By
From the * Section of Immunobiology and Section of Pulmonary and Critical Care Medicine, § Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520;
Pathology and Laboratory Medicine Service and ¶ Section of Pulmonary Medicine, Veterans
Administration, Connecticut Health Care System, West Haven, Connecticut 06516; and the ** Howard Hughes Medical Institute, New Haven, Connecticut 06536
Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased
mucus secretion is an important clinical symptom and contributes to airway obstruction in
asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic
for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked
increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in
mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4-deficient mice
were not recruited to the lung and did not induce mucus production. When this defect in
homing was overcome by administration of TNF-, IL-4
/
Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is
crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.
Asthma is a chronic inflammatory disease of the bronchial airways defined by intermittent episodes of airway obstruction. Patients with asthma present with wheezing and cough productive of mucous secretions. Mucus
hypersecretion is an important cause of airway obstruction
in asthma (1, 2). Airway biopsies show infiltration of the
mucosa and submucosa with lymphocytes and eosinophils
and hyperplasia of goblet cells and submucosal glands. In
autopsy specimens from patients who died in status asthmaticus, obstructing plugs of mucus and cellular debris
have been identified in the small airways.
In asthma, mucus hypersecretion is believed to result, at
least in part, from inflammation, although the mechanisms
by which mucus is induced have not been detailed. Studies
have shown that the volume of expectorated sputum in
asthmatics correlates with the number of eosinophils in the
sputum (3). In addition, sputum DNA levels correlate with
mucus production (4). A variety of inflammatory mediators
have been shown to stimulate mucus secretion including
histamine, prostaglandins, leukotrienes, platelet activating factor, and eosinophil cationic protein (5, 6). The cytokines IL-4 and IL-5 have recently been shown to effect airway
epithelial mucus production. Transgenic mice that overexpress IL-4 or IL-5 in the lung each showed a marked increase in mucus in the airway epithelium (7, 8). It is unclear
if IL-4 and/or IL-5 act directly on the airway epithelium or
if these cytokines are some of many inflammatory mediators involved in mucus production.
The regulation of airway epithelial cell mucus production by CD4 T cells has not been studied, although CD4 T
cells are thought to play a primary role in initiating the inflammatory cascade that leads to asthma. Activated CD4 T
cells have been identified in bronchial biopsies and bronchoalveolar lavage fluid of asthmatic patients (9), and
animal models have shown that depletion of CD4 T cells
inhibits airway inflammation (12, 13). Antigen-activated CD4
T cells can differentiate into effector cells with distinct functional properties (14). Th1 cells are a subset of
CD4 T cells which secrete the potent macrophage activating cytokine, IFN- Th2 cells secreting IL-4 and IL-5 have been shown to be
present and activated in the bronchial wall of asthmatic individuals (9, 23). The presence of activated Th2 cells and
eosinophils in the airways of asthmatics have been associated with more severe airway hyperresponsiveness (24, 25).
Despite considerable correlative evidence supporting a role
for Th2 cells in the pathogenesis of asthma, a cause and effect relationship has not been shown. Furthermore, IFN- To better understand the role of CD4 T cells in the development of airway pathology in asthma, we developed an
in vivo system in which antigen-specific Th1 or Th2 cells
were produced, and their ability to influence airway inflammation and airway epithelial mucus production was investigated. From these studies we show that Th2, but not
Th1 cells, induce an inflammatory response which is strikingly similar to the pathologic features observed in human asthmatics. Th2 cells, but not Th1 cells induce mucus hypersecretion in the bronchial epithelium. Furthermore, using Th2 cells deficient in IL-4, we show that IL-4 is necessary for Th2-induced airway inflammation. Although current
evidence suggests that IL-4 has a role in inducing mucus
production by bronchial epithelial cells, we show that Th2-like cells can stimulate mucus production in the absence of
IL-4.
Mice.
DO11.10 mice, which are transgenic for the TCR recognizing OVA peptide 323-339 (pOVA323-339) (30), were provided to us on BALB/c background by Ken Murphy (Washington University, St. Louis, MO) and were bred in our facilities. IL-4-deficient BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were bred in our facilities. Transfer recipients and immunizations were performed on 6-12-wk-old BALB/c mice (Jackson
ImmunoResearch Labs., West Grove, PA).
Generation of Th1 or Th2 Cells.
To generate Th1 or Th2 cells
from DO11.10 mice, CD4 T cells were isolated by negative selection as previously described (31) using mAbs to CD8 (clone
53-6.72, clone 2.43 [32]), Class II MHC I-Ad (212.A1 [33]) and
anti-Ig-coated magnetic beads (Advanced Magnetics, Inc. Cambridge, MA). Syngeneic T-depleted splenocytes were used as
APC and prepared by negative selection using antibodies to CD4
(GK1.5 [34]), anti-CD8, anti-Thy1 (35) and treatment with rabbit complement. APCs were mitomycin-C treated. To induce
Th2 cells from nontransgenic mice, IL-4-deficient mice and
BALB/c mice were injected intraperitoneally with 100 µg of
OVA (Sigma Chemical Co., St. Louis, MO) in 4.5 mg of alum
(Immuject; Sigma). 4 d after immunization, spleens and local
draining lymph nodes were harvested and CD4 T cells were isolated. All cultures were set up in flasks containing equal numbers
of CD4 T cells and APCs at a concentration of 2-4 × 106 cells/
ml. To generate Th1 cells, cultures contained pOVA323-339 at 5 µg/ml, IL-12 at 5 ng/ml (Genetics Institute, Cambridge, MA), IL-2
at 10 U/ml (Collaborative Research Inc., Waltham, MA), and anti-
IL-4 (11B11 [36]) at inhibitory concentration. To generate Th2
cells, cultures contained pOVA323-339 at 5 µg/ml for DO11.10 cells
or OVA at 50 µg/ml for nontransgenic CD4 T cells, IL-4 at 200 U/ml (Collaborative Research, Inc.), IL-2 at 10 U/ml, and anti-
IFN- Transfer of Cells and Aerosol Administration of OVA.
