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Production: Defining a Novel Role for IFN- in the Regulation of Allergic Airway Inflammation1
*
Sections of Pulmonary and Critical Care Medicine and
Immunobiology,
Department of Dermatology and
§
Department of Pathology, Yale University School of Medicine, New Haven, CT 06520; and
¶
Pathology and Laboratory Medicine Service, Veterans Affairs Connecticut Health Care System, West Haven, CT 06516
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R-/- mice, indicating that IFN- was
responsible for reducing airway eosinophils in the absence of IL-4.
Intranasal administration of IFN- to mice after IL-4+/+
Th2 cell transfer also caused a reduction in airway, but not lung
parenchymal, eosinophils. These studies show that IL-4 indirectly
promotes airway eosinophilia by suppressing the production of IFN-.
IFN- reduces airway eosinophils by engaging its receptor on
hemopoietic cells, possibly the eosinophil itself. These studies
capitalize on the complex counterregulatory effects of Th1 and Th2
cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We
define a new regulatory role for IFN-, demonstrating that
eosinophilic inflammation is differentially regulated at distinct sites
within the respiratory tract. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ag challenge of allergic asthmatics leads to Th2 cell activation, IL-4 and IL-5 production, and eosinophilia in the peripheral blood and the lung (7, 8, 9, 10, 11). IL-5 regulates eosinophilia through expansion and release of eosinophils from the bone marrow, enhances chemotactic signals in the respiratory tract, and inhibits apoptosis (12, 13, 14). In response to chemoattractants, including IL-5, eotaxin, macrophage chemoattractant protein-1, and leukotrienes, eosinophils enter the lung tissue from the vascular space through interactions with adhesion molecules, like VCAM-1, accumulate in perivascular and peribronchial spaces in lung tissue and then migrate into the airways (15). In IL-5-deficient mice eosinophil accumulation in the lung and airways is markedly reduced (16, 17).
IL-4 has also been shown to regulate airway eosinophilia, but the precise mechanism by which this occurs has not been defined. In animal studies in which IL-4 is absent or blocked, airway eosinophils were reduced (18, 19, 20, 21). Because there is defective Th2 cell generation in the absence of IL-4 with reduced IL-5 production (21), a reduction in Th2 cells could be responsible for the decrease in airway eosinophils. Yet, in vitro-generated IL-4-/- Th2 cells that produced high levels of IL-5 also showed a reduction in airway eosinophils (19), suggesting that IL-4 also had an independent effector function in the development of eosinophilia. IL-4 is also known to stimulate eotaxin production and regulate VCAM-1 expression (22, 23). Thus, it has been theorized that IL-4 can regulate eosinophilia by multiple potential mechanisms.
In this manuscript, we show that IL-4 regulates airway eosinophilia but
does not affect the accumulation of eosinophils in the lung parenchyma,
indicating that different mechanisms regulate these two compartments
within the respiratory tract. We show that IL-4 controls airway
eosinophilia through its ability to suppress IFN- production and not
by direct actions on eosinophils. These studies show the complex
counterregulatory effects of Th1 and Th2 cytokines in vivo and define a
novel role for IFN- in the regulation of airway, but not lung
tissue, eosinophilia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, IL-4 deficient (IL-4-/-) BALB/c,
and IFN--deficient (IFN--/-) BALB/c mice
6–12 wk of age (The Jackson Laboratory, Bar Harbor, ME) were
sensitized with OVA and/or used as transfer recipients. DO11.10 mice,
which are transgenic for the TCR recognizing OVA peptide 323–339
(pOVA323–339)3
(24), were kindly provided to us on the BALB/c background
by Ken Murphy (Washington University, St. Louis, MO) and were bred in
our facilities. DO11.10 were crossed to IL-4-/-
BALB/c mice. IFN-R-/- mice were kindly
provided by J. Aguet (25) and were backcrossed at least
six generations onto BALB/c. To generate bone marrow chimeras, female
BALB/c (IFN-R+/+) and
IFN-R-/- mice were treated with 1000 cGy of
ionizing radiation followed by i.v. transfer of 5 x
106 bone marrow cells isolated from BALB/c
IFN-R+/+ or
IFN-R-/- male mice. Donor cell engraftment
was confirmed in previous experiments by fluorescence in situ
hybridization for the Y chromosome on PBMC as described
(26). Male cells made up 
90% of the transplanted PBMC,
and this was similar to fluorescence in situ hybridization results with
nontransplanted male PBMC.
