and nitric oxide from macrophages via IFN-
induction
Department of Pathology and Molecular Medicine, and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada
Correspondence: Dr. Zhou Xing, Rm. 4H19, Health Science Centre, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. E-mail: xingz{at}fhs.mcmaster.ca
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and nitric oxide (NO) release by using an
in vitro model of intracellular infection. IL-12 alone
released relatively little TNF- and NO, whereas live mycobacteria
alone released TNF- markedly but little NO from murine alveolar
macrophages. However, IL-12 and mycobacteria together enhanced TNF-
and NO release synergistically. Because IL-12 and mycobacteria also
released IFN-
from macrophages synergistically, and exogenous
IFN- with mycobacteria enhanced TNF- and NO release
synergistically, we examined the role of endogenous IFN- in
IL-12/mycobacteria-stimulated macrophage activation. Using macrophages
from mice deficient in IFN-, we found that
IL-12/mycobacteria-enhanced macrophage TNF- and NO release was
mediated through endogenous IFN-. We further demonstrated that
IFN- and mycobacteria together had a selective effect on macrophage
cytokine release because they released TNF- synergistically but not
macrophage chemotactic protein-1 (MCP-1). These findings reveal that
IL-12 can activate macrophages potently during intracellular infection,
and this activating effect is mediated primarily through its effect on
macrophage IFN- release.
Key Words: nitric oxide • MCP-1 • cytokine • tuberculosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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(IFN-), and
tumor necrosis factor (TNF-), play an important role in the
development of cell-mediated immune responses to intracellular
infections, particularly those caused by intracellular bacteria
[1
2
3
4
5
]. Among these, IL-12 is released primarily by
antigen-presenting cells and acts as a linker between the innate and
acquired immunities by inducing the differentiation of antigen-specific
T cells of Th1 phenotype and the release of IFN- from activated T
cells as well as natural killer (NK) cells [6
7
8
9
]. T
cell-derived IFN- is considered crucial to the adequate activation
of macrophages, the ultimate effector cells in host defense against
intracellular infection [2
, 5
].
IFN--activated macrophages demonstrate increased release of nitric
oxide (NO), which accounts for subsequently enhanced mycobactericidal
activities in macrophages [4
, 10
]. Recent
evidence from us and others has, however, revealed that IL-12 is
capable of a broader spectrum of biologic activities in the
cell-mediated immunity by acting on multiple immune cell types
including macrophages [11
12
13
].
TNF- is primarily a macrophage-derived cytokine [3
].
TNF-, together with IL-12 and IFN-, is markedly induced during
mycobacterial infection [9
, 11
,
14
15
16
17
]. Studies using anti-TNF- antibodies or gene
knock-out mice have demonstrated a critical role of TNF- in the
optimal expression of type 1 cell-mediated immune responses to
intracellular infections [14
, 15
,
18
, 19
]. In contrast to the tight control of
macrophage IFN response, macrophages appear to release TNF- under
a variety of in vitro stimulatory conditions
[3
, 11
, 20
]. However, the
mechanisms by which macrophage TNF- release is regulated during
intracellular infection remain to be completely understood, and, in
particular, the role of IL-12 in macrophage TNF- and NO responses is
still unclear. In our current study, we used a model of intracellular
infection to study TNF- and NO responses in macrophages upon the
interaction with type 1 cytokines and intracellular pathogens. We have
examined the effect of IL-12, IFN-, and live mycobacteria, alone or
in combination, on macrophage TNF- and NO release and dissected the
mechanisms by which IL-12 modulates macrophage TNF- and NO release.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-/- mice were purchased from the Jackson
Laboratory (West Grove, PA; C57BL/6-ifngtmlTs-stock
002287).
Reagents
An attenuated live strain of Mycobacterium bovis
(BCG) was obtained from Connaught Laboratories (North York, Ontario,
Canada). RPMI 1640 medium was supplemented with 10% fetal calf serum
(FCS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Murine IL-12
or IFN- was used as cytokine-containing supernatants generated by
transducing an A549 epithelial cell line with a gene-transfer vector
expressing murine IL-12 or IFN- [22
,
23
]. The high concentration of murine IL-12 or IFN- in
these supernatants was determined by enzyme-linked immunosorbent assay
(ELISA). Approximately 60% of IL-12 protein was found to be in the
bioactive form of IL-12 p70. A control empty vector was used to
generate control supernatants, which contained no IL-12 or IFN- and
were shown to have no effect on macrophage activation at all. In some
experiments, recombinant murine IL-12 and IFN- proteins (R&D
Systems, Minneapolis, MN) were also used. An antimurine macrophage
chemotactic protein-1 (MCP-1) antibody [purified immunoglobulin G
(IgG)] was purchased from R&D Systems.
