Immunology 1977 32 609
The effects of halothane anaesthesia on antibody-dependent cellular cytotoxicity in rats
B. M. VO SE &
I.
KI M BER Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
Received 11 March 1976; acceptedfor publication 5 August 1976
Summary. Lymphocytes from rats killed while anaesthetized with 1 5 per cent halothane showed a significantly reduced capacity to induce lysis of antibody-coated target cells compared with those from untreated rats. This effect was short-lived however, being no longer apparent in lymphoid cells taken from rats 2 h after their recovery from such anaesthesia. Surgical procedures were not effective in extending the duration of reduced ADCC activity. The implication of these findings is that the post-operative depression of this in vitro assay of immunocompetence results from the influence of the anaesthetic agent per se or its metabolites with surgical trauma having little r6le.
reaction (ADCC) are particularly susceptible to this depression in patients undergoing surgery (Vose & Moudgil, 1976). It is difficult in clinical studies to separate the effects of duration and depth of anaesthesia, surgical trauma and the degrees of tissue damage and stress each of which correlates with post-surgical depression of immunocompetence (Berenbaum, Fluck & Hurst, 1973; Cullen & van Belle, 1975). The objective of this present study was to isolate, in an animal model, the effects of surgery and anaesthesia in order to clarify their respective r6les in the post-operatively reduced capacity of leucocytes to induce lysis of antibody-coated target cells in the ADCC assay.
INTRODUCTION
MATERIALS AND METHODS
Leucocytes taken in the immediate post-operative period from patients receiving surgery under general anaesthesia have a diminished response to phytohaemagglutinin (PHA) (Riddle & Berenbaum, 1967), pokeweed mitogen, PPD (Berenbaum, Fluck & Hurst, 1973) and tumour antigens (Cochran, Spilg, Mackie & Thomas, 1972; Vose & Moudgil, 1975). It has also been demonstrated that the cells effecting the antibody-dependent cellular cytotoxicity
Animals Rats of the Wistar strain maintained in this laboratory and weighing between 200 and 350 g were used. Anaesthesia Animals were induced by an atmosphere of 4 per cent halothane delivered from a Fluotec Mark II vapourizer in oxygen (3 I/min) and nitrous oxide (6 I/min). They were maintained in 1-5 per cent halothane in the same gas mixture. The duration of anaesthesia was, in each case, 40 min. Under the conditions of anaesthesia no consistent changes in rectal temperature were noted.
Correspondence: Dr B. M. Vose, Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX.
609
610
B. M. Vose & I. Kimber
Surgical procedures Surgical procedures consisted either of the removal of a small (1 x 2 cm) area of skin from the right flank or partial hepatectomy. Effector cells Four groups of animals were examined: (1) untreated rats killed by cervical dislocation (2) rats killed whilst under halothane anaesthesia (3) rats killed 2 h after recovery from anaesthesia and (4) rats killed 2 h after recovery from anaesthesia and surgery. All animals were matched for weight and sex. Splenic lymphoid cells were suspended in Hanks's balanced salt solution (HBSS) (Oxoid) by gently teasing finely chopped spleens through a 200 mesh stainless steel gauze. Following sedimentation (200 g, 5 min) contaminating erythrocytes were removed by exposure to Tris-buffered NH4CI. Remaining cells were washed three times in HBSS. Target cells Chang cells, originally derived from normal human adult liver, and maintained in continuous tissue cultures were employed as target cells. Lines were cultured in Eagle's modified essential medium adapted for suspension culture (Flow Laboratories) supplemented with 10 per cent heat-inactivated foetal bovine serum (FBS) (Flow Laboratories). Cells were harvested from the monolayer by exposure to 025 Trypsin (Sigma Chemical Company) in HBSS for 5 min at 37°. The resulting cell suspension was washed twice in HBSS and resuspended at a concentration of 1 x 106/ml in Eagle's modified essential medium buffered with 40 mm HEPES and supplemented with 10 per cent heat-inactivated FBS. The cells were labelled by drop-wise addition of 100 PCi sodium (5I Cr) chromate (Radiochemical Centre, Amersham) per 1 x 106 cells and incubated with occasional gentle agitation at 370 for 1 J h. The
cells were then washed three times in HBSS and resuspended in MEM HEPES supplemented with 10 per cent FBS.
