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*
Medical Research Council Unit Immunology in Infectious Diseases, Department of Immunology, University of Cape Town, Cape Town, South Africa; and
Institute of Immunology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
(2, 4, 5).
These cytokines stimulate the antimicrobial activity of infected
macrophages, allowing intracellular mycobacterial killing. The key
factor for the release of IFN- by NK and Th1 cells is IL-12
(6, 7). It is produced by APCs upon interaction with
mycobacteria (8). In addition, IL-12 has direct
stimulatory effects on effector functions of CD8+
T cells (9), which are critical for an effective control
of mycobacterial infection (2, 10). It has been shown that
in the absence of endogenous IL-12, mice are highly susceptible to
mycobacteria due to impaired type 1 cytokine responses and granuloma
formation (11, 12).
IL-12, originally called NK cell stimulatory factor (13)
or CTL maturation factor (14), is a 70- to 75-kDa
heterodimer (IL-12p70) consisting of disulfide-bonded 35-kDa (p35) and
40-kDa (p40) subunits (13, 14). The biological activities
of IL-12p70 are mediated through the high-affinity IL-12R, which is
composed of IL-12R
1 and IL-12R2 chains (15). The
biological activity of heterodimeric IL-12p70 requires the interaction
of p40 with IL-12R1 and the interaction of p35 with IL-12R2
(15). In contrast, p40 homodimer
(p(40)2),4
which binds to the IL-12R1, has been shown to exert an antagonistic
activity on IL-12R1 in both in vitro and in vivo systems by
inhibiting the biological activity of IL-12p70 (16, 17).
Because secretion of IL-12p70 is associated with excess production of
p(40)2 (14, 18), it has been
proposed that excess p40 may down-regulate IL-12-mediated immune
responses (19, 20). However, phenotypic differences
between IL-12p35-/- and
IL-12p40-/- mice have indicated an agonistic
role of homodimeric IL-12p40 in some systems, including cardiac
allograft rejection (21) and cryptococcal infection
(22).
In the present study, IL-12p35-/- mice, which secrete normal concentrations of the p40 subunit (23), and IL-12p35-/-p40-/- mice were infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or M. tuberculosis to evaluate the role of endogenously produced IL-12p40 in mycobacterial infection. We report a protective and agonistic function of IL-12p40 in mycobacterial infections with a therapeutic effect of IL-12p(40)2 in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12p35-/- (23) and IL-12p40-/- mice (24) on a 129Sv/Ev background, were backcrossed five times to C57BL/6. Mutant and C57BL/6 wild-type mice were maintained under specific pathogen-free conditions. For generation of homozygous double-deficient mice, IL-12p35-/- and IL-12p40-/- mice were intercrossed and genotyped by PCR. For experiments, animals were kept in autoclaved cages with autoclaved bedding, food, water, and microfilter lids. All experiments performed were in accordance with the guidelines of the Animal Research Ethics Board of the University of Cape Town (Cape Town, South Africa).
Bacteria
Mycobacteria were grown in Middlebrook 7H9 broth (Difco,
Detroit, MI) supplemented with Middlebrook oleic acid, dextrose, and
catalase enrichment medium (Life Technologies, Gaithersburg,
MD), 0.002% glycerol, and 0.05% Tween 80. Midlog phase
cultures were harvested, aliquoted, and frozen at -80°C. After
thawing, viable cell counts were determined by plating serial dilutions
of the cultures on Middlebrook 7H10 agar plates followed by incubation
at 37°C. Mycobacterial strains M. bovis BCG (Pasteur) and
M. tuberculosis (H37Rv) were used and experiments were
performed in level II and level III biohazard facilities. Before
infection with M. bovis BCG or M. tuberculosis,
stock solutions of mycobacteria were diluted in PBS, and the
preparation was sonicated to ensure proper dispersion of mycobacteria.
For i.v. infection with M. bovis BCG, mice were infected
with 2 x 106 CFU mycobacteria via a lateral
tail vein. Pulmonary infection with M. tuberculosis was
performed in an inhalation exposure system (Glas-Col, Terre-Haute, IN).
Mice were exposed for 40 min to an aerosol generated by nebulizing
5.5 ml of a suspension containing 107 live
bacteria. Inoculum size was checked 24 h after infection by
determining the bacterial load in the lung of infected mice.
