| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-T Cells Are Critical for Survival and Early Proinflammatory Cytokine Gene Expression During Murine Klebsiella Pneumonia1
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain were
intratracheally inoculated with Klebsiella pneumoniae.
T cell knockout mice displayed increased mortality at both early
and late time points. In contrast, mice specifically lacking only
ß-T cells were no more susceptible than wild-type mice. Pulmonary
bacterial clearance in -T cell knockout mice was unimpaired.
Interestingly, these mice displayed increased peripheral blood
dissemination. Rapid up-regulation of IFN- and TNF- gene
expression, critical during bacterial infections, was markedly impaired
in lung and liver tissue from -T cell-deficient mice 24 h
postinfection. The increased peripheral blood bacterial dissemination
correlated with impaired hepatic bacterial clearance following
pulmonary infection and increased hepatic injury as measured by plasma
aspartate aminotransferase activity. Combined, these data suggest that
mice lacking -T cells have an impaired ability to resolve
disseminated bacterial infections subsequent to the initial pulmonary
infection. These data indicate that -T cells comprise a critical
component of the acute inflammatory response toward extracellular
Gram-negative bacterial infections and are vital for the early
production of the proinflammatory cytokines IFN- and
TNF-. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An emerging field of interest is the interaction between cells of the
innate and acquired immune response during pathogenic insult
(7, 8, 9, 10). T cells expressing the ß-TCR complex
comprise the majority of peripheral T cells (
90%), whereas
-TCR-expressing cells are in the minority (10%). ß-T
cell-mediated immunity has been defined in recent years
according to the profile of cytokines produced and the corresponding
immune response generated (11, 12). Th1 cells produce
IL-2, IL-12, and IFN- but not IL-4, IL-5, or IL-10. Th1 responses
result in cell-mediated immunity such as delayed-type hypersensitivity
and macrophage activation. In contrast, Th2 T cells produce IL-4, IL-5,
and IL-10 but not IL-2, IL-12, or IFN-. The resultant immune
response promotes humoral immune responses. Interestingly, recent
studies have indicated that -T cell clones can also be segregated
into "Th1" or "Th2" classifications, with a bias toward
production of Th1 cytokines (13, 14).
Although ß-T cells have been shown to be important in a variety of
infection models (15, 16, 17, 18), less is known about the role of
-T cells during infection. The role of -T cells during
infection appears to vary depending on the pathogenic model studied. A
protective role for -T cells has been shown in several models,
particularly in the setting of intracellular pathogens such as
Toxoplasma (19) and Listeria
infection (20, 21). Conversely, mice deficient in -T
cells have increased resistance to i.p. infection with Salmonella
choleraesuis (22).
To study the role of specific T cell populations in Gram-negative
bacterial pneumonia, mice deleted of their TCR ß- and/or -chain by
homologous recombination were intratracheally inoculated with K.
pneumoniae. Mice specifically lacking -T cells have
increased susceptibility to pulmonary bacterial challenge when compared
with ß-T cell knockout and wild-type mice. -T cell knockout
mice have significantly impaired early expression of pulmonary and
hepatic IFN- and TNF- mRNA following K. pneumoniae
infection, increased peripheral blood bacterial dissemination, and
increased hepatic bacterial burden subsequent to the initial pulmonary
infection. These data detail a heretofore unrecognized critical role
for -T cells during acute, extracellular bacterial infections and
further support the concept that -T cells bridge host innate and
acquired immune responses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6J-Tcrb (ß-T cell deficient), C57BL/6J-Tcrd (-T
cell deficient), C57BL/6J-TcrbTcrd (ß/-T cell deficient),
and C57BL/6J wild-type mice were purchased from The Jackson Laboratory
(Bar Harbor, ME) and housed in specific pathogen-free conditions within
the animal care facility at the University of Michigan (Unit for
Laboratory Animal Medicine) until the day of sacrifice.
K. pneumoniae inoculation
K. pneumoniae strain 43816 serotype 2 (American Type Culture Collection, Manassas, VA) was grown in trypic soy broth (Difco, Detroit, MI) overnight at 37°C. Bacterial concentration was determined by measuring the amount of absorbance at 600 nm and compared with a predetermined standard curve. Bacteria were then diluted to the desired concentration for intratracheal inoculation. Mice were anesthetized with pentobarbital (diluted 1:7 in saline). The trachea was exposed, and 30 µl inoculum or saline was administered via a sterile 26-gauge needle. An aliquot of the inoculated K. pneumoniae suspension was serially diluted onto blood agar plates to determine actual dose of intratracheally injected bacteria.
Whole lung or liver homogenization for CFU and myeloperoxidase (MPO)3 analyses
At designated time points, the mice were euthanized by inhalation of CO2. The lungs or liver were perfused with 1–2 ml PBS/5 mM EDTA and removed for analyses as previously described (23). Briefly, organs were homogenized using a tissue homogenizer (Biospec Products, Bartlesville, OK) in 1 ml PBS/complete protease inhibitor mixture (Boehringer Mannheim Biochemical, Chicago, IL). For organ CFU determination, a small aliquot of tissue homogenate was serially diluted and plated on blood agar plates, incubated at 37°C, and colonies counted.
