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Spontaneous cytotoxicity against viral-infected cells: Effects of leucocytes and sera from older mice
CELLULAR
IMMUNOLOGY
70, 180-187 (1982)
SHORT COMMUNICATIONS Spontaneous Cytotoxicity against Viral-Infected Cells: Effects of Leucocytes and Sera from Older Mice H. WATANABEAND J.S. MACKENZIE Department
of Microbiology,
University of Western Australia, Western Australia, 6009
Received November
17, 1981: accepted March
NEDLANDS,
30, 1982
Splenic leucocyte effector cells from 3-month-old C3H/HeJ mice which were antibody negative for influenza A virus were found to be spontaneously cytotoxic for mouse L929 fibroblasts infected with influenza virus, but not for uninfected cells. The cytotoxic activity was significantly enhanced if the effector cells were preincubated with splenic leucocytes from older 6- or 9-month-old antibody-negative mice before being added to the target cells. Enhancement appeared to be due to a cooperative effect between leucocytes from mice of different ages. Preincubation of effector cells with autologous sera markedly suppressed spontaneous cytotoxicity, but the suppression was significantly less with sera from 7- or lo-month-old mice than with sera from 3-month-old mice. Most cytotoxic activity was observed with a nylon wool-adherent subpopulation of leucocytes, although some cytotoxicity remained in the nonadherent fraction. The results suggested that spontaneous cytotoxicity was due to two subpopulations of cells, possibly adherent monocyte-macrophages and nonadherent natural killer cells.
INTRODUCTION Lymphocytes from normal donors have been shown to be spontaneously cytotoxic in vitro to target cells infected with various viruses (l-7). In some studies, an association was observed between spontaneous cytotoxic activity and the presence of antiviral antibodies (l-4), whereas in other studies, antiviral antibodies could not be detected (5-8). Spontaneous cytotoxicity has also been demonstrated by monocyte macrophages against human fibroblasts infected with herpes simplex virus in the absence of antibody (9). In this report, we describe spontaneous cytotoxic activity by splenic leucocytes obtained from C3H/HeJ mice which were antibody negative for influenza A virus against mouse L929 fibroblasts infected with influenza, but not against uninfected target cells. The effect on spontaneous cytotoxicity of preincubating effector cells with splenic leucocytes and sera from older mice were examined. MATERIALS AND METHODS Animals. Male C3H/HeJ inbred mice were obtained from the Small Animal Breeding Centre, University of Western Australia. Unless stated otherwise in the text, mice were used at 3 months of age. 180 OOOS-8749/82/090180-08$02OO/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
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Virus. Influenza A/England/42/72 (H3N2) virus stocks were grown in embryonated chicken eggs as described previously (10). Cell cultures. Mouse L929 fibroblasts were employed as target cells in cytotoxicity assays. The cells were grown in RPM1 1640 medium supplemented with 10% FCS.’ Enzyme-linked immunosorbent assay. An enzyme-linked immunosorbent assay was used to ensure that mice were antibody negative for influenza A/England/42/ 72 virus and to measure the presence of viral antigens on infected target cell surfaces. The assay has been described in detail elsewhere for serological (10) and cell-surface ( 11) titrations. Spontaneous cytotoxicity assay. Splenic leucocytes were obtained from groups of six influenza antibody-negative 3-month-old mice, and the cell suspension separated by centrifugation at 250g for 10 min over Ficoll-Paque (Pharmacia Fine Chemicals, Sweden). The leucocytes were washed and resuspended to 2 X 10’ cells/ ml in RPM1 1640 supplemented with 10% inactivated FCS as effector cell preparations. Target L929 fibroblasts (1 .O X 10’ cells) were infected and labeled simultaneously with 2.5 X lo3 hemagglutinating units of virus and with 150 &i “Cr (sodium chromate; Radiochemical Centre Ltd., Amersham, England) dissolved in serum-free RPM1 1640, and incubated for 1 hr at 37°C. The cells were then washed and overlaid with fresh medium and the incubation continued for a further 7 hr, at which time large quantities of hemagglutinin were detected at the surface of infected cells by enzyme immunoassay (11). The cells were then trypsinized, washed, and resuspended to 5 X lo5 cells/ml in RPM1 1640 supplemented with 10% inactivated FCS. Uninfected control cells were treated similarly. Aliquots of 100 ~1 of both effector leucocytes and target cells at a ratio of 4O:l were added to wells of microculture plates in replicates of 5, and the plates incubated for 11 hr at 37°C. Appropriate controls were included. The total releasable “Cr was determined by dissolving target cells with 1% Triton X-100 in distilled water. After the 11-hr incubation period, the percentage spontaneous Wr release was calculated as cpm wells containing leucocytes - cpm wells containing medium 100 x cpm total releasable “Cr - cpm wells containing medium ’ where all wells contained labeled target cells. Experiments were undertaken to determine the effects of premixing effector leucocytes from 3-month-old mice with sera or splenic leucocytes from older mice. A similar cytotoxic assay system was employed except that the effector cells were mixed with either sera from 3-, 7-, or lo-month-old mice diluted to a final concentration of 10 or 1% or splenic leucocytes from 3-, 6-, or 9-month-old mice at a 1:l cell ratio, and the effector-sera and effector-leucocyte mixtures incubated for 2 hr at 37°C with occasional shaking before being added to the target cells at a 2O:l effector-to-target ratio. The sera had been inactivated at 56°C for 30 min, dialyzed overnight against RPM1 1640 medium supplemented with 10% inactivated FCS at 4°C and centrifuged at 600g for 20 min before being mixed with the effector cells. After the 11-hr incubation period, comparative spontaneous “Cr release was calculated as age indices, defined as net cpm of effector cells mixed with sera from 3-, 7-, or lo-month-old mice (effector-sera mixture) or effector cells ’ Abbreviations
used: FCS, fetal calf serum; cpm, counts per minute.
