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-Dependent Mechanism1
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Asthma Research Group, Department of Medicine, McMaster University, Hamilton, Ontario, Canada; and
Department of Cell Biology and Immunology Faculty of Medicine, Vrije University, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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)
and more Th2 (IL-4 and IL-5) cytokines, leading to more severe airway
hyperreactivity and inflammation after intranasal Ag challenge. After
OVA pulsing and adoptive transfer, lung macrophages selectively
promoted a Th1 response in Ag-sensitized recipients and did not induce
pulmonary eosinophilia. By contrast, OVA pulsing and adoptive transfer
of a lung cell preparation, consisting of dendritic cells, B cells, and
macrophages, promoted a Th2 response with an associated inflammatory
response that was suppressed when macrophages were present and
pretreated with IFN-, but exacerbated when macrophages were depleted
before IFN- treatment. In addition, Th1-promoting activity of lung
macrophages was not related to the autocrine production of IL-12p40.
These results suggest that the Th1-promoting APC activity may be an
inherent property of the lung macrophage population, and may play an
important role, upon stimulation by IFN-, in antagonizing an ongoing
Th2 immunity and Th2-dependent allergic responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expansion of memory Th2-type cells during the secondary exposure to
allergen, in addition to the commitment of naive T cells to this
phenotype in the primary response, is required for the development of
the Th2 immunity in allergic asthma. A critical step in triggering the
secondary immune responses in the lung is the presentation of inhaled
allergen to the memory T cells by local APCs. Increasing evidence
suggests that an already deviated T cell response can be reversed or
further augmented depending on the type of APCs responsible for
restimulation and the ensuing secondary immune response (1, 12, 13). Dendritic cells (DCs) in the respiratory tracts have been
specialized for mobilizing a default Th2 immunity at the mucosal sites
(14). Indeed, studies on allergic murine models of asthma,
primarily using gene suicide or disruption techniques, have
demonstrated that DCs are essential for the presentation of inhaled
allergen to previously activated Th2 cells in the lung and critical for
the subsequent development of chronic allergic airway inflammation
(1). By contrast, B cells do not appear to be essential
for Ag presentation in the airways, even though they are also
characterized as Th2-promoting APCs (13, 15). One key
question, not yet answered, is whether the resident macrophages are
involved in the development of a lung mucosal allergic immunity. The
importance of this issue is emphasized by studies showing that the APC
activity of macrophages is associated with the development of Th1 cells
and the attenuation of Th2 responses in other systems (10, 16, 17, 18). This effect is believed to occur through macrophage
IL-12 production (19), since IL-12, as well as IFN-, is
effective for directing a primary immunity through Th1 pathways
(20, 21). However, resident DCs are also competent in
IL-12 secretion (22), and both IL-12-producing APC types
appear to have differing effects on the cellular and humoral immunology
in the allergic process (1, 12, 14). Furthermore, IL-12
has been reported to enhance rather than to suppress ongoing Th2-type
responses in certain circumstances (23, 24, 25, 26). Together,
these results indicate that IL-12 production may not be able to account
completely for the Th1-promoting activity of macrophages. In addition,
the potential in vivo effects of the resident macrophages on
antagonizing Th2-mediated allergic inflammation in the lung have not
been described.
This study aimed to determine the role of macrophages in the lung
allergic immunity in a mouse asthma model, previously characterized to
develop eosinophilic airway inflammation and AHR (3, 13).
Initially, we use alveolar macrophage (AM)-depleted mice to demonstrate
that lung macrophages play a protective role against allergen airway
challenge, at least partially through a mechanism of control Th1/Th2
immune responses. In a series of adoptive transfers, we characterize
the activity of lung macrophages in local Ag presentation that is Th1
oriented, IL-12 independent, and subjected to regulation by IFN-.
