Brain Research, 524 (1990) 297-302
297
Elsevier BRES 15759
Effect of cerebral hemisphere decortication on the cytotoxic activity of natural killer and natural cytotoxic lymphocytes in the mouse Natale Belluardo 1, Giuseppa Mud61, Silvano Celia 2 and Mauro Bindoni 1 1Institute of Human Physiology, University of Catania, Catania (Italy) and 2Department of Pharmacology, University of Milan, Milan (Italy) (Accepted 20 February 1990)
Key words: Cerebral cortex; Hemisphere lateralization; Natural immunity; Natural killer lymphocyte; Natural cytotoxic lymphocyte; Cytotoxicity
A comparison was made of the effects of left and right cerebral decortication on cytotoxic activity of natural killer and natural cytotoxic lymphocytes in the mouse. Natural killer cytotoxic activity was significantly reduced after right decortication, whereas left decortication led to a less pronounced, though still significant fall. The cytotoxic activity of natural cytotoxic cells, on the other hand, was significantly increased, particularly 15 days after left decortication. These findings mirror the results of previously published personal findings following electrothermocoagulation of the hypothalamus. The suggestion is made that the cortex and the hypothalamus form an integrated system for the control of certain aspects of natural immunity. INTRODUCTION The extensive data gathered thus far from animals and man provide compelling evidence that the immune and nervous systems communicate by means of humoral messages and constitute an integrated system of control against tumor cells and infectious agents 9'1°'18'2°'22'3°. Two major cellular immune mechanisms play a role in cancer defense. These are represented by induced antigen-specific cell-mediated immunity and natural cellmediated immunity. Natural killer (NK) and natural cytotoxic (NC) lymphocytes are the most clearly identified cell subpopulations with a natural immunity. Both lines possess a broad spectrum of recognition of antigen determinants, do not require presensitization, are not limited by the major histocompatibility complex (MHC) and do not display an immunological m e m o r y H'12'24' 29,41,42 While certain characteristics are c o m m o n to these two lines, they can nonetheless be distinguished from each other by some of their m e m b r a n e markers, by their cytotoxic effects on the target cells, and by their responsiveness to regulation factors ~2'32'4t. Our previous work has shown that intactness of the nuclei of the median region of the hypothalamus is a sine qua non for the efficient maturation of NK lymphocytes 5A7, whereas injury to the hypothalamus results in significant enhancement of NC lymphocyte activity 5. Other workers have shown that the cerebral cortex also exerts a significant influence on some aspects of immune system function in
the mouse. Hemispheric lateralization of control is evident, since partial ablation of the left hemisphere cortex results in a significant decrease in the number of T lymphocytes and N K cytotoxic activity 2'37. This paper presents a comparative study of left and right hemisphere decortication on NK and NC activity in the mouse. NK cytotoxicity was particularly depressed after right decortication, whereas a significant, though transient, increase in NC activity was primarily evident after left decortication. These changes in NK and NC activity mirror our observations after injury to the hypothalamus. MATERIALS AND METHODS
Animals Male C57BL/6 mice aged 6-7 weeks (Charles River Breeding Laboratory, Calco, Italy) were used. They were housed in conditioned air (24-25 °C) and under controlled lighting (06.00-18.00 h). They were given a standard diet and water ad libitum. Body weights and temperatures were checked at various times during the experiments.
Decortication Anesthesia was induced with a mixture of ketamine-HCl (Bristol Myers, Syracuse, NY) and absolute alcohol. The scalp was turned back. The skullcap covering the cerebral cortex was trepanned and removed without damage to the sagittal sinus. Part of the right or left cortex was then resected with a suitably modified bistoury without injuring the underlying structures. A small quantity of a penicillin-streptomycin solution was poured over the wound. The piece of skullcap was replaced and the soft tissues were sutured. A sham operation (SO) consisting of removal and replacement of the skullcap only served as control. Electrothermocoagulation (ETC) of part of the hind right paw was performed with an electrode
Correspondence: M. Bindoni, Istituto di Fisiologia umana, v.le A. Doria, 6, 95125 Catania, Italy. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
298 connected to a radiofrequency oscillator as another type of control.
Target cells (TC) Yac-1 and WEHI 164 lines were exponentially grown on RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 1% glutamine, penicillin, streptomycin and amphotericin B (Mycostatine, Eurobios, Paris), and 5 x 10-5 M 2-mercaptoethanol.