Epicutaneous sensitization and inhaled OVA challenge
BALB/c IL-4+/+ and IL-4-/- BALB/c were sensitized with OVA by epicutaneous exposure, as previously described (27). OVA (grade V; Sigma, St. Louis, MO), 100 µl of 1.0 mg/ml solution in PBS, or PBS alone was applied to a gauze in the center of an occlusive skin patch (DuoDERM Extra Thin; ConvaTec, Princeton, NJ). Patches were affixed to the mice and left intact for 4 days, with application of a second patch on day 14. On days 28, 29, 32, and 33 mice were given intranasal OVA, 25 µg in 50 µl of PBS, and were sacrificed 24 h after the final OVA challenge.
Generation of Th2 cells
To generate Th2 cells from IL-4+/+ or
IL-4-/- DO11.10 mice, CD4 T cells were isolated
by negative selection as previously described (28) using
mAbs to CD8 (clone 53-6.72, clone 2.43; Ref. 29), class II
MHC I-Ad (212.A1; Ref. 30), and
anti-Ig-coated magnetic beads (Collaborative Research, Bedford,
MA). Naive CD4 T cells were further isolated from this population by
positive selection with a biotinylated anti-L-selectin Ab (Mel-14;
BD PharMingen, San Diego, CA) and strepatividin microbeads using MACS
(Miltenyi, Auburn, CA). Syngeneic T-depleted splenocytes were used as
APCs and prepared by negative selection using Abs to CD4 (GK1.5; Ref.
31), anti-CD8, anti-Thy1 (32), and
treatment with rabbit complement. APCs were mitomycin-C treated. To
generate Th2 cells, CD4 T cells were stimulated with
pOVA323–339 (5 µg/ml), IL-4 (200 U/ml) (Becton
Dickinson, Franklin Lakes, NJ), and anti-IFN- (XMG1.2; Ref.
33) at inhibitory concentration. All cultures were set up
in flasks containing a 1:2 ratio of CD4 T cells and APCs at a
concentration of 2.5 x 105 CD4 T cells/ml
and were maintained for 4 days.
Transfer of cells and aerosol administration of OVA
Cultured Th2-like cells were harvested after 4 days, washed with
PBS, and 2.5 or 5 x 106 cells were injected
i.v. into syngeneic recipient mice. One day after transfer of cells,
mice were challenged with inhaled 1% OVA in PBS as previously
described (34), for 20 min daily for a total of 7 days
over a period of 9 days (4 consecutive days exposed, 2 days rested, 3
consecutive days exposed). Control mice received inhaled OVA only.
IFN- (2500 U; Life Technologies, Rockville, MD) was administered
intranasally to mice every other day during OVA exposure. Mice were
sacrificed 24 h after the final OVA exposure.
Bronchoalveolar lavage (BAL) and lung digestion
BAL was performed by cannulation of the trachea and lavage with 1 ml of PBS. Isolation of lung leukocytes was performed after BAL and perfusion of blood from lungs. Lung tissue was minced and digested with collagenase type IV 150 U/ml (Worthington Biochemical, Freehold, NJ) and DNase 10 U/ml (Sigma) for 1 h at 37°C and passed again through a wire mesh to dissociate cells. Cytospin preparations of BAL and lung cells were stained with Diff-Quik (Baxter Healthcare, Miami, FL), and differentials were performed on 200 cells based on morphology and staining characteristics.