Cultured Th1
or Th2-like cells were harvested after 4 d, washed with PBS and
5 × 106 cells were injected intravenously into syngeneic BALB/c
recipients. Control mice received freshly isolated CD4 T cells
from naive BALB/c mice or in some experiments no cells were
transferred. In experiments in which mice received TNF- Lymph Node Isolation and Bronchoalveolar Lavage.
Lung draining
lymph nodes (peribronchial and parathymic), were isolated and
single cell suspensions prepared for FACS®. BAL was performed
by cannulation of the trachea and lavage with 1 ml of PBS.
Cytospin preparations of BAL cells were stained with Dif-Quik
(Baxter Healthcare Corp., Miami, FL) and differentials were performed based on morphology and staining characteristics. Isolation of lung lymphocytes was performed after BAL and perfusion of blood from lungs. Lung tissue was passed through a wire mesh, digested with collagenase Type IV 150 U/ml (Worthington Biochemical Corp., Freehold, NJ), elastase 10 U/ml (Sigma), and
DNase 10 U/ml (Sigma) for 1 h at 37°C and passed again through
a wire mesh to dissociate cells. Cell preparations were subjected
to FACS® analysis.
Cytokine Production by Th1 or Th2 Cells.
At the time of transfer, an aliquot of Th1- or Th2-like cells or naive CD4 T cells
were retained for restimulation. 1 × 106 CD4 T cells/ml and 1 × 106/ml freshly isolated BALB/c APCs were cultured with OVA
(50 µg/ml) or pOVA (5 µg/ml). Supernatants were collected at
48 h. BAL cells obtained from individual mice were restimulated
in vitro at 2 × 106 cells/ml in the presence of pOVA (5 µg/ml).
FACS® analysis was performed on BAL cells to determine the
percentage of D011.10 transgenic CD4 T cells and the amount of
cytokine per ml per 106 transgenic T cells was calculated.
Cytokine Assays.
IFN- FACS® Analysis.
At the time of transfer, FACS® analysis was
performed on Th1, Th2, and naive CD4 T 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 antibody, KJ1-26 (38), and fluorescein isothiocyanate-avidin D (Vector Laboratories, Burlingame, CA). KJ1-26 is specific for the transgenic T cell receptor in the DO11.10 mice. After
a period of inhalational exposure, PBMC, LN, lung, and BAL
cells were also analyzed by FACS® using these antibodies.
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 and stained with hematoxylin and eosin (H & E), Giemsa,
diastase-periodic acid-Schiff (DPAS), mucicarmine, and alcian blue.
Immunohistochemistry was performed on lungs perfused and fixed
with periodate-lysine-pyrophosphate as previously described (39).
Sections were stained with antibodies to MHC Class II, CD4,
and the transgenic TCR using KJ1-26.
Histological Mucus Index.
Formalin-fixed, paraffin embedded
lungs were sectioned in the coronal plane at 5 microns. Lungs were
sectioned to ensure that central airways were visible. After staining with DPAS, marker dots in a grid with 2-mm spacing were
placed over the entire lung section. The slide was examined at
100× final magnification on an Olympus BH-2 microscope
(Olympus, Tokyo, Japan) with a rectangular 10-mm square reticule grid (American Optical Corp. Buffalo, NY) inserted in one
eyepiece. Each marker dot was placed in the lower left corner of
the field, and all intersections of airway epithelium with the reticle grid were counted in that field, distinguishing mucus containing or normal epithelium. Approximately 25% of the total lung
section was scored. The ratio of total number of mucus positive
intersections and the total of all intersections, which we call the
histologic mucus index (HMI), is equivalent to the linear percent
of epithelium positive for mucus (40). This index was calculated
for each mouse lung and then the mean of the HMI was calculated for each experimental group.
To investigate the effects of different T cell
subsets on airway pathology, Th1 and Th2 cells were generated from CD4 T cells isolated from DO11.10 mice
which are transgenic for the TCR recognizing pOVA 323-339.
Using a standard procedure to polarize CD4 T cell responses (41), splenic CD4 T cells were stimulated by
pOVA323-339 in the presence of IL-12, IL-2, and anti-IL-4
to induce Th1-like cells or IL-4, IL-2, and anti-IFN-
To determine whether Th1 or Th2 CD4 T cells
could be recruited to the lung as committed effector cells,
DO11.10 Th1 or Th2 cells were transferred intravenously
into syngeneic BALB/c mice. A control group of mice received unprimed naive CD4 T cells. OVA was then administered to the mice by aerosol inhalation to recruit OVA-specific cells to the respiratory tract and to activate
the cells locally. A control group of mice received aerosolized PBS. After 7 d of aerosol exposure, leukocytes from
peripheral blood, lung-draining lymph nodes, BAL, and
lung tissue were isolated and analyzed by FACS® to determine the contribution to inflammation of the transferred population of transgenic DO11.10 CD4 T cells (Table 1).
While the donor TCR transgenic cells were not detectable
in PBMC or uninvolved lymph nodes after aerosolized
OVA, the transgenic TCR was expressed on CD4 T cells
of the draining LN (8 and 13%, Th1 and Th2). Even more
notable were the cells recovered from BAL and lung which
expressed the donor transgenic TCR on a majority of CD4
T cells after aerosolized OVA administration. However, TCR transgenic cells were not seen in the lung, BAL, or
lung draining LN after PBS administration. Thus, in the
mice that received donor DO11.10 CD4 T cells and exposure to inhaled OVA, Th1 and Th2 cells were both recruited selectively to the lung and local LN with similar efficiency.
Table 1.
FACS® Staining of Mononuclear Cells from Mice that
Received TCR Transgenic CD4 T cells
. CD4 Th2 cells make a different panel
of cytokines, including IL-4, IL-5, and IL-10 (17, 18). Th2
cells are potent in activating B cells to secrete antibody,
particularly IgE (19). IL-5 secretion by Th2 cells also
influences eosinophil differentiation, maturation and endothelial adherence (22).
has been identified in bronchoalveolar lavage (BAL)1 fluid
and serum of asthmatic patients suggesting that Th1-like cells may contribute to pathology in this disease (26).
