Mechanisms of Ageing and Development 112 (1999) 197 – 215 www.elsevier.com/locate/mechagedev Genetic differences in the age-associated decrease in inducibility of natural killer cells by interferon-a/b Artur Plett, Donna M. Murasko * Department of Microbiology and Immunology, MCP Hahnemann Uni6ersity School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA Received 8 June 1999; accepted 1 October 1999 Abstract Natural killer (NK) cells, which are important in viral infections and anti-tumor activity, show reduced cytotoxicity in aged mice. The mechanism(s) for this age-related decline in NK activity has not been clearly established. We assessed changes in NK cytotoxicity in splenocytes and peripheral blood mononuclear cells after interferon (IFN)-a/b stimulation in adult (6 months) and aged (22–26 months) C57Bl/6, Balb/c, and (Balb/c×C57Bl/6)F1 mice. Aged C57Bl/6 and Balb/c mice had a significantly reduced IFN-a/b-stimulated NK cytotoxicity compared to adult mice. In contrast, adult and aged F1 mice showed similar NK cytotoxicity after IFN-a/b induction. The decreased ability of NK cells of aged mice to respond to induction by IFN-a/b was not due to a requirement for an increased amount of IFN or for a longer period of treatment with IFN. Further, this decreased response did not appear to be the result of suppressive activity of adherent cells or T cells. While the percentage of NK cells (NK1.1 +) was similar in adult and aged mice, the (CD8 + NK1.1+ ) subset of NK cells was significantly increased in aged mice. Importantly, the percentage of CD8+ NK1.1+ cells was inversely related to the cytotoxicity observed after IFN-a/b treatment. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: NK cells; Aging; Interferon * Corresponding author. Tel.: +1-215-991-8357; fax: +1-215-848-2271. E-mail address: murasko@mcphu.edu (D.M. Murasko) 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 9 1 - 3 198 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 1. Introduction Natural killer (NK) cells, which are an important component of the innate immune system, play a critical role in defense against viral infections and cancer. They are large granular lymphocytes (LGL) that are cytotoxic without the need for prior activation, display no immunologic memory, and are not restricted by the major histocompatibility complex (MHC). In addition, cytokines, such as interleukin (IL)-2, IL-12, and interferons (IFN) type I (IFN-a/b) and II (IFN-g), can enhance NK cell activity (Reiter, 1993; Kutza and Murasko, 1994). Although initially defined for its antiviral activity (Dianzani and Antonelli, 1989), IFN-a/b is now known as a pleotrophic cytokine that has a major impact on the proliferation of a wide range of cells, including tumor cells (Fleischmann and Fleischmann, 1992). In addition, IFN-a/b can modify various aspects of the immune response, including increasing expression of MHC Class I antigens (Minato et al., 1980), modifying recirculation of lymphocytes (Korngold et al., 1983; Mann et al., 1989), and augmenting cytotoxic responses of both NK cells and CD8 + T cells (Minato et al., 1980). IFN-a/b has been used in anti-cancer therapy because of both its anti-proliferative and immunomodulatory activities. As an anti-cancer agent, IFN-a/b has been shown to protect from tumor development and metastatic dissemination in animals (Markovic and Murasko, 1990), as well as to be effective in some human cancers, including head and neck carcinoma and lymphomas (Tyring, 1992). Since the incidence of cancers increases with age, the effectiveness of IFN-a/b as an anti-cancer therapy in the elderly needs to be carefully evaluated. Using a murine melanoma model in which we have shown that IFN can prevent metastatic dissemination and mortality in 50% of young mice (Markovic and Murasko, 1990), we have obtained preliminary data demonstrating that IFN has no effect on tumor dissemination in aged mice. The effectiveness of IFN-a/b treatment in this model in young mice is dependent on enhancement of NK activity (Markovic and Murasko, 1991). Thus it is possible that the lack of anti-tumor activity of IFN-a/b in aged mice is due to the inability of IFN-a/b to augment NK activity in aged mice. Age-related alterations in immune function have been described in both human and in animals. Although decreased T cell proliferative responses to both mitogenic and antigenic stimulation have been the most consistent and profound changes that occur with age (Murasko and Goonewardene, 1990; Miller, 1996), decreased cytotoxic T cell function (Effros and Walford, 1983) and impaired antibody responses have also been reported. However, the effects of age on NK activity have been explored less extensively. Although initial reports in Balb/c mice suggested that basal NK activity decreased significantly with age (Kiessling et al., 1975), additional studies indicated that basal NK activity actually peaks at about 2–3 months of age, decreases by 6 months of age and then remains fairly constant from 6 months through death (Albright and Albright, 1983; Provinciali et al., 1989). NK activity can be enhanced after treatment with either IL-2 or IFN-a/b in 6 month, but not 24 month, old Balb/c mice (Provinciali et al., 1989). These limited data suggest that aging has a preferential, deleterious effect on inducible, but not basal, NK cell activity in the murine model. A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 199 Since the melanoma model utilized by our laboratory was originally established in C57Bl/6 (B6) mice, the current study addressed whether IFN-a/b can augment NK activity in adult and aged B6 and (Balb/cByJNia × C57Bl/6JNia)F1 (CB6F1) mice. The age-related requirements for enhancement of NK by IFN-a/b in both amount and length of treatment were evaluated, as well as the possible suppressive roles of T cells or adherent cells. Finally, the possibility that there is an age-associated decrease in the total number of NK (NK1.1+ ) cells, was explored. 