Isolation of lung macrophages and culture conditions
A bronchoalveolar lavage procedure was carried out to isolate
lung macrophages from naive C57BL/6, IL-12-/-, or IFN--/- mice,
as previously described [11
]. Briefly, the mouse lung
was lavaged with a total of 2.6 ml phosphate-buffered saline (PBS) in
six aliquots (0.3 mlx2 and 0.5 mlx4) through a polyethylene tube
(Becton Dickinson, Sparks, MD) cannulated into the mouse trachea.
Collected lavage fluids were centrifuged to pellet cells. Approximately
0.6 to 1 x 106 cells per mouse were obtained, and
>98% of these cells were alveolar macrophages identified by
differential staining. For each experiment, cells isolated from three
to four mice of each strain were pooled. These cells were resuspended
in RPMI culture media and cultured in 96-well plates at a density of
0.1 x 106 cells/well without or with various stimuli,
including 50 cfu/cell of live M. bovis BCG
[11
], various concentrations of IL-12 or IFN-, or a
combination of BCG and IL-12 or IFN- in a total volume of 0.25 ml
for 72 h at 37°C. The supernatants were collected and stored at
-20°C until cytokine measurement.
Cytokine measurements
The level of cytokines in culture supernatants was determined by
using ELISA kits for murine TNF-, IFN-, MCP-1, or IL-12p70,
purchased from R&D Systems. The sensitivity of detection for all of
these ELISA kits was <5 pg/ml. Total IL-12 protein (IL-12p40 and p70)
was measured by using an ELISA kit purchased from Biosource
International (Montreal, Canada).
Measurement of NO production
The release of NO by macrophages was determined by measuring the
concentration in culture supernatants of the end product of NO, nitrite
[24
]. Briefly, diluted supernatants were mixed at a 1:1
ratio with Griess reagent buffer (Sigma Chemical Co., St. Louis, MO).
The absorbance was determined at 540 nm by using a spectrophotometer.
The final concentrations of nitrite were calculated from a standard
curve derived from prepared solutions of NaNO2 of known
concentrations.
Data analysis
Whenever applicable, the difference comparison was made by using
a Student’s t-test. The difference was considered
statistically significant when p 
0.05.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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increased
total IL-12 release, the combined stimulation by mycobacteria and
IFN- had a synergistic effect on total IL-12 release (Table 1 ). Because only IL-12p70 is the bioactive form of IL-12
[1
], we also measured the content of IL-12p70 under
these conditions. Of interest, minimal amounts of IL-12p70 were
detected under all of these conditions studied (Table 1)
. This finding
is in agreement with recent studies that demonstrate macrophage release
of IL-12p70 was under a much tighter control than IL-12p40, and
intracellular infection of murine macrophages by B. abortus
released only IL-12p40 but not IL-12p70 [25
,
26
]. |
View this table: [in a new window] |
Table 1. Release of IL-12 by Macrophages (pg/ml)
|
release by macrophages release, we
used bioactive IL-12 to stimulate macrophages because macrophages
themselves released little IL-12p70 (Table 1)
. To this end, alveolar
macrophages from naive C57BL/6 mice were isolated and cultured without
or with IL-12 or live mycobacterial BCG. Freshly isolated alveolar
macrophages released a small amount of TNF- spontaneously during
culture (120 pg/ml; Fig. 1
). (No effect on TNF- release was observed when macrophages were
cultured with the control supernatant generated with the empty vector.)
However, IL-12, when used at different concentrations, enhanced only
marginally TNF- release (200 pg/ml). This finding reveals that IL-12
by itself is not a strong inducer of macrophage TNF- release. In
contrast, upon stimulation with mycobacteria alone, macrophages
released significant amounts of TNF- (Fig. 1)
, supporting the
finding that macrophages release this cytokine in response to
stimulation by a variety of infectious agents [3
].
