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Infection and Immunity, December 1999, p. 6242-6248, Vol. 67, No. 12
Veterinary Molecular Biology, Montana State
University, Bozeman, Montana 59717-3610
Received 16 March 1999/Returned for modification 21 May
1999/Accepted 1 September 1999
The stimulation of gamma interferon (IFN- Protection against intracellular
pathogens depends upon the stimulation of a T helper 1 (Th1)-type
immunity (15, 34). Interleukin-12 (IL-12) has been shown to
be essential in the clearance of intracellular pathogens, e.g.,
Salmonella typhimurium (4-6, 17, 24, 30). IL-12
is a 70-kDa heterodimeric cytokine consisting of two subunits, p35 and
p40, produced by phagocytic cells in response to bacteria, bacterial
products, and intracellular parasites (40). IL-12 induces
IFN- IL-18 was first isolated and cloned from livers of mice infected with
Propionibacterium acnes and challenged with
lipopolysaccharide (31). IL-18 is produced as a precursor
protein of 192 amino acids which is cleaved into a mature protein of
152 amino acids or approximately 18 kDa by the IL-1 IL-18 has recently been shown to be important in the clearance of
several intracellular pathogens, including Yersinia
enterocolitica, a gram-negative, rod-shaped bacterium
(3), and the fungal pathogen Cryptococcus
neoformans (16, 46). Although IL-18 and IL-12 share
many properties, including the induction of IFN- While IFN- Reagents.
Cells were cultured in RPMI 1640 medium with
L-glutamine (BioWhittaker, Walkersville, Md.) supplemented
with 10 mM HEPES buffer, 1 mM sodium pyruvate solution, 1×
nonessential amino acids (Cellgro; Mediatech, Inc., Herndon, Va.), 100 IU of penicillin per ml, and 100 µg of streptomycin per ml (Cellgro;
Mediatech). Fetal bovine serum was supplied by HyClone Laboratories,
Inc. (Logan, Utah). Antibodies and cytokines included murine IL-12 (R&D
Systems, Inc., Minneapolis, Minn.), murine IL-12p40 (PharMingen, San
Diego, Calif.), murine IL-18 (PreproTech, Inc., Rocky Hill, N.J.),
murine IFN- Bacterial strains.
Wild-type S. typhimurium H71
was kindly provided by David M. Hone (Institute of Human Virology,
Medical Biotechnology Center, University of Maryland at Baltimore).
Attenuated S. typhimurium Molecular cloning of murine IL-18.
Murine IL-18 mRNA was
isolated from activated RAW 264.7 cells, a murine macrophage cell line
(ATCC TIB-71; American Type Cell Culture, Manassas, Va.). The RAW 264.7 cells were infected with the attenuated S. typhimurium H647
for 24 h at 37°C, and mRNA was phenol-chloroform extracted with
Tri-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio). The
entire coding region for IL-18 protein was amplified by reverse
transcriptase PCR and cloned into the
BamHI-HindIII site of the pmal-c2 plasmid
(New England BioLabs, Beverly, Mass.). The IL-18 cDNA sequence
(31) was confirmed by DNA sequencing. The IL-18 gene was
inserted downstream from the maltose-binding protein (MBP), and
recombinant murine IL-18 was expressed as a maltose-binding fusion
protein. Expression of recombinant MBP-IL-18 was induced by the
addition of 0.6 mM IPTG to a mid-log-phase bacterial culture. Cells
were harvested 4 h later, and the recombinant MBP-IL-18 fusion
protein was purified on an amylose affinity column according to the
manufacturer's protocol. In addition, a thioredoxin IL-18 fusion
protein was also generated by cloning into the pET32a vector (Novagen,
Inc., Milwaukee, Wis.) at the EcoRI and
HindIII sites. This fusion protein was expressed and
isolated according to manufacturer's protocol.
Rabbit anti-mouse IL-18 antiserum.
