1 Divisions of Hematology/Oncology and 2 Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259; and 3 Pharmaceutical Division, Department of Biotechnology, Bayer Corporation, Berkeley, California 94701-1986
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The potential role of airway
interleukin-5 (IL-5) expression in eliciting mucus production was
demonstrated in a pulmonary IL-5 transgenic mouse model (NJ.1726) in
which naive transgenic mice display comparable levels of airway mucus
relative to allergen-sensitized and -challenged wild-type mice.
The intrinsic mucus accumulation of NJ.1726 was abolished in compound
transgenic-gene knockout mice deficient of either CD4+
cells [NJ.1726/CD4(/
)] or
T cell receptor-positive
(TCR+) cells [NJ.1726/
TCR(
/
)]. In addition,
mucus production in naive NJ.1726 was inhibited by >90% after
administration of the soluble anti-IL-4 receptor
-subunit
antagonist. The loss of mucus production in NJ.1726/CD4(
/
),
NJ.1726/
TCR(
/
), and anti-IL-4 receptor
-subunit
antagonist-treated mice occurred notwithstanding the significant
pulmonary eosinophilia and expansion of airway B cells induced by
ectopic IL-5 expression. Furthermore, the loss of mucus accumulation
occurred in these mice despite elevated levels of airway and peripheral
IL-5, indicating that IL-5 does not directly induce goblet cell
metaplasia and mucus production. Thus pulmonary expression of IL-5
alone is capable of inducing CD4+ T cell-dependent goblet
cell metaplasia, apparently mediated by IL-4 receptor
-subunit-ligand interactions, and represents a previously
unrecognized novel pathway for augmenting allergen-induced mucus production.
asthma; goblet cell; transgenic; gene knockout
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INCREASED MUCUS
PRODUCTION and secretion is a common characteristic of human
asthma and is believed to be partly responsible for the development of
airway obstruction, a pathophysiological manifestation associated with
this disease (23). Although mice do not have
epithelium-associated mucous glands in the lung and thus mucus plugging
of bronchioles is infrequently observed, allergen-mediated inflammation
is associated with the induction of goblet cell metaplasia regardless
of the specific model utilized (e.g., see Refs. 2, 14). The proliferation of these mucus-containing
epithelial cells has been shown (1) to be intimately
linked to T helper type 2 (Th2)-mediated inflammation in response
to aeroallergen exposure, and studies have implicated specific
roles for the Th2 cytokines interleukin-4 (IL-4) (7), IL-9
(40), and IL-13 (18) as proinflammatory
mediators linked to the induction of mucus production. Moreover, recent
studies (5) have also implicated the Th1 cytokine
interferon- (IFN-
) as an important downregulator of
allergen-induced mucus overproduction in the lung, suggesting that this
response results from a complex interplay of multiple inflammatory signals.
Transgenic/gene knockout mice have been particularly instrumental to the characterization of the specific signaling pathways contributing to increased mucus production after allergen challenge. Overexpression of either IL-9 (12) or IL-13 (44) in the lungs of transgenic mice elicits allergen-independent goblet cell metaplasia and mucus overproduction. Adoptive transfer studies (5, 6) using gene knockout mice and ovalbumin (OVA) T cell receptor (TCR) transgenic animals have demonstrated that CD4+ T cells and IL-4 receptor expression are each necessary (and possibly sufficient) for allergen-induced mucus overproduction. In addition, studies utilizing IL-5-deficient mice showed that allergen-induced mucus production can occur in the absence of this cytokine and extensive pulmonary eosinophilia (6). Collectively, however, these earlier studies demonstrate only that mucus production is causatively linked to IL-4/IL-13 expression or can occur in the absence of IL-5 and pulmonary eosinophils. The unresolved issue is whether IL-5 and/or eosinophils themselves are capable of eliciting pulmonary mucus overproduction. Indeed, constitutive ectopic expression of IL-5 from Clara cells in naive transgenic mice (line NJ.1726; Ref. 26) is sufficient to induce a pulmonary eosinophilia. More significantly, these naive animals exhibit mucus production approaching levels observed in wild-type allergen-challenged mice. However, the mechanism responsible for this IL-5-dependent, allergen-independent mucus production is currently undefined.