Because macrophages are exposed not only to mycobacteria but also to
IL-12 during pulmonary mycobacterial infection [16
], we
examined the effect of combined stimulation by mycobacterial infection
and IL-12. There was a remarkable synergistic effect on TNF- release
by mycobacteria and IL-12 (Fig. 1)
. The level of induced TNF- was
100–400% higher than by mycobacteria alone, depending on the dose of
IL-12. These data provide strong evidence that IL-12 plays an active
role in modulation of macrophage TNF- responses during mycobacterial
infection.
 View larger version (18K): [in a new window] |
Figure 1. Synergistic effect on macrophage TNF- release by IL-12 and
mycobacteria. Alveolar macrophages were isolated from the lung of
wild-type C57BL/6 mice and cultured in the absence or presence of IL-12
(800 and 2000 pg/ml) alone, live mycobacteria BCG alone, or BCG with
IL-12-containing supernatant for three days. The resultant supernatants
were measured for TNF- by ELISA. Cells cultured with BCG and control
supernatant (C) released similar amounts of TNF- as those cultured
with BCG. Results for most conditions are expressed as mean ±
SE from triplicate wells. The difference between no treat
vs. BCG and BCG vs. BCG + IL-12 (2 ng/ml) is statistically
significant (p<0.05). The results are representative of at
least two to three independent experiments.
|
release, we used the same doses of recombinant
murine IL-12 (rIL-12) protein also to stimulate macrophages. Similar to
our findings obtained by using IL-12-containing supernatants, although
rIL-12 released little TNF-, rIL-12 and mycobacteria had a
synergistic effect on TNF- release (Table 2
). |
View this table: [in a new window] |
Table 2. TNF- Release by Macrophages (pg/ml)
|
and mycobacteria on TNF- release by
macrophages under appropriate stimulatory conditions
[11
12
13
], the synergistic effect on TNF- release by
IL-12 and mycobacteria may be mediated through endogenous IFN-. To
address this issue, we examined first the content of IFN- in the
supernatants from IL-12- or IL-12/mycobacteria-stimulated macrophages
of C57BL/6 mice. Indeed, although mycobacteria and IL-12 alone released
little or small amounts of IFN-, respectively, IL-12 and
mycobacteria together synergistically released significant amounts of
IFN- (Fig. 2A
). We then examined the effect of exogenously added IFN- in the
presence or absence of mycobacteria on TNF- release from alveolar
macrophages isolated from naive C57BL/6 mice. IFN- alone, as
compared with IL-12 alone, appeared to be more potent in releasing
TNF- (300–450 pg/ml), although this difference was not
statistically significant (p=0.09; Figs. 1
and 2B
).
Furthermore, we examined whether there was a synergistic effect on
TNF- release when macrophages were stimulated with IFN- and
mycobacteria. Indeed, IFN- and mycobacteria together enhanced
synergistically TNF- release by 200–500% compared with that by
mycobacteria alone (Fig. 2B) .
 View larger version (15K): [in a new window] |
Figure 2. (A) Synergistic effect on macrophage IFN- release by IL-12 and
mycobacteria. Alveolar macrophages were obtained from the lung of
wild-type C57BL/6 mice and cultured in the absence or presence of IL-12
alone, BCG alone, BCG with control supernatant (C), or BCG with
IL-12-containing supernatant for three days. The supernatants were
measured for IFN- by ELISA. (B) Synergistic effect on macrophage
TNF- release by IFN- and mycobacteria. Alveolar macrophages were
obtained from the lung of wild-type C57BL/6 mice and cultured in the
absence or presence of IFN- alone (800 and 2000 pg/ml), BCG alone,
BCG with control supernatant (C), or BCG with IFN--containing
supernatant. The supernatants were measured for TNF- by ELISA.
Results for most conditions are expressed mean ± SE
from triplicate wells. The difference between no treat vs. BCG and BCG
vs. BCG + IFN- (2 ng/ml) is statistically significant
(p<0.05). The results are representative of at least two to
three independent experiments.
|
-containing supernatant on
TNF- release by macrophages, we tested the effect of rIFN-. Very
similar to our findings presented in Fig. 2B
, rIFN- by itself
enhanced macrophage TNF- release only moderately, and rIFN- + mycobacteria enhanced TNF- release markedly in a synergistic
manner (Table 2) .
