1 Division of Hematology/Oncology and 2 Pulmonary Medicine, Department of Biochemistry and Molecular Biology, S. C. Johnson Medical Research Building, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259; and 3 Department of Respiratory Medicine, Second Hospital, Zhejiang University College of Medicine, HangZhou 310009, People's Republic of China
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ABSTRACT |
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A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.
mouse; lung; inflammation; transgenic/knockout; interleukin-5
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INTRODUCTION |
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THE PULMONARY EOSINOPHILIA accompanying the pathological changes associated with asthma has been a correlative feature recognized even in the earliest studies investigating this disease (see for example Ref. 26). Since these early 20th century studies, innumerable investigations have confirmed and detailed this relationship, demonstrating that the presence of eosinophils is predictive of disease severity and occurs even in mild cases (4). The recruitment of eosinophils also occurs in animal models of allergen-mediated respiratory inflammation; the mouse, in particular, has been extensively studied. Despite the abundance of clinical studies and the availability of mouse models that correlate pulmonary eosinophilia with lung dysfunction, eosinophil effector functions are poorly understood. Indeed, the role of these cells in allergic respiratory disease has recently been questioned (31).
Attempts to define a specific causative relationship between the recruitment of eosinophils and onset/progression of pulmonary pathologies [e.g., mucus overproduction and airway hyperresponsiveness (AHR)] in the mouse have been equivocal and in some cases even suggest the lack of such a relationship (see for example Refs. 8, 9, 24, 25). Conversely, the most compelling data suggesting that eosinophils are linked to allergen-induced pulmonary pathologies arise from studies manipulating in vivo IL-5 levels, including IL-5 knockout mice (18), antibody neutralization studies (see for example Ref. 30), and IL-5 overexpression in transgenic mice (32). These studies each relied on the premise that IL-5 activities in the mouse are limited to proliferative and survival effects on eosinophils, activities that elicit B cell maturation, and potential agonist effects directly on airway smooth muscle (22, 34). However, because ovalbumin (OVA)-induced pulmonary pathologies are not diminished in B cell-deficient mice (29), the loss of pulmonary pathologies in IL-5-deficient animals has been assumed to be a consequence of IL-5-mediated effects on eosinophils alone. For example, studies using IL-5 knockout mice (18) or neutralization studies using the TRFK-5 antibody (23) each demonstrated that the loss of allergen-induced pulmonary eosinophilia was accompanied by the elimination of AHR. Moreover, a study investigating allergen provocation in the mouse also demonstrated that IL-5 was necessary for the late phase of allergen-mediated bronchoconstriction (6). Interestingly, the effects of abolishing IL-5 were not necessarily limited to pulmonary pathophysiology as Kung and colleagues (30) demonstrated that treatment of mice with TRFK-5 antibodies significantly reduced goblet cell metaplasia/mucus production.
In an attempt to avoid the pleiotropic effects of neutralizing IL-5, eosinophils were uniquely ablated in the lungs of allergen-sensitized/challenged mice through a strategy using a depleting rat monoclonal antibody specific for mouse CCR3. CCR3 is a chemokine receptor whose principle ligands (i.e., eotaxin-1 and -2) are potent chemoattractants displaying a unique specificity for eosinophils (44). In the mouse, CCR3 expression appears to be restricted to eosinophils. Moreover, systemic administration of a depleting anti-CCR3 monoclonal antibody was capable of inducing a targeted reduction in peripheral blood, although potential effects on all leukocytes were not examined (20). In contrast to the apparent eosinophil specificity of CCR3 antibodies, the loss of CCR3 function in knockout mice led to a 4- to 12-fold increase in lung tissue mast cells/basophils following OVA sensitization/aerosol challenge, suggesting to the authors that CCR3 expression may also occur on these granulocytes (27). The specificity and utility of depleting eosinophils using an anti-CCR3 antibody strategy were determined in this study by modifying the previously published protocol using this reagent to yield a methodology that abolishes eosinophils from the airway lumen of OVA-sensitized/challenged mice and reduces the perivascular/peribronchial eosinophilia to levels indistinguishable from naive saline-challenged animals. Significantly, the data show that no other cell types, including mast cells/basophils, are affected as a consequence of antibody treatment. In particular, lymphocyte activities responsible for the underlying inflammatory responses associated with allergen challenge were intact, including allergen-specific immunoglobulin production (B cell functions), T cell-mediated elaboration of pulmonary Th2 cytokines, and ex vivo allergen-dependent memory responses of isolated T cells. Despite leaving T cell functions apparently intact, the selective ablation of eosinophils attenuates allergen-induced goblet cell metaplasia/mucus production and eliminates AHR in response to cholinergic receptor agonist. These data suggest a causative relationship exists between pulmonary eosinophils and allergen-induced pulmonary pathologies and potentially highlight a synergy between CD4+ T cell activities and eosinophil effector functions (reviewed in Ref. 33), both of which are apparently required for the development of the pathologies associated with allergic respiratory inflammation.
