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*
Laboratory of Experimental Internal Medicine, Departments of
Pathology and
Infectious Diseases, Tropical Medicine, and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and
Department of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mycobacteria are intracellular pathogens that are taken up by host
alveolar macrophages
(AMs),3 in which they
either are killed or survive. Surviving bacilli start to proliferate
and are released, leading to infection of additional host cells.
Apoptosis of AMs could be an effective weapon to kill or inhibit the
growth of intracellular mycobacteria. Several findings suggest that AM
apoptosis plays an important role in tuberculosis. Infection of human
AMs with M. tuberculosis has been shown to induce apoptosis
in vitro (2). Furthermore, extensive apoptosis (50–70%)
was found within tuberculous granulomas in lungs of tuberculosis
patients (2), and a significant increase in the number of
apoptotic AMs was observed in bronchoalveolar lavage fluid (BALF) from
patients with active pulmonary tuberculosis (3, 4).
Despite these observations, it is not clear which role AM apoptosis
plays in the pathobiology of this disease and whether it increases or
decreases the mycobacterial load in vivo. In vitro studies suggest that
apoptosis may be a macrophage defense mechanism to infection by
mycobacteria. Indeed, apoptosis of human monocytes limited the growth
of Mycobacterium avium (5), Mycobacterium
bovis bacillus Calmette-Guérin (6), and
M. tuberculosis (7). However, in vitro studies
are not adequate to determine the net effect of AM depletion on the
host response to tuberculosis. AMs have important phagocytic and immune
functions that could be disturbed by the apoptotic process. Clearance
of microorganisms that reach the alveolar space relies partly on
phagocytic AMs. Furthermore, macrophages present mycobacterial Ags to
CD4+ T lymphocytes that are central in the
acquired resistance to M. tuberculosis. Macrophages are a
significant source of type 1 cytokines during mycobacterial infection
(8), which are known to be important for the development
of protective immunity (9). In addition, AMs produce
IFN-
in response to M. tuberculosis (10),
which is a pivotal mediator in host resistance to tuberculosis
(11, 12). Finally, mononuclear cells are involved in the
formation of granulomas, which are critical in restricting
mycobacterial growth and dissemination (13). Hence,
theoretically AM depletion could have beneficial and detrimental
effects during tuberculosis in vivo.
In the present study, we determined the role of AM depletion in M. tuberculosis infection in mice, using the well-validated method of intrapulmonary delivery of liposome-encapsulated dichloromethylene diphosphonate (clodronic-acid disodium salt tetrahydrate, CL2MBP). Intratracheal administration of liposome-encapsulated CL2MBP selectively depletes AMs (14) by apoptosis (15, 16) without damaging other cell types in the lung (17). In this work, we present the first evidence that AM depletion in vivo leads to improved clearance of M. tuberculosis bacilli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathogen-free 6-wk-old female BALB/c mice were obtained from Harlan Sprague-Dawley (Horst, The Netherlands) and were maintained in biosafety level 3 facilities. The Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands) approved all experiments.
Experimental infection
A virulent laboratory strain of M. tuberculosis H37Rv was grown in liquid Dubos medium containing 0.01% Tween 80 for 4 days. A replicate culture was incubated at 37°C, harvested at mid-log phase, and stored in aliquots at -70°C. For each experiment, a vial was thawed and washed twice with sterile 0.9% NaCl. Mice were anesthetized by inhalation with isoflurane (Abbott Laboratories, Kent, U.K.) and infected with 1 x 105 live bacilli in 50 µl saline, as determined by viable counts on 7H11 Middlebrook agar plates. Bacterial administration was performed intranasally (i.n.), as described previously (18, 19, 20). Groups of eight mice per time point were sacrificed 2 or 5 wk postinfection, and lungs and one lobus of the liver were removed aseptically. Organs were homogenized with a tissue homogenizer (Biospec Products, Bartlesville, OK) in 5 vol of sterile 0.9% NaCl, and 10-fold serial dilutions were plated on Middlebrook 7H11 agar plates to determine bacterial loads. Colonies were counted after 21-day incubation at 37°C. Numbers of CFUs are provided as total in the lungs or as total per gram liver. For cytokine measurements, lung homogenates were diluted 1/1 in lysis buffer (150 mM NaCl, 15 mM Tris, 1 mM MgCl.H2O, 1 mM CaCl2, 1% Triton X-100, 100 µg/ml pepstatin A, leupeptin, and aprotinin), and incubated on ice for 30 min. Supernatants were sterilized using a 0.22-µm filter (Corning, Corning, NY) and frozen at -20°C until assays were performed.
