Conditioned immunosuppression in young versus aged mice: Differences in cells and responses to environmental stimuli lead to altered conditioning in aged animals

BRAIN,

BEHAVIOR,

AND

IMMUNITY

1,

306-317 (1987)

Conditioned lmmunosuppression in Young versus Aged Mice: Differences in Cells and Responses to Environmental Stimuli Lead to Altered Conditioning in Aged Animals REGINALD Departments

M. GORCZYNSKI

of Sttrgery und Immunology. University of Toronto, Mt. Sinai Research Room 853, 600 University Avenue, Toronto M5G 1x5. Ontario, Canada

Institute.

Aged mice (>20 months of age) show a decreased immune response after antigen challenge compared to their young counterparts. In this study aged mice were also found to show a diminished conditioned immunosuppression after associative learning trials with cyclophosphamide and saccharin. followed by immune stimulation in the presence of saccharin, when compared to young (IO weeks) syngeneic mice. Adoptive transfer experiments in which cells from nonconditioned or conditioned young or aged mice were injected into irradiated conditioned young or aged syngeneic mice (exposed or not exposed to conditioned stimuli) revealed the following: (I) There was an altered responsiveness of normal cells injected into conditioned aged mice (reexposed to cues) compared to the response in young recipients: (2) Cells from conditioned young mice failed to show conditioned immunosuppression on adoptive transfer to irradiated conditioned aged mice: (3) Cells from conditioned aged mice failed to show conditioned immunosuppression on adoptive transfer to irradiated conditioned young mice; (4) The changes seen in spleen cells from conditioned aged mice (relative to similar cells from young mice) were to be found in the T cell population of these animals. These data are consistent with the idea that during aging changes in both the responding cells and the conditioned environment, along with the interaction of these, produce a decreased ability to document conditioned immunosuppression of antibody responses. fc 1987 Academic Preht. Inc.

INTRODUCTION

Data from a variety of studies argue convincingly that classical conditioning can be used to modify both antibody and cell-mediated immune responses (Ader & Cohen, 1975, 1985; Bovbjerg, Ader, & Cohen, 1982; Gorczynski, MacRae, & Kennedy, 1982; Gorczynski & Kennedy, 1984: Gorczynski, Kennedy, & Ciampi, 1985; Gorczynski, 1987). There are also more recent data to suggest that the converse may also be true: that immune processes may alter conditioning in experimental animals (Ader, Grota, & Cohen, 1987). To date, however, there are few studies that have explored the mechanism(s) by which this CNS-immune system interaction may be mediated (Gorczynski et al., 1985; Gorczynski, 1987). Moreover, while the immune response in aged mice declines after direct antigen challenge compared to the response in younger animals (Kay and Makinodan. 19811, the effect of classical conditioning in aged mice has been studied in only a cursory manner (Gorczynski, MacRae, and Kennedy, 1983). It is known that lymphoid tissue is innervated by both P-adrenergic and cholinergic neurons (Felten, Overhage, Felten, & Schmedtje, 1981; Bullock & Pomerantz. 1984). In addition, lymphocytes have been shown to possess receptors for neurotransmitters and neuroendocrine substances which in turn can modify im306 0889-1591187 $3.00 Copyright All right\

!’ 1987 hy Academic Pro\. of reproduction tn anv fkm

Inc reserved

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mune responses in culture (Hall & Goldstein, 1981; Johnson, Torres, Smith, Dion, & Blalock, 1984). Intraventricular application of lymphokines can alter immunity (Hall et al., 1985). Consistent with lesioning studies in animals are other data which show that during an immune response changes in neuronal firing occur in the hypothalamic area of the CNS (Cross, Markesberry, Brooks, & Roszman, 1980; Besedovsky, Felix, & Haas, 1977). From such observations one could make a case for investigating changes in lymphokines during conditioning studies as a means to analyze the interconnections between the CNS and the immune system. The approach used in this report continues a series of studies reported earlier in which the role of environment: cell interactions in conditioning phenomena were explored by adoptive transfer experiments (Gorczynski, 1987). The additional question(s) asked in this study concerns the nature, if any, of the differences in classical conditioning of immune responses in young animals versus aged animals. This analysis developed naturally from our earlier reported failure to produce conditioned immunosuppression using aged mice in the same studies in which young animals consistently showed (after repeated associative trials with cyclophosphamide and saccharin) a conditioned immunosuppression on challenge with sheep erythrocytes given along with cues (saccharin) (Gorczynski et al., 1983). MATERIALS