Cytokine assays
At the time of transfer, an aliquot of Th2-like cells was
retained for restimulation. A total of 2.5 x
105 CD4 T cells/ml, 2.5 x
105/ml freshly isolated APCs, and pOVA (5
µg/ml) were cultured, and supernatants were collected at 24 h.
BAL cells obtained from individual mice were restimulated in vitro at
2 x 106 cells/ml in the presence of pOVA (5
µg/ml). IFN-, IL-4, IL-5, and IL-13 levels from cell supernatants
were determined by ELISA (Endogen, Cambridge, MA). The lower limit of
sensitivity for each of the ELISAs was 0.6 ng/ml for IFN-, 10 pg/ml
for IL-4, 0.010 ng/ml for IL-5, and 0.025 ng/ml for IL-13. FACS
analysis was performed on BAL cells to determine the percentage of
DO11.10-transgenic CD4 T cells and the amount of cytokine per
milliliter was adjusted for 2.5 x 105 CD4 T
cells/ml.
FACS analysis
At the time of transfer, FACS (Becton Dickinson) analysis was
performed on Th2 cell preparations to determine the purity of
transferred cell populations. Cells were stained with anti-CD4
(Quantum Red-L3T4; Sigma) and, in mice that received DO11.10-transgenic
CD4 cells, the biotinylated anticlonotypic Ab, KJ1.26
(35), and FITC-avidin D (Vector Laboratories, Burlingame,
CA). KJ1.26 is specific for the transgenic TCR in the DO11.10 mice.
Transferred cells were uniformly >96% CD4 positive. Intracytoplasmic
cytokine staining was performed on lung and BAL cells stimulated for
6 h with PMA (Sigma) and ionomycin (Sigma), 4 h after
treatment with brefeldin A (Epicenter Technologies, Chicago, IL). After
activation, the cells were stained with anti-CD4 and KJ1.26 and
then permeablized, fixed (Fix’n Perm; Caltag Laboratories, Burlingame,
CA), and stained with PE-labeled anti-IFN- or an isotype control
Ab (BD PharMingen).
Measurement of OVA-specific Ab in serum
OVA-specific serum Abs (IgG1, IgG2a, IgE) were measured by ELISA as previously described (27) using biotin-labeled rat anti-mouse Abs (anti-IgG1 (Biosource International, Camarillo, CA), anti-IgG2a (BD PharMingen), and anti-IgE (Biosource International)). Serum OVA-specific Ab concentrations were calculated by comparison to the following standards: 1) monoclonal anti-OVA mouse IgG1 (Sigma); 2) monoclonal anti-OVA mouse IgE (kindly provided by Dr. E. Gelfand, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO); and 3) IgG2a, pooled hyperimmune serum generated by repeated i.p. injection of BALB/c IL-4-/- mice with OVA in alum (concentration arbitrarily set at 200,000 U/ml).
Lung histology
Lungs were prepared for histology by perfusing the animal via the right ventricle with 20 ml of PBS. Lungs were then inflated with 1.0 ml of fixative instilled through a tracheostomy tube. Samples for paraffin sectioning were formalin fixed, sectioned in the coronal plane at 5 µm, and stained with hematoxylin and eosin and a modified Congo red to detect eosinophils. In most cases, eosinophils were quantified as the number of eosinophils/mm2 of bronchovascular area, as previously described (36). For the skin-sensitized, intranasally challenged mice, total inflammatory infiltrate per bronchovascular area was highly variable from region to region. To control for this, eosinophils were quantified as a percentage of total inflammatory infiltrate in the bronchovascular area.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the course of investigating the role of IL-4 in allergic lung
inflammation, we identified conditions in which lung tissue
eosinophilia was induced but there was a reduction in eosinophil
accumulation in the airways. IL-4+/+ and
IL-4-/- BALB/c mice were sensitized with OVA by
an occlusive skin patch and then challenged with inhaled OVA. Previous
studies showed that epicutaneous sensitization with Ag led to polarized
Th2 responses (27). IL-4+/+ mice
exhibited a marked influx of eosinophils in the lung tissue and in
cells recovered from the airway by BAL (Fig. 1
). In contrast, while
IL-4-/- mice showed eosinophilic lung
inflammation, the number of eosinophils in the BAL was reduced (Fig. 1A). Eosinophils made up an equivalent proportion of
infiltrating cells in the lung interstitium in
IL-4+/+ and IL-4-/- mice
after OVA sensitization and challenge (Fig. 1B).