C.CORTEX OLFACTORY BULB
CEREBELLUM
Preparation of effector cells (EC) Mice from each group were sacrificed by rapid decapitation 7, 15 and 30 days after decortication, SO and ETC, and the blood was collected for plasma hormone determination. Spleen cells (SC) were prepared as described by de Landazuri et al. 15. Cells from 4 to 6 animals were used for each experiment. Adhering cells were removed by incubating 2-5 x 108 SC on 1.8 g nylon wool columns in plastic syringes 28. Cells were added to 2 ml complete medium and incubated at 37 °C for 45 min. Non-adhering cells were eluted with 40 ml of medium at 37 °C.
Centrifugation on discontinuous density Percoll gradients SC free of adhering cells were fractionated by centrifugation on a discontinuous density Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden) according to Timonen et al. 43. Briefly, the medium and the Percoll were brought to 290 mosmol/kg H20 with sterile distilled water and PBS 10x, respectively. Seven discontinuous Percoll gradients ranging from 38.6 to 70.1% were prepared in the culture medium. After stratification of the gradient in conical centrifuge tubes, 5 x 107 lymphocytes were placed on the gradient and the tubes were centrifuged at room temperature for 45 min at 350 g. The cells at the interfaces were recovered with a Pasteur pipette and washed with RPM! 1640 admixed with 5% FCS. Recovery exceeded 80%. Cell vitality was >95% as shown by the Trypan blue exclusion test. EC morphology was studied by centrifuging 6 x 104 iymphocytes in 0.2 ml PBS for 8 min at 550 rpm on microscope slides in a Cytospin 2 centrifuge (Shandon Southern Products, Runcorn, Cheshire, U.K.). The slides were dried, fixed in methanol and stained for 20 min with Giemsa 10% (Fisher Scientific, Fairlawn, N J) diluted in pH 7.4 phosphate buffer. The morphology of the cells was observed using an immersion objective.
NK cytotoxicity assay Yac-1 cells (5 × 106) were labelled with 200 pCi of Naz51CrO4 (1 mCi/ml, Amersham, Buckinghamshire, U.K.) at 37 °C in 5% CO;~ for ! h. Various concentrations of EC were cultured with 5 × l0 s labelled TC for 4 h at 37 °C in 5% CO 2 in plates with roundbottomed microwells (cat. no. 63320; Nunc, Kamptrup, Denmark). The plates were then centrifuged at 400 g for 10 min. The supernatant was collected from each well and counted in a gamma counter (1275 LKB Wallac, Oy, Turku, Finland). Each sample was tested in triplicate. The supernatant from the wells containing TC only in 0.2 ml medium was used as the control of the spontaneous release of 51Cr. This ranged from 5-10% of the total TC 51Cr uptake in all cases. 51Cr-labelled TC (5 x 103) were also counted to determine the total uptake. Percentage cytotoxicity was calculated as follows:
.,-
total uptake cpm
Fig. 1. Schematic pattern of hemispheric decorticated area.
NC cytotoxicity assay Unfractionated spleen cell suspensions were employed. WEHI164 TC (5 x 106) were labelled with 200/~Ci of Na251CrO4 for 1 h at 37 °C and then washed 3 times with complete medium. TC (1 x 104) were cultured with SC for 16 h at various EC:TC ratios in U-shaped microwells. Cell collection and the isotope count and release percentage were performed as in the NK assay. Spontaneous release was 10-20%. The tested groups were assayed in triplicate. In each of 4 independent experiments, spleens of 5-6 mice/group were tested separately. Part of the lymphocytes from the animals employed for the NK test were taken before pooling. The means of all values were used for statistical purpose and to compile the graphs.
Growth hormone (GH), prolactin (PRL) and corticosterone
Evaluation of the cell population
% cytotoxicity = cpm test group - cpm spontaneous release
AREA OF C E R E B R A L C O R T E X R E M O V E D
x
The mice were habituated to handling to minimize transient variations in plasma hormone levels usually induced by stress. The blood of mice sacrificed 15 days after decortication, SO or ETC, was collected from the neck vessels in slightly heparinized test tubes. Plasma samples obtained by centrifugation were stored at -80 °C and assayed within 1 month. GH and PRL levels were determined by the double-antibody method of Schalch et al. 38 and Niswender et al. 31, using materials supplied through the courtesy of Dr. Parlow (Pituitary Hormones and Antisera Center, Torrance, CA). The results were expressed in ng hormone/ml according to the NIH standards (potencies about 0.6 IU/mg for mGH and 30 IU/mg for mPRL). The sensitivity of the method is 0.5 ng/ml for mGH and 1.0 ng/ml for mPRL. Interassay variability (5%) was avoided by running a single RIA for all samples. Plasma level of corticosterone was determined by the fluorimetric method of Guillemin et al. 2a.
