Allergen-Induced Pulmonary Eosinophilia Signaling Is Required for qG

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Jeffrey Crosby,† Melvin I. Simon,‡ Nancy A. Lee,† and James J. Lee2*. The complexity and .... E-mail address: [email protected] Copyright © 2002 by The ...

Gq Signaling Is Required for Allergen-Induced Pulmonary Eosinophilia1 Michael T. Borchers,* Paul J. Justice,† Tracy Ansay,† Valeria Mancino,‡ Michael P. McGarry,* Jeffrey Crosby,† Melvin I. Simon,‡ Nancy A. Lee,† and James J. Lee2* The complexity and magnitude of interactions leading to the selective infiltration of eosinophils in response to inhaled allergens are formidable obstacles to a larger understanding of the pulmonary pathology associated with allergic asthma. This study uses knockout mice to demonstrate a novel function for the heterotrimeric G protein, Gq, in the regulation of pulmonary eosinophil recruitment. In the absence of Gq signaling, eosinophils failed to accumulate in the lungs following allergen challenge. These studies demonstrate that the inhibition of eosinophil accumulation in the airways is attributed to the failure of hemopoietically derived cells to elaborate GM-CSF in the airways. The data suggest that activation of a Gq-coupled receptor(s) on resident leukocytes in the lung elicits expression of GM-CSF, which, in turn, is required for allergen-induced pulmonary eosinophilia, identifying a novel pathway of eosinophil-associated effector functions leading to pulmonary pathology in diseases such as asthma. The Journal of Immunology, 2002, 168: 3543–3549.

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ntigen-induced recruitment/activation of proinflammatory leukocytes to the lung as well as activation of resident leukocytes are invariant features of allergic respiratory inflammation. In particular, cytokines, T cells, and T cellassociated secretagogues appear to be contributors to pulmonary pathology resulting in the selective recruitment of eosinophils to the lung (1, 2). This vectorial migration of eosinophils results from both receptor-ligand-mediated activation as well as a series of dynamic interactions between adhesion molecules expressed on eosinophils and the vascular endothelium (3). In addition, the generation of chemoattractant gradients within the lung appear to be critical to mediate the selective movement of eosinophils (4). 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, G␣q, G␣i, G␣s, and G␣12. G protein signal transduction mediates cellular responses by regulating second messenger

Divisions of *Pulmonary Medicine and †Hematology and Oncology, Mayo Clinic, Scottsdale AZ 85259; and ‡Division of Biology, California Institute of Technology, Pasadena, CA 91125 Received for publication October 22, 2001. Accepted for publication January 25, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported by funds from the Mayo Foundation, National Heart, Lung, and Blood Institute Award HL60793 (to N.A.L.), National Heart, Lung, and Blood Institute Training Grant HL07897 (to M.T.B.), and an individual National Research Service Award (to M.T.B.). 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: [email protected]

Copyright © 2002 by The American Association of Immunologists

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 G␣q 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 marrowderived cell(s), but was independent of T cell responsiveness to Ag and eosinophil chemotaxis.

Materials and Methods Mice 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. 0022-1767/02/$02.00

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Assessment of allergic inflammation

Cytokine assays

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 ␮l) of 20 ␮g 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).

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.

Immunohistochemistry and assessment of peribronchial eosinophils

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.

Immunohistochemistry was performed using a rabbit polyclonal Ab against mouse major basic protein (MBP). MBP Ag-Ab complexes were detected in 4-␮m 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 ⫻ 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), 1⫻ 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 ⫻ 106) in 96-well round-bottom plates alone or with 20 ␮g/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 ␮g/ml) was allowed to adhere to plates overnight at 4°C. Splenocytes (1 ⫻ 106) were added in a volume of 250 ␮l medium containing anti-CD28 Ab (1 ␮g/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 ⫻ 106) from wild-type and Gq⫺/⫺ mice that had been previously sensitized with OVA were restimulated in vitro with 200 ␮g/ml OVA. Supernatants were collected at 72 h, and cytokine levels of IFN-␥ and IL-4 were determined by ELISA. 3

Abbreviations used in this paper: BAL, bronchoalveolar lavage; MBP, major basic protein.

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

Results Allergen-induced airway eosinophilia is significantly lower in Gq⫺/⫺ mice 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. 1A) 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). Histological examination of the lungs from OVA-challenged mice demonstrated a similar lack of peribronchial eosinophil accumulation in Gq⫺/⫺ mice compared with wild-type animals (Fig. 1, 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 wildtype 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). Gq signaling in a marrow-derived cell type(s) is required for the development of OVA-induced pulmonary eosinophilia 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

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FIGURE 2. Levels of marrow-derived and circulating leukocytes are unaffected in Gq⫺/⫺ mice. No significant differences (p ⬍ 0.05) was observed in the composition of femoral marrow-derived (A) or peripheral blood (B; viz, the tail vasculature) leukocytes from wild-type vs Gq⫺/⫺ mice following saline or OVA challenge (day 28). Values presented are the mean ⫾ SE (n ⫽ 4 –5 mice/group).