AND METHODS

Mice. C57BL/6 mice were purchased from the Jackson Laboratories, Bar Harbor, Maine. Mice were housed five per cage and allowed food and water nd libitum (except where on restricted water schedule; see text). Zrrcldiution. Mice to be irradiated were given 900 R of y-irradiation from a ‘j’Cs source at a dose of 80 R per min. All mice were reconstituted with spleen cells within 2 h of irradiation. Cell prepuration. Spleen cells were prepared aseptically in PBS containing 0.3% bovine serum albumin as described elsewhere (Gorczynski & Cunningham, 1978). Nylon wool purified T cells, and anti-thy 1.2 treated spleen (B) cells were obtained as before (Gorczynski, 1987), the latter using a monoclonal antibody and rabbit complement obtained from Cedarlane Laboratories (Hornby, Ontario), Sheep erythrocytes (SRBC) were obtained every 2 weeks from Woodland Farms (Guelph), and were washed three times in PBS prior to use in AFC assays or as immunogen in mice. Assay for antibody-forming cells (AFC). This assay, using modified Cunningham chambers, is described elsewhere (Gorczynski & Cunningham, 1978). IgGAFC were determined as before using a rabbit anti-mouse IgG antibody. Conditioning of mice. Mice were maintained on a daily watering schedule in which water was available for only a 30-min period (8:OO AM to 8:30 AM). After 10 days, animals were exposed once to (0.1%) saccharin (conditioned stimulus, CS) in their drinking water and immediately thereafter received an injection of cyclophosphamide (100 mg/kg) in 0.5 ml PBS given intraperitoneally (unconditioned stimulus, US). Reexposure (CS + US) to cyclophosphamide (Cy) and saccharin (Sacc) was repeated on two further occasions at 21-day intervals. Animals were used in the experiments described 21 days after the last exposure to (CS + US), When taste aversion was assessed a preference test was used. Animals were

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offered water ( = NCS) and saccharin ( = CS) at the same time (in separate bottles) and preference (for Sacc) calculated as (saccharin-flavored H,O consumed)-(HO consumed)/(total fluid consumed). This measure varied from + 1 (total Sacc preference) to - 1 (total water preference, i.e., taste aversion). Statistical analysis. Differences in antibody responses between groups were initially assessed by one-way analysis of variance (ANOVA). Thereafter, pairwise comparison of groups used a nonparametric (Wilcoxon rank sum) test (AFC responses in any group followed a nonnormal distribution). RESULTS Failure to Demonstrate Conditioned Responses in Aged Mice

