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Max Planck Institut für Immunbiologie, Freiburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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response to micro-organisms.
As a consequence, unlike other Lpsd mouse
strains, they do not acquire a partial LPS susceptibility when treated
with sensitizing bacteria. Because IL-12 is important for the microbial
induction of IFN-, we investigated whether the production or
function of IL-12 might be defective in Cr mice. IL-12 mRNA (p35 and
p40) was present in the spleen of untreated Cr mice, IL-12p40 mRNA was
inducible in mice injected with live or killed Salmonella
typhimurium, and IL-12 (p70) was inducible in macrophages by
bacteria. Thus, Cr mice exhibit normal IL-12 responses. In functional
tests, splenocytes of untreated or of S.
typhimurium-infected mice failed to produce IFN- when
stimulated with murine rIL-12 or with a combination of IL-12 and murine
rIL-18 or Con A. Furthermore, Cr mice were identical with IL-12p35/p40
and IL-12 receptor 
1 knockout mice in their impaired in
vivo and in vitro IFN- responses to bacteria. Thus, Cr mice carry a
second genetic defect unrelated to the Lps/tlr4 mutation
that underlies the IL-12 unresponsiveness and contributes to the LPS
resistance and impaired innate immune response in this
strain. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The LPS susceptibility of mice may increase considerably during
infection or after treatment with a variety of killed bacteria, such as
Calmette-Guérin bacillus, Propionibacterium acnes,
Coxiella burnetii, and Salmonella typhimurium
(12, 13, 14, 15, 16). The bacteria-induced sensitization toward LPS is
mediated by endogenously produced IFN- (13, 17). Upon
LPS challenge, sensitized Lpsn mice produce
enhanced amounts of proinflammatory cytokines, such as TNF-
, IL-6,
or IFN-, and are hypersensitive to the lethal activity of LPS and
TNF-. Sensitization to LPS proceeds also in the
Lpsd C3H/HeJ and BALB/c/l mice, leading to
a change of phenotype from LPS resistant to partially LPS sensitive
(15, 18, 19). However, a similar sensitization is lacking
in Cr mice, which remain LPS resistant after treatment with sensitizing
bacteria (20). The absence of sensitization in Cr mice is
due to a defect in IFN- production in response to bacteria and
parasites (13, 20, 21, 22). The impaired IFN- production is
not due to a general defect in IFN- response, because Cr mice are
capable of producing IFN- when stimulated with the T cell mitogen
Con A and CD3 mAbs (20, 21, 22).
Different cell types and soluble factors are involved in the induction
of IFN-. Thus, in addition to IFN--producing cells (T cells, NK
cells, and macrophages) and accessory cells (macrophages and dendritic
cells) a number of cytokines are known to participate directly or
indirectly in the induction of IFN- (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). It has
been shown that IFN- is not produced in bacteria-treated Cr mice
(22). However, it was recently shown that this lack of
IFN- production is due to their LPS unresponsiveness and cannot
explain the IFN- defect of Cr mice (33). Furthermore,
we demonstrated that the production of IL-1, IL-10, and TNF-
(33), as well as of IL-2, IL-4, IL-15, IL-18, TGF-, and
the membrane-associated proteins B7-1, B7-2, and CD40 (our unpublished
observations), which are all known to be involved in IFN- production
(34, 35), are normal in Cr mice. Therefore, the IFN-
defect of Cr mice does not seem to be related to a defective production
of one of the above factors.
Among the soluble factors, IL-12 is of special interest, being capable
of directly inducing IFN- in T and NK cells and promoting T cell
differentiation in the IFN--producing Th1 subset (reviewed in Ref.
