Divisions of 1 Hematology/Oncology and 3 Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259; and 2 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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CD4+ T cells have a critical role in the development
of allergic pulmonary inflammation, including the recruitment of
eosinophils to the airway lumen and interstitium. The expression of
interleukin (IL)-5 by CD4+ cells has, in particular, often
been lionized as the central link between allergic inflammation and the
concomitant expansion or recruitment of eosinophils. The mechanism(s)
by which CD4+ T cells mediates eosinophil recruitment was
assessed with gene knockout mice deficient for T cells or T cell
subtypes and a unique IL-5 transgenic mouse (line NJ.1726) that
constitutively overexpresses this cytokine in the lung epithelium.
Pulmonary IL-5 expression is significantly attenuated in T cell- and
CD4+ but not CD8+ cell-deficient animals,
suggesting an obvious explanation for the lack of eosinophils in the
lungs of T cell-deficient and CD4(/
) mice. However, although the
constitutive expression of IL-5 in the lung epithelium of NJ.1726 mice
elicited an eosinophilia in the airway lumen of both naive and
ovalbumin-treated mice, in the absence of CD4+ cells,
allergen-mediated eosinophil recruitment to the bronchoalveolar lavage
fluid was abolished. Moreover, intranasal instillation of the potent
eosinophil-specific chemokine eotaxin-2 was incapable of eliciting
eosinophil recruitment in naive and ovalbumin-treated NJ.1726
CD4(
/
) mice, suggesting that eosinophil trafficking during allergic
inflammatory responses is a consequence of a CD4+
cell-mediated event(s) in addition to IL-5 expression and the establishment of a pulmonary chemokine gradient.
asthma; allergy; mouse model; transgenic; gene knockout; interleukin-5
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INTRODUCTION |
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STUDIES OF ALLERGIC RESPIRATORY inflammation have centered on the relative roles of both resident pulmonary leukocytes (e.g., mast cells, natural killer cells, alveolar macrophages, dendritic cells) and leukocytes actively recruited to the lung as a consequence of antigen challenge (e.g., T lymphocytes and eosinophils). The recruitment of CD4+ T cells, and eosinophils in particular, correlates with the onset and progression of pulmonary dysfunction even in nominal cases of asthma, and the magnitude of the influx of these infiltrates is generally proportional to disease severity (5). Remarkably, this significant airway eosinophil recruitment has been a recognized marker of asthma even in the earliest clinical studies of this disease (see, for example, Ref. 25). Although debate exists regarding eosinophil effector activities occurring in the lung, at least five independent mechanisms potentially allow the eosinophil to modulate the intensity of pulmonary inflammation. 1) Eosinophils are immunoregulative cells capable of secreting both T helper (Th) type 1 and Th2 cytokines (54) as well as several chemokines (see, for example, Ref. 34). 2) Activated eosinophils generate and release copious amounts of reactive oxygen intermediates (10). 3) Recruited eosinophils are a source of small-molecule lipid mediators of inflammation [e.g., cysteinyl leukotrienes (24, 52)] and platelet-activating factor (PAF) (33). 4) Eosinophils secrete a series of degradative enzymes [e.g., lysophospholipase (39)] that may have significant effects on lung structure, including pulmonary surfactant activity. 5) The toxic granule proteins called eosinophil secondary granule proteins found in eosinophils recruited to the lung are toxic to epithelial cells at the concentrations present in respiratory secretions from asthma patients (15) and may increase vascular permeability (36). In addition, eosinophil secondary granule proteins potentially induce airway smooth muscle contraction (20) and activate several cell types, including epithelial goblet cells [see review by Gleich and Adolphson (19)].
Despite the commonality of CD4+ T cell and eosinophil pulmonary recruitment, the hypothesized activities of CD4+ T cells are substantively different and are thought to orchestrate the underlying allergic inflammation primarily through the release of cytokine inflammatory signals. Particular focus has centered on the elaboration of a series of cytokines collectively characteristic of Th2-type inflammation. Th2-associated cytokines have pleiotropic inflammatory effects, eliciting mucus overproduction and goblet cell metaplasia [e.g., interleukin (IL)-4, -9, -13 (7, 8, 50)], eosinophil proliferation [e.g., IL-5 (48)], airway expression of chemokines with eosinophil agonist activities (57), immunoglobulin class switching [e.g., IL-4, -13 (27)], Th2 T cell differentiation [e.g., IL-4, -13 (2)], upregulation or activation of specific cell adhesion molecules [e.g., IL-4, -13 (31, 56)], and, in mice, B cell proliferation [e.g., IL-5 (45)]. The specific contributory activities of CD4+ cells during allergen sensitization or challenge that lead to pulmonary pathology have not been resolved because several of these CD4+ cell-dependent changes directly contribute to pulmonary pathology (e.g., mucus overproduction), whereas their effects on B cells [i.e., antibody production (29)] and mast cells (46) do not appear to be necessary.