When the APC ability of adoptively transferred lung macrophages is
up-regulated by IFN- before allergen exposure, the allergen-induced
T cell response shifts to a predominant Th1 phenotype and the allergic
airway inflammation is completely suppressed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed in a specific pathogen-free facility. All mice were 10–12 wk old at the time of the experiments. This study was approved by the animal ethics committee of McMaster University (Hamilton, Ontario, Canada).
Immunization and airway challenge
Mice received primary and booster immunization with i. p. injection of 10 µg soluble OVA, adsorbed to 2 mg of alum in 0.2 ml of saline on days 0 and 7. On day 15, airway challenge was performed with intranasal injection of 50 µl of a 1.5% solution of OVA in PBS or PBS alone. Allergic responses were measured 3 days after intranasal challenge.
Depletion of AM before intranasal challenge
The liposome-mediated macrophage depletion technique is based on the intracellular delivery of dichloromethylene-bisphosphonate (CL2MDP or clodronate was a generous gift of Boehringer Mannheim, Mannheim, Germany), which induces apoptosis in AM, having no effect on interstitial macrophages or DCs (27, 28, 29). CL2MDP liposomes (60 µl) were intranasally injected to Ag-sensitized mice 4 and 2 days prior to airway challenge after light i.p. anesthesia using a mixture of ketamine and xylazine (45 and 8 mg/kg, respectively). Optimal depletion of AM was achieved with this procedure, which was shown by BAL cell differential counting and immunohistochemistry staining of lung tissue with F4/80 (a specific marker for mature macrophages) mAb (Serotec, Raleigh, NC). The control mice were administered the equal volume of PBS liposome.
Measurement of airway responsiveness
Airway responsiveness to i.v. methacholine challenge was measured as previously described (30). Briefly, mice were anesthetized with i. p. injection of avertin (240 mg/kg; Aldrich Chemical, Milwaukee, WI), intubated with an 18-gauge tracheal cannula, and ventilated at a rate of 90 breaths/min with a tidal volume of air (0.1 ml/kg). Muscle paralysis was achieved by i.v. administration of pancuronium (0.03 mg/kg). After stabilization for a few minutes, incremental doses of methacholine (10–330 µg/kg) were given via i.v. injection. Ventilatory frequency was reduced to 27 breaths/min for the first 30 s after each methacholine delivery, as discussed previously (30). Total respiratory system resistance (RRS) was measured, and airway reactivity was expressed as the slope of the straight line regression between peak RRS and the log10 of the methacholine dose (30).
BAL fluid collection
BAL fluid was obtained by injecting and recovering of two 0.5-ml aliquots of PBS via a tracheal cannula. Cells in the lavage fluids were counted using a hemocytometer, and the differentials were determined by utilizing light microscopy to count 300 cells on cytospin preparations. The supernatants of BAL fluid were stored at -70°C for ELISA analyses.
Lung histology
The lungs were inflated by injecting into the tracheal a 1-ml solution of optimum cutter temperature compound (OCT; Somagen, Edmonton, Alberta, Canada) in PBS (1:1). Blocks of the tissue samples were embed in OCT, snap frozen in liquid nitrogen, and stored at -70°C until use. Sections (6-µm) were fixed in acetone before staining. Cyanide-resistant eosinophilic peroxidase activity, using potassium cyanide, 3, 3'-diaminobenzidine (Sigma, St. Louis, MO), and hydrogen peroxide, was applied in eosinophil staining (31). For detection of AM in the alveolar spaces, F4/80 Ab and biotinylated rabbit anti-rat IgG (Dako, Carpenteria, CA) were utilized, followed by the addition of avidin-biotin complex/HRP (Dako) and diaminobenzidine substrate. Positive cells from 10 airways/per mouse were enumerated in 1 µm of bronchial mucosa, and results were expressed as numbers of cells per millimeter of bronchial epithelium.
Lung APC preparations
Lung cells from OVA-sensitized mice were prepared as previously
described, with slight modifications (14, 32, 33).
