in graphic illustration; Tom Kelleher, Gwyneth Jones Lamont, and. Laura Mattes for expert technical assistance; and Theresa Grigsby and Catherine Redden for ...
Vol. 56, No. 5
INFECTION AND IMMUNITY, May 1988, P. 1162-1166 0019-9567/88/051162-05$02.00/0 Copyright ©D 1988, American Society for Microbiology
Killing of Human Myelomonocytic Leukemia and Lymphocytic Cell Lines by Actinobacillus actinomycetemcomitans Leukotoxin DAVID L.
SIMPSON,'* PETER BERTHOLD,2 AND NORTON S. TAICHMAN'
Departments of Pathology1 and Restorative Dentistry,2 and the Periodontal Diseases Research Center, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6002 Received 13 October 1987/Accepted 28 January 1988
The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin.
We are interested in the pathogenic determinants of Actinobacillus actinomycetemcomitans infection in human periodontal disease. A. actinomycetemcomitans is currently the subject of widespread interest as a possible etiologic agent in localized juvenile periodontitis because it is isolated with high frequency in individuals suffering from this disorder (18). While A. actinomycetemcomitans shares an arsenal of potential virulence factors with certain other suspected periodontal pathogens, it produces a unique leukotoxin which may enable it to subvert host antibacterial defense systems (12). In its action upon target cells, the leukotoxin demonstrates striking cellular and species specificities, killing isolated blood polymorphonuclear leukocytes (PMNs) and monocytes (MNs) of humans and certain nonhuman primates (13) but not comparable cells of other animals (11, 12, 14). In contrast to PMNs and MNs, human blood lymphocytes, erythrocytes, and platelets, as well as fibroblasts, endothelial cells, and epithelial cells, are resistant to the effects of the leukotoxin. The molecular bases of the cellular and species specificities of the leukotoxin are not presently understood. Our interest in the mechanism of target cell selectivity and our wish to identify a continuous cell line that would serve as a model target cell for such studies led us to a more detailed examination of cytotoxic specificity. Zambon and colleagues (19) first reported that intact A. actinomycetemcomitans destroyed human leukemic HL-60 cells. In this investigation we report that purified leukotoxin kills a number of human hematopoietic tumor cell lines and demonstrate the suitability of these cell lines as models with which to study the mechanism of action of the actinobacillus leukotoxin.
Health supplied SVG and PC-12 cells, respectively. HL-60 was supplied by Philip Simon of SmithKline Beckman Corp., Philadelphia, Pa. All other cells or cell lines were obtained from either the Cell and Molecular Genetics Center of the University of Pennsylvania or the American Type Culture Collection in Rockville, Md. In most instances, cells were cultured in plastic T-flasks (75 cm2; Corning Glass Works, Corning, N.Y.) as suspension cultures in RPMI 1640 (GIBCO Laboratories, Grand Island, N.Y.) or an appropriate medium. The medium was supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), 5 mM L-glutamine, a penicillin-streptomycin mixture (GIBCO), and 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Sigma Chemical Co., St. Louis, Mo.). Cultures were maintained at 37°C in a humidified chamber in 5% C02-95% air and were subcultured twice weekly by serial dilution. Prior to being tested, cells were washed twice in cold phosphate-buffered saline (pH 7.2), and cell viability was checked by trypan blue dye exclusion. All cell lines used in these studies were free of
mycoplasma contamination. Human PMNs. PMNs were isolated from heparinized venous blood of healthy volunteers (18 to 35 years old) on Ficoll-Hypaque (Bionetics, Kensington, Md.) and dextran sedimentation as previously described (15). Cells were suspended in Hanks balanced salt solution with 0.1% gelatin (HBSSG). More than 95% of the cells were PMNs with a viability of more than 95% as determined by trypan blue dye
exclusion. Measurement of leukotoxic activity. Killing of various cells was routinely measured by quantitating extracellular release of 51Cr from prelabeled cells. Test cells (5 x 107 cells) were washed by centrifugation (17) and labeled with 0.5 mCi of 51Cr in the form of sodium chromate (Dupont, NEN Research Products, Boston, Mass.) for 50 min at 37°C in a total final volume of 1 ml of HBSSG. After a wash in HBSSG by centrifugation (1,000 x g, 5 min, 4°C), the cell pellet was gently suspended in 3 ml of HBSSG and reincubated for 3 min at 37°C with shaking to remove bound label and to lower background counts. Cells were then washed and centrifuged as before, and cell concentrations were adjusted to 5 x 106 cells per ml of HBSSG. Cytotoxicity assays were run in triplicate in 96-well round-