36). Its IFN--inducing activity can be enhanced further
by a synergistic action with accessory cytokines, such as IL-18, or
with other stimuli, such as Con A (37). Early in vitro
studies showed that bacteria, bacterial products (including LPS), and
intracellular parasites were among the most powerful inducers of IL-12
in accessory cells (38, 39). Several in vivo studies have
shown that IL-12 is the main factor responsible for the IFN-
production during various bacterial, parasitic, and viral infections
(reviewed in Ref. 40). In the present study it is shown
that the IFN- defect of Cr mice is caused by their inability to
respond to IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacteria S. typhimurium (C5), Staphylococcus aureus, Listeria monocytogenes, and P. acnes (strain ATCC 12930; American Type Culture Collection, Manassas, VA) were grown and killed as described previously (33). For use, the bacteria were suspended in pyrogen-free PBS, pH 7.2. LPS of Salmonella abortus equi in its uniform triethylamine salt form was obtained as described previously (41). A sterile aqueous stock solution (10 mg/ml) was prepared and stored at 4°C. Before use, the LPS was diluted further with pyrogen-free PBS to the desired concentration. Murine rIL-12 (mrIL-12) was purchased from PharMingen (San Diego, CA), and mrIL-18 was obtained from PANSYSTEMS (Aidenbach, Germany). Con A was purchased from Pharmacia (Freiburg, Germany).
Animals
Lpsn mice, C57BL/10ScSn (Sn), BALB/c, 129
SvPas, IL-12R1-/- 129 Sv
background, and IL-12p35/p40-/- on BALB/c
background, and Lpsd mice, Cr and BALB/c/l, were
bred under specific pathogen-free conditions in the animal facilities
of the Max Planck Institut für Immunbiologie. Breeding pairs of
IL-12R1-/- and
IL-12p35/p40-/- were provided by Dr. M. K.
Gately (Hoffmann-La Roche, Nutley, NJ), Lpsd ScN
breeding pairs were purchased from the Animal Production Program
(Frederick Cancer Research and Development Center, Frederick, MD) and
bred under conventional clean conditions in the animal facilities of
the Max Planck Institute. Five- to 10-wk-old animals of both sexes were
used in this study.
P. acnes sensitization and TNF- induction
Groups of mice received 625 µg of P. acnes in 0.2 ml of PBS/25 g body weight i.v. Seven days later, the animals received an i.v. injection of LPS (in 0.2 ml of PBS/25 g). One hour after challenge, the animals were exsanguinated under ether anesthesia. The blood was collected in heparinized tubes and centrifuged at 4°C. The resulting plasma was aliquoted and stored at -80°C.
TNF- bioassay
TNF- in plasma was measured in a cytotoxicity test using a
TNF--sensitive L929 cell line in the presence of actinomycin D as
described previously (42, 43). The detection limit of the
assay was 60 pg of TNF/ml plasma. Rabbit anti-mouse TNF-
(Genzyme, Boston, MA) was used as an inhibitor to test the specificity
of the assay.
Infection
For infection, S. typhimurium (C5), previously passaged through Sn mouse spleens to warrant its virulence, was grown overnight at 37°C on Luria Bertoni agar (Difco, Detroit, MI). Bacteria were suspended in PBS to a concentration of 1500 CFU/ml estimated turbidometrically, and 300 CFU/mouse was administrated i.v. The actual number of CFU in the suspension was determined by plating on Luria Bertoni agar plates and counting the CFU after overnight culture.
Macrophages
Macrophages were derived from bone marrow precursor cells of 5- to 6-wk-old mice after 10 days of culture in the presence of L cell-conditioned medium, as previously described (33). Cells were centrifuged, washed twice, and suspended in a serum-free, high glucose formulation of DMEM at a concentration of 1 x 106/ml. The cells (2 x 105/well) were placed in 96-well plates (Nunc, Roskilde, Denmark) and cultured at 37°C in a humidified atmosphere containing 8% CO2 for 24 h. Thereafter, the macrophage supernatants were replaced by fresh medium, and 10 µl/well of the stimulating agent being tested was added. Cultivation then continued for an additional 24 h. Culture supernatants for IL-12 measurement were collected and stored in aliquots at -80°C.
Splenocytes
Splenocyte suspensions were prepared from spleens of 6- to
8-wk-old mice by pressing spleens through a wire grid. Pooled cells
from three or four animals were suspended in serum-free DMEM, adjusted
to a concentration of 107/ml, and placed (2
x 106/well) in 96-well plates (Nunc, Roskilde,
Denmark). They were then cultured in the presence or the absence of
stimulating agents (10 µl/well) at 37°C in a humidified atmosphere
containing 8% CO2 for 24 h. Culture
supernatants for determination of IFN- were stored in aliquots at
-80°C until use.
