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IMMUNOLOGICAL COMMUNICATIONS, 4 (5), 453-463 (1975)
Apollonj, C. Vasile, A. F l o r i d i and F.C.
L1.C.
Laboratorio d i Imnunologia.
Natali
I s t i t u t o Regina Zlena. Rome.
Italy. Abstract Euman p e r i p h e r a l blood lymphocytes (PBL) stimulated by phytohenoagglutinin (PEIA) manifest augmented g l y c o l y s i s and o x i d a t i o n of g l u ~ o s e - l - ~ 4 C ,i n d i c a t i n g an increased u t i l i z a t i o n of the pentose pathway. L a c t i c aci d production, a s index of increased g l y c o l y s i s , follows t h e same k i n e t i c of thymidine incorporation and can be e a s i l y q u a n t i t a t e d by an enzymatic assay.
-in vitro
Introduction
The e f f e c t o f mitogens on lymphocytes has been extens i v e l y i n v e s t i g a t e d d u r i n g the p a s t y e a r s because of valuable a i d i n understanding a c t i v a t i o n of i m u n o c y t e s by a n t i g e n s and c e l l cooperation. D i f f e r e n t mitogens so f a r used appear t o a c t s e l e c t i v e l y on lymphocytes s u b c l a s s e s and t o induce s e v e r a l changes i n the biochemical a c t i v i t i e s of lymphoid
cells. I n p a r t i c u l a r , v a r i a t i o n s i n n u c l e i c acid (1),phosphol i p i d s (2, 3 ) , cAbP (4) and c e l l u l a r s u b f r a c t i o n netaboliarn
( 5 , 6) o f lymphocytes have i n f a c t been described folloiving t h e i r c o n t a c t with l e c t i n s .
This study has been performed
454
APOLLONJ ET AL
with two aims i. t o gain more information on t h e e x t e n t and types of changes i n t h e carbohydrate metabolism occurring i n
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/06/15 For personal use only.
PHA stimulated PBL, ii. t o attempt a q u a n t i t a t i v e e s t i m a t i o n of such changes.
The r e s u l t s obtained i n d i c a t e t h a t increased
glycolysis
i n PBL even a f t e r s h o r t c o n t a c t ( 5 min.)
OCCUTS
with PHA. These changes whioh precede tho l a t e events of b l a s t transformation and DNA s y n t h e s i s may be q u a n t i t a t i v e l y estimated by means of l a c t a t e production. Llaterials and Methods
Human p e r i p h e r a l blood mononuclear o e l l s (average 90$ lymphocytes and l e s s than lC$ monocytes) were i s o l a t e d from heparinized blood of apparently healthy donors by oentrifugat i o n on F i c o l l (Pharmacia)-Urovison (Shering) gradient.
The
i s o l a t e d c e l l s were mashed twice with medium WEdI 1640
sup
plenented with 5% C O ~ , containing 100 pd.1
FCS, 2 x
mtr/ml glutamine and
of Gentamicin (Shering). Lymphocytes
were incubated with PHA f o r d i f f e r e n t periods of time and divided i n two a l i q u o t s f o r examination of n u c l e i c a c i d and carbohydrates metabolism r e s p e c t i v e l y . DNA s t i m u l a t i o n was asBayed i n f i c r o t i t e r t r a y s (Falcon IS-FB-96) i n which 6 x 105 c e l l s p e r w e l l , were p l a t e d i n t r i p l i c a t e i n presence of 1020 pl of ?HA (Difco 25 mg/ml). Optimal concentration of PHA inducing maximal tiiymidine incorporation was chosen according t o dose response experiments. Cells were c u l t u r e d i n presence of PHA f o r 72 hours i n a humidified atmosphere c o n t a i n i n g
5%
C02. Each c e l l c u l t u r e was l a b e l l e d f o r 16 hours with 0.5 uCi of t r i t i n t e d t i m i d i n e . L a b e l l i n g was stopped by a d d i t i o n of
5 ml of cold calinc. C e l l s were f i l t e r e d on ‘.‘Fhatnann 3X: paper
CARBOHYDRATE METABOLISM IN LYMPHOCYTES
455
and thoroughly washed with an excess of
lo$
TCA, 95$ e t h a n o l ,
and e t h e r . The d r i e d f i l t e r s were counted i n 10 ml toluene-?ermafluor 25 x (Dackard) s c i n t i l l a t i o n fluid i n a IIuclear Chicago
MKI s c i n t i l l a t i o n counter.