NJ.1726 mice were observed to exhibit significant expansion of B cell
and the CD4+ and CD8+ T cell populations
independent of allergen exposure. Therefore, we assessed the potential
role of IL-5 and/or pulmonary eosinophils in eliciting mucus
overproduction in mice alone or synergistically with individual
lymphocyte subsets in allergen-challenged wild-type mice and naive IL-5
transgenic mice, as well as compound IL-5 transgenic/gene knockout
animals deficient of B cells or specific T cell subtypes (e.g.,
CD4+, CD8+, TCR+, and
TCR+). These studies demonstrate that allergen-induced
mucus production occurred in C57BL/6J mice deficient of
CD8+ or
TCR+ T cells. However, in the
absence of CD4+ or
TCR+ T cells, mucus
production was obviated, confirming earlier studies (5, 6)
showing the necessity of these cells. Interestingly, the mucus
production observed in naive IL-5 transgenic mice was also abolished in
mice that were deficient of either CD4+ or
TCR+ T cells. The elimination of mucus production in these
compound transgenic-gene knockout mice occurred despite a profound
pulmonary eosinophilia and elevated levels of peripheral and lung IL-5. This suggests that a previously unknown IL-5-mediated effect on T cells
exists that is capable of eliciting mucus production independent of
IL-5 activities on eosinophils and potential effects on airway epithelium.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice.
Transgenic mice constitutively expressing murine IL-5 from the lung
epithelium (line NJ.1726) were generated as previously described
(26) and maintained by continual backcross to C57BL/6J. NJ.1726 animals were bred with gene knockout mice (Jackson
Laboratories, Bar Harbor, ME) lacking either B cells
[C57BL/6-Igh-6tm1Cgn (24)], T cells
(/
) [C57BL/6J-Tcr
tm1Mom
Tcr
tm1Mom (31)],
TCR+
cells [C57BL/6J-Tcr
tm1Mom (31)],
TCR+ cells [C57BL/6J-Tcr
tm1Mom
(22)], CD4+ cells
[C57BL/6J-Cd4tm1Knw (30)], or
CD8+ cells [C57BL/6-Cd8atm1Mak
(13)] to generate compound IL-5 transgenic-gene knockout
mice deficient in B, T,
TCR+,
TCR+, CD4+, and CD8+ cells,
respectively. Genotypes of mice derived from these crosses were
determined by the presence of the IL-5 transgene (PCR of tail DNA) and
loss of B cells or T cells or T cell subtypes as assayed by flow
cytometry on peripheral blood using conjugated antibodies against B220
(B cells), TCR-
, TCR-
, CD4, and CD8, as previously described
(3). Control C57BL/6J mice were obtained from Jackson
Laboratories, and all procedures were conducted on mice 8-12 wk of
age maintained in microisolator cages housed in a specific
pathogen-free animal facility. The sentinel cages within this animal
colony were negative for viral antibodies and the presence of known
mouse pathogens. Protocols and studies involving animals were conducted
in accordance with National Institutes of Health and Mayo Clinic
Foundation guidelines.
OVA sensitization and challenge. Mice were sensitized and challenged with chicken OVA as previously described (11). Briefly, mice were sensitized by an intraperitoneal injection (100 µl) of 20 µg chicken OVA (Sigma, St. Louis, MO) emulsified in 2 mg Imject Alum [Al(OH)3/Mg(OH)2; Pierce, Rockfield, IL] on days 0 and 14. Mice were subsequently challenged with an aerosol generated from 1% OVA in saline or saline alone for 20 min by ultrasonic nebulization (DeVilbiss, Somerset, PA) on days 24, 25, and 26. Assessments of goblet cell metaplasia and mucus overproduction and parenchyma eosinophils were performed on day 28. All NJ.1726 strains were placed on a similar protocol with the exception of intraperitoneal saline injection (without Imject Alum) on days 0 and 14 and administration of nebulized saline on days 24, 25, and 26.
Assessment of goblet cell metaplasia and mucus overproduction. On day 25 of the OVA sensitization and challenge protocol, mice were euthanized with ketamine (2 mg/kg body wt). Lungs were perfused with 10% formalin (Biochemical Sciences, Swedensboro, NJ), excised, and bathed in 10% formalin overnight before embedding in paraffin. Mucus cell development along the airway epithelium was quantified in paraffin-embedded tissue sections (4-8 µm) stained with periodic acid-Schiff's reagent (PAS). Parasaggital sections (n = 5 mice/group) were analyzed by bright-field microscopy using an image analysis software program (ImagePro Plus, Media Cybernetics, Silver Spring, MD) to derive an airway mucus index (MI) reflective of both the amount of mucus per airway and the number of airways affected. The mucus content of all the airways per section (20-30, proximal to distal) was measured from groups of four to five animals. An imaging program (Image ProPlus, Media Cybernetics) was used to quantify the area and intensity of PAS staining per airway. The data were quantified as follows: MI = (average PAS staining intensity of airway epithelium) × (area of airway epithelium staining with PAS)/(total area of conducting airway epithelium) × (total number of airways assessed).