Immunosuppression

of Antibody

A number of groups have shown that animals receiving pretreatment schedules in which Cy, a nonspecific immunosuppressive drug, is given in association with a novel taste in the drinking supply (saccharin), will subsequently show a diminished antibody response on challenge with SRBC if that challenge is given along with the “cues” for immunosuppression (Sacc) (for review see Ader & Cohen, 1985). The first 8 rows of Table 1 present typical data for such a study, examining IgG-AFC 7 days post-SRBC challenge in groups of eight lo-week-old mice given the treatments shown. Note that in the experimental group (CS + US) reexposed to Sacc (CS) after challenge with SRBC, Sacc was also given on Days 2,4, and 6 after SRBC. All mice previously exposed to Cy gave decreased AFC relative to non-Cy treated mice (rows 3, 4, 7, and 8). However, only the group in which Cy exposure initially occurred in the context of Sacc (CS + US) showed suppression when reexposed to Sacc (CS + US:CS) at the time of antigen challenge (compare groups 3, 4 and 7, 8). In contrast to the data obtained from mice 10 weeks of age, rows 9 through 16 contain the data from an identical experiment with mice initially conditioned at 84 weeks of age. Once again, pretreatment of mice with Cy led to a generally decreased response compared to non-Cy treated animals (rows 11, 12, 15, and 16). However, even when this pretreatment with Cy was in the context of Sacc (rows 15, 16) challenge with SRBC in the presence of Sacc (CS + US:CS) gave no less of an antibody response than that seen in the presence of water (compare rows 1.5 and 16), and was similar to that seen with nonconditioned mice (rows 11 and 12). By these criteria, there was no statistically significant conditioned immunosuppression in aged mice, a result consistent with an earlier preliminary report (Gorczynski et al. 1983). These data are not explainable simply in terms of the generally lower AFC responses in aged versus young mice. The response in US and (CS + US:CS) groups for aged mice is the same, and thus quite distinct from the finding in young mice (rows 3,4; 7,s). The “background” response in the US and (CS + US) groups of young mice can be brought to the lower levels seen in aged animals by using greater doses of cyclophosphamide at 21-day intervals. Nevertheless,

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TABLE 1 Immunosuppression in Normal or Aged Mice after Repeated Associative-Learning Trials of Saccharin:Cyclophosphamide

___

Pretreatment”

Age of mice” (weeks)

H,O:PBS WC) H,O:Cy (US)

H20:PBS WC)

Sacc:Cy (CS + US)

IgG AFC per spleend (range)

Row

NCS cs

0.71 + 0.20

30,300(26,970-34,200) 32,580(25.35&41,780)

NCS cs

0.58 + 0.19

18.650(11,690-29.410) 17.120(12,450-23.620)

A

NCS cs

0.63 + 0.21

33,620(26,120-43,040) 30.470(25.310-36,320)

6

16.160(11,690-22.250) 7,330(2,45&18,030)*

7 8

17,290(9,690-30,950) 14,370(9,23&22,250)

9 10

11,450(7,370--17,850)

NCS cs

Sacc:Cy (CS + US)

Sacc:PBS (CS)

Preference’ (for saccharin)

1 2

3

IO

Sacc:PBS (CS)

H,O:Cy (US)

Experimental treatmentA (test trial)

-0.40

+ 0.25

5

NCS cs

0.65 + 0.21

NCS cs

0.60 + 0.23

9.690(6.45&14,620)

I1 12

NCS cs

0.68 + 0.20

16,610(9,160-30,330) 17.270( 10.470-28.570)

13 14

10,220(7,690-13,490) 8,170(5.22&12,840)

I5 16

84

NCS cs

-0.19

+ 0.31

” Groups of IO- or 84-week-old C57BLi6 mice (16 mice per group) on a restricted daily water intake were given three trials (at 21-day intervals) of the indicated treatments. Cyclophoshamide (Cy) was injected at a dose of 100 mgikg. NC = nonconditioned. ’ 21 days after the last treatment, subgroups of 8 mice were exposed to water alone (HZ0 = NCS) or given saccharin (Sacc = CS) and HZ0 in a Sacc preference test. Consumption of each was assessed independently (see footnote c). 30 min after fluid intake all animals were injected ip with 5 x lOa SRBC in 0.5 ml PBS. ( Taste aversion in groups offered Sacc and H,O (see Materials and Methods for more details). ’ Geometric mean IgG AFUgroup at 7 days. * p < .05. Wilcoxon rank sum test.

