INT. J. HYPERTHERMIA,
1992,
VOL.
8,
NO.
1, 87-97
Effects of hyperthermia on natural killer cells: inhibition of lytic function and microtubule organization H. YANG, W. LAUZON and I. LEMAIREt Laboratory of Immunopharmacology , Department of Pharmacology, Faculty of Medicine. University of Ottawa. Ottawa, Ontario, Canada KIH 8MS
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(Received 28 January 1991; revised 16 April 1991; uccepted 23 April 1991) Cells with natural killer activity (NK) may play an important role in host defence against turnour cells. The lytic function of NK cells is very sensitive to hyperthermic inactivation. However, cells with NK activity isolated from rat spleen and exposed to 41-42.S"C for 30 min could partially recover their cytotoxic activity after incubation at 37°C. The recovered cytotoxicity was still NK-specific, as it only resulted in the lysis of YAC-I sensitive targets, and could not lyse NK-resistant P815 mastocytoma cells. Conjugate formation assay using NK cells labelled with specific monoclonal antibody (mAb) 3.2.3 indicated that the binding of NK cells to targets was not significantly affected by heat treatment. Compared to controls, however, microtubule organizing centre (MTOC) reorientation towards the region of intercellular contact was reduced by 40% in heated effector cells. This was accompanied by a greater inhibition (62-77%) of NK lytic activity. Kinetic analysis indicated that MTOC reorientation capacity recovered following incubation at 37°C. MTOC recovery was maximal 4 h after treatment whereas that of lytic activity peaked at 6 h. These data indicate that NK cells recover NK-specific lytic activity after heat inactivation. Moreover, our study demonstrates that hyperthermia interferes with post-binding MTOC reorientation, and further supports a role for microtubule in secretory processes involved in NK-mediated cytolysis. Key words: Spleen lymphocytes, natural killer cell, cytotoxicity, hyperthermia, microtubule organizing centre.
1. Introduction NK cells are a subpopulation of lymphocytes which are able to lyse certain tumour cells and virus-infected cells without previous sensitization and MHC restriction (Herberman et al. 1979, Hanna 1983, Ortaldo and Herberman 1984). Therefore, cells with NK activity may play an important role in surveillance against tumour invasion and in host protection from virus infection. In addition to peripheral blood lymphocytes, lymphocytes from lung, spleen, lymph nodes and liver exhibit NK cell activity (Wiltrout et al. 1985, Fukui et al. 1987, Yron et al. 1988, Lauzon et al. 1990). Although the molecular mechanisms of target cell lysis by NK cells remain to be elucidated, it is generally accepted that NK cell-mediated cytotoxicity consists of at least three steps, including binding of effector to targets, triggering of effector cells and target cell lysis (Young 1989). Various events are thought to be involved in the triggering step (Herberman et al. 1986, Prochazka et al. 1986). Among these, it has been suggested that microtubule assembly (Carpen 1987) and reorientation of microtubule organizing centre (MTOC) towards the region of intercellular contact may be requisite for killing (Groscurth 1989). Clinically, hyperthermia treatment has attracted much interest as a useful tool for cancer therapy. The combination of hyperthermia with radiotherapy or chemotherapy has been found to be potentially effective in treating tumours (Dewey 1984, Robins 1984, Bull 1984). However, it was reported that hyperthermia in vitro inhibited NK cell activity (Kalland and Dahlquist 1983, Onsrud 1983, Yoshioka et al. 1989). Such disadvantage would impede +To whom correspondence should be addressed 0265-6736/92 $ 3 . 0 0 01992 Taylor & Francis Ltd
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
88
H . Yang et al.
the application of hyperthermia as a modality for cancer treatment since cells with NK cell activity may be important in tumour cell killing. In a previous work, we observed that hyperthermia-treated human peripheral blood cells could recover their lytic activity after incubation at 37°C (Yang and Mitchel 1991). Based on experimental evidence that organ-associated NK activity may be more relevant than systemic NK activity with regard to metastasis (Wiltrout et al. 1985) we investigated the functional response of spleen NK cells to hyperthermia treatment. We report here that NK cells from spleen are as sensitive as blood NK cells hyperthermia inactivation. We confirm our previous observations that NK cells partially recover their activity following hyperthermia, and we extend these findings by showing that spleen NK cells retain their selectivity of killing. We further demonstrate that hyperthermia suppression of NK activity is partly associated with inhibition of MTOC reorientation towards the target, thus supporting a role for microtubule and MTOC in the NK-lytic process.