The pleiotropic nature of CD4+ T cell function has been a
significant obstacle to defining specific mechanisms by which these cells elicit eosinophil recruitment to the lung. That is, potential CD4+ cell-mediated effects range from attenuation of
eosinophil proliferation in the marrow and inhibition of eosinophil
recruitment from the circulation to survival of mature eosinophils in
the airway lumen. For example, if CD4+ T cells are the
prominent cell type expressing IL-5 and, in turn, are therefore
necessary for eosinophil proliferation, the loss of pulmonary
eosinophilia is a likely outcome of the depletion of CD4+
cells. Indeed, antibody depletion of CD4+ cells eliminated
allergen-induced pulmonary eosinophilia to an extent similar to that
seen in depletion studies of IL-5 itself (see, for example, Ref.
18 vs. Ref. 49). CD4+
T cell-mediated activities also appear necessary for the establishment of some chemokine gradients within the lung (57) that are
presumably necessary, in part, for airway eosinophil recruitment.
Alternatively, CD4+ T cell expression of IL-4 and/or IL-13
mediates changes in endothelial expression of cell surface adhesion
molecules during inflammatory responses. Many of these changes appear
to be necessary for the recruitment of eosinophils to the airways,
including the upregulation of E-selectin (13) and the
4-integrin receptor ligand vascular cell adhesion
molecule-1 (see, for example, Ref. 26). In addition, a
series of elegant studies published by Cohn and colleagues (6, 8) suggested that IL-4 expression may have effects on eosinophil recruitment and survival through the upregulation of another T cell-derived cytokine, interferon (IFN)-
. These studies suggest that
increased levels of IFN-
may elicit eosinophil apoptosis and
thus abolish accumulation of airway eosinophils. Moreover, CD4+ T cell-mediated increases in granulocyte-macrophage
colony-stimulating factor are also likely to have significant effects
on eosinophil survival and, therefore, airway accumulation of these
leukocytes (44).
The constitutive ectopic expression of IL-5 in the airway epithelium associated with the transgenic mouse line NJ.1726 (32) was used, together with gene knockout mice deficient for specific T cell subtypes, to better define the specific CD4+ T cell-dependent mechanisms necessary for eosinophil recruitment to the lung. In particular, the issues of allergen-induced CD4+ T cell expression of IL-5 and the establishment of a chemokine gradient were assessed. The data presented show that although reconstitution of IL-5 expression in CD4+ T cell-deficient mice induced an eosinophilia in the pulmonary interstitium of allergen-challenged mice, eosinophils were nonetheless incapable of migrating to the airway lumen. Furthermore, eosinophil recruitment to the lumen was not recoverable through the instillation of the potent eosinophil chemokine eotaxin-2, demonstrating that CD4+ T cell-mediated accumulation of eosinophils in the lungs is the result of a concurrent mechanism(s) that acts in addition to cell proliferation and vectorial movement in response to a chemoattractant.
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MATERIALS AND METHODS |
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Mice.
Transgenic mice constitutively expressing murine IL-5 from the
lung epithelium (line NJ.1726) were generated as previously described
(32) and were maintained by continual backcross to C57BL/6J. NJ.1726 animals were bred with gene knockout mice (Jackson Laboratory, Bar Harbor, ME) lacking either T cells [(/
);
C57BL/6J TCR-
tm1Mom/TCR-
tm1Mom
(37)], CD4+ cells [C57BL/6J
CD4tm1Knw (35)], or CD8+ cells
[C57BL/6 CD8atm1Mak (16)] to generate
compound IL-5 transgenic gene knockout mice deficient in T,
CD4+, and CD8+ cells, respectively. The
genotypes of mice derived from these crosses were determined by the
presence of the IL-5 transgene (PCR of tail DNA) and loss of T cells or
T cell subtypes as assayed by flow cytometry on peripheral blood with
conjugated antibodies against mouse T cell receptor (TCR)-
, TCR-
,
CD4+, and CD8+ as previously described
(4). Control C57BL/6J mice were obtained from the Jackson
Laboratory, 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.
