Yamanashi Med. J. 6(1), 39--43, 199I
The Effect of Ageing on Cell Cycle Kinetics
in Mouse Spleen Lymphocytes
Hitoshi HosmNo, Tatsuya TAKEsmTA, Zentaro YAMAGATA, Sumio ImMA,
Akio AsAKA, and Makoto HiGuRAsHii)
Dopartment ofHealth Sciences, Yamanashi Medical College, i)Department ofMaternal and Child Health, Uniwersity ofTok>)o
School ofMedicine
Abs￡ract: We examined the inffuence of ageing on mouse spleen lymphocytes. Spleen
lymphocytes from young and old mice were cultured, and cell cycle kinetics were analyzed by the
method of sister chromatid differential staining (SCD). Proliferation index and mitotic index of
spleen lymphocytes were significantly lower in old mice than in young mice at several culture
incubation times. These findings shoNsr that age-related changes of cell cycle kinetics in spleen
lymphocytes are consistent with those of' in vitro studies using human lymphocytes. Thus, the
findings of" the present study strongly suggest that the present culture system using mouse spleen
lymphocytes is useful as an altemative model for studying age-related changes.
Key words: Ageing, Lymphocytes, Sister chromatid exchange, CeH cycle kinetics, Spleen
INTRODUCTION
SenesceRce is characterized by a general
reduction in the capacity of the ageing
organ-ism to carry out a wide variety of physiological
functioRs. AmoRg these reduced physiological
functions, that of the immune response in
ageing animals has been reported in several
studiesi)rm3). A study of cell proliferation of T
and B lymphocytes upoR stimula￡ion with
mitogens, such as Con A, phytohemagglutinin
(PHA), LPS and pokeweed mitogeB, showed
age-related declilte in RNA synthesis,3H-let}cine or 3I-I-thymidiRe incorporatioR4). There are only two studies, however, reporting
fiRdings for age-related reduction of cell liferatioR; Tice et al.5) reported impaired
pro-liferative response of peripheral lymphocytes Tamaho, Nakakoma, Yamanashi 409-38,Japan
Received October3,1990
Accepted January 8, 1991
from aged humans, aRd Schneider et al.6)
found similar findings in mouse boRe marrow
cells in vivo, using the sis￡er chromatid
dif-ferential staining (SCD) method. There are,
however, no reports examining age-related
chaRges of cell proliferation in mouse spleen lymphocytes using SCD. In the present st}.idy
we investigated cell cycle kinetics in cultured
spleen lymphocytes from young and old mice
and aRalyzed variations in kinetics over time during mitogenic stimulatioR.
MATERIALs AND METHeDs
Animals
BDFi male mice aged 100 weeks and l4
weeks were utilized. Mitogens
Concanavalin A (CoB A, 6 nglml) and lipo-polysaccharide (LPS, 30 paglml), respectively, were used as mitogens to establish cultt}res
40 H. Hoshine et al.
Proparation of cecltztres
The animals were sacrificed by cervical
dislocation and the spleens were removed. A cell suspensioB was prepared by gently teasing
the spleen in PBS (pH 6.8). The cells were
washed and resuspended in RPMI 1640
medium with L-glutamine (Gibco).
For the analysis of cell cycle kiRetics, 2×106
cells were added to 2 ml RPMI l640 containing 20% fetal bovine serum (FBS, Gibco)
inacti-vated with heat (560C 30 min.), O.02 mM
2-mercapto-e￡haRol (2ME), 1% penicil}in and s￡reptomycin, and LPS or Con A. The medium
used during the entire culture period a}so contained 3 paM bromodeoxyuridine (BrdUrd,
Sigma). The cultures were incubated for the
intervalsof12,18,24,30,36,42,48,54and60
hours at 370C in complete darkRess. Threehours before fixation, colcemid (final concen-tration 0.5 psgfml, Wako) was added to each
culture. The cells were theR collected by
centrifugation, exposed to IO mM Na-citrate
and 5e mM KCI hypotonic solu￡ion for l5
min., and fixed 3 times in ethanol: acetic acid (8:1).Sister chromatid dij[farential staining
Air dried chromosorne preparations were
made, and a modification of the
fluorescence-p}us-Giemsa method7) was applied to obtain
harlequin chromosomes. With the SCD
method, first, secoRd, third aRd later metaph-ases were clearly identified. The SCD method
used was the techRique developed by Latt8) and modified by Perry and Wolff9).