2. Materials and methods 2.1. Animals and muterials Wistar rats derived from a pathogen-free colony were purchased from Charles River Canada Inc. (St-Constant, Quebec). The rats were housed in an isolated temperaturecontrolled quarters in an animal isolator unit (Upjohns Scientific, Toronto, Ontario). Sixto 10-week-old male rats were used. Monoclonal antibody (mAb) 3.2.3 that recognizes rat NK cells (Chambers et al. 1989) was generously provided by Dr J. Hiserodt, University of Pittsburgh, Pen. Affinity-purified mouse 5A6 mAb to chick tubulin (Aitchison and Brown 1986) were gifts from Dr D. Brown, University of Ottawa. FITC-conjugated goat antimouse IgG (Fab, fragment) was purchased from Sigma (St Louis, Miss.). 2.2. Preparation of effector cells The rats were anaesthetized with sodium pentobarbital and the spleen was surgically obtained. The cell suspension was made by gently pressing the spleen through a stainless steel screen into 20 ml RPMI-1640 medium (Gibco Laboratories, Grand Island, NY) and washed once. After sedimentation and centrifugation, the pellet was resuspended in phosphate-buffered saline (PBS) and loaded onto a Ficoll-Hypaque gradient (specific density, 1.077) for isolation of spleen lymphocytes (SL). After 45 rnin centrifugation at 750 g, cells at the interface were collected and washed three times in PBS. To remove adherent cells, the cells were further passed through a nylon wood fibre column (Wako Chemicals, Dallas, Tex.). The resultant cell suspension contained only 0- 1 % macrophages, as evidenced by Wright-Giemsa staining. The viability of cells, measured by trypan blue exclusion, was more than 98 % . 2.3. Membrane labelling and conjugate formation Non-adherent SL preparation (1 x 106/ml) was reacted with mouse mAb 3.2.3 (1:500) for 30 min at 4°C. The cell suspension was washed three times in PBS, resuspended at I x 10h/ml and incubated with FITC-conjugated goat anti-mouse IgG (Fab, fragment, 1 :200 dilution) for 30 rnin at 4°C. The cells were washed three times in PBS and labelled cells were layered onto poly-L-lysine-coated coverslips for 10 min at room temperature. The coverslips were then washed in PBS and fixed with 3 . 7 % formaldehyde in PEM buffer (80 mM pipes, 5 mM EGTA, 1 mM MgCl, pH 6.8) for 10 min. The coverslips were washed again and mounted onto glass slides. Lymphocytes positively stained were determined by fluorescence microscopy. At least 200 lymphocytes were counted in each sample and the percentage of positive cells calculated. For conjugate experiments, 1 x lo5 labelled cells (in 0.1 ml) were mixed with YAC-1 cells (105/0. 1 mi) (1:l ratio). The
Inhibition of NK activity and MTOC orientation
89
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
mixture was centrifuged for 5 min at 50 g and incubated at 4°C for 15 min to allow conjugate formation. The pellets were then gently resuspended and layered cnto poly-~lysine-coated coverslips. Based on their size, YAC- 1 cells were readily distinguishable from lymphocytes by microscopic examination. Positively stained SL conjugates were detected with immunofluorescence microscopy and the percentage of positive cells was calculated after counting more than 200 lymphocytes. 2.4. MTOC labelling Non-adherent SL were mixed with YAC-1 target cells (1:l ratio) for conjugate formation. The mixture was then added onto poly-L-lysine-coated coverslips for 10 rnin at room temperature. Cells were washed in PBS, permeabilized with 1 % Triton-X-100 for visualization of microtubule (MT) and reduced in NaBH, (1 mg/ml in PBS) for 7 min. MT were first reacted with mouse anti-tubulin mAb (1gG) for 30 min at room temperature, washed and incubated with FITC-conjugated goat anti-mouse IgG (Fab, fragment). Stained effector-target conjugates were examined by immunofluorescence microscopy. At least 200 conjugates were counted and the location of MTOC was determined. The orientation of MTOC towards the target was expressed as a percentage of conjugates examined.
