Developmentaland ComparativeImmunology,Vol. 14, pp. 359-365, 1990 Printed in the USA. All rights reserved.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
VARIABILITY OF NATURAL KILLER CELL ACTIVITY IN ANURAN AMPHIBIANS M a m d o o h G h o n e u m , * E d w i n L. C o o p e r , t and Ismail Sadek~: *Department of Otolaryngology, Charles R Drew, University of Medicine and Science, Los Angeles, CA 90059; *.tDepartment of Anatomy and Cell Biology, UCLA School of Medicine. Los Angeles, CA 90024; :l:Department of Zoology, Faculty of Science, Alexandria University, Egypt
(Submitted March 1987;Accepted April 1989) [~Keywords--NK cell activity; frogs; anuran
amphibians; conjugates; lytic units; spleen; peripheral blood; bone marrow.
Introduction Our recent studies have demonstrated that frogs possess cytotoxic cells which appear functionally similar to mammalian NK cells (6-8,16,26,27,29-31). We have shown that N K cells of ectothermic vertebrates cause the destruction of allogeneic target cells and xenogeneic tumor cells in vitro. Frog (6,7) and mammalian NK cells (5,11-13) respond similarly to certain inhibitory drugs, such as EDTA, DMSO, glutaraldehyde and trypsin. Furthermore, EM studies have revealed similarities between frog and mammalian NK with respect to cell structure and intracellular changes after they bind to targets (31). This report is designed to compare patterns of NK activity with respect to amphibian species and tissue distribution.
Materials and Methods
N A S C O West, M o d e s t o , CA. The average weight per animal was 20g, 100g, and 270g, respectively. Frogs and toads were kept at 22°C in tilted plastic pans with water at the lower end which was changed twice weekly. They were fed meal worms twice per week when the water was changed and allowed to adapt to laboratory conditions for 2 weeks prior to experimentation.
Harvesting Putative Effector Cells After a n e s t h e t i z a t i o n with ether, spleens were dissected and teased in Amphibian Ringer Solution (ARS). Bone m a r r o w was r e m o v e d in ARS by flushing it several times using a 1 mL syringe with a 27 gauge needle, then washed twice in ARS. Heparinized peripheral blood was obtained by cardiac puncture and washed twice in ARS. All effector cells were adjusted to 10 x 106 cells/mE
Target Cells
Animals and Maintenance Adult frogs and toads of both sexes, Rana pipiens, Rana catesbeiana and Bufo americanus, were obtained from Address c o r r e s p o n d e n c e to Dr. M. Ghoneum, Department of Otolaryngology, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059.
The YAC-1 tumor cell line, a Maloney Leukemia virus induced mouse T-cell lymphoma of A/Sn mouse origin, was maintained in our laboratory in complete m e d i u m ( C M ) . CM c o n s i s t e d o f RPMI-1640, supplemented with 10% h e a t - i n a c t i v a t e d fetal bovine serum (FBS) and 1% antibiotics (Grand Island Biologicals, Santa Clara, CA).
359
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Cytotoxicity Assay YAC-1 target cells (5 x 106 cells) cultured for 24 h were labeled with 100 i~Ci Na2CrO 4 solution (New England, Nuclear, Boston, MA) for 1 h and washed four times in 5 mL Hanks balanced salt solution (HBSS) as described before (Ghoneum et al. 1987c). Cells (1 x 104) in 0.1 mL CM were pipetted into 96 well-rounded b o t t o m Linbro plates (Linbro Chemical Co., Hamden, CT). Effector cells were suspended at 10 × 106 mL ARS, and pipetted into quadruplicate wells to give effector target (E:T) cell ratios of 100:1, 50:1, and 25:1. Following incubation for 4 h at room temperature, plates were centrifuged at 1400 rpm for 5 min, the supernatant (0.1 mL) from each well was c o l l e c t e d and counted in triplicate in a gamma counter. The percentage of isotope released was calculated using the following formula: % lysis =
(LUX Scientific Corporation, Thousand Oaks, CA) which were precoated with agarose. Later, 1 mL CM was added to each well to avoid dehydration and control wells were prepared without using effector cells. The plates were incubated for 3 h at 22°C and 5% CO2, then stained with 0.1% trypan blue for 5 min. The percentages of leukocytes binding to target cells and those lysing their targets were determined by the same investigator using light microscopy. Viability of target cells was not less than 98% before beginning the experiments.
