Acta Anaesthesia1 Scand 1990: 34: 241-248
Cell-mediated and humoral immune responses to total hip replacement under spinal or general anaesthesia M. SALOand M. NISSILA Departments of Anaesthesiology and Medical Microbiology, University of Turku, Turku, Finland
Regional anaesthesia has many advantages over general anaesthesia in hip surgery. When the effects of total hip replacement under spinal or general anaesthesia were compared in 22 patients, the only difference between the groups occurred in PHA-induced lymphocyte proliferative responses. I n contrast, no differences between the groups were observed in leucocyte or differential counts, lymphocyte or subtype counts, most mitogen-induced lymphocyte proliferative responses, NK-cell activity, IgG, IgM or IgA production by unstimnlated or PWM-stimulated lymphocytes, proliferative responses of control lymphocytes with 15% patient serum, or chemiluminescence values in phagocytosis of zymosan opsonized with patient serum. Thus, regional anaesthesia is indicated in total hip replacement for reasons other than the immune response. Received I October, accepted for publication 7 November 1989
Key words: Cell-mediated immunity; general anesthesia; hip prosthesis; hip replacement; humoral immunity; immune response; spinal anesthesia.
Regional anaesthesia modulates metabolic and endocrine responses to an operation (1 ) . It has also been reported to affect some immune responses during an operation (2-4), although controversial results have also been reported (5). Most of these effects have been observed in lower abdominal and lower extremity operations under epidural anaesthesia. In this study, the effects of total hip replacement on cell-mediated and humoral immunity were studied in patients undergoing operation under spinal or general anaesthesia. Effective host defences are important in these patients, since infectious complications may severely impair operative results (6).
PATIENTS AND METHODS Twenty-two patients admitted for total hip replacement were randomly divided into those operated on under spinal (the spinal anaesthesia group) or general anaesthesia (the general anaesthesia group). The groups showed no differences in the background data of the patients (Table 1j . The patients belonged to anaesthesia risk groups ASA 1 or 2. The study was approved by the joint Ethics Committee of the Turku University Medical Faculty and the University Central Hospital. The patients gave their informed consent to participate in the study. Both groups received a premedication of 0.5 mg atropine and 50 mg pethidine intramuscularly 45 min before operation. For spinal anaesthesia the patients received 4.3 f 0.4 ml ( m e a n t s.d.) ofisobaric bupivacaine hcl (Marcain" 5 mg/ml, Astra, Sodertalje, Sweden) slowly injected into the subarachnoid space using a midline approach
through the L3-L4 or L2-L3 intervertebral space with the patient lying on the non-surgical side. Cranial spread of analgesia determined by the pin-prick test 10-15 min after injection was between dermatomes T h 4 and ThlO on the surgical side. Oxygen 4 I/min was administered through a Ventimask during operation and in the recovery room. General anaesthesia was induced with fentanyl 127 f 79 pg (mean f s.d.), thiopental 260 & 80 mg and suxamethonium (10 patients) or pancuronium ( I patient). For maintenance of anaesthesia, the patients received 350 f 126 pg of fentanyl, 0.2-1 .O% v/v of enflurane and 70% nitrous oxide in oxygen. Pancuronium bromide 7.45 f 1.29 mg was used for muscle relaxation during anaesthesia. To maintain normocapnia, pulmonary ventilation was controlled by Normocap (Datex, Helsinki, Finland). During operation a Lubinus prosthesis in 20 patients and an Exeter and a Lord prosthesis, each in one patient, was inserted via the postero-lateral approach. Two patients in the spinal anaesthesia group underwent operation for loosened prosthesis, and the other patients had primary operations for osteoarthrosis. The prostheses were fixed with methylmethacrylate cement, and dicloxacillin was used for antibiotic prophylaxis. Blood loss during operation was 1300 f 830 ml in the spinal anaesthesia group and I 160 f 410 ml in the general anaesthesia group. Blood loss was replaced with crystalloid solutions, plasma expanders and blood transfusion. During operation and the recovery room period, the patients in the spinal anaesthesia group received 2600 f 380 ml of Ringer's lactate solution, 550 f 270 ml of plasma expanders and 3.5 f 1.4 units of packed red blood cells or whole blood. Patients in the general anaesthesia group received 2400 f 300 ml of Ringer's lactate solution, 640 f 230 ml of plasma expanders and 3.1 f 1.3 units of blood products. For thrombosis prophylaxis, both groups received 500 ml Macrodexm (Pharmacia, Uppsala, Sweden) on the day ofoperation and on 3 postoperative days. For postoperative pain relief, 10 mg of oxycodone was given intra-
242
M. SALO AND M. NISSILX
Table 1 Background data of patients undergoing total hip replacement under spinal or general anaesthesia. Means f s.d. No differences between groups.
