Expression of interleukin 2 and the interleukin 2 receptor in aging rats

CELLULAR

IMMUNOLOGY

120, l-9 ( 1989)

Expression of lnterleukin 2 and the lnterleukin 2 Receptor in Aging Rats’ NIKIU J. HOLBROOK,*~~RAJESHK.CHOPRA,-~MICHAEL T.McCou,* JAMESE.NAGEL,~DOUGLASC.POWERS,-~WILLIAMH.ADLER,~ AND EDWARDL. SCHNEIDER* *Laboratory of Molecular Genetics and tClinical Immunology Section, Laboratory of Clinical Physiology, Gerontology Research Center, National Institute on Aging, 4940 Eastern Avenue, Baltimore, Maryland 21224 ReceivedMay27.1988; acceptedDecember 12, 1988 Lymphocytes of aged animals exhibit a marked decreasein proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreasedexpression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and ILZR expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23 187 and phorbol my&ate acetate (PMA). Proliferation was significantly decreasedin aged lymphocytes (30-50s) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23 187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation. 0 1989 Academic Press,Inc.

INTRODUCTION One of the most striking and consistent observations with mammalian aging is a decline in immune competency (l-3). Impaired proliferation of cultured T cells following stimulation with mitogens and antigens has been found in humans, mice, and rats (4-6). In both humans and mice, the decreasedproliferation of lymphocytes from aged donors in response to mitogen or antigen is correlated with a decreased ability to produce interleukin 2 (IL-2), a lymphokine required for T cell proliferation (7-10). In addition, in humans and mice, decreasedproliferation is associated with decreasedexpression of the receptor for IL-2 (IL-2R) (11). In rats the situation is less clear. Two studies examining IL-2 synthesis in response to mitogen have produced opposing results (6, 12) and IL-2R expression in aged rats has not been explored. In this study we examined proliferation and IL-2 and IL-2R expression in cultures of splenocytes from young and aged rats using two different activation stimuli: (i) ’ This work was supported in part by the MacArthur Foundation ResearchProgram on SuccessfulAging. ’ To whom all correspondence should be addressed.

000%8749/89 $3.00 Copyright 0 1989 by Academic Press,Inc. All rights of reproduction in any form reserved.

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concanavalin A (Con A), a plant lectin which acts nonspecifically through the surface T cell receptor complex, and (ii) dual treatment with the calcium ionophore, A23 187, and phorbol myristate acetate (PMA) which mimic the action of mitogen but bypass the need for T cell surface receptors ( 13). Although proliferation in response to these stimuli was significantly decreasedin spleen cells from aged rats compared to their younger counterparts, we observed no age-related decreasein either IL-2 synthesis or IL-2 mRNA levels. In addition, no decrease in lymphocyte IL-2R expression occurred with aging. These results indicate that the reduced proliferative capacity of lymphocytes from aged rats is not the result of impaired ability to genetically express IL-2 or IL-2R. MATERIALS AND METHODS Animals. Barrier reared male Fischer 344 rats 4 to 5 and 22 to 24 months of age were obtained from the animal colony maintained by Charles River Breeding Laboratories (Wilmington, MA) for the National Institute on Aging. Animals were housed at our facility for at least 2 weeks before being used in experiments. Male Wistar rats 4 to 5 and 22 to 24 months of age were obtained from the Gerontology Research Center Animal Colony. This colony is fully accredited by the American Association for Accreditation of Laboratory Animal Care and is monitored for viral, bacterial, and parasitic infections. Isolation of spleen lymphocytes. Rats were killed by ether inhalation and the spleens aseptically removed, teased apart, and minced in RPM1 1640 medium (GIBCO, Grand Island, NY) containing 2 mM glutamine, 20 mZl4Hepes, 5 X 10d5M 2-mercaptoethanol, 25 pg/ml gentamicin, and 10%fetal bovine serum (complete medium). Single-cell suspensions were obtained by sequential passagethrough 18-, 20-, and 22gauge needles. Erythrocytes were lysed by treatment with Tris-buffered ammonium chloride (17 mM Tris-0.73% NH&l, pH 7.4), and the cells were washed twice with medium. Viability was greater than 90% by trypan blue dye exclusion. Proliferation. Rat spleen cells (105/well) were cultured in 96-well flat-bottomed microculture plates (COSTAR, Cambridge, MA) in a total volume of 0.2 ml complete medium. Cultures were treated with either 5 pg/ml Con A (Sigma Chemical Co., St. Louis, MO) or 0.1 PM A23187 and 10 rig/ml PMA (Sigma) as indicated. Cultures were incubated at 37°C for 24 to 72 hr in a humidified atmosphere with 5% CO2 in air. Cell proliferation was quantitated by [3H]thymidine (1 @i/well, sp act 2 Ci/ mmol, DuPont, New England Nuclear, Wilmington, DE) incorporation during the final 18 hr of culture. Cells were harvested onto glassfiber filters, washed in succession with water and methanol, and air-dried prior to addition of scintillation fluid and analysis by scintillation counting. Assayfor IL-2 production. The amount of IL-2 in the cell cultures was quantitated by the ability of the culture supernatants to stimulate [3H]thymidine incorporation by IL-Zdependent CTLL-2 cells ( 14). For this bioassay 100 ~1of culture supernatant was twofold serially diluted in complete medium. Five thousand CTLL in a volume of 100 ~1were added to each well and plates were incubated at 37°C for 24 hr. For the last 4 hr of culture 1 PC1of [3H]thymidine was added to each well and [3H]thymidine incorporation into DNA was measured by liquid scintillation counting. Partially purified laboratory standard IL-2 was titrated in each experiment to provide a standard curve. The units of IL-2 were calculated by probit analysis and expressed in halfmaximal units ( 14).