Lytic Units (LU) LUs were calculated from effector titration curves according to previous procedures (9). One LU was defined as the number of effector cells required to achieve 15% percent lysis. Absolute cy-
Exp. release - Sp. release Max. release - Sp. release
Spontaneous release (Sp) from target cells was always 8-10% of the maximum release (Max). Maximum release was measured by adding 0.1 mL Triton X-100 to design~/ted wells.
X
100
totoxicity was also calculated, that is, LUs per total number of leukocytes from spleen, peripheral blood, or bone marrow.
Statistical Analysis Single Cell Cytotoxicity Assay in Agarose A single cell assay developed by Grimm and Bonavida (1979) was employed using our own slight modification. Briefly, leukocytes were mixed with equal numbers of target cells (0.5 × 106) in 0.5 mL CM. Effector and target cells were incubated for 5 min at room temperature (22°C), centrifuged at 800 rpm for 5 min, the pellets were resuspended gently and added to 2.5 mL 0.5% agarose (Type A, Sigma, St. Louis, MO), precooled to 41°C. Small aliquots were then pipetted to multiplate 8 wells
The results are based on leukocytes recovered from 4-5 frogs and toads in each group. A two-tailed Student's t test was used to determine the significance of differences in relative and absolute NK cell activity and percentage of effector: target cell binding and killing.
Results
Relative NK Cell Activity Relative NK activity refers to the percentage lysis of target cells by a specific
Variability of NK activity
361
120 IIO E)O
Spleen
PBL
OM
;90 80 ~- 7 0
.~ 6 0
~
50 40 30 20 IO
R~.
R.r-
B.a.
R.p.
Re.
ito.
nnm
R.p.
n..c.
e.=.
Figure 1. Relative NK cell activity in spleen, peripheral blood (PBL), and bone marrow (BM). Leukocytes were derived from Rana pipiens--R.p. ([7), Rana catesbiena--R.c. ([]), and Bufo arnericanus--B.a. (i). Activity was calculated per 1 x 107 effector cells. Data represent Mean _+ SD of 4 - 5 animals examined separately. [*P < 0.025, **P < 0.05 compared with R.p.]
number of effector cells (1 × 107) in spleens, PBL, and BM from three species (Fig. 1). Splenic NK cell levels varied considerably. R. pipiens showed the highest (39 LU), followed by R. catesbeiana (29 LU), while B. americanus had the lowest (10 LU). Peripheral blood NK activity revealed 34, 23, and 17 LU in R. pipiens, R. catesbeiana, and B. americanus respectively. Bone marrow NK activity showed similar patterns of species dependent values.
Absolute N K Cell Activity
Absolute values were calculated by multiplying values obtained from relative NK activity by the total number of cells in each tissue examined. Absolute values were in contrast to relative ones. For example, absolute splenic activity was the reverse in all three species: B. americanus > R. catesbeiana > R. pipiens. Similarly, PBL from B. americanus had the highest activity, followed by R. catesbeiana and R. pipiens which
had the lowest. NK activity of bone marrow cells was higher in B. americanus in comparison with that of R. catesbeiana, and again, lowest levels were detected in R. pipiens (Fig. 2).
Effector: Target Cell Binding and Killing
To analyze possible mechanisms responsible for differences in NK activity among the three species, a single cell assay in agarose was employed. This assay can distinguish between binding capacity of effector cells to their targets and subsequent lysis. Figure 3 shows insignificant changes in the binding capacity of effector cells: (i) by tissue distribution within the same species, and (ii) among different species. These values expressing binding capacity ranged between 10-17%. In contrast, the lytic effect varied significantly among the three species. Effector cells from B. americanus had significantly lower values (12-20%) when compared
362
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150-
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B.
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_'=~_ 80 =. ro -] 60
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B.a.
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R.~
B.o.
Figure 2. Absolute NK activity in spleen, peripheral blood (PBL), and bone marrow (BM). Leukocytes were isolated from R.p. ([-]), R.c. ([]), and B.a. (1). Activity was calculated per total leukocytes in each tissue. Mean - SD of 4 - 5 animals examined separately. [*P < 0.001, **P < 0.025 compared with R.p.]
to R. pipiens (35-40%). Those for R. catesbeiana showed values between the other two species.
