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*
Department of Medicine, Division of Pulmonary and Critical Care Medicine,
Department of Pathology,
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Microbiology, Toho University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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100-fold more susceptible to
lethal pneumonia induced by L. pneumophila. However,
this treatment did not alter early bacterial clearance, despite a
substantial decrease in neutrophil influx at this time point. Cytokine
profiles in the lungs of control mice demonstrated strong T1 responses,
characterized by an increase of IFN-
and IL-12. In contrast,
neutrophil-depleted mice exhibited significantly lower levels of
IFN- and IL-12, and elevation of T2 cytokines, IL-4 and IL-10.
Immunohistochemistry of bronchoalveolar lavage cells demonstrated the
presence of IL-12 in neutrophils, but not alveolar macrophages.
Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was
paralleled to neutrophil number. However, intratracheal administration
of recombinant murine IL-12 did not restore resistance, whereas
reconstitution of IFN- drastically improved bacterial clearance and
survival in neutrophil-depleted mice. Taken together, these data
demonstrated that neutrophils play crucial roles in primary L.
pneumophila infection, not via direct killing but more
immunomodulatory effects. Our results suggest that the early
recruitment of neutrophils may contribute to T1 polarization in a
murine model of L. pneumophila
pneumonia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Legionella organisms usually infect humans via inhalation of
contaminated aerosols from waterborne environmental sources. In the
lungs, bacteria infect cells through binding to complement receptors on
the surface, and multiplies predominantly in monocytes/macrophages
(6, 7, 8, 9). The development of the A/J mouse model of L.
pneumophila pneumonia has provided a valuable tool to analyze
pathogenesis of this disease (10, 11). Macrophages of A/J
mice are believed to be specifically permissive for L.
pneumophila and pneumonia induced in these animals resembles human
disease in both pathological findings and cytokine responses. Previous
studies have demonstrated protective roles of T1 cytokines, such as
IFN- and IL-12, in L. pneumophila pneumonia model
(12, 13, 14). In contrast, the T2 cytokine, IL-10, facilitates
growth of this organism in macrophages, due in part to IL-10-mediated
suppression of T1 cytokines (15). T1-polarized cytokine
productions may be a critical event for resistance to intracellular
pathogens including Legionella, although how T1/T2 balance
is organized in vivo, or which cell types are crucial to determine the
course of disease, T1-directed self-limiting or T2-directed
progressive, is still poorly understood.
Early accumulation of neutrophils to sites of infection is a consistent observation in Legionella pneumonia in both animal models and humans (11, 16, 17), and neutropenia is known to be an important risk factors for this disease (18). However, neutrophils have been regarded as a minor cell type for protection against Legionella infection, largely because previous data demonstrated that Legionella organisms are resistant to killing effects by neutrophils, even in well opsonized condition (19, 20). The current progress in this field has shed light on novel functions of neutrophils as immunoregulatory cells in several infection models, such as Listeria and Mycobacterium (21, 22, 23, 24, 25). Specifically, accumulating data indicates that neutrophils have the ability to synthesize and release immunoregulatory cytokines/chemokines, including IL-12, which may affect T1/T2 host responses (26, 27, 28, 29, 30). These data suggest that neutrophils may be a crucial cell population not only for innate resistance, but also for polarization of T1/T2 balance against intracellular organisms. However, how neutrophils are involved in determining T1/T2 cytokine balance, or the significance of neutrophils as T1-driving cells in vivo, remains to be determined.
In the present study, we examined immunomodulatory roles of neutrophils in an A/J mouse model of L. pneumophila pneumonia. Our data demonstrated a marked increase in susceptibility to L. pneumophila in neutrophil-depleted A/J mice. Interestingly, this was accompanied with a shift of cytokine balance from a T1- to T2-phenotype response in the lungs, but not with increase of bacterial burden during the acute phase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine rIFN-, rIL-4, rIL-10, rIL-12, and mAbs to these
cytokines were purchased from R&D Systems (Minneapolis, MN). Polyclonal
anti-murine IFN-, IL-4, IL-10, and IL-12 Abs used in the ELISA
were produced by immunization of rabbits with these murine recombinant
cytokines in multiple intradermal sites with CFA.
Animals
Female specific pathogen-free 6- to 8-wk-old A/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in specific pathogen-free conditions within the animal care facility at the University of Michigan (Ann Arbor, MI) until the day of sacrifice.
L. pneumophila inoculation
We used a clinical isolate of L. pneumophila suzuki
strain (serogroup-1) for animal experiments.
N-(2-acetamido)-2-aminoethanesulfonic acid (Sigma, St.