Briefly, the lungs were perfused via the right ventricle with 5 ml of
PBS containing 100 U heparin to remove blood and intravascular cells.
Then the tissue was minced and incubated for 1 h at 37°C on a
rocker in complete RPMI (cRPMI) 1640 that was supplemented with 10%
FCS, L-glutamine (2 mM), 2-ME (50 µM), sodium pyruvate (1
µM), HEPES (10 mM), penicillin (100 U/ml), and streptomycin (100
µg/ml) (Sigma), containing collagenase A (0.7 mg/ml) and DNase (50
U/ml; Sigma). The enzyme-digested tissue was tapped through a wire
screen to obtain single cells, and erythrocytes were lysed by treatment
with NH4CL-Tris buffer. The cells were washed
twice with PBS, overlaid on Lymphoprep (Nycomed Pharma AS, Oslo,
Norway), and centrifuged at 1800 rpm for 20 min. Three APC preparations
were used in experiments: 1) Lung cells, which referred to the enriched
mononuclear cells recovered from the Lymphoprep interface after
centrifuging and washed with PBS. 2) Lung macrophages, freshly isolated
lung cells, were incubated at a concentration of 2 x
106 cells/ml in tissue culture dishes for 2
h at 37°C, 5% CO2, followed by extensive
washing with prewarmed cRPMI to remove nonadherent cells. The adherent
cells were continuously incubated for 2 h. After washing with warm
medium again, macrophages were harvested from adherent cultures by
incubating for 10 min with a cell dissociation solution (Sigma). This
procedure yielded a purity of >85% macrophages confirmed by FACS
analysis with F4/80 staining. 3) Macrophage-depleted lung cells:
freshly isolated lung cells were resuspended in a solution of 11 mM
D-glucose, 5.5 mM KCL, 137 mM NaCl, 25 mM
Na2HPO4, and 5.5 mM
Na2HPO4 x
2H2O (GKN), supplemented with 5% FCS. The cell
suspension was then eluted through nylon wool to remove macrophages and
washed by GKN supplemented with 5% BSA (13). Depletion of
macrophages was confirmed by FACS analysis with a <15% of
F4/80-positive cells in this preparation. Both lung cells and
macrophage-depleted lung cells were irradiated at 2000 rad, and the
three cell preparations were incubated overnight, with or without OVA
(1 mg/ml) pulsing, in 50-ml polypropylene conical centrifuge tubes. In
some cases, lung APC preparations were exposed to IFN- (5 ng/ml)
2 h before incubation with OVA.
Lung cell transfer
Prepared APCs were washed three times in protein-free PBS after overnight incubation. A total of 3 x 106 irradiated lung cells or 1.5 x 106 lung macrophages or 5 x 105 irradiated macrophage-depleted lung cells, in 0.05 ml PBS, was immediately transferred by intranasal injection into the lungs of the sensitized recipients, after a light i.p. anesthesia, as described above. Pulmonary immunologic and inflammatory responses were measured 3 days after transfer. To confirm the persistence of the transferred APCs in the lung for the duration of the protocol, the cells were labeled with 4,6-diamidino-2-phenylindole (DAPI; Sigma), as previously described (3). Two or three days after cell transfer, the recipient lungs were fixed in 10% Formalin, and 10-µm frozen sections were examined by fluorescence microscopy.