(XMG1.2 [37]) at inhibitory concentration. Cultures were
maintained for 4 d.
, 2 µg
of TNF-
(R&D Systems, Minneapolis, MN) was administered
intranasally in 50 µl PBS to anesthetized animals at the time of
cell transfer. 1 d after transfer of cells, mice were challenged with
inhaled 1% OVA in PBS or PBS alone for 20 min daily for 7-10 d.
Mice were placed in a plastic chamber (27 × 20 × 10 cm) fitted
with an attachment to allow entry of the aerosol from an ultrasonic nebulizer (1-5-µm particles by manufacturer's specifications, OMRON; UltraAir NE-U07, Vernon Hills, IL). A small
hole on the opposite end of the chamber ensured continuous airflow.
, IL-4, IL-5, and IL-10 levels from cell
supernatants were determined by ELISA (Endogen, Cambridge,
MA). Assays were standardized with recombinant IFN-
, IL-5, IL-10
(Endogen), and IL-4 (Collaborative Research, Inc.). The lower
limit of sensitivity for each of the ELISAs was 0.6 ng/ml (IFN-
),
5 pg/ml (IL-4), 0.010 ng/ml (IL-5), and 200 pg/ml (IL-10).
Generation and Characterization of OVA-specific Th1 and
Th2 Cells.
to
induce Th2-like cells. 99% of the resulting activated Th1
or Th2 effector cells expressed CD4 and the DO11.10
TCR, recognized by the clonotypic monoclonal antibody,
KJ1-26. An aliquot of cells from each culture was restimulated in vitro in the presence of pOVA323-339 and APCs,
and supernatants were assayed for IFN-
, IL-4, IL-5 (Fig. 1
A), and IL-10 (data not shown). The CD4 T cells stimulated to differentiate into Th1 cells produced high levels of
IFN-
and undetectable IL-4, IL-5, and low levels of IL-10,
while the cells stimulated to differentiate into Th2 cells secreted high levels of IL-4, IL-5, IL-10, and minimal IFN-
. Thus, we generated CD4 Th1 and Th2 effector cells responsive to OVA peptide. Naive CD4 T cells (N) freshly
isolated from BALB/c mice did not secrete cytokines in response to OVA.
Fig. 1.
Cytokine production by Th1 or Th2-like cells before and after transfer. (A) At the
time of transfer into recipient
mice in vitro generated
DO11.10 CD4 Th1, Th2 or
freshly isolated CD4 T cells from
naive BALB/c mice (N) were
cultured with antigen presenting cells (2 × 106 cells/ml) in the
presence of pOVA323-339. (B) After 7 d of exposure to inhaled
OVA, BAL was performed on
individual mice that received
Th1 (Th1-OVA), Th2 (Th2-OVA), or naive (N) CD4 T
cells. Total leukocytes recovered
from BAL were restimulated in vitro with pOVA323-339. BAL cells from mice that received naive CD4 T cells (N) and inhaled OVA contained <3% lymphocytes and were insufficient for cytokine analysis. Supernatants were collected at 48 h and cytokine ELISAs were performed. Cytokines production in
BAL was adjusted for 106 DO11.10 CD4 T cells per ml as determined by FACS® analysis. Mean cytokine levels (±SEM) are shown (n = 4-5 mice per
group). One experiment is shown and is representative of three experiments.
[View Larger Version of this Image (16K GIF file)]
Aerosol
exposure
Source of cells
Donor cells*
Th1
Th2
N
% KJ1-26+ CD4
cells (range)
In vitro primed cells
99
99
at time of transfer
PBS
Lung-draining LN
<1
<1
<1
PBS
BAL
OVA
PBMC
<1
<1
<1
OVA
Inguinal, mesenteric LN
Not tested
<1
<1
OVA
Lung-draining LN
13 (8-16)
8 (4-10)
<1
OVA
BAL
69 (61-77)
57 (50-60)
<1
OVA
Lung
74
73
<1
*
DO11.10 TCR transgenic Th1, Th2, or naive (N) CD4 T cells were
transferred into BALB/c mice, and the mice were exposed to 7 d of
aerosolized OVA or PBS. FACS® analysis was performed after aerosol
exposure to PBS or OVA, as denoted. Cells were stained with anti-CD4 and KJ1-26 antibodies. Values are the mean percent (range) for
individual Th1 or Th2 mice (3-5 mice/group). Lung lymphocytes
were isolated and pooled from three mice.
After transfer of CD4 Th1, Th2, or naive cells and administration of
PBS, BAL fluid contained too few CD4 T cells for these studies.
To determine if the donor cells retained the polarized
cytokine profiles they exhibited at the time of transfer,
BAL was performed on mice after 7 d of exposure to aerosolized OVA, and recovered cells were restimulated with
OVA. As seen in Fig. 1 B, BAL cells from the primed mice
that received Th1 cells (Th1-OVA) produced high levels
of IFN- and minimal IL-4, IL-5, and IL-10 (data not shown) upon restimulation, while BAL cells from OVA-primed mice that received Th2 donor cells (Th2-OVA)
produced IL-4, IL-5, and IL-10. BAL cells from control
mice that received naive CD4 T cells and were exposed to
inhaled OVA contained too few lymphocytes for restimulation.
Thus, after transfer and exposure to aerosolized OVA, OVA-reactive CD4 Th1 or Th2 cells were recruited to the respiratory tract and were reactivated to secrete the cytokines they exhibited at the time of transfer. The transferred cells maintained their commitment to the secretion of Th1 or Th2 cytokines in response to OVA as has been described previously (41).