Louis, MO)-buffered yeast extract broth supplemented with 0.4 mg/ml
L-cysteine and 0.135 mg/ml ferric nitrate was used as
liquid medium
(BYE-broth)3
(31). To prepare solid medium, 2 mg/ml activated charcoal
and 15 mg/ml agar were added to liquid medium (BCYE-agar). Bacteria
were incubated on BCYE-agar for 4 days at 37°C. Single colony was
transferred to 3 ml of BYE-broth, and then incubated overnight at
37°C with constant shaking. Bacterial suspension was again
transferred to fresh BYE-broth and incubated overnight in the same
condition. The concentration of bacteria in broth was determined by
measuring the amount of absorbance at 600 nm. Postexponential-phase
bacteria were used as challenging organisms (31). Animals
were anesthetized with 90–100 mg pentobarbital per kilogram of
animal i.p. The trachea was exposed, and 30 µl inoculum or saline was
administered via a sterile 26 gauge needle. The skin incision was
closed with surgical staples.
Lung harvesting for determination of bacterial number and cytokine analysis
At designated time points, the mice were sacrificed by CO2 asphyxia. Before lung removal, the pulmonary vasculature was perfused with 1 ml of PBS containing 5 mM EDTA, via the right ventricle. Whole lungs were then harvested for assessment of bacterial number and cytokine protein expression. After removal, whole lungs were homogenized in 1.0 ml PBS with protease inhibitor (Boehringer Mannheim, Indianapolis, IN) using a tissue homogenizer (Biospec Products, Bartlesville, OK) under a vented hood. Portions of homogenates (10 µl) were inoculated on BCYE-agar after serial 1:10 dilutions with PBS. The remaining homogenates were incubated on ice for 30 min, then centrifuged at 2500 rpm for 10 min. Supernatants were collected, passed through a 0.45-µm filter (Gelman Sciences, Ann Arbor, MI), then stored at -20°C for assessment of cytokine levels.
Murine cytokine ELISAs
Murine cytokines were quantitated using a modification of a
double ligand method as previously described (32).
Standards were 1/2 log dilutions of murine recombinant cytokine from 1
pg/ml to 100 ng/ml. This ELISA method consistently detected murine
IFN-, IL-4, IL-10, and IL-12 concentrations above 20–50 pg/ml. The
ELISA did not cross-react with other cytokines, such as IL-1, IL-2,
IL-6, or TNF-
. In addition, the ELISA did not cross react with
members of the murine chemokine family, including murine
keratinocyte-derived chemokine, macrophage inflammatory protein-2,
monocyte chemoattractant protein-1, macrophage inflammatory
protein-1, or RANTES. In some experiments, levels of IL-12 p70 in
the lungs were determined using a commercially available ELISA kit
(DuoSet, ELISA development system; R&D Systems), according to the
manufacturer’s directions. The p70 heterodimer was measured because
this complex represents the biologically active form of IL-12.
Granulocyte depletion
To characterize the role of granulocytes during Legionella infection, mice were depleted of granulocytes before Legionella challenge. For depletion, we used RB6-8C5 mAb, a rat anti-mouse IgG2b, directed against Ly-6G, previously known as Gr-1, an Ag on the surface of murine granulocytes (33, 34). The Ab was produced by TSD BioServices (Germantown, NY) by i.p. injection of RB6-8C5 hybridoma into nude mice and subsequent ascites collection. A total of 100 µg of RB6-8C5 was administered i.p. 1 day before challenge with organism. This resulted in peripheral blood neutropenia (absolute circulating neutrophil count <50 cells/µl) by days 1 and 3 after Ab administration in both infected and control animals, with a return of peripheral counts to pretreatment levels by day 5 (35).
Lung digest for preparation of total lung cells
Total lung leukocytes were isolated 2 days after infection from control or neutrophil-depleted mice. Lungs were removed from euthanized animals, and leukocytes were prepared as previously described (35). Cell counts and viability were determined by trypan blue exclusion counting on a hemacytometer. Cytospin slides were prepared and stained with Diff-Quick to perform cell differentials.
Bronchoalveolar lavage (BAL) and immunohistochemistry
Mice were sacrificed 2 days after inoculation with bacteria for the performance of BAL. The trachea was exposed and intubated using a 1.7-mm OD polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. Approximately 5 ml of lavage fluid was retrieved per mouse. Cytospins were subsequently prepared from BAL cells for immunohistochemistry. Cells were fixed in 100% ethanol for 10 min. Immunostaining was conducted using Dako EnVision System (Dako, Carpinteria, CA) according to the manufacturer’s instructions. Endogenous peroxidase activity was blocked with 0.3% H2O2 in 50% methanol for 20 min, and smear slides were incubated for 30 min with 1000x diluted anti-murine IL-12 rabbit serum at room temperature. The specimens were rinsed and then incubated for 30 min with diluted biotinylated anti-rabbit Ig Ab (1:500). Then, the slides were rinsed and incubated for 30 min with peroxidase-labeled polymer (Dako) at room temperature. As a chromogen, diaminobenzidine (Dako) was used. Counterstaining was performed with hematoxylin. Diluted preimmune serum (1000x) was used as a control. In some experiments, BAL cells were lysed with PBS containing 0.1% Triton X-100 (Sigma), and then IL-12 in lysates was examined.
