By
From the * Institute of Experimental Immunology, University Hospital, CH-8091 Zürich,
Switzerland; Department of Internal Medicine, University Hospital, CH-8091 Zürich,
Switzerland; § Department of Pathology, University Hospital, CH-8091 Zürich, Switzerland;
Department of Molecular Genetics, Research Institute of Molecular Pharmacology, D-12207 Berlin,
Germany; and ¶ Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario, M4X1K9,
Canada
Listeria monocytogenes is widely used as a model to study immune responses against intracellular
bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon- (IFN-
) and tumor necrosis factor-
(TNF-
) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This
study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF)1 or 2 for
their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive
nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages.
ICSBP
/
and to a lesser degree also IRF2
/
mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage
(PEM) cultures stimulated with IFN-
in vitro; in addition these cultures showed reduced and
delayed oxidative burst upon IFN-
stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently
controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-
-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of
macrophages, whereas IRF1-induced RNI are not limiting.
Listeria monocytogenes, a grampositive facultative intracellular bacterium, infects macrophages and hepatocytes
in mice and has been used as a classic model to study immune responses against intracellular bacteria (1). Neutrophile
granulocytes (2), Much information has accumulated about molecular and
in vivo biological function of IFNs over the past 15 years
(for review: see 24, 25). Two different pathways can be
distinguished: IFN- A variety of IFN-induced transcription factors have now
been described, most of them belonging to the structurally
related family of the interferon regulatory factors (IRFs) and
some being identical with signal transducers and activators of
transcription (STATs; Table 1). It has been revealed that
there is an overlap between the two IFN systems at the level
of transcription. Whereas some components of the interferon-stimulated gene factor (ISGF) 3 T cells (3), and above all macrophages
(4) are important during the early phase of the immune response. In SCID mice lacking mature B and T lymphocytes, NK cells activated by macrophage-derived TNF-
have been shown to activate the listericidal effector mechanisms of macrophages via secretion of IFN-
(5). These cells
are able to restrict initial replication of Listeria in murine
liver and spleen, since IFN-
inhibits evasion of Listeria
from phagosomes into the cytoplasm (6). Specific T cells
are needed for final elimination of the pathogen (7) and also
for protection against secondary infection (8). Studies of
Listeria infection in mice deficient for IFN-
(11), IFN-
receptor (12) or TNF receptor 1 (13) have shown that the
two cytokines IFN-
and TNF-
are crucial for survival.
However, the involved signaling pathways are not known,
and the effector mechanisms used by macrophages for killing of Listeria are still debated. The role of reactive oxygen
intermediates (ROI)1 (14) as well as reactive nitrogen
intermediates (RNI) (20) has been analyzed repeatedly;
these experiments revealed variations between different experimental setups and analyzed species.
and -
are binding to the type I IFN
receptor, whereas IFN-
binds to the type II IFN receptor.
Analysis of gene-targeted mice deficient for only one (12, 26)
or both of these receptors (27) have revealed that depending upon the type of the pathogen these two systems are either redundant or complementary in their antimicrobial activity (for review see 28).
are only induced by
type I IFN (29), IRF1 (30, 31) and STAT1 (32, 33) can be
upregulated via both IFN receptors or by viruses directly
(31), and interferon consensus sequence binding protein
(ICSBP) is the prototype of a type II IFN-induced factor
(34, 35). IRF2 is omitted from Table 1, because the way of
its induction has not been clearly elucidated so far. The fact
that IRF2 is lacking in ICSBP
/
mice (36) suggests induction via IFN-
pathway. In vitro transfection systems
with reporter genes have revealed that IRF1 (37) and ISGF3 (29) are activating transcription of genes containing the interferon-stimulated response element (ISRE) in their
promotor sequence, whereas ICSBP (38) and IRF2 (37)
have repressor activity for ISRE-containing genes.
The generation of gene-targeted mice for the transcription factors IRF1 (39, 40), IRF2 (39) and ICSBP (36) allows to test for their biological role and their induction in
different infectious disease models, especially for activation
of macrophages. This study therefore evaluated the susceptibility of these mouse strains to Listeria infection in vivo
and compared it to some macrophage effector functions
upon IFN- stimulation in vitro.
Mice.
Mice deficient for ICSBP (background C57BL6×
129Sv), IRF1(129Sv), IRF2 (C57BL/6), IFN I and II receptor
(both 129Sv) were generated by gene targeting in embryonic
stem cells as described (12, 26, 36, 39). IRF1-deficient mice were
kindly provided by Prof. Charles Weissmann (Institute for Molecular Biology I, University of Zürich, Switzerland). IFN type I
receptor/
(A129) and IFN type II receptor
/
(G129) mice
were obtained from the breeding colony of Prof. M. Aguet (Institute for Molecular Biology I, University of Zürich, Switzerland).
Control C57BL/6 or 129Sv mice as well as RAG2
/
mice were
obtained from the Institute for Laboratory Animals (Veterinary
Hospital, Zürich, Switzerland). Mice were used at 6-10 wk of
age. The different breedings (except A129 and G129) and all the
experiments were performed under conventional (non-SPF) conditions.
Listeria Culture and Infection. Listeria monocytogenes was originally obtained from B. Blanden (Canberra, Australia). It was cultured in trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD), and overnight cultures were titrated on tryptose blood agar plates (Difco Laboratories, Detroit, MI). For injection, the original culture was diluted in BSS to inject the indicated dose in 200 µl for i.v. or 30 µl for injection into the footpad (i.f.).
