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The Journal of Immunology, 2005, 175: 1820-1826.
Copyright © 2005 by The American Association of Immunologists

Depletion of T Cells by Type I Interferon: Differences between Young and Aged Mice1

Jiu Jiang*, Diara Gross*, Shoko Nogusa{dagger}, Philip Elbaum* and Donna M. Murasko2,*,{dagger}

* Department of Microbiology and Immunology and {dagger} Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type I IFN (IFN-I or IFN-{alpha}{beta}) plays an important role in the innate immune response against viral infection. Here we report that a potent inducer of IFN-{alpha}{beta}, polyinosinic-polycytidylic acid [poly(I:C)], led to the depletion of T cells in young, but not aged mice, and that this depletion was limited to central memory, but not effector memory, T cells. Although early activation of T cells in vivo by poly(I:C), as demonstrated by CD69, was not impaired with aging, the expression of active caspase-3 was higher in young compared with aged mice. This depletion of T cells and induction of active caspase-3 in young mice and of CD69 in both young and aged mice by poly(I:C) were blocked by anti-IFN-{alpha}{beta} Ab. Although poly(I:C) stimulated lower circulating levels of IFN-{alpha}{beta} in aged mice, administration of IFN-{alpha}{beta} after poly(I:C) did not induce depletion of T cells in aged mice. These results indicate that IFN-{alpha}{beta} plays a critical role in the depletion of T cells of young mice, and further suggest that the lower level of functional IFN-{alpha}{beta} and decreased induction of active caspase-3 in T cells of aged mice after poly(I:C) may be responsible for the increased resistance of T cells of aged mice to depletion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Aging is associated with a decline in immune responses (1, 2, 3). Although many aspects of the immune system demonstrate age-associated alterations, T cells show the greatest and most consistent changes (4). The T cell response of both aged mice and humans has been characterized by decreased proliferation and cytotoxic activity, as well as altered cytokine production (4, 5). The increase in morbidity and mortality in the elderly due to infections has been attributed to this decrease in immune response (6).

Several investigations have observed a depletion of nonspecific cells early after infection (7, 8). They postulated that this depletion of nonresponding T cells may be necessary for the subsequent proliferation and expansion of specific T cells required for clearance of the infectious agents. Based on this information, we previously performed studies that demonstrated early after E55+murine leukemia virus (E55+MuLV)3 infection there is a nonspecific depletion of both naive and memory T cells in young mice that occurs via apoptosis (9). This depletion does not occur in aged mice after virus infection (9). We hypothesize that this lack of "space" in the lymphocytic compartment may be responsible for the decreased specific T cell response of aged mice to virus infections. However, the mechanism of this depletion has not been elucidated.

During the host defense against viral and bacterial infections, the innate immune response interacts with an adaptive immune response to eliminate pathogens. In the context of host defense against viral infection, one of the first discovered and most well studied cytokines is type I IFN (IFN-I) (10, 11), which includes IFN-{alpha} and IFN-{beta}. IFN-{alpha}{beta} is induced quickly and efficiently in many types of cells upon infection by various viruses (12, 13), and limits virus replication by establishing an antiviral state in uninfected cells (14). Mice deficient in IFN-{alpha}{beta} receptor (IFN-{alpha}{beta}R) are much more susceptible to viral infection (15, 16); replication and virulence of profoundly attenuated viruses are largely restored to levels of wild-type virus in mice lacking the IFN-{alpha}{beta}R (17).

To explore whether or not IFN-{alpha}{beta} could be involved in the depletion of T cells early during viral infection, we used polyinosinic-polycytidylic acid [poly(I:C)], a powerful inducer of IFN-{alpha}{beta} (18, 19), and Ab specific to IFN-{alpha}{beta}. Our results demonstrate that IFN-{alpha}{beta} plays an important role in the depletion of T cells of young mice after stimulation with poly(I:C). Furthermore, lower levels of functional IFN-{alpha}{beta} in plasma and decreased induction of active caspase-3 in T cells after poly(I:C) administration may be involved in the resistance of T cells of aged mice to depletion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Six- and 22-mo-old C57BL/6 male mice were purchased from the National Institute on Aging at Harlan Sprague Dawley. Eight-week-old C57BL/6 male mice were purchased from The Jackson Laboratory. All mice were maintained in microisolators in American Association of Laboratory Animal Care-approved facilities at the Drexel University College of Medicine (Philadelphia, PA) and were provided food and water ad libitum. Mice were allowed to acclimate for at least 1 wk in our facilities before use. Mice exhibiting enlarged spleens or tumors were eliminated from the study.