Lymphocytes used f o r carbohydrate
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metabolism d e t e r n i n a t i o n a f t e r PHA c o n t a c t $:ere xished twice with Krebs-Ringer bicarbonate b u f f e r . In order t o determine glucose oxidation, c e l l s were incubated i n d u p l i c a t e i n 3 m l of Krebs-Ringer bicarbonate b u f f e r c o n t a i n i n g 0.25 pCi of l a b e l l e d s u b s t r a t e . Incubations were c a r r i e d out i n stoppered f l a s k s with a removable c e n t e r w e l l , a t 37°C i n
95$
5% C02
02.
a t shaking r a t e of 100/min. A f t e r one hour 1 n l of Hyarnine (Packard) was i n j e c t e d i n t o the c e n t e r !:re11 and the r e a c t i o n
was stopped by a d d i t i o n of 0.3 m l of 4
The f l a s k s 4' were shaken an a d d i t i o n a l hour i n order t o c o l l e c t I4CO2. The HClO
Hyamine w a s then q u a n t i t a t i v e l y t r a n s f e r r e d t o s c i n t i l l a t i o n v i a l s and counted i n a Nuclear Chicago LXI l i q u i d s c i n t i l l a t i o n spectrometer. Determinations of anerobic i;lycolysis were c a r r i e d out i n 25 ml Erlenmeyer f l a s k s a t 3 7 O C for 60 min. i n a Dubnoff b a t h a t shaking r a t e of 100/min. ICrebs-Rinmr b i c a r bonate with 10 mM glucose was used a s r e a c t i o n nedium and t h e gas phase was
95% N2, 5% C02.
A t the end o f the incubation
time the r e a c t i o n was stopped with 0.25 m l of 2.5
12 H C l O
4
the c o n t e n t of the flasks c e n t r i f u g e d a t 16.000 rpm f o r
and
15 min.
The n e u t r a l i z e d supernatant were used for c n z p a t i c determinat i o n of lactate (7). Results I n o r d e r t o e v a l u a t e the e f f e c t s of PHA on Ijmphocytes, carbohydrate metabolism, t h e a c t i o n of the l e c t i r . t i o n of g l ~ c o s e - 6 - ~ ~ Cglucose-l-14C, ,
m oxitla-
and anaerobic g l y c o l y s i s
456
APOLLONJ ET AL.
has been studied. As shown i n Table 1, PRA does not modify the
-
production r a t e of 14C0, from e ; l ~ c o s e - 6 - ~ ~ Cthus , indicating t h a t under these experimental conditions the oxidative metabolism i s not a f f e c t e d . Since i t is w e l l IC~OQM t h a t
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PHA increases
3H thymidine incorporation i n t o nucleic
acid, the e f f e c t of PHA on g l u c o ~ e - l - ~ ~ u tci l i z a t i o n was tested. This is j u s t i f i e d by the f a c t t h a t g l u c ~ s e - l ~ ~ C e n t e r s pentose pathway supplying metabolites f o r nucleic acid synthesia. As shown i n Fig. 1 the oxidation of glucosei e g r e a t l y increased by PEA and, as observed f o r t h e
l-'C
glycolysis, the e f f e c t is already appreciable a f t e r t e n minutes.
O f i n t e r e s t is the d i f f e r e n t k i n e t i c s of 14C0 produc2 t i o n folloming PHA contact. I n f a c t i n unstimulated c e l l s , inoreases as YCO 2 lymphocytes LQCO
2
l i n e a r function of time, whereas i n stimulated
production is higher d u r i n g the first minutes
and becomes l i n e a r only a f t e r t h i s period. On the b a s i s of these data i t was possible t o perform a q u a n t i t a t i v e estimation of
TABLE 1 Effect of PK4 on glucose-6- 14C oxidation by PBL TIME
UNSTDRLATED
STIMULATED
138 + 12
10'
135 f 8
20'
265 2 1 5
270 f 9
40 '
495
+ 28
520 + 35
60'
720 + 51
768 + 49
(O)
Cpm/lO 7 c e l l s . Each value
(O)
standard deviation i s averaged
from three d i f f e r e n t experiments performed i n duplicate.
