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Previous Article | Next Article 
Infection and Immunity, June 2006, p. 3180-3189, Vol. 74, No. 6
0019-9567/06/$08.00+0 doi:10.1128/IAI.02004-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Differential Requirements for Soluble and Transmembrane Tumor Necrosis Factor in the Immunological Control of Primary and Secondary Listeria monocytogenes Infection
Korana Musicki,1
Helen Briscoe,2
Stephen Tran,1
Warwick J. Britton,1,2 and
Bernadette M. Saunders1,2*
Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6,
Newtown, NSW 2042, Australia,1
Discipline of Medicine, Central Clinical School, University of Sydney, Sydney, Australia2
Received 12 December 2005/
Returned for modification 2 February 2006/
Accepted 7 March 2006
 |
ABSTRACT
|
|---|
The relative contributions of transmembrane tumor necrosis factor (memTNF)
and soluble tumor necrosis factor (solTNF) in innate and adaptive
immunity are poorly defined. We examined the capacities of wild-type
(WT) mice, TNF–/– mice, and memTNF mice,
which express only transmembrane TNF, to control primary and secondary
Listeria monocytogenes infections. Soluble TNF was not
required for induction or maintenance of protective immunity against a
low-dose (200-CFU) Listeria infection. In contrast to
TNF–/– mice, both WT and memTNF mice cleared
the bacilli within 10 days and were fully protected against rechallenge
with a lethal infective dose. Furthermore, T cells transferred from
immune mice, but not from naïve, WT, and memTNF mice, protected
TNF–/– recipients against an otherwise
lethal infection. By contrast, infection with a higher dose of
Listeria (2,000 CFU) clearly demonstrated that solTNF is
required to coordinate an optimal protective inflammatory response.
memTNF mice were more susceptible to a high-dose infection, and they
exhibited delayed bacterial clearance, increased inflammation, and
necrosis in the liver that resulted in 55% mortality. The dysregulated
inflammation was accompanied by prolonged elevated expression of mRNAs
for several chemokines as well as the macrophage effector molecules
inducible nitric oxide synthase and LRG-47 in the livers of memTNF mice
but not in the livers of WT mice. These data demonstrated that memTNF
is sufficient for establishing protective immunity against a primary
low-dose Listeria infection but that solTNF is required for
optimal control of cellular inflammation and resistance to a primary
high-dose infection. By contrast, memTNF alone is sufficient for
resolution of a secondary, high-dose infection and for the transfer of
protective immunity with memory T
cells.
|
INTRODUCTION
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Listeria monocytogenes is a ubiquitous, facultative
intracellular coccobacillus that is associated with septic abortion in
pregnant women and disease in immunocompromised patients
(31). Immunity to
listerial infection is due to rapid activation of neutrophils and
macrophages (6,
18,
25), in addition to
Listeria-specific CD4+ and
CD8+ T cells, particularly those targeting the
pore-forming exotoxin listeriolysin O
(1,
11,
13,
23,
28). Tumor necrosis
factor (TNF), a pleiotropic cytokine that mediates a broad range of
proinflammatory activities, is expressed predominantly by activated
macrophages and T cells and has an essential protective role in
Listeria infection. TNF–/– mice, as
well as animals deficient in TNFRI, are highly susceptible to
listeriosis (24,
26). In contrast,
administration of recombinant TNF rescued animals from an otherwise
lethal dose of Listeria
(12,
19,
24,
26).
There
are two forms of TNF, transmembrane TNF (memTNF) and soluble
TNF (solTNF), which function physiologically by interacting with the
receptors TNFRI and TNFRII. TNF is initially synthesized as a
nonglycosylated, transmembrane protein, which exists as a homotrimer.
Membrane-bound TNF may be cleaved by the matrix metalloprotease, tumor
necrosis factor alpha-converting enzyme, which leads to the
release of soluble TNF
(16). The two TNF
receptors are expressed on a diverse range of cell types, but they have
distinct downstream effects
(22). Furthermore,
identification of a casein kinase I motif in the intracellular domain
of memTNF suggests that memTNF itself is also able to transduce signals
as a receptor (14,
36).
The relative
contributions of memTNF and solTNF to inflammation during infection or
autoimmune disease have been difficult to elucidate. The availability
of mice which express only memTNF on a
TNF–/– or
TNF–/–/lymphotoxin–/–
background permits analysis of this question. Mice which express memTNF
in the absence of solTNF
(27) or in the absence of
both solTNF and lymphotoxin
(20) were protected
against acute Mycobacterium tuberculosis infection, but they
succumbed to late progressive infection
(21,
29). Furthermore, in a
model of autoimmune encephalomyelitis, the disease progression in
memTNF mice was indistinguishable from the disease progression in
wild-type (WT) mice, whereas TNF–/– mice
showed delayed disease onset
(27).
In
this study, we used memTNF mice to determine the relative contributions
of soluble and memTNF to the control of primary and
secondary L. monocytogenes infections. memTNF mice cleared a
low-dose Listeria infection with kinetics similar to those of
WT mice, and they were fully protected against an otherwise lethal
secondary challenge. However, following primary infection with a higher
dose of Listeria, memTNF mice exhibited delayed clearance of
bacteria with slower T-cell accumulation and increased inflammation and
mortality, indicating that soluble TNF is required for optimal control
of a primary infection. In contrast, expression of memTNF alone by
immune T cells was sufficient to transfer immunity to Listeria
infection.
|
MATERIALS AND METHODS
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Animals.
C57BL/6 mice that were 6 to 10 weeks
old were purchased from the Animal Resource Centre (Perth, Australia).
TNF-deficient mice (TNF–/–) were generated
by targeted disruption of TNF in C57BL/6 mice as previously described
(15). memTNF mice, in
which the gene expressing memTNF was placed into
TNF–/– mice, were generated at DNAX Research
Institute, Palo Alto, CA
(27). All mice were kept
in specific-pathogen-free conditions at the Centenary Institute Animal
Facility. All experiments were undertaken with the approval of the
University of Sydney Animal Ethics
Committee.
Experimental infection.
L.
monocytogenes strain EGD was prepared as previously described
(24). WT,
memTNF, and TNF–/– mice were infected with
Listeria intravenously via the lateral tail vain. At specified
times, the numbers of viable bacteria in the spleen and liver were
determined by plating serial dilutions of organ homogenates on tryptic
soy agar (Difco, Detroit, MI) and incubating the preparations
overnight. Heat-killed L. monocytogenes was prepared by
incubating Listeria cells at 80°C for 2 h.
