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Divisions of
*
Pulmonary Medicine and
Hematology and Oncology, Mayo Clinic, Scottsdale AZ 85259; and
Division of Biology, California Institute of Technology, Pasadena, CA 91125
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many receptor-ligand interactions are necessary to elicit eosinophil
recruitment (as well as other inflammatory responses). In addition,
there are a variety of intracellular signaling events required to
transduce these receptor-mediated interactions. Heptahelical
transmembrane receptors coupled to G proteins (the largest family of
cell surface proteins in the human genome (5)) are capable
of responding to many forms of stimuli, including, as is the case for
proinflammatory leukocytes, gradients of chemoattractants leading to
activation and tissue-specific recruitment (6). The
function of these cell surface receptors is controlled by 16 GTP
binding G
proteins that can be classified into four subfamilies of G
proteins, Gq, Gi,
Gs, and G12. G
protein signal transduction mediates cellular responses by regulating
second messenger activities, including phospholipases, adenylyl
cyclases, phosphodiesterases, and ion channels (7). Thus,
the combinatorial interaction of multiple receptors generates a large
number of permutated signaling pathways, leading to unique
stimulus-response reactions.
The Gq family, includes four subtypes
Gq, G11,
G14, and G15/16.
Of particular interest is one member of this family,
Gq, that is expressed in many of the
leukocytes/tissues involved in allergic inflammatory reactions, such as
the thymus and spleen (all hemopoietic cell types examined)
(8), lung epithelium (9), and endothelial
cells (10). In addition, it is noteworthy that
Gq-coupled receptors have been linked to the
induction of the NFAT family of transcription factors
(11), potential regulators of early immune response
cytokine expression (e.g., IL-4) involved in T cell
differentiation/activation and the development allergic inflammation
(12, 13). Moreover, Gq protein
expression is increased in guinea pig lungs following Ag challenge
(14); however, the potential function(s) of
Gq in allergen-induced pulmonary inflammation
have not been investigated.
In this study mice deficient in the Gq subunit
were used to investigate its potential role(s) in allergen-induced
recruitment of eosinophils to the lung. The data demonstrate a required
role for Gq signaling in the development of
allergen-induced airway eosinophilia. This effect appears to be
mediated by failure of the knockout mice to elaborate pulmonary levels
of GM-CSF in response to allergen. The Gq
signaling defect is limited to a marrow-derived cell(s), but was
independent of T cell responsiveness to Ag and eosinophil
chemotaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gq-deficient mice were generated by homologous recombination as previously described (15). Compound Gq-/-/IL-5 transgenic mice were obtained by crossing Gq-/- animals with mice constitutively expressing IL-5 from peripheral T cells (16). All procedures were conducted on specific pathogen-free mice 8–12 wk of age maintained in ventilated microisolator cages housed in an American Association for Accreditation of Laboratory Animal Care-accredited animal facility. Protocols and studies involving animals were conducted in accordance with National Institutes of Health and Mayo Clinic Foundation guidelines.
Assessment of allergic inflammation
The OVA model of allergic pulmonary inflammation has been previously described (17). Briefly, mice (20–30 g) were sensitized by an i.p. injection (100 µl) of 20 µg chicken OVA (Sigma-Aldrich, St. Louis, MO) emulsified in 2 mg Imject Alum (Al(OH)3/Mg(OH)2; Pierce, Rockford, IL) on days 0 and 14. Mice were subsequently challenged with an aerosol of 1% OVA in saline or saline alone on days 24, 25, and 26. Eosinophil accumulation was assessed on days 27, 28, and 29 by enumerating bronchoalveolar lavage (BAL)3 leukocytes as previously described (17). Cell-free BAL fluids and serum were flash-frozen in liquid nitrogen and stored at -80°C before cytokine level determination by ELISA. Assessments of blood leukocytes were performed on day 28 on both peripheral blood (viz, the tail vasculature) and femoral bone marrow as previously described (16).
