- Email: [email protected]
[28] Induction and characterization of tortoise interferon
196
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
Induction
and Characterization Interferon
[28]
of Tortoise
B y A N G E L S. GALABOV
Methods for the study of the interferon system in the cells of tortoise are described in this chapter. Numerous data show that the interferon system is phylogenetically very old and of ubiquitous character: it is found in all classes of vertebrates, and the presence of interferon-like substances is proved also in the cells of insects and higher plants and in bacteria. 1-3 This mechanism of cell defense precedes not only the humoral forms of immunity, but perhaps phagocytosis as well. 4,5 The processes of interferon induction as well as the basic physicochemical characteristics and biological effects of interferon were studied almost exclusively in cell systems from birds and mammals. 6,7 However, convincing evidence exists that interferon production occurs also in the cells of lower (cold-blooded) vertebrates--fishes s-14 and reptiles. 15-21 The functioning of the interferon system in the cells of these animals shows some peculiar features that disclose more completely the interferon mechanisms. Tortoises are the most suitable among the reptiles as a source for preparing cell cultures for interferon studies. Methods for interferon induction in different types of tortoise cells are described below. Induction and Characterization of Interferon in Cell Culture of Tortoise Kidney
Preparation of Primary Cell Cultures of Kidney and Lung of Tortoise Primary cell cultures of tortoise kidney were prepared originally by Shindarov 22,zz and by Fauconnier and Pachopos. 24 According to the Shindarov's technique the kidney of an adult tortoise (Testudo graeca, Tes-
1 A. S. Galabov, Bull. Inst. Pasteur (Paris) 71, 233 (1973). 2 j. Vil~ek, "Interferons," pp. 30-31. Springer-Verlag, Vienna and New York, 1%9. 3 K. H. Fantes, in "Interferons and Interferon Inducers" (N. B. Finter, ed.), pp. 263-265. North-Holland Publ., Amsterdam, 1973. 4 N. N. Sirotinin, in "Manual of Microbiology, Clinics, and Epidemiology of Infectious Diseases" (G. B. Vygodtchikov, ed.), pp. 274-2%. Medizina Publ. House, Moscow, 1964. 5 L. A. Zilber, in "Basic Immunology" (V. I. Satchkov, ed.), pp. 173-183. Medgiz State Publ. House of Med. Liter., Moscow, 1958.
METHODS IN ENZYMOLOGY, VOL. 78
Copyright© 1981by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181978-7
[28]
TORTOISE INTERFERON
197
tudo hermani, etc.) are excised through a wide opening made in the plastron (the ventral part of the carapace). After decapsulation, the kidneys are cut into small pieces and disaggregated by fractional trypsinization at 35° with 0.2-0.25% trypsin (1 : 250, Difco) solution. A suspension of 1.5 to 3 × 105 cells/ml is prepared in growth medium containing 15% heated calf serum, 0.5% lactalbumin hydrolyzate, penicillin, and streptomycin (100 units/ml each) in saline solution (pH 7.2): Hanks' saline or saline prepared by dissolving 6.5 g of NaCI, 0.14 g of KCI, 0.12 g of CaCI2,0.2 g of MgSO4, and 0.02 g of phenol red in 1000 ml of double-distilled water. The cells ofTestudo graeca kidney (TGK cells) grow optimally at 37° and form a confluent layer within 3-5 days. This culture possesses a high stability, being preserved as an intact monolayer for at least 1 month if kept at 4 °. Its ability to grow is maintained up to 4 months under such conditions of storage.2~ Analogous is the method of preparing a primary cell culture of tortoise lung, 26a7 except that the trypsinization is performed at 32°. The cells are suspended at 2 × 10~ per milliliter in the growth medium. At 37° in 3 - 4 days they form a complete monolayer, consisting of epithelial and fibroblast types of cells. The suspension of trypsinized cells may be stored for 2 months at 4° while preserving its ability to growY 5
Interferon Induction in TGK Cells by Viruses Choice of lnducing Virus and Multiplicity of lnfection. This cell culture is susceptible to infection by a variety of animal viruses of almost all
6 N. B. Finter, ed., "Interferons and Interferon Inducers," pp. 1-262, 267-480. NorthHolland Publ., Amsterdam, 1973. 7 W. E. Stewart II, "The Interferon System," pp. 1-323. Springer-Verlag, Vienna and New York, 1979. 8 M. Gravell and R. G. Malsberger, Ann. N.Y. Acad. Sci. 126, 555 (1%5). 9 A. R. Beasly, M. M. Sigel, and L. W. Clem, Proc. Soc. Exp. Biol. Med. 121, 1169 (1%6). 10 H. K. Oie and P. C. Loh, Proc. Soc. Exp. Biol. Med. 136, 369 (1971). 11 R. K. Kelly and P. C. Loh, In Vitro 9, 73 (1973). 12 p. Kinkelin and M. Leberre, C. R. Hebd. Seances Acad. Sci., Ser. D. 279, 445 (1974). lz p. Kinkelin and M. Dorson, J. Gen. Virol. 19, 125 (1973). 1~ j. DeSena, and G. J. Rio, Infect. lmmun. 11,815 (1975). 1~ E. Falcoff and B. Fauconnier, Proc. Soc. Exp. Biol. Med. 118, 609 (1965). 16 A. Galabov, Z. Savov, and V. Vassileva, Acta Virol. (Prague) 17, 1 (1973). lr A. Galabov, S. Petrunova, and Z. Savov, Experientia 29, 900 (1973). 18 A. S. Galabov and Z. A. Savoy, Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. 1: Orig. Reihe A 225, 1 (1973). 19 A. S. Galabov, E. H. Velichkova, and S. S. Petrunova, in "Interferon and Interferon Inducers" (I. FOldes and M. Talas, eds.), pp. 136-159. Publ. House Hung. Acad. Sci., Budapest, 1975.