For transfer experiments, TNF–/– mice were
irradiated with 500 rads prior to infection. Groups of mice were
injected intravenously with 200 bacilli and 7.5 x
106 purified T cells. Bone marrow-derived macrophages were
cultured with 15% L929 supernatant for 6 days before overnight
prestimulation with combinations of 200 U/ml gamma interferon
(IFN-
) and 10 ng/ml of lipopolysaccharide. Macrophages were
infected for 1 h with Listeria at a multiplicity of
infection of 1, they were washed, and bacterial loads were determined
after 4 h of
incubation.
Cytokine production and phenotypic analysis of cellular infiltration.
Single-cell suspensions were prepared
from mouse livers perfused with phosphate-buffered saline (PBS)
containing heparin (10 U/ml; Sigma, St. Louis, MO) and from
splenocytes. Erythrocytes were lysed, and cells were suspended in RPMI
medium (Cytosystem, Sydney, Australia) containing 10% fetal calf serum
(Trace, Sydney, Australia), 2 mM L-glutamine, 0.5 µM
2-mercaptoethanol (Sigma), 100 U/ml penicillin (Trace), and 100
µg/ml streptomycin (CSL, Melbourne, Australia). Leukocytes were
incubated on ice with anti-CD16/CD32 monoclonal antibodies (MAbs) (BD
Pharmingen, San Diego, CA) and then stained with fluorescently labeled
MAbs. In addition, some leukocytes were cultured overnight on anti-CD3
MAb-coated plates (BD Pharmingen) to which brefeldin A (Sigma) was
added for the final 4 h; the surface markers were stained,
and the cells were permeabilized for intracellular staining. The
fluorescent MAbs used for phenotypic analysis with a FACSCalibur
(Becton Dickinson, San Jose, CA) were CD4-allophycocyanin,
CD8a-peridinin-chlorophyll protein (PerCP),CD8b.2-phycoerythrin, Gr-1-fluorescein isothiocyanate,
Mac-1-allophycocyanin, NK1.1-phycoerythrin, CD62L-phycoerythrin
(BD Pharmingen), IFN--fluorescein isothiocyanate, and
isotype controls (Caltag, Burlingame, CA). The percentage of apoptosis
was determined by annexin-V staining (BD Pharmingen), performed
according to the manufacturer's instructions. Splenocytes were cultured
with heat-killed L. monocytogenes or medium (control) for
72 h. IFN- production in the culture supernatant was
determined by a capture enzyme-linked immunosorbent assay, as
previously described
(29).
T-cell purification.
T cells were
enriched from single-cell spleen suspensions by magnetic cell sorting
with indirect microbeads (Miltenyi Biotec, Gladbach, Germany). Briefly,
cells were incubated with a combination of phycoerythrin-conjugated
anti-B220 and anti-major histocompatibility complex class II MAb,
followed by anti-phycoerythrin microbeads, before negative selection
using an autoMACs (Miltenyi Biotec). Purification was
confirmed by staining for the leukocyte markers CD3, CD4, CD8, Mac-1,
and B220, using fluorescently labeled antibodies (BD Pharmingen) and
analysis with a FACSCalibur. This procedure resulted in acquisition of
T-cell populations whose purity was greater than
93%.
RNA purification and RTQ-PCR.
Liver
tissue was homogenized in 1 ml RNAzol trireagent (Sigma) and
stored at –70°C. Extraction of total RNA, RNA
purification, reverse transcription, and real-time quantitative PCR
(RTQ-PCR) were performed as previously described
(29). Primers for all
target genes (Table
1) were designed using the Primer Express 1.5 software (Applied
Biosystems, Foster City, CA) and were made by Proligo (Sydney,
Australia). RTQ-PCR was performed with a PE Applied Biosystems model
7700 sequence detector. The identity and purity of the PCR product were
confirmed by melting curve analysis. All data were analyzed using the
PE Applied Systems Sequence Detector 1.7 software and were plotted as
the increase in fluorescence intensity of the SYBR green reporter dye
versus the cycle number. The threshold cycle number was used to
quantify the target gene expression for each sample, using the
comparative threshold cycle method. The results represented the
expression of the target gene relative to the expression in WT
uninfected mice.
Histology.
Liver tissue samples were perfused
and fixed in 10% neutral buffered formalin (Fronine, Sydney, Australia)
and embedded in paraffin blocks, and 5-µm sections were cut.
The sections were stained with hematoxylin and eosin for
histopathological
examination.
Immunofluorescence.
Perfused liver tissue samples were
snap frozen in optimal cutting temperature compound
(Tissue-Tek, Sakura, Japan). Then 5-µm sections of the tissue
were cut with a Cryostat (Microm, Walldorf, Germany),
adhered to poly-L-lysine-coated (Sigma) slides, and fixed in
acetone (BDH, Melbourne, Australia) for 10 min. The samples were
rehydrated in PBS and then kept in a humidity chamber at 37°C,
with two PBS washes between steps. Samples were blocked with 30% horse
serum for 10 min before polyclonal rabbit anti-mouse inducible nitric
oxide synthase (iNOS) (Upstate Biotech, Lake Placid, NY) was added for
30 min. Anti-rabbit immunoglobulin G-fluorescein isothiocyanate
(Silenus, Melbourne, Australia) was added for 30 min, and then antifade
mountant [0.3% 1,4-diazabicyclo(2,2,2)octan (Merck, Darmstadt,
Germany), 90% glycerol (BDH)] was
applied.
Statistical analysis.
Statistical analyses of the results
of immunological assays and log-transformed bacterial counts were
conducted using analysis of variance (ANOVA) or Student's t
test. Fisher's least protected significance difference post hoc test
was used for pairwise comparison of multigroup data sets. Survival was
calculated with a Kaplan-Meier nonparametric survival plot, and
significance was assessed by the log rank Mantel-Cox test. A P
value of <0.05 was considered
significant.
|
RESULTS
|
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Delayed bacterial clearance and increased mortality in memTNF mice following high-dose, but not low-dose, primary Listeria infection.
In order to
determine if transmembrane TNF alone is sufficient to induce protective
immunity against a primary Listeria infection, WT,
TNF–/–, and memTNF mice were infected
intravenously with a high dose of L. monocytogenes (2,000
CFU). The bacterial loads increased initially in the WT mice, peaking
at days 3 to 5, before rapid clearance by day 10 (Fig.
1A and
B).
In contrast, TNF–/– mice were unable to
control the growth of Listeria; these mice became moribund
(mean survival time, 4.5 days; P < 0.0001) (Fig.