Colony enumeration assay
Bacterial loads in the lung, liver, and spleen of infected mice were evaluated at different time points after infection with M. bovis BCG or M. tuberculosis. Organs were weighed and defined aliquots were homogenized in 0.04% Tween 80 saline. Tenfold serial dilutions of organ homogenates were plated in duplicates onto Middlebrook 7H10 agar plates containing 10% oleic acid, dextrose, and catalase and incubated at 37°C for 19–21 days. Colonies on plates were enumerated and results are expressed as log10 CFU per organ.
Histopathological analysis
For analysis mice were killed by cervical dislocation. Organs were fixed in 4% buffered formalin and paraffin-embedded. Two- to 3-µm sections were stained with H&E and a modified Ziehl-Nielsen method. The latter involved staining in a prewarmed (60°C) carbol-fuchsin solution for 30 min followed by 30 min at room temperature; destaining was performed for 1 min in 0.5% HCl-ethanol before counterstaining with hematoxylin for 4 min.
Immunohistochemistry
Frozen tissue sections were prepared using a cryostat (Leica CM 1850) and 3-aminopropyltriethoxysilane-coated slides. Seven-micrometer sections were air-dried and fixed in 75% acetone and 25% ethanol for 5 min before storage at -80°C. Slides were washed in PBS and incubated with a primary Ab against CD4 (clone GK 1.5) for 90 min at room temperature. After incubation with appropriately diluted rabbit anti-rat mAb coupled with Texas Red (Jackson Immuno-Research Laboratories, West Grove, PA) for 30 min at room temperature, sections were mounted in Fluoromount (DAKO, Carpinteria, CA) and assessed by immunofluorescence microscopy (Nikon, Randburg, South Africa).
Delayed-type hypersensitivity
Infected mice were challenged in the right footpad with 10 µg of purified protein derivative (PPD; State Vaccine Institute, Pinelands, South Africa) in 50 µl of PBS and in the left footpad with 50 µl of PBS alone. Swelling in each footpad was measured after 24, 48, and 72 h using a Mitutoyo micrometer caliper (Brütsch, Zürich, Switzerland), and the difference was taken as the amount of Ag-specific delayed-type hypersensitivity (DTH). The PPD preparation did not induce swelling in noninfected animals.
Ag-specific IFN- production
For measuring Ag-specific production of IFN-, single cell
suspension of peripheral lymph nodes were prepared from mice 8 wk after
infection with M. bovis BCG. Cells were resuspended in IMDM
(Life Technologies, Paisley, U.K.) supplemented with 10% FBS (Life
Technologies), 0.05 mM 2-ME (Sigma, Cape Town, South Africa), and
penicillin and streptomycin (100 U/ml and 100 µg/ml; Life
Technologies). Lymph node cells (2 x 106)
were incubated with 2 x 104 peritoneal
macrophages that were pulsed for 4 h with 2 x
105 M. bovis BCG and 1 µg of PPD in
antibiotic-free IMDM. Resident peritoneal macrophages were obtained 1
day before the experiment by peritoneal lavage of uninfected C57BL/6
mice and incubated in 96-well flat-bottom microplates (Nunc,
Naperville, IL) in complete IMDM.
To determine mycobacterial Ag-specific IFN- production, 100 µl of
supernatants from cultures were taken and kept frozen at -80°C.
IFN- levels were analyzed in 3-fold serial dilutions using a
sandwich ELISA with coating and biotinylated anti-IFN- mAbs (BD
PharMingen, San Diego, CA), as previously described (25).
The detection limit was 200 pg/ml.
CD4+ and CD8+ T cell enrichment
Peripheral lymph nodes were obtained 8 wk after infection with M. bovis BCG and single cell suspensions were prepared in IMDM. Enrichment of CD4+ T cells was performed by positive selection with magnetic mouse CD4 Dynabeads and mouse CD4 DETACHaBEAD (Dynal, Lake Success, NY). CD8+ cells were enriched by incubation of the CD4+-depleted cell suspension with anti-B220-specific Dynabeads. Positively selected CD4+ T cells from lymph nodes contained >90% CD4+ cells, and negatively enriched CD8+ T cell suspensions contained >90% CD8+ cells, as determined by flow cytometry analysis (26).
Functional CD4+ T cell assays
In vitro responses to mycobacterial Ags were measured as described previously (27), with slight modifications: 4 x 105 CD4+ enriched lymph node cells obtained from infected mice were cultured with 2 x 104 peritoneal macrophages pulsed with 2 x 105 M. bovis BCG and 1 µg of PPD for 4 h in antibiotic-free IMDM. Resident peritoneal macrophages were obtained 1 day before the experiment from peritoneal lavages of uninfected C57BL/6 mice and incubated in 96-well flat-bottom microplates (Nunc) in complete IMDM. CD4+ T cells and pulsed macrophages were incubated for 72 h at 37°C and 5% CO2. For the proliferation assay, 1 µCi of [3H]thymidine was added for the last 10 h of culture. After harvesting, thymidine incorporation was measured in a liquid scintillation counter.