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MATERIALS AND METHODS |
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Animals. C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Experimental protocols were performed on mice 8-12 wk old maintained in ventilated microisolator cages housed in a specific pathogen-free animal facility. Procedures and studies involving animals were conducted in accordance with National Institutes of Health and Mayo institutional guidelines.
OVA sensitization and challenge. Mice were sensitized and challenged with chicken OVA as previously described (12). Briefly, on days 0 and 14, all animals were injected (100 µl ip) with OVA (20 µg crude grade IV; Sigma, St. Louis, MO) emulsified in 2.25 mg of aluminum hydroxide/magnesium hydroxide (Pierce, Rockford, IL). On days 24-26, animals were exposed for 20 min to an OVA aerosol (1% wt/vol OVA in saline) generated with an ultrasonic nebulizer (DeVilbiss, Somerset, PA). Control animals received a saline-only aerosol. The mice were assessed for pulmonary cellular infiltrates, histopathologies, and lung function on day 28.
Administration of anti-mouse CCR3 monoclonal antibodies. On days 24-26 of the OVA protocol, animals were administered rat anti-mouse CCR3 antibodies by two independent routes. Before each aerosol OVA challenge, mice were injected (intraperitoneally) with 150 µg of rat anti-mouse CCR3 monoclonal antibodies [6S2-19-4; IgG2b (a kind gift of Dr. R. Coffman, DNAX, Palo Alto, CA)]. In addition, 50 µg of anti-mouse CCR3 antibodies were mixed with the OVA solution generating the nebulant (final concentration: 1 µg/ml) and administered with each of the three OVA challenges. Control mice were exposed to equivalent amounts of nonspecific rat IgG2b (Pharmingen, Torrence, CA) by both routes of administration.
Collection of bronchoalveolar lavage fluid and serum, isolation
of splenocytes and marrow-derived cells, and the enumeration of
leukocytes.
On day 28 of the OVA protocol, animals were killed, the
tracheas were cannulated, and the lungs were lavaged (3 × 0.5 ml) with ice-cold PBS/2% fetal calf serum. The methods and assessments of
bronchoalveolar lavage (BAL) cells have been described previously (32). Cell-free BAL fluid samples were stored at 80°C
before assessment of cytokine levels by ELISA.
Allergen-mediated histological changes in the lung and
immunohistochemical detection of eosinophils in paraffin-embedded
tissue.
Pulmonary histology, including mucus cell content of the airway
epithelium, was assessed from tissues excised and fixed in 10%
formalin [lungs were inflated with a fixed volume (0.5 ml) of
fixative]. The lung samples (day 28) were washed free of
formalin and subsequently dehydrated through an ethanol series before
equilibration in xylene and embedding in paraffin. Sections (4 µm)
were stained with 0.1% toluidine blue to identify and localize
pulmonary mast cells/basophils. In addition, lung sections were also
stained with periodic acid-Schiff (PAS) and counterstained with
hematoxylin/methylgreen to assess goblet cell metaplasia/ mucus
production. Parasagittal sections were analyzed by bright-field
microscopy, and analysis of the mucus content of the airway epithelium
of mice from different groups was based on the evaluation of airways
(both proximal and distal)/mouse (n = 5
animals/group). Relative comparisons of mucus content between cohorts
of animals were preformed using the imaging program Image ProPlus
(Media Cybernetics, Silver Spring, MD). The data were quantified as an
airway mucus index: [(average PAS staining intensity of the airway
epithelium) × (area of airway epithelium staining with
PAS)]/[(total area of the conducting airway epithelium) × (total number of airways assessed)].