2.5. Culture conditions RPMI-1640 was used as growth medium for YAC-1 cells, a mouse lymphoma cell line sensitive to rat NK cell-mediated lysis and DMEM (Gibco) for P815 cells, a mouse mastocytoma cell line resistant to rat NK cell killing. Media were supplemented with 10% dialysed fetal calf serum (FCS) (Gibco), HEPES (20 mM/ml) and antibiotics. The culture media were changed three times weekly. Target cells in exponential growth were used in all experiments. 2.6. N K cytotoxicity assay NK cytotoxicity was measured by a standard "Cr release assay as reported previously (Lemaire and St-Jean 1990). YAC-1 or P815 target cells (lo6 in 0.1 ml) were labelled with 100 pCi of "Cr (sodium chromate in isotonic saline, Amersham, Oakville, Ontario) for 60 min. After washing (three times) in medium supplemented with 10% FCS, cells were adjusted to 10"/ml and labelled targets (104/0. 1 ml) were distributed into 96-well microtitre plates. Various concentrations of effector cells in 100 p1 medium were added, giving different ratios (100:1 to 12.5). After incubation for 4 h the plates were centrifuged at 200 g for 10 min, 100 pl supernate was collected from each well and the released radioactivity (cpm) was determined in a well-type gamma counter (Raytest PW8000). The results were calculated as percentage cytotoxicity obtained from the mean counts of triplicate samples using the following formula: Percentage specific lysis= ER-SR x 100 TR-SR where ER represents cpm released in the presence of effector cells and SR represents cpm due to spontaneous release. TR represents total releasable cpm as measured by adding 1 %I sodium dodecylsulphate (SDS). In all experiments, SR was less than 10%of TR. NK cytotoxicity was also expressed as lytic units (LU,,) calculated by a computer program based on cytotoxic percentage of four different effector:target ratios (Pross et al. 198I). One LUz0was defined as the number of effector cells required to achieve 20% specific release from lo6 target cells. In some cases the results were expressed as a percentage of control, which was calculated as follows:
90
H. Yang et al.
LU2, from experimental x 100 LU2(,from control
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
2.7. Hyperthermia treatment SL were suspended at a concentration of 3-5 x 10h/ml in medium. Less than 2 ml of cell suspension was heated at various temperatures from 41 "C to 42.5"C by water bath immersion for 30 min. Control samples were maintained at 37°C. After hyperthermia exposure the samples were washed once and distributed into the microtitre plates for immediate or recovery measurement of NK lytic activity. 2.8. Stutistical analysis Data are expressed as mean values from at least three separate experiments fSEM. Statistical significance was evaluated by Student's unpaired t test as calculated by T-EASE (IS1 Software) program (p
1992,
VOL.
8,
NO.
1, 87-97
Effects of hyperthermia on natural killer cells: inhibition of lytic function and microtubule organization H. YANG, W. LAUZON and I. LEMAIREt Laboratory of Immunopharmacology , Department of Pharmacology, Faculty of Medicine. University of Ottawa. Ottawa, Ontario, Canada KIH 8MS
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
(Received 28 January 1991; revised 16 April 1991; uccepted 23 April 1991) Cells with natural killer activity (NK) may play an important role in host defence against turnour cells. The lytic function of NK cells is very sensitive to hyperthermic inactivation. However, cells with NK activity isolated from rat spleen and exposed to 41-42.S"C for 30 min could partially recover their cytotoxic activity after incubation at 37°C. The recovered cytotoxicity was still NK-specific, as it only resulted in the lysis of YAC-I sensitive targets, and could not lyse NK-resistant P815 mastocytoma cells. Conjugate formation assay using NK cells labelled with specific monoclonal antibody (mAb) 3.2.3 indicated that the binding of NK cells to targets was not significantly affected by heat treatment. Compared to controls, however, microtubule organizing centre (MTOC) reorientation towards the region of intercellular contact was reduced by 40% in heated effector cells. This was accompanied by a greater inhibition (62-77%) of NK lytic activity. Kinetic analysis indicated that MTOC reorientation capacity recovered following incubation at 37°C. MTOC recovery was maximal 4 h after treatment whereas that of lytic activity peaked at 6 h. These data indicate that NK cells recover NK-specific lytic activity after heat inactivation. Moreover, our study demonstrates that hyperthermia interferes with post-binding MTOC reorientation, and further supports a role for microtubule in secretory processes involved in NK-mediated cytolysis. Key words: Spleen lymphocytes, natural killer cell, cytotoxicity, hyperthermia, microtubule organizing centre.
