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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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While signal transducer and activator of transcription protein 6 (STAT6) is important in interleukin-4
(IL-4)-induced commitment of CD4+ T cells to the T helper cell, type 2 (Th2) phenotype and IgE isotype switching in B cells, its role in other IL-4-mediated events and their impact upon the allergic
response is less evident. In the present study we demonstrate the critical role of STAT6 in the development of allergic airway eosinophilia and airway hyperresponsiveness (AHR) after allergen sensitization and challenge. STAT6-deficient (STAT6
/) mice did not develop a Th2 cytokine response or
an allergen-specific IgE response. Further, STAT6/ mice had a reduced constitutive and allergen-induced expression of CD23 as well as lower mucus production in the airway epithelium. Critically,
we show that IL-5 alone can reconstitute airway eosinophilia and AHR in sensitized and challenged
STAT6/ mice. This emphasizes the essential nature of the IL-4-dependent signaling of T cells to
the Th2 phenotype and secretion of IL-5, resulting in the airway eosinophilia and AHR. These observations underscore the importance of targeting this pathway in new antiallergic asthma drug development.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Allergic asthma is a complex disease associated with airway hyperresponsiveness (AHR) and chronic airways inflammation, as well as elevated levels of immunoglobulin E (IgE) in the serum. The airway mucosal inflammation in allergic asthma is characterized by an influx of activated eosinophils and T and B lymphocytes. The eosinophil is thought to be the major effector cell in the pathogenesis of the allergic AHR by releasing cytotoxic proteins that damage the airway epithelium and cause structural changes (1). However, it is now increasingly clear that T cells orchestrate the inflammatory response. Increased numbers of CD4+ T cells have been identified in the bronchial mucosa of allergic asthmatics expressing elevated levels of messenger RNA (mRNA) for interleukin-4 (IL-4) and IL-5 consistent with the T helper cell, type 2 (Th2) phenotype (2).
Clinical and experimental evidence suggests that IL-4 and
IL-5 are central to the development and maintenance of the
allergic AHR. IL-5 is known to regulate the growth, differentiation, and activation of eosinophils, whereas IL-4 is critical
for the commitment of T cells to the CD4+ Th2 phenotype
and is essential for IgE isotype switching in B cells (3). In
addition, IL-4 upregulates the expression of major histocompatibility complex (MHC) class II and the low-affinity IgE receptor (CD23, Fc
RII) on B cells enhancing antigen presentation, and the vascular cell adhesion molecule-1 (VCAM-1) on
endothelial cells facilitating eosinophil recruitment to the airways (6, 7). IL-4 may also increase airway mucus secretion (8).
The role of IL-4 in the pathogenesis of AHR has been further elucidated in murine models of allergic AHR and airway eosinophilia. Using either neutralizing antibody to IL-4, administered during sensitization, or mice deficient in IL-4, the development of airway eosinophilia, AHR, and the increase in serum IgE normally seen after sensitization and allergen provocation are reduced or abolished (9). Furthermore, because the synthesis of the Th2 cytokine, IL-5, was shown to be attenuated, the sequential involvement of IL-4 committing T cells to the Th2 phenotype that secretes IL-5 and that induces airway eosinophilia and AHR has been proposed, although it has also been suggested that the role of IL-4 in allergic AHR may also be independent of IL-5 and eosinophils (9).
IL-4 exerts its biological effects by binding to the IL-4 receptor complex consisting of the IL-4/IL-13 receptor 
chain
and the common 
chain of the type I cytokine receptor superfamily. Signal transduction may occur by two separate pathways: The phosphorylation and activation of signal transducer
and activator of transcription protein 6 (STAT6) by Janus kinase 1 (JAK1) and JAK3, which once activated, dimerizes,
translocates to the nucleus, and binds to specific promoter
regions to regulate gene transcription. Ligation of the IL-4
receptor may also lead to activation of the insulin receptor
substrate-2 (IRS-2) which can associate with proteins like phosphatidylinositol 3-kinase and may be important in the proliferative response to IL-4 (12). Although IL-4 may activate both
signaling pathways, it is clear from recent experiments with STAT6-deficient (STAT6/) mice that the STAT6 pathway
is the principal signaling pathway involved in commitment of
CD4+ T cells to the Th2 phenotype and IgE isotype switching
in B cells (13, 14). Thus it appears that the STAT6 pathway is
an integral part of the allergic response.