In vivo AM depletion
CL2MBP was a gift from Roche Diagnostics (Mannheim, Germany). Preparation of liposomes containing CL2MBP was done as described previously (17). For assessment of AM depletion, five uninfected mice per group were i.n. inoculated with 100 µl of 0.9% NaCl, PBS liposomes, or CL2MBP liposomes. Two days later, AMs were quantified in the BALF. For the tuberculosis experiments, 100 µl saline, PBS liposomes, or CL2MBP liposomes were instilled 2 days before and 6, 14, and 25 days after M. tuberculosis challenge.
Detection of apoptotic cells
To confirm apoptotic cell death induced by CL2MBP liposomes, a cleavage of poly(ADP-ribose) polymerase (PARP) was determined, as described previously (21). Briefly, 7 h after i.n. instillation of CL2MBP liposomes, tissue Tek OTC compound (Miles Scientific, Naperville, IL) was instilled intratracheally into lungs, which were then snap frozen and stored at -70°C. Cryostat sections (7 µm) of frozen lungs were fixed in cold acetone for 10 min, incubated with 0.3% H2O2 in methanol for 15 min, blocked for nonspecific Ig binding by incubation for 30 min with a 1/10 dilution of normal goat serum, and incubated overnight with rabbit anti-PARP cleavage site (214/215)-specific Ab (Biosource International, Camarillo, CA; 5 µg/ml). This was followed by a 30-min incubation with poly-HRP goat anti-rabbit IgG (Immunovision, Springdale, AZ). The peroxidase activity was revealed by adding AEC substrate (3-amino-9-ethyl-carbazole; Sigma, Buchs, Switzerland) and H2O2. Sections were counterstained with hematoxylin. Negative controls were established by adding nonspecific isotype controls as primary Abs.
Assessment of in vitro effect of CL2MDP liposomes on M. tuberculosis
A total of 2.5 x 103 CFUs was incubated in octuplicate in 96-well round-bottom culture plates in the presence of Lowenstein-Jensen medium (Becton Dickinson, Franklin Lakes, NJ) with either 0.9% NaCl, PBS liposomes, or CL2MBP liposomes. After 48-h incubation at 37°C in 5% CO2, colonies were counted.
Lung lavage
Bronchoalveolar lavage was performed to obtain intraalveolar cells. Briefly, mice were anesthetized, and the trachea was exposed through a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter (Abbott, Sligo, Ireland). The lungs were then lavaged with two 0.5-ml aliquots of sterile 0.9% NaCl. A total of 0.9–1 ml of lavage fluid was retrieved per mouse, and total leukocyte count was determined using a hemacytometer and TÜRK’s solution (Merck, Gibbstown, NJ). BALFs from infected mice were fixed with 2% paraformaldehyde. The number of AMs, polymorphonuclear cells (PMNs), and lymphocytes were calculated from these totals, using cytospin preparations stained with modified Giemsa stain (Diff-Quick; Baxter, McGaw Park, IL).
Histological analyses
The left lungs were removed 2 or 5 wk after inoculation with M. tuberculosis and fixed in 4% paraformaldehyde in PBS for 24 h. One lobus of the liver of noninfected mice was removed 2 days after CL2MBP liposome treatment. After embedding in paraffin, 4-µm-thick sections were stained with eosin hematoxylin-eosin or the Ziehl-Neelsen (ZN) stain for acid fast bacilli. All slides were coded and semiquantitatively scored for the total area of inflammation (percentage of surface of the slide) and granuloma format by a pathologist.
FACS analysis
Lung cells from mice 2 and 5 wk postinfection (eight mice per group) were analyzed by FACS (Becton Dickinson). Pulmonary cell suspension was obtained using an automated disaggregation device (Medimachine System; Dako, Glostrup, Denmark) and resuspended in medium (RPMI 1640 (BioWhittaker, Belgium), 10% FCS, 1% antibiotic-antimycotic (Life Technologies, Rockville, MD)). Cells from two mice per group were pooled for each time point (yielding four samples for FACS analysis per group) and were brought to a concentration of 4 x 106 cells/ml FACS buffer (PBS supplement with 0.5% BSA, 0.01% NaN3, and 100 mM EDTA). Immunostaining for cell surface molecules was performed for 30 min at 4°C using directly labeled Abs against CD3 (anti-CD3 PE), CD4 (anti-CD4 CyChrome), CD8 (anti-CD8 FITC, anti-CD8 PerCP), CD25 (anti-CD25 FITC), CD69 (anti-CD69 FITC), and Gr-1 (anti-Gr-1 FITC). All Abs were used in concentrations recommended by the manufacturer (PharMingen, San Diego, CA). To correct for aspecific staining, an appropriate control Ab (rat IgG2; PharMingen) was used. Cells were fixed with 2% paraformaldehyde, T cells were analyzed by gating the CD3+ population, and granulocytes by gating the forward and side angle scatter-gated PMN population. The number of positive cells was obtained by setting a quadrant marker for nonspecific staining.