Flow cytometry
To determine the phenotype of lung APCs prepared as described
above, 106 lung cells, macrophage-depleted lung
cells, or lung macrophages were incubated after overnight culture for
30 min at 4°C with FITC-conjugated anti-F4/80, PE-conjugated
anti-CD11c (Serotec)/CD19 (PharMingen, Mississauga, Ontario,
Canada), and anti-DEC-205 (NLDC-145) with the secondary Ab
PE-conjugated anti-rat IgG (Serotec), then washed with PBS
containing 2% FCS. Expression of MHC class II and accessory
molecules on these cell preparations, with and without exposure to
IFN- (5 ng/ml), was also assessed by staining with FITC-conjugated
anti-I-Abd/B7-1 and PE-conjugated
anti-B7-2 (Serotec). Presently, no specific marker is available for
DC, which were therefore identified by negative expression of
F4/80/CD19 (a B cell Ag) combined with positive expression of CD11c (a
marker highly expressed on DC, but also on macrophage) and DEC 205 (a
marker highly expressed on DC) (34). A FACScan flow
cytometer (Becton Dickinson, Mississauga, Ontario, Canada) and
CellQuest software were used for analysis.
In vitro T cell responses to Ag-pulsed lung APCs
Spleen cells were prepared from Ag-sensitized mice, and T cells
were purified using a T cell column (R&D Systems, Minneapolis, MN).
Freshly isolated lung macrophages or macrophage-depleted lung cells
were incubated in 50-ml polypropylene conical centrifuge tubes at
37°C, 5% CO2, with or without OVA pulsing (1
mg/ml) in the absence or presence of IFN- (5 ng/ml) overnight. After
washing with prewarmed PBS, the cultured APCs (1 x
105 cells) were cocultured with T cells (1
x 105 cells) in triplicate in 96-well tissue
culture plates for 96 h. For proliferation assay, methyl
[3H]thymidine (Amersham, Arlington Heights, IL)
was added to the 96-well plates (0.5 µCi/well) 18 h before
harvesting. The harvested cells were analyzed in a Beckman liquid
scintillation counter (Beckman Instruments, Fullerton, CA). Results
were expressed as mean cpm for triplicate wells. Supernatants collected
from the parallel cultures (48 h) were kept at -80°C for ELISA
analysis.
Cytokine ELISA
IL-4, IL-5, IL-12p40, and IFN- levels in BAL fluid and cell
culture supernatants were assayed by ELISA according to the procedure
recommended by the manufacturer (PharMingen, San Diego, CA).
Statistical analysis
ANOVA and paired t test were applied to determine differences in various parameters (Statistica; Statsoft Com, Tulsa, OK). A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the effects of AM on the development of airway allergic
responses, CL2MDP liposome that specifically
depleted AM in the lung (28, 29) was intranasally injected
to OVA-sensitized mice 4 and 2 days prior to Ag or PBS challenge. This
procedure caused a more than 85% reduction in the number of AM/ml of
BAL compared with the control administration of PBS liposome
(p < 0.01) (Fig. 1
). AM depletion from
CL2MDP liposome-treated lungs was further
confirmed by the absence of F4/80+ cells in the
alveolar spaces, as assessed by immunohistochemistry.
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A). Consistent
with the allergic nature of the inflammation, the increase was most
pronounced for eosinophils, with a 2-fold higher number/ml of BAL than
that achieved by Ag challenge alone (Fig. 1A). The number of
eosinophils in the bronchial mucosa was also significantly increased in
AM-deleted allergen-challenged mice compared with mice with the
challenge alone (p < 0.05) (Figs. 1B and 2). Furthermore,
allergen-induced methacholine AHR was further enhanced in AM-depleted
mice (p < 0.05) (Fig. 1C).
Th1/Th2 cytokine assay in the BAL indicated that the allergic responses
exacerbated by AM depletion were related to alterations in the T
cell-mediated immunity. Intranasal allergen challenge to Ag-sensitized
mice provoked higher levels of the Th2-type cytokines IL-4
(p = 0.06), IL-5 (p <
0.05), and the Th1-type cytokine IFN- (p <
0.05) in BAL than those achieved by PBS challenge (Fig. 3A). AM depletion further
increased BAL levels of IL-4 (p < 0.05) and
IL-5 (p < 0.01) in this model, but, by
contrast, the levels of IFN- were reduced (p
< 0.05). The BAL levels of the three cytokines were not altered by AM
depletion in PBS-challenged mice (Fig. 3A). Consistent with
previous reports (12, 19), these results may reflect the
Ag-specific Th1-stimulating activity of AM, which might be able to
antagonize Th2-mediated allergic responses.