Very recently the role of STAT6 in the development of AHR was demonstrated (15, 16). Both studies demonstrate the role for STAT6 in the development of AHR to intravenous methacholine (MCh). However, Kuperman and colleagues using STAT6-deficient mice on a BALB/c background demonstrated only partial inhibition (~ 50%) of bronchoalveolar lavage fluid (BALF) eosinophilia, questioning the role of the eosinophil in the development of AHR in their model (15). This contrasts with the observations of Akimoto and coworkers who using STAT6-deficient mice on a C57BL/6 background demonstrated almost complete inhibition of BALF eosinophilia (> 90%) (16).
In the present study the role of STAT6 in mediating allergic AHR and airway eosinophilia was investigated using B6/
129 mice deficient in STAT6. Furthermore, to distinguish
among the many functions of IL-4 on IgE production, CD23
and VCAM-1 expression, mucus production, and the commitment of T cells for IL-5 production, which may contribute to
AHR, sensitized STAT6/ mice were reconstituted with IL-5
alone during allergen challenge.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animals
Male and female STAT6/ mice on a B6/129 background were obtained from Dr. J. N. Ihle (St. Jude Children's Research Hospital, Memphis, TN) and bred at National Jewish Medical and Research Center (13). Congenic (B6/129) STAT6-sufficient (STAT6+/+) mice were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were maintained on ovalbumin (OVA)-free diets. All experimental animals used in this study were under a protocol approved by
the Institutional Animal Care and Use Committee of the National
Jewish Medical and Research Center.
Sensitization and Airway Challenge
Groups of mice, 10 to 12 wk of age, were sensitized by intraperitoneal injection of 20 µg OVA (Grade V; Sigma Chemical Co., St. Louis, MO) emulsified in 2.25 mg aluminium hydroxide (AlumImuject; Pierce, Rockford, IL) in a total volume of 100 µl on Days 1 and 14. Mice were challenged (20 min) via the airways, twice daily (morning and afternoon) with OVA (1% in saline) for 5 d (Days 28, 29, 30, 31, and 32) using ultrasonic nebulization (AeroSonic ultrasonic nebulizer; DeVilbiss, Somerset, PA). Forty-eight hours after the last OVA challenge (Day 34) AHR was assessed and tissues obtained for further analysis. Control mice groups received OVA challenge alone.
Administration of Murine Recombinant IL-5 (rIL-5)
In studies performed with mice reconstituted with IL-5, mice received a single intravenous injection of murine rIL-5 (50 ng in 200 µl phosphate-buffered saline [PBS]) 2 h before the first OVA challenge on Days 28, 30, and 32.
Determination of Airway Responsiveness
Airway responsiveness was assessed as a change in airway function after challenge with aerosolized MCh. Anesthetized (pentobarbital sodium, intraperitoneally, 70 to 90 mg/kg), tracheostomized (stainless steel cannula, 18-gauge) mice were mechanically ventilated and lung function was assessed using methods described by Takeda and colleagues (17). Mice were placed in a whole body plethysmograph and ventilated (model 683; Harvard Apparatus, South Natick, MA) via the tracheostomy tube at 160 breaths/min and a tidal volume of 150 µl with a positive end-expiratory pressure of 2 to 4 cm H2O. Transpulmonary pressure, lung volume, and flow were determined. Lung resistance (RL) and dynamic compliance (Cdyn) were continuously computed (Labview; National Instruments, TX) by fitting flow, volume, and pressure to an equation of motion. MCh aerosol was administered for 10 s (60 breaths/min, 500 µl tidal volume) in increasing concentrations (6.25, 12.5, 25, 50, 100 mg/ml). Maximal values of RL and minimal values of Cdyn were taken and expressed as a percentage change from baseline following saline aerosol.
Bronchoalveolar Lavage (BAL)
Immediately after assessment of AHR, lungs were lavaged via the tracheal tube with Hanks' balanced salt solution (HBSS, 1 × 1 ml 37° C). Total leukocyte numbers were measured (Coulter Counter; Coulter Corporation, Hialeah, FL). Differential cell counts were performed by counting at least 300 cells on cytocentrifuged preparations (Cytospin 2; Shandon Ltd., Runcorn, Cheshire, UK), stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and differentiated by standard hematological procedures.