The MBP-IL-18 fusion
protein was conjugated to keyhole limpet hemocyanin (KLH; Calbiochem,
La Jolla, Calif.) with 10 mM glutaraldehyde (Sigma) as previously
described (33). New Zealand White rabbits were immunized
with the KLH-MBP-IL-18 conjugate (200 µg of IL-18 fusion protein)
emulsified in complete Freund adjuvant (Sigma), followed with booster
immunizations (100 µg of IL-18 fusion protein) with incomplete Freund
adjuvant (Sigma) for the production of polyclonal anti-mouse IL-18
antibodies. To test the specificity of the rabbit anti-IL-18 antisera,
microtiter wells (35) were coated overnight with 1.0 µg of
recombinant mouse IL-18 thioredoxin (Trx)-IL-18 fusion protein,
recombinant mouse IFN- Mice.
BALB/c mice were obtained from The National Cancer
Institute (Frederick, Md.). BALB/c mice containing a disruption in the gene encoding the p40 subunit of IL-12 (22, 23, 27) were obtained from Jackson Laboratories (Bar Harbor, Maine). Breeding pairs
of the IL-12p40 knockout (IL-12KO) mice, stock mice, and infected
experimental mice were maintained under microisolator conditions.
Sterile food and water were provided ad libitum.
Salmonella infections.
Groups of 5- to
8-week-old BALB/c mice and IL-12KO mice were given intraperitoneal
(i.p.) injections of either vehicle (phosphate-buffered saline [PBS])
or rabbit anti-mouse IL-18 antiserum prior to and after oral challenge.
On the day of infection, mice were pretreated with 50% saturated
sodium bicarbonate solution (2) and then orally infected
with 5 × 107 CFU/0.2 ml of wild-type S. typhimurium H71 strain. Mice were observed twice daily, and the
extent of survival was recorded.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role of Endogenous Interleukin-18 in Resolving
Wild-Type and Attenuated Salmonella typhimurium
Infections
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) has been shown to be
essential in resolving infections by intracellular pathogens. As such,
several different cytokines including, interleukin-12 (IL-12) and
IL-18, can induce IFN-. To resolve Salmonella
infections, the stimulation of IL-12 and IFN- are important for
mediating its clearance. In this present study, the relevance of IL-18
in protection against oral challenge with Salmonella
typhimurium was investigated to determine the role of this
IFN--promoting cytokine. Rabbit anti-murine IL-18 antisera was
generated and administered prior to the oral challenge of BALB/c and
IL-12p40-deficient knockout (IL-12KO) mice with a wild-type S. typhimurium strain. The median survival time was reduced by 2 days for the anti-IL-18-treated BALB/c mice, while no significant
reduction in survival rate for the anti-IL-18-treated IL-12KO mice was
observed compared to vehicle-treated mice. To investigate the
contribution of IL-18 to resolving Salmonella infections,
an attenuated aro-negative mutant (H647) was orally administered to BALB/c mice. This Salmonella infection
induced both IL-12 and IFN- in both the Peyer's patches and the
spleens. In vehicle-treated mice, Peyer's patch IL-12 peaked by
24 h, while IL-18 levels peaked at 3 days, suggesting sequential
support by these cytokines for IFN-. Anti-IL-18 treatment exerted
its greatest effect upon the mucosal compartment, limiting early
IFN- production. However, anti-IL-18 treatment had little effect
upon splenic IFN- levels until late in the response. Infection of
IL-12KO mice with H647 strain induced IFN-, but it was not supported
by IL-18, although IL-18 levels were reduced by this treatment. These
results suggest that IL-18 does contribute to the clearance of S. typhimurium and that endogenously induced IL-18 could not
substitute for IL-12.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
cytokine production from natural killer (NK) and T cells and
drives Th1 cell development (39, 40). A recently identified
cytokine, gamma interferon (IFN-)-inducing factor (IGIF) or IL-18
has been shown to share many properties with IL-12 (29, 32,
42).