Determination of Bacterial Titers. On the indicated days after infection the whole spleen and one lobe of the liver were taken out and homogenized. Bacterial titers were determined by plating out four serial 10-fold dilutions of organ suspensions on tryptose blood agar plates.
Adoptive Transfer of Spleen Cells.
On day 0, spleen single cell
suspensions were let to adhere to plastic to deplete them from
macrophages. After 2 h 3 × 107 splenocytes were transferred into
nonirradiated RAG2/
recipients. On day 1 the recipients were
infected with 2 × 105 CFU of Listeria, and on day 10 liver and
spleen were taken out to determine bacterial titers.
Peritoneal Macrophage Cultures.
Peritoneal macrophages (PEM)
of different strains were elicited by injection of 2 ml of a starch solution (2%; Merck, Darmstadt, Germany) intraperitoneally on day
5 and harvested on day 0 by rinsing the peritoneal cavity with
10 ml of cold BSS. The macrophages were washed three times
with BSS supplemented with albumin to prevent clumping and
then plated on cover slips in 24-well plates. Cells were cultured in
IMDM (Gibco, Basel, Switzerland) supplemented with 10% FCS,
glutamine, and 50 µg/ml gentamicin, an only extracellularly effective antibiotic. After 2 h of adherence the cover slips were washed twice and put in 1 ml IMDM. The cultures were stimulated with 200 ng/ml LPS, with 200 U/ml recombinant murine
IFN-
(Genzyme, Cambridge, MA) or a combination of both for
42 h and then used for determination of nitric oxide (NO) production, of respiratory burst or of Listeria killing in vitro. In those
cultures used for killing assays, the medium was changed to antibiotic-free after 24 h.
Determination of NO and Respiratory Burst. NO production was measured by determination of nitrite accumulation in PEM cultures with Griess reagent (0.05% N-1-naphthyl-ethylene-diamine-dihydrochloride/0.5% sulfanilamide/2.5% phosphoric acid; all from Fluka, Buchs, Switzerland) as described (41). In brief, 50 µl cell culture supernatant was added to 150 µl Griess reagent in 96well plates and incubated at room temperature for 10 min. Absorption was read with an ELISA reader at 570 and 630 nm.
Respiratory burst was measured as H2O2 production by cultured PEM upon PMA (Sigma, Buchs, Switzerland) stimulation as described (18). In brief, H2O2 secretion of macrophages was quantified by chemiluminescence under presence of horseradish peroxidase type I (Sigma) and 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol; Sigma) after triggering with 50 ng/ml PMA. Light emission was discontinuously measured over 15 min in a LKB 1251 luminometer (LKB, Bromma, Sweden). Values in mV were converted into pmol H2O2 after calibration by the scopoletin method (42). The cells on the cover slips were counted, and values for NO and H2O2 calculated as nmol/105 cells. In Fig. 4 B stimulation index of stimulated versus unstimulated cultures is shown, because absolute values of respiratory burst varied between the experiments. PMA was used to trigger respiratory burst because, as a chemically defined substance, it is the most reliable burst trigger. Also opsonized Listeria, BCG or zymosan could be used with similar capacities to trigger burst (23, 43), but more variability. Because we investigated mouse strains deficient for various IFNdependent transcription factors, the induction phase of NADPH oxidase during 2 d under IFN-
In Vitro Killing Assay. PEM cultures in antibiotic-free medium as described above were infected with 107 CFU of Listeria from an overnight culture, washed three times and opsonized with normal human serum. After 15 min of phagocytosis the infected cultures were washed thoroughly, and gentamicin-containing medium and the respective stimulators were added. To determine the infection rate at time point t0, three cover slips were taken out. The remaining ones were further cultured for 7 h to allow digestion of Listeria by macrophages. After 7 h the cover slips were taken out, dried, and then stained according to May-Grünwald-Giemsa. For each mouse strain and each stimulation a total of 600 macrophages were counted under the microscope to determine the number of Listeria-infected cells. The change of infected macrophages was calculated in percentage of the infection rate at t0. For details, see reference 18.
Immunohistochemistry. Mice infected with 5 × 103 CFU of Listeria i.v. were sacrificed on day 5 or 6. Organs were immersed in Hank's BSS and frozen in liquid nitrogen. 5-µm cryosections were fixed with acetone for 10 min, immunostained for Listeria with a polyclonal rabbit anti-Listeria serum (diluted 1/2,000; kindly provided by Professor J. Bille, Institute of Microbiology, University Hospital of Lausanne, Switzerland) and for iNOS with a polyclonal rabbit anti-iNOS serum (diluted 1/1,500; Biomol, Plymouth, PA). Bound primary antibodies were detected using a sandwich staining procedure. Sections were incubated with alkaline phosphatase-labeled goat anti-rabbit Ig (diluted 1/80; Jackson Laboratories, Bar Harbor, Maine) followed by alkaline phosphataselabeled donkey anti-goat Ig (diluted 1/80; Tago). Dilutions of secondary reagents were made in TBS containing 5% normal mouse serum. All incubation steps were done for 30 min at room temperature. Alkaline phosphatase was visualized using naphthol ASBI phosphate and New Fuchsin (Sigma) as substrate, which yields a red color reaction product. Endogenous alkaline phosphatase was blocked by levamisole. Sections were counterstained with hemalum, and cover slips were mounted with glycerol/gelatin.