Splenocyte stimulation
Single cell suspensions were obtained by crushing spleens through a 40-µm cell strainer (Becton Dickinson). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA, pH 7.4), and the remaining cells were washed twice. Splenocytes were suspended in medium (RPMI 1640 (BioWhittaker), 10% FCS, 1% antibiotic-antimycotic (Life Technologies)), seeded in 96-well round-bottom culture plates at a cell density of 5 x 105 cells in triplicate, and stimulated with 20 µg/ml tuberculin-purified protein derivative (PPD; Statens Seruminstitut, Copenhagen, Denmark). Supernatants were harvested after a 48-h incubation at 37°C in 5% CO2, and cytokine levels were analyzed by ELISA.
Splenocyte proliferation assay
Proliferation of splenocytes was measured by the MTT assay, which measures reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to formasan in mitochondria of viable cells (22). Splenocytes were seeded in triplicate at a density of 5 x 105 cells/well in flat-bottom 96-well plates and stimulated with 20 µg/ml PPD. After 42 h at 37°C in 5% CO2, cells were incubated with 5 mg/ml MTT (Sigma, St. Louis, MO) in PBS (pH 7.2) for an additional 6 h. Supernatants were decanted, and the formazan precipitates were solubilized by the addition of 0.04 N HCl in isopropanol and placed on a plate shaker for 10 min, after which cells were dissolved in 2% paraformaldehyde. Cell proliferation was quantified using an ELISA reader at 570 nm. The absorbance of the untreated cultures was set at 100%.
Cytokine measurements
Cytokines were measured in lung homogenates and spleen cell
supernatants by specific ELISAs using matched Ab pairs according to the
manufacturer’s instructions: IFN-, IL-2, IL-4 (R&D Systems,
Minneapolis, MN), and IL-10 (PharMingen).
Statistical analysis
All values are expressed as mean ± SEM. Comparisons were
done with Mann-Whitney U tests. For comparison of survival
curves, Kaplan-Meier analysis with a log rank test was used. Values of
p 
0.05 were considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intranasal administration of liposome-encapsulated
CL2MBP resulted in >70% AM depletion in BALF of
uninfected mice after 2 days (Fig. 1
a). Liposome treatment showed
no effect on macrophage numbers. Lungs of CL2MBP
liposome-treated mice presented large areas of degenerated macrophages
with cell debris and apoptotic bodies in the alveolar spaces. This
result is in line with previous reports on the capacity of
intratracheally administered CL2MBP liposomes or
CL2MBP liposomes given by aerosol to deplete AMs
(23, 24). Induction of AM apoptosis by
CL2MBP liposome treatment was confirmed by the
detection of cleaved PARP in lung tissue (Fig. 1b).
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, survival in AM+ (saline) mice decreased
extensively from day 35 onward, resulting in 90% mortality after 5 mo.
In sharp contrast, all AM- mice controlled the
same infectious dose and survived the 5-mo follow-up
(p < 0.0001). In addition, a significant
decrease in survival was found in the AM+
(liposomes) mice compared with AM- animals
(p = 0.029).
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a). At 5
wk postinfection, significant differences in tissue content of M.
tuberculosis bacilli were observed between
AM+ and AM- mice. The
lungs of AM- mice contained 9.7-fold less viable
mycobacteria than those of AM+ (saline) animals
(p = 0.021, Fig. 3a) and 7.1-fold
less than of AM+ (liposomes) mice
(p = 0.035).
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At 2 wk postinfection, the mycobacterial load in the liver of
AM- mice was increased in comparison with
AM+ (saline) mice (p =
0.02) and AM+ (liposomes) mice
(p = 0.004, Fig. 3
b). At 5 wk
postinfection, the number of organisms in the liver of
AM- mice was 3.6 times lower than that in
AM+ (saline) mice (p =
0.011, Fig. 3b) and 2.7 times lower than in
AM+ (liposomes) animals (not significantly
different). Bacterial counts in lungs and liver of the control groups
treated with either saline or liposomes mice were not significantly
different at either time point.