Discussion The role of NK cells in host surveillance against malignancy has been established. Talmadge et al. 1980 found that an NK susceptible syngeneic melanoma cell line grew more rapidly in beige mice which have lower N K activity than normal mice. With respect to anuran amphibians, we have recently demonstrated susceptibility of Bufo regularis to tumor induction after treatment with chemical carcinogens and cocarcinogens (25). Other models have also been examined, such as R. pipiens (23,24), and X e n o p u s laevis (2,15), revealing different susceptibilities to tumor development. Therefore, for the future it seems of interest to examine whether susceptibility to tumor development in anurans is dependent upon variations in NK activity.
In the present study, we found that relative N K activity is species dependent: R. pipiens > R. catesbeiana > B. americanus. These results are apparently similar to levels of mammalian NK activity which also vary according to mouse strains (14), it is an autosomal dominant trait (19). The mechanism for lower N K activity in B. a m e r i c a n u s when compared with R. pipiens is unknown, however, it might be explained in the following manner. NK-tumor cell interaction proceeds through several discrete stages which include: (i) eff e c t o r - t a r g e t cell r e c o g n i t i o n and binding, (ii) triggering and activation of N K cells, (iii) release of lytic factor ( N K C F ) , and (iv) target cell death (11,33). Results of our single cell assays revealed similar species binding capacity of NK cells to YAC-1 targets, but the lytic effect is different: R. pipiens had at least 2 - 3 fold higher values than B. americanus. With respect to absolute NK activity, the values were in contrast to those calculated for relative activity: B. americanus > R. catesbeiana > R.
Variability of NK activity
363
45"
SPLEEN
0-
..k.
T
35"
=3025-
o5i I0-
R.p.
Re.
B.a.
R~.
Ba.
--l-R.p.
Rc.
B.a.
Rp.
Rc.
I
._~p.
PBL
35" 308 25-
g zo.
g
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o
15-
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.
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R.c
B.a. BM
4035-
0~25-
g
~'20o
15-
IO. Rp.
R.c
B.a.
B.a.
Figure 3. Percentage of binding between leukocytes and YAC-I target ceils and percentage of bound leukoc~es lysing their targets. Leukocytes were isolated from spleen, peripheral blood (PBL), and bone marrow (BM): R.p. (r-l), R.c. (1), and B.a. (1). Mean _ SD of 4-5 animals examined separately. [*P < 0.001 compared with R.p.].
pipiens. Of course, this difference is a reflection of increased cellularity of spleen, PBL, and BM in B. americanus when compared to R. pipiens.
We have found high levels of NK activity in spleen, PBL, and bone marrow. The unique result relevant to previous work is demonstrating NK activity in
364
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bone marrow since anuran amphibians are the first terrestrial vertebrates to possess it. We already know from previous studies that frog bone marrow may be a source of effector cells for transplantation immunity and antibody synthesis in heavily irradiated animals, suggesting the presence of T and B cells or their precursors (4,21,22). As for mammalian NK cells, several studies have indicated that spleen and PBL are rich sources of N K activity, while bone
marrow exhibits only negligible levels of cytotoxicity (1,14,20). Nevertheless, it has been suggested by Lanier et al. (17) and Lanier and Phillips (18) that true NK cells which are non-MHC restricted, in contrast to CTL, require the bone marrow microenvironment for full differentiation. This supports an interesting phylogenetic speculation concerning the origin of NK cells since amphibians occupy a crucial point in the evolution of terrestial vertebrates (3,28).
References 1. Antonelli, P.; Stewart, W., II; Dupont, B. Distribution of natural killer cell activity in peripheral blood, cord blood, thymus, lymph nodes and spleen and the effect of in vitro treatment with interferon preparation. Clin. Immunoi. Immunopathol. 19:161-169; 1981. 2. Balls, M.; Ruben, L. N. The transmission of lymphosarcoma in Xenopus laevis, the South African clawed toad. Cancer Res. 27:654-659; 1967. 3. Cooper, E. L. Phylogeny of cytotoxicity. Endeavor. 4:160-165; 1980. 4. Cooper, E. L.; Schaefer, D. W. Bone marrow restoration of transplantation immunity in the leopard frog Rana pipiens. Proc. Soc. Exp. Biol. Med. 135:406-411; 1970. 5. Deem, R. L.; Targan, S. R. Evidence of a dynamic role of the target cell membrane during the early stages of the natural killer cell lethal hit. J. Immunol. 133:72-77; 1984. 6. Ghoneum, M.; Cooper, E. L. Inhibition of frog SK effector-target cell binding. Dev. Comp. Immunol. 11:167-178; 1987. 7. Ghoneum, M.; Cooper, E. L.; Smith, C. Inhibition of SK cell activity in frogs by certain drugs and sugars. Dev. Comp. Immunol. I 1:363-373; 1987a. 8. Ghoneum, M.; Smith, C. T.; Cooper E. L. Membrane markers and mitogenic responses of cytotoxic cells in anuran amphibians. Cell. Mol. Biol. 33:807-817; 1987b. 9. Ghoneum, M.; Gill, G.; Wojdani, A.; Payne, C.; Alfred, L. J. Suppression of basal and Corynebacterium parvum-augmented NK activity during chemically induced tumor development. Int. J. Immunopharmacol. 9:71-79; 1987c. 10. Grimm, E.; Bonavida, B. Mechanism of cellmediated cytotoxicity at the single cell level. I. Estimation of cytotoxic T lymphocyte frequency and relative lytic efficiency. J. Immunol. 123:2861-2869; 1979. 11. Hiserodt, J. C.; Britvan, L.; Targan, S. R. Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, program-
12.
