Immunology 1979 36 367
Lymphocyte emperipolesis revisited II.
FURTHER CHARACTERIZATION OF THE LYMPHOCYTE SUBPOPULATION INVOLVED
FIONA M. REID, G. P. SANDILANDS, KATHLEEN G. GRAY & J. R. ANDERSON University Department of Pathology, Western Infirmary, Glasgow
Received 15 May 1978; acceptedfor publication 15 June 1978
Summary. We have previously shown that lymphocyte emperipolesis, the phenomenon whereby lymphocytes wander freely within the cytoplasm of larger host cells, is almost totally dependent on host cell pre-sensitization with IgG type antibody. In addition, the particular lymphocyte subpopulation involved was partially characterized as an Fc receptor bearing, non-T cell. In this study we have attempted to further define the emperipoletic lymphocyte subpopulation using cell fractionation procedures and correlation analysis. Significant correlation was found between emperipolesis and both Fc receptor bearing, surface immunoglobulin negative (Fc+, SIg-) cells and antibody dependent lymphocyte mediated cytotoxicity; observations which suggest that the socalled K cells are the emperipoletic effector cells. The significance of these findings in relation to antibody dependent cytolysis is discussed.
penetrate the cytoplasm of larger 'host' cells and wander freely therein, i.e. emperipolesis, remains an aspect of lymphocyte behaviour which is little understood. In a previous publication we have reinvestigated this phenomenon and established a standard in vitro assay using human peripheral blood lymphocytes (PBL) as effector cells and Chang liver cells as host cells (Sandilands, Reid, Gray & Anderson, 1978). Emperipolesis was found to be virtually dependent on pre-sensitization of the host cells with rabbit antibody (IgG) and good correlation was demonstrated between the proportion of Fc receptor bearing PBL and the percentage of Chang cells with one or more intracytoplasmic lymphocyte (EMP). It is now well established that there are at least two major functional Fc receptor positive (Fc+) lymphocyte subpopulations: B cells, also characterized by the possession of surface stable immunoglobulin (SIg) and K cells (antibody dependent cytotoxic effector cells) which are SIg negative (Fc+, SIg-) (Sandilands, Gray, Cooney, Froebel & Anderson, 1976). More recently Moretta, Ferrarini, Mingari, Moretta & Webb (1976) have suggested that some resting T cells have receptors for the Fc portion of IgG (T y cells), the relationship of which to B and K cells remains uncertain. In the previous publication (Sandilands et al., 1978) it was shown that selective
INTRODUCTION The phenomenon whereby lymphocytes actively Correspondence: Dr Fiona M. Reid, University Department of Pathology, Western Infirmary, Glasgow G11 6NT.
0019-2805/79/0200-0367S02.00 © 1979 Blackwell Scientific Publications
367
368
Fiona M. Reid et al.
depletion of PBL capable of forming rosettes with sheep erythrocytes (i.e. T cells) failed to diminish emperipolesis, and human thymocyte suspensions were found to be non-emperipoletic. It would therefore seem likely that the majority of T cells do not participate in emperipolesis under the conditions used. In the present study we have investigated the role of B and K cells in emperipolesis. Several approaches were employed to investigate the behaviour of B cells in the emperipolesis assay. (1) The selective removal of SIg+ cells by adherence to nylon wool (2) the use of B cells derived from patients with chronic lymphatic leukaemia (CLL) and (3) pretreatment of effector cells with anti-Ig serum. In addition correlations were sought by conducting membrane marker tests for SIg and Fc receptors (EA rosette test) in parallel with the emperipolesis assay. The possible involvement of K cells in emperipolesis was investigated by conducting the assays for emperipolesis and antibody dependent lymphocyte cytotoxicity (ADC) in parallel.
MATERIALS AND METHODS Separation of human peripheral blood lymphocytes (PBL) Human PBL were separated from heparinized whole blood on a Ficoll-metrizoate column of specific gravity 1 077 as described by Boyum (1968). Aspirated PBL were washed twice in HEPES-buffered Eagle's medium (HEM), pH 6-9 (Gibco Laboratories, Glasgow) and adjusted to the required concentration in HEM supplemented with 10%0 foetal calf serum (FCS) and 1% glutamine (HEM + S). Unless otherwise stated the property of monocytes to adhere to the surface of a plastic dish was used for their depletion. Monocytes remaining in these preparations were identified by morphology and by use of the esterase stain as described by Horwitz, Allison, Ward & Kight (1977). The viability of these virtually pure lymphocyte suspensions, as assessed by the exclusion of trypan blue, was > 98%.
