| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institutes of
*
Immunology and
Pathology, College of Veterinary Medicine, University of Leipzig, Leipzig, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF-
levels. In contrast,
IL-12p40-/- mice showed reduced IFN- production, which
was associated with significantly elevated serum IgE levels. Early
during infection (days 3 and 4 postinfection), as well as late (day 20
postinfection), the number of infected phagocytes was strongly
increased in the absence of IL-12p40 indicating impaired bactericidal
activity when IL-12p40 was missing. Liver histopathology revealed a
decreased number of mononuclear granulomas in IL-12p40-/-
mice. Depletion of CD4+ or CD8+ T
lymphocytes in vivo suggested that both T cell subpopulations
contribute to the IL-12p40-dependent protective functions. Analysis of
IL-12p40 vs IL-23p19 mRNA expression revealed an up-regulation of only
IL-12p40 mRNA during Salmonella infection. Together
these data indicate that IL-12p40 can induce protective mechanisms
during both the innate and the adaptive type 1 immune response in
Salmonella infection. This novel activity of IL-12p40
complements the well described dominant and essential role of IL-12p75
in protective immunity to Salmonella
infection. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-12 represents a heterodimeric protein consisting of two glycosylated subunits (35 and 40 kDa) that are linked covalently by a disulfide bond (5, 6). The p40 subunit is strongly induced in APCs by bacterial and viral Ags, whereas the p35 subunit is constitutively transcribed (7, 8). Within the endoplasmic reticulum, both subunits are joined together to form the bioactive IL-12p75 molecule (9). IL-12p75 has previously been shown to be the key factor for the induction of a Th1 immune response, which is essential to control infections with intracellular pathogens (10, 11, 12).
Besides the heterodimeric molecule IL-12p75, the p40 subunit can also
form homodimers (IL-12(p40)2). An excess of
IL-12(p40)2 was found to act antagonistically to
IL-12p75 in vitro and in vivo, because this molecule is able to compete
with IL-12p75 for the IL-12R
1 binding site (13, 14, 15, 16).
But unlike the IL-12p75, IL-12(p40)2 cannot
bind to the IL-12R2 subunit of the IL-12 receptor, which is
responsible for signal transduction leading to STAT-4 activation
(17). Recently, a new cytokine designated IL-23 was
identified, which consists of the IL-12p40 subunit covalently linked to
a newly discovered 19-kDa polypeptide chain (18). Like the
IL-12(p40)2, this new molecule is also capable of
binding only to IL-12R1 but not IL-12R2 (18). So far
it is not known whether IL-23 can antagonize IL-12p75-induced
effects.
Interestingly, an IL-12p40-dependent activity was found to contribute to the protective immune response against the fungal pathogen Cryptococcus neoformans, a result that was previously published by our group (19). IL-12(p40)2 was also shown to act agonistically with IL-12 in a murine transplantation model via the activation of CD8+ T cells (20). Here we present for the first time data which show that an IL-12p40-dependent activity is able to contribute to protective immunity against S.Enteritidis independently of IL-12p75.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Breeding pairs of BALB/c and 129Sv/Ev mice of three different genotypes (wild-type; IL-12p35-/-, Ref. 21 ; and IL-12p40-/- or IL-12p35/40-/-, Ref. 22) were provided by Dr. H. Mossmann (Max-Planck-Institute for Immunobiology, Freiburg, Germany). Mutant mice had been backcrossed five times on a BALB/c background (23) or were on a genetically pure 129Sv/Ev background. Animals were housed under specific pathogen-free conditions in filter-topped cages within a Maxi-Miser Caging system (Thoren Caging Systems, Hazleton, PA) containing sterile bedding. Mice were fed sterile food and water. For most experiments wild-type and mutant BALB/c mice were used unless otherwise stated. To study the role of the Ity gene Itys BALB/c mice were compared with Ityr 129Sv/Ev mice. Animals were used at 8–12 wk of age.
Mice were infected with an attenuated vaccination strain of S. enterica serovar Enteritidis (S. Enteritidis; ade-, his-; SALMOVAC SE; Impfstoffwerk Dessau-Tornau, Rosslau, Germany) (4, 24) and, in indicated cases, challenged with one of two different virulent wild-type strains of S. Enteritidis (147 Nalr, high virulent or 64/03 Nalr, low virulent; provided by Dr. S. Springer, Impfstoffwerk Dessau-Tornau).
Infection model
The animals (3–8/group) were inoculated i.p. with different doses (5 x 101 to 1 x 107 CFU) (4, 25). Different standard inocula were used to study the early (1 x 106 CFU) or the late (2.5 x 103 CFU) infection stage in both BALB/c and 129Sv/Ev background. Using an inoculum of 2.5 x 103 CFU in IL-12p40-deficient mice clinical symptoms occurred starting with day 16 postinfection (p.i.)4 on the BALB/c or with day 42 p.i. in the 129Sv/Ev mice. If not otherwise stated, wild-type and mutant mice on the BALB/c background were used in the experiments described.
Preparation of S. Enteritidis Ag
Heat-killed S. enteritidis (hk S.E.) were prepared by incubating bacteria at 60°C for 60 min in a water bath. Killed salmonellae were stored at 1 x 1010/ml in PBS at -75°C. This preparation was used as Ag for ex vivo stimulation of splenocytes.
Bacterial count in spleen and liver
After different time points (2, 3, 4, 20, 42, or 48 days p.i.), three to six mice per group were sacrificed. The thorax and the peritoneum of the animals were opened under sterile conditions, and they were bled by cardiac puncture, followed by the isolation of spleen and liver. After the determination of the spleen and liver weights, both organs were homogenized. Then the spleen and liver homogenates were diluted 1/3 or 1/10 (w/v), respectively, in PBS. Log10 serial dilutions of the homogenates in PBS were plated onto selective Xylose-Lysine-Desoxicholate agar (Merck, Darmstadt, Germany). Colonies were counted after 24- and 48-h incubation periods at 37°C. In cases in which only a part of the organ was homogenized, counts were corrected for the whole organ weights.