Cytotoxic CD8+ T cell assay
The cytotoxic activity of CD8+ T cell-enriched lymph node cells was performed as previously described in a 4-h 51Cr release assay (28). Effector cells were harvested and cultured in complete IMDM with 10 µg/ml PPD at 37°C and 5% CO2. For a source of target cells, macrophages were obtained from peritoneal lavages of uninfected C57BL/6 mice, seeded at 1 x 104 cells/well in 96-well round-bottom microplates (Nunc) in antibiotic-free IMDM. Cells were then pulsed with 1 x 105 M. bovis BCG, 1 µg of PPD, and 2 µCi of 51Cr (Amersham Pharmacia Biotech, Piscataway, NJ). After washing of target cells, effector cells were added at different E:T ratios and plates were incubated for 4 h at 37°C. A 100-µl volume of supernatant was then collected from each well, and radioactivity release was measured in a Packard gamma counter (Cobra III; Packard Intrument, Downers Grove, IL). The percentage of specific lysis was calculated taking into account spontaneous and maximum release of target cells incubated with medium alone or 100% Triton X-100 as follows: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.
IL-12p(40)2 administration and immunoblotting
Homodimeric IL-12p40 was produced by Chinese hamster ovary cells stably transfected with mouse IL-12p40 cDNA and purified as described previously (29). Purified IL-12p(40)2 was >95% pure, as assessed by SDS-PAGE. Briefly, samples were boiled in SDS-PAGE buffer for 2 min and separated on a 5–15% polyacrylamide gradient gel (Life Technologies). Gels were stained in Coomassie brilliant blue (Sigma-Aldrich, St. Louis, MO) for 6 h and destained overnight before visualization of protein bands. For immunoblotting, procedures were conducted at room temperature and Abs were diluted in blocking buffer (PBS/5% skim milk powder). Proteins were transferred (semidry in-house blotting apparatus) onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked for 4 h and incubated in a 1/1500 dilution of rat anti-mouse IL-12p40 mAb (C15.6, 2 mg/ml; BD PharMingen) for 3 h. After three washes in PBS/0.05% Tween 20, membranes were incubated for 1 h in a 1/1000 dilution of rabbit anti-rat IgG (H and L chains) alkaline phosphatase-conjugated Ab (1 mg/ml; Southern Biotechnology Associates, Birmingham, AL). Washing was repeated and membranes were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate tablets (Roche Diagnostic Systems, Indianapolis, IN) for 15 min. SDS-PAGE and results were verified using IL-12 anti-sera. Endotoxin contamination was <10 IU/mg IL-12p(40)2, as measured by the Limulus amebocyte lysate assay (M. Gately, unpublished observation). M. bovis BCG-infected mice received either 60 µg of rIL-12p(40)2 i.p. or PBS as control daily for 28 days.
Quantification of IL-12p40, IL-23p19, and IL-18 by real-time PCR
Total RNA was extracted from homogenized mouse spleen, liver,
and lung using TriReagent (Molecular Research Center, Cincinnati, OH),
and DNA contamination was digested by molecular grade RNase-free DNaseI
(Promega, Madison, WI) as per the manufacturer’s instructions. The
purity and integrity of the RNA was controlled by absorbance at
A260/280 and gel electrophoresis.
Absence of contaminating genomic DNA was verified by real-time PCR
amplification of the RNA using the -actin primers. Reverse
transcription was performed in 30-µl total volume, including 10 ng of
total RNA in 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM
MgCl2, 110 mM DTT, 0.5 mM each dNTPs, 0.7 U/µl
RNasin (Promega), and 16 U/µl Moloney murine leukemia virus
reverse transcriptase (Life Technologies), and incubated at 40°C for
2.5 h before inactivation of enzyme activity at 75°C for 15
min.