Emperipolesis assay This assay has been described previously in detail (Sandilands et al., 1978). Five million PBL, in 2 ml HEM + S were incubated at 370 for 2 h on a monolayer of antibody sensitized Chang liver cells (Gibco
Laboratories, Glasgow). Monolayers were prepared on the surface of 60 x 15 mm plastic tissue culture plates by incubating 1 x 106 Chang cells in 2 ml HEM + S at 370 for 2 h in an atmosphere of 5 % CO2. One hundred microlitres of heat inactivated rabbit anti-Chang cell antiserum (1/10 was added to each plate and following incubation at 370 for 1 h the sensitized cells were rinsed several times in HEM + S to remove unbound antibody. Non-adherent PBL were removed by gently washing the monolayer with HEM and the adherent cells were stained with Leishman's. The wet preparations were examined microscopically ( x 400) under a coverslip sealed with vaseline. Emperipolesis (EMP) was expressed as the mean percentage of Chang cells with one or more intracytoplasmic lymphocytes. These lymphocytes were in the same focal plane as the Chang cell cytoplasm and were surrounded by the characteristic clear unstainable halo previously described by Trowell (1949). Intracytoplasmic PBL were readily discernible from PBL attached to the side of a Chang cell or to its upper surface.
Chicken EA rosette test Equal volumes (250 ,1) of suspensions of chicken E sensitized with rabbit 7S antibody (1/500) and of PBL (4 x 106 cells/ml HEM) were mixed and centrifuged at 200 g for 5 min. The pelleted cells were gently resuspended by pipette, fixed with glutaraldehyde and stained with trypan blue as described elsewhere (Sandilands et al., 1976). The final stained cell suspensions were examined as wet preparations. At least 200 lymphocytes on each slide count and the mean percentage of lymphocytes forming rossettes, i.e. binding two or more chicken EA was determined.
Surface immunoglobulin (SIg) SIg was detected by the indirect immunofluorescence method as described by Brown & Greaves (1974). Lymphocytes were treated with rabbit anti-human immunoglobulin (RAHG) serum, (Blood Transfusion Service, Law Hospital, Carluke) previously absorbed with human thymocytes, followed by fluorescein-labelled goat anti-rabbit immunoglobulin serum (Behring AG-Marburg). At least 200 cells per slide were inspected for SIg using alternate white light to detect the cells and incident UV illumination to examine them for fluorescence. These examinations were performed on a Leitz Orthoplan microscope.
369
Lymphocyte emperipolesis revisited Removal of SIg-bearing cells by adherence to nylon wool Depletion of SIg-bearing cells was accomplished by incubating PBL suspended in 2 ml HEM for 1 h at 370 in the barrel of a 5 ml syringe containing 0-4 g of lightly packed nylon wool. Non-adherent cells were eluted from the column and tested in the SIg and emperipolesis assays. Pretreatment of PBL with anti-Ig serum PBL were incubated at room temperature for 30 min with polyvalent RAHG (1/5) which had previously been absorbed with human thymocytes. Following two washes in HEM the PBL suspension was adjusted to a concentration of 5 x 106 in 2 ml HEM+ S for testing in the emperipolesis assay.
Antibody dependent lymphocyte cytotoxicity (ADC) This test has been described in detail elsewhere (Sandilands et al., 1976). Briefly 1 x 104 Chang cells labelled with 5'Cr and sensitized with rabbit antibody (1 in 5000) were incubated at 370 for 20 h with 1 x 10 PBL in HEM +S. Tubes were then centrifuged at 400 g for O min and half of the supernate was removed to a second tube. From gamma counts on both tubes the percentage net lysis of Chang cells was calculated as previously described. In those experiments where ADC and emperipolesis assays were conducted in parallel 1 x 106 of 51Cr-treated Chang cells were incubated with 100 Aul rabbit anti-Chang cell serum (1/10) for 1 h at 37° then washed twice in HEM+ S. These Chang cells were then used in the ADC test described above using a PBL: Chang cell ratio of 5: 1.
Table 1. The effect of monocyte depletion on EMP Monocyte depleted
Control Expt no.