ELISAs
IFN- in supernatants of splenocyte cultures and in murine
plasma was estimated by a previously described ELISA (44).
The limit of IFN- detection was 60 pg/ml. IL-12 (p70) in
supernatants of macrophage cultures was estimated by ELISA using
anti-IL-12 mAb (C17.8) and biotin-labeled anti-IL-12 mAb
(C15.6; PharMingen, Hamburg, Germany) as described previously
(45). The limit of detection was 15 pg/ml.
RNA extraction
Total RNA was isolated from freshly removed spleens or from cultured splenocytes by a guanidinium isothiocyanate-phenol-chloroform-isoamyl alcohol procedure (46) as described previously (33). The RNA concentration in RNase-free H2O was determined by absorbance at 260 nm.
RT-PCR
Expression of IL-12p35, IL-12p40, and
2-microglobulin mRNA was determined by RT-PCR.
One microgram of total RNA was reverse transcribed using the Moloney
leukemia virus reverse transcriptase from Life Technologies
(Eggenstein, Germany) using random-pd(T)12–18
primers (Pharmacia). The products were appropriately diluted in
H2O and used for qualitative PCR analysis, using
34 cycles of amplification in a Biometra (Gottingen, Germany) Thermal
Cycler UNO-Thermoblock. RT-PCR primers for IL-12p35 (sense,
5'-GATGACATGGTGAAGACGGCC; antisense, 5'-GGAGGTTTCTGGCGCAGAGT), IL-12p40
(sense, 5'-CTGGCCAGTACACCTGCCAC; antisense,
5'-GTGCTTCCAACGCCAGTTCA), and 2-microglobulin
(sense, 5'-TGACCGGCTTGTATGCTATC; antisense, 5'-CAGTGTGAGCCAGGATATAG)
were synthesized by BIG-Biotech (Freiburg, Germany). The exon sequences
to which all primer pairs anneal contain at least one intron between
them, which permits the identification of products derived from
contaminating genomic DNA. The annealing temperatures used were 58°C
for IL-12p35 and IL-12p40 mRNA and 55°C for
2-microglobulin mRNA detection.
Northern blot analysis
RNA samples (
15 µg) were fractionated on 1.2% denaturing
agarose-formaldehyde gel and transferred to Nytran filters as described
previously (47). RNAs were hybridized overnight at 65°C
with random primed 32P-labeled cDNA probes as
described earlier (22). The IFN- probe was a 440-bp
cDNA fragment of mouse IFN- (EcoRI/EcoRV
digest of pMugPl plasmid) provided by D. Stüber (Hoffmann-La
Roche, Basel, Switzerland). The IL-12p40 probe was an RT-PCR-amplified
DNA fragment using the above-described specific primer pairs. The IL-18
probe was a RT-PCR-amplified DNA fragment using specific primers
(sense, 5'-ACTGTACAACCGCAGTAATACGG; antisense,
5'-AGTGAACATTACAGATT TATCCC) at a 62°C annealing temperature.
The amounts of total RNA applied to the electrophoresis gel for each
sample were visualized by the intensity of the ethidium bromide-stained
band (18S rRNA) on the Nytran filter (Schleicher & Schuell, Keene, NH)
used for hybridization.
RNase protection assay (RPA)
IL-12R1 and -2
mRNA were detected by RPA using a RiboQuant MultiProbe RPA system for
the detection of cytokine receptors mRNA (PharMingen, San Diego, CA),
as described in the provider’s protocol. Briefly, the template set
(mCR-3) was used for a T7 RNA polymerase-dependent synthesis of
32P-labeled antisense RNA probes. The RNA samples
were hybridized overnight with an excess of labeled probes. After
treatment with RNase A and T1 and with proteinase K, the samples were
loaded on a 6% polyacrylamide-Tris-borate-EDTA-urea gel and run at
1900 V with 1x Tris-borate-EDTA electrophoresis buffer, pH 8.3. The
gel was dried and exposed on film (BIOMAX MS; Eastman Kodak, Rochester,
NY) using a cassette with an intensifying screen (Cronex Lightening
Plus; DuPont, Wilmington, DE) and kept at -70°C until film
development.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 is a heterodimer of two polypeptide chains, IL-12p35 (p35)
and IL-12p40 (p40). The presence of p35 and p40 mRNA in
IFN--defective, Cr and IFN--normal, Sn mice was tested for by
analysis of the total spleen RNA using RT-PCR. As shown in Fig. 1
, p35 and p40 mRNA were present in
unstimulated splenocytes of both mouse strains. Thus, both IL-12 genes
can be transcribed in Cr mice.
