Production of Lymphokine-Like Factors (Cytokines) by Simian Virus 40-Infected and Simian Virus 40-Transformed Cells Plerluigi E. Bigazzi, MD, Takeshi Yoshida, MD, Peter A. Ward, MD, and Stanley Cohen, MD
Macrophage migration inhibitory (MIF-like) activity was demonstrated in the supernatant fluids from primary cultures of African green monkey kidney cells infected with simian virus 40 (SV 40) virus. Kidney cell cultures not infected by virus had no MIF activity. Supernatant fluids from continuous cultures of nontransformed and SV 40-transformed human fibroblasts contained MIF-like activity. Productive infection with SV 40 virus results in the production of a lymphokine-like factor, as previously observed in other virus-cell systems, involving mumps virus and Newcastle disease virus. However, while infection with these paramyxoviruses causes the production of macrophage and neutrophil chemotactic agents as well as an MIF, SV 40 infection does not induce chemotactic factors. The results reported here, taken in conjunction with previous observations by ourselves and others, suggest that the production of lymphokine-like factors (cytokines) may represent a general biologic phenomenon, and that many, if not all, cell types, when appropriately stimulated, may be capable of such activity. (Am J Pathol 80:69-78, 1975)
VIRAL INFECTION of variouis nonlymphoid cells has been shown to resuilt in the produiction of factors which modify the behavior of different kinds of inflammatory cells. The viruses uised were Newcastle disease viruis and muimps virus, the infected cells were chick embryo, monkey kidney, and monkey parotid cells, in in vivo or in vitro systems." The suibstances released by infected cells appear to be distinct from interferon and incltude a factor which inhibits macrophage migration in vitro and factors which are chemotactic for neutrophils and macrophages. It has been suggested that the production of substances which modify inflammatory cell properties may represent a general biologic phenomenon, capable of being triggered by different agents in different cells, and that this phenomenon might play a role in host defense. Thuis, in the case of viral infection, mechanisms of resistance would be threefold, namely, interferon produiction, the immune response, incluiding both antibody and lymphokine production, and the generation of lymphokine-like From the Department of Microbiology and The Center for Immtnology, State University of New York at Bulffalo, Buffalo, New York, and the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticuit. Supported by Grants CA-02357, AI-12225, and AI-12477 from the National Institutes of Health. Accepted for ptublication March 6, 1975. Address reprint reqtuests to Dr. P. E. Bigazzi, Department of Microbiology, School of Medicine, 327 Sherman Hall, State University of New York at Btuffalo, Btuffalo, NY 14214. 69
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suibstances by the infected cells themselves. These latter suibstances have been defined as cytokines.4' Cytokine produiction was first investigated with respect to infection by paramyxovirtises. SV 40 virtus was then chosen because of its specific interest as an oncogenic virus and because of its possible relationship with viruises isolated from patients with progressive multifocal leuikoencephalopathy.6'8 Preliminary investigations demonstrated the production of an MIF-like suibstance by infected monkey cells.9 Recently, Hammond et al.10 have reported the presence of macrophage migration inhibitory activity in suipernatants from SV 40-transformed mouse fibroblasts. The present study was designed to further explore the ability of SV 40 virus to induce the production of cytokines in productively infected and in transformed cells. Materials and Methods Cell Cultures Primary African green monkey kidney (AGMK) cells (GIBCO, Grand Island, N.Y.) were grown in 75 sq cm plastic flasks (Falcon Plastics, Oxnard, Calif.), using 25 ml/flask of Eagle's minimuim essential mediuim (MEM) in Earle's saline, suipplemented with 10% fetal calf seruim (FCS, GIBCO), 100 units/ml penicillin, and 100 ,g/ml streptomycin. AGMK cells were also grown in 25 sq cm plastic flasks (Falcon) uising 5 ml/flask of MEM and in 16 by 125 mm tuibes uising 1.0 ml/tube of the same medium. Normal WL-38 and SV 40-transformed WL-38 (WI-38VA13A) obtained, respectively, from Drs. Hayflick and Girardi, were grown in 25 sq cm plastic flasks uising 5 ml/flask of Eagle's MEM with 10% FCS. Virus Preparations SV 40 viruis (VR239) was obtained from the American Type Cultuire Collection and passaged twice in primary AGMK cells. Cultuires were evaluated for characteristic cytopathic effects, that is, vacuiolization, rotunding, pyknosis, and detachment of cells. Infectivity assays were performed by inoculating 0.1 ml of viruis suspension at tenfold dillutions into tuibe cuiltuires of primary AGMK cells, observing the cuiltuires for cytopathic effects for a period of 14 days and then calcuilating titers by the method of Reed and Muench." After the second passage, the virus titer was 105 8 TCID50/0.1 ml. Harvesting of Media From Cell Cultures Normal Primary AGMK Cells When cell monolayers had become confluent, the mediuim was changed. After 24 houirs of inctubation the mediuim from one flask was removed, irradiated with uiltraviolet light uising a germicidal lamp (Spectroderm International Co., Fairfax, Va.) at a distance of 15 cm for 15 minuites, centrifuiged at 1085g for 30 mintutes in the cold (Sorvall Suiperspeed RC2B) and then stored at -85 C. After 48 houirs the medium from another flask was removed and similarly treated, and so on for 7 days. SV 40-Infected Cultures
Complete monolayers of primary AGMK cells in 75 sq cm flasks were inocuilated with 0.5 ml of tndiluited SV 40 virus, gently rocked to insure maximtum adsorption, and in-
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ctibated at room temperatture for 20 mintutes. After this period, 25 ml of Eagle's MEM stupplemented with 2% FCS were added, and the bottles were incuibated at 37 C. Control flasks were inoctulated with 0.5 ml meditum or 0.5 ml of noninfected AGMK cell Iysates, and then treated in a similar way. After 24 hoturs of inctubation, the media from one infected and one control flask were removed, irradiated with uiltraviolet light, etc., as described above. Before harvesting the meditum, cuiltuires were observed for cytopathic effects, and the percentage of cells showing CPE was recorded. All materials were stored at -85 C after collection. WI-38 and WI-38VA13A