subsequent reexposure to CS (in the (CS + US) group) still causes a significant conditioned immunosuppression (unpublished data), which is not seen in aged mice. It is important to note that the aged animals did show a taste aversion analagous to that seen with young mice (rows 7, 8 and 15, 16 in column 3). This failure to correlate taste aversion with conditioned immunity has been discussed elsewhere (Ader & Cohen, 1985; Gorczynski & Kennedy, 1987; Gorczynski, 1987). A final point worthy of note, and also discussed in an earlier report, is the greater range in immune responses seen in mice showing conditioned immunity. Thus, in the

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group shown in row 8 above, two of the eight animals demonstrated a response within the range of the control group (row 7) and were thus nonconditioned (for immunosuppressionthey did show equivalent taste aversion to other animals in this group). Adoptive

Transfer

of Cells into Syngeneic

Irradiated

Conditioned

Mice

In a previous study the role of cells/environment in producing the phenomenon of conditioned suppression of antibody responses was investigated by adoptive transfer experiments (Gorczynski, 1987). In order to explore whether the changes seen in conditioning between young and aged mice were in turn due to changes in the cells/environment of the responsive animals, the same approach was used. In the first of such experiments, cells from normal mice were injected into irradiated conditioned or nonconditioned young or aged mice and the effect of subsequent reexposure to conditioning cues was assessed. It is clear from Table 2 that irradiated conditioned young and aged mice retained their taste aversion to the CS (see rows 8 and 16). Also, as discussed elsewhere (Gorczynski, 1987), reexposure of conditioned young mice to cues (CS + US:CS) after transfer of naive cells led to increased IgG-AFC responses (rows 7, 8; compare with rows 3, 4). This effect was diminished when transfer was into conditioned aged mice (rows 15, 16), though the trend was in the same general direction. The next series of studies was designed to investigate the results of adoptive transfer of cells from conditioned mice into irradiated conditioned young (Table 3) or aged (Table 4) mice. Groups of mice received the standard pretreatment protocol outlined previously (see first column of Table 3) and were subsequently injected with cells from conditioned young mice which had (CS + US:CS; rows 1-6; 9-14) or had not (CS + US:NCS; rows 7, 8; 15, 16) been reexposed to CS before sacrifice. Rows l-8 in this table show data from responses in young recipients; rows 9-16 are the responses in aged mice. As noted earlier, there was no difference in taste aversion in the conditioned young/aged mice (rows 5-8; 13-16). Conditioned recipient animals receiving cells from (CS + US:NCS) mice developed enhanced IgG-AFC responses after CS exposure (rows 7,8; this effect was less pronounced with aged recipients, rows 15, 16: see also Table 2). When the response of spleen ceils from (CS + US:CS; CS + US:NCS) mice was compared in conditioned animals not exposed to CS (rows 5,7; 13, 1.5) it was apparent that responses seen in young recipients were equivalent to those seen in aged mice. Moreover, in both sets of recipients, cells from (CS + US:CS) mice gave greater responses than cells from (CS + US:NCS) mice. This somewhat anomalous finding has also been reported previously (Gorczynski, 1987). Finally, comparison of rows 5, 6 and 13, 14 emphasizes the observation made in Table 1. Adoptive transfer of cells from (CS + US:CS) mice to irradiated conditioned young, but not aged, recipients led to reexpression of conditioned immunosuppression if those recipients were reexposed to the CS after antigen challenge. Table 4 represents an experiment equivalent to that in Table 3 except that the donor cells in this case were from conditioned aged mice. There are a number of features of this Table which should be compared with

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TABLE 2 IgG-AFC in Irradiated Conditioned Young or Aged Mice Adoptively Transferred with Spleen Lymphocytes from Nonconditioned Mice

_ ---

Recipients”

Age Pretreatment

(weeks)

Experimental treatmen@ (post-transplant)

Taste aversion’

IgG AFC per spleen“ (range)

Row

NC

NCS CS

0.63 + 0.23

3360(3610-4320) 3150(269C-k3500)

I ?