198
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
principal groups, including: alpha- (Sindbis 15'2s and Semliki Forest virus2a), flavi- (West Nile virusl6), orthomyxo- (influenza A/H2N2/57 and B3°), paramyxo- (Sendai 23'24, Newcastle disease virus, 3~ mumps, 3~ respiratory syncytial virus33), rhabdo- (vesicular stomatitis virus3~), adeno(adenovirus 52s), herpes- (herpes simplex virus2S'35), and pox- (vaccinia 36) viruses. Therefore, TGK cells can be used to study induction of interferon by viruses as well as for the interferon assay. As known, alpha-, flavi-, and paramyxoviruses are among the most potent interferon inducers in cultured homeothermic cells. T M The optimal multiplicity of infection (MOI) for highest interferon production by West Nile virus (WNV) (grown intracerebrally in mice) is 2 LD~o per cell. TGK monolayer cells (7 to 8 days old) are infected by 60-120 min adsorption at room temperature, wfished three times with saline, and covered with fresh maintenance medium without serum (medium 199 or Eagle's MEM with lactalbumin hydrolyzate, 0.05% and 0.2%, respectively, and antibiotics). An important advantage of this virus as inducer is that it stimulates a relatively high production of interferon without causing a cytopathic effect (CPE) up to day 10 after virus inoculation. At determined intervals the culture medium is harvested and adjusted with 2 N perchloric acid to pH 2.0. After 18 hr at 4 ° the liquid is centrifuged at 2500 rpm for 60 min, and the supernatant is brought to pH 7.0
20 A. S. Galabov and E. H. Velichkova, J. Gen. Viroi. 28, 259 (1975). 21 V. Vassileva and A. Galabov, Acta Microbiol. Acad. Sci. Hung. 22, 323 (1975). 22 L. Shindarov, Dokl. Bolg. Akad. Nauk 15, 539 (1962). 23 L. Shindarov, Acta Virol. (Prague) 6, 540 (1962). 24 B. Fauconnier and M. Pachopos, Ann. Inst. Pasteur, Paris 102, 661 (1962). 25 L. Shindarov, in "'Proceedings of First National Congress of Microbiology" (Sofia, 1965), pp. 693-696. Publ. House Acad. Bulg. Sci., Sofia, 1967. 26 L. Shindarov and V. Vassileva, Epidemiol. Mikrobiol. lnfekts. Boles. 3, 271 (1965). 27 L. Schindarov, and W. Wassileva, Arch. Gesamte Virusforsch. 19, 250 (1966). 2s L. Shindarov, Z. Savov, and V. Vassileva, Acta Med. Bulg. 6, 54 (1979). 29 L. Shindarov, Z. Savoy, S. Todorov, and B. Iwanov, Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. 1: Orig. Reihe A 222, 168 (1972). 30 L. Shindarov and S. Todorov, Khigiena 6(6), 24 (1963). 3x L. Schindarov and S. Todorov, Zentralbl. Bakteriol. Parasitenk., Infektionskr. Hyg. Abt. 1: Orig. Reihe A 186, 495 (1961). 32 B. Fauconnier, Ann. Inst. Pasteur, Paris 105, 439 (1963). 33 L. Shindarov, E. Velichkova, and N. Runewski, Acta Med. Bulg. 3, 7 (1975). 34 L. Shindarov, and Z. Savov, Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 1: Orig. Reihe A 192, 420 (1964). 35 B. Fauconnier, Ann. Inst. Pasteur, Paris 105, 444 (1963). 3e L. Shindarov, Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. 1: Orig. Reihe A 187, 285 (1962).
[28]
TORTOISE INTERFERON
199
1o24-1 512 256-: /2,9 64•
32-
~ f6-
/ i
t
i
I
i
i
i
I
i
I
24 48 72 .96 120 144 168 192 216 -','zO Houz'.s rjtc/e ~• v//'us / Hz~cu/u //2wJ
FIG. 1. Interferon production kinetics at 37° (Q) and 20° (©) in T e s t u d o g r a e c a kidney cells infected with West Nile virus. Data are from Galabov et al. 16
with 1 N NaOH. The titer of so-called " c r u d e " interferon thus obtained 1n,37 is determined (see below). At an incubation temperature of 37° the kinetics of interferon production in TGK cells shows a characteristic pattern (Fig. 1). The production begins after the sixth hour and reaches maximum at 48-72 hr after virus inoculation. Usually, in the next 24 hr a sharp reduction of the interferon titer occurs. 16,18 When Semliki Forest virus (SFV) is used as inducer (stock virus with 1-2 passages in TGK cells), the optimal multiplicity of infection is 1-3 (60-min adsorption at room temperature). A comparatively fast development of characteristic CPE up to a complete destruction of the cell monolayer at 72-96 hr is observed. The interferon liter reaches its maximum by 48 hr, with a markedly expressed CPE, but the cell monolayer is still preserved. 16 Newcastle disease virus (NDV) can also be used as an inducer. The inoculation of the cultures is carried out with undiluted UV-inactivated allantoic fluid (106 EID~ per milliliter of the stock virus) by 60-min adsorption at room temperature. The interferon production in this case begins by 12 hr and reaches a maximum by 48 hr. 21 Sendai virus (MOI = 1) induces a marked interferon production at 48 hr simultaneously with the appearance of a marked CPE. For this tea37 G. P. Lampson, A. A. TyteU, M. M. Nemes, and M. R. Hilleman, P r o c . S o c . E x p . Biol. M e d . 112, 468 (1%3).
200
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
son it is recommended to clarify the culture medium harvested by centrifugation (10 min at 3000 rpm) and then to sediment viral particles by a second centrifugation (2 hr at 50,000-100,000 g). The upper two-thirds of the latter supernatant are taken as a " c r u d e " interferon. ~ Optimal Age of the Inducible Cell Culture. The highest interferon production by virus inducers is obtained when "aged" TGK cell cultures are inoculated. For instance, the quantity of interferon induced by WNV in 7to 8-day-old cultures is about 16 times higher in comparison with that in 4-day-old cultures.~n'18 Stimulation of interferon production is known also in "aged" human, mouse, and chick cells a8-4° and is ascribed to the synthesis of a cell protein that stimulates interferon induction. 4~ Incubation Temperature. The optimal incubation temperature for virus-induced interferon production in TGK cells is 37°. ~n,a8 This coincides with the temperature optima both for the metabolic processes in TGK cells 29 and for their growth. 22,25,2a This cell system offers the possibility to study interferon induction at relatively low incubation temperatures. For example, when WNV-infected TGK cells are incubated at 20 °, a marked, although much delayed, interferon production is established (Fig. 1). Trace amounts of interferon can be detected not earlier than the 144th hour, and the maximum titers are still lower than that at 37 °. A relatively earlier establishment of interferon production at 20° is found after alphavirus (SFV, Sindbis) inoculation. The interferon level in the culture fluid persists for a comparatively longer period at 20° than at 37°, probably because of heat inactivation in the latter case. Interferon production in " y o u n g " and "aged" cell monolayers is approximately equal. 1n'~8 Of particular interest is that interferon production occurs in TGK cells even at 8 °, although the titers are rather low (4-8 units/ml), measured at 5-12 days after virus (SFV, Sindbis virus, WNV) inoculation. ~s This phenomenon of interferon production at low temperatures is due to the fact that at 8° some metabolic activity in TGK cells and alphavirus growth are still retainedY s'29
CharacteriZation of Virus-Induced Interferon in TGK Cells The following tests can be used for identification of the interferon in the culture fluids obtained as described above [treated culture fluid (TCF) = " c r u d e " interferon].
38 C. McLaren, Arch. Gesamte Virusforsch. 32, 13 (1970). 39 H. Liblkova and E. Henslov~i, Acta Virol. (Prague) 13, 16 (1969). 40 M. Ho and J.-F. Enders, Virology 9, 446 (1959). 41 N. Kato and H. J. Eggers, Virology 37, 545 (1969).
[28]
TORTOISE INTERFERON
201
Biological Tests I. Contact test for lack of virocidal effect. A suspension of vesicular stomatitis virus (VSV) or Sindbis virus, 100 TCIDs0/ml, is treated with TCF for 24 hr at 37°, and the residual infectivity is assayed. 15.10 2. Species specificity test. A parallel titration of TCF and mouse or human interferons is carried out on both TGK and L cells (or on human cells), challenged by VSV (100 TCIDso, see below) or Sindbis virus (1000 TCID~0), etc. 15'16 3. Lack of antiviral specificity. TCF is assayed in TGK cells, challenged by VSV, SFV, NDV, Sendai, mumps, and vaccinia viruses, HSV, etc. ls,16 4. Lack of effect of antiviral antibody. A mixture of equal volumes of TCF and virus-inducer specific antiserum are incubated for 60 min at 37° and the interferon titer of TCF is determined. 15
Effect of Physical and Chemical Agents 1. Stability at pH 2.0. The TCF is adjusted to pH 2.0 with 2 N perchloric acid and left for 48 hr at 4o. TM Alternatively, TCF can be subjected to dialysis for 24 hr at pH 2.0 and 24 hr at pH 7.0. TM 2. Heat stability test. A series of TCF samples are incubated for 30 or 60 min at 56°, 65°, and 75 °. The tortoise interferon is completely stable for 60 rain at 56° and is inactivated only partially at 65-750. TM 3. Ether treatment. TCF is mixed with ether (1 : 1, v/v), shaken for 10 min at 4°, and then centrifuged at 3000 rpm for 10 min. The aqueous phase is freed from residual ether by nitrogen. No clear reduction of interferon activity after ether treatment has been observed. 15 4. Trypsin treatment. Twice-crystallized trypsin (100-250/zg) is dissolved in 1 ml of TCF. After a 60-min incubation at 37°, 200/zg of purified trypsin soybean inhibitor are added. 15,1e Complete loss of interferon activity is seen. 5. Ribonuclease treatment. Add 100 ~g of RNase per milliliter of TCF for 2 hr at 37° and pH 7.0.15 6. Deoxyribonuclease treatment. Exactly as above but with DNase. The interferon is stable to both RNase and DNase.15,16
Molecular Weight Determination. The molecular weight of the tortoise interferon (virus-induced in TGK cells) can be determined 17 by gel filtration according to Andrews. 42 TCF are first dialyzed against 30 vol42 p. Andrews, Biochem. J. 91,222 (1964).