Enumeration of peritoneal cells or splenocytes containing viable salmonellae
To evaluate the number of peritoneal or splenic phagocytes that were positive for viable intracellular salmonellae, cells were freshly isolated from the peritoneal cavity by lavage with 5 ml of ice-cold PBS or from the spleen by passage through a 100-µm mesh cell strainer (Falcon; BD Biosciences, Heidelberg, Germany). Before plating, cells were washed three times in PBS, then incubated for 1 h in RPMI 1640 containing 100 µg/ml gentamicin (Life Technologies, Karlsruhe, Germany) to completely eliminate extracellular bacteria. After two additional washing steps in PBS, the cell number was adjusted to 1 x 105/ml or 1 x 106/ml, and 1 ml of the peritoneal cell or splenocyte suspension was plated onto Xylose-Lysine-Desoxicholate agar (Merck) and air-dried under sterile conditions. The density of the plated cells was checked microscopically to verify that single nonconfluent bacterial colonies could develop from the seeded cells. Growing colonies were counted after 24- and 48-h incubation periods at 37°C. Each single colony represented one Salmonella-positive phagocyte.
Detection of intracellular Salmonella Ag-positive cells by flow cytometry
Intracellular Salmonella Ag (comprising viable and killed/processed intracellular salmonellae) was indirectly fluorochrome-stained using a polyclonal rabbit anti-S. Enteritidis antiserum as primary Ab and a FITC-labeled goat anti-rabbit Ig (Jackson Immunoresearch Laboratories, West Grove, PA) as secondary Ab. The antiserum was obtained after s.c. immunization, and two intramuscular booster applications of an Ag suspension containing heat-killed and freeze-thawed S. enteritidis bacteria combined with Al(OH)3 (Eurobio, Raunheim, Germany). Phagocytes were identified by staining with the PE-labeled anti-CD11b mAb M1/70.15 (Caltag Laboratories, Hamburg, Germany) simultaneously to the secondary staining of the Salmonella Ag.
Determination of total and Ag-specific serum IgE
Total IgE serum levels were measured with a sandwich ELISA according to a standard protocol by using mAbs and a murine IgE standard from BD PharMingen (Heidelberg, Germany). Briefly, 96-well microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) were coated overnight at 4°C with the anti-IgE mAb R35-118 in 0.1 M carbonate buffer (pH 9.6). Plates were blocked and incubated with the 1/5 diluted serum samples and the mouse IgE standard. Then plate-bound IgE was detected by incubation with the biotinylated anti-IgE mAb R35-72 for 1 h followed by a 30-min incubation at room temperature with streptavidin-peroxidase (Southern Biotechnology Associates, Birmingham, AL) and the ABTS substrate reaction (Sigma, Deisenhofen, Germany). The detection limit was 8 ng/ml.
For the detection of Ag-specific IgE microtiter plates (Polysorp; Nunc) were coated overnight at 4°C with whole-cell extracts prepared by repeated freeze-thaw cycles of a fresh bulk culture of S. Enteritidis. Plates were blocked and then incubated with undiluted and 1/5 diluted mouse sera for 2 h at room temperature, followed by detection of Ag-bound specific IgE serum Abs using the biotinylated anti-IgE mAb R35-72 and streptavidin-peroxidase as described above.
Isolation of splenocytes and ex vivo stimulation
Single cell suspensions of the spleens were prepared by mincing and passing the tissue through a 100-µm mesh cell strainer (Falcon). The erythrocytes were lysed by using ammonium chloride buffer (0.15 M NH4Cl, 0.1 mM EDTA disodium salt dihydrate, 10 mM NaHCO3; Merck). The splenocytes were washed three times in cold PBS and resuspended in RPMI 1640 culture medium (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine (Biochrom, Berlin, Germany), 10 mM HEPES buffer, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME (Sigma). Following adjustment of the cell number to 5 x 106/ml, 1 ml of splenocyte suspension was dispensed into 24-well plates and stimulated with either mitogen (5 µg/ml Con A; Sigma) or Ag (106-108 hk S.E.) or left unstimulated. After 48 h of culture at 37°C in a humidified atmosphere containing 5% CO2, cell-free culture supernatants were harvested and stored in aliquots at -75°C until cytokine measurements.
Cytokine determination in sera and splenocyte supernatants
Cytokines (IFN-, IL-12p40, TNF-, IL-4, IL-6, IL-10) in
sera and cell culture supernatants were measured by sandwich ELISA
according to standard protocols by using matching pairs of mAbs.
Briefly, IFN- was assessed by using the mAb AN-18 (5 µg/ml;
American Type Culture Collection (ATCC), Manassas, VA) as capture and
the peroxidase-conjugated mAb XMG1.2 (ATCC) as detection Ab (both mAbs
were provided by Dr. H. Gallati, Hoffmann-LaRoche, Basel, Switzerland),
with recombinant mouse IFN- (provided by Dr. G. R. Adolf, Ernst
Boehringer Institute, Vienna, Austria) as the standard (detection
limit: 16 pg/ml). IL-12p40 was measured by using the mAb 5C3 (5
µg/ml; ATCC) as capture and biotinylated polyclonal goat
anti-mouse IL-12 as detection Ab (both Abs were provided by Dr. H.
Gallati, F. Hoffmann-LaRoche) combined with streptavidin-peroxidase
(Southern Biotechnology Associates), with recombinant mouse IL-12
(provided by Dr. M. Gately, Hoffmann-LaRoche, Nutley, NJ) as the
standard (detection limit: 156 pg/ml). IL-6 was measured using the mAb
MP5-20F3 (5 µg/ml) as capture and the biotinylated mAb MP5-32C11 (5
µg/ml) as detection Ab (both obtained from BD PharMingen) combined
with streptavidin-peroxidase (Southern Biotechnology Associates), with
recombinant mouse IL-6 (BD PharMingen) as standard (detection limit: 32
pg/ml).