The levels of -actin, IL-12p40, p19, and IL-18 mRNA in the cDNA were
quantified by real-time PCR on a Lightcycler (Roche Diagnostic
Systems). Hot start PCR was performed in 20 µl containing 50 mM
Tris-HCL (pH 8.3), 4 mM MgCl2, 500 µg/ml BSA, 200 µM dNTPs,
0.125 U/µl Taq, 0.175 µM Taqstart Abs, 0.25 µM
primers, and 2 µl of cDNA template and monitored in real time with
the fluorescent DNA binding dye SYBR green (Roche Diagnostic
Systems). The following primer pairs were used: -actin (forward
5'-ACCCACACTGTGCCCATCTA-3', reverse 5'-ATCGGAACCGCTCGTTGC;
321-bp product), IL-12p40 (forward 5'-CAGCTCGCAGCAAAGCAA-3',
reverse 5'-GACGCCATTCCACATGTCACT3'; 530-bp product), IL-18 (forward
5'-TGGTTCCATGCTTTCTGG-3', reverse 5'-TCCGTATTACTGCGGTTGT-3'; 279-bp
product), and p19 (forward 5'-TGCTGGATTGCAGAGCAGTAA-3', reverse
5'-GCATG CAGAGATTCCGAGAGA-3'; 121-bp product). Standard curves for
-actin, IL-12p40, IL-18, and p19 were generated by performing 2-fold
dilutions of positive samples. The mRNA level for each sample was
normalized by dividing the calculated value by the housekeeping
-actin value. The various mRNA levels in the mutant mice were then
relatively quantified in comparison to the respective levels in
wild-type mice using the relative standard curve method (Applied
Biosystems, Foster City, CA).
Statistical analysis
Data are expressed as the means of individual determinations and SDs. Statistical analysis was performed using the Student’s t test or the Mann-Whitney rank sum test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of IL-12p40 during
infection with M. bovis BCG, we compared
IL-12p35-/-p40-/-,
IL-12p35-/-, and C57BL/6 wild-type mice.
IL-12p35-/- mice produce normal levels of
endogenous IL-12p40 (23). Wild-type as well as
IL-12p35-/- mice were able to clear bacterial
burden in infected organs, shown by decreased bacterial loads in
spleen, liver, and lung during the monitored 17 wk of infection (Fig. 1
a). In contrast,
IL-12p35-/-p40-/- mice
showed 3- to 4-log higher CFUs relative to
IL-12p35-/- and wild-type mice, resulting in an
inability to control infection
(107–108 CFU/organ). Of
importance, all
IL-12p35-/-p40-/- mice
survived infection but were chronically infected, with multiple
bacterial and inflammatory infiltrates resulting in severe pathology in
spleen (splenomegaly), liver, and lung (data not shown), 7 mo
postinfection. Four weeks postinfection, T cell-driven Ag-specific
immune responses were present in
IL-12p35-/- mice, which also exhibited a
similar DTH reaction to wild types, using the Ag-specific footpad
rechallenge model by injection of PPD of M. tuberculosis,
measured 24, 48, and 72 h postinjection. In striking contrast,
IL-12p35-/-p40-/- mice
were unable to mount a DTH response 4 (Fig. 1b) or 6 wk
(data not shown) postinfection. These results suggest a role of
IL-12p40 in mediating specific immune responses during infection with
M. bovis BCG, which is independent of bioactive
IL-12p70.
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Protective immunity during mycobacterial infection is dependent on
granuloma formation leading to control and containment of the
infection, which is closely associated with T cell responses. In
agreement with the bacterial burden, histological examination of acid
fast bacilli in hepatic granulomas 9 wk postinfection revealed only few
bacilli in wild-type (Fig. 2, a and d) or IL-12p35-/-
mice (Fig. 2, b and d) but abundant bacilli in
IL-12p35-/-p40-/- mice
(Fig. 2, c and d). Increased numbers of
multibacillary granulomas were also observed in
IL-12p35-/-p40-/- mice
at 5 wk postinfection when CFUs between mutant strains were still
comparable (Table I). This indicates an
impairment of granuloma function in double-deficient mice. Subsequent
cellular analysis by immunohistochemistry showed sparsely infiltrated
CD8+ T cells within the granulomas of all strains
of mice (data not shown). In IL-12p35-/- mice
(Fig. 2f) similar numbers of infiltrating
CD4+ T cells were found compared with C57BL/6
controls (Fig. 2, e and h). In contrast,
IL-12p35-/-p40-/- mice
showed significant reduced CD4+ T cell
infiltrates (Fig. 2, g and h) with diffuse
formation within the granuloma comprising large areas with foamy
macrophages (Fig. 2g) due to the presence of many bacilli
(Fig. 2c). Together, these data show that granuloma
formation and function is present in
IL-12p35-/- mice but impaired in
IL-12p35-/-p40-/-
mice.