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SHORT COMMUNICATIONS TABLE 1 Spontaneous Cytotoxicity by Splenic Leucocyte Effector Cells from Seronegative Mice against Influenza A Virus-Infected Target Cells Experiment No.
Spontaneous 5’Cr release (%)
Influenza infected
1 2 3
37.2 16.3 27.2 26.9 f 10.5”
Uninfected control
1 2 3
1.1 2.0 1.0 1.4 + 0.6”
Target cells
a Mean + one standard deviation of the mean.
mixed with additional splenic leucocytes from 3-, 6-, or 9-month-old mice (effectorleucocyte mixture), over net cpm of effecters mixed with other sera or leucocytes from 3-month-old mice (effector-standard mixture), using the formula cpm effector-sera or effector-leucocyte mixture - cpm medium alone , cpm effector-standard mixture - cpm medium alone where labeled target cells were present in all wells. Separation of eflector cells. Splenic leucocyte effector cells from 3-month-old mice were separated into adherent and nonadherent subpopulations by passage through a nylon wool column as described elsewhere ( 12). The subpopulations were then mixed with sera from 3- or lo-month-old mice, incubated for 2 hr at 37°C and added to the cytotoxicity assay as described above, except that the effector-totarget cell ratio was 10:1. RESULTS Spontaneous Cytotoxicity Cells
by Splenic Leucocytes against Virus-Znfected Target
Splenic leucocytes were prepared from 3-month-old C3H/HeJ mice which had been shown to be antibody negative for influenza A/England/42/72 virus, and examined for their spontaneous cytotoxicity against uninfected and influenza-infected L929 target cells in an 11-hr assay. The percentage spontaneous cytotoxicity observed in three experiments, done over a 6-month period, is shown in Table 1. The mean cytotoxicity against infected target cells was found to be significantly greater (P < 0.05) than against uninfected control cells. Similar results were obtained using effector cells from 7- and lo-month-old mice. The conditions necessary for this high spontaneous cytotoxicity against virusinfected target cells were determined, and were found to include a requirement for the presence of a large amount of viral antigens on the surface of target cells, a high ratio of effector-to-target cells, and a cytotoxic assay with an incubation time of at least 11 hr (results not shown). In the latter instance, cytotoxic assays with
SHORT
TABLE Enhancement
2
of Spontaneous Cytotoxicity by Preincubation with Splenic Leucocytes from Older Mice
Age of mice (months) from which additional leucocytes were obtained
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Effector-toleucocyte ratio
of Effector
Cells
Age indices of cytotoxicity” f SEM’
3 6 9
1:l 1:l 1:l
1.00 f 0.02 1.57 f 0.05’ 1.64 f 0.03’
3 6 9
1O:l 1O:l 1O:l
1.00 -I 0.01 1.00 f 0.03 1.07 f 0.01
3 6 9
0:lOO 0:lOO 0:lOO
1.00 f 0.02 1.01 f 0.01 1.07 f 0.02
’ Age indices are defined as cpm of 5’Cr release in the presence of leucocytes from 3-, 6-, or 9-monthold mice over the cpm of “Cr release in the presence of leucocytes from 3-month-old mice. b Standard error of the mean. ’ Significant (P < 0.001) compared with leucocytes from 3-month-old mice.
incubation times of 4 or 7 hr were insufficient to clearly exhibit significant spontaneous cytotoxicity. The Eflect on Spontaneous Cytotoxicity of Premixing E$ector Cells with Splenic Leucocytes from Older Mice An increase in spontaneous cytotoxicity was found when effector cells were premixed with splenic leucocytes from older mice. Effector cells from 3-month-old mice were mixed with an equal number of splenic leucocytes from 3-, 6-, or 9month-old antibody-negative mice, and incubated for 2 hr at 37°C before being added to the infected target cells. The results are shown in Table 2 as age indices, defined as the ratio between cpm of “Cr released by effector cells in the presence of splenic leucocytes from 3-, 6-, or 9-month-old mice over cpm of “Cr released by effector cells in the presence of splenic leucocytes from 3-month-old mice. A significantly higher (P < 0.001) spontaneous cytotoxicity was observed when the effector cells were preincubated with leucocytes from 6- or 9-month-old mice than with leucocytes from 3-month-old mice. However, if the number of additional splenic leucocytes was reduced to 10% of the number of effector cells while retaining the same effector-to-target ratio, no significant increase in cytotoxicity was detected. These results suggested that leucocytes from older mice either contained more cytotoxic cells, or that their cytotoxic activity was greater than leucocytes from younger 3-month-old mice. Additional controls were included in the above experiments in which the effector cells were replaced with medium. The age indices in these controls were therefore calculated as the ratio of cpm released by splenic leucocytes from 3-, 6-, or 9month-old mice over cpm released by splenic leucocytes from 3-month-old mice. No enhancement of spontaneous cytotoxicity, however, was observed with the leu-