IL-4, IL-10, and TNF- were measured by using the respective DuoSet
ELISA development systems (R&D Systems, Wiesbaden, Germany) following
the manufacturers instructions.
In vivo neutralization of IL-12p40
In indicated experiments IL-12p40 was neutralized in IL-12p35-/- mice by i.p. injection of 100 µg of anti-mouse IL-12p40 mAb 10F6 (ATCC, provided by Dr. H. Gallati) per mouse at days -1 and 1 p.i.
In vivo depletion of CD4+ and CD8+ cells
To deplete CD4+ or CD8+ T cells, groups of five animals of each genotype (wild-type, IL-12p35-/-, IL-12p40-/-) were treated with anti-mouse CD4 (anti-L3T4, clone GK 1.5, rat IgG2b; a gift from Dr. S. Ehlers, Research Center Borstel, Borstel, Germany) or with anti-mouse CD8 (anti-Lyt2.2, clone 2.43, rat IgG2b; a gift from Dr. S. Ehlers; in combination with anti-Lyt2.1/Lyt2.2, clone 53-6.7, rat IgG2a; BD PharMingen). Both CD4 and CD8 Abs were administered i.p. at total doses of 0.5 mg/mouse on three consecutive days (days -6, -5, and -4). Six days after the first mAb injection the degree of depletion (reaching 99.9% for CD4+ and 99.6% for CD8+) was analyzed by FACS analysis (26). Then the depleted animals were infected with 2.5 x 103 CFU S. enteritidis.
Histopathological examination
Spleen and liver tissue samples from sacrificed mice were immediately fixed in 5% phosphate-buffered formalin and embedded in paraffin. Tissue sections (5 µm) were stained with H&E and analyzed for the tissue granulomatous response using light microscopy. Randomly selected (n = 8) high power fields of tissue sections from both organs were examined from each animal.
Quantification of IL-12p40, IL-23p19, and -actin mRNA levels by
real-time PCR
Total RNA was extracted from 10 mg of homogenized mouse spleen using the RNeasy minikit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions combined with a DNA digestion step (DNase, Roche Diagnostics, Mannheim, Germany). Reverse transcription was performed with 500 ng of total RNA, peqGOLD dNTP-Mix (Peqlab, Erlangen, Germany), oligo(dT) primer, and Expand Reverse Transcriptase (both obtained from Roche Diagnostics) according to the manufacturer’s instructions using a conventional thermocycler (GeneAmp PCR System 2400; Applied Biosystems, Weiterstadt, Germany) in a total volume of 20 µl.
For relative quantification of IL-12p40 and IL-23p19 mRNA, 5 µl of
each sample cDNA was used for real-time PCR, which was conducted on the
LightCycler Instrument (Roche Diagnostics). The amplification of cDNA
was monitored with the fluorescent DNA binding dye SYBR Green using the
LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics),
according to the manufacturer’s instructions. Specific primers were
designed using Primer Express software (Applied Biosystems). The
following primer pairs were used for -actin (forward primer, 5'-AGA
GGG AAA TCG TGC GTG AC-3'; reverse primer, 5'-CAA TAG TGA TGA CCT GGC
CGT-3'; product size 148 bp), IL-12p40 (forward primer, 5'-GGA AGC ACG
GCA GCA GAA TA-3'; reverse primer, 5'-AAC TTG AGG GAG AAG TAG GAA
TGG-3'; product size 180 bp), IL-23p19 (forward primer, 5'-TGC TGG ATT
GCA GAG CAG TAA-3'; reverse primer, 5'-GCA TGC AGA GAT TCC GAG AGA-3';
product size 121 bp). The cytokine mRNA levels of infected animals were
relatively quantitated in comparison to the respective level of naive
animals by using the comparative CT (threshold
cycle) method (27). Briefly, the amount of target,
normalized to an endogenous reference (-actin) and relative to a
calibrator (cytokine mRNA expression in naive animals), is given by:
2-
CT, where
CT = CT,X -
CT,R is the difference in threshold cycles for
target and reference and CT =
CT,q - CT,cb is the
difference in the threshold cycles for any sample q and the calibrator
cb. Before using the CT method for relative
quantitation, a validation experiment was performed to demonstrate that
efficiencies of target amplification and reference amplification are
approximately equal. For validation of IL-12p40 and IL-23p19 mRNA
expression total cDNAs prepared from murine primary bone marrow
macrophages stimulated with IFN- (100 U/ml) + LPS (100 ng/ml) or
RAW264.7 macrophages (ATCC) stimulated with hk S.E.
(107/ml) were used as model systems to generate
standard curves for IL-12p40 or IL-23p19, respectively. The crossing
point for each reaction was determined using the Second Derivative
Maximum algorithm and the arithmetic baseline adjustment using
LightCycler software (Roche Diagnostics).