1C), and the
bacterial loads were 2 and 4 log10 higher in the
spleen and liver, respectively, by day 5 (P
< 0.0001) (Fig. 1A and
B). In memTNF mice, the bacterial growth initially
resembled that in their WT counterparts. From day 4, however, the
memTNF mice diverged into two phenotypes: those that were able to
control and clear the Listeria infection, albeit with a
significant delay (P < 0.02) compared to WT mice, and
those that developed an overwhelming infection and became moribund
between 4 and 8 days postinfection. This resulted in an overall
mortality rate of 55% (P < 0.009) (Fig.
1C). The listerial burdens
in moribund mice (7.5 log10 in the spleen and 8.5
log10 in the liver) were markedly increased and similar to
those in TNF–/– mice (7.5 log10
and 9.0 log10, respectively). When mice were infected with a
10-fold-lower dose of Listeria (200 CFU), WT and
TNF–/– mice responded with patterns similar
to those observed with the higher dose. WT mice cleared the infection,
while TNF–/– mice became moribund (Fig.
1D). The majority of
memTNF mice also survived a low-dose infection (Fig.
1D) and cleared the
bacilli without the delay seen in memTNF mice given 2,000 CFU (data not
shown).
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FIG. 1. Delayed
bacterial clearance and increased susceptibility of memTNF mice to
Listeria infection. WT ( ), memTNF ( ), and
TNF–/– ( ) mice were infected with
2,000 (A to C) or 200 (D) CFU of Listeria
intravenously. The bacterial loads in the spleen (A) and
liver (B) were determined by serial dilution of organ
homogenates at different times. The dotted line indicates the limit of
detection. The data are the means and standard errors for five mice per
group from one of three representative experiments. Significance was
determined by ANOVA. An asterisk indicates that the P value
was <0.02 for a comparison of memTNF and WT mice, and a dagger
indicates that the P value was <0.0001 for a
comparison of TNF–/– and WT mice. Additional
groups of mice were monitored twice daily and euthanized if they
displayed signs of declining health. (C) Time to euthanasia
in three experiments following infection of WT (n =
19), memTNF (n = 19), and TNF–/– (n = 11) mice with 2,000 CFU of Listeria. (D) Time to euthanasia in two
experiments following infection of WT (n = 21), memTNF
(n = 29), and TNF–/– (n = 19) mice with
200 CFU of Listeria. Significance was determined by the log
rank Mantel-Cox test. A double dagger indicates that the P
value was <0.009 for a comparison of memTNF and WT mice; a
section sign indicates that the P value was
<0.0001 for a comparison of TNF–/–
and WT mice; and a paragraph sign indicates that the P value
was 0.0001 for a comparison of memTNF and
TNF–/– mice. ns, not
significant.
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Delayed accumulation of leukocytes in memTNF mice during primary Listeria infection.
The primary site of Listeria
infection is the liver. To determine if the increased susceptibility of
the memTNF mice to a high-dose infection was due to alterations in the
inflammatory response, the influx of leukocytes into the liver was
measured throughout the course of infection. The numbers of macrophages
and neutrophils increased in all groups by day 3 and peaked in WT and
memTNF mice at day 7 (Fig.
2). In WT mice, despite the early development of small inflammatory foci
(described below), the total numbers of leukocytes did not increase
significantly until day 5, when they rapidly expanded,
reaching a peak at day 7 before decreasing (Fig.
2). Leukocyte recruitment
to the livers of memTNF mice, however, was delayed (P
< 0.04 at day 7), and the population did not reach the maximal
size until day 14, 1 week after the peak of infection. These
differences were largely due to a delay in the recruitment of T cells
into the liver. In WT mice, the number of CD4+ T
cells rose sharply after day 5 and remained elevated from day 7 to day
28, while the number of CD8+ T cells increased
greatly beginning on day 5, peaked at day 10, and fell to preinfection
levels by day 14. In contrast, the maximal increases in the numbers of
CD4+ and CD8+ T cells in memTNF
mice did not occur until days 10 to 14 (Fig.
2 and
3).
At day 7, significantly fewer T cells were isolated from the livers of
memTNF mice than from the livers of WT mice (for
CD4+ T cells, P < 0.002; for
CD8+ T cells, P < 0.03). Despite
the differences in the total numbers of T cells isolated from the
liver, the percentages of T cells that were positive for the apoptotic
marker annexin-V were not significantly different between the WT and
memTNF mice (Table
2).
Delayed T-cell responses in memTNF mice during listerial infection.
Both CD4+ and
CD8+ T cells contribute to protective antilisterial
immunity. The numbers of CD4+ and
CD8+ T cells in the spleens and livers of infected
WT mice increased rapidly beginning on day 5. These cells displayed the
activated phenotypes CD62Llo (Fig.
3A) and CD44hi
(data not shown). Elevated numbers of activated CD4+
T cells were still detectable in the liver at day 28, but the numbers
had declined to preinfection levels in the spleen by day 14. The
recruitment of activated CD4+ T cells to the livers
of memTNF mice was delayed to day 10 (P < 0.02) and
then followed the pattern of the WT response. CD4+
T-cell activation was significantly delayed in the spleens of memTNF
mice (at day 7, P < 0.003). Furthermore, the numbers
of activated CD8+ T cells, which are required for
protection against Listeria infection, peaked at day 10 in the
livers of both memTNF and WT mice and then declined to preinfection
levels in WT mice but remained elevated in memTNF mice at 28 days
postinfection. In the spleen, the numbers of activated
CD8+ T cells peaked between days 7 and 10 in both WT
and memTNF mice.
IFN- production is the hallmark of an
activated Th1 response. In the spleens of both WT and memTNF mice, the
peak number of IFN--producing T cells occurred by day 7 (Fig.
3B), although beginning on
day 5, cultures of spleen cells, which contained similar numbers of T
cells, from the two strains of mice produced similar levels of
IFN- in response to heat-killed L. monocytogenes
(Fig. 3C). Examination of
the livers showed that while in WT mice the number of
IFN--secreting CD4+ T cells had risen
beginning on day 5 and remained elevated, in memTNF mice the expansion
of this population was delayed to day 7 postinfection (P
< 0.0004 at day 7). Infiltration of IFN--secreting
CD8+ T cells was comparable in the livers and
spleens of WT and memTNF
mice.
Enhanced and prolonged macrophage activation in memTNF mice.
Activation of macrophage antibacterial
functions is a key requirement for successful elimination of the
invading pathogen. It has previously been demonstrated that the enzymes
iNOS and LRG-47 are crucial for the development of protective immunity
against Listeria
(4,
17). The mRNA expression
in the livers of WT and memTNF Listeria-infected mice revealed
that the levels of both iNOS and LRG-47 increased in WT and memTNF mice
by 3 days postinfection and remained elevated in memTNF mice but not in
WT mice at day 7 (Fig.