Ovalbumin sensitization and challenge. Wild-type mice were sensitized by an intraperitoneal injection (100 µl) of 20 µg of chicken ovalbumin (Ova; Sigma, St. Louis, MO) emulsified in 2 mg of 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 bronchoalveolar lavage (BAL) fluid IL-5 levels and airway or alveolar tissue eosinophils were performed on day 28.
BAL, eosinophil counts, and cytokine level
determinations.
Lungs were lavaged three times with 0.5 ml of PBS containing 2% FCS.
The recovered BAL fluids were pooled and centrifuged, generating a BAL
cell pellet and a cell-free supernatant. Total cell counts were
determined with a hemacytometer, and eosinophil counts were performed
on Wright-stained cytospin slides (Cytospin 3, Shandon Scientific,
Pittsburgh, PA) by counting 300 cells. Cell-free lavage fluid was
frozen on dry ice and stored at
80°C until used. Murine IL-5 levels
were measured with a sandwich ELISA assay as described by the supplier
of the anti-IL-5 antibodies (PharMingen, San Diego, CA).
Immunohistochemical identification of lung eosinophils. Immunohistochemistry was performed with a rabbit polyclonal antibody against murine 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). Briefly, endogenous peroxidase was quenched in the lung sections with 3% hydrogen peroxide in methanol for 20 min at 25°C. Subsequently, the sections were pepsin digested (Zymed, San Francisco, CA) for 30 min at 25°C, washed in PBS (GIBCO BRL) for 15 min, and blocked with PBS containing 1.0% normal goat serum and 1% BSA for 30 min at 25°C before incubation with primary antibody (1:3,000 dilution, 60 min at 25°C). Antibody-bound slides were washed in PBS-1.0% BSA and incubated (30 min, 25°C) with biotinylated goat anti-rabbit antibody (1:500 dilution). Sections were incubated first with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 min at 25°C and then with diaminobenzidine (3 min, 25°C) for the development of a colored reaction product before being counterstained with methyl green. Tissue eosinophils were quantified by counting the total number of MBP-positive cells in five randomly selected high-power fields per section and expressed as eosinophils per square millimeter of lung tissue (n = 6 mice/group).
Eosinophil transmigration assay. Polycarbonate membrane (5 µm) Transwell inserts in 24-well tissue culture polystyrene plates (Costar, Corning, NY) were preincubated with medium (RPMI 1640 containing 5% FCS) for 1 h. The medium was removed, and the eosinophils (1 × 106 cells) and recombinant mouse IL-5 (30 ng/ml) in a total volume of 200 µl of medium were placed in the inserts, and, in turn, the inserts were subsequently seated in the wells of the plate, each of which contained 500 µl of medium alone or medium containing mouse eotaxin-2. The plates were incubated at 37°C in 5% CO2 for 90 min. Eosinophil movement is expressed as a migration index reflecting the ratio of the number of cells migrating in response to the chemokine relative to the number of cells migrating in response to the medium alone.
Intranasal instillation of eotaxin-2.
Lyophilized recombinant murine eotaxin-2 (PeproTech, Rocky Hill, NJ)
was resuspended in PBS-0.1% BSA at a concentration of 10 µg/µl,
and individual aliquots were stored at 80°C. Before being used, the
aliquots were thawed on ice and diluted to 0.25 µg/µl with 0.1%
BSA-PBS (vehicle). Experimental groups of mice were slightly
anesthetized with isoflurane with the use of a Veterinary Anesthesia
Systems machine (Ohio Medical Products, Columbus, OH), and 5 µg of
eotaxin-2 (total volume 20 µl) or vehicle alone were administered by
intranasal instillation. Eotaxin-2 administration of Ova-sensitized and
aerosol-challenged mice occurred on day 28 of the
experimental timeline noted in Eosinophil transmigration assay. Assessments of eosinophils in the BAL fluids from
all groups of mice were performed, as described above, 6 h
postadministration (i.e., at the observed maxima of airway eosinophil
recruitment occurring in naive IL-5 transgenic mice).
