J. PERTTILA, O.-P. LEHTONEN, M. SALO AND R. TERTTI. Many host defence mechanisms are affected by open-heart surgery. For example, cell-mediated.
Br. J. Anaesth. (1986), 58, 1027-1030
EFFECTS OF CORONARY BYPASS SURGERY UNDER HIGH-DOSE FENTANYL ANAESTHESIA ON GRANULOCYTE CHEMILUMINESCENCE J. PERTTILA, O.-P. LEHTONEN, M. SALO AND R. TERTTI Many host defence mechanisms are affected by open-heart surgery. For example, cell-mediated (Roth et al., 1981) and humoral immunity (Eskola et al., 1984) and natural killer cell activity (Ryhanen, Huttunen and Ilonen, 1984) are depressed. In addition, complement activation has been documented (Haslam, Townsend and Branthwaite, 1980). However, the most important single defence mechanism against invading bacterial infections is phagocytosis with intracellular killing. Measurement of chemiluminescence is a new, rapid method for the evaluation of bactericidal related oxidative mechanisms of phagocytically activated granulocytes (Horan, English and McPherson, 1982). The present study evaluated the changes in granulocyte chemiluminescence activity against zymosan, 5. aureus, E. coli and N-formylmethionyl-leucylphenylalanine (FMLP) in patients after coronary artery bypass surgery. FMLP is a synthetic tripeptide, an analogue of bacterial chemotactic factors (Snyderman and Goetzl, 1981). Zymosan is a suspension of yeast particles. S. aureus and E. coli were chosen because they are responsible for the majority of the postoperative infections in our hospital. PATIENTS AND METHODS
The study was undertaken in 12 patients admitted for elective coronary bypass surgery involving extracorporeal circulation (mean ± S D age 51 ± 6 yr, weight 77 ± 9 kg and height 170 ± 8 cm). No patient had a history of malignancy, and none was receiving immunosuppressive drugs. The J. PERTTIIA. MJ>., M. SALO, M.D. (Department of Anaesthesiology); O.-P. LEHTONBN, MJJ., R. TERTTI, MJJ. (Department
of Medical Microbiology); University of Turku, SF-20520 Turku, Finland.
SUMMARY Phagocytosis provides one of the body's first-line defences against invading bacteria. The present study evaluated granulocyte microbicidal-related oxidative mechanisms by measurement of chemiluminescence responses in the phagocytosis of zymosan, S. aureus, E. coli and N-formylmethionyl-leucylphenylalanine (FMLP) in 12 patients undergoing coronary bypass surgery under high-dose fentanyl anaesthesia. With preoperative values as a baseline, a significant depression of in vitro responses to zymosan was seen on days 1, 3-4 after operation and to S. aureus and E. coli on days 3-4 after operation, with recovery by days 6-7. Responses to FMLP were unaffected. operation was performed under high-dose fentanyl anaesthesia (induction dose of fentanyl 75 ug kg"1, total dose during operation 108 ± 6 jig kg"1) with 40 % oxygen in air. Surgery was performed under moderate (30 °C) generalized hypothermia. The heart-lung machine with a bubble oxygenator was primed with 1500 ml of crystalloid solution, 1000 ml of gelatin solution and 3-4 units of packed red blood cells. The flow rate was 2.4 litre min'Ym 1 body surface area. Patients received cefuroxime 1.5 g i.v. during the surgical procedure, and 750 mg three times daily for the 3 days following operation. As antithrombotic drugs, acetylsalicylic acid 250 mg once daily and dipyridamole 75 mg three times daily were given before and after the operation. The mean duration of surgery was 328 ± 59 min, and of extracorporeal circulation was 116 ± 26 min. After operation the lungs of all patients were ventilated artificially until the following morning. One patient with an urinary catheter had an urinary tract infection (S. aureus), which was
1028 recognized 5 days after operation. No other complications were observed. Blood samples Samples of heparinized peripheral blood were taken between 7.30 and 8.00 a.m. on the day before and on the day of operation, and on days 1, 3 or 4 and 6 or 7 after operation. The samples were processed and measured immediately. At least 200 cells were counted for leucocyte and differential counts, using May—Grunwald— Giemsa-stained smears. Isolation of granulocytes Granulocytes were isolated by a modification of the one-step. Percoll density gradient centrifugation method (Hjorth, Jonsson and Vretblad, 1981). An isotonic Percoll stock solution was made by diluting 9 vol of Percoll (Pharmacia, Fine Chemicals, Uppsala, Sweden) with 1 vol of NaCl 1.5 mol litre-1. The 74% and 55% (v/v) Percoll solutions were made by diluting the isotonic stock solution appropriately with NaCl 0.15 mol litre"1. Heparinized blood 4 ml was carefully layered on top of the discontinuous Percoll gradient prepared with 55 % isotonic Percoll 4 ml and, under it, 74% isotonic Percoll 4 ml. The gradients were centrifuged at 350 x^ for 20 min at 4 °C. A clear band of granulocytes formed in the lower part of the gradient just above the band of erythrocytes. The cells were removed carefully with a Pasteur pipette, washed twice with Hanks' balanced salt solution without phenol red (HBSS, Flow laboratories Ltd, Irvine, Scotland) with opsonizing plasma added. Finally, the granulocytes were resuspended to the final concentration of 2 x 10* cells ml"1. The purity of the granulocyte pool was evaluated on spread smears stained with MayGriinwald—Giemsa stain. The viability of the cells (over 95%) was confirmed with the trypan blue exclusion method.
