- Email: [email protected]
Increase of vulnerability to lymphotoxin in cells infected by vesicular stomatitis virus and its further augmentation by interferon
CELLULAR lMMUNOLOCY
95 2 18-225 ( 1985)
Increase of Vulnerability to Lymphotoxin in Cells Infected by Vesicular Stomatitis Virus and Its Further Augmentation by Interferon’ D. ADERKA, D. NOVICK, T. HAHN, D. G. FISCHER, AND D. WALLACH~ Department of Virology, The Weizmann Instituteof Science, Rehovot, 76100 Israel Received September 24, 1984; accepted December 22, 1984 The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assess4 in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-y, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect. Q 1985 Academic m hc. INTRODUCTION
A central question in the study of lymphotoxin (LT),3 which has remained unresolved, is the nature of the physiological targets for cytotoxicity of these proteins. Cells of only a few lines were found to be killed effectively by LT under normal growth conditions. However, in the presence of metabolic blockers such as cycloheximide (CHI), dinitrophenol, actinomycin D, or mitomycin C there is a significant increase in the effectiveness of cell killing by LT and cells which appear resistant to LT can become vulnerable to its cytotoxic effect (l-5). It therefore appears that the effectiveness of cell killing by LT can be subjected to modulation depending on the activity of some metabolic functions in the target cell. We recently observed that HeLa cells, which can be killed by LT in the presence of CHI but not in its absence, also exhibit a cytotoxic effect of LT when they are infected by vesicular stomatitis virus (VSV) (4). This study shows that such sensitization to LT upon infection by VSV can be observed in cells of several other cell lines as well, i This study was supported by a grant from the Fund of Basic Research, administered by the Israel Academy of Sciences and Humanities and by a grant from the Leukemia Research Foundation, Chicago, Ill. * To whom correspondence should be addressed. 3 Abbreviations used: CHI-cycloheximide; IFN-interferon; LT-lymphotoxin; PFU-plaque forming unit; PHA-phytohemagglutinin; VSV-Vesicular Stomatitis Virus. 218 OQO8-8749185$3.00 Copyright0 1985 by Academic Press, Inc. All ri& of reproductionin any form resewed.
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECTION
219
and that interferon (IFN) can potentiate the destruction of virus-infected cells by LT even though it does not sensitize uninfected cells. Infection by VSV might thus render cells as targets for selective destruction by LT. MATERIALS
AND METHODS
Cells and viruses. The human HeLa cells (ATCC CCL 2.1 (6)), WISH cells (ATCC CCL 25 (7)), SV-80 cells (8), and the African green monkey Vero cells (ATCC CCL 81) were all grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum. Stocks of VSV (Indiana type, three times plaque purified) were prepared by infecting BSC-1 cells at 0.02 PFU/cell and allowing new viruses to be produced for 20 hr. Lymphotoxin and interferons. Lymphotoxin was induced in human peripheral blood lymphocytes with phytohemagglutininP (PHA) and partially-purified by adsorption to controlled pore glass as described previously (5) except that the pretreatment of lymphocytes with IPN, prior to their stimulation by PHA was omitted. IFN-7 present in the preparations of LT was eliminated using a monoclonal antibody against the IPN (hybridoma No. 166 (9)). This was done by applying all LT preparations on immunoadsorbents constructed by coupling the monoclonal antibody to glutaraldehyde-treated agarose polyacrylic hydrazide (Bio-Yeda, Israel) (10). The LT eluted from the immunoadsorbents exhibited no antiviral activity (on
Time
(hn.1
FIG. I. Cytotoxicity of LT in VSV-infected HeLa (A), WISH (B), Vera (C), and SV-80 cells (D). Cell killing at various time-sfollowing infection by VSV (0 - 0) and its enhancement when applying LT (30 U/ml) 2 hr following infection (O---O) is shown in comparison to the absence of any cytotoxic effect of LT on cells which were not infected (0 - Cl). Also shown in D is the further enhancement of cell killing when applying IFN-7 (100 U/ml) on the SV-80 cells prior to infection by VSV and application of LT
(A-A).
220
ADERKA ET AL.