Antibody Rabbits were immunized by an initial i.m. injection of Chang cells in Freund's complete adjuvant followed by three weekly i.v. injections. Serum was obtained from the rabbit 10 days following the last immunization, inactivated by heating to 560 for 30 min and diluted with MEM HEPES and 10 per cent FBS.
Antibody-dependent cellular cytotoxicity (ADCC) test Two hundred microlitres of Chang cells (25 x 104/ml) were mixed with antiserum or normal rabbit serum in 2-ml capacity stoppered plastic tubes (Luckhams Ltd). The antibody/target cell mixture was incubated for 1 h at room temperature before aliquots (200 p1) of lymphoid cells in HEPESbuffered MEM supplemented with 10 per cent FBS were added to give the desired effector: target cell ratio. The tubes were incubated for 16 h at 370, centrifuged at 200g for 5 min and 200p1 of the supernatant removed. The supernatant sample and pellets were counted in a gamma counter (Searle). Spontaneous release of 5 'Cr by Chang cells was measured in tubes containing Chang cells and medium alone and maximum 5 Cr release by lysis with a 1:100 dilution of the detergent Triton x-100. All tests were carried out in groups of five tubes. Percentage 5'Cr release for each tube was then calculated from the formula: per cent 51Cr release = 3 x supernatant counts 100 x-. supernatant and pellet counts 1 Cytotoxicity was then derived by the following formula: cytotoxicity = per cent s 1 Cr release - spontaneous 5 1 Cr release maximum 5 1 Cr release - spontaneous 5 1Cr release x100 ADCC = (cytotoxicity in presence of rabbit antiChang cell antiserum) - (cytotoxicity in presence of normal rabbit serum). Removal of adherent cells The effector cell population was depleted of adherent cells by incubation on glass. Spleen cells prepared as described above were resuspended in HEPESbuffered MEM supplemented with 10 per cent FBS. This suspension was incubated for 30 min at 370 in a 30 cm2 glass flask placed horizontally. Non adherent lymphoid cells were collected by gentle agitation of the flask and removal of the supernatant medium.
Removal ofphagocytic cells Phagocytes were removed from the splenocytes by incubation of the cells with 0-1 g of heated- sterilized carbonyl iron. The mixture was incubated for 1 h at 370 with occasional gentle agitation. Cells which
611
Halothane anaesth esia and ADCC phagocytosed the iron were pelleted with a magnet and the supernatant suspension collected.
35r
Carrageenan treatment Inhibition of cells of the monocyte: macrophage series with carrageenan. Spleen cells were incubated with carrageenan (Marine Colloids Incorporated USA) at a final concentration of 200 mg/ml in HEPES-buffered MEM supplemented with 10 per cent FBS at 370 for 2 h. Following incubation the cells were washed three times in a large volume of Hanks BSS.
30k
25k 20k
0
15k
0 0
Characterization of the effector population Morphological differentiation of leucocytes present in the effector population was performed on cytocentrifuge preparations stained with Jenner Giemsa. A minimum of 200 cells were counted. RESULTS Optimization of ADCC in rats In order to determine the optimum conditions for the detection of ADCC activity in rats a number of parallel experiments were carried out in which the effect of increasing effector cell: target cell ratios and antibody dilution on the cytotoxicity of rat spleen cells for antibody-coated Chang target cells was measured (Figs 1 and 2). It was found that the rabbit anti-Chang cell antiserum showed a sharp peak in its capacity to facilitate target cell lysis at a final dilution of 1 x 10-3 in the presence of spleen cells at an effector: target cell ratio of 1000: 1 (Fig. 1). By contrast, the effect of increasing effector cell: target cell ratios was much less marked with cytotoxic potential being constant between the ratios 200:1 and 1000:1 in the presence of the optimum concentration of antiserum. Normal rabbit serum was generally ineffective in facilitating target cell lysis showing only low levels of cytotoxicity at a range of dilutions and in the presence of varying numbers of effector cells. However, it was recognized that this represented a valuable control and was incorporated into subsequent tests which were standardized such that the final dilution of both rabbit anti-Chang cell antiserum and normal rabbit serum in the tubes was 1 x 10-3 and an effector: target cell ratio was 1000: 1. Under these conditions no difference in cytotoxic potential was noted between spleen cells and lymph node cells from the cervical lymph nodes.