Histochemistry
Lungs were fixed by inflation (2 ml) and immersion in 10% formalin. Cells containing eosinophilic major basic protein (MBP) were identified by immunohistochemical staining as previously described using rabbit-anti-mouse MBP (kindly provided by Dr. J. Lee, Mayo Clinic, Scottsdale, AZ) (18). The slides were examined in a blinded fashion with a Nikon microscope equipped with a fluorescein filter system. Numbers of eosinophils in the perivascular, peribronchial, and peripheral tissues were evaluated using the IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh counting 6 to 8 different sections per animal. For detection of mucus containing cells in formalin-fixed airway tissue, sections (10 µm) were cut and stained with periodic acid-Schiff (PAS), hematoxylin and eosin.
Measurement of BALF Cytokines
Cytokine concentrations in the BALF supernates were measured by
ELISA. Briefly, 96-well plates (Immulon 2; Dynatech, Chantilly, VA)
were coated with either anti-interferon gamma (anti-IFN; R4-6A2),
anti-IL-4 (11B11), or anti-IL-5 (TRFK-5) (all Pharmingen, San Diego,
CA) and blocked with PBS/10% fetal calf serum (FCS) overnight.
Samples were added: biotinylated anti-IFN- (XMG 1.2), anti-IL-4
(BVD6-24G2), or anti-IL-5 (TRFK-4) were used as detection antibodies (all Pharmingen); and the reaction was amplified with avidin-
horseradish peroxidase (Sigma). Cytokine concentrations were determined by comparison with the known cytokine standards (Pharmingen). The limit of detection was 4 pg/ml.
Measurement of Total and OVA-specific Antibody
Serum levels of total IgE and OVA-specific IgE, IgG1, and IgG2a were measured by ELISA. Briefly, 96-well plates (Immulon 2; Dynatech, Chantilly, VA) were coated with either OVA (5 µg/ml) or purified anti-IgE (02111D; Pharmingen). After addition of serum samples, a biotinylated anti-IgE antibody (02122D; Pharmingen) was used as detecting antibody, and the reaction amplified with avidin-horseradish peroxidase (Sigma). IgG1 and IgG2a were detected using alkaline phosphatase-labeled anti-IgG1 (02003 E) and anti-IgG2a (02013 E) (both from Pharmingen). The OVA-specific antibody titers of the samples were related to pooled standards that were generated in the laboratory and expressed as ELISA units per ml (EU/ml). Total IgE levels were calculated by comparison with known mouse IgE standards (Pharmingen). The limit of detection was 100 pg/ml for total IgE.
Isolation of Spleen Mononuclear Cells and Fluorescent-activated Cell Sorter (FACS) Analysis
Spleens were harvested and mononuclear cells were purified by passing the tissue through a stainless steel mesh, followed by density gradient centrifugation (Organon Teknika, Durham, NC). The cells were washed three times in PBS and plated in 96-well round-bottom plates at 400,000 cells/well. After preincubation with mouse serum, cells were incubated with either fluorescein isothiocyanate (FITC)-conjugated (anti-CD3 clone 145-2C11, anti-B220 clone RA3-6B2, anti-CD23 clone B3B4) or phychoerythrin (PE)-conjugated antibodies (anti-CD4 clone RM4-5, anti-CD8 clone 53-6.7; Pharmingen) or isotype controls in staining buffer (PBS, 2% FCS, 0.1% sodium azide) for 30 min on ice. After washing, cells were examined (10,000 gated events were analyzed) using an EPICS XL analyzer (Coulter Electronics, Hialeah, FL). Results are expressed as the percentage of cells expressing a given surface marker.
Statistical Analysis
Analysis of variance (ANOVA) was used to determine the levels of difference between all groups. Comparisons for all pairs were performed by Tukey-Kramer honest significant difference (HSD) test. p Values for significance were set to 0.05. Values for all measurements are expressed as mean ± SEM.