Flow cytometry of leukocytes recovered from total lung digests. Leukocytes within the lung parenchyma were assessed by collagenase digestion of perfused lungs. Isolation of lung cells was performed as previously described (3). Briefly, perfused lungs were removed and diced into pieces <300 µl in volume. Hanks' balanced salt solution (HBSS; 4 ml; GIBCO, Gaithersburg, MD) containing 175 U/ml collagenase (Sigma), 10% FCS (GIBCO), 100 U/ml penicillin, and 100 µg/ml streptomycin was added to the tissue and incubated for 60 min at 37°C in an orbital shaker. The digested lungs were sheared with a 20-gauge needle and filtered through 45- and 20-µm filters. Cells were washed three times and resuspended in HBSS before counting with a hemacytometer. Lymphocyte populations in the lung were subsequently identified and expressed as the product of the total cell count and the percentage of total cells analyzed (1 × 105) by flow cytometry. CD3+/CD4+ double-positive, CD3+/CD8+ double-positive, and B cells were identified or quantified by staining with the following conjugated antibodies; phycoerytherin-anti-mouse CD3 (Caltag, Burlingame, CA), FITC-anti-mouse CD4 (Pharmingen, San Diego, CA), FITC-anti-mouse CD8 (Pharmingen), and phycoerytherin-anti-mouse B220 (Caltag). Flow cytometry was performed on a FACScan flowcytometer (Becton Dickinson, Franklin Lakes, NJ). Data acquisition and analysis were performed using CellQuest software (Becton Dickinson).
Immunocytochemistry of lung sections and detection of airway eosinophils. Eosinophils within the lung parenchyma were identified by immunohistochemistry using a rabbit polyclonal antibody against mouse major basic protein (MBP). MBP antigen-antibody complexes were detected in 4-µm sections of formalin-fixed, paraffin-embedded sections of mouse lungs as previously described (11). Tissue eosinophils were assessed by determining the total number of MBP-positive cells in five randomly selected high-power fields, with data expressed as eosinophils per square millimeter of lung tissue (n = 4-5 mice/group).
Treatment of NJ.1726 mice with IL-4 receptor antagonist. The "QY" IL-4 mutant binds the IL-4 receptor with an affinity similar to wild-type IL-4. However, unlike IL-4, the QY mutant is unable to mediate receptor signaling (16). NJ.1726 mice were administered QY according to a previously published protocol (17). Briefly, NJ.1726 mice (~2 mo of age) were injected intraperitoneally two times on day 0 with 50 µg of QY. On days 1 through 10 of this protocol, mice were injected one time intraperitoneally with 10 µg of QY. Control mice received injections of diluent (i.e., PBS) throughout the protocol. On day 10, the lungs from experimental and control groups were removed and prepared for histology and mucus quantitation as described in Assessment of goblet cell metaplasia and mucus overproduction.
Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed on parametric data using t-tests with differences between means considered to be significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allergen-induced mucus production is dependent on
TCR+ T cells and not B cells or
TCR+ T cells.
OVA challenge of C57BL/6J mice induces airway inflammation
characterized by increases in CD3+, CD4+, and
B220+ cell populations (Table
1). Furthermore, OVA sensitization and aerosol challenge elicits a pronounced increase in airway eosinophils 48 h after the final allergen challenge (data not shown). Figure 1, A, B, and
F, shows that the sensitization and challenge regime also
results in goblet cell metaplasia and the production of mucus relative
to saline-treated control groups (MI = 9.9 ± 1.1 vs. 0.4 ± 0.02, respectively). OVA sensitization and aerosol
challenge of mice deficient in specific lymphocyte cell types
demonstrated that allergen-induced mucus production was independent of
B cells (Fig. 1, C and F; MI = 9.7 ± 0.8) and
TCR+ T cells (Fig. 1, E and
F; MI = 8.7 ± 1.7). In contrast, goblet cell
metaplasia and mucus production in OVA-treated
TCR+
T cell-deficient mice was abolished (Fig. 1, D and
F; MI = 1.2 ± 0.98).
|
|
OVA-induced mucus production requires
CD4+ T cells.
A comparison of mucus production in OVA-sensitized and
aerosol-challenged CD4+ T cell-deficient and
CD8+ T cell-deficient mice demonstrates that
CD4+ and not CD8+ T cells are required for
OVA-induced goblet cell metaplasia (Fig. 2; MI = 0.69 ± 0.39 vs.
8.1 ± 1.1, respectively).
|
Constitutive ectopic expression of IL-5 in respiratory epithelial
cells results in mucus production in naive transgenic mice.
Pulmonary IL-5 expression induces an inflammatory-type condition in
naive NJ.1726 mice, including the accumulation of inflammatory leukocytes (i.e., eosinophils, T cells, and B cells) in the lung and
changes in the airway epithelium (26; Table 1). Among the changes
occurring in the airways of these transgenic mice is a significant
goblet cell metaplasia and accompanying mucus production. The MI of
naive (i.e., saline-challenged) NJ.1726 mice is more than 10-fold
higher relative to saline-challenged wild-type mice (Fig.
3, A and E, vs.