13.
14.
15.
16.
17.
18.
19. 20. 21. 22.
ming and killer cell-independent steps. J. Immunol. 129:1782-1787; 1982a. Hiserodt, J. C.; Britvan, L.; Targan, S. R. Differential effects of various pharmacologic agents on the cytolytic reaction mechanism of the human natural killer lymphocytes: further resolution of programming for iysis and KCIL into discrete steps. J. Immunol. 129:22662270; 1982b. Hiserodt, J. C.; Britvan, L.; Targan, S. R. Studies on the mechanism of the human natural killer cell lethal hit: analysis of the mechanism of protease inhibition of the lethal hit. J. Immunol. 131:2705-2709; 1983. Itoh, K.; Suzuki, R.; Umezu, Y.; Hanaumi, K.; Kumagai, K. Studies of murine large granular lymphocytes, lI. Tissue, strain and age distribution of LGL and LAL. J. Immunol. 129:395-405; 1982. Khudoley, V. V.; Picard, J. J. Liver and kidney tumors induced by N-nitrose-diemethylamine in Xenopus borealis (Parker). Int. J. Cancer. 25:679-683, 1980. Klempau, A. E.; Cooper, E. L. T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians. III: Assessment and indentification of iducible killer T-lymphocytes (IKTL) and spontaneous killer T-lymphocytes (SKTL). Dev. Comp. Immunol. 8:649-661; 1984. Lanier, L. L.; Phillips, J. H.; Hackett, J.; Tutt, M.; Kumar, V. Natural killer cells: definition of cell type rather than a function. J. Immunol. 137:2735-2739; 1986. Lanier, L. L.; Phillips, J. H. What are natural killer cells? ISI atlas of science: immunology. Philadelphia, PA: Institute of Scientific Information; 1988:p. 15. Petranyi, G. G.; Kiessling, R.; Klein, G. Genetic control of natural killer lymphocytes in the mouse. Immunogenetics. 2:53-61; 1975. Potter, M. R.; Moore, M. Organ distribution of natural cytotoxicity in the rat. Clin. Exp. Immunol. 34:78-86; 1978. Ramirez, J. A.; Cooper, E. L. Detecting plaque forming cells in frog bone marrow. Dev. Comp. Immunol. 4:173-175; 1980. Ramirez, J. A.; Wright, R. K.; Cooper, E. L.
Variability of NK activity
23.
24.
25.
26.
27.
Further evidence for the role of bone marrow from Rana pipiens in immune responses. Dev. Comp. Immunol. 7:303-312; 1983. Rolfins-Smith, L. A.; Cohen, N. Effect of thymectomy on development of Lucke renal adenocarcinoma in virus-infected leopard frog tadpoles. J. Natl. Cancer Inst. 68:133-138; 1982. Rollins-Smith, L. A.; Cohen, N. Effect of thyroxine on induction of Lucke renal adenocarcinomas in Lucke tumor herpes virus-infected leopard frog tadpoles. J. Natl. Cancer Inst. 73:717, 1986. Sadek, I. A. The Egyptian toad as a sensitive model to show the effect of corn oil on liver tumor induced by DMBA. Nutr. Res. 6:333335; 1986. Sadek, I. A.; Ghoneum, M.; Cooper, E. L. Effect of 20-methylcholanthrene on amphibian natural killer cells. Dis. Aquat. Org. 3:155158, 1987a. Sadek, I. A.; Ghoneum, M.; Cooper, E. L. Retinoic acid and its effect on natural killer cells in toads. J. Nutr. Growth Can. 4:191197; 1987b.