IL-2 AND

IL-2R

EXPRESSION

IN AGING

RATS

3

Flow cytometric quantitation of IL-2R. The expression of membrane IL-2R on cultured splenocytes was examined with a murine IgG, monoclonal anti-rat IL-2R antibody (clone ART 18; Boehringer-Mannheim Biochemicals, Indianapolis, IN). The culture conditions were the same as described in the proliferation assay.After 72 hr of stimulation, cells were washed with phosphate-buffered saline (PBS) containing 1% fetal bovine serum and incubated for 30 min on ice with the anti-rat IL-2R antibody. The cells were again washed and incubated for 30 min with optimally diluted FITC conjugated rat anti-mouse IgG antisera (Boehringer-Mannheim). The cells were then washed, resuspended in a 1 pg/ml propidium iodide (PI) solution, and analyzed on a flow cytometer (FACScan Becton Dickinson, Mountain View, CA). Histograms were generated from live cells (i.e., those not stained by PI) as determined by forward scatter and fluorescein fluorescence. Preparation and analysis ofRNA. Spleen cells were cultured 24 hr in 150 cm2 flasks at a density of lo6 cells/ml ( lo8 total cells) with either Con A or A23187 and PMA. Lymphocytes were collected by centrifugation and washed in ice-cold PBS, pH 7.4. Total cellular RNA was extracted in 4 A4 guanidinium isothiocyanate and pelleted through a cesium chloride cushion (15). For Northern blot analysis 20 pg of total RNA, denatured by heating to 55°C in 50% formamide, was electrophoresed on a 6% formaldehyde gel and transferred to a Gene Screen Plus membrane (DuPont, New England Nuclear, Boston, MA). Membranes were hybridized to mouse IL-2 (provided by Dr. Tadatsugu Taniguchi, Japan Foundation for Cancer Research, Tokyo, Japan) (16) and human actin (provided by Dr. Laurence H. Kedes, Stanford University School of Medicine, Palo Alto, CA) cDNA probes labeled with [32P]CTP using the random primer method (17). Hybridization was carried out at 42°C for 24 hr in 50% formamide, 10% sodium dodecyl sulfate (SDS), 1 A4 sodium chloride, 10%dextran sulfate, and 200 pg/ml salmon sperm DNA. After hybridization the blots were washed at room temperature for 5 min in 2X SSC( 1X SSCis 0.15 M NaCl, 0.0 15 M sodium citrate, pH 7.2) followed by two washesat 60°C for 30 min in 2X SSC-0. 1% SDS and two more washes at room temperature in 0.1 X SSC. Damp filters were exposed at -70°C to Kodak XAR film (Eastman Kodak, Rochester, NY) with intensifying screens.Quantitative analysis of autoradiograms from Northern blot analysis was performed using a scanning densitometer with the results presented as arbitrary units corresponding to the relative areasunder the peaks. IL-2 binding assay.Splenocytes were extensively washed with RPM1 1640 containing 2% FBS, incubated for 2-3 hr in IL-2-free complete medium at 37°C and then again washed twice. This procedure has previously been demonstrated to remove all membrane-bound IL-2 (18). [1251]IL-2 binding to the cells was assayed using the method of Robb et al. (19) with minor modification. Briefly, 50-~1 serial dilutions ( 1.4-400 PM) of ice-cold [ 1251]IL-2(DuPont, New England Nuclear) was mixed with 5 X lo5 splenocytes that had been suspended in 50 ~1 of ice-cold complete medium with 10 mM Hepes and placed in 1.5-ml conical microcentrifuge tubes (Sarstedt, Inc., Princeton, NJ). The tubes were gently vortexed and then mechanically rotated at 4°C about their longitudinal axis for 30 min. Following this incubation, I ml of ice-cold medium was added to each tube and they were centrifuged at 9000g for 20 sec. The supernatant was carefully removed and saved for determination of the amount of unbound IL-2. The cell pellet was resuspended in 100 ~1ice-cold medium, layered over 300 ~1of dioctyl phthalate:dibutyl phthalate oil mixture (1: 1.1, v/v; Aldrich Chemical, Milwaukee, WI), and centrifuged at 9000g for 90 sec.The supernatant