2. Materials and methods 2.1. Animals Specific pathogen-free C57Bl/6JNia (B6), Balb/cNia (Balb/c) and (Balb/cByJNia× C57Bl/6JNia)F1 (CB6F1) mice were obtained from the NIA colony maintained by Charles River (Kingston, NY) at 2 months (young), 6–8 months (adult) and 22 – 26 months (aged) of age. All mice were maintained in AAALAC certified barrier facilities at MCP Hahnemann University. All mice were given food and water ad libitum. Experiments were conducted after mice had acclimated to our facilities for at least 2 weeks. Mice that demonstrated enlarged spleens or tumors were eliminated from the study. 2.2. Cell isolation and preparation Peripheral blood was obtained from the retro-orbital sinus and collected into heparanized blood collection tubes (Fisher Scientific, Pittsburgh, PA). Spleens were removed, placed in RPMI-1640 (Biowhittaker, Walkersville, MD), and homogenized to obtain single cell suspensions. Mononuclear cells were isolated as described previously (Kutza and Murasko, 1994). Briefly, spleen cell homogenates or diluted whole blood was layered onto ficoll hypaque-1083 (Sigma, St. Louis, MO) to isolate mononuclear cells by density gradient centrifugation (2500 rpm for 20 min at RT). Cells were washed twice in RPMI-1640 (1000) rpm 10 min at RT) and then resuspended at 1×106 cells/ml in complete media containing RPMI-1640, 10% fetal bovine serum (FBS) (BioWhittaker, Walkersville, MD) and 1% glutamine (BioWhittaker). 2.3. NK cytotoxicity assay A standard 4 h51Cr-release assay with YAC-1 cells as targets to assess NK cell activity was utilized (Albright and Albright, 1983). Briefly, 1 × 106 YAC-1 cells were incubated with 200 mCi Na 51CrO4 (ICN, Costa Mesa, CA) for 2 h at 37°C. During this incubation, cells were mixed every 20 min by gentle tapping to ensure maximal uptake of Na51 CrO4. The cells were then washed twice with RPMI-1640, resuspended in complete media, and then rotated for 1 h at RT. After the final wash, YAC-1 cells were resuspended at 1× 104 cells/ml in complete media and 200 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 plated in round bottom 96 well microtiter plates (ICN, Costa Mesa, CA). Effector cell preparations from spleen or blood were then added to wells at effector to target (E:T) ratios of 100, 50, or 25:1. All samples were assayed in triplicate. Target cells were incubated with either media alone or 5% Triton X-100 to quantitate spontaneous and maximum release, respectively. After a 4 h incubation at 37°C, supernatants were harvested using the Skatron harvesting system (Skatron, Sterling, VA) and radioactivity in supernatants was quantitated using a gamma counter (Packard, Sterling, VA). Percent cytotoxicity was calculated as follows: % Cytotoxicity = [CPM(sample)− CPM(spontaneous release)] [CPM(maximum release)− CPM(spontaneous release)] ×100 Spontaneous release was always less than 10% of maximal release. Maximal release was always between 1200 and 6000 cpm. 2.4. Interferon treatment 2.4.1. In 6itro Effector cells (2× 106cells/ml) were incubated with varying doses of IFN-a/b (ranging from 1× 103 – 1 × 105 units per 106 cells) for 1–8 h at 37°C in 5% CO2. Preliminary experiments (see Section 3) established that maximal cytotoxicity occurred after a 4 h incubation with IFN-a/b (Western Biomedical and Diagnostics, San Diego, CA); therefore, this exposure time was used in all subsequent experiments. After IFN treatment, microtiter plates were centrifuged to pellet the cells and the media containing IFN-a/b was discarded. Cells were then plated at the appropriate E:T ratios and NK cytotoxicity was assayed as described above. 2.4.2. In 6i6o IFN-a/b (ranging from 2×103 –2× 105 units/ml) was diluted in pyrogen-free saline. Mice were given a 0.5 ml intraperitoneal inoculation. Spleens were harvested 24 h later and NK cytotoxicity was evaluated as described above. Results are presented as percent cytotoxicity or as fold induction. Fold induction is calculated according to the following formula: (% IFN-a/b induced cytotoxicity } % basal cytotoxicity), where a fold induction of one equals no induction over basal. 2.5. Assessment of surface phenotype Splenocytes were washed with phosphate buffered saline (PBS) and resuspended in staining buffer (1% FBS, 0.1% sodium azide-PBS) at 5× 106 cells/ml. Cells were then incubated with mouse-anti-NK1.1 antibody conjugated to phycoerythrin (PE), and rat-anti-CD8 antibody conjugated to allophycocyanin (APC) (Pharmingen, San Diego, CA) for 30 min at 4°C in the dark. Cells were then washed with staining buffer and fixed with 1% paraformaldehyde. 10 000–30 000 lymphocyte gated events were acquired for each sample on a FACScan or FACSCalibur flow A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 201 cytometer using LYSYS II software and analyzed using CellQuest software (Becton Dickinson, Mountain View, CA). 2.6. Depletion of cells 2.6.1. Separation of adherent and non-adherent cells Adherent cells were isolated using methods that have been described previously (Brunda et al., 1983). Briefly, pooled mononuclear splenocytes from adult (6–8 months) or aged (22 – 26 month) mice were incubated for 2 h at 37°C in 5% CO2 in 96 well flat bottom microtiter plates (Falcon, Lincoln Park, NJ). Non-adherent cells were then carefully removed to minimize dislodging of the adherent cells. Both non-adherent and adherent cells were washed three times with complete media. Non-adherent splenocytes from adult mice were added to adherent cells from either adult or aged mice or were incubated alone as non-adherent controls. These same combinations were used for non-adherent splenocytes of aged mice. All treatment groups were then stimulated with IFN-a/b as described above. After a 4 h incubation with IFN-a/b, non-adherent cells were transferred to round bottom 96 well microtiter plates and mixed with 51Cr-labeled YAC-1 targets to assess cytotoxic activity. 