Airway Inflammation in Mice that Received Th1 or Th2 Cells.To investigate how different effector CD4 T cells
influence inflammation in the respiratory tract, mice that
received Th1, Th2, or unprimed naive CD4 T cells were
compared. Mice that received Th1 or Th2 cells and were
exposed to aerosolized OVA had moderate inflammation
in the respiratory tract (Fig. 2, A1 and B1). The lungs from
both groups of mice showed inflammation in a predominantly peribronchial and perivascular pattern. Despite similar degrees of inflammation, the two groups of mice had
strikingly different inflammatory processes as determined by
lung histology and analysis of BAL cells. The inflammatory
infiltrates in the lungs of mice that received Th1 cells consisted of neutrophils, small mononuclear cells and macrophages (Fig. 2 B1). Differential counts performed on cells
recovered from BAL confirmed these findings (Fig. 3). Immunohistochemical analysis of lung tissue showed that many
infiltrating inflammatory cells stained with an anti-CD4 antibody (Fig. 4 A), the majority of these cells also stained with
the TCR anticlonotypic antibody, KJ1-26 (Fig. 4 B). MHC
Class II expression in the lungs of these mice was increased
on bronchial epithelial cells (Fig. 4 C) when compared to
mice that received Th2 cells (Fig. 4 D), as expected from
the effects of IFN- on airway epithelial cells (42).
In mice that received Th2 cells, the inflammation showed a large proportion of infiltrating eosinophils (Fig. 2 B2). By immunohistochemistry, CD4 positive cells were seen as the other predominant cell population in the inflammatory infiltrate, and a majority of CD4 expressing cells stained with the KJ1-26 antibody (data not shown). BAL cell counts show that eosinophils and lymphocytes were the predominant inflammatory cells present (Fig. 3). A minority of cells in the BAL fluid were neutrophils after 7 d of exposure to inhaled OVA. When exposures were carried out for 10 d, the percentage of neutrophils in BAL fluid decreased to zero, while mice that received Th1 cells and inhaled OVA had persistence of neutrophilia (data not shown).
Mice that received Th1 or Th2 cells and were exposed to aerosolized PBS had no lung pathology (data not shown). Mice that received naive CD4 T cells and aerosolized OVA had no significant lung inflammation (Fig. 2 D). Mice that received Th1 or Th2 cells and inhaled OVA did not exhibit histopathologic evidence of inflammation in other organs (data not shown).
Increased Mucus Staining and Secretion in Lungs of Mice that Received Th2 but Not Th1 Cells.Bronchial epithelial cells
in mice that received OVA-specific Th2 cells and inhaled
OVA showed hyperplasia and extensive DPAS positive
staining indicating the presence of mucin collections (Fig. 2
B3). These findings were most striking in the central airways, although peripheral airways were also involved. Histological sections also showed increased amounts of mucus
within the airway lumena of mice that received Th2 cells
and aerosolized OVA. The material also stained positive for
mucicarmine and alcian blue (data not shown). Mice that
received Th1 cells and inhaled OVA had minimal to absent
mucus staining (Fig. 2 A3). An HMI performed on lung
sections from mice that received Th2 cells and inhaled OVA
showed that 65% of airway epithelial cells were mucinous, while mice that received Th1 cells and inhaled OVA or naive CD4 T cells and inhaled OVA had <5% of mucinous
cells in the airways (Fig. 5).
Thus, mice that received transfer of OVA-specific Th2 cells and exposure to inhaled OVA had markedly increased mucus staining in the bronchial epithelium. Transfer of Th1 or Th2 cells and exposure to inhaled OVA resulted in a comparable level of inflammation, albeit comprising different cell populations, but only Th2 cells stimulated bronchial epithelial mucus production.
IL-4 Production by Donor T Cells Is Critical for Th2 Cell-induced Lung Inflammation.Previous studies had shown
that IL-4 overexpression in the airways resulted in mucus
hypersecretion (7). To investigate the precise mechanism
by which Th2 cells induced increased mucus staining, we
began by studying the role of IL-4 in these processes. OVA-specific Th2 cells from IL-4-deficient (IL-4 /
)
mice or wild-type (IL-4 +/+) BALB/c mice were generated as has been described previously (41, 43). Mice were
immunized with OVA in alum, and primed CD4 T cells
were isolated and stimulated in vitro for 4 d with OVA in
the presence of IL-4 and anti-IFN-
. An aliquot of cells
from each culture was restimulated in vitro in the presence of OVA and APCs, and supernatants were assayed for IFN-
,
IL-4, IL-5, and IL-10 (Table 2). IL-4
/
OVA-specific
Th2 cells produced comparable levels of IL-5 and IL-10
when compared to IL-4 +/+ OVA-specific Th2 cells, but
IL-4 was produced only by IL-4 +/+ Th2 cells. CD4 Th2
cells were then transferred into BALB/c IL-4 +/+ recipients and the mice were exposed to inhaled OVA for 7 d.
Control mice received no transferred cells and were exposed to inhaled OVA. In contrast to mice that received
IL-4 +/+ Th2 cells, IL-4
/
Th2 cells did not induce
significant lung inflammation or mucus production (Fig. 6).
Mice that received IL-4
/
Th2 cells and inhaled OVA
had a 10-fold reduction in total BAL cell recovery compared to mice that received IL-4 +/+ Th2 cells. Furthermore, eosinophils and lymphocytes were strikingly reduced
in the BAL fluid. These data suggested that IL-4
/
Th2
cells had either died or that production of IL-4 by the donor Th2 cell was necessary for its entry into the lung. Since
staining of tissue sections revealed that VCAM-1 expression
was markedly reduced in mice that received IL-4
/
Th2
cells and inhaled OVA (data not shown; Cohn, L., manuscript in preparation) and since VCAM-1 expression on lung endothelium has previously been shown to be critical
for both lymphocyte and eosinophil recruitment to the
lung (44), these experiments suggested that the transferred
IL-4
/
Th2 cells were not recruited to the lung because
of a defect in lymphocyte-endothelial adhesion.
IL-4 Is not Critical for Mucus Induction Once Cells Have Been Recruited to the Lung.