.2x
a-
10k-
0
5
Ix10-2
Ix10-3
IxIo4
Serum dilution Figure 1. Characterization of ADCC in rats, effect of increasing serum dilution to effector cell: target cell ratio = 1000: 1. (0) Rabbit anti-Chang cell antiserum; (0) normal rabbit serum.
35r30k 25S ._
x 0
0
cL 0
20k
15-
L.
10o
*
5
*
.
S
1000:1 500:1 2000:1 200:1 Effector: target cell ratio Figure 2. Characterization of ADCC in rats, effect of increasing effector:target cell ratios. (0) Antibody dilution, 1 x 10-3; (0) rabbit anti-Chang cell antiserum.
100:1
B. M. Vose & L Kimber
612
Effect of anaesthesia and surgery on ADCC in rats Leucocytes from control, untreated rats showed a wide variation in their capacity to induce lysis of antibody-coated Chang cell targets (Figs 3-5). To some extent this could be explained by differences in the spontaneous release of 'ICr inasmuch as groups of animals in a single test showed a more confined distribution of cytotoxicities. Experiments were therefore designed so that comparisons of reactivities between test and control groups was performed only within single experiments and not between those performed at different times. By this criterion rats killed by cervical dislocation whilst under halothane-induced anaesthesia showed a significantly reduced potential (P < 0-01) for cytotoxicity compared with control animals which were killed by cervical dislocation alone (Fig. 3). Such a depression of ADCC activity was not evident (P e c
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P
The effects of halothane anaesthesia on antibody-dependent cellular cytotoxicity in rats
B. M. VO SE &
I.
KI M BER Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
Received 11 March 1976; acceptedfor publication 5 August 1976
Summary. Lymphocytes from rats killed while anaesthetized with 1 5 per cent halothane showed a significantly reduced capacity to induce lysis of antibody-coated target cells compared with those from untreated rats. This effect was short-lived however, being no longer apparent in lymphoid cells taken from rats 2 h after their recovery from such anaesthesia. Surgical procedures were not effective in extending the duration of reduced ADCC activity. The implication of these findings is that the post-operative depression of this in vitro assay of immunocompetence results from the influence of the anaesthetic agent per se or its metabolites with surgical trauma having little r6le.
reaction (ADCC) are particularly susceptible to this depression in patients undergoing surgery (Vose & Moudgil, 1976). It is difficult in clinical studies to separate the effects of duration and depth of anaesthesia, surgical trauma and the degrees of tissue damage and stress each of which correlates with post-surgical depression of immunocompetence (Berenbaum, Fluck & Hurst, 1973; Cullen & van Belle, 1975). The objective of this present study was to isolate, in an animal model, the effects of surgery and anaesthesia in order to clarify their respective r6les in the post-operatively reduced capacity of leucocytes to induce lysis of antibody-coated target cells in the ADCC assay.
INTRODUCTION
MATERIALS AND METHODS
Leucocytes taken in the immediate post-operative period from patients receiving surgery under general anaesthesia have a diminished response to phytohaemagglutinin (PHA) (Riddle & Berenbaum, 1967), pokeweed mitogen, PPD (Berenbaum, Fluck & Hurst, 1973) and tumour antigens (Cochran, Spilg, Mackie & Thomas, 1972; Vose & Moudgil, 1975). It has also been demonstrated that the cells effecting the antibody-dependent cellular cytotoxicity
Animals Rats of the Wistar strain maintained in this laboratory and weighing between 200 and 350 g were used. Anaesthesia Animals were induced by an atmosphere of 4 per cent halothane delivered from a Fluotec Mark II vapourizer in oxygen (3 I/min) and nitrous oxide (6 I/min). They were maintained in 1-5 per cent halothane in the same gas mixture. The duration of anaesthesia was, in each case, 40 min. Under the conditions of anaesthesia no consistent changes in rectal temperature were noted.
