Immunology and Cell Biology (1992) 70, 309-313
In vitro metabolic inhibition of the human lymphocyte:
Influence on the expression of interleukin-2 receptors
H. KARLSSON and L. N A S S B E R G E R
Department of Medical Microbiology, Section of Clinical Immunology, Solvegatan 23, Lund, Sweden
Summary We have investigated the effects of energy depletion in human lymphocytes on the
expression of the membrane-bound interleukin-2 receptor {IL-2R) and the release of soluble IL-2R.
Concanavalin-A (Con-A) and IL-2-transformed lymphocytes were incubated with sodium fluoride,
inhibiting the Embden-Meyerhof pathway, rotenone and 2,4-dinitrophenol (2,4-DNP), which are
known to disturb mitochondria 1 high energy production. Rotenone was found to be the most potent
inhibitor, with a half maximal effect at 10 nmol/L. 2,4-DNP and sodium fluoride, although not as
potent as rotenone, showed marked inhibitory effects in higher concentrations, with half maximal
effects at 50 jj^mol/L and 5 mmol/L respectively. No difference in the inhibition pattern for
membrane-bound IL-2R compared with siL-2R was observed. It is concluded that intracellular
.synthesis, transport and subsequent membrane insertion or release of receptors important for
immunoregulation require high energy phosphate compounds and are sensitive to disturbances in
intracellular energy levels.
Introduction
Lymphocytes proliferate when they are exposed to interleukin-2 (IL-2). This effect is
mediated by IL-2 binding to its high affinity
receptor, consisting of at least two subunits, a
75 kl)a (P) and a 55 kDa (a) cham. The a
chain also exists in a soluble form, a 45 kDa
glycoprotein that binds IL-2 with low
affinity.'-''
Normal metabolism is required for optimal
cell function. Most of the energy is derived
from mitochondria! oxidative phosphorylation and some from the glycolytic pathway,
This study aimed to investigate how the expression of membrane-bound IL-2 receptors
and release of its soluble counterpart were
affected by the downregulation of mitochondrial and glycolytic energy generation. Furthermore, we aimed to investigate if there
was any difference in the expression and
release of the membrane-bound soluble receptors, respectively, due to energy deprivation.
We also compared the inhibition of receptor
expression and release with the inhibition of
blastoid transformation, and monitored the
intraceliular levels of adenosine trisphosphate
(ATP) to verify that the observed effects on
the other parameters were actually caused by a
decrease in intracellular ATP.
Materials and methods
Hnman lymphocytes from healthy blood
donors were isolated by density centrifugation
on Lymphoprep (Nycomed, Oslo, Norway)
and grown in RPMI-1640 medium (Flow
Laboratories, VI, USA), supplemented with
2 mmol/L L-glutamine and 10|.ig/inL gentamicin. The mitogen Concanavalin-A (Pharmacia, Uppsala, Sweden) and human
recombinant lL-2 (Boehringer, Mannheim,
Germany) were used in final concentrations of
lOng/mL and 20 U/mL, respectively, to
stimulate the cells and to exclude any secondary effects due to inhibited IL-2 synthesis.
The following two metabolic inhibitors
Correspondence: Lennart Nassberger, Department of Medical Microbiology, Section of Clinical
Immunology, Sblvcgacan 23, 223 62 Lund, Sweden.
Accepted for publication 2 July 1992.
310
H. Karhson and L. Nassherger
that affect mitochondrial energy generation
were used: 2,4-dinitrophenol {2,4-DNP),
an uncoLipler of oxidative phosphorylation,
and rotenone, which complexes with NADH
dehydrogenase and thus inhibits electron
transport beginning with NAD-coupled
dehydrogenases. Sodium fluoride was used for
inhibiting the glycolytic pathway. The compounds were dissolved in RPMI-1640 (NaF)
or ethanol (rotenone and 2,4-DNP). Cells
were never exposed to ethanol in higher
concentrations than 1%; control cells incubated with 1% ethanol remained unaffected.
Experimental design
Isolated cells were allowed to recover for 24 h
from the density centrifugation before experiments were initiated. Thereafter cells were
incubated with Con-A, IL-2 and the respective compounds in parallel, in the following
final concentrations: 2,4-DNP (0.1 ^imol/L,
1 |imol/L, lOnmoI/L, 0.1 mmol/L and
1 mmol/L); rotenone (1 pmoI/L, 10 pmol/L.
0.1 nmol/L, 1 nmol/L, 10nmol/L,0.1 fimol/
L, 1 |imol/L and 10^moI/L); and NaF
(lOnmol/L, 0.1 mmol/L, 1 mmol/L and
10 mmol/L).
