β-Arrestin-2 regulates the development of allergic asthma

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking β-arrestin-2, a G protein–coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the β-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking β-arrestin-2. β-arrestin-2–deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine–mediated CD4⁺ T cell migration to the lung. This report provides the first evidence that β-arrestin-2 is required for the manifestation of allergic asthma. Because β-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Asthma is a complex inflammatory disease that afflicts nearly 15 million Americans. Despite research advances, the worldwide prevalence, morbidity, and mortality of asthma have increased over the last two decades (1–3). In humans, the hallmark feature of allergic asthma is the abnormal expansion in the lung of Th cells that produce Th2 cytokines. This pathological event leads to the symptoms of asthma including airway inflammation, airway hyperresponsiveness, reversible airflow obstruction, and airway remodeling.

Like other immune cells, T cells are functionally dependent on their ability to migrate, localize within tissues, and interact with other immune cells (4). Chemotaxis, the process by which immune cells migrate, is mediated by chemokine activation of chemokine receptors (5).

Chemokine receptors are part of the enormous family of heptahelical cell surface receptors known as G protein–coupled receptors (GPCRs) (6). These receptors transduce extracellular signals into intracellular events by activating heterotrimeric G proteins. The dissociation of these G protein subunits activates cell signaling systems such as adenylate cyclases, phospholipases, and ion channels, which ultimately results in a physiological response. In the case of chemokine receptors, at least one of these physiological responses is cell migration.

Like other GPCRs, chemokine receptor function is regulated by β-arrestin proteins. β-arrestins, members of the arrestin family of proteins, are designated β-arrestin-1 or β-arrestin-2, are ubiquitously expressed, and regulate GPCR function through multiple mechanisms (7–9). As their name suggests, β-arrestin proteins were originally discovered to “arrest” G protein–mediated cell signaling events (10). Since that time, our understanding of the mechanisms by which β-arrestin modulates GPCR function has expanded considerably. In addition to their classical role, β-arrestin proteins also act as adapters that couple GPCRs to a clathrin-coated pit endocytic mechanism and as scaffolds that link GPCRs to a second wave of cell signaling via MAPK and other signaling pathways.

In vitro studies have shown that lymphocytes devoid of β-arrestin-2 and human embryonic kidney 293 cells with suppressed expression of β-arrestin-2 demonstrate impaired migration toward the chemotactic factor stromal cell–derived factor-1α (SDF-1α), also known as CXCL12 (11, 12). Although β-arrestin-2 is essential to the normal migration of immune cells in vitro, the ability of β-arrestin-2 to mediate immune cell chemotaxis in vivo has not been tested.

Because chemotaxis is crucial to the process of inflammation, we theorized that mice lacking β-arrestin-2 might be protected from developing allergic-asthmatic inflammation. To model allergic asthma in mice we used a standard method consisting of sensitization and challenge to OVA (13). This mouse model of allergic asthma mimics several features of human asthma.

To examine the role of β-arrestin-2 in the pathogenesis of asthma, we used an OVA model of allergic asthma and an endotoxin model of nonallergic asthma. We measured physiological and biological variables including airway responsiveness, airway inflammatory cell infiltration, T cell chemotaxis, cytokine levels in whole-lung lavage fluid, and serum Ig levels.

Airway responsiveness. OVA treatment resulted in a dramatic increase in airway responsiveness in WT mice (Figure 1) as measured by airway pressure time index. In marked contrast, βarr2–/– mice treated with OVA (βarr2–/–-OVA mice) showed no such increase in airway responsiveness compared with either WT-alum or βarr2–/–-alum (control) mice.

Figure 1

Effect of OVA treatment on airway responsiveness. Airway responsiveness to methacholine, defined by the time-integrated change in peak airway pressure, or airway pressure time index (APTI), was measured for βarr2^–/– (circles) and WT (squares) mice treated with either alum (filled symbols) or OVA (open symbols). Data are mean ± SEM; n = 9–12 mice per group. *Effect of OVA treatment was significantly different between WT and βarr2^–/– mice. P < 0.05.

