Br. J. Surg. Vol. 66 (1979) 507-509
The effect of levamisole on postoperative immunosuppression R . W I N D L E , R . F. M . W O O D A N D P. R . F . BELL* SUMMARY
Cell mediated immunity was studied before and after surgery in patients with colorectal cancer. Thymusderived ( T ) lymphocytes were enumerated and their non-specific activity was measured by assessing phytohaemagglutinin (PHA)-induced blastogenesis. Specific T lymphocyte activity was measured by leucocyte migration inhibition in response to purified protein derivative of tuberculin (PPD) and autochthonous tumour extract. One group of patients received levamisole postoperatively and the other group consisted of controls. There was a more rapid return to preoperative levels of leucocyte migration inhibition to both antigens by the levamisole group. However, levamisole administration had only a marginal efect on the postoperative depression of cell counts and did not restore depressed lymphoblast transformation.
FOLLOWING surgery there is a depression of cell mediated immunity. This has been demonstrated by a reduction in the numbers of circulating thymus-derived (T) lymphocytes (Miller et al., 1976). In addition, lymphoblast transformation in response to phytohaemagglutinin (PHA) (Riddle and Berenbaum, 1967; Park et a]., 1971) and leucocyte migration inhibition (Cochran et al., 1972) are both depressed after operation. Intraoperative manipulation of a malignant lesion may cause dissemination of cells. A reduction in cell mediated immunity at such a time could therefore detrimentally affect prognosis. Levamisole, an anthelmintic agent, has been shown to restore cell mediated immunity depressed by age (Verhaegen et al., 1973) or cancer (Tripodi et al., 1973). The effect of levamisole on cell mediated immunity depressed following surgery has not to our knowledge been reported. We have investigated cell mediated immunity before and after surgery for colorectal cancer and have assessed the effect of the postoperative administration of levamisole. Cell mediated immunity was studied using three techniques : 1 . Enumeration of T lymphocytes. 2. The leucocyte migration inhibition test in response to purified protein derivative of tuberculin (PPD) and autochthonous tumour. 3. The degree of blastogenesis in response to phytohaemagglutinin (PHA). Patients and methods Two groups of 10 patients with colorectal cancer were studied. One group of patients received 50 mg of levamisole three times per day orally on the first 3 postoperative days. The other group of patients acted as controls and were not given levamisole. The groups were age-matched with 8 males and 2 females in each group. In 7 patients in each group the tumour was confined to the bowel and a curative resection was performed. Three oatients in each group had extensive disease with lymph node ~nvolvementor distant metastases. Of these 6 patients,-4 had a palliative resection and 1 patient in each group was inoperable
and underwent colostomy alone. Anaesthesia and medication were identical for both groups with the exception of blood transfusion. Four control and 6 levamisole patients were transfused during surgery. Blood was taken for immunological assessment immediately before surgery and at 3 and 7 days after operation for T cell estimation, and at 1, 14 and 28 days after operation for the tests of lymphocyte function. Further tests were performed at 2 months, if there was persistent immunosuppression. Immunological assessment Enumeration of lymphocytes: A total white cell count was performed and lymphocytes were enumerated by a differential white cell count on a Giemsa-stained, air-dried blood film. T cells were enumerated by assessing E rosette formation by the method of Strong et al. (1975). Heparinized blood was centrifuged on a Ficoll-Hypaque gradient and lymphocytes at the interphase were collected, washed in Hanks' solution and counted. Next, 100 p1 of lymphocyte suspension with 4 x lo8 cells/ml were mixed with 100 p1 of heat-inactivated fetal calf serum and 100 pI of 0.5 per cent sheep red cells. The mixture was incubated a t 37 "C for 1 h, centrifuged at 200 g for 5 min and finally refrigerated at 4 "C for 16 h. The cell button was resuspended by gentle tapping of the tube. The proportion of lymphocytes without rosettes and of lymphocytes with three or more attached sheep red cells was assessed. One hundred rosettes were counted and each experiment was performed in quadruplicate. Pre- and postoperative results were compared using the Wilcoxon matched pairs signed ranks test and have been expressed as counts &one standard error. Antigenic exrracts: Extracts rich in large membranous cell fragments were prepared for each patient from normal colonic mucosa taken at least 10 cm from the tumour site and from a portion of the resected tumour using a modification of the method of Jones and Turnbull (1975). The homogenized tumour was centrifuged at 1000 g and an aliquot was removed and subjected to further centrifugation at 3000 g. The residue was resuspended in half the volume of supernatant. The protein concentration of this suspension was measured using a ultraviolet spectrophotometer and the specimen was stored at -20 "C for use as antigen in the leucocyte migration inhibition test. Leucocyte migration inhibition test: The method used was that of Serborg and Bendixen (1967). The patient's cells were migrated in plastic wells containing medium alone for controls and medium containing PPD (200 pg/ml) to test tuberculin sensitivity. To test tumour-directed cell mediated immunity the wells were filled with medium containing either normal colonic extract or colonic tumour extract. The tumour extract was used at the maximum concentration which had been shown to be non-inhibitory to the migration of leucocytes from a normal patient. This was 100 pg/ml for each patient. The medium used was Eagles MEM with 10 per cent fetal calf serum. Six wells were used for each test and after incubation at 37 "C for 16 h the migration areas were drawn using a microscope with a camera lucida attachment. The areas were then measured by planimetry. Significant migration inhibition was assessed by the MannWhitney U test of ranking. The results have been expressed as the number of patients in each group who, following surgery, had a leucocyte migration inhibition test significantly depressed below that found preoperatively. Comparison between the rates of return of the treatment groups was also made using the Mann-Whitney U test.
