By
From the Max-Planck-Institut für Immunbiologie, D-79108 Freiburg, Germany
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Abstract |
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Interferon (IFN)-, a key immunoregulatory cytokine, has been thought to be produced solely
by activated T cells and natural killer cells. In this study, we show that murine bone marrow- derived macrophages (BMM
) secrete large amounts of IFN-
upon appropriate stimulation.
Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-
mRNA transcripts,
the combined stimulation of BMM
with both cytokines leads to the efficient production of
IFN-
protein. The macrophage-derived IFN-
is biologically active as shown by induction of
inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings
uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-
but also a potent IFN-
-producing
cell.
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Introduction |
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Interferon (IFN)- regulates a variety of important immunological programs. It is the predominant cytokine
during Th1-dominated immune reactions, participates importantly during antigen presentation, and is the prototypical macrophage-activating cytokine. Consequently, a pivotal role of IFN-
in the clearance of various intracellular pathogens has been amply demonstrated (1). One of the
key events during innate immune reactions is the production of IL-12 mainly by macrophages (2). IL-12 induces
NK cells to rapidly secrete IFN-
, which then acts back to
activate macrophages early in an immune response. Furthermore, IL-12 induces IFN-
production by T cells and
is the key cytokine driving Th1 cell differentiation.
A recently discovered novel cytokine, IL-18, shares many
functional properties with IL-12. IL-18 was described originally as a Kupffer cell-derived costimulating factor essential for the production of IFN- in a murine LPS-induced
shock model (3). IL-18 has been cloned recently (4), and
was shown subsequently to induce IFN-
in human (5)
and murine (6) T cells. Moreover, strong synergistic effects
between IL-12 and IL-18 in the induction of IFN-
secretion of T cells (5, 6) or NK cells (7) were described.
A few reports described the secretion of low levels of
IFN- by murine (8, 9) or human (10) macrophages stimulated with IFN-
itself (8), IL-12 (9), or Mycobacterium tuberculosis (10). Because these findings are at variance with
the widely accepted view that T cells and NK cells are the
sole producers of IFN-
, the general significance of these
observations remained uncertain. A recent report demonstrated the secretion of IFN-
even by B cells upon combined stimulation with IL-12 and IL-18 (11). In this study,
we demonstrate that these two cytokines synergistically induce macrophages to secrete large amounts of IFN-
which is biologically active in an autocrine fashion. The data suggest that macrophage activation may operate by an
autocrine positive feedback loop involving multiple cytokines, including IFN-
.
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Materials and Methods |
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Medium and Reagents.
All cell culture was performed in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 60 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 1× nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL, Paisley, UK). Recombinant murine IFN-Animals and Generation of Bone Marrow-derived Macrophages.
Mice of strains AKR/N, C57BL/6, and 129Sv and mice homozygous for a targeted mutation of the IFN-IFN- Assay, Nitric Oxide Measurement, and Analysis of CD40
Expression by FACS®.
Reverse Transcription PCR.
5 × 106 BMMRNA In Situ Hybridization.
In situ hybridization was performed using a digoxigenin-labeled riboprobe corresponding to the region between nucleotides 371 and 1075 of the mouse IFN- ![]() |
Results |
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AKR/N-BMM were stimulated with various
concentrations of IL-12 or IL-18 alone or in combination
(Fig. 1). At different time points, the IFN-
concentrations in
the supernatants were determined by ELISA. The stimulation
of the macrophages with IL-12 or IL-18 alone did not result
in detectable IFN-
secretion. In sharp contrast, both cytokines synergized strongly to induce readily detectable levels
of IFN-
in the supernatants at all time points tested. The
cytokine accumulated from 3.7 ng/ml per 105 cells at 24 h
to 42 ng/ml per 105 cells IFN-
at 96 h. Stimulation of the
macrophages with IL-12 and IL-18 at 96 h was still suboptimal, as a plateau of IFN-
secretion has not been reached.
The induced levels of IFN-
are comparable to those
reached upon anti-CD3 stimulation of the prototypical Th1 T cell clone AE7 (81 ng/ml per 105 cells, data not
shown). The ability of the macrophages to secrete IFN-
is
not restricted to the AKR/N strain of mice. We also tested BMM
of C57BL/6 (see also Fig. 3 A) and BALB/c (data
not shown) mice with similar results. To demonstrate the
purity of our BMM
population and to exclude the possibility of contaminating NK, T, or B cells in our assay, we
analyzed the BMM
by FACS®. The population was consistently found to be homogeneously positive for F4/80,
MAC-1, I-Ak, B7.1, and CD16/32. T cell antigens (CD3,
CD4, and CD8), NK cell marker (DX5, and also NK1.1
for C57BL/6 mice) or B cell antigens (B220, CD19, and
CD5) were uniformly negative (data not shown).