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To determine whether the observed differences between
IL-12p35-/- and
IL-12p35-/-p40-/- mice
were directly mediated by IL-12p40, M. bovis BCG-infected
IL-12p35-/-p40-/- mice
were supplemented in vivo by administering
rIL-12p(40)2, produced by a transfected Chinese
hamster ovary cell line (kindly provided by M. Gately,
Hoffmann-LaRoche, Nutley, NJ). Immunoblotting of purified
IL-12p(40)2 with anti-IL-12p40 mAbs revealed
a purity of 99% IL-12p40 homodimer and 1% IL-12p40 monomer (Fig. 3a). After 9 wk postinfection,
increased resistance in p40-supplemented
IL-12p35-/-p40-/- mice
was observed, resulting in a 10- to 15-fold reduction of bacterial
burden in infected organs compared with nonsupplemented but infected
IL-12p35-/-p40-/- mice.
Similar CFUs in liver and lung were found in
IL-12p35-/- mice as compared with wild-type
mice (Fig. 3b). Importantly, supplementation with
IL-12p40 was able to fully restore the Ag-specific recall response in
infected
IL-12p35-/-p40-/-
mice, demonstrated by a comparable DTH response to infected
IL-12p35-/- and C57BL/6 mice (Fig. 3c). In summary, these results demonstrate that exogenously
administrated p(40)2 is able to restore some of the
defective functions observed in
IL-12p35-/-p40-/- mice,
which suggests an agonistic role for IL-12p40 in the absence of
bioactive IL-12p70.
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IL-12p70 promotes Th1 differentiation leading to IFN-
production and a type 1 cellular immune response. Therefore, we
compared Ag-specific T cell functions in response to BCG infection.
Lymph nodes from infected mice were isolated 9 wk postinfection and
cells were restimulated with Ag-presenting peritoneal macrophages in
vitro to measure IFN- responses by T cells. The ratio and percentage
of CD4+ and CD8+ T cells
were similar in all three strains as determined by FACS analysis (data
not shown). Lymphocytes from wild-type as well as from
IL-12p35-/- mice showed a marked and comparable
IFN- response (Fig. 4a). In
contrast, this response was abolished in lymphocytes from
IL-12p35-/-p40-/- mice.
Next, the Ag-specific proliferative response of
CD4+ T cells was determined by coculturing
enriched CD4+ T cells from infected mice with
M. bovis BCG-infected peritoneal macrophages (Fig. 4b). In comparison to the Ag-specific proliferative response
of wild-type mice, proliferation of CD4+ T cells
was reduced in IL-12p35-/- mice. However,
proliferation was completely abrogated in
IL-12p35-/-p40-/- mice.
These results demonstrate a defective Ag-specific proliferative
response in the absence of endogenous IL-12p40. Finally,
CD8+ T cells were analyzed for their Ag-specific
cytotoxic activity. Syngenic peritoneal macrophages pulsed with
M. bovis BCG and PPD were used as target cells for
CD8+ T cells isolated from infected mice.
Cytotoxic activity was present in IL-12p35-/-
mice, albeit clearly lower than that observed in
CD8+ cells from wild-type mice. The cytotoxic
activity of CD8+ T cells from
IL-12p35-/-p40-/- mice
was further reduced to 32% of the maximum killing observed in
wild-type mice. Collectively, these data show striking evidence for an
agonistic and protective role of IL-12p40 by promoting Ag-specific Th1
and CTL effector functions.
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Because the agonistic IL-12p40-dependent activities described
above leave open whether homodimeric IL-12p40 or the recently
discovered p40p19 heterodimer (designated as IL-23) are responsible for
protection we quantified expression levels by real-time
RT-PCR in spleen from M. bovis
BCG-infected mice 2 mo postinfection. In comparison to wild-type
controls, IL-12p35-/- and
IL-12p35-/-p40-/-
mice showed a 3- and 8-fold increase in p19 mRNA levels (Fig. 5 and
Table II). Neither IL-12p40 nor IL-18 was significantly increased in
mutant mice compared to infected controls at this time point of
infection (Table II).
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Airborne infection with M. tuberculosis is the most
prevalent infectious disease for humans worldwide. Consequently, we
addressed whether the protective and agonistic role of IL-12p40, which
we observed after infection with M. bovis BCG, has relevance
for infection with virulent M. tuberculosis. To mimic the
natural route of infection in humans, mice were infected with 100 CFU
of the virulent M. tuberculosis strain H37Rv in an
inhalation exposure system to induce pulmonary tuberculosis. In the
absence of bioactive IL-12p70 all mutant mice died, whereas wild-type
mice survived infection, confirming an essential protective role for
IL-12p70 in experimental tuberculosis (Fig. 6a).