Incuhations were then allowed to proceed
for 72 h. The difference in the expression of
membrane-bound IL-2R between stimulated
and unstimulated cells was found to be maximal after 72 h (data not shown). At the end of
each experiment the cells were sediniented
and the supernatant was collected and stored
at -IQ'C until analysis for soluble IL-2R
was carried out. The remaining pellet was
then washed three times, and then further
analysed for the IL-2R* population by flow
cytometry (Coulter Electronics, USA) using
an R-phycoerythrin-conjugated anti-CD25
monoclonal antibody (Dakopatts, A/S,
Denmark). For measuring the amount of soluble IL-2R present in the cell culture supernatant, an enzyme-linked immunosorbent
assay (T-cell Sciences, USA) was used. Briefly,
the wells were coated with a murine monoclonal antibody to sIL-2R. The test sample
and a second HRP-conjugated monoclonal
antibody were incubated together for 3 h. Cell
growth was measured by [ H]-thymidine incorporation (1 \xC\ per well) during the last
4 h ot the 72 h incubations in 96-well
microtitre plates. The uptake was then calculated relative to control cells incubated with
Con-A and IL-2 only. Values presented are
means of at least three and up to five independent experiments. The ATP contents of the
cells at the end of the incubation was monitored using an LKB Wallac 1251 luminometer and an ATP-monitoring reagent
(Bio-Orbit, Turku, Finland). Before ATP
monitoring, cells were washed twice in
Dulbecco-bovine serum albumin, and then
the number of cells in each sample was
counted. Cells were then spun down and
resuspended in 200 jiL of 2.5% trichloracetic
acid and put on a rotary shaker for 45 min.
The lysate was then pelleted and the ATP
containing supernatant removed. Reagent
(10 )iL) and a 10 |iL test sample were added to
0.1 mol/L Tris-acetate and 2 mmoI/L ethylenediamine tetra-acetic acid buffer (pH 7.75)
to a final volume of 300 jiL in a disposable
4 mL polystyrene cuvette. Using an ATP
standard (Bio-Orbit. Turku, Finland), the ATP
concentration per 10"^ cells was calculated.
Cell viability at the end of the incubations
was > 80%, as estimated by Trypan Blue
exclusion.
Results
Due to biological variations the results for all
parameters are shown as the per cent change
relative to control cells incubated with Con- A
and IL-2 only.
The membrane-bound IL-2R was most
affected by rotenone, which induced a dosedependent reduction of the expression
whereby maximal inhibition was achieved at
0.1 mmol/L (Fig. 1). Both 2,4-DNP and
NaF showed a different inhibitory pattern. In
the concentration ranges 0.1 |jmol/L to
lOpmol/L for 2,4-DNP and 10 nmol/L to
1 mmoI/L for NaF there was only a minor
reduction in theexpression of membrane-bound
receptors. A break point at lO^moI/L and
1 mmoI/L was observed for 2,4-DNP and NaF,
respectively, where a small increase in concentration caused a major decrease i n the expression
of membrane-bound IL-2 receptors (Fig. 1).
The average value for the membrane-bound
^ population in controls was 70%.
311
Metabolic regulation oflL-2R expression
10"'"
10'^
10"^
Concentration (mol/L)
Fig. 1. Effect on expression of the membranebound p55 IL-2 receptor on Con-A and IL-2treated human lymphocytes by rotenone {•), 2.4DNP {•) and NaF {D). Values are means ± s.e.m.
(M = 3-5 for each value).
An almost identical result was observed for
the release of sIL-2R (Fig. 2). Control cells
accumulated at 2500-3500 U/mL of sIL-2R.
[•'Hj-Thymidine incorporation was maximally
inhibited by the metabolic inhibitors in the
foilowing order: rotenone, 2,4-DNP and NaF
(Fig. 3). Sodium fluoride was the only inhibitor that showed a pattern where inhibition
of the receptors paralleled the inhibition of
cell growth. 2,4-DNP reduced the [^H]thymidine incorporation gradually in the
range 0.1 nmol/L to 10)imol/L where the
expression of receptors was unaffected. Rotenone inhibited [' H]-thymidine incorporation to
50% at 0.5 |amol/L but inhibited the expression of and release of membrane-bound and
10
Coficentration (mol/L)
Fig. 2.
tor from
cytes by
(H - 3-5
Effect on release of the soluble lL-2 recepCon-A and IL-2-treated human lymphorotenone (•), 2,4-DNP (•) and NaF (D)
for each value).
Conceniration (mol/L)
Fig. 3. Effect on pHj-thymidine incorporation in
Con-A and IL-2-treated human lymphocytes by
rotenone (•), 2,4-DNP (•) and NaF (D) (« = 3-5
for each value).
soluble receptors to 50% in a concentration as
low as 10 nmol/L. Control cells incorporated
150 000-250 000 ct/min.
Figure 4 shows that the three compounds
perform inhibitory effects on the generation
of intraeellular ATP. These effects parallel the
effects on [" H]-thymidine incorporation to a
large extent. The intraeellular ATP concentration in control cells ranged between 1 and
10 ^mol/L/10*^ cells.
Discussion
The expression of membrane-bound IL-2R is
important for the proliferation and differentiation of T lymphocytes and is regulated by
IL-2.'-'' When IL-2 is bound to its high
Concenifaiion (mo(/L)
Fig. 4. Influence on ATP levels at the end of the
incubations by rotenone (•), 2,4-DNP (•) and
NaF (D). Values arc means from two independent
incubations.