202
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
umes of PBS 43 (pH 7.4) for 72 hr and concentrated 15-30 times by the use of polyethylene glycol 6000 (Carbowax 20M, Serva). A column (110 × 1.5 cm) Sephadex G-100 (Pharmacia) is equilibrated with PBS, pH 7.4, containing 0.02% sodium azide. Calibration of the column can be performed with the following proteins used as standard markers: human serum albumin (69,000), bovine serum albumin (64,000, dimer 134,000), ovalbumin (45,000), chymotrypsinogen (25,000), soybean trypsin inhibitor (21,500), cytochrome c (13,000). Proteins (2-3 mg each) together with 0.2% Blue Dextran 2000 (Pharmacia) are dissolved in PBS and applied in a 2-ml volume. The flow rate of the column is 7-10 ml/cmZ/hr, and 4-ml fractions are collected. The optical density of the fractions is read spectrophotometrically at a wavelength of 280 nm; Blue Dextran is additionally read at 625 nm, and cytochrome c at 412 nm. To determine the molecular weight of the interferon, 2 ml of the concentrated TCF, containing Blue Dextran and cytochrome c, are applied to the column. The interferon titer of the 4-ml effluent fractions is determined (see below). The molecular weight is estimated by plotting the elution volume of the interferon peak on the calibration curve of the column. A typical elution profile of virus-induced tortoise interferon is presented in Fig. 2; the molecular weight is 33,500.17
Interferon Assay The Indiana strain of VSV can be recommended as a suitable challenge virus. It grows easily in TGK monolayer cell cultures, attaining a titer of 105 TCIDs0/ml or about 2.2 × 106 PFU/ml. VSV reproduces and forms plaques even at a suboptimal incubation temperature of 20°, although its reproductive cycle is markedly delayed, a0,44 The following methods for interferon assay can be recommended. 1. CPE inhibition method, an Tube monolayer TGK cell cultures are treated for 24 hr at 37° with serial dilutions of the TCF tested. The dilutions are done with medium 199 (Difco) supplemented with 0.05% lactalbumin hydrolyzate and antibiotics. Then the cells are washed twice and inoculated with 100 TCIDs0 of VSV. 2. Plaque-inhibition method, a6 Monolayer cultures in 50-100 ml flasks are inoculated with 100-150 P F U of VSV after being previously treated with diluted TCF as above. The cultures are covered with an agar overlay (4-8 ml) consisting of 1.5% agar (Difco) in Eagle's MEM (Difco), 10% heated calf serum, 2.25 mg of sodium bicarbonate per milliliter, and antibiotics. 43 R. Dulbecco and M. Vogt, J. Exp. Med. 99, 167 (1954). 44 A. S. Galabov, Z. Savov, and L. Shindarov, in "Abstracts of Third National Congress of Microbiology" (Sofia, 1973), p. 102. Publ. House Acad. Bulg. Sci., Sofia, 1973.
[28]
TORTOISE INTERFERON
203
Io ~_
b _-t05 ~ S e r u m albumin in
n Iryl~in inhiL~i~or
/04
t.
,,b
I
/0 J
] '
'
' / o ' o
'
'
1~o
'
'
'
rn I
Elutiofl volurrJe FIG. 2. Chromatographic pattern of Semliki Forest virus-induced interferon in Testudo graeca kidney cells on Sephadex G-100. A, Calibration curve; B, elution profile of concentrated interferon preparation, 1 ml. Data are from Galabov et al.t7
204
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
The interferon titer in both cases is determined after a 48-hr incubation at 37° and is expressed in interferon units per milliliter; i.e., the reciprocal of the highest dilution of the tested samples that prevents the CPE in at least half of the tube cultures (method 1) or results in a 50% reduction of the plaque number (method 2). The interferon can be assayed also by measuring the reduction of the challenge virus yield (e.g., Sindbis virus, MOI = 0.25) in TGK cells pretreated with diluted 1 : 4 TCF tested. 15
Antiviral Effect of Tortoise Interferon The effect of interferon on the reproduction of different viruses in TGK cells can be determined by doing one-step growth cycle or multicycle studies. Three-day-old monolayer cultures are treated for 18-24 hr at 37° with 50- I00 units per milliliter of tortoise interferon. After two washings with saline, the cultures are infected (by adsorption for 1-2 hr at 4° or at room temperature) at high MOI in order to obtain a one-step growth cycle. For instance, the MOI used is 10 for SFV and 1 for VSV and vaccinia virus, respectively. The maintenance medium consists of Eagle's MEM with lactalbumin hydrolyzate and 5% heated calf serum. At the end of the one-step cycle, whose duration has to be preliminarily determined, two flasks (or four test tubes) per sample are frozen for measuring the infectious virus yield. The antiviral effect of interferon can be expressed as inhibition of virus yield, zl,44,45 The duration of the one-step cycle in TGK cells of SFV, Sindbis virus, and VSV is 12 hr21aa; of vaccinia virus, 15 hr21; herpes simplex virus 1 and respiratory syncytial virus, 18-20 hr2S; Sendai virus and NDV, 20-24 h#S; and adenovirus 5, 24-30 hr. 2s The viruses tested can be arranged according to their sensitivity to interferon as follows: SFV(S-) (A logs = 3.3-4.0) > herpes simplex virus 1 (2.8 logs) > SFV(S+), VSV, and NDV (2.0-2.5 logs) > vaccinia virus (1.2-1.7 logs) > EMC virus and adenovirus 5 (0.7-1.0 l o g ) . 21'44"45 This cell system allows the antiviral effect of interferon to be studied at 20°. Moreover, both the treatment of the cells by interferon preparations and the reproduction of different viruses can be carried out at this incubation temperature? 8"28a9"44"45It is necessary to take into account the increase of the virus growth cycle time at this temperature. 28,29 For instance, the duration of the SFV growth cycle at 37° is 12 hr, whereas at 20° it is 72 hr. 29 The duration of the growth cycle at 20° of Sindbis virus is 96 hr; of adeno 5 and Sendai, 120 hr; of NDV, 144 hr; of herpes simplex virus 1,168 hr. 2s It is worth noting that the antiviral effect of interferon is approximately the same at 20° and 37° against most of the viruses tested. 18.44.45 45 A. S. Galabov and Z. Savov, unpublished data.