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RESULTS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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AHR Is Abolished in STAT6/ Mice
To determine the effect of STAT6 deficiency on the development of AHR, OVA-sensitized and nonsensitized, STAT6+/+
and STAT6/ mice were challenged with an aerosol of
OVA. Forty-eight hours after allergen provocation, airway responsiveness (RL, Cdyn) to aerosolized MCh was measured
(17). Baseline RL and Cdyn measurements after saline challenge of nonsensitized OVA-challenged STAT6+/+ mice
were 0.88 ± 0.08 cm H2O · ml
1 · s (n = 10) and 0.045 ± 0.004 ml · cm H2O1 (n = 10), respectively, and did not differ significantly (p > 0.05) across the groups. STAT6+/+ mice sensitized and challenged with OVA developed AHR when compared with STAT6+/+ mice that received OVA challenge
alone (Figure 1). In contrast, STAT6/ mice that were sensitized and challenged with OVA developed no AHR when compared with STAT6/ mice challenged only, indicating
that STAT6 signaling was an absolute requirement for the development of allergic AHR in this model.
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Airway Eosinophilic Inflammation Is
Inhibited in STAT6/ Mice
The number and type of inflammatory cells in the airways of
STAT6+/+ and STAT6/ mice sensitized and challenged
with OVA were assessed in BALF (Figure 2). Total cell numbers recovered in BALF were essentially the same in all
groups of mice (OVA challenge only: STAT6+/+ 278 ± 53 × 103 cells/ml, n = 17; STAT6/ 295 ± 24 × 103 cells/ml, n = 19; OVA sensitization and challenge: STAT6+/+ 396 ± 60 × 103 cells/ml, n = 23; STAT6/ 234 ± 27 × 103 cells/ml, n = 15; p > 0.05). The inflammatory cell type recovered in the
BALF of the STAT6+/+ and STAT6/ mice that received
OVA challenge alone consisted almost entirely of macrophages. After OVA sensitization and challenge, STAT6+/+
mice developed a predominant eosinophilia (approximately
77% of total cells), with reduced macrophage number, and
only small changes in BALF lymphocyte and neutrophil numbers seen. STAT6/ mice, after OVA sensitization and
challenge, like STAT6+/+ mice, demonstrated a reduced
macrophage number with small increases in BALF lymphocyte and neutrophil numbers. However, in contrast to the
STAT6+/+ mice, only a small eosinophilic response was seen
(approximately 7% of total cells).
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Eosinophilic inflammation was further assessed in airway
tissues (Figure 3) using an antibody directed against MBP.
STAT6+/+ and STAT6/ mice that received OVA challenge
alone demonstrated little tissue eosinophilia (Figures 3a, 3b,
and 3e). OVA sensitization and challenge of STAT6+/+ mice
(n = 7) resulted in a striking perivascular (1,105 ± 36 cells/
mm2), peribronchial (933 ± 41 cells/mm2), and peripheral (811 ± 41 cells/mm2) eosinophilia (Figures 3c and 3e). In marked contrast, STAT6/ mice (n = 10) receiving OVA sensitization
and challenge developed only a small, nonsignificant (p > 0.05) perivascular (83 ± 8 cells/mm2), peribronchial (97 ± 10 cells/mm2), and peripheral (128 ± 34 cells/mm2) eosinophilia
(Figsures 3d and 3e). These results are consistent with our findings in the BALF and confirm the importance of STAT6 in the
development of the allergic airway eosinophilic inflammation.
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STAT6/ Mice Do Not Develop a Th2 Response in
the Lung After OVA Sensitization and Challenge
Concentrations of IL-4, IL-5, and interferon gamma (IFN-)
proteins in BALF were measured. Low levels of IL-5 and
IFN- but not IL-4 were present in both STAT6+/+ mice (IL-4: < 4 pg/ml, n = 15; IL-5: 24 ± 9 pg/ml, n = 15; IFN-: 137 ± 80 pg/ml, n = 9) and STAT6/ mice (IL-4: < 4 pg/ml, n = 13;
IL-5: 43 ± 18 pg/ml, n = 14; IFN-: 103 ± 45 pg/ml, n = 9)
that received OVA challenge alone (Figures 4a, 4b, and 4c).
After OVA sensitization and challenge of STAT6+/+ mice,
significantly (p < 0.05) elevated levels of IL-4 (37 ± 4 pg/ml,
n = 14) and IL-5 (356 ± 90 pg/ml, n = 19) but not IFN- (136 ± 50 pg/ml, n = 14), were present in BALF (Figures 4a, 4b, and
4c). This would suggest the development of a Th2-type response associated with the allergic AHR. After OVA sensitization and challenge of STAT6/ mice, only concentrations
of IFN- were significantly increased (627 ± 156 pg/ml, n = 16) indicative of a Th1 response. These observations denote
the profound effect STAT6 deficiency has on the generation of
Th2 cytokines, IL-4 and IL-5, in the airways after antigen sensitization and challenge and are consistent with the significant
inhibition of AHR and airway eosinophilia seen in STAT6/
mice.