On the third day of cuiltture the meditum from all bottles was removed, centriftuged, and frozen. After 5 days of cuiltture, when a complete cell monolayer was formed, the meditum from one flask was removed and treated as previotusly described, and so on from the sixth to twelfth days. Macrophage Migration Inhibition
Normal Hartley gtuinea pigs weighing 350 to 400 g were injected with 15 ml sterile light mineral oil. Fouir days later, peritoneal extudate cells were collected, washed twice with Hanks' balanced salt soltution (GIBCO) and packed into capillary ttubes as described previoutsly.12 Two capillary ttubes were ctulttured in a Sykes-Moore chamber containing test ctultture fltuid stupplemented with normal gtuinea pig sertum. The migration from at least fotur capillary tthbes was meastured for each test sample. The procedtures uised to calcuilate migration index (MI) and percent inhibition were also described previouisly.12 The MI was calctilated as the ratio of the migration area of the experimental to the control ttibes. In accordance with uisuial practice, an MI of 80 or less was considered as significant activity. Assays for Chemotactic Activity
A modified Boyden chamber was uised as previouisly described.1"' The chamber was divided by a micropore filter in an uipper compartment containing netitrophils or macrophages, and a lower compartment containing 100 ,ul of the fluiid to be assayed for chemotactic activity. Neuitrophils were obtained from rabbits injected intraperitoneally with 0. I% glycogen in saline 4 houirs previouisly 14 and macrophages from rabbits injected intraperitoneally with mineral oil 4 days previouisly.'5 All cells were suispended in mediuim 199 with 10% rabbit seruim or 0.1 % bovine seruim albuimin. When neuitrophils were uised, filters of pore size 0.65 u were uitilized in the chambers; with macrophages, filters of pore size 5 ,u were uitilized. Cell migration was qtuantitated by counting the nuimber of migrating cells in five high-power fields tinder a light microscope. Fractionation of Fluids Containing MIF-Like Activity
Fluiids were concentrated as previouisly described 2 and then applied to a Sephadex GI00 coluimn equiilibrated with 0.01 M phosphate-buiffered saline, pH 7.4. Eluiates were collected at a flow rate of 0.1 ml/min in 3.0-ml samples. Fractions near the void voluime (which contain proteins of high molecuilar weight) were pooled as Fraction I, while fractions containing the phenol red marker were pooled as Fraction VI. Intermediate fractions of e(quial voluime were pooled as Fractions II to V (approximately 30 ml/fraction). All fractions were lyophilized, redissolved into 3 ml of buiffer, dialyzed against RPMI 1640 mediuim, and then tested for MIF activity.
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Table 1-Failure of Supernatants From Noninfected AGMK Cell Cultures to Inhibit Macrophage Migration
Percent inhibition -7.7 -0.8 5.9 -10.9 -1.5 -4.4 0.6
Migration index* Day 107.7 ± 7.4 1 2 100.8 ± 8.2 3 94.1 ± 5.6 4 110.9 ± 7.8 5 101.5± 9.1 104.4 ± 13.6 6 7 99.4 ± 5.6 * With respect to medium as control.
Results
SV 40-Infected AGMK Cells
As shown in Table 1, there was no inhibition of macrophage migration by media from uninfected AGMK cells cultuired for a period of 7 days. If anything, there was a slight stimulation of the migration of guinea pig macrophages. On the other hand, when media from SV 40-infected cuiltuires were tested, using as controls media from noninfected cells, there was definite inhibition of migration. This was most noticeable with media obtained 24, 48, and 72 hours after infection (Text-figure 1). No significant inhibition was observed with media obtained after the third day of infection. When cells were observed for cytopathic effects, none were seen during the first 2 days of infection, but in the following days there was a steady increase in the percentage of affected cells. To ruile ouit the possibility that migration was inhibited by a factor cytotoxic for macrophages, the viability of the inhibited macrophages was tested by trypan blue exclusion and by continued cultivation for over 48 houirs. No difference in viability was observed between macrophages treated with control supernatant and those treated with supernatant from 50 40 C 0 a
30-
c
3 20 . 10
K
I
IL
v
1
2
3
4
DAYS
5
6
7
100 _
80 S._ 40 v 20
it
TFXT-FIGURE 1-Macrophage migration inhibitory activity (solid columns) in suipernatants of SV 40infected AGMK cell ctiltures. The percentage of cells in these cuiltures showing cytopathic effects (line), as defined in the text, is plotted on the same time scale.
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infected cells. To rtile ouit the possibility that residuial virus in the fluiids interfered with macrophage migration even in the absence of gross cytotoxicity, doses of virus identical to those used to infect AGMK cell ctultuires were added to cell-free medium, which was then centrifuged and irradiated with uiltraviolet light in the same manner as the supernatants from infected cultures. The resulting preparation was devoid of migration inhibitory activity. In fact, even nonirradiated control preparations prepared in this way were devoid of activity. In contrast to our previous observations with mumps and Newcastle disease virus, no evidence of chemotactic activity for neutrophils or macrophages was found in any of the supernatants from the SV 40-infected cultures. Characterization of MIF-Like Substance
Media obtained from SV 40-infected AGMK ctulttures at 24 hours after infection, i.e., when the MIF-like activity is at its maximum, were subjected to fractionation by Sephadex G-100 chromatography. The MIF-like activity of each fraction was tested and was found to be greatest in a fraction corresponding to a molecular weight range of 45,000 to 65,000 and in another fraction corresponding to an approximate molecular weight of 12,000 (Text-figuire 2). WI-38 and WI-38VA13A Cells
Media from complete monolayers of normal and SV 40-transformed WI-38 cells were tested on guinea pig macrophages using as control pooled media from the first 3 days of cultuire, which were uiniformly devoid of activity. Inhibition of macrophage migration was obtained with media from both types of cell cultuires over a period of several days (Table 2). Althouigh both normal and SV 40-transformed cells were capable of prodtucing MIF activity, the supernatants from SV 40-transformed fibroblasts harvested on the sixth and seventh days had significantly greater activity than corresponding fluids from normal cell cuiltures.