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28. Savary, C. A.; Lotzova, E. Phylogeny and ontogeny of NK cells. In: Immunobiology of natural killer cells. Lotzova, E.; Herberman, R. B.; eds. Boca Raton, FL: CRC Press, Inc.; 1986:45. 29. Smith, C. T.; Cooper, E. L. Identification of cytotoxic cells in Rana. Fed. Proc. 42:937; 1983. 30. Smith, C. T.; Cooper, E. L. Characteristics of putative NK and CTL in frogs. Fed. Proc. 44:1921; 1985. 31. Smith, C. T.; Ghoneum, M.; Cooper, E. L. Ultrastructure of cytotoxic cells in frogs. Acta Zool. 69:125-133; 1988. 32. Talmadge, J. E.; Meyers, K. M.; Prieur, D. J.; Starkey, J. R. Role of NK cells in tumor growth and metastases in beige mice. Nature. 284:622-624; 1980. 33. Wright, S. C.; Bonavida, B. Studies on the mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC). I. Release of cytotoxic factors specific for NK-sensitive target cells (NKCF) during co-culture of NK effector cells with NK target cells. J. Immunol. 129:433-439; 1982.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
VARIABILITY OF NATURAL KILLER CELL ACTIVITY IN ANURAN AMPHIBIANS M a m d o o h G h o n e u m , * E d w i n L. C o o p e r , t and Ismail Sadek~: *Department of Otolaryngology, Charles R Drew, University of Medicine and Science, Los Angeles, CA 90059; *.tDepartment of Anatomy and Cell Biology, UCLA School of Medicine. Los Angeles, CA 90024; :l:Department of Zoology, Faculty of Science, Alexandria University, Egypt
(Submitted March 1987;Accepted April 1989) [~Keywords--NK cell activity; frogs; anuran
amphibians; conjugates; lytic units; spleen; peripheral blood; bone marrow.
Introduction Our recent studies have demonstrated that frogs possess cytotoxic cells which appear functionally similar to mammalian NK cells (6-8,16,26,27,29-31). We have shown that N K cells of ectothermic vertebrates cause the destruction of allogeneic target cells and xenogeneic tumor cells in vitro. Frog (6,7) and mammalian NK cells (5,11-13) respond similarly to certain inhibitory drugs, such as EDTA, DMSO, glutaraldehyde and trypsin. Furthermore, EM studies have revealed similarities between frog and mammalian NK with respect to cell structure and intracellular changes after they bind to targets (31). This report is designed to compare patterns of NK activity with respect to amphibian species and tissue distribution.
Materials and Methods
N A S C O West, M o d e s t o , CA. The average weight per animal was 20g, 100g, and 270g, respectively. Frogs and toads were kept at 22°C in tilted plastic pans with water at the lower end which was changed twice weekly. They were fed meal worms twice per week when the water was changed and allowed to adapt to laboratory conditions for 2 weeks prior to experimentation.
Harvesting Putative Effector Cells After a n e s t h e t i z a t i o n with ether, spleens were dissected and teased in Amphibian Ringer Solution (ARS). Bone m a r r o w was r e m o v e d in ARS by flushing it several times using a 1 mL syringe with a 27 gauge needle, then washed twice in ARS. Heparinized peripheral blood was obtained by cardiac puncture and washed twice in ARS. All effector cells were adjusted to 10 x 106 cells/mE
Target Cells
Animals and Maintenance Adult frogs and toads of both sexes, Rana pipiens, Rana catesbeiana and Bufo americanus, were obtained from Address c o r r e s p o n d e n c e to Dr. M. Ghoneum, Department of Otolaryngology, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059.
The YAC-1 tumor cell line, a Maloney Leukemia virus induced mouse T-cell lymphoma of A/Sn mouse origin, was maintained in our laboratory in complete m e d i u m ( C M ) . CM c o n s i s t e d o f RPMI-1640, supplemented with 10% h e a t - i n a c t i v a t e d fetal bovine serum (FBS) and 1% antibiotics (Grand Island Biologicals, Santa Clara, CA).