-converting
enzyme (10, 12). IL-18 promotes the production of IFN-
and granulocyte-macrophage colony-stimulating factor and inhibits IL-10
production (38). IL-18 also enhances NK cell activity and
proliferation of T cells (28, 29, 37) and upregulates Fas
ligand expression (36, 41).
, it appears that
IL-18 and IL-12 are independently regulated and are functionally different with respect to receptor binding and signal transduction pathways (28). The interaction of IL-18 and IL-12 in the
clearance of intracellular pathogens is not yet fully understood.
is essential for the clearance of Salmonella
(4-6, 17, 24, 30) and supposably attenuated
Salmonella vectors can behave lethally in mice deficient of
IFN- (14, 43), it is unclear whether IL-12 is solely
responsible for IFN- stimulation or whether other IFN--promoting
cytokines contribute to the clearance of Salmonella. In this
present study, we investigated the role of IL-18 in protection against
S. typhimurium.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(Genzyme Corp., Cambridge, Mass.), hamster and rat
anti-mouse IL-12p35 cocktail (PharMingen), rat anti-mouse IL-12p40
(clone C15.6; PharMingen), biotinylated rat anti-mouse IL-12p40 (clone C17.8; PharMingen), anti-mouse IFN- (clone R4-6A2; PharMingen), biotinylated rat anti-mouse IFN- (clone XMG1.2; PharMingen), horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin G (IgG;
Southern Biotechnology Associates, Birmingham, Ala.), alkaline phosphatase (AP)-goat anti-biotin (Vector Laboratories, Inc., Burlingame, Calif.), and HRP-goat anti-biotin (Vector Laboratories).
aroA asd mutant
strain H647 was previously described (2, 9, 44). Bacteria
strains were streaked on Luria broth (LB) agar plates and incubated
overnight at 37°C. A single colony was used to inoculate LB cultures.
Bacterial colony forming units were quantified by reading the
absorbance at an optical density at 600 nm on a Spectronic 20 (Bausch & Lomb) spectrometer and interpolating from a standard curve for the
respective bacterial strain. Bacterial counts were verified by plating
serial dilutions of bacterial suspensions on LB agar plates.
, recombinant mouse IL-12p70, or Trx (Sigma)
per ml. After the blocking step, various dilutions of immune rabbit
anti-IL-18 antisera were added, followed by incubation for 3 h at
37°C. After a wash step, a 1:1,000 dilution of HRP-goat anti-rabbit
IgG (heavy-chain-specific) antibody was added for 90 min at 37°C.
After a washing step, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium (ABTS; Moss, Inc., Pasadena, Calif.) was added, and the
absorbance was measured at 415 nm with a Kinetics Reader model EL312
(Bio-Tek Instruments, Winooski, Vt.).
after oral exposure to
Salmonella, BALB/c mice received an oral gavage with a 50% saturated sodium bicarbonate solution and were then orally challenged with 5 × 109 CFU of the attenuated
Salmonella sp. strain H647 (2, 44). On days 
1,
2, 4, and 6 postinfection (p.i.), mice received i.p. injections of PBS
(or normal rabbit serum) or rabbit anti-mouse IL-18 antisera that had
been previously dialyzed against PBS. On days 0.5, 1, 3, and 7 p.i., spleens and Peyer's patches were harvested. Tissues from
individual mice (three animals/group) were Dounce homogenized in 1 ml
of PBS. Cellular debris was removed by centrifugation, and the
supernatants were stored at 20°C until analysis for cytokine
levels. This experiment was repeated three times.
in the absence of IL-12, IL-12KO
mice were orally gavaged with S. typhimurium H647. On days
0.5, 1, 3, and 7 days p.i., spleens and Peyer's patches were harvested
as described above and stored for cytokine analysis.
Cytokine-specific ELISAs.