Detection of serum IgG1 and IgE. Serum concentrations of total IgE were determined using an immunoassay kit (Endogen, Cambridge, MA) according to the manufacturer's instructions. In addition, OVA-specific IgG1 levels were determined as previously described (39). The limits of detection associated with each assay are 2 ng/ml and 0.2 A410 OD U/ml, respectively.
Cytokine assays.
Cytokine levels in BAL fluid were determined by ELISA. Mouse IL-4,
IL-5, and IL-13 levels were assessed using immunoassay kits (Endogen)
as per the manufacturer's instructions. The limits of detection for
each assay were: IL-4 10 pg/ml, IL-5
10 pg/ml, and IL-13
30 pg/ml.
Ex vivo cytokine production by allergen-stimulated splenocytes.
Mice were sensitized and either aerosol challenged with OVA or
challenged with OVA concurrent with the administration of rat anti-mouse (rm) CCR3 mAb as described above. On day 28 (2 days following the last aerosol challenge), spleens were removed and cells were suspended in RPMI-1640 media containing 5% FCS, penicillin (100 U/ml), and streptomycin (100 U/ml) and cultured in 96-well microtiter plates at a concentration of 5 × 106
cells/ml in the presence of 20 µg/ml of OVA. The capacity of lymphocytes in the spleen suspensions to produce both Th1 (e.g., IFN-
) and Th2 (e.g., IL-4 and IL-5) cytokines in response to OVA
stimulation was assessed 48 h later by centrifuging the microtiter plates (600 g, 10 min) and removing aliquots of each well
supernatant for determination of cytokine levels by ELISA.
Assessments of AHR. AHR was measured in response to incremental provocation with aerosolized methacholine on day 28 of the OVA protocol by whole body plethysmography (Buxco Electronics, Troy, NY) as previously described (13). Dose-response data were plotted as enhanced pause (Penh) vs. the log10 of the methacholine solution (mg/ml) used to generate the aerosol. There were no statistically significant differences (Tukey-Kramer honestly significant difference test) observed among the baseline Penh values (±SE) of the different animal groups used in these studies.
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RESULTS |
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Rat anti-mouse CCR3 monoclonal antibodies selectively ablate
eosinophils.
A preliminary assessment of the cell-depleting specificity of an
rmCCR3 mAb in vivo was performed using a protocol similar to the
antibody-dependent complement-mediated cell lysis-depleting strategy of
Grimaldi and colleagues (20). R
mCCR3 mAb (150 µg) was administered (intraperitoneally) to naive mice on 3 successive days; control animals received nonspecific rat
IgG2b. These data showed that 24 h following the final
antibody administration eosinophils were reduced in the peripheral
blood by >90% (Fig. 1). In contrast to
the significant decrease of blood eosinophils, administration of
r
mCCR3 mAb had no effect on the numbers of the remaining leukocytes (i.e., lymphocytes, monocytes, and neutrophils), demonstrating the
specificity of r
mCCR3 mAb treatment for only eosinophils.
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A strategy of systemic and local administration of rmCCR3 mAb
uniquely abolishes allergen-induced airway eosinophil recruitment.
Although circulating eosinophils were effectively eliminated in
OVA-sensitized mice systemically administered (intraperitoneally) r
mCCR3 mAb concurrently with the OVA aerosol challenges, this methodology only decreased, i.e., did not abolish, eosinophil numbers
in the lung (data not shown). Investigations of alternative administration strategies, in contrast, showed that it was possible to
completely block the allergen-induced recruitment of eosinophils to the
lungs of mice through a dual-administration strategy in which r
mCCR3
mAb was delivered as an aerosol to the lung (local) in conjunction with
systemic administration to the peritoneal cavity. Systemic/local
delivery of r
mCCR3 mAb, similar to systemic administration alone,
resulted in an ~80% reduction of bone marrow eosinophils (relative
to nonspecific IgG-treated mice) 48 h following the last OVA
aerosol challenge [1.75 ± 0.29 (% of marrow) vs. 10.25 ± 1.44, respectively]. However, this dual administration of r
mCCR3
mAb eliminated spleen and peripheral blood eosinophils without any
significant effects on the numbers of lymphocytes, monocytes/macrophages, or neutrophils in these peripheral compartments (Fig. 2). Enumeration of the BAL
cellularity following OVA challenge showed that this depletion of
eosinophils also occurred in the airway lumen, again without any
effects on other leukocytes in the airways (Fig.