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cocytes from older mice (Table 2). These results were unexpected if leucocytes from older mice exhibit greater cytotoxic activity, as was suggested above from age indices after they were preincubated with effector cells from young mice. It would appear, therefore, that the enhancement of spontaneous cytotoxicity shown by preincubating leucocytes from 6- and 9-month-old mice with effector cells was due to some cooperative effect between the effector cells and the older splenic leucocytes, and that this cooperative effect does not occur between cells of the same age. These results were highly reproducible in repeated experiments. The Efiect on Spontaneous Cytotoxicity of Preincubating Effector Cells with Sera from Older Mice To determine the effect of the addition of serum on spontaneous cytotoxicity, sera were collected from 3-, 7-, and lo-month-old mice which were antibody negative for influenza virus, dialyzed overnight against RPM1 1640 medium, and added at a final concentration of 10 or 1% (v/v) to effector cells from 3-month-old mice. After incubation for 2 hr at 37°C the effector cell-sera mixtures were added to infected target cells in the 11-hr cytotoxicity assay. The results are shown in Table 3 as age indices, calculated as described earlier for additional splenic leucocytes. The spontaneous cytotoxicity by effector cells preincubated with sera from 7- or lo-month-old mice was found to be significantly higher (P < 0.05) than with sera from 3-month-old mice when the sera were used at a final concentration of 10%. However, when sera were employed at a final concentration of l%, no significant increase was detected. These results were also highly reproducible in repeated experiments. To investigate the effect of serum on cytotoxicity further, and in order to obtain more information on the cells responsible for cytotoxic activity, the effector cells were separated into adherent and nonadherent subpopulations on a nylon wool column (12), and the percentage cytotoxicity of the two subpopulations was determined with and without preincubation with sera from 3- and lo-month-old antibody-negative mice. The results are shown in Table 4. The adherent cells, TABLE
3
Effect on Spontaneous Cytotoxicity of Preincubating Effector Cells with Sera from Antibody-Negative Mice of Various Ages
Dilution
of sera
Age of mice (months) from which sera were collected
Age indices of cytotoxicitqP + SEM*
1:lO
3 I 10
1.00 * 0.21 1.50 + 0.13’ 1.55 f 0.17’
1:lOO
3 I 10
1.00 + 0.23 1.12 f 0.20 1.11 + 0.13
a Age indices are defined as cpm of “Cr release in the presence of sera from 3-, 7-, or IO-month-old mice over cpm of 5’Cr release in the presence of sera from 3-month-old mice. b Standard error of the mean. c Significant (P < 0.05) compared with sera from 3-month-old mice.
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TABLE Effect of Nylon Wool-Adherent Cytotoxicity
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and Nonadherent Effector Cell Fractions on Spontaneous in the Presence and Absence of Sera
Nylon wool fraction
Effector cells preincubated with
Spontaneous “Cr release (o/o) k SEM”
Adherent cells (monocyte-macrophages)
Medium 3-Month-old IO-Month-old
sera sera
24.3 -t 0.9’ 10.6 f 0.6’ 13.8 + 0.9
Nonadherent
Medium 3-Month-old IO-Month-old
sera sera
13.6 zk 0.4’ 6.0 k 0.6d 7.5 + 0.8“
cells
’ Standard error of the mean. b Significantly different (P < 0.01). ‘Significantly different (P < 0.05). d Difference not significant (P > 0.1).
probably monocyte macrophages, exhibited significantly greater (P < 0.01) spontaneous cytotoxicity than did the nonadherent cells in the absenceof sera. However, significant levels of cytotoxicity were found in the nonadherent fraction. Preincubation of both effector subpopulations with sera, used at a final concentration of lo%, markedly suppressed their cytotoxic activity, but the amount of cytotoxic activity by the adherent fraction was significantly higher (P < 0.01) than that of the nonadherent subpopulation, regardless of the age of the mice from which the sera had been collected. Furthermore, the spontaneous cytotoxicity of the adherent cells was significantly enhanced (P < 0.05) by preincubation with sera from lomonth-old mice compared to sera from 3-month-old mice, whereas no significant increase was observed when nonadherent cells were preincubated with the older sera. These results indicate that although serum is strongly suppressive on spontaneous cytotoxic activity, the amount of suppression decreases with age. DISCUSSION In this report, splenic leucocytes from influenza virus antibody-negative mice were found to be spontaneously cytotoxic in vitro for influenza virus-infected target L929 cells, but not for uninfected control cells. Other studies have also described spontaneous cytotoxicity for target cells infected with various viruses in the absence of antiviral antibody, including influenza (5), Sendai (6), mumps (7), and herpes simplex (9) viruses. It was also found that spontaneous cytotoxicity was enhanced by preincubating effector cells with splenic leucocytes from older, influenza-virus antibody-negative mice, and although preincubating effector cells with serum markedly reduced cytotoxic activity, the amount of suppression was significantly less with sera from older mice. Similar findings have not been reported previously for spontaneous cytotoxicity against virus-infected target cells. Since antibodies to influenza virus could not be detected in the sera of mice used in this study by a highly sensitive enzyme immunoassay (10, 13), it was unlikely that the spontaneous cytotoxicity for influenza-infected target cells was due to antibody-dependent cellular cytotoxicity or to antibody plus complement cytotox-