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HOLBROOK ET AL.

Con A

A2a3,87 PMA

FIG. 1. Con A- and A23 1Wand-PMA-induced proliferation in splenocyte cultures of young and aged Fischer 344 rats. [‘H]Thymidine incorporation was measured 48 hr after stimulation of lymphocytes with Con A or A23 187 and PMA.

was discarded and the tips of the microcentrifuge tubes containing the cell pellet were cut off and used to determine cell-bound radiolabeled IL-2. The high-affinity IL-2R/ cell were determined from the binding curve and equilibrium dissociation constant (&) by Scatchard analysis ( 19). RESULTS

ProliferativeResponsesof SpleenCells of Young and Aged Fischer 344 Rats Preliminary studies indicated that there were no differences in the optimal stimulus dose or the kinetics of the response between young and aged rats. Levels of [3H]thymidine incorporation by spleen cells from young and aged Fischer 344 rats in response to 5 wg/ml Con A or 0.1 PM A23 187 plus 10 rig/ml PMA are shown in Fig. 1. The results are expressed as the mean & SEM for six separate experiments for each age group. Consistent with previous reports the proliferative response of spleen lymphocytes from the aged rats to Con A was decreased (30-50s) relative to the levels seenwith lymphocytes from young rats.

IL-2 Production and IL-2 mRNA Expression in Young and Aged Fischer 344 Rats Stimulated with A2318 7 and PMA Since previous studies suggestedthat the decreasedproliferative capacity of mitogen-stimulated lymphocytes from aged rats is due, at least in part, to their inability to produce sufficient IL-2 we examined IL-2 synthesis by spleen cell cultures from young and aged rats stimulated with either Con A or A23 187 and PMA. Preliminary experiments determined the optimal dosesof A23 187 and PMA for IL-2 production and IL-Zspecific mRNA accumulation to be 0.5 PM and 10 rig/ml, respectively, for cells from both aged and young animals. Maximum IL-2 mRNA levels were seen after 18-24 hr of stimulation while IL-2 activity in the media peaked between 24 and 48 hr of culture. Therefore, supernatants for IL-2 activity and cells for RNA isolation were harvested 24 hr after stimulation with A23 187 and PMA. In Fig. 2 the results of 13 separateexperiments comparing IL-2 production and IL2 mRNA expression in spleen lymphocytes from young and aged Fischer 344 rats

5

IL-2 AND IL-2R EXPRESSION IN AGING RATS ”

500

c07 c

400

2

300

A c .?