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We also assayed the commonly used mouse macrophage
cell lines RAW 264.7, J774, RAW 309Cr, P388D1, and
IC 21. In contrast to BMM, none of the five cell lines
produced IFN-
upon stimulation with IL-12 and IL-18
(data not shown).
T o
address the level of regulation of IFN- synthesis, we stimulated BMM
with IL-12 (10 ng/ml) or IL-18 (50 ng/
ml), alone or in combination, and prepared mRNA after 2, 8, and 24 h of stimulation. IFN-
mRNA expression was
then evaluated by reverse transcription (RT)-PCR (Fig. 2
A). In the case of BMM
stimulated with IL-12 plus IL-18, a prominent band of IFN-
mRNA appeared as early
as 2 h after addition of the cytokines, and increased further
at later time points after stimulation. The PCR product
was proven to result from IFN-
mRNA by sequencing
(data not shown). Surprisingly, although no secreted IFN-
was detectable in the supernatant of BMM
stimulated
with IL-12 alone or IL-18 alone, IFN-
mRNA was clearly induced. This induction appears to be more prominent in the case of stimulation with IL-12 than with IL-18
and increases over time during the culture period. Nevertheless, as shown semiquantitatively by titrating the input
cDNA of the PCR reaction, the induction of IFN-
mRNA by combined stimulation with IL-12 and IL-18 is clearly far more efficient, consistent with the synergism observed at the protein level. To estimate the frequency of
IFN-
-producing macrophages within the whole population, we performed RNA in situ hybridization experiments
(Fig. 2, B and C). After 18 h of stimulation with IL-12 plus
IL-18, ~25-30% of the BMM
stained strongly positive for IFN-
mRNA (Fig. 2 C), whereas unstimulated control BMM
were homogeneously negative (Fig. 2 B).
Control stainings with the riboprobe in sense orientation
also yielded completely negative results (data not shown).
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To test the biological activity of IFN- produced
endogenously by BMM
stimulated with IL-12 plus IL-18, we tested two different indicators of IFN-
-mediated
macrophage activation. First, IFN-
is known to synergize
with TNF-
in the induction of inducible NO synthase
(iNOS) with concomitant production of nitric oxide (NO)
in macrophages (16). Therefore, we stimulated BMM
of
AKR/N and C57BL/6 mice with optimal concentrations
of IL-12, IL-18, or IL-12 plus IL-18 and titrated increasing
concentrations of TNF-
into these cultures. Control cultures received IFN-
. As a readout for iNOS induction,
we determined nitrite, the stable end product of NO, in
the supernatants after 96 h of stimulation (Fig. 3 A). IFN-
was measured simultaneously in the supernatants. In macrophages exposed to IL-12 plus IL-18, increasing concentrations of TNF-
induced iNOS very efficiently, although
somewhat less than upon optimal stimulation with exogenously added IFN-
. Experiments with mice lacking the
IFN-
receptor (IFN-
R
/
) proved that endogenously
produced IFN-
is responsible for the induction of iNOS:
although IFN-
R
/
-BMM
stimulated with IL-12 plus
IL-18 plus TNF-
secreted IFN-
comparable to control
IFN-
R+/+-BMM
(129Sv), no nitrites were detectable
in the supernatant of the IFN-
R
/
-BMM
, reflecting a
failure of iNOS induction when IFN-
is unable to signal
via its receptor (Fig. 3 B). Indeed, nitrite accumulation reflected iNOS induction, as demonstrated by the addition of
the iNOS inhibitor L-monomethyl-L-arginine, which completely abolished detectable nitrites without influencing
IFN-
induction (data not shown).
Two additional interesting observations were consistently made. First, with increasing concentrations of TNF-,
IFN-
levels stimulated with IL-12 plus IL-18 decrease
(Fig. 3, A and B). IFN-
R
/
-BMM
are an exception to
this rule (Fig. 3 B), pointing to a possible role of enhanced
consumption of IFN-
upon increasing concentrations of
TNF-
. The phenomenon is unrelated to the simultaneously produced NO, known to be potentially autotoxic
to the secreting macrophage itself (16), because L-monomethyl-L-arginine had no influence on the observed decrease of IFN-
(data not shown). Second, in the case of
the C57BL/6-BMM
(Fig. 3 A), IL-12 alone synergizes
with TNF-
to induce IFN-
as well as iNOS. In AKR/N mice, a different and/or less efficient synergism seems to
operate: in five independent experiments, a slight induction
of nitrites was noted consistently upon costimulation with
50 ng/ml TNF-
, whereas IFN-
in the supernatant was
either undetectable or only marginally elevated (e.g., 1.5 ng/ml in Fig. 3 A).