No. of patients Male/female Age, years Weight, kg Length, m Duration of anaesthesia, min Duration of operation, min Haemoglobin conc., g/l - preoperative - postoperative Hospital stay, days _
*
_
_
~
~~
Spinal anaesthesia
General anaesthesia
11 1/10 65k7 69+ 12 1.59 f 0.06 187 53* 131 f 50
11 1/10 59f 12 70f 12 1.62 f 0.06 163 f 13 103k 9
135f 10 116+9 15.7 + 3.6
139 f 8 123f 16 16.3 f 4.0
~~
From injection of the local anaesthetic to the end of surgery.
muscularly as needed. No infectious or other complications were observed, except for postoperative confusion in one patient in the general anaesthesia group. Blood samples Peripheral venous blood samples were obtained preoperatively on the day of operation and on postoperative days 1 , 3-4 and 6-7 between 07.30 and 08.00 a.m. Processing of the samples was started immediately thereafter. One of the authors (MS) gave all the spinal or general anaesthesias except for one, and the same two orthopaedic surgeons did all the operations. Blood samples were processed by one laboratory nurse (PJ). Lymphocyte subpopulations were identified, and measurements of proliferative responses as well as immunoglobulin production were conducted by one author (MS), whereas natural killer cell function was measured by the other author (MN). Isolation of &nphocytes Mononuclear cells were separated on Ficoll-Isopaque (Ficoll-Paque, Pharmacia Fine Chemicals, Uppsala, Sweden) by centrifugation at 400 g for 40 min at 4°C (7). After separation the cells were washed twice with RPMI-1640 medium (Grand Island Biological Company, Grand Island, NY) containing 50 p g / d gentamicin, and finally with the same medium supplemented with 10% heat-inactivated foetal calf serum (FCS, Gibco Biocult, Glasgow, Scotland). The purity of the lymphocyte yield was evaluated, and the viability of the lymphocytes was confirmed as >95% by the trypan blue exclusion method. IdentSfication of lymphocytes and their subpopulations Leucocyte count was done on a chamber and differential count was done by counting at least 200 cells on May-Griinwald-Giemsa-stained smears. T-lymphocyte numbers were determined with monoclonal antibody OKT,, T-helperlinducer lymphocytes with OKT, and Tsuppressor/cytotoxiclymphocytes with OKT, (Ortho Diagnostic System, Raritan, NJ, USA) and natural killer cells (NK-cells) with Leu, (Becton Dickinson Monoclonal Center, Mountain View, Ca, USA) by the indirect immunofluorescence method (8) using fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Cappel Laboratories, Cochranville, PA, USA). B-lymphocytes were determined by their surface membrane Ig (sIg) using direct immunofluorescence (9). On each occasion, at least 200 cells were counted under a Leitz Orthoplan microscope. Monocytes in the preparations were identifed with latex particles (Bacto-Latex, Difco Laboratories, Detroit, Mich., USA).
