[CANCER RESEARCH 50. 2949-2952. May 15. 1990]
Influences of Interleukins 2 and 4 on Tumor Necrosis Factor Production by Murine
Mononuclear Phagocytes1
William H. McBride,2 James S. Economou, Ramin Nayersina, Sian Comora, and Richard Essner
Department of Radiation Oncology [W. H. M., R. A/., S. C.J, Jonsson Comprehensive Cancer Center [W. H. M., J. S. E.], and Division of Surgical Oncology
[J. S. E., R. E.], UCLA Medical Center, Los Angeles, California 90024
ABSTRACT
Administration of rccombinant human interleukin 2 (IL-2) to mice
gave rise to peritoneal macrophages and blood monocytes that were
primed to produce large amounts of tumor necrosis factor (TNF). Mac
rophages from IL-2-treated athymic mice responded less well than those
from euthymic mice. In addition to its in vivo priming effect, IL-2 was
able to directly stimulate TNF production in vitro by purified monocytes.
Macrophages responded to IL-2 generally less well than monocytes both
in vitro and in vivo.
In contrast to IL-2, recombinant murine interleukin 4 (11.-4) downregulated TNF synthesis by macrophages. In vitro pretreatment of
macrophages with IL-4 largely abolished their ability to synthesize TNF
in response to IL-2 or lipopolysaccharide. Also, administration of IL-4
to mice blocked the ability of IL-2 and lipopolysaccharide to prime
macrophages in vivo for TNF production. Overall, these results demon
strate that IL-2 and IL-4 can act antagonistically to regulate TNF
production by macrophages.
In spite of its down-regulatory action on TNF production, IL-4 was
unable to protect mice against the lethal toxic effects of lipopolysaccha
ride or IL-2.
INTRODUCTION
The interleukins IL-23 and IL-4 are autocrine T-cell growth
factors (1-3). In mice, IL-4 stimulates predominantly CDS* Tcells and CD4+ cells of the Th2 subset, whereas IL-2 stimulates
the Thl subset (4, 5). These subsets are defined by the array of
lymphokines they secrete (5). Thl-cells produce IL-2, tumor
necrosis factor ß,
and 7 interferon, while Th2-cells produce IL4 and interleukin 5. In humans, although different T-cell pop
ulations can produce different lymphokines, such differences
seem less well defined, perhaps because, unlike in mice, IL-4
appears to stimulate T-cell proliferation by both IL-2-independent and IL-2-related pathways (6).
IL-2 and IL-4 influence the behavior of B-cells and macro
phages as well as of T-cells. IL-4 is B-cell stimulatory factor 1
(7, 8) and IL-2 supports immunoglobulin secretion by B-cells
(9). Macrophage cytotoxicity for tumors has been reported to
be enhanced by IL-4 treatment (10), as have expression of
MHC class 1, MHC class II, and adherence molecules (10-13).
Human monocytes produce tumor necrosis factor «and IL-1
in response to IL-2 treatment both in vitro (14-16) and in vivo
(14), while we have recently demonstrated that IL-4 treatment
in vitro has a down-regulatory effect on the production of these
monokines (17). Since IL-2 and IL-4 are both generated during
Received 8/4/89; revised 2/8/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This investigation was supported by USHHS Grants CA-44384 (W. H. M.)
and CA-50780 (J. S. E.) awarded by the National Cancer Institute. DHHS.
J. S. E. is a National Cancer Institute Clinical Investigator, Award IKO8-01360.
2To whom requests for reprints should be addressed, at Department of
Radiation Oncology. UCLA School of Medicine. 10833 Leconte Avenue, Los
Angeles. CA 90024.
3The abbreviations used are: IL-2. interleukin 2; IL-4 interleukin 4; LPS,
lipopolysaccharide; TNF, tumor necrosis factor; MHC, major histocompatibility
complex; FCS. fetal calf serum; rMuIL-4. recombinant murine interleukin 4; IL1. interleukin I; Th2. T-helper 2; Thl, T-helper I.
immune responses, the question arises as to how they interact
to regulate responses.
IL-4 has been reported to interfere with certain IL-2-mediated responses. It has been found to suppress IL-2-driven
lymphokine-activated killer cell activation in human (18-20)
but not murine (21) systems, while having diverse effects on
cytotoxic T-cell responses (20, 22, 23). IL-4 has also been shown
to interfere with IL-2 and Staphylococcus aureus-induced acti
vation of human B-cells (9, 24).