To test if induction of adhesion molecules on the vascular endothelium would facilitate recruitment of OVA-specific IL-4 /
Th2 cells to
the lung, we transferred OVA-specific IL-4 +/+, IL-4
/
Th2 cells or naive CD4 T cells into mice and treated the
mice with inhaled TNF-
. TNF-
has previously been
shown to increase both VCAM-1 and E-selectin expression
on lung endothelium (45). Mice were then exposed to 7 d
of inhaled OVA. As seen in Fig. 7 B, inflammation, as indicated by increased numbers of cells in the BAL, was observed in the mice that received IL-4 +/+ and IL-4
/
Th2 cells, whereas mice that received naive CD4 T cells and TNF-
had minimal inflammation. Mucus staining
was increased in the bronchial epithelium of mice that received IL-4 +/+ or IL-4
/
Th2 cells and exposure to
inhaled OVA (Fig. 7 A). Mice that received transfer of naive CD4 T cells, TNF-
and inhaled OVA did not show
mucus staining in the bronchial epithelium.
OVA-specific IL-4 +/+ Th2 cells and IL-4 /
Th2
cells both induced eosinophilic inflammation, although IL-4
+/+ Th2 cells induced a greater degree of airway eosinophilia (Fig. 7 B). This may the result of more effective entry
into the lung of IL-4- and IL-5-producing Th2 cells or to
a greater stability of this Th2 population compared to IL-4
/
Th2 cells, since it is known that IL-4 is important
for persistence of Th2 cell populations early after polarization (46).
These results show clearly that donor IL-4-deficient Th2
cells can stimulate mucus production in the airway. However, to rule out a possible role of endogenous IL-4 secretion in mucus production, we transferred IL-4 +/+ and
IL-4 /
Th2 cells into IL-4-deficient recipient mice.
Mice were exposed to TNF-
and then aerosolized OVA
for 7 d. As seen in Fig. 8, there was persistent induction of
mucus in the airway epithelium in mice that received IL-4
/
Th2 cells in the absence of any recipient IL-4.
Clearly, IL-4 secreted by CD4 T cells or by other cells recruited in the inflammatory process is not necessary to
stimulate mucus production in the airway epithelium.
Th2 cells, through the production of cytokines after specific antigen stimulation, have been hypothesized to initiate a cascade of events that leads to asthma. Although less widely acknowledged, BAL cells from asthmatic subjects have been shown to produce Th1-like cytokines (26). Our objective was to determine whether CD4 Th2 or Th1 cells in isolation could reproduce some of the inflammatory changes associated with asthma in a direct transfer model, and whether these inflammatory change would influence other important pathologic processes in asthma, specifically mucus hypersecretion. To do this we set up in vitro cultures designed to generate populations of cells containing a high frequency of antigen-specific Th1 or Th2 cells. The populations of CD4 effector cells we generated secreted cytokines in patterns that define them as Th1 or Th2. By biasing the generation towards Th1 or Th2 cells, this technique limits the potential for inducing mixed Th1 and Th2 populations typically generated by systemic immunization with antigen. Once CD4 Th1 or Th2 cells were generated, they were transferred into recipient mice that were then challenged with inhaled antigen.
We demonstrate that OVA-specific CD4 Th1 or Th2 cells can be recruited and activated in the respiratory tract and their activation results in different inflammatory pathology. Th2 cells activated in the respiratory tract result in pulmonary eosinophilia and mucus hypersecretion. Mice that received CD4 Th1 cells and inhaled OVA have comparable degrees of inflammation, but do not show significant changes in mucus production or eosinophilia. The histological findings in mice that received Th2 cells have a striking resemblance to human asthmatics.
The function of CD4 Th2 cells in modulating mucus
production has not been studied previously. We show that
Th2 cells specifically stimulate mucus hypersecretion. The
striking difference in mucus induction by Th1 and Th2
cells and the previous finding that IL-4 over-expression induced mucus hypersecretion (7) suggested that this was due
to IL-4. When Th2 cells from IL-4 /
mice were transferred into mice and exposed to inhaled antigen there was no increase in mucus production, however, the cells were
not recruited to the lung. These studies show the critical role
of inflammation in mucus hypersecretion and confirms observations in a variety of human diseases, including asthma,
that lung inflammation is necessary for mucus production
(47). Others have investigated the role of IL-4 in lung inflammation using antigen immunized IL-4-deficient mice
(48). They concluded that the lack of lymphocytes and
eosinophils in the lung after antigen challenge related to an
inability to generate Th2 cells. Recent studies show that
with prolonged antigen challenge, some airway inflammation can be induced in IL-4-deficient mice (49). In these
systemically immunized IL-4-deficient mice, inflammation
may result from activation of a mixed population of Th1
and Th2 cells, since IFN-
produced by Th1 cells can activate different inflammatory pathways. Our work shows
that IL-4 is required by Th2 cells to home to the lung and
this function of IL-4 is distinct from its effects on Th2 cell
development since in our study Th2 cells were generated
in vitro. Thus, lung inflammation induced by activated
CD4 Th2 cells is dependent on their secretion of IL-4.
The precise function of IL-4 in regulating Th2 cell-
induced inflammation in the lung is not clear. It has been
shown that IL-4 upregulates VCAM-1 expression on lung
endothelial cells (45) and the interaction of VCAM-1 and
VLA-4 on lymphocytes and eosinophils is critical for transendothelial migration of these cells to lung after antigen challenge in previously sensitized mice (44). In addition,
chemokines including RANTES, MIP-1, MCP-1, and
eotaxin, have been shown to have important roles in recruitment of lymphocytes and eosinophils to the lung (50,
51). The interplay of cytokines and chemokines in recruitment of leukocytes to the lung after antigen challenge is
complex. Although our studies point to a reduction in
VCAM-1 expression to explain the inhibition of inflammatory cell recruitment in mice that received IL-4-deficient
Th2 cells, the precise effector function of IL-4 in these processes has not yet been detailed.
Once Th2 IL-4 /
cells were activated and recruited
to the lung after administration of TNF-
to the recipient
mice, we showed that mucus was still induced in the bronchial epithelium. Therefore, Th2 secretion of IL-4 did not
directly induce airway epithelial mucus. Furthermore, IL-4
production by non-CD4 T cells was not directly responsible for induction of mucus.