US

NCS cs

0.69 + 0.26

3090(265&3500) 3330(282&3530)

3 4

cs

NCS CS

0.75 + 0.21

3670(2510-5380) 3240(272@-3530)

5 6

cs + us

NCS cs

+ 0.31

3070(2630-3500) 4880(3YlO-6060~"

I 8

NC

NCS cs

0.60 + 0.22

367OC3100-4360) 3520(2960-3570)

9 IO

us

NCS cs

0.54 + 0.19

3260(2870-3390) 3890(2750-5540)

II 12

NCS cs

0.68 + 0.26

3480(296&3500) 3360(272C-3680)

13 14

18

-0.33

94 CS

3670t2960-4580) I5 4520(3410-6060) 16 -~_-~ ’ Groups of 16 mice received three treatments as in Table 1 (see also Materials and Methods). Animals were given 900 R whole body irradiation 20 days after the last treatment and transplanted (iv) within 2 h with 20 x IO6 spleen cells pooled from 20 normal IO-week-old syngeneic mice, along with 5 x 10' SRBC (given ip). h-rl As for b-d of Table I. IgG-AFC (8 mice/group) were assayed at I2 days post-transplant. * p < .05. Wilcoxon rank sum test. cs + us

NCS cs

-0.18+

0.22

Table 3. First note that taste aversion is similar in aged and young animals (see also Tables l-3). However, using cells from aged (CS t- US:CS) mice, no significant conditioned immunosuppression after challenge with antigen and exposure to CS was seen. even in young recipients (rows 5, 6; see also rows 13. 14). There was a reduced tendency for cells obtained from (CS + US:NCS) mice to produce enhanced responses in conditioned recipients compared to similar cells from young (Table 3) mice (rows 7, 8; 15, 16). There was also only an insignificant increase seen in the IgG-AFC response in recipients not reexposed to the CS using cells of (CS + US:CS) mice compared with cells from (CS + US:NCS) mice (rows 13, 15: 5, 7). Altered Response of Lymphocytes from “Cued” Conditiorzed Young versus Aged Mice in Conditioned und Nonconditioned Environments 1s a Function of T Cell Subpopulations Tables 3 and 4 indicate that cells from (C’S + US:CS) mice gave an enhanced

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TABLE 3 IgG-AFC in Irradiated Conditioned Young or Aged Mice Adoptiveiy Transferred with Spleen Lymphocytes from Conditioned Young Mice Recipients”

4% Pretreatment

(weeks)

Experimental treatmentb (post transplant)

Taste aversion’

IgG-AFC per spleen (range)

Row

NC

NCS cs

0.63 + 0.23

4510(3530-5710) 4760(4100-5540)

1 2

us

NCS cs

0.61 + 0.19

4630(3970-5380) 4070(3350-4970)

3 4

18 cs + us

NCS cs

-0.36

cs + us***

NCS cs

-0.33 + 0.24

NC

NCS cs

0.61 t 0.29

4030(3 15~5250) 4390(359@-5380)

9 10

US

NCS cs

0.72 + 0.21

4140(337@-5120) 4660(3 13&6970)

II 12

+ 0.29

4810(4250-5490) 2540(196&3290)

*

2620(2070-3290) II 4970-(4260-5770) *

*

5 6 7 8

94 cs + us

NCS cs

-0.23

cs + us***

NCS cs

-0.31 + 0.27

+ 0.33

4560(377@-5660) 3970(296&5380) 2570( 1720-3830) I 3850(3290-4540)

*

13 14 15 16

-____ -Note. See footnotes u-d. Table 2. All 900-R recipient mice received 20 x IO6 spleen cells from (Cy:Sacc) conditioned mice (see text) which had been injected with PBS and reexposed to saccharin (CS) at 3,2, and 1 day before sacrifice. The group marked *** received cells from similarly conditioned mice not exposed to the CS before sacrifice. Spleen cell recovery in these two groups was equivalent (106 2 23 x 10’). IgG-AFC are geometric means for 8 mice/group. * p < .05, Wilcoxon rank sum test.