[28]
TORTOISE INTERFERON
205
In experiments on interferon assay, a clear stimulation of CPE by the challenge virus (VSV) is observed in the cell cultures treated with the lower dilutions (1:2-1 : 16) of TCF during 72 hr of induction (both at 20° and 37°)? ~a8 This phenomenon may be ascribed 1~a8 to a substance, synthesized as a component of the interferon system, like "antagonist, ''4~ "interferon antagonist,"47 o r " stimulon."48 The antagonist content can be expressed in interferon antagonist units (IAU) per milliliter, reciprocals of the highest dilution of TCF sample tested that stimulates clearly the CPE of VSV (100 TCIDso per test tube). There are clear-cut kinetic differences between the production of interferon and of antagonist in TGK cells, especially at 20°? 6a8 The "antagonist" possesses the following characteristics: stability at pH 2.0, inactivation by trypsin, and insensitivity to RNase and DNase? n Induction and Characterization of Interferon in Explanted Tortoise Peritoneal Leukocytes
Preparation of Explanted Tortoise Peritoneal Leukocytes Figure 3 schematically represents a technique for easy access to the peritoneum of the tortoise via an opening excised in the plastron of a decapitated adult animal. The peritoneal cavity is washed with 30-40 ml of cooled PBS. The wash is centrifuged in the cold at 1800-2000 rpm for 5 rain. The cell pellet is resuspended in medium 199 containing 10% heated calf serum and antibiotics, pH 7.3. The suspension is diluted to 2.5 × 106 cells/ml. The cells thus obtained (Testudo graeca peritoneal cells, TGP cells) tga° are mainly mononuclear leukocytes, namely: lymphocytes (90-95%), large mononuclear cells of the macrophage type (3-6%), and monocytes (2%). A small number of polymorphonuclear leukocytes are also present (1%). ~9,z°
Interferon Induction in TGP Cells by Viruses The TGP cell suspension is distributed into 1-ml portions in test tubes and immediately inoculated with NDV at an MOI of about 0.4 EIDso per cell, which is optimal for interferon production. 19,20 At 37° the interferon production is established as early as hour 2, increases until hour 8, and reaches relatively high titers (> 4000 units/ml). About a 16-fold decrease in production is found at 100 times greater MOI (40 EID~ per cell). At lower incubation temperatures, interferon production is strongly delayed. 46 j. L. Truden, M. M. Sigel, and L. S. Dietrich, Virology 33, 95 (1967). 47 E. T. Sheaff and R. B. Stewart, Can. J. Microbiol. 14, %5 (1%8). 4~ C. Chany and C. Brailovsky, C. R. Hebd. Seances Acad. Sci. Ser. D 261, 4282 (1965).
206
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
F~G. 3. Excisingan opening m the plastron (ventral part of the carapace) of a tortoise (Testudo graeca) for access to the peritoneum. For instance, at 26° interferon titers attain a normal level only by hour 20, a level that is attained by 6 hr at 37° (Fig. 4A). B y contrast with the observations with TGK cells, no interferon production in response to virus inducer occurs at 20° or 80.19,2° " C r u d e " interferon (TCF) samples are prepared by single freezing and thawing of the suspensions of TGP cells and subsequent adjustment with 6 N HCI to pH 2.0. After 2 or 3 days at 4° the suspension is centrifuged and the pH of the supernatant is adjusted to 7.0 with 5 N NaOH. Interferon is assayed with TGK cells challenged by VSV as described previously.
Interferon Induction in TGP Cells by Poly(I).Poly(C) Interferon production in TGP cells can be induced by a 30-min poly(I).poly(C) treatment (e.g., 30/~g per 2.5 × I06 cells). The cells are then centrifuged (1800 rpm for 5 min at 4°), washed once with PBS, resuspended in fresh medium, and incubated at 26° for 24 hr. ~9
Interferon Induction in TGP Cells by Enterobacterial Endotoxins The TGP cells release interferon also after treatment with endotoxintype inducers, e.g., O antigens, lipopolysaccharide, and glycolipid of Enterobacteriaceae. The optimal incubation temperature for interferon production in this
TORTOISE I N T E R F E R O N
[28]
2s6 /
. . . . . .
207 ,..,
! A
.~
64
Z
KTGPC
2
5
4
5
6 7 HOURS
8
20
8
20
256 ~E
128
o'J I.-
64
Z D
32
Z 0 n-W LL 13:7
w I---
B
HJ
16 8 4
2
3
4
5
6 7 HOURS
F]G. 4. Interferon production kinetics at 37° (stippled bar), 26 ° (filled bar); 20° (hatched bar), and 8° (open bar) in Testudo graeca peritoneal cells. (A) Inoculated with Newcastle disease virus (H strain; MOI = 40). (B) Treated with Serratia marcescens endotoxin (1 /,¢g per 2.5 × 106 cells). KTGPC = noninoculated cells (control). Data are from Galabov and Velichkova. z°
case is 26°. 19,2° Similarly to the peritoneal cells of mammals, 49,5° the doseresponse curve for bacterial endotoxins (e.g., O antigens) in TGP cells shows a maximum at 1/zg/ml. The kinetics of interferon production is typical: interferon is released after hour 4 and reaches its maximum at hour 6 (Fig. 4B). At 37 ° no interferon can be detected at least up to hour 8. At 20 ° interferon production is lower as compared to production at 26 ° , the maximum being achieved not earlier than at about 20 hr. It is of partic49 L. Borecky, V. Lackovi~, and K. Waschke, in " L ' l n t e r f ~ r o n " (C. Chany, and P. De Somer, eds.), Colloq. Inst. Natl. Sante Rech. Med. 6, 37 (1969). 50 A. S. Galabov and S. M. Galabov, Acta Virol. (Prague) 17, 493 (1973).
208
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
ular interest that interferon production is found even at 8°, although greatly reduced and delayed? %z° Under the conditions described, especially when the explanted cells are rapidly prepared and immediately used, the spontaneously released interferon in TGP cells shows very low titers (4 units/ml) at 26° and is practically absent at 20 ° and 8°.
Characterization of TGP Cell Interferon The antiviral substance produced in TGP cells, irrespective of the type of inducer used, displays all the basic properties of interferon, as determined by the methods described above: lack of virocidal effect and antiviral specificity, species specificity, stability at pH 2.0, a relatively high thermostability (30-60 min at 56°), a high sensitivity to trypsin, and resistance to RNase, DNase, and ether treatment. 19 The molecular weight of TGP cell interferon can be determined by Sephadex gel chromatography. The interferon samples are dialyzed for 3 days in citrate buffer (0.098 M citric acid, 0.004 M NazHPO4), pH 2.15, and 2 days in PBS, pH 7.4, then concentrated 10-fold with polyethylene glycol 6000. The following molecular species of interferon produced in TGP cells has been found19: (a) in NDV-induced cells, a single peak with molecular weight of 58,500; (b) in endotoxin-induced cells, two species with molecular weights of I02,000 and 43,500; (c) in poly(I).poly(C)-induced cells, two species with molecular weights of 144,000 and 63,000 Conclusion An analysis of the experimental data on induction and characteristics of tortoise interferon demonstrates the functioning of a complete interferon system, analogous to that in mammals and birds. The large temperature range (more than 30 °) throughout which the interferon system functions is together with some other metabolic activities 2a in tortoise cells (TGK, TGP) is a definite advantage. By stepwise decrease of the incubation temperature it has been possible to characterize in more detail the separate components of the interferon system. As demonstrated, 18by choosing a suitable incubation temperature a selective synthesis of one or more components of the interferon system may be achieved. In fact, by this approach data have been obtained that the interferon system is multicomponent.