2.6.2. T cell depletions T cells were depleted by negative selection using immunomagnetic beads from DYNAL Laboratories (Lake Success, NY), with minor modifications of the manufacturer’s instructions. Briefly, 2×107 cells were incubated with purified rat anti-mouse CD4 antibody, rat-anti-mouse CD8 antibody (Pharmingen, San Diego, CA), or both for 30 min on ice. The cells were washed with RPMI-1640 and resuspended in 1% cold bovine serum albumin (BSA) in RPMI-1640 containing 4 × 107 DYNAL magnetic beads coated with goat-anti-rat-IgG antibodies. The tubes were resuspended by gentle tapping every minute for 30 min, after which they were placed onto a magnet and the supernatant aspirated. To ensure that NK cell numbers were constant despite the enrichment of NK cells in T cell depleted samples, control and depleted samples were resuspended to the same volume. Cells were then plated in 96 well round-bottom microtiter plates and assayed for NK activity as described above. These samples were also analyzed by flow cytometry to determine the effectiveness of depletion. Staining showed that \ 95% of CD8 and \85% of CD4 cells were removed by this depletion protocol. 2.7. Statistics Differences between basal and IFN-a/b-induced cytotoxicity in young, adult and aged animals were evaluated using a Students’ t-test. The significance of differences in the kinetics and dose response experiments was determined using ANOVA followed by a Bonferroni multiple-comparison post-hoc test. Statistics were done using the SPSS statistics package (SPSS, Chicago, IL). 202 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 3. Results 3.1. Age-associated changes in induction of NK acti6ity in B6 mice: effects of IFN-h/i concentration Previous reports indicated that while maximal NK activity is induced after a 4 h stimulation with IFN-a/b in vitro in both young and aged Balb/c mice, the level of activity in aged mice was lower than that of young mice (Provinciali et al., 1989). We questioned whether B6 mice also demonstrated an age-associated change in IFN-induced NK activity similar to that observed in Balb/c mice. To examine this question, IFN-induced NK activity was measured in 2- (young), 6- (adult), and 24-month old (aged) B6 mice. As shown in Fig. 1, IFN-a/b-induced NK cytotoxicity was significantly higher than basal NK activity (PB 0.001) in B6 mice of all three ages. However, several points concerning this induction became apparent: (1) Both young and adult mice showed a 3- to 3.5-fold increase in NK activity, whereas aged mice showed only a 1.5- to 2-fold increase. (2) The level of NK activity after stimulation with all doses of IFN-a/b was significantly higher in both 2- and 6-month old mice compared to 24-month old mice. (3) Basal NK activity of 2-month old mice was significantly higher than basal NK activity of both 6- and 24-month old B6 mice; basal NK activities of 6- and 24-month old mice were comparable. Since 2-month old mice had higher basal NK activity, which could Fig. 1. NK activity in young, adult and aged B6 mice after in vitro IFN-a/b treatment. Splenocytes from three animals in each age group were stimulated with IFN-a/b for 4 h at the doses indicated and assayed separately for in vitro cytotoxicity. The figure shows representative data from one of three experiments at E:T = 100:1. Bars reflect mean + SD. IFN-a/b-induced activity was significantly increased over basal 6 activity with all IFN concentrations (P B0.001; c). IFN-a/b-induced cytotoxicity of both the 2 and 6 month groups were significantly higher (P B0.05; *) than the 24 month group at every dose of IFN-a/b tested. Basal cytotoxicity of 2 month animals was significantly higher (P = 0.002; $) than basal cytotoxicity of both 6 and 24 month old mice. A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 203 Fig. 2. NK activity in adult and aged B6 mice after in vivo stimulation with IFN-a/b and Poly I:C. Adult (6 month) and aged (24 month) B6 mice (3 – 4 per age group) were injected with 0.5 ml saline containing 100 mg of Poly I:C or the doses of IFN-a/b indicated. In vitro NK activity was assayed 24 h later. Bars represent mean percent cytotoxicity 9SEM (E:T= 100:1). NK cytotoxicity of adult mice was significantly enhanced relative to basal levels (PB 0.02; *) and to cytotoxicity of aged mice (P B0.01; c ), after treatment with Poly I:C or 105 Units of IFN-a/b. Cytotoxicity in aged animals was not significantly increased after treatment with Poly I:C or any of the IFN-a/b doses. Basal (saline treated) cytotoxicity was not significantly different between adult and aged animals. account for differences in the absolute level of NK activity after treatment with IFN, all subsequent comparisons were made between 6–8 month (adult) and 22– 26 month (aged) animals. Collectively, these data suggest that the defect in induction of NK activity in aged B6 mice cannot be overcome by treatment with higher doses of IFN. To determine whether the effects on NK activity after treatment with IFN-a/b in vitro reflect the in vivo circumstance, mice were given intraperitoneal inoculations of either IFN-a/b, (103 – 105 U) or 100 mg Poly I:C. Splenocytes were harvested and NK cytotoxic activity was assessed 24 h after treatment. As seen in Fig. 2, although treatment with 105 units of IFN-a/b increased NK activity significantly in 6-month old mice, this amount of IFN-a/b did not alter NK activity in 24-month old B6 mice. Further, treatment with Poly I:C, which induces the production of endogenous IFN-a/b, significantly increased NK activity of adult, but not aged, B6 mice. The inability of IFN-a/b or Poly I:C to stimulate NK activity suggests that aged mice do not respond to IFN given exogenously or produced endogenously. These results confirm previous studies in Balb/c mice (Provinciali et al., 1989) showing that NK activity in aged mice cannot be augmented significantly by treatment with exogenous IFN-a/b. 204 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 3.2. Aged-associated changes in induction of NK acti6ity by IFN-h/i in B6 mice: kinetics The above data clearly demonstrated that the augmentation of NK activity by IFN-a/b was diminished in aged B6 mice and could not be restored by higher concentrations of IFN-a/b. However, it was still possible that this decrease in inducible NK activity of aged mice could either be enhanced or restored after a longer exposure to IFN-a/b in vitro. To test this possibility, splenocytes from adult (6 months) and aged (24 months) B6 mice were incubated with 103 units of IFN-a/b per 106 cells for 1, 2, 4, or 8 h. As seen in Fig. 3, NK activity of adult mice increased significantly after 1 h of treatment with IFN-a/b, and after