without ( ) and with GTN (•). The data are presented as the median of the number of S. aureus CFU/mL whole blood with range (n ¼ 5). Thoracic and Cardiovascular Surgeon
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Fig. 2 Changes in levels of leukocyte Fcγ receptors during SECC, S. aureus infested without ( ) and with GTN (•). Expression of (A) granulocyte CD32 and (B) monocyte CD32. Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5).
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC
Results GTN Has No Effect on Staphylococcus aureus Growth during SECC
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of leukocyte CD16 and CD64. Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD32.
Staphylococcus aureus affects the Expression of Key Proteins Involved in Leukocyte Adhesion
Staphylococcus aureus were introduced into parallel SECC circuits as described in methods. GTN was added to one of these parallel circuits. We performed five independent parallel SECC runs, each lasted 240 minutes. In both groups, bacterial counts were significantly reduced (p 0.05) over time. Addition of GTN did not generate any intergroup differences (►Fig. 1). To explore possible immunological mechanisms involved in the potential reductive effect of GTN on S. aureus growth, we analyzed a panel of markers of inflammatory response.
Expression of key molecules involved in cell adhesion was determined by measuring the level of CD11b (adhesion and C3bi receptor), CD65 (E-selectin ligand), and CD162 (Pselectin ligand). Addition of S. aureus induced significant increases in expression of granulocyte and monocyte CD11b, monocyte CD65, and monocyte CD162 over time (p 0.01) (►Fig. 3A–D), indicating an activation of these cells. Granulocyte expression of CD162 was though reduced over time. Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD11b, CD65, and CD162.
Staphylococcus aureus Reduces the Expression of Granulocyte Fcγ-Receptor CD32 but Stimulates the Expression of Monocyte CD32
Staphylococcus aureus Stimulates the Levels of Lipopolysaccharide Receptor, C3b Receptors, and Leukocyte Activation Marker
Staphylococcus aureus significantly reduced the expression of granulocyte CD32 and stimulated the expression of monocyte CD32 (p 0.05) over time (►Fig. 2A, B). Theoretically, the reduced expression of granulocyte CD32 may be due to an internalization of the receptor. There were no significant changes, with or without addition of GTN, for the expression
To screen for other potential effects of GTN on other components of the innate immune system, we also analyzed the levels of CD14 (lipopolysaccharide receptor), CD35 (C3b receptor), and CD66b (a marker of granulocyte activation). Addition of S. aureus induced significant increases in monocyte CD14, leukocyte CD35, and granulocyte CD66b levels
Fig. 3 Changes in expression of leukocyte key proteins, involved in cell adhesion, during SECC, S. aureus infested without () and with GTN (•). Expression of (A) granulocyte CD11b, (B) monocyte CD11b, (C) monocyte CD65, and (D) monocyte CD162. Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5). Thoracic and Cardiovascular Surgeon
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performed with the Statistica 10.0 software for Windows (StatSoft, Tulsa, Oklahoma, United States).
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC (p 0.01) over time (►Fig. 4A–D). Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD14, CD35, and CD66b.
Staphylococcus aureus Stimulates Expression of Lysosomal Granule Membrane Protein The levels of granulocyte CD63 (lysosomal granule protein) were analyzed. Addition of S. aureus resulted in a significant increase in monocyte CD63 levels (p 0.01) over time. Addition of GTN to the circuits did not generate any significant intergroup changes in the expression of CD63 (►Fig. 5).
Staphylococcus aureus Increases the Levels of TCC, C3a, and MPO Staphylococcus aureus induced a significant increase of TCC, C3a, and MPO levels (p 0.01) over time. Addition of GTN to the circuits did not generate any significant intergroup changes for TCC, C3a, and MPO.