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by killed S.
typhimurium in the spleen of Cr and Sn mice
The detection of mRNA for the highly regulated p40 gene is a good
indicator for IL-12 production (40). We investigated the
induction of p40 mRNA in the spleen of Cr and Sn mice, stimulated with
heat-killed S. typhimurium, by Northern blot analysis. As
shown in Fig. 2a, only very
faint bands of p40 mRNA were detectable in the spleen of unstimulated
mice of either mouse strain. Stimulation with bacteria led to a strong
induction of p40 mRNA in animals of both strains. The levels were
highest at 1 and 2 h of stimulation and decreased 4 h after
stimulation (Fig. 2a). However, while S.
typhimurium-treated mice of both strains exhibited a similar
induction of p40 mRNA, only Sn mice exhibited a strong induction of
IFN- mRNA (Fig. 2a). In accordance, circulating IFN-
was detectable only in Sn, but not in Cr, mice (Fig. 2b).
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transcripts in
Lpsn and Lpsd mice by
S. typhimurium infection
Induction of p40 mRNA was investigated also in the spleen of Sn
and Cr mice, during the first 4 days of infection with S.
typhimurium. In addition, induction of mRNAs for IL-18 and IFN-
was followed in the infected mice. Because S. typhimurium
contains LPS, and the two mouse strains, Lpsd Cr
and Lpsn Sn, differ in their responsiveness to
LPS, the induction of the above cytokines was also investigated in
infected Lpsn and Lpsd
mice on BALB/c background (BALB/c and BALB/c/l). Control noninfected
mice of all four strains used exhibited no IFN- mRNA and no, or only
a very weak, expression of p40 mRNA in the spleen. However, the mice
showed a constitutive expression of IL-18 mRNA in this organ. All mice,
especially those of the Lpsd strains, exhibited
a detectable induction of p40 mRNA and a significant up-regulation of
IL-18 mRNA on day 4 of infection. Interestingly, p40 mRNA bands induced
in the spleen of Cr and BALB/c/l mice were always visibly stronger than
those in Sn or BALB/c mice, respectively. This finding correlates with
the faster progression of S. typhimurium infection observed
in Lpsd mice and may be explained by the higher
bacterial load present in the spleen of these animals (Table I). However, induction of IFN- mRNA
became detectable on day 2 only in Sn, BALB/c, and BALB/c/l, and
increased further on days 3 and 4 of infection (in Fig. 3, only expression on day 4 is shown). In
Cr mice, IFN- mRNA was completely absent on days 2 and 3 (data not
shown), and only a weak expression appeared first on day 4 (Fig. 3). In
agreement, plasma levels of IFN- were detectable in infected Sn,
BALB/c, and BALB/c/l, but not in infected Cr mice (Table I).
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To test the capability of Cr mice to produce and secrete IL-12,
macrophages of Cr, Sn, BALB/c, and BALB/c/l mice were prepared and
stimulated with killed S. typhimurium and S.
aureus and with LPS. IL-12 in macrophage supernatants was
determined by a specific ELISA for IL-12p70. As shown in Fig. 4, unstimulated macrophages of all mouse
strains used, including Cr, already exhibited a low constitutive
production of IL-12, which increased by a factor of 10 or more upon
addition of bacteria to the cultures. Addition of LPS, as expected,
induced an IL-12 response only in macrophages of
Lpsn Sn and BALB/c, but not of
Lpsd Cr and BALB/c/l, mice.