To exclude the possibility that liposome-encapsulated CL2MBP had a direct effect on mycobacteria, M. tuberculosis was incubated in vitro in the presence or absence of this agent for 2 days. Bacterial counting demonstrated no direct antimycobacterial effect of liposome-encapsulated CL2MBP (data not shown).
Together these findings suggest that AM depletion by apoptosis can play an important role in controlling M. tuberculosis infection.
Histology
Two weeks after M. tuberculosis inoculation, lungs of
AM+ (saline/lipsomes) mice exhibited more or less
well-defined granulomas comprising a majority of epithelioid and foamy
cells and a small number of lymphocytes throughout the parenchyma (Fig. 4a). Dense lymphocytic
infiltrates were also present around small vessels. Lungs of
AM- mice showed a relatively diffuse infiltrate
of granulocytes with prominent perivascular lymphocytic infiltrates.
Well-defined granulomas were not present (Fig. 4b). The
percentage of inflamed parenchyma was similar in all groups
(AM+ (saline) mice, 21.25 ± 3.5%;
AM+ (liposomes) mice, 16.9 ± 3.5%;
AM- mice, 18.8 ± 2.9%).
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, c and
d). Cell subsets
CD4+ T cells have an established role in
protective immunity against M. tuberculosis infection
(25, 26, 27), and must be stimulated with specific ligands on
the surface of APCs. To study whether AMs are important for the
induction of CD4+ T cell-mediated immunity, we
investigated the phenotypes of immune cells in total lungs by FACS
analysis. As shown in Table I, the
percentages of CD4+ T cells did not differ
between AM+ and AM- mice 5
wk postinfection and were slightly reduced in AM- mice 2
wk postinfection. Two weeks after infection, CD4+
lymphocytes of AM- mice were demonstrated to be
more activated than CD4+ lymphocytes of
AM+ mice, as assessed by the activation markers
CD69 and CD25.
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To obtain more insight into the leukocyte influx into the alveolar
compartment, lungs were lavaged, cells were counted, and cytospin
preparations were stained with eosin hematoxylin-eosin (Table II). Two weeks postinfection, the number
of leukocytes was higher in AM- mice than in
AM+ (saline/liposomes) mice. In line with the
numbers of leukocytes in total lungs at 5 wk postinfection, cell
numbers in BALFs were lower in AM- mice than in
AM+ (saline/liposomes) mice. As could be
expected, CL2MBP liposome-treated animals had 2
times less AMs in their BALFs than AM+
(saline/liposomes) mice. The amount of PMNs and lymphocytes in
AM- mice was however increased 2 wk
postinfection in comparison with AM+
(saline/liposomes) mice. As a consequence of lower leukocyte numbers in
AM- mice 5 wk postinfection, numbers of PMNs and
lymphocytes were decreased in this group compared with
AM+ (saline/liposomes) mice.
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Since development of early Th1 cellular immunity is essential for
the elimination of M. tuberculosis (9), we
investigated whether the improved outcome of tuberculosis seen in the
AM- mice was associated with a shift in cytokine
production early in the infection. We therefore measured the
concentrations of Th1 (IFN- and IL-2) and Th2 (IL-4, IL-10)
cytokines in the lung. As shown in Fig. 5, all cytokines were reduced in
AM- mice compared with AM+
(saline/liposomes) mice 2 wk postinfection. Importantly, when compared
with AM+ (saline/liposomes) mice, Th2 cytokine
concentrations were relatively more reduced than the levels of Th1
cytokines in AM- mice. As a consequence, a more
profound Th1 response was found in lungs of
AM- mice.
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The ability of spleen cells, harvested 2 wk postinfection with
M. tuberculosis to produce cytokines ex vivo upon
stimulation with PPD, was investigated as another measure of Th1 vs Th2
response. Spleen cells from AM- mice secreted
3.5-fold higher levels of IFN- than splenocytes from
AM+ (saline) mice and 2-fold higher levels than
splenocytes from AM+ (liposomes) mice (Fig. 6). IL-4 was not detectable in
supernatants of PPD-stimulated splenocytes in all groups. When
stimulated with coated anti-CD3 and anti-CD28 Abs, splenocytes
from AM- mice secreted higher levels of IFN-
and significantly lower levels of IL-4 compared with
AM+ (saline) animals. In addition, the
proliferation responses of splenocytes to PPD were estimated using the
MTT incorporation assay. We found that splenocytes from
AM- mice induced the strongest proliferative
response to PPD, although not statistically significant
(AM+ (saline) mice, 155 ± 17%;
AM+ (liposomes) mice, 158 ± 6%; and
AM- mice, 195 ± 31%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and toxic
effector molecules (reactive oxygen intermediates and reactive nitrogen
intermediates), and the deprivation of the intracellular iron
availability. On the other hand, mycobacteria may in part rely on the
intracellular environment of AMs for their multiplication. We
demonstrate in this study that depletion of AMs in vivo improves the
outcome of pulmonary tuberculosis, as indicated by a total protection
against lethality and an attenuated outgrowth of mycobacteria in lungs.