Proliferation iRdex (PI) and mitotic index
(MI) were Bsed to investigate cell cycle kinetics.
`
PI was calculated as fo11ows: PI==(1 × % metaphases at the first dividion + 2 × %me￡aphases at the second divisien + 3 × %
metaphases at the third or later division)1100.
MI was calculated as follows: MI== nttmber of metaphases per 1,OOO cells. ORe hundred cells
undergoing metaphase were examiRed to
ca}culate PI aRd 3,OOO cells were examined to
calculate MI. Statistical evaluation of data was by Student's t-test.
REsuLTs
Figures 1 and 2 show cell cycle kinetics in spleeB cells frorr} young and old mice spleeR stimulated with LPS and Con A, respectively.
It was found that the proportioRs of cells
tmdergoing Xi (first metaphase), X2 (secend r}etaphase) and X3ntF (third aRd later
metaph-ase) changed with ageing for both LPS and Con A stimulation. The proportion of ceils
undergoing Xi was significantly higher in the
spleens from old mice than iR ,those from
young mice, and was particularly higher at 30-,
36- and 42-hour incubation times. On the
other haRd, the proportion ofcells undergoing
Xs+ was significantly lower in the spleens from
old mice than in those from young mice at 42-, 48- and 54-hour incubation times.
Table l shows PI values for lymphocytes from young and old mice at all incubation
times, for LPS stimulatio'n, aRd Table 2 shows
those for Con A stimulation. PI values for both
LPS and Con A of lymphocytes from both
yotmg and old mice increased with iRcreasing incubatioR time. IR LPS-stimtdated cultures the PI va}ue of lymphocytes from yom}g mice was significantly higher than that of
lympho-cytes from old mice at 24, 42 aRd 54 hours
(p<O.Ol). In Con A-stimu}ated cultures, the differences between PI values of lymphocytes
from yoLmg and oJd mice are more
pro-Rounced thaR those fouBd in LPS-stimulated
cultures.
Figures 3 and 4 show changes in MI with
culture time for young and old rnice iB LPS-and Con A-stirr}ulated lyrnphocytes, respec-tively. No differeBce betweeR MI at 12 hours and that at I8 hours was observed for
lyinpho-cytes from either young or old rr}ice. After 24
hours, MI increased, and in LPS-stimulated
lymphocytes there were significant diffe}'ences
between lymphocytes from young aRd old
mice at 24, 42 and 48 hours (p<O.05). IR Con A-stimulated cultures MI values for lympho-cytes from young aRd old mice showed
slgni-co J` us o ur co < x tu < N us } =< Fo -ta o * too 80 60 40 20
o
1OO 80 60 40 20o
lOO 80 60 40 20o
The Effect of Ageing on the Cell Cycle Kinetics in
×I CELLS IOO
'Q t't. OYOUNG
Nt LYNxx･q- 40
'--..N.s..o 20
×2 CELLS9o
[rt o 100 t.t.E co 80<
x
cu 60<
F
ue 40=
t 20
b
9o
L O 100*
80 60 40 20 oMouse Spleen Lymphocytes
qi= :"--O't f1 .O-o. H N N N N b. xx ss NN O-xx s ssH"ON s Xl CELLS
e oLD
OYOliNG
41 --- "iK'pt
'o- .. ･O --,ts "- t ' o"" .･ 'e .d tt t. te
×3.CELLS ..t.."..$ ."'O ft.･･o e
"' e-'Fig. 1. t"'eH-s X2 CELLS o..'' -t t .o'' x -oeng.pt..-mu-tt8 24 30 36
CUL`rURECell cyGle Kinetics spleen lymphocytes mlce. Table 1. Proliferation (Mean±S.E.)