BRITISH JOURNAL OF ANAESTHESIA was boiled for 30 min. It was then opsonized by incubating it with opsonizing plasma at 37 °C for 30 min. After washing, zymosan was centrifuged and resuspended in PBS to a concentration of 20 mg ml"1. Preparation of FMLP Chemotactic peptide, N-formylmethionylleucylphenylalanine (FMLP, Sigma Chemical Co.) was dissolved in dimethylsulphoxide (Sigma Chemical Co.) and diluted with HBSS to 7 x 10"' mol litre-1. Preparation of bacteria Staphylococcus aureus (strain 25923, American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A.) and Escherichia coli (strain 25922, ATCC) bacteria were grown for 6 h in brain-heart infusion broth (Difco, Detroit, U.S.A.). The bacteria were then harvested in tryptone soya broth (Difco) containing 5% dimethylsulphoxide and stored in small aliquots at — 70 °C. After thawing, the amounts of viable bacteria were measured by the limiting dilution method described by Koch (1981). The suspension was washed three times with HBSS and its concentration adjusted to 1 x 10* bacteria ml"1 with HBSS.
Chemiluminescence assay To each test vial, 0.5 ml of granulocyte suspension (1 x 10s cells) in 1.4% opsonizing plasma was added and the mixture was incubated for 30 min at 37 °C. After incubation, 100 (j.1 of luminol 0.001 mol litre"1 (Sigma Chemical Co.) was pipetted and the solution was incubated for 30 min at 37 °C. Immediately before measurement, 100 ul of the microorganisms (10' bacteria), zymosan, FMLP or HBSS as blank solution was added to the appropriate vials. The vials were placed immediately in the light-proof chamber of Luminometer 1251 (LKB Wallac, Finland) and Opsonizing plasma measurements started. Chemiluminescence in Plasma from a blood group O Rh-negative FMLP vials was measured at 10-s intervals and in healthy person without any medication was other vials at 5-min intervals for 70 min. The collected to opsonize zymosan and the micro- resulting light output was measured in millivolts organisms. The plasma was stored at — 70 °C in (mV). The results were read at peak activity, which was found to be comparable to the area 2 ml volumes until required. under the chemiluminescence curve. Preparation of opsonized zymosan Zymosan (Sigma Chemical Co., St Louis, Statistical analysis Missouri, U.S.A.) suspended in phosphate buffChanges in leucocyte and granulocyte counts, ered saline (PBS) to a concentration of 20 mg ml"1 and in chemiluminescence values, were studied by
GRANULOCYTE CHEMILUMINESCENCE AFTER BYPASS SURGERY
1029
the analysis of variance for repeated measurements blood transfusion; most of these can affect the and by Dunnett's test. Logarithmic transformation function of granulocytes. Indeed the granulocyte was used for normalization of the distribution of chemiluminescence responses to zymosan, 5. chemiluminescence values. aureus and E. coli were depressed in this study following surgery. Depression of the metabolic activity of granulocytes has also been shown by RESULTS the nitroblue tetrazolium dye test during and after Leucocyte and granulocyte counts were increased anaesthesia and surgery (Bardosi and Tekeres, on the first day after operation (P < 0.05) and on 1985), and depressed chemiluminescence resdays 6-7 (P < 0.01), but not on days 3-4 (table I). ponses have also been observed after general The chemiluminescence responses of the granu- anaesthesia (Lippa et al., 1983). However, since locytes to zymosan were decreased on day 1 the most marked depression of chemiluminescence (P < 0.01) and on days 3-4 (P < 0.01); and those in this study occurred on days 3 and 4 of the to 5. aureus (P < 0.01) and E. coli (P < 0.05) were postoperative period, anaesthesia could not have decreased on days 3-4 (table II). The values had been the only cause. returned to preoperative values by days 6—7 in the Of the drugs received by the patients, antipostoperative period. No changes were