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icity. In addition, no spontaneous cytotoxicity was observed with either sera or conditioned medium in the absence of effector cells. Natural killer (NK) cells have been implicated in the spontaneous lysis of target cells infected with mumps (7), Sendai (6, 8), and possibly influenza (5) viruses. However, the peak activity of NK cells has been shown to occur in young mice ( 14) rather than older mice, and NK cytotoxicity has been found to be markedly stimulated by interferon (15). In this study, the age indices described in Table 2 in the absence of effector cells demonstrated that leucocytes from older mice exhibited levels of cytotoxic activity similar to those of leucocytes from younger mice. However, the long incubation time (11 hr) required for significant spontaneous cytotoxicity could be indicative of NK activity since influenza viruses are known to be effective inducers of interferon. Interferon assays were not undertaken during the effector-target incubation period. Fractionation of the effector cells into adherent and nonadherent subpopulations on a nylon wool column showed that the principal mediators of cytotoxicity were in the adherent fraction. This method of fractionation had been described previously by Gordon and Shore (12) who had shown that 30% of the nylon wooladherent cells were phagocytic monocytes (latex-positive), a threefold increase compared to unfractionated cells. They also found that 10% of the T lymphocytes in the unfractionated cells were adherent, and the surface Ig-positive B lymphocytes increased fivefold in the adherent fraction. However, the adherent subclass of T lymphocytes, Ly-123+ cells, have not been shown to be spontaneously cytotoxic for virus-infected cells, and B lymphocytes are noncytotoxic. It would seem likely, therefore, that mediators of cytotoxicity in the adherent fraction were monocytemacrophages. Not only have monocyte-macrophages been found to be spontaneously cytotoxic for herpes simplex virus-infected target cells in the absence of antibody (9), but the phagocytic activity of macrophages have been shown to be unaltered or even enhanced with age ( 16). The spontaneous cytotoxicity remaining in the nonadherent fraction, however, may be due to NK cell activity. The age indices shown in Table 2 indicate that splenic leucocytes from 3-, 6-, and 9-month-old mice exhibited similar cytotoxic activities against infected target cells. An increased spontaneous cytotoxicity was observed, however, when 3-monthold effector cells were preincubated with leucocytes from older antibody-negative mice. These results suggest that the enhanced cytotoxic activity was due to a cooperative effect between the effector cells and the leucocytes from older mice. Whether this effect was the result of cell-to-cell contact or soluble factors produced by either or both of the leucocyte populations generating increased cytotoxic activity was not determined. However, the effect was almost completely abrogated if the number of additional leucocytes from older animals was reduced from 50 to 10% of the effector cell number. Preincubation of effector cells with sera from 3-month-old and lo-month-old mice at a final concentration of 10% markedly reduced the amount of spontaneous cytotoxic activity for infected target cells. Nevertheless, despite this general suppression, the relative levels of cytotoxic activity were significantly higher when effector cells were preincubated with sera from 7- and lo-month-old mice than with sera from 3-month-old mice, as shown by age indices in Table 3 and by percentage spontaneous cytotoxicity (with adherent cells only) in Table 4. This effect was not observed, however, when the sera were diluted to a final concentration of 1% (Table 3). A number of possible explanations can be advanced for the
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increased spontaneous cytotoxicity in the presence of sera from older animals. Thus suppressor factors specific for monocyte macrophages (or the adherent cells in Table 4) might decrease in older animals; alternatively active effecters might be amplified by soluble factors in sera, which increase with age; or autoantibodies, which usually increase with age, might be directed against the animal’s own cellsurface antigens resulting in an antibody-mediated cytolysis. The results obtained by preincubating effector cells with splenic leucocytes and sera from older mice were in contrast to our other findings with influenza-immune cytotoxic T lymphocytes. The specific cytotoxicity by T lymphocytes was significantly depressed by preincubation of the lymphocytes with splenic leucocytes or sera from older influenza virus antibody-negative mice, or with conditioned medium from Con A-activated splenic leucocytes from older mice (Watanabe and Mackenzie, submitted for publication). Since an enhancement of spontaneous cytotoxicity can occur with age under certain conditions, albeit abnormal, these results may provide an additional avenue for investigation of the increased incidence of autoimmune phenomena in older animals, despite the apparently contradictory reports of the appearance of suppressor cells in old mice ( 17- 19). ACKNOWLEDGMENTS We thank P. Hodgkin and J. Flexman for their help in sections of this study. The work was supported by the National Health and Medical Research Council of Australia.