Lung MPO activity, as an indirect measurement of total neutrophil numbers, was quantitated by a method as described previously (23). Briefly, 100 µl lung homogenate was mixed with 100 µl MPO homogenization buffer (0.5% hexadecyltrimethylammonium bromide and 5 mM EDTA) and vortexed. The mixture was sonicated and centrifuged at 12,000 x g for 15 min. The supernatant was then mixed 1:15 with assay buffer and read at 490 nm. MPO units were calculated as the change in absorbance over time.
Peripheral blood CFU analyses
For determination of peripheral blood dissemination, heparinized blood was collected by cardiac puncture at the indicated time points. Serial dilutions were plated onto blood agar plates, incubated at 37°C, and colonies counted.
Total lung and liver leukocyte isolation
Total lung and liver leukocytes were isolated as previously described (24). Briefly, tissue was minced with scissors to a fine slurry in 15 ml/lung digestion buffer (RPMI 1640/5% FCS/1 mg/ml collagenase (Boehringer Mannheim Biochemical)/30 µg/ml DNase (Sigma, St. Louis, MO). Tissue slurries were enzymatically digested for 30 min at 37°C. Any undigested fragments were further dispersed by drawing the solution up and down through the bore of a 10-ml syringe. The total lung cell suspension was pelleted, resuspended, and spun (3000 rpm) through a 20% Percoll gradiant to enrich for leukocytes before further analysis. Liver leukocytes were prepared following the same procedure as for lung leukocytes with the following modification: cells were spun at a lower speed (1500 rpm) through a 35% Percoll gradient. Cell counts and viability were determined using trypan blue exclusion counting on a hemacytometer. Cytospin slides were prepared and stained with a modified Wright-Giemsa stain.
Multiparameter flow cytometric analyses
Total lung and liver leukocytes were isolated as described
above. For analyses of T cell subsets, isolated leukocytes were stained
with biotinylated anti--TCR or anti-ß-TCR and
anti-CD4-FITC plus anti-CD8-FITC. TCR expression was detected
by the addition of streptavidin-PE (all reagents from PharMingen, San
Diego, CA, unless otherwise noted). In addition, cells were stained
with anti-CD45-Tricolor (Caltag Laboratories, South San Francisco,
CA), allowing discrimination of leukocytes from nonleukocytes and thus
eliminating any nonspecific binding of T cell surface markers on
nonleukocytes. T cell subsets were analyzed by first gating
on CD45-positive "lymphocyte sized" leukocytes, then examined for
FL1 and FL2 fluorescence expression. Cells were collected on a FACScan
or FACScalibur cytometer (Becton Dickinson, San Jose, CA) using
CellQuest software (Becton Dickinson). Analyses of data were performed
using the CellQuest software package. Percent positive cells indicated
in histogram plots represent the percentage of positive cells
back-calculated to total leukocytes.
Isolation and RT-PCR amplification of whole lung mRNA
Whole lung or liver (2 lobes) was harvested at the indicated
time points, immediately "snap frozen" in liquid nitrogen, then
stored at -70°C for further analyses. Total cellular RNA from the
frozen tissue was isolated by homogenizing in 3 ml TRIzol Reagent (Life
Technologies, Gaithersburg, MD) following the TRIzol protocol. Total
RNA was determined by spectrometric analysis at 260 nm wavelength.
IFN-, TNF-, and ß-actin mRNA expression was determined by
RT-PCR using the Access RT-PCR system kit from Promega (Madison, WI)
following the manufacturer’s protocol. The following primer pairs (all
primers 5' 
3') were used for specific mRNA amplification: mouse (m)
IFN- sense, GGC TGT TTC TGG CTG TTA CTG CCA CG; mIFN- antisense,
GAC AAT CTC TTC CCC ACC CCG AAT CAG; mTNF- sense, CCT GTA GCC CAC
GTC GTA GC; mTNF- antisense, AGC AAT GAC TCC AAA GTA GAC C;
mß-actin sense, CTT CTA CAA TGA GCT GCG TGT G; mß-actin antisense,
GAT TCC ATA CCC AAG AAG GAA GG. cDNA products were detected on a 2%
agarose gel containing ethidium bromide and bands visualized and
photographed using UV transillumination.
Southern hybridization analyses
RT-PCR agarose gels were transferred in 0.4N NaOH onto Zetaprobe
membrane (Bio-Rad, Richmond, CA). The following antisense internal
probes specific for amplified cDNA products were used: mIFN-, GAG
ATA ATC TGG CTC TGC AGG; mTNF-, GCT CAG CCA CTC CAG CTG CTC C;
mß-actin, GCC TGG ATG GCT ACG TAC ATG GC. Southern filter
hybridization was performed by incubating membranes with
32P end-labeled oligonucleotide internal probes
diluted in hybridization buffer (6x SSC, 0.5% SDS, 5x Denhardts) for
2–3 h. Membranes were then washed twice with 2x SSC/0.1% SDS buffer
followed by two washes with 0.1x SSC/0.1% SDS buffer. Membranes were
exposed to autoradiographic film and developed after adequate exposure
time. A digital picture of each autoradiograph was taken and band
intensities analyzed using NIH Image public domain software (developed
at the Research Services Branch of the National Institute of Mental
Health; available for download at http://rsb.info.nih.gov/nih-image).