Statistical analysis
Statistical analyses were performed with the Mann-Whitney rank sum test or Student’s t test if the samples passed the normality and the equal variance analyses by using SigmaStat for Windows software (Jandel, Erkrath, Germany).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To characterize the course of S. Enteritidis infection
in mice in the absence of either IL-12p75 or IL-12p40, the survival
rates as well as the survival times of
IL-12p35-/-,
IL-12p40-/-, and wild-type mice were
determined. Wild-type mice that were able to produce IL-12p75 as well
as IL-12p40 survived inocula of up to 107 CFU of
S. Enteritidis (LD50 = 3 x
107) and showed clinical symptoms following
inoculation with 
106 CFU. In contrast, both
IL-12p35- and IL-12p40-deficient mice were more sensitive to
Salmonella infection. Mice of both strains died when
infected with 5 x 103 CFU. However, at
doses lower than 5 x 103 CFU the survival
rates and the survival times differed between IL-12p35- and
IL-12p40-deficient mice in a dose-dependent fashion (Fig. 1
). At an inoculum of 50 CFU
IL-12p35-/- mice completely survived up to day
50 p.i., whereas 50% of the IL-12p40-/-
mice died starting at day 18 p.i. These data indicate that
IL-12p40 contributes to the control of S. Enteritidis
infection at relatively low infection doses.
|
IL-12p35-/-,
IL-12p40-/-, and wild-type mice were infected
with S. Enteritidis and analyzed for the bacterial load
in the major visceral target organs spleen and liver. Different
bacterial inocula were used to study the early stage of infection on
days 3 and 4 p.i. (1 x 106 CFU) or the
late stage of infection on day 20 p.i. (2.5 x
103 CFU). Interestingly, in both the early (Fig. 2A) and the late phase of the
infection (Fig. 2B) the recovered bacterial numbers from the
spleen or liver were significantly lower in
IL-12p35-/- than in
IL-12p40-/- mice. These data indicate an
agonistic role of IL-12p40 in innate immunity and also in adaptive
immunity. Similar results were obtained for
Itys BALB/c and
Ityr 129Sv/Ev mice indicating
Nramp1 (Ity)-independent agonistic activity of
IL-12p40. Following inoculation with 2.5 x
103 CFU salmonellae were detectable at only low
levels in wild-type mice of both genetic backgrounds at day
20 p.i.
|
and TNF- in
IL-12p35-/- than in IL-12p40-/- mice
Two days p.i., higher levels of IFN- and TNF- were
detectable in the sera from IL-12p35-/- mice
compared with those from IL-12p40-/- mice or
from IL-12p35-/- mice that were treated with
the neutralizing anti-IL-12p40 mAb 10F6 (Fig. 3). This pattern of IFN- and TNF-
production was accompanied by serum IL-12p40 in S.Enteritidis-infected IL-12p35-deficient mice at similar
levels as in infected wild-type mice (Fig. 3). None of the three
cytokines was detectable in naive mice of either genotype (data not
shown).
|
To study the underlying IL-12p40-dependent protective mechanism(s)
in S. Enteritidis-infected wild-type, IL-12p35-, and
IL-12p40-deficient mice, the bactericidal capacity of splenic
phagocytes was determined by plating and subsequently counting
Salmonella-positive splenocytes. Using this assay it was
possible to detect single intracellularly infected phagocytes. As shown
in Fig. 4 the number of S.
Enteritidis-infected phagocytes was significantly elevated in
IL-12p40-/- compared with
IL-12p35-/- mice as early as day 4 p.i. in
the peritoneal cavity (Fig. 4A) as well as later on (i.e.,
days 20, 42, and 48 p.i.) in the spleen (Fig. 4B).
|
A). At the later time points (i.e., days 20, 42, and
48 p.i.) viable Salmonella-positive cells were no more
detectable in the spleens from wild-type mice, at detection limits of
92 and 60 cells/spleen (Fig. 4B).
The data obtained for viable Salmonella-positive splenic
phagocytes were confirmed by intracellular staining of
Salmonella Ag in splenic macrophages derived from wild-type,
IL-12p35-/-, and
IL-12p40-/- mice (day 20 p.i.) and
analysis by flow cytometry. In IL-12p40-/- mice
the percentages of S. Enteritidis Ag-positive
macrophages were 3.6-fold higher than in wild-type mice, and 3.5-fold
higher than in IL-12p35-/- mice. In addition,
the total number of Ag-positive splenic macrophages was 1.9-fold
higher compared with wild-type mice, and 2.4-fold higher compared with
IL-12p35-/- mice; this shows that the total
cell number was lower in wild-type than in
IL-12p35-/- mice (Fig. 5). These data indicate a correlation
between the high levels of intracellular Ag and the number of live
intracellular salmonellae shown in Fig. 4 in IL-12p40-deficient mice.
Interestingly, this analysis demonstrated also the presence of residual
Salmonella Ag in wild-type splenic macrophages at a time
point where no viable bacteria could be found anymore in
wild-type mice.
|
To differentiate the immune responses induced by S.Enteritidis in wild-type, IL-12p35-, and IL-12p40-deficient mice
into Th1- or Th2-biased responses, IFN- and TNF- vs IL-4 levels
in mitogen- or Ag-induced splenocyte culture supernatants, and serum
IgE levels from S. Enteritidis-infected animals were
analyzed after 20 (BALB/c) or 48 (129Sv/Ev) days of infection. As
mentioned in Materials and Methods the symptomatic stages of
the S. Enteritidis infection occurred between days 16
and 20 p.i. in BALB/c mice and between days 42 and 48 p.i. in
129Sv/Ev mice when infected with 2.5 x 103
CFU. Therefore, we analyzed T cell responses at day 20 p.i. in
BALB/c and day 48 p.i. in 129Sv/Ev mice.