Gene targeted mice deficient for ICSBP, IRF1, or IRF2
were infected with various doses of Listeria intravenously or
peripherally i.f., and survival was monitored daily (Table 2).
All ICSBP/
mice died after injection of a dose as low as
50 CFU of Listeria, whereas five of six IRF2
/
mice succumbed to a dose of 5 × 103 CFU within 12 d. In contrast,
IRF1
/
and wild-type mice resisted to a dose of 5 × 103
CFU injected intravenously. However, IRF1
/
mice on
C57BL/6 background and held under strict SPF conditions also showed enhanced susceptibility to Listeria, when injected with a 5-10-times higher dose intraperitoneally
(Ferrick, D., and H.W. Mittrücker, personal communication). Listeria titers in liver and spleen were determined 24 h
after a high dose (2 × 105 CFU) and 5 d after an intermediate dose (5 × 103 CFU) of Listeria injected intravenously.
In the first 24 h, when neutrophils seem to play an important role (2), there was almost no titer difference between
the three strains and only a 10-fold difference compared to
control mice (data not shown). However, after 5 d when
activated macrophages are essential for control of Listeria
infection, ICSBP
/
and IRF2
/
showed between 102-
and 106-fold higher titers in liver and spleen, whereas
IRF1
/
mice controlled Listeria replication comparable to
controls (Fig. 1 A). In vitro gene regulation studies have revealed that ICSBP and IRF2 form complexes which then
have a markedly enhanced DNA binding capacity to ISRE
compared to the single factors (44). In contrast to IRF1, they
are both negative regulators of classical IFN-induced genes.
However, both transcription factors are obviously of major
importance for early anti-Listeria immune responses. Since
it has been shown that ICSBP
/
mice do not express
IRF2 (although the gene is intact [36]), this can explain the
even more drastic phenotype of ICSBP
/
compared to
IRF2
/
mice, because they represent functionally a double knock-out phenotype.
|
Competition of different transcription factors of the IRF
family at the DNA binding level has been demonstrated in
in vitro studies (45). It was therefore possible that lack of
IFN type I-induced transcription factors would lead to increased activity of IFN type II-induced factors. To test this
in vivo, we infected mice deficient for the type II (G129)
or the type I (A129) IFN receptor and control mice (wt129)
with 5 × 103 CFU of Listeria and determined bacterial titers in liver and spleen on day 5 (Fig. 1 B). As demonstrated
earlier (12), G129 mice showed drastically enhanced bacterial replication and lethality (Table 2), whereas A129 eliminated the pathogen even more efficiently than wt129 mice.
This result suggests that competition between the two signaling pathways at the transcription factor level occurs. IFN
type II-induced transcription factors (and among them especially ICSBP) may compensate for the lack of IFN type
I-induced factors in the A129 mouse, thereby conferring
even higher resistance to Listeria infection than in control
mice. Because early Listeria clearance in nude mice (46) has
been shown to be more efficient than in immunocompetent
controls because their macrophages are preactivated (probably by LPS derived from normal intestinal bacteria leaking
into circulation), this may be an additional factor explaining the results in A129 mice and also the difference between
IRF1/
mice held under conventional versus SPF conditions.
Our results of
anti-Listeria immune response in ICSBP/
mice suggested
a major defect of IFN-
-induced macrophage function, because lymphocytes, especially cytotoxic T cells, but also
B cells, had been shown to function almost normally after
viral infections (36, 39). Therefore macrophage functions
were tested in vivo by adoptive transfer experiments and in
vitro by PEM cultures.
RAG2/
mice are devoid of functional T and B cells,
but have normal macrophages and natural killer cells (47).
When infected with an intermediate dose of Listeria, they are
able to control bacterial replication comparable to nude mice
(46), but cannot eliminate the pathogen. To test whether
ICSBP-deficient T cells could develop normal specific anti-
Listeria immunity, we transferred on day 0 macrophage-
depleted ICSBP
/
spleen cells into RAG2
/
mice, challenged them with a high dose of Listeria (2 × 105 CFU i.v.)
on day 1 and evaluated Listeria titers in liver and spleen on
day 10 to look for efficiency of the specific immune response. As a positive control normal spleen cells and as a
negative control no spleen cells were transferred. The result
(Fig. 2) revealed no difference of Listeria counts between
recipients of ICSBP
/
and ICSBP+/+ spleen cells; in contrast RAG2
/
mice that did not receive spleen cells exhibited 100- (spleen) to 1,000-fold (liver) higher bacterial
counts. This result indicates that ICSBP
/
splenocytes (especially the mutant T cells) were able to promote elimination of Listeria as successfully as normal lymphocytes in cooperation with the intact macrophage compartment of the
RAG2
/
mouse.
Analysis of Listeria Killing in an In Vitro PEM Culture.