Mucus hypersecretion was recently described in transgenic mice that overexpress IL-4 selectively in the lung (7). At an early age these mice had a marked peribronchial cellular infiltration with eosinophils, lymphocytes and neutrophils. In these transgenic mice, the precise effects of IL-4 could not be separated from the effects of other inflammatory cells found in the lungs. Others have shown a temporal correlation of mucus production with lung IL-4 mRNA levels (52). Our data show that IL-4 is crucial for mucus induction. IL-4 appears to function predominantly as an effector cytokine for recruitment of inflammatory cells to the lung to sites of antigen delivery.
The factors that directly induce mucus are still not clear. In our studies, airway eosinophilia is associated with increased airway epithelial mucus collections. Other studies suggest that eosinophils may have a role in mucus secretion. Recent studies of mice that overexpress IL-5 in the lung epithelium show increased mucus staining in the airways (8). These mice also have dramatic peribronchial infiltration with eosinophils. Furthermore, eosinophil cationic protein has been shown in the guinea pig to be a mucus secretagogue (5). Hogan et al. (49) recently showed that mice can be induced to develop some peribronchial eosinophilic inflammation, along with airway epithelial cell damage and mucosal edema in the absence of IL-4. When these mice were treated with anti-IL-5, these pathological findings were inhibited, suggesting an important role for IL-5 and eosinophilia in some of the pathological changes associated with asthma. It has also been suggested that eosinophils, by damaging the bronchial epithelium and exposing the bronchial wall to more chemical stimuli, increase neural-mediated mechanisms of mucus secretion (47). We are currently studying the role of Th2 cell secretion of IL-5 in mucus production. CD4 Th2 cells also produce other known factors that are distinct from Th1 cells, such as IL-10 and IL-6, that may modulate mucus production. Henderson et al. (53) showed that leukotriene inhibition reduced mucus accumulation and eosinophilia in the airways of antigen stimulated mice. Leukotrienes are secreted predominantly by mast cells after IgE engagement with antigen. Thus, these findings suggest a Th2-mediated mechanism of mucus control. We measured equivalent levels of leukotrienes in the BAL fluid of both Th1 and Th2 mice after exposure to inhaled OVA despite the striking differences in mucus production (data not shown). Serum OVA-specific IgE levels were undetectable in mice that received Th2 cells and inhaled OVA at the time mice were sacrificed, also indicating that mast cells were not activated in our system. Thus, these differences suggest that mucus may be induced by a variety of inflammatory mediators. It is not clear at present if these mediators function through a final common pathway, perhaps the eosinophil.
In summary, these studies examine the direct effects of T cell subsets in airway inflammation. We have shown that Th2 cells influence some of the pathological processes that we associate with human asthma. Despite similar degrees and localization of lung inflammation, Th2 cells, and not Th1 cells, induce airway epithelial mucus production. We have further dissected the effector functions of CD4 Th2 cells using IL-4-deficient mice. Although some recent studies suggest that IL-4 mediates the induction of mucus, we have shown that IL-4 is critical only for the primary phase of mucus induction, lung inflammation. Once lung inflammation is initiated, CD4 Th2-like cells stimulate airway epithelial mucus production in the absence of IL-4.
Address correspondence to Dr. L. Cohn, Section of Immunobiology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208011, New Haven, CT 06520-8011. Tel.: (203) 785-5391; FAX: (203) 737-1764; e-mail lcohn{at}biomed.med.yale.edu
Received for publication 17 July 1997 and in revised form 9 September 1997.
1 Abbreviations used in this paper: BAL, bronchoalveolar lavage; DPAS, diastase-periodic acid-Schiff; HMI, histologic mucus index; pOVA323-339, OVA peptide 323-339.The authors would like to thank J. Elias, J. Pober and A. Ray for helpful discussion, and P. Ranney and I. Visintin for technical assistance.
This work was supported by the Yale Cancer Center, the Howard Hughes Medical Institute, and the National Institutes of Health grants R01-HL54450 (K. Bottomly), P50-HL56389 (K. Bottomly, R.J. Homer), and K08-HL03308 (L. Cohn).
1. |
James, A., and
N. Carroll.
1995.
Theoretical effects of mucus
gland discharge on airway resistance in asthma.
Chest.
107:
110S
|
2. | Moreno, R.H., J.C. Hogg, and P.D. Pare. 1986. Mechanics of airway narrowing. Am. Rev. Respir. Dis. 133: 1171-1180 [Medline]. |
3. | Tanizaki, Y., H. Kitani, M. Okazaki, T. Mifune, F. Mitsunobu, and I. Kimura. 1993. Mucus hypersecretion and eosiophils in bronchoalveolar lavage fluid in adult patients with bronchial asthma. J. Asthma. 30: 257-262 [Medline]. |
4. | Fahy, J.V., D.J. Steiger, J. Liu, C.B. Basbaum, W.E. Finkbeiner, and H.A. Boushey. 1993. Markers of mucus secretion and DNA levels in induced sputum from asthmatics and from healthy subjects. Am. Rev. Respir. Dis. 147: 1132-1137 [Medline]. |
5. | Lundgren, J.D., R.T. Davey, B. Lundgren, J. Mullol, Z. Marom, C. Logun, J. Baraniuk, M.A. Kaliner, and J.H. Shelhamer. 1991. Eosinphil cationic protein stimulates and major basic protein inhibits airway mucus secretion. J. Allergy Clin. Immunol. 87: 689-698 [Medline]. |
6. | Wanner, A., M. Salathe, and T. O'Riordan. 1996. Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154: 1868-1902 [Medline]. |
7. | Temann, U.A., B.G. Prasad, M.W., C. Basbaum, S.B. Ho, R.A. Flavell, and J.A. Rankin. 1997. A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion. Am. J. Respir. Cell Mol. Biol. 16:471-478. |
8. |
Lee, J.J.,
M.P. McGarry,
S.C. Farmer,
K.L. Denzler,
K.A. Larson,
P.E. Carrigan,
I.E. Brenneise,
M.A. Horton,
A. Haczku,
E.W. Gelfand, et al
.
1997.
Interleukin-5 expression in
the lung epithelium of transgenic mice leads to pulmonary
changes pathognomonic of asthma.