TEXT-FIGURE 2-Sephadex G100 coltmn chromatography of active stipernatant from SV 40infected AGMK cell colltoires. (OD, line; inhibition, solid columns, ovalbtomin marker, a; cytochrome C marker, b)I
b
a
4.0 a
E 3.0 Go
2.0
01.0
50 I
II-
0
30 10 C
TUBE NUMBER
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Table 2-Macrophage Migration Inhibition Activity of Supernatants From Normal and SV 40-Transformed (WI-38VA13A) Human Fibroblasts
(WI-38)
Migration index Day
Normal*
SV 40-transformed*
39.3 ± 2.7 56.3 ± 3.5 6 7 64.1 ± 2.3 43.0 ± 3.1 48.0 ± 1.8 8 38.3 ± 1.5 46.9 ± 2.5 39.7 ± 3.0 10 32.8 ± 2.1 11 39.6 ± 1.6 42.4 ± 2.6 12 35.6 ± 2.0 * Migration index calculated with respect to pooled (negative) supernatants from Days 1 to 3; this index reflects absolute activity of each supernatant.
Discussion
The present stuidy shows that media from monkey kidney cells infected with SV 40 contain a suibstance capable of inhibiting the migration of guiinea pig peritoneal macrophages. This suibstance is not toxic for macrophages, as demonstrated by vital staining and migration of these cells after continuiouis cuiltivation for more than 48 houirs. We have also excluided the possibility that residuial viruis in the fluiids couild interfere with macrophage migration even in the absence of gross cytotoxicity. The MIF-like activity can be localized by Sephadex chromatography in two fractions, one corresponding to a molecular weight of 45,000 and the other to 12,000. Classic MIF derived from antigen-stimulated lymphocyte cuiltuires has also been localized in two fractions of similar molecuilar weight.16 These results are somewhat different than those reported in stuidies of muimps or Newcastle disease viruis infections, where only the larger molecuilar weight migration inhibition factor could be demonstrated.2 The other difference between the results of the present study and those we have previotusly reported is that infection by mumps or Newcastle disease viruises leads to the generation of chemotactic factors for nelutrophils and macrophages as well as MIF activity. Fuirther work with a variety of viruis-cell systems is necessary to determine the biologic significance of this distinction. In any case, the dissociation of MIF and chemotactic factor produiction reported here may have important practical considerations, as this could facilitate the isolation and characterization of these factors, first in virus-infected cell systems and subsequiently in antigenstimulated lymphocyte cultuires. As one example, the preparation of monospecific antisera directed against single lymphokines requires starting material with restricted activities. To date we have obtained only
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antisera with generalized antilymphokine activity using lymphocytederived, MIF-rich preparations for immunizations.'7 If similarities of biologic function of the various MIF-like factors imply chemical and antigenic similarity, then suitable preparations of virus-induiced MIF could be used to prepare antisera which might then be of use in the study of the more complicated lymphocyte systems. The present results extend our previous studies on cytokine produiction by muimps and Newcastle disease virus.'3 Lytic infection with SV 40 leads to the appearance of a migration inhibition factor in cultuires of monkey kidney cells which otherwise do not release such factors. We have also shown that continuouis lines of human fibroblasts, both normal and SV 40transformed, release suibstances with MIF activity into the mediuim. A comparison between MIF activity of the two types of cells indicates that in the early stages of cultuire, SV 40-transformed cells may release more MIF-like substance than normal cells. However, it is also possible that uinavoidable differences in the metabolism of the two different cell types may have affected relative migration inhibitory activity in a nonspecific manner. A similar production of MIF had been previously observed in lymphoid and nonlymphoid cell lines,'8"19 while recently Hammond et al.10 have reported that SV 40-transformed mouse fibroblasts elaborate an MIF-like substance. In contrast to our observations, these latter auithors found that their untransformed fibroblast line (3T3) had no background MIF activity. In our previous investigations we demonstrated that release of MIF induced by mumps virus was independent of the production of interferon.2 Even though, in the present study, we have not tested for interferon produiction, the evidence available seems to show that SV 40 is a poor interferon induicer. Plummer 20 and Oxman 21 have not observed any interferon production by monkey cells infected with this virus. Diderholm 22 reported the production of a substance inhibitory for pseudorabies and polymyelitis viruses, with the highest titers late in the course of SV 40 infection (from the eighth to the twentieth day). On the other hand, the MIF-like activity we have observed in the infected monkey cell system is present only from the first to the third day after infection. In recent years a variety of activities have been attributed to interferon, in addition to its ability to induce resistance to viral infections.23'24 These activities include the inhibition of transformation of cells by oncogenic viruses 25,26; the inhibition of growth of malignant cells, both in vitro and in vivo 27,28; the enhancement of phagocytosis and lymphocyte-mediated cytotoxicity 29,30; and the activation or the inhibition of antibody-forming cells.3' Since preparations of interferon are rather crude, all these activities might not