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in response to
mrIL-12
As shown in the foregoing experiments, Cr mice exhibit a normal
production of IL-12 in response to bacteria. In view of the fact that
IL-12 is a direct inducer of IFN-, we investigated whether the
inability of Cr mice to produce IFN- when treated with bacteria may
be related to IL-12 unresponsiveness. Splenocytes of Cr, Sn, BALB/c,
and BALB/c/l mice were stimulated in parallel with different amounts of
mrIL-12, and IFN- levels in culture supernatants were estimated by
ELISA. As shown in Fig. 5, IL-12 induced
a dose-dependent production of IFN- in the Sn, BALB/c, and BALB/c/l
cultures, but not in Cr cultures. Furthermore, in Sn splenocytes, the
IFN- responses to IL-12 were enhanced by addition of rIL-18 or
suboptimal (low) amounts of Con A, both of which are known to synergize
with IL-12 in the induction of IFN- (Fig. 6, top). The IFN- responses
to IL-12 alone or in combination with IL-18 or Con A were still higher
when instead of normal splenocytes, splenocytes of S.
typhimurium-infected Sn mice were used (Fig. 6, bottom). In contrast, in splenocytes of noninfected or
infected Cr mice an IFN- response to IL-12 alone or to a combination
of IL-12 with IL-18 or Con A was absent (Figs. 5 and 6). The IFN-
response was completely absent even when the amount of IL-12 used in
this experiment was increased to 20 ng/ml.
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responses
From the above data we assumed that IL-12 unresponsiveness is the
most likely reason for the defective IFN- response of Cr mice to
bacteria. To confirm this, we extended the studies to
IL-12R1-deficient
(IL-12R1-/-) and
IL-12p35/p40-/- mice, both lacking endogenous
IL-12 activity. A comparison of the
IL-12R1-/-,
IL-12p35/p40-/-, and Cr mice revealed that all
three types of mice were identical in all respects tested. Splenocytes
from all types of mice exhibited no IFN- response to killed bacteria
(S. typhimurium, S. aureus, P. acnes,
and L. monocytogenes in concentrations up to 100 µg/ml).
Furthermore, no circulating IFN- was detectable in plasma of mice
infected with 3 x 102 or 3 x
104 CFU of S. typhimurium (measured up
to day 5 after infection; Tables I
and II). Finally, splenocytes of
all three mouse strains exhibited easily detectable IFN- responses
when stimulated in vitro with high amounts of Con A (5 µg/ml).
However, these responses were often 2–3 times lower than the responses
obtained in cultures of closely related mice with normal IL-12 activity
(data not shown). From these results it is concluded that the inability
of Cr mice to produce IFN- when stimulated with bacteria is due to
IL-12 unresponsiveness.
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From the above results, it is evident that Cr mice exhibit, in
addition to their known LPS response defect, a second phenotypic
abnormality, namely IL-12 unresponsiveness. The question arose of
whether the two defects were caused by a single mutational event or
whether they are based on two independent mutations. For this reason,
we compared the ability of mrIL-12 to induce IFN- in splenocytes
from ScN and Sn mice, which are closely related to Cr mice. The ScN
strain is the progenitor to Cr and was shown in the past to also be
unresponsive to LPS. Contrary to Lpsn Sn
mice, both Lpsd mice, Cr and ScN, fail to
express the tlr4 gene (S. N. Vogel and M. A.
Freudenberg, unpublished observations). Splenocytes of Cr, ScN, and Sn
mice were stimulated in parallel with IL-12 (6.25 ng/ml) for 24 h.
Stimulation with Con A (5 µg/ml) was conducted as a positive control.
Although splenocytes of Cr mice produced no detectable IFN- in
response to IL-12, those of ScN and Sn mice produced comparable amounts
of this cytokine (Fig. 7). Thus, although
Cr mice are defective in IL-12 responsiveness, their progenitor ScN,
like Sn mice, have normal IL-12 responsiveness. Therefore, it is
concluded that the IL-12 unresponsiveness of Cr mice represents a later
mutational event that occurred independently of the mutation of
tlr4.
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Pretreatment of mice with heat-killed P. acnes elicits
an IFN--dependent sensitization toward LPS (13, 48). In
the following experiments we compared the LPS susceptibility of control
and P. acnes-pretreated mice with either a functional (Sn,
ScN, 129 SvPas, BALB/c, and BALB/c/l) or a defective (Cr,
IL-12p35/p40-/-, and
IL-12R1-/-) IL-12 system.