Lung inflammation. Histological analysis showed that only mild extravasation of inflammatory cells, including CD3⁺ T lymphocytes, occurred in the airways of βarr2–/– mice treated with OVA, whereas severe cellular infiltration occurred in the interstitium of WT-OVA mice (Figure 2, a and b). Quantitation of CD3⁺ cells per mm of basement membrane or CD4⁺ cells harvested from lung show that βarr2–/– mice experience significantly reduced T lymphocyte infiltration into the lung (Table 1). Differential cell counts from lavage fluid supported the histological data revealing that lymphocyte and eosinophil infiltration into the lungs of βarr2–/–-OVA mice was markedly reduced compared with WT-OVA mice and was not different from that in alum-treated control mice of either genotype (Figure 2c). The lack of T lymphocytes in the lung of βarr2–/–-OVA mice suggests that the migration of T cells to the lung is impaired in the absence of β-arrestin-2.

Figure 2

Effect of OVA treatment on airway inflammation. (a) Effect of genotype and OVA treatment on lung inflammation evaluated by histological analysis of lung sections. Lung sections from WT-alum (lower left) and βarr2^–/–-alum mice (lower right) appeared normal with no inflammatory cell infiltration. Lung sections from WT-OVA mice (upper left) showed severe cellular infiltration in the interstitium. Lung sections from βarr2^–/–-OVA mice (upper right) showed mild extravasation of inflammatory cells in the interstitium. Representative histological sections are shown (n = 8–11 mice per group). (b) Representative images of cross-sectioned airways together with peribronchovascular connective tissue are shown. CD3⁺ T cells were counted in the subtended bronchovascular interstitium. OVA-treated WT mice showed an increased number of CD3⁺ T cells in the peribronchovascular zone relative to alum-treated mice. No such infiltration of CD3⁺ T cells was observed in OVA-treated βarr2^–/– mice. (c) Effect of genotype and OVA treatment on lung inflammation assessed by identification of cells harvested from whole-lung lavage. Black bars represent WT-OVA mice; white bars represent βarr2^–/–-OVA mice; light gray bars represent WT-alum mice; dark gray bars represent βarr2^–/–-alum mice. Data are mean ± SEM; n = 9–12 mice per group. *P < 0.05 versus all other groups.

Table 1

Quantitation of T lymphocytes in the lung

Cytokine levels. Activated T cells migrate to the lungs and release inflammatory cytokines to orchestrate the allergic inflammatory response. Consistent with the demonstrated lack of lymphocytes in the airways of βarr2–/–-OVA mice was their diminished level of Th2 cytokines. Analysis of lavage fluid from WT-OVA mice showed a significant increase in the levels of IL-4, IL-5, and IL-13 relative to the respective cytokine level in lavage fluid from βarr2–/–-OVA mice and alum controls (Figure 3a). To determine whether βarr2–/– mice have a Th1-skewed response that might inhibit the development of Th2 lymphocytes, we measured Th1-type cytokines in lung lavage fluid (18). Levels of IL-12 and IFN-γ were not different between WT and βarr2–/– mice, and the level of these cytokines was unaffected by OVA treatment (Figure 3b).

Figure 3

Effect of genotype and OVA treatment on lung cytokine release in whole-lung lavage fluid. (a) Cytokines associated with a Th2-type response were significantly elevated in WT-OVA mice relative to βarr2^–/–-OVA mice and alum-treated mice of either genotype. Black bars represent WT-OVA mice; white bars represent βarr2^–/–-OVA mice. Cytokine levels in alum-treated WT and alum-treated βarr2^–/– mice were not different and therefore were combined as shown by gray bars. Data are mean ± SEM calculated from three independent experiments; n = 11–19 mice per group. *P < 0.05 versus all other groups. (b) Cytokines associated with a Th1-type response were not significantly elevated by OVA treatment and were similar for WT and βarr2^–/– mice. Black bars represent WT-OVA mice; white bars represent βarr2^–/–-OVA mice. Light gray bars represent alum-treated WT mice; dark gray bars represent βarr2^–/– mice. Data are mean ± SEM calculated from two to three independent experiments; n = 7–13 mice per group.