200

2 l-4 I A

B 6000 I

A

I

4000 a: 5 ‘; 3000

EE

N ; 2000

100

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0

3 1

2

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4

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6 7 6 Rat Pair

9

10 1112

13

z 1000 4 G 01

1

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6 Rat

7

6

9 10 1, 12 13

Pair

FIG. 2. IL-2 expression in A23 187-and-PMA-stimulated lymphocyte cultures of young and aged Fischer 344 rats. (A) IL-2 activity was measured in supernatants of cultured lymphocytes as described in the text. (B) IL-2 mRNA expression was determined by densitometric tracing of Northern blots for each of the animal pairs. The units reflect relative differences in mRNA levels among the 13 animal pairs.

stimulated with A23 187 and PMA are shown. For each experiment the values shown are derived from a single animal. Although the overall mean IL-2 activity in culture supernatants was somewhat higher for supernatants of cells from aged rats than from younger rats, no significant differences were seen between the two groups (Fig. 2A). Densitometric analysis of IL-2 mRNA levels in the two groups produced similar results (Fig. 2B). Considerable variation in the amount of IL-2 synthesized was observed for both the young and the aged groups. However, this variation was not attributable to differences in overall gene expression. Densitometric analysis of Northern blots (Fig. 3) revealed that while there was a 6-fold variation in IL-2 mRNA levels among the 13 rats, there was less than a 1Sfold variation for actin mRNA expression in the same mRNA samples (except for the lane designated 3-Oin which only 10 pg rather than 20 pg RNA was loaded onto the gel). IL-2 Production and IL-2 mRNA Expression by Con A-Stimulated Cellsfrom Young and Aged Fischer 344 Rats The levels of IL-2 activity and IL-2 mRNA expression in Con A-stimulated young and aged rat spleen cells were 5- to IO-fold lower than levels seen using A23 187 and PMA (Fig. 4). Young and old animal pairs l-7 used in these studies were the same as those utilized for A23 187 and PMA stimulation in Fig. 2. The relative differences seen among specific animal pairs with Con A stimulation were similar to those observed with A23 187 and PMA. As with A23 187 and PMA treatment we observed no defect in IL-2 bioactivity (Fig. 4A) or IL-2 mRNA expression (Fig. 4B) in Con Astimulated cells from aged rats relative to young rats. Since strain differences in the age-related decline in immune function of mice have been reported (20), we also examined cells from a second rat strain, Wistar, for their ability to expressIL-2 upon stimulation with either A23 187 and PMA or Con A. We did not observe any age-related impairment in IL-2 expression in Wistar rat cells with either A23 187 and PMA or Con A stimulation. IL-2R Expression on the Surfaces of Splenocytesfrom Young and Old Rats We examined IL-2R expression on the surface of splenocytes from old and young rats utilizing a monoclonal antibody to the rat IG2R and flow cytometry. There was

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HOLBROOK ET AL.

FIG. 3. Northern blot for IL-2 and actin mRNA expression in selected animal pairs. Lanes A and B are mRNA from unstimulated lymphocytes from a young and an aged rat, respectively; lanes 1-Oto 6-Oare mRNA from stimulated aged rat lymphocytes; lanes 1-Y to 6-Y are mRNA from stimulated young rat lymphocytes.

a significantly lower number of cells recovered from old rat splenic cell cultures than from young rats with both A23 187 and PMA (57%) and Con A (48%) stimulation (Fig. 5). However, the percentage of IG2R positive cells in elderly rat cell cultures was slightly (18%) but not significantly decreasedwhen compared to young rat cell cultures (Fig. 6). We next examined, by [ ‘*‘I]IL-2 binding, the expression of high-affinity IL-2R on the surface of stimulated cells from old and young rats. Scatchard analysis revealed that the receptors on stimulated lymphocytes of old and young rats had similar affin-

2

3

4

Rat Pair

5

6

7

5

6

Rat Pair

FIG. 4. IL-2 expression in Con A-stimulated lymphocyte cultures from young and aged Fischer 344 rats. IL2 activity and mRNA units were determined as described for Fig. 2. Animal pairs 1 to 7 correspond to the same animal pairs in Fig. 2. (A) IL-2 activity in supematants. (B) IL-2 mRNA expression in treated cultures.