4). Staining for iNOS expression in the liver demonstrated that in WT mouse
livers, iNOS expression was confined to a few discrete foci at day 3
and that there was slight enlargement by day 7 (Fig.
5A and
B).
However, iNOS was present throughout several larger lesions in memTNF
mice at day 3 and remained prevalent in larger regions of tissue at day
7 (Fig. 5C and D). In
vitro killing assays demonstrated that IFN--stimulated bone
marrow-derived macrophages from memTNF mice were as effective as WT
macrophages in killing Listeria (for WT mouse macrophages,
36.3% killing compared to unstimulated macrophages; for memTNF mouse
macrophages, 35.0% killing compared to unstimulated
macrophages).
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FIG. 4. Enhanced
macrophage activation in livers of infected memTNF mice. WT (black
bars) and memTNF (gray bars) mice were infected with 2,000 CFU of
Listeria intravenously. mRNA expression in the livers from
uninfected mice and infected mice was measured by RTQ-PCR to determine
the relative (rel.) expression of LRG-47 and iNOS. The data are the
means and standard errors for five mice per group from one of two
representative experiments. Significance was determined by ANOVA. An
asterisk indicates that the P value was <0.05 for a
comparison of memTNF and WT
mice.
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FIG. 5. Increased
inflammation, necrosis, and iNOS expression in memTNF mice. WT, memTNF,
and TNF–/– mice were infected with 2,000 CFU
of L. monocytogenes intravenously. The photographs show
typical sections from one of five mice in two representative
experiments. (A to D) Perfused liver sections were frozen in optimal
cutting temperature compound, and 5-µm sections were stained
for iNOS expression and examined at a magnification of x200. (A
and B) WT livers on day 3 (A) and day 7 (B). (C and D) memTNF
livers on day 3 (C) and day 7 (D). (E to L) Perfused liver
sections were fixed in neutral buffered formalin, embedded in paraffin,
sectioned, and stained with hematoxylin and eosin. (E, G, I, and K)
Magnification, x25. (F and H) Magnification, x400. (J and L)
Magnification, x200. (E and F) WT mice 5 days postinfection.
(E) Inflammatory foci, scattered throughout the tissue, were mostly
discrete and localized and consisted of tightly apposed mononuclear
cells. Several necrotic lesions also were evident. (F) The necrotic
lesions mostly consisted of a central core of degraded tissue
surrounded by neutrophils and mononuclear cells. (G and H)
TNF–/– mice 5 days postinfection. (G)
Overwhelming hepatic destruction with intense inflammatory involvement
was observed throughout the preparation. Darkly staining neutrophils
appeared to radiate out from a central region of tissue degradation
through the remaining viable liver. (H) High-power view of the
interface (arrow in panel G) revealed disintegrated tissue, which was
surrounded by cellular debris and pyknotic matter. (I and J)
Nonmoribund memTNF mice 5 days postinfection. (I) Inflammatory foci
mostly consisted of mononuclear leukocytes and neutrophils, but they
were more frequent, larger, less organized, and less compact in memTNF
mice than in their WT counterparts. Most necrotic lesions comprised a
small area of purulent matter circumscribed by neutrophils and
mononuclear cells. Other necrotic lesions consisted of a central core
of degraded cells, predominantly neutrophils, surrounded by a region of
disintegrated tissue, which was in turn encircled by inflammatory
cells. (J) Enlargement (arrow in panel I) showing a central core of
dying inflammatory cells and hepatocytes surrounded by completely
necrotized tissue and a thin outer layer of leukocytes. (K and L)
Moribund memTNF mice 5 days postinfection. (K) Rampant cellular
infiltration and hepatic destruction. Darkly staining collections of
cells, scattered throughout the tissue, were composed primarily of
apoptotic bodies, neutrophils, and necrotic matter. (L) Enlargement of
a section (arrow in panel K) showing areas of complete tissue
destruction apparent throughout the
preparation.
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Increased cellular inflammation and necrosis in livers of memTNF mice.
The livers of
Listeria-infected WT mice contained small discrete
inflammatory foci, in addition to the occasional (<one
lesion/section) small necrotic lesion at day 5 (Fig.
5E). The foci were
predominantly composed of tight aggregates of macrophages and
lymphocytes, with the occasional necrotic lesion containing primarily
neutrophils, circumscribing the area of necrosis (Fig.
5F). The number and size
of inflammatory foci were greatest at day 5, and the lesions
progressively cleared throughout the course of infection (data not
shown). By day 14, only very occasional small cellular foci were still
evident, and the rest of the liver appeared normal. In contrast, in the
livers of infected TNF–/– mice there were
large areas of cellular infiltration and extensive tissue destruction,
and neutrophils were the dominant cell type (Fig.
5G and H). Analysis at a
higher magnification revealed that the cells at the circumference of
necrosis were heavily infected with coccobacilli (data not
shown).
In contrast to the distinct pathological phenotypes of
infected WT and TNF–/– mice, memTNF mice
infected with 2,000 CFU of Listeria exhibited a spectrum of
pathologies, particularly at day 5. Mice that appeared to be healthy
exhibited an immunological response similar to that of the WT mice but
showed increased pathology. The livers contained increased numbers of
small compact foci per section, along with more frequent larger
necrotic lesions (Fig. 5I and
J). In addition, mice that appeared to be physically
moribund (with ruffled fur, reduced mobility, and weight loss) showed
liver pathology similar to that of the
TNF–/– mice (Fig.
5K and L). The memTNF mice
that survived the early infection proceeded to clear the bacilli, and
there was a progressive reduction in inflammatory lesions. Overall,
clearance of the inflammatory foci was delayed in the memTNF mice
compared with the WT mice, and larger numbers of inflammatory foci were
still evident in memTNF mouse livers at 14 days postinfection (data not
shown).
memTNF mice that received a low dose of Listeria
also showed increased inflammatory responses in the liver, even though
there was no delay in the clearance of bacteria. The inflammatory
response in WT mice peaked at 5 to 7 days postinfection and was
resolved by day 10. In TNF–/– mice,
extensive necrosis occurred in low-dose infections, as well as in
high-dose infections (data not shown). The inflammatory response in the
memTNF mice resembled that in the livers of memTNF mice that survived a
high-dose infection. The inflammatory foci were discrete and localized
but more extensive than those in WT mice. Moreover, the inflammatory
response resolved more slowly in the memTNF mice than in the WT mice,
and there were increased numbers of inflammatory foci in the liver at
10 days postinfection (data not
shown).