Antibody-mediated depletion of CD4+ T cells. CD4+ T cells were depleted in some groups of mice with a rat anti-mouse CD4+ monoclonal antibody (GK1.5). The protocol used was first described by Gavett and colleagues (18) and includes intraperitoneal administration of 0.5 mg of GK1.5 (or control rat IgG; Sigma) to naive NJ.1726 mice 1 wk before eotaxin-2 instillation. BAL fluid leukocytes were enumerated 6 h after chemokine instillation.
Statistical analysis. Data presented are means ± SE. Statistical analysis was performed on parametric data with Student's t-tests, with differences between means considered significant when P < 0.05.
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RESULTS |
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CD4+ T
cells are necessary for the elaboration of airway IL-5
levels in allergen-sensitized and -challenged mice.
The pulmonary recruitment of eosinophils that occurs as a consequence
of an allergic inflammatory response depends on the elaboration of
eosinophil proliferative signals such as IL-5 (14). This
is exemplified in Ova-sensitized and aerosol-challenged mice (see
MATERIALS AND METHODS for details of allergen protocol)
because IL-5 levels increased dramatically over the undetectable levels in control saline-challenged animals (Fig.
1). This allergen-induced IL-5 increase
was a T cell-dependent phenomenon and was abolished in
Ova-treated T cell-deficient [(
/
)/
(
/
)] mice.
Moreover, assessments of knockout mice with deficiencies of specific T
cell subtypes demonstrated that the presence of CD4+ and
not CD8+ cells is required for IL-5 expression with this
sensitization and challenge protocol.
|
Allergen-induced pulmonary eosinophilia does not occur in
CD4(/
) mice.
A direct and predictable consequence of eliminating IL-5 expression was
the abolition of allergen-induced pulmonary eosinophilia. Immunohistochemical staining with a rabbit anti-mouse eosinophil MBP
polyclonal antiserum demonstrated that eosinophil infiltration of the
lung did not occur in Ova-treated T cell-deficient and CD4(
/
) mice
but did occur in CD8(
/
) mice (Fig.
2). Moreover, recruitment of
eosinophils to the airways was similarly affected because BAL fluid
eosinophil numbers after Ova challenge were abolished only in T
cell-deficient and CD4(
/
) mice (Fig.
3). It is noteworthy that this effect was
restricted to eosinophils because the numbers of other cell types
(i.e., macrophages, lymphocytes, and neutrophils) in the BAL fluid were
similar among the different knockout mice; the percentage of each of
these cells reflected only the presence or absence of an
allergen-induced pulmonary eosinophilia (data not shown).
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|
Ectopic overexpression of IL-5 in the lung
epithelium results in a pulmonary eosinophilia that is independent of
the presence or absence of T cell subtypes.
Our laboratory (22) previously reported the
creation of a transgenic mouse line (NJ.1726) that constitutively
expresses IL-5 from lung epithelium with a rat CC10 promoter fragment.
This earlier study showed that homeostatic baseline pulmonary
eosinophil numbers are greatly elevated relative to wild-type control
animals. The data presented in Fig. 4
demonstrate that these elevated levels of pulmonary eosinophils
did not increase further in response to Ova sensitization and aerosol
challenge of NJ.1726 mice. In addition, the pulmonary eosinophil levels
in Ova-treated NJ.1726 mice were also independent of the presence of T
cells or T cell subtypes. Eosinophil numbers per unit area of the lung
were equivalent in Ova-treated NJ.1726 mice and compound NJ.1726/gene
knockout mice devoid of T lymphocytes [(
/
)/
(
/
)],
CD4+ cells, or CD8+ cells (Fig. 4). In
particular, Ova sensitization and aerosol challenge of compound NJ.1726
CD4(
/
) mice resulted only in a nominal decrease in eosinophil
infiltration around the airway submucosa relative to
Ova-treated NJ.1726 animals (Fig. 5).
However, the high-magnification views presented in Fig. 5 demonstrate
that, compared with NJ.1726 mice, eosinophils appear less frequently in
the alveolar spaces of NJ.1726 CD4(
/
) animals.
|
|
CD4+ T
cells are required for allergen-mediated eosinophil recruitment to the
airways of IL-5 transgenic mice.
Although the pulmonary eosinophilia of NJ.1726 mice was not
substantively affected by Ova sensitization and aerosol challenge, this
was not true of the allergen-induced airway eosinophilia. Ova treatment
of NJ.1726 mice resulted in a robust BAL fluid eosinophilia (Fig.