Specific IFN- and TNF- band intensities were normalized to
ß-actin to account for differences in total RNA loading in each
sample.
Plasma aspartate aminotransferase (AST) analyses
Plasma levels of AST, as an indication of hepatic cellular injury, was determined on plasma samples collected 2 days post K. pneumoniae inoculation. AST activity was quantitated by the Clinical Chemistry Laboratory at the University of Michigan Medical Center using an automated spectrophotometric assay.
Statistical analyses
Statistical significance was determined using the unpaired, two-tailed Alternate Welsh t test and nonparametric Mann-Whitney test. Calculations were performed using Instat for Macintosh (GraphPad Software, San Diego, CA). Statistical analyses of survival curves were performed by the logrank test using the Prism software program (GraphPad Software).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-T cells are more susceptible to
K. pneumoniae-induced mortality
The importance of T cells during acute Gram-negative bacterial
pneumonia was examined using mice completely lacking all subsets of
mature T cells as a result of TCR ß- and -chain deletions.
Intratracheal inoculation of 5 x 103K. pneumoniae into C57BL/6 control mice induced mortality within
3–4 days postinfection and resulted in an overall mortality rate of
40–50% by day 10. Interestingly, C57BL/6J-TcrbTcrd (ß/-T
cell-deficient) mice inoculated with the same dose of K.
pneumoniae displayed increased mortality at both early and late
time points (Fig. 1A
, p
< 0.05), indicating a protective role for T cells from
Klebsiella-induced mortality.
|
ß-T cells or -T
cells was responsible for this observed increase in mortality,
C57BL/6J-Tcrb (ß-T cell knockout) and C57BL/6J-Tcrd (-T cell
knockout) were infected with K. pneumoniae. Interestingly,
the increased sensitivity of T cell knockout mice was due to the
absence of -T cells rather than ß-T cells (Fig. 1
B, p < .05). In contrast, ß-T cell
knockout mice were no more susceptible to K. pneumoniae
induced mortality than their wild-type control littermates.
-T cells represent a small subset of total pulmonary
leukocytes
-T cells have been shown to constitute a minority subset of
the total T cell population in most peripheral lymphoid organs;
however, less is known about -T cell distribution within the
lung. To determine the frequency of -T cells, total lung
leukocytes were harvested from C57BL/6J wild-type mice by enzymatic
dissociation and subjected to multiparameter flow cytometric analyses.
Fewer than 1% of lung leukocytes expressed the -TCR (Fig. 2). Essentially all of these pulmonary
-T cells lacked CD4 and CD8 expression (data not shown). In
contrast, ß-T cells represent 15–20% of total lung leukocytes,
with >90% of these expressing CD4 or CD8 (data not shown).
Interestingly, -T cells (percentage and total) were increased in
lungs of ß-T cell knockout mice, representing 4–5% of total lung
leukocytes. As expected, these animals were devoid of any
ß-TCR-expressing cells.
|
-T cell knockout mice have unimpaired pulmonary bacterial
clearance but display elevated peripheral blood dissemination
The most plausible explanation for the increased mortality of
-T cell knockout mice following intratracheal inoculation of
K. pneumoniae would be impaired clearance of bacteria from
the pulmonary airspace. To address this, lung bacterial burden in
-T cell knockout and ß-T cell knockout mice was examined 1
and 2 days postintratracheal infection. Bacterial counts increased to a
similar degree in lungs of ß-T cell knockout, -T cell
knockout, and wild-type mice by 1-day postinoculation (data not shown).
Pulmonary bacterial numbers continued to increase in all three groups
of animals by day 2, but again with no significant differences between
the T cell subset-deficient animals and wild-type mice (Fig. 3A). Furthermore, neutrophil
recruitment, as measured by lung MPO activity, was similar between
-T cell knockout, ß-T cell knockout, and wild-type mice at
these time points, indicating that pulmonary neutrophil influx could
occur in the absence of -T cells or ß-T cells (data not
shown).
|
-T cell knockout mice when
compared with mice lacking ß-T cells by 2 days postinoculation.
(Fig. 3B). Although not quite reaching the level of
statistical significance, the trend in all individual experiments was
for -T cell knockout mice to have elevated blood bacterial counts
when compared with B6 wild-type mice.
-T cell knockout mice have impaired early expression of
IFN- and TNF- mRNA in lung following K. pneumoniae
infection
IFN- and TNF- have been shown to be critically important in
resolution of pulmonary bacterial infections. To determine whether
impaired localized expression of these two cytokines could contribute
to the increased peripheral blood bacterial burden seen in -T
cell knockout mice, IFN- and TNF- mRNA expression in lung and
liver was examined by RT-PCR. IFN- mRNA was rapidly induced in the
lungs of both B6 wild-type mice and ß-T cell knockout mice within
1 day of infection. Most interestingly, -T cell knockout mice had
a 5-fold reduction in IFN- mRNA when compared with B6 mice and a
7-fold reduction vs ß-T cell knockout mice (Fig. 4, A and C). This
impaired IFN- production by -T cell knockout mice was
transient; by day 2, IFN- mRNA levels had increased to that seen in
infected B6 control mice. TNF- mRNA production was similarly
impaired in -T cell knockout mice 1 day postinfection, albeit
less dramatically than seen with IFN- (2-fold reduction vs B6;
3-fold vs ß-T cell knockout mice; Fig. 4, B and
C). As with IFN-, this reduction in TNF- was
restricted to the first day of infection; by day 2, levels had
increased to that seen in control mice. Mice lacking ß-T cells
were unimpaired in their production of pulmonary IFN- and TNF-
mRNA 1 day postinfection. By day 2, these mice expressed increased
cytokine message when compared with wild-type mice.