Enhanced and prolonged expression of chemokine mRNA in memTNF mice.
In order to determine whether the
differences in accumulation of leukocytes observed in the
Listeria-infected livers were due to altered chemokine
expression, mRNAs for the monocyte/lymphocyte-attracting chemokines
CCL3 and CCL4 and the neutrophil attractant CXCL1 were examined. Early
neutrophil recruitment is essential for optimal protective immunity
against Listeria infection
(5), so initially we
examined the response in the liver after only 24 h of
infection. At this time, WT mice expressed 3-fold more CXCL1 mRNA than
memTNF mice expressed (for WT mice, 15.56-fold increase compared with
uninfected mice [range, 7.9- to 19.3-fold]; for memTNF mice, 5.48-fold
increase [range, 4.3- to 6.3-fold]), although there was no early
increase in CCL3 expression (for WT mice, 0.51-fold increase; for
memTNF mice, 1.3-fold increase). We also examined expression of the
chemokines CXCL1, CCL3, and CCL4 over the course of the listerial
infection (Fig.
6). Expression of these three chemokines was elevated in all three strains
of mice at day 3 and returned to the basal levels by day 7
in only the WT mice. In the memTNF mice, mRNA expression
remained elevated at day 7 and did not return to the basal
levels until day 14.
T cells from immune memTNF mice protect against secondary Listeria infection.
To determine
whether memTNF is sufficient for the development and expression of
memory responses, WT and memTNF mice were infected with 200 CFU of
Listeria and 8 weeks later challenged with 105
Listeria CFU/mouse. This dose was lethal for both WT and
memTNF naïve mice, which had highly elevated bacterial loads at
day 3 (Fig.
7) and succumbed to infection between days 2 and 4 (Fig.
8A). Both WT and memTNF mice which had previously cleared a low-dose
infection controlled this otherwise lethal infection; there were
4-log10-fewer bacteria the liver and spleen at day 3 than in
naïve mice (Fig. 7),
and the level of survival was 100% (Fig.
8A). Furthermore, we
examined the rate of proliferation of immune T cells from both WT and
memTNF mice following restimulation. T cells from immune mice were
purified, labeled with carboxyfluorescein diacetate succinimidyl ester,
and transferred into WT and memTNF naïve recipients, which were
then challenged with a lethal dose of Listeria. There was no
difference in the patterns of migration or rates of proliferation of
immune T cells from WT and memTNF mice (data not shown). At
72 h postinfection, we found that immune T cells from both
strains of mice had preferentially migrated to the liver, and
CD8+ T cells in the liver were the only cells that
underwent more than one round of division (data not
shown).
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|
FIG. 7. Transmembrane
TNF is sufficient to control bacterial growth following a secondary
challenge with a lethal dose of Listeria. WT (black bars) and
memTNF (gray bars) mice were infected with 200 CFU of Listeria
intravenously. Eight weeks after the initial infection, naïve and
immune mice were infected with 105 CFU of Listeria.
The bacterial loads in the spleen (A) and liver (B)
were determined at 3 and 5 days postinfection. The dotted line
indicates the limit of detection. The data are the means and standard
errors for five mice per group from one of two representative
experiments.
|
|
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|
FIG. 8. Protection
against secondary Listeria infection does not require soluble
TNF. (A) WT and memTNF mice were infected with 200 CFU of
Listeria intravenously, and 8 weeks after the initial
infection, naïve and immune mice were infected with
105 CFU of Listeria. The data are the time to
euthanasia for between 8 and 10 mice/group. Significance was determined
by the log rank Mantel-Cox test. The asterisk indicates that the
P value was <0.001 for a comparison of immune and
naïve mice. (B) WT and memTNF mice were infected with
200 CFU of Listeria intravenously, and 14 days later,
single-spleen-cell suspensions were prepared from immune and
naïve mice, and T cells were isolated by negative selection
(CD3+, >90%). Two hundred colony-forming
units of Listeria and 5 x 106 purified T
cells were injected intravenously into
TNF–/– mice that had been irradiated
24 h previously with 500 rads. The data are the time to
euthanasia for 11 to 15 mice/group. Significance was determined by the
log rank Mantel-Cox test. The dagger indicates that the P
value was <0.002 for comparisons of WT immune and WT
naïve mice and memTNF immune and memTNF naïvemice.
|
|
T cells from immune memTNF mice transfer protection to TNF-deficient mice.
Finally, we examined the role of memTNF
expressed on T cells in conferring protection to
TNF–/– mice. T cells from naïve or
immune mice that had been infected with 200 CFU of Listeria 14
days previously were transferred into TNF–/–
recipients at the time of challenge with 200 CFU of Listeria.
TNF–/– mice that received naïve T
cells from either WT or memTNF mice succumbed to infection after 4 to 5
days (Fig. 8B), whereas
over 70% of TNF–/– mice that received immune
T cells from either WT or memTNF mice survived infection. When these
mice were culled 28 days after infection, they had completely
eradicated the bacterial infection, their livers were normal, with no
discernible inflammation remaining, and they exhibited strong
antigen-specific IFN- responses (data not
shown).
|
DISCUSSION
|
|---|
This study
established the differential requirements for soluble and transmembrane
TNF for the control of primary and secondary Listeria
infections. TNF was required for optimal control of a primary
Listeria infection, acting principally through coordination of
the inflammatory response. Transmembrane TNF was sufficient to resolve
a low-dose infection, although there was increased hepatic
inflammation, but at a higher infective dose of L.
monocytogenes, a bimodal response to infection developed between
days 3 and 6. While the initial bacterial loads in WT and memTNF mice
were comparable, differences in cell recruitment, with more diffuse
cellular influx and increased necrosis in the livers of the memTNF
mice, were visible by day 3. About one-half of the memTNF mice
developed a rapidly progressive course of infection with florid hepatic
destruction (Fig. 5I and
K) and subsequent mortality. The surviving memTNF mice
exhibited delayed bacterial clearance (Fig.
1A and B) and a
dysregulated inflammatory response with delayed recruitment of T cells
and increased pathology. Indeed, the inflammation in the memTNF mice
was more severe than that in the WT mice at all stages of infection.
This inflammation resolved more slowly after clearance of the bacteria
in mice that survived a primary infection, indicating that soluble TNF
may contribute to restoration of the normal homeostatic mechanisms
after bacterial clearance.