312
H. Karhson and L. Nassherger
affmity receptor it is internalized by a
receptor-mediated process.^"' Little information is available about this process of IL-2
signal transmittance from the cell surface
to the interior of the cell. However, the
mechanisms involved require energy. All biological processes involving pinocytosis and
endocytosis require energy, which has been
illustrated by demonstrating that there is a
pronounced reduction in these activities at low
temperatures. • It has aiso been demonstrated,
by direct measurements, that there is an increase in heat production during pinocytosis.'"
Others have shown that there is a reduction in
fluid endocytosis in liver cells incubated with
NaF and dinitrophenol.'' Transport of the
receptor in the opposite direction from the site
of synthesis to insertion in the plasma membrane is also an energy-dependent process.'^
The mechanism of exocytosis, the morphological reciprocal of endocytosis, was first
described in the adrenal medulla in 1957.
From a biochemical point of view exocytosis
requires the intracellular elevation of Ca^ *
and the production of high energy compounds
such as ATP. Thus exocytosis is an energydependent process and signifies a second
energy-requiring step in the secretory
process.'"^
This study demonstrates that the proliferation and subsequent expression of membranebound and soluble IL-2R are both clearly
energy-dependent processes. Beside transport
through the cytoplasm there are other energyrequiring processes that have to be carried out
before the receptor can enter the plasma membrane. The IL-2 receptors are synthesized
as precursors that undergo several steps of
modifications before membrane insertion.^''"^
Deprivation of the energy supply will lead to
one or several of these mechanisms being
inhibited. It was not surprising to find that
mitochondrial inhibitors exerted a more pronounced effect compared with NaF. Sodium
fluoride inhibits the Embden-Meyerhof pathway; the ATP yield via this mechanism is
much less than via the oxidative phosphorylation (2 and 36 molecules of ATP per glucose
molecule, respectively) during the subsequent
mitochondrial oxidations.'^
When the cells were incubated with NaF, a
high concentration was needed before inhibi-
tion was observed compared with 2,4-DNP
and rotenone. Even though the glycolytic
pathway was inhibited there will be enough
energy available in the eel! to carry out different intracellular processes. The drastic change
in the expression of membrane-bound and
soluble receptors that was observed at rather
high concentrations of NaF may be due to a
general depressive effect on the cell rather
than a lack of available energy, as further
underlined by the effect observed for [^H]thymidine incorporation.
When mitochondrial oxidative phosphorylation is blocked there is still a contribution,
although small, from the glycolytic pathway
that may compensate for the loss of mitochondrial ATP. 2,4-DNP caused a gradual decrease
in [" Hj-thymidine incorporation up to
10)imol/L, after which a rapid decline was
seen; the membrane-bound and soluble receptors however, remained unaffected up to
10 pmol/L but were highly inhibited at
higher concentrations.
Rotenone, on the other hand, appeared to
inhibit celllular proliferation to a lesser extent
than the expression of receptors. The difference in the inhibition of membrane-bound
as well as soluble IL-2 receptors observed
between 2,4-DNP and rotenone may be due
to the fact that 2,4-DNP causes an increase
in glucose utilization. It has been reported that
2,4-DNP in concentrations similar to those
used in this experiment can produce a marked
increase in glucose utilization in lymphocytes."' Rotenone conplexes with NADH
dehydrogenase, which is partially located in
the lipid core oi the inner mitochondrial
membrane, and due to its high hydrophobicity
rotenone quickly gains access to this compartment. This enzyme complex is situated in the
beginning of the respiratory chain.'^
This study indicates that changes in the
metabolic rate may influence receptor expression by cells of the immune system. It is also
shown that expression of the membranebound IL-2R and release of the sIL-2R are
processes equally sensitive to disturbances in
ATP levels, indicating that release of the
receptors does not require more or less ATP
than membrane insertion. Furthermore, we
have demonstrated that rotenone has a higher
Metabolic regulation ofIL-2R expression
potency than 2,4-DNP in inhibiting mitochondrial ATP generation.
What may the cHnical consequences be?
Could it perhaps be of any importance in the
treatment of disease to slow down the metabolic activity of a cell? In experimental work it
has been recently demonstrated that chronic
energy intake restriction prolongs life span in
genetically short-lived autoimmune-prone
strains of mice.'^ Furthermore, depletion of
ATP may occur as a result of ischaemia and
shock. Secondary effects due to such a condition are an increase in cell size that stretches
the membrane and the activation of deleterious processes such as the stimulation of phospholipases degrading the phospholipids of
the plasma membrane. This may also contribute in viva to reduced numbers of membrane
receptors.^*' Even a minute decrease of intracellular ATP levels may have a greater influence on the immunoregulation than is
generally believed.
Acknowledgements
This work was suported by grants from the
Medical Faculty, University of Lund, and
Kungliga Fysiografiska Sallskapet, University
of Lund.
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