As shown in Fig. 6A, the
mitogen-induced IFN- concentrations in splenic culture supernatants
from IL-12p35-/- 129Sv/Ev and from
IL-12p35-/- BALB/c mice were 2.5- and 9.8-fold
higher than from IL-12p40-deficient mice, respectively. Similar data
were obtained for Ag-induced IFN- (data not shown). In
IL-12p40-deficient 129Sv/Ev mice, lower IFN- amounts corresponded
with elevated IL-4 amounts. However, IL-4 was almost undetectable in
culture supernatants derived from IL-12p40-deficient BALB/c
splenocytes. Moreover, the total IgE serum level was significantly
elevated in both IL-12p40-deficient 129Sv/Ev and even in BALB/c mice
where no IL-4 was detectable ex vivo compared with wild-type or
IL-12p35-deficient mice (Fig. 6B). The pattern of the total
IgE response corresponded to that of Salmonella Ag-specific
IgE in the different genotypes (data not shown). Together these data
indicate a residual type 1 immune response (in the absence of IL-12p75)
that depends on IL-12p40, whereas in the absence of IL-12p40 a stronger
type 2 response develops.
|
Because our data not only indicated early innate immunity defects
in the absence of IL-12p40 but also later potentially T cell-dependent
defects, we were interested in evaluating the impact of
CD4+ or CD8+ T cells as
potential targets for IL-12p40 in S. Enteritidis
infection. Both T cell subpopulations were alternatively depleted in
vivo. In IL-12p35-/- mice the depletion of both
CD4+ and CD8+ T cells
resulted in 107- and 43-fold higher numbers of infected splenic
phagocytes, respectively (Fig. 7), and
significantly elevated bacterial numbers in the liver (mean ± SD
log10 CFU control: 4.503 ± 1.017; mean
± SD log10 CFU CD4-depletion: 6.049 ±
1.082 p = 0.048; mean ± SD
log10 CFU CD8 depletion: 6.319 ± 0.742
p = 0.012). This indicates that the p40-dependent
bactericidal activity involves both T cell subpopulations. In contrast,
in wild-type mice only the depletion of CD8+
cells led to an increase (9.4-fold) of infected splenic phagocytes
(Fig. 7) and significantly increased bacterial burden in liver
(mean ± SD log10 CFU control: 2.496 ±
0.760; mean ± SD log10 CFU CD8 depletion:
3.862 ± 0.344, p = 0.006) and spleen (mean
± SD log10 CFU control: 2.188 ± 0.252;
mean ± SD log10 CFU CD8 depletion:
2.892 ± 0.408, p = 0.011). Therefore, in
wild-type mice with a functional IL-12 system
CD8+ T cells appear to be a central effector
population. This is somewhat more pronounced than reported by other
authors who observed only a marginal contribution of
CD8+ T cells to immunity against
Salmonella (28, 29), and may depend on the time
point for the analysis of the Salmonella infection and/or
the use of different Salmonella strains and/or the infection
route.
|
The tissue granuloma response is impaired in IL-12p40-/- mice
Semiquantitative histopathological examination revealed an early
onset of mononuclear granuloma formation in the presence of IL-12p40
(day 4 p.i.) (Table I). At day
20 p.i. the histopathological response in liver and spleen of
IL-12p40-/- mice was characterized by reduced
numbers of mononuclear granulomas but a higher number of mixed cell
granulomas, consisting predominantly of neutrophils compared with
IL-12p35-/- mice (Table I). Characteristically,
the mononuclear liver granulomas were much less condensed in
IL-12p35-/- than in
IL-12p40-/- mice, which points to the beginning
disintegration of granulomas in the absence of IL-12p40 (Fig. 8, middle and lower
panels). Moreover, large nondemarcated necroses were only
detectable in IL-12p40-/- mice. The liver
tissue response in wild-type mice was similar at days 4 and 20
p.i. showing only very few small mononuclear granulomas that were
obviously sufficient to control the infection (Table I, and Fig. 8, upper panel).
|
|
) comparable to those
seen in IL-12p40-/- mice (Fig. 8).
|
IL-12p40 mRNA but not IL-23p19 mRNA is expressed in S. Enteritidis-infected mice
Because the agonistic IL-12p40-dependent activities described
above leave open whether homodimeric IL-12p40 (as shown in a murine
transplantation model, Ref. 20) and/or the newly
discovered p40-p19 heterodimer designated IL-23 (18) are
responsible for protection we wished to clarify the contribution of
IL-12p40 and/or IL-23. Therefore, real-time PCR was performed to
quantitate the up-regulation of IL-12p40 and IL-23p19 mRNA in spleen
from S. Enteritidis-infected mice at day 20 p.i. in comparison to
naive control mice (Fig. 10). This
analysis revealed that only the IL-12p40 but not IL-23p19
message was induced in the spleens of S.Enteritidis-infected BALB/c mice. The IL-12p40 mRNA level was
increased 192-fold in wild-type mice and 89-fold in
IL-12p35-/- mice (Fig. 10, C–F).
There was also a slight increase of IL-12p40 mRNA in
IL-12p40-/- mice, which results from a
technical problem in construction of the knockout strain
(22). As explained by Magram et al. (22) the
inactivation of the IL-12p40 gene was mediated by the
replacement of exon 3 with a neo cassette. Later it was
shown that a transcript resulted from removal of the mutated exon 3 by
a precise splice. That transcript encodes a mutated p40 protein
(with a deletion of 89 amino acids) that was shown to be immunoreactive
but biologically inactive (22). The primers used by us for
real-time RT-PCR hybridized in that region. Expression of p35 mRNA was
found in infected wild-type and IL-12p40-/-
mice but not in IL-12p35-/- mice as one would
expect (data not shown). Together with the induction of IL-12p40 mRNA,
this suggests a physiological role of IL-12p75 and potentially of
homodimeric IL-12p40 but not of IL-23.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Following inoculation of bacterial doses between 104 and 107 CFU both IL-12p35-/- and IL-12p40-/- mice died completely within 8–20 days p.i. (data not shown). Such inocula are sublethal in wild-type mice and are capable of mediating adoptive immunity to challenge with wild-type S. enteritidis (strains 147Nalr, Ref. 4 ; or 64/03Nalr, J. Lehmann, unpublished results). These data confirm previously reported results, which highlighted the importance of IL-12 in S. Typhimurium (30) and S. Dublin infection (31) in mice by neutralizing IL-12 in vivo using anti-murine IL-12 polyclonal antibodies or mAbs, respectively. It has to be noted that the commonly used anti-IL-12 Abs recognize all presently known expression forms of p40: IL-12p75, IL-12p40, and IL-23. In accordance with the present study, IL-12p40 and IL-23 can be excluded to be crucial for infection with higher doses of Salmonella because there was no phenotypic difference at those infective doses between mice lacking IL-12p75 and mice lacking IL-12p40 and IL-23.