In this study, we show that IL-4 down-regulates IL-2- and
LPS-induced TNF production by murine macrophages, both in
vitro and in vivo, and that IL-2 and IL-4 have antagonistic roles
in the generation of macrophage effector cells.
MATERIALS
AND METHODS
Mice. C3Hf/Sed//Kam euthymic and athymic female mice, 10-14
weeks of age, that were bred and maintained in our specific pathogenfree mouse colony were used in experiments. The endotoxin-resistant
CjH/HeJ mice were obtained from The Jackson Laboratory.
Reagents. RPMI 1640 medium was purchased from GIBCO (Grand
Island, NY), FCS, <1 ng/ml endotoxin) and D-galactosamine from
Sigma, Nutridoma serum-free culture supplement from BoehringerMannheim (Indianapolis, IN), and LPS of Escherichia coli B026:B6
from Difco (Detroit, MI). Recombinant human IL-2 was from Cetus
(Emeryville, CA); it had an activity of 3 x IO6 unit/mg protein and
contained no more than 0.05 ng endotoxin/mg protein, as determined
in the Limulus assay. rMuIL-4 was a kind gift from Immunex (Seattle,
Washington) and had an activity of 10s units/mg. In a series of previous
experiments (14) and in pilot experiments, we used polymyxin B (5 ^g/
ml) to inhibit any possible effect of contaminating endotoxin. The
results clearly demonstrated that contaminating endotoxin does not
contribute significantly to the findings presented here.
In Vivo Experiments. Mice were given i.p. injections of 5 x IO4units
recombinant human IL-2, IO5units rMuIL-4, and/or saline twice daily
for 2-2.5 days. A multiple injection schedule was chosen because this
is how IL-2 is most commonly administered to humans. Mice receiving
combined treatment were first given one initial injection of IL-4 alone,
because earlier in vitro data suggested that IL-4 pretreatment was most
effective at inhibiting TNF responses to LPS (17). Cells were removed
from mice 18 h after the last injection.
Macrophages. Peritoneal macrophages were isolated by adherence
from cells removed from the peritoneum by lavage. Peritoneal cell
counts were increased 2-3-fold in the IL-2- and IL-4-treated groups
but there was no obvious difference between control and treated groups
in the number of macrophages or monocytes that adhered. Monocytes
were obtained by adherence of mononuclear cells after Ficoll-Hypaque
separation of heparinized blood. Macrophage and monocyte monolayers were obtained by plating cells at 2 x IO6 cells/well, in 24-well
plates (Costar), in RPMI medium with 20% fetal calf serum (endotoxinscreened; Sigma) for 30 min at 37°C,with vigorous shaking every 10
min. The high FCS concentration inhibits lymphocyte adherence. The
wells were washed 4 times with 0.5 ml Hanks' balanced salt solution
and then cultured in 0.5 ml RPMI 1640 medium supplemented with
antibiotics and 1% serum-free Nutridoma (Boehringer Mannheim). The
adherent cells were >95% macrophages. Certain wells received 1 ug/
ml endotoxin, 10' units/ml IL-2, or 400 units/ml IL-4, concentrations
shown to be optimal in pilot experiments. After a 20-h incubation,
supernatants were removed and tested for the presence of TNF. Im-
2949
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EFFECTS OF IL-2 AND IL-4 ON TNF PRODUCTION
(Table 2). If the monocytes came from IL-2-treated mice, they
were even more responsive. They produced higher baseline
levels of TNF and produced large amounts of TNF when
stimulated by IL-2 and, in particular, by LPS.
Monocytes from IL-2-treated athymic mice were again less
responsive to LPS than were those from euthymic mice, sug
gesting an additional T-cell-dependent pathway. C^H/HeJ
monocytes responded poorly to both IL-2 and LPS.
Effect of IL-4 on TNF Production by Macrophages. rMuIL-4
added to normal peritoneal macrophages in vitro did not induce
TNF production (Table 3). In fact, it appeared to down-regulate
TNF production, inasmuch as incubation of normal peritoneal
macrophages with IL-4 for 6 h rendered the macrophages
unresponsive to LPS, as judged by subsequent TNF production.
This finding is in agreement with what we have observed with
human monocytes (17).