Levels of IFN- were measured by
an antigen-capture enzyme-linked immunosorbent assay (ELISA) as
previously described (35). This ELISA procedure was adapted
to measure IL-12p70 and IL-12p40 with microtiter plates coated at 16 µg of anti-IL-12p35 antibody and 6 µg of anti-IL-12p40 (clone
C15.6) antibody per ml, respectively. IL-12p70 and IL-12p40 were
detected by using the biotinylated rat anti-mouse IL-12p40 (clone
C17.8) antibody and AP-goat anti-biotin antibody. To measure IL-18
levels, a rat monoclonal anti-IL-18 antibody was used to coat
microtiter wells at 2.0 µg/ml in sterile PBS overnight at room
temperature. After blocking with PBS plus 1% bovine serum albumin for
1 h at 37°C, tissue homogenates in PBS and recombinant IL-18
(PreproTech, Inc.) were added to the wells and incubated overnight at
4°C. After a washing, a biotinylated goat anti-mouse IL-18 antibody
(200 ng/ml) was added to the wells for 2 h at 37°C. After a
washing, a 1:1,000 dilution of HRP-goat anti-biotin antibody was added
to the wells and incubated for 1 h at room temperature. After a
washing, ABTS substrate was added, and the absorbance was measured at
415 nm on a Kinetics Reader model EL312.
Statistical analysis. The Kaplan-Meier method (GraphPad Prism; GraphPad Software, Inc., San Diego, Calif.) was applied to obtain the survival fractions after infection with a lethal dose of wild-type S. typhimurium. By using the Mantel-Haenszel log rank test, the P values for the statistical differences between vehicle and anti-IL-18 antibody treatments were discerned at the 95% confidence interval. All other data were analyzed by Student t test, and significant values were recorded at a P value of <0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Anti-IL-18 sera diminishes survival against wild-type S. typhimurium infection.
To assess the relevance of IL-18 in
protection against oral challenge with S. typhimurium, a
recombinant IL-18 was generated as an MBP fusion which allowed for the
production of an IL-18-specific, neutralizing antiserum. This antiserum
recognized commercially available recombinant murine IL-18 but not
recombinant murine IL-12 or IFN-. The antiserum was also able to
inhibit IL-18-induced IFN- by anti-CD3-stimulated T cells (data not
shown). To block endogenous IL-18 production, the rabbit anti-mouse
IL-18 serum was administered to BALB/c mice 1 day prior to and on the
day of oral challenge with wild-type S. typhimurium. Such
anti-IL-18 treatment did result in a significant reduction
(P < 0.005) in the survival rate compared to the
normal serum or to PBS-treated mice (Fig.
1). The median survival rate for the
anti-IL-18-treated group (n = 19) was 6 days compared
to the vehicle-treated group (n = 21), and the median
survival rate was 8 days. Thus, the anti-IL-18 treatment accelerated
death by approximately 2 days, implicating the importance of IL-18 in
resolving infection with wild-type S. typhimurium. The
extent of colonization in the two subject groups was also examined. No
statistical difference in the extent of colonization in the Peyer's
patches and spleens was noted at 3 days p.i. with the wild-type
S. typhimurium.
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, BALB/c; 
, anti-IL-18-treated BALB/c; 
, IL-12KO; and
, anti-IL-18-treated IL-12KO.
Role of IL-18 subsequent infection with an avirulent
Salmonella vector.
Diminished survival was evident
with the anti-IL-18-treated BALB/c mice after oral challenge with
wild-type S. typhimurium, suggesting that IL-18 contributes
to the development of protective immunity against
Salmonella. To consider how IFN- is induced by IL-18, we
selected to use an avirulent, aro-negative
Salmonella vaccine vector for infection in order to monitor
the early events in the mucosal and systemic compartments. Such
experimentation would permit the study of how IFN- is induced
ultimately for protection, eliminating the concerns attributed to
Salmonella virulence.