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STAT6 Is Required for the Development of a Total IgE Response and the Production of Antigen-specific IgE and IgG1
Serum total IgE and OVA-specific IgE, IgG1, and IgG2a concentrations were measured. STAT6+/+ mice after challenge
alone had serum levels of total IgE of 853 ± 129 pg/ml (n = 12). After sensitization and challenge serum levels of total IgE
rose significantly to 2,701 ± 106 pg/ml (n = 18, p < 0.05). In
contrast, no significant concentrations of total IgE were detectable in STAT6/ mice challenged alone (n = 10) or
after sensitization and challenge (n = 16). No demonstrable
levels of OVA-IgE or OVA-IgG1 were found in the serum of
STAT6+/+ mice and STAT6/ mice that were OVA challenged only (Table 1). After OVA sensitization and challenge,
however, STAT6+/+ mice developed significant (p < 0.05)
serum levels of OVA-IgE and OVA-IgG1 (Table 1), consistent with our observations of a Th2 (elevated BALF IL-4, IL-5)
response in these mice. In contrast, OVA-sensitized and challenged STAT6/ mice did not develop an OVA-IgE response and only a small OVA-IgG1 response was seen (Table
1). However, STAT6/ mice, unlike STAT6+/+ mice, following sensitization and challenge, developed a striking IgG2a response (Table 1), consistent with increased amounts of IFN- in the BALF of these mice. Again these results highlight the
importance of STAT6 in the development of the Th2 response
and the impact of this on isotype switching of B cells.
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Expression of the Low-affinity IgE Receptor
(FcRII, CD23) Is Diminished in STAT6/ Mice
To examine the role of STAT6 on CD23 expression, the expression of CD23 on spleen mononuclear cells was assessed
(Figure 5). After OVA challenge only, spleen mononuclear
cells from STAT6+/+ mice demonstrated a low level (14 ± 3% total events, n = 7) of CD23 expression. In sensitized and
challenged STAT6+/+ mice, CD23 expression was increased
significantly (32 ± 2% total events, n = 6, p < 0.05). In contrast, the expression of CD23 on spleen mononuclear cells
from STAT6/ receiving OVA challenge alone (4 ± 1% total events, n = 6) was very low and did not increase after
OVA sensitization and challenge (6 ± 2% total events, n = 5).
Differences in CD23 expression did not seem to reflect a change in the relative numbers of T cells or B cells as the number of cells expressing the surface markers, CD3+, CD4+,
CD8+, and B220+ did not change significantly between groups.
This also highlights the normal development of T and B cells
in the STAT6/ mice.
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STAT6 Is Essential in the Development of Mucus Production
Lung sections were stained in order to identify mucus-containing cells in the airway epithelium of STAT6+/+ and STAT6/
mice. After sensitization and challenge, there were a large
number of cells staining positive for mucus in STAT6+/+
mice compared with control animals (Figures 6a and 6b). In
contrast, the airway epithelium of STAT6/ mice was devoid of cells staining positive for mucus (Figures 6c and 6d),
even in the sensitized and challenged group, indicating the importance of STAT6 in airway mucus production.
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Reconstitution of STAT6/ Mice with IL-5 Induces
Airways Eosinophilia and AHR
STAT6+/+ and STAT6/ mice were given murine rIL-5
(intravenously) on Days 1, 3, and 5 of OVA challenge.
STAT6/ mice that received OVA challenge alone did not
develop airway eosinophilia. In contrast, STAT6/ mice
that were sensitized and challenged with OVA and given IL-5
developed a marked AHR (Figure 7) and airway eosinophilia
in BALF (173 ± 52 ×103 cells/ml, n = 9; Figure 8). After reconstitution with IL-5, the degree of AHR was similar to that
observed in STAT6+/+ mice reconstituted with IL-5 (Figure
7). The number of eosinophils in the BALF was significantly
less than in STAT6+/+ mice reconstituted with IL-5 (Figure
8) although not significantly different from the number of
eosinophils in the BALF of STAT6+/+ mice without IL-5
(Figure 2). No significant differences in AHR or BALF eosinophilia were seen between STAT6+/+ mice with or without
IL-5 reconstitution.