42 48 54 60
TIME (hr) in LPS-stimulated from youRg and oldIndex in N
o
x Ns sO.x s t x"-o- '" -+--'O LPS-stimulated .o"x' !/ 1/ ptxx x1 t1 ×3.CELLS .-'-O t-o" xY '(>"24 30 36 42 48 54 60
CULTURE TIME (hr)Fig. 2. Cell cycle Kinetics in Con A-stimulated
spleen lymphocytes from young and old mlce.
spleen lymphocytes from young and old mice
Incubation time (hr) 18 24 30 36 42 48 54 60
YOUNG
OLD
1.07±O.04 l.O4,!O.O1 I.l8±O.Ol:k* l.O7ri O.O2** 1.48±O.02 1.37±O.05 1.93±O.Ol 1.76±O.07 2.20±O.03** 1.91±O.04*}ge2.45±e.e3 2.66±O.02** 2.81±O.06 2.33±e.02 2.50±O.03** 2.76±O.e5
**: p<O.Ol.
"rable 2･ Proliferation Index mice (Mean±S.E.)
in ConA-stimulated spleen lymhpocytes from young and old
Incubation time (hr) 24 30 36 42 48 54 60
YOUNG
OLD
1.23±O.Ol 1.08±O.02:i:* 1.48±O.e5a:* I.}3±O.O2;t::÷: 1.77±O.02* 1.51±O.07* 2.25±O.06 2.oo±o.oe32.62±e.04* 2.75±O.03lj':* 2.81±O.04
2.37!O.Otl:t: 2.53±O.Oi** 2.70±O.O03
42 H. Hoshino et al. × us at
K
9
F
o
tE
75 50 25 o e OLD O YOUNG12 18 24 30 36 42 48
CULTURE TIME (hr)Fig. 3. Mitotic Index in LPS-stimulated spleen
lymphocytes from young and old mice.
× t.k.l
o
!
o
i:o
t
E
75 50 25 o e OLD o YOUNG Fig. 4.12 l8 24 30 36 42 48
CULTURE TIME (hr)Mitotic IRdex in Con A-stimulated spleeR lymphocytes from youRg and old mice.
ficant differences at 24 (p<O.O}), 86 (p<O.Ol)
and 48 hours (p<O.Ol).
DIscussloN
The observed age-related decliAes in PI and MI values after mitogenic stimulation accord
well with previous reports5)'6), indicating that in aged subjects a reduction ofthe responses to mitogens can be generally recognized. It is still
ambigueus, however, whe￡her this decline is due to qualitative andlor quantitative changes
of Iymphocy￡es, i.e., to a delayed cell cycle, to a
lengthened duratioR before entry into the first cell division, or to a decrease in the number of
responding cells. As early as at 24-hours
incubation, sigltifican￡ differences iR PI and
MI were observed between lymphocytes from
young and old mice, These results indicate that
in Iymphocytes from old mice there is
pro-longation ofthe time before eRtry into the first cell cycle after mitogen stimulatioR. It is also possible that there is a decrease in the number
of responding cells, as there were highly
significant differences in MI for lymphocytes
from y'eung and old mice. However, the
relative difference iR PI for lymphocyte,s fromthe two age groups did not seem to increase with culture time. This fiBding does not
sup-port the hypothesis that there is a delayed cell
cycle in lymphocytes from old mice. These
findiltgs showing age-related changes in cell
proliferation in spleen lymphocytes are
consis-tent with the changes reported in an in witro study using human lymphocytes5). The culture system usiRg mouse spleen lymphocytes in vitro described here is less complicated and more
easily managed thaB an in vitro culture sys￡em
using humaR lymphocytes. We therefore
strongly suggest that the system described here
is useful as an altema￡ive model for studying age-related changes. Comparison of responses to Con A and to LPS showed that for both PI
and MI the decrease with age was more
pronounced in CoR A-stimulated cultures thafi in LPS-stimulated cultures. LPS is reported to be the mitogen activating mainly B-cells}O),
while Con A activates rflainly T-cellsii). Thus, the present results support the ceRclusion that
ageing has a greater effect on T-cells than on
B-cells.
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related changes in Go-Gi transition and
The Effect of Ageing on the Ce}1 Cycle Kinetics in Mouse Sp}een Lymphocytes 43
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