WISH cells) and induced no increase in synthesis of HLA A, B, C proteins when applied to SV-80 cells, confirming that EN-y was effectively eliminated. Concentrations of LT are given in terms of cytotoxic activity in CHI-treated SV80 cells where a unit of LT is defined as the concentration which, when applied on the cells simultaneously with CHI, (50 &ml) caused killing of 50% of the cells in 12 hr of incubation. IFN-cu (induced in leukocytes of chronic myelogenous leukemia with Sendai virus), IFN-@ (induced in cultures of foreskin cells with poly(rI:rC)), and IFN-7 (induced in human peripheral blood mononuclear cells with PHA and phorbol-l2O-myristate- 13-acetate) were affinity purified to homogeneity using immunoadsorbents prepared with monoclonal antibodies against the respective IPN and their concentrations were determined by comparison to N.I.H. standards.
Examination of the cytotoxic eflectsof LT on VSV-infectedand uninfectedcells. Cells were seededin 9-mm microwells at 4 X lo4 cells/well. Thirty-six to forty-eight hours later VSV ( lo7 plaque-forming units (PFU)/ml) was applied at aliquots of 5 X lo5 PFU/well and, following 2 hr of virus-adsorption, the virus-containing medium was replaced with fresh growth medium. IFN was applied on the cells 16 hr prior to infection; LT was applied 2 hr following application of the virus. Cell killing was quantitated by measuring uptake of neutral red by the cells (5, 11) and +LT vsv
vsv
Hela
WISH
Vero
FIG. 2. Morphology of HeLa, WISH, and Vero cells (left column) compared to the cytopathic effect observed following infection of those cells by VSV (middle column) and its further enhancement in applying LT (30 U/ml) 2 hr following infection (right column). Photographs were taken 15 hr following infection of VSV (X 120).
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECTION
221
its extent is presented as a percentage of the cells which were incubated for the same period in the absence of VSV, LT, or IFN. RESULTS In cells of several lines, increase in vulnerability to the cytotoxic effect of LT could be observed following infection by VSV. Thus, in VSV-infected HeLa cells, WISH, Vero, and SV-80 cells, LT exerted a cytotoxic effect which was significantly beyond that of the virus itself (Fig. 1, circles, and Fig. 2), even though in the absence of viral infection these same cells appeared completely resistant to LT (Fig. 1, rectangles, and Fig. 4, compare A and B). A more detailed analysis of the effect of LT on SV-80 cells (Figs. 3 and 4) showed that LT cytotoxicity could first be observed at about 12 hr following infection by the virus, at the time when viral cytopathy also first became apparent. Killing of all cells in the culture was completed in the presence of LT much earlier than in its absence (Fig. 1C and D and Fig. 3: VSV + 30 U/ml LT compared to VSV alone). The effectiveness of LT cytotoxicity on the infected cells was proportional to its concentration (Fig. 3) yet even at a hundred times the concentration at which LT caused measurable killing of infected cells it still failed to exert any cytotoxic effect on uninfected ones (Fig. 3, squares). Exposing cells to LT for a longer period, by applying it 12 hr prior to infection by virus, further increased the effectiveness of cell destruction. However, even under these conditions cell killing started only at about 12 hr following infection (not shown). Treating the cells with IFN prior to infection by VSV affected their vulnerability to LT in a complex fashion. When applied at concentrations which protected cells from cytopathy of the virus, IFN also prevented the killing of cells by LT (Fig. 5, compare solid and empty symbols). On the other hand, below the antiviral range of concentrations IFN had the opposite effect, namely, it enhanced the cytotoxic
‘*) VSV+ LT 6u/ml
vSv+LT
15u/ml
VSV+ LT 30 u/ml
Time
(Iin.)
FIG. 3. Kinetic examination of LT cytotoxicity, in VSV-infected SV-80 cells, at various LT concentrations compared to the lack of cytotoxic effect of LT in the uninfected cells (m).
222
ADERKA ET AL.