MATERIALS AND METHODS Sources of cell cultures. Cell cultures of the following types were kindly provided by the following individuals at the University of Pennsylvania, Philadelphia: U937, Steven Douglas; KG-1 and J774A.1, Edward Lally; Vero and COS1, Gary Cohen; P8-15, Tibor Keler; NK, Jacki Kornbluth; JURKAT, Elaine DeFreitas; GM1056a and GM1056b, Jonnie Moore; and chondrocytes, Irving Shapiro. Joseph Bressler and Gordon Guroff of the National Institutes of *
Corresponding author. 1162
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TABLE 1. Cytotoxic specificity of the actinobacillus leukotoxin Description
Cell type or line
Human monocytic and myelocytic leukemic HL-60 U937 KG-1 KG-la HEL K-562
Promyelocytic Monocytic Myelogenous leukemia Less mature subclone (KG-1) Human erythroleukemic Human erythroleukemic
Susceptibility to actinobacillus leukotoxin (ED50 [ng]) 2 9 16 50 4
>500a
Human lymphocytic and lymphocyte derived
Daudi Raji JURKAT MOLT-4 NK All-1 All-2 GM1056a GM1056b GM4025b Neural SVG PC-12 NB212
Miscellaneous SP2 EL4.IL-2 P8-15 J774A.1 M1 EL4 L-929 3T3
Fibroblasts Vero COS-1 Endothelial cells
Chondrocytes a
b
B cell B cell T cell T lymphoma Non-T, non-B lymphoid Pre-B lymphoma Pre-B lymphoma Myeloma Epstein-Barr virus-transformed lymphoblast Human lymphoblast
20 10 8 3 3 3 28 40 30 33
Human SV40b_transfected glial cell Rat pheochromocytoma Human neuroblastoma
>500a >5Wa >500a
Murine myeloma Murine thymoma Murine mastocytoma BALB/c murine macrophage Murine myeloblast Murine leukemia Murine fibroblasts Murine embryo fibroblasts Human gingiva African green monkey (kidney) SV-40-transfected african green monkey (kidney) Human umbilical vein or bovine aorta (primary culture) Chicken (primary culture)
>50 >50 >500a >500a >500a >50ja >500a >50a >50ja
>50Ja >500a >500a >500
Cultures not susceptible to 500 ng of leukotoxin, the maximum dose tested. SV40, Simian virus 40.
bottomed microculture plates (Linbro/Titertek) containing 75 ,u1 of human serum (blood group AB; preheated at 56°C for 30 min and diluted 1/32 in HBSSG), 50 ,ul of various test
were incubated at in a humidified were terminated
titis) by conventional (17) or immunoaffinity (E. T. Lally, D. L. Simpson, and N. S. Taichman, Abstr. Int. Assoc. Dent. Res., abstr. no. 504, 1987) chromatography. Protein concentration was determined by the micro-Bradford procedure (2). Under standard experimental conditions, labeled cells were exposed to various doses (0 to 500 ng of protein) of
release from labeled cells in HBSSG alone ranged from 4 to 8% of the total available label (released from cultures incubated in 0.05% Triton X-100). Cytotoxicity was usually expressed as the 50% effective dose (ED50); that is, the dose of leukotoxin (in nanograms) required to cause the release of 50% of 51Cr from prelabeled target cells. Actinobacillus leukotoxin. The leukotoxin used in these experiments was purified from A. actinomycetemcomitans JP2 (isolated from a patient with localized juvenile periodon-
pure leukotoxin. Controls included cells in HBSS alone as well as cells incubated with heat-inactivated (56°C, 30 min) leukotoxin. The effects of time (0 to 120 min) and temperature (4 to 37°C) on the kinetics of the cytotoxic response were assessed by modifying the standard assay system. Electron microscopy. Cells were exposed to pure leukotoxin (ED90) under standard assay conditions for 10 min at 37°C. Experimental and control cells (which were exposed to heat-inactivated leukotoxin) were fixed in 1% OS04 in Tyrode solution for 60 min at 4°C. The specimens were dehydrated and embedded in Epon. Ultrathin sections were placed on Formvar-carbon-coated grids, double stained with uranyl acetate and lead citrate, and examined in a transmis-
RI1 of labeled cells (5 x 105). The cultures 37°C for various periods (usually 45 min) atmosphere (5% C02-95% air). Experiments by centrifugation (1,500 x g, 5 min, 4C), and samples (100 ,ul) of the culture supernatant were counted ind a gamma spectrometer (Beckman Gamma 5500; Beckman Instruments, Inc., Fullerton, Calif.). Background 51Cr samples,
and 100
SIMPSON ET AL.