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To characterize the APC activity of lung macrophages, three lung
APC preparations, with and without Ag pulsing, were intranasally
transferred into the lungs of Ag-sensitized mice. They were 1)
irradiated enriched lung mononuclear cells (lung cells), 2) irradiated
macrophage-depleted lung cells, and 3) lung macrophages. Sixty-five
percent of lung cells were positive for F4/80 (a marker for mature
macrophages), and 65% also expressed CD11c (Fig. 4). After removal of macrophages from
lung cells, 88% of the cell preparation was F4/80-negative cells, but
47% were still positive for CD11c, and 25% for CD19 (Fig. 4).
Following enrichment for macrophages, the F4/80-positive cells
accounted for 86% of the cell population. In addition, 50% of
macrophage-depleted lung cells expressed DCE-205, whereas only 3% of
DCE-205-positive cells were detected in lung macrophages. Both lung
macrophages and macrophage-depleted lung cells significantly expressed
MHC class II (I-Abd) and costimulatory
(B7-1/B7-2) molecules, but the level of I-Abd
expression was significantly higher on macrophage-depleted lung cells
(Table I).
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Adoptive transfer of 1.5 x 106
OVA-pulsed lung macrophages or 3 x 106
OVA-pulsed lung cells intranasally resulted in these cells being
deposited in the mice lungs (Fig. 5), and
enhanced BAL cytokine levels in the recipient mice compared with the
mice that received unpulsed lung macrophages or unpulsed lung cells
(Fig. 3B). OVA-pulsed lung macrophages selectively enhanced
IFN- levels in the BAL of the recipient mice
(p < 0.05), whereas the pulsed lung cells
increased the concentrations of all three cytokines, but only IL-4 and
IL-5 reached statistical significance (p <
0.05 and p < 0.01, respectively). Transfer of either
of the unpulsed cell preparations did not increase BAL cytokine
production compared with that induced by intranasal PBS injection (Fig. 3A). The magnitude of the increases in the IL-4 and IL-5
production induced by transfer of OVA-pulsed lung cells was similar to
that achieved by intranasal Ag challenge (Fig. 3, A and
B). Consistent with our in vivo results, OVA-pulsed lung
macrophages significantly increased IFN- production by spleen T
cells from Ag-sensitized mice in vitro, whereas the IL-4 production was
amplified by OVA-pulsed macrophage-depleted lung cells (Table II).
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). The number of
neutrophils and eosinophils after transfer of OVA-pulsed lung cells was
3.9-fold and 7.4-fold higher, respectively, than that seen with
OVA-pulsed lung macrophages (p < 0.05 and
p < 0.001). As expected, sensitized mice receiving
either unpulsed lung macrophages or unpulsed lung cells showed similar
BAL cell differential counts as mice with intranasal PBS injection
(Fig. 1A).
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). This was associated with significantly higher
levels of IL-4 and IL-5 (p < 0.05 for both),
and significantly lower levels of IFN- (p <
0.05) in the same mice (Fig. 3B). Th1-promoting activity of lung macrophages is not related to IL-12p40 production
IL-12 has been reported to be a contributor to the Th1-promoting
activity of macrophages (12, 19, 34). To evaluate this
mechanism, endogenous IL-12p40 production was measured in the BAL of
the recipient mice following transfer of three lung APC preparations.