Administration of reagents

Mice were given poly(I:C) (Sigma-Aldrich) at the indicated dose in pyrogen-free saline i.v. In some experiments, mice were injected i.v. with 2 x 105 neutralizing units of sheep anti-mouse IFN-{alpha}{beta} Ab or control Ab 2 h before administration of poly(I:C). Anti-mouse IFN-{alpha}{beta} serum was obtained from a sheep bled 21 wk after the start of immunization with repeated doses of mouse L cell IFN-{alpha}{beta} (20, 21). The serum had a titer of 1 x 107 U/ml against mouse {alpha} and {beta} IFN, as determined by neutralization against 8 U of IFN as described (22). Some mice were injected i.v. with 2 x 106 U of IFN-{alpha}{beta} (Lee Biomedical Research) 3 h after poly(I:C).

Measurement of IFN-{alpha}{beta} in plasma

The plasma of mice were obtained 6 h after inoculation of poly(I:C). IFN-{alpha}{beta} levels were determined by bioassay, as previously described (23). Briefly, serial dilutions of plasma were incubated with L-929 cells overnight at 37°C with 5% CO2 and humidity. The next day, EMC virus was added, incubated overnight, and the level of overall cytopathology was determined. One unit of IFN-{alpha}{beta} was defined as the amount that resulted in 50% reduction in cytopathology. IFN-{alpha}{beta} levels were also determined by ELISA. ELISA kit was purchased from PBL Biomedical Laboratories.

Flow cytometry

Spleens and other tissues were removed from individual mice, and lymphocytes were prepared and resuspended in 1% BSA in PBS at a concentration of 1 x 106 cells/well in a 96-well plate. Cells were stained for surface markers using mAbs (anti-CD4, CD8, CD69, CD44, and CD62L) purchased from BD Pharmingen. Purified rabbit anti-mouse IFN-{alpha}{beta}R-{alpha} (IFN-{alpha}{beta}R{alpha}) Ab was purchased from Santa Cruz Biotechnology and PE-conjugated goat anti-mouse IgG Ab from Jackson ImmunoResearch Laboratories. To analyze Bcl-2 and active caspase-3 expression in T cells, splenocytes were resuspended in RPMI 1640, and cells were surface stained with {alpha}-CD8 mAb in 1% BSA/PBS. Cells were permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) for 20 min on ice, and intracellular staining was performed for 1 h on ice with either an anti-Bcl-2 or anti-active caspase-3 mAbs (BD Pharmingen). Cells were then fixed with 1% paraformaldehyde in PBS. Flow cytometry was performed with a FACSCalibur (BD Biosciences), and data were analyzed with FlowJo software 4.2 version (Tree Star).

Statistical analysis

The analysis was performed using Student’s t test. Multiple treatment groups within individual experiments were compared by ANOVA, followed by Tukey’s post hoc test. Correlation between two parameters was analyzed by linear regression analysis, and Spearman/Pearson correlation using GraphPad InStat version 3.0 (GraphPad Software). Significant differences were determined at the level of p < 0.05. Results are expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Poly(I:C) depletes T cells of young, but not aged, mice

In our previous studies, we reported that CD8 and CD4 T cells of young, but not aged, mice were depleted by apoptosis during the early stage of virus infection (9). To investigate whether IFN-{alpha}{beta} is involved in T cell depletion in young and aged mice, we used a strong inducer of IFN-{alpha}{beta}, poly(I:C), which is considered an agent that mimics virus infection (24). After i.v. administration of 250 µg of poly(I:C), depletion of splenic CD8 and CD4 T cells was observed in young but not aged mice (Fig. 1). Significant depletion of both CD8 and CD4 T cells occurred at 12 h (46% for CD8 T cells and 36% for CD4 T cells) and 24 h (20% for both CD8 and CD4 T cells) in young mice. A higher dosage of poly(I:C) (400 µg) resulted in greater depletion at 12 h (62% of CD8 and 50% of CD4 T cells; unpublished data). In aged mice, no depletion of either CD8 T cells or CD4 T cells was observed at any time. No depletion in either age group occurred at 3 or 6 h, and numbers of CD8 and CD4 T cells in young mice returned to baseline by 72 h post treatment with poly(I:C) (unpublished data). These data demonstrate that T cells of aged mice are more resistant to depletion by poly(I:C) than those of young mice.