an 8-h exposure to IFN-a/b was enhanced five-fold. In contrast, NK activity of aged mice did not increase significantly over basal activity until 4 h of treatment with IFN-a/b. However, even at its peak, inducible NK activity of aged mice was still only 2.5-fold higher than basal NK activity. Fig. 3 also shows that NK activity was significantly higher in adult compared to aged mice after IFN-a/b treatment at all time points, although basal NK activity did not differ significantly between adult and aged animals (3 and 4%, respectively). Collectively, these two sets of experiments clearly indicate that the limited induction of NK cytotoxicity by IFN-a/b in aged B6 mice, relative to the induction achieved in adult B6 mice, cannot be attributed to differences in the dose of, or time of exposure to, IFN-a/b. Fig. 3 suggests that inducible NK activity did not plateau in either adult or aged mice even after treatment with IFN-a/b for 8 h. Despite this, longer time periods of Fig. 3. In vitro kinetics of IFN-a/b stimulated NK activity in adult and aged B6 mice. Splenocytes (three to four mice per age group) were stimulated with IFN-a/b at 103U/106 cells for the time indicated and assayed individually. Results show the mean fold induction 9 SEM relative to basal cytotoxicity. Level of induction in adult (6 month) animals is significantly higher than induction in aged (24 month) mice (P B 0.01; *) at every time-point. Significant (PB 0.05; c ) increase in activity relative to basal activity was apparent by 1 h of stimulation in adult mice, but not until 4 h in aged mice. Data represent one of two experiments. A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 205 Fig. 4. NK activity of adult and aged CB6F1 and Balb/c mice. Splenocytes from 5 – 6 animals of each age and each strain were assayed separately after 4 h stimulation with IFN-a/b (103U/106 cells). Bars indicate mean cytotoxicity (E:T = 100:1)9SEM. IFN-a/b-induced cytotoxicity was significantly increased relative to basal cytotoxicity for CB6F1 and Balb/c mice, in both adult (PB 0.001; $) and aged (P B 0.04; c ) groups. In Balb/c mice, IFN-a/b-induced cytotoxicity was significantly lower in aged compared to adult mice (PB 0.02; *). Levels of cytotoxicity after IFN-a/b treatment were comparable in adult and aged CB6F1 mice. Basal cytotoxicity was not significantly different between adult and aged animals of either strain. IFN-a/b treatment were not explored for several reasons. First, a preliminary experiment indicated that NK activity did not increase significantly when splenocytes from young or aged mice were treated with IFN-a/b from 8–12 h (data not shown). Second, NK activity increased, but not significantly, from 4–8 h in both young and aged mice, suggesting a plateau in augmentation. Most importantly, lymphokine activated killer (LAK) cell activity is also induced after treatment with IFN-a/b for 12 h. Thus, at later time points, the activities of NK and LAK cells overlap, making it difficult to clearly delineate the responding cell. In all subsequent studies, splenocytes were treated with 103 units of IFN-a/b per 106 cells for 4 h because these conditions resulted in a significant level of difference between young and aged animals. In addition, our results could be easily compared with previous reports in Balb/c mice that measured NK activity at the time point. 3.3. Age-associated changes in NK acti6ity after IFN-h/i stimulation: genetics The above results clearly demonstrate that NK activity is lower after treatment with IFN-a/b in aged compared to adult B6 mice. Since we have shown that the anti-metastatic effects of IFN-a/b treatment are maximal in our melanoma model in young CB6F1 mice (unpublished data), we next questioned whether CB6F1 mice would demonstrate age-related changes in inducible NK activity similar to that seen in B6 mice. Thus, we compared induction of NK activity by IFN-a/b in adult and aged CB6F1 mice. Fig. 4 shows that the induction of NK cytotoxicity by IFN-a/b in aged CB6F1 mice was comparable to that achieved in adult mice. 206 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 The differential effects of age on IFN-induced NK activity in B6 compared to CB6F1 mice were surprising. Previous studies had shown that the other parental strain of the CB6F1 mice, the Balb/c strain, also demonstrates age-associated decreases in NK activity after treatment with IFN-a/b (Provinciali et al., 1989). Although it was possible that different age-associated defects in induction of NK activity were operative in B6 and Balb/c mice and the results of the CB6F1 mice represent genetic complementation, it was imperative to determine whether the Balb/c parent of the CB6F1 mice used in the present studies exhibited the age-associated defect in inducible NK activity that had been reported previously. Fig. 4 shows that although IFN-a/b enhanced NK activity in both adult and aged Balb/c mice, the level of induction was significantly lower in aged compared to adult Balb/c mice. These data strongly suggest that the similar augmentation of NK cell activity by IFN-a/b in aged and adult CB6F1 mice cannot be directly attributed to a contribution of either parental strain, since NK cells from aged animals in both Balb/c and B6 strains show a reduced induction by IFN-a/b. 3.4. Age-associated changes in IFN-induced NK acti6ity: organ distribution It has been reported that IFN-a/b-induced NK activity does not decrease with age in humans (Kutza et al., 1995), which is in direct contrast to our findings with aged B6 and Balb/c mice. These differences could reflect a species difference in the pattern of decline in IFN-a/b-inducible NK activity. Alternatively, the differences could be organ specific since NK activity was assessed in peripheral blood mononuclear cells (PBMC) in humans, but in splenocytes in our studies. To address this possibility, IFN-a/b-induced NK activity of PBMC from adult and aged B6 and CB6F1 mice was assessed. As shown in Fig. 5, PBMC from both adult and aged CB6F1 mice, and from adult B6 mice, demonstrated enhanced NK activity after IFN-a/b treatment, reflecting about a 2.5-fold increase in all three groups. However, NK activity only increased 1.5-fold relative to basal activity after IFN-a/b treatment in aged B6 mice. This induced level of NK activity in PBMC of aged B6 mice was significantly lower than adult B6 mice (P= 0.03). These data clearly indicate that the same pattern of inducibility by IFN-a/b was observed in PBMC and splenocytes from both B6 and CB6F1 mice. These data suggest that differences in the ability of IFN-a/b to stimulate NK activity in mice and humans may reflect not only variation between species, but also variation among strains within the same species. 