These results suggest that AMs facilitate the growth of M.
tuberculosis in the pulmonary compartment, and that AM apoptosis
may be part of the host defense mechanisms during tuberculosis.
Interestingly, AMs do seem to have a significant role in the initial
capturing of mycobacteria, as indicated by the observation that 2 wk
postinfection AM- mice had more mycobacteria in
their livers. Host cell apoptosis has already been demonstrated to be a defense strategy to limit the growth of viruses, which like mycobacteria live intracellularly (28, 29, 30). The fact that AM apoptosis might contribute to host defense is further supported by observations of an inverse relationship between apoptosis and virulence, i.e., the virulent M. tuberculosis strain H37Rv induced less apoptosis upon human AM infection than the attenuated H37Ra strain (2). Hence, mycobacteria seem to have developed ways to modulate the protective apoptotic process of AMs, and pathogen-induced suppression of the host cell-death pathway may serve to evade host defenses that can act to limit the infection. It should be noted that the role of AMs in respiratory infections by extracellularly growing pathogens is opposite. Indeed, induction of AM apoptosis during Klebsiella pneumonia impaired host defense mechanisms (31).
The most straightforward interpretation of the improved tuberculosis
outcome in AM- mice is that AM depletion reduces
the viability of M. tuberculosis because the environment for
intracellular replication and hiding is destroyed (32).
Furthermore, apoptotic bodies maintain their plasma membrane integrity
so that bacilli are contained from the extracellular environment and
can be engulfed by newly recruited AMs (5). A further
explanation for the protection observed with AM depletion may be that
the early immune response was dominated by a Th1-type profile that is
essential for resistance to mycobacteria (9). The
predominance of Th1-type cytokines in AM- mice
existed both in lung tissue, in which especially the concentrations of
Th2 cytokines were decreased, and in supernatants of PPD-stimulated
splenocytes. Wang et al. (8) recently reported that lung
macrophages harvested during mycobacterial infection release
significant amounts of type 1 cytokines. In line with these
observations, we found lower levels of IFN- and IL-2 in lung
homogenates 2 wk postinfection. However, the net in vivo effect of AM
depletion was a relative type 1 dominance in the lung. It is unlikely
that this type 1 shift was the consequence of a milder inflammatory
response in AM- mice, which, if anything, showed
slightly more evidence of inflammation (Tables I and II). It is
conceivable that the depletion of AMs was involved in this shift. AMs
are typical macrophage populations, which are known to induce
differentiation of naive T cells into Th2 type cells, and to exert
Th2-associated effector functions (33, 34). It is
furthermore reasonable that the reduced IL-10 levels in
AM- mice were involved in this shift,
considering that IL-10 is derived from AMs (35, 36) and
that this cytokine down-regulates type 1 cytokine production
(37). Another reason for the better outcome in
AM- mice could be the enhanced influx of PMNs
and activated lymphocytes into the lung of AM-
mice (Tables I and II). T cells have a prominent role in the protective
immunity against M. tuberculosis (38), and
therefore increased numbers of these cells most likely contribute to
resistance. In support of a role for PMNs in mycobacterial infections,
a protective function of a neutrophilic response was demonstrated by
increased susceptibility of mice depleted of neutrophils to M.
tuberculosis (39). It is conceivable that with the
lack of phagocytosing AMs in AM- mice, PMNs get
more signals to migrate to the lung to ingest and eliminate the
apoptotic bodies derived from AMs.
AM- mice were fully or even more capable of attraction and activation of T cells in the pulmonary compartment. In this context, it should be noted that AMs are poor APCs (40, 41, 42, 43) and that dendritic cells (44, 45) and interstitial macrophages (46) are considered the most efficient APCs in the lung. In vitro experiments even point out that AMs are highly T cell suppressive (47, 48, 49). It is conceivable that the suppressive effects of AMs on the pulmonary immune response may serve to limit damage caused by severe immune responses in lung tissue, but at the same time may impair host defense during tuberculosis.