IL-4 appeared to have similar effects in vivo. Injection of IL4 i.p. (twice daily for 2.5 days) into mice yielded peritoneal
macrophages that were unable to respond to LPS stimulation
in vitro, and this could not be counteracted by injection of 50
/ugLPS just after the last IL-4 injection. Interestingly, IL-4 did
inhibit the "macrophage disappearance" reaction that followed
munohistology for class II antigen was performed using monoclonal
antibody ATCC HB15 with a biotinylated second antibody and streptavidin-peroxidase developing system (Biogenex, Dublin, CA).
TNF Assay. A cytotoxicity assayed based on the release of radioac
tivity from "Cr-labeled Actinomycin D (5 Mg/ml)-treated L929 cells
was used, as described earlier (14, 17). Labeled L929 cells, in RPMI
1640 medium with 10% PCS, were plated at 5 x IO4 cells/well in 96
well flat-bottomed plates (Falcon), along with an equal volume (O.I ml)
of TNF-containing supernatants or dilutions thereof. After 18 h at
37°Cin a humidified 5% CO2 incubator, 0.1 ml of the supernatant was
retrieved and counted in a gamma counter. The percentage of specific
cytotoxicity was calculated [(experimental counts —spontaneous
counts/total counts - spontaneous counts) x 100%]. Standard concen
trations of recombinant human TNF (Cetus) were run to provide a
dose-response curve and cytotoxicity was expressed in units/ml super
natant by extrapolation onto this standard curve.
Statistics. The data presented are representative of at least three
independent sets of experiments, involving 3-5 mice per group. Statis
tical analysis was by Student's t test.
RESULTS
Effect of IL-2 on TNF Production by Peritoneal Macrophages
and Monocytes. Normal peritoneal macrophages in the absence
of any deliberately added exogenous stimulus produced little
TNF over a 24-h period of culture (Table 1). TNF secretion
could be stimulated by LPS, although responses were generally
poor. In contrast, peritoneal macrophages from IL-2-treated
mice (5 x IO4units twice daily for 2.5 days) were very responsive
to LPS, indicating that they had been primed in vivo. IL-2induced priming was seen with doses as low as 5 x 10* units
IL-2 twice daily but 5 x IO4units was optimal (data not shown).
Peritoneal macrophages from IL-2-treated athymic nude
mice also responded to in vitro LPS stimulation better than did
those from saline-treated controls, although not nearly as well
as those from IL-2-treated euthymic mice, indicating that in
vivo priming has a T-cell-dependent component. Macrophages
from saline-treated athymic and euthymic mice gave very sim
ilar i/i vitro responses (data not shown). Macrophages from the
IL-2-treated endotoxin-resistant CjH/HeJ strain of mice were,
as expected, unresponsive to LPS. In all these experiments, the
addition of IL-2 directly to peritoneal macrophages in vitro had
little, if any, effect on TNF production, even if the macrophages
were from IL-2-treated mice and primed for TNF production.
Peritoneal macrophages from IL-2-treated C3Hf/Sed//Kam
athymic and euthymic mice displayed morphological signs of
activation even without in vitro stimulation. They spread rapidly
and had an increased number of processes. Over 80% were
positive for class II MHC expression by immunohistology,
compared with 8-10% in controls.
In contrast to peritoneal macrophages that responded poorly
to in vitro stimulation, normal monocytes produced considera
ble quantities of TNF when stimulated with either IL-2 or LPS
injection of this high dose of LPS and that prevented us from
retrieving sufficient cells from the control group.
Because we had shown that treatment of mice with injections
of IL-2 primed macrophages for TNF production, the effect of
IL-4 on this process was evaluated. One injection of IL-4 was
given before concomitant i.p. administration of IL-2 and IL-4
twice daily for 2 days. Table 4 shows that IL-4 abrogated the
ability of IL-2 to prime macrophages for TNF production, as
judged by their in vitro response to LPS. In this experiment,
IL-4 was again shown to inhibit the priming effect of LPS
administered in vivo. This time a lower dose of LPS (0.5 ¿¿g,
i.p.) was used, to enable us to get a reasonable cell yield from
the LPS-treated control mice.