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Of particular interest, IL-5-reconstituted, STAT6/ mice
still developed a Th1 response after sensitization and challenge, indicated by BALF levels of IL-4 (< 4 pg/ml, n = 7) and IFN- (647 ± 110 pg/ml, n = 8). Similarly, these mice did not demonstrate detectable concentrations of total IgE (n = 8), OVA-specific IgE, or OVA-specific IgG1 (n = 8) after sensitization
and challenge, but did develop a significant OVA-specific
IgG2a response (718 ± 110 EU/ml, n = 6). In addition, CD23
on spleen mononuclear cells from STAT6/ mice did not increase after OVA sensitization and challenge (5 ± 2% total
events, n = 6). Furthermore, after administration of IL-5, the
airway epithelium did not show significant staining for mucus-containing cells following sensitization and challenge despite
the demonstration of AHR (Figures 6e and 6f).
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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STAT proteins have been identified as a class of transcription
factors important in mediating cytokine-induced responses. Of those identified to date, only STAT6 is activated in response to IL-4 (12). Given the clinical and experimental relevance of this cytokine to the development of allergic asthma,
allergic airways inflammation, and AHR, we have investigated the role of STAT6 in mediating the development of
allergic airways inflammation and AHR using STAT6/
mice (4). After allergen (OVA) sensitization and challenge,
STAT6+/+ mice developed AHR and airway eosinophilic inflammation. These changes were associated with elevated serum levels of OVA-specific IgE and IgG1, and increased levels of IL-4 and IL-5 in BALF, indicative of a Th2-mediated
allergic response.
Association between eosinophil accumulation and activation
in the airways, disease severity, and AHR has led to the belief that the eosinophil is a major effector cell in allergic asthma. In contrast to their STAT6+/+ counterparts, STAT6/ mice
developed only a small, nonsignificant BALF and tissue eosinophilia, concordant with the lack of AHR in these animals.
Macrophage numbers were significantly reduced in both STAT6-sufficient and -deficient mice. The reason for this is not clear
but may reflect increased expression of intercellular adhesion
molecule-1 (ICAM-1) on the surface of activated macrophages
leading to increased adhesion to the airway epithelium (19).
These observations further support the role of the eosinophil
in the development of AHR in this model and more importantly, establish the critical role of STAT6 in these responses.
It is now apparent that CD4+ and CD8+ T cells, and the
Th2 cytokines IL-4 and IL-5 play an important role in the
pathogenesis of airway eosinophilia and AHR (2, 3, 7, 18,
20). The significant increase in BALF IL-4 and IL-5 concentrations, but not IFN- concentrations, in STAT6+/+
mice after OVA sensitization and challenge suggests the development of a Th2-mediated allergic response in these mice.
In contrast, STAT6-/- mice that did not develop airway eosinophilia and AHR after OVA sensitization and challenge,
showed little elevation of IL-4 and IL-5 levels in BALF, demonstrating the role of STAT6 in the generation of Th2 cytokines. The lack of a Th2 response is consistent with recent in vitro experiments with STAT6/ mice that suggest the
STAT6 pathway is the principal signaling pathway involved in
the IL-4 commitment of CD4+ T cells to the Th2 phenotype
(13, 14). Previous studies with IL-4-deficient mice have identified the importance of this cytokine in the commitment of T
cells to the IL-4-, IL-5-producing CD4+ Th2 phenotype and
the subsequent development of allergic airway eosinophilia
and/or AHR (5, 9, 11, 23). Administration of IL-4 neutralizing
antibody during sensitization but not during challenge prevents the development of the allergic airway eosinophilia and
AHR, indicating the primary role of IL-4 during the initial priming of T cells for commitment to the Th2 phenotype (9-
11). Our observations highlight the critical role of the STAT6
pathway in this process. In contrast, STAT6/ mice developed a Th1 response to allergen sensitization and challenge,
shown by the marked increase in BALF IFN- levels which is
itself known to downregulate the Th2 phenotype and the development of airways eosinophilia and AHR (24).