J. Exp. Med.
185:
2143-2156
|
9. | Robinson, D.S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A.M. Bentley, C. Corrigan, S.R. Durham, and A.B. Kay. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326: 298-304 [Abstract]. |
10. | Wilson, J.W., R. Djukanovic, P.H. Howarth, and S.T. Holgate. 1992. Lymphocyte activation in bronchoalveolar lavage and peripheral blood in atopic asthma. Am. Rev. Respir. Dis. 145: 958-960 [Medline]. |
11. | Bentley, A.M., Q. Meng, D.S. Robinson, Q. Hamid, A.B. Kay, and S.R. Durham. 1993. Increases in activated T lymphocytes, eosinophils, and cytokine mRNA expression for interleukin-5 and granulocyte/macrophage colony-stimulating factor in bronchial biopsies after allergen inhalation challenge in atopic asthmatics. Am. J. Respir. Cell Mol. Biol. 8: 35-42 [Medline]. |
12. | Olivenstein, R., P.M. Renzi, L.J. Xu, J.P. Yang, and J.G. Martin. 1994. Effects of W3/25 monoclonal antibody on pulmonary inflammation and the late airway response in Brown-Norway rats. Am. Rev. Respir. Dis. 149: 528a . (Abstr.) . |
13. | Nakajima, H., I. Iwamoto, S. Tomoe, R. Matsumara, H. Tomioka, K. Takatsu, and S. Yoshida. 1992. CD4+ T-lymphocytes and interleukin-5 mediate antigen-induced eosinophil infiltration into mouse trachea. Am. Rev. Respir. Dis. 146: 374-377 [Medline]. |
14. |
Cher, D., and
T. Mosmann.
1987.
Two types of murine
helper T cell clone: II. Delayed type hypersensitivity is mediated by Th1 clones.
J. Immunol.
138:
3688-3694
|
15. |
Stout, R., and
K. Bottomly.
1989.
Antigen-specific activation of effector macrophages by IFN-![]() |
16. | Kim, J., A. Woods, E. Becker-Dunn, and K. Bottomly. 1985. Distinct functional phenotypes of cloned Ia-restricted helper T cells. J. Exp. Med. 162: 188-201 [Abstract]. |
17. |
Killar, L.,
G. MacDonald,
J. West,
A. Woods, and
K. Bottomly.
1987.
Cloned Ia restricted T cells that do not produce
IL4/BSF-1 fail to help antigen specific B cells.
J. Immunol.
138:
1674-1679
|
18. |
Mosmann, T.R.,
H. Cherwinski,
M.W. Bond,
M.A. Giedlin, and
R.L. Coffman.
1986.
Two types of murine helper T cell
clone. I. Definition according to profiles of lymphokine activities and secreted proteins.
J. Immunol.
136:
2348-2357
|
19. | Kuhn, R., K. Rajewsky, and W. Muller. 1991. Generation and analysis of IL4 deficient mice. Science (Wash. DC). 254: 707-710 [Medline]. |
20. | Finkelman, F.D., I.M. Katona, J.F. Urban, C.M. Snapper Jr., J. Ohara, and P. W.E. . 1986. Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B cell stimulatory factor 1. Proc. Natl. Acad. Sci. USA. 83: 9675-9683 [Abstract]. |
21. | Stevens, T.L., A. Bossie, V.M. Sanders, R. Fernandez-Botran, R. Coffman, T.R. Mosmann, and E. Vitetta. 1988. Regulation of antibody isotypic secretion by subsets of antigen-specific helper T cells. Nature (Lond.). 334: 255-258 [Medline]. |
22. | Resnick, M.B., and P.F. Weller. 1993. Mechanisms of eosinophil recruitment. Am. J. Respir. Cell Mol. Biol. 8: 349-355 [Medline]. |
23. | Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A.J. Wardlaw, C.J. Corrigan, B. Bradley, S.R. Durham, J.V. Collins, P.K. Jeffery, et al . 1991. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J. Clin. Invest. 87: 1541-1546 [Medline]. |
24. | Bradley, B.L., M. Azzawi, M. Jacobson, B. Assoufi, J.V. Collins, A.M. Irani, L.B. Schwartz, S.R. Durham, P.K. Jeffery, and A.B. Kay. 1991. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J. Allergy Clin. Immunol. 88: 661-674 [Medline]. |
25. | Poulter, L.W., C. Power, and C. Burke. 1990. The relationship between bronchial immunopathology and hyperresponsiveness in asthma. Eur. Respir. Journal 3: 792-799 . |
26. | Calhoun, W.J., K.L. Hinton, and R.E. Friedenheim. 1995. Evidence for simultaneous Th1 and Th2 lymphocyte activation in allergic asthma following segmental antigen challenge. Am. J. Respir. Crit. Care Med. 151: 778a .(Abstr.) . |
27. | Cembrzynska-Nowak, M., E. Szklarz, A.D. Inglot, and J.A. Teodorczyk-Injeyan. 1993. Elevated release of tumor necrosis factor-alpha and interferon-gamma by bronchoalveolar leukocytes from patients with bronchial asthma. Am. Rev. Respir. Dis. 147: 291-295 [Medline]. |
28. | Corrigan, C.J., and A.B. Kay. 1990. CD4 T-lymphocyte activation in acute severe asthma. Relationship to disease severity and atopic status. Am. Rev. Respir. Dis. 141: 970-977 [Medline]. |
29. | Krug, N., J. Madden, A.E. Redington, P. Lackie, R. Dhukanovic, U. Schauer, S.T. Holgate, A.J. Frew, and P.H. Howarth. 1996. T cell cytokine profile evaluated at the single cells level in BAL and blood in allergic asthma. Am. J. Respir. Cell Mol. Biol. 14: 319-326 [Abstract]. |
30. | Murphy, K.M., A.B. Heimberger, and D.Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+ CD8+TCR lo thymocytes in vivo. Science (Wash. DC). 250: 1720-1723 [Medline]. |
31. |
Levin, D.,
S. Constant,
T. Pasqualini,
R. Flavell, and
K. Bottomly.
1993.
Role of dendritic cells in the priming of CD4+
T lymphocytes to peptide antigen in vivo.