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necessarily be carried by the same molecuilar species S2 btut instead might represent different produicts released by infected cells. The present sttudy and ouir previous observations demonstrate that muiltiple suibstances with diverse biologic properties are generated as a consequience of viruis infection. Many of these have featuires in common with certain of the known lymphokines. For this reason, we defined a new class of biologic mediator substances, the cytokines.4' These are substances produiced by nonlymphoid as well as lymphoid cells when those cells are triggered by a variety of agents (incluiding viral infections). These suibstances modify the behavior of other cells (most notably inflammatory cells). In this view, lymphokines, the mediator substances produiced by lymphocytes (triggered by specific antigen or by mitogens), represent a restricted set of cytokines made by one class of cells, activated in certain uinique ways. It is tempting to postulate that cytokine produiction represents one of the most primitive cellular self-defense systems and that cellular immuinity, dependent on lymphokine production, and humoral immunity represent a suibsequient evoluition of this system. References 1. Ward PA, Cohen S, Flanagan TD: Leukotactic factors elaborated by viruis-infected tissuies. J Exp Med 135:1095-1103, 1972 2. Flanagan TD, Yoshida T, Cohen S: Produiction of macrophage migration inhibition factors by viruis-infected cell cuiltuires. Infect Immun 8:145-150, 1973 3. Yoshida T, Flanagan TD, Genco RJ, Cohen S: Viruis-induiced migration inhibitory activity in experimental muimps infection. Clin Immuinol Immuinopathol 2:472-480, 1974 4. Cohen S, Ward PA, Bigazzi PE: Cell cooperation in cell-mediated immuinity. Mechanisms of Cell-Mediated Immunity. Edited by RT McClulskey, S Cohen. New York, John Wiley and Sons, 1974, pp 331-358 5. Cohen S, Bigazzi PE, Yoshida T: Similarities of T cell fuinction in cell-mediated immulnity and antibody prodtuction. Cell Immunol 12:150-159, 1974 6. Weiner LP, Herndon RM, Narayan 0, Johnson RT, Shah K, Ruibenstein LJ, Preziosi TJ, Conley FK: Isolation of viruis related to SV40 from patients with progressive mtultifocal letukoencephalopathy. N Engl J Med 286:385-390, 1972 7. Mtullarkey MF, Hrtuska JF, Takemoto KK: Comparison of two huiman papovavirtuses with simian viruis 40 by strtuetuiral protein and antigenic analysis. J Virol 13:1014-1019, 1974 8. Martin MA, Gelb LD, Garon C, Takemoto KK, Lee TNH, Sack GH Jr, Nathans D: Characterization of "heavy" and "light" SV40-like particles from a patient with PML. Virology 59:179-189, 1974 9. Bigazzi PE, Cohen S, Ward PA, Yoshida T: Produiction of lymphokine-like factors by SV40-infected cell cuiltuires. Fed Proc 33:640, 1974 (Abstr) 10. Hammond ME, Roblin RO, Dvorak AM, Selvaggio SS, Black PH, Dvorak HF: MIF-like activity in simian virtus 40-transformed 3T3 fibroblast cuiltuires Science 185:955-957, 1974 11. Reed LJ, Mutench H: A simple method of estimating 50 per cent endpoints. Amer J Hyg 27:493-497, 1938
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12. Yoshida T, Janeway CA Jr, Pauil WE: Activity of migration inhibitory factor in the absence of antigen. J Immuinol 109:201-206, 1972 13. Ward PA, Cochrane CG, Miiller-Eberhard HJ: Fturther stuidies on the chemotactic factor of complement and its formation in vivo. Immuinology 11:141-153, 1966 14. Hill JH, Ward PA: C3 letikotactic factors produiced by a tissuie protease. J Exp Med 130:505-518, 1969 15. Ward PA: Chemotaxis of mononuiclear cells. J Exp Med 128:1201-1221, 1968 16. Yoshida T, Sonozaki H, Cohen S: The produiction of migration inhibition factor by B and T cells of the guiinea pig. J Exp Med 138:784-797, 1973 17. Yoshida T, Bigazzi, PE, Cohen S: The produiction of anti-guinea pig lymphokine antibody. J Immunol 114:688-691, 1975 18. Papageorgioui PS, Henley WL, Glade PR: Produiction and characterization of migration inhibitory factor(s) (MIF) of established lymphoid and non-lymphoid cell lines. J Immuinol 108:494-504, 1972 19. Tuibergen DG, Feldman JD, Pollack EM, Lerner RA: Produiction of macrophage migration inhibition factor by continuiouis cell lines. J Exp Med 135:255-266, 1972 20. Pluimmer G: Interfering properties of simian viruses. Br J Exp Pathol 44:58-65, 1963 21. Oxman MN: Interferon, tuimors and tuimor viruises. Interferons and Interferon Induicers. Edited by NB Finter. New York, North-Holland/American Elsevier, 1973, pp 391-480 22. Diderholm H: Produiction of interferon by monkey kidney cells infected with simian viruis 40. Arch Ges Viruisforsch 14:39-44, 1963 23. Grossberg SE: The interferons and their inducers: molecuilar and therapeuitic considerations (third of three parts). N Engl J Med 287:122-128, 1972 24. Acton JD: The lymphoreticuilar system and interferon produiction. J Reticuiloendothel Soc 14:449-461, 1973 25. Todaro GJ, Baron S: The role of interferon in the inhibition of SV40 transformation of mouise cell line 3T3. Proc Natl Acad Sci USA 54:752-756, 1965 26. Oxman MN, Levin MJ: Interferon and transcription of early viruis-specific RNA in cells infected with simian viruis 40. Proc Natl Acad Sci USA 68:299-302, 1971 27. Gresser I, Bouirali-Matiry C: The antituimor effect of interferon in lymphocyte- and macrophage-depressed mice. Proc Soc Exp Med Biol 144:896-900, 1973 28. Gaffney EV, Picciano PT, Grant CA: Inhibition of growth and transformation of huiman cells by interferon. J Natl Cancer Inst 50:871-878, 1973 29. Huiang K-Y, Donahoe RM, Gordon FB, Dressler HR: Enhancement of phagocytosis by interferon-containing preparations. Infect Immlun 4:581-588, 1971 30. Lindahl P, Leary P, Gresser I: Enhancement by interferon of the specific cytotoxicity of sensitized lymphocytes. Proc Natl Acad Sci USA 69:721-725, 1972 31. Bratun W, Levy HB: Interferon preparations as modifiers of immuine responses. Proc Soc Exp Biol Med 141:769-773, 1972 32. Borecky L, Ftuchsberger N, Hajnicka V, Stancek D, Zemla J: Distribtution of antiviral and cell-inhibitory activity in interferon preparations. Acta Virol (Praha) 16:356-358, 1972
Acknowledgments We thank Ann Marie Least and Dorothy Osmak for their skilled technical assistance and Caroline Maltbie for her excellent secretarial assistance in the preparation of the mantuscript.