IL-12p35-/- mice survived up to 20 wk
postinfection, whereas the life span of infected
IL-12p35-/-p40-/- mice
did not exceed the first 10 wk postinfection.
IL-12p35-/- mice were also able to mount a DTH
response similar to wild-type controls (Fig. 6b) and similar
IL-12p40 serum levels as wild-type controls (5 and 5.5 ng/ml,
respectively) 16 wk postinfection. Infected
IL-12p35-/-p40-/- mice
were completely defective in their DTH response, suggesting impaired
cell-mediated responses as demonstrated in the BCG model. Indeed,
lymphocyte infiltration to the site of infection during early pulmonary
tuberculosis was comparable to that observed in wild-type (Fig. 6c) and IL-12p35-/- mice (Fig. 6d), but appeared to be reduced in granulomas of
IL-12p35-/-p40-/- mice
(Fig. 6e). Together, these results demonstrate residual
resistance to M. tuberculosis in
IL-12p35-/- mice which is dependent on the
presence of endogenous IL-12p40.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(32, 33) or TNF- (34)
responsiveness, demonstrating some residual resistance in the absence
of endogenous IL-12p70. Surprisingly,
IL-12p35-/- mice were able to control and clear
infection with M. bovis BCG with only slightly higher CFU
than wild-type controls, showing that effective immunity to M.
bovis BCG is not dependent on bioactive IL-12p70. Moreover,
IL-12p35-/- mice were less susceptible to
pulmonary infection with M. tuberculosis than
IL-12p35-/-p40-/- mice.
These striking phenotypical differences in resistance to mycobacterial
infection suggest a protective role for IL-12p40, independent of
bioactive IL-12p70. In vivo therapy with IL-12p(40)2
reversed the susceptible phenotype of M. bovis BCG-infected
double- deficient mice to a more resistant phenotype, providing
direct evidence for a protective function of IL-12p40. Previous
studies observing phenotypical differences in survival time and
organ burden of Cryptococcus neoformans-infected
IL-12p35-/- and
IL-12p40-/- mice (22) have already
indicated a protective role of IL-12p40. Moreover, in a cardiac
allograft rejection model using IL-12p35-/- or
IL-12p40-/- mice as recipients, other
investigators could demonstrate the presence of alloreactive Th1
responses in the absence of IL-12p70 but presence of IL-12p40
(21, 35). Recently, an agonistic function of IL-12p40
produced by lung epithelial cells was also observed in a viral
infection as well as in Salmonella enteritidis
infection using IL-12-deficient mice (36, 37). Together
with our mycobacterial infection studies and the successful in vivo
therapy with IL-12p(40)2, we present striking evidence for
an agonistic role of endogenous and exogenous IL-12p40.
Subsequently, we investigated the cellular mechanism underlying the
IL-12p40-mediated protection in mycobacterial infection. With respect
to an agonistic role of IL-12p40 in mediating Th1 immune responses, we
found comparable Ag-specific IFN- production by restimulated
lymphocytes and Ag-specific proliferation of CD4+
T cell in BCG-infected IL-12p35-/- mice as in
wild-type mice. In addition, in vivo T cell activation, as measured by
DTH responses after injection of PPD, was unimpaired in M.
bovis BCG- and M. tuberculosis-infected
IL-12p35-/- mice. All these functions were
severely impaired in infected double-deficient mice. Indeed,
supplementation with IL-12p(40)2 was able to
fully restore DTH responses in M. bovis BCG-infected
IL-12p35-/- p40-/-
mice, demonstrating a direct role of IL-12p40 in these T cell
functions. The relationship between protection and DTH is not clear and
may involve two separate mechanisms in the immune response to
tuberculosis as discussed in a recent review (38). A DTH
reaction in the skin after injection of PPD may be a similar reaction
to granuloma formation in infected organs, involving the recruitment of
large numbers of macrophages and sensitized T cells. In their review,
Orme and Cooper (38) propose that protection is
preferentially cytokine driven and DTH chemokine driven, the latter
triggered by TNF-. In this report we have shown that IL-12p40
appears a crucial factor for PPD-induced DTH. A possible explanation
could be its chemotactic and activating properties on macrophages,
which have been recently demonstrated in several studies (36, 39, 40).