To evaluate listericidal activity of macrophages of the different mutant mouse strains, we tested PEM in an in vitro
killing assay. ICSBP/
, IRF1
/
, and IRF2
/
PEM were
elicited by starch injection intraperitoneally, plated onto cover
slips and cultured as described in Materials and Methods. After 42 h the cultures were infected with Listeria in vitro, and the number of infected cells determined at time point
t0 and after 7 h of infection. The results (Fig. 3) show that
PEM of ICSBP
/
and, to a lesser degree, IRF2
/
mice
allowed enhanced replication of Listeria, whereas PEM of IRF1
/
and normal mice were able to reduce the bacterial load in these macrophage cultures. Also the number of
bacteria per macrophage was higher in ICSBP
/
mice
(mostly more than 10 bacteria/cell) compared to their controls (0-4 bacteria/cell), revealing some macrophages with
plenty of Listeria and typical comet tails (18). This finding
confirms the defect in macrophage effector function, which
correlates with the in vivo susceptibility of these mouse
strains to Listeria (ICSBP
/
>IRF2
/
>IRF1
/
).
The effector mechansim responsible for listericidal properties of macrophages is
widely studied and still not clearly defined. ROI (14) as
well as RNI (20) have been proposed to be of major importance. Therefore NO production (measured as nitrite
accumulation in culture medium) and respiratory burst
upon PMA stimulation (H2O2 production measured by
chemiluminescence) were tested in PEM cultures stimulated with LPS and/or IFN- as described in Materials and
Methods. NO production (Fig. 4 A) was absent in IRF1
/
mice confirming earlier results that iNOS cannot be induced by IFN type I or II combined with LPS and/or
TNF in the absence of IRF1 (48, 49). In contrast, iNOS
activity was normal in ICSBP
/
and in IRF2
/
mice as
well as in G129 mice (50). This finding in IRF2
/
mice
differs from recently published results (51). The high susceptibility of the ICSBP
/
and G129 strains to Listeria infection in vivo and in vitro indicates that the NO effector
mechanism does not play a limiting role in Listeria clearance.
The phenotype of IRF1
/
mice found here is compatible
to the published results of Listeria infection in iNOS-deficient
mice (23). These mice showed also a slightly enhanced
bacterial replication (10-100-fold) and a higher lethality to
Listeria infection, but only after injection of 6 × 104 CFU
i.v. The LD50 of iNOS-deficient mice was only a factor 10 lower compared to their normal littermates, whereas in the
case of ICSBP
/
mice this difference is more than 10,000fold (Table 1; LD50 for C57BL/6 mice is ~3 × 105 CFU
[52]). The fact that iNOS induction in the susceptible
strains (ICSBP
/
, IRF2
/
) was higher than in controls
(Figs. 4 A, and 5) could even indicate that NO may have a
toxic effect on infected cells during murine listeriosis.
With respect to respiratory burst upon PMA challenge,
all mouse strains showed comparable basal activity of unstimulated cultures; however, in ICSBP/
and IRF2
/
mice ROI production could not be stimulated by IFN-
(Fig. 4 B) and was 3-5 min delayed compared to control
mice (data not shown). This finding suggests that deficient
ROI production might be partially responsible for the high
susceptibility of ICSBP
/
mice to Listeria, but it cannot
fully explain the drastic phenotype of these mice. At least a
third effector pathway not yet known may have to be
evoked to explain this phenotype (see Discussion). In addition, the fact that LPS-induced respiratory burst was enhanced in IRF2
/
mice correlates inversely with a recently
published finding of high IRF2 levels in LPS-hyporesponsive mouse strains (53). Thus, IRF2 may mediate the macrophage deactivating effect of LPS (42).
Apart from macrophages hepatocytes are a major target cell
in murine listeriosis. They are infected by direct cell-to-cell spread of the pathogen that is able to associate with actin
filaments of the cytoskeleton (54). Hepatocytes can produce NO. Therefore, to see whether the phenotype of
ICSBP/
and IRF2
/
mice is due to a localized inability
of iNOS expression in the liver, immunohistological analysis of iNOS expression in liver (Fig. 5) and spleen (not shown)
after Listeria infection was performed. Mice of the three
gene-targeted and control strains were infected with Listeria
(5 × 103 CFU i.v.). On day 5 or 6 liver and spleen were
taken, cryosectioned, and then immunostained for Listeria
and for iNOS with an appropriate polyclonal rabbit antiserum. Induction of iNOS comparable to wild-type mice
could be demonstrated in all strains except IRF1
/
(Fig.
5, C, F, K, M, O). It was abundant in regions where Listeria and abcesses were found (detail shown in Fig. 5, G and H).
In addition fulminant Listeria proliferation in liver (Fig. 5 E) and spleen of ICSBP
/
mice was found with accompanying tissue destruction (Fig. 5 D) correlating with the high
bacterial titers (Fig 1). This analysis also shows that the susceptibility of ICSBP
/
and IRF2
/
mice to Listeria is not
due to inefficient NO production in the liver. In contrast,
IRF1
/
mice were well protected and did not express
iNOS in the liver. This cannot be explained by earlier decline of the bacterial load because these mice had equal
Listeria titers in the liver as wild-type mice 5 d after infection (Fig. 1 A). The same analysis was performed on spleen
sections with comparable results (not shown).