FIG. 4. Morphology of SV-80 cells under normal growth conditions (A) compared to the morphology of cells which were treated by LT (B) infected by VSV (C), infected by VSV and treated by LT (D), or infected by VSV and treated with both IFN-7 and LT (E). IFN--y (100 U/ml) was applied on the cells 16 hr prior to infection and LT (30 U/ml) 2 hr following infection. Photographs were all taken 20 hr following infection by VSV (X 150).
effect of LT against virus-infected cells (Figs. 5 and 6). Cells which were not infected by VSV remained resistant to LT even in the presence of IFN (Fig. 5, diamonds, and Fig. 6, squares). In comparing the response of cells to the three types of IFN (Fig. 4) the stimulatory effect on cell destruction was found to be induced with EN--y at a significantly wider range of concentrations than with IFN-(I! or -8. Thus, in SV-80 cells enhancement of cell destruction by IF’Ny could be observed at IFN concentrations
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECTION
0.01 IFN
(units/ml)
IFN
0.1
I
223
IO
(units/ml)
FIG. 5. IFN effect on the cytotoxicity of LT in SV-80 cells (A) and in HeLa cells (B). IFN-cu (0, l ), IFN-@ @, q ), and IFN-7 (A, A) were applied at the indicated concentrations prior to infection by VSV (solid symbols) or infection by VSV followed by application of LT (6 U/ml, empty symbols), or application of LT alone (6 U/ml, for the three types of IFN, 0). Note the enhancement of cell killing at low concentrations of IFN and the protective effect of IFN at higher concentrations.
as high as 100 U/ml (Fig. 6), while with IFN-(w and -/3 such enhancement could be observed only at IFN concentrations lower than 1 U/ml (Fig. 5A, empty circles and rectangles). Similar differences between the responsesto the differing IFN, although to a lesser extent, could also be observed with HeLa cells (Fig. 5B). DISCUSSION The cytotoxicity of LT is not restricted to target cells which exhibit foreign antigens on their surface nor does it seem to be affected by the nature of the major histocompatibility antigens in these cells. There is therefore much interest in the fact that, nevertheless, some kind of selectivity in its function can be observed: It is shown to exert a cytotoxic effect on VSV-infected cells, even though in the absence of viral infection these same cells exhibit resistance to its cytotoxicity. Preliminary observations indicate that a number of other viruses besides VSV can also sensitize cells to LT (unpublished observations). What mechanisms underlie this distinction between uninfected and virus-infected cells is unknown. As increase in vulnerability to LT can also be observed in cells following treatment by inhibitors of RNA or of protein synthesis (2, 5) it seems possible that the sensitization to LT in cells infected by VSV is, at least in part, a consequence of the shut-off in synthesis of cellular RNA and proteins, which this virus induces. IFN is shown to potentiate the cytotoxicity of LT against the infected cells and has also been reported to increase the cytotoxic effect of LT in cells which were sensitized to its effect by mitomycin C (12) or CHI (4). However, under normal growth conditions we found cells to be resistant to LT even in the presence of IFN. Moreover, cells infected by VSV are shown to become resistant to LT when treated with IFN at concentrations which inhibit replication of the virus. The protection of
224
ADERKA ET AL.
0
2
4
6
I6
32
LT lunih/ml) FIG. 6. Enhancement by IlW-y of LT cytotoxicity in VW-infected SV-80 cells. The cytotoxic effect of LT at various concentrations in VSV-infected cells (0) and its further enhancement by treating these cells with IFN-7 (10 U/ml, 16 hr prior to infection (0) or 100 U/ml, prior to infection (A)) is shown in comparison to the resistance to LT observed in uninfected cells (m) even when they are also treated with IFN-y at 100 U/ml (0).