FIGURE 1. The recruitment of eosinophils to the airways of OVA-sensitized/challenged Gq⫺/⫺ mice is significantly reduced compared with that in wild-type animals. A, BAL-derived eosinophils were assessed as a function of time after OVA challenge (control groups received a saline-only challenge) in wild-type vs Gq⫺/⫺ mice. ⴱ, Significantly different (p ⬍ 0.05) from wild-type mice. Eosinophils comprise ⬍1% of leukocytes in the airways of saline-challenged mice of either genotype. Values presented are the mean ⫾ SE (n ⫽ 8 –10 mice/group). B–E, Trafficking of eosinophils to the peribronchial regions of wild-type and Gq⫺/⫺ lungs was assessed by immunocytochemistry using a rabbit polyclonal Ab specific for mouse MBP. Saline control groups: B, wild-type; D, Gq⫺/⫺. OVA-challenged groups: C, wild-type; E, Gq⫺/⫺. Photomicrographs are from representative sections taken from five mice per group. F, Quantification of peribronchial eosinophil accumulation in wild-type and Gq⫺/⫺ mice. No differences in the number or specific location of eosinophils recruited to the lung were observed between similarly treated mice of either genotype. Scale bar ⫽ 100 ␮m.

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 re-

sponse 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.

Eosinophils deficient of Gq migrate in response to chemoattractants 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 OVAinduced pulmonary eosinophil recruitment.

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FIGURE 3. Absence of Gq signaling in a marrow-derived cell(s) accounts for the inhibition of allergen-induced pulmonary eosinophil accumulation. Bone marrow chimeric mice were generated by adoptive engraftment of either wild-type or Gq⫺/⫺ marrow into wild-type recipients. Following recovery, mice were sensitized and aerosol-challenged with OVA (control animals received a saline-only challenge), and BAL eosinophils were enumerated 24 h after the last challenge (i.e., day 28). Nonirradiated wild-type mice are included for comparison. Values presented are the mean ⫾ SE (n ⫽ 6 – 8 mice/group). ⴱ, Significantly different (p ⬍ 0.05) from wild-type donor mice.

Systemic T cell responses occur in OVA-treated Gq⫺/⫺ mice 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 crosslinking 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. Twentyfour 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. The loss of local GM-CSF production was demonstrated as being fundamental to the inhibition of eosinophil accumulation in Gq⫺/⫺ mice by instillation of recombinant cytokine. Intranasal administration of 1 ng mouse rGM-CSF into Gq⫺/⫺ mice immediately following each OVA challenge (i.e., days 24, 25, and 26), recovered the ability to develop airway eosinophil accumulation; this eosinophilia was similar to levels observed in wild-type mice

FIGURE 4. Gq signaling is not required for eosinophil migration or T lymphocyte activation/Ag recognition. A, Pure populations of Gq-deficient peripheral blood eosinophils were isolated from compound IL-5 transgenic/Gq⫺/⫺ mice. Vectorial movement of wild-type vs Gq⫺/⫺ eosinophils in response to representative G protein-coupled receptor-ligand interactions was determined using an in vitro Transwell migration assay. No significant differences (p ⬍ 0.05) were observed between eosinophils from either genotype. Baseline migration in all assays (i.e., nonspecific movement) was also not significantly different (⬃1 ⫻ 104) among the different cohorts of animals examined. B–F, Splenocytes from wild-type and Gq⫺/⫺ mice were cultured in the presence of Con A (B and C), anti-CD3 and anti-CD28 TCR Abs (D and E), or OVA (F), and the production of IL-4 and IFN-␥ was determined by ELISA of the culture supernatants. No significant differences (p ⬍ 0.05) were observed between mice of either genotype. Values presented are the mean ⫾ SE of duplicate determinations conducted on three separate occasions.

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FIGURE 6. Administration of GM-CSF into the airways of Gq⫺/⫺ mice induces airway eosinophil accumulation. OVA-sensitized/challenged (controls received saline-only challenge) wild-type vs Gq⫺/⫺ mice were administered (intranasally) recombinant mouse GM-CSF (days 24, 25, and 26) before assessment of BAL (A) and peribronchial eosinophilia (B; day 28). ⴱ, Significantly different (p ⬍ 0.05) from the wild-type OVA-treated group.

FIGURE 5. Pulmonary production of GM-CSF, but not Th2 cytokines, is attenuated in Gq⫺/⫺ mice. BAL levels of IL-4 (A), IL-5 (B), and GMCSF (C) were determined in wild-type vs Gq⫺/⫺ mice 3 h following the initial OVA challenge (day 24). Values presented are the mean ⫾ SE (n ⫽ 7 mice/group). N.D., Not detected.

(Fig. 6, A and B). The effect of mouse rGM-CSF instillation was specific to OVA-treated Gq⫺/⫺ mice as the introduction of rmGMCSF into the airways of saline-challenged mice of either genotype or of OVA-treated wild-type animals had no effect on eosinophil levels.

Discussion Gq expression (and presumably Gq signaling) is widely distributed in the mouse, occurring in nearly every tissue examined (8). Surprisingly, despite this tissue/cell distribution, effects of Gq deficiency on allergen-induced recruitment of eosinophils to the lung were limited to a marrow-derived cell(s). However, the identification of a responsible cell-type linking Gq signaling and the loss of allergen-induced pulmonary eosinophilia remains problematic. 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 in-

volved 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 –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

3548 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 Th2specific 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 Th1skewed 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 (33–35), 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.

Acknowledgments We thank the histology core facility at the Mayo Clinic Scottsdale as well as the Mayo Clinic Scottsdale Graphic Arts Core Facility (Director: Marv Ruona). We gratefully acknowledge the assistance of Bonnie Broadhead and our research program assistant, Linda Mardel, whose efforts often go unnoticed.

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