REFERENCES 1. Steele, R. W., Hensen, S. A., Vincent, M. M., Fucillo, D. A., and Bellanti, J. A., J. Immunol. 110, 1502, 1973. 2. Greenberg, S. B., Criswell, B. S., and Couch, R. B., J. Immunol. 115, 601, 1975. 3. Greenberg, S. B., Criswell, B. S., Six, H. R., and Couch, R. B., J. Immunol. 119, 2100, 1977. 4. Msller-Larsen, A., Heron, I., and Haahr S., Infect. Immun. 16, 43, 1977. 5. Santoli, D., Trinchieri, G., and Lief, F. S., J. fmmunol. 121, 526, 1978. 6. Weston, P. A., Levy, N. L., and Koren, H. S., J. Immunol. 125, 1387, 1980. 7. H&fast, B., Andersson, T., and Perlmann, P., J. Immunol. 121, 755, 1978. 8. Weston, P. A., Jensen, P. J., Levy, N. L., and Koren, H. S., J. Immunol. 126, 1220, 1981. 9. Stanwick, T. L., Campbell, D. E., and Nahmias, A. J., Cell. Immunol. 53, 413, 1980. 10. Watanabe, H., Pollett, M., and Mackenzie, J. S., Aust. J. Exp. Biol. Med. Sci. 58, 189, 1980. 11. Watanabe, H., and Mackenzie, J. S., Arch. Viral. 67, 31, 1981. 12. Gordon, D. S., and Shore, S. L., Cell. Immunol. 50, 19, 1980. 13. Watanabe, H., Pollett, M., and Mackenzie, J. S., Aust. J. Exp. Biol. Med. Sci. 57, 303, 1979. 14. Gudkowicz, G., and Hochmen, P. S., Immunol. Rev. 44, 13, 1979. 15. Santoli, D., Trinchieri, G., and Koprowski, H., J. Immunol. 121, 542, 1978. 16. Makinodan, T., and Adler, W. H., Fed. Proc. 34, 153, 1975. 17. Goide, E. A., Innes, J. B., and Weksler, M. E., J. Exp. Med. 144, 1037, 1976. 18. Segre, D., and Segre, M., J. Immunol. 116, 735, 1976. 19. Callard, R. E., and Basten, A., Eur. J. Immunol. 8, 552, 1978.
IMMUNOLOGY
70, 180-187 (1982)
SHORT COMMUNICATIONS Spontaneous Cytotoxicity against Viral-Infected Cells: Effects of Leucocytes and Sera from Older Mice H. WATANABEAND J.S. MACKENZIE Department
of Microbiology,
University of Western Australia, Western Australia, 6009
Received November
17, 1981: accepted March
NEDLANDS,
30, 1982
Splenic leucocyte effector cells from 3-month-old C3H/HeJ mice which were antibody negative for influenza A virus were found to be spontaneously cytotoxic for mouse L929 fibroblasts infected with influenza virus, but not for uninfected cells. The cytotoxic activity was significantly enhanced if the effector cells were preincubated with splenic leucocytes from older 6- or 9-month-old antibody-negative mice before being added to the target cells. Enhancement appeared to be due to a cooperative effect between leucocytes from mice of different ages. Preincubation of effector cells with autologous sera markedly suppressed spontaneous cytotoxicity, but the suppression was significantly less with sera from 7- or lo-month-old mice than with sera from 3-month-old mice. Most cytotoxic activity was observed with a nylon wool-adherent subpopulation of leucocytes, although some cytotoxicity remained in the nonadherent fraction. The results suggested that spontaneous cytotoxicity was due to two subpopulations of cells, possibly adherent monocyte-macrophages and nonadherent natural killer cells.
INTRODUCTION Lymphocytes from normal donors have been shown to be spontaneously cytotoxic in vitro to target cells infected with various viruses (l-7). In some studies, an association was observed between spontaneous cytotoxic activity and the presence of antiviral antibodies (l-4), whereas in other studies, antiviral antibodies could not be detected (5-8). Spontaneous cytotoxicity has also been demonstrated by monocyte macrophages against human fibroblasts infected with herpes simplex virus in the absence of antibody (9). In this report, we describe spontaneous cytotoxic activity by splenic leucocytes obtained from C3H/HeJ mice which were antibody negative for influenza A virus against mouse L929 fibroblasts infected with influenza, but not against uninfected target cells. The effect on spontaneous cytotoxicity of preincubating effector cells with splenic leucocytes and sera from older mice were examined. MATERIALS AND METHODS Animals. Male C3H/HeJ inbred mice were obtained from the Small Animal Breeding Centre, University of Western Australia. Unless stated otherwise in the text, mice were used at 3 months of age. 180 OOOS-8749/82/090180-08$02OO/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
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Virus. Influenza A/England/42/72 (H3N2) virus stocks were grown in embryonated chicken eggs as described previously (10). Cell cultures. Mouse L929 fibroblasts were employed as target cells in cytotoxicity assays. The cells were grown in RPM1 1640 medium supplemented with 10% FCS.’ Enzyme-linked immunosorbent assay. An enzyme-linked immunosorbent assay was used to ensure that mice were antibody negative for influenza A/England/42/ 72 virus and to measure the presence of viral antigens on infected target cell surfaces. The assay has been described in detail elsewhere for serological (10) and cell-surface ( 11) titrations. Spontaneous cytotoxicity assay. Splenic leucocytes were obtained from groups of six influenza antibody-negative 3-month-old mice, and the cell suspension separated by centrifugation at 250g for 10 min over Ficoll-Paque (Pharmacia Fine Chemicals, Sweden). The leucocytes were washed and resuspended to 2 X 10’ cells/ ml in RPM1 1640 supplemented with 10% inactivated FCS as effector cell preparations. Target L929 fibroblasts (1 .O X 10’ cells) were infected and labeled simultaneously with 2.5 X lo3 hemagglutinating units of virus and with 150 &i “Cr (sodium chromate; Radiochemical Centre Ltd., Amersham, England) dissolved in serum-free RPM1 1640, and incubated for 1 hr at 37°C. The cells were then washed and overlaid with fresh medium and the incubation continued for a further 7 hr, at which time large quantities of hemagglutinin were detected at the surface of infected cells by enzyme immunoassay (11). The cells were then trypsinized, washed, and resuspended to 5 X lo5 cells/ml in RPM1 1640 supplemented with 10% inactivated FCS. Uninfected control cells were treated similarly. Aliquots of 100 ~1 of both effector leucocytes and target cells at a ratio of 4O:l were added to wells of microculture plates in replicates of 5, and the plates incubated for 11 hr at 37°C. Appropriate controls were included. The total releasable “Cr was determined by dissolving target cells with 1% Triton X-100 in distilled water. After the 11-hr incubation period, the percentage spontaneous Wr release was calculated as cpm wells containing leucocytes - cpm wells containing medium 100 x cpm total releasable “Cr - cpm wells containing medium ’ where all wells contained labeled target cells. Experiments were undertaken to determine the effects of premixing effector leucocytes from 3-month-old mice with sera or splenic leucocytes from older mice. A similar cytotoxic assay system was employed except that the effector cells were mixed with either sera from 3-, 7-, or lo-month-old mice diluted to a final concentration of 10 or 1% or splenic leucocytes from 3-, 6-, or 9-month-old mice at a 1:l cell ratio, and the effector-sera and effector-leucocyte mixtures incubated for 2 hr at 37°C with occasional shaking before being added to the target cells at a 2O:l effector-to-target ratio. The sera had been inactivated at 56°C for 30 min, dialyzed overnight against RPM1 1640 medium supplemented with 10% inactivated FCS at 4°C and centrifuged at 600g for 20 min before being mixed with the effector cells. After the 11-hr incubation period, comparative spontaneous “Cr release was calculated as age indices, defined as net cpm of effector cells mixed with sera from 3-, 7-, or lo-month-old mice (effector-sera mixture) or effector cells ’ Abbreviations
used: FCS, fetal calf serum; cpm, counts per minute.