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  		FIGURE 1. Poly(I:C) depletes T cells of young but not aged mice. Young (6-month-old) and aged (22-mo-old) C57BL/6 mice were inoculated i.v. with poly(I:C) (250 µg/mouse). At different times postinjection (12 and 24 h), splenocytes were isolated and stained with anti-CD4 and CD8 Abs. CD4 and CD8 T cells were quantitated by FACS. Bars represent the mean of absolute number of T cells in spleens. At each time point, n = 3; error bars represent ± SD. *, p < 0.05 by ANOVA test followed by Tukey’s post hoc test for a comparison of mice treated with poly(I:C) and PBS. Similar results were obtained in three separate experiments.

 
Depletion of T cells by poly(I:C) is due to apoptosis

Our previous results have shown that the depletion of T cells in young mice early during viral and bacterial infections is due to apoptosis (7, 9). To examine whether the depletion of T cells in young mice after stimulation with poly(I:C) is related to apoptosis, we examined two questions: 1) Is comparable depletion seen in other tissues, therefore eliminating the possibility that the decreased numbers in the spleen reflects migration rather than apoptosis? 2) Is there up-regulation of markers associated with apoptosis after poly(I:C) administration? Comparable to our previous data with E55+MuLV (9), the number of both CD4 and CD8 T cells did not increase, but decreased although not significantly in mesenteric lymph nodes and blood 24 h after inoculation of poly(I:C) in young mice (Fig. 2).



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  		FIGURE 2. Depletion of splenic T cells is not due to migration. Young and aged C57BL/6 mice were inoculated i.v. with poly(I:C) (250 µg/mouse). Lymphocytes were isolated from different tissues (spleen, mesenteric lymph nodes (MLN), and peripheral blood) 24 h postinoculation. T cells in each compartment were quantitated by FACS. Bars represent the mean of absolute number of T cells in each tissue. At each time point, n = 3; error bars represent ± SD. *, p < 0.05 by Student’s t test for a comparison of mice of the same age treated with poly(I:C) or with PBS. Similar results were obtained in two separate experiments.

 
We also examined markers associated with apoptosis. Administration of poly(I:C) induced active caspase-3, an important enzyme for cells undergoing apoptosis (25). Assessed by intracellular staining with anti-active caspase-3 Ab as described (26), the intracellular expression of active caspase-3 in CD8 T cells was significantly increased 24 h after poly(I:C) in young mice, while only minimal up-regulation of active caspase-3 was observed in aged mice (Fig. 3, A and B). This difference was not due to a shift in kinetics because limited up-regulation of active caspase-3 was found in both groups at other time points examined (1.5, 3, and 6 h; unpublished data).



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  		FIGURE 3. Induction of apoptosis-associated markers by poly(I:C). Young and aged C57BL/6 mice were inoculated i.v. with poly(I:C) (250 µg/mouse). At 24 h postinjection, splenocytes were isolated and surface stained with anti-CD8 Ab and intracellularly stained with either anti-active caspase-3 or anti-Bcl-2 Abs. The expression of these molecules was examined by FACS (gated on CD8 T cells). A and B, Active caspase-3 and C and D, Bcl-2 expression in CD8 T cells 24 h after poly(I:C). In each group, n = 3; error bars represent ± SD. *, p < 0.05 by Student’s t test for a comparison of mice of the same age treated with poly(I:C) or with PBS. The experiment was performed three times with similar results.

 
Because Bcl-2 is considered to be a protooncogene that prevents apoptosis in many cell types (27, 28), we explored the possibility that there was an increase of Bcl-2 in aged mice after poly(I:C). Although aged mice demonstrated significantly higher basal expression of Bcl-2 in CD8 T cells than young mice (p < 0.05, Fig. 3, C and D), poly(I:C) did not affect Bcl-2 expression in CD8 T cells (Fig. 3D) in either young or aged mice, which correlated with the results in vitro that have shown that IFN-{alpha}{beta} does not raise Bcl-2 levels in activated T cells (29). These results demonstrate that the depletion of T cells in young mice by poly(I:C) is due to apoptosis, rather than migration.