Effect of IL-4 on LPS and IL-2 Toxicity. TNF is thought to
play a role in the toxicity associated with endotoxemia (25-27)
and IL-2 administration (14). Because IL-4 down-regulated
TNF production by macrophages stimulated with LPS and IL2, we examined its effect on their toxicity in mice. In prelimi-
Table 1 TNF production by peritoneal macrophages in response to IL-2 in vitro
and in vivo
xSourceC,H/Sed//Kam
in vivo"
TNF production (units
Control3.4
vitro*3.6
Table 2 TNF production by monocytes in response to IL-2 in vitro and in vivo
IO~Vml)SourceC3H/Sed//Kam
vivo"
TNF production
(units x
Control4.7 vitro016.9
in
vitro'198.0±
±2.5"
59.0"
±0.6
+
11.1 ±1.8 64.3 ±2.9" 11.766.0 ±548.0"
CjH/Sed//KamCjH/HeJIL-2in
ND'
687.0 ±29.0"
+
48.2 ±4.7
+
2.6 ±0.1IL-28.1 ±0.9"LPSin 9.3 ±0.3"
"5x10" units of IL-2 twice daily for 2.5 days in vivo.
b IO3units IL-2/ml in vitro.
c 1 (jg/ml LPS in vitro.
a P < 0.05.
' ND, not done.
Table 3 Effect of IL-4, in vivo and in vitro, on LPS-stimulated TNF production
10~!/ml)In
TNF production (units x
vitro'9.5
vivo
treatmentControl
±1.9"
±1.0
±0.2
+
2.3 ±0.3
3.2 ±0.5 7424.0 ±308.0a
C3H/Sed//Kam
24.4 + 8.2"
ND'
+
1.6 + 0.1
CjH/Kam/nunu
+
5.1+0.6IL-2in6.4 ±0.710-2/ml)LPSin
5.0 ±0.3
CjH/HeJIL-2
"5x10* units IL-2 twice daily for 2.5 days in vivo.
b IO3units IL-2/ml in vitro.
' 1 Mg/ml LPS.
d P < 0.05.
' ND, not done.
+
LPS*0.8
±0.7
±0.9
84.0
55.04.7
±
±0.8
IL-4C
±0.2
4.7 ±0.7
1.3 ±1.0
3.6 ±1.0
IL-4 + LPS"Control2.3
3.6 ±0.4LPS*1 2.9 ±0.4IL-4" 2.8 ±0.5
1.5 ±0.2IL-4"2.4
" 400 units/ml IL-4 in vitro.
LPS in vitro.
1
c 10' units IL-4 in vivo twice daily for 2.5 days.
" 50 MgLPS in vivo.
2950
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EFFECTS OF 1L-2 AND IL-4 ON TNF PRODUCTION
Table 4 Effect of IL-4 and IL-2 in vivo on LPS-stimulated TNF production
production
lO'Vml)-LPS1.3
(units x
In vivo
treatmentSaline
IL-4"
IL-2C
IL-4* + IL-2C
LPS*
IL-4* + LPSCTNF
LPS"1.2
±0.4
0.8 ±0.6
0.7 ±0.8
0.9 ±0.6
1.0 ±0.3
0.8 ±0.6+
±0.1
1.0 + 0.6
1141.8 + 59.1
2.6 ±0.1
8.4 ±0.9
0.9 ±0.3
" 1 Mg/ml in vitro.
* 10* units IL-4 in vivo twice daily for 2.5 days.
' 5 X IO4units IL-2 in vivo twice daily for 2 days.
' 0.5 MgLPS in vivo.
Table 5 Effect of IL-4 on LPS and IL-2 toxicity
treatment"
Dead/total15/15+
Treatment150Mg/LPS150„g/LPS0.1
5/520/20-1-
days1.
dead at 2
2.2.2All
1.
dayAll
dead at <1
10/1015/15+
dayAll
dead at <1
D-galactosamine0.1
f¡gLPS plus 20 mg
mgo-galactosamineIL-2IL-2IL-4
,jg LPS plus 20
death*All
of
betweendays
dead
86.
5 and
5/5Day
6. 6. 6. 6
" 10' units rMu-IL-4 or saline were injected twice daily. LPS and LPS plus Dgalactosamine were injected immediately after the fourth injection of IL-4 (or
saline); no further injections were given. Human IL-2 (10' units) was injected
along with the second IL-4 (or saline) injection and concomitantly with IL-4 (or
saline) until death.
* Time from the initiation of IL-2 or LPS injections.
nary experiments, the LD50 for LPS injected i.p. into normal
mice was found to be 98 ±30 ^g (95% confidence limits of
mean); mice died within 48 h. IL-4 (IO5 units) administered
twice daily for 2 days did not protect mice against a single
challenge with 150 ^g LPS (Table 5).