6), dwarfing by >10-fold the eosinophil
numbers recruited to the BAL fluid of
allergen-sensitized/aerosol-challenged wild-type mice (compare Figs. 3
and 6). This dramatic increase was obviously due in part to the large
number of available eosinophils in the perivascular/peribronchial
regions of NJ.1726 mice. However, allergen sensitization and aerosol
challenge of compound NJ.1726 gene knockout mice deficient for specific
T cells or T cell subtypes demonstrated that the simple availability of
eosinophils is insufficient to elicit allergen-induced recruitment to
the BAL fluid. Allergen sensitization and aerosol challenge of compound
transgenic/gene knockout mice deficient for T cells or cells of the
CD4+ subtype, but not the CD8+ subtype, failed
to induce the recruitment of eosinophils to the BAL fluid despite the
availability of exaggerated levels of pulmonary eosinophils (Fig. 6).
|
Eosinophils from NJ.1726 CD4(/
)
mice are able to respond to mouse eotaxin-2.
The inability of eosinophils from NJ.1726 CD4(
/
) mice to
migrate to the airway lumen in response to Ova treatment was
potentially a consequence of a previously unknown yet significant
perturbation(s) associated with eosinophils (i.e., a cell autonomous
effect caused by the CD4+ deficiency). However, these
effects appear unlikely. Migration of eosinophils from NJ.1726
CD4(
/
) mice (assessed by in vitro Transwell assay) in response to
eotaxin-2, a CCR3-binding chemokine with eosinophil agonist activity
(57), is equivalent to the migration exhibited by
eosinophils derived from NJ.1726 mice (Fig. 7).
|
Intranasal instillation of eotaxin-2 elicits the recruitment of
eosinophils to the airways of IL-5 transgenic but not
IL-5 transgenic CD4(/
) mice.
CD4+ T cells have been hypothesized to elicit airway
recruitment of eosinophils during allergic inflammatory responses
through the induced expression of eosinophil agonist chemokines and the establishment of a vectorial gradient (41). In particular,
recent data (57) have suggested that eotaxin-2 represents
an archetype signal characterizing this potential mechanism. These
studies demonstrated that eotaxin-2 is a powerful and specific
eosinophil chemoattractant whose expression in the lung is induced as a
consequence of allergen sensitization and aerosol challenge. The
ability of eotaxin-2 alone to mediate vectorial movement of eosinophils
to the airway lumen is demonstrated in Fig.
8A. Intranasal
instillation of eotaxin-2 into the lungs of naive NJ.1726 mice was
sufficient to elicit the recruitment of eosinophils to the BAL fluid
relative to unmanipulated or vehicle-instilled animal groups. In
contrast, the instillation of eotaxin-2 failed to elicit eosinophil
recruitment in naive compound NJ.1726 CD4(
/
) mice (Fig.
8A). Moreover, eotaxin-2 instillation in Ova-sensitized and
aerosol-challenged NJ.1726 CD4(
/
) mice also failed to elicit
eosinophil recruitment, demonstrating that eotaxin-2-initiated movement
does not require an allergen-mediated costimulatory signal. The unique
necessity for the presence of CD4+ cells for eosinophil
trafficking in response to eotaxin-2 instillation is demonstrated in
the antibody depletion study presented in Fig. 8B. Treatment
of naive NJ.1726 mice [i.e., CD4+(+/+) animals] with
anti-mouse CD4+ monoclonal antibodies (GK1.5) before
eotaxin-2 instillation abolished the ability of this chemokine to
elicit eosinophil recruitment to the lumen. These data show that
CD4+ cells are a critical component of eosinophil
trafficking within the lung, independent of potential developmental
defects associated with CD4-deficient knockout mice.
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DISCUSSION |
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The hypothesis that CD4+ T cells mediate much of the pulmonary pathology associated with allergic inflammation is supported by extensive studies of both human asthma patients (28) and the characterization of several mouse models of respiratory inflammation (17). In particular, adoptive transfer of CD4+ cells to syngeneic hosts (53), the use of neutralizing antibodies (18), and, more recently, the availability of gene knockout mice have demonstrated that these cells are necessary (and possibly sufficient) to elicit the recruitment of eosinophils to the lung (21). The CD4+ cell-mediated activity commonly suggested to be primarily responsible for pulmonary eosinophil recruitment is the elaboration of the eosinophilopoietic Th2 cytokine IL-5. The underlying mechanism hypothesized is that after antigen provocation, CD4+ cells are required to generate IL-5, which, in turn, is necessary for the release of the reservoir of eosinophils stored in the bone marrow (30), the proliferation of marrow-derived eosinophil lineage-committed progenitors (48), and the subsequent recruitment of these cells to the lung (38). Moreover, IL-5 has direct effects on eosinophil maturation, survival, and activation (1, 3, 55), suggesting that elaboration of IL-5 levels potentially elicit multiple mechanisms leading to the accumulation of pulmonary eosinophils.