Central memory, but not effector memory, phenotype CD8 T cells are depleted in young mice by poly(I:C)

Because aged mice have a higher percentage of memory T cells (30), a possible explanation for differences in susceptibility to depletion of T cells in aged mice was a differential resistance of memory cells to depletion. Comparable to data in the virus system (9), memory cells of young but not aged mice were susceptible to depletion after poly(I:C) (Fig. 4A). Due to the recent emphasis on further classification of memory cells, we extended our previous studies to examine differences in susceptibility to poly(I:C) between central memory cells (CCR7+, CD62Lhigh, and CD44high) and effector memory cells (CCR7–, CD62Llow, and CD44high) (31, 32). CD8 T cells in spleens were analyzed 12 and 24 h posttreatment with poly(I:C). In young mice, poly(I:C) depleted 70% and 47% of central memory CD8 T cells at 12 and 24 h posttreatment, respectively. Minimal depletion of effector memory CD8 T cells was observed. No depletion of either CD62LhighCD44high or CD62LlowCD44high CD8 T cells occurred in aged mice at either time (Fig. 4, B and C). These results demonstrate that effector memory CD8 T cells are more resistant than central memory CD8 T cells to depletion by poly(I:C) in young mice, whereas all CD8 T cells of aged mice are resistant to depletion.



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  		FIGURE 4. Central memory, but not effector memory, phenotype CD8 T cells are depleted in young mice by poly(I:C). Young and aged C57BL/6 mice were inoculated i.v. with poly(I:C) (250 µg/mouse). The splenocytes were prepared and stained with anti-CD4, CD8, CD44, and CD62L Abs 12 h and 24 h after injection. A, Total numbers of CD44highCD4 and CD44highCD8 T cells in spleens were determined. B, Percentages of central memory (CD44highCD62Lhigh) and effector memory (CD44highCD62Llow) phenotype CD8 T cells were determined by FACS (gated on CD8+ cells). C, Total numbers of CD44highCD62Lhigh and CD44highCD62Llow CD8 T cells in spleens were determined. At each time point, n = 3; error bars represent ± SD. *, p < 0.05 by ANOVA test followed by Tukey’s post hoc test for a comparison of mice treated with poly(I:C) and PBS. Data is representative of four independent experiments with similar results.

 
IFN-{alpha}{beta} plays a critical role in the depletion of T cells by poly(I:C)

The above results demonstrate that T cells of young, but not aged, mice are depleted 12 to 24 h after administration of poly(I:C). To investigate whether IFN-{alpha}{beta} is an essential component of the poly(I:C) induced depletion of T cells, the ability of Ab specific for IFN-{alpha}{beta} to inhibit this depletion was explored. Eight-week-old mice were injected i.v. with anti-IFN-{alpha}{beta} or control Abs 2 h before administration of poly(I:C) (250 µg, i.v.). The number of splenic CD8 T cells decreased ~43% 24 h after poly(I:C). Anti-IFN-{alpha}{beta} Ab was able to prevent this depletion of CD8 T cells, whereas administration of control Ab had little effect on depletion (Fig. 5A). Furthermore, anti-IFN-{alpha}{beta} Ab abrogated the induction of active caspase-3 in T cells by poly(I:C) (Fig. 5B). These data strongly suggest that IFN-{alpha}{beta} is involved in the depletion of T cells after stimulation with poly(I:C) and mediates this depletion via apoptosis.



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  		FIGURE 5. IFN-{alpha}{beta} plays a critical role in the depletion of T cells by poly(I:C). Eight-week-old young C57BL/6 male mice were inoculated i.v. with poly(I:C) (250 µg/mouse). In some groups, each mouse was injected i.v. with 2 x 105 U of anti-IFN-{alpha}{beta} Ab (Ab) or control Ab (cAb) 2 h before poly(I:C). Splenocytes were prepared and surface stained with anti-CD8 Ab and intracellularly stained with anti-active caspase-3 Ab 24 h after poly(I:C). A, The absolute numbers of CD8 T cells in spleens of young mice. B, Active caspase-3 expression in CD8 T cells of young mice after poly(I:C) (gated on CD8+ cells). n = 3; error bars represent ± SD. *, p < 0.05 by ANOVA test followed by Tukey’s post hoc test for a comparison of mice treated with poly(I:C) and PBS. Data are representative of two separate experiments with similar results.

 
Aged mice produce less functional IFN-{alpha}{beta} after inoculation with poly(I:C) than young mice

To examine whether age-associated differences in induction of IFN-{alpha}{beta} by poly(I:C) could be responsible for differences in depletion of T cells, mice were treated i.v. with different doses of poly(I:C). Peripheral blood was obtained 6 h later, and IFN-{alpha}{beta} levels in plasma were determined by bioassay. As shown in Fig. 6A, administration of 100 µg of poly(I:C) induced similar levels of IFN-{alpha}{beta} in plasma of young and aged mice (~25 U/ml). However, young mice produced 3.5-fold more IFN-{alpha}{beta} than aged mice after 200 µg of poly(I:C) (~86 vs 24.6 U/ml, p < 0.05). The IFN-{alpha}{beta} in plasma reached even higher levels (~190 U/ml) in young mice when 400 µg of poly(I:C) was injected, although no aged mice survived at this dose. These results indicate that more IFN-{alpha}{beta} was produced in young than in aged mice after poly(I:C) stimulation.