1. Introduction NK cells are a subpopulation of lymphocytes which are able to lyse certain tumour cells and virus-infected cells without previous sensitization and MHC restriction (Herberman et al. 1979, Hanna 1983, Ortaldo and Herberman 1984). Therefore, cells with NK activity may play an important role in surveillance against tumour invasion and in host protection from virus infection. In addition to peripheral blood lymphocytes, lymphocytes from lung, spleen, lymph nodes and liver exhibit NK cell activity (Wiltrout et al. 1985, Fukui et al. 1987, Yron et al. 1988, Lauzon et al. 1990). Although the molecular mechanisms of target cell lysis by NK cells remain to be elucidated, it is generally accepted that NK cell-mediated cytotoxicity consists of at least three steps, including binding of effector to targets, triggering of effector cells and target cell lysis (Young 1989). Various events are thought to be involved in the triggering step (Herberman et al. 1986, Prochazka et al. 1986). Among these, it has been suggested that microtubule assembly (Carpen 1987) and reorientation of microtubule organizing centre (MTOC) towards the region of intercellular contact may be requisite for killing (Groscurth 1989). Clinically, hyperthermia treatment has attracted much interest as a useful tool for cancer therapy. The combination of hyperthermia with radiotherapy or chemotherapy has been found to be potentially effective in treating tumours (Dewey 1984, Robins 1984, Bull 1984). However, it was reported that hyperthermia in vitro inhibited NK cell activity (Kalland and Dahlquist 1983, Onsrud 1983, Yoshioka et al. 1989). Such disadvantage would impede +To whom correspondence should be addressed 0265-6736/92 $ 3 . 0 0 01992 Taylor & Francis Ltd
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
88
H . Yang et al.
the application of hyperthermia as a modality for cancer treatment since cells with NK cell activity may be important in tumour cell killing. In a previous work, we observed that hyperthermia-treated human peripheral blood cells could recover their lytic activity after incubation at 37°C (Yang and Mitchel 1991). Based on experimental evidence that organ-associated NK activity may be more relevant than systemic NK activity with regard to metastasis (Wiltrout et al. 1985) we investigated the functional response of spleen NK cells to hyperthermia treatment. We report here that NK cells from spleen are as sensitive as blood NK cells hyperthermia inactivation. We confirm our previous observations that NK cells partially recover their activity following hyperthermia, and we extend these findings by showing that spleen NK cells retain their selectivity of killing. We further demonstrate that hyperthermia suppression of NK activity is partly associated with inhibition of MTOC reorientation towards the target, thus supporting a role for microtubule and MTOC in the NK-lytic process.
2. Materials and methods 2.1. Animals and muterials Wistar rats derived from a pathogen-free colony were purchased from Charles River Canada Inc. (St-Constant, Quebec). The rats were housed in an isolated temperaturecontrolled quarters in an animal isolator unit (Upjohns Scientific, Toronto, Ontario). Sixto 10-week-old male rats were used. Monoclonal antibody (mAb) 3.2.3 that recognizes rat NK cells (Chambers et al. 1989) was generously provided by Dr J. Hiserodt, University of Pittsburgh, Pen. Affinity-purified mouse 5A6 mAb to chick tubulin (Aitchison and Brown 1986) were gifts from Dr D. Brown, University of Ottawa. FITC-conjugated goat antimouse IgG (Fab, fragment) was purchased from Sigma (St Louis, Miss.). 2.2. Preparation of effector cells The rats were anaesthetized with sodium pentobarbital and the spleen was surgically obtained. The cell suspension was made by gently pressing the spleen through a stainless steel screen into 20 ml RPMI-1640 medium (Gibco Laboratories, Grand Island, NY) and washed once. After sedimentation and centrifugation, the pellet was resuspended in phosphate-buffered saline (PBS) and loaded onto a Ficoll-Hypaque gradient (specific density, 1.077) for isolation of spleen lymphocytes (SL). After 45 rnin centrifugation at 750 g, cells at the interface were collected and washed three times in PBS. To remove adherent cells, the cells were further passed through a nylon wood fibre column (Wako Chemicals, Dallas, Tex.). The resultant cell suspension contained only 0- 1 % macrophages, as evidenced by Wright-Giemsa staining. The viability of cells, measured by trypan blue exclusion, was more than 98 % . 2.3. Membrane labelling and conjugate formation Non-adherent SL preparation (1 x 106/ml) was reacted with mouse mAb 3.2.3 (1:500) for 30 min at 4°C. The cell suspension was washed three times in PBS, resuspended at I x 10h/ml and incubated with FITC-conjugated goat anti-mouse IgG (Fab, fragment, 1 :200 dilution) for 30 rnin at 4°C. The cells were washed three times in PBS and labelled cells were layered onto poly-L-lysine-coated coverslips for 10 min at room temperature. The coverslips were then washed in PBS and fixed with 3 . 7 % formaldehyde in PEM buffer (80 mM pipes, 5 mM EGTA, 1 mM MgCl, pH 6.8) for 10 min. The coverslips were washed again and mounted onto glass slides. Lymphocytes positively stained were determined by fluorescence microscopy. At least 200 lymphocytes were counted in each sample and the percentage of positive cells calculated. For conjugate experiments, 1 x lo5 labelled cells (in 0.1 ml) were mixed with YAC-1 cells (105/0. 1 mi) (1:l ratio). The