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CARBOHYDRATE METABOLISM I N LYMPHOCYTES
45 7
S t i m u l a t i o n time ( m i n ) FIGURE 1 Oxidation of s1u~ose-1-14~ i n PHA stimulated and n o n s t i n u l a t e d (M ) PBL.
(-1
Each value 2 standard devia-
t i o n is averaged from t h r e e d i f f e r e n t experiments performed i n duplicate.
the pentose cycle i n stimulated and unstimulated lymphocytes. production r a t e from ~ l u c o s e - l - ~ ~ C 2 gives i n f a c t t h e r a t e of pentose oxidation
The d i f f e r e n c e between 1 4 C 0 and glucose-6-14C directly.
Although t h i s method allows only a semi-quantitative
estimation, i t m u s t be s t r e s s e d t h a t under the present
ex-
peri:nental c o n d i t i o n s r e s u l t s were h i g l y reproducible. A 8
APOLLONJ ET AL.
458
3hOn i n fig. 2 PHA g r e a t l y s t i m u l a t e s pentose pathway i n a m merit
with the increased s y n t h e s i s o f n u c l e i c a c i d . The higher
g l ~ c o s e - l - ~ ~oxidation C can be t e n t a t i v e l y r e l a t e d t o a g r e a t e r request of metabolites needed for the n u c l e i c acid synthesis,
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r a t h e r than a d i r e c t e f f e c t of PEL4 on enzyme of pentose cycle. The anaerobic production of l a c t i o acid by lymphocytes under
3
2
-
0 I
0 X
E
n v
1
10
20
30
40
50
00
Stimulation t i m e ( m i n ) FICURZ 2
Quantitative evaluation of pentoso cycle i n stimulated (O-----o) and unstimulated ( 1 ) PBL. The f i g u r e s have
been c a l c u l a t e d on the d a t a of Table 1 and Fig. 1.
CARBOHYDRATE METABOLISM IN LYMPHOCYTES
459
PHA s t i m u l a t i o n i s reported i n Fig. 3. As it nay be seen g l y c o l y s i s i s a l r e a d y double a f t e r t e n minutes.
The percent
i n c r e a s e is independent from s t i m u l a t i o n time, being the same a l s o a f t e r 60 min.
A comparison between l a c t i c a c i d produc-
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t i o n and thymidine i n c o r p o r a t i o n i s shown i n Fig.
4,
where a
f i x e d number of lymphoid c e l l s was stimulated f o r 20 minutes with i n c r e a s i n g
I
/
PHA concentrations. Percent i n c r e a s e i n l a c t i c
.
Stimulation t i m e (mini FIGURE 3 Anaerobic production of l a c t i c aoid by PW s t i m u l a t e d
(M)
and unstimulated (-)
PBL.
Each value 2 standard
d e v i a t i o n i s a.vera@d from t h r e e d i f f e r e n t experiments performed i n d u p l i c a t e .
AE'OLLONJ ET AL.
460
1
8000. I
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6000
50 40
O/o
increase
lactate production
30
20
I
10
I
5
2o
PHA
-Thymidine
___
pl
100
uptake
L a c t a t e production FIGURE 4
Effect of PHA concentration on thymldine uptake and l a c t a t e production by PBL.
acid production and thymidine uptake
followed the same response.
R e l a t i v e l y hich concentrations of l e c t i n d i d n o t induce appreciable chances i n DNA and carbohydrate metabolism. Discussion Lynphoid c e l l s from d i f f e r e n t s p e c i e s have been shown t o be stimulated i n v i t r o by non-clonal rnito,-ns
such a s PHA, and
t o undergo trannnfomation followed by DIJA s y n t h e s i s ( 8 ) . These e f f e c t s have inclicstcd some d e p e e of s p e c i f i c i t y f o r e i t h e r 3
CARBOHYDRATE METABOLISM I N LYMPHOCYTES
461
o r T lymphocytes (9, lo) and have provided a great d e a l of knowledge i n t o a c t i v a t i o n mechanisms which may be involved i n the immune response.
Uethods of d e t e c t i o n of lymphocytes
s t i m u l a t i o n a r e based upon q u a n t i t a t i v e e v a l u a t i o n o f radio-
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l a b e l l e d DNA and p r o t e i n precursors uptake by lymphoid c e l l s
(11, 1 2 , 13,
14).
