The Journal of Allergy and Clinical Immunology
Volume 125, Issue 1 , Pages 257-263.e5, January 2010

Counterregulation of β2-adrenoceptor function in human mast cells by stem cell factor

  • Glenn Cruse, PhD

      Affiliations

    • Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
    • Corresponding Author InformationReprint requests: Glenn Cruse, PhD, Institute for Lung Health, Department of Respiratory Medicine, Clinical Sciences Building, Glenfield Hospital, Groby Rd, Leicester, LE3 9QP, United Kingdom.
    • ,
  • Weidong Yang, PhD

      Affiliations

    • Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
    • ,
  • S. Mark Duffy, PhD

      Affiliations

    • Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
    • ,
  • Latifah Chachi, BSc

      Affiliations

    • Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
    • ,
  • Mark Leyland, PhD

      Affiliations

    • Department of Biochemistry, University of Leicester, Leicester, United Kingdom
    • ,
  • Yassine Amrani, PhD

      Affiliations

    • Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
    • ,
  • Peter Bradding, DM

      Affiliations

    • Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom

Received 12 January 2009; received in revised form 10 July 2009; accepted 5 August 2009. published online 28 October 2009.

Article Outline

Background

Mast cells contribute to the pathophysiology of asthma with the sustained release of both preformed and newly generated mediators in response to allergens and other diverse stimuli. Stem cell factor (SCF) is the key human mast cell growth factor, but also primes mast cells for mediator release. SCF expression is markedly increased in asthmatic airways. Short-acting β2-adrenoceptor drugs such as albuterol inhibit human lung mast cell (HLMC) degranulation in vitro in the absence of SCF, but their effect in the presence of SCF is not known.

Objective

The aim of this study was to elucidate the effects of albuterol on HLMC function in the presence of SCF.

Methods

Mediator release and KCa3.1 ion channel activity were analyzed in purified HLMC. Intracellular signalling and β2-adrenoceptor phosphorylation and internalization were analyzed in the HMC-1 human mast cell line.

Results

β2-Adrenoceptor agonist-dependent inhibition of KCa3.1 ion channels and HLMC mediator release was markedly attenuated in the presence of SCF. Remarkably, albuterol actually potentiated IgE-induced histamine release in a dose-dependent manner when both SCF and IgE were present. These effects were related to the SCF-dependent phosphorylation of Tyr350 on the β2-adrenoceptor with immediate uncoupling of the receptor followed by receptor internalization.

Conclusion

The potentially beneficial effects of β2-adrenoceptor agonists in asthmatic airways may be blunted as a result of the high concentrations of SCF present.

Key words: β2-Adrenoceptor, albuterol, asthma, mast cell, desensitization

Abbreviations used: β2-AR, β2-Adrenoceptor, cAMP, Cyclic AMP, 1-EBIO, 1-Ethyl-