Ig production. The absence of the classic features of a Th2-mediated asthmatic response in OVA-treated βarr2–/– mice indicates that β-arrestin-2 exerts its regulatory effect early in the progression of allergic asthma. β-Arrestin-2 may regulate the handling of aeroallergen, a series of events that occurs prior to, and is necessary for, T cell stimulation and allergen-specific Ig production. However, the serum levels of OVA-specific IgE and OVA-specific IgG₁ were significantly elevated in βarr2–/–-OVA mice, and the magnitude of this antigen-specific antibody production was not significantly different from the response observed in WT-OVA mice (Figure 4, a and b). Thus, βarr2–/– mice are competent in their ability to present antigen, to generate antigen-specific T cell responses, and to undergo Ig isotype switching. Measurement of IgG_2a, a serum Ig typical of a Th1-type response, was not different between WT and βarr2–/– mice (Figure 5, a and b). These data show that the lack of a Th2-type response in βarr2–/– mice is not due to enhanced induction of the Th1 arm of the Th cell pathway for development.

Figure 4

Effect of OVA treatment on serum Ig production. Although OVA-specific IgE (a) and IgG₁ (b) levels changed significantly over time, serum Ig levels in WT-OVA mice (black bars) were not significantly different from those in βarr2^–/–-OVA mice (white bars) when compared at each timepoint (days 10, 17, and 24). These OVA-specific Ig’s were not detected in alum-treated mice of either genotype. Measurements were made using the endpoint titer method. Data are mean ± SD calculated from two independent experiments; n = 3–8 mice per group. One-way ANOVA and the Tukey HSD post-hoc test were used.

Figure 5

IgG_2a production. (a) Total serum IgG_2a levels were not different between WT-OVA mice (black bars) and βarr2^–/–-OVA mice (white bars) at day 10, 17, or 24. Similarly, there was no difference in IgG_2a levels in serum from alum-treated WT (light gray bars) and alum-treated βarr2^–/– mice (dark gray bars). Data are mean ± SEM calculated from three independent experiments; n = 4–14 mice per group. (b) OVA-specific IgG_2a measurements were not different between WT-OVA mice (black bars) and βarr2^–/–-OVA mice (white bars) at day 24. Measurements were made using the endpoint titer method. Data are mean ± SD calculated from two experiments; n = 3 mice per group. ND, non-detectable.

CD4+ T cell chemotaxis. In allergic asthma, MDC is a potent Th2 cell chemoattractant produced by activated lung macrophages, airway smooth muscle cells, bronchiolar epithelial cells, and by Th2 cells themselves (5). To investigate whether CD4⁺ T cell migration to the lung is impaired in βarr2–/– mice, we tested the ability of splenocytes, thoracic lymph node cells, and lung lymphocytes from OVA-treated WT and βarr2–/– mice to respond to MDC. Only lung lymphocytes, and not spleen or lymph node T cells (data not shown), migrated toward MDC. While CD4⁺ T cells harvested from lung of OVA-treated WT mice migrated well toward MDC, CD4⁺ T cells lacking β-arrestin-2 were significantly impaired in their migration toward MDC (Figure 6a). MDC levels in lavage fluid were significantly elevated in OVA-treated WT mice, but not in similarly treated βarr2–/– mice (Figure 6b). These data support the notion that β-arrestin-2 positively regulates MDC-mediated CD4⁺ T cell migration to the lung in OVA-treated mice. However, β-arrestin-2 may alternatively, or additionally, regulate CD4⁺ T cell migration in response to a chemokine other than MDC.