3). It is noteworthy that the loss of
eosinophils in six or eight r
mCCR3 mAb-treated mice was absolute as
these cells were absent even from comprehensive surveys of entire
cytospin preparations. Treatment of OVA-sensitized/challenged mice with r
mCCR3 mAb also eliminated the allergen-induced increase of tissue eosinophils in the perivascular and peribronchial regions as well as
the airway submucosa (Fig. 4). Complete
surveys of several (i.e., 2-4) parasagittal lung sections from
r
mCCR3 mAb-treated mice (n = 5) revealed that
eosinophil levels in these mice were reduced to levels
indistinguishable from naive saline-challenged animals (Fig.
5). Significantly, the treatment of
OVA-sensitized/challenged mice with r
mCCR3 mAb did not lead to a
loss of lung tissue mast cells/basophils (Fig.
6), demonstrating a lack of significant expression of CCR3 on these granulocytes. Moreover, the ablation of
eosinophils following treatment with r
mCCR3 mAb was also not accompanied by changes in the number of pulmonary mast cells/basophils relative to OVA-treated mice administered nonspecific control antibody
(data not shown).
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Ablation of circulating and pulmonary eosinophils does not affect
lymphocyte-mediated acquired immune responses to OVA, including
immunoglobulin and Th2 cytokine production.
The potential expression of CCR3 on other leukocytes, particularly
lymphocytes (1), held open the possibility that the loss
of allergen-induced pulmonary eosinophilia in mice treated with
anti-CCR3 antibodies resulted from effects on underlying immune
responses and not through the direct targeting of eosinophils. However,
two measures of lymphocyte function demonstrated that administration of
rmCCR3 mAb had no discernable effect(s) on either allergen-induced B
cell functions or T cell activities. Assessments of serum
immunoglobulin production (i.e., B cell/CD4+ T cell
functions) showed that administration of r
mCCR3 mAb concurrent with
OVA aerosol challenge did not affect either the significant rise in
total IgE occurring in allergen-treated mice (Fig.
7A) or the appearance of
OVA-specific IgG1 (Fig. 7B). Allergen-mediated pulmonary production of Th2 cytokines (i.e., CD4+ T cell
activities) was also unaffected by administration of r
mCCR3 mAb. The
BAL levels of IL-4, IL-5, and IL-13 in r
mCCR3 mAb-treated mice
following the last OVA challenge were indistinguishable from control
animals receiving nonspecific IgG2b (Fig.
8A). In addition, Th1
cytokines such as IFN-
remained at low/undetectable levels in the
BAL of all the groups examined (data not shown). Moreover, ex vivo
restimulation of splenocytes with OVA (i.e., memory responses) was also
unaffected as equivalent levels of Th2 cytokines (i.e., IL-4 and IL-5),
as well as IFN-
, were observed from splenocytes of wild-type and
r
mCCR3 mAb-treated mice (Fig. 8B).
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The selective loss of airway eosinophils significantly
attenuates pulmonary histopathologies, including
OVA-induced goblet cell metaplasia/mucus production.
RmCCR3 mAb-treated mice displayed a generalized reduction in airway
inflammation typified by a decrease in airway epithelial hypertrophy
(see for example Fig. 4, right). In addition, mucous levels
in the airways of r
mCCR3 mAb-treated mice displayed a 75% decrease
relative to OVA-treated animals receiving nonspecific rat
IgG2b (Fig. 9). However,
despite the inhibition of goblet cell metaplasia/mucus production in
r
mCCR3 mAb-treated mice, in absolute terms the observed
metaplasia/mucus production was nonetheless elevated relative to
saline-challenged control mice, demonstrating that eosinophil-dependent
and -independent mechanisms are necessary for the levels of goblet cell
metaplasia/mucus production observed in OVA-treated wild-type mice.