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American Journal of Pathology
Macrophage migration inhibitory (MIF-like) activity was demonstrated in the supernatant fluids from primary cultures of African green monkey kidney cells infected with simian virus 40 (SV 40) virus. Kidney cell cultures not infected by virus had no MIF activity. Supernatant fluids from continuous cultures of nontransformed and SV 40-transformed human fibroblasts contained MIF-like activity. Productive infection with SV 40 virus results in the production of a lymphokine-like factor, as previously observed in other virus-cell systems, involving mumps virus and Newcastle disease virus. However, while infection with these paramyxoviruses causes the production of macrophage and neutrophil chemotactic agents as well as an MIF, SV 40 infection does not induce chemotactic factors. The results reported here, taken in conjunction with previous observations by ourselves and others, suggest that the production of lymphokine-like factors (cytokines) may represent a general biologic phenomenon, and that many, if not all, cell types, when appropriately stimulated, may be capable of such activity. (Am J Pathol 80:69-78, 1975)
VIRAL INFECTION of variouis nonlymphoid cells has been shown to resuilt in the produiction of factors which modify the behavior of different kinds of inflammatory cells. The viruses uised were Newcastle disease viruis and muimps virus, the infected cells were chick embryo, monkey kidney, and monkey parotid cells, in in vivo or in vitro systems." The suibstances released by infected cells appear to be distinct from interferon and incltude a factor which inhibits macrophage migration in vitro and factors which are chemotactic for neutrophils and macrophages. It has been suggested that the production of substances which modify inflammatory cell properties may represent a general biologic phenomenon, capable of being triggered by different agents in different cells, and that this phenomenon might play a role in host defense. Thuis, in the case of viral infection, mechanisms of resistance would be threefold, namely, interferon produiction, the immune response, incluiding both antibody and lymphokine production, and the generation of lymphokine-like From the Department of Microbiology and The Center for Immtnology, State University of New York at Bulffalo, Buffalo, New York, and the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticuit. Supported by Grants CA-02357, AI-12225, and AI-12477 from the National Institutes of Health. Accepted for ptublication March 6, 1975. Address reprint reqtuests to Dr. P. E. Bigazzi, Department of Microbiology, School of Medicine, 327 Sherman Hall, State University of New York at Btuffalo, Btuffalo, NY 14214. 69
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suibstances by the infected cells themselves. These latter suibstances have been defined as cytokines.4' Cytokine produiction was first investigated with respect to infection by paramyxovirtises. SV 40 virtus was then chosen because of its specific interest as an oncogenic virus and because of its possible relationship with viruises isolated from patients with progressive multifocal leuikoencephalopathy.6'8 Preliminary investigations demonstrated the production of an MIF-like suibstance by infected monkey cells.9 Recently, Hammond et al.10 have reported the presence of macrophage migration inhibitory activity in suipernatants from SV 40-transformed mouse fibroblasts. The present study was designed to further explore the ability of SV 40 virus to induce the production of cytokines in productively infected and in transformed cells. Materials and Methods Cell Cultures Primary African green monkey kidney (AGMK) cells (GIBCO, Grand Island, N.Y.) were grown in 75 sq cm plastic flasks (Falcon Plastics, Oxnard, Calif.), using 25 ml/flask of Eagle's minimuim essential mediuim (MEM) in Earle's saline, suipplemented with 10% fetal calf seruim (FCS, GIBCO), 100 units/ml penicillin, and 100 ,g/ml streptomycin. AGMK cells were also grown in 25 sq cm plastic flasks (Falcon) uising 5 ml/flask of MEM and in 16 by 125 mm tuibes uising 1.0 ml/tube of the same medium. Normal WL-38 and SV 40-transformed WL-38 (WI-38VA13A) obtained, respectively, from Drs. Hayflick and Girardi, were grown in 25 sq cm plastic flasks uising 5 ml/flask of Eagle's MEM with 10% FCS. Virus Preparations SV 40 viruis (VR239) was obtained from the American Type Cultuire Collection and passaged twice in primary AGMK cells. Cultuires were evaluated for characteristic cytopathic effects, that is, vacuiolization, rotunding, pyknosis, and detachment of cells. Infectivity assays were performed by inoculating 0.1 ml of viruis suspension at tenfold dillutions into tuibe cuiltuires of primary AGMK cells, observing the cuiltuires for cytopathic effects for a period of 14 days and then calcuilating titers by the method of Reed and Muench." After the second passage, the virus titer was 105 8 TCID50/0.1 ml. Harvesting of Media From Cell Cultures Normal Primary AGMK Cells When cell monolayers had become confluent, the mediuim was changed. After 24 houirs of inctubation the mediuim from one flask was removed, irradiated with uiltraviolet light uising a germicidal lamp (Spectroderm International Co., Fairfax, Va.) at a distance of 15 cm for 15 minuites, centrifuiged at 1085g for 30 mintutes in the cold (Sorvall Suiperspeed RC2B) and then stored at -85 C. After 48 houirs the medium from another flask was removed and similarly treated, and so on for 7 days. SV 40-Infected Cultures
Complete monolayers of primary AGMK cells in 75 sq cm flasks were inocuilated with 0.5 ml of tndiluited SV 40 virus, gently rocked to insure maximtum adsorption, and in-
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ctibated at room temperatture for 20 mintutes. After this period, 25 ml of Eagle's MEM stupplemented with 2% FCS were added, and the bottles were incuibated at 37 C. Control flasks were inoctulated with 0.5 ml meditum or 0.5 ml of noninfected AGMK cell Iysates, and then treated in a similar way. After 24 hoturs of inctubation, the media from one infected and one control flask were removed, irradiated with uiltraviolet light, etc., as described above. Before harvesting the meditum, cuiltuires were observed for cytopathic effects, and the percentage of cells showing CPE was recorded. All materials were stored at -85 C after collection. WI-38 and WI-38VA13A