As a second system to demonstrate the functional potential
of the BMM-derived IFN-
, we assayed the IFN-
-mediated upregulation of CD40. Resting BMM
are negative
or only slightly positive for CD40, whereas stimulation
with IFN-
for 48 h leads to an upregulation of this costimulatory molecule (M. Munder, unpublished observation). The same induction was noted on IFN-
R+/+-BMM
(129Sv) stimulated with IL-12 plus IL-18. Again,
endogenously produced IFN-
was responsible for this effect, as demonstrated by the lack of CD40 induction on
IFN-
R
/
-BMM
stimulated with IL-12 plus IL-18
(Fig. 4). BMM
of mice with 129 background are definitively less efficient in IFN-
production upon IL-12/IL-18
stimulation (Fig. 3 B, and Fig. 4) than BMM
of strains
AKR/N or C57BL/6 (Fig. 1, and Fig. 3 A). Nevertheless, even these lower amounts of induced IFN-
clearly autoactivate the macrophages to upregulate iNOS and CD40.
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Discussion |
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These experiments were stimulated by the results of several authors showing synergism between IL-12 and IL-18
in the induction of IFN- by human (5) and murine (6) T
cells and murine NK cells (7). Remarkably, both cytokines
were shown recently to cooperatively induce IFN-
secretion of murine B cells (11). We now extend these observations by demonstrating that macrophages become efficient
IFN-
-producing cells upon combined stimulation with
IL-12 and IL-18. The levels of IFN-
were of a similar
magnitude as that produced by T cells, and we observe efficient autocrine macrophage activation by endogenously
generated IFN-
in vitro.
We found that a considerable proportion but not all
BMM express IFN-
mRNA under our conditions of in
vitro stimulation. Moreover, five established macrophage
cell lines failed to produce IFN-
under these conditions.
A detailed comparison (17) of various IFN-
-induced activation parameters between immortalized macrophage cell
lines and ex vivo-derived peritoneal macrophages demonstrated that each immortalized cell line exhibited only a part of the spectrum of the determined activation markers,
whereas all were detected in normal macrophages. It is not
clear whether these and our findings reflect a genuine
quantitative or qualitative macrophage heterogeneity in the
response to cytokines, and/or a loss of functional potency
associated with prolonged tissue culture maintenance.
Although the combined stimulation of the BMM with
IL-12 and IL-18 is required to stimulate IFN-
secretion,
both cytokines alone suffice in inducing detectable IFN-
mRNA. Whether this difference reflects merely a limitation of the ELISA sensitivity or hints towards unknown
posttranscriptional regulatory mechanisms remains to be
elucidated. Furthermore, Fultz et al. described the detection of IFN-
mRNA without secreted protein upon LPS stimulation of various types of murine macrophages, including BMM
(18), a finding we were unable to reproduce in the present study (not shown).
An interesting side-aspect of our work is the finding that
IL-12 and TNF-, similar to their synergistic effects in the
induction of IFN-
by NK cells (19), also cooperate in the induction of IFN-
secretion by BMM
. Among the three
strains tested, this effect is seen only in C57BL/6-BMM
,
possibly reflecting a genetic polymorphism related to the
known genetic differences between inbred mice in the
ability to generate Th1- or Th2-dominated immune responses. Thus, a detailed comparison of this phenomenon
between different mouse strains might prove fruitful.
Our work demonstrates for the first time that macrophages
secrete high levels of IFN- upon combined stimulation
with IL-12 and IL-18. Upon appropriate activation by pathogens or LPS, macrophages are known to be important producers of IL-12 (2) or IL-18 (3). Thus, our findings unravel
a novel potential pathway of autocrine macrophage activation involving endogenously produced IFN-
. This pathway might play a pivotal role during early innate immune
reactions, i.e., before the development of adaptive immunity, in infectious diseases (7, 10), in septic shock (3), as
well as during autoimmune reactions (20).
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Footnotes |
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Address correspondence to Manuel Modolell, Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany. Phone: 49-761-5108-535; Fax: 49-761-5108-534; E-mail: modolell{at}immunbio.mpg.de
Received for publication 12 March 1998 and in revised form 6 April 1998.
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