Lymphocyte proliferative responses Twenty-five pl of heparinized blood (whole blood culture) or 1 x lo5 mononuclear cells (separated lymphocyte culture) were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with gentamicin 50 pg/ml and the mitogens or antigen in wells offlat-bottomed microtiter plates (A/S Nunc, Roskilde, Denmark) in humidified air and 5% CO, at 37°C for 96 h (10). The volume of each culture was 200 pl. The mitogens in cultures with whole blood were phytohaemagglutinin M (PHA, Difco) 625 pg/ml, concanavalin A (Con A, Pharmacia Fine Chemicals) 25 pg/ml and pokeweed rnitogen (PWM, Gibco) 25 pl/ml. In cultures with separated lymphocytes the mitogens were PHA 25 p l / d and PWM 5 pl/ml, and the antigen was formalinized Staphylococcus aureus strain Cowan, (StaCw, Department of Medical Microbiology, University of Turku) 0.1% vjv, all as final concentrations. Eighteen hours before harvesting, 0.25 pCi of Iz5Ilabelled 5-iodo-2'-deoxyuridine ( ' W J d R , specific activity 9&110 mCi/mg (Radiochemical Centre, Amersham, England) in 20 pI was added to each microculture together with 5-fluoro-2-deoxyuridine (FUdR, Fluka, Buchs, Switzerland) to a final concentration of mol/l. Radioactivity was measured with an automatic well-type gamma counter (LKB-Wallac, Turku, Finland) as counts per minute (cpm) of Iz5IUdRincorporation into DNA. All cultures were made in triplicate. Natural killer cell function Five x 1035'Cr-labelledtarget cells of the human erythroleukemic cell line K562 were mixed with different concentrations of mononuclear cells from patients as effector cells to obtain effector to target cell ratios 100:1, 50:1, 25:1, 12.5:1, 6.25:l and 3.125:l as described earlier (1 1). The cell suspensions were incubated in round-bottomed wells of microtiter plates (Nunc, Roskilde, Denmark) in triplicate in a volume of 200 pl at 37°C in humidified air and 5% CO, for 4 h. After incubation, the radioactivity of 100 pl of the cell-free supernatant was counted with an automatic well-type gamma counter (LKB-Wallac). Specific 5'Cr release as a measure of natural killer (NK) cell function was: [(experimentalreleasespontaneous release)/ (maximum releasespontaneous release)] x 100. Spontaneous 5'Crrelease was measured by incubating target cells with assay medium, and maximum release was measured by incubating target cells in water containing 5% sodium dodecyl sulphate (Merck, Darmstadt, West Germany). Polyclonal Ig production Five x lo5 mononuclear cells were cultured in 1 ml of RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with gentamicin 50 FCS, 0.2% sodium bicarbonate and PWM 2.5 pl/ml as pg/ml, lymphocyte activator in the absence and presence of hydrocortisone mol/l or Con A 0.75 pg/ml in 12x 75 mm round-bottomed tissue culture tubes (Falcon 2058, Falcon, Oxnard, Ca, USA) (12). A pretested lot of FCS without mitogenic activity was used throughout the study. The cultures were kept in humidified air and 15% CO, at 37°C for 14 days. After culture, supernatants were collected and their IgG, IgM and IgA concentrations were measured with the trapping antibody enzyme-linked immunosorbent assay (ELISA) (13). The supernatants from lymphocyte cultures were added in proper dilutions to wells of microtiter plates (Linbro/Titertek, Flow Laboratories Inc., Hamden, CT, USA), which had previously been coated with rabbit anti-human IgG, IgM or IgA (Dako-immunoglobulins a/s, Copenhagen, Denmark). Bound IgG, IgM and IgA were detected with peroxidase-conjugated rabbit anti-human IgG, IgM and IgA (Dako-immunoglobulins a/s), respectively. Cytophilic immunoglobulins were not removed, since their amount was below the detection limit 10 ng/ml of the method. Absorbance was measured at 492 nm with a Titertek Multiskan photometer (Eflab Oy, Helsinki, Finland). The
243
ANAESTHESIA AND IMMUNE RESPONSE
Table 2 Leucocyte and differential counts. Means f s.d. * = P< 0.05, ** = P < 0.01 compared with preoperative values. No differences between groups.
Leucocytes x i09/l Neutrophils
spinal general spinal general spinal general spinal general spinal general spinal general
yo
Eosinophils ’-’/, Basophils
yo
Lymphocytes o/n Monocytes yo
Postoperative day
Preoperative day
1
5.1 f 1.3 6.2 _+ 1.2 55.5 f 11.7 59.9 5 8.3 3.5 f 2.6 3.4 i 2.8 0.2 +_ 0.5 0.0 2 0.0 39.5 k 11.3 35.7 k 9.7 1.2 2 0.8 1.1 f 0 . 8
8.1 f 2.4** 7.82 2.2** 73.0 2 9.8** 74.6 f 9.2** 0.7 f 0.8** 0.5 0.7** 0.1 f 0.2 0.0 f 0.0 2 5 . 5 8.8** ~ 23.5 f 9.4** 0.6 2 0.8 1 . 5 1.4 ~
amount of class-specific Ig was determined from standard curves of samples with known amounts of Ig (Behringwerke AG, Marburg, West Germany). All tests were done in duplicate.