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SHORT COMMUNICATIONS TABLE 1 Spontaneous Cytotoxicity by Splenic Leucocyte Effector Cells from Seronegative Mice against Influenza A Virus-Infected Target Cells Experiment No.
Spontaneous 5’Cr release (%)
Influenza infected
1 2 3
37.2 16.3 27.2 26.9 f 10.5”
Uninfected control
1 2 3
1.1 2.0 1.0 1.4 + 0.6”
Target cells
a Mean + one standard deviation of the mean.
mixed with additional splenic leucocytes from 3-, 6-, or 9-month-old mice (effectorleucocyte mixture), over net cpm of effecters mixed with other sera or leucocytes from 3-month-old mice (effector-standard mixture), using the formula cpm effector-sera or effector-leucocyte mixture - cpm medium alone , cpm effector-standard mixture - cpm medium alone where labeled target cells were present in all wells. Separation of eflector cells. Splenic leucocyte effector cells from 3-month-old mice were separated into adherent and nonadherent subpopulations by passage through a nylon wool column as described elsewhere ( 12). The subpopulations were then mixed with sera from 3- or lo-month-old mice, incubated for 2 hr at 37°C and added to the cytotoxicity assay as described above, except that the effector-totarget cell ratio was 10:1. RESULTS Spontaneous Cytotoxicity Cells
by Splenic Leucocytes against Virus-Znfected Target
Splenic leucocytes were prepared from 3-month-old C3H/HeJ mice which had been shown to be antibody negative for influenza A/England/42/72 virus, and examined for their spontaneous cytotoxicity against uninfected and influenza-infected L929 target cells in an 11-hr assay. The percentage spontaneous cytotoxicity observed in three experiments, done over a 6-month period, is shown in Table 1. The mean cytotoxicity against infected target cells was found to be significantly greater (P < 0.05) than against uninfected control cells. Similar results were obtained using effector cells from 7- and lo-month-old mice. The conditions necessary for this high spontaneous cytotoxicity against virusinfected target cells were determined, and were found to include a requirement for the presence of a large amount of viral antigens on the surface of target cells, a high ratio of effector-to-target cells, and a cytotoxic assay with an incubation time of at least 11 hr (results not shown). In the latter instance, cytotoxic assays with
SHORT
TABLE Enhancement
2
of Spontaneous Cytotoxicity by Preincubation with Splenic Leucocytes from Older Mice
Age of mice (months) from which additional leucocytes were obtained
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Effector-toleucocyte ratio
of Effector
Cells
Age indices of cytotoxicity” f SEM’
3 6 9
1:l 1:l 1:l
1.00 f 0.02 1.57 f 0.05’ 1.64 f 0.03’
3 6 9
1O:l 1O:l 1O:l
1.00 -I 0.01 1.00 f 0.03 1.07 f 0.01
3 6 9
0:lOO 0:lOO 0:lOO
1.00 f 0.02 1.01 f 0.01 1.07 f 0.02
’ Age indices are defined as cpm of 5’Cr release in the presence of leucocytes from 3-, 6-, or 9-monthold mice over the cpm of “Cr release in the presence of leucocytes from 3-month-old mice. b Standard error of the mean. ’ Significant (P < 0.001) compared with leucocytes from 3-month-old mice.