|
T cell knockout mice have increased hepatic cellular injury
following K. pneumoniae infection
Data thus far indicate that -T cell knockout mice had
increased peripheral blood bacterial dissemination in conjunction with
impaired early expression of IFN- and TNF-. To determine whether
this increased blood bacterial burden may lead to increased hepatic
injury, plasma AST levels were measured as an indication of hepatic
cellular injury. All three groups of mice, -T cell knockout,
ß-T cell knockout, and wild-type mice had increased plasma AST
levels 2 days postintratracheal inoculation of bacteria when compared
with saline-injected control mice (p < 0.05).
Of interest, -T cell knockout mice had significantly elevated AST
levels when compared with ß-T cell knockout mice (Fig. 5, p < 0.05).
|
T cell knockout mice display elevated liver bacterial burden
following intratracheal inoculation with K. pneumoniae
The increased peripheral blood bacterial burden seen in -T
cell knockout mice suggested an impaired ability of these mice to
resolve the disseminated bacterial infection. As blood borne bacteria
are predominantly cleared within the liver (25, 26, 27), we
analyzed total liver bacterial burden 2 days following intratracheal
inoculation of K. pneumoniae. Total liver bacterial burden
was significantly increased in -T cell knockout mice when
compared with ß-T cell knockout mice (Fig. 6, p <
0.01).
|
-T cell knockout mice have impaired early expression of
IFN- and TNF- mRNA in liver following K.
pneumoniae infection
The increased hepatic bacterial burden seen in -T cell
knockout mice suggests an impaired liver response to Gram-negative
bacteria. We examined hepatic expression of IFN- and TNF- mRNA to
determine whether production was impaired. Like the lung, liver IFN-
and TNF- mRNA levels 24 h post infection were markedly impaired
in -T cell knockout mice when compared with both ß-T cell
knockout and wild-type mice (Fig. 7).
Interestingly, reduction in TNF- mRNA was more pronounced in liver
than in lung whereas the reduction in IFN- mRNA was similar in both
organs. As with the lung, impaired liver TNF- and IFN- mRNA
expression was transient, with cytokine mRNA levels returning to that
seen in ß-T cell knockout and wild-type mice by day 2 (data not
shown).
|
T cells represent a small subset of total liver leukocytes
T cell knockout mice appear to have an impaired ability to
resolve K. pneumoniae infection once dissemination beyond
the primary pulmonary site of infection has occurred. To determine the
frequency of -T cells in the liver, cells were isolated and
examined for TCR expression. Similar to the lung, -T cells
represented a small fraction of total liver leukocytes in C57BL/6 mice.
Liver T cell numbers were increased in ß-T cell
knockout mice, although not as dramatically as seen in lung leukocytes
(Fig. 8).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain of the TCR complex. Mice lacking both T cell subsets were
found to exhibit increased mortality at both early and late time points
following intratracheal inoculation of K. pneumoniae. Of
interest, this enhanced susceptibility was exclusively due to the
specific absence of the minority -T cells rather than the
majority ß-T cell population. The absence of -T cells
resulted in increased peripheral blood bacterial dissemination 2 days
postinfection, which correlated with increased hepatic bacterial burden
and cellular injury. Rapid expression of the proinflammatory cytokines
IFN- and TNF- has been shown to be critical for an effective host
inflammatory response in several bacterial pneumonia models (6, 28, 29). We observed that mice lacking -T cells had
pronounced defects in IFN- and TNF- mRNA expression 1 day
postinfection, particularly in the liver. Combined, these data indicate
that the absence of -T cells results in decreased pulmonary and
hepatic IFN- and TNF- production immediately following
intratracheal inoculation of K. pneumoniae. This decreased
proinflammatory cytokine production likely contributes to increased
blood and liver bacterial burden, resulting in increased liver
damage.
Our data clearly indicate a critical role for -T cells in
effective host responses to Gram-negative bacterial pneumonia. The
profound effects seen in the absence of a cell population comprising
<1% of lung or liver leukocytes are intriguing. Production of the
proinflammatory cytokines TNF- and IFN-, known to be critical
during bacterial pneumonia, was severely impaired immediately following
infection in mice lacking -T cells. The absolute number of lung
-T cells remained constant during the first 3 days of infection
(data not shown), suggesting that resident rather than recruited
-T cells are necessary for early TNF- and IFN- mRNA
expression. However, it is worth noting that there was not an absolute
absence of either cytokine within the first 24 h following
infection, and, by day 2 postinfection, cytokine production was normal.
This suggests that cells other than -T cells, such as NK cells or
ß-T cells, likely produce these cytokines albeit with slower
kinetics. This is supported by the recent observation that -T
cells responded more strongly than ß-T cells to systemic bacterial
infections or LPS stimulation (30).