Synergistic effect on TNF- release by
IL-12/mycobacteria is dependent on endogenous IFN-
Having established that IL-12 and mycobacteria release
synergistically not only TNF- but also IFN- and that exogenous
IFN- together with mycobacteria stimulate synergistically TNF-
release in macrophages, we set out to examine whether the potentiated
release of TNF- by IL-12/mycobacteria required endogenous IFN-.
To this end, we isolated alveolar macrophages from the lung of naive
mice deficient in the gene coding for IFN- and cultured them under
different conditions. IL-12 alone stimulated little TNF- release,
whereas IFN- alone stimulated a higher level of TNF- release by
IFN--/- macrophages (Fig. 3A
). Mycobacteria alone released significant amounts of TNF- from
these cells, comparable with those by cells from wild-type C57BL/6 mice
(Fig. 1)
. However, stimulation by IL-12 and mycobacteria failed to
enhance TNF- release further in the absence of endogenous IFN-
and as a positive control, exogenously added IFN- with mycobacteria
released synergistically TNF- from these cells (Fig. 3A)
. These
findings reveal that IFN- is required for
IL-12/mycobacteria-enhanced TNF- responses in macrophages.
 View larger version (17K): [in a new window] |
Figure 3. (A) Requirement of endogenous IFN- for
IL-12/mycobacteria-enhanced TNF- response in macrophages. Alveolar
macrophages were isolated from the lung of IFN- gene knock-out mice
and cultured in the absence or presence of IL-12, IFN-, or BCG
alone, or BCG with IL-12 or IFN-. In the absence of IFN-,
IL-12/mycobacteria failed to enhance TNF- release synergistically
from macrophages. The difference between IL-12 + BCG and
IFN- + BCG is statistically significant (p<0.01).
(B) Independence of endogenous IL-12 for IFN-/mycobacteria-enhanced
TNF- response in macrophages. Alveolar macrophages were isolated
from the lung of IL-12p40 gene knock-out mice and cultured in the
absence or presence of IFN- or BCG alone or BCG with IFN-. The
supernatants were measured for TNF- by ELISA. The difference between
BCG and BCG + IFN- (800 pg or 2 ng/ml) is statistically
significant (p<0.01). Results (A and B) are expressed as
mean ± SE from triplicate wells/condition.
|
and mycobacteria on macrophage
TNF- release is IL-12-independent for IL-12/mycobacteria-enhanced TNF- release. Although we
have shown that macrophages under the current stimulatory conditions
released little bioactive IL-12p70 (Table 1)
, it cannot be ruled out
that endogenous IL-12p70, which may present in a membrane-bound form on
macrophages [27
], may potentiate the effect by IFN-
and mycobacteria. To this end, we examined the response in alveolar
macrophages isolated from mice deficient in IL-12. We found that
mycobacteria alone released increased amounts of TNF- in the absence
of endogenous IL-12, similar to those by macrophages from wild-type
C57BL/6 (Fig. 1
; Fig. 3B
), and the lack of IL-12 did not compromise at
all the synergistic effect of IFN- and mycobacteria on TNF-
release (Fig. 3B)
. These findings have thus established a cascade of
macrophage type 1 cytokine responses during intracellular mycobacterial
infection: 1) Infection of macrophages by mycobacteria triggers basal
levels of TNF- release, which is IL-12- or IFN--independent. 2)
IL-12 and mycobacteria are required for macrophage IFN- release. 3)
It is IFN- that, together with intracellular mycobacteria, enhances
the level of TNF- release synergistically; importantly, this
enhanced TNF- release results from direct stimulation by
IFN-/mycobacteria, which is independent of IL-12.
IFN- and mycobacteria do not have a synergistic effect on
MCP-1 release from macrophages
It was important to examine also whether IFN- and mycobacteria
have a synergistic effect on the release of cytokines other than
TNF- in macrophages. To this end, we have chosen to examine the
release of MCP-1, a C-C mononuclear cell chemokine shown to play a role
during mycobacterial infection [28
]. IFN- or
mycobacteria alone only moderately enhanced MCP-1 release (Fig. 4
). However, IFN- and mycobacteria together did not impose a
synergistic effect on MCP-1 release in macrophages obtained from naive
C57BL/6 mice. These results suggest a restricted synergistic effect on
macrophage cytokine responses by IFN- and intracellular pathogens.