incubation times of 4 or 7 hr were insufficient to clearly exhibit significant spontaneous cytotoxicity. The Eflect on Spontaneous Cytotoxicity of Premixing E$ector Cells with Splenic Leucocytes from Older Mice An increase in spontaneous cytotoxicity was found when effector cells were premixed with splenic leucocytes from older mice. Effector cells from 3-month-old mice were mixed with an equal number of splenic leucocytes from 3-, 6-, or 9month-old antibody-negative mice, and incubated for 2 hr at 37°C before being added to the infected target cells. The results are shown in Table 2 as age indices, defined as the ratio between cpm of “Cr released by effector cells in the presence of splenic leucocytes from 3-, 6-, or 9-month-old mice over cpm of “Cr released by effector cells in the presence of splenic leucocytes from 3-month-old mice. A significantly higher (P < 0.001) spontaneous cytotoxicity was observed when the effector cells were preincubated with leucocytes from 6- or 9-month-old mice than with leucocytes from 3-month-old mice. However, if the number of additional splenic leucocytes was reduced to 10% of the number of effector cells while retaining the same effector-to-target ratio, no significant increase in cytotoxicity was detected. These results suggested that leucocytes from older mice either contained more cytotoxic cells, or that their cytotoxic activity was greater than leucocytes from younger 3-month-old mice. Additional controls were included in the above experiments in which the effector cells were replaced with medium. The age indices in these controls were therefore calculated as the ratio of cpm released by splenic leucocytes from 3-, 6-, or 9month-old mice over cpm released by splenic leucocytes from 3-month-old mice. No enhancement of spontaneous cytotoxicity, however, was observed with the leu-
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cocytes from older mice (Table 2). These results were unexpected if leucocytes from older mice exhibit greater cytotoxic activity, as was suggested above from age indices after they were preincubated with effector cells from young mice. It would appear, therefore, that the enhancement of spontaneous cytotoxicity shown by preincubating leucocytes from 6- and 9-month-old mice with effector cells was due to some cooperative effect between the effector cells and the older splenic leucocytes, and that this cooperative effect does not occur between cells of the same age. These results were highly reproducible in repeated experiments. The Efiect on Spontaneous Cytotoxicity of Preincubating Effector Cells with Sera from Older Mice To determine the effect of the addition of serum on spontaneous cytotoxicity, sera were collected from 3-, 7-, and lo-month-old mice which were antibody negative for influenza virus, dialyzed overnight against RPM1 1640 medium, and added at a final concentration of 10 or 1% (v/v) to effector cells from 3-month-old mice. After incubation for 2 hr at 37°C the effector cell-sera mixtures were added to infected target cells in the 11-hr cytotoxicity assay. The results are shown in Table 3 as age indices, calculated as described earlier for additional splenic leucocytes. The spontaneous cytotoxicity by effector cells preincubated with sera from 7- or lo-month-old mice was found to be significantly higher (P < 0.05) than with sera from 3-month-old mice when the sera were used at a final concentration of 10%. However, when sera were employed at a final concentration of l%, no significant increase was detected. These results were also highly reproducible in repeated experiments. To investigate the effect of serum on cytotoxicity further, and in order to obtain more information on the cells responsible for cytotoxic activity, the effector cells were separated into adherent and nonadherent subpopulations on a nylon wool column (12), and the percentage cytotoxicity of the two subpopulations was determined with and without preincubation with sera from 3- and lo-month-old antibody-negative mice. The results are shown in Table 4. The adherent cells, TABLE
3
Effect on Spontaneous Cytotoxicity of Preincubating Effector Cells with Sera from Antibody-Negative Mice of Various Ages
Dilution
of sera
Age of mice (months) from which sera were collected
Age indices of cytotoxicitqP + SEM*
1:lO
3 I 10
1.00 * 0.21 1.50 + 0.13’ 1.55 f 0.17’
1:lOO
3 I 10
1.00 + 0.23 1.12 f 0.20 1.11 + 0.13
a Age indices are defined as cpm of “Cr release in the presence of sera from 3-, 7-, or IO-month-old mice over cpm of 5’Cr release in the presence of sera from 3-month-old mice. b Standard error of the mean. c Significant (P < 0.05) compared with sera from 3-month-old mice.
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TABLE Effect of Nylon Wool-Adherent Cytotoxicity
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and Nonadherent Effector Cell Fractions on Spontaneous in the Presence and Absence of Sera
Nylon wool fraction
Effector cells preincubated with
Spontaneous “Cr release (o/o) k SEM”
Adherent cells (monocyte-macrophages)
Medium 3-Month-old IO-Month-old
sera sera
24.3 -t 0.9’ 10.6 f 0.6’ 13.8 + 0.9
Nonadherent
Medium 3-Month-old IO-Month-old
sera sera
13.6 zk 0.4’ 6.0 k 0.6d 7.5 + 0.8“
cells
’ Standard error of the mean. b Significantly different (P < 0.01). ‘Significantly different (P < 0.05). d Difference not significant (P > 0.1).