The increase in lung eosinophil levels as a consequence of CD4+ T cell-dependent elaboration of pulmonary IL-5 levels confirms the importance of this CD4+ T cell activity; i.e., the loss of Ova-induced eosinophil recruitment to the BAL fluid of T cell- and CD4+ cell-deficient mice, relative to wild type, parallels a similar loss of allergen-induced pulmonary IL-5 levels. However, it is noteworthy that IL-5 levels were not extinguished in either T cell- or CD4+ cell-deficient mice. Previous studies (23) have demonstrated that under specific circumstances, CD8+ cells are a potential source of IL-5. In addition, studies (51) with an intraperitoneal sensitization model system have demonstrated that NK cells are a prodigious source of IL-5. Nonetheless, these data suggest that although alternative sources of IL-5 may exist, CD4+ cells are the predominant source of IL-5 during allergic inflammatory responses in the lung.
Several lines of evidence now suggest, however, that IL-5 expression by
CD4+ cells, although an important activity leading to
allergic pulmonary eosinophilia, is but one activity necessary for the
recruitment of eosinophils to the airway lumen. Collectively, the
implicit conclusion of this evidence is that CD4+
cell-mediated eosinophil production is not solely responsible for the
dramatic increase in pulmonary eosinophils associated with allergic
inflammation. 1) Little difference is observed in the number
of circulating peripheral blood eosinophils between Ova-sensitized and
aerosol-challenged wild-type mice and gene knockout mice deficient in T
cells or T cell subtypes (21) (data not shown).
2) Adoptive transfer of Ova-specific IL-4-deficient Th2
cells before an aerosol challenge failed to induce an airway eosinophilia despite increased levels of IL-5 and an induced pulmonary eosinophilia (6). 3) Constitutive ectopic
expression of IL-5 from the lung epithelium induces a dramatic
pulmonary eosinophilia independent of CD4+ T cells;
however, in the absence of these cells, allergen-induced eosinophil
trafficking to the airways is abolished. Interestingly, the loss of
eosinophil recruitment occurring in mice deficient for this T cell was
equivalent to the loss observed in mice devoid of all T cells (i.e.,
/
TCR-deficient mice), suggesting a necessary and possibly
sufficient role for CD4+ cells alone in this model system.
A concurrent CD4+ cell-mediated activity, apparently critical for the recruitment of eosinophils to the airways during allergen challenge, is the establishment of multiple chemokine gradients (for review, see Ref. 41). In particular, the abundance of the chemokine receptor CCR3 on eosinophils and the exclusive nature of signaling by eotaxins through this receptor have led to hypotheses of eosinophil trafficking being dependent on these receptor-ligand interactions. Moreover, in vivo mouse studies (38) have shown that IL-5 and eotaxin-1 cooperatively mediates the significant eosinophil recruitment occurring in the airways of allergen-challenged mice. Recent data demonstrate that another member of this functional chemokine group, eotaxin-2, displays even greater abilities to attract eosinophils (data not shown). Moreover, Zimmermann and colleagues (57) have shown that allergen sensitization and challenge result in a marked induction of eotaxin-2 expression in the lung. This study further suggests that induction of eotaxin-2 may be linked to IL-4 expression by activated T cells, thus creating a causative link between T cell activity and the establishment of a pulmonary chemokine gradient. The eotaxin-2 instillation experiments presented in this study that used IL-5 transgenic mice demonstrate that in an otherwise wild-type animal, the establishment of an eotaxin-2 pulmonary gradient elicits eosinophil airway recruitment. Surprisingly, this recruitment was absolutely dependent on the presence of CD4+ cells even in naive mice; i.e., despite the constitutive levels of IL-5 and the resulting ubiquitous presence of eosinophils throughout the pulmonary interstitium, eotaxin-2 did not elicit eosinophil trafficking to the airways of naive compound transgenic/gene knockout mice. Interestingly, a concurrent Ova sensitization and challenge did not recover eosinophil recruitment in these eotaxin-2 reconstituted mice, suggesting that allergen-mediated eosinophil trafficking is also dependent on CD4+ cell-mediated signals independent of IL-5 expression and the establishment of a pulmonary chemokine gradient.
The inability to elicit eosinophil recruitment in CD4+
cell-deficient mice (knockout or antibody-mediated depletion) by
reconstituting the dominant eosinophil agonist activities associated
with these cells (i.e., IL-5 expression and, therefore, eosinophil
proliferation as well as the establishment of an eotaxin pulmonary
gradient) suggests that an additional CD4+ cell-dependent
mechanism(s) is required for eosinophil trafficking. Hypotheses of
CD4+ cell-mediated eosinophil recruitment that require
direct physical interactions between T cells and eosinophils are
limited because of the logistical constraints of pulmonary eosinophil
numbers relative to resident CD4+ cells as well as their
relative location within the lung (i.e., there are too many eosinophils
relative to T cells, and the two cell types are often not found
together). However, the recent demonstration (42) that
eosinophils act as antigen presentation cells in the lung suggests that
at some levels, direct physical interactions between T cells and
eosinophils are occurring and may contribute to the activation state of
a subpopulation of either or both cell types. The identity of
additional CD4+ cell-mediated mechanisms is likely
associated with either direct T cell-eosinophil interactions through a
soluble intermediate(s) or T cell-induced changes in the lung on which
eosinophil trafficking is dependent. Studies in support of either
potential mechanism have been described in the literature. Adoptive
transfer experiments with Th2-differentiated lymphocytes derived from
gene knockout mice have implicated IFN- as a potential signal with
direct eosinophil agonist effects (6, 8). These studies
delineate a T cell regulatory network in which IL-4/IL-13 expression
downregulates the levels of T cell-derived IFN-
. In turn, IFN-
appears to directly modulate eosinophil accumulation in the lung
through a mechanism restricted to activities on hematopoietic cells,
with eosinophils being a likely target (6). In this
paradigm, the loss of CD4+ cells leads to decreased levels
of IL-4/IL-13 and concomitant increases in IFN-
, resulting in either
an inhibition of eosinophil recruitment to the airway or an increase in
eosinophil turnover (e.g., apoptosis). In either case, the net
effect is a substantive decrease in steady-state levels of eosinophils
in the lumen that is not recoverable by reconstitution of IL-5 or an
eotaxin gradient. Alternatively, several studies (reviewed in Ref.
43) have shown that inflammatory signals released by
CD4+ cells lead to priming/activation of both effector
leukocytes and capillary endothelial cells. In particular, this
interaction leads to changes in cell surface adhesion molecule (and/or
receptor) expression on eosinophils (9, 12, 47) and
endothelial cells (40) and thus may have significant
effects directly on adhesion molecule-mediated eosinophil recruitment
or on the activation necessary for eosinophil survival. The loss of
CD4+ cells in this model prevents the required cell
adhesion molecule (and/or receptor) changes, thus preventing eosinophil
airway accumulation. Similar to the paradigm described above, the
CD4+ cell signals required for these cell surface changes
are not recoverable by simply reconstituting IL-5 expression or the
pulmonary chemokine gradient (i.e., eosinophils are produced and
attracted to the lung but are unable to move from the interstitium).
The commonality of either explanation is the description of potential CD4+ cell activities independent of IL-5-mediated
eosinophil proliferation or chemokine-mediated recruitment,
highlighting the importance of additional CD4+ T
cell-dependent mechanisms in the establishment of the pulmonary eosinophilia associated with allergic respiratory inflammation.
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ACKNOWLEDGEMENTS |
---|
We thank Edith Hines for assistance with mouse genotyping, the Mayo Clinic Scottsdale Histology Facility (Anita Jennings, Director), and the Mayo Clinic Scottsdale Graphic Arts Department (Marv Ruona). We also thank Linda Mardel whose contribution to our productivity is often overlooked.
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FOOTNOTES |
---|
This study was supported by funds from the Mayo Clinic Foundation; National Heart, Lung, and Blood Institute (NHLBI) Individual Investigator Award HL-60793-01S (to N. A. Lee); NHLBI Training Grant HL-07897; and NHLBI National Research Service Awards HL-10361 (to M. T. Borchers) and HL-10176JC (to J. R. Crosby).
Address for reprint requests and other correspondence: N. A. Lee, Div. 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.
Received 29 May 2001; accepted in final form 10 September 2001.
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