Mono
1 2 3 Mean
14 13 18 15
EMP
Mono
EMP
(%)
(%)
13 21 16 16-6
0 6 4 3-3
16 20 13 16-3
Table 2. Effect of passing lymphocytes through nylon wool columns on the percentage of surface immunoglobulin (SIg) bearing cells and emperipolesis (EMP). Mean+ S.D. of 3 experiments
Lymphocyte suspension Non-adherent cells Control
14-3±4-7 4-6+ 5-4
Sig (%) EMP (%)
3-322 7-7± 7-3
Table 3. The effect of pretreatment of lymphocytes with rabbit anti-human immunogolbulin (RAHG) serum on
emperipolesis (EMP)
Exp no. EMP (%Y)
1 2 3
Mean± S.D.
Lymphocyte suspension RAHG treated Control 10 20 7 12-3+ 6-8
10-5 19 5 10-5 13-5+ 5
RESULTS
(a) The relationship between EMP and monocytes As shown in Table 1 depletion of monocytes from the effector cell suspension by adherence to plastic had no effect on EMP. (b) The relationship between EMP and B cells (i) Selective depletion of SIg+ (B) cells. PBL suspensions recovered from nylon wool columns were tested for EMP and SIg. While it was apparent that non-adherent PBL were significantly depleted
of SIg+ cells (77% reduction), emperipolesis actually increased (Table 2).
was
(ii) Pretreatment of PBL with rabbit anti-human immunoglobulin (RAHG) serum. This was found to have no effect on EMP (Table 3).
(iii) Chronic lymphatic leukaemia (CLL) 'B' cells. In three separate experiments SIg+ PBL obtained from patients with CLL were found to be totally
non-emperipoletic.
Fiona M. Reid et al.
370 50 r
Table 4. Effect of incubation on antibody sensitized Chang cells on the ability of PBL to form EA-rosettes and on their ADC activity.
0
Net lysis (%) at PBL: Chang cell ratio of
40 V Expt no.
$ 30h *
0'
e
(I
U)
0 0
20k
IL
Control PBL before emperipolesis Mean+ S.D. PBL removed from emperipolesis plate Mean+S.D.
0
. 0 0
0
0
-
10j
1 2 3
1 2 3
EA+
(%)
10 :1
30 :1
17 38 33 29+ 10 9 4 6 55 5-2+1
0 32 27 19-7+ 17 0 0 0 0
16 58 65 46+ 26-5 9 0 0 3+5-2
0
0
0
10
20 EMP (%)
30
40
Figure 1. Correlation between the proportion of Fc+, SIg7 cells and emperipolesis (EMP). n=21; r=0-83; P=
Lymphocyte emperipolesis revisited II.
FURTHER CHARACTERIZATION OF THE LYMPHOCYTE SUBPOPULATION INVOLVED
FIONA M. REID, G. P. SANDILANDS, KATHLEEN G. GRAY & J. R. ANDERSON University Department of Pathology, Western Infirmary, Glasgow
Received 15 May 1978; acceptedfor publication 15 June 1978
Summary. We have previously shown that lymphocyte emperipolesis, the phenomenon whereby lymphocytes wander freely within the cytoplasm of larger host cells, is almost totally dependent on host cell pre-sensitization with IgG type antibody. In addition, the particular lymphocyte subpopulation involved was partially characterized as an Fc receptor bearing, non-T cell. In this study we have attempted to further define the emperipoletic lymphocyte subpopulation using cell fractionation procedures and correlation analysis. Significant correlation was found between emperipolesis and both Fc receptor bearing, surface immunoglobulin negative (Fc+, SIg-) cells and antibody dependent lymphocyte mediated cytotoxicity; observations which suggest that the socalled K cells are the emperipoletic effector cells. The significance of these findings in relation to antibody dependent cytolysis is discussed.
penetrate the cytoplasm of larger 'host' cells and wander freely therein, i.e. emperipolesis, remains an aspect of lymphocyte behaviour which is little understood. In a previous publication we have reinvestigated this phenomenon and established a standard in vitro assay using human peripheral blood lymphocytes (PBL) as effector cells and Chang liver cells as host cells (Sandilands, Reid, Gray & Anderson, 1978). Emperipolesis was found to be virtually dependent on pre-sensitization of the host cells with rabbit antibody (IgG) and good correlation was demonstrated between the proportion of Fc receptor bearing PBL and the percentage of Chang cells with one or more intracytoplasmic lymphocyte (EMP). It is now well established that there are at least two major functional Fc receptor positive (Fc+) lymphocyte subpopulations: B cells, also characterized by the possession of surface stable immunoglobulin (SIg) and K cells (antibody dependent cytotoxic effector cells) which are SIg negative (Fc+, SIg-) (Sandilands, Gray, Cooney, Froebel & Anderson, 1976). More recently Moretta, Ferrarini, Mingari, Moretta & Webb (1976) have suggested that some resting T cells have receptors for the Fc portion of IgG (T y cells), the relationship of which to B and K cells remains uncertain. In the previous publication (Sandilands et al., 1978) it was shown that selective
INTRODUCTION The phenomenon whereby lymphocytes actively Correspondence: Dr Fiona M. Reid, University Department of Pathology, Western Infirmary, Glasgow G11 6NT.