In contrast to higher inocula, at relatively low bacterial infection
doses between 50 and 5000 CFU an IL-12p40-dependent activity (i.e.,
IL-12p40 and/or IL-23) seems to contribute to the control of
Salmonella infection, because survival rate and survival
time were significantly higher and the bacterial burden in spleen and
liver significantly lower in IL-12p35-/-
compared with IL-12p40-/- mice. Because total
organ burden and the frequency of infected phagocytes were elevated in
the absence of IL-12p40 already at days 3 and 4 (Figs. 2A
and 4A), IL-12p40 appears to play a role already during the
early innate immune response to activate macrophages or other
phagocytes for Salmonella killing. The
IL-12p40-dependent stimulatory effect on innate immunity can be
observed for a wide range of infective doses of even up to
107 CFU (data not shown). However, at those high
doses this early IL-12p40-dependent protective effect does not lead to
long-term survival (see Fig. 1) suggesting that at those high
infective doses IL-12p75 plays a major role for long-term protection.
It has still to be investigated whether IL-12p40 is able to stimulate
the phagocytes directly or indirectly for secretion of other
proinflammatory cytokines such as TNF- or IFN-. Of particular
interest within this frame appears the role of macrophage-derived
IFN-, which was recently described in other systems (32, 33). Our data support the hypothesis that the IL-12p40-dependent
activity is biologically relevant in immune responses toward low-dose
infections (e.g., critical in persistent Salmonella
infection) and that high-dose infections also require IL-12p75.
The IL-12p40-dependent promoting activity on innate cellular immune mechanisms is not related to the Nramp1 genotype. Mice of both the resistant and the susceptible genetic background developed a Th2-biased immune response against S. Enteritidis in the absence of IL-12p40. This is in agreement with previously published data showing similar cytokine response profiles in Ityr and Itys congenic mouse strains following infection with S.Dublin (34).
IL-12p75 has previously been shown to be the key factor for Th1 cell
differentiation (10, 11, 12). Intracellular pathogens such as
Salmonella induce predominantly type 1 immune responses,
which include differentiation and activation of pathogen-specific Th1
and/or Tc1 cells (3). Therefore, type 1 response-inducing
cytokines (e.g., IL-12p75) or effector cytokines (e.g., IFN-,
TNF-) are often crucial in intracellular infections. This was
confirmed for IL-12 (30, 31), IFN- (29, 35, 36, 37, 38), and TNF- (29, 37, 39, 40, 41) in murine
salmonellosis. To study a potential promoting effect of IL-12p40 on the
development of the S. Enteritidis-induced type 1
response we have analyzed the concentrations of IFN- and TNF- vs
IL-4 or IgE in splenocyte supernatants and sera. The results
demonstrate a significant Th2 shift in S.
Enteritidis-infected IL-12p40-deficient BALB/c and even more
pronounced in 129Sv/Ev mice, characterized by higher in vivo IgE but
lower in vivo and ex vivo IFN- levels. Thus, not only IL-12p75
contributes to Th1 induction during salmonellosis but also IL-12p40.
This is in agreement with results from a murine CMV model showing that
endogenous IL-12p40 in the absence of IL-12p75 appears to stimulate
early IFN- and TNF- responses (42).
Histopathological analysis also provided evidence for early and late
effects mediated by IL-12p40. At day 4 p.i. no mononuclear
granulomas were formed in the absence of IL-12p40. This is reminiscent
of our previously published finding in IL-12p40-deficient mice infected
with C. neoformans (19). In infection with this
fungal pathogen granuloma formation was found to depend on IL-12p40
(19). Moreover, homodimeric IL-12p40 was recently shown to
exert chemotactic activity on macrophages in vitro (43).
In S. Enteritidis infection later neutrophils appear to
compensate for the defect in formation of mononuclear granulomas. In
addition, T cell depletion in infected
IL-12p35-/- mice, especially the depletion of
CD8+ T cells, leads to loss of structured
mononuclear granulomas, which is followed by the appearance of
condensed necrotic foci (see Fig. 9) very similar to
IL-12p40-/- mice (see Fig. 8). This suggests
that T cells are important sources for cytokines (e.g., TNF-)
required for the recruitment of mononuclear cells and granuloma
formation.
There are three derivatives of the "p40 cytokine family" described
so far: 1) IL-12p75 (
p40 + p35) (5, 6), 2)
IL-12(p40)2 (13, 14, 15, 19), and 3)
IL-23 ( p40 + p19) (18). It was previously shown in
vitro that mouse IL-12(p40)2 is 25- to 50-fold
more potent as an IL-12p75 antagonist than mouse IL-12p40 monomer
(14). Mice lacking the p35 gene are deficient
in IL-12p75 production but are capable of producing
(p40)2 and IL-23. In contrast, mice lacking the
p40 gene are unable to produce IL-12p75,
IL-12(p40)2, and IL-23. Thus, the results from
this study do not allow a differentiation between IL-12p40- (monomer or
homodimer) and IL-23-mediated functions. However, real-time RT-PCR
measurement showed that neither in wild-type nor in
IL-12p35-/- mice IL-23p19 is induced at the
mRNA level, whereas IL-12p40 mRNA is strongly up-regulated in both
wild-type and IL-12p35-/- mice at day 20
p.i. This points to IL-12p40 as the critical immunostimulatory
cytokine and does not support an involvement of IL-23. Data from a
murine transplantation model (20) and a murine
paramyxoviral bronchitis model (44) also point to
homodimeric IL-12p40 as the active compound. Considering these studies
and the higher affinity of IL-12(p40)2 vs
IL-12p40, it is likely that homodimeric IL-12p40 but not monomeric
IL-12p40 is the active molecule that mediates the protective effects in
Salmonella-infected IL-12p35-/-
mice. To define the active p40-dependent compound experiments are
planned to treat infected IL-12p35/40-/- mice
with rIL-12(p40)2 and, for comparison, with
rIL-23.