Because both DNA and p r o t e i n s y n t h e s i s
a r e two energy r e q u i r i n g metabolic a c t i v i t i e s ~ v a r i a t i o n so f carbohydrates metabolism i n stimulated lymphocytes is expected t o preceed changw i n p r o t e i n and DNA synthesis. To t e s t t h i s hypothesis w e have analyzed the e x t e n t and type of changes i n energy metabolism i n human FBL stimulated by PEA concentrat i o n s which induce maxinal thymidine incorporation. PHA stimulat i o n of lymphocytes determines an i n c r e a s e o f both energy and s y n t h e s i s metabolisms. Owing t o t h e former, t h e h i g h e r l a c t a t e production should presumably be r e l a t e d t o t h e f a c t t h a t PEA decreases ATP-ADP l e v o l s (15).
The i n f l u e n c e of PHA on l a c t a t e
production has a l r e a d y been reported (16, 17). Xowever, t h e f i n d i n g s of Polgar (18) who showed t h a t l a c t a t e production is h i g h e r i n absence of PEA than i n presence of t h i s mitogen d u r i n g the first hour of incubation, could n o t be confirmed. O u r d a t a a r e i n agreement with those of Roos and Loos (15) who demonstrated an i n c r e a s e i n l a c t a t e production w i t h i n 15-30 minutes a f t e r the a d d i t i o n of PHA.
These authors, c a r r i e d out t h e i r determinations
i n continuous presence o f PHA, while i n the p r e s e n t study carbohydrate metabolism has been analyzed a f t e r removal of l e c t i n from stimulated lymphocytes.
These d i f f e r e n t c o n d i t i o n s may
ex-
p l a i n the lower degree of s t i m u l a t i o n and t h e d i f f e r e n t k i n e t i c i n l a c t a t e production observed i n our experiments. Regardless
APOLLONJ ET AL.
462
of the d i f f e r e n t experimental conditions, i n c r e a s e of l a c t a t e
production could be already appreciated five minutes a f t e r incubation with PHA.
Because l a c t a t e production appears t o
be stimulated so rapidly, i t i s highly suggestive t h a t
the
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glycolysis is increased t o meet energy demand both for a c i d s nucleic and p r o t e i n s synthesis. by two observations.
This hypothesis i s supported
F i r s t , following PHA stimulation, both
thymidine incorporation and u t i l i z a t i o n of pentose pathway a r e increased.
Secondly, l a c t a t e production and thymidine in-
corporation follow t h e same response with regard t o PHA
con-
c e n t r a t i o n s . The highest l a c t a t e production w a s obtained
at
P a concentrations which were a b l e t o s t i m u l a t e the g r e a t e s t thymidine incorporation. The possible use of l a c t a t e a c i d q u a n t i t a t i o n as simple measurement of lymphoid c e l l s s t i m u l a t i o n is a t the p r e s e n t
under study. dcknowledments This work was p a r t i a l l y supported by Lab. Tecnologia Biomedica. C.N.R.
Italy. References and Rubin, A.D.,
1.
Cooper, H.L.
2.
Resh, K. and Ferber, E.,
Lancet,
Europ. J. Bioohem.,
1972.
3.
Fisher, I). B. and Elueller, G . L ,
4.
Smith, J.:V.,
c.x.,
S t e i n e r , A.L.,
J. Clin. Invest.,
2: 723,
BBA,
*.
1971.
3:153,
434, 1971.
Newberry, 'II.C.Jr.
2:442,
1965.
Bnd Parker,
46 3
CARBOHYDRATE METABOLISM IN LYMPHOCYTES
5.
Hirshhorn, I:.,
and Ilirshhorn, R.,
6.
Hirshhorn, R.,
B r i t t i n g e r , C.,
C.,
7.
J. C e l l Biol.,
EIirshhorn, K.
1965.
and 'hissman,
1968.
Uohorst, 11. J., i n Methods of enzymatic a n a l y s i s e d i t e d by H.U.
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/06/15 For personal use only.
21: 412,
2: 10.46,
Icmcet,
Bergineyer, p.
266
h a d . Press, Uew York, 1965.
8.
Naspits, K.K.
9.
Greaves, 111. and Janossy, G.,
2:1, 1968. Rev., 11: 87,
and Richter, lA., Prog. 9 1 e r 3 , Transplant.
1972 10.
Andersson, J., LIoller, C.,
4: 381, 11.
C e l l Immunol.,
and Sjoherg, O.,
1972.