cells by the antiviral effect of IFN, on one hand, and the enhancement of destruction of the infected cells as a result of their sensitization to LT by IFN, on the other hand, is reflected in a bimodal pattern of the concentration dependence of the effect of IFN on cell viability; enhancement of destruction by LT, at low, sub-antiviral IFN concentrations, and protection from killing at higher concentrations. The ranges of IFN concentrations at which enhancement and suppression of cell destruction were observed turned out to be dependent on the type of IFN applied. EN-7 which, with regard to several kinds of IFN effects, is a more effective immunoregulatory agent than the other interherons (cf., e.g., (13-15)) showed the enhancement of cell response to LT at a wider range of concentrations than IFN-a and -8. Paradoxically, killing of virus-infected cells, which is often an undesirable effect of the virus itself, is also one of the major modes of immune defense against viral infection. These two aspects of the death of virus-infected cells can well be demonstrated in cells infected by VSV: The virus itself is highly cytopathic. However, production and release of virions can only take place during that time at which the infected cell is still alive. Thus, even though the virus itself eventually kills the cell, any enhancement of cell killing, by immune cytolytic mechanisms, can be highly beneficial in reducing the yield of new virions and thus decreasing the chance that other cells will become infected. There are several mechanisms of immune cytolysis which can be exerted selectively against virus-infected cells; some-such as the cytotoxicity of cytotoxic T lymphocytes-are directed by specific recognition of viral antigens on the surface of infected cells, while others-such as the activity of NK cells-by other mechanisms of distinction whose nature is yet unknown. This study indicates that LT can also
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECT ION
225
destroy selectively cells which are infected by viruses and that IF’N can augment this cytotoxic activity. There are also other indications implicating the cytotoxicity of LT in the response to viral infection. We have recently observed that Sendai virus is a potent inducer of LT formation in cultures of mononuclear leukocytes (Aderka and Wallach, manuscript in preparation), while IFN has been found to increase the responsiveness of these cells to inducers of LT formation (3). Although it is difficult to infer from such in vitro observations the way LT functions in vivo, they do raise the possibility that LT has a role in the cytotoxic activities which cells of the immune system exert against virus-infected cells. ACKNOWLEDGMENT We thank Mrs. Sylvia Budilovsky for skillful technical assistance. Note added in proof: The preparations of LT applied in this study were far from pure. We therefore find it important to note that we lately reproduced the presented findings with homogenously purified preparations of such a protein. We recently isolated a major species of human cytotoxin with an M, of about 17,500, using a monoclonal antibody against that protein (Hahn et al., Proc. Natl. Acad. Sci. USA, in press). By its molecular size that cytotoxin seemed to be. identical to the one designated by others as “tumor-necrosis-factor” (Pennica et al., Nature (London) 312, 724, 1974). The protein was produced most effectively by monocytes, upon their infection by Sendai virus or their treatment with phorbol-12O-myristate- 13-acetate,but could also be detected, at a significant level, in lectin-induced LT preparations of the kind applied in this study (unpublished). When testing the effect of the isolated 17.5~kDacytotoxin on VSV-infected and uninfected SV-80 cells we observed the same kind of selectivity as that found with crude preparations of lymphotoxins, namely, marked cytotoxicity against the virus-infected cells (as well as against CHI-treated, uninfected cells) and further potentiation of that cytotoxicity by treatment with recombinant IFNy, but no cytotoxic effect of the protein against uninfected normally growing cells.
REFERENCES 1. 2. 3. 4.
Williams, T. W., and Granger, G. A., Cell. Immunol. 6, 171, 1973. Rosenau, W., Goldberg, M. L., and Burke, G. C., J. Immunol. 111, 1128, 1973. Wallach, D., and Hahn, T., Cell. Immunol. 76, 390, 1983. Wallach, D., Aderka, D., Budilovsky, S., and Hahn, T., In “The Biology of the Interferon System” (E. De Maeyer and H. Schellekens, Eds.), pp. 293-303. Elsevier, Amsterdam. 5. Wallach, D., J. Immunol. 132, 2464, 1984. 6. Gey, G. O., CoBman, W. D., and Jubicek, M. J., Cancer Rex 12, 264, 1952. 7. HayIhck, L., Exp. Cell Res. 23, 14, 1961. 8. Todaro, J. G., Green, H., and Swift, M. R., Science (Washington, D.C.) 153, 1252, 1966. 9. Novick, D., Eshhar, Z., Fischer, D. G., Friedlander, J., and Rubinstein, M., EMBO J. 2, 1527, 1983. 10. Wilchek, M., and Miron, T., In “Methods in Enzymology,” Vol. 34, “Affinity Techniques (Enzyme Purification: Part B)” (W. B. Jakoby and M. Wilchek, eds.), p. 72. Academic Press, New York, 1974. I 1. Finter, N. B., J. Gen. Virol. 5, 419, 1969. 12. Williams, T. W., and Bellanti, J. A., J. Immunol. 130, 5 18, 1983. 13. Sonnenfeld, G., Mandel, A. D., and Me&an, T. C., Cell. Immunol. 34, 193, 1977. 14. Wallach, D., J. Interjkon Res. 2, 329, 1982. 15. Wallach, D., Fellous, M., and Revel, M., Nature (London) 299, 833, 1982.