Isolation and RT-PCR amplification of whole-lung mRNA
Whole lungs were harvested, immediately "snap frozen" in liquid nitrogen, and stored at -70°C, and RT-PCR was performed as previously described (32). Briefly, total cellular RNA from frozen lungs was isolated, reversed transcribed into cDNA and then amplified as previously described. After amplification, the samples (20 µl) were separated on a 2% agarose gel containing 0.3 mg/ml (0.003%) of ethidium bromide and bands were visualized and photographed using UV transillumination.
Statistical analysis
Statistical significance was determined using the unpaired, two-tailed alternate Welsh t test and the nonparametric Mann-Whitney test. Calculations were performed using InStat for Macintosh (GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo studies were performed to determine the effect of
neutrophil depletion on survival after administration of L.
pneumophila. As shown in Table I
,
control mice challenged with 3.6 x 106 CFU
survived, whereas an increase in the bacterial inoculum by 5-fold
killed all control mice by day 6. In contrast, when A/J mice were
treated with neutrophil-depleting RB6-8C5 Ab 1 day before infection, a
drastic increase in lethal sensitivity was observed. All mice
challenged with 1.4 x 105 CFU died and
death of mice was still observed using a challenge dose as low as
2.8 x 104 CFU. In contrast, we could not
observe any change of lethal sensitivity in nonpermissive C57BL/6 mice
when these mice were kept neutropenic by RB6-8C5 Ab (data not shown).
These results indicated that neutrophil depletion sensitized permissive
A/J mice to Legionella-induced lethality by 100-fold,
suggesting that neutrophils are a critical cell population for
resistance to replicative L. pneumophila infection.
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After intratracheal (i.t.) administration of
104, 105, and
106 CFU of L. pneumophila, bacterial
number in the lungs was compared in control and neutrophil-depleted
mice (Fig. 1). In control mice, bacterial
number in the lungs increased 10- to 100-fold by day 2. In
neutrophil-depleted mice, increase of bacterial number in the lungs was
quite similar to those of control mice on day 1 and day 2 post
challenge. However, significant differences were observed by day 3. In
control mice, a greater than 10-fold reduction in bacterial number was
demonstrated at this time point, whereas no such clearance was observed
in neutrophil-depleted mice.
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To confirm effects of RB6-8C5 (anti-Gr1) Ab, total cell number
and leukocyte differentials in the lungs were examined on day 2 after
infection (Fig. 2). A slight decrease in
total cell number was observed in anti-Gr-1 Ab-treated mice,
although this decrease was not statistically significant. As expected,
a near complete reduction of neutrophil influx was demonstrated in
anti-Gr1 Ab-treated mice. In control mice, granulocytes,
monocytes/macrophages, and lymphocytes constituted 52, 30, and 17%,
respectively, while those in anti-Gr1 Ab-treated mice were 4, 67,
and 29%, respectively. Eosinophils were 0.5–1% in control mice, and
there was no increase during the course of L. pneumophila
pneumonia in A/J mice. Interestingly, we observed an increase in the
number of macrophages/monocytes and lymphocytes in anti-Gr1
Ab-treated mice. This data demonstrated that the anti-Gr1 Ab used
strongly suppressed early accumulation of neutrophils.
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To further characterize enhanced susceptibility of
neutrophil-depleted mice to L. pneumophila, we examined the
expression of T1 (IL-12 and IFN-) and T2 phenotype cytokines (IL-4
and IL-10) in the lungs on days 1, 2, and 3 after inoculation with
105 CFU of bacteria (Fig. 3 and Fig. 4). In control mice, increases in IL-12
and IFN- were demonstrated during the first 2 days after infection,
which gradually decreased thereafter. Surprisingly, substantial
suppression of these cytokines was observed in neutrophil-depleted
mice. Specifically, a 40 and 66% reduction in IL-12 was noted in
neutrophil-depleted mice at 1 and 2 days after Legionella
administration, respectively, as compared with control animals
(p < 0.05 and p < 0.01,
respectively). Similarly, a significant reduction of IFN- was
detected in the lungs of neutrophil-depleted mice on days 2 and 3 after
infection. Conversely, early elevations of T2 cytokine IL-4 and IL-10
were observed in the lungs of neutrophil-depleted mice, whereas no
appreciable induction of these cytokines was noted in control mice
challenged with L. pneumophila (Fig. 4). These results
suggested that neutrophil depletion resulted in a shift of cytokine
balance from T1-dominant to T2-dominant response in the lungs of mice
infected with L. pneumophila. To confirm a shift of cytokine
balance in neutrophil-depleted mice, we next examined mRNA expression
of IFN- and IL-10 in the lungs of infected animals. Consistent with
the protein data, we observed a clear reduction of IFN- message and
up-regulation of IL-10 message in neutrophil-depleted mice (data not
shown). These data further suggest a shift of cytokine balance in
neutrophil-depleted mice that occurs at the level of mRNA
expression/accumulation.