* Department of Surgery, The General Hospital, Leicester.
508
R. Windle et al.
Table I: PATIENT LYMPHOCYTE COUNTS Lymphocytes x 103/mm3 (mean +s.e. mean) Days postop. Patients Preoo. 3 7 Controls ( n = 10) Levamisole ( n = 10)
1.494 0.12 1.181r0.15* 1.30 ~ 0 . 1 3 0.92*0.20
* Significant difference from
1.3510.25 1.20f0.18
preoperative count, P 28 Controls ( n = 9) Levamisole ( n = 8)
9 I
7 0
4
2
0
P < 0.001.
Table 1V: PATIENTS WITH A DEPRESSED LEUCOCYTE MIGRATION INHIBITION TEST FOLLOWING SURGERY (TUMOUR EXTRACT) Days 3 7 14 28 > 28 Patients Controls ( n = 4) Levamisole ( n = 3)
4 2
4 0
4
0
4
P < 0.05. Table V : PATIENTS WITH DEPRESSED LYMPHOCYTE BLASTOGENESIS (PHA-INDUCED) FOLLOWING SURGERY Days No depression Patients following surgery 3 7 14 28 >28 Controls ( n = 10) Levamisole ( n = 10)
Results Lymphocyte and E rosette counts There was a depression of both total lymphocyte and E rosette counts 3 days following surgery in both groups. This reached significance (P
The effect of levamisole on postoperative immunosuppression R . W I N D L E , R . F. M . W O O D A N D P. R . F . BELL* SUMMARY
Cell mediated immunity was studied before and after surgery in patients with colorectal cancer. Thymusderived ( T ) lymphocytes were enumerated and their non-specific activity was measured by assessing phytohaemagglutinin (PHA)-induced blastogenesis. Specific T lymphocyte activity was measured by leucocyte migration inhibition in response to purified protein derivative of tuberculin (PPD) and autochthonous tumour extract. One group of patients received levamisole postoperatively and the other group consisted of controls. There was a more rapid return to preoperative levels of leucocyte migration inhibition to both antigens by the levamisole group. However, levamisole administration had only a marginal efect on the postoperative depression of cell counts and did not restore depressed lymphoblast transformation.
FOLLOWING surgery there is a depression of cell mediated immunity. This has been demonstrated by a reduction in the numbers of circulating thymus-derived (T) lymphocytes (Miller et al., 1976). In addition, lymphoblast transformation in response to phytohaemagglutinin (PHA) (Riddle and Berenbaum, 1967; Park et a]., 1971) and leucocyte migration inhibition (Cochran et al., 1972) are both depressed after operation. Intraoperative manipulation of a malignant lesion may cause dissemination of cells. A reduction in cell mediated immunity at such a time could therefore detrimentally affect prognosis. Levamisole, an anthelmintic agent, has been shown to restore cell mediated immunity depressed by age (Verhaegen et al., 1973) or cancer (Tripodi et al., 1973). The effect of levamisole on cell mediated immunity depressed following surgery has not to our knowledge been reported. We have investigated cell mediated immunity before and after surgery for colorectal cancer and have assessed the effect of the postoperative administration of levamisole. Cell mediated immunity was studied using three techniques : 1 . Enumeration of T lymphocytes. 2. The leucocyte migration inhibition test in response to purified protein derivative of tuberculin (PPD) and autochthonous tumour. 3. The degree of blastogenesis in response to phytohaemagglutinin (PHA). Patients and methods Two groups of 10 patients with colorectal cancer were studied. One group of patients received 50 mg of levamisole three times per day orally on the first 3 postoperative days. The other group of patients acted as controls and were not given levamisole. The groups were age-matched with 8 males and 2 females in each group. In 7 patients in each group the tumour was confined to the bowel and a curative resection was performed. Three oatients in each group had extensive disease with lymph node ~nvolvementor distant metastases. Of these 6 patients,-4 had a palliative resection and 1 patient in each group was inoperable