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INFECT. IMMUN.
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Dose of Leukotoxin (ng) Dose of Leukotoxin (ng) FIG. 1. Susceptibility of hematopoietic cell lines to leukotoxin. Dose-response curves for HL-60 and U937 cells (A) and Raji and JURKAT cells (B) compared with those for human blood PMNs (A) and HL-60 cells (B).
sion electron microscope (Philips EM 300, operated at 80 kV; Philips Electronic Instruments, Mahwah, N.J.). RESULTS AND DISCUSSION Susceptibilities of various cells to killing by actinobacillus leukotoxin are presented in Table 1. The cells include myelocytic and monocytic leukemia cell lines (6) as well as lymphoblastic and more mature lymphocytic T- and B-cell lines (7). Several human promyelocytic and monocytic leukemia cell lines, including HL-60, U937, and KG-1, are susceptible to killing by the actinobacillus leukotoxin. In contrast, K-562, a human erythroleukemic cell line, is not susceptible to the toxin. Despite the fact that freshly isolated peripheral blood lymphocytes were not susceptible to the toxin (12), a number of human lymphoblastic, mature Tlymphocytic (JURKAT and MOLT-4), and B-lymphocytic (Raji and Daudi) cell lines were susceptible to the actinobacillus leukotoxin. In general, the less mature lymphoblastic lines were not as susceptible to the toxin as were the mature T- and B-cell lines. Nonhematopoietic human cell lines and cell lines from other species were not affected when exposed to high doses of toxin (.500 ng) under comparable conditions. Killing of susceptible cells by the purified leukotoxin was a dose-dependent phenomenon (Fig. 1). The ED50s of most susceptible human tumor cells were similar (within a 20-fold dosage range of toxin) to those of human blood PMNs (Fig. 1). Blood PMNs were the most susceptible target cells, with an ED50 of 0.85 ng. ED50s for four representative hematopoietic tumor cells lines were 2.2 ng for HL-60, 9.25 ng for U937, 8.5 ng for JURKAT, and 10 ng for Raji. ED50s for other susceptible hematopoietic tumor cells lines are listed in Table 1. Dose-dependent killing of susceptible target cells was also observed upon incubation with either freshly harvested whole leukotoxic A. actinomycetemcomitans strains or with sonic extracts prepared from these organisms (16) (data not shown). Target cell destruction by the leukotoxin was also confirmed by trypan blue staining of dead cells or by extracellular release of lactic dehydrogenase (data not shown). Destruction of target cells by actinobacillus leukotoxin was a time- and temperature-dependent phenomenon. When
exposed to an ED%0 of leukotoxin, maximal release of 51Cr occurred within 20 to 40 min in all susceptible cell lines. In contrast, nonsusceptible cells failed to release label even after 120 min of incubation with the toxin. In addition, label was not released from susceptible cells exposed to toxin at 4°C instead of 37°C. Leukotoxin-mediated cell death was inhibited by monoclonal antibodies to the toxin (11) and by sera obtained from patients with juvenile periodontitis (15) (Fig. 2). The latter contained immunoglobulin G antibodies directed against the leukotoxin (8). In contrast, normal human serum had no inhibitory effect on the reaction (Fig. 2). Heat-inactivated r
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FIG. 2. Antibody inhibition of leukotoxin-mediated cytotoxicity. The ability of antileukotoxin antibodies to inhibit cell killing was determined with murine monoclonal antibodies against the leukotoxin (3) and with sera from humans with juvenile periodontitis (8, 15) as test reagents. Twofold serial dilutions of sera or purified monoclonal antibody in 25 p.l of HBSSG were preincubated for 5 min at room temperature with leukotoxin (ED95) in 50 ,ul of HBSSG and then with 100 Iul of 51Cr-labeled HL-60 (ILii) or JURKAT (= ) for 45 min at 37°C. Controls included incubations carried out in the presence of various dilutions of normal human serum. For illustration, the highest dilutions of monoclonal antibody (1:1,000) and human juvenile periodontitis serum (1:200) which completely neutralized the toxin are presented. Normal human serum (1:5) failed to neutralize the bioactivity of the leukotoxin.
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LEUKOTOXIN KILLING OF HUMAN HEMATOPOIETIC TUMOR CELLS
3a
1165
3b
3d FIG. 3. Electron microscopy of the cytotoxic response. The effects of pure leukotoxin (ED90) on U937 (a [control] and b [experimental]) and JURKAT (c [control] and d [experimental]) cells were evaluated by transmission electron microscopy as previously described (12). Cells were exposed to toxin at 37 C for 10 min In control experiments, cells were exposed to heat-inactivated toxin and appear to be intact (a and c). Cultures exposed to leukotoxin exhibit various degrees of degeneration and in extreme instances appear to be disintegrating (b and d).