Two weeks postinfection, more M. tuberculosis CFUs were recovered from livers of AM- mice than from AM+ mice. Our study does not elucidate the mechanisms contributing to this observation. Possibly, bacilli that remain extracellularly (such as in AM- mice) gain access to the blood and lymphatic circulation more easily. It is unlikely that CL2MBP liposome inhalation influenced the number and/or function of Kupffer cells in the liver, considering that liposomes are not able to cross capillary walls and other vascular barriers (17). The absence of an effect of inhaled CL2MBP liposomes on Kupffer cells is supported by the fact that liver histology did not differ between different treatment groups (data not shown).
Liposomes were used to encapsulate Cl2MBP, because these phospholipid spheres are eagerly taken up by macrophages. Liposomes can reduce the phagocytic and migratory behavior of AMs (50) and may therefore influence host defense against M. tuberculosis. In accordance, animals treated i.n. with liposomes only (i.e., without CL2MBP), displayed an enhanced survival and a slight (not significant) reduction in M. tuberculosis CFU in lungs and liver compared with AM+ (saline) mice. Since we sought to determine the role of AMs in pulmonary tuberculosis, control mice should have normal, nonsuppressed AMs (17), and in this way we consider AM+ (saline) animals better controls than AM+ (liposome) mice. Physical depletion of AMs with CL2MBP liposomes ensures that all functions of the macrophages that have ingested this compound are abrogated. AMs that phagocytosed the liposomes alone are expected to have some functional disabilities. Nonetheless, AM+ liposome-treated mice differed significantly from AM- mice with respect to all responses measured.
This study is the first to show that AM depletion in vivo is protective in M. tuberculosis infection and that it is associated with an enhanced Th1-mediated immune response. AM apoptosis as observed in patients with tuberculosis could therefore be an important antimycobacterial defense process. The present results not only provide new insights into possible macrophage antimicrobial defense mechanisms, but also reveal potentially new therapeutic strategies to manage intracellular bacterial diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jaklien Leemans, Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, G2-105, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
3 Abbreviations used in this paper: AM, alveolar macrophage; BALF, bronchoalveolar lavage fluid; Cl2MBP, dichloromethylene bisphosphonate; i.n., intranasal; PARP, poly(ADP-ribose) polymerase; PMN, polymorphonuclear cell; PPD, purified protein derivative.
Received for publication April 14, 2000. Accepted for publication January 22, 2001.
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 A. S. Borders, M. A. Hersh, M. L. Getchell, N. van Rooijen, D. A. Cohen, A. J. Stromberg, and T. V. Getchell Macrophage-mediated neuroprotection and neurogenesis in the olfactory epithelium Physiol Genomics, November 14, 2007; 31(3): 531 - 543. [Abstract] [Full Text] [PDF]
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 A. Forbes, M. Pickell, M. Foroughian, L.-J. Yao, J. Lewis, and R. Veldhuizen Alveolar macrophage depletion is associated with increased surfactant pool sizes in adult rats J Appl Physiol, August 1, 2007; 103(2): 637 - 645. [Abstract] [Full Text] [PDF]
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M. P. O'Sullivan, S. O'Leary, D. M. Kelly, and J. Keane A Caspase-Independent Pathway Mediates Macrophage Cell Death in Response to Mycobacterium tuberculosis Infection Infect. Immun., April 1, 2007; 75(4): 1984 - 1993. [Abstract] [Full Text] [PDF]
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 C. W. Wieland, M. E. Kerver, S. Florquin, M. A. Nolte, J. Borst, R. van Lier, M. H. J. van Oers, and T. van der Poll CD27 contributes to the early systemic immune response to Mycobacterium tuberculosis infection but does not affect outcome Int. Immunol., November 1, 2006; 18(11): 1531 - 1539. [Abstract] [Full Text] [PDF]
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J. M. Shea, T. B. Kechichian, C. Luberto, and M. Del Poeta The cryptococcal enzyme inositol phosphosphingolipid-phospholipase C confers resistance to the antifungal effects of macrophages and promotes fungal dissemination to the central nervous system. Infect. Immun., October 1, 2006; 74(10): 5977 - 5988. [Abstract] [Full Text] [PDF]