100
All tests were performed with 4 EC:TC ratios ranging from 100:1 to 12.5:1. The results in the graph are for the 50:1 ratio and comparable with those for the other ratios. All tests were run in triplicate. The standard error between the replicates was < 5 % of the mean. In each of 4 independent experiments, spleens of 5-6 mice per group were pooled. The means of 4 experiments were used for statistical tests and compiling the graphs.
Fig. 2. Histologic section of partial decorticated cerebral hemisphere. No signs of damage are evident in subcortical areas. Corpus callosum is not involved by surgery.
299 TABLE I
Percentage of NK cytotoxicity 7, 15 and30 days after decortication, SO and E TC Fvalues from two-way ANOVA.
Source
Days after surgery
Animal groups Percoli fractions Animal groups x Percoll fractions
7
15
30
F3,9o = 9.7* F5,9o = 309.1" F~5,9o = 1.9 ns
F3,97 ---- 52.8*
F3,97 = 12.1"
F5.97 = 176.3"
F5,97 = 239.7*
F~5,97 = 11.1"
Fts,97 = 1.6 ns
*P < 0.0001.
Statistical analysis The data were analyzed by a two-way analysis of variance (ANOVA) using group of animals and fractions (NK tests) or ratios (NC tests) as the main effects, with repeated measures on the first
factor. Corticosterone-level differences were analyzed by one-way ANOVA. Following a significant overall ANOVA, comparisons among group means were made by two-tailed Student's t-test.
a
-SHAM OPERATION ......... MUSCLE E LECTROTHERMO= COAGULATION
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LEFT OECORT,CATION
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297
Elsevier BRES 15759
Effect of cerebral hemisphere decortication on the cytotoxic activity of natural killer and natural cytotoxic lymphocytes in the mouse Natale Belluardo 1, Giuseppa Mud61, Silvano Celia 2 and Mauro Bindoni 1 1Institute of Human Physiology, University of Catania, Catania (Italy) and 2Department of Pharmacology, University of Milan, Milan (Italy) (Accepted 20 February 1990)
Key words: Cerebral cortex; Hemisphere lateralization; Natural immunity; Natural killer lymphocyte; Natural cytotoxic lymphocyte; Cytotoxicity
A comparison was made of the effects of left and right cerebral decortication on cytotoxic activity of natural killer and natural cytotoxic lymphocytes in the mouse. Natural killer cytotoxic activity was significantly reduced after right decortication, whereas left decortication led to a less pronounced, though still significant fall. The cytotoxic activity of natural cytotoxic cells, on the other hand, was significantly increased, particularly 15 days after left decortication. These findings mirror the results of previously published personal findings following electrothermocoagulation of the hypothalamus. The suggestion is made that the cortex and the hypothalamus form an integrated system for the control of certain aspects of natural immunity. INTRODUCTION The extensive data gathered thus far from animals and man provide compelling evidence that the immune and nervous systems communicate by means of humoral messages and constitute an integrated system of control against tumor cells and infectious agents 9'1°'18'2°'22'3°. Two major cellular immune mechanisms play a role in cancer defense. These are represented by induced antigen-specific cell-mediated immunity and natural cellmediated immunity. Natural killer (NK) and natural cytotoxic (NC) lymphocytes are the most clearly identified cell subpopulations with a natural immunity. Both lines possess a broad spectrum of recognition of antigen determinants, do not require presensitization, are not limited by the major histocompatibility complex (MHC) and do not display an immunological m e m o r y H'12'24' 29,41,42 While certain characteristics are c o m m o n to these two lines, they can nonetheless be distinguished from each other by some of their m e m b r a n e markers, by their cytotoxic effects on the target cells, and by their responsiveness to regulation factors ~2'32'4t. Our previous work has shown that intactness of the nuclei of the median region of the hypothalamus is a sine qua non for the efficient maturation of NK lymphocytes 5A7, whereas injury to the hypothalamus results in significant enhancement of NC lymphocyte activity 5. Other workers have shown that the cerebral cortex also exerts a significant influence on some aspects of immune system function in
the mouse. Hemispheric lateralization of control is evident, since partial ablation of the left hemisphere cortex results in a significant decrease in the number of T lymphocytes and N K cytotoxic activity 2'37. This paper presents a comparative study of left and right hemisphere decortication on NK and NC activity in the mouse. NK cytotoxicity was particularly depressed after right decortication, whereas a significant, though transient, increase in NC activity was primarily evident after left decortication. These changes in NK and NC activity mirror our observations after injury to the hypothalamus. MATERIALS AND METHODS
Animals Male C57BL/6 mice aged 6-7 weeks (Charles River Breeding Laboratory, Calco, Italy) were used. They were housed in conditioned air (24-25 °C) and under controlled lighting (06.00-18.00 h). They were given a standard diet and water ad libitum. Body weights and temperatures were checked at various times during the experiments.