Effects of serum factors Lymphogte prolijieratiue responses. One x lo5 mononuclear cells from healthy controls were cultured with 15% patient serum as described above. The mitogens were PHA 25 pg/ml, Con A 5 pg/ml and PWM 5 pl/ml as final concentrations. The test were done in triplicate. Chemilumznescence in phagoqtosis of zymosan opsonized with patient serum. Zymosan (Sigma Chemical Co., St. Louis, Missouri, USA) boiled in phosphated buffered saline (PBS) in a concentration of 20 mg/ml for 30 min was opsonized by incubating it with patient serum 1:100 at 37°C for 30 min. In the chemiluminescence assay (14), I x lo6 granulocytes (0.5 ml suspension) isolated from healthy donors by Percoll density gradient centrifugation (15) and incubated at 37°C for 30 min were incubated with luminol (Sigma) at 37°C for 30 min. The suspension was added to vials containing 100 p1 of opsonized zymosan or HBSS as blank solution. Chemiluminescence emission was measured at 37°C in millivolts with a Luminometer 1251 (Wallac) at 5-min intervals for 70 min. The results were read as peak activity. Each test was done in duplicate. Cortisol concentration Serum cortisol concentrations were measured by using a radioimmunoassay kit (IZ5I)from Farmos Diagnostica (Turku, Finland).
3-1 6.2 f 2.6 6.6 f 2.0 68.2 f 6.8** 71.2f 11.0** 3.9 2.3 4.5 f 1.9 0.2 f 0.3 0.1 kO.2 26.2 f 5.7** 23.2 f 9.8** 1.320.8 1.020.9
6-7 6.6 5 1.6 5.3f 1.7 60.9 f 6.8 62.5 f 9.0 4.4 f 3.0 5.0 f 3.4 0.0 f 0.2 0.1 k0.2 32.8 2 6.0* 31.1 f 7.3* 1.8f 1.1 1.3f0.6
Statistics The repeated measures analysis of variance was used to detect overall changes between measurements. If a significant overall effect occurred, individual events at different time points were compared with Dunnett’s test. Student’s t-test was used to compare patient background data and preoperative values between the groups. Logarithmic transformation was used to normalise the distribution of lymphocyte proliferative responses and immunoglobulin values.
RESULTS After total hip replacement, leucocytosis and increased percentages of neutrophils and decreased percentages of eosinophils and lymphocytes were observed (P
Cell-mediated and humoral immune responses to total hip replacement under spinal or general anaesthesia M. SALOand M. NISSILA Departments of Anaesthesiology and Medical Microbiology, University of Turku, Turku, Finland
Regional anaesthesia has many advantages over general anaesthesia in hip surgery. When the effects of total hip replacement under spinal or general anaesthesia were compared in 22 patients, the only difference between the groups occurred in PHA-induced lymphocyte proliferative responses. I n contrast, no differences between the groups were observed in leucocyte or differential counts, lymphocyte or subtype counts, most mitogen-induced lymphocyte proliferative responses, NK-cell activity, IgG, IgM or IgA production by unstimnlated or PWM-stimulated lymphocytes, proliferative responses of control lymphocytes with 15% patient serum, or chemiluminescence values in phagocytosis of zymosan opsonized with patient serum. Thus, regional anaesthesia is indicated in total hip replacement for reasons other than the immune response. Received I October, accepted for publication 7 November 1989
Key words: Cell-mediated immunity; general anesthesia; hip prosthesis; hip replacement; humoral immunity; immune response; spinal anesthesia.
Regional anaesthesia modulates metabolic and endocrine responses to an operation (1 ) . It has also been reported to affect some immune responses during an operation (2-4), although controversial results have also been reported (5). Most of these effects have been observed in lower abdominal and lower extremity operations under epidural anaesthesia. In this study, the effects of total hip replacement on cell-mediated and humoral immunity were studied in patients undergoing operation under spinal or general anaesthesia. Effective host defences are important in these patients, since infectious complications may severely impair operative results (6).