Fig. 1, A and F, MI = 5.9 ± 1.5 vs.
0.4 ± 0.02, respectively). Moreover, this naive transgenic MI is
only nominally larger than the MI observed in OVA-sensitized and
aerosol-challenged wild-type mice (Fig. 1F, MI = 9.9 ± 1.1), demonstrating that pulmonary IL-5 expression alone
(i.e., independent of allergen-induced Th2 inflammation) is capable of
eliciting airway mucus production.
|
IL-5-associated mucus production in naive transgenic mice is
dependent on presence of CD4+ T cells.
In addition to IL-5 expression and airway inflammation (Table 1), the
intrinsic mucus production in naive NJ.1726 mice was also dependent on
the presence of CD4+ TCR+ T cells (Fig.
3). Specifically, despite similar levels of serum IL-5
(3), the MI of naive compound transgenic-gene knockout mice deficient in CD4+ T cells [NJ.1726/ CD4(
/
)] was
reduced by 80% relative to naive NJ.1726 mice (Fig. 3E,
MI = 2.03 ± 0.58 and 5.9 ± 1.5, respectively). Interestingly, despite the dramatic increase in both pulmonary CD4+ T cells and B cells associated with these IL-5
transgenic mice, mucus expression remained elevated in B cell-deficient
NJ.1726 animals (Fig. 3, C and E, MI = 5.2 ± 0.35).
Increased mucus production in NJ.1726 mice requires IL-4
receptor -subunit-mediated signaling events.
We used the IL-4 receptor antagonist QY (16) to
block IL-4 and IL-13 signaling through this receptor to assess the
potential contribution these cytokines have in mucus production
associated with naive NJ.1726 mice. Blockade of IL-4 receptor
-subunit demonstrated that signaling events through this receptor
were necessary for the mucus production associated with naive
NJ.1726 mice (Fig. 4). Treatment of
NJ.1726 mice with QY reduced the MI to levels comparable with
naive wild-type animals (0.27 ± 0.17 vs. 0.40 ± 0.02, respectively).
|
T cell-mediated production of mucus induced by ectopic IL-5
expression occurs independent of airway eosinophils.
In addition to increased mucus production in naive NJ.1726 mice,
ectopic expression of IL-5 in the airways elicits eosinophil recruitment to the lung parenchyma (26), raising the
possibility that the induced eosinophilia was responsible for the
intrinsic goblet cell metaplasia and mucus production associated
with these mice. Eosinophil infiltration of the lung was assessed
in naive NJ.1726 mice and compound transgenic-gene knockout animals to determine if a correlative relationship exists in naive NJ.1726 mice
between goblet cell metaplasia and mucus production and the development
of a pulmonary eosinophilia. Both parameters were assessed in cohorts
of animals representing naive NJ.1726 mice and compound transgenic-gene
knockout mice deficient in B cells [NJ.1726/B(/
)] or
CD4+ T cells [NJ.1726/CD4(
/
)]. Goblet cell metaplasia
and mucus production was observed only in naive NJ.1726 and
NJ.1726/B(
/
) mice, yet no differences were observed in the number
of eosinophils per square millimeter of basement membrane found in all
three naive cohorts (Fig. 5).
Interestingly, each group of naive animals had tissue eosinophil
numbers equivalent to OVA-sensitized and aerosol-challenged C57BL/6J
mice (a cohort with elevated mucus production).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Airway inflammation characterized by infiltration by eosinophils and T cells and an accompanying increase in expression of Th2 cytokines (e.g., IL-4, IL-5, and IL-13) are common characteristics of both asthma patients and mouse respiratory inflammation models (27, 42). One particularly striking feature common to both humans and mice exposed to aerosolized allergens is the development of goblet cell metaplasia and the concomitant expression of mucus glycoproteins (14). The observed mucus production after allergen challenge can lead to narrowing of midsized airways and in severe cases precipitate mucus-plugging of the airways (23), which undoubtedly leads, in part, to airway narrowing and subsequent dyspnea.
The strong correlation between mucus overproduction and Th2 inflammation after allergen exposure has led to the common speculation that there is a cause and effect relationship in which T cells are responsible for the observed changes in mucus levels in the airways in asthma. However, Blyth et al. (2) have demonstrated that induction of mucus expression and goblet cell metaplasia precedes both lymphocytic and eosinophilic inflammation and can persist for 7 days after airway inflammation has returned to resting levels. Although the increase in mucus expression does not correlate with the timing of the inflammatory cell infiltrate, it does correlate with the observed time course of Th2 cytokine production after a single exposure to allergen challenge (32). In addition, Henderson and colleagues (21) have demonstrated that inhibition of either 5-lipoxygenase or the 5-lipoxygenase-activating gene blocks airway mucus overproduction after intranasal OVA challenge. Given the rapid manner in which leukotriene synthesis and release occurs (19), the aforementioned data suggest that cells associated with the acute response to allergen exposure (i.e., macrophages or airway epithelium) are critical for the induction of mucus production. These seemingly contrary reports raise the question as to whether cells of the acquired immune response (i.e., CD4+ T cells) and the cytokines associated with a Th2 immune reaction (i.e., IL-4, IL-5, and IL-13) are the sole inflammatory pathways leading to allergen-induced mucus overproduction.