95 2 18-225 ( 1985)
Increase of Vulnerability to Lymphotoxin in Cells Infected by Vesicular Stomatitis Virus and Its Further Augmentation by Interferon’ D. ADERKA, D. NOVICK, T. HAHN, D. G. FISCHER, AND D. WALLACH~ Department of Virology, The Weizmann Instituteof Science, Rehovot, 76100 Israel Received September 24, 1984; accepted December 22, 1984 The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assess4 in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-y, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect. Q 1985 Academic m hc. INTRODUCTION
A central question in the study of lymphotoxin (LT),3 which has remained unresolved, is the nature of the physiological targets for cytotoxicity of these proteins. Cells of only a few lines were found to be killed effectively by LT under normal growth conditions. However, in the presence of metabolic blockers such as cycloheximide (CHI), dinitrophenol, actinomycin D, or mitomycin C there is a significant increase in the effectiveness of cell killing by LT and cells which appear resistant to LT can become vulnerable to its cytotoxic effect (l-5). It therefore appears that the effectiveness of cell killing by LT can be subjected to modulation depending on the activity of some metabolic functions in the target cell. We recently observed that HeLa cells, which can be killed by LT in the presence of CHI but not in its absence, also exhibit a cytotoxic effect of LT when they are infected by vesicular stomatitis virus (VSV) (4). This study shows that such sensitization to LT upon infection by VSV can be observed in cells of several other cell lines as well, i This study was supported by a grant from the Fund of Basic Research, administered by the Israel Academy of Sciences and Humanities and by a grant from the Leukemia Research Foundation, Chicago, Ill. * To whom correspondence should be addressed. 3 Abbreviations used: CHI-cycloheximide; IFN-interferon; LT-lymphotoxin; PFU-plaque forming unit; PHA-phytohemagglutinin; VSV-Vesicular Stomatitis Virus. 218 OQO8-8749185$3.00 Copyright0 1985 by Academic Press, Inc. All ri& of reproductionin any form resewed.
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECTION
219
and that interferon (IFN) can potentiate the destruction of virus-infected cells by LT even though it does not sensitize uninfected cells. Infection by VSV might thus render cells as targets for selective destruction by LT. MATERIALS
AND METHODS
Cells and viruses. The human HeLa cells (ATCC CCL 2.1 (6)), WISH cells (ATCC CCL 25 (7)), SV-80 cells (8), and the African green monkey Vero cells (ATCC CCL 81) were all grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum. Stocks of VSV (Indiana type, three times plaque purified) were prepared by infecting BSC-1 cells at 0.02 PFU/cell and allowing new viruses to be produced for 20 hr. Lymphotoxin and interferons. Lymphotoxin was induced in human peripheral blood lymphocytes with phytohemagglutininP (PHA) and partially-purified by adsorption to controlled pore glass as described previously (5) except that the pretreatment of lymphocytes with IPN, prior to their stimulation by PHA was omitted. IFN-7 present in the preparations of LT was eliminated using a monoclonal antibody against the IPN (hybridoma No. 166 (9)). This was done by applying all LT preparations on immunoadsorbents constructed by coupling the monoclonal antibody to glutaraldehyde-treated agarose polyacrylic hydrazide (Bio-Yeda, Israel) (10). The LT eluted from the immunoadsorbents exhibited no antiviral activity (on
Time
(hn.1
FIG. I. Cytotoxicity of LT in VSV-infected HeLa (A), WISH (B), Vera (C), and SV-80 cells (D). Cell killing at various time-sfollowing infection by VSV (0 - 0) and its enhancement when applying LT (30 U/ml) 2 hr following infection (O---O) is shown in comparison to the absence of any cytotoxic effect of LT on cells which were not infected (0 - Cl). Also shown in D is the further enhancement of cell killing when applying IFN-7 (100 U/ml) on the SV-80 cells prior to infection by VSV and application of LT
(A-A).
220
ADERKA ET AL.