The absence of detectable p19 mRNA expression as described above is not sufficient for final exclusion of a potential role of IL-23 in immunity to Salmonella infection. In further investigations we have to neutralize p19 or specifically IL-23 or use p19-/- mice to address the question more directly, whether the IL-12p40-dependent protective mechanism(s) on immunity against S. Enteritidis are mediated by IL-12(p40)2 or IL-23.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Landesveterinär- und Lebensmitteluntersuchungsamt Sachsen- Anhalt, Stendal, Germany.
3 Address correspondence and reprint requests to Dr. Gottfried Alber, Institute of Immunology, College of Veterinary Medicine, University of Leipzig, An den Tierkliniken 11, D-04103 Leipzig, Germany. E-mail address: alber{at}rz.uni-leipzig.de
4 Abbreviations used in this paper: p.i., postinfection; hk S.E., heat-killed S. Enteritidis.
Received for publication June 4, 2001. Accepted for publication August 23, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent shock. Infect. Immun. 65:4734.[Abstract]
2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817.-producing cells. J. Immunol. 158:643.[Abstract]
production and type 1 cytokine responses. Immunity 4:471.[Medline]
and IFN- in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro- Salmonella vaccines. Microb. Pathog. 13:477.[Medline]
interferon production in mouse typhoid. Infect. Immun. 64:189.[Abstract]
upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187:2103. and nitric oxide from macrophages via IFN- induction. J. Leukocyte Biol. 68:897. production in murine splenocytes: role of natural killer cells and macrophages. J. Immunol. 150:3973.[Abstract]
interferon in late stages of murine salmonellosis. Infect. Immun. 61:4248. interferon and tumor necrosis factor- in resistance to Salmonella typhimurium infection. Infect. Immun. 60:450. antibodies on histopathology of primary Salmonella infections. Infect. Immun. 63:3674.[Abstract]
p55 receptor. Infect. Immun. 66:3355.This article has been cited by other articles:
|
|
 |
|
  Z. Mikhak, A. Farsidjani, and A. D. Luster Endotoxin Augmented Antigen-Induced Th1 Cell Trafficking Amplifies Airway Neutrophilic Inflammation J. Immunol., June 15, 2009; 182(12): 7946 - 7956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Siegemund, N. Schutze, S. Schulz, K. Wolk, K. Nasilowska, R. K. Straubinger, R. Sabat, and G. Alber Differential IL-23 requirement for IL-22 and IL-17A production during innate immunity against Salmonella enterica serovar Enteritidis Int. Immunol., May 1, 2009; 21(5): 555 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. van de Wetering, R. A. de Paus, J. T. van Dissel, and E. van de Vosse IL-23 modulates CD56+/CD3- NK Cell and CD56+/CD3+ NK-like T Cell function differentially from IL-12 Int. Immunol., February 1, 2009; 21(2): 145 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Schulz, G. Kohler, N. Schutze, J. Knauer, R. K. Straubinger, A. A. Chackerian, E. Witte, K. Wolk, R. Sabat, Y. Iwakura, et al. Protective Immunity to Systemic Infection with Attenuated Salmonella enterica serovar Enteritidis in the Absence of IL-12 Is Associated with IL-23-Dependent IL-22, but Not IL-17 J. Immunol., December 1, 2008; 181(11): 7891 - 7901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Schulz, G. Kohler, C. Holscher, Y. Iwakura, and G. Alber IL-17A is produced by Th17, {gamma}{delta} T cells and other CD4- lymphocytes during infection with Salmonella enterica serovar Enteritidis and has a mild effect in bacterial clearance Int. Immunol., September 1, 2008; 20(9): 1129 - 1138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, M. Komai-Koma, D. S. Gilchrist, D. K. Hsu, F.-T. Liu, T. Springall, and D. Xu Galectin-3 Is a Negative Regulator of Lipopolysaccharide-Mediated Inflammation J. Immunol., August 15, 2008; 181(4): 2781 - 2789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Price, K. R. Simpfendorfer, R. R. Mantena, J. Holden, W. R. Heath, N. van Rooijen, R. A. Strugnell, and O. L. C. Wijburg Gamma Interferon-Independent Effects of Interleukin-12 on Immunity to Salmonella enterica Serovar Typhimurium Infect. Immun., December 1, 2007; 75(12): 5753 - 5762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wan, M. A. Versnel, W. y. Cheung, P. J. M. Leenen, N. A. Khan, R. Benner, and R. C. M. Kiekens Chorionic gonadotropin can enhance innate immunity by stimulating macrophage function J. Leukoc. Biol., October 1, 2007; 82(4): 926 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. van Duivenvoorde, W. G. H. Han, A. M. Bakker, P. Louis-Plence, L.-M. Charbonnier, F. Apparailly, E. I. H. van der Voort, C. Jorgensen, T. W. J. Huizinga, and R. E. M. Toes Immunomodulatory Dendritic Cells Inhibit Th1 Responses and Arthritis via Different Mechanisms J. Immunol., August 1, 2007; 179(3): 1506 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. L. Rudner, K. I. Happel, E. A. Young, and J. E. Shellito Interleukin-23 (IL-23)-IL-17 Cytokine Axis in Murine Pneumocystis carinii Infection Infect. Immun., June 1, 2007; 75(6): 3055 - 3061. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Chackerian, S.-J. Chen, S. J. Brodie, J. D. Mattson, T. K. McClanahan, R. A. Kastelein, and E. P. Bowman Neutralization or Absence of the Interleukin-23 Pathway Does Not Compromise Immunity to Mycobacterial Infection Infect. Immun., November 1, 2006; 74(11): 6092 - 6099. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Khader, S. Partida-Sanchez, G. Bell, D. M. Jelley-Gibbs, S. Swain, J. E. Pearl, N. Ghilardi, F. J. deSauvage, F. E. Lund, and A. M. Cooper Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection J. Exp. Med., July 10, 2006; 203(7): 1805 - 1815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Kleinschek, U. Muller, S. J. Brodie, W. Stenzel, G. Kohler, W. M. Blumenschein, R. K. Straubinger, T. McClanahan, R. A. Kastelein, and G. Alber IL-23 Enhances the Inflammatory Cell Response in Cryptococcus neoformans Infection and Induces a Cytokine Pattern Distinct from IL-12 J. Immunol., January 15, 2006; 176(2): 1098 - 1106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Wozniak, A. A. Ryan, J. A. Triccas, and W. J. Britton Plasmid Interleukin-23 (IL-23), but Not Plasmid IL-27, Enhances the Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection Infect. Immun., January 1, 2006; 74(1): 557 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. W. DePaolo, R. Lathan, B. J. Rollins, and W. J. Karpus The Chemokine CCL2 Is Required for Control of Murine Gastric Salmonella enterica Infection Infect. Immun., October 1, 2005; 73(10): 6514 - 6522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. I. Happel, P. J. Dubin, M. Zheng, N. Ghilardi, C. Lockhart, L. J. Quinton, A. R. Odden, J. E. Shellito, G. J. Bagby, S. Nelson, et al. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae J. Exp. Med., September 19, 2005; 202(6): 761 - 769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. I. Happel, E. A. Lockhart, C. M. Mason, E. Porretta, E. Keoshkerian, A. R. Odden, S. Nelson, and A. J. Ramsay Pulmonary Interleukin-23 Gene Delivery Increases Local T-Cell Immunity and Controls Growth of Mycobacterium tuberculosis in the Lungs Infect. Immun., September 1, 2005; 73(9): 5782 - 5788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Noone, E. A. Lewis, A. B. Frawely, R. W. Newman, B. P. Mahon, K. H. Mills, and P. A. Johnson Novel mechanism of immunosuppression by influenza virus haemagglutinin: selective suppression of interleukin 12 p35 transcription in murine bone marrow-derived dendritic cells J. Gen. Virol., July 1, 2005; 86(7): 1885 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Schuetze, S. Schoeneberger, U. Mueller, M. A. Freudenberg, G. Alber, and R. K. Straubinger IL-12 family members: differential kinetics of their TLR4-mediated induction by Salmonella Enteritidis and the impact of IL-10 in bone marrow-derived macrophages Int. Immunol., May 1, 2005; 17(5): 649 - 659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Holscher, A. Holscher, D. Ruckerl, T. Yoshimoto, H. Yoshida, T. Mak, C. Saris, and S. Ehlers The IL-27 Receptor Chain WSX-1 Differentially Regulates Antibacterial Immunity and Survival during Experimental Tuberculosis J. Immunol., March 15, 2005; 174(6): 3534 - 3544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Lieberman, F. Cardillo, A. M. Owyang, D. M. Rennick, D. J. Cua, R. A. Kastelein, and C. A. Hunter IL-23 Provides a Limited Mechanism of Resistance to Acute Toxoplasmosis in the Absence of IL-12 J. Immunol., August 1, 2004; 173(3): 1887 - 1893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Johnson Murine Oviduct Epithelial Cell Cytokine Responses to Chlamydia muridarum Infection Include Interleukin-12-p70 Secretion Infect. Immun., July 1, 2004; 72(7): 3951 - 3960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Ghilardi, N. Kljavin, Q. Chen, S. Lucas, A. L. Gurney, and F. J. de Sauvage Compromised Humoral and Delayed-Type Hypersensitivity Responses in IL-23-Deficient Mice J. Immunol., March 1, 2004; 172(5): 2827 - 2833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fieschi, S. Dupuis, E. Catherinot, J. Feinberg, J. Bustamante, A. Breiman, F. Altare, R. Baretto, F. Le Deist, S. Kayal, et al. Low Penetrance, Broad Resistance, and Favorable Outcome of Interleukin 12 Receptor {beta}1 Deficiency: Medical and Immunological Implications J. Exp. Med., February 17, 2003; 197(4): 527 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. R. Lankford and D. M. Frucht A unique role for IL-23 in promoting cellular immunity J. Leukoc. Biol., January 1, 2003; 73(1): 49 - 56. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Gran, G.-X. Zhang, S. Yu, J. Li, X.-H. Chen, E. S. Ventura, M. Kamoun, and A. Rostami IL-12p35-Deficient Mice Are Susceptible to Experimental Autoimmune Encephalomyelitis: Evidence for Redundancy in the IL-12 System in the Induction of Central Nervous System Autoimmune Demyelination J. Immunol., December 15, 2002; 169(12): 7104 - 7110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Huaux, M. Arras, D. Tomasi, V. Barbarin, M. Delos, J.-P. Coutelier, A. Vink, S. H. Phan, J.-C. Renauld, and D. Lison A Profibrotic Function of IL-12p40 in Experimental Pulmonary Fibrosis J. Immunol., September 1, 2002; 169(5): 2653 - 2661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Yrlid and M. J. Wick Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter J. Immunol., July 1, 2002; 169(1): 108 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Holscher, R. A. Atkinson, B. Arendse, N. Brown, E. Myburgh, G. Alber, and F. Brombacher A Protective and Agonistic Function of IL-12p40 in Mycobacterial Infection J. Immunol., December 15, 2001; 167(12): 6957 - 6966. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