Rosenbere, S . B . ,
and Levy, R.,
J. Immol.,
108: 1080,
1971. 12.
Terry, B.D.,
13.
Levy, R.,
Rosenbbrg, S.A.,
Transplantation,
Ling, N.R.,
Ficker, S. and
Schechter, R.,
2:541,
1972.
Lymphocytes stimulation. Rorth Holland,
Amsterdam, 1968.
14
Oppenheim, J.J.,
15.
Roos, D.,
Fed. Proc.,
and Loos, J.A.,
3: 21, 1968.
Biochem. Biophys. hcta, 2221
565, 1970. 16.
Hedeskov, C . J .
17
Hedeskov, C.J.,
18.
Polgar, P.R.,
Cell Res.,
19
and Esmann, V., Biochem.
J.,
Blood,
2.163,
x:373,
&pt.
1968.
Rabinmritz, U . , Schimo, I., and !'lilhite,
of Hematology,
1960.
Foster, J.X. and Cooperband, S.Z.,
42: 455,
1966.
a:455,
1968.
B.A.,
Brit.,
J.
IMMUNOLOGICAL COMMUNICATIONS, 4 (5), 453-463 (1975)
Apollonj, C. Vasile, A. F l o r i d i and F.C.
L1.C.
Laboratorio d i Imnunologia.
Natali
I s t i t u t o Regina Zlena. Rome.
Italy. Abstract Euman p e r i p h e r a l blood lymphocytes (PBL) stimulated by phytohenoagglutinin (PEIA) manifest augmented g l y c o l y s i s and o x i d a t i o n of g l u ~ o s e - l - ~ 4 C ,i n d i c a t i n g an increased u t i l i z a t i o n of the pentose pathway. L a c t i c aci d production, a s index of increased g l y c o l y s i s , follows t h e same k i n e t i c of thymidine incorporation and can be e a s i l y q u a n t i t a t e d by an enzymatic assay.
-in vitro
Introduction
The e f f e c t o f mitogens on lymphocytes has been extens i v e l y i n v e s t i g a t e d d u r i n g the p a s t y e a r s because of valuable a i d i n understanding a c t i v a t i o n of i m u n o c y t e s by a n t i g e n s and c e l l cooperation. D i f f e r e n t mitogens so f a r used appear t o a c t s e l e c t i v e l y on lymphocytes s u b c l a s s e s and t o induce s e v e r a l changes i n the biochemical a c t i v i t i e s of lymphoid
cells. I n p a r t i c u l a r , v a r i a t i o n s i n n u c l e i c acid (1),phosphol i p i d s (2, 3 ) , cAbP (4) and c e l l u l a r s u b f r a c t i o n netaboliarn
( 5 , 6) o f lymphocytes have i n f a c t been described folloiving t h e i r c o n t a c t with l e c t i n s .
This study has been performed
454
APOLLONJ ET AL
with two aims i. t o gain more information on t h e e x t e n t and types of changes i n t h e carbohydrate metabolism occurring i n
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/06/15 For personal use only.
PHA stimulated PBL, ii. t o attempt a q u a n t i t a t i v e e s t i m a t i o n of such changes.
The r e s u l t s obtained i n d i c a t e t h a t increased
glycolysis
i n PBL even a f t e r s h o r t c o n t a c t ( 5 min.)
OCCUTS
with PHA. These changes whioh precede tho l a t e events of b l a s t transformation and DNA s y n t h e s i s may be q u a n t i t a t i v e l y estimated by means of l a c t a t e production. Llaterials and Methods
Human p e r i p h e r a l blood mononuclear o e l l s (average 90$ lymphocytes and l e s s than lC$ monocytes) were i s o l a t e d from heparinized blood of apparently healthy donors by oentrifugat i o n on F i c o l l (Pharmacia)-Urovison (Shering) gradient.