WISH cells) and induced no increase in synthesis of HLA A, B, C proteins when applied to SV-80 cells, confirming that EN-y was effectively eliminated. Concentrations of LT are given in terms of cytotoxic activity in CHI-treated SV80 cells where a unit of LT is defined as the concentration which, when applied on the cells simultaneously with CHI, (50 &ml) caused killing of 50% of the cells in 12 hr of incubation. IFN-cu (induced in leukocytes of chronic myelogenous leukemia with Sendai virus), IFN-@ (induced in cultures of foreskin cells with poly(rI:rC)), and IFN-7 (induced in human peripheral blood mononuclear cells with PHA and phorbol-l2O-myristate- 13-acetate) were affinity purified to homogeneity using immunoadsorbents prepared with monoclonal antibodies against the respective IPN and their concentrations were determined by comparison to N.I.H. standards.
Examination of the cytotoxic eflectsof LT on VSV-infectedand uninfectedcells. Cells were seededin 9-mm microwells at 4 X lo4 cells/well. Thirty-six to forty-eight hours later VSV ( lo7 plaque-forming units (PFU)/ml) was applied at aliquots of 5 X lo5 PFU/well and, following 2 hr of virus-adsorption, the virus-containing medium was replaced with fresh growth medium. IFN was applied on the cells 16 hr prior to infection; LT was applied 2 hr following application of the virus. Cell killing was quantitated by measuring uptake of neutral red by the cells (5, 11) and +LT vsv
vsv
Hela
WISH
Vero
FIG. 2. Morphology of HeLa, WISH, and Vero cells (left column) compared to the cytopathic effect observed following infection of those cells by VSV (middle column) and its further enhancement in applying LT (30 U/ml) 2 hr following infection (right column). Photographs were taken 15 hr following infection of VSV (X 120).
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECTION
221
its extent is presented as a percentage of the cells which were incubated for the same period in the absence of VSV, LT, or IFN. RESULTS In cells of several lines, increase in vulnerability to the cytotoxic effect of LT could be observed following infection by VSV. Thus, in VSV-infected HeLa cells, WISH, Vero, and SV-80 cells, LT exerted a cytotoxic effect which was significantly beyond that of the virus itself (Fig. 1, circles, and Fig. 2), even though in the absence of viral infection these same cells appeared completely resistant to LT (Fig. 1, rectangles, and Fig. 4, compare A and B). A more detailed analysis of the effect of LT on SV-80 cells (Figs. 3 and 4) showed that LT cytotoxicity could first be observed at about 12 hr following infection by the virus, at the time when viral cytopathy also first became apparent. Killing of all cells in the culture was completed in the presence of LT much earlier than in its absence (Fig. 1C and D and Fig. 3: VSV + 30 U/ml LT compared to VSV alone). The effectiveness of LT cytotoxicity on the infected cells was proportional to its concentration (Fig. 3) yet even at a hundred times the concentration at which LT caused measurable killing of infected cells it still failed to exert any cytotoxic effect on uninfected ones (Fig. 3, squares). Exposing cells to LT for a longer period, by applying it 12 hr prior to infection by virus, further increased the effectiveness of cell destruction. However, even under these conditions cell killing started only at about 12 hr following infection (not shown). Treating the cells with IFN prior to infection by VSV affected their vulnerability to LT in a complex fashion. When applied at concentrations which protected cells from cytopathy of the virus, IFN also prevented the killing of cells by LT (Fig. 5, compare solid and empty symbols). On the other hand, below the antiviral range of concentrations IFN had the opposite effect, namely, it enhanced the cytotoxic
‘*) VSV+ LT 6u/ml
vSv+LT
15u/ml
VSV+ LT 30 u/ml
Time
(Iin.)
FIG. 3. Kinetic examination of LT cytotoxicity, in VSV-infected SV-80 cells, at various LT concentrations compared to the lack of cytotoxic effect of LT in the uninfected cells (m).
222
ADERKA ET AL.