PATIENTS AND METHODS Twenty-two patients admitted for total hip replacement were randomly divided into those operated on under spinal (the spinal anaesthesia group) or general anaesthesia (the general anaesthesia group). The groups showed no differences in the background data of the patients (Table 1j . The patients belonged to anaesthesia risk groups ASA 1 or 2. The study was approved by the joint Ethics Committee of the Turku University Medical Faculty and the University Central Hospital. The patients gave their informed consent to participate in the study. Both groups received a premedication of 0.5 mg atropine and 50 mg pethidine intramuscularly 45 min before operation. For spinal anaesthesia the patients received 4.3 f 0.4 ml ( m e a n t s.d.) ofisobaric bupivacaine hcl (Marcain" 5 mg/ml, Astra, Sodertalje, Sweden) slowly injected into the subarachnoid space using a midline approach
through the L3-L4 or L2-L3 intervertebral space with the patient lying on the non-surgical side. Cranial spread of analgesia determined by the pin-prick test 10-15 min after injection was between dermatomes T h 4 and ThlO on the surgical side. Oxygen 4 I/min was administered through a Ventimask during operation and in the recovery room. General anaesthesia was induced with fentanyl 127 f 79 pg (mean f s.d.), thiopental 260 & 80 mg and suxamethonium (10 patients) or pancuronium ( I patient). For maintenance of anaesthesia, the patients received 350 f 126 pg of fentanyl, 0.2-1 .O% v/v of enflurane and 70% nitrous oxide in oxygen. Pancuronium bromide 7.45 f 1.29 mg was used for muscle relaxation during anaesthesia. To maintain normocapnia, pulmonary ventilation was controlled by Normocap (Datex, Helsinki, Finland). During operation a Lubinus prosthesis in 20 patients and an Exeter and a Lord prosthesis, each in one patient, was inserted via the postero-lateral approach. Two patients in the spinal anaesthesia group underwent operation for loosened prosthesis, and the other patients had primary operations for osteoarthrosis. The prostheses were fixed with methylmethacrylate cement, and dicloxacillin was used for antibiotic prophylaxis. Blood loss during operation was 1300 f 830 ml in the spinal anaesthesia group and I 160 f 410 ml in the general anaesthesia group. Blood loss was replaced with crystalloid solutions, plasma expanders and blood transfusion. During operation and the recovery room period, the patients in the spinal anaesthesia group received 2600 f 380 ml of Ringer's lactate solution, 550 f 270 ml of plasma expanders and 3.5 f 1.4 units of packed red blood cells or whole blood. Patients in the general anaesthesia group received 2400 f 300 ml of Ringer's lactate solution, 640 f 230 ml of plasma expanders and 3.1 f 1.3 units of blood products. For thrombosis prophylaxis, both groups received 500 ml Macrodexm (Pharmacia, Uppsala, Sweden) on the day ofoperation and on 3 postoperative days. For postoperative pain relief, 10 mg of oxycodone was given intra-
242
M. SALO AND M. NISSILX
Table 1 Background data of patients undergoing total hip replacement under spinal or general anaesthesia. Means f s.d. No differences between groups.
No. of patients Male/female Age, years Weight, kg Length, m Duration of anaesthesia, min Duration of operation, min Haemoglobin conc., g/l - preoperative - postoperative Hospital stay, days _
*
_
_
~
~~
Spinal anaesthesia
General anaesthesia
11 1/10 65k7 69+ 12 1.59 f 0.06 187 53* 131 f 50
11 1/10 59f 12 70f 12 1.62 f 0.06 163 f 13 103k 9
135f 10 116+9 15.7 + 3.6
139 f 8 123f 16 16.3 f 4.0
~~
From injection of the local anaesthetic to the end of surgery.
muscularly as needed. No infectious or other complications were observed, except for postoperative confusion in one patient in the general anaesthesia group. Blood samples Peripheral venous blood samples were obtained preoperatively on the day of operation and on postoperative days 1 , 3-4 and 6-7 between 07.30 and 08.00 a.m. Processing of the samples was started immediately thereafter. One of the authors (MS) gave all the spinal or general anaesthesias except for one, and the same two orthopaedic surgeons did all the operations. Blood samples were processed by one laboratory nurse (PJ). Lymphocyte subpopulations were identified, and measurements of proliferative responses as well as immunoglobulin production were conducted by one author (MS), whereas natural killer cell function was measured by the other author (MN). Isolation of &nphocytes Mononuclear cells were separated on Ficoll-Isopaque (Ficoll-Paque, Pharmacia Fine Chemicals, Uppsala, Sweden) by centrifugation at 400 g for 40 min at 4°C (7). After separation the cells were washed twice with RPMI-1640 medium (Grand Island Biological Company, Grand Island, NY) containing 50 p g / d gentamicin, and finally with the same medium supplemented with 10% heat-inactivated foetal calf serum (FCS, Gibco Biocult, Glasgow, Scotland). The purity of the lymphocyte yield was evaluated, and the viability of the lymphocytes was confirmed as >95% by the trypan blue exclusion method. IdentSfication of lymphocytes and their subpopulations Leucocyte count was done on a chamber and differential count was done by counting at least 200 cells on May-Griinwald-Giemsa-stained smears. T-lymphocyte numbers were determined with monoclonal antibody OKT,, T-helperlinducer lymphocytes with OKT, and Tsuppressor/cytotoxiclymphocytes with OKT, (Ortho Diagnostic System, Raritan, NJ, USA) and natural killer cells (NK-cells) with Leu, (Becton Dickinson Monoclonal Center, Mountain View, Ca, USA) by the indirect immunofluorescence method (8) using fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Cappel Laboratories, Cochranville, PA, USA). B-lymphocytes were determined by their surface membrane Ig (sIg) using direct immunofluorescence (9). On each occasion, at least 200 cells were counted under a Leitz Orthoplan microscope. Monocytes in the preparations were identifed with latex particles (Bacto-Latex, Difco Laboratories, Detroit, Mich., USA).