response on adoptive transfer to (CS + US:NCS) recipients. This effect was especially marked with cells from young mice. Data in Table 5 suggest that this activity in the T cell effect is explainable in terms of an increased “helper” population in these cells. Spleen cells were pooled from groups of 15 young or aged mice conditioned as described under Materials and Methods, and T and B cells were prepared from each pool. These cells were then adoptively transferred into irradiated young or aged nonconditioned mice in the combinations shown in Table 5 and antibody responses were measured at 12 days post-transplant. Regardless of the source of B cells used, T cells from (CS + US:CS) young mice gave a greater T-helper response than did T cells from (CS + US:NCS) young mice (e.g., rows 7-9, compared with 4-6; rows 16-18 compared with 1315). The T-helper response seen was in fact now equivalent to that obtained from

ALTERED

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IgG-AFC in Irradiated Conditioned Recipients” Pretreatment

Age

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TABLE 4 Young or Aged Mice Adoptively Transferred with Spleen Cells from Conditioned Aged Mice

Experimental treatment’ (post transplant)

Taste aversion’

IgC-AFC per spleend (range)

NC

NCS CS

0.67

+

0.29

3760(3010-3790) 3190(252&3790)

US

NCS cs

0.69

+

0.24

3350(28lCdO20) 3830(2960-4960)

Row

18

cs + us

NCS cs

-0.28

+

0.21

3610(2750-4720) 2670(1620-4400)

cs + us***

NCS cs

-0.33

+

0.28

3260(2310-4580) 4140(335&5250)

NC

NCS cs

0.65

+

0.21

3390(2710-4270) 3760(2820-5010)

us

NCS cs

0.54

+

0.19

3070(2570-3680) 3520(289&4320)

94

cs + us

NCS cs

-0.21

+

0.20

3810(3390-4320) 3320(2670-4150)

13 14

cs

NCS cs

-0.30

+

0.28

2720(210&3530) 3860(3310-4550)

15 16

+ us***

Note. Footnotes are as for Table 3.

NC mice (rows 1-3; 10-12) which were never exposed to cyclophosphamide. This diminished T-helper response of cells derived from (CS + US:NCS) mice is presumably the cause of the decreased AFC response seen with adoptive transfer of unfractionated spleen cells (rows 5, 7; 13, 15 in Table 3). When T cells were derived from aged mice, the increased T-helper effect seen with cells from (CS + US:CS) mice was less pronounced (see rows 22-24 and 19-21). This effect was independent of the host in which the response was measured (data not shown; see Table 6 later and 10-18). In a final study the response of mixtures of T and B cells from conditioned young or aged mice in conditioned young or aged recipients was measured (Table 6). Data for taste aversion in these animals is not shown but (see Tables l-4) there was no significant difference in taste aversion in the conditioned mice. T cells from young (CS + US:NCS) mice gave enhanced responses in irradiated conditioned young mice on reexposure to the CS regardless of the B cell source (rows 14). In contrast similar studies with T cells from young (CS + US:CS) mice showed immunosuppression in conditioned young mice on reexposure to the CS, again regardless of the B cell source (rows 5-10). Adoptive transfer into

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T Cells from Conditioned

__~

---

TABLE 5 Young Mice Confer More Help to B Cells Than Do Similar Cells from Conditioned Age Mice

-

Donor T cells”

Age (weeks)

Treatment

Donor B cell?