The type II IFN system has been shown to be of crucial
importance for immunity against Listeria, because mice deficient for IFN- (11) or the type II IFN receptor (12) are
highly susceptible to this bacterium. However, the intracellular signalling pathway and the final effector mechanisms
involved in Listeria clearance are only incompletely understood. The presented analysis of three gene-targeted mouse
strains deficient of ICSBP, IRF1, or IRF2 with respect to
their capacity to survive and eliminate Listeria in vivo and
in vitro and to the ability of their macrophages to respond
with ROI or RNI production upon IFN-
stimulation
suggests three major conclusions: (a) ICSBP/IRF2 complex (but not IRF1) is of crucial importance for murine innate immunity to Listeria in vivo and in vitro, (b) iNOS
induction and NO synthesis play no limiting role for anti-
Listeria activity of macrophages upon IFN-
stimulation, (c)
stimulation of ROI production by IFN-
together with a
postulated third yet unknown effector pathway in macrophages may be responsible for protection in the early phase of primary Listeria infection. The role of NO for antimicrobial activity of macrophages has been tested in other
infectious model systems. It seems to play an important role
in leishmaniasis (55, 56) and tuberculosis (48), but has no
limiting effect in toxoplasmosis (57) and listeriosis (this study,
references 58, 59).
Our results of the analysis of mice deficient for IFN type I or II receptors revealed surprisingly that the type I IFN receptor-deficient mice (A129) were better protected than their normal littermates. This finding may reveal in vivo competition of transcription factors of both signaling pathways at the DNA binding level (45) suggesting that absence of the type I system enhances function of the type II system and concurrently Listeria protection. A potentiating effect of LPS leaking through from intestinal bacteria leading to macrophage preactivation may be involved.
From the analysis of TNF receptor 1-deficient (13) mice
it is known that TNF- is a second important cytokine for
protection against Listeria. It is produced by macrophages
upon infection with Listeria and may act via the following
two pathways: (a) the SCID model revealed that TNF-
is
necessary for activation of NK cells that then produce IFN-
to further induce TNF receptor 1 and TNF-
expression
(60, 61) and macrophage effector functions (4); (b) macrophage- or
T cell-derived TNF-
may act in an autocrine or paracrine fashion directly on macrophages to activate anti-Listeria effector molecules. Involvement of the ICSBP/
IRF2 complex in the signaling cascade of the TNF receptor could theoretically explain the described in vivo findings, but this has not been formally demonstrated so far. In
addition, another transcription factor, NF-IL6, which can
be upregulated by LPS/CD14 and also by TNF-
(62), is
important for clearance of Listeria as demonstrated in the
NF-IL6-deficient mice (63).
From our findings in three different mouse strains and
from the published literature, the following model of signaling events in activation of anti-Listeria immunity may be
proposed (Fig. 6): after activation of IFN receptors various
tyrosine kinases are induced and STAT proteins phosphorylated (Table 1); they regulate the induction and activation
of transcription factors of the IRF family among which the
exclusively IFN--dependent ICSBP mediates protection
against Listeria. Two major questions remain open: (a)
What molecules are involved in the signalling of the TNF
receptor that could explain its importance for anti-Listeria immunity (ICSBP, NF-IL6, other transcription factors, indirect effect via NK cell activation)? (b) How do macrophages kill Listeria? Our results, but also the published ones
on IFN type II receptor- and iNOS-deficient mice, rather
argue against RNI production being a limiting factor. ROI
may be involved since ICSBP- and NF-IL6-deficient mice
had reduced respiratory burst, and this correlated with high susceptibility to Listeria infection. But still there may be a potential third mechanism involved to explain the drastic
phenotype of ICSBP
/
mice. Studies on iron metabolism
of peritoneal macrophages (64) and murine
-thalassemia
(65) suggested that iron scavengers lead to enhanced, and
iron overload to reduced, resistance to Listeria by direct interference with the essential bacterial iron metabolism.
Whether IFN-
- and/or TNF-
-mediated enhancement
of iron-binding proteins can explain resistance to murine
listeriosis remains to be investigated.
Address correspondence to Thomas Fehr, Institute of Experimental Immunology, University Hospital, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland.
Received for publication 21 October 1996 and in revised form 17 December 1996.
1Abbreviations used in this paper: ICSBP, interferon consensus sequence binding protein; i.f., into the footpad; iNOS, inducible nitric oxide synthase; IRF1/2, interferon regulatory factor 1/2; ISGF, interferon-stimulated gene factor; ISRE, interferon-stimulated response element; NO, nitric oxide; PEM, peritoneal macrophage; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; STAT, signal transducers and activators of transcription.We would like to thank A. Schaffner and H. Hengartner for expert advice and helpful discussion; C. Weissmann and M. Aguet for mutant mouse strains; J. Bille for anti-Listeria serum; A. Althage, L. Vlk, and H. Haber for excellent technical support; H. Neff and N. Wey for photographs; and E. Hörhager, and S. Kläusli for secretarial help.
This work was supported by the Swiss National Science Foundation grant no. 31-32195.91 to R.M. Zinkernagel, and grants no. 31-4577.95 and 32-42536.94 to G. Schoedon), the Kanton of Zürich and the German Science Foundation (DFG).