Immunohistochemistry and assessment of peribronchial eosinophils
Immunohistochemistry was performed using a rabbit polyclonal Ab against mouse major basic protein (MBP). MBP Ag-Ab complexes were detected in 4-µm sections of formalin-fixed, paraffin-embedded lungs (n = 5 mice/group) on day 28 using methodologies previously described (17). Eosinophils surrounding the airways were quantified by counting the number of MBP-positive cells per square millimeter of submucosal tissue (n = 5 mice/group) with an image analysis software program (ImagePro Plus; Media Cybernetics, Silver Spring, MD).
Bone marrow transfer
Bone marrow chimeras were generated by exposing female wild-type mice to 1100 cGy whole body lethal irradiation. Bone marrow cells (1 x 107) from wild-type or Gq-/- male donors were transferred by tail vein injection. Mice were used in experiments following a 60-day recovery period. Donor cell engraftment of >90% was achieved in all recipients as determined by a PCR assay designed to quantify X vs Y chromosome-specific sequences (18).
Eosinophil isolation and in vitro migration assays
Eosinophils were isolated and purified from tail
vasculature-derived blood of IL-5 transgenic mice (16) and
compound IL-5 transgenic/Gq-/-
mice. Heparinized blood was layered onto a Percoll gradient (60%
Percoll (
= 1.084), 1x HBSS, 15 mM HEPES (pH 7.4), and 0.003 N
HCl) and centrifuged (45 min, 3000 rpm, 4°C). The buffy coat was
recovered and washed twice in PBS containing 2% FCS. Eosinophils were
isolated using MACS (Miltenyi Biotech, Auburn, CA). B cells and T cells
were removed by positive selection with Ab-conjugated magnetic beads
specific for CD45-R (B220) and CD90 (Thy 1.2), respectively. Eosinophil
migration was determined using a modified method of Okada et al.
(19). Migration is expressed as a migration index,
assessing the number of cells migrating in response to chemoattractant
relative to the number of cells migrating in response to medium
alone.
Isolation and stimulation of splenocytes in vitro
Wild-type and Gq-/-
mice were sensitized twice with OVA/alum on days 0 and 14 as described
above. Spleens were removed (day 24), and cells were isolated in RPMI
1640 medium containing 10% FCS, penicillin (100 U/ml), and
streptomycin (100 U/ml). The capacity of lymphocytes to produce both
Th1 (e.g., IFN-
) and Th2 (e.g., IL-4) cytokines was assessed by
culturing splenocytes (
1 x 106) in
96-well round-bottom plates alone or with 20 µg/ml Con A for 24
h at 37 °C in 5% CO2. The ability of T cells
to produce these cytokines in response to TCR binding was examined
using mAbs to CD3 and CD28 (BD PharMingen, San Diego, CA). Anti-CD3 Ab
(10 µg/ml) was allowed to adhere to plates overnight at 4°C.
Splenocytes (1 x 106) were added in a
volume of 250 µl medium containing anti-CD28 Ab (1 µg/ml) and
incubated for 72 h at 37 °C in 5% CO2.
Aliquots of the supernatant were assayed for cytokine levels by
ELISA.
OVA recall assay
Isolated splenocytes (1 x 106) from
wild-type and Gq-/- mice that
had been previously sensitized with OVA were restimulated in vitro with
200 µg/ml OVA. Supernatants were collected at 72 h, and cytokine
levels of IFN- and IL-4 were determined by ELISA.
Cytokine assays
Cytokine levels in serum, lavage fluid, and culture medium were
determined by ELISA. Mouse IL-4, IL-5, IFN-, and GM-CSF ELISA kits
from R&D Systems (Minneapolis, MN) were used according to the
manufacturer’s protocol. The limits of detection for each assay were:
IFN-, 30 pg/ml; IL-4, 10 pg/ml; IL-5, 10 pg/ml; and GM-CSF,
5 pg/ml.