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AHR that occurs as a consequence of OVA sensitization/aerosol
challenge is abolished in the absence of pulmonary eosinophils.
We employed whole body plethysmography to determine if allergen-induced
AHR was a concomitant pathophysiological response to the infiltration
of the lung and airway lumen by eosinophils. The methacholine
dose-response curves (means of single-animal measurements) showed that
OVA-sensitized/challenged mice receiving rmCCR3 mAb were not
hyperresponsive to methacholine provocation (Fig.
10). In contrast, the cohort of mice
receiving nonspecific rat IgG2b showed a methacholine dose
response equivalent to OVA-sensitized/challenged wild-type mice,
demonstrating the existence of a causative relationship between
allergen-mediated AHR and the presence of pulmonary eosinophils.
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DISCUSSION |
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The link between the recruitment of eosinophils to the lung and
the onset/progression of allergic respiratory pathology has remained
correlative despite repeated attempts to establish causative mechanisms. For example, anti-IL-5 strategies to ablate eosinophils have inherent ambiguities deriving from observations that IL-5 has
potential activities on other cell types (34). The advent of a strategy to specifically ablate eosinophils in the mouse, however,
now provides evidence of a causative link. A review of the available
evidence shows that the value of this strategy is uniquely a function
of the eosinophil specificity of targeting CCR3+ cells
without identifiable effects on any other cell types or inflammatory
responses: 1) Grimaldi and colleagues (20)
demonstrated by FACS that rmCCR3 mAb reactivity was restricted to
eosinophils. 2) Administration of r
mCCR3 mAb led to the
depletion of only eosinophils from circulation with no effects on other
leukocytes. This effect was also extended to analyses of peripheral
lymphoid tissue such as the spleen, where effects were restricted to
eosinophils. 3) Dual systemic/local administration of
r
mCCR3 mAb to mice during the OVA challenge phase of a
sensitization/challenge protocol abolished eosinophils from the airway
lumen with no observable effects on other cell types present in BAL
fluid. In addition, the administration of r
mCCR3 mAb had effects
only on lung tissue eosinophils and did not lead to the ablation of any
other infiltrating/resident leukocyte. Moreover, unlike observations
from OVA-sensitized/challenged CCR3-deficient mice (27),
r
mCCR3 mAb treatment elicited effects in the lung only on eosinophil
numbers and did not modulate pulmonary mast cell/basophil numbers.
4) Administration of r
mCCR3 mAb to OVA-treated mice had
no effects on the underlying mechanisms leading to the induced Th2
inflammatory responses. In particular, administration of r
mCCR3 mAb
had no effects on B or T cell activities. 5) Administration of r
mCCR3 mAb had no apparent effects on lung structure and did not
affect the morbidity/mortality of the mice, suggesting that "structural" cell types in the mouse were unaffected by this
depleting antibody.
In addition to the eosinophil specificity of the protocol employed, the ability of this strategy to effectively abolish eosinophils in the BAL and reduce levels in peribronchial/perivascular regions to levels equivalent to saline-challenged control mice is essential to the conclusions drawn about the roles of these cells in allergic respiratory inflammation. Previous studies purporting to dissociate pulmonary pathology from the recruitment of eosinophils, including studies demonstrating that AHR occurs in the absence of a significant airway eosinophilia, have likely overlooked the potential role(s) of eosinophils in these respective model systems. For example, methodologies depleting allergen-induced pulmonary eosinophils through cytokine/chemokine deficiencies also have potential consequences on cell types other than eosinophils. The data presented in other studies (see for example Refs. 8, 9, 25) also do not show that AHR occurs in the absence of an airway eosinophilia. Rather, they show that AHR occurs in mice even though the induced airway eosinophilia had been dramatically reduced (i.e., in each of these studies, eosinophil numbers in the experimental mice are low but elevated relative to control animals). The small numbers of eosinophils present may actually be sufficient to elicit AHR. The final unresolved issue is the relative activation state of infiltrating eosinophils. The eosinophils recruited to the lungs in previous studies noted above, albeit few in number, may all be primed and competent to execute effector functions. Thus, it may not be the specific number of eosinophils recruited to the lung that is critical but their activation state and their location within the lung [i.e., perhaps strategically located small subpopulations of activated eosinophils near airway smooth muscle or pulmonary nerve cells are the actual effector cells recruited to the lungs in these models (see for example Ref. 10)].