seen in inflammatory agents are able to inhibit granulocyte chemiluminescence values for responses to chemiluminescence (Van Dyke et al., 1979). FMLP or in unstimulated controls, nor were any However, the dose of acetylsalicylic acid was low differences observed between the two preoperative and medication continued throughout the period values in any of the responses. of observation. Although the patients received prophylactic cefuroxime, cephalotin has been the only cephalosporin shown to inhibit chemiluminDISCUSSION escence activity at high plasma concentrations A coronary bypass operation involving extracorp- (Welch, Davis and Thrupp, 1981; Milatovic, oreal circulation is a procedure characterized by 1983). Any effect of serum suppressive factors extensive surgical trauma, mechanical manipula- such as those found in septic patients (Lanser et tion of blood, deep anaesthesia, hypothermia and al., 1985) is also unlikely because, in our study, the granulocytes were washed several times during TABLE I. Peripheral blood leucocyte and granulocyte counts harvesting and we always used the same opson(means ± SD) before and after coronary bypass operation. izing plasma. One explanation might be a lack of *P < 0.05; **P < 0.01, compared vrith values 1 day before some opsonic receptors on the surfaces of granulooperation cytes, since different opsonic receptors (Fc and C3b receptors) are needed for phagocytosis of Leucocyte Granulocyte zymosan, 5. aureus and E. coli (Hed and Stendahl, count count (xlO* (xl0» 1982; Horwitz, 1982). Another explanation may litre"1) litre"') be a discharge of myeloperoxidase-containing granules and exhaustion of the granulocytes, 1 day before op. 7.42±2.15 4.59 ±1.81 because phagocytosis-induced and luminol-enOp.day, before op. 7.93 ±2.43 5.46 ±2.09 After operation hanced chemiluminescence depends on a myelo1 day 9.78 ±2.34* 8.28 ±2.09** peroxidase-mediated reaction of the granulocytes 3-4 days 6.90 ±1.46 5.61 ±1.37 (De Chatelet et al., 1982). Indeed, such granulo6-7 days 11.13±3.17** 7.60±2.40** cyte degranulation has been found after surgery TABLE II. Chemiluminescence responses of granulocytes in phagocytosis of zymosan, S. aureus, E. coli and FMLP ("log means (mV)±SD). *P < 0.05; **P < 0.01, compared with values 1 day before operation
1 day before op. Op.day, before op. After operation 1 day 3-^1 days 6-7dayi
Zymosan
5. aureus
3.29±0.17 3.26 ±0.14
3.04 ±0.20 2.99 ±0.23
3.05 ±0.23** 2.97 ±0.31** 3.12±0.19
2.77 ±0.44 2.85 ±0.54 2.90±037 1.26 ±0.40 2.60±0.41** 2.68 ±0.52* 2.76 ±0.43 .32 ±0.32 2.75 ±0.31 2.85 ±0.40 2.63 ±0.26 .31 ±0.32
E. coli
FMLP
Control
3.11 ±0.30 2.85±0.21 L .35 ±0.21 3.07 ±0.29 2.93 ±0.28 l.36±0.41
1030 (El-Maallem and Fletcher, 1981; Davies, Sheppard and Fletcher, 1983). Responses to FMLP, the chemoattractant, were unaffected in the present study. Granulocytes have specific high-affinity receptors for FMLP-like molecules (Snyderman and Goetzl, 1981). Since the chemiluminescence-producing chemoattractant response is mediated by proteins on the surface of the granulocyte cell (Allfred and Hill, 1978), their activity seems to remain unaffected following surgery. However, there are some reports of depressed chemotaxis after surgery and anaesthesia (Stanley et al., 1976; Wandall, 1982). The depression of granulocyte chemiluminescence activity that occurred in this study after coronary bypass surgery reflects a disturbance in granulocyte microbicidal functions. This depression might contribute to postoperative infection, especially in critically ill patients. Such an infection was not, however, observed in any of the patients studied. The postoperative granulocytosis possibly compensated for the disturbance in granulocyte function. ACKNOWLEDGEMENTS We wish to thank Mrs Marjo-Riitta Terasjarvi for expert technical assistance. This study was supported by a grant from the University of Turku (M.S.).