359
360
M. Ghoneum et al.
Cytotoxicity Assay YAC-1 target cells (5 x 106 cells) cultured for 24 h were labeled with 100 i~Ci Na2CrO 4 solution (New England, Nuclear, Boston, MA) for 1 h and washed four times in 5 mL Hanks balanced salt solution (HBSS) as described before (Ghoneum et al. 1987c). Cells (1 x 104) in 0.1 mL CM were pipetted into 96 well-rounded b o t t o m Linbro plates (Linbro Chemical Co., Hamden, CT). Effector cells were suspended at 10 × 106 mL ARS, and pipetted into quadruplicate wells to give effector target (E:T) cell ratios of 100:1, 50:1, and 25:1. Following incubation for 4 h at room temperature, plates were centrifuged at 1400 rpm for 5 min, the supernatant (0.1 mL) from each well was c o l l e c t e d and counted in triplicate in a gamma counter. The percentage of isotope released was calculated using the following formula: % lysis =
(LUX Scientific Corporation, Thousand Oaks, CA) which were precoated with agarose. Later, 1 mL CM was added to each well to avoid dehydration and control wells were prepared without using effector cells. The plates were incubated for 3 h at 22°C and 5% CO2, then stained with 0.1% trypan blue for 5 min. The percentages of leukocytes binding to target cells and those lysing their targets were determined by the same investigator using light microscopy. Viability of target cells was not less than 98% before beginning the experiments.
Lytic Units (LU) LUs were calculated from effector titration curves according to previous procedures (9). One LU was defined as the number of effector cells required to achieve 15% percent lysis. Absolute cy-
Exp. release - Sp. release Max. release - Sp. release
Spontaneous release (Sp) from target cells was always 8-10% of the maximum release (Max). Maximum release was measured by adding 0.1 mL Triton X-100 to design~/ted wells.
X
100
totoxicity was also calculated, that is, LUs per total number of leukocytes from spleen, peripheral blood, or bone marrow.
Statistical Analysis Single Cell Cytotoxicity Assay in Agarose A single cell assay developed by Grimm and Bonavida (1979) was employed using our own slight modification. Briefly, leukocytes were mixed with equal numbers of target cells (0.5 × 106) in 0.5 mL CM. Effector and target cells were incubated for 5 min at room temperature (22°C), centrifuged at 800 rpm for 5 min, the pellets were resuspended gently and added to 2.5 mL 0.5% agarose (Type A, Sigma, St. Louis, MO), precooled to 41°C. Small aliquots were then pipetted to multiplate 8 wells
The results are based on leukocytes recovered from 4-5 frogs and toads in each group. A two-tailed Student's t test was used to determine the significance of differences in relative and absolute NK cell activity and percentage of effector: target cell binding and killing.
Results
Relative NK Cell Activity Relative NK activity refers to the percentage lysis of target cells by a specific
Variability of NK activity
361
120 IIO E)O
Spleen
PBL
OM
;90 80 ~- 7 0
.~ 6 0
~
50 40 30 20 IO
R~.
R.r-
B.a.
R.p.
Re.
ito.
nnm
R.p.
n..c.
e.=.
Figure 1. Relative NK cell activity in spleen, peripheral blood (PBL), and bone marrow (BM). Leukocytes were derived from Rana pipiens--R.p. ([7), Rana catesbiena--R.c. ([]), and Bufo arnericanus--B.a. (i). Activity was calculated per 1 x 107 effector cells. Data represent Mean _+ SD of 4 - 5 animals examined separately. [*P < 0.025, **P < 0.05 compared with R.p.]
number of effector cells (1 × 107) in spleens, PBL, and BM from three species (Fig. 1). Splenic NK cell levels varied considerably. R. pipiens showed the highest (39 LU), followed by R. catesbeiana (29 LU), while B. americanus had the lowest (10 LU). Peripheral blood NK activity revealed 34, 23, and 17 LU in R. pipiens, R. catesbeiana, and B. americanus respectively. Bone marrow NK activity showed similar patterns of species dependent values.
Absolute N K Cell Activity
Absolute values were calculated by multiplying values obtained from relative NK activity by the total number of cells in each tissue examined. Absolute values were in contrast to relative ones. For example, absolute splenic activity was the reverse in all three species: B. americanus > R. catesbeiana > R. pipiens. Similarly, PBL from B. americanus had the highest activity, followed by R. catesbeiana and R. pipiens which
had the lowest. NK activity of bone marrow cells was higher in B. americanus in comparison with that of R. catesbeiana, and again, lowest levels were detected in R. pipiens (Fig. 2).