The link of eosinophils not only to AHR but also to allergen-mediated goblet cell metaplasia/mucus production implies that activities from this cell type are causative of a wide range of pulmonary pathologies. Similar to observations following administration of anti-IL-5 antibodies (30), OVA-induced mucus production decreased following the selective ablation of pulmonary eosinophils. This observation suggests the existence of both eosinophil-independent and -dependent pathways leading to allergen-mediated goblet cell metaplasia/mucus production. The link of this histopathology with eosinophils, however, is surprising as Th2/Th1 BAL cytokine levels were seemingly unaffected, relative to OVA-treated wild-type mice, in the absence of eosinophils. In addition, a number of previous studies has suggested that such a link does not exist. For example, adoptive transfer studies using OVA T cell receptor transgenic animals and IL-5-deficient mice showed that allergen-induced mucus production can occur in the absence of both IL-5 and an extensive pulmonary eosinophilia (7). These studies suggest that CD4+ T cells and IL-4 receptor expression are each necessary (and possibly sufficient) for allergen-induced mucus overproduction (7) with particular focus centering on IL-13 as the relevant cytokine ligand (21). We would suggest that the resolution of the quandary as to whether eosinophils participate in mucus production is simply that these previous studies demonstrated that mucus production was possible in a relative absence of eosinophils, not that eosinophils have no role in this histopathology. Indeed, the mucus production occurring in eosinophil-depleted mice demonstrates that this response, in part, occurs independent of eosinophils. Nonetheless, the absolute magnitude of goblet cell metaplasia/mucus production in the absence of eosinophils was significantly lower, implying that an activity(ies) of eosinophils augments the levels of pathology induced by IL-4/IL-13-dependent mechanisms.
The demonstration that eosinophils contribute to multiple allergen-induced pulmonary pathologies suggests that upon, and/or concomitant with, recruitment to the lung, eosinophils undergo changes resulting in the execution of effector functions. Several lines of evidence implicate IL-4/IL-13 as potential factors mediating mechanisms accounting for the seemingly independent, and yet dependent, behavior of allergic pulmonary pathologies on eosinophil effector functions. IL-4/IL-13 receptor ligand interactions occur on many different cell types in the lung, including T (19) and B lymphocytes (5), mast cells (41), lung endothelial cells (3), lung epithelial cells (42), pulmonary alveolar macrophages (15), and eosinophils (14). Any one of these interactions, particularly events associated with the expression of IL-13, is likely necessary, but not sufficient, to elicit allergic pulmonary pathologies (17). Moreover, the end point levels of observed pathologies are also likely the result of both the linear sum of each pathway's contribution and also a result of synergistic effects between multiple pathways. For example, IL-4/IL-13-mediated effects on T cells may have downstream consequences on other cell types in the lung contributing to pulmonary pathology. The possibility exists that IL-4/IL-13 receptor ligand interactions directly on eosinophils may signal changes that generally have been described in the literature as "activation." In this model, IL-4/IL-13 receptor ligand interactions elicit both eosinophil-independent events and the execution of eosinophil effector function(s), the sum of which is responsible for the development of pulmonary pathologies following allergen challenge (43). The immunoregulative capacity of eosinophils themselves may even contribute to this process. Pulmonary eosinophils following allergen challenge have been shown to express GATA-3 resulting in the de novo activation of Th2 cytokine genes (28). Moreover, Bandeira-Melo and colleagues (2) demonstrated that eotaxin, through CCR3-mediated signaling pathways, rapidly mobilizes preformed stores of IL-4 in eosinophils through vesicular transport mechanisms. Although the release of IL-4/IL-13 by eosinophils themselves appears insufficient to significantly affect total BAL cytokine levels, localized release within pulmonary microenvironments may modulate immune responses in areas where eosinophils have concentrated (e.g., peribronchial regions) by autocrine/paracrine regulatory mechanisms.