Discussion Intravenous preparation of GTN has previously been shown to effectively reduce the growth of various bacterial strains, including S. aureus. GTN in different physiological saline solutions was bactericidal.7 In this study, though, for the first
Melki et al. time, we have shown that GTN does not affect S. aureus growth in whole blood during SECC. During our experiment, we also screened several important proteins and markers involved in the recruitment and activation of leukocytes in response to S. aureus infection, which has allowed us to address the question whether or not GTN addition may affect the activity of the innate immune system. Our study confirms that SECC with whole blood infested with S. aureus, induces an activation of both granulocytes and monocytes, as reflected by increases in the majority of measured parameters at most time points. Addition of GTN did not significantly alter expression of the investigated cell surface receptors. A weakness of this study is the low number of experiments performed, and also that the experiments were performed at only one GTN dose. The GTN dose delivered in this study was correlated to clinically established levels. We intend though to perform additional studies including longer SECC times and GTN dose response correlation. On the contrary, the hypothesis is new and, since GTN is an important NO donor widely used in clinical practice for patients with angina pectoris, pulmonary hypertension, and systemic blood pressure control, the results could be of major clinical importance, and further investigations are therefore desirable. NO has been shown to facilitate the killing of the intracellular pathogen
Fig. 4 Changes in levels of components of the innate immune system during SECC, S. aureus infested without ( ) and with GTN (•). Expression of (A) monocyte CD14, (B) granulocyte CD35, (C) monocyte CD35, and (D) granulocyte CD66b. Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5). Thoracic and Cardiovascular Surgeon
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Fig. 5 Changes in leukocyte expression of lysosomal granule membrane protein CD63 during SECC, S. aureus infested without () and with GTN (•). Data are presented as median fluorescence intensity (MFI) value with range (n ¼ 5).
Leishmania donovani, the causative agent of visceral leishmaniasis, by stimulating phagosomal maturation.13 In a wider perspective, our results may promote further studies in patients with leishmaniasis and/or other bacterial infections. One may question whether our observed changes are not only due to the time effect of SECC. To clarify this, we have performed further experiments with similar settings. Preliminary results from these unpublished experiments indicate that SECC by itself induces increased expression of leukocyte activation markers CD11b, CD14, CD35, and CD162, but seem to have no effect on CD16, CD32, CD65, CD63, and CD66b. An interesting issue is whether GTN had any effect at all on the innate immune system. Cartwright et al stated that physiologically relevant NO levels did not affect lymphocyte adhesion to endothelium and expression of proteins, such as cell adhesion molecule-1 (C-CAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin.14 ECC always leads to activation of the innate immune system as a part of systemic inflammatory response in which the excessive oxidative stress plays an important role.15 The interpretation of our results may be complicated by the fact that the ECC-induced activation of different cascade systems alters the pro- and anti-inflammatory balance of the body many times during postoperative period. In this process, NO is deeply involved, although its role is still not fully understood. NO may temporarily move this inflammatory balance in beneficial or harmful direction depending on actual status in biochemical and/ or redox milieu.16 Worth considering is also that the ability of S. aureus to develop resistance to innate immunity by S. aureus adaptive response to nitrosative stress.17 Also, other recent studies of host–pathogen interaction give us reasons to revise our understanding of the nature of S. aureus infection. The bacteria produce several factors to evade neutrophil response. These include chemotaxis inhibitory protein of staphylococci, staphylococcal complement inhibitor, clump-
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ing factor A, and extracellular adherence protein.18,19 For survival, S. aureus can in fact hide inside neutrophils.20 The very presence of granulocytes may therefore generate better S. aureus survival. In vitro studies have shown that GTN have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. GTN induces an antimitogenic effect and inhibits cytokine production and expression, cell-mediated cytotoxicity, and antibody production.21 Since our observed changes were mostly detected at the late stage of SECC and, due to the short viability of neutrophils, therefore make them difficult to relate to settings of more time prolonged ECC such as extracorporeal membrane oxygenation or dialysis, where also there is a continuous release of new neutrophils from the bone marrow. In our model, we observed a steadily decreasing number of neutrophils, probably due to a combination of adhesion to the foreign surfaces, mechanical destruction, and apoptosis. What role apoptosis of immune cells play in our settings is unclear. Therefore, it would be of interest to perform further studies to find out mechanisms behind it and elucidate potential clinical impact.
Acknowledgments This study was supported by Swedish Heart-Lung Foundation (V.M.), and Sellanders’ Family Fund and the Heart Lung Fund, Uppsala University Hospital (J.B.).
Note This study was approved by the appropriate ethics committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Conflict of Interest None.
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Glyceryl Trinitrate Effect on S. aureus Growth during SECC
Glyceryl Trinitrate Effect on S. aureus Growth during SECC
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6 Kobzik L. Translating NO biology into clinical advances: still
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