3.5. Age-associated changes in induction of NK cell acti6ity by IFN-h/i: mechanism 3.5.1. Percentage of NK (NK1.1+) cells It is possible that a decreased percentage of NK cells may account for reduced inducibility of NK activity by IFN-a/b in aged B6 mice. To address this possibility, splenocytes from adult and aged B6 mice were stained with the NK-specific monoclonal antibody, NK1.1. Representative staining patterns for adult and aged A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 207 B6 mice are shown in Fig. 6. The pattern of staining (Fig. 6, panels A and B) and the percentage of NK1.1 cells (Table 1) in spleen and PBMC were similar in adult and aged B6 mice. In addition, there was no correlation between the percentage of NK1.1 + cells and the level of cytotoxicity after IFN-a/b treatment (R= 0.09, r 2 =0.04, P =0.14) for 24 adult and 24 aged mice. The similar percentages of NK cells in adult and aged animals suggest that the difference in induction of NK activity by IFN-a/b is not simply due to a reduced percentage of NK1.1 cells in the mononuclear cell population of aged mice. 3.5.2. Percentage of NK1.1+ CD8+ cells Although the percentage of NK1.1+ cells was not significantly different between adult and aged B6 mice, the percentage NK1.1+ CD8+ cells within the population of NK1.1 + cells was significantly increased in aged compared to adult B6 mice (Fig. 6, panels C and D). Table 1 shows that the percentage of NK1.1+ CD8+ cells was two-fold higher in aged B6 mice. Importantly, the percentage of NK1.1 +CD8 + cells was negatively correlated with cytotoxicity after IFN-a/b treatment (R = − 0.86, r 2 =0.75, P= 0.02; n= 6 adult and 6 aged mice). The correlation is even observed when results from multiple experiments are considered (R= − 0.48, r 2 = − 0.21, P =0.005,n = 29 adult and 29 aged), which suggests a robust correlation considering the high interassay variability of the 51Cr release assay. These results suggest that the increased percentage of NK cells expressing the CD8 marker may be at least partially responsible for the reduced induction of NK activity by IFN-a/b in aged B6 mice. Fig. 5. NK activity in PBMC of adult and aged CB6F1 and B6 mice after IFN-a/b treatment. PBMCs from six to eight animals of each age group in each strain were assayed separately for cytotoxicity after a 4 h stimulation with IFN-a/b (103U/106 cells). Bars show the mean fold induction 9 SEM relative to basal levels of cytotoxicity. While the fold increase in CB6F1 mice is not significantly different between adult and aged, fold induction in PBMC of aged B6 animals is significantly lower compared to adult mice (PB 0.03; *). 208 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 Fig. 6. Phenotypic profile of B6 splenocytes. Splenocytes were stained with a PE-conjugated anti-NK1.1 antibody and/or APC-conjugated anti-CD8 antibody. 10 000 – 30 000 lymphocyte-gated events were acquired. Panels A and B show a representative staining profile for NK1.1 alone in adult and aged mice, respectively. Panels C and D show a representative profile for NK1.1 and CD8 two-color staining in adult and aged mice respectively. Quadrants were set based on isotype controls. 3.5.3. Suppression of NK cell cytotoxicity by adherent cells Several reports in mice have indicated that splenic and peritoneal adherent cells suppress both basal and IFN-induced NK activity (Brunda et al., 1983; Irimajiri et al., 1985; Riccardi et al., 1986). To determine if adherent cells were responsible for the limited effect of IFN-a/b on NK activity in aged mice, adherent and non-adherent splenocytes from adult and aged B6 mice were separated prior to IFN-a/b treatment. Cells were then combined as follows: non-adherent cells from adult mice were added to adherent cells from adult or aged mice, and similarly, non-adherent cells from aged mice were added to adherent cells from aged or adult mice. Non-adherent cells alone or non-adherent cells mixed with adherent cells were stimulated with 103 units of IFN-a/b for 4 h. As shown in Fig. 7, IFN-a/b-induced cytotoxicity in adult animals showed higher cytotoxicity in the absence of adherent A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 209 Table 1 Percentages of NK1.1 cells in spleen and peripheral blood of adult and aged B6 micea,b Spleenc Peripheral bloodd %NK1.1e Adult Aged %NK1.1+CD8+f %NK1.1 %NK1.1+CD8+ 4.229 0.35 4.589 0.56 12.39 9 1.62 29.349 3.21g 5.65 90.27 6.61 91.12 ND ND a Mononuclear cells of spleen or peripheral blood (5×105)were stained with NK1.1-PE and CD8APC, followed by acquisition of 10 000 to 30 000 lymhocyt-gated events. b There was no significant different in percent of NK1.1+ cells between adult and aged mice in either spleen or peripheral blood (P=0.2) c Represents the mean of 33 adult and 33 aged B6 mice. d Represents the mean of 5 adult and 5 aged mice. e Percent of total lymphocytes. f Percent of total NK1.1+ cells. g Percent of NK1.1+CD8+ cells was significantly elevated in aged compard to adult mice (PB 0.0001). cells, although this increase was not significant. In contrast, removal of non-adherent cells had no effect on inducible NK activity in aged mice. Further, IFN-a/b induction of NK cytotoxicity in non-adherent cells of adult and aged mice was comparable in the presence of either aged or adult adherent cells. This suggests that whatever suppressive effect adherent cells may have, is not different between adherent cells from adult or aged animals. Fig. 7. Effect of adherent cells on IFN-a/b-induced NK activity in adult and aged B6 mice. Pooled splenocytes from two mice of the same age were assessed for NK activity with and without adherent cells. Solid bars indicate mean IFN induced cytotoxicity 9 SD of triplicate samples. Basal cytotoxicity of unseparated splenocytes of adult and aged groups (hatched bars), were not significantly different. IFN-a/b-induced cytotoxicity of adult mice was significantly higher in all three treatment groups compared to aged mice treated comparably (PB 0.01; *). Figure shows a representative experiment in B6 mice, two additional experiments were done in both B6 and Balb/c mice, with similar results. 