An enhanced TNF- response to LPS served as an indicator of
sensitization. As shown in Fig. 8
pretreatment with P. acnes induced a strong sensitization
toward LPS in all Lpsn mouse strains with
functional IL-12. A sensitization toward LPS was also induced in both
P. acnes-treated Lpsd mice belonging
to this group, as shown by a partial phenotype change from strictly
LPS-nonresponsive to partially LPS-sensitive (Fig. 8). In contrast,
sensitization was absent in Lpsn
IL-12p35/p40-/-,
IL-12R1-/-, and
Lpsd Cr mice, showing that sensitization to LPS
by P. acnes requires a functional IL-12 system. Thus,
independently of the differences in the tlr4 gene, Cr mice
share a similar phenotype with IL-12p35/p40-/-
and IL-12R1-/- mice, being
resistant to the sensitizing effect of P. acnes.
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The biological activity of IL-12 on cells is mediated via the
IL-12R, composed of two subunits, 1 and
2. In this study we compared the inducibility
of IL-121 and 2 mRNA
by Con A in splenocytes of Cr with that of other mouse strains (Sn,
BALB/c, and BALB/c/l). As shown in Fig. 9a, Con A induced a strong,
long-lasting induction of mRNA of both receptor chains in splenocytes
of all four mouse strains tested, including Cr. The expression of
IL-12R1 and 2 mRNA in
Cr and other mouse strains was also analyzed in the spleen of S.
typhimurium-infected animals. As shown in Fig. 9b, on
day 4 of infection, IL-121 and
2 mRNA expression was induced in all mouse
strains to a comparable degree. These experiments revealed that both
known IL-12R genes are present and normally regulated in Cr mice. The
production of functionally normal protein chains remains to be
determined and is currently under study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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response to microorganisms is impaired, and
this is responsible for the absence of sensitization to LPS in these
mice during infection. Because the microbial induction of IFN- is
predominantly IL-12 dependent, we investigated whether a defect in the
production or function of IL-12 may be present in these mice. The
present results provide evidence that bacterially induced IL-12
production in Cr mice is intact, being comparable to that of other
mouse strains tested, especially to the responses of the
Lpsd BALB/c/l mice.
The induction of IFN- in resting and activated splenocytes by rIL-12
was used as a measure of the IL-12 responsiveness in these cells. In
complete contrast to splenocytes of Sn, BALB/c and BALB/c/l mice,
neither splenocytes from healthy nor those from infected Cr mice
responded to IL-12 alone or to a combination of IL-12 and IL-18 or Con
A. Thus, the impaired IFN- response of Cr mice to bacteria and other
micro-organisms investigated previously (13, 20, 21, 22) is
explained by a lack of IL-12 responsiveness, and this is the underlying
reason for the absence of sensitization of these mice to LPS by
micro-organisms. The importance of IL-12 for the development of LPS
hypersensitivity is evidenced further by the results obtained with
IL-12-/- or
IL-12R1-/- knockout mice.
These mice are phenotypically similar to Cr mice, exhibiting impaired
IFN- responses to bacteria and no sensitization to LPS after
P. acnes treatment. These results are in accordance with
those of an earlier study that demonstrated that IL-12 plays an
important role in LPS-induced pathology in Calmette-Guérin
bacillus-primed mice (49).
The IL-12 unresponsiveness of Cr mice is not related to their
tlr4 gene defect. As shown here, ScN mice, the progenitor
strain of Cr, while carrying an identical tlr4 mutation
(S. N. Vogel, unpublished observations; A. Poltorak and M. A.
Freudenberg, unpublished observations), exhibit intact IL-12
responsiveness, indicating that this second mutation must have occurred
after separation of Cr mice from the progenitor colony. As a result,
ScN mice exhibit normal IFN- responses and, like
Lpsd C3H/HeJ and BALB/c/l mice, become partially
LPS responding, when pretreated with P. acnes. Further
evidence that in Cr mice the defect underlying IL-12 unresponsiveness
is not related to the tlr4 mutation was obtained very
recently in our laboratory. By intercrossing Cr and Sn mice, there was
an independent assortment of the IL-12 unresponsiveness and
tlr4 defect in the second filial generation (unpublished
observations). Therefore, Cr mice carry two independent mutations, both
determining LPS resistance. In addition to the mutation of
tlr4, the defect in the IL-12 response exacerbates their LPS
resistance. For this reason Cr mice are highly resistant to LPS under
conditions (e.g., during infection) during which all other
Lpsd mouse strains (C3H/HeJ, BALB/c/l, and ScN)
become partially responsive to LPS.