Figure 6

Chemotactic responses to MDC and release of MDC in lavage fluid. (a) Lung CD4⁺ T cells from OVA-treated βarr2^–/– mice (white bars) exhibit decreased migration toward MDC. CD4⁺ T cells were isolated on day 24 of the OVA treatment protocol and tested for their ability to move chemotactically toward 100 nM MDC. Shown is the mean chemotactic index and SE from three independent experiments. *P < 0.05 vs. βarr2^–/–-OVA mice. (b) Release of MDC into whole-lung lavage fluid was significantly induced in OVA-treated WT mice (black bars) but not OVA-treated βarr2^–/– mice (white bars). MDC levels in alum-treated WT and alum-treated βarr2^–/– mice were not different and therefore were combined (gray bars). Data are mean ± SEM calculated from two independent experiments; n = 9 mice per group. **P < 0.05 versus combined alum-treated mice.

Nonallergic asthma model. To further investigate the specificity of the impaired asthmatic response in βarr2–/– mice, we subjected mice to a model of endotoxin-mediated asthma, or nonallergic asthma. Endotoxin, an LPS, is a major component of the outer cell membrane of all gram-negative bacteria, and even small amounts of this molecule are immune-stimulatory (19). Subjects with endotoxic asthma demonstrate lung neutrophilic inflammation and airway hyperresponsiveness, and this response is coordinated by alveolar macrophages and epithelial cells rather than T cells (20). Both WT and βarr2–/– mice exposed to aerosolized LPS developed lung neutrophilic inflammation and increased airway responsiveness (Figure 7, a and b). Thus, there is no general impairment of lung inflammatory processes or in the ability of airways to respond to methacholine in βarr2–/– mice.

Figure 7

Effect of LPS treatment on airway responsiveness and inflammation. (a) Effect of genotype and LPS treatment on lung inflammation assessed by identification of cells harvested from whole-lung lavage. Black bars represent WT LPS-treated mice; white bars represent βarr2^–/– LPS-treated mice. Data are mean ± SEM; n = 13–15 mice per group. (b) Airway responsiveness to methacholine, defined by the time-integrated change in peak airway pressure was measured for βarr2^–/– (circles) and WT (squares) mice treated with LPS (open symbols) or untreated (filled symbols). Data are mean ± SEM; n = 13–15 mice per group. *LPS treatment caused a similar significant increase in APTI in WT and βarr2^–/– mice. P < 0.05.

When treated with OVA, WT mice develop symptoms of allergic asthma. In stark contrast, these symptoms, including airway inflammation and airway hyperresponsiveness, do not appear in similarly treated βarr2–/– mice. These results suggest that β-arrestin-2 is essential to the development of allergic asthma and that it exerts its regulatory effect at a proximal step in the inflammatory cascade.

Numerous immune cell types, including APCs, T lymphocytes, B lymphocytes, eosinophils, and mast cells, interact in a highly coordinated fashion to respond to allergens. Although the nature and sequence of these interactions are not entirely delineated, the infiltration of activated Th2 cells into the lung is a primary pathological event underlying allergic asthma. Without these cells, the symptoms of asthma, including airway inflammation, airway hyperresponsiveness, and reversible airflow obstruction, do not occur (21–23).

The initial event in the development of allergic asthma is the processing of inhaled allergens. This involves the capture, modification, and presentation of allergen to T and B cells (24). Once activated, T cells proliferate and differentiate to a Th2 phenotype defined by the release of type 2 cytokines (IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13). Release of these cytokines at germinal centers within peripheral lymphoid tissue supports B cell–mediated production of allergen-specific antibodies (25, 26). In addition, type 2 cytokines released from T cells that have migrated to the lung activate and recruit mast cells and eosinophils, the primary effector cells of the allergic asthmatic response (1).

Our study shows that antigen processing, presentation, and activation of T cells are intact in βarr2–/– mice since their serum levels of OVA-specific IgE and IgG₁ are comparable to those in WT mice and are significantly elevated compared with alum-treated mice. Although there is a trend for a slight delay in the kinetics of the Ig response in OVA-treated βarr2–/– mice, the impact of this on the physiological and biological measurements made on day 24, when OVA-IgE levels are the same, is unknown. Despite the significant production of OVA-IgE and OVA-IgG₁ in OVA-treated βarr2–/– mice, the classic features of a Th2-mediated asthmatic response, including airway inflammation, airway hyperresponsiveness, and elevated Th2 cytokines, are absent in these mice. Therefore, the defect in the allergic asthmatic response in βarr2–/– mice lies between the generation of a Th2 response and airway inflammation. Our data showing defective CD4⁺ T cell migration to the Th2 chemoattractant MDC and reduced levels of MDC in lavage fluid suggests that the defect is present at the level of Th2 cell trafficking to the lung.