We also cultured macrophages in the presence of IFN-, mycobacteria,
and an antimurine MCP-1 antibody, and measured TNF- response. We
found that MCP-1 antibody only minimally reduced TNF- release,
suggesting that MCP-1 is unlikely involved in IFN- and
mycobacteria-stimulated TNF- release (unpublished results).
 View larger version (19K): [in a new window] |
Figure 4. IFN- and mycobacteria cannot enhance MCP-1 response
in macrophages synergistically. Alveolar macrophages were obtained
from the lung of wild-type C57BL/6 mice and cultured in the absence or
presence of IFN- or BCG alone or BCG with IFN- for three days.
The supernatants were measured for MCP-1 by ELISA. Results are
expressed as mean ± SE from triplicate wells. The
difference among no treat vs. BCG, no treat vs. BCG + IFN-, and
BCG vs. BCG + IFN- is all statistically significant
(p<0.05, p<0.01, and p<0.05,
respectively).
|
and mycobacteria on macrophage NO
release alone
enhanced NO release moderately (Fig. 5
). The effect of IL-12 on macrophage NO release had not been
investigated previously. It has been found that only IFN- but not
any other macrophage-activating cytokines including IL-1, TNF-, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) can release
NO from naive macrophages in vitro [31
]. Our
finding that IL-12 alone is unable to release NO from macrophages
supports such highly selected activity of proimmune cytokines in
macrophage NO release. In contrast, we observed a synergistic effect on
NO release by combined stimulation with mycobacteria and IL-12 or
IFN-. However, in the absence of intrinsic IFN-, mycobacteria and
IL-12 failed to induce NO production, in sharp contrast to NO induction
by mycobacteria and exogenously added IFN- (Fig. 5)
. These findings
indicate that IL-12 and mycobacteria synergized to induce NO via the
effect of endogenously released IFN- and extend the previous
observation that a maximum of NO release from macrophages requires
IFN- and lipopolysaccharide (LPS) [32
]. Our data
suggest also that intracellular pathogens and IFN- together form a
unique combination leading to the full activation of macrophages, which
is characterized by markedly enhanced release of TNF- and NO.
 View larger version (24K): [in a new window] |
Figure 5. Synergistic effect of mycobacteria and IL-12 or IFN- on NO release
by macrophages. Alveolar macrophages were obtained from the lung of
C57BL/6 or IFN--deficient (IFN--/-) mice and cultured in the
absence or presence of BCG, IL-12 (800 pg/ml), or IFN- (800 pg/ml),
or a combination of BCG and IL-12 or IFN-. The concentration of NO
was determined by measuring nitrite in culture supernatants. Results
are expressed as average value from two to three wells/condition.
|
are released from antigen-presenting cells. The initial macrophage
TNF- release is IFN--independent. IL-12, together with
intracellular bacteria, is an ignitor of macrophage activation.
IL-12-stimulated IFN- release from macrophages and lymphocytes is an
ultimate macrophage activator, which, together with intracellular
pathogens, is capable of markedly potentiating macrophage release of
TNF- and NO, molecules crucial to mycobactericidal activities
(Fig. 6
).
 View larger version (17K): [in a new window] |
Figure 6. Schematic presentation of cytokine cascade during pulmonary
mycobacterial infection. Intracellular pathogens release IL-12 and
TNF- from antigen-presenting alveolar macrophages (AM) and dendritic
cells (DC). TNF- release by these cells is IFN--independent.
IL-12 acts on antigen-specific T cells and together with pathogens,
acts on alveolar macrophages to release IFN-. Macrophage IFN-
release requires intracellular pathogens and IL-12. IFN- and
mycobacteria synergistically activate macrophages to release
antimicrobial molecules including TNF- and NO.
|
was kindly provided by Dr. Jay Kolls. An adenoviral
gene-transfer vector for murine IL-12 was constructed by Drs. Jonathan
Bramson and Mary Hitt at McMaster University. Received April 6, 2000; revised June 29, 2000; accepted June 30, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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