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Given that decreases in IL-12 were noted in neutrophil-depleted
mice, we next performed immunohistochemistry of BAL cells to determine
whether neutrophils were potential cellular sources of IL-12. Cytospin
samples were prepared from BAL cells of nonneutropenic mice 2 days
after infection, which were stained with rabbit preimmune or
anti-IL-12 antiserum (Fig. 5).
Compared with control staining (Fig. 5A), there were many
cells containing cell-associated IL-12 (Fig. 5B).
Importantly, the vast majority of cells staining positively for IL-12
were neutrophils. In contrast, few macrophages contained
cell-associated IL-12. Immunohistochemical analysis for the presence of
cell-associated IFN- was also performed. As expected, no IFN- was
detected in any BAL cells (data not shown).
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We next examined IL-12 protein levels in BAL cell lysates. BAL
cells were collected from control and neutrophil-depleted mice 2 days
after infection. Recovered cells were lysed, and IL-12 levels in cell
lysates were examined by ELISA (Fig. 6).
Neutrophil numbers in BAL of control and anti-Gr1-treated mice were
8.16 x 105 and 1.13 x
105, respectively. Although IL-12 was detected in
BAL cell lysates from both control and neutrophil-depleted mice, levels
in control mice were 2-fold higher than that of anti-Gr1-treated
group (Fig. 6A). In addition, a positive correlation between
IL-12 levels and neutrophil number was demonstrated (Fig. 6B). Taken together, these data suggested that neutrophils
serve as an important source of IL-12 in the air space of mice infected
with L. pneumophila.
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on survival and
bacterial number in the lungs
We hypothesized that reductions of T1 cytokines may be responsible
for the increased lethality observed in neutrophil-depleted mice.
Therefore, we determined the effects of i.t. administration of
recombinant murine IL-12 or IFN- on the ability to reconstitute
immunity in neutrophil-depleted mice. As shown in Fig. 7, a marked improvement in survival was
demonstrated in mice administered 500 ng murine rIFN- i.t. at the
time of Legionella challenge. In contrast, no beneficial
effects were observed after i.t. murine rIL-12 administration. IL-12
failed to improve survival regardless of route of administration (i.t.
or i.p.) or amount of IL-12 (20, 100, or 500 ng) administered. The
protective effects of exogenous IFN- were accompanied with
significant reduction of bacterial number in the lungs of
neutrophil-depleted animals (Fig. 8).
Conversely, IL-12 administration did not alter bacterial number in
neutropenic mice at any of the time points examined (days 1–3, data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T1-phenotype cytokine responses play a crucial role in host resistance
to intracellular pathogens, including bacteria, parasites, and viruses.
It has been well recognized that IL-12 released from cells of the
innate immune system contributes to the development of T1 host
responses (36, 37). This cytokine functions as a growth
factor to activate T lymphocytes and NK cells, and induces the
secretion of IFN- from these cells (38, 39). In the
present study, significant reduction of the T1 cytokines IL-12 and
IFN- was demonstrated in mice depleted of neutrophils. Because
neutrophils were shown to be a major source of IL-12 in bronchoalveolar
lavage cells, our data indicated a role for neutrophils in driving
T1-type host responses, which may be mediated, in part, through the
expression of IL-12. The fact that neutrophil depletion results in only
a partial decrease in IL-12 suggests that other cells produce IL-12 in
response to intrapulmonary Legionella infection. Although
alveolar macrophages did not express IL-12 by immunohistochemistry,
other cells including interstitial macrophages/monocytes and dendritic
cells may be relevant sources of IL-12.
The mAb RB6-8C5 recognizes an Ag present on cell surface of mature eosinophils and neutrophils, and specifically depletes these granulocytes in vivo. Additionally, other investigators have reported that RB6-8C5 can bind to a small population of CD8+ and CD4+ T cells (40). However, it is likely that contribution of these T cells to RB6-8C5 Ab-induced sensitization is minimal because total depletion of CD4+ or CD8+ T cells induced only minor change of lethal sensitivity in Legionella pneumonia (data not shown). Another possible cell-type responsible for in vivo RB6-8C5 Ab effect is eosinophils, but its population was at most 1% during the course of infection. Interestingly, we observed a significant increase in the number of macrophages/monocytes in Gr1-treated mice, although the cause and physiologic relevance of this finding remains unclear. Therefore, these observations indicate that the specific lack of neutrophils after RB6-8C5 mAb administration accounts for the dramatic increase of lethal sensitivity in A/J mice model of L. pneumophila pneumonia.
Because bacterial numbers in blood and spleen were not detectable even in neutropenic mice, we believe that mice died from localized pulmonary infection. Pathological analysis in neutropenic animals demonstrated persistent inflammation, such as mononuclear cell infiltration, into the lung airspaces and interstitium, as well as exudation into alveolar (data not shown), which was well correlated with the lack of bacterial clearance. From these observations, we conclude that mice died from pulmonary failure due to Legionella infection, although exact mechanisms involved, such as cytotoxicity of Legionella to pulmonary cells, remained to be clarified. In preliminary studies, we observed acceleration of apoptosis in the lungs of mice infected with Legionella. The role of apoptosis in pathogenesis of Legionella pneumonia are the focus of ongoing studies in our laboratory.