Instillation of GM-CSF into OVA-challenged mice
Immediately following each OVA challenge (i.e., days 24, 25, and 26), 1 ng recombinant mouse GM-CSF (R&D Systems) in PBS/0.1% BSA or vehicle alone was instilled intranasally into lightly anesthetized mice.
Statistical analysis
Data presented are the mean ± SE. Statistical analysis was performed on parametric data using t tests with differences between means considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Airway and peribronchial eosinophil accumulations are hallmark
features of allergic pulmonary models in the mouse, with peak
eosinophil accumulation in the lung typically occurring 24–48 h
following the last allergen challenge. In
Gq-/- mice, however, the
numbers of eosinophils in the BAL (Fig. 1
A) following OVA challenge
was significantly lower compared with wild-type mice. The inhibition of
BAL eosinophil accumulation was observed at all time points examined
over 72 h, precluding the likelihood of a transient early or
delayed increase in Gq-/-
mice. This effect on pulmonary eosinophil accumulation is reflective of
the total number of cells recovered in the BAL of
Gq-/- mice, which was
consistently lower than that in wild-type mice (30–40% reduction).
Significant numbers of eosinophils were not recovered from the lungs of
saline-challenged mice of either genotype (data not shown).
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, B–F). However, this reduction in airway
eosinophils was not associated with decreased levels of circulating Th2
cytokines. Serum IL-4 and IL-5 levels following Ag challenge were not
significantly different between wild-type and
Gq-/- mice (IL-4, 64.7 ±
12.6 and 68.7 ± 18.2 pg/ml, respectively; IL-5, 82.8 ± 27.5
and 99 ± 23.1, respectively). Serum cytokine levels in
saline-challenged control mice of either genotype were below the limit
of detection. The cell number and composition of bone marrow and peripheral blood are unaffected in Gq-/- mice
The expression of Gq in a wide array of
leukocytes (8) suggested that the loss of this signaling
pathway would lead to perturbations in hemopoietic compartments and
thus account for the absence of a pulmonary eosinophilia in
Gq-/- mice. However, no
significant differences in the percentages of granulocytes,
lymphocytes, or mononuclear cells were observed in
Gq-/- mice relative to
wild-type controls following OVA challenge (Fig. 2, A and B).
Moreover, the total number of cells recovered from the bone marrow or
peripheral blood was not different among mice of either genotype (data
not shown).
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The expression of Gq in hemopoietically
derived cells as well as structural cells of the lung (8)
necessitated an initial assessment of contributing
Gq signaling events from either compartment. Bone
marrow engraftment studies were preformed, adoptively transferring
either wild-type or Gq-/-
marrow into wild-type recipients. Engrafted mice were subsequently
sensitized/challenged with OVA to determine whether the reduction in
airway eosinophila was primarily a consequence of a marrow-derived cell
or defects associated with one or more nonhemopoietic lineages. The
response to OVA challenge in wild-type mice receiving wild-type marrow
was indistinguishable from that in nonirradiated wild-type animals.
However, adoptive engraftment of
Gq-/- marrow into wild-type
mice resulted in a significant decrease in airway eosinophil
accumulation following OVA challenge (Fig. 3). These data demonstrate that
perturbations of intracellular signaling in one or more
lympho-hemopoietic cell types are responsible for the observed decrease
in airway eosinophilia.
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Eosinophil chemotaxis following allergen challenge is controlled
by concurrent G protein-coupled receptor-ligand interactions
(3), suggesting that potential signaling deficiencies lie
within the eosinophil itself. In vitro Transwell migration assays were
used to assess potential cell autonomous effects of
Gq signaling on eosinophil migration. No
differences were observed in eotaxin-1-mediated chemotaxis of wild-type
vs Gq-/- eosinophils (Fig. 4A). Furthermore, the
migration of Gq-deficient eosinophils was
unaffected (relative to wild-type) in response to other
chemoattractants shown to bind, and signal through, distinct receptors
on these cells (e.g., platelet-activating factor and complement factor
C5a (our unpublished observations); Fig. 4A). Collectively,
these data show that Gq signaling is an unlikely
causative event(s) leading to OVA-induced pulmonary eosinophil
recruitment.