and underwent colostomy alone. Anaesthesia and medication were identical for both groups with the exception of blood transfusion. Four control and 6 levamisole patients were transfused during surgery. Blood was taken for immunological assessment immediately before surgery and at 3 and 7 days after operation for T cell estimation, and at 1, 14 and 28 days after operation for the tests of lymphocyte function. Further tests were performed at 2 months, if there was persistent immunosuppression. Immunological assessment Enumeration of lymphocytes: A total white cell count was performed and lymphocytes were enumerated by a differential white cell count on a Giemsa-stained, air-dried blood film. T cells were enumerated by assessing E rosette formation by the method of Strong et al. (1975). Heparinized blood was centrifuged on a Ficoll-Hypaque gradient and lymphocytes at the interphase were collected, washed in Hanks' solution and counted. Next, 100 p1 of lymphocyte suspension with 4 x lo8 cells/ml were mixed with 100 p1 of heat-inactivated fetal calf serum and 100 pI of 0.5 per cent sheep red cells. The mixture was incubated a t 37 "C for 1 h, centrifuged at 200 g for 5 min and finally refrigerated at 4 "C for 16 h. The cell button was resuspended by gentle tapping of the tube. The proportion of lymphocytes without rosettes and of lymphocytes with three or more attached sheep red cells was assessed. One hundred rosettes were counted and each experiment was performed in quadruplicate. Pre- and postoperative results were compared using the Wilcoxon matched pairs signed ranks test and have been expressed as counts &one standard error. Antigenic exrracts: Extracts rich in large membranous cell fragments were prepared for each patient from normal colonic mucosa taken at least 10 cm from the tumour site and from a portion of the resected tumour using a modification of the method of Jones and Turnbull (1975). The homogenized tumour was centrifuged at 1000 g and an aliquot was removed and subjected to further centrifugation at 3000 g. The residue was resuspended in half the volume of supernatant. The protein concentration of this suspension was measured using a ultraviolet spectrophotometer and the specimen was stored at -20 "C for use as antigen in the leucocyte migration inhibition test. Leucocyte migration inhibition test: The method used was that of Serborg and Bendixen (1967). The patient's cells were migrated in plastic wells containing medium alone for controls and medium containing PPD (200 pg/ml) to test tuberculin sensitivity. To test tumour-directed cell mediated immunity the wells were filled with medium containing either normal colonic extract or colonic tumour extract. The tumour extract was used at the maximum concentration which had been shown to be non-inhibitory to the migration of leucocytes from a normal patient. This was 100 pg/ml for each patient. The medium used was Eagles MEM with 10 per cent fetal calf serum. Six wells were used for each test and after incubation at 37 "C for 16 h the migration areas were drawn using a microscope with a camera lucida attachment. The areas were then measured by planimetry. Significant migration inhibition was assessed by the MannWhitney U test of ranking. The results have been expressed as the number of patients in each group who, following surgery, had a leucocyte migration inhibition test significantly depressed below that found preoperatively. Comparison between the rates of return of the treatment groups was also made using the Mann-Whitney U test.
* Department of Surgery, The General Hospital, Leicester.
508
R. Windle et al.
Table I: PATIENT LYMPHOCYTE COUNTS Lymphocytes x 103/mm3 (mean +s.e. mean) Days postop. Patients Preoo. 3 7 Controls ( n = 10) Levamisole ( n = 10)
1.494 0.12 1.181r0.15* 1.30 ~ 0 . 1 3 0.92*0.20
* Significant difference from
1.3510.25 1.20f0.18
preoperative count, P 28 Controls ( n = 9) Levamisole ( n = 8)
9 I
7 0
4
2
0
P < 0.001.
Table 1V: PATIENTS WITH A DEPRESSED LEUCOCYTE MIGRATION INHIBITION TEST FOLLOWING SURGERY (TUMOUR EXTRACT) Days 3 7 14 28 > 28 Patients Controls ( n = 4) Levamisole ( n = 3)
4 2
4 0
4
0
4
P < 0.05. Table V : PATIENTS WITH DEPRESSED LYMPHOCYTE BLASTOGENESIS (PHA-INDUCED) FOLLOWING SURGERY Days No depression Patients following surgery 3 7 14 28 >28 Controls ( n = 10) Levamisole ( n = 10)
Results Lymphocyte and E rosette counts There was a depression of both total lymphocyte and E rosette counts 3 days following surgery in both groups. This reached significance (P