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
Induction
and Characterization Interferon
[28]
of Tortoise
B y A N G E L S. GALABOV
Methods for the study of the interferon system in the cells of tortoise are described in this chapter. Numerous data show that the interferon system is phylogenetically very old and of ubiquitous character: it is found in all classes of vertebrates, and the presence of interferon-like substances is proved also in the cells of insects and higher plants and in bacteria. 1-3 This mechanism of cell defense precedes not only the humoral forms of immunity, but perhaps phagocytosis as well. 4,5 The processes of interferon induction as well as the basic physicochemical characteristics and biological effects of interferon were studied almost exclusively in cell systems from birds and mammals. 6,7 However, convincing evidence exists that interferon production occurs also in the cells of lower (cold-blooded) vertebrates--fishes s-14 and reptiles. 15-21 The functioning of the interferon system in the cells of these animals shows some peculiar features that disclose more completely the interferon mechanisms. Tortoises are the most suitable among the reptiles as a source for preparing cell cultures for interferon studies. Methods for interferon induction in different types of tortoise cells are described below. Induction and Characterization of Interferon in Cell Culture of Tortoise Kidney
Preparation of Primary Cell Cultures of Kidney and Lung of Tortoise Primary cell cultures of tortoise kidney were prepared originally by Shindarov 22,zz and by Fauconnier and Pachopos. 24 According to the Shindarov's technique the kidney of an adult tortoise (Testudo graeca, Tes-
1 A. S. Galabov, Bull. Inst. Pasteur (Paris) 71, 233 (1973). 2 j. Vil~ek, "Interferons," pp. 30-31. Springer-Verlag, Vienna and New York, 1%9. 3 K. H. Fantes, in "Interferons and Interferon Inducers" (N. B. Finter, ed.), pp. 263-265. North-Holland Publ., Amsterdam, 1973. 4 N. N. Sirotinin, in "Manual of Microbiology, Clinics, and Epidemiology of Infectious Diseases" (G. B. Vygodtchikov, ed.), pp. 274-2%. Medizina Publ. House, Moscow, 1964. 5 L. A. Zilber, in "Basic Immunology" (V. I. Satchkov, ed.), pp. 173-183. Medgiz State Publ. House of Med. Liter., Moscow, 1958.
METHODS IN ENZYMOLOGY, VOL. 78
Copyright© 1981by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181978-7
[28]
TORTOISE INTERFERON
197
tudo hermani, etc.) are excised through a wide opening made in the plastron (the ventral part of the carapace). After decapsulation, the kidneys are cut into small pieces and disaggregated by fractional trypsinization at 35° with 0.2-0.25% trypsin (1 : 250, Difco) solution. A suspension of 1.5 to 3 × 105 cells/ml is prepared in growth medium containing 15% heated calf serum, 0.5% lactalbumin hydrolyzate, penicillin, and streptomycin (100 units/ml each) in saline solution (pH 7.2): Hanks' saline or saline prepared by dissolving 6.5 g of NaCI, 0.14 g of KCI, 0.12 g of CaCI2,0.2 g of MgSO4, and 0.02 g of phenol red in 1000 ml of double-distilled water. The cells ofTestudo graeca kidney (TGK cells) grow optimally at 37° and form a confluent layer within 3-5 days. This culture possesses a high stability, being preserved as an intact monolayer for at least 1 month if kept at 4 °. Its ability to grow is maintained up to 4 months under such conditions of storage.2~ Analogous is the method of preparing a primary cell culture of tortoise lung, 26a7 except that the trypsinization is performed at 32°. The cells are suspended at 2 × 10~ per milliliter in the growth medium. At 37° in 3 - 4 days they form a complete monolayer, consisting of epithelial and fibroblast types of cells. The suspension of trypsinized cells may be stored for 2 months at 4° while preserving its ability to growY 5
Interferon Induction in TGK Cells by Viruses Choice of lnducing Virus and Multiplicity of lnfection. This cell culture is susceptible to infection by a variety of animal viruses of almost all
6 N. B. Finter, ed., "Interferons and Interferon Inducers," pp. 1-262, 267-480. NorthHolland Publ., Amsterdam, 1973. 7 W. E. Stewart II, "The Interferon System," pp. 1-323. Springer-Verlag, Vienna and New York, 1979. 8 M. Gravell and R. G. Malsberger, Ann. N.Y. Acad. Sci. 126, 555 (1%5). 9 A. R. Beasly, M. M. Sigel, and L. W. Clem, Proc. Soc. Exp. Biol. Med. 121, 1169 (1%6). 10 H. K. Oie and P. C. Loh, Proc. Soc. Exp. Biol. Med. 136, 369 (1971). 11 R. K. Kelly and P. C. Loh, In Vitro 9, 73 (1973). 12 p. Kinkelin and M. Leberre, C. R. Hebd. Seances Acad. Sci., Ser. D. 279, 445 (1974). lz p. Kinkelin and M. Dorson, J. Gen. Virol. 19, 125 (1973). 1~ j. DeSena, and G. J. Rio, Infect. lmmun. 11,815 (1975). 1~ E. Falcoff and B. Fauconnier, Proc. Soc. Exp. Biol. Med. 118, 609 (1965). 16 A. Galabov, Z. Savov, and V. Vassileva, Acta Virol. (Prague) 17, 1 (1973). lr A. Galabov, S. Petrunova, and Z. Savov, Experientia 29, 900 (1973). 18 A. S. Galabov and Z. A. Savoy, Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. 1: Orig. Reihe A 225, 1 (1973). 19 A. S. Galabov, E. H. Velichkova, and S. S. Petrunova, in "Interferon and Interferon Inducers" (I. FOldes and M. Talas, eds.), pp. 136-159. Publ. House Hung. Acad. Sci., Budapest, 1975.
198
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
principal groups, including: alpha- (Sindbis 15'2s and Semliki Forest virus2a), flavi- (West Nile virusl6), orthomyxo- (influenza A/H2N2/57 and B3°), paramyxo- (Sendai 23'24, Newcastle disease virus, 3~ mumps, 3~ respiratory syncytial virus33), rhabdo- (vesicular stomatitis virus3~), adeno(adenovirus 52s), herpes- (herpes simplex virus2S'35), and pox- (vaccinia 36) viruses. Therefore, TGK cells can be used to study induction of interferon by viruses as well as for the interferon assay. As known, alpha-, flavi-, and paramyxoviruses are among the most potent interferon inducers in cultured homeothermic cells. T M The optimal multiplicity of infection (MOI) for highest interferon production by West Nile virus (WNV) (grown intracerebrally in mice) is 2 LD~o per cell. TGK monolayer cells (7 to 8 days old) are infected by 60-120 min adsorption at room temperature, wfished three times with saline, and covered with fresh maintenance medium without serum (medium 199 or Eagle's MEM with lactalbumin hydrolyzate, 0.05% and 0.2%, respectively, and antibiotics). An important advantage of this virus as inducer is that it stimulates a relatively high production of interferon without causing a cytopathic effect (CPE) up to day 10 after virus inoculation. At determined intervals the culture medium is harvested and adjusted with 2 N perchloric acid to pH 2.0. After 18 hr at 4 ° the liquid is centrifuged at 2500 rpm for 60 min, and the supernatant is brought to pH 7.0
20 A. S. Galabov and E. H. Velichkova, J. Gen. Viroi. 28, 259 (1975). 21 V. Vassileva and A. Galabov, Acta Microbiol. Acad. Sci. Hung. 22, 323 (1975). 22 L. Shindarov, Dokl. Bolg. Akad. Nauk 15, 539 (1962). 23 L. Shindarov, Acta Virol. (Prague) 6, 540 (1962). 24 B. Fauconnier and M. Pachopos, Ann. Inst. Pasteur, Paris 102, 661 (1962). 25 L. Shindarov, in "'Proceedings of First National Congress of Microbiology" (Sofia, 1965), pp. 693-696. Publ. House Acad. Bulg. Sci., Sofia, 1967. 26 L. Shindarov and V. Vassileva, Epidemiol. Mikrobiol. lnfekts. Boles. 3, 271 (1965). 27 L. Schindarov, and W. Wassileva, Arch. Gesamte Virusforsch. 19, 250 (1966). 2s L. Shindarov, Z. Savov, and V. Vassileva, Acta Med. Bulg. 6, 54 (1979). 29 L. Shindarov, Z. Savoy, S. Todorov, and B. Iwanov, Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. 1: Orig. Reihe A 222, 168 (1972). 30 L. Shindarov and S. Todorov, Khigiena 6(6), 24 (1963). 3x L. Schindarov and S. Todorov, Zentralbl. Bakteriol. Parasitenk., Infektionskr. Hyg. Abt. 1: Orig. Reihe A 186, 495 (1961). 32 B. Fauconnier, Ann. Inst. Pasteur, Paris 105, 439 (1963). 33 L. Shindarov, E. Velichkova, and N. Runewski, Acta Med. Bulg. 3, 7 (1975). 34 L. Shindarov, and Z. Savov, Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 1: Orig. Reihe A 192, 420 (1964). 35 B. Fauconnier, Ann. Inst. Pasteur, Paris 105, 444 (1963). 3e L. Shindarov, Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. 1: Orig. Reihe A 187, 285 (1962).