0019-2805/79/0200-0367S02.00 © 1979 Blackwell Scientific Publications
367
368
Fiona M. Reid et al.
depletion of PBL capable of forming rosettes with sheep erythrocytes (i.e. T cells) failed to diminish emperipolesis, and human thymocyte suspensions were found to be non-emperipoletic. It would therefore seem likely that the majority of T cells do not participate in emperipolesis under the conditions used. In the present study we have investigated the role of B and K cells in emperipolesis. Several approaches were employed to investigate the behaviour of B cells in the emperipolesis assay. (1) The selective removal of SIg+ cells by adherence to nylon wool (2) the use of B cells derived from patients with chronic lymphatic leukaemia (CLL) and (3) pretreatment of effector cells with anti-Ig serum. In addition correlations were sought by conducting membrane marker tests for SIg and Fc receptors (EA rosette test) in parallel with the emperipolesis assay. The possible involvement of K cells in emperipolesis was investigated by conducting the assays for emperipolesis and antibody dependent lymphocyte cytotoxicity (ADC) in parallel.
MATERIALS AND METHODS Separation of human peripheral blood lymphocytes (PBL) Human PBL were separated from heparinized whole blood on a Ficoll-metrizoate column of specific gravity 1 077 as described by Boyum (1968). Aspirated PBL were washed twice in HEPES-buffered Eagle's medium (HEM), pH 6-9 (Gibco Laboratories, Glasgow) and adjusted to the required concentration in HEM supplemented with 10%0 foetal calf serum (FCS) and 1% glutamine (HEM + S). Unless otherwise stated the property of monocytes to adhere to the surface of a plastic dish was used for their depletion. Monocytes remaining in these preparations were identified by morphology and by use of the esterase stain as described by Horwitz, Allison, Ward & Kight (1977). The viability of these virtually pure lymphocyte suspensions, as assessed by the exclusion of trypan blue, was > 98%.
Emperipolesis assay This assay has been described previously in detail (Sandilands et al., 1978). Five million PBL, in 2 ml HEM + S were incubated at 370 for 2 h on a monolayer of antibody sensitized Chang liver cells (Gibco
Laboratories, Glasgow). Monolayers were prepared on the surface of 60 x 15 mm plastic tissue culture plates by incubating 1 x 106 Chang cells in 2 ml HEM + S at 370 for 2 h in an atmosphere of 5 % CO2. One hundred microlitres of heat inactivated rabbit anti-Chang cell antiserum (1/10 was added to each plate and following incubation at 370 for 1 h the sensitized cells were rinsed several times in HEM + S to remove unbound antibody. Non-adherent PBL were removed by gently washing the monolayer with HEM and the adherent cells were stained with Leishman's. The wet preparations were examined microscopically ( x 400) under a coverslip sealed with vaseline. Emperipolesis (EMP) was expressed as the mean percentage of Chang cells with one or more intracytoplasmic lymphocytes. These lymphocytes were in the same focal plane as the Chang cell cytoplasm and were surrounded by the characteristic clear unstainable halo previously described by Trowell (1949). Intracytoplasmic PBL were readily discernible from PBL attached to the side of a Chang cell or to its upper surface.
Chicken EA rosette test Equal volumes (250 ,1) of suspensions of chicken E sensitized with rabbit 7S antibody (1/500) and of PBL (4 x 106 cells/ml HEM) were mixed and centrifuged at 200 g for 5 min. The pelleted cells were gently resuspended by pipette, fixed with glutaraldehyde and stained with trypan blue as described elsewhere (Sandilands et al., 1976). The final stained cell suspensions were examined as wet preparations. At least 200 lymphocytes on each slide count and the mean percentage of lymphocytes forming rossettes, i.e. binding two or more chicken EA was determined.
Surface immunoglobulin (SIg) SIg was detected by the indirect immunofluorescence method as described by Brown & Greaves (1974). Lymphocytes were treated with rabbit anti-human immunoglobulin (RAHG) serum, (Blood Transfusion Service, Law Hospital, Carluke) previously absorbed with human thymocytes, followed by fluorescein-labelled goat anti-rabbit immunoglobulin serum (Behring AG-Marburg). At least 200 cells per slide were inspected for SIg using alternate white light to detect the cells and incident UV illumination to examine them for fluorescence. These examinations were performed on a Leitz Orthoplan microscope.