CS + US:NCS

18

NC

18

CS + US:NCS

94

cs + us:cs

CS + US:NCS 94

Treatment

IgG AFC per spleen’ (range)

NC CS + US:NCS cs + us:cs

5360(42106840) 4810(396@-5880) 5070(3910-6570)

I 2 3

NC CS + US:NCS cs + us:cs

3170(208&4820) 2650(210&3330) 2590( 156&4320)

4 5 6

NC CS + US:NCS cs + us:cs _~- -.~ NC CS + US:NCS cs + us:cs

5160(42106310)* 5090(3870-6770)* 4650(3260-6570)*

7 8 9

4970(4260-5770) 4960(3760-6570) 5350(4130-6840)

IO 11 12

NC CS + US:NCS cs + us:cs

3220(205&5060) 2960(212041 IO) 2630(221&3100)

13 14 I5

NC CS + US:NCS cs + us:cs ~~-__NC CS + US:NCS cs + us:cs

5350(4280-6630)* 4780(3660-6190)* 5 170(3970-6700)*

16 17 I8

1890(940-3750) 2320(1120-4820) 1760(870-3570)

19 20 21

Age (weeks) _~~

NC

18

M. GORCZYNSKI

Row

94

NC 2860( 167@4910) 22 CS + US:NCS 2750(2010-3750) 23 cs + us:cs 2820( 193@4060) 24 ~~ ~~ ~ -~ -rr.6 T cells were obtained by passage two times through nylon wool of cells from conditioned (3 X Sacc:Cy; see Materials and Methods) young (rows 4-9; 13-18) or aged (rows 19-24) mice reexposed to H,O (rows 4-6; 13-15, 19-21) or Sacc (rows 7-9; 16-18; 22-24). T cells were transferred iv into each of 6 900-R syngeneic IO-week recipient mice along with IO x lo6 B cells (anti-thy 1.2 treated spleen cells) of similar mice (see second column). All mice were challenged with 5 X 10” SRBC ip after spleen cell transfer. ’ As for d of Tables 2-4. Control animals (not shown) given only one of the five T cell preparations or one of the six B cell preparations gave a mean IgG-AFC response (over the 51 survivors of all I I groups) of 131(33-523). There was no obvious difference between any of the control groups. * p < .05. Wilcoxon rank sum test. cs + us:cs

conditioned aged mice reexposed to CS failed to reveal evidence for conditioned immunosuppression (rows I 1, 12). When T cells from (CS + US:CS) aged mice were transferred to conditioned young recipients with/without subsequent CS exposure, there was a minimal im-

ALTERED

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AGE

TABLE 6 Role of T or B Cells in Differences in Adoptive Transfer of Conditioned Responses Using Cells from Young or Aged Mice Transferred into Young or Aged Conditioned Recipients Donor

T cells”

‘he

_______-

Donor

B cellsb

Age Treatment

(weeks)

Recipients’

(weeks)

Treatment

(weeks)

18 -.__--

IgG-AFC per spleen” (range )

Row

2650(189&37101 4310(312&5940)*

I 2

18 cs

NCS cs --____ NCS cs

+ us:cs __-

CS + US:NCS 18

Treatment NCS CS

CS + US:NCS CS + US:NCS

__.-

As

2390( 165&3460) 4170(3040-5880)*

13 4

4860(361&6570) 2320(165@3360)*

5 6 7 8

18 cs

+ us:cs

18

NCS cs

4560(389&5380) 1970( 127lL30701*

cs

+ us:cs

NCS CS

4080(2860-5770) 1670(97CL2860)*

cs

+ us:cs

NCS cs

4250(338&5270) 3510(2820-4360)

cs + us:cs 94 _--~----.--18

94

9 10 II I? -

CS + US:NCS

--.~

NCS cs

3050(237lL3900) ?060( 153M780)

13 I4

NCS cs

3180(231M320) 2490( 197~31301

15 16

18

- -----94 94

cs

cs

+ us:cs

18

cs

t

us:cs

NCS cs

2890(217C-3870) ?050( 1460-2890)

17 I8

CS

+ US:NCS

NC.5 cs

2760(209&3640) 2210~1730-2810)

19 20

cs

+ us:cs --.