The finding of increased Klebsiella-induced mortality in
-T cell knockout mice in the absence of increased lung bacterial
burden was unexpected. In addition, pulmonary neutrophil recruitment
was unimpaired in -T cell knockout mice. These findings are in
contrast to a recent study of nocardial pneumonia in -T cell
knockout mice, where it was reported that these animals had unimpeded
bacterial growth that correlated with a paucity of inflammatory
neutrophil recruitment (31). The observed lack of
differences in pulmonary bacterial clearance is particularly surprising
given the delay in TNF- and IFN- mRNA expression in the lung.
These two cytokines in particular have been shown to be critical for
effective lung anti-bacterial host defenses (6, 28, 29). Combined, these data suggest that -T cell knockout
mice are not succumbing to overwhelming pulmonary bacterial
infection.
Despite similar pulmonary bacterial burdens in -T and ß-T
cell knockout mice, a significantly greater bacterial burden was
observed in the blood of mice lacking -T cells. There are several
possible explanations for this increase in blood bacterial burden in
the absence of elevated pulmonary bacterial counts. One possibility is
increased pulmonary architectural damage in -T cell knockout mice
following bacterial infection leading to increased bacterial
"leakage" into the peripheral blood. However, histological
examination of lung sections obtained 1 or 2 days postinfection
revealed no overt differences between -T cell knockout mice and
wild-type mice or ß-T cell knockout mice (data not shown). A
second possibility we favor is that bacterial seeding of the blood
occurs at a similar rate in both -T cell knockout and ß-T
cell knockout mice. However, mice lacking -T cells may have an
impaired ability to clear bacteria from the blood stream, resulting in
increased bacterial burden and mortality.
The observation of increased liver bacterial numbers and cellular
injury supports the hypothesis that mice lacking -T cells have an
altered or impaired response to Gram-negative bacteremia, resulting in
increased bacterial growth in liver and/or blood. It is interesting to
note that early induction of hepatic proinflammatory cytokines in
response to blood-borne bacteria is markedly impaired in -T cell
deficient mice, further supporting an impaired host inflammatory
response. Studies examining the role of proinflammatory cytokines, in
particular TNF-, in sepsis or endotoxemia models indicate that a
dampened proinflammatory response results in less end-organ injury,
including hepatic injury (32, 33, 34). However, these models
generally use a bolus injection of bacteria or LPS and thus likely
reflect a different pathogenic challenge than seen in our model of
continual bacterial "leakage" from the primary pulmonary site of
infection into peripheral blood. Another possible explanation for
increased hepatic injury is that the presence of -T cells may
protect against hepatic injury, and that the absence of these cells
predisposes the liver to injury in the setting of sepsis. Experimental
evidence to dispute this possibility was seen in a model of S.
choleraesuis bacteremia where the absence of -T cells
protected mice from hepatic cellular injury (35). A final
and more likely explanation for increased liver injury may simply be a
reflection of the increased bacterial burden seen in the blood liver of
-T cell knockout mice, resulting in increased endotoxin exposure
and enhanced LPS-induced hepatic injury (36).
Mice lacking ß-T cells, a major component of the T cell
compartment in both lung and liver, appear to have compensated in part
for the lack of ß-T cells by increasing the number of -T
cells in the lymphoid organs. Although not reaching the level of
statistical significance, the consistent trend was for ß-T cell
knockout mice to have decreased blood and liver bacterial numbers, less
hepatic injury, and increased proinflammatory cytokine mRNA induction
than their corresponding infected wild-type littermates. Combined,
these data support our hypothesis that -T cells are a critical
component of the host immune response to Gram-negative bacterial
infections. It is worth noting that increased numbers of -T cells
in the lungs of ß-T cell knockout mice did not result in lower
pulmonary bacterial numbers, in contrast to the trend for improved
systemic responses by these mice. However, with these observations in
mind, the increased number of -T cells in ß-T cell knockout
mice did not result in enhanced survival when compared with wild-type
infected mice. These data suggest that our intratracheal inoculation
model of Klebsiella pneumonia results in two potentially
different types of infections, localized and systemic, and that
-T cells may play different roles in these two types of
infections. To better study the role of -T cells in disseminated,
systemic bacterial infections, we have begun studies characterizing
host responses to i.v. inoculation K.
pneumoniae.
The decrease in early IFN- production in -T cell knockout mice
suggests that -T cells themselves are the cellular source of
IFN-. However, it is possible that -T cells stimulate
production of IFN-, from NK cells for example, so that their absence
would result in decreased IFN- production. Indeed, this scenario has
been shown in Listeria-infected animals (20).
Rapid IFN- production, necessary for clearance of
Listeria, has been shown to be NK cell-derived. However, in
the absence of -T cells, there is a marked decrease in NK cell
production of IFN-. Although we cannot exclude this scenario in our
model, we favor the possibility that -T cells themselves are the
cellular source of IFN-. Mice rendered genetically deficient in
IFN- production display increased mortality following
intratracheal K. pneumoniae inoculation when compared with
their IFN- competent littermates (data not shown). Interestingly,
the survival curves for IFN- knockout and -T cell knockout
mice were essentially identical, suggesting that IFN- production is
-T cell-derived. Additionally, in vivo NK cell depletion by
anti-NK1.1 Ab treatment had no detrimental effect on survival (data
not shown). One would predict that if the IFN- was NK cell-derived
rather than -T cell-derived, then NK cell-depleted mice would
display an increased susceptibility to pulmonary bacterial
challenge similar to -T cell knockout and IFN- knockout mice.