210 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 Fig. 8. Effect of T cell depletion on IFN-a/b-induced NK activity in adult and aged B6 mice. Spleen cells were treated with magnetic beads alone (CTRL), or specifically depleted of T cell subsets. ‘ − 4’ indicates depletion of CD4 + cells, ‘ −8’ indicates depletion of CD8 + cells, and ‘ −4/8’ indicates depletion of both CD4 + and CD8 + cells. All groups were stimulated with IFN-a/b (103U/106 cells) for 4 h. Bars show the mean percent cytotoxicity9 SEM of two identical experiments done with pools of three adult and three aged B6 mice in each age group. Depletion does not induce any statistically significant changes in basal or IFN-induced cytotoxicity in adult or aged groups. 3.5.4. Suppression of NK cell cytotoxicity by T cells T cells have been shown to modify NK cell responses in vivo and in vitro after IFN-a/b stimulation in young mice (Markovic and Murasko, 1991, 1993). To determine if T cells influence the age-associated decrease in IFN-a/b-induced NK activity, CD4 and CD8 cells were depleted separately and together before or after IFN-a/b stimulation of splenocytes. As seen in Fig. 8, depletion of CD4 and/or CD8 cells before IFN-a/b stimulation did not affect basal or IFN-a/b-induced cytotoxicity in aged animals. Although in adult animals there is a small trend for increased basal and IFN-a/b-stimulated cytotoxicity after CD8 cell depletion, the increase is not statistically significant. Depleting T cells after IFN-a/b stimulation had no effect on either basal or induced NK activity in adult or aged mice (data not shown). These data indicate that neither CD8 nor CD4 cells directly suppress the in vitro induction of NK cells by IFN-a/b in aged mice. 4. Discussion Since there is an increase in the incidence of cancer in the elderly, the validation of the effectiveness of various cancer therapies must be extended to this population. One treatment modality has used IFN-a/b. This cytokine has been successful as an immunotherapeutic agent for some human cancers (Tyring, 1992), as well as in animal models (Markovic and Murasko, 1991). It is clear that at least some of this anti-tumor activity of IFN-a/b dependent on enhancement of NK cytotoxicity. Based on these observations, the ability of IFN-a/b to enhance NK cytotoxicity in aged and adult mice was evaluated in this study. A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 211 The results presented have not only confirmed previous data, but also have extended the information currently available on age-associated changes in IFN-a/binducible NK activity in mice. Our data confirmed previous results from Balb/c mice indicating that the decrease in basal NK activity is not an aging phenomenon per se, but rather a phenomenon of maturation (Provinciali et al., 1989). The most dramatic decrease in basal NK activity occurs between 2–6 months of age, with the level of basal NK activity remaining fairly constant from 6 months through the rest of the lifespan. Therefore, any useful assessment of age-associated modification in NK activity in mice must compare animals at least 6 months old to mice at or about their median lifespan. One study reported that enhancement of NK cytotoxicity after IFN-a/b treatment showed a gradual decrease with increasing age, with 6 month old Balb/c mice maintaining a greater IFN-a/b-inducibility of NK activity compared to 24-month old mice (Provinciali et al., 1989). The current results extend the previous findings to B6 mice, indicating that while basal NK activity is stable from 6 through 28 months of age, there is an age-associated decrease in the ability of NK cells of aged mice to be augmented by IFN-a/b. Since both B6 and Balb/c mice demonstrate an age-associated decrease in inducibility of NK cells after stimulation with IFN-a/b, it was assumed that this decrease would also be seen in their F1 progeny. In contrast to our prediction, CB6F1 mice showed no age-associated decrease in induction of NK cytotoxicity by IFN-a/b. Although this may be a random genetic difference, it is possible that this represents a complementation in different defects in the Balb/c and B6 parents. Since the CB6F1 mouse does not show the decline, it may hold genetic clues to the maintenance of an augmentable NK response in aged animals. This would not only help elucidate NK cell function in aging mice, but could also be useful in investigations of IFN therapy in elderly humans. Although most elderly do not show a decline in induction of NK activity by cytokines with increasing age, it has been reported that there is a subset of individuals who demonstrate a decreased response of NK cells after IFN induction (Mysliwska et al., 1992). Since the human population is very heterogeneous, it is possible that most individuals represent the complementation of the appropriate genes to maintain NK activity. The subset of individuals who show decreased inducibility may reflect homozygosity at the genes that are responsible for the decreased response to IFN of inbred mouse strains. It is important to note, however, that our results demonstrate that the differences seen between mice and humans is not due to the source of cells that are being utilized, since NK activity of PBMC reflects the NK activity of splenocytes. While one previous report showed that basal NK activity of PBMC did not decrease with age in CBA/J and C3H/HeN mice (Lanza and Djeu, 1982), it did not assess inducibility of NK activity in PBMC. Our results demonstrate that B6 mice show an age-related decline in inducibility of NK activity in PBMC that is comparable to the decrease seen in splenocytes, while the CB6F1 mouse shows comparable