FIG. 4. Morphology of SV-80 cells under normal growth conditions (A) compared to the morphology of cells which were treated by LT (B) infected by VSV (C), infected by VSV and treated by LT (D), or infected by VSV and treated with both IFN-7 and LT (E). IFN--y (100 U/ml) was applied on the cells 16 hr prior to infection and LT (30 U/ml) 2 hr following infection. Photographs were all taken 20 hr following infection by VSV (X 150).
effect of LT against virus-infected cells (Figs. 5 and 6). Cells which were not infected by VSV remained resistant to LT even in the presence of IFN (Fig. 5, diamonds, and Fig. 6, squares). In comparing the response of cells to the three types of IFN (Fig. 4) the stimulatory effect on cell destruction was found to be induced with EN--y at a significantly wider range of concentrations than with IFN-(I! or -8. Thus, in SV-80 cells enhancement of cell destruction by IF’Ny could be observed at IFN concentrations
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECTION
0.01 IFN
(units/ml)
IFN
0.1
I
223
IO
(units/ml)
FIG. 5. IFN effect on the cytotoxicity of LT in SV-80 cells (A) and in HeLa cells (B). IFN-cu (0, l ), IFN-@ @, q ), and IFN-7 (A, A) were applied at the indicated concentrations prior to infection by VSV (solid symbols) or infection by VSV followed by application of LT (6 U/ml, empty symbols), or application of LT alone (6 U/ml, for the three types of IFN, 0). Note the enhancement of cell killing at low concentrations of IFN and the protective effect of IFN at higher concentrations.
as high as 100 U/ml (Fig. 6), while with IFN-(w and -/3 such enhancement could be observed only at IFN concentrations lower than 1 U/ml (Fig. 5A, empty circles and rectangles). Similar differences between the responsesto the differing IFN, although to a lesser extent, could also be observed with HeLa cells (Fig. 5B). DISCUSSION The cytotoxicity of LT is not restricted to target cells which exhibit foreign antigens on their surface nor does it seem to be affected by the nature of the major histocompatibility antigens in these cells. There is therefore much interest in the fact that, nevertheless, some kind of selectivity in its function can be observed: It is shown to exert a cytotoxic effect on VSV-infected cells, even though in the absence of viral infection these same cells exhibit resistance to its cytotoxicity. Preliminary observations indicate that a number of other viruses besides VSV can also sensitize cells to LT (unpublished observations). What mechanisms underlie this distinction between uninfected and virus-infected cells is unknown. As increase in vulnerability to LT can also be observed in cells following treatment by inhibitors of RNA or of protein synthesis (2, 5) it seems possible that the sensitization to LT in cells infected by VSV is, at least in part, a consequence of the shut-off in synthesis of cellular RNA and proteins, which this virus induces. IFN is shown to potentiate the cytotoxicity of LT against the infected cells and has also been reported to increase the cytotoxic effect of LT in cells which were sensitized to its effect by mitomycin C (12) or CHI (4). However, under normal growth conditions we found cells to be resistant to LT even in the presence of IFN. Moreover, cells infected by VSV are shown to become resistant to LT when treated with IFN at concentrations which inhibit replication of the virus. The protection of
224
ADERKA ET AL.
0
2
4
6
I6
32
LT lunih/ml) FIG. 6. Enhancement by IlW-y of LT cytotoxicity in VW-infected SV-80 cells. The cytotoxic effect of LT at various concentrations in VSV-infected cells (0) and its further enhancement by treating these cells with IFN-7 (10 U/ml, 16 hr prior to infection (0) or 100 U/ml, prior to infection (A)) is shown in comparison to the resistance to LT observed in uninfected cells (m) even when they are also treated with IFN-y at 100 U/ml (0).