Recently, several investigators have reported that neutrophils may
serve a variety of protective roles in the immune response to infection
through synthesizing immunomodulatory cytokines and chemokines
(26, 27, 28, 29, 30). The ability of neutrophils to release these
factors may confer an important role on these cells in shaping
subsequent immune processes. Romani et al. (28) have
reported that in murine Candida infection, neutrophils
secreted IL-12 and IL-10, correlating with the respective development
of self-limiting and progressive disease. More recently, Bliss et al.
(29) have reported that in vitro stimulation of murine
neutrophils with T. gondii Ag induced IL-12 and TNF-
production in an IFN- independent manner. We detected a significant
amount of IL-12 in neutrophils of BAL cells from
Legionella-infected mice. These data suggested that
neutrophils may be a major source of IL-12 in the air spaces, where
Legionella organisms infect and multiply. Their ability to
rapidly migrate to a focus of infection, as well as their large
numbers, suggested that neutrophils may play an important role as a
cytokine source during early infection.
The reconstitution experiments demonstrated that administration of
IFN- was extremely effective in restoring immunity in
neutrophil-depleted mice with Legionella pneumonia. These
data are consistent with previous reports that IFN- is a critical
factor for host defense against Legionella infection
(12, 14), and further suggested its potential application
to immunocompromised individuals. In contrast, we could not observe
beneficial effects of exogenous IL-12 in neutrophil-depleted mice with
Legionella infection. These data are in contrast to a
previous report indicating that administration of IL-12 could
completely restore resistance to Candida infection in
neutropenic mice, although several experimental conditions, such as
timing and frequency of treatment, were different (28). In
contrast, other investigators have reported limitations of exogenous
IL-12 to drive T1 host responses in vivo (41, 42). There
are several possible explanations for the lack of effectiveness of
IL-12 in neutropenic mice with Legionella pneumonia.
Firstly, the intermittent administration of IL-12 may not provide a
sufficient duration of biologic activity to provide for adequate
development of T1-phenotype responses. In this regard, sustained
expression using IL-12 cDNA-containing adenovirus may be a more
promising strategy, as we have reported previously (43).
However, preliminary studies suggest that transient expression of the
IL-12 transgenes (p35 and p40) failed to reconstitute immunity in
neutrophil-depleted mice with Legionella pneumonia (data not
shown). A second possibility is that T1-promoting activity of exogenous
IL-12 was blunted by the coordinate induction of the T2 cytokines IL-4
and IL-10. This is a distinct possibility given that these T2 cytokines
possess strong suppressive effects on T1 cytokine production (44, 45). In fact, we observed a 4-fold reduction of bacterial number
in neutropenic IL-12-reconstituted mice when anti-IL-4 serum was
simultaneously administered, as compared with neutropenic animals
treated with IL-12 alone (data not shown). A third and most plausible
explanation is that neutrophils are a source of additional factor(s),
which may work alone or in concert with IL-12 to trigger T1-type host
responses. In particular, IL-18 and IL-15 have been shown to
synergistically interact with IL-12 to enhance IFN- production
(46, 47, 48). It is not known at present whether neutrophils
represent an important cellular source of these cytokines. Other
candidate IFN--inducing molecules that are made by neutrophils
include TNF- and the CXC chemokine IFN--inducible protein-10
(49, 50). To this end, we observed a decrease in
IFN--inducible protein-10 in BAL cell lysates collected from
neutropenic mice as compared with control animals challenged with
L. pneumophila (data not shown). The contribution of these
cytokines/chemokines to neutrophil-driven T1 responses is the focus of
ongoing studies.
We observed no change of lethal sensitivity to Legionella in C57BL/6 mice (a nonpermissive mouse strain) when these animals were made neutropenic. This is in distinct contrast to that observed in A/J mice (a permissive mouse strain), in which a critical role of neutrophils was demonstrated. The reason for the permissive nature of A/J mice to Legionella infection has not been clearly defined, but is believed to be attributable to attenuated macrophage killing of ingested bacteria (10, 11, 12, 13). Importantly, human macrophages are also permissive for intracellular growth of L. pneumophila (6, 7, 8, 9). Enhanced "permissiveness" in this mouse strain when made neutropenic further supports an important immunomodulatory effect of neutrophils on macrophage function (rather than by direct neutrophil mediated ingestion and killing).
In conclusion, the present data suggests that the early recruitment of neutrophils contributes to T1 polarization in a murine model of L. pneumophila pneumonia. These data support the recent concept that neutrophils may be a crucial cell population bridging innate resistance and acquired cell-mediated immune responses, and further pointed a role for neutrophils in driving T1-type host responses against the intracellular organism L. pneumophila.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kazuhiro Tateda, 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: BYE-broth, N-(2-acetamido)-2-aminoethanesulfonic acid-buffered yeast extract broth supplemented with 0.4 mg/ml L-cysteine and 0.135 mg/ml ferric nitrate; BCYE-agar, 2 mg/ml activated charcoal and 15 mg/ml agar added to liquid medium; i.t., intratracheal; BAL, bronchoalveolar lavage.