On the third day of cuiltture the meditum from all bottles was removed, centriftuged, and frozen. After 5 days of cuiltture, when a complete cell monolayer was formed, the meditum from one flask was removed and treated as previotusly described, and so on from the sixth to twelfth days. Macrophage Migration Inhibition
Normal Hartley gtuinea pigs weighing 350 to 400 g were injected with 15 ml sterile light mineral oil. Fouir days later, peritoneal extudate cells were collected, washed twice with Hanks' balanced salt soltution (GIBCO) and packed into capillary ttubes as described previoutsly.12 Two capillary ttubes were ctulttured in a Sykes-Moore chamber containing test ctultture fltuid stupplemented with normal gtuinea pig sertum. The migration from at least fotur capillary tthbes was meastured for each test sample. The procedtures uised to calcuilate migration index (MI) and percent inhibition were also described previouisly.12 The MI was calctilated as the ratio of the migration area of the experimental to the control ttibes. In accordance with uisuial practice, an MI of 80 or less was considered as significant activity. Assays for Chemotactic Activity
A modified Boyden chamber was uised as previouisly described.1"' The chamber was divided by a micropore filter in an uipper compartment containing netitrophils or macrophages, and a lower compartment containing 100 ,ul of the fluiid to be assayed for chemotactic activity. Neuitrophils were obtained from rabbits injected intraperitoneally with 0. I% glycogen in saline 4 houirs previouisly 14 and macrophages from rabbits injected intraperitoneally with mineral oil 4 days previouisly.'5 All cells were suispended in mediuim 199 with 10% rabbit seruim or 0.1 % bovine seruim albuimin. When neuitrophils were uised, filters of pore size 0.65 u were uitilized in the chambers; with macrophages, filters of pore size 5 ,u were uitilized. Cell migration was qtuantitated by counting the nuimber of migrating cells in five high-power fields tinder a light microscope. Fractionation of Fluids Containing MIF-Like Activity
Fluiids were concentrated as previouisly described 2 and then applied to a Sephadex GI00 coluimn equiilibrated with 0.01 M phosphate-buiffered saline, pH 7.4. Eluiates were collected at a flow rate of 0.1 ml/min in 3.0-ml samples. Fractions near the void voluime (which contain proteins of high molecuilar weight) were pooled as Fraction I, while fractions containing the phenol red marker were pooled as Fraction VI. Intermediate fractions of e(quial voluime were pooled as Fractions II to V (approximately 30 ml/fraction). All fractions were lyophilized, redissolved into 3 ml of buiffer, dialyzed against RPMI 1640 mediuim, and then tested for MIF activity.
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Table 1-Failure of Supernatants From Noninfected AGMK Cell Cultures to Inhibit Macrophage Migration
Percent inhibition -7.7 -0.8 5.9 -10.9 -1.5 -4.4 0.6
Migration index* Day 107.7 ± 7.4 1 2 100.8 ± 8.2 3 94.1 ± 5.6 4 110.9 ± 7.8 5 101.5± 9.1 104.4 ± 13.6 6 7 99.4 ± 5.6 * With respect to medium as control.
Results
SV 40-Infected AGMK Cells
As shown in Table 1, there was no inhibition of macrophage migration by media from uninfected AGMK cells cultuired for a period of 7 days. If anything, there was a slight stimulation of the migration of guinea pig macrophages. On the other hand, when media from SV 40-infected cuiltuires were tested, using as controls media from noninfected cells, there was definite inhibition of migration. This was most noticeable with media obtained 24, 48, and 72 hours after infection (Text-figure 1). No significant inhibition was observed with media obtained after the third day of infection. When cells were observed for cytopathic effects, none were seen during the first 2 days of infection, but in the following days there was a steady increase in the percentage of affected cells. To ruile ouit the possibility that migration was inhibited by a factor cytotoxic for macrophages, the viability of the inhibited macrophages was tested by trypan blue exclusion and by continued cultivation for over 48 houirs. No difference in viability was observed between macrophages treated with control supernatant and those treated with supernatant from 50 40 C 0 a
30-
c
3 20 . 10
K
I
IL
v
1
2
3
4
DAYS
5
6
7
100 _
80 S._ 40 v 20
it
TFXT-FIGURE 1-Macrophage migration inhibitory activity (solid columns) in suipernatants of SV 40infected AGMK cell ctiltures. The percentage of cells in these cuiltures showing cytopathic effects (line), as defined in the text, is plotted on the same time scale.
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infected cells. To rtile ouit the possibility that residuial virus in the fluiids interfered with macrophage migration even in the absence of gross cytotoxicity, doses of virus identical to those used to infect AGMK cell ctultuires were added to cell-free medium, which was then centrifuged and irradiated with uiltraviolet light in the same manner as the supernatants from infected cultures. The resulting preparation was devoid of migration inhibitory activity. In fact, even nonirradiated control preparations prepared in this way were devoid of activity. In contrast to our previous observations with mumps and Newcastle disease virus, no evidence of chemotactic activity for neutrophils or macrophages was found in any of the supernatants from the SV 40-infected cultures. Characterization of MIF-Like Substance
Media obtained from SV 40-infected AGMK ctulttures at 24 hours after infection, i.e., when the MIF-like activity is at its maximum, were subjected to fractionation by Sephadex G-100 chromatography. The MIF-like activity of each fraction was tested and was found to be greatest in a fraction corresponding to a molecular weight range of 45,000 to 65,000 and in another fraction corresponding to an approximate molecular weight of 12,000 (Text-figuire 2). WI-38 and WI-38VA13A Cells
Media from complete monolayers of normal and SV 40-transformed WI-38 cells were tested on guinea pig macrophages using as control pooled media from the first 3 days of cultuire, which were uiniformly devoid of activity. Inhibition of macrophage migration was obtained with media from both types of cell cultuires over a period of several days (Table 2). Althouigh both normal and SV 40-transformed cells were capable of prodtucing MIF activity, the supernatants from SV 40-transformed fibroblasts harvested on the sixth and seventh days had significantly greater activity than corresponding fluids from normal cell cuiltures.