IL-4 is essential for immunoglobulin isotype switching in B
cells to IgE and IgG1, whereas IFN- stimulates IgG2a isotype switching (4, 5, 25). In the present study, substantiating the development of the Th2 response, sensitized and challenged STAT6+/+ mice developed allergen (OVA)-specific
IgE and IgG1 responses. In contrast, STAT6/ sensitized
and challenged mice produced no allergen-specific IgE but did
develop a significant IgG2a response consistent with the Th1
response seen in these mice. These observations again delineate the important role of STAT6 in the development of the
Th2 response and the impact on isotype switching in B cells. The role of allergen-specific IgE in airway eosinophilia and
AHR is nevertheless controversial. Studies in IL-4-deficient
mice and with IL-4 neutralizing antibody, as in the present
study, demonstrate an absence or reduced levels of serum IgE
as well as abrogated airways eosinophilia and AHR (9, 10, 23). One mechanism could be absence of IgE-mediated mast cell
activation which may lead to bronchoconstriction and enhanced airway responsiveness (26). However, a number of
studies have shown that mast cell-deficient mice develop normal airway eosinophilia and AHR in response to allergen sensitization and challenge (17). Further, it has been shown that
after systemic sensitization and repeated airway challenge, B
cell-deficient mice and CD40-deficient mice develop airways eosinophilia and AHR without antibody production (11, 27).
IL-4 is important in regulating CD23 expression on B cells,
antigen presenting cells, and eosinophils at least in humans. CD23 can regulate IL-4-induced IgE synthesis, and has been
implicated in cellular adhesion as well as antigen presentation
(28). In the present study, spleen mononuclear cells from
STAT6+/+ mice demonstrated a low constitutive expression
of CD23 which significantly increased after allergen sensitization and challenge. In contrast, constitutive expression of
CD23 in STAT6/ mice was very low and did not increase
after sensitization and challenge. As anti-CD23 antibody has
been shown to inhibit allergen-induced airways eosinophilia and AHR, it may be postulated that the downregulation of
CD23 in STAT6/ mice contributed to the lack of airway
eosinophilia and AHR (29). However, this is unlikely because
in a similar model CD23-deficient mice show normal (even increased) airway eosinophilia and AHR after sensitization and
challenge (30).
IL-4, and by implication STAT6, may affect other functions
that may be involved in the development of AHR. IL-4 is
known to upregulate the expression of the cellular adhesion
molecule, VCAM-1 facilitating eosinophil recruitment (7).
The importance of STAT6 in this process was not assessed in
the present study. Previous observations with anti-IL-4 antibody suggest only a partial inhibition of VCAM-1 expression
after allergen sensitization and challenge, suggesting the presence of IL-4-independent pathways in the upregulation of
VCAM-1, and thus may not account for the abrogation of airways eosinophilia seen in STAT6/ mice (31). More recent
observations have confirmed that STAT6 is not essential for
the allergen-induced expression of VCAM-1 (15). There is evidence to suggest that IL-4 regulates mucus cell hyperplasia
(8). Increased mucus secretion seen in the asthmatic airways
may contribute to AHR (32). Increased mucus cell staining
was observed after sensitization and challenge of STAT6+/+
mice. In contrast, no mucus cell staining was seen in the airway epithelium of STAT6/ mice, demonstrating the importance of IL-4 and STAT6 in this process.
STAT6 appears to be essential for many of the IL-4-mediated responses that may impact on the development of AHR.
The importance of STAT6 is further highlighted by its role in
IL-13-mediated responses. STAT6 is known to be activated
by IL-13, and IL-13-mediated responses are inhibited in
STAT6/ mice (14, 33). This cytokine, which shares the IL-4
receptor chain, shares many of the biological functions of
IL-4, including enhanced CD23 and MHC class II expression on B cells and IgE isotype switching in B cells (14, 34, 35).
The key to defining the major events affected by STAT6 deficiency accounting for the absence of airway eosinophilia and AHR lies in the reconstitution experiments with IL-5. The importance of IL-5 in the differentiation, activation, and survival of eosinophils has been clearly identified (3). In addition, several in vivo studies using either IL-5-deficient mice or neutralizing antibody to IL-5 have also identified the crucial role of IL-5 in the development of airway eosinophilia and, in some studies, the subsequent development of AHR after allergen sensitization and challenge (10, 11, 18, 22).