Assessments of airway anatomy and the rate of metaplastic airway epithelial cell responses in the mouse, however, argue against an acute response to allergen exposure. Secretory serous cells and mucus-producing cells (i.e., goblet cells) are rarely observed in naive mice and epithelial-associated mucous glands are virtually nonexistent (33). Instead, airway Clara cells are the predominant cell type, comprising ~60% of the airway epithelium in the mouse (34, 39). After exposure to allergens and/or toxicants, the secretory cells undergo metaplasia/hyperplasia and concurrently accumulate mucus within cytoplasmic granules. However, the time course of these events is quite prolonged. It has been shown that increased expression of MUC2 mucin transcripts takes ~24 h, whereas the presence of the fully glycosylated protein requires at least 48 h after toxicant exposure (38). A similar time course has been suggested for the other major respiratory mucin MUC5A/C (41). Therefore, the anatomic data from mice concerning mucus-producing cell types and the biochemical data concerning the rate of mucus production suggest that this phenomenon is associated with events that occur over a period of days, much like the time course of Th2 inflammation in the airways after allergen exposure.
Previous studies (35) in the mouse have shown that IL-5 effector functions are predominantly exerted on eosinophils and B cells. In both leukocytes, IL-5 is primarily a proliferative signal for lineage-committed precursors; however, substantive effects have also been demonstrated on mature cells in the periphery. For example, B cell differentiation into plasma cells appears to be mediated, in part, by IL-5 effector function(s) (36), and IL-5 has been shown (4, 9, 37) to enhance eosinophil chemotaxis and survival. IL-5 has also been shown (29) to elicit bone deposition in the spleen, indicating that IL-5 has broad effects on various cell types previously not associated with IL-5 effector functions. The data presented in this study suggest that IL-5 has additional activities that are mediated by (through?) CD4+ T cells as pulmonary expression of IL-5, in the absence of allergen sensitization and aerosol challenge, induces goblet cell metaplasia and mucus production.
The necessity of CD4+ T cells in an IL-5-dependent model
system complements previous studies that have implicated
CD4+ T cells and Th2 inflammatory responses as critical
components of this phenomenon. For example, OVA-induced mucus
production is absent in RAG(/
) mice (T cell and B cell deficient)
and adoptive transfer of wild-type CD4+ T cells into these
mice reconstitutes this parameter (10). In addition, Cohn
et al. (6, 7) have demonstrated, through a series of
adoptive transfer studies, that CD4+ T cells of the Th2
subset and signaling through IL-4 receptor
-subunit have significant
roles in mucus production, although IL-4 expression is not required.
Likewise, Gavett et al. (15) have demonstrated that the
administration of an IL-4 receptor antagonist blocks allergen-induced
mucus production, suggesting a role for IL-13 in this process (6,
20). Consistent with this finding, Grunig and colleagues
(18) have demonstrated that selective neutralization of
IL-13 inhibits allergen-induced mucus overproduction and expression of
IL-13 from the lung epithelium of transgenic mice induces goblet cell
metaplasia and mucus overproduction in the absence of an allergen
challenge (44). Additional studies (8), however, have also demonstrated that adoptively
transferred Th1 polarized T cells are as efficient as Th2 committed
cells at inducing mucus production in IFN-
receptor-deficient
recipient mice. These data suggest that mucus production after allergen exposure is complex and is likely a consequence of interactions between
several T cell-derived factors.
Previous studies (e.g., see Ref. 6) have demonstrated that goblet cell metaplasia and mucus production can occur in the absence of IL-5 and significant airway eosinophilia. Our data support this conclusion by demonstrating that increased IL-5 levels and robust airway eosinophilia are insufficient to induce mucus. This indicates that IL-5 does not have a direct effect on airway epithelium to induce goblet cell metaplasia and mucus production. However, the ability of IL-5 to elicit mucus production through a CD4+ T cell-dependent mechanism suggests that pulmonary expression of IL-5 represents an additional mechanism during an allergen provocation capable of eliciting mucus production. The demonstration that this IL-5-mediated effect is dependent on the presence of CD4+ T cells suggests a previously unknown activity associated with this cytokine. In particular, does IL-5 mediate the proliferation of specific T cell subpopulations and/or does IL-5 signaling activate T cells in such a way as to elicit, among other events, goblet cell metaplasia and mucus production? The dramatic increase in pulmonary CD4+ T cells in naive NJ.1726 mice, as well as the dramatic increase in circulating T cells associated with another IL-5 transgenic line (28), implies a previously unknown IL-5 effector function on T cells, although an indirect mechanism mediated by IL-5 effects on a third cell population cannot be ruled out.