The
i s o l a t e d c e l l s were mashed twice with medium WEdI 1640
sup
plenented with 5% C O ~ , containing 100 pd.1
FCS, 2 x
mtr/ml glutamine and
of Gentamicin (Shering). Lymphocytes
were incubated with PHA f o r d i f f e r e n t periods of time and divided i n two a l i q u o t s f o r examination of n u c l e i c a c i d and carbohydrates metabolism r e s p e c t i v e l y . DNA s t i m u l a t i o n was asBayed i n f i c r o t i t e r t r a y s (Falcon IS-FB-96) i n which 6 x 105 c e l l s p e r w e l l , were p l a t e d i n t r i p l i c a t e i n presence of 1020 pl of ?HA (Difco 25 mg/ml). Optimal concentration of PHA inducing maximal tiiymidine incorporation was chosen according t o dose response experiments. Cells were c u l t u r e d i n presence of PHA f o r 72 hours i n a humidified atmosphere c o n t a i n i n g
5%
C02. Each c e l l c u l t u r e was l a b e l l e d f o r 16 hours with 0.5 uCi of t r i t i n t e d t i m i d i n e . L a b e l l i n g was stopped by a d d i t i o n of
5 ml of cold calinc. C e l l s were f i l t e r e d on ‘.‘Fhatnann 3X: paper
CARBOHYDRATE METABOLISM IN LYMPHOCYTES
455
and thoroughly washed with an excess of
lo$
TCA, 95$ e t h a n o l ,
and e t h e r . The d r i e d f i l t e r s were counted i n 10 ml toluene-?ermafluor 25 x (Dackard) s c i n t i l l a t i o n fluid i n a IIuclear Chicago
MKI s c i n t i l l a t i o n counter.
Lymphocytes used f o r carbohydrate
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/06/15 For personal use only.
metabolism d e t e r n i n a t i o n a f t e r PHA c o n t a c t $:ere xished twice with Krebs-Ringer bicarbonate b u f f e r . In order t o determine glucose oxidation, c e l l s were incubated i n d u p l i c a t e i n 3 m l of Krebs-Ringer bicarbonate b u f f e r c o n t a i n i n g 0.25 pCi of l a b e l l e d s u b s t r a t e . Incubations were c a r r i e d out i n stoppered f l a s k s with a removable c e n t e r w e l l , a t 37°C i n
95$
5% C02
02.
a t shaking r a t e of 100/min. A f t e r one hour 1 n l of Hyarnine (Packard) was i n j e c t e d i n t o the c e n t e r !:re11 and the r e a c t i o n
was stopped by a d d i t i o n of 0.3 m l of 4
The f l a s k s 4' were shaken an a d d i t i o n a l hour i n order t o c o l l e c t I4CO2. The HClO
Hyamine w a s then q u a n t i t a t i v e l y t r a n s f e r r e d t o s c i n t i l l a t i o n v i a l s and counted i n a Nuclear Chicago LXI l i q u i d s c i n t i l l a t i o n spectrometer. Determinations of anerobic i;lycolysis were c a r r i e d out i n 25 ml Erlenmeyer f l a s k s a t 3 7 O C for 60 min. i n a Dubnoff b a t h a t shaking r a t e of 100/min. ICrebs-Rinmr b i c a r bonate with 10 mM glucose was used a s r e a c t i o n nedium and t h e gas phase was
95% N2, 5% C02.
A t the end o f the incubation
time the r e a c t i o n was stopped with 0.25 m l of 2.5
12 H C l O
4
the c o n t e n t of the flasks c e n t r i f u g e d a t 16.000 rpm f o r
and
15 min.
The n e u t r a l i z e d supernatant were used for c n z p a t i c determinat i o n of lactate (7). Results I n o r d e r t o e v a l u a t e the e f f e c t s of PHA on Ijmphocytes, carbohydrate metabolism, t h e a c t i o n of the l e c t i r . t i o n of g l ~ c o s e - 6 - ~ ~ Cglucose-l-14C, ,
m oxitla-
and anaerobic g l y c o l y s i s
456
APOLLONJ ET AL.
has been studied. As shown i n Table 1, PRA does not modify the
-
production r a t e of 14C0, from e ; l ~ c o s e - 6 - ~ ~ Cthus , indicating t h a t under these experimental conditions the oxidative metabolism i s not a f f e c t e d . Since i t is w e l l IC~OQM t h a t
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PHA increases
3H thymidine incorporation i n t o nucleic
acid, the e f f e c t of PHA on g l u c o ~ e - l - ~ ~ u tci l i z a t i o n was tested. This is j u s t i f i e d by the f a c t t h a t g l u c ~ s e - l ~ ~ C e n t e r s pentose pathway supplying metabolites f o r nucleic acid synthesia. As shown i n Fig. 1 the oxidation of glucosei e g r e a t l y increased by PEA and, as observed f o r t h e
l-'C
glycolysis, the e f f e c t is already appreciable a f t e r t e n minutes.