D-Galactosamine is known to sensitize mice to the effects of
LPS (28, 29). In preliminary experiments, 20 mg D-galactosamine injected i.p. immediately before LPS lowered the LD50 to
0.06 ±0.04 ¿<g.
Mice died within 24 h. IL-4 did not protect
mice against challenge with 0.1 ^g LPS administered concom
itantly with D-galactosamine (Table 5).
Toxicity of IL-2 for mice can be cumulative and death occurs
after repeated administration of sufficiently high doses. Mice
given 10s units IL-2 twice daily died 6 to 7 days after initiation
of treatment. Concomitant administration of IL-4 with IL-2
did not influence this predictable mortality (Table 5).
DISCUSSION
TNF is an important cytokine with a wide range of immunoregulatory and effector functions. These include cytotoxicity
for tumor cells (30-32), augmentation of cellular immune re
sponses (33), and activation of phagocytic cells (32. 34). It has
been implicated as a mediator in endotoxic shock (25-27). in a
variety of immunopathological reactions (35-37). and circum
stantially in the toxicity of IL-2-based immunotherapy regimens
(14).
In this study we have shown that IL-2 can have direct and
indirect effects on macrophage activation. The presence of a
direct pathway can be inferred from the fact that IL-2 directly
stimulated murine monocytes to produce TNF, confirming our
findings with human monocytes (14). However, this depended
upon the source of macrophages. Peritoneal macrophages were
less responsive, even if the macrophages were from IL-2-treated
mice and were highly responsive to LPS. The reasons for the
differences between the macrophage populations in their re
sponses to IL-2 and to LPS are not clear. They may be associ
ated with the differential expression of specific receptors such
as the IL-2 receptor, which could change with differentiation.
In addition to the direct effects of IL-2 on monocytes, it can
be inferred from our data with athymic and euthymic mice that
IL-2 can, in addition, stimulate macrophages indirectly via a Tcell-dependent pathway that most probably involves a cytokine
cascade. A lymphokine pathway whereby antigen-responsive
helper T-cells induce TNF release from mononuclear cells has
been described which involves 7 interferon and a "cytotoxicitytriggering factor," which may act on macrophages (38). These
in vivo effects of IL-2 are also seen in patients receiving IL-2
immunotherapy. Their monocytes have an increased capacity
for TNF production (14) and increased levels of TNF can be
measured in patients' sera.4
IL-4 has been reported to induce differentiation of certain
macrophage populations, as judged by an increase in expression
of MHC class I and II and adhesion antigens (10-13). De
creased production of factors responsible for tumor cytostasis
and for chemotaxis has been shown for IL-4-treated human
monocytes, the former possibly being associated with decreased
IL-1 production, although increased macrophage cytotoxicity
has also been reported (10). We have reported that IL-4 in vitro
inhibits TNF and IL-1 production by human monocytes (17).
The down-regulatory effect of IL-4, added in vitro, on macro
phage TNF production seen with human monocytes was con
firmed in this study, using murine macrophages. Further, injec
tion of IL-4 prevented IL-2 and LPS from priming macro
phages in vivo for TNF production.
In spite of its strong down-regulatory action, IL-4 was unable
to protect mice against lethal doses of LPS and IL-2. Evidence
has been presented that TNF is an important mediator of
endotoxic shock (25-27); however other factors may play a role
in LPS and IL-2 toxicity or at least in pathways that lead to
death. Also, our studies do not address the production of TNF
in what may be organs critical for the toxic effects of LPS or
IL-2. Further investigations into this area of research are re
quired to resolve some of these questions.
We have shown that IL-2 and IL-4 have distinct antagonistic
effects on macrophage function, as evidenced by TNF produc
tion. This is all the more interesting because, at least in mice,
they are expressed in a mutually exclusive fashion by Thl- and
Th2-cells and appear to dictate the array of lymphokines that
are coordinately expressed. In so doing, they determine the
balance of cellular to humoral immunity. Our data support the
view that macrophages and their products are an intimate part
of the complex regulatory interactions of cells and cytokines
that determine the ultimate effector outcome. Further elucida
tion of the mechanisms underlying these interactions may lead
to more sophisticated cytokine-based immunotherapy regimens
than are presently available.
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Influences of Interleukins 2 and 4 on Tumor Necrosis Factor
Production by Murine Mononuclear Phagocytes
William H. McBride, James S. Economou, Ramin Nayersina, et al.
Cancer Res 1990;50:2949-2952.
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