Received for publication October 11, 2000. Accepted for publication December 12, 2000.
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N. Kesteman, G. Vansanten, B. Pajak, S. M. Goyert, and M. Moser Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines J. Leukoc. Biol., March 1, 2008; 83(3): 640 - 647. [Abstract] [Full Text] [PDF]
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 J. P. Mizgerd and S. J. Skerrett Animal models of human pneumonia Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L387 - L398. [Abstract] [Full Text] [PDF]
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 J. P. Mizgerd Acute Lower Respiratory Tract Infection N. Engl. J. Med., February 14, 2008; 358(7): 716 - 727. [Full Text] [PDF]
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E. McFarlane, C. Perez, M. Charmoy, C. Allenbach, K. C. Carter, J. Alexander, and F. Tacchini-Cottier Neutrophils Contribute to Development of a Protective Immune Response during Onset of Infection with Leishmania donovani Infect. Immun., February 1, 2008; 76(2): 532 - 541. [Abstract] [Full Text] [PDF]
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J. M. Daley, A. A. Thomay, M. D. Connolly, J. S. Reichner, and J. E. Albina Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice J. Leukoc. Biol., January 1, 2008; 83(1): 64 - 70. [Abstract] [Full Text] [PDF]
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H. van Faassen, R. KuoLee, G. Harris, X. Zhao, J. W. Conlan, and W. Chen Neutrophils Play an Important Role in Host Resistance to Respiratory Infection with Acinetobacter baumannii in Mice Infect. Immun., December 1, 2007; 75(12): 5597 - 5608. [Abstract] [Full Text] [PDF]
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 P. C. Kousis, B. W. Henderson, P. G. Maier, and S. O. Gollnick Photodynamic Therapy Enhancement of Antitumor Immunity Is Regulated by Neutrophils Cancer Res., November 1, 2007; 67(21): 10501 - 10510. [Abstract] [Full Text] [PDF]
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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]
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F. L. Ribeiro-Gomes, M. C. A. Moniz-de-Souza, M. S. Alexandre-Moreira, W. B. Dias, M. F. Lopes, M. P. Nunes, G. Lungarella, and G. A. DosReis Neutrophils Activate Macrophages for Intracellular Killing of Leishmania major through Recruitment of TLR4 by Neutrophil Elastase J. Immunol., September 15, 2007; 179(6): 3988 - 3994. [Abstract] [Full Text] [PDF]
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T. Yoshimura and M. Takahashi IFN-{gamma}-Mediated Survival Enables Human Neutrophils to Produce MCP-1/CCL2 in Response to Activation by TLR Ligands J. Immunol., August 1, 2007; 179(3): 1942 - 1949. [Abstract] [Full Text] [PDF]
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M. Marks, T. Burns, M. Abadi, B. Seyoum, J. Thornton, E. Tuomanen, and L.-a. Pirofski Influence of Neutropenia on the Course of Serotype 8 Pneumococcal Pneumonia in Mice Infect. Immun., April 1, 2007; 75(4): 1586 - 1597. [Abstract] [Full Text] [PDF]
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E. T. Fuse, K. Tateda, Y. Kikuchi, T. Matsumoto, F. Gondaira, A. Azuma, S. Kudoh, T. J. Standiford, and K. Yamaguchi Role of Toll-like receptor 2 in recognition of Legionella pneumophila in a murine pneumonia model J. Med. Microbiol., March 1, 2007; 56(3): 305 - 312. [Abstract] [Full Text] [PDF]
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 B. A. Maletto, A. S. Ropolo, D. O. Alignani, M. V. Liscovsky, R. P. Ranocchia, V. G. Moron, and M. C. Pistoresi-Palencia Presence of neutrophil-bearing antigen in lymphoid organs of immune mice Blood, November 1, 2006; 108(9): 3094 - 3102. [Abstract] [Full Text] [PDF]
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T. Suzuki, Y. Yoshikawa, H. Ashida, H. Iwai, T. Toyotome, H. Matsui, and C. Sasakawa High Vaccine Efficacy against Shigellosis of Recombinant Noninvasive Shigella Mutant That Expresses Yersinia Invasin J. Immunol., October 1, 2006; 177(7): 4709 - 4717. [Abstract] [Full Text] [PDF]
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Y. Aratani, F. Kura, H. Watanabe, H. Akagawa, Y. Takano, A. Ishida-Okawara, K. Suzuki, N. Maeda, and H. Koyama Contribution of the myeloperoxidase-dependent oxidative system to host defence against Cryptococcus neoformans. J. Med. Microbiol., September 1, 2006; 55(Pt 9): 1291 - 1299. [Abstract] [Full Text] [PDF]
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R. Sporri, N. Joller, U. Albers, H. Hilbi, and A. Oxenius MyD88-Dependent IFN-{gamma} Production by NK Cells Is Key for Control of Legionella pneumophila Infection J. Immunol., May 15, 2006; 176(10): 6162 - 6171. [Abstract] [Full Text] [PDF]