probably monocyte macrophages, exhibited significantly greater (P < 0.01) spontaneous cytotoxicity than did the nonadherent cells in the absenceof sera. However, significant levels of cytotoxicity were found in the nonadherent fraction. Preincubation of both effector subpopulations with sera, used at a final concentration of lo%, markedly suppressed their cytotoxic activity, but the amount of cytotoxic activity by the adherent fraction was significantly higher (P < 0.01) than that of the nonadherent subpopulation, regardless of the age of the mice from which the sera had been collected. Furthermore, the spontaneous cytotoxicity of the adherent cells was significantly enhanced (P < 0.05) by preincubation with sera from lomonth-old mice compared to sera from 3-month-old mice, whereas no significant increase was observed when nonadherent cells were preincubated with the older sera. These results indicate that although serum is strongly suppressive on spontaneous cytotoxic activity, the amount of suppression decreases with age. DISCUSSION In this report, splenic leucocytes from influenza virus antibody-negative mice were found to be spontaneously cytotoxic in vitro for influenza virus-infected target L929 cells, but not for uninfected control cells. Other studies have also described spontaneous cytotoxicity for target cells infected with various viruses in the absence of antiviral antibody, including influenza (5), Sendai (6), mumps (7), and herpes simplex (9) viruses. It was also found that spontaneous cytotoxicity was enhanced by preincubating effector cells with splenic leucocytes from older, influenza-virus antibody-negative mice, and although preincubating effector cells with serum markedly reduced cytotoxic activity, the amount of suppression was significantly less with sera from older mice. Similar findings have not been reported previously for spontaneous cytotoxicity against virus-infected target cells. Since antibodies to influenza virus could not be detected in the sera of mice used in this study by a highly sensitive enzyme immunoassay (10, 13), it was unlikely that the spontaneous cytotoxicity for influenza-infected target cells was due to antibody-dependent cellular cytotoxicity or to antibody plus complement cytotox-
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icity. In addition, no spontaneous cytotoxicity was observed with either sera or conditioned medium in the absence of effector cells. Natural killer (NK) cells have been implicated in the spontaneous lysis of target cells infected with mumps (7), Sendai (6, 8), and possibly influenza (5) viruses. However, the peak activity of NK cells has been shown to occur in young mice ( 14) rather than older mice, and NK cytotoxicity has been found to be markedly stimulated by interferon (15). In this study, the age indices described in Table 2 in the absence of effector cells demonstrated that leucocytes from older mice exhibited levels of cytotoxic activity similar to those of leucocytes from younger mice. However, the long incubation time (11 hr) required for significant spontaneous cytotoxicity could be indicative of NK activity since influenza viruses are known to be effective inducers of interferon. Interferon assays were not undertaken during the effector-target incubation period. Fractionation of the effector cells into adherent and nonadherent subpopulations on a nylon wool column showed that the principal mediators of cytotoxicity were in the adherent fraction. This method of fractionation had been described previously by Gordon and Shore (12) who had shown that 30% of the nylon wooladherent cells were phagocytic monocytes (latex-positive), a threefold increase compared to unfractionated cells. They also found that 10% of the T lymphocytes in the unfractionated cells were adherent, and the surface Ig-positive B lymphocytes increased fivefold in the adherent fraction. However, the adherent subclass of T lymphocytes, Ly-123+ cells, have not been shown to be spontaneously cytotoxic for virus-infected cells, and B lymphocytes are noncytotoxic. It would seem likely, therefore, that mediators of cytotoxicity in the adherent fraction were monocytemacrophages. Not only have monocyte-macrophages been found to be spontaneously cytotoxic for herpes simplex virus-infected target cells in the absence of antibody (9), but the phagocytic activity of macrophages have been shown to be unaltered or even enhanced with age ( 16). The spontaneous cytotoxicity remaining in the nonadherent fraction, however, may be due to NK cell activity. The age indices shown in Table 2 indicate that splenic leucocytes from 3-, 6-, and 9-month-old mice exhibited similar cytotoxic activities against infected target cells. An increased spontaneous cytotoxicity was observed, however, when 3-monthold effector cells were preincubated with leucocytes from older antibody-negative mice. These results suggest that the enhanced cytotoxic activity was due to a cooperative effect between the effector cells and the leucocytes from older mice. Whether this effect was the result of cell-to-cell contact or soluble factors produced by either or both of the leucocyte populations generating increased cytotoxic activity was not determined. However, the effect was almost completely abrogated if the number of additional leucocytes from older animals was reduced from 50 to 10% of the effector cell number. Preincubation of effector cells with sera from 3-month-old and lo-month-old mice at a final concentration of 10% markedly reduced the amount of spontaneous cytotoxic activity for infected target cells. Nevertheless, despite this general suppression, the relative levels of cytotoxic activity were significantly higher when effector cells were preincubated with sera from 7- and lo-month-old mice than with sera from 3-month-old mice, as shown by age indices in Table 3 and by percentage spontaneous cytotoxicity (with adherent cells only) in Table 4. This effect was not observed, however, when the sera were diluted to a final concentration of 1% (Table 3). A number of possible explanations can be advanced for the
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increased spontaneous cytotoxicity in the presence of sera from older animals. Thus suppressor factors specific for monocyte macrophages (or the adherent cells in Table 4) might decrease in older animals; alternatively active effecters might be amplified by soluble factors in sera, which increase with age; or autoantibodies, which usually increase with age, might be directed against the animal’s own cellsurface antigens resulting in an antibody-mediated cytolysis. The results obtained by preincubating effector cells with splenic leucocytes and sera from older mice were in contrast to our other findings with influenza-immune cytotoxic T lymphocytes. The specific cytotoxicity by T lymphocytes was significantly depressed by preincubation of the lymphocytes with splenic leucocytes or sera from older influenza virus antibody-negative mice, or with conditioned medium from Con A-activated splenic leucocytes from older mice (Watanabe and Mackenzie, submitted for publication). Since an enhancement of spontaneous cytotoxicity can occur with age under certain conditions, albeit abnormal, these results may provide an additional avenue for investigation of the increased incidence of autoimmune phenomena in older animals, despite the apparently contradictory reports of the appearance of suppressor cells in old mice ( 17- 19). ACKNOWLEDGMENTS We thank P. Hodgkin and J. Flexman for their help in sections of this study. The work was supported by the National Health and Medical Research Council of Australia.
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