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  		FIGURE 6. Aged mice produce less functionally active IFN-{alpha}{beta} after inoculation with poly(I:C) than young mice. A, Young and aged C57BL/6 male mice were injected i.v. with different doses of poly(I:C) (100, 200, and 400 µg per mouse). The level of IFN-{alpha}{beta} in plasma was examined by bioassay 6 h post injection. Bars represent the mean IFN-{alpha}{beta} levels (± SD). *, p < 0.05 by ANOVA test and Tukey’s post hoc test for a comparison of mice treated with different doses of poly(I:C) and PBS. N represents the number of mice examined. Dotted line indicates the limit of detection. Data is the combination of four independent experiments with similar results. B, Young and aged mice were inoculated i.v. with poly(I:C) (250 µg/mouse). Mice were bled to detect IFN-{alpha}{beta} levels in plasma via bioassay and ELISA 6 h postinjection. Data points indicated actual amount in units per milliliter (U/ml) and in picograms per milliliter (pg/ml) IFN-{alpha}{beta} produced by each individual mouse via bioassay and ELISA, respectively. Linear regression of data points from both assays retrieved lines displayed for young and aged mice (n = 6). r2 value indicates Pearson correlation comparing results from both assays within each group. Statistical posttest was used to determine significance (p < 0.05) of correlation between two assays. Similar results were obtained in two separate experiments.

 
Interestingly, these results contrasted with a previous report indicating that levels of IFN-{alpha}{beta} in aged mice was increased compared with young mice after administration of poly(I:C) (33). Review of these data indicated that the primary difference between the two studies was the method of assessment of IFN-{alpha}{beta}: bioassay vs ELISA. To address this difference in methodology, mice were inoculated with 250 µg of poly(I:C) and their plasma was assessed by both methods. The bioassay still reflected lower levels of IFN-{alpha}{beta} in aged mice compared with young mice (120 ± 32.2 U/ml vs 160 ± 26.8 U/ml); however, the ELISA indicated a similar decrease (2458 ± 381 pg/ml vs 3040 ± 146 pg/ml). Importantly, correlation of the data generated by the two methods indicated that although there was an excellent correlation between the results of the two assays in young mice (r2 = 0.97, p = 0.004), there was minimal correlation in aged mice (r2 = 0.001, p = 0.96) (Fig. 6B), suggesting that a portion of the IFN may not be functionally active in at least some aged mice.

Aged mice demonstrate higher IFN-{alpha}{beta}R on T cells than young mice

In addition to producing less functional IFN-{alpha}{beta}, the decreased response of aged mice to poly(I:C) may reflect altered expression of IFN-{alpha}{beta}R. To examine the differences in expression of IFN-{alpha}{beta}R on subsets of CD8 T cells in young and aged mice, splenocytes were isolated and stained with anti-IFN-{alpha}{beta}R Ab. As shown in Fig. 7, IFN-{alpha}{beta}R expression on both total CD8 T cells and naive (CD44low) CD8 T cells was higher in aged than young mice (p = 0.004 and 0.02, respectively). In contrast, no significant difference of IFN-{alpha}{beta}R expression on either central or effector memory CD8 cells was observed between young and aged mice. Treatment with poly(I:C) neither induced greater IFN-{alpha}{beta}R expression nor shifted the basal pattern of expression (data not shown). These results suggest that differences in the expression of IFN-{alpha}{beta}R do not appear to be responsible for the decreased response of aged mice to poly(I:C).



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  		FIGURE 7. IFN-{alpha}{beta}R{alpha} expression on T cells. Splenocytes of young and aged mice (n = 3/age group) were prepared and stained with anti-IFN-{alpha}{beta}R{alpha} Ab. T cell populations were identified using anti-CD8, CD44, and CD62L Abs. CD8N = CD8+CD44low; CD8EM = CD8+CD44highCD62Llow; CD8CE = CD8+CD44highCD62Lhigh. Mean fluorescence intensity (MFI) of IFN-{alpha}{beta}R{alpha} expression on T cells was determined by FACS. *, MFI of IFN-{alpha}{beta}R{alpha} on cells of aged mice is significantly higher than that on cells of young mice (p < 0.05).