The recipient mice transferred with OVA-pulsed lung APC preparations,
irrespective of their phenotypes, had significantly increased IL-12p40
levels in the BAL (p < 0.05 or 0.01), whereas
IL-12p40 levels were just detectable in the BAL of the mice that
received either unpulsed lung macrophages or unpulsed lung cells (Fig. 7). While OVA-pulsed macrophage-depleted
lung cells induced the most severe Th2/inflammatory responses in the
recipient airways, their BAL IL-12p40 concentrations were similar to
those seen in the airways challenged by OVA-pulsed lung macrophages
that had a predominant Th1 response and almost no inflammation. In
addition, OVA-pulsed lung cells invoked the highest IL-12p40 production
in the recipient airways, but this did not reduce the magnitude of the
Th2 responses and allergic inflammation. Thus, Th1-promoting activity
of lung macrophages occurred through Ag presentation appeared to be
independent of IL-12p40 production.
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selectively up-regulates the APC activity of lung
macrophages in vitro
IFN- is a crucial mediator in the induction of Th1-type
responses and capable of selectively up-regulating the APC activity in
monocytes/macrophages (12, 17, 18). Such effect of IFN-
on lung macrophages was tested in vitro and compared with the effect on
macrophage-depleted lung cells. Upon exposure to IFN- before OVA
pulsing, MHC class II (I-Abd) expression was
significantly increased in lung macrophages, but not
macrophage-depleted lung cells (p < 0.05)
(Fig. 8). In coculture with spleen T
cells obtained from Ag-sensitized mice, IFN--treated OVA-pulsed lung
macrophages enhanced the T cell proliferation and IFN- but not IL-4
production (Tables II and III) compared
with the same untreated cells. In contrast, the T cell-stimulating
ability of OVA-pulsed macrophage-depleted lung cells was not
significantly altered by IFN-.
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-treated lung macrophages prevent allergen-induced
eosinophilic inflammation in vivo
The in vivo effect of IFN- on the APC activity of lung
macrophages was evaluated via transferring lung cells and
macrophage-depleted lung cells, both being treated by IFN- before Ag
pulsing, to Ag-sensitized mice. IFN- pretreatment in OVA-pulsed lung
cells inhibited the transfer-induced recruitment of eosinophils into
the recipient lungs by 98% (p < 0.001),
macrophages by 60% (p < 0.01), lymphocytes by
83% (p < 0.05), and neutrophils by 94%
(p < 0.05). In contrast, this effect was not
seen in OVA-pulsed macrophage-depleted lung cells with IFN-
treatment, in which the degree of the inflammatory responses after the
transfer was similar to that invoked by transfer of the same untreated
cells (Fig. 9A). The parallel
changes following transfer of IFN--treated OVA-pulsed lung cells
were a decrease in the BAL levels of IL-4 and IL-5 in the mice
recipients, which approached, but did not reach, statistical
significance, and a significant increase in the concentrations of
IFN- (p < 0.05). In addition, the BAL
cytokine levels in the mice recipients were comparable after transfer
of OVA-pulsed macrophage-depleted lung cells with and without IFN-
pretreatment (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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after OVA
challenge. These observations suggest that AM exerted a protective
effect against allergy in the lung partially through a Th1-stimulating
mechanism.
We next attempted to evaluate whether lung macrophages are able to
selectively trigger a Th1 response at the bronchial mucosal sites
through processing and presentation of Ag to the T cells. This was done
by instilling into the lungs of OVA-sensitized mice, OVA-pulsed lung
macrophages, or OVA-pulsed lung cells with and without macrophage
depletion. OVA-pulsed lung macrophages significantly increased IFN-
production in the airways of the recipient mice, whereas OVA-pulsed
lung cells, consisting of DCs, B cells, and macrophages, significantly
enhanced the production not only of IFN-, but also of IL-4 and IL-5.
However, when macrophages were removed from lung cells before OVA
pulsing, a more biased Th2 immunity, as indicated by further increased
production of IL-4 and IL-5, was observed in the recipient lungs after
the transfer, which was coupled with a significant decrease in the
IFN- production. These results clearly indicate that like spleen
macrophages (12, 19, 34), lung macrophages are capable of
converting to Th1-oriented APC upon exposure to allergen, and that the
participation of these cells in Ag presentation inhibits, to some
degree, the development of Th2 immune responses in the bronchial
mucosa.