As optimal TNF- production has been shown to be IFN--dependent,
the absence of -T cell-derived IFN- could explain the
reduction in TNF-. Current experiments are ongoing to determine the
exact cellular source(s) of IFN- and TNF-.
Other studies have suggested a link between -T cells and the
production of TNF- and IFN-. Macrophages from -T cell
knockout mice display impaired TNF- production when stimulated with
LPS in vitro (37). Preincubation of these macrophages with
wild-type -T cells restored LPS-induced TNF- production. This
priming activity of -T cells was partially inhibited by
anti-IFN- Abs, suggesting that -T cell derived IFN- was
required for optimal TNF- secretion by macrophages challenged with
LPS in vitro (and possibly Gram-negative bacteria in vivo). Similar
findings were seen in an in vivo model of S. choleraesuis
sepsis (22). In a model of listerosis, IFN- production
by NK cells was shown to be markedly reduced in -T cell-deficient
animals (20). Spleen cells harvested from infected
-T cell-deficient animals were transiently impaired in their
TNF- production following in vitro stimulation, having decreased
production on day 1 postinfection but not by day 4.
Taken together, these data indicate that of the three major
IFN--producing populations (-T cells, ß-T cells, NK
cells), only mice deficient in -T cells exhibit increased
mortality to K. pneumonia. The markedly decreased
expression of IFN- and TNF- mRNA seen in K.
pneumoniae-infected -T cell knockout mice suggests that
-T cells, although comprising a small percentage of lung and
liver leukocytes, are critically important for production of
proinflammatory cytokines vital for the resolution of Gram-negative
bacterial infections. Furthermore, our data suggest that -T cell
knockout mice succumb due to an impaired ability to clear disseminated
bacteria from the bloodstream and liver rather than due to an inability
to clear the organism from the primary site of infection in the lung.
Most importantly, our data indicate that -T cells comprise a
critical component of the acute inflammatory response toward
extracellular Gram-negative bacterial infections and are vital for the
early production of the proinflammatory cytokines IFN- and
TNF-.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Thomas A. Moore, University of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: MPO, myeloperoxidase; AST, aspartate aminotransferase; m, mouse.
Received for publication January 11, 2000. Accepted for publication June 7, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T-cell bridge: linking innate and acquired immunity. Nat. Med. 4:764.[Medline]
T cells. Adv. Immunol. 71:77.[Medline]
and other unconventional T lymphocytes: what do they see and what do they do?. Proc. Natl. Acad. Sci. USA 93:2272. T cell clones. Eur. J. Immunol. 27:1704.[Medline]
cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation. J. Immunol. 160:1965. T lymphocytes. Infect. Immun. 64:1744.[Abstract]
ß and T cells in immunity against an intracellular bacterial pathogen. Nature 365:53.[Medline]
T cells are resistant to lethal infection with Salmonella choleraesuis. Infect. Immun. 63:3736.
T cells by TNF-. J. Immunol. 160:5221. T lymphocytes. J. Immunol. 162:5033. and tumor necrosis factor production during experimental endotoxemia. Eur. J. Immunol. 24:1167.[Medline]
-T cells for liver injury induced by Salmonella infection in mouse. Microbiol. Immunol. 43:461.[Medline]
T cells in priming macrophages to produce tumor necrosis factor-. Eur. J. Immunol. 25:1465.[Medline]
This article has been cited by other articles:
|
|
 |
|
  F. Venet, C.-S. Chung, X. Huang, J. Lomas-Neira, Y. Chen, and A. Ayala Lymphocytes in the Development of Lung Inflammation: A Role for Regulatory CD4+ T Cells in Indirect Pulmonary Lung Injury J. Immunol., September 1, 2009; 183(5): 3472 - 3480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. O'Brien, M. A. Taylor, J. Hartley, T. Nuhsbaum, S. Dugan, K. Lahmers, M. K. Aydintug, J. M. Wands, C. L. Roark, and W. K. Born Protective Role of {gamma}{delta} T Cells in Spontaneous Ocular Inflammation Invest. Ophthalmol. Vis. Sci., July 1, 2009; 50(7): 3266 - 3274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Cogen and T. A. Moore {beta}2-Microglobulin-Dependent Bacterial Clearance and Survival during Murine Klebsiella pneumoniae Bacteremia Infect. Immun., January 1, 2009; 77(1): 360 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Hirsh and W. G. Junger Roles of Heat Shock Proteins and {gamma}{delta}T Cells in Inflammation Am. J. Respir. Cell Mol. Biol., November 1, 2008; 39(5): 509 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Sutherland, J. S. Olsen, A. McKinstry, S. A. Villalta, and P. J. Wolters Mast Cell IL-6 Improves Survival from Klebsiella Pneumonia and Sepsis by Enhancing Neutrophil Killing J. Immunol., October 15, 2008; 181(8): 5598 - 5605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-L. Small, S. McCormick, N. Gill, K. Kugathasan, M. Santosuosso, N. Donaldson, D. E. Heinrichs, A. Ashkar, and