Other potential mechanisms underlying the phenotype observed in βarr2–/– mice have been evaluated but ruled out. Other studies have shown that enhanced induction of Th1-type cytokines may inhibit the development of a Th2-type response (18). In our study we find no evidence that βarr2–/– mice experience Th1-type inhibition of their Th2-type response since IgG_2a, IL-12, and IFN-γ (factors indicative of a Th1 response) are not elevated in βarr2–/– mice relative to WT mice.

Another possible explanation for the failure of βarr2–/– mice to develop asthma is mast cell dysfunction. Antigen crosslinking of cell-surface IgE receptors stimulates mast cell release of mediators including histamine, prostaglandins, leukotrienes, and cytokines. Collectively, these agents perpetuate airway inflammation and airway hyperresponsiveness and thus appear pivotal to the development of allergic asthma (27). However, the ability to respond to OVA and to develop allergic asthma is preserved in mast cell–deficient mice (28, 29). Thus, the failure of βarr2–/– mice to develop asthma cannot be explained by mast cell dysfunction.

T cell function is crucial to the development of allergic asthma. When T cells are nonfunctional or absent, allergic inflammation, airway hyperresponsiveness, and lung cytokine production are prevented in response to OVA treatment (21–23). These results are nearly identical to the dramatically impaired allergic asthmatic response we observed in βarr2–/– mice. Because βarr2–/– mice have normal levels of CD3⁺ T splenocytes (11) and normal architecture of spleen follicles (data not shown), we suggest that T cell dysfunction, rather than reduction in T cell number, is at the root of the impaired allergic asthmatic response in βarr2–/– mice. Since the manifestation of allergic asthma requires T cell proliferation, differentiation, and migration, any one of these processes may be regulated by β-arrestin-2 and thus dysregulated in mice completely lacking that protein.

We hypothesize that T cell migration is the process regulated by β-arrestin-2 in allergic asthma. Multiple in vitro reports demonstrate that β-arrestin regulates chemokine receptor signaling and internalization (28, 30, 31). Recently, others in our group used the chemokine SDF-1α (or CXCL12) to show that CXCR4-mediated in vitro migration of nonactivated splenic T cells is impaired in cells lacking β-arrestin-2 (11). In two separate studies, Gonzalo et al. showed that lung inflammation and airway hyperresponsiveness are significantly reduced when SDF-1α or MDC are neutralized by antibodies (32, 33). Thus, reduced migration of activated T cells to SDF-1α may contribute to the phenotype we observed in βarr2–/– mice. However, in our study, lung SDF-1α was not significantly induced by OVA treatment in either βarr2–/– or WT mice (data not shown). Thus, MDC is more likely to play a significant role in the development of asthma in our model. Indeed, we showed that not only is the MDC signal reduced in βarr2–/– mice, but the chemotactic response to this signal is also impaired. Thus, β-arrestin-2 positively regulates MDC-mediated Th2 cell migration in the context of allergic asthma. We hypothesize that diminished Th2 cell migration to the lung is the primary impairment in βarr2–/– mice and is largely responsible for their protection against the development of allergic asthma.

The mechanisms by which β-arrestin-2 may regulate T cell chemotaxis in vivo or in vitro are not presently defined. β-Arrestin proteins act as scaffolds to link GPCR activation to at least three MAPK cascades: the extracellular signal–regulated kinase (ERK) cascade (34), the JNK-3 cascade (35), and the p38 MAPK cascade (12). A recent report indicates that β-arrestin-2 activation of the p38 MAPK cascade is required for CXCR4-mediated migration of human embryonic kidney 293 cells to SDF-1α (12). β-Arrestin-2 may similarly regulate CCR4, the receptor for MDC, through mechanisms involving MAPK or other signaling pathways. Although MAPKs regulate the phosphorylation of nuclear transcription factors, recent studies indicate that β-arrestin–MAPK scaffolds are preferentially targeted to cytosolic substrates (34–36). Such cytosolic substrates may be proteins involved in cell chemotaxis. Thus, β-arrestin-2, through its action as a scaffold protein, may positively regulate T cell chemotaxis.