O f i n t e r e s t is the d i f f e r e n t k i n e t i c s of 14C0 produc2 t i o n folloming PHA contact. I n f a c t i n unstimulated c e l l s , inoreases as YCO 2 lymphocytes LQCO
2
l i n e a r function of time, whereas i n stimulated
production is higher d u r i n g the first minutes
and becomes l i n e a r only a f t e r t h i s period. On the b a s i s of these data i t was possible t o perform a q u a n t i t a t i v e estimation of
TABLE 1 Effect of PK4 on glucose-6- 14C oxidation by PBL TIME
UNSTDRLATED
STIMULATED
138 + 12
10'
135 f 8
20'
265 2 1 5
270 f 9
40 '
495
+ 28
520 + 35
60'
720 + 51
768 + 49
(O)
Cpm/lO 7 c e l l s . Each value
(O)
standard deviation i s averaged
from three d i f f e r e n t experiments performed i n duplicate.
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CARBOHYDRATE METABOLISM I N LYMPHOCYTES
45 7
S t i m u l a t i o n time ( m i n ) FIGURE 1 Oxidation of s1u~ose-1-14~ i n PHA stimulated and n o n s t i n u l a t e d (M ) PBL.
(-1
Each value 2 standard devia-
t i o n is averaged from t h r e e d i f f e r e n t experiments performed i n duplicate.
the pentose cycle i n stimulated and unstimulated lymphocytes. production r a t e from ~ l u c o s e - l - ~ ~ C 2 gives i n f a c t t h e r a t e of pentose oxidation
The d i f f e r e n c e between 1 4 C 0 and glucose-6-14C directly.
Although t h i s method allows only a semi-quantitative
estimation, i t m u s t be s t r e s s e d t h a t under the present
ex-
peri:nental c o n d i t i o n s r e s u l t s were h i g l y reproducible. A 8
APOLLONJ ET AL.
458
3hOn i n fig. 2 PHA g r e a t l y s t i m u l a t e s pentose pathway i n a m merit
with the increased s y n t h e s i s o f n u c l e i c a c i d . The higher
g l ~ c o s e - l - ~ ~oxidation C can be t e n t a t i v e l y r e l a t e d t o a g r e a t e r request of metabolites needed for the n u c l e i c acid synthesis,
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r a t h e r than a d i r e c t e f f e c t of PEL4 on enzyme of pentose cycle. The anaerobic production of l a c t i o acid by lymphocytes under
3
2
-
0 I
0 X
E
n v
1
10
20
30
40
50
00
Stimulation t i m e ( m i n ) FICURZ 2
Quantitative evaluation of pentoso cycle i n stimulated (O-----o) and unstimulated ( 1 ) PBL. The f i g u r e s have
been c a l c u l a t e d on the d a t a of Table 1 and Fig. 1.
CARBOHYDRATE METABOLISM IN LYMPHOCYTES
459
PHA s t i m u l a t i o n i s reported i n Fig. 3. As it nay be seen g l y c o l y s i s i s a l r e a d y double a f t e r t e n minutes.
The percent
i n c r e a s e is independent from s t i m u l a t i o n time, being the same a l s o a f t e r 60 min.
A comparison between l a c t i c a c i d produc-
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t i o n and thymidine i n c o r p o r a t i o n i s shown i n Fig.
4,
where a
f i x e d number of lymphoid c e l l s was stimulated f o r 20 minutes with i n c r e a s i n g
I
/
PHA concentrations. Percent i n c r e a s e i n l a c t i c
.
Stimulation t i m e (mini FIGURE 3 Anaerobic production of l a c t i c aoid by PW s t i m u l a t e d
(M)
and unstimulated (-)
PBL.
Each value 2 standard
d e v i a t i o n i s a.vera@d from t h r e e d i f f e r e n t experiments performed i n d u p l i c a t e .
AE'OLLONJ ET AL.
460
1
8000. I
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6000
50 40
O/o
increase
lactate production
30
20
I
10
I
5
2o
PHA
-Thymidine
___
pl
100
uptake
L a c t a t e production FIGURE 4
Effect of PHA concentration on thymldine uptake and l a c t a t e production by PBL.
acid production and thymidine uptake
followed the same response.