Z. Xing NK Cells Play a Critical Protective Role in Host Defense against Acute Extracellular Staphylococcus aureus Bacterial Infection in the Lung J. Immunol., April 15, 2008; 180(8): 5558 - 5568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Tschop, A. Martignoni, H. S. Goetzman, L. G. Choi, Q. Wang, J. G. Noel, C. K. Ogle, T. A. Pritts, J. A. Johannigman, A. B. Lentsch, et al. {gamma}{delta} T cells mitigate the organ injury and mortality of sepsis J. Leukoc. Biol., March 1, 2008; 83(3): 581 - 588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. R. Karaolis, M. W. Newstead, X. Zeng, M. Hyodo, Y. Hayakawa, U. Bhan, H. Liang, and T. J. Standiford Cyclic Di-GMP Stimulates Protective Innate Immunity in Bacterial Pneumonia Infect. Immun., October 1, 2007; 75(10): 4942 - 4950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Bhan, N. W. Lukacs, J. J. Osterholzer, M. W. Newstead, X. Zeng, T. A. Moore, T. R. McMillan, A. M. Krieg, S. Akira, and T. J. Standiford TLR9 Is Required for Protective Innate Immunity in Gram-Negative Bacterial Pneumonia: Role of Dendritic Cells J. Immunol., September 15, 2007; 179(6): 3937 - 3946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Li, A. R. Burns, R. E. Rumbaut, and C. W. Smith {gamma}{delta} T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion Am. J. Pathol., September 1, 2007; 171(3): 838 - 845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Borghesi and C. Milcarek Innate versus Adaptive Immunity: A Paradigm Past Its Prime? Cancer Res., May 1, 2007; 67(9): 3989 - 3993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Kuhl, N. N. Pawlowski, K. Grollich, C. Loddenkemper, M. Zeitz, and J. C. Hoffmann Aggravation of intestinal inflammation by depletion/deficiency of {gamma}{delta} T cells in different types of IBD animal models J. Leukoc. Biol., January 1, 2007; 81(1): 168 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-S. Chung, L. Watkins, A. Funches, J. Lomas-Neira, W. G. Cioffi, and A. Ayala Deficiency of {gamma}{delta} T lymphocytes contributes to mortality and immunosuppression in sepsis Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2006; 291(5): R1338 - R1343. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Egan, J. E. Dalton, E. M. Andrew, J. E. Smith, M.-J. Gubbels, B. Striepen, and S. R. Carding A Requirement for the V{gamma}1+ Subset of Peripheral {gamma}{delta} T Cells in the Control of the Systemic Growth of Toxoplasma gondii and Infection-Induced Pathology J. Immunol., December 15, 2005; 175(12): 8191 - 8199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zeng, T. A. Moore, M. W. Newstead, J. C. Deng, S. L. Kunkel, A. D. Luster, and T. J. Standiford Interferon-Inducible Protein 10, but Not Monokine Induced by Gamma Interferon, Promotes Protective Type 1 Immunity in Murine Klebsiella pneumoniae Pneumonia Infect. Immun., December 1, 2005; 73(12): 8226 - 8236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Curtis Cell-mediated Adaptive Immune Defense of the Lungs Proceedings of the ATS, December 1, 2005; 2(5): 412 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Wands, C. L. Roark, M. K. Aydintug, N. Jin, Y.-S. Hahn, L. Cook, X. Yin, J. Dal Porto, M. Lahn, D. M. Hyde, et al. Distribution and leukocyte contacts of {gamma}{delta} T cells in the lung J. Leukoc. Biol., November 1, 2005; 78(5): 1086 - 1096. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Maeda, P. Reddy, K. P. Lowler, C. Liu, D. K. Bishop, and J. L. M. Ferrara Critical role of host {gamma}{delta} T cells in experimental acute graft-versus-host disease Blood, July 15, 2005; 106(2): 749 - 755. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Deng, T. A. Moore, M. W. Newstead, X. Zeng, A. M. Krieg, and T. J. Standiford CpG Oligodeoxynucleotides Stimulate Protective Innate Immunity against Pulmonary Klebsiella Infection J. Immunol., October 15, 2004; 173(8): 5148 - 5155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Deng, X. Zeng, M. Newstead, T. A. Moore, W. C. Tsai, V. J. Thannickal, and T. J. Standiford STAT4 Is a Critical Mediator of Early Innate Immune Responses against Pulmonary Klebsiella Infection J. Immunol., September 15, 2004; 173(6): 4075 - 4083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Ponomarev, M. Novikova, M. Yassai, M. Szczepanik, J. Gorski, and B. N. Dittel {gamma}{delta} T Cell Regulation of IFN-{gamma} Production by Central Nervous System-Infiltrating Encephalitogenic T Cells: Correlation with Recovery from Experimental Autoimmune Encephalomyelitis J. Immunol., August 1, 2004; 173(3): 1587 - 1595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Uezu, K. Kawakami, K. Miyagi, Y. Kinjo, T. Kinjo, H. Ishikawa, and A. Saito Accumulation of {gamma}{delta} T Cells in the Lungs and Their Regulatory Roles in Th1 Response and Host Defense against Pulmonary Infection with Cryptococcus neoformans J. Immunol., June 15, 2004; 