Decortication Anesthesia was induced with a mixture of ketamine-HCl (Bristol Myers, Syracuse, NY) and absolute alcohol. The scalp was turned back. The skullcap covering the cerebral cortex was trepanned and removed without damage to the sagittal sinus. Part of the right or left cortex was then resected with a suitably modified bistoury without injuring the underlying structures. A small quantity of a penicillin-streptomycin solution was poured over the wound. The piece of skullcap was replaced and the soft tissues were sutured. A sham operation (SO) consisting of removal and replacement of the skullcap only served as control. Electrothermocoagulation (ETC) of part of the hind right paw was performed with an electrode
Correspondence: M. Bindoni, Istituto di Fisiologia umana, v.le A. Doria, 6, 95125 Catania, Italy. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
298 connected to a radiofrequency oscillator as another type of control.
Target cells (TC) Yac-1 and WEHI 164 lines were exponentially grown on RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 1% glutamine, penicillin, streptomycin and amphotericin B (Mycostatine, Eurobios, Paris), and 5 x 10-5 M 2-mercaptoethanol.
C.CORTEX OLFACTORY BULB
CEREBELLUM
Preparation of effector cells (EC) Mice from each group were sacrificed by rapid decapitation 7, 15 and 30 days after decortication, SO and ETC, and the blood was collected for plasma hormone determination. Spleen cells (SC) were prepared as described by de Landazuri et al. 15. Cells from 4 to 6 animals were used for each experiment. Adhering cells were removed by incubating 2-5 x 108 SC on 1.8 g nylon wool columns in plastic syringes 28. Cells were added to 2 ml complete medium and incubated at 37 °C for 45 min. Non-adhering cells were eluted with 40 ml of medium at 37 °C.
Centrifugation on discontinuous density Percoll gradients SC free of adhering cells were fractionated by centrifugation on a discontinuous density Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden) according to Timonen et al. 43. Briefly, the medium and the Percoll were brought to 290 mosmol/kg H20 with sterile distilled water and PBS 10x, respectively. Seven discontinuous Percoll gradients ranging from 38.6 to 70.1% were prepared in the culture medium. After stratification of the gradient in conical centrifuge tubes, 5 x 107 lymphocytes were placed on the gradient and the tubes were centrifuged at room temperature for 45 min at 350 g. The cells at the interfaces were recovered with a Pasteur pipette and washed with RPM! 1640 admixed with 5% FCS. Recovery exceeded 80%. Cell vitality was >95% as shown by the Trypan blue exclusion test. EC morphology was studied by centrifuging 6 x 104 iymphocytes in 0.2 ml PBS for 8 min at 550 rpm on microscope slides in a Cytospin 2 centrifuge (Shandon Southern Products, Runcorn, Cheshire, U.K.). The slides were dried, fixed in methanol and stained for 20 min with Giemsa 10% (Fisher Scientific, Fairlawn, N J) diluted in pH 7.4 phosphate buffer. The morphology of the cells was observed using an immersion objective.