1. | Kaufmann, S.H.E.. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11: 129-163 [Medline] . |
2. | Conlan, J.W., and R.J. North. 1991. Neutrophil-mediated dissolution of infected host cells as a defense strategy against a facultative intracellular bacterium. J. Exp. Med. 174: 741-744 [Abstract] . |
3. | Hiromatsu, K., Y. Yoshikai, G. Matsuzaki, S. Ohga, K. Muramori, K. Matsumoto, J.A. Bluestone, and K. Nomoto. 1992. A protective role of gamma/delta T cells in primary infection with Listeria monocytogenes in mice. J. Exp. Med. 175: 49-56 [Abstract] . |
4. | Bancroft, G.J., R.D. Schreiber, and E.R. Unanue. 1991. Natural immunity: a T-cell-independent pathway of macrophage activation, defined in the scid mouse. Immunol. Rev. 124: 5-24 [Medline] . |
5. | Wherry, J.C., R.D. Schreiber, and E.R. Unanue. 1991. Regulation of gamma interferon production by natural killer cells in scid mice: roles of tumor necrosis factor and bacterial stimuli. Infect. Immun. 59: 1709-1715 [Medline] . |
6. | Portnoy, D.A., R.D. Schreiber, P. Connelly, and L.G. Tilney. 1989. Gamma interferon limits access of Listeria monocytogenes to the macrophage cytoplasm. J. Exp. Med. 170: 2141-2146 [Abstract] . |
7. |
Bancroft, G.J.,
R.D. Schreiber,
G.C. Bosma,
M.J. Bosma, and
E.R. Unanue.
1987.
A T cell-independent mechanism of
macrophage activation by interferon-gamma.
J. Immunol.
139:
1104-1107
|
8. | Mielke, M.E.A., S. Ehlers, and H. Hahn. 1988. T-cell subsets in delayed-type hypersensitivity, protection, and granuloma formation in primary and secondary Listeria infection in mice: superior role of Lyt-2+ cells in acquired immunity. Infect. Immun. 56: 1920-1925 [Medline] . |
9. | Harty, J.T., R.D. Schreiber, and M.J. Bevan. 1992. CD8 T cells can protect against an intracellular bacterium in an interferon gamma-independent fashion. Proc. Natl. Acad. Sci. USA. 89: 11612-11616 [Abstract] . |
10. | Kägi, D., B. Ledermann, K. Bürki, H. Hengartner, and R.M. Zinkernagel. 1994. CD8+ T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur. J. Immunol. 24: 3068-3072 [Medline] . |
11. | Dalton, D.K., S. Pitts-Meek, S. Keshav, I.S. Figari, A. Bradley, and T.A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science (Wash. DC). 259: 1739-1742 [Medline] . |
12. | Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R.M. Zinkernagel, and M. Aguet. 1993. Immune response in mice that lack the interferon-gamma receptor [see comments]. Science (Wash. DC). 259: 1742-1745 [Medline] . |
13. | Rothe, J., W. Lesslauer, H. Lotscher, Y. Lang, P. Koebel, F. Kontgen, A. Althage, R. Zinkernagel, M. Steinmetz, and H. Bluethmann. 1993. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature (Lond.). 364: 798-802 [Medline] . |
14. | Lepay, D.A., R.M. Steinman, C.F. Nathan, H.W. Murray, and Z.A. Cohn. 1985. Liver macrophages in murine listeriosis. J. Exp. Med. 161: 1503-1512 [Abstract] . |
15. | Peck, R.. 1989. Gamma interferon induces monocyte killing of Listeria monocytogenes by an oxygen-dependent pathway; alpha- or beta-interferons by oxygen-independent pathways. J. Leukoc. Biol. 46: 434-440 [Abstract] . |
16. | van Dissel, J.T., J.J.M. Stikkelbroek, and R. van Furth. 1993. Differences in the rate of intracellular killing of catalase-negative and catalase-positive Listeria moncytogenes by normal and interferon-gamma-activated macrophages. Scand. J Immunol. 37: 443-446 [Medline] . |
17. |
Leenen, P.J.M.,
B.P. Canono,
D.A. Drevets,
J.S.A. Voerman, and
P.A. Campbell.
1994.
TNF-![]() ![]() |
18. | Bläuer, F., P. Groscurth, M. Schneemann, G. Schoedon, and A. Schaffner. 1995. Modulation of the antilisterial activity of human blood-derived macrophages by activating and deactivating cytokines. J. Interferon Cytokine. Res. 15: 105-114 [Medline] . |
19. | Inoue, S., S.-I. Itagaki, and F. Amano. 1995. Intracellular killing of Listeria monocytogenes in the J774.1 macrophagelike cell line and the lipopolysaccharide (LPS)-resistant mutant LPS1916 cell line defective in the generation of reactive oxygen intermediates after LPS treatment. Infect. Immun. 63: 1876-1886 [Abstract] . |
20. |
Beckerman, K.P.,
H.W. Rogers,
J.A. Corbett,
R.D. Schreiber,
M.L. McDaniel, and
E.R. Unanue.
1993.
Release
of nitric oxide during the T cell-independent pathway of
macrophage activation.
J. Immunol.
150:
888-895
|
21. | Flesch, I.E.A., J.H. Hess, and S.H.E. Kaufmann. 1994. NADPH diaphorase staining suggests a transient and localized contribution of nitric oxide to host defence against an intracellular pathogen in situ. Int. Immunol. 6: 1751-1757 [Abstract] . |
22. | Boockvar, K.S., D.L. Granger, R.M. Poston, M. Maybodi, M.K. Washington, J.B. Hibbs Jr., and R.L. Kurlander. 1994. Nitric oxide produced during murine listeriosis is protective. Infect. Immun. 62: 1089-1100 [Abstract] . |
23. | MacMicking, J.D., C. Nathan, G. Hom, N. Chartrain, D.S. Fletcher, M. Trumbauer, K. Stevens, Q.-w. Xie, K. Sokol, N. Hutchinson, et al . 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 81: 641-650 [Medline] . |
24. | Williams, B.R.G.. 1991. Transcriptional regulation of interferon-stimulated genes. Eur. J Biochem. 200: 1-11 [Abstract] . |
25. |
Sen, G.C., and
P. Lengyel.
1992.