172(12): 7629 - 7634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Workalemahu, M. Foerster, and C. Kroegel Expression and synthesis of fibroblast growth factor-9 in human {gamma}{delta} T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-{beta}1/IL-15 J. Leukoc. Biol., April 1, 2004; 75(4): 657 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ramsburg, R. Tigelaar, J. Craft, and A. Hayday Age-dependent Requirement for {gamma}{delta} T Cells in the Primary but Not Secondary Protective Immune Response against an Intestinal Parasite J. Exp. Med., November 3, 2003; 198(9): 1403 - 1414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Prass, C. Meisel, C. Hoflich, J. Braun, E. Halle, T. Wolf, K. Ruscher, I. V. Victorov, J. Priller, U. Dirnagl, et al. Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by Sympathetic Activation Reversal by Poststroke T Helper Cell Type 1-like Immunostimulation J. Exp. Med., September 2, 2003; 198(5): 725 - 736. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Moore, M. L. Perry, A. G. Getsoian, C. L. Monteleon, A. L. Cogen, and T. J. Standiford Increased Mortality and Dysregulated Cytokine Production in Tumor Necrosis Factor Receptor 1-Deficient Mice following Systemic Klebsiella pneumoniae Infection Infect. Immun., September 1, 2003; 71(9): 4891 - 4900. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Gao, W. Yang, M. Pan, E. Scully, M. Girardi, L. H. Augenlicht, J. Craft, and Z. Yin {gamma}{delta} T Cells Provide an Early Source of Interferon {gamma} in Tumor Immunity J. Exp. Med., August 4, 2003; 198(3): 433 - 442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, H. K. Lee, J. F. Bukowski, H. Li, R. A. Mariuzza, Z. W. Chen, K.-H. Nam, and C. T. Morita Conservation of Nonpeptide Antigen Recognition by Rhesus Monkey V{gamma}2V{delta}2 T Cells J. Immunol., April 1, 2003; 170(7): 3696 - 3706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zeng, T. A. Moore, M. W. Newstead, R. Hernandez-Alcoceba, W. C. Tsai, and T. J. Standiford Intrapulmonary Expression of Macrophage Inflammatory Protein 1{alpha} (CCL3) Induces Neutrophil and NK Cell Accumulation and Stimulates Innate Immunity in Murine Bacterial Pneumonia Infect. Immun., March 1, 2003; 71(3): 1306 - 1315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Moore, M. L. Perry, A. G. Getsoian, M. W. Newstead, and T. J. Standiford Divergent Role of Gamma Interferon in a Murine Model of Pulmonary versus Systemic Klebsiella pneumoniae Infection Infect. Immun., November 1, 2002; 70(11): 6310 - 6318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Steele, M. Zheng, E. Young, L. Marrero, J. E. Shellito, and J. K. Kolls Increased Host Resistance against Pneumocystis carinii Pneumonia in {gamma}{delta} T-Cell-Deficient Mice: Protective Role of Gamma Interferon and CD8+ T Cells Infect. Immun., September 1, 2002; 70(9): 5208 - 5215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Chackerian, J. M. Alt, T. V. Perera, C. C. Dascher, and S. M. Behar Dissemination of Mycobacterium tuberculosis Is Influenced by Host Factors and Precedes the Initiation of T-Cell Immunity Infect. Immun., August 1, 2002; 70(8): 4501 - 4509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Mancuso, A. Gottschalk, S. M. Phare, M. Peters-Golden, N. W. Lukacs, and G. B. Huffnagle Leptin-Deficient Mice Exhibit Impaired Host Defense in Gram-Negative Pneumonia J. Immunol., April 15, 2002; 168(8): 4018 - 4024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Shen, D. Zhou, L. Qiu, X. Lai, M. Simon, L. Shen, Z. Kou, Q. Wang, L. Jiang, J. Estep, et al. Adaptive Immune Response of Vgamma 2Vdelta 2+ T Cells During Mycobacterial Infections Science, March 22, 2002; 295(5563): 2255 - 2258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J Boyton and P. J Openshaw Pulmonary defences to acute respiratory infection Br. Med. Bull., March 1, 2002; 61(1): 1 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.B. Moore, T.A. Moore, and G.B. Toews Role of T- and B-;lymphocytes in pulmonary host defences Eur. Respir. J., November 1, 2001; 18(5): 846 - 856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. L. Wormley Jr., C. Steele, K. Wozniak, K. Fujihashi, J. R. McGhee, and P. L. Fidel Jr. Resistance of T-Cell Receptor delta -Chain-Deficient Mice to Experimental Candidaalbicans Vaginitis Infect. Immun., November 1, 2001; 69(11): 7162 - 7164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Deng, K. Tateda, X. Zeng, and T. J. Standiford Transient Transgenic Expression of Gamma Interferon Promotes Legionella pneumophila Clearance in Immunocompetent Hosts Infect. Immun., October 1, 2001; 69(10): 6382 - 6390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. K. Selin, P. A. Santolucito, A. K. Pinto, E. Szomolanyi-Tsuda, and R. M. Welsh Innate Immunity to Viruses: Control of Vaccinia Virus Infection by {{gamma}}{{delta}} T Cells J. Immunol., June 1, 2001; 166(11): 6784 - 6794. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