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, IL-5, and IL-13 were produced, while BAL cells from
IL-4+/+ mice produced only Th2 cytokines, IL-4,
IL-5, and IL-13 (Table I). OVA-specific
IgG2a was increased in IL-4-/- mice, but was
not detectable in IL-4+/+ mice (Table I). These
findings indicated that Th2 cell priming was impaired in
IL-4-/- mice leading to a mixed population of
Th1 and Th2 cells, thus confirming previous studies showing increased
Th1 immune responses in immunized, Ag-challenged
IL-4-/- mice (18, 21, 37).
Therefore, it was possible that the reduction in BAL eosinophils in
IL-4-/- mice could be explained by a reduction
in Th2 cells and reduced IL-5 production in the lung.
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We next investigated if this specific defect affecting airway
eosinophil accumulation, but not tissue eosinophilia, could be a result
of reduced Th2 cell generation and fewer activated Th2 cells in the
lung. To bypass the problem of Th2 cell development that might occur in
vivo in the absence of IL-4, we generated Th2 cells in vitro from
TCR-transgenic, DO11.10 IL-4+/+ and
IL-4-/- mice by providing exogenous IL-4.
DO11.10 IL-4+/+ and
IL-4-/- CD4 cells were stimulated with APCs,
pOVA323–339, and IL-4 for 4 days, as previously
described (19). The Th2 population generated from
IL-4-/- mice produced no IL-4, but secreted
high levels of IL-5 and IL-13 (Fig. 2A). These cytokines were
comparable to the levels secreted by IL-4+/+
DO11.10 Th2 cells except for the production of IL-4. Both populations
of Th2 cells produced low levels of IFN- when compared with Th1
cells generated concurrently, which typically produced between 300 and
500 ng/ml of IFN- (data not shown, Ref. 19).
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A), as previously shown
(19). An histologic assessment of tissue eosinophils
showed similar numbers of eosinophils in the lungs of mice that
received IL-4+/+ or
IL-4-/- Th2 cells (Figs. 3B and
4). In both groups of animals,
eosinophils infiltrated the peribronchial and perivascular regions. In
addition, equal numbers of eosinophils were isolated from lung tissue
of mice that received IL-4+/+ or
IL-4-/- Th2 cells (Fig. 3C).
Furthermore, in a system completely deficient in IL-4, when
IL-4-/- Th2 cells were transferred into
IL-4-/- BALB/c recipient mice, a similar
differential reduction in BAL eosinophils was observed (data not
shown). This differential in airway eosinophilia did not change when
the exposure to inhaled OVA was prolonged for 13 days (3.1 x
105 (±0.26) vs 0.5 x
105 (±0.1) BAL eosinophils in mice that received
IL-4+/+ vs IL-4-/- Th2
cells, p = 0.01).
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To insure that the transferred DO11.10
IL-4-/- Th2 cell population had retained a Th2
cytokine profile throughout the period of inhaled Ag challenge, we
harvested BAL cells from mice that received
IL-4+/+ or IL-4-/- cells
after exposure to inhaled OVA and restimulated the cells in vitro with
pOVA323–339 (Fig. 2
B). Both
populations of BAL cells produced similar levels of IL-5 and IL-13,
and, as expected, IL-4 production by BAL cells of mice that received
IL-4-/- Th2 cells was markedly decreased.
Although, IFN- production was minimally increased in BAL cells from
mice that received IL-4-/- Th2 cells vs mice
that received IL-4+/+ Th2 cells, this level of
IFN- was still very low compared with restimulated BAL cells from
mice that received Th1 cells, which typically produce >1000 ng/ml of
IFN- (34, 38). These data indicate that both the
DO11.10 IL-4+/+ Th2 and
IL-4-/- Th2 cell populations retained a Th2
profile in vivo.