The interferon system; a
bird's eye view of its biochemistry.
J. Biol. Chem.
267:
5017-5020
|
26. | Müller, U., U. Steinhoff, L.F. Reis, S. Hemmi, J. Pavlovic, R.M. Zinkernagel, and M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science (Wash. DC). 264: 1918-1921 [Medline] . |
27. |
van den Broek, M.F.,
U. Müller,
S. Huang,
M. Aguet, and
R.M. Zinkernagel.
1995.
Antiviral defence in mice lacking
both ![]() ![]() ![]() |
28. | van den Broek, M.F., U. Müller, S. Huang, R.M. Zinkernagel, and M. Aguet. 1995. Immune defence in mice lacking type I and/or type II interferon receptors. Immunol. Rev. 148: 5-18 [Medline] . |
29. | Levy, D.E., D.J. Lew, T. Decker, D.S. Kessler, and J.E. Darnell Jr.. 1990. Synergistc interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3. EMBO (Eur. Mol. Biol. Organ.) J. 9: 1105-1111 [Abstract] . |
30. | Coccia, E.M., G. Marziali, E. Stellacci, E. Perrotti, R. Ilari, R. Orsatti, and A. Battistini. 1995. Cells resistant to interferon-beta respond to interferon-gamma via the Stat1-IRF-1 pathway. Virology. 211: 113-122 [Medline] . |
31. | Miyamoto, M., T. Fujita, Y. Kimura, M. Maruyama, H. Harada, Y. Sudo, T. Miyata, and T. Taniguchi. 1988. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell. 54: 903-913 [Medline] . |
32. | Meraz, M.A., J.M. White, K.C.F. Sheehan, E.A. Bach, S.J. Rodig, A.S. Dighe, D.H. Kaplan, J.K. Riley, A.C. Greenlund, D. Campbell, et al . 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell. 84: 431-442 [Medline] . |
33. | Durbin, J.E., R. Hackenmiller, M.C. Simon, and D.E. Levy. 1996. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell. 84: 443-450 [Medline] . |
34. | Driggers, P.H., D.L. Ennist, S.L. Gleason, W.-H. Mak, M.S. Marks, B.-Z. Levi, J.R. Flanagan, E. Appella, and K. Ozato. 1990. An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes. Proc. Natl. Acad. Sci. USA. 87: 3743-3747 [Abstract] . |
35. |
Politis, A.D.,
J. Sivo,
P.H. Driggers,
K. Ozato, and
S.N. Vogel.
1992.
Modulation of interferon consensus sequence binding protein mRNA in murine peritoneal macrophages.
J. Immunol.
148:
801-807
|
36. | Holtschke, T., J. Löhler, Y. Kanno, T. Fehr, N. Giese, F. Rosenbauer, J. Lou, K.-P. Knobeloch, L. Gabriele, J.F. Waring, et al . 1996. Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell. 87: 307-317 [Medline] . |
37. | Harada, H., T. Fujita, M. Miyamoto, Y. Kimura, M. Maruyama, A. Furia, T. Miyata, and T. Taniguchi. 1989. Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell. 58: 729-739 [Medline] . |
38. | Nelson, N., M.S. Marks, P.H. Driggers, and K. Ozato. 1993. Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferoninduced gene transcription. Mol. Cell Biol. 13: 588-599 [Abstract] . |
39. | Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T.M. Kündig, R. Amakawa, K. Kishihara, A. Wakeham, et al . 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell. 75: 83-97 [Medline] . |
40. | Kimura, T., K. Nakayama, J. Penninger, M. Kitagawa, H. Harada, T. Matsuyama, N. Tanaka, R. Kamijo, J. Vilcek, T.W. Mak, and T. Taniguchi. 1994. Involvement of the IRF-1 transcription factor in antiviral responses to interferons. Science (Wash. DC). 264: 1921-1923 [Medline] . |
41. | Schoedon, G., M. Schneemann, S. Hofer, L. Guerrero, N. Blau, and A. Schaffner. 1993. Regulation of the L-argininedependent and tetrahydrobiopterin-dependent biosynthesis of nitric oxide in murine macrophages. Eur. J. Biochem. 213: 833-839 [Abstract] . |
42. |
Rellstab, P., and
A. Schaffner.
1989.
Endotoxin suppresses
the generation of O2- and H2O2 by "resting" and lymphokine-activated human blood-derived macrophages.