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J. Lee, H. G. Remold, M. H. Ieong, and H. Kornfeld Macrophage Apoptosis in Response to High Intracellular Burden of Mycobacterium tuberculosis Is Mediated by a Novel Caspase-Independent Pathway J. Immunol., April 1, 2006; 176(7): 4267 - 4274. [Abstract] [Full Text] [PDF]
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M. Chen, H. Gan, and H. G. Remold A Mechanism of Virulence: Virulent Mycobacterium tuberculosis Strain H37Rv, but Not Attenuated H37Ra, Causes Significant Mitochondrial Inner Membrane Disruption in Macrophages Leading to Necrosis J. Immunol., March 15, 2006; 176(6): 3707 - 3716. [Abstract] [Full Text] [PDF]
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C. W. Wieland, S. Florquin, J. M. Pater, S. Weijer, and T. van der Poll CD4+ Cells Play a Limited Role in Murine Lung Infection with Mycobacterium kansasii Am. J. Respir. Cell Mol. Biol., February 1, 2006; 34(2): 167 - 173. [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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C. M. Bosio, A. W. Goodyear, and S. W. Dow Early Interaction of Yersinia pestis with APCs in the Lung J. Immunol., November 15, 2005; 175(10): 6750 - 6756. [Abstract] [Full Text] [PDF]
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C. W. Wieland, S. Florquin, E. D. Chan, J. C. Leemans, S. Weijer, A. Verbon, G. Fantuzzi, and T. van der Poll Pulmonary Mycobacterium tuberculosis infection in leptin-deficient ob/ob mice Int. Immunol., November 1, 2005; 17(11): 1399 - 1408. [Abstract] [Full Text] [PDF]
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 S. Weijer, C. W. Wieland, S. Florquin, and T. van der Poll A thrombomodulin mutation that impairs activated protein C generation results in uncontrolled lung inflammation during murine tuberculosis Blood, October 15, 2005; 106(8): 2761 - 2768. [Abstract] [Full Text] [PDF]
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X. Shao, A. Mednick, M. Alvarez, N. van Rooijen, A. Casadevall, and D. L. Goldman An Innate Immune System Cell Is a Major Determinant of Species-Related Susceptibility Differences to Fungal Pneumonia J. Immunol., September 1, 2005; 175(5): 3244 - 3251. [Abstract] [Full Text] [PDF]
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P. M. Gray, S. Arimilli, E. M. Palmer, G. D. Parks, and M. A. Alexander-Miller Altered Function in CD8+ T Cells following Paramyxovirus Infection of the Respiratory Tract J. Virol., March 15, 2005; 79(6): 3339 - 3349. [Abstract] [Full Text] [PDF]
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E. A. Murphy, J. M. Davis, A. S. Brown, M. D. Carmichael, N. Van Rooijen, A. Ghaffar, and E. P. Mayer Role of lung macrophages on susceptibility to respiratory infection following short-term moderate exercise training Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2004; 287(6): R1354 - R1358. [Abstract] [Full Text] [PDF]
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J. Branger, S. Florquin, S. Knapp, J. C. Leemans, J. M. Pater, P. Speelman, D. T. Golenbock, and T. van der Poll LPS-binding protein-deficient mice have an impaired defense against Gram-negative but not Gram-positive pneumonia Int. Immunol., November 1, 2004; 16(11): 1605 - 1611. [Abstract] [Full Text] [PDF]
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J. S. Ferguson, J. J. Weis, J. L. Martin, and L. S. Schlesinger Complement Protein C3 Binding to Mycobacterium tuberculosis Is Initiated by the Classical Pathway in Human Bronchoalveolar Lavage Fluid Infect. Immun., May 1, 2004; 72(5): 2564 - 2573. [Abstract] [Full Text] [PDF]
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S. A. Fulton, S. M. Reba, R. K. Pai, M. Pennini, M. Torres, C. V. Harding, and W. H. Boom Inhibition of Major Histocompatibility Complex II Expression and Antigen Processing in Murine Alveolar Macrophages by Mycobacterium bovis BCG and the 19-Kilodalton Mycobacterial Lipoprotein Infect. Immun., April 1, 2004; 72(4): 2101 - 2110. [Abstract] [Full Text] [PDF]