In summary, two populations of CD4 Th cells that differ predominantly in the production of IL-4, when activated in the respiratory tract, have different effects on airway eosinophilia. IL-5, a critical cytokine regulating eosinophilia, was produced at equivalent levels by these cell populations at the time of transfer and upon recovery of BAL cells. Therefore, the block in eosinophil accumulation in the airway lumen that occurs upon transfer of IL-4-/- Th2 cells cannot be explained by a defect in Th2 generation and IL-5 production and must be explained by an effector function of IL-4.
IFN- inhibits airway eosinophil accumulation and is enhanced in
the IL-4-/- Th2 cell population
Because IFN- production was increased in OVA epicutanously
sensitized, OVA-challenged IL-4-/- mice (Table I), and by BAL cells from mice that received
IL-4-/- Th2 cells (Fig. 2B), we next
investigated whether, in the absence of IL-4, IFN- was responsible
for reducing airway eosinophilia. We transferred DO11.10
IL-4+/+ or IL-4-/- Th2
cells into IFN-R-/- or wild-type
(IFN-R+/+) mice and exposed mice to inhaled
OVA. Any IFN- produced in IFN-R-/-
recipient mice would have no effect (25).
IFN-R-/- mice that received DO11.10
IL-4+/+ or IL-4-/- Th2
cells showed comparable, high numbers of eosinophils in the BAL fluid
(Fig. 5). Lung tissue eosinophils were
assessed histologically, and the numbers were similar in
IFN-R-/- mice that received
IL-4+/+ or IL-4-/- Th2
cells (2118 (±36) vs 2162 (±93) per mm2). Mice
that were only exposed to inhaled OVA did not exhibit significant
eosinophilic lung or airway inflammation. The marked increase in
eosinophilia in IFN-R-/- mice, even after
transfer of highly skewed Th2 cells, has been noted previously
(38). Thus, in the absence of IFN-R signaling,
IL-4-/- Th2 cells induce tissue and airway
eosinophilia comparable to levels induced by
IL-4+/+ Th2 cells. This shows that IFN-R
engagement leads to the reduction in eosinophils in the BAL after
transfer of IL-4-/- Th2 cells into wild-type
(IFN-R+/+) mice.
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production or by blocking an inhibitory
function of IFN-. If IL-4 blocks an effect of IFN-, then this
effect should persist when IL-4 and IFN- are both secreted. To
determine whether IFN- could inhibit airway eosinophilia when IL-4
was present in the lung, we administered IFN- intranasally to mice
that received IL-4+/+ Th2 cells. Intranasal
IFN- was given to mice daily during inhaled OVA challenge. Mice that
received IL-4+/+ Th2 cells and IFN- had a
reduction in BAL eosinophils, while there was no difference in the
number of lung tissue eosinophils whether or not the mice received
IFN- (Table II) This shows that
IFN- only inhibits the accumulation of airway eosinophils. Because
this effect can be induced in the presence of IL-4-secreting Th2 cells,
IL-4 appears to regulate eosinophilia by suppressing IFN-
production.
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, either
the transferred, transgenic Th cell population or endogenous cells in
recipient mice, we performed intracytoplasmic staining on cells
isolated from lung tissue of BALB/c
(IL-4+/+/IFN-R+/+) mice
that received either IL-4+/+ or
IL-4-/- Th2 cells and inhaled OVA. After
stimulation with PMA and ionomycin, IFN- was produced predominantly
in the CD4-expressing population (Fig. 6). A significantly higher proportion of
CD4 T cells produced IFN- in mice that received
IL-4-/- Th2 cells compared with
IL-4+/+ Th2 cells (30% (±1) vs 9.5% (±0.5),
p < 0.003) (Fig. 6, A and B).