R e l a t i v e l y hich concentrations of l e c t i n d i d n o t induce appreciable chances i n DNA and carbohydrate metabolism. Discussion Lynphoid c e l l s from d i f f e r e n t s p e c i e s have been shown t o be stimulated i n v i t r o by non-clonal rnito,-ns
such a s PHA, and
t o undergo trannnfomation followed by DIJA s y n t h e s i s ( 8 ) . These e f f e c t s have inclicstcd some d e p e e of s p e c i f i c i t y f o r e i t h e r 3
CARBOHYDRATE METABOLISM I N LYMPHOCYTES
461
o r T lymphocytes (9, lo) and have provided a great d e a l of knowledge i n t o a c t i v a t i o n mechanisms which may be involved i n the immune response.
Uethods of d e t e c t i o n of lymphocytes
s t i m u l a t i o n a r e based upon q u a n t i t a t i v e e v a l u a t i o n o f radio-
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l a b e l l e d DNA and p r o t e i n precursors uptake by lymphoid c e l l s
(11, 1 2 , 13,
14).
Because both DNA and p r o t e i n s y n t h e s i s
a r e two energy r e q u i r i n g metabolic a c t i v i t i e s ~ v a r i a t i o n so f carbohydrates metabolism i n stimulated lymphocytes is expected t o preceed changw i n p r o t e i n and DNA synthesis. To t e s t t h i s hypothesis w e have analyzed the e x t e n t and type of changes i n energy metabolism i n human FBL stimulated by PEA concentrat i o n s which induce maxinal thymidine incorporation. PHA stimulat i o n of lymphocytes determines an i n c r e a s e o f both energy and s y n t h e s i s metabolisms. Owing t o t h e former, t h e h i g h e r l a c t a t e production should presumably be r e l a t e d t o t h e f a c t t h a t PEA decreases ATP-ADP l e v o l s (15).
The i n f l u e n c e of PHA on l a c t a t e
production has a l r e a d y been reported (16, 17). Xowever, t h e f i n d i n g s of Polgar (18) who showed t h a t l a c t a t e production is h i g h e r i n absence of PEA than i n presence of t h i s mitogen d u r i n g the first hour of incubation, could n o t be confirmed. O u r d a t a a r e i n agreement with those of Roos and Loos (15) who demonstrated an i n c r e a s e i n l a c t a t e production w i t h i n 15-30 minutes a f t e r the a d d i t i o n of PHA.
These authors, c a r r i e d out t h e i r determinations
i n continuous presence o f PHA, while i n the p r e s e n t study carbohydrate metabolism has been analyzed a f t e r removal of l e c t i n from stimulated lymphocytes.
These d i f f e r e n t c o n d i t i o n s may
ex-
p l a i n the lower degree of s t i m u l a t i o n and t h e d i f f e r e n t k i n e t i c i n l a c t a t e production observed i n our experiments. Regardless
APOLLONJ ET AL.
462
of the d i f f e r e n t experimental conditions, i n c r e a s e of l a c t a t e
production could be already appreciated five minutes a f t e r incubation with PHA.
Because l a c t a t e production appears t o
be stimulated so rapidly, i t i s highly suggestive t h a t
the
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glycolysis is increased t o meet energy demand both for a c i d s nucleic and p r o t e i n s synthesis. by two observations.
This hypothesis i s supported
F i r s t , following PHA stimulation, both
thymidine incorporation and u t i l i z a t i o n of pentose pathway a r e increased.
Secondly, l a c t a t e production and thymidine in-
corporation follow t h e same response with regard t o PHA
con-
c e n t r a t i o n s . The highest l a c t a t e production w a s obtained
at
P a concentrations which were a b l e t o s t i m u l a t e the g r e a t e s t thymidine incorporation. The possible use of l a c t a t e a c i d q u a n t i t a t i o n as simple measurement of lymphoid c e l l s s t i m u l a t i o n is a t the p r e s e n t
under study. dcknowledments This work was p a r t i a l l y supported by Lab. Tecnologia Biomedica. C.N.R.
Italy. References and Rubin, A.D.,
1.
Cooper, H.L.
2.
Resh, K. and Ferber, E.,
Lancet,
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Fisher, I). B. and Elueller, G . L ,
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Smith, J.:V.,
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CARBOHYDRATE METABOLISM IN LYMPHOCYTES
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Hirshhorn, I:.,
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Hirshhorn, R.,
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