Concerning an IL-12p40-mediated CTL response, we showed residual
cytotoxic activity of primed CD8+ T cells from
M. bovis BCG-infected IL-12p35-/-
mice on syngenic targets, albeit to a low level considering the
relatively low maximum CTL response achieved in wild-type mice. In
contrast, the cytotoxic activity of corresponding cells from
IL-12p35-/-p40-/- mice
was completely impaired. In IL-12p35-/- mice
primary CTL responses against vaccinia virus were strikingly reduced;
however, comparison with
IL-12p35-/-p40-/- mice
was not performed (41). How do these IL-12p40-dependent
stimulatory effects on T cell responses explain the observed resistance
in mycobacterial infection? Both CD4+ and
CD8+ T cells are known to be responsible for
protection against mycobacterial infection (42). The
mycobacteriostatic and mycobactericidal effector mechanisms involved in
protective granulomatous responses are known to be strictly dependent
on a Th1 immune response (2, 4, 5) because IFN-
produced by Th1 cells (43) activates the mycobactericidal
effector functions of macrophages (44, 45). These
responses are normally promoted by IL-12p70 (6). Moreover,
MHC-I-restricted cytotoxic CD8+ T cells are able
to lyse infected macrophages directly (46, 47). These T
cell effector functions were present in M. bovis BCG- or
M. tuberculosis-infected IL-12p35-/-
mice; therefore, granuloma formation and function were rather normal in
these mutant mice, suggesting that endogenous IL-12p40 is involved in
stimulating mycobacterial-specific T cell effector functions. Previous
studies using IL-12p40-/- mice showed
exacerbated susceptibility to M. bovis BCG (11)
or M. tuberculosis (12), with defects similar
to those observed by us in
IL-12p35-/-p40-/- mice.
The residual resistance, albeit on a high infective and chronic burden
in M. bovis BCG-infected IL-12p40-/-
mice (11) or
IL-12p35-/-p40-/-
mice, demonstrates the presence of a residual and protective immune
response in the absence of IL-12p40 and IL-12p70. This residual
protection could be attributed to IL-18, because IL-18 mRNA was present
in the spleen of M. bovis BCG-infected
IL-12p35-/- and
IL-12p35-/-p40-/- mice
(Table II
). The recovery of macrophage activation in M.
tuberculosis-infected IL-12p40-/- mice has
previously been shown to be mediated by IL-18 (12).
However, 7 mo postinfection, double-deficient mice showed severe
histopathology in infected organs, demonstrating the limited role for
IL-18 and other protective responses in the absence of IL-12p40 and
p70. From our studies it is also evident that IL-12p40 is able to
effectively substitute the bioactive functions of IL-12p70 in M.
bovis BCG-infected IL-12p35-/- mice in an
agonistic fashion, but only partially in M.
tuberculosis-infected IL-12p35-/- mice.
These gradual differences in protection may be due to the higher
virulence of M. tuberculosis H37Rv compared with M.
bovis BCG but nonetheless highlight the crucial importance of
bioactive IL-12p70 in M. tuberculosis infection. Currently,
we are investigating whether IL-12p(40)2 may be
used as an immune adjuvant to optimize BCG vaccination. IL-12p70 has
recently been reported to be effective as an adjuvant
(48).
It is not clear why IL-12p(40)2 functions as an
IL-12p70 agonist in some systems but as an antagonist in most others.
Mechanistically, we seem to understand the antagonistic function
of homodimeric p40 on natural killer and T cells (16, 29, 49, 50, 51). The p40 subunit of the IL-12p70 heterodimer binds to
the IL-12R1 chain, and signal transduction by IL-12p70 is mediated
by interaction of the IL-12p35 subunit with the IL-12R2 chain of the
IL-12R. Homodimeric IL-12p40 competes with IL-12p70 for binding to the
IL-12R1 chain of the heterodimeric IL-12R (15). The
observed stimulatory effects of IL-12p40 on T cells are likely to be
mediated through this receptor component in an agonistic fashion. Zou
and colleagues (52) reported that IL-12R1 and
IL-12R2 associate with different Janus kinases and therefore may
contribute to distinct signaling pathways: the cytoplasmic domain of
IL-12R1 associates with tyrosine kinase 2, while the
cytoplasmic domain of IL-12R2 interacts with Janus kinase 2. Hence,
IL-12R1 may be capable of transducing IL-12p40 signals via tyrosine
kinase 2, a possibility that remains to be investigated. An
alternative, perhaps more likely, explanation is that IL-12R1
associates with an as yet unknown component of the IL-12R. A hitherto
unidentified third component associating with the IL-12R1 subunit
was recently reported (53). More recently, a p19 protein
has been identified which combines with IL-12p40 to form a novel,
biologically active cytokine, designated as IL-23, with similar but
discrete functions from IL-12p70 (54). It has been shown
that IL-23 binds to IL-12R1 but not to IL-12R2, and a potential
new signal transducing transmembrane receptor subunit has recently been
identified (R. A. Kastelein, unpublished observation).