The majority of IFN--producing cells were in the CD4-expressing,
TCR-transgenic (KJ1.26+) cell population. The
frequency of IFN--staining, KJ1.26-positive cells was higher in
cells isolated from the lungs of mice that received
IL-4-/- Th2 cells compared with
IL-4+/+ Th2 cells (49.5 (±1.5) vs 30 (±1);
p < 0.005) (Fig. 6, C and D). In
both groups of mice, cells that stained positive for IFN- did not
stain positive for IL-4 (data not shown). The same intracytoplasmic
staining results were obtained using BAL cells isolated from these mice
(data not shown).
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. We transferred
IL-4+/+ or IL-4-/-
(IFN-+/+) Th2 cells into
IFN-+/+ or IFN--/-
mice. Note that, in these mice, recipient endogenous lymphocytes lack
the ability to produce IFN-, therefore only the transferred,
transgenic IL-4-/- Th cells are capable of
producing IFN-. After exposure to inhaled OVA, eosinophils were
reduced in the BAL of IFN-+/+ or
IFN--/- mice that received
IL-4-/- Th2 cells compared with mice that
received IL-4+/+ Th2 cells (Fig. 7). IFN--/-
mice that were only exposed to inhaled OVA had no eosinophils in the
BAL (data not shown). These studies confirm that IFN- produced by
the transferred, TCR-transgenic IL-4-/- Th2
cell population inhibits BAL eosinophilia.
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R on hemopoietic cells blocks eosinophil accumulation in the
airway
We hypothesized two possible mechanisms by which IFN- might
inhibit BAL eosinophil accumulation: IFN- might effect the
eosinophil directly or, alternatively, it might block airway
eosinophilia indirectly by modulating the airway epithelium. Studies
have shown that IFN- can affect eosinophils directly in vitro
(39), and a variety of chemokines secreted by and adhesion
molecules present on airway epithelial cells are important for
promoting eosinophil migration in the respiratory tract
(15). To determine whether this IFN- effect was
mediated by hemopoietic cells or nonhemopoietic cells, we generated
bone marrow chimeric mice with IFN-R+/+ and
IFN-R-/- mice.
IFN-R+/+ and
IFN-R-/- mice were irradiated and received
bone marrow from either IFN-R+/+ or
IFN-R-/- mice
(IFN-R+/+
IFN-R+/+,
IFN-R-/-IFN-R+/+,
IFN-R+/+IFN-R-/-,
IFN-R-/-IFN-R-/-).
Three months after bone marrow reconstitution, DO11.10
IL-4-/- Th2 cells were transferred into each of
the recipient chimeras and mice were exposed to inhaled OVA. As
expected, BAL eosinophil migration was blocked in
IFN-R+/+IFN-R+/+
mice (Fig. 8). In
IFN-R+/+IFN-R-/-
mice, eosinophilia was still reduced. But, in
IFN-R-/-IFN-R+/+
and
IFN-R-/-IFN-R-/-
recipient mice, IL-4-/- Th2 cells induced BAL
eosinophilia. Bone marrow chimeras that received inhaled OVA only had
no significant lung inflammation (data not shown). These data indicate
that inhibition of eosinophil migration from the lung tissue to the
airway lumen is mediated by IFN-R on hemopoietic cells. The slight
reduction in eosinophils in
IFN-R-/-IFN-R+/+
mice may result from subtotal reconstitution of mice with
IFN-R-/- bone marrow, because chromosomal
analysis of transplanted animals typically reveals that after
reconstitution 10% or less of PBL may be of host origin (see
Materials and Methods). Alternatively, nonhemopoietic cells
may respond to IFN- to provide a minimal inhibitory signal to
eosinophils. When IFN-R is absent on nonhemopoietic cells, including
lung epithelium, vascular, and stromal cells, IFN- still exerts its
inhibitory effects on eosinophil accumulation in the airway lumen.
Thus, IFN- reduces airway eosinophils either by a direct action on
eosinophils or indirectly through effects on other bone
marrow-derived hemopoietic cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. IFN- produced at low levels in the lung blocks the
accumulation of eosinophils in the airway, but not in the lung tissue
through its action on hemopoietic cells, possibly through effects on
the eosinophil itself.