 
Early activation of T cells in young and aged mice after stimulation with poly(I:C)

Because the level of IFN-{alpha}{beta}R expression is not altered by aging, the question remained whether or not signal transduction through the IFN-{alpha}{beta}R was modified in aged mice. To examine whether early activation of T cells by poly(I:C) is altered in aging, we compared the early T cell activation markers CD69 at various times (0, 3, 6, 12, 24, and 72 h) after administration of poly(I:C). The kinetics and level of expression of CD69 were comparable in young and aged mice on CD8 T cells (Fig. 8A). Expression of CD69 was apparent 3 h after poly(I:C), reached peak expression at 12 h, and returned to control levels by 72 h. These results show that early activation of T cells by poly(I:C) is not affected by aging.



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  		FIGURE 8. Kinetics of early activation of T cells in young and aged mice after stimulation with poly(I:C). A, Young and aged C57BL/6 mice were inoculated i.v. with poly(I:C) (250 µg/mouse). At different times postinjection (3, 6, 12, 24, and 72 h), splenocytes were isolated and stained with anti-CD8 and CD69 Abs. The expression of CD69 on CD8 T cells was examined by FACS (gated on CD8 T cells). B, Expression of CD69 on CD8 T cells of young and aged mice (gated on CD8 T cells) and the experiment was the same as described in Fig. 5. At each time point, n = 3; error bars represent ± SD, p < 0.05 by ANOVA test followed by Tukey’s post hoc test for a comparison of young and aged mice at each time treated with poly(I:C). The experiment was performed twice with similar results.

 
However, it is possible that the induction of CD69 was not mediated through IFN-{alpha}{beta}. To answer this question, young and aged mice were injected i.v. with anti-IFN-{alpha}{beta} Ab 2 h before administration of poly(I:C). As shown in Fig. 8B, although induction of CD69 on CD8 T cells was apparent in both young and aged mice 24 h after inoculation of poly(I:C), anti-IFN-{alpha}{beta} Ab completely abrogated the up-regulation of CD69 in both groups. These data clearly indicate that IFN-{alpha}{beta} is involved in the induction of CD69 on CD8 T cells by poly(I:C) in both young and aged mice and suggest that IFN-{alpha}{beta}R is functionally active in aged mice.

Administration of exogenous IFN-{alpha}{beta} after poly(I:C) fails to induce depletion of T cells in aged mice

Because the above data suggest that: 1) IFN-{alpha}{beta} is an essential component of depletion of T cells in young mice (Fig. 5A); 2) poly(I:C) induces less functional IFN-{alpha}{beta} in aged mice (Fig. 6); and 3) IFN-{alpha}{beta}R appears intact in aged mice (Figs. 7 and 8), we wanted to determine whether inoculation of exogenous IFN-{alpha}{beta} in addition to poly(I:C) could induce depletion of T cells in aged mice. Young and aged mice were injected i.v. with IFN-{alpha}{beta} (2 x 106 U) 3 h after poly(I:C). Twenty-four hours after poly(I:C), the depletion of splenic T cells was examined. Our results show that inoculation of exogenous IFN-{alpha}{beta} after poly(I:C) neither induced depletion of T cells in aged mice nor induced additional depletion of T cells in young mice (Fig. 9). However, 24 h after exogenous IFN-{alpha}{beta} was administrated alone to young mice, 26% and 21% depletion of splenic CD4 and CD8 T cells, respectively, was observed. Twenty-six percent depletion of central memory CD8 T cells occurred, although no depletion of effector memory CD8 T cells was found in spleens (unpublished data).



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  		FIGURE 9. Administration of exogenous IFN-{alpha}{beta} after poly(I:C) does not induce depletion of T cells in aged mice. Young and aged C57BL/6 mice were inoculated i.v. with poly(I:C) (250 µg/mouse). In one group, mice were injected i.v. with IFN-{alpha}{beta} (2 x 106 U) 3 h after poly(I:C). Splenocytes were prepared and stained with anti-CD4 and CD8 Abs 24 h after poly(I:C). CD8 and CD4 T cells were quantitated by FACS. Bars represent the mean of absolute number of T cells. In each group, n = 3; error bars represent ± SD. *, p < 0.05 by ANOVA test and Tukey’s post hoc test for a comparison of mice treated with poly(I:C) or poly(I:C) plus IFN-I to mice treated with PBS. The experiment was performed two times with similar results.