[28]
TORTOISE INTERFERON
199
1o24-1 512 256-: /2,9 64•
32-
~ f6-
/ i
t
i
I
i
i
i
I
i
I
24 48 72 .96 120 144 168 192 216 -','zO Houz'.s rjtc/e ~• v//'us / Hz~cu/u //2wJ
FIG. 1. Interferon production kinetics at 37° (Q) and 20° (©) in T e s t u d o g r a e c a kidney cells infected with West Nile virus. Data are from Galabov et al. 16
with 1 N NaOH. The titer of so-called " c r u d e " interferon thus obtained 1n,37 is determined (see below). At an incubation temperature of 37° the kinetics of interferon production in TGK cells shows a characteristic pattern (Fig. 1). The production begins after the sixth hour and reaches maximum at 48-72 hr after virus inoculation. Usually, in the next 24 hr a sharp reduction of the interferon titer occurs. 16,18 When Semliki Forest virus (SFV) is used as inducer (stock virus with 1-2 passages in TGK cells), the optimal multiplicity of infection is 1-3 (60-min adsorption at room temperature). A comparatively fast development of characteristic CPE up to a complete destruction of the cell monolayer at 72-96 hr is observed. The interferon liter reaches its maximum by 48 hr, with a markedly expressed CPE, but the cell monolayer is still preserved. 16 Newcastle disease virus (NDV) can also be used as an inducer. The inoculation of the cultures is carried out with undiluted UV-inactivated allantoic fluid (106 EID~ per milliliter of the stock virus) by 60-min adsorption at room temperature. The interferon production in this case begins by 12 hr and reaches a maximum by 48 hr. 21 Sendai virus (MOI = 1) induces a marked interferon production at 48 hr simultaneously with the appearance of a marked CPE. For this tea37 G. P. Lampson, A. A. TyteU, M. M. Nemes, and M. R. Hilleman, P r o c . S o c . E x p . Biol. M e d . 112, 468 (1%3).
200
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
son it is recommended to clarify the culture medium harvested by centrifugation (10 min at 3000 rpm) and then to sediment viral particles by a second centrifugation (2 hr at 50,000-100,000 g). The upper two-thirds of the latter supernatant are taken as a " c r u d e " interferon. ~ Optimal Age of the Inducible Cell Culture. The highest interferon production by virus inducers is obtained when "aged" TGK cell cultures are inoculated. For instance, the quantity of interferon induced by WNV in 7to 8-day-old cultures is about 16 times higher in comparison with that in 4-day-old cultures.~n'18 Stimulation of interferon production is known also in "aged" human, mouse, and chick cells a8-4° and is ascribed to the synthesis of a cell protein that stimulates interferon induction. 4~ Incubation Temperature. The optimal incubation temperature for virus-induced interferon production in TGK cells is 37°. ~n,a8 This coincides with the temperature optima both for the metabolic processes in TGK cells 29 and for their growth. 22,25,2a This cell system offers the possibility to study interferon induction at relatively low incubation temperatures. For example, when WNV-infected TGK cells are incubated at 20 °, a marked, although much delayed, interferon production is established (Fig. 1). Trace amounts of interferon can be detected not earlier than the 144th hour, and the maximum titers are still lower than that at 37 °. A relatively earlier establishment of interferon production at 20° is found after alphavirus (SFV, Sindbis) inoculation. The interferon level in the culture fluid persists for a comparatively longer period at 20° than at 37°, probably because of heat inactivation in the latter case. Interferon production in " y o u n g " and "aged" cell monolayers is approximately equal. 1n'~8 Of particular interest is that interferon production occurs in TGK cells even at 8 °, although the titers are rather low (4-8 units/ml), measured at 5-12 days after virus (SFV, Sindbis virus, WNV) inoculation. ~s This phenomenon of interferon production at low temperatures is due to the fact that at 8° some metabolic activity in TGK cells and alphavirus growth are still retainedY s'29
CharacteriZation of Virus-Induced Interferon in TGK Cells The following tests can be used for identification of the interferon in the culture fluids obtained as described above [treated culture fluid (TCF) = " c r u d e " interferon].
38 C. McLaren, Arch. Gesamte Virusforsch. 32, 13 (1970). 39 H. Liblkova and E. Henslov~i, Acta Virol. (Prague) 13, 16 (1969). 40 M. Ho and J.-F. Enders, Virology 9, 446 (1959). 41 N. Kato and H. J. Eggers, Virology 37, 545 (1969).
[28]
TORTOISE INTERFERON
201
Biological Tests I. Contact test for lack of virocidal effect. A suspension of vesicular stomatitis virus (VSV) or Sindbis virus, 100 TCIDs0/ml, is treated with TCF for 24 hr at 37°, and the residual infectivity is assayed. 15.10 2. Species specificity test. A parallel titration of TCF and mouse or human interferons is carried out on both TGK and L cells (or on human cells), challenged by VSV (100 TCIDso, see below) or Sindbis virus (1000 TCID~0), etc. 15'16 3. Lack of antiviral specificity. TCF is assayed in TGK cells, challenged by VSV, SFV, NDV, Sendai, mumps, and vaccinia viruses, HSV, etc. ls,16 4. Lack of effect of antiviral antibody. A mixture of equal volumes of TCF and virus-inducer specific antiserum are incubated for 60 min at 37° and the interferon titer of TCF is determined. 15
Effect of Physical and Chemical Agents 1. Stability at pH 2.0. The TCF is adjusted to pH 2.0 with 2 N perchloric acid and left for 48 hr at 4o. TM Alternatively, TCF can be subjected to dialysis for 24 hr at pH 2.0 and 24 hr at pH 7.0. TM 2. Heat stability test. A series of TCF samples are incubated for 30 or 60 min at 56°, 65°, and 75 °. The tortoise interferon is completely stable for 60 rain at 56° and is inactivated only partially at 65-750. TM 3. Ether treatment. TCF is mixed with ether (1 : 1, v/v), shaken for 10 min at 4°, and then centrifuged at 3000 rpm for 10 min. The aqueous phase is freed from residual ether by nitrogen. No clear reduction of interferon activity after ether treatment has been observed. 15 4. Trypsin treatment. Twice-crystallized trypsin (100-250/zg) is dissolved in 1 ml of TCF. After a 60-min incubation at 37°, 200/zg of purified trypsin soybean inhibitor are added. 15,1e Complete loss of interferon activity is seen. 5. Ribonuclease treatment. Add 100 ~g of RNase per milliliter of TCF for 2 hr at 37° and pH 7.0.15 6. Deoxyribonuclease treatment. Exactly as above but with DNase. The interferon is stable to both RNase and DNase.15,16
Molecular Weight Determination. The molecular weight of the tortoise interferon (virus-induced in TGK cells) can be determined 17 by gel filtration according to Andrews. 42 TCF are first dialyzed against 30 vol42 p. Andrews, Biochem. J. 91,222 (1964).