Activation of CD4+ T cells through TCR ligation requires interaction with cells that present the antigenic peptide in association with MHC molecules and that additionally express the necessary costimulatory ligands. Thus, the APC ability of lung macrophages to activate the Th1 immune response in our model system would be related to the cells’ capacity to express both MHC class II and costimulatory molecules. Based on our phenotyping data, F4/80+ lung macrophages significantly expressed MHC class II (I-Abd) and also CD80/CD86 after overnight culture. I-Abd expression on lung macrophages was lower compared with macrophage-depleted lung cells, which mainly contained F4/80- CD11c+ (DC) and F4/80- CD19+ (B cell) cells. However, CD80 and CD86 expression were comparable between the two lung APC preparations. These data further confirmed the T cell-activating properties of lung macrophages. The difference in the expression of I-Abd between the two lung APC preparations might reflect the different APC potency between lung DCs and lung macrophages. Indeed, DCs have been shown to be superior to other APC types at stimulating T cell proliferation (33, 35).
Our results are consistent with previous studies, which have characterized the ability of DCs on mucosal surfaces, such as the murine lung and gut, to stimulate Th2 responses (1, 14). By contrast, the APC activity of macrophages in other organ systems, in which the cells served as the exclusive APC, has been specialized for promoting the Th1 development (12, 19, 34). However, the role of macrophages, in an immune process involving other APC populations, remains unclear. In this study, we evaluated the relative importance of macrophage Th1-promoting activity in Ag-challenged murine airways, in which DCs are known to be critically important in Ag presentation (1, 14). We monitored the inflammatory process following transfer of three OVA-pulsed lung APC preparations. Transfer of OVA-pulsed lung macrophages alone resulted in negligible increases in the number of BAL inflammatory cells. In contrast, transfer of OVA-pulsed lung cells elicited airway eosinophilia in the mice recipients, whereas the most severe and extensive airway inflammation was induced via transfer of OVA-pulsed macrophage-depleted lung cells. These results suggest that competition for Ag presentation between lung macrophages and DCs, in the secondary immune response, influences the magnitude of the allergic inflammatory responses. However, the activity of macrophages in this regard is insufficient to completely prevent allergen-induced Th2 cell-mediated responses.
Another question posed in this study was whether the APC potency of
lung macrophages could be enhanced before adoptive transfer to a
sufficient level to prevent the development of the Th2-mediated
allergic responses in the mice recipients. It has previously been
reported that the functional activity of APCs is subjected to
regulation by cytokine signals and that Th1-priming capacity of
macrophages can be markedly up-regulated by treatment with IFN-
(12, 14, 17, 18, 36). We therefore treated lung cells,
with and without macrophage depletion, with IFN- before Ag
pulsing and transferred these two-cell preparations to the lungs of
Ag-sensitized mice. Notably, the effect of IFN- pretreatment
depended on the presence of lung macrophages. The inflammatory
responses in the recipient airways caused by transfer of OVA-pulsed
lung cells were suppressed when macrophages were present and treated by
IFN-. In striking contrast, when macrophages were removed, IFN-
treatment of the cell preparation before transfer failed to show any
beneficial effects on the subsequent inflammatory events. In vitro,
IFN- pretreatment significantly elevated the expression of MHC class
II molecule on OVA-pulsed lung macrophages, but not pulsed
macrophage-depleted lung cells, with increased T cell proliferation and
increased production of IFN- rather than IL-4 in the T
cell/macrophage cocultures. These suggest that the type of T cell
immune responses in the airways of the mice recipients shifted toward a
Th1 dominance following transfer of IFN--treated
macrophage-containing lung cells with Ag pulsing, but remained Th2
polarized when macrophages were depleted before exposing the lung cell
preparation to IFN-. The mechanism underlying this effect has not
yet been investigated, but appears to be related to an inherent
property of macrophages. It has been shown that the APC ability of DCs
is selectively modified by GM-CSF (14, 36); therefore, the
different obligatory signals for the functional activity of DCs and
macrophages may also reflect the different effector properties of these
two APC types.