 
To determine whether exogenous IFN-{alpha}{beta} could elicit any response in aged mice, NK activity was evaluated in splenocytes. Consistent with previously published data (34), IFN-{alpha}{beta} induced a 3-fold increase in basal NK activity of young mice, and only a 50% increase in basal NK activity of aged mice (unpublished data). These results suggest that aged mice can respond to IFN-{alpha}{beta} but at a lower level than young mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been reported that the depletion of T cells of young mice occurs early during bacterial and viral infections (7, 8, 9, 35). In addition, it was demonstrated that depletion of T cells in young mice was due to apoptosis and not migration of cells from spleen to other organs. In contrast, our recent studies have shown that T cells of aged mice are resistant to depletion early during viral infection (9). Furthermore, adoptive transfer experiments indicated that both the environment of the aged mouse and properties intrinsic to the T cells of aged mice contribute to the limited depletion of T cells of aged mice after virus infection (9).

IFN-{alpha}{beta} is produced by the host in response to many types of viral infections, and mediates antiviral activities through the induction of cellular proteins (13). These cytokines provide an early line of defense against viral infections, acting hours to days before adaptive immune responses are induced (36). IFN-{alpha}{beta} may serve, directly or indirectly, as either a growth factor or an apoptosis factor (37, 38, 39). Indeed, IFN-{alpha} is able to induce or sensitize cells to apoptosis (40, 41).

To address the role of IFN-{alpha}{beta} in T cell depletion, we used poly(I:C), which is a strong inducer of IFN-{alpha}{beta} (18, 19, 23). Our results confirmed other studies that showed that poly(I:C) can deplete T cells of young mice by apoptosis (8), and extending these studies clearly showed that IFN-{alpha}{beta} plays a critical role in T cell depletion by poly(I:C) because anti-IFN-{alpha}{beta} Ab is able to block this depletion. Our data further demonstrate that central, but not effector, memory CD8 T cells are depleted by poly(I:C) in young mice. This difference in depletion of central vs effector memory CD8 T cells is also apparent in young mice early after infection with several different viruses (unpublished data). This depletion of nonspecific naive, as well as central memory, T cells by IFN-{alpha}{beta} may provide "space" for the maximum expansion of specific T cells and subsequent elimination of the virus.

Similar to our results with virus infection (9), T cells of aged mice were not depleted after administration of poly(I:C). A simple explanation for this lack of depletion by poly(I:C) could be that aged mice do not make IFN-{alpha}{beta} in response to poly(I:C). Since a previous report (33) indicated that aged mice produce more IFN-{alpha}{beta} in response to poly(I:C), we did not think this was a viable explanation. However, our data clearly indicated that aged mice make less IFN-{alpha}{beta} than young mice (Fig. 6A). The differences in the two results could have been due to the route of administration (i.p. vs i.v.), the assay used (ELISA vs bioassay), the amount of poly(I:C) used (100 µg vs 200 µg) or the time when IFN-{alpha}{beta} was assessed (4 h vs 6 h). Because we find: 1) no difference in the level of IFN-{alpha}{beta} production by young and aged mice when 100 µg is administered, and 2) no detectable levels of IFN-{alpha}{beta} 3 h post administration in either young or aged mice, it is unlikely that either of the latter two options are viable. We, therefore, assessed IFN-{alpha}{beta} levels in the same plasma samples by both ELISA and bioassay. Using plasma from mice obtained 6 h after inoculation with 250 µg of poly(I:C), IFN-{alpha}{beta} levels were lower in aged mice by both bioassay and ELISA. Interestingly, there was a strong positive correlation between the bioassay and ELISA in young mice (r2 = 0.97, p = 0.0004), but not in aged mice (r2 = 0.001, p = 0.96) (Fig. 6B). These results suggest that the aged mice may be producing IFN that has lost functional activity.

Our results are consistent with studies on the expression of TLR3 in aged mice. poly(I:C) has been shown to be a ligand for TLR3 (42, 43). Stimulation of TLR3 by poly(I:C) leads directly or indirectly to the activation of NF-{kappa}B and the production of IFN-{alpha}{beta} (42). Little induction of IFN-{alpha} or IFN-{beta} was observed in TLR3-deficient macrophages after poly(I:C) treatment compared with wild-type cells, which indicates that transcriptional induction by poly(I:C) of genes for IFN-{alpha} or IFN-{beta} is dependent on TLR3 (42). In aged mice, the expression and function of TLR3 is severely impaired (44), which may result in lower circulating levels of IFN-{alpha}{beta} in aged mice than in young mice after stimulation with poly(I:C).