However, it should be noted that Cr mice when treated with exogenous
IFN- acquire partial LPS sensitivity. This raises the question of
how a response to LPS, however low it may be, is possible in the
complete absence of Tlr4. Two possibilities arise. One of these has
been proposed by Vogel and coworkers, attributing the weak responses of
sensitized Lpsd C3H/HeJ mice to bacterial
contaminants (LPS-associated protein) present in the LPS preparations
(50) that act synergistically with LPS (51).
Although such an explanation is plausible, it is still not clear how
the synergizing activity of LPS is expressed in
Lpsd mice. Another explanation is that LPS
may stimulate Lpsd mice via a
Tlr4-independent pathway(s), however to a low degree that becomes
clearly detectable only after sensitization with IFN-. Such a
pathway(s) may involve other members of the Tlr protein family, for
example Tlr2 (52).
A large amount of information exists on IL-12 recognition and signaling
in the mouse (40). We do not know yet whether the IL-12R
itself or components of the signal transduction pathway are defective
in Cr mice. However, because responses to other cytokines including
TNF- (53), IFN-, IFN- (22), and
IFN- (13) are intact in these mice, we assume that the
defect concerns a component(s) involved more specifically in the IL-12
response. Components specific for recognition and signaling by IL-12,
such as the two subunits of IL-12R (1 and
2) (54, 55) or the transcription
factor STAT4, have been demonstrated (56). We showed in
this study that the induction of transcripts for both IL-12R subunits
does not differ from that seen in other IL-12 responder mouse strains
investigated. However, this does not prove that functional receptor
proteins are expressed on the surface of Cr cells.
In this study the indicator of IL-12 responsiveness in Cr mice was
IFN- production. In further investigations other activities of
IL-12, such as proliferative activity on T cells and NK cell
cytotoxicity, should also be addressed. All these activities contribute
to the natural resistance to infection. It should be mentioned in this
connection that Cr mice were already shown to be highly susceptible to
infection with L. major (21), exhibiting a
similar nonhealing phenotype as IL-12p40 and p35 knockout mice
(57, 58).
Another important factor involved in antibacterial defense is the
Lps/tlr4 gene controlling LPS susceptibility, which is
defective in BALB/c/l and Cr mice (7, 9, 10, 11). As shown in
this study both mouse strains exhibited higher numbers of S.
typhimurium in their spleens and were more susceptible to
infection than their related Lpsn BALB/c
and Sn mice. The high number of bacteria in infected BALB/c/l mice is
also a likely explanation for the high levels of IFN- appearing in
the animals after the third day of infection. A similar observation was
made earlier in S. typhimurium-infected
Lpsd C3H/HeJ mice (20).
Evidently the inability of Lpsd mice to
sense LPS abrogates their ability to detect and react against invading
Gram-negative bacteria at the early stages of infection.
Because both LPS and IL-12 responsiveness, which are necessary for the integrity of an antimicrobial defense, are impaired in Cr mice, it is postulated that Cr mice are highly sensitive to different pathogens.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marina A. Freudenberg, Max Planck Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany.
3 Abbreviations used in this paper: Cr, C57BL/10ScCr; ScN, C57BL/10ScN; BALB/c/l, LPS-resistant BALB/c; mrIL, murine recombinant IL; Sn, C57BL/10ScSn; RPA, RNase protection assay.
Received for publication May 16, 2000. Accepted for publication October 6, 2000.
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References |
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interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice. Infect. Immun. 60:1994.-receptor deficient mice. J. Endotox. Res. 3:291.
interferon production in endotoxin-responder and -nonresponder mice during infection. Infect. Immun. 59:3484., a cofactor in the interferon production induced by Gram-negative bacteria in mice. J. Exp. Med. 181:953. upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187:2103. in macrophages by lipopolysaccharide. Int. Immunol. 5:1383.-interferon production: participation of macrophages and NK-like cells. J. Immunol. 131:288.[Abstract]
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4000
pg/ml.