The provocative consequence of IL-4/IL-13 receptor ligand-mediated events on eosinophils is the identification of a mechanism linking eosinophils with T cell-derived signals. This suggests that the apparent codependence of OVA-induced pathologies on CD4+ T cells is the result of T cell-eosinophil interactions leading to the execution of eosinophil effector functions and, therefore, to the onset/progression of pulmonary pathology. Recent studies demonstrate a potential role of eosinophils as antigen presentation cells (APC) capable of interacting with T cells in the lymph nodes of the lung (40). In addition, MacKenzie and colleagues (36) suggest that this APC function is a critical/prominent effector function of recruited eosinophils. Thus, a simplistic explanation for the lack, or attenuation, of pulmonary pathologies in the absence of eosinophils is that the APC functions of these cells are necessary for the underlying inflammation. However, it is noteworthy that the anti-CCR3-mediated loss of eosinophils from the lung did not have a demonstrable effect on T cell activities, demonstrating that while eosinophils are capable of functioning as APCs, this activity does not appear to be a significant component of the immune responses leading to allergic respiratory inflammation. Instead, "action at a distance" paradigms, in which T cells and eosinophils communicate through secreted cellular signals, are likely mechanisms resulting in the activation of one or both cell types and, in turn, the development of pulmonary pathologies. Interestingly, the lack of phenotypic effects (relative to wild type) in OVA-treated knockout mice deficient of eosinophil secondary granule protein genes [e.g., MBP-1 (13) or EPO (12)] and electron microscopy studies failing to find evidence of eosinophil degranulation in the mouse (37) suggest that the relevant eosinophil effector functions contributing to mucus production and/or AHR do not include release of secondary granule proteins. The implicit conclusion is that eosinophil effector functions are mediated by a previously overlooked mechanism(s) and/or a heretofore unknown pathway(s).
Collectively, observations suggesting that eosinophils are necessary, but not sufficient, to elicit pulmonary pathologies are consistent with most of the available data in the literature. A notable exception is a clinical study of asthma patients using an anti-IL-5 antibody treatment regime, in which it is concluded that eosinophils are not necessary for allergen-mediated pulmonary pathology (31). These data, however, have since been called into question on the basis of methodological limitations (38) and the demonstration that the antibody treatment had little effect on lung tissue eosinophils (16). In addition, the conclusion that eosinophils did not contribute to airway pathologies was also predicated, in part, by defining the origins of disease in terms of single cellular/molecular events (i.e., so-called "all or none" theses of causation), a criterion that no one cell type, or molecule, is likely to fulfill as an explanation for a polygenic syndrome (11) such as asthma. The demonstration in this study that eosinophils are required for allergen-induced pulmonary pathologies in the mouse provides evidence of a direct causative relationship. Moreover, the data support an expanded view of eosinophil activities in the lung and suggest that potential interactions with T cells are underlying mechanisms leading to allergic respiratory inflammation and lung dysfunction.
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ACKNOWLEDGEMENTS |
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It is with gratitude that we thank Dr. G. Gleich for ongoing discussions and advice as the study progressed. Special thanks go to the Mayo Clinic Scottsdale Graphic Arts Facility (Director: M. Ruona) as well as research program assistant L. Mardel and colleagues J. Ford and P. McGarry; without this administrative staff, we could not function as an integrated group or a productive laboratory.
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FOOTNOTES |
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These studies represent a considerable effort on the part of many individuals in addition to the cited authors. In particular, we acknowledge Dr. R. (Bob) Coffman for sharing with us the rat anti-mouse CCR3 monoclonal antibody used in this study and for encouragement to develop methodologies using this reagent. We also acknowledge the efforts of J. Caplette, the Mayo Clinic Scottsdale basic/clinical engineer, for a devotion to detail and for ensuring that equipment reproducibly functions with precision.
This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grants HL-58723 (N. Lee) and HL-60793 (N. Lee) and the Arizona Chapter of the American Lung Association (J. Crosby).
Address for reprint requests and other correspondence: J. J. Lee, Division of Pulmonary Medicine, Dept. of Biochemistry and Molecular Biology, S. C. Johnson Medical Research Bldg., Mayo Clinic Scottsdale, 13400 E. Shea Boulevard, Scottsdale, AZ 85259 (E-mail address: jjl11{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.
September 13, 2002;10.1152/ajplung.00260.2002
Received 2 August 2002; accepted in final form 12 September 2002.
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