It is possible that IL-5 may induce T cell expression of various factors (e.g., release of eicosanoids directly by T cells or indirectly by T cell activation of eosinophils), leading to increased mucus production independent of Th2-associated inflammatory signals. However, IL-5-mediated secretion of IL-4/IL-13 by T cells would be a parsimonious explanation linking IL-5 effector function and goblet cell metaplasia and mucus production. Alternatively, because eosinophils are capable of releasing Th2 cytokines (43), the pulmonary eosinophilia induced by IL-5 expression may play a significant role via T cell-mediated activation. Although IL-4 and IL-13 expression was not elevated above basal levels in the NJ.1726 mice (data not shown), this does not rule out the possibility that IL-5 may lead to the release of these cytokines at low (i.e., undetectable) levels or in a time-dependent fashion by activating T cells and/or, in turn, possibly eosinophils. Indeed, the administration of an IL-4 receptor antagonist inhibited the mucus production apparently mediated by IL-5, demonstrating a role for IL-4/IL-13 in the observed mucus production associated with NJ.1726 mice. Provocatively, similar data are found in a mouse study of OVA-induced respiratory inflammation utilizing anti-IL-5 antibody (25). In this study, administration of an anti-IL-5 monoclonal (TRFK-5) to OVA-treated wild-type mice led to a significant decrease in mucus production relative to control antibody-treated mice. These data suggest that IL-5 expression in allergen-treated wild-type mice also uniquely augments mucus production and may represent an important component of signaling pathways effecting the absolute levels of mucus production in the lung. Thus in addition to effects primarily on eosinophils and eosinophil progenitor cells, IL-5 apparently has a novel function(s) in allergic pulmonary inflammation that leads to mucus production. This conclusion would imply the existence of a previously unrecognized IL-5 activity (activities) on T cells critical to the development of allergen-induced pulmonary pathology.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the Mayo Clinic Scottsdale Histology Facility (A. Jennings, Director) and Graphic Arts Department (M. Ruona) for technical assistance. We also thank our research program assistant, L. Mardel, whose contribution to our productivity is often overlooked.
![]() |
FOOTNOTES |
---|
This work was supported by the Mayo Clinic Foundation; National Heart, Lung, and Blood Institute (NHLBI) Individual Investigator Award HL-60793-01S (N. A. Lee); NHLBI Training Grant HL-07897; and National Research Service Awards HL-10361 (M. T. Borchers) and HL-10176 (J. Crosby).
Address for reprint requests and other correspondence: N. A. Lee, Division of Hematology/Oncology, Mayo Clinic Scottsdale, 13400 E. Shea Blvd., Scottsdale, AZ 85259 (E-mail: nlee{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 21, 2001;10.1152/ajplung.00195.2001
Received 1 June 2001; accepted in final form 2 December 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arm, JP,
and
Lee TH.
The pathobiology of bronchial asthma (review).
Adv Immunol
51:
323-382,
1992[ISI][Medline].
2.
Blyth, DI,
Pedrick MS,
Savage TJ,
Hessel EM,
and
Fattah D.
Lung inflammation and epithelial changes in a murine model of atopic asthma.
Am J Respir Cell Mol Biol
14:
425-438,
1996[Abstract].
3.
Borchers, MT,
Crosby J,
Justice P,
Farmer S,
Hines E,
Lee JJ,
and
Lee NA.
Intrinsic AHR in IL-5 transgenic mice is dependent on CD4+ cells and CD49d-mediated signaling.
Am J Physiol Lung Cell Mol Physiol
281:
L653-L659,
2001
4.
Broide, DH,
Hoffman H,
and
Sriramarao P.
Genes that regulate eosinophilic inflammation.
Am J Hum Genet
65:
302-307,
1999[ISI][Medline].
5.
Cohn, L,
Herrick C,
Niu N,
Homer RJ,
and
Bottomly K.
IL-4 promotes airway eosinophilia by suppressing IFN-gamma production: defining a novel role for IFN-gamma in the regulation of allergic airway inflammation.
J Immunol
166:
2760-2767,
2001
6.
Cohn, L,
Homer RJ,
MacLeod H,
Mohrs M,
Brombacher F,
and
Bottomly K.
Th2-induced airway mucus production is dependent on IL-4R alpha, but not on eosinophils.
J Immunol
162:
6178-6183,
1999
7.
Cohn, L,
Homer RJ,
Marinov A,
Rankin J,
and
Bottomly K.
Induction of airway mucus production by T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production.
J Exp Med
186:
1737-1747,
1997
8.
Cohn, L,
Homer RJ,
Niu N,
and
Bottomly K.