Therefore to distinguish among the many functions of IL-4
on IgE production, CD23 and VCAM-1 expression, mucus
production, and the commitment of T cells for IL-5 production, which may contribute to AHR, sensitized STAT6/
mice were reconstituted with IL-5 alone during allergen challenge. IL-5-reconstituted STAT6/ mice developed a marked
airway eosinophilia and AHR. No such responses occurred in
nonsensitized STAT6/ mice reconstituted with IL-5. These
observations suggest that independently of an allergen-specific IgE response, mucus production, or CD23 upregulation,
airways eosinophilia and AHR could develop after IL-5 administration alone. These observations highlight the importance of STAT6 in the IL-4-dependent signaling of T cells to
the Th2 phenotype and secretion of IL-5, resulting in airway
eosinophilia and AHR.
Interestingly, IL-5 alone was not sufficient to induce airway
eosinophilia and AHR in the absence of prior sensitization, implying that prior antigen presentation and perhaps T-cell
priming were necessary. This would suggest that a second signal is required for the development of the response which is
not IgE but which may be provided directly or indirectly by
activated T cells. In STAT6+/+ mice, a Th2 response developed after sensitization and challenge which generates IL-5 to
drive the eosinophilic response. However, in STAT6/
mice, sensitization and challenge resulted in a Th1, IFN-, and
IgG2a response. Nevertheless, in these mice in the presence of
exogenous IL-5 this was sufficient to generate airway eosinophilia and AHR.
During the preparation of this report two studies outlining the role of STAT6 in the development of AHR were published (15, 16). Both studies demonstrate an important role for STAT6 in the development of AHR to intravenous acetylcholine, consistent with our own observations with aerosolized MCh. However, Kuperman and colleagues using STAT6-deficient mice on a BALB/c background demonstrated only partial inhibition (approximately 50%) of BALF eosinophilia, questioning the role of the eosinophil in the development of AHR in their model (15). Lung eosinophilia or eosinophil activation were not examined. These results are in direct contrast to our own and those of Akimoto and coworkers who using STAT6-deficient mice on a C57BL/6 background demonstrated almost complete inhibition of BALF eosinophilia (> 90%) (16). The differences seen in these studies may reflect both differences in sensitization and challenge and/or strain differences. Similar reasons may explain the apparent discrepancies in the literature as to the role of IL-4, IL-5, and eosinophils in the development of AHR (9, 10).
Importantly, our studies on reconstitution of STAT6-deficient mice with IL-5 extend previous observations in STAT6/
mice much further and distinguish among the contributions of
IgE/IgG1, mucin production, CD23, and IL-5/airway eosinophilia in the development of AHR.
In summary, these data demonstrate the critical role of
STAT6 in the development of IL-5 production and subsequent airway eosinophilia and AHR. The finding that IL-5
alone can reconstitute airway eosinophilia and AHR in sensitized and challenged STAT6/ mice, emphasizes the essential nature of the IL-4/IL-13-dependent signaling pathway in
these responses. It is therefore apparent that this signaling
pathway may be important in the regulation of many of the
key features of allergic asthma, particularly AHR and chronic
airways eosinophilic inflammation. It appears that IL-4/13 are
central to these processes as they are critical to the development of a Th2 type T-cell response that will produce IL-5, crucial for the development of an airways eosinophilic inflammation, that in turn will lead to epithelial damage and enhanced
airway responsiveness. In our own studies it would appear that
IgE was less important in modulating the development of airways eosinophilia and AHR, however, IgE may well be important in acute disease exacerbations of asthma, and could also
contribute to the ongoing chronic inflammation. As our data
show that STAT6 is critical for the IgE response, this highlights the importance of targeting the STAT6 pathway in the
development of new antiallergic asthma drugs.
| |
Footnotes |
|---|
Correspondence and requests for reprints should be addressed to Erwin W. Gelfand, M.D., Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: gelfande{at}njc.org
(Received in original form September 14, 1998 and in revised form January 27, 1999).
Acknowledgments: The authors thank Dr. J. N. Ihle (St. Jude Children's Research Hospital, Memphis, TN) who provided the STAT6-deficient mice and Dr. J. J. Lee (Mayo Clinic, Scottsdale, AZ) for providing the anti-MBP antibody.
Supported by NIH Grant HL-36577 and a grant from the Allergy and Immunology Institute of the International Life Sciences Institute Research Foundation. The opinions expressed herein are those of the authors and do not necessarily represent the views of the International Life Sciences Institute Research Foundation.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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