Correspondence: Dr B. M. Vose, Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX.
609
610
B. M. Vose & I. Kimber
Surgical procedures Surgical procedures consisted either of the removal of a small (1 x 2 cm) area of skin from the right flank or partial hepatectomy. Effector cells Four groups of animals were examined: (1) untreated rats killed by cervical dislocation (2) rats killed whilst under halothane anaesthesia (3) rats killed 2 h after recovery from anaesthesia and (4) rats killed 2 h after recovery from anaesthesia and surgery. All animals were matched for weight and sex. Splenic lymphoid cells were suspended in Hanks's balanced salt solution (HBSS) (Oxoid) by gently teasing finely chopped spleens through a 200 mesh stainless steel gauze. Following sedimentation (200 g, 5 min) contaminating erythrocytes were removed by exposure to Tris-buffered NH4CI. Remaining cells were washed three times in HBSS. Target cells Chang cells, originally derived from normal human adult liver, and maintained in continuous tissue cultures were employed as target cells. Lines were cultured in Eagle's modified essential medium adapted for suspension culture (Flow Laboratories) supplemented with 10 per cent heat-inactivated foetal bovine serum (FBS) (Flow Laboratories). Cells were harvested from the monolayer by exposure to 025 Trypsin (Sigma Chemical Company) in HBSS for 5 min at 37°. The resulting cell suspension was washed twice in HBSS and resuspended at a concentration of 1 x 106/ml in Eagle's modified essential medium buffered with 40 mm HEPES and supplemented with 10 per cent heat-inactivated FBS. The cells were labelled by drop-wise addition of 100 PCi sodium (5I Cr) chromate (Radiochemical Centre, Amersham) per 1 x 106 cells and incubated with occasional gentle agitation at 370 for 1 J h. The
cells were then washed three times in HBSS and resuspended in MEM HEPES supplemented with 10 per cent FBS.
Antibody Rabbits were immunized by an initial i.m. injection of Chang cells in Freund's complete adjuvant followed by three weekly i.v. injections. Serum was obtained from the rabbit 10 days following the last immunization, inactivated by heating to 560 for 30 min and diluted with MEM HEPES and 10 per cent FBS.
Antibody-dependent cellular cytotoxicity (ADCC) test Two hundred microlitres of Chang cells (25 x 104/ml) were mixed with antiserum or normal rabbit serum in 2-ml capacity stoppered plastic tubes (Luckhams Ltd). The antibody/target cell mixture was incubated for 1 h at room temperature before aliquots (200 p1) of lymphoid cells in HEPESbuffered MEM supplemented with 10 per cent FBS were added to give the desired effector: target cell ratio. The tubes were incubated for 16 h at 370, centrifuged at 200g for 5 min and 200p1 of the supernatant removed. The supernatant sample and pellets were counted in a gamma counter (Searle). Spontaneous release of 5 'Cr by Chang cells was measured in tubes containing Chang cells and medium alone and maximum 5 Cr release by lysis with a 1:100 dilution of the detergent Triton x-100. All tests were carried out in groups of five tubes. Percentage 5'Cr release for each tube was then calculated from the formula: per cent 51Cr release = 3 x supernatant counts 100 x-. supernatant and pellet counts 1 Cytotoxicity was then derived by the following formula: cytotoxicity = per cent s 1 Cr release - spontaneous 5 1 Cr release maximum 5 1 Cr release - spontaneous 5 1Cr release x100 ADCC = (cytotoxicity in presence of rabbit antiChang cell antiserum) - (cytotoxicity in presence of normal rabbit serum). Removal of adherent cells The effector cell population was depleted of adherent cells by incubation on glass. Spleen cells prepared as described above were resuspended in HEPESbuffered MEM supplemented with 10 per cent FBS. This suspension was incubated for 30 min at 370 in a 30 cm2 glass flask placed horizontally. Non adherent lymphoid cells were collected by gentle agitation of the flask and removal of the supernatant medium.