NCS cs

2520(186&3390) 2130(139~3230)

21 22

NCS cs

2690(207&3500) 2?10(166C~2950)

+ us:cs

18 -.--~94

cs

94

+ us:cs

Note. Footnotes u,h as for Table 5. c.d As for b.d of Table 3. Again (see also Table 5) mice receiving at sacrifice (Day 12: mean over all groups 86(33-221 j). * p < .OS, Wilcoxon rank sum test.

only

T or B cells produced

__-

no significant

-

23 24

IpG-AFC

munosuppressive effect seen, apparently independent of the B cell source (rows 13-18). Adoptive transfer into aged recipients failed to reveal any evidence for conditioned immunosuppression (rows 19-24). DISCUSSION

Results from a preliminary study indicated that aged mice were less able to demonstrate a conditioned immune response than normal (10 weeks old) animals (Gorczynski et al., 1983). This effect was independent of the documented poorer immune responses in aged mice (Kay & Makinodan, 1981). The present analysis attempted to document whether this decreased conditioned immunity is itself an inherent property of the cells in aged mice or is in some way due to the changes

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M.

GORCZYNSKI

in the local environment during the aging process. Given the recent data indicating that taste-aversion learning was itself a function of immune status in a model of autoimmune disease in mice, it was important to examine initially for any differences in taste aversion in the mice under study (Ader et al., 1987). As reported in Tables 1-4, there is no demonstrable difference in taste aversion in young or aged mice conditioned using the protocol described. In keeping with an earlier study (Gorczynski, 1987) it was found that conditioned young recipients reexposed to cues (CS + US:CS) and adoptively transferred with naive cells showed an enhanced not depressed immune response (Table 2). This predisposition of the host environment in conditioned mice to immune enhancement (the opponent process) (Eikelboom & Stewart, 1982; Greenberg, Dyck, & Sandler, 1984) is not seen as markedly in aged mice (Table 2; see also rows 7, 8 and 15, 16 in Table 3). In a similar vein, cells from (CS + US:CS) mice gave greater responses in NCS mice than cells from (CS + US:NCS) animals (rows 5, 7 and 13, 15 or Table 3; once again this difference was significantly blunted using cells from aged mice, Table 4). This effect was independent of the host environment. These data also imply some kind of “opponent process” occurring when cells from (CS + US:CS) mice are studied in a “normal” environment. Only when interactions occurred within a conditioned environment was conditioned immunosuppression seen when cells from (CS + US:CS) mice were transferred to (CS + US:CS) hosts-and this immunosuppression, in turn, was only documented when both cells and host mice were “young” (rows 5, 6 and 7, 8 of Tables 3 and 4). The data of Tables 5 and 6 suggest that the environmental differences documented, along with the enhanced or decreased response seen with cells of conditioned young/aged mice (seen in NCS or CS mice respectively), operate on, and are a function of, T cells (not B cells) in the transferred populations. These data also suggest that aged conditioned mice develop less augmented T-helper function than their young counterparts (Table 5); that there is a decreased ability of the aged environment to “signal” conditioned immunosuppression to “acceptor” cells (Table 3, 6); and that there is a decreased “acceptor” population in conditioned aged mice able to receive the appropriate signal (for immunosuppression) in a conditioned environment (Table 4, 6). The rationale for using opponent process theory as an explanation for the data described has been explained elsewhere (Gorczynski, 1987). In brief, a working hypothesis is that at least two opponent processes occur in conditioned mice: one leads to a greater splenic T-helper function after repeated conditioning trials, and the other produces an environment leading to more IgG-AFC when normal cells are transferred but less when cells from conditioned mice are transferred. The latter is suggested to be due to a cell (suppressor T-cell? See, for instance, Gorczynski et al., 1985) which is optimally activated only by the neurohormonal milieu of the conditioned environment, This hypothesis can adequately explain the differences between the young and aged animals described above. Whether such postulated differences exist in the cells/ environment of conditioned aged mice (versus similar young mice) remains to be tested. ACKNOWLEDGMENT This

work

has been

supported

by the MRC

of Canada

(Grand

MA-5440).

ALTERED

CONDITIONED

IMMUNOSUPPRESSION

WITH

AGE

317

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