T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production.
J Exp Med
190:
1309-1318,
1999
9.
Collins, PD,
Marleau S,
Griffiths-Johnson DA,
Jose PJ,
and
Williams TJ.
Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.
J Exp Med
182:
1169-1174,
1995[Abstract].
10.
Corry, DB,
Grunig G,
Hadeiba H,
Kurup VP,
Warnock ML,
Sheppard D,
Rennick DM,
and
Locksley RM.
Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice.
Mol Med
4:
344-355,
1998[ISI][Medline].
11.
Denzler, KL,
Farmer SC,
Crosby JR,
Borchers MT,
Cieslewicz G,
Larson KA,
Cormier-Regard S,
Lee NA,
and
Lee JJ.
Eosinophil major basic protein-1 does not contribute to allergen-induced airway pathologies in mouse models of asthma.
J Immunol
165:
5509-5517,
2000
12.
Dong, Q,
Louahed J,
Vink A,
Sullivan CD,
Messler CJ,
Zhou Y,
Haczku A,
Huaux F,
Arras M,
Holroyd KJ,
Renauld JC,
Levitt RC,
and
Nicolaides NC.
IL-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice.
Eur J Immunol
29:
2130-2139,
1999[ISI][Medline].
13.
Fung-Leung, WP,
Schilham MW,
Rahemtulla A,
Kundig TM,
Vollenweider M,
Potter J,
van Ewijk W,
and
Mak TW.
CD8 is needed for development of cytotoxic T cells but not helper T cells.
Cell
65:
443-449,
1991[ISI][Medline].
14.
Garlisi, CG,
Falcone A,
Hey JA,
Paster TM,
Fernandez X,
Rizzo CA,
Minnicozzi M,
Jones H,
Billah MM,
Egan RW,
and
Umland SP.
Airway eosinophils, T cells, Th2-type cytokine mRNA, and hyperreactivity in response to aerosol challenge of allergic mice with previously established pulmonary inflammation.
Am J Respir Cell Mol Biol
17:
642-651,
1997
15.
Gavett, SH,
Chen X,
Finkelman F,
and
Wills-Karp M.
Depletion of murine CD4+ T lymphocytes prevents antigen-induced airway hyperreactivity and pulmonary eosinophilia.
Am J Respir Cell Mol Biol
10:
587-593,
1994[Abstract].
16.
Grunewald, SM,
Kunzmann S,
Schnarr B,
Ezernieks J,
Sebald W,
and
Duschl A.
A murine interleukin-4 antagonistic mutant protein completely inhibits interleukin-4-induced cell proliferation, differentiation, and signal transduction.
J Biol Chem
272:
1480-1483,
1997
17.
Grunewald, SM,
Werthmann A,
Schnarr B,
Klein CE,
Brocker EB,
Mohrs M,
Brombacher F,
Sebald W,
and
Duschl A.
An antagonistic IL-4 mutant prevents type I allergy in the mouse: inhibition of the IL-4/IL-13 receptor system completely abrogates humoral immune response to allergen and development of allergic symptoms in vivo.
J Immunol
160:
4004-4009,
1998
18.
Grunig, G,
Warnock M,
Wakil AE,
Venkayya R,
Brombacher F,
Rennick DM,
Sheppard D,
Mohrs M,
Donaldson DD,
Locksley RM,
and
Corry DB.
Requirement for IL-13 independently of IL-4 in experimental asthma.
Science
282:
2261-2263,
1998
19.
Henderson, W, Jr.
Lipid-derived and other chemical mediators of inflammation in the lung (review).
J Allergy Clin Immunol
79:
543-553,
1987[ISI][Medline].
20.
Henderson, WR, Jr,
Chi EY,
and
Maliszewski CR.
Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse model of asthma.
J Immunol
164:
1086-1095,
2000
21.
Henderson, WR, Jr,
Lewis DB,
Albert RK,
Zhang Y,
Lamm WJ,
Chiang GK,
Jones F,
Eriksen P,
Tien YT,
Jonas M,
and
Chi EY.
The importance of leukotrienes in airway inflammation in a mouse model of asthma.
J Exp Med
184:
1483-1494,
1996[Abstract].
22.
Itohara, S,
Mombaerts P,
Lafaille J,
Iacomini J,
Nelson A,
Clarke AR,
Hooper ML,
Farr A,
and
Tonegawa S.
T cell receptor delta gene mutant mice: independent generation of alpha beta T cells and programmed rearrangements of gamma delta TCR genes.
Cell
72:
337-348,
1993[ISI][Medline].
23.
Jeffery, PK.
Morphology of the airway wall in asthma and in chronic obstructive pulmonary disease.
Am Rev Respir Dis
143:
1152-1161,
1991[ISI][Medline].
24.