369
Lymphocyte emperipolesis revisited Removal of SIg-bearing cells by adherence to nylon wool Depletion of SIg-bearing cells was accomplished by incubating PBL suspended in 2 ml HEM for 1 h at 370 in the barrel of a 5 ml syringe containing 0-4 g of lightly packed nylon wool. Non-adherent cells were eluted from the column and tested in the SIg and emperipolesis assays. Pretreatment of PBL with anti-Ig serum PBL were incubated at room temperature for 30 min with polyvalent RAHG (1/5) which had previously been absorbed with human thymocytes. Following two washes in HEM the PBL suspension was adjusted to a concentration of 5 x 106 in 2 ml HEM+ S for testing in the emperipolesis assay.
Antibody dependent lymphocyte cytotoxicity (ADC) This test has been described in detail elsewhere (Sandilands et al., 1976). Briefly 1 x 104 Chang cells labelled with 5'Cr and sensitized with rabbit antibody (1 in 5000) were incubated at 370 for 20 h with 1 x 10 PBL in HEM +S. Tubes were then centrifuged at 400 g for O min and half of the supernate was removed to a second tube. From gamma counts on both tubes the percentage net lysis of Chang cells was calculated as previously described. In those experiments where ADC and emperipolesis assays were conducted in parallel 1 x 106 of 51Cr-treated Chang cells were incubated with 100 Aul rabbit anti-Chang cell serum (1/10) for 1 h at 37° then washed twice in HEM+ S. These Chang cells were then used in the ADC test described above using a PBL: Chang cell ratio of 5: 1.
Table 1. The effect of monocyte depletion on EMP Monocyte depleted
Control Expt no.
Mono
1 2 3 Mean
14 13 18 15
EMP
Mono
EMP
(%)
(%)
13 21 16 16-6
0 6 4 3-3
16 20 13 16-3
Table 2. Effect of passing lymphocytes through nylon wool columns on the percentage of surface immunoglobulin (SIg) bearing cells and emperipolesis (EMP). Mean+ S.D. of 3 experiments
Lymphocyte suspension Non-adherent cells Control
14-3±4-7 4-6+ 5-4
Sig (%) EMP (%)
3-322 7-7± 7-3
Table 3. The effect of pretreatment of lymphocytes with rabbit anti-human immunogolbulin (RAHG) serum on
emperipolesis (EMP)
Exp no. EMP (%Y)
1 2 3
Mean± S.D.
Lymphocyte suspension RAHG treated Control 10 20 7 12-3+ 6-8
10-5 19 5 10-5 13-5+ 5
RESULTS
(a) The relationship between EMP and monocytes As shown in Table 1 depletion of monocytes from the effector cell suspension by adherence to plastic had no effect on EMP. (b) The relationship between EMP and B cells (i) Selective depletion of SIg+ (B) cells. PBL suspensions recovered from nylon wool columns were tested for EMP and SIg. While it was apparent that non-adherent PBL were significantly depleted
of SIg+ cells (77% reduction), emperipolesis actually increased (Table 2).
was
(ii) Pretreatment of PBL with rabbit anti-human immunoglobulin (RAHG) serum. This was found to have no effect on EMP (Table 3).
(iii) Chronic lymphatic leukaemia (CLL) 'B' cells. In three separate experiments SIg+ PBL obtained from patients with CLL were found to be totally
non-emperipoletic.
Fiona M. Reid et al.
370 50 r
Table 4. Effect of incubation on antibody sensitized Chang cells on the ability of PBL to form EA-rosettes and on their ADC activity.
0
Net lysis (%) at PBL: Chang cell ratio of
40 V Expt no.
$ 30h *
0'
e
(I
U)
0 0
20k
IL
Control PBL before emperipolesis Mean+ S.D. PBL removed from emperipolesis plate Mean+S.D.
0
. 0 0
0
0
-
10j
1 2 3
1 2 3
EA+
(%)
10 :1
30 :1
17 38 33 29+ 10 9 4 6 55 5-2+1
0 32 27 19-7+ 17 0 0 0 0
16 58 65 46+ 26-5 9 0 0 3+5-2
0
0
0
10
20 EMP (%)
30
40
Figure 1. Correlation between the proportion of Fc+, SIg7 cells and emperipolesis (EMP). n=21; r=0-83; P=