augmentation of NK activity by IFN-a/b in both peripheral blood and spleen throughout the ages examined. The mechanism of decreased inducibility of NK activity with increasing age was explored. The microenvironment of the NK cell could influence the level of 212 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 cytotoxicity achieved in aged mice. A suppressive interaction with monocytes is one possibility, since suppression of basal NK activity by adherent cells has been reported in aged humans and mice (Brunda et al., 1983; Irimajiri et al., 1985; Mysliwska et al., 1992; Riccardi et al., 1986). In addition, the ability of splenic adherent cells to suppress basal NK cytotoxicity was observed to be more pronounced in aged (24 months) compared to young (2 months) C3H/HeN mice (Riccardi et al., 1986). Our results, however, show no effect of adherent cells on IFN-a/b-induced NK activity in either adult or aged mice, suggesting that after the maturational change in NK has happened, the adherent cells from adult or aged mice do not have different suppressive abilities on NK cytotoxicity. Although there is limited information on the ability of T cells to suppress NK cytotoxicity in aged mice, there are several studies suggesting that T cells can regulate NK activity. One study reported that CD8 cells of some humans suppress basal NK activity in vitro (Garcia-Penarrubia et al., 1989). In addition, several ˜ reports have indicated that anesthesia inhibits IFN-a/b induction of NK cytotoxicity in mice (Markovic et al., 1993) and humans (Kutza et al., 1997). In the mouse model, depletion of CD8 cells after anesthesia, but prior to in vitro stimulation with IFN-a/b, was able to restore anesthesia-mediated inhibition of NK cell activity (Markovic and Murasko, 1993). Our mouse model of immunotherapy using B16 melanoma cells also demonstrates regulation by T cells. The effectiveness of IFN-a/b in this model is dependent on IFN-a/b augmentation of NK activity, since depletion of NK cells completely abrogates any protection. However, depletion of only CD4 cells also abrogates protection, while depletion of both CD4 and CD8 cells results in protection comparable to that seen in intact animals. One interpretation of these results is that in this melanoma model CD8 cells inhibit IFN induction of NK activity, while CD4 cells counterbalance this CD8 suppression (Markovic and Murasko, 1991). In contrast, our current results suggest that the age-associated decrease in IFN-a/b-inducibility of NK cells in vitro is not affected by the presence of T cells. Innate changes to NK cells could also be responsible for the decrease response of NK to IFN-a/b. Our data clearly demonstrate that the deficiency is not related to a requirement of the NK cells of aged mice for more IFN-a/b or for an extended incubation with IFN-a/b to allow the activation to occur. The possibility that NK cells of aged mice demonstrate a decreased number or affinity of receptors for IFN-a/b is currently being explored. Another simple explanation could be that aged mice have a reduced number of NK cells compared to adult mice. While one report showed that the percentage of splenic NK cells (determined by reactivity with anti-asialo GM1 antibody) was reduced in young (2-month old) compared to aged (10–13 month old) DBA mice (Dussault and Miller, 1994), another study using a more specific indicator of NK cells (reactivity with anti-NK1.1 antibody) found that the percentage of splenic NK cells was not different between young (3–4 month) and aged (22–26 month) B6 mice (Mikael et al., 1994). Evaluation of the percentage of NK1.1+ cells in our studies also indicated that a decrease in NK cell number was not responsible for the age-associated decrease in NK activity. However, upon further evaluation, we A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 213 found that aged mice demonstrate an increase in the percent of NK cells that express the T cell marker, CD8. Aged mice had a two-fold higher percentage of CD8 +NK1.1 + cells than adult mice, representing about a third of all NK cells in aged mice. Importantly, the level of cytotoxicity observed after induction with IFN-a/b was inversely related to the percentage of NK1.1 + CD8+ cells. Presently, the functions and activity of CD8+ NK1.1+ cells have not been extensively explored. They are thought to be LAK cell precursors in mice (Lee et al., 1996). In humans, an equivalent population that is CD8+ CD57+ is increased in disease conditions such as rheumatoid arthritis and are proposed to have a role in autoimmunity (Imberti et al., 1997). Interestingly, the CD8+ CD57 + cell population increases in elderly people (Miyaji et al., 1997), indicating some similarity in the generation of these cells in mice and humans with aging. Since removal of CD8+ cells, which would include the CD8+ NK cells, did not affect cytotoxicity in the depletion studies (Fig. 8), it is possible that CD8 + NK1.1 + cells may not be cytotoxically active in the short-term NK assay. Techniques are being developed to isolate this small percentage of splenic lymphocytes to address this question more directly. It will be interesting to see whether NK cytotoxic activity is limited to the NK1.1+ CD8− population, and whether IFN-a/b will have differential effects on the CD8+ and CD8− NK subpopulations. There are other mechanisms in the activation pathway of NK cell activity that must be evaluated for their contribution in the age-associated decrease of IFN-a/ b-induced NK cytotoxicity. Reduced cytotoxicity by NK cells of aged mice could reflect defective binding of IFN-a/b or impaired signal transduction after interaction with the cytokine. Alternatively, NK cells of aged mice might exhibit a reduced binding to the target cells; this has been reported aged DBA/2 mice (Dussault and Miller, 1994). Additionally, decreased production and/or release of cytotoxic effectors such as granzymes, perforins, and Fas ligand could result in decreased cytotoxicity. Further evaluation of these mechanisms in adult and aged mice in relation to IFN-a/b-induced NK cytotoxicity will provide important clues to the age-related decrease in NK activity in B6 mice. The mechanisms in B6 animals could then be evaluated in CB6F1 mice to identify possible genetic differences linked to the defect and possible application in treatment of elderly humans. Acknowledgements This manuscript was supported by NIH Grant AG13542. We would like to thank Dr Elizabeth Gardner for her assistant with both the interpretation of the flow cytometry data and with editorial revisions of this manuscript, and Ms Christine Kinsinger for assistance in preparation of this manuscript. 