TEXT-FIGURE 2-Sephadex G100 coltmn chromatography of active stipernatant from SV 40infected AGMK cell colltoires. (OD, line; inhibition, solid columns, ovalbtomin marker, a; cytochrome C marker, b)I
b
a
4.0 a
E 3.0 Go
2.0
01.0
50 I
II-
0
30 10 C
TUBE NUMBER
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BIGAZZI ET AL
Table 2-Macrophage Migration Inhibition Activity of Supernatants From Normal and SV 40-Transformed (WI-38VA13A) Human Fibroblasts
(WI-38)
Migration index Day
Normal*
SV 40-transformed*
39.3 ± 2.7 56.3 ± 3.5 6 7 64.1 ± 2.3 43.0 ± 3.1 48.0 ± 1.8 8 38.3 ± 1.5 46.9 ± 2.5 39.7 ± 3.0 10 32.8 ± 2.1 11 39.6 ± 1.6 42.4 ± 2.6 12 35.6 ± 2.0 * Migration index calculated with respect to pooled (negative) supernatants from Days 1 to 3; this index reflects absolute activity of each supernatant.
Discussion
The present stuidy shows that media from monkey kidney cells infected with SV 40 contain a suibstance capable of inhibiting the migration of guiinea pig peritoneal macrophages. This suibstance is not toxic for macrophages, as demonstrated by vital staining and migration of these cells after continuiouis cuiltivation for more than 48 houirs. We have also excluided the possibility that residuial viruis in the fluiids couild interfere with macrophage migration even in the absence of gross cytotoxicity. The MIF-like activity can be localized by Sephadex chromatography in two fractions, one corresponding to a molecular weight of 45,000 and the other to 12,000. Classic MIF derived from antigen-stimulated lymphocyte cuiltuires has also been localized in two fractions of similar molecuilar weight.16 These results are somewhat different than those reported in stuidies of muimps or Newcastle disease viruis infections, where only the larger molecuilar weight migration inhibition factor could be demonstrated.2 The other difference between the results of the present study and those we have previotusly reported is that infection by mumps or Newcastle disease viruises leads to the generation of chemotactic factors for nelutrophils and macrophages as well as MIF activity. Fuirther work with a variety of viruis-cell systems is necessary to determine the biologic significance of this distinction. In any case, the dissociation of MIF and chemotactic factor produiction reported here may have important practical considerations, as this could facilitate the isolation and characterization of these factors, first in virus-infected cell systems and subsequiently in antigenstimulated lymphocyte cultuires. As one example, the preparation of monospecific antisera directed against single lymphokines requires starting material with restricted activities. To date we have obtained only
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antisera with generalized antilymphokine activity using lymphocytederived, MIF-rich preparations for immunizations.'7 If similarities of biologic function of the various MIF-like factors imply chemical and antigenic similarity, then suitable preparations of virus-induiced MIF could be used to prepare antisera which might then be of use in the study of the more complicated lymphocyte systems. The present results extend our previous studies on cytokine produiction by muimps and Newcastle disease virus.'3 Lytic infection with SV 40 leads to the appearance of a migration inhibition factor in cultuires of monkey kidney cells which otherwise do not release such factors. We have also shown that continuouis lines of human fibroblasts, both normal and SV 40transformed, release suibstances with MIF activity into the mediuim. A comparison between MIF activity of the two types of cells indicates that in the early stages of cultuire, SV 40-transformed cells may release more MIF-like substance than normal cells. However, it is also possible that uinavoidable differences in the metabolism of the two different cell types may have affected relative migration inhibitory activity in a nonspecific manner. A similar production of MIF had been previously observed in lymphoid and nonlymphoid cell lines,'8"19 while recently Hammond et al.10 have reported that SV 40-transformed mouse fibroblasts elaborate an MIF-like substance. In contrast to our observations, these latter auithors found that their untransformed fibroblast line (3T3) had no background MIF activity. In our previous investigations we demonstrated that release of MIF induced by mumps virus was independent of the production of interferon.2 Even though, in the present study, we have not tested for interferon produiction, the evidence available seems to show that SV 40 is a poor interferon induicer. Plummer 20 and Oxman 21 have not observed any interferon production by monkey cells infected with this virus. Diderholm 22 reported the production of a substance inhibitory for pseudorabies and polymyelitis viruses, with the highest titers late in the course of SV 40 infection (from the eighth to the twentieth day). On the other hand, the MIF-like activity we have observed in the infected monkey cell system is present only from the first to the third day after infection. In recent years a variety of activities have been attributed to interferon, in addition to its ability to induce resistance to viral infections.23'24 These activities include the inhibition of transformation of cells by oncogenic viruses 25,26; the inhibition of growth of malignant cells, both in vitro and in vivo 27,28; the enhancement of phagocytosis and lymphocyte-mediated cytotoxicity 29,30; and the activation or the inhibition of antibody-forming cells.3' Since preparations of interferon are rather crude, all these activities might not