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 A. B. Molofsky, B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse, K. Tateda, and M. S. Swanson Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection J. Exp. Med., April 17, 2006; 203(4): 1093 - 1104. [Abstract] [Full Text] [PDF]
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C. K. Cote, N. Van Rooijen, and S. L. Welkos Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection Infect. Immun., January 1, 2006; 74(1): 469 - 480. [Abstract] [Full Text] [PDF]
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T. Iyoda, K. Nagata, M. Akashi, and Y. Kobayashi Neutrophils Accelerate Macrophage-Mediated Digestion of Apoptotic Cells In Vivo as Well as In Vitro J. Immunol., September 15, 2005; 175(6): 3475 - 3483. [Abstract] [Full Text] [PDF]
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S. Teske, A. A. Bohn, J. F. Regal, J. J. Neumiller, and B. P. Lawrence Activation of the aryl hydrocarbon receptor increases pulmonary neutrophilia and diminishes host resistance to influenza A virus Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L111 - L124. [Abstract] [Full Text] [PDF]
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S. Yoshizawa, K. Tateda, T. Matsumoto, F. Gondaira, S. Miyazaki, T. J. Standiford, and K. Yamaguchi Legionella pneumophila Evades Gamma Interferon-Mediated Growth Suppression through Interleukin-10 Induction in Bone Marrow-Derived Macrophages Infect. Immun., May 1, 2005; 73(5): 2709 - 2717. [Abstract] [Full Text] [PDF]
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K. P.J.M. van Gisbergen, M. Sanchez-Hernandez, T. B.H. Geijtenbeek, and Y. van Kooyk Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN J. Exp. Med., April 18, 2005; 201(8): 1281 - 1292. [Abstract] [Full Text] [PDF]
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A. Y. Koh, G. P. Priebe, and G. B. Pier Virulence of Pseudomonas aeruginosa in a Murine Model of Gastrointestinal Colonization and Dissemination in Neutropenia Infect. Immun., April 1, 2005; 73(4): 2262 - 2272. [Abstract] [Full Text] [PDF]
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E. B. Eruslanov, I. V. Lyadova, T. K. Kondratieva, K. B. Majorov, I. V. Scheglov, M. O. Orlova, and A. S. Apt Neutrophil Responses to Mycobacterium tuberculosis Infection in Genetically Susceptible and Resistant Mice Infect. Immun., March 1, 2005; 73(3): 1744 - 1753. [Abstract] [Full Text] [PDF]
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A. C. Herring, N. R. Falkowski, G.-H. Chen, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Transient Neutralization of Tumor Necrosis Factor Alpha Can Produce a Chronic Fungal Infection in an Immunocompetent Host: Potential Role of Immature Dendritic Cells Infect. Immun., January 1, 2005; 73(1): 39 - 49. [Abstract] [Full Text] [PDF]
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M. Akamine, F. Higa, N. Arakaki, K. Kawakami, K. Takeda, S. Akira, and A. Saito Differential Roles of Toll-Like Receptors 2 and 4 in In Vitro Responses of Macrophages to Legionella pneumophila Infect. Immun., January 1, 2005; 73(1): 352 - 361. [Abstract] [Full Text] [PDF]
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S. D. Swain, T. W. Wright, P. M. Degel, F. Gigliotti, and A. G. Harmsen Neither Neutrophils nor Reactive Oxygen Species Contribute to Tissue Damage during Pneumocystis Pneumonia in Mice Infect. Immun., October 1, 2004; 72(10): 5722 - 5732. [Abstract] [Full Text] [PDF]
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C. R. Brown, V. A. Blaho, and C. M. Loiacono Treatment of Mice with the Neutrophil-Depleting Antibody RB6-8C5 Results in Early Development of Experimental Lyme Arthritis via the Recruitment of Gr-1- Polymorphonuclear Leukocyte-Like Cells Infect. Immun., September 1, 2004; 72(9): 4956 - 4965. [Abstract] [Full Text] [PDF]
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C. Nara, K. Tateda, T. Matsumoto, A. Ohara, S. Miyazaki, T. J. Standiford, and K. Yamaguchi Legionella-induced acute lung injury in the setting of hyperoxia: protective role of tumour necrosis factor-{alpha} J. Med. Microbiol., August 1, 2004; 53(8): 727 - 733. [Abstract] [Full Text] [PDF]
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L. Del Rio, B. A. Butcher, S. Bennouna, S. Hieny, A. Sher, and E. Y. Denkers Toxoplasma gondii Triggers Myeloid Differentiation Factor 88-Dependent IL-12 and Chemokine Ligand 2 (Monocyte Chemoattractant Protein 1) Responses Using Distinct Parasite Molecules and Host Receptors J. Immunol., June 1, 2004; 172(11): 6954 - 6960. [Abstract] [Full Text] [PDF]