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T cell activity was assessed in
Gq-/- mice to determine
whether the loss of allergen-induced pulmonary eosinophilia was a
consequence of Gq-dependent effects on T cell
function. No differences were observed in the ability of splenocytes
isolated from wild-type or
Gq-/- mice to elaborate IL-4
and IFN- in response to the mitogen Con A or nonspecific T cell
activation, viz, the cross-linking of TCRs (i.e., anti-CD3; Fig. 4, B–E). In addition, Ag recall assays demonstrated that
lymphocytes and APC from Gq-/-
mice were able to elicit Th2 cytokine production in vitro upon exposure
to OVA. Splenocytes from Gq-/-
mice generate equivalent amounts of IL-4 in response to Ag stimulation
(Fig. 4F), showing that Gq is not
required to generate a memory response toward a particular Ag.
Pulmonary production of GM-CSF, but not Th2 cytokines, is dependent on Gq signaling
Local immune responses (i.e., BAL cytokine levels) potentially
leading to, and/or augmenting, eosinophil accumulation in the lung
following OVA challenge were assessed in
Gq-/- mice. Twenty-four hours
following the first (day 24) OVA aerosol challenge (i.e., the kinetic
maxima of cytokine levels in this protocol (20)) the
production of lymphocyte-derived Th2 cytokines (e.g., IL-4 and IL-5)
was unaffected in Gq-/- mice
(Fig. 5). However, local production of
GM-CSF was significantly reduced as a consequence of the
Gq deficiency. These data show that the pulmonary
level of GM-CSF increases in OVA-treated wild-type mice from an
undetectable level (before OVA challenge) to 40 pg/ml. In contrast,
OVA treatment of Gq-/- mice led only to an
nominal increase in GM-CSF levels (5 pg/ml), representing an 88%
reduction relative to OVA-treated wild-type animals.
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, A and B). The
effect of mouse rGM-CSF instillation was specific to OVA-treated
Gq-/- mice as the introduction of
rmGM-CSF into the airways of saline-challenged mice of either genotype
or of OVA-treated wild-type animals had no effect on eosinophil
levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eosinophils themselves express several receptors that are coupled to
heterotrimeric G proteins, many of which are involved in the migration
and activation of these cells in response to inflammatory mediators.
For example, G protein-coupled receptors involved in eosinophil
migration include receptors responsible for the binding and signaling
of a diverse group of mediators, including chemokines (21, 22), leukotriene B4 (23), platelet-activating
factor (23), complement factor 5a (23),
PGD2 (24), and neuropeptides
(25). Interestingly, the majority of the responses
reported for these receptors have been demonstrated to be pertussis
toxin-sensitive, indicating the involvement of the
Gi or Go family of
heterotrimeric G proteins. The ability of
Gq-/- eosinophils to migrate
with equal potency as wild-type eosinophils in response to several
ligands with eosinophil agonist activities (i.e., eotaxin-1, C5a, and
platelet-activating factor) supports this apparent independence of
Gq signaling. Gq expression
has been detected in many mouse tissues and leukocytes, including
macrophage, T cells, and B cells (8). However, the
expression of Gq in mouse (or human) eosinophils
has not been previously examined. Significantly, we were unable to
detect Gq expression in mouse eosinophils by
Western blot or sequencing of RT-PCR products using degenerate primers
for G subunits (data not shown). This would suggest that the absence
of Gq signaling in eosinophils does not account
for the failure of Gq-/- mice
to develop pulmonary eosinophilia.