J. Immunol.
151:
6742-6750
|
32. | Ledbetter, J.A., and L.A. Herzenberg. 1979. Xenogenic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47: 63-90 [Medline]. |
33. |
Landais, D.,
B.N. Beck,
J.-M. Buerstedde,
S. Degraw,
D. Klein,
N. Koch,
D. Murphy,
M. Pierres,
T. Tada,
K. Yamamoto, et al
.
1987.
The assignment of chain specificities for
anti-Ia monoclonal antibodies using L cell transfectants.
J. Immunol.
137:
3002-3005
|
34. | Dialynas, D.P., D.B. Wilde, P. Marrack, A. Pierres, K.A. Wall, W. Havran, G. Otten, M.R. Loken, M. Pierres, J. Kappler, and F.W. Fitch. 1983. Characterization of the murine antigenic determinant, L3T4a, recognized by a monoclonal antibody GK1.5: Expression of L3T4a by functional T cell clones appears to correlate primarily with Class II MHC antigen-reactivity. Immunol. Rev. 74: 29-56 [Medline]. |
35. | Jones, B.. 1983. Evidence that the Thy-1 molecule is a target for T cell mitogenic antibody against brain-associated antigens. Eur. J. Immunol. 13: 678-684 [Medline]. |
36. | Ohara, J., and W.E. Paul. 1985. Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1. Nature (Lond.). 315: 333-336 [Medline]. |
37. | Cherwinski, H.M., J.H. Schumacher, K.D. Brown, and T.R. Mosmann. 1987. Two types of mouse helper T cell clone: III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166: 1229-1244 [Abstract]. |
38. | Marrack, P., R. Shimonkevitz, C. Hannum, K. Haskins, and J. Kappler. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. J. Exp. Med. 158: 1635-1646 [Abstract]. |
39. | Wong, S., S. Guerder, I. Visintin, E. Reich, K.E. Swenson, R.A. Flavell, and C.A. Janeway. 1995. Expression of the co-stimulator molecule B7-1 in pancreatic B-cells accelerates diabetes in the NOD mouse. Diabetes. 44: 326-329 [Abstract]. |
40. | Weibel, E.R. 1979. Sterologic Methods. Academic Press, London. pp. 9-159. |
41. | Swain, S.L.. 1994. Generation and in vivo persistence of polarized Th1 and Th2 memory cells. Immunity. 1: 543-552 [Medline]. |
42. | Rossi, G.A., O. Sacco, B. Balbi, S. Oddera, T. Mattioni, S. Corte, C. Ravazzoni, and L. Allegra. 1990. Human ciliated bronchial epithelial cells: expression of the HLA-DR antigens and of the alpha gene, modulation of the HLA-DR antigens by gamma interferon and antigen presenting cell function in the MLR. Am. J. Respir. Cell Mol. Biol. 3: 431-439 [Medline]. |
43. | Noben-Trauth, N., P. Kropf, and I. Muller. 1996. Susceptibility to Leishmania major infection in interleukin-4-deficient mice. Science (Wash. DC). 271: 987-990 [Abstract]. |
44. | Nakajima, H., H. Sano, T. Nishimura, S. Yoshida, and I. Iwamoto. 1994. Role of VCAM-1/VLA-4 and ICAM-1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue. J. Exp. Med. 179: 1145-1154 [Abstract]. |
45. | Neumann, B., T. Machleidt, A. Lifka, K. Pfeffer, V. Deitmar, T.W. Mak, B. Holzmann, and M. Kronke. 1996. Crucial role of 55-kilodalton TNF receptor in TNF-induced adhesion molecule expression and leukocyte organ infiltration. J. Immunol 156: 1587-1593 [Abstract]. |
46. | Murphy, E., K. Shibuya, N. Hosken, P. Openshaw, V. Maino, K. Davis, K. Murphy, and A. O'Garra. 1996. Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J. Immunol. 158: 2648-2653 [Abstract]. |
47. | Lundgren, J.D., and J.H. Shelhamer. 1990. Pathogenesis of airway mucus hypersecretion. J. Allergy Clin. Immunol. 85: 399-417 [Medline]. |
48. | Coyle, A.J., G. Le Gros, C. Bertrand, S. Tsuyuki, C.H. Heusser, M. Kopf, and G.P. Anderson. 1995. Interleukin-4 is required for the induction of lung Th2 mucosal immunity. Am. J. Respir. Cell Mol. Biol. 13: 54-59 [Abstract]. |
49. | Hogan, S.P., A. Mould, H. Kikutani, A.J. Ramsay, and P.S. Foster. 1997. Aeroallergen- induced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-specific immunoglobulins. J. Clin. Invest. 99: 1326-1339 . |
50. |
Gonzalo, J.A.,
C.M. Lloyd,
L. Kremer,
E. Finger,
C. Martinez-A.,
M.H. Siegelman,
M. Cybulsky, and
J.C. Gutierrez-Ramos.
1996.
Eosinophil recruitment to the lung in a murine
model of allergic inflammation: the role of T cells, chemokines, and adhesion molecules.
J. Clin. Invest.
98:
2332-2345
|
51. | Lukacs, N.W., R.M. Streiter, K. Warmington, P. Lincoln, S.W. Chensue, and S.L. Kunkel. 1997. Differential recruitment of leukocyte populations and alteration of airway hyperreactivity by C-C family chemokines in allergic airway inflammation. J. Immunol. 158: 4398-4404 [Abstract]. |
52. | Budhecha, S., R.K. Albert, E. Chi, and D.B. Lewis. 1997. Kinetic relationship between IL-4 and muc5 gene expression with presence of airway mucus in a murine asthma model. Am. J. Respir. Crit. Care Med. 155: 756a . (Abstr.) . |
53. | Henderson, W.R., D.B. Lewis, R.K. Albert, Y. Zhang, W.J.E. Lamm, G.K.S. Chiang, F. Jones, P. Eriksen, Y.T. Tien, M. Jonas, and E. Chi. 1996. The importance of leukotrienes in airway inflammation in a mouse model of asthma. J. Exp. Med. 184: 1483-1494 [Abstract]. |