214 A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 References Albright, J.W., Albright, J.F., 1983. Age-associated impairment of murine natural killer activity. Proc. Natl. Acad. Sci. 80, 6371–6375. Brunda, M.J., Taramelli, D., Holden, H.T., Varesio, L., 1983. Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice. J. Immunol. 130, 1974– 1979. Dianzani, F., Antonelli, G., 1989. Physiologic mechanisms of production and action of interferon in response to viral infections. Adv. Exp. Med. Biol. 257, 47 – 59. Dussault, I., Miller, S.C., 1994. Decline in natural killer cell-mediated immunosurveillance in aging mice—a consequence of reduced cell production and tumor binding capacity. Mech. Ageing Dev. 75, 115–129. Effros, R.B., Walford, R.L., 1983. Diminished T-cell response to influenza virus in aged mice. Immunology 49, 387–391. Fleischmann, W.R., Fleischmann, C.M., 1992. Mechanisms of interferons antitumor actions. In: Baron, S., et al. (Eds.), Interferon: Principles and Medical Applications. University Texas Medical Branch at Galveston, Galveston, TX, pp. 299 – 310. Garcia-Penarrubia, P., Bankhurst, A.D., Koster, F.T., 1989. Prostaglandins from human T suppressor/ ˜ cytotoxic cells modulate natural killer antibacterial activity. J. Exp. Med. 170, 601 – 606. Imberti, L., Sottini, A., Signorini, S., Gorla, R., Primi, D., 1997. Oligoclonal CD4 + CD57 + T-cell expansions contribute to the imbalanced T-cell receptor repertoire of rheumatoid arthritis patients. Blood 89, 2822–2832. Irimajiri, N., Bloom, E.T., Makinodan, T., 1985. Suppression of murine natural killer cell activity by adherent cells from aging mice. Mech. Ageing Dev. 31, 155 – 162. Kiessling, R., Klein, E., Pross, H., Wigzell, H., 1975. Natural killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell. Eur. J. Immunol. 5, 117–121. Korngold, R., Blank, K.J., Murasko, D.M., 1983. Effect of interferon on thoracic duct lymphocyte output: induction with either polyI: C or vaccinia virus. J. Immunol. 130, 2236. Kutza, J., Murasko, D.M., 1994. Effects of aging on natural killer cell activity and activation by interleukin-2 and IFN-a. Cell. Immunol. 155, 195 – 204. Kutza, J., Kaye, D., Murasko, D.M., 1995. Basal natural killer cell activity of young versus elderly humans. J. Gerontol.: Biol. Sci. 50A, B110 – B116. Kutza, J., Murasko, D.M., Gratz, I., Afshar, M., 1997. The effects of general anesthesia and surgery on basal and interferon stimulated natural killer (NK) cell activity of humans. Anesth. Analg. 85, 918. Lanza, E., Djeu, J.Y., 1982. Age-independent natural killer cell activity in murine peripheral blood. In: Heberman, R. (Ed.), NK Cells and Other Natural Effector Cells. Academic Press, New York, pp. 335–340. Lee, U., Santa, K., Habu, S., Nishimura, T., 1996. Murine asialo GM1 +CD8 +T cells as novel interleukin-12-responsive killer T cell precursors. Jap. J. Cancer Res. 87, 429 – 432. Mann, E., Markovic, S.N., Murasko, D.M., 1989. Inhibition of lymphocyte recirculation by murine interferon:effects of various interferon preparations and timing of administration. J. Interfer. Res. 9, 35–51. Markovic, S.N., Murasko, D.M., 1990. Neoadjuvant immunotherapy with interferon of the spontaneously metastasizing murine B16F10L melanoma. Int. J. Cancer 77, 788 – 794. Markovic, S.N., Murasko, D.M., 1991. Role of natural killer and T-cells in interferon induced inhibition of spontaneous metastases of the B16F10L murine melanoma. Cancer Res. 51, 1124 – 1128. Markovic, S.N., Murasko, D.M., 1993. Anesthesia inhibits interferon-induced natural killer cell cytotoxicity via Induction of CD8 + suppressor cells. Cell. Immunol. 151, 474 – 480. Markovic, S.N., Knight, P.R., Murasko, D.M., 1993. Inhibition of interferon stimulation of natural killer cell activity in mice anesthesized with halothane or isoflurane. Anesthesiology 78, 700 – 706. Mikael, N., Mirza, N.M, Zaharian, B.I., Deulofeut, H., Salazar, M., Yunis, E.J., Dubey, D.P., 1994. Genetic control of the decline of natural killer cell activity in aging mice. Growth Dev. Aging 58, 3–12. A. Plett, D.M. Murasko / Mechanisms of Ageing and De6elopment 112 (2000) 197–215 215 Miller, R.A., 1996. The aging immune system: primer and prospectus. Science 273, 70 – 74. Minato, N., Reid, L., Cantor, H., Lengyel, P., Bloom, B.R., 1980. Mode of regulation of natural killer cell activity by interferon. J. Exp. Med. 152, 124 – 137. Miyaji, C., Watanabe, H., Minagawa, M., Toma, H., Kawamura, T., Nohara, Y., Nozaki, H., Sato, Y., Abo, T., 1997. Numerical and functional characteristics of lymphocyte subsets in centenarians. J. Clin. Immunol. 17, 420–429. Murasko, D., Goonewardene, I.M., 1990. T-cell function in aging mechanisms of decline. Ann. Rev. Gerontol. Geriatr. 10, 71–96. Mysliwska, J., Mysliwski, A., Romanowski, P., Digda, J., Sosnowska, D., Foerster, J., 1992. Monocytes are responsible for depressed natural killer (NK) activity in both young and elderly low NK responders. Gerontology 38, 41–49. Provinciali, M., Muzzioli, M., Fabris, N., 1989. Timing of appearance and disappearance of IFN and IL-2 induced natural immunity during ontogenetic development and aging. Exp. Gerontol. 24, 227–236. Reiter, Z., 1993. Interferon—a major regulator of natural killer cell-mediated cytotoxicity. J. Interfer. Res. 13, 247–257. Riccardi, C., Giampietri, A., Migliorati, G., Frati, L., Herberman, R.B., 1986. Studies on the mechanism of low natural killer cell activity in infant and aged mice. Nat. Immunol. Cell Growth Regul. 5, 238–249. Tyring, S.K., 1992. Introduction to clinical uses of interferons. In: Baron, S., et al. (Eds.), Interferon: Principles and Medical Applications. University of Texas Medical Branch at Galveston, Galveston, TX, pp. 399–408. .