Magnification, x 6,500.
toxin (Fig. 2), extracts of nonleukotoxic A. actinomycetemcomitans strains, and various other bacteria (e.g., Bacteroides gingivalis, Streptococcus mutans, Actinomyces viscosus, and Treponema denticola) failed to destroy target cells. Cell destruction by the leukotoxin was also verified by transmission electron microscopy (Fig. 3). After exposure to toxin, both U937 and JURKAT cell lines exhibited profound morphological changes. The cells were swollen, and their nuclei appeared pyknotic and exhibited clumping of the chromatin material around the nuclear membrane. The cytoplasm was heavily vacuolated, granular in texture, and
devoid of most granules and other organelles. The plasma membrane of some cells appeared to be disintegrating. Other myelomonocytic and lymphocytic cells underwent analogous morphological changes upon exposure to the toxin (not shown). Leukemic cells incubated with identical amounts of heat-treated (56°C, 30 min) toxin showed no obvious signs of ultrastructural damage. The results of these studies indicate that a number of human hematopoietic cell lines may serve as model systems for the study of the mechanisms of actinobacillus leukotoxinmediated killing of target cells. In previous experiments we established that human as well as certain nonhuman primate
1166
SIMPSON ET AL.
PMNs and MNs are susceptible to the leukotoxin (11, 13, 14). On the other hand, other primate cells and leukocytes from nonprimate species are not killed by the leukotoxin. The results of the present investigation indicate that susceptibility to the leukotoxin is similarly conserved in various hematopoietic tumor cell lines. Thus, human promyelocytic, monocytic, and lymphocytic cell lines were affected by the toxin, but other human or animal cell lines showed no untoward effects when treated in a similar manner. These studies strongly suggest that the interaction(s) of leukotoxin with HL-60, U937, JURKAT, and Raji cells is very similar to its interaction(s) with blood PMNs and MNs. In determining how the leukotoxin kills susceptible cells, susceptible tumor cell lines offer distinct advantages over peripheral PMNs or MNs; they are relatively homogeneous and are easily established and maintained in culture. Consequently, data obtained with these cells should be more consistent and reproducible than that obtained with PMNs or MNs. These tumor cells exist in an arrested yet pliant state of maturation (6, 7); it should be possible to manipulate their genetic and phenotypic properties to, for example, select and isolate somatic cell variants which are resistant to leukotoxin-mediated killing. We suspect that leukotoxin, like many other cytotoxins (1, 4, 10), binds to cell surface receptors and injures the plasma membrane, because leukotoxic activity and associated leukotoxin polypeptides can be concomitantly and selectively adsorbed from crude polymyxin B extracts of A. actinomycetemcomitans by glutaraldehyde-fixed susceptible target cells but not by other fixed cells (13). Further, certain mono- and disaccharides with a mannose and galactose configuration protect target cells from the toxin to various degrees (11; D. L. Simpson and N. S. Taichman, Abstr. Int. Assoc. Dent. Res., abstr. no. 1647, 1987). Finally, certain lectins, such as wheat germ agglutinin and Phaseolus vulgaris erythroagglutinin, protect leukemia cells from destruction by leukotoxin (D. L. Simpson and N. S. Taichman, Abstr. Soc. Complex Carbohydr., abstr. no. 106, 1987). Cellular resistance to the toxin in nonsusceptible cells may be due to the absence of or a decrease in the number or availability of leukotoxin receptors on the cell membrane. Alternatively, resistance to leukotoxin could also be a consequence of an impairment of cytopathic events that occur subsequent to toxin binding and membrane perturbation. To elucidate the molecular mode of action of the toxin, we are currently generating genetically stable, toxin-resistant variant cell lines. Such variants have been described for a variety of other plant and bacterial toxins (5, 9) and should similarly be instrumental in dissecting the membrane and subcellular events in the cytotoxic process. Understanding the selective killing of a number of leukemia cell lines and the molecular basis of the marked cellular and species specificities of the leukotoxin may reveal new insights into the unique discriminatory capabilities of native toxins and may also provide a useful probe with which to study hematopoietic tumor cell biology. ACKNOWLEDGMENTS We thank W. Terrence Donohue for his assistance and expertise in graphic illustration; Tom Kelleher, Gwyneth Jones Lamont, and Laura Mattes for expert technical assistance; and Theresa Grigsby and Catherine Redden for help in preparing the manuscript. These studies were supported by Public Health Service grants DE-02623, DE-03995, DE-07118, and NS-23564 from the National Institutes of Health.