REFERENCES Allfred, C , and Hill, H. (1978). Effect of chemoattractants on chemiluminescence. Infect. Immun., 19, 833. Bardosi, L., and Tekeres, M. (1985). Impaired metabolic activity of phagocytk cells after anaesthesia and surgery. Br. J. Anaesth., 57, 520. Davies, J., Sheppard, K., and Fletcher, J. (1983). The effects of surgery on the activity of neutrophil granule proteins. Br. J. Haematol., 53, 5. De Chatelet, L., Long, G., Shirley, P., Bass, D., Thomas, M., Henderson, F., and Cohen, M. (1982). Mechanism of the luminol-dependent chemiluminescence of human neutrophils. J. Immunol, 129, 1589. El-Maallem, H., and Fletcher, J. (1981). Effects of surgery on neutrophil granulocyte function. Infect. Immun., 32, 38. Eskola, J., Salo, M., Viljanen, M. K., and Ruuskanen, O. (1984). Impaired B lymphocyte function during open-heart surgery. Br. J. Anatsth., 56, 333.
BRITISH JOURNAL OF ANAESTHESIA Haslam, P. L., Townsend, P. J., and Branthwaite, M. A. (1980). Complement activation during pulmonary bypass. Anaesthesia, 35, 22. Hed, J., and Stendahl, O. (1982). Differences in the ingestion mechanisms of IgG and C3b particles in phagocytosis by neutrophils. Immunology, 45, 727. Hjorth, R., Jonsson, A.-K., and Vretblad, P. (1981). A rapid method for purification of human granulocytes using Percoll. A comparison with dextran sedimentation. J. Immunol. Meth., 43, 95. Horan, T. D., English, D., and McPherson, T. A. (1982). Association of neutrophil chemiluminescence with microbicidal activity. Ctin. Immunol. Immunopathol., 22, 259. Horwitz, M. A. (1982). Phagocytosis of microorganisms. Rev. Infect. Dis., 4, 104. Koch, A. L. (1981). Growth measurement; in Manual of Methods for General Bacteriology, p. 188. Washington DC: American Society for Microbiology. Lanser, M. E., Mao, P., Brown, G., Coleman, B., and Siegel, J. H. (1985). Serum-mediated depression of neutrophil chemiluminescence following blunt trauma. Arm. Surg., 202,111. Lippa, S., De Sole, P., Meucci, E., Littarru, G. P., De Francisci, G., and Magalini, S. I. (1983). Effect of general anesthetics on human granulocyte chemiluminescence. Experientia, 39, 1386. Milatovic, D. (1983). Antibiotics and phagocytosis. Review. Eur. J. Clin. Microbiol., 2, 414. Roth, J. A., Golub, S. H., Cukingnan, R. A., Brazier, J., and Morton, D. L. (1981). Cell-mediated immunity is depressed following cardiopulmonary bypass. Am. Thorac. Surg., 31, 350. Ryhanen, P., Huttunen, K., and Ilonen, J. (1984). Natural killer cell activity after open-heart surgery. Acta Anaesthesiol. Scand., 28, 490. Snyderman, R., and Goetzl, E. J. (1981). Molecular and cellular mechanisms of leukocyte chemotaxis. Science, 213, 830. Stanley, T., Hill, G., Portas, M., Hogan, N., and Hill, H. (1976). Neutrophil chemotaxis during and after general anesthesia and operation. Anesth. Analg., 55, 668. Van Dyke, K., Van Dyke, C , Udeinya, J., Blister, C , and Wilson, M. (1979). A new screening system for nonsteroidal anti-inflammatory drugs based upon inhibition of chemiluminescence produced from human cells (granulocytes). Clin. Chem., 25, 1655. Wandall, J. (1982). Leucocyte mobilization and function in vitro of blood and exudative leucocytes after inguinal hemiotomy. Br. J. Surg., 69, 669. Welch, W. D., Davis, D., and Thrupp, L. D. (1981). Effect of antimicrobial agents on human polymorphonudear leukocyte microbicidal function. Anwmcrob. Agents Chemother., 20, 15.