This study is the first study to
demonstrate that protective immunity to Listeria infection can
be conferred by memTNF-expressing T lymphocytes. Thus, T-cell
expression of transmembrane TNF is sufficient to permit effective
T-cell migration and activation of infected macrophages with resolution
of infection.
TNF plays an important role in establishing
coordinated immune responses, in part through regulated recruitment of
inflammatory cells, stimulation of adhesion molecule expression on
endothelial cells, and induction of chemotaxis
(32,
33). Our data confirm
that neither soluble nor transmembrane TNF is essential for the
upregulation of chemokine expression, but the regulation of chemokine
mRNA is altered in the absence of soluble TNF. The initial expression
of the neutrophil-recruiting chemokine CXCL1 was higher in WT mice at
24 h postinfection than in memTNF mice. As neutrophils play a
major protective role in resistance to Listeria infection, the
capacity of WT mice to recruit neutrophils quickly to the site of
infection may be an important survival advantage. Furthermore, the
relative expression of both CXC and CC chemokines was elevated in the
liver 3 days postinfection, at the time of maximum listerial burden,
but returned to basal levels in WT mice by day 7, when maximal
recruitment of monocytes and granulocytes occurred. However, memTNF
mice had increased levels of CCL3, CCL4, and CXCL1 mRNAs in the liver,
which remained elevated at day 7 and did not return to basal levels
until day 14 (Fig. 6).
This increased chemokine expression in memTNF mice may have been a
manifestation of a perturbed feedback loop, in which chemokine
secretion was sustained until inflammatory stimuli were removed from
infected cells by recruited leukocytes. Indeed, there was delayed
listerial clearance in memTNF mice during a high-dose primary
infection, and at day 7 postinfection memTNF mice showed a 40- to
50-fold increase in mRNA expression for the lymphocyte- and
macrophage-recruiting chemokines CCL3 and CCL4. Overall, memTNF alone
was less efficient than the combination of soluble and transmembrane
TNF for initiating cell recruitment. Thus, while neither soluble nor
transmembrane TNF is essential for chemokine induction, these molecules
may instead be involved in establishing the multiple overlapping
chemokine gradients in the extracellular matrix that tightly regulate
the orientation of leukocytes
(33). While soluble TNF
can rapidly diffuse to reach target cells and stimulate production of
chemotactic gradients, transmembrane TNF relies on cell-cell contact,
which is unlikely to be as efficient on its own.
In
addition to recruiting leukocytes, TNF is required for organization of
the inflammatory infiltrate into discrete organized lesions
(32). Consistent with
previous reports (12),
most inflammatory foci in TNF–/– mice
consisted of groups of heavily infected hepatocytes with predominantly
neutrophilic involvement and rampant necrosis by day 4. memTNF mice
also exhibited greater necrosis and had larger, more frequent and
diffuse inflammatory lesions than WT mice, even though the total
leukocyte numbers from digested livers were similar for the two
strains. This may have been a reflection of the diffuse structure of
inflammatory lesions in memTNF mice. The increased number of
neutrophils, which was evident histologically in the lesions of memTNF
and TNF–/– mice, was not confirmed by the
cytometric analysis, but this may have been due to the death of
activated neutrophils sensitive to the mechanical stress of
purification. Indeed, in our flow cytometric analysis, forward/side
scatter gating on cellular infiltrate indicated that there was a higher
percentage of dead cells in memTNF and
TNF–/– mice than in their WT
counterparts.
In WT mice, a reduction in the bacterial load led
to contraction of the response with a decline in the numbers of
splenocytes and leukocytes in the liver. This homeostatic mechanism is
crucial for minimizing immunopathology. By contrast, in memTNF mice,
there was a delay in the clearance of CD4+ and
CD8+ T cells and in resolution of inflammatory
lesions in the liver. Depletion of CD4+ or
CD8+ T cells from TNF–/–
mice leads to reduced immunopathology and necrosis and to prolonged
survival following Mycobacterium avium or Mycobacterium
bovis BCG infection
(7,
37). This suggests that
soluble TNF has an immunoregulatory function that limits excessive
Th1-type inflammatory responses.
The differential requirements
for soluble TNF and transmembrane TNF may also be partially due to a
signaling bias for either of the two TNF receptors. memTNF is thought
to signal predominantly through the proinflammatory receptor
TNFRII and not through the proapoptotic signaling receptor
TNFRI (10).
Furthermore, memTNF can also function as a receptor. Studies
have demonstrated that macrophages become refractory to
lipopolysaccharide stimulation and unresponsive to proapoptotic signals
following reverse signaling through memTNF
(14). However, the
percentages of apoptotic T cells isolated from the liver during
infection did not differ significantly for the WT and memTNF mice. This
suggests that apoptotic signaling through TNFRI either was not reduced
in the memTNF mice or was adequately compensated for by other available
apoptotic pathways. Along with regulating inflammation, TNF also acts
at other stages in the response to intracellular infection to provide
optimal activation of macrophages
(22,
37). TNF, in synergy with
IFN-, induces macrophage activation with consequent increased
expression of iNOS and reactive nitrogen intermediates (Fig.
4)
(3,
8). These data demonstrate
that T-cell expression of memTNF alone is sufficient for T-cell
migration and subsequent macrophage activation. Furthermore, these data
demonstrate that macrophages do not need to express TNF to control an
otherwise lethal infection but instead can be adequately activated by
surface TNF expression on immune T cells alone.
Finally, the
differential requirements for soluble and transmembrane TNF provide a
potential explanation for the increase in infectious diseases,
especially tuberculosis, reported in individuals who receive anti-TNF
therapy. The recently introduced anti-TNF therapies, which have proven
to be highly successful in the treatment of chronic inflammatory
processes, including Crohn's disease, rheumatoid arthritis, and
psoriasis (30), have been
hampered by their interference with host cell immunity. The two
commonly used TNF antagonists are the monoclonal antibody infliximab
and the soluble TNFRII fusion protein etanercept
(30). Infliximab, which
has high affinity for both solTNF and memTNF, has been associated with
a higher frequency of infectious complications, such as tuberculosis
reactivation and listerial meningitis, than etanercept, which binds
predominantly to solTNF
(2,
9,
35). Indeed, this is
consistent with our findings that memTNF is sufficient to orchestrate
protective immunity against a low-dose Listeria infection but
cannot fully compensate for the lack of solTNF during a high-dose
infection. The development of inhibitors of soluble TNF which leave
functional memTNF may allow TNF inhibition of inflammatory disease
while sufficient protective immunity is maintained by memTNF
(34).
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by
the National Health and Medical Research Council of Australia and by
the New South Wales Department of Health through a research
infrastructure grant to the Centenary Institute of Cancer Medicine and
Cell Biology.