INFECT. IMMUN. LITERATURE CITED 1. Arbuthnott, J. P. 1982. Bacterial cytolysins: membrane damaging toxins, p. 107-129. In P. Cohen and S. van Heyningen (ed.), Molecular action of toxins and viruses. Elsevier Biomedical
Press, Amsterdam. 2. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. DiRienzo, J. M., C.-C. Tsai, B. J. Shenker, N. S. Taichman, and E. T. Lally. 1985. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans. Infect. Immun. 47:31-36. 4. Eidels, L., R. L. Proia, and D. A. Hart. 1983. Membrane receptors for bacterial toxins. Microbiol. Rev. 47:596-620. 5. Goldmacher, V. S., J. Anderson, M.-L. Schultz, W. A. Blattler, and J. M. Lambert. 1987. Somatic cell mutants resistant to ricin, diphtheria toxin and immunotoxins. J. Biol. Chem. 262: 3205-3209. 6. Harris, P., and R. Ralph. 1985. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J. Leukocyte Biol. 37:407-422. 7. Hozumi, M. 1985. Established leukemia cell lines: their role in the understanding and control of leukemia differentiation. Crit. Rev. Oncol. Hematol. 3:235-277. 8. McArthur, W. P., C.-C. Tsai, P. C. Baehni, R. J. Genco, and N. S. Taichman. 1981. Leukotoxic effects of Actinobacillus actinomycetemcomitans: modulation by serum components. J. Periodontal Res. 16:159-170. 9. Olsnes, S., and A. Phil. 1982. Toxic lectins and related proteins, p. 51-105. In P. Cohen and S. van Heyningen (ed.), Molecular action of toxins and viruses. Elsevier Biomedical Press, Amsterdam. 10. Shier, W. T. 1982. Cytolytic mechanisms: self-destruction of mammalian cells by activation of endogenous hydrolytic enzymes. J. Toxicol. Toxin Rev. 1:1-32. 11. Taichman, N. S., R. T. Dean, and C. J. Sanderson. 1980. Biochemical and morphological characterization of the killing of human monocytes by a leukotoxin derived from Actinobacillus actinomycetemcomitans. Infect. Immun. 28:258-268. 12. Taichman, N. S., W. P. McArthur, C.-C. Tsai, P. C. Baehni, B. J. Shenker, P. Berthold, C. Evian, and R. Stevens. 1982. Leukocidal mechanisms of Actinobacillus actinomycetemcomitans, p. 261-269. In R. J. Genco and S. E. Mergenhagen (ed.), Host-parasite interactions in periodontal diseases. American Society for Microbiology, Washington, D.C. 13. Taichman, N. S., D. L. Simpson, S. Sakurada, M. Cranfield, J. DiRienzo, and J. Slots. 1987. Comparative studies on the biology of Actinobacillus actinomycetemcomitans leukotoxin in primates. Oral Microbiol. Immunol. 2:97-104. 14. Taichman, N. S., and J. M. A. Wilton. 1981. Leukotoxicity of an extract from Actinobacillus actinomycetemcomitans for gingival polymorphonuclear leukocytes. Inflammation 5:1-12. 15. Tsai, C.-C., W. P. McArthur, P. C. Baehni, C. Evian, R. J. Genco, and N. S. Taichman. 1981. Serum neutralizing activity against Actinobacillus actinomycetemcomitans leukotoxin in juvenile periodontitis. J. Clin. Periodontol. 8:338-348. 16. Tsai, C.-C., W. P. McArthur, P. C. Baehni, B. F. Hammond, and N. S. Taichman. 1979. Extraction and partial characterization of a leukotoxin from a plaque-derived gram-negative microorganism. Infect. Immun. 25:427-439. 17. Tsai, C.-C., B. J. Shenker, J. M. DiRienzo, D. Malamud, and N. S. Taichman. 1979. Extraction and isolation of a leukotoxin from Actinobacillus actinomycetemcomitans with polymyxin B. Infect. Immun. 43:700-705. 18. Zambon, J. J. 1985. Actinobacillus actinomycetemcomitans in human periodontal disease. J. Clin. Periodontol. 12:1-20. 19. Zambon, J. J., C. Deluca, J. Slots, and R. J. Genco. 1983. Studies of the leukotoxin from Actinobacillus actinomycetemcomitans using the promyelocytic HL-60 cell line. Infect. Immun. 40:205-212.