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necessarily be carried by the same molecuilar species S2 btut instead might represent different produicts released by infected cells. The present sttudy and ouir previous observations demonstrate that muiltiple suibstances with diverse biologic properties are generated as a consequience of viruis infection. Many of these have featuires in common with certain of the known lymphokines. For this reason, we defined a new class of biologic mediator substances, the cytokines.4' These are substances produiced by nonlymphoid as well as lymphoid cells when those cells are triggered by a variety of agents (incluiding viral infections). These suibstances modify the behavior of other cells (most notably inflammatory cells). In this view, lymphokines, the mediator substances produiced by lymphocytes (triggered by specific antigen or by mitogens), represent a restricted set of cytokines made by one class of cells, activated in certain uinique ways. It is tempting to postulate that cytokine produiction represents one of the most primitive cellular self-defense systems and that cellular immuinity, dependent on lymphokine production, and humoral immunity represent a suibsequient evoluition of this system. References 1. Ward PA, Cohen S, Flanagan TD: Leukotactic factors elaborated by viruis-infected tissuies. J Exp Med 135:1095-1103, 1972 2. Flanagan TD, Yoshida T, Cohen S: Produiction of macrophage migration inhibition factors by viruis-infected cell cuiltuires. Infect Immun 8:145-150, 1973 3. Yoshida T, Flanagan TD, Genco RJ, Cohen S: Viruis-induiced migration inhibitory activity in experimental muimps infection. Clin Immuinol Immuinopathol 2:472-480, 1974 4. Cohen S, Ward PA, Bigazzi PE: Cell cooperation in cell-mediated immuinity. Mechanisms of Cell-Mediated Immunity. Edited by RT McClulskey, S Cohen. New York, John Wiley and Sons, 1974, pp 331-358 5. Cohen S, Bigazzi PE, Yoshida T: Similarities of T cell fuinction in cell-mediated immulnity and antibody prodtuction. Cell Immunol 12:150-159, 1974 6. Weiner LP, Herndon RM, Narayan 0, Johnson RT, Shah K, Ruibenstein LJ, Preziosi TJ, Conley FK: Isolation of viruis related to SV40 from patients with progressive mtultifocal letukoencephalopathy. N Engl J Med 286:385-390, 1972 7. Mtullarkey MF, Hrtuska JF, Takemoto KK: Comparison of two huiman papovavirtuses with simian viruis 40 by strtuetuiral protein and antigenic analysis. J Virol 13:1014-1019, 1974 8. Martin MA, Gelb LD, Garon C, Takemoto KK, Lee TNH, Sack GH Jr, Nathans D: Characterization of "heavy" and "light" SV40-like particles from a patient with PML. Virology 59:179-189, 1974 9. Bigazzi PE, Cohen S, Ward PA, Yoshida T: Produiction of lymphokine-like factors by SV40-infected cell cuiltuires. Fed Proc 33:640, 1974 (Abstr) 10. Hammond ME, Roblin RO, Dvorak AM, Selvaggio SS, Black PH, Dvorak HF: MIF-like activity in simian virtus 40-transformed 3T3 fibroblast cuiltuires Science 185:955-957, 1974 11. Reed LJ, Mutench H: A simple method of estimating 50 per cent endpoints. Amer J Hyg 27:493-497, 1938
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12. Yoshida T, Janeway CA Jr, Pauil WE: Activity of migration inhibitory factor in the absence of antigen. J Immuinol 109:201-206, 1972 13. Ward PA, Cochrane CG, Miiller-Eberhard HJ: Fturther stuidies on the chemotactic factor of complement and its formation in vivo. Immuinology 11:141-153, 1966 14. Hill JH, Ward PA: C3 letikotactic factors produiced by a tissuie protease. J Exp Med 130:505-518, 1969 15. Ward PA: Chemotaxis of mononuiclear cells. J Exp Med 128:1201-1221, 1968 16. Yoshida T, Sonozaki H, Cohen S: The produiction of migration inhibition factor by B and T cells of the guiinea pig. J Exp Med 138:784-797, 1973 17. Yoshida T, Bigazzi, PE, Cohen S: The produiction of anti-guinea pig lymphokine antibody. J Immunol 114:688-691, 1975 18. Papageorgioui PS, Henley WL, Glade PR: Produiction and characterization of migration inhibitory factor(s) (MIF) of established lymphoid and non-lymphoid cell lines. J Immuinol 108:494-504, 1972 19. Tuibergen DG, Feldman JD, Pollack EM, Lerner RA: Produiction of macrophage migration inhibition factor by continuiouis cell lines. J Exp Med 135:255-266, 1972 20. Pluimmer G: Interfering properties of simian viruses. Br J Exp Pathol 44:58-65, 1963 21. Oxman MN: Interferon, tuimors and tuimor viruises. Interferons and Interferon Induicers. Edited by NB Finter. New York, North-Holland/American Elsevier, 1973, pp 391-480 22. Diderholm H: Produiction of interferon by monkey kidney cells infected with simian viruis 40. Arch Ges Viruisforsch 14:39-44, 1963 23. Grossberg SE: The interferons and their inducers: molecuilar and therapeuitic considerations (third of three parts). N Engl J Med 287:122-128, 1972 24. Acton JD: The lymphoreticuilar system and interferon produiction. J Reticuiloendothel Soc 14:449-461, 1973 25. Todaro GJ, Baron S: The role of interferon in the inhibition of SV40 transformation of mouise cell line 3T3. Proc Natl Acad Sci USA 54:752-756, 1965 26. Oxman MN, Levin MJ: Interferon and transcription of early viruis-specific RNA in cells infected with simian viruis 40. Proc Natl Acad Sci USA 68:299-302, 1971 27. Gresser I, Bouirali-Matiry C: The antituimor effect of interferon in lymphocyte- and macrophage-depressed mice. Proc Soc Exp Med Biol 144:896-900, 1973 28. Gaffney EV, Picciano PT, Grant CA: Inhibition of growth and transformation of huiman cells by interferon. J Natl Cancer Inst 50:871-878, 1973 29. Huiang K-Y, Donahoe RM, Gordon FB, Dressler HR: Enhancement of phagocytosis by interferon-containing preparations. Infect Immlun 4:581-588, 1971 30. Lindahl P, Leary P, Gresser I: Enhancement by interferon of the specific cytotoxicity of sensitized lymphocytes. Proc Natl Acad Sci USA 69:721-725, 1972 31. Bratun W, Levy HB: Interferon preparations as modifiers of immuine responses. Proc Soc Exp Biol Med 141:769-773, 1972 32. Borecky L, Ftuchsberger N, Hajnicka V, Stancek D, Zemla J: Distribtution of antiviral and cell-inhibitory activity in interferon preparations. Acta Virol (Praha) 16:356-358, 1972
Acknowledgments We thank Ann Marie Least and Dorothy Osmak for their skilled technical assistance and Caroline Maltbie for her excellent secretarial assistance in the preparation of the mantuscript.
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