We thank Jenny Kingham and her staff for excellent
animal care and Nathan Field and Katie Hall for technical
assistance.
|
FOOTNOTES
|
|---|
* Corresponding
author. Mailing address: Centenary Institute of Cancer Medicine and
Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia. Phone:
61-2-9565-6114. Fax: 61-2-9565-6103. E-mail:
b.saunders{at}centenary.usyd.edu.au. 
Editor:
J. L. Flynn
|
REFERENCES
|
|---|
| 1. | Bouwer,
H. G., R. A. Barry, and D. J.
Hinrichs. 1997. Acquired immunity to an intracellular
pathogen: immunologic recognition of L. monocytogenes-infected
cells. Immunol. Rev.
158:137-146.[CrossRef][Medline] |
| 2. | Bowie,
V. L., K. A. Snella, A. S. Gopalachar,
and P. Bharadwaj. 2004. Listeria meningitis associated
with infliximab. Ann. Pharmacother.
38:58-61.[Abstract/Free Full Text] |
| 3. | Britton,
W. J., N. Meadows, D. A. Rathjen, D. R.
Roach, and H. Briscoe. 1998. A tumor necrosis factor
mimetic peptide activates a murine macrophage cell line to inhibit
mycobacterial growth in a nitric oxide-dependent fashion.Infect. Immun.
66:2122-2127.[Abstract/Free Full Text] |
| 4. | Collazo,
C. M., G. S. Yap, G. D. Sempowski,
K. C. Lusby, L. Tessarollo, G. F. Woude, A. Sher,
and G. A. Taylor. 2001. Inactivation of
LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes
with essential, pathogen-specific roles in resistance to infection.J. Exp. Med.
194:181-188.[Abstract/Free Full Text] |
| 5. | Czuprynski,
C. J., J. F. Brown, N. Maroushek, R. D.
Wagner, and H. Steinberg. 1994. Administration of
anti-granulocyte mAb RB6-8C5 impairs the resistance of mice to
Listeria monocytogenes infection. J.
Immunol.
152:1836-1846.[Abstract] |
| 6. | Czuprynski,
C. J., C. Theisen, and J. F. Brown.1996
. Treatment with the antigranulocyte monoclonal
antibody RB6-8C5 impairs resistance of mice to gastrointestinal
infection with Listeria monocytogenes. Infect.
Immun.
64:3946-3949.[Abstract] |
| 7. | Ehlers,
S., S. Kutsch, E. M. Ehlers, J. Benini, and K. Pfeffer.2000
. Lethal granuloma disintegration in
mycobacteria-infected TNFRp55–/– mice is
dependent on T cells and IL-12. J. Immunol.
165:483-492.[Abstract/Free Full Text] |
| 8. | Fang,
F. C. 2004. Antimicrobial reactive oxygen
and nitrogen species: concepts and controversies. Nat. Rev.
Microbiol.
2:820-832.[CrossRef][Medline] |
| 9. | Gardam,
M. A., E. C. Keystone, R. M. Menzies,
S. E. Skamene, R. Long, and D. C. Vinh.2003
. Anti-tumour necrosis factor agents and tuberculosis
risk: mechanisms of action and clinical management. Lancet
Infect. Dis.
3:148-155.[CrossRef][Medline] |
| 10. | Grell,
M., E. Douni, H. Wajant, M. Lohden, M. M. Clauss,
B. S. Georgopoulos, W. Lesslauer, G. Kollias, K. Pfizenmaier,
and P. Scheurich. 1995. The transmembrane form of
tumor necrosis factor is the prime activating ligand of the 80 kDa
tumor necrosis factor receptor. Cell
83:793-802.[CrossRef][Medline] |
| 11. | Harty,
J. T., and M. J. Bevan. 1995.
Specific immunity to Listeria monocytogenes in the
absence of IFN gamma. Immunity
3:109-117.[CrossRef][Medline] |
| 12. | Havell,
E. A. 1989. Evidence that tumor necrosis
factor has an important role in antibacterial resistance.J. Immunol.
143:2894-2899.[Abstract] |
| 13. | Kagi,
D., B. Ledermann, K. Burki, H. Hengartner, and R. M.
Zinkernagel. 1994. CD8+ T
cell-mediated protection against an intracellular bacterium by
perforin-dependent cytotoxicity. Eur. J.
Immunol.
24:3068-3072.[Medline] |
| 14. | Kirchner,
S., S. Boldt, W. Kolch, S. Haffner, S. Kazak, P. Janosch, E. Holler, R.
Andreesen, and G. Eissner. 2004. LPS resistance in
monocytic cells caused by reverse signaling through transmembrane TNF
(mTNF) is mediated by the MAPK/ERK pathway. J. Leukoc.
Biol.
75:324-331.[Abstract/Free Full Text] |
| 15. | Korner,
H., D. S. Riminton, D. H. Strickland, F.
A. Lemckert, J. D. Pollard, and J. D. Sedgwick.1997
. Critical points of tumor necrosis factor action in
central nervous system autoimmune inflammation defined by gene
targeting. J. Exp. Med.
186:1585-1590.[Abstract/Free Full Text] |
| 16. | Locksley,
R. M., N. Killeen, and M. J. Lenardo.2001
. The TNF and TNF receptor superfamilies: integrating
mammalian biology. Cell
104:487-501.[CrossRef][Medline] |
| 17. | MacMicking,
J. D., C. Nathan, G. Hom, N. Chartrain, D. S.
Fletcher, M. Trumbauer, K. Stevens, Q. W. Xie, K. Sokol, N.
Hutchinson, et al. 1995. Altered responses to
bacterial infection and endotoxic shock in mice lacking inducible
nitric oxide synthase. Cell
81:641-650.[CrossRef][Medline] |
| 18. | Mocci,
S., S. A. Dalrymple, R. Nishinakamura, and R. Murray.1997
. The cytokine stew and innate resistance to L.
monocytogenes. Immunol. Rev.
158:107-114.[CrossRef][Medline] |
| 19. | Nakane,
A., T. Minagawa, and K. Kato. 1988. Endogenous tumor
necrosis factor (cachectin) is essential to host resistance against
Listeria monocytogenes infection. Infect.
Immun.
56:2563-2569.[Abstract/Free Full Text] |
| 20. | Olleros,
M. L., R. Guler, N. Corazza, D. Vesin, H. P.
Eugster, G. Marchal, P. Chavarot, C. Mueller, and I. Garcia.2002
. Transmembrane TNF induces an efficient cell-mediated
immunity and resistance to Mycobacterium bovis bacillus
Calmette-Guerin infection in the absence of secreted TNF and
lymphotoxin-alpha. J. Immunol.