Removal ofphagocytic cells Phagocytes were removed from the splenocytes by incubation of the cells with 0-1 g of heated- sterilized carbonyl iron. The mixture was incubated for 1 h at 370 with occasional gentle agitation. Cells which
611
Halothane anaesth esia and ADCC phagocytosed the iron were pelleted with a magnet and the supernatant suspension collected.
35r
Carrageenan treatment Inhibition of cells of the monocyte: macrophage series with carrageenan. Spleen cells were incubated with carrageenan (Marine Colloids Incorporated USA) at a final concentration of 200 mg/ml in HEPES-buffered MEM supplemented with 10 per cent FBS at 370 for 2 h. Following incubation the cells were washed three times in a large volume of Hanks BSS.
30k
25k 20k
0
15k
0 0
Characterization of the effector population Morphological differentiation of leucocytes present in the effector population was performed on cytocentrifuge preparations stained with Jenner Giemsa. A minimum of 200 cells were counted. RESULTS Optimization of ADCC in rats In order to determine the optimum conditions for the detection of ADCC activity in rats a number of parallel experiments were carried out in which the effect of increasing effector cell: target cell ratios and antibody dilution on the cytotoxicity of rat spleen cells for antibody-coated Chang target cells was measured (Figs 1 and 2). It was found that the rabbit anti-Chang cell antiserum showed a sharp peak in its capacity to facilitate target cell lysis at a final dilution of 1 x 10-3 in the presence of spleen cells at an effector: target cell ratio of 1000: 1 (Fig. 1). By contrast, the effect of increasing effector cell: target cell ratios was much less marked with cytotoxic potential being constant between the ratios 200:1 and 1000:1 in the presence of the optimum concentration of antiserum. Normal rabbit serum was generally ineffective in facilitating target cell lysis showing only low levels of cytotoxicity at a range of dilutions and in the presence of varying numbers of effector cells. However, it was recognized that this represented a valuable control and was incorporated into subsequent tests which were standardized such that the final dilution of both rabbit anti-Chang cell antiserum and normal rabbit serum in the tubes was 1 x 10-3 and an effector: target cell ratio was 1000: 1. Under these conditions no difference in cytotoxic potential was noted between spleen cells and lymph node cells from the cervical lymph nodes.
.2x
a-
10k-
0
5
Ix10-2
Ix10-3
IxIo4
Serum dilution Figure 1. Characterization of ADCC in rats, effect of increasing serum dilution to effector cell: target cell ratio = 1000: 1. (0) Rabbit anti-Chang cell antiserum; (0) normal rabbit serum.
35r30k 25S ._
x 0
0
cL 0
20k
15-
L.
10o
*
5
*
.
S
1000:1 500:1 2000:1 200:1 Effector: target cell ratio Figure 2. Characterization of ADCC in rats, effect of increasing effector:target cell ratios. (0) Antibody dilution, 1 x 10-3; (0) rabbit anti-Chang cell antiserum.
100:1
B. M. Vose & L Kimber
612
Effect of anaesthesia and surgery on ADCC in rats Leucocytes from control, untreated rats showed a wide variation in their capacity to induce lysis of antibody-coated Chang cell targets (Figs 3-5). To some extent this could be explained by differences in the spontaneous release of 'ICr inasmuch as groups of animals in a single test showed a more confined distribution of cytotoxicities. Experiments were therefore designed so that comparisons of reactivities between test and control groups was performed only within single experiments and not between those performed at different times. By this criterion rats killed by cervical dislocation whilst under halothane-induced anaesthesia showed a significantly reduced potential (P < 0-01) for cytotoxicity compared with control animals which were killed by cervical dislocation alone (Fig. 3). Such a depression of ADCC activity was not evident (P e c
30k 0 0 0
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20L
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:130±5
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