Effector: Target Cell Binding and Killing
To analyze possible mechanisms responsible for differences in NK activity among the three species, a single cell assay in agarose was employed. This assay can distinguish between binding capacity of effector cells to their targets and subsequent lysis. Figure 3 shows insignificant changes in the binding capacity of effector cells: (i) by tissue distribution within the same species, and (ii) among different species. These values expressing binding capacity ranged between 10-17%. In contrast, the lytic effect varied significantly among the three species. Effector cells from B. americanus had significantly lower values (12-20%) when compared
362
M. Ghoneum et al.
150-
,0,-°
B.
II I g I
,oo-
_'=~_ 80 =. ro -] 60
Iq.l~
Rr=
8A
R.p.
R.~
B.a.
Rp.
R.~
B.o.
Figure 2. Absolute NK activity in spleen, peripheral blood (PBL), and bone marrow (BM). Leukocytes were isolated from R.p. ([-]), R.c. ([]), and B.a. (1). Activity was calculated per total leukocytes in each tissue. Mean - SD of 4 - 5 animals examined separately. [*P < 0.001, **P < 0.025 compared with R.p.]
to R. pipiens (35-40%). Those for R. catesbeiana showed values between the other two species.
Discussion The role of NK cells in host surveillance against malignancy has been established. Talmadge et al. 1980 found that an NK susceptible syngeneic melanoma cell line grew more rapidly in beige mice which have lower N K activity than normal mice. With respect to anuran amphibians, we have recently demonstrated susceptibility of Bufo regularis to tumor induction after treatment with chemical carcinogens and cocarcinogens (25). Other models have also been examined, such as R. pipiens (23,24), and X e n o p u s laevis (2,15), revealing different susceptibilities to tumor development. Therefore, for the future it seems of interest to examine whether susceptibility to tumor development in anurans is dependent upon variations in NK activity.
In the present study, we found that relative N K activity is species dependent: R. pipiens > R. catesbeiana > B. americanus. These results are apparently similar to levels of mammalian NK activity which also vary according to mouse strains (14), it is an autosomal dominant trait (19). The mechanism for lower N K activity in B. a m e r i c a n u s when compared with R. pipiens is unknown, however, it might be explained in the following manner. NK-tumor cell interaction proceeds through several discrete stages which include: (i) eff e c t o r - t a r g e t cell r e c o g n i t i o n and binding, (ii) triggering and activation of N K cells, (iii) release of lytic factor ( N K C F ) , and (iv) target cell death (11,33). Results of our single cell assays revealed similar species binding capacity of NK cells to YAC-1 targets, but the lytic effect is different: R. pipiens had at least 2 - 3 fold higher values than B. americanus. With respect to absolute NK activity, the values were in contrast to those calculated for relative activity: B. americanus > R. catesbeiana > R.
Variability of NK activity
363
45"
SPLEEN
0-
..k.
T
35"
=3025-
o5i I0-
R.p.
Re.
B.a.
R~.
Ba.
--l-R.p.
Rc.
B.a.
Rp.
Rc.
I
._~p.
PBL
35" 308 25-
g zo.
g
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o
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.
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B.a.
B.a.
Figure 3. Percentage of binding between leukocytes and YAC-I target ceils and percentage of bound leukoc~es lysing their targets. Leukocytes were isolated from spleen, peripheral blood (PBL), and bone marrow (BM): R.p. (r-l), R.c. (1), and B.a. (1). Mean _ SD of 4-5 animals examined separately. [*P < 0.001 compared with R.p.].
pipiens. Of course, this difference is a reflection of increased cellularity of spleen, PBL, and BM in B. americanus when compared to R. pipiens.
We have found high levels of NK activity in spleen, PBL, and bone marrow. The unique result relevant to previous work is demonstrating NK activity in
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bone marrow since anuran amphibians are the first terrestrial vertebrates to possess it. We already know from previous studies that frog bone marrow may be a source of effector cells for transplantation immunity and antibody synthesis in heavily irradiated animals, suggesting the presence of T and B cells or their precursors (4,21,22). As for mammalian NK cells, several studies have indicated that spleen and PBL are rich sources of N K activity, while bone
marrow exhibits only negligible levels of cytotoxicity (1,14,20). Nevertheless, it has been suggested by Lanier et al. (17) and Lanier and Phillips (18) that true NK cells which are non-MHC restricted, in contrast to CTL, require the bone marrow microenvironment for full differentiation. This supports an interesting phylogenetic speculation concerning the origin of NK cells since amphibians occupy a crucial point in the evolution of terrestial vertebrates (3,28).