168:3394-3401.[Abstract/Free Full Text] |
| 21. | Olleros,
M. L., R. Guler, D. Vesin, R. Parapanov, G. Marchal, E.
Martinez-Soria, N. Corazza, J. C. Pache, C. Mueller, and I.
Garcia. 2005. Contribution of transmembrane tumor
necrosis factor to host defense against Mycobacterium bovis
bacillus Calmette-Guerin and Mycobacterium tuberculosis
infections. Am. J. Pathol.
166:1109-1120.[Abstract/Free Full Text] |
| 22. | Pfeffer,
K. 2003. Biological functions of tumor necrosis factor
cytokines and their receptors. Cytokine Growth Factor
Rev.
14:185-191.[CrossRef][Medline] |
| 23. | Portnoy,
D. A., P. S. Jacks, and D. J.
Hinrichs. 1988. Role of hemolysin for the
intracellular growth of Listeria monocytogenes. J. Exp.
Med.
167:1459-1471.[Abstract/Free Full Text] |
| 24. | Roach,
D. R., H. Briscoe, B. M. Saunders, and W.
J. Britton. 2005. Independent protective effects for
tumor necrosis factor and lymphotoxin alpha in the host response to
Listeria monocytogenes infection. Infect.
Immun.
73:4787-4792.[Abstract/Free Full Text] |
| 25. | Rogers,
H. W., and E. R. Unanue. 1993.
Neutrophils are involved in acute, nonspecific resistance to
Listeria monocytogenes in mice. Infect. Immun.
61:5090-5096.[Abstract/Free Full Text] |
| 26. | Rothe,
J., W. Lesslauer, H. Lotscher, Y. Lang, P. Koebel, F. Kontgen, A.
Althage, R. Zinkernagel, M. Steinmetz, and H. Bluethmann.1993
. Mice lacking the tumour necrosis factor receptor 1
are resistant to TNF-mediated toxicity but highly susceptible to
infection by Listeria monocytogenes. Nature
364:798-802.[CrossRef][Medline] |
| 27. | Ruuls,
S. R., R. M. Hoek, V. N. Ngo, T. McNeil,
L. A. Lucian, M. J. Janatpour, H. Korner, H.
Scheerens, E. M. Hessel, J. G. Cyster, L.
M. McEvoy, and J. D. Sedgwick. 2001.
Membrane-bound TNF supports secondary lymphoid organ structure but is
subservient to secreted TNF in driving autoimmune inflammation.Immunity
15:533-543.[CrossRef][Medline] |
| 28. | Sasaki,
T., M. Mieno, H. Udono, K. Yamaguchi, T. Usui, K. Hara, H. Shiku, and
E. Nakayama. 1990. Roles of CD4+
and CD8+ cells, and the effect of administration of
recombinant murine interferon gamma in listerial infection. J.
Exp. Med.
171:1141-1154.[Abstract/Free Full Text] |
| 29. | Saunders,
B. M., S. Tran, S. Ruuls, J. D. Sedgwick, H.
Briscoe, and W. J. Britton. 2005.
Transmembrane TNF is sufficient to initiate cell migration and
granuloma formation and provide acute, but not long-term, control of
Mycobacterium tuberculosis infection.J. Immunol.
174:4852-4859.[Abstract/Free Full Text] |
| 30. | Scallon,
B., A. Cai, N. Solowski, A. Rosenberg, X. Y. Song, D. Shealy,
and C. Wagner. 2002. Binding and functional
comparisons of two types of tumor necrosis factor antagonists.J. Pharmacol. Exp. Ther.
301:418-426.[Abstract/Free Full Text] |
| 31. | Schuchat,
A., B. Swaminathan, and C. V. Broome. 1991.
Epidemiology of human listeriosis. Clin. Microbiol. Rev.
4:169-183.[Abstract/Free Full Text] |
| 32. | Sedgwick,
J. D., D. S. Riminton, J. G. Cyster, and
H. Korner. 2000. Tumor necrosis factor: a
master-regulator of leukocyte movement. Immunol. Today
21:110-113.[CrossRef][Medline] |
| 33. | Springer,
T. A. 1994. Traffic signals for lymphocyte
recirculation and leukocyte emigration: the multistep paradigm.Cell
76:301-314.[CrossRef][Medline] |
| 34. | Steed,
P. M., M. G. Tansey, J. Zalevsky, E. A.
Zhukovsky, J. R. Desjarlais, D. E. Szymkowski, C.
Abbott, D. Carmichael, C. Chan, L. Cherry, P. Cheung, A. J.
Chirino, H. H. Chung, S. K. Doberstein, A. Eivazi,
A. V. Filikov, S. X. Gao, R. S. Hubert,
M. Hwang, L. Hyun, S. Kashi, A. Kim, E. Kim, J. Kung, S. P.
Martinez, U. S. Muchhal, D. H. Nguyen, C. O'Brien,
D. O'Keefe, K. Singer, O. Vafa, J. Vielmetter, S. C. Yoder,
and B. I. Dahiyat. 2003. Inactivation of TNF
signaling by rationally designed dominant-negative TNF variants.Science
301:1895-1898.[Abstract/Free Full Text] |
| 35. | Wallis,
R. S., M. S. Broder, J. Y. Wong,
M. E. Hanson, and D. O. Beenhouwer.2004
. Granulomatous infectious diseases associated with
tumor necrosis factor antagonists. Clin. Infect. Dis.
38:1261-1265.[CrossRef][Medline] |
| 36. | Watts,
A. D., N. H. Hunt, Y. Wanigasekara, G. Bloomfield,
D. Wallach, B. D. Roufogalis, and G. Chaudhri.1999
. A casein kinase I motif present in the cytoplasmic
domain of members of the tumour necrosis factor ligand family is
implicated in ‘reverse signalling.’ EMBO
J.
18:2119-2126.[CrossRef][Medline] |
| 37. | Zganiacz,
A., M. Santosuosso, J. Wang, T. Yang, L. Chen, M. Anzulovic, S.
Alexander, B. Gicquel, Y. Wan, J. Bramson, M. Inman, and Z. Xing.2004
. TNF-alpha is a critical negative regulator of type 1
immune activation during intracellular bacterial infection.J. Clin. Investig.
113:401-413.[CrossRef][Medline] |
Infection and Immunity, June 2006, p. 3180-3189, Vol. 74, No. 6
0019-9567/06/$08.00+0 doi:10.1128/IAI.02004-05
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Abstract
Introduction