Lymphocyte proliferative responses Twenty-five pl of heparinized blood (whole blood culture) or 1 x lo5 mononuclear cells (separated lymphocyte culture) were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with gentamicin 50 pg/ml and the mitogens or antigen in wells offlat-bottomed microtiter plates (A/S Nunc, Roskilde, Denmark) in humidified air and 5% CO, at 37°C for 96 h (10). The volume of each culture was 200 pl. The mitogens in cultures with whole blood were phytohaemagglutinin M (PHA, Difco) 625 pg/ml, concanavalin A (Con A, Pharmacia Fine Chemicals) 25 pg/ml and pokeweed rnitogen (PWM, Gibco) 25 pl/ml. In cultures with separated lymphocytes the mitogens were PHA 25 p l / d and PWM 5 pl/ml, and the antigen was formalinized Staphylococcus aureus strain Cowan, (StaCw, Department of Medical Microbiology, University of Turku) 0.1% vjv, all as final concentrations. Eighteen hours before harvesting, 0.25 pCi of Iz5Ilabelled 5-iodo-2'-deoxyuridine ( ' W J d R , specific activity 9&110 mCi/mg (Radiochemical Centre, Amersham, England) in 20 pI was added to each microculture together with 5-fluoro-2-deoxyuridine (FUdR, Fluka, Buchs, Switzerland) to a final concentration of mol/l. Radioactivity was measured with an automatic well-type gamma counter (LKB-Wallac, Turku, Finland) as counts per minute (cpm) of Iz5IUdRincorporation into DNA. All cultures were made in triplicate. Natural killer cell function Five x 1035'Cr-labelledtarget cells of the human erythroleukemic cell line K562 were mixed with different concentrations of mononuclear cells from patients as effector cells to obtain effector to target cell ratios 100:1, 50:1, 25:1, 12.5:1, 6.25:l and 3.125:l as described earlier (1 1). The cell suspensions were incubated in round-bottomed wells of microtiter plates (Nunc, Roskilde, Denmark) in triplicate in a volume of 200 pl at 37°C in humidified air and 5% CO, for 4 h. After incubation, the radioactivity of 100 pl of the cell-free supernatant was counted with an automatic well-type gamma counter (LKB-Wallac). Specific 5'Cr release as a measure of natural killer (NK) cell function was: [(experimentalreleasespontaneous release)/ (maximum releasespontaneous release)] x 100. Spontaneous 5'Crrelease was measured by incubating target cells with assay medium, and maximum release was measured by incubating target cells in water containing 5% sodium dodecyl sulphate (Merck, Darmstadt, West Germany). Polyclonal Ig production Five x lo5 mononuclear cells were cultured in 1 ml of RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with gentamicin 50 FCS, 0.2% sodium bicarbonate and PWM 2.5 pl/ml as pg/ml, lymphocyte activator in the absence and presence of hydrocortisone mol/l or Con A 0.75 pg/ml in 12x 75 mm round-bottomed tissue culture tubes (Falcon 2058, Falcon, Oxnard, Ca, USA) (12). A pretested lot of FCS without mitogenic activity was used throughout the study. The cultures were kept in humidified air and 15% CO, at 37°C for 14 days. After culture, supernatants were collected and their IgG, IgM and IgA concentrations were measured with the trapping antibody enzyme-linked immunosorbent assay (ELISA) (13). The supernatants from lymphocyte cultures were added in proper dilutions to wells of microtiter plates (Linbro/Titertek, Flow Laboratories Inc., Hamden, CT, USA), which had previously been coated with rabbit anti-human IgG, IgM or IgA (Dako-immunoglobulins a/s, Copenhagen, Denmark). Bound IgG, IgM and IgA were detected with peroxidase-conjugated rabbit anti-human IgG, IgM and IgA (Dako-immunoglobulins a/s), respectively. Cytophilic immunoglobulins were not removed, since their amount was below the detection limit 10 ng/ml of the method. Absorbance was measured at 492 nm with a Titertek Multiskan photometer (Eflab Oy, Helsinki, Finland). The
243
ANAESTHESIA AND IMMUNE RESPONSE
Table 2 Leucocyte and differential counts. Means f s.d. * = P< 0.05, ** = P < 0.01 compared with preoperative values. No differences between groups.