References 1. Antonelli, P.; Stewart, W., II; Dupont, B. Distribution of natural killer cell activity in peripheral blood, cord blood, thymus, lymph nodes and spleen and the effect of in vitro treatment with interferon preparation. Clin. Immunoi. Immunopathol. 19:161-169; 1981. 2. Balls, M.; Ruben, L. N. The transmission of lymphosarcoma in Xenopus laevis, the South African clawed toad. Cancer Res. 27:654-659; 1967. 3. Cooper, E. L. Phylogeny of cytotoxicity. Endeavor. 4:160-165; 1980. 4. Cooper, E. L.; Schaefer, D. W. Bone marrow restoration of transplantation immunity in the leopard frog Rana pipiens. Proc. Soc. Exp. Biol. Med. 135:406-411; 1970. 5. Deem, R. L.; Targan, S. R. Evidence of a dynamic role of the target cell membrane during the early stages of the natural killer cell lethal hit. J. Immunol. 133:72-77; 1984. 6. Ghoneum, M.; Cooper, E. L. Inhibition of frog SK effector-target cell binding. Dev. Comp. Immunol. 11:167-178; 1987. 7. Ghoneum, M.; Cooper, E. L.; Smith, C. Inhibition of SK cell activity in frogs by certain drugs and sugars. Dev. Comp. Immunol. I 1:363-373; 1987a. 8. Ghoneum, M.; Smith, C. T.; Cooper E. L. Membrane markers and mitogenic responses of cytotoxic cells in anuran amphibians. Cell. Mol. Biol. 33:807-817; 1987b. 9. Ghoneum, M.; Gill, G.; Wojdani, A.; Payne, C.; Alfred, L. J. Suppression of basal and Corynebacterium parvum-augmented NK activity during chemically induced tumor development. Int. J. Immunopharmacol. 9:71-79; 1987c. 10. Grimm, E.; Bonavida, B. Mechanism of cellmediated cytotoxicity at the single cell level. I. Estimation of cytotoxic T lymphocyte frequency and relative lytic efficiency. J. Immunol. 123:2861-2869; 1979. 11. Hiserodt, J. C.; Britvan, L.; Targan, S. R. Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, program-
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Further evidence for the role of bone marrow from Rana pipiens in immune responses. Dev. Comp. Immunol. 7:303-312; 1983. Rolfins-Smith, L. A.; Cohen, N. Effect of thymectomy on development of Lucke renal adenocarcinoma in virus-infected leopard frog tadpoles. J. Natl. Cancer Inst. 68:133-138; 1982. Rollins-Smith, L. A.; Cohen, N. Effect of thyroxine on induction of Lucke renal adenocarcinomas in Lucke tumor herpes virus-infected leopard frog tadpoles. J. Natl. Cancer Inst. 73:717, 1986. Sadek, I. A. The Egyptian toad as a sensitive model to show the effect of corn oil on liver tumor induced by DMBA. Nutr. Res. 6:333335; 1986. Sadek, I. A.; Ghoneum, M.; Cooper, E. L. Effect of 20-methylcholanthrene on amphibian natural killer cells. Dis. Aquat. Org. 3:155158, 1987a. Sadek, I. A.; Ghoneum, M.; Cooper, E. L. Retinoic acid and its effect on natural killer cells in toads. J. Nutr. Growth Can. 4:191197; 1987b.
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28. Savary, C. A.; Lotzova, E. Phylogeny and ontogeny of NK cells. In: Immunobiology of natural killer cells. Lotzova, E.; Herberman, R. B.; eds. Boca Raton, FL: CRC Press, Inc.; 1986:45. 29. Smith, C. T.; Cooper, E. L. Identification of cytotoxic cells in Rana. Fed. Proc. 42:937; 1983. 30. Smith, C. T.; Cooper, E. L. Characteristics of putative NK and CTL in frogs. Fed. Proc. 44:1921; 1985. 31. Smith, C. T.; Ghoneum, M.; Cooper, E. L. Ultrastructure of cytotoxic cells in frogs. Acta Zool. 69:125-133; 1988. 32. Talmadge, J. E.; Meyers, K. M.; Prieur, D. J.; Starkey, J. R. Role of NK cells in tumor growth and metastases in beige mice. Nature. 284:622-624; 1980. 33. Wright, S. C.; Bonavida, B. Studies on the mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC). I. Release of cytotoxic factors specific for NK-sensitive target cells (NKCF) during co-culture of NK effector cells with NK target cells. J. Immunol. 129:433-439; 1982.