cytokine levels. Administration of anti-mouse
IL-18 antisera prior to and during oral challenge with attenuated
Salmonella sp. strain H647 resulted in a lesser change in
Peyer's patch weight at 12 h after infection compared to
vehicle-treated mice (Fig. 2A) but, by
24 h p.i., was dramatically enhanced (Fig. 2A). Upon analysis for
cytokine levels in the Peyer's patch homogenates, we observed
induction of IFN- as early as 12 h, with maximal expression by
7 days p.i. Anti-IL-18 treatment showed enhanced IFN- production by
12 h, and this was significantly diminished (P < 0.001) up to 3 days after infection (Fig. 2B). Between 3 and 7 days
p.i., significant increases (P < 0.021) in IFN-
levels between the two groups were evident (Fig. 2B). These IFN-
levels were supported by concomitant increases in IL-12 (Fig. 2C). As evident at 7 days p.i., significantly higher levels of IL-12 were observed in the anti-IL-18 treatment group compared to the
vehicle-treated group (Fig. 2C). Low levels of IL-12p40 were observed
in the Peyer's patches throughout the course of infection, with minor
but significant differences (P < 0.001) in the
IL-12p40 levels between the two groups (Fig. 2D).
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0.001; Fig. 3A).
Unlike the Peyer's patches, no significant differences in IFN-
levels in spleens were observed between vehicle-treated and
anti-IL-18-treated groups until 7 days p.i., at which point
significantly less (P 0.001) IFN- was observed
with the anti-IL-18 group (Fig. 3B). Anti-IL-18 treatment of orally
challenged BALB/c mice with attenuated Salmonella sp. strain
H647 strain also resulted in significant decreases in IL-12 levels at
each day tested (P 0.001 and P = 0.011; Fig. 3C). Although an initial rise in IL-12p40 levels was
observed at 12 h p.i., the IL-12p40 levels were reduced on days 3 and 7 p.i. for the anti-IL-18-treated group compared to the
vehicle-treated group (P 0.001; Fig. 3D). Thus,
these data suggest that the mucosal and systemic immune compartments
behave differently as a result of the anti-IL-18 treatment during oral
challenge with an attenuated Salmonella strain.
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Anti-IL-18 treatment lowers in vivo levels of IL-18 in BALB/c
mice.
To assess whether IL-18 is induced after oral infection with
H647 strain, Peyer's patch and splenic homogenates were assessed for
the presence of IL-18. Except for the 12-h time point, the anti-IL-18
treatment significantly reduced IL-18 levels in the Peyer's patches
(Fig. 4A). Peak induction of IL-18 was
evident 3 days after oral infection with the attenuated
Salmonella sp. strain H647, which corresponded to decreases
in IL-12 levels in vehicle-treated mice (Fig. 2C). This reduction of
IL-18 also coincided with the observed reduction in Peyer's patch
IFN- levels (Fig. 2B), suggesting that the mucosally induced IFN-
was supported in part by IL-18.
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Role of IL-18 subsequent infection of IL-12KO mice with an
attenuated Salmonella.
In the absence of IL-12, we sought to
determine whether IL-18 could be induced and whether it could
contribute to IFN- production. Thus, H647-infected IL-12KO mice were
examined at 3 days p.i., at which point IL-18 appeared to be induced to
its greatest extent. Anti-IL-18 treatment of IL-12KO mice resulted in
no significant change in Peyer's patch weight when compared to
vehicle-treated mice (Fig. 5A). In
contrast, anti-IL-18-treated IL-12KO mice did show significant
increases in splenic weights by 3 days p.i. with H647 strain
(P < 0.001; Fig. 5A). Although these mice were
deficient in bioactive IL-12, IFN- was still in induced (Fig. 5B);
however, anti-IL-18 treatment exerted no significant effect upon this
IFN- generation (Fig. 5B) in contrast to the effect observed with
the Peyer's patches from normal BALB/c mice (Fig. 2B). To determine whether IL-18 could be induced in the absence of bioactive IL-12, both
the Peyer's patches and the spleen were able to generate IL-18 (Fig.