IL2 AND IL2R EXPRESSION IN AGING RATS

G- 70 b 60 5 50 e 40 P 8 30 P 20 = 0” 10 -I

0

7

r-t

l-h

A231 87

Con A

and

PMA

FIG. 5. Cell recovery in 72-hr Con A- and A23 187-and-PMA-stimulated cell cultures as measured by trypan blue dye exclusion.

ities as determined from the equilibrium dissociation constant &. No difference in the number of high-affinity IL-2R per cell was seen between old and young A23 187and-PMA-stimulated rat lymphocytes. In fact, the Con A-stimulated old rat lymphocytes had an approximately twofold greater number of high-affinity IL-2R/cell than levels found on young rat lymphocytes (Fig. 7). DISCUSSION IL-2 plays a key role in the regulation of many T cell functions (l-3). In mice, decreased cellular proliferation in response to mitogen or antigen stimulation is clearly correlated with the decreasedsynthesis of IL-2 (8). Addition of exogenous IL2 restores the proliferative capacity as well as a variety of other immune functions in mice, but not in rat or human cells (7,8). In an earlier study Gilman et al. (6) found that unlike cells from old humans and mice, cells from aged rats produced normal levels of IL-2 in responseto the mitogens Con A and phytohemagglutinin. However, these same cells were deficient in their ability to produce IL-2 in response to allogeneic or antigenic stimulation. Such differences in responsiveness suggest that altered cell surface receptors for antigen may account for the decreasedIL-2 production by cells from aged animals. However,

VI = 3

60

f E

60

: P

40

s

Con A

3

A231 87 and

PMA

FIG. 6. IL-2R expression in Con A- and A23 187-plus-PMA-stimulated lymphocyte cultures of young and aged Fischer 344 rats.

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HOLBROOK ET AL. 1200

O4-6monfh. I2 2-24mmlh.

= f

1000

I

\ Kl

600

.! A 2

600

E

400

2 c .P I -Illi

200 0

Con A

A231 87 and

PMA

FIG. 7. High-affinity IL-2R expression in Con A- and A23 187-and-PMA-stimulated cell cultures.

more recently, Cheung et al. ( 12) reported that IL-2 production was significantly decreased in aged rats stimulated with Con A. They found that following stimulation with Con A, production of bioactive IL-2 decreased67% between 5 and 29 months of age. This decline paralleled an 85% decline in IL-2 mRNA expression, leading them to conclude that lymphocytes from aged rats had a decreasedability to genetically expressIL-2 (2 1). Our results contrast sharply with those of Cheung and colleagues ( 12,2 1). Despite a significant reduction in mitogen-induced proliferation of spleen cells from aged rats relative to young ones, we observed no impairment of IL-2 expression in cells from aged rats stimulated with Con A. Furthermore, no age-related alterations in IL-2 biologic activity and mRNA expression were observed in two different strains of rats and with two separate stimuli, Con A and A23 187 plus PMA. In fact, in our studies and those of Gilman et al. (6) Con A-stimulated lymphocytes from older rats actually accumulated higher levels of IL-2 activity in cultured supernatants than those found with cells from young rats. Furthermore, lymphocytes of both aged and young rats were equally capable of producing up to lo-fold higher levels of IL-2 when stimulated with A23 187 and PMA. Thus, we conclude that aged rat splenocytes do not appear to have any defect in producing either IL-2 or mRNA for IL-2. Gilman et al. (6) found that mitogen-activated “old” cells do not absorb IL-2 as efficiently as mitogen-activated “young” cells, thus causing IL-2 to accumulate in lymphocyte cultures of cells from older rats. On the basis of these findings they further hypothesized that mitogenic stimulation does not induce “old” T cells to produce a sufficient number of surface IL-2R required for subsequent proliferation. However, they did not actually measure the IL-2R levels directly. Our studies suggestthat the mechanism responsible for the decreasedproliferative capacity of cells from old rats is more complicated. We observed only a slight decreasein the total number of IL2R positive lymphocytes from old rats relative to younger animals. However, it was not of the magnitude that might account for the substantial age-related decline in proliferation. There was also no decreasein high-affinity IL-2R expression on cells from the old rats. In fact, with Con A stimulation, there was an increase in highaffinity IL-2R expression. There are many other potential qualitative differences which could explain the decline in mitogen-stimulated proliferation seen with aging in rat lymphocytes. For example, there might be an age-related defect in IL-2R function at the post-IL-2 binding stage(i.e., internalization and signal transduction). Further studies are needed to addressthis possibility as well as other factors involved in the processof mitogen stimulation of cell proliferation.