Alternatively, in keeping with their classically described role, β-arrestins may regulate chemotaxis through termination of chemokine receptor signaling (9). This signal termination, or chemokine receptor desensitization, may be crucial to the directional sensing, and thus migration, of T cells.

Although we feel that β-arrestin-2 regulation of T cell migration is the major mechanism responsible for the profound protection against allergic asthma observed in the βarr2–/– mice, β-arrestin-2 regulation of other T cell functions, or modulation of cells other than lymphocytes, may make a contribution to the dramatic phenotype observed in these mice.

β-arrestin-2 could regulate nonhematopoietic cells in the lung such as airway smooth muscle or airway epithelial cells. Indeed, levels of MDC in lavage fluid are reduced in βarr2–/– mice. This may indicate a positive role for β-arrestin-2 in regulating chemokine production/secretion by either of these lung cell types. Alternatively, reduced MDC levels in lavage fluid may be explained by altered aeroallergen handling in βarr2–/– mice or may be a by-product of impaired T cell migration, since Th2 cells are known to produce MDC. Airway responses to methacholine challenge were not different between βarr2–/– and WT mice when treated with alum or LPS, suggesting that β-arrestin-2 does not regulate airway smooth muscle cell contraction.

β-arrestin-2 may regulate T cell differentiation. Yamashita et al. showed that T cell antigen receptor–mediated activation of the Ras-ERK1/2 pathway enhances IL-4 receptor signaling and is required for the differentiation of naive T cells into Th2 cells (37). Although neither T cell receptors nor IL-4 receptors are GPCRs, there is evidence in the literature of β-arrestin-2 modulation of non–GPCR-mediated events through transactivation of nonheptahelical receptors or through intracellular modulation of downstream signaling pathways resulting from antigen or cytokine receptor stimulation (38). Although differentiation of T cells to the Th2 lineage may be reduced in βarr2–/– mice, our results suggest that this does not occur during the antigen sensitization phase since the release of IL-4 from Th2 cells is required for the production of allergen-specific IgE by B cells (39) and this function is not impaired in βarr2–/– mice.

The fact that LPS-exposed βarr2–/– mice developed neutrophilic lung inflammation and increased airway responsiveness comparable to similarly treated WT mice demonstrates that there is no universal impairment in inflammatory cell migration in βarr2–/– mice. This demonstration of a functionally intact innate immune system is critical to the pursuit of β-arrestin-2 as a potential therapeutic target for treating allergic asthma.

The absence of β-arrestin-2 prevents the development of allergic asthma without compromising the function of the innate immune system. Regulation of T cell migration to the lung by β-arrestin-2 is thought to be the primary mechanism underlying this protection. Because β-arrestin-2 exerts its regulatory effect proximal to the recruitment of activated Th2 cells into the lung it is an attractive therapeutic target for treating allergic asthma.

We thank Kris Riebe and Humphrey Kendall for managing the mouse colony. We thank Mike Foster for instruction on aerosol exposure and a critical review of the manuscript. We thank Herman Staats for guidance with respect to the endpoint titer measurement of Ig’s. We thank Don Burdick from the Duke University Statistical Consulting Center for advice on statistical analysis. R.J. Lefkowitz is an investigator of the Howard Hughes Medical Institute. These studies were supported by an award to R.J. Lefkowitz from The Sandler Family Supporting Foundation and by NIH grants CA-098110 to D.D. Patel and P50 ES-09607, R01 ES-07498, and U01 HL-66611 to D.A. Schwartz.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: G protein–coupled receptor (GPCR); stromal cell–derived factor-1α (SDF-1α); β-arrestin-2–deficient (βarr2–/–) macrophage-derived chemokine (MDC); honestly significant difference (HSD).

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