Formation of biologically active p19p40 heterodimer seems to require
intracellular synthesis of both subunits because it is a
disulfide-linked dimer. However, some mouse p19 is also secreted in the
absence of p40, at least in transient expression experiments
(54). If complex formation with monomeric p40 to IL-23 is
also possible extracellularly, we cannot exclude the possibility that
the minor fraction of monomeric p40 present in the administered
p(40)2 used for supplementation studies may have
caused the therapeutic effect by IL-23 complex formation with
endogenous p19. However, because there is a 100:1 molar excess of
homodimeric p40 which may compete with newly formed p19p40 complexes
for binding with IL-12R1, we would further need to speculate that
p19p40 has a far greater affinity than the p40 homodimer, to explain
the therapeutic effects. Indeed, p19 mRNA was hardly detectable in all
strains, with only a 3-fold increase of p19 mRNA found in M.
bovis BCG-infected IL-12p35-/- mice
compared with wild-type controls. Unfortunately, no information on
p19p40 affinities to IL-12R1 is currently available (R. A.
Kastelein, unpublished observation). Moreover, neutralizing Abs to
block IL-19 in IL-12p35-/- mice or
IL-12p35-/-p40-/- mice
are currently not available (R. A. Kastelein, unpublished
observation) which would allow us to test this hypothesis directly in
vivo. However, a p19 gene-deficient mouse strain has been recently
generated and is in the process of characterization (R. A.
Kastelein, unpublished observation). Planned mycobacterial studies,
including comparative infections with intercrossed
p19-/-p35-/- mice and
p19-/-p35-/-p40-/-
mice will univocally clarify the contribution of endogenous p40
independently of p19 (i.e., IL-23). Furthermore, supplementation of
p35-/-p40-/- mice vs
p19-/-p35-/-p40-/-
mice with p40 homodimer will determine whether IL-23 complex formation
is possible and responsible for the agonistic and protective role of
p40, demonstrated in this study.
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Acknowledgments |
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Footnotes |
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2 Current address: Molecular Infection Biology, Research Center Borstel, Borstel, Germany.
3 Address correspondence and reprint requests to Dr. Frank Brombacher, Medical Research Council Unit Immunology in Infectious Diseases, Department of Immunology, University of Cape Town, Health Science Faculty, GSH OMB H47, Observatory 7925, South Africa. E-mail address: fbrombac{at}mweb.co.za
4 Abbreviations used in this paper: p(40)2, p40 homodimer; DTH, delayed-type hypersensitivity; PPD, purified protein derivative; BCG, bacillus Calmette-Guérin.
Received for publication June 11, 2001. Accepted for publication October 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-type cytokine receptor subunits. Proc. Natl. Acad. Sci. USA 93:14002. production and type 1 cytokine responses. Immunity 4:471.[Medline]
receptor-deficient mice. J. Immunol. 158:5297.[Abstract]
-interferon receptor or inducible nitric-oxide synthase. Infect. Immun. 66:1208. genes. Science 259:1739. receptor have profoundly altered responses to infection with bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide. J. Exp. Med. 178:1435.-producing cells. J. Immunol. 158:643.[Abstract]
B by interleukin-12 p40 in microglial cells. J. Biol. Chem. 276:7899. and nitric oxide synthetase 2. J. Immunol. 164:371. in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249.1 and 2 and JAK kinases. J. Biol. Chem. 272:6073.1 subunit. Cell. Immunol. 186:39.[Medline]
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), and
IL-12p35-/-p40-/- mice (
) at 5, 9, and
17 wk postinfection. Differences between IL-12p35-/- and
IL-12p35-/-p40-/- mice were statistically
significant (*, p < 0.01; **,
p < 0.001). b, For measuring DTH,
C57BL/6 (filled bar), IL-12p35-/- (shaded bar), and
IL-12p35-/-p40-/- mice (open bar) were
challenged in the left hind footpad with PBS and the right footpad with
10 µg of PPD of M. tuberculosis at wk 4 after
infection. Differences in footpad swelling between
IL-12p35-/- and
IL-12p35-/-p40-/- mice were highly
significant (p < 0.0001). These results are
representative of four experiments.