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F. L. Ribeiro-Gomes, A. C. Otero, N. A. Gomes, M. C. A. Moniz-de-Souza, L. Cysne-Finkelstein, A. C. Arnholdt, V. L. Calich, S. G. Coutinho, M. F. Lopes, and G. A. DosReis Macrophage Interactions with Neutrophils Regulate Leishmania major Infection J. Immunol., April 1, 2004; 172(7): 4454 - 4462. [Abstract] [Full Text] [PDF]
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T. Kikuchi, T. Kobayashi, K. Gomi, T. Suzuki, Y. Tokue, A. Watanabe, and T. Nukiwa Dendritic Cells Pulsed with Live and Dead Legionella pneumophila Elicit Distinct Immune Responses J. Immunol., February 1, 2004; 172(3): 1727 - 1734. [Abstract] [Full Text] [PDF]
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S. Bennouna, S. K. Bliss, T. J. Curiel, and E. Y. Denkers Cross-Talk in the Innate Immune System: Neutrophils Instruct Recruitment and Activation of Dendritic Cells during Microbial Infection J. Immunol., December 1, 2003; 171(11): 6052 - 6058. [Abstract] [Full Text] [PDF]
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 R. F. Silver, L. Zukowski, S. Kotake, Q. Li, F. Pozuelo, A. Krywiak, and R. Larkin Recruitment of Antigen-Specific Th1-Like Responses to the Human Lung following Bronchoscopic Segmental Challenge with Purified Protein Derivative of Mycobacterium tuberculosis Am. J. Respir. Cell Mol. Biol., July 1, 2003; 29(1): 117 - 123. [Abstract] [Full Text] [PDF]
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H. F. Ismail, P. Fick, J. Zhang, R. G. Lynch, and D. J. Berg Depletion of Neutrophils in IL-10-/- Mice Delays Clearance of Gastric Helicobacter Infection and Decreases the Th1 Immune Response to Helicobacter J. Immunol., April 1, 2003; 170(7): 3782 - 3789. [Abstract] [Full Text] [PDF]
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S. Buonocore, F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, and V. Flamand Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo Blood, February 15, 2003; 101(4): 1469 - 1476. [Abstract] [Full Text] [PDF]
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 K D Lettinga, S Weijer, P Speelman, J M Prins, T van der Poll, and A Verbon Reduced interferon-{gamma} release in patients recovered from Legionnaires' disease Thorax, January 1, 2003; 58(1): 63 - 67. [Abstract] [Full Text] [PDF]
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 B. Mehrad, M. Wiekowski, B. E. Morrison, S.-C. Chen, E. C. Coronel, D. J. Manfra, and S. A. Lira Transient Lung-Specific Expression of the Chemokine KC Improves Outcome in Invasive Aspergillosis Am. J. Respir. Crit. Care Med., November 1, 2002; 166(9): 1263 - 1268. [Abstract] [Full Text] [PDF]
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M. J. Lyons, T. Yoshimura, and D. N. McMurray Mycobacterium bovis BCG Vaccination Augments Interleukin-8 mRNA Expression and Protein Production in Guinea Pig Alveolar Macrophages Infected with Mycobacterium tuberculosis Infect. Immun., October 1, 2002; 70(10): 5471 - 5478. [Abstract] [Full Text] [PDF]
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M. Feldmesser, A. Mednick, and A. Casadevall Antibody-Mediated Protection in Murine Cryptococcus neoformans Infection Is Associated with Pleotrophic Effects on Cytokine and Leukocyte Responses Infect. Immun., March 1, 2002; 70(3): 1571 - 1580. [Abstract] [Full Text] [PDF]
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M. Dalod, T. P. Salazar-Mather, L. Malmgaard, C. Lewis, C. Asselin-Paturel, F. Briere, G. Trinchieri, and C. A. Biron Interferon {alpha}/{beta} and Interleukin 12 Responses to Viral Infections: Pathways Regulating Dendritic Cell Cytokine Expression In Vivo J. Exp. Med., February 19, 2002; 195(4): 517 - 528. [Abstract] [Full Text] [PDF]
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L. Del Rio, S. Bennouna, J. Salinas, and E. Y. Denkers CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility During Toxoplasma gondii Infection J. Immunol., December 1, 2001; 167(11): 6503 - 6509. [Abstract] [Full Text] [PDF]
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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]
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B. T. Wipke and P. M. Allen Essential Role of Neutrophils in the Initiation and Progression of a Murine Model of Rheumatoid Arthritis J. Immunol., August 1, 2001; 167(3): 1601 - 1608. [Abstract] [Full Text] [PDF]
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) or saline (
)
1 day before infection, and then mice were i.t. infected with L.
pneumophila (1.7 x 105 CFU). Two days later,
mice were sacrificed for analysis of cell numbers and differentiation
in the lungs (n = 5). *, p <
0.05; **, p < 0.01.
, saline
control.