Kitamura, D,
Roes J,
Kuhn R,
and
Rajewsky K.
A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene.
Nature
350:
423-426,
1991[ISI][Medline].
25.
Kung, TT,
Stelts DM,
Zurcher JA,
Adams GK,
Egan RW,
Kreutner W,
Watnick AS,
Jones H,
and
Chapman RW.
Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody.
Am J Respir Cell Mol Biol
13:
360-365,
1995[Abstract].
26.
Lee, JJ,
McGarry MP,
Farmer SC,
Denzler KL,
Larson KA,
Carrigan T,
Brenneise IE,
Horton MA,
Haczku A,
Gelfand EW,
Leikauf GD,
and
Lee NA.
Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma.
J Exp Med
185:
2143-2156,
1997
27.
Lee, NA,
Gelfand EW,
and
Lee JJ.
Pulmonary T cells and eosinophils: coconspirators or independent triggers of allergic respiratory pathology?
J Allergy Clin Immunol
107:
945-957,
2001[ISI][Medline].
28.
Lee, NA,
McGarry MP,
Larson KA,
Horton MA,
Kristensen AB,
and
Lee JJ.
Expression of IL-5 in thymocytes/T cells leads to the development of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies.
J Immunol
158:
1332-1344,
1997[Abstract].
29.
Macias, MP,
Fitzpatrick LA,
Brenneise I,
McGarry MP,
Lee JJ,
and
Lee NA.
Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice.
J Clin Invest
107:
949-959,
2000
30.
McCarrick, JW,
Parnes JR,
Seong RH,
Solter D,
and
Knowles BB.
Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells.
Transgenic Res
2:
183-190,
1993[ISI][Medline].
31.
Mombaerts, P,
Clarke AR,
Rudnicki MA,
Iacomini J,
Itohara S,
Lafaille JJ,
Wang L,
Ichikawa Y,
Jaenisch R,
Hooper ML,
and
Tonegawa S.
Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages.
Nature
360:
225-231,
1992[ISI][Medline]. [published erratum appears in Nature 1992 Dec 3, 360: 491]
32.
Ohkawara, Y,
Lei XF,
Stampfli MR,
Marshall JS,
Xing Z,
and
Jordana M.
Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation.
Am J Respir Cell Mol Biol
16:
510-520,
1997[Abstract].
33.
Pack, RJ,
Al-Ugaily LH,
and
Morris G.
The cells of the tracheobronchial epithelium of the mouse: a quantitative light and electron microscope study.
J Anat
132:
71-84,
1981[ISI][Medline].
34.
Plopper, CG,
Hill LH,
and
Mariassy AT.
Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A study of man with comparison of 15 mammalian species.
Exp Lung Res
1:
171-180,
1980[ISI][Medline].
35.
Sanderson, CJ.
The biological role of interleukin 5.
Int J Cell Clon
8:
147-154,
1990.
36.
Sanderson, CJ,
Campbell HD,
and
Young IG.
Molecular and cellular biology of eosinophil differentiation factor (interleukin-5) and its effects on human and mouse B cells.
Immunol Rev
102:
29-50,
1988[ISI][Medline].
37.
Sehmi, R,
Wardlaw AJ,
Cromwell O,
Kurihara K,
Waltmann P,
and
Kay AB.
Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects.
Blood
79:
2952-2959,
1992[Abstract].
38.
Sheehan, JK,
Thornton DJ,
Howard M,
Carlstedt I,
Corfield AP,
and
Paraskeva C.
Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fully glycosylated units.
Biochem J
315:
1055-1060,
1996[ISI][Medline].
39.
Smith, MN,
Greenberg SD,
and
Spjut HJ.
The Clara cell: a comparative ultrastructural study in mammals.
Am J Anat
155:
15-30,
1979[ISI][Medline].
40.
Townsend, JM,
Fallon GP,
Matthews JD,
Smith P,
Jolin EH,
and
McKenzie NA.
IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development.
Immunity
13:
573-583,
2000[ISI][Medline].
41.
Voynow, JA,
Young LR,
Wang Y,
Horger T,
Rose MC,
and
Fischer BM.
Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.
Am J Physiol Lung Cell Mol Physiol
276:
L835-L843,
1999
42.
Wills-Karp, M.
Immunologic basis of antigen-induced airway hyperresponsiveness.
Annu Rev Immunol
17:
255-281,
1999[ISI][Medline].
43.
Woerly, G,
Roger N,
Loiseau S,
and
Capron M.
Expression of Th1 and Th2 immunoregulatory cytokines by human eosinophils.
Int Arch Allergy Immunol
118:
95-97,
1999[ISI][Medline].
44.
Zhu, Z,
Homer RJ,
Wang Z,
Chen Q,
Geba GP,
Wang J,
Zhang Y,
and
Elias JA.
Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.
J Clin Invest
103:
779-788,
1999