Inhibition of NK activity and MTOC orientation
89
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
mixture was centrifuged for 5 min at 50 g and incubated at 4°C for 15 min to allow conjugate formation. The pellets were then gently resuspended and layered cnto poly-~lysine-coated coverslips. Based on their size, YAC- 1 cells were readily distinguishable from lymphocytes by microscopic examination. Positively stained SL conjugates were detected with immunofluorescence microscopy and the percentage of positive cells was calculated after counting more than 200 lymphocytes. 2.4. MTOC labelling Non-adherent SL were mixed with YAC-1 target cells (1:l ratio) for conjugate formation. The mixture was then added onto poly-L-lysine-coated coverslips for 10 rnin at room temperature. Cells were washed in PBS, permeabilized with 1 % Triton-X-100 for visualization of microtubule (MT) and reduced in NaBH, (1 mg/ml in PBS) for 7 min. MT were first reacted with mouse anti-tubulin mAb (1gG) for 30 min at room temperature, washed and incubated with FITC-conjugated goat anti-mouse IgG (Fab, fragment). Stained effector-target conjugates were examined by immunofluorescence microscopy. At least 200 conjugates were counted and the location of MTOC was determined. The orientation of MTOC towards the target was expressed as a percentage of conjugates examined.
2.5. Culture conditions RPMI-1640 was used as growth medium for YAC-1 cells, a mouse lymphoma cell line sensitive to rat NK cell-mediated lysis and DMEM (Gibco) for P815 cells, a mouse mastocytoma cell line resistant to rat NK cell killing. Media were supplemented with 10% dialysed fetal calf serum (FCS) (Gibco), HEPES (20 mM/ml) and antibiotics. The culture media were changed three times weekly. Target cells in exponential growth were used in all experiments. 2.6. N K cytotoxicity assay NK cytotoxicity was measured by a standard "Cr release assay as reported previously (Lemaire and St-Jean 1990). YAC-1 or P815 target cells (lo6 in 0.1 ml) were labelled with 100 pCi of "Cr (sodium chromate in isotonic saline, Amersham, Oakville, Ontario) for 60 min. After washing (three times) in medium supplemented with 10% FCS, cells were adjusted to 10"/ml and labelled targets (104/0. 1 ml) were distributed into 96-well microtitre plates. Various concentrations of effector cells in 100 p1 medium were added, giving different ratios (100:1 to 12.5). After incubation for 4 h the plates were centrifuged at 200 g for 10 min, 100 pl supernate was collected from each well and the released radioactivity (cpm) was determined in a well-type gamma counter (Raytest PW8000). The results were calculated as percentage cytotoxicity obtained from the mean counts of triplicate samples using the following formula: Percentage specific lysis= ER-SR x 100 TR-SR where ER represents cpm released in the presence of effector cells and SR represents cpm due to spontaneous release. TR represents total releasable cpm as measured by adding 1 %I sodium dodecylsulphate (SDS). In all experiments, SR was less than 10%of TR. NK cytotoxicity was also expressed as lytic units (LU,,) calculated by a computer program based on cytotoxic percentage of four different effector:target ratios (Pross et al. 198I). One LUz0was defined as the number of effector cells required to achieve 20% specific release from lo6 target cells. In some cases the results were expressed as a percentage of control, which was calculated as follows:
90
H. Yang et al.
LU2, from experimental x 100 LU2(,from control
Int J Hyperthermia Downloaded from informahealthcare.com by UB Kiel on 10/26/14 For personal use only.
2.7. Hyperthermia treatment SL were suspended at a concentration of 3-5 x 10h/ml in medium. Less than 2 ml of cell suspension was heated at various temperatures from 41 "C to 42.5"C by water bath immersion for 30 min. Control samples were maintained at 37°C. After hyperthermia exposure the samples were washed once and distributed into the microtitre plates for immediate or recovery measurement of NK lytic activity. 2.8. Stutistical analysis Data are expressed as mean values from at least three separate experiments fSEM. Statistical significance was evaluated by Student's unpaired t test as calculated by T-EASE (IS1 Software) program (p