IL-2 AND IL-2R EXPRESSION IN AGING RATS

9

Of interest in our studies was the finding that while Con A stimulation resulted in 2-fold greater proliferation relative to A23 187 and PMA (Fig. I), A23 187 and PMA induction produced 5- to IO-fold higher levels of IL-2 and IL-2 mRNA relative to Con A (Figs. 2 and 4). However, the two treatments resulted in similar IL-2R expressions (Fig. 6). These findings indicate that while mitogen-induced IL-2 and IL-2R synthesis and mitogen-induced proliferation are all related they respond differently to diverse stimuli. In summary, our results showed that lymphocytes of aged rats responded similarly to those of young rats in the expression of IL-2 and IL-2R following mitogenic stimulation. Thus, the defect responsible for decreasedproliferative capacity of mitogeninduced aged lymphocytes is probably related to other aspectsof T cell activation. REFERENCES 1. Makinodan, T., Fed. Proc. 31, 1239, 1978. 2. Kay, M., and Makinodan, T., Prog. Allergy 29, 134, 1981. 3. Thoman, M. L., J. Amer. Geriatrics Sot. 33,78 1, 1985. 4. Weksler, M., and Hutteroth, T. H., J. Clin. Invest. 53,99, 1974. 5. Adler, W. H., Takiguchi, T., and Smith, R. T., J. Zmmunol. 107, 1357, 1971. 6. Gilman, S. C., Rosenberg, J. S., and Feldman, J. D., J. Zmmunol. 128,644, 1982. 7. Gillis, S., Kozak, R., Durante, M., and Weksler, M. E., J. Clin. Invest. 67,937, 1981. 8. Thoman, M. L., and We&e, W. O., J. Zmmunol. 128,2358, 1982. 9. Chang, M., Makinodan, T., Peterson, W. J., and Strehler, B. L., J. Zmmunol. 129,2426, 1982. 10. Nagel, J. E., Chopra, R. K., Chrest, F. J., McCoy, M. T., Schneider, E. L., Holbrook, N. J., and Adler, W. H., J. Clin. Invest. 81, 1096, 1988. 11. Vie, H., and Miller, R. A., Mech. Ageing Dev. 33,3 13, 1986. 12. Cheung, H. T., Twu, J. S., and Richardson, A., Exp. Gerontof. 18,451, 1983. 13. Truneh, A., Albert, F., Golstein, P., and Schmitt-Verhulst, A. M., Nature (London) 313,3 18, 1985. 14. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A., J. Zmmunol. 120,2027, 1978. 15. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J., Rutter, W. J., Biochemistry l&5294, 1979. 16. Kashima, N., Nishi-Takaoka, C., Fuji@ T., Taki, S., Yamada, G., Hamuro, J., and Taniguchi, T., Nature (London) 313,402, 1985. 17. Feinberg, A. P., and Vogelstein, B., Anal. Biochem. 132,6, 1983. 18. Cantrell, D. A., andsmith, K. A., J. Exp. Med. 158, 1895, 1983. 19. Robb, R. J., Mayer, P. C., and Garlick, R., J. Zmmunol. Methods 81, 15, 1985. 20. Licastro, F., and Walford, R. L., Mech. Ageing Dev. 31(2), 171, 1985. 21. Wu, W., Pahlavani, M., Cheung, H. T., and Richardson, A., Cell. Zmmunol. 100,224, 1986.