Many previous reports have shown that IL-4 regulates airway
eosinophilia. In studies in immunized IL-4-/-
mice or animals that were treated with anti-IL-4 during
sensitization with Ag, eosinophilia was reduced and so were Th2
cytokines (21, 37). These studies capitalize on the
important role of IL-4 in directing Th2 differentiation. In the absence
of IL-4, Th2 cell generation and IL-5 production were reduced and there
was a reduction in both airway and lung tissue eosinophils. Because Th2
cell generation was affected, it was unclear if IL-4 had other effector
functions in controlling eosinophilia. Previous work in our laboratory
using adoptively transferred, in vitro-differentiated
IL-4-/- Th2 cells also showed a reduction in
airway eosinophils and suggested that IL-4 also played an effector role
in this process (19). The data we now present show that
IL-4 controls airway eosinophilia independently of IL-5 by
down-regulating IFN- secretion. Thus, IL-4 has at least two roles in
promoting airway eosinophilia, one in directing Th2 cell generation and
IL-5 production, the other in suppressing IFN- production. Neither
of these effects results from the direct action of IL-4 on the
eosinophil.
IL-4 regulates IFN- production in two different ways. First, IL-4
promotes Th2 cell generation and in its absence, IFN--producing Th1
cells develop (37). Second, IL-4 suppresses IFN-
production in already differentiated Th1 cells through an, as yet,
undefined mechanism (40, 41, 42). In these studies, it is not
exactly clear which mechanism leads to the IFN- production in
IL-4-/- Th2 cells. We show that the population
of IL-4-/- Th2 cells, upon activation in vivo
with inhaled Ag, produce more IFN- compared with the population of
IL-4+/+ Th2 cells due to a larger subset of Th1
cells. Yet, both DO11.10 IL-4+/+ and
IL-4-/- Th2 populations remained heavily skewed
toward Th2, given their high and equivalent levels of IL-4, IL-5, and
IL-13. It is difficult to determine whether the increase in IFN-
production in IL-4-/- Th2 cells reflects a
requirement for IL-4 to suppress IFN- production and maintain the
cells as Th2 or if there was expansion of a small pool of memory Th1
cells that resulted from the primary stimulation with Ag. In either
case, the absence of IL-4 and the resulting increase in IFN- after
Ag challenge leads to a reduction in airway eosinophils.
Airway eosinophilia is a hallmark of the inflammatory response in asthma. Eosinophils are believed to exert toxic and proinflammatory effects when they are activated to secrete eosinophil-specific cationic proteins, lipid mediators, and cytokines. These products of degranulation are believed to cause airway hyper-responsiveness by damage to the epithelium, stimulation of nerve endings, or by direct effects on smooth muscle (15, 43). The initiation of an eosinophilic response in the lung requires Th2 cell activation and IL-5 production (16, 44). Once induced to differentiate in the bone marrow, eosinophils cross from the vascular space into the lung through interactions with VCAM-1 and ICAM-1 (45) in response to different chemotactic signals. Eosinophils first appear to migrate into the lung parenchyma from the postcapillary venules and later begin to accumulate in the airway lumen (46). Once eosinophils have reached the airway lumen, they may be activated or die and release granular proteins that damage the airway epithelium. Recent studies also indicate that eosinophils can migrate from the airway lumen, where they take up inhaled Ag, to local lymph nodes and present Ag to T cells (47).
Most inflammatory stimuli of eosinophils control the accumulation of
eosinophils in both the lung parenchyma and the airway, as shown in
studies in which IL-5, eotaxin, or leukotrienes were absent in vivo
(16, 46, 48). However, ICAM-2 differentially regulates
eosinophil migration within the lung (49). When mice
deficient in ICAM-2 were challenged with inhaled Ag, there was reduced
eosinophil migration from the lung tissue into the airway. Our studies
show that there are additional mechanisms that regulate eosinophilia in
these two compartments within the respiratory tract. Similar to a
deficiency in ICAM-2, low doses of locally produced IFN- only reduce
airway eosinophils, while tissue eosinophils accumulate. Yet, IFN-
inhibits by a different mechanism than ICAM-2 deficiency. IFN-
causes a persistent reduction in airway eosinophils even after
prolonged Ag exposure, in contrast to ICAM-2-/-
mice that developed BAL eosinophilia at a later time point.