J. Immunol.
142:
2813-2820
|
43. | Schaffner, A., and P. Rellstab. 1988. Gamma-interferon restores listericidal activity and concurrently enhances release of reactive oxygen metabolites in dexamethasone-treated human monocytes. J. Clin. Invest. 82: 913-919 [Medline] . |
44. | Bovolenta, C., P.H. Driggers, M.S. Marks, J.A. Medin, A.D. Politis, S.N. Vogel, D.E. Levy, K. Sakaguchi, E. Appella, J.E. Coligan, et al . 1994. Molecular interactions between interferon consensus sequence binding protein and members of the interferon regulatory factor family. Proc. Natl. Acad. Sci. USA. 91: 5046-5050 [Abstract] . |
45. | Weisz, A., S. Kirchhoff, and B.-Z. Levi. 1994. IFN consensus sequence binding protein (ICSBP) is a conditional repressor of IFN inducible promoters. Int. Immunol. 6: 1125-1131 [Abstract] . |
46. | Emmerling, P., H. Finger, and H. Hof. 1977. Cell-mediated resistance to infection with Listeria monocytogenes in nude mice. Infect. Immun. 15: 382-385 [Medline] . |
47. | Shinkai, Y., G. Rathbun, K.P. Lam, E.M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, and A.M. Stall. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 68: 855-867 [Medline] . |
48. | Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, S.I. Koh, T. Kimura, S.J. Green, et al . 1994. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science (Wash. DC). 263: 1612-1615 [Medline] . |
49. | Martin, E., C. Nathan, and Q.-w. Xie. 1994. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med. 180: 977-984 [Abstract] . |
50. | Kamijo, R., D. Shapiro, J. Le, S. Huang, M. Aguet, and J. Vilcek. 1993. Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor. Proc. Natl. Acad. Sci. USA. 90: 6626-6630 [Abstract] . |
51. | Salkowski, C.A., S.A. Barber, G.R. Detore, and S.N. Vogel. 1996. Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes. J. Immunol. 157: 3107-3110 . |
52. | Berche, P.A.. 1985. Resistance to listeriosis in two lines of mice genetically selected for high and low antibody production. Immunology. 56: 707-715 [Medline] . |
53. | Barber, S.A., M.J. Fultz, C.A. Salkowski, and S.N. Vogel. 1995. Differential expression of interferon regulatory factor 1 (IRF-1), IRF-2, and interferon consensus sequence binding protein genes in lipopolysaccharide (LPS)-responsive and LPS-hyporesponsive macrophages. Infect. Immun. 63: 601-608 [Abstract] . |
54. | Dabiri, G.A., J.M. Sanger, D.A. Portnoy, and F.S. Southwick. 1990. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc. Natl. Acad. Sci. USA. 87: 6068-6072 [Abstract] . |
55. | Stenger, S., H. Thüring, M. Röllinghoff, and C. Bogdan. 1994. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J. Exp. Med. 180: 783-793 [Abstract] . |
56. | Wei, X.Q., I.G. Charles, A. Smith, J. Ure, G.J. Feng, F.P. Huang, D. Xu, W. Muller, S. Moncada, and F.Y. Liew. 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature (Lond.). 375: 408-411 [Medline] . |
57. |
Khan, I.A.,
T. Matsuura,
S. Fonseka, and
L.H. Kasper.
1996.
Production of nitric oxide (NO) is not essential for protection against acute Toxoplasma gondii infection in IRF-1![]() ![]() |
58. | Samsom, J.N., J.A. Langermans, P.H. Groeneveld, and R. van Furth. 1996. Acquired resistance against a secondary infection with Listeria monocytogenes in mice is not dependent on reactive nitrogen intermediates. Infect. Immun. 64: 1197-1202 [Abstract] . |
59. |
Gregory, S.H.,
E.J. Wing,
R.A. Hoffman, and
R.L. Simmons.
1993.
Reactive nitrogen intermediates suppress the
primary immunologic response to Listeria.
J. Immunol.
150:
2901-2909
|
60. | Aggarwal, B.B., T.E. Eessalu, and P.E. Hass. 1985. Characterization of receptors for human tumor necrosis factor and their regulation by gamma-interferon. Nature (Lond.) 318: 665-667 [Medline] . |
61. | Collart, M.A., D. Belin, J.-D. Vassalli, S. de Kossodo, and P. Vassalli. 1986. Gamma-interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors. J. Exp. Med. 164: 2113-2118 [Abstract] . |
62. | Akira, S., K. Yoshida, T. Tanaka, T. Taga, and T. Kishimoto. 1995. Targeted disruption of the IL-6 related genes: gp130 and NF-IL-6. Immunol. Rev. 148: 221-253 [Medline] . |
63. | Tanaka, T., S. Akira, K. Yoshida, M. Umemoto, Y. Yoneda, N. Shirafuji, H. Fujiwara, S. Suematsu, N. Yoshida, and T. Kishimoto. 1995. Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell. 80: 353-361 [Medline] . |
64. | Alford, C.E., T.E. King Jr., and P.A. Campbell. 1991. Role of transferrin, transferrin receptors, and iron in macrophage listericidal activity. J. Exp. Med. 174: 459-466 [Abstract] . |
65. | Ampel, N.M., D.B. Van Wyck, M.L. Aguirre, D.G. Willis, and R.A. Popp. 1989. Resistance to infection in murine beta-Thalassemia. Infect. Immun. 57: 1011-1017 [Medline] . |
66. |
Lieschke, G.J.,
D. Grail,
G. Hodgson,
D. Metcalf,
E. Stanley,
C. Cheers,
K.J. Fowler,
S. Basu,
Y.F. Zhan, and
A.R. Dunn.
1994.
Mice lacking granulocyte colony-stimulating factor
have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.
Blood.
84:
1737-1746
|
67. | Fujita, T., L.F. Reis, N. Watanabe, Y. Kimura, T. Taniguchi, and J. Vilcek. 1989. Induction of the transcription factor IRF-1 and interferon-beta mRNAs by cytokines and activators of second-messenger pathways. Proc. Natl. Acad. Sci. USA. 86: 9936-9940 [Abstract] . |