202
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
umes of PBS 43 (pH 7.4) for 72 hr and concentrated 15-30 times by the use of polyethylene glycol 6000 (Carbowax 20M, Serva). A column (110 × 1.5 cm) Sephadex G-100 (Pharmacia) is equilibrated with PBS, pH 7.4, containing 0.02% sodium azide. Calibration of the column can be performed with the following proteins used as standard markers: human serum albumin (69,000), bovine serum albumin (64,000, dimer 134,000), ovalbumin (45,000), chymotrypsinogen (25,000), soybean trypsin inhibitor (21,500), cytochrome c (13,000). Proteins (2-3 mg each) together with 0.2% Blue Dextran 2000 (Pharmacia) are dissolved in PBS and applied in a 2-ml volume. The flow rate of the column is 7-10 ml/cmZ/hr, and 4-ml fractions are collected. The optical density of the fractions is read spectrophotometrically at a wavelength of 280 nm; Blue Dextran is additionally read at 625 nm, and cytochrome c at 412 nm. To determine the molecular weight of the interferon, 2 ml of the concentrated TCF, containing Blue Dextran and cytochrome c, are applied to the column. The interferon titer of the 4-ml effluent fractions is determined (see below). The molecular weight is estimated by plotting the elution volume of the interferon peak on the calibration curve of the column. A typical elution profile of virus-induced tortoise interferon is presented in Fig. 2; the molecular weight is 33,500.17
Interferon Assay The Indiana strain of VSV can be recommended as a suitable challenge virus. It grows easily in TGK monolayer cell cultures, attaining a titer of 105 TCIDs0/ml or about 2.2 × 106 PFU/ml. VSV reproduces and forms plaques even at a suboptimal incubation temperature of 20°, although its reproductive cycle is markedly delayed, a0,44 The following methods for interferon assay can be recommended. 1. CPE inhibition method, an Tube monolayer TGK cell cultures are treated for 24 hr at 37° with serial dilutions of the TCF tested. The dilutions are done with medium 199 (Difco) supplemented with 0.05% lactalbumin hydrolyzate and antibiotics. Then the cells are washed twice and inoculated with 100 TCIDs0 of VSV. 2. Plaque-inhibition method, a6 Monolayer cultures in 50-100 ml flasks are inoculated with 100-150 P F U of VSV after being previously treated with diluted TCF as above. The cultures are covered with an agar overlay (4-8 ml) consisting of 1.5% agar (Difco) in Eagle's MEM (Difco), 10% heated calf serum, 2.25 mg of sodium bicarbonate per milliliter, and antibiotics. 43 R. Dulbecco and M. Vogt, J. Exp. Med. 99, 167 (1954). 44 A. S. Galabov, Z. Savov, and L. Shindarov, in "Abstracts of Third National Congress of Microbiology" (Sofia, 1973), p. 102. Publ. House Acad. Bulg. Sci., Sofia, 1973.
[28]
TORTOISE INTERFERON
203
Io ~_
b _-t05 ~ S e r u m albumin in
n Iryl~in inhiL~i~or
/04
t.
,,b
I
/0 J
] '
'
' / o ' o
'
'
1~o
'
'
'
rn I
Elutiofl volurrJe FIG. 2. Chromatographic pattern of Semliki Forest virus-induced interferon in Testudo graeca kidney cells on Sephadex G-100. A, Calibration curve; B, elution profile of concentrated interferon preparation, 1 ml. Data are from Galabov et al.t7
204
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
The interferon titer in both cases is determined after a 48-hr incubation at 37° and is expressed in interferon units per milliliter; i.e., the reciprocal of the highest dilution of the tested samples that prevents the CPE in at least half of the tube cultures (method 1) or results in a 50% reduction of the plaque number (method 2). The interferon can be assayed also by measuring the reduction of the challenge virus yield (e.g., Sindbis virus, MOI = 0.25) in TGK cells pretreated with diluted 1 : 4 TCF tested. 15
Antiviral Effect of Tortoise Interferon The effect of interferon on the reproduction of different viruses in TGK cells can be determined by doing one-step growth cycle or multicycle studies. Three-day-old monolayer cultures are treated for 18-24 hr at 37° with 50- I00 units per milliliter of tortoise interferon. After two washings with saline, the cultures are infected (by adsorption for 1-2 hr at 4° or at room temperature) at high MOI in order to obtain a one-step growth cycle. For instance, the MOI used is 10 for SFV and 1 for VSV and vaccinia virus, respectively. The maintenance medium consists of Eagle's MEM with lactalbumin hydrolyzate and 5% heated calf serum. At the end of the one-step cycle, whose duration has to be preliminarily determined, two flasks (or four test tubes) per sample are frozen for measuring the infectious virus yield. The antiviral effect of interferon can be expressed as inhibition of virus yield, zl,44,45 The duration of the one-step cycle in TGK cells of SFV, Sindbis virus, and VSV is 12 hr21aa; of vaccinia virus, 15 hr21; herpes simplex virus 1 and respiratory syncytial virus, 18-20 hr2S; Sendai virus and NDV, 20-24 h#S; and adenovirus 5, 24-30 hr. 2s The viruses tested can be arranged according to their sensitivity to interferon as follows: SFV(S-) (A logs = 3.3-4.0) > herpes simplex virus 1 (2.8 logs) > SFV(S+), VSV, and NDV (2.0-2.5 logs) > vaccinia virus (1.2-1.7 logs) > EMC virus and adenovirus 5 (0.7-1.0 l o g ) . 21'44"45 This cell system allows the antiviral effect of interferon to be studied at 20°. Moreover, both the treatment of the cells by interferon preparations and the reproduction of different viruses can be carried out at this incubation temperature? 8"28a9"44"45It is necessary to take into account the increase of the virus growth cycle time at this temperature. 28,29 For instance, the duration of the SFV growth cycle at 37° is 12 hr, whereas at 20° it is 72 hr. 29 The duration of the growth cycle at 20° of Sindbis virus is 96 hr; of adeno 5 and Sendai, 120 hr; of NDV, 144 hr; of herpes simplex virus 1,168 hr. 2s It is worth noting that the antiviral effect of interferon is approximately the same at 20° and 37° against most of the viruses tested. 18.44.45 45 A. S. Galabov and Z. Savov, unpublished data.