5C). The Peyer's patch tissue IL-18 levels were similar to that
observed in normal BALB/c mice but were approximately one-third less
than that observed for splenic tissue IL-18 levels (Fig. 4). Anti-IL-18
treatment did result in significant reduction of Peyer's patch and
splenic tissue IL-18 (P = 0.011 and P = 0.02, respectively; Fig. 5C). Thus, endogenously induced IL-18 did not compensate for the absence of IL-12 in IFN- production.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The cytokine IFN- produced by T and NK cells plays an important
role in the inhibition of bacterial growth and in stimulating early
host defenses against virulent S. typhimurium
(15). IL-12, an important inducer of IFN-, is essential
in the clearance of intracellular pathogens, including S. typhimurium (17). A recently identified cytokine, IGIF
or IL-18, has been shown to have properties similar to those of IL-12
and together with IL-12 in synergistically induced IFN-
(28). In the present study, the role of IL-18 in eliciting a
protective immune response against oral challenge with S. typhimurium was investigated.
IL-18 did contribute to protection against oral challenge with
wild-type S. typhimurium, as evidenced by these studies
inhibiting IL-18 action. Comparisons of the extent of survival showed
that the anti-IL-18 treatment reduced the median survival rate by 2 days compared to vehicle-treated mice. Our results are in agreement with a recent study in which mice treated in vivo with recombinant IL-18 were protected upon systemic challenge with S. typhimurium (25). As with Salmonella, it
appears that IL-18 contributes to protective immunity against a number
of intracellular pathogens but to varying degrees (1, 3, 7, 8, 16,
41, 45). Kawakami et al. (16) showed that in vivo
treatment with IL-18 could augment survival after pulmonary challenge
with C. neoformans. Similarly, IL-18 participated in the
susceptibility to infection with Y. enterocolitica when
infected mice were treated with an anti-IL-18 antibody but failed to
augment colonization upon treatment with recombinant IL-18
(3). Similarly, we were unable to notice any significant
change in colonization caused by the anti-IL-18 treatment of mice
infected with wild-type S. typhimurium at 3 days p.i.
Likewise, only a minor change in Salmonella colonization was
observed when mice were treated in vivo with recombinant IL-18 (25). Collectively, these studies suggest that IL-18
contributes to resistance, perhaps by enhancing IL-12 or other
IFN--promoting cytokines. As such, the role for IL-18 may be
secondary to IL-12, serving in a cofactor capacity, since IL-18 is
dependent upon secreted IL-12 for activation. IL-12 regulates IL-18 by
directing the expression of the IL-18 receptor (1, 45). Bohn
et al. (3) observed that IL-18-induced IFN- stimulation
is IL-12 dependent, with minimal IL-18 activity observed in the absence of functional IL-12. In contrast, IL-12 does not rely upon IL-18, since
IL-12-induced IFN- was only slightly reduced in the presence of
anti-IL-18 antibodies (3). While these studies implicate IL-18 as a cofactor, a recent study showed that indeed IL-18 could contribute to resistance, as evidenced by reversing death due to herpes
simplex virus type 1 infection (8). Since IL-18 upregulates Fas ligand expression (7, 41), this may enhance
cytotoxic-T-cell activity or augment IFNs to resolve HSV-1 infections.
It is important to note that immunity to such viruses may rely more on
specific rather than on innate immunity.
To assess how IL-18 contributes to innate immunity after infection with
an attenuated Salmonella vector, cytokine analyses of
isolated mucosal and systemic lymphoid tissues were performed. Such
information should enhance our understanding regarding how IFN- is
induced by subsequent infection with such vaccine vectors. To this end,
we investigated the presence of IFN-, IL-12, and IL-18 at specific
time points during the first week of infection with the attenuated
S. typhimurium strain H647. Anti-IL-18 treatment enhanced
the early production of IFN- evident by 12 h, production then
declined by 24 h for at least 3 days and, by 7 days p.i., slightly
enhanced IFN- levels were detected. A portion of the early IFN-
does appear to be supported by IL-12, since anti-IL-18 treatment did
induce greater levels of IL-12. Likewise, IL-18 levels were also
increased by 12 h by the anti-IL-18 treatment, but it is unclear
how much of this cytokine was being released. Clearly, the levels of
IL-12 in the Peyer's patches were important during the early phase of
infection, with maximal levels evident at 24 h p.i. Subsequently,
Peyer's patch IL-12 levels begin to decline for 7 days p.i., with a
concomitant rise in IL-18. IL-18 reached maximal levels by 3 days p.i.