NK cytotoxicity assay Yac-1 cells (5 × 106) were labelled with 200 pCi of Naz51CrO4 (1 mCi/ml, Amersham, Buckinghamshire, U.K.) at 37 °C in 5% CO;~ for ! h. Various concentrations of EC were cultured with 5 × l0 s labelled TC for 4 h at 37 °C in 5% CO 2 in plates with roundbottomed microwells (cat. no. 63320; Nunc, Kamptrup, Denmark). The plates were then centrifuged at 400 g for 10 min. The supernatant was collected from each well and counted in a gamma counter (1275 LKB Wallac, Oy, Turku, Finland). Each sample was tested in triplicate. The supernatant from the wells containing TC only in 0.2 ml medium was used as the control of the spontaneous release of 51Cr. This ranged from 5-10% of the total TC 51Cr uptake in all cases. 51Cr-labelled TC (5 x 103) were also counted to determine the total uptake. Percentage cytotoxicity was calculated as follows:
.,-
total uptake cpm
Fig. 1. Schematic pattern of hemispheric decorticated area.
NC cytotoxicity assay Unfractionated spleen cell suspensions were employed. WEHI164 TC (5 x 106) were labelled with 200/~Ci of Na251CrO4 for 1 h at 37 °C and then washed 3 times with complete medium. TC (1 x 104) were cultured with SC for 16 h at various EC:TC ratios in U-shaped microwells. Cell collection and the isotope count and release percentage were performed as in the NK assay. Spontaneous release was 10-20%. The tested groups were assayed in triplicate. In each of 4 independent experiments, spleens of 5-6 mice/group were tested separately. Part of the lymphocytes from the animals employed for the NK test were taken before pooling. The means of all values were used for statistical purpose and to compile the graphs.
Growth hormone (GH), prolactin (PRL) and corticosterone
Evaluation of the cell population
% cytotoxicity = cpm test group - cpm spontaneous release
AREA OF C E R E B R A L C O R T E X R E M O V E D
x
The mice were habituated to handling to minimize transient variations in plasma hormone levels usually induced by stress. The blood of mice sacrificed 15 days after decortication, SO or ETC, was collected from the neck vessels in slightly heparinized test tubes. Plasma samples obtained by centrifugation were stored at -80 °C and assayed within 1 month. GH and PRL levels were determined by the double-antibody method of Schalch et al. 38 and Niswender et al. 31, using materials supplied through the courtesy of Dr. Parlow (Pituitary Hormones and Antisera Center, Torrance, CA). The results were expressed in ng hormone/ml according to the NIH standards (potencies about 0.6 IU/mg for mGH and 30 IU/mg for mPRL). The sensitivity of the method is 0.5 ng/ml for mGH and 1.0 ng/ml for mPRL. Interassay variability (5%) was avoided by running a single RIA for all samples. Plasma level of corticosterone was determined by the fluorimetric method of Guillemin et al. 2a.
100
All tests were performed with 4 EC:TC ratios ranging from 100:1 to 12.5:1. The results in the graph are for the 50:1 ratio and comparable with those for the other ratios. All tests were run in triplicate. The standard error between the replicates was < 5 % of the mean. In each of 4 independent experiments, spleens of 5-6 mice per group were pooled. The means of 4 experiments were used for statistical tests and compiling the graphs.
Fig. 2. Histologic section of partial decorticated cerebral hemisphere. No signs of damage are evident in subcortical areas. Corpus callosum is not involved by surgery.
299 TABLE I
Percentage of NK cytotoxicity 7, 15 and30 days after decortication, SO and E TC Fvalues from two-way ANOVA.
Source
Days after surgery
Animal groups Percoli fractions Animal groups x Percoll fractions
7
15
30
F3,9o = 9.7* F5,9o = 309.1" F~5,9o = 1.9 ns
F3,97 ---- 52.8*
F3,97 = 12.1"
F5.97 = 176.3"
F5,97 = 239.7*
F~5,97 = 11.1"
Fts,97 = 1.6 ns
*P < 0.0001.
Statistical analysis The data were analyzed by a two-way analysis of variance (ANOVA) using group of animals and fractions (NK tests) or ratios (NC tests) as the main effects, with repeated measures on the first
factor. Corticosterone-level differences were analyzed by one-way ANOVA. Following a significant overall ANOVA, comparisons among group means were made by two-tailed Student's t-test.
a
-SHAM OPERATION ......... MUSCLE E LECTROTHERMO= COAGULATION
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.... -
J7 i " ~://
-
40
LEFT OECORT,CATION
-
.
.
.
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SHAM OPERATION .......... MUSCLE ELECTROTHERMO= COAGULATION ee . . . . LEFT DECORTICATION ~-,J, RIGHT . . . .
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