Leucocytes x i09/l Neutrophils
spinal general spinal general spinal general spinal general spinal general spinal general
yo
Eosinophils ’-’/, Basophils
yo
Lymphocytes o/n Monocytes yo
Postoperative day
Preoperative day
1
5.1 f 1.3 6.2 _+ 1.2 55.5 f 11.7 59.9 5 8.3 3.5 f 2.6 3.4 i 2.8 0.2 +_ 0.5 0.0 2 0.0 39.5 k 11.3 35.7 k 9.7 1.2 2 0.8 1.1 f 0 . 8
8.1 f 2.4** 7.82 2.2** 73.0 2 9.8** 74.6 f 9.2** 0.7 f 0.8** 0.5 0.7** 0.1 f 0.2 0.0 f 0.0 2 5 . 5 8.8** ~ 23.5 f 9.4** 0.6 2 0.8 1 . 5 1.4 ~
amount of class-specific Ig was determined from standard curves of samples with known amounts of Ig (Behringwerke AG, Marburg, West Germany). All tests were done in duplicate.
Effects of serum factors Lymphogte prolijieratiue responses. One x lo5 mononuclear cells from healthy controls were cultured with 15% patient serum as described above. The mitogens were PHA 25 pg/ml, Con A 5 pg/ml and PWM 5 pl/ml as final concentrations. The test were done in triplicate. Chemilumznescence in phagoqtosis of zymosan opsonized with patient serum. Zymosan (Sigma Chemical Co., St. Louis, Missouri, USA) boiled in phosphated buffered saline (PBS) in a concentration of 20 mg/ml for 30 min was opsonized by incubating it with patient serum 1:100 at 37°C for 30 min. In the chemiluminescence assay (14), I x lo6 granulocytes (0.5 ml suspension) isolated from healthy donors by Percoll density gradient centrifugation (15) and incubated at 37°C for 30 min were incubated with luminol (Sigma) at 37°C for 30 min. The suspension was added to vials containing 100 p1 of opsonized zymosan or HBSS as blank solution. Chemiluminescence emission was measured at 37°C in millivolts with a Luminometer 1251 (Wallac) at 5-min intervals for 70 min. The results were read as peak activity. Each test was done in duplicate. Cortisol concentration Serum cortisol concentrations were measured by using a radioimmunoassay kit (IZ5I)from Farmos Diagnostica (Turku, Finland).
3-1 6.2 f 2.6 6.6 f 2.0 68.2 f 6.8** 71.2f 11.0** 3.9 2.3 4.5 f 1.9 0.2 f 0.3 0.1 kO.2 26.2 f 5.7** 23.2 f 9.8** 1.320.8 1.020.9
6-7 6.6 5 1.6 5.3f 1.7 60.9 f 6.8 62.5 f 9.0 4.4 f 3.0 5.0 f 3.4 0.0 f 0.2 0.1 k0.2 32.8 2 6.0* 31.1 f 7.3* 1.8f 1.1 1.3f0.6
Statistics The repeated measures analysis of variance was used to detect overall changes between measurements. If a significant overall effect occurred, individual events at different time points were compared with Dunnett’s test. Student’s t-test was used to compare patient background data and preoperative values between the groups. Logarithmic transformation was used to normalise the distribution of lymphocyte proliferative responses and immunoglobulin values.
RESULTS After total hip replacement, leucocytosis and increased percentages of neutrophils and decreased percentages of eosinophils and lymphocytes were observed (P