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J. Branger, J. C. Leemans, S. Florquin, S. Weijer, P. Speelman, and T. van der Poll Toll-like receptor 4 plays a protective role in pulmonary tuberculosis in mice Int. Immunol., March 1, 2004; 16(3): 509 - 516. [Abstract] [Full Text] [PDF]
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T. D. Russell, Q. Yan, G. Fan, A. P. Khalifah, D. K. Bishop, S. L. Brody, and M. J. Walter IL-12 p40 Homodimer-Dependent Macrophage Chemotaxis and Respiratory Viral Inflammation Are Mediated through IL-12 Receptor {beta}1 J. Immunol., December 15, 2003; 171(12): 6866 - 6874. [Abstract] [Full Text] [PDF]
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D. H. Dockrell, H. M. Marriott, L. R. Prince, V. C. Ridger, P. G. Ince, P. G. Hellewell, and M. K. B. Whyte Alveolar Macrophage Apoptosis Contributes to Pneumococcal Clearance in a Resolving Model of Pulmonary Infection J. Immunol., November 15, 2003; 171(10): 5380 - 5388. [Abstract] [Full Text] [PDF]
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A. Spira, J. D. Carroll, G. Liu, Z. Aziz, V. Shah, H. Kornfeld, and J. Keane Apoptosis Genes in Human Alveolar Macrophages Infected with Virulent or Attenuated Mycobacterium tuberculosis: A Pivotal Role for Tumor Necrosis Factor Am. J. Respir. Cell Mol. Biol., November 1, 2003; 29(5): 545 - 551. [Abstract] [Full Text]
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M. L. Hart, D. A. Mosier, and S. K. Chapes Toll-Like Receptor 4-Positive Macrophages Protect Mice from Pasteurella pneumotropica-Induced Pneumonia Infect. Immun., February 1, 2003; 71(2): 663 - 670. [Abstract] [Full Text] [PDF]
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 S. Knapp, J. C. Leemans, S. Florquin, J. Branger, N. A. Maris, J. Pater, N. van Rooijen, and T. van der Poll Alveolar Macrophages Have a Protective Antiinflammatory Role during Murine Pneumococcal Pneumonia Am. J. Respir. Crit. Care Med., January 15, 2003; 167(2): 171 - 179. [Abstract] [Full Text] [PDF]
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M. B. Jordan, N. van Rooijen, S. Izui, J. Kappler, and P. Marrack Liposomal clodronate as a novel agent for treating autoimmune hemolytic anemia in a mouse model Blood, January 15, 2003; 101(2): 594 - 601. [Abstract] [Full Text] [PDF]
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C. J. Riendeau and H. Kornfeld THP-1 Cell Apoptosis in Response to Mycobacterial Infection Infect. Immun., January 1, 2003; 71(1): 254 - 259. [Abstract] [Full Text] [PDF]
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R. Pasula, T. Weaver, M. A. Martinez, and W. J. Martin II Morphologic Detection and Functional Assessment of Reconstituted Normal Alveolar Macrophages in the Lungs of SCID Mice J. Immunol., October 15, 2002; 169(8): 4504 - 4510. [Abstract] [Full Text] [PDF]
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J. C. Leemans, M. J. B. M. Vervoordeldonk, S. Florquin, K. P. van Kessel, and T. van der Poll Differential Role of Interleukin-6 in Lung Inflammation Induced by Lipoteichoic Acid and Peptidoglycan from Staphylococcus aureus Am. J. Respir. Crit. Care Med., May 15, 2002; 165(10): 1445 - 1450. [Abstract] [Full Text] [PDF]
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M. Engele, K. Castiglione, N. Schwerdtner, M. Wagner, P. Bolcskei, M. Rollinghoff, and S. Stenger Induction of TNF in Human Alveolar Macrophages As a Potential Evasion Mechanism of Virulent Mycobacterium tuberculosis J. Immunol., February 1, 2002; 168(3): 1328 - 1337. [Abstract] [Full Text] [PDF]
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N. P. Juffermans, J. C. Leemans, S. Florquin, A. Verbon, A. H. Kolk, P. Speelman, S. J. H. van Deventer, and T. van der Poll CpG Oligodeoxynucleotides Enhance Host Defense during Murine Tuberculosis Infect. Immun., January 1, 2002; 70(1): 147 - 152. [Abstract] [Full Text] [PDF]
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U. A. Maus, M. A. Koay, T. Delbeck, M. Mack, M. Ermert, L. Ermert, T. S. Blackwell, J. W. Christman, D. Schlondorff, W. Seeger, et al. Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1245 - L1252. [Abstract] [Full Text] [PDF]
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) or AM+
(liposomes) mice (
) compared to mice receiving saline (
; 100%).
The data are presented as mean and SEM from five mice per group. *,
p < 0.05 AM- mice vs AM+
(saline) mice; 