[28]
TORTOISE INTERFERON
205
In experiments on interferon assay, a clear stimulation of CPE by the challenge virus (VSV) is observed in the cell cultures treated with the lower dilutions (1:2-1 : 16) of TCF during 72 hr of induction (both at 20° and 37°)? ~a8 This phenomenon may be ascribed 1~a8 to a substance, synthesized as a component of the interferon system, like "antagonist, ''4~ "interferon antagonist,"47 o r " stimulon."48 The antagonist content can be expressed in interferon antagonist units (IAU) per milliliter, reciprocals of the highest dilution of TCF sample tested that stimulates clearly the CPE of VSV (100 TCIDso per test tube). There are clear-cut kinetic differences between the production of interferon and of antagonist in TGK cells, especially at 20°? 6a8 The "antagonist" possesses the following characteristics: stability at pH 2.0, inactivation by trypsin, and insensitivity to RNase and DNase? n Induction and Characterization of Interferon in Explanted Tortoise Peritoneal Leukocytes
Preparation of Explanted Tortoise Peritoneal Leukocytes Figure 3 schematically represents a technique for easy access to the peritoneum of the tortoise via an opening excised in the plastron of a decapitated adult animal. The peritoneal cavity is washed with 30-40 ml of cooled PBS. The wash is centrifuged in the cold at 1800-2000 rpm for 5 rain. The cell pellet is resuspended in medium 199 containing 10% heated calf serum and antibiotics, pH 7.3. The suspension is diluted to 2.5 × 106 cells/ml. The cells thus obtained (Testudo graeca peritoneal cells, TGP cells) tga° are mainly mononuclear leukocytes, namely: lymphocytes (90-95%), large mononuclear cells of the macrophage type (3-6%), and monocytes (2%). A small number of polymorphonuclear leukocytes are also present (1%). ~9,z°
Interferon Induction in TGP Cells by Viruses The TGP cell suspension is distributed into 1-ml portions in test tubes and immediately inoculated with NDV at an MOI of about 0.4 EIDso per cell, which is optimal for interferon production. 19,20 At 37° the interferon production is established as early as hour 2, increases until hour 8, and reaches relatively high titers (> 4000 units/ml). About a 16-fold decrease in production is found at 100 times greater MOI (40 EID~ per cell). At lower incubation temperatures, interferon production is strongly delayed. 46 j. L. Truden, M. M. Sigel, and L. S. Dietrich, Virology 33, 95 (1967). 47 E. T. Sheaff and R. B. Stewart, Can. J. Microbiol. 14, %5 (1%8). 4~ C. Chany and C. Brailovsky, C. R. Hebd. Seances Acad. Sci. Ser. D 261, 4282 (1965).
206
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
F~G. 3. Excisingan opening m the plastron (ventral part of the carapace) of a tortoise (Testudo graeca) for access to the peritoneum. For instance, at 26° interferon titers attain a normal level only by hour 20, a level that is attained by 6 hr at 37° (Fig. 4A). B y contrast with the observations with TGK cells, no interferon production in response to virus inducer occurs at 20° or 80.19,2° " C r u d e " interferon (TCF) samples are prepared by single freezing and thawing of the suspensions of TGP cells and subsequent adjustment with 6 N HCI to pH 2.0. After 2 or 3 days at 4° the suspension is centrifuged and the pH of the supernatant is adjusted to 7.0 with 5 N NaOH. Interferon is assayed with TGK cells challenged by VSV as described previously.
Interferon Induction in TGP Cells by Poly(I).Poly(C) Interferon production in TGP cells can be induced by a 30-min poly(I).poly(C) treatment (e.g., 30/~g per 2.5 × I06 cells). The cells are then centrifuged (1800 rpm for 5 min at 4°), washed once with PBS, resuspended in fresh medium, and incubated at 26° for 24 hr. ~9
Interferon Induction in TGP Cells by Enterobacterial Endotoxins The TGP cells release interferon also after treatment with endotoxintype inducers, e.g., O antigens, lipopolysaccharide, and glycolipid of Enterobacteriaceae. The optimal incubation temperature for interferon production in this
TORTOISE I N T E R F E R O N
[28]
2s6 /
. . . . . .
207 ,..,
! A
.~
64
Z
KTGPC
2
5
4
5
6 7 HOURS
8
20
8
20
256 ~E
128
o'J I.-
64
Z D
32
Z 0 n-W LL 13:7
w I---
B
HJ
16 8 4
2
3
4
5
6 7 HOURS
F]G. 4. Interferon production kinetics at 37° (stippled bar), 26 ° (filled bar); 20° (hatched bar), and 8° (open bar) in Testudo graeca peritoneal cells. (A) Inoculated with Newcastle disease virus (H strain; MOI = 40). (B) Treated with Serratia marcescens endotoxin (1 /,¢g per 2.5 × 106 cells). KTGPC = noninoculated cells (control). Data are from Galabov and Velichkova. z°
case is 26°. 19,2° Similarly to the peritoneal cells of mammals, 49,5° the doseresponse curve for bacterial endotoxins (e.g., O antigens) in TGP cells shows a maximum at 1/zg/ml. The kinetics of interferon production is typical: interferon is released after hour 4 and reaches its maximum at hour 6 (Fig. 4B). At 37 ° no interferon can be detected at least up to hour 8. At 20 ° interferon production is lower as compared to production at 26 ° , the maximum being achieved not earlier than at about 20 hr. It is of partic49 L. Borecky, V. Lackovi~, and K. Waschke, in " L ' l n t e r f ~ r o n " (C. Chany, and P. De Somer, eds.), Colloq. Inst. Natl. Sante Rech. Med. 6, 37 (1969). 50 A. S. Galabov and S. M. Galabov, Acta Virol. (Prague) 17, 493 (1973).
208
INDUCTION AND PRODUCTION OF INTERFERONS
[28]
ular interest that interferon production is found even at 8°, although greatly reduced and delayed? %z° Under the conditions described, especially when the explanted cells are rapidly prepared and immediately used, the spontaneously released interferon in TGP cells shows very low titers (4 units/ml) at 26° and is practically absent at 20 ° and 8°.
Characterization of TGP Cell Interferon The antiviral substance produced in TGP cells, irrespective of the type of inducer used, displays all the basic properties of interferon, as determined by the methods described above: lack of virocidal effect and antiviral specificity, species specificity, stability at pH 2.0, a relatively high thermostability (30-60 min at 56°), a high sensitivity to trypsin, and resistance to RNase, DNase, and ether treatment. 19 The molecular weight of TGP cell interferon can be determined by Sephadex gel chromatography. The interferon samples are dialyzed for 3 days in citrate buffer (0.098 M citric acid, 0.004 M NazHPO4), pH 2.15, and 2 days in PBS, pH 7.4, then concentrated 10-fold with polyethylene glycol 6000. The following molecular species of interferon produced in TGP cells has been found19: (a) in NDV-induced cells, a single peak with molecular weight of 58,500; (b) in endotoxin-induced cells, two species with molecular weights of I02,000 and 43,500; (c) in poly(I).poly(C)-induced cells, two species with molecular weights of 144,000 and 63,000 Conclusion An analysis of the experimental data on induction and characteristics of tortoise interferon demonstrates the functioning of a complete interferon system, analogous to that in mammals and birds. The large temperature range (more than 30 °) throughout which the interferon system functions is together with some other metabolic activities 2a in tortoise cells (TGK, TGP) is a definite advantage. By stepwise decrease of the incubation temperature it has been possible to characterize in more detail the separate components of the interferon system. As demonstrated, 18by choosing a suitable incubation temperature a selective synthesis of one or more components of the interferon system may be achieved. In fact, by this approach data have been obtained that the interferon system is multicomponent.