Thus, the Peyer's patch IFN- may be more dependent on IL-18 later
in the infection or, alternatively, IL-18 may have an additive effect
on the IL-12 dependence.
For the later stage of the mucosal infection, Peyer's patch IL-12
levels dramatically increased in the anti-IL-18-treated BALB/c mice,
and this may be due to a compensation for the limiting IL-18. This
compensation mechanism was affected by the absence of IL-12. In IL-12KO
mice, the anti-IL-18 treatment greatly reduced Peyer's patch levels of
IL-18, although significant levels of IFN- were still induced. In
the IL-12KO spleen, IL-18 levels were slightly but significantly
reduced by the anti-IL-18 treatment, but IFN- levels were not
affected. Thus, in the absence of IL-12, the reduction in splenic IL-18
did not correspond to a change in IFN- levels, suggesting other
compensating mechanisms for supporting IFN- generation in the
systemic compartment.
By using an IL-12p70-specific ELISA, the IL-12 detected in the mucosal and systemic compartments is the IL-12p70 heterodimer. IL-12p40, implicated as an IL-12 antagonist (11, 13, 21, 26), and previous studies that relied solely on the detection of p40 (19, 20) may have inadvertently overestimated the amounts of p70 heterodimer. This is particularly important in Salmonella infections, where substantial amounts of IL-12 p40 homodimer are induced during the early phase of an S. dublin infection. In fact, the IL-12p40 was expressed to concentrations exceeding the IL-12p70 by more than a 100-fold (5). As shown in our study, the IL-12p40 levels in the Peyer's patches reflected the changes of IL-12 concentrations. In the systemic compartment, this also was true, except at 12 h, when more IL-12p40 was detected for the anti-IL-18-treated mice. This slight increase in IL-12p40 over IL-12p70 levels as a consequence of anti-IL18 treatment could be due to the early events associated with the regulation of IL-12p40, since endogenous IL-12p40 could be detected in normal BALB/c spleens. Moreover, IL-12p40 is more sensitive to upregulation than IL-12p70, as has been previously shown (5).
The regulation of IL-18 and IL-12 is not fully understood. IL-18 may
induce IL-12 indirectly by inducing low amounts of IFN-, which in
turn activates macrophages to produce IL-12, and IL-12, together with
IL-18, synergistically induces higher levels of IFN- (3).
IL-18, unlike IL-12, is incapable of inducing naive CD4+ T
cells to develop into Th1 cells; however, IL-18 is a costimulatory factor that enhances IFN- production by stimulated Th1 clones (18, 28).
IL-18 and IL-12 are both important for protection against S. typhimurium. It is not known why these two similar cytokines contribute to immunity against intracellular pathogens, but our evidence suggests that they exhibit an overlapping effect which may be time dependent. Further studies are needed to understand how IL-18 and IL-12 interact and regulate each other and how they direct cellular immunity.
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ACKNOWLEDGMENTS |
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We thank Peter Hillemeyer and Karyn Carlson for their expert technical assistance.
This work was in part supported by NIH-NIAID grant AI-41123 and NRI-CRGP/USDA grant 9602195.
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FOOTNOTES |
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* Corresponding author. Mailing address: Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610. Phone: (406) 994-6244. Fax: (406) 994-4303. E-mail: dpascual{at}montana.edu.
Present address: U.S. Department of Agriculture, Agriculture
Research Service, Southeast Poultry Research Laboratory, Athens, GA 30605.
Editor: R. N. Moore
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, December 1999, p. 6242-6248, Vol. 67, No. 12
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