cells by the antiviral effect of IFN, on one hand, and the enhancement of destruction of the infected cells as a result of their sensitization to LT by IFN, on the other hand, is reflected in a bimodal pattern of the concentration dependence of the effect of IFN on cell viability; enhancement of destruction by LT, at low, sub-antiviral IFN concentrations, and protection from killing at higher concentrations. The ranges of IFN concentrations at which enhancement and suppression of cell destruction were observed turned out to be dependent on the type of IFN applied. EN-7 which, with regard to several kinds of IFN effects, is a more effective immunoregulatory agent than the other interherons (cf., e.g., (13-15)) showed the enhancement of cell response to LT at a wider range of concentrations than IFN-a and -8. Paradoxically, killing of virus-infected cells, which is often an undesirable effect of the virus itself, is also one of the major modes of immune defense against viral infection. These two aspects of the death of virus-infected cells can well be demonstrated in cells infected by VSV: The virus itself is highly cytopathic. However, production and release of virions can only take place during that time at which the infected cell is still alive. Thus, even though the virus itself eventually kills the cell, any enhancement of cell killing, by immune cytolytic mechanisms, can be highly beneficial in reducing the yield of new virions and thus decreasing the chance that other cells will become infected. There are several mechanisms of immune cytolysis which can be exerted selectively against virus-infected cells; some-such as the cytotoxicity of cytotoxic T lymphocytes-are directed by specific recognition of viral antigens on the surface of infected cells, while others-such as the activity of NK cells-by other mechanisms of distinction whose nature is yet unknown. This study indicates that LT can also
LYMPHOTOXIN
IN VESICULAR STOMATITIS
VIRUS INFECT ION
225
destroy selectively cells which are infected by viruses and that IF’N can augment this cytotoxic activity. There are also other indications implicating the cytotoxicity of LT in the response to viral infection. We have recently observed that Sendai virus is a potent inducer of LT formation in cultures of mononuclear leukocytes (Aderka and Wallach, manuscript in preparation), while IFN has been found to increase the responsiveness of these cells to inducers of LT formation (3). Although it is difficult to infer from such in vitro observations the way LT functions in vivo, they do raise the possibility that LT has a role in the cytotoxic activities which cells of the immune system exert against virus-infected cells. ACKNOWLEDGMENT We thank Mrs. Sylvia Budilovsky for skillful technical assistance. Note added in proof: The preparations of LT applied in this study were far from pure. We therefore find it important to note that we lately reproduced the presented findings with homogenously purified preparations of such a protein. We recently isolated a major species of human cytotoxin with an M, of about 17,500, using a monoclonal antibody against that protein (Hahn et al., Proc. Natl. Acad. Sci. USA, in press). By its molecular size that cytotoxin seemed to be. identical to the one designated by others as “tumor-necrosis-factor” (Pennica et al., Nature (London) 312, 724, 1974). The protein was produced most effectively by monocytes, upon their infection by Sendai virus or their treatment with phorbol-12O-myristate- 13-acetate,but could also be detected, at a significant level, in lectin-induced LT preparations of the kind applied in this study (unpublished). When testing the effect of the isolated 17.5~kDacytotoxin on VSV-infected and uninfected SV-80 cells we observed the same kind of selectivity as that found with crude preparations of lymphotoxins, namely, marked cytotoxicity against the virus-infected cells (as well as against CHI-treated, uninfected cells) and further potentiation of that cytotoxicity by treatment with recombinant IFNy, but no cytotoxic effect of the protein against uninfected normally growing cells.
REFERENCES 1. 2. 3. 4.
Williams, T. W., and Granger, G. A., Cell. Immunol. 6, 171, 1973. Rosenau, W., Goldberg, M. L., and Burke, G. C., J. Immunol. 111, 1128, 1973. Wallach, D., and Hahn, T., Cell. Immunol. 76, 390, 1983. Wallach, D., Aderka, D., Budilovsky, S., and Hahn, T., In “The Biology of the Interferon System” (E. De Maeyer and H. Schellekens, Eds.), pp. 293-303. Elsevier, Amsterdam. 5. Wallach, D., J. Immunol. 132, 2464, 1984. 6. Gey, G. O., CoBman, W. D., and Jubicek, M. J., Cancer Rex 12, 264, 1952. 7. HayIhck, L., Exp. Cell Res. 23, 14, 1961. 8. Todaro, J. G., Green, H., and Swift, M. R., Science (Washington, D.C.) 153, 1252, 1966. 9. Novick, D., Eshhar, Z., Fischer, D. G., Friedlander, J., and Rubinstein, M., EMBO J. 2, 1527, 1983. 10. Wilchek, M., and Miron, T., In “Methods in Enzymology,” Vol. 34, “Affinity Techniques (Enzyme Purification: Part B)” (W. B. Jakoby and M. Wilchek, eds.), p. 72. Academic Press, New York, 1974. I 1. Finter, N. B., J. Gen. Virol. 5, 419, 1969. 12. Williams, T. W., and Bellanti, J. A., J. Immunol. 130, 5 18, 1983. 13. Sonnenfeld, G., Mandel, A. D., and Me&an, T. C., Cell. Immunol. 34, 193, 1977. 14. Wallach, D., J. Interjkon Res. 2, 329, 1982. 15. Wallach, D., Fellous, M., and Revel, M., Nature (London) 299, 833, 1982.