Furthermore, IFN- effects are mediated by receptors on hemopoietic
cells, while eosinophils required ICAM-2 on nonhemopoietic cells for
early migration into the airway lumen.
IFN- appears to inhibit airway eosinophilia by directly binding its
receptor on eosinophils or by activating an intermediate bone
marrow-derived cell that has an inhibitory function. Although we had
postulated that IFN- might be acting on the airway epithelium to
inhibit eosinophil migration from the lung to the airway, IFN-R
chimeras show that this is improbable. It is also unlikely that IFN-
inhibits eosinophil migration into the airway, because we did not
observe eosinophil accumulation with an increase in lung tissue
eosinophils in concert with their reduction in the airway. Recent
reports show that IFN- may enhance eosinophil apoptosis
(50). Alternatively, IFN- may enhance eosinophil
recirculation out of the airway lumen and into the lymph nodes
(47). The precise mechanism of the action of IFN- on
airway eosinophils is currently under investigation in our
laboratory.
The localized effects of IFN- that we describe here are different
from our recent studies in which Th1 and Th2 cells were cotransferred
into mice exposed to inhaled OVA (38). Those data showed
that IFN- inhibited both Th2-induced lung tissue and airway
eosinophilia despite high levels of IL-5. IFN- production by BAL
cells in those mice was 10-fold higher than the levels we measured in
Fig. 2B (our unpublished data). At high levels, IFN-
appears to affect both airway and lung parenchymal eosinophils,
possibly by reducing the number of circulating blood eosinophils,
whereas at low levels only the accumulation of eosinophils in the
bronchial airways is affected. Therefore, in addition to the distinct
effects of IFN- at different sites within the lung, eosinophils in
other tissues may also be influenced by IFN- depending on the level
produced in the lung.
We define now with certainty that differential regulation of eosinophils occurs within the lung, yet the functional role of compartmentalization in the respiratory tract is not known. In asthma, eosinophils in BAL and biopsies have been shown to correlate with disease severity (6, 51). Inflammatory mediators released by eosinophils have been identified in both the submucosa and the airway lumen (6, 52). Thus, it appears that asthma pathology results from eosinophil degranulation in different sites. Major basic protein applied intranasally to animals led to epithelial damage and airway hyperresponsiveness (53, 54), suggesting that eosinophil degranulation in the airway can cause asthma. Therefore, reducing airway eosinophils in asthmatics may help to control disease. Most importantly, recognizing that eosinophils have different regulatory controls within the lung, investigators should now consider sampling both lung tissue and the airway in asthmatics and in animal models of disease to determine how new therapies affect eosinophils within the lung.
IFN--induced regulation of the allergic inflammatory response has
potentially important implications for immune modulation in asthma.
Drugs that block IL-4 may increase IFN- levels in vivo, despite the
differentiated nature of the Th2 cells, and inhibit eosinophils.
Evidence now suggests that immunotherapy stimulates Th1 responses,
enhances IFN- levels in allergic disease, and correlates with
improved symptoms, possibly leading to long-term modification of Th2
responses (55, 56, 57). A more precise understanding of the
mechanisms of action of IFN- in the respiratory tract may help to
focus our efforts in devising new treatment strategies for asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lauren Cohn, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208057, New Haven, CT 06520-8057.
3 Abbreviations used in this paper: pOVA323–339, OVA peptide 323–339; BAL, bronchoalveolar lavage.
Received for publication September 11, 2000. Accepted for publication December 1, 2000.
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0.002 compared with eosinophils in
IFN-