The Th1-promoting property of macrophages has been attributed to the
cell production of IL-12, a cytokine highly effective in inhibiting
IL-4 and stimulating IFN- synthesis in both unprimed as well as
resting memory T cells (19, 21, 37, 38, 39). However, in this
study, transfer of both lung macrophages and macrophage-depleted
DC-containing lung cell preparation significantly induced IL-12p40
production in an allergen-dependent manner in the airways of the mice
recipients. Although this finding is in accordance with previous
reports on constitutive IL-12 secretion by DCs (22, 40, 41), the dissociation between the APC-stimulated T cell
phenotypes and their IL-12p40 production in the mice lungs after
adoptive transfer was still surprising. We interpret these results in
four possible ways: first, with comparable IL-12p40 production, there
may be a difference in the synthesis and release of IL-12p70, assembled
by a 35-kDa subunit (p35) and a 40-kDa subunit (p40) within the same
cell (42), by the two transferred APC preparations.
Second, the T cell may be triggered directly by the OVA peptide-MHC
complex in macrophage (43). Third, IL-18 or an unknown
cytokine aside from IL-12 may induce the cells to produce IFN-
(44). And last, a potential lack in the production of
IL-10, a cytokine necessary for Th2 proliferation, by the murine lung
macrophages (45) may further amplify the inclination of
Th1 development.
Although we show that lung macrophages are capable of MHC class
II-dependent Ag presentation, they have been previously viewed as an
immunosuppressor, inhibiting T cell proliferation via producing NO and
PGE2 (46, 47). Such paradoxical
activities have also been addressed on macrophages from other organ
systems (15, 16, 17, 43, 48), reflecting the heterogeneity
in these cells. It has been suggested that suppressor activity is
restricted to specific macrophage phenotype, with other phenotypes
supporting normal T cell activation (49). Macrophages that
differentiate in vitro under the influence of IFN- acquire the
ability to stimulate Th1 phenotype development (12), but
conversely become immunosuppressive APCs under the influence of M-CSF
(49). This functional conversion in activated macrophages
has also been found in vivo through the course of
Mycobacterium tuberculosis infection from the
acute phase to the chronic stage (50). Our results are
likely to reflect the immunostimulatory activity of lung macrophages
upon acute allergen and IFN- exposure, which might switch toward
immunosuppression after chronic and repeated allergen exposure
(47). These characteristics point to a possibility that
the protective Th1-promoting effect of lung macrophages could be
subsequently reinforced by their suppressive effects on T cell
responses in an allergic inflammatory process. The latter property of
lung macrophages may play an important role in down-regulating the
chronic pulmonary lymphoproliferative responses.
In summary, this study demonstrates, for the first time, the ability of lung macrophages to attenuate allergic inflammation and AHR in a murine model of allergic asthma, by mounting Th1 responses in the bronchial mucosa that antagonized Th2 responses to inhaled allergen. Increasing our understanding of macrophage regulation may allow us to increase their activity in Ag presentation, thereby preventing the development of secondary allergic responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul M. O’Byrne, Firestone Regional Chest and Allergy Clinic, St. Joseph’s Hospital, 50 Charlton Avenue East, Hamilton, Ontario, Canada.
3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; AM, alveolar macrophage; BAL, bronchoalveolar lavage; CL2MDP, dichloromethylene-bisphosphonate; DAPI, 4,6-diamidino-2-phenylindole; DC, dendritic cell; cRPMI, complete RPMI.
Received for publication April 20, 2000. Accepted for publication October 31, 2000.
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