The depletion of T cells by poly(I:C) does not seem related to the early activation of T cells, because early activation of T cells by poly(I:C) as indicated by CD69 expression is not impaired with aging (Fig. 8A). Although these results indicate that induction of CD69 is not an appropriate marker for subsequent apoptosis, they do indicate that T cells of aged mice are responsive to IFN-{alpha}{beta}, because the induction of CD69 on T cells of both young and aged mice can be blocked by anti-IFN-{alpha}{beta} Ab (Fig. 8B). This response of aged mice to IFN-{alpha}{beta} as demonstrated by induction of CD69 is consistent with our current (Fig. 7) and previous (45) results indicating that the level of IFN-{alpha}{beta}R1 on CD4, CD8, and NK cells was higher in aged than young mice. Similar to the decreased depletion of T cells by poly(I:C) in this study, inoculation of either poly(I:C) or IFN-{alpha}{beta} resulted in less induction of NK activity in aged, compared with young, mice (34). The comparable binding of IFN-{alpha}{beta} to T cells (Fig. 7) and NK cells (45) but lower functional outcomes (i.e., depletion and cytotoxicity, respectively), may suggest that the affinity of these receptors is decreased. The inability of poly(I:C) to deplete T cells (Fig. 1) and to induce NK activity (34) in aged mice while inducing expression of activation markers (e.g., CD69) suggests either that different pathways exist for induction of activation markers (e.g., CD69) and of apoptosis and cytotoxicity, or that activation reflects early events in the pathway with later events required for apoptosis and cytotoxicity being blocked. If the triggering for apoptosis requires a higher saturation of IFN-{alpha}{beta}R binding, administration of additional exogenous IFN-{alpha}{beta} should have been able to induce depletion. However, no depletion occurred after inoculation of additional IFN-{alpha}{beta}. The aged mice did respond to the IFN-{alpha}{beta} as demonstrated by induction of NK cytotoxicity, albeit at a lower level than in young mice. This suggests either that a different amount of IFN-{alpha}{beta} is required for apoptosis or that another signal (e.g., TNF-{alpha}) may also be required for apoptosis and it, too, is altered with age.

Our data indicate that apoptosis-associated active caspase-3 is differentially increased in young and aged mice. Activation of caspase-3 is regarded as a common step in all apoptotic pathways. Higher expression of active caspase-3 in T cells of young compared with aged mice after poly(I:C) may at least partially explain the differential sensitivity to T cell-depletion in young and aged mice induced by poly(I:C). Although this difference in the activation of caspase-3 may reflect a major component of the resistance of T cells of aged mice to apoptosis, differences in expression of Bcl-2 in T cells of aged mice may also contribute to this resistance (33). Bcl-2 is considered to be a protooncogene that blocks apoptosis in many cell types (28). Whereas our data is consistent with previous reports that IFN-{alpha}{beta} does not raise Bcl-2 levels in activated T cells in vitro (29), we found that basal levels of Bcl-2 in CD8 T cells were significantly higher in aged than in young mice (Fig. 3, C and D). A similar increase in basal levels of Bcl-2 in T cells of aged mice has previously been reported (33). Although it is possible that higher basal levels of Bcl-2 expression contribute to the resistance of aged CD8 T cells to apoptosis, the similar level of basal Bcl-2 expression on CD4 T cells of aged and young mice, but an increased resistance of CD4 T cells of aged mice to depletion (unpublished data), suggest that either a different mechanism of resistance to depletion exist in CD4 and CD8 T cells or that Bcl-2 is not involved in this resistance.

In summary, our results show that IFN-{alpha}{beta} is responsible for the depletion of T cells from young, but not aged, mice. This depletion is limited to central memory, but not effector memory, T cells. The differences in IFN-{alpha}{beta} mediated depletion of T cells between young and aged mice might contribute to immune impairment with aging. This has important implications for understanding the mechanisms of differences in the immune response between young and aged individuals during early infection, which will help to develop vaccine strategies for elderly people.


   Acknowledgments
 
We thank Lisa L. Lau, Lauren A. Zenewicz, and Amy Troy for critical reading of the manuscript, and Yongquan Sun for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AG14913. Back

2 Address correspondence and reprint requests to Dr. Donna M. Murasko, Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104. E-mail address: donna.murasko{at}drexel.edu Back

3 Abbreviations used in this paper: MuLV, murine leukemia virus; poly(I:C), polyinosinic-polycytidylic acid. Back

Received for publication July 26, 2004. Accepted for publication May 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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