Allergic pulmonary inflammation, including the specific accumulation of
eosinophils in the lung, is a process regulated by T cells
(26). In particular, the expression of Th1/Th2 cytokines
have been implicated as a root cause of eosinophil accumulation,
eliciting both effects directly on eosinophil proliferation and/or
survival (e.g., IL-5 (27) and IFN- (28))
as well as indirect mechanisms enhancing pulmonary eosinophil
recruitment (e.g., IL-4/IL-13 (29, 30, 31)). Moreover, in
vitro studies have implicated Gq signaling in the
induction of NFAT (11), a family of transcription factors
potentially involved in the regulation of cytokine expression following
stimulation of the TCR complex (32). However, the loss of
allergen-induced pulmonary eosinophilia in
Gq-/- mice is probably not a
consequence of an impaired memory response or the ability to generate a
Th2 response. T cells from
Gq-/- mice were able to
produce IL-4 and IFN- in response to either Con A stimulation or TCR
activation. In addition, T cells isolated from OVA-sensitized knockout
mice were also able to initiate a Th2-specific immune response
following OVA restimulation in vitro. Interestingly, IFN- was not
detected above baseline values (data not shown) following in vitro OVA
restimulation, indicating that the lack of an airway eosinophilia is
also not the result of a Th1-skewed cytokine balance that would inhibit
the accumulation of eosinophils (28).
The resolution of this quandary regarding a cell autonomous defect associated with the Gq deficiency probably resides in the unique loss of GM-CSF production in the lungs of Gq-/- mice. The link between local GM-CSF production and allergen-induced eosinophil accumulation in the lung is multifaceted, including increased effectiveness of Ag presentation to T cells (33, 34, 35), increased eosinophil survival (36, 37), and enhanced eosinophil migration (38, 39). Local production of GM-CSF in the lungs of asthma patients is primarily confined to macrophages (40), T cells (41, 42), eosinophils (43), and epithelial cells (44). Similar increases in pulmonary GM-CSF levels have also been demonstrated in the lungs of mice following allergen sensitization/challenge (20). The allergen challenge studies of mice following adoptive engraftment of wild-type vs Gq marrow eliminate epithelial cells as a prominent contributor of pulmonary GM-CSF.
The identity of the cellular source of GM-CSF (and presumably the
target cell of the Gq deficiency) is unresolved,
but may include T cells and/or alveolar macrophages. Macrophages, in
particular, are a prodigious source of GM-CSF (45); they
are a predominant resident cells in the lung (i.e., alveolar
macrophages are present before allergen provocation), and evidence in
the literature suggests potential Gq-dependent
pathways in the macrophage that may lead to the elaboration of GM-CSF.
For example, endothelins are small peptides released into the lung
during the initial phase of allergic pulmonary inflammation
(46). These mediators signal through receptors coupled to
Gq (47) and are potent agonists of
GM-CSF production by monocytes in vitro (48). Moreover,
antagonism of endothelin receptors (primarily the A subtype) reduces
eosinophil accumulation by 70% in animal models of airway
inflammation (49, 50). Evidence also exists for a
potential macrophage-dependent mechanism of neurogenic inflammation of
the airways induced by substance P as another possible
Gq-mediated pathway leading to GM-CSF production
and eosinophil accumulation (51, 52).
The narrow effects observed in Gq-/- mice suggest a unique role for Gq signaling in the regulation of allergen-induced pulmonary inflammation. The apparent requirement of Gq signaling during these responses implies that although leukocytes expresses multiple G protein family members with potentially overlapping and redundant activities, a specificity of function for individual G proteins exists in a given cell. These studies identify Gq signaling pathways as critical regulators of allergic inflammation and further elucidate the mechanisms mediating the eosinophil accumulation that occurs in diseases such as asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James J. Lee, Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Samuel C. Johnson Medical Research Building-Research, Mayo Clinic Scottsdale, 13400 East Shea Boulevard, Scottsdale, AZ 85259. E-mail address: jlee{at}mayo.edu
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; MBP, major basic protein.
Received for publication October 22, 2001. Accepted for publication January 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-8 production by monocytes and macrophages of asthmatic subjects. Eur. Respir. J. 7:1096.[Abstract]
q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. J. Biol. Chem. 274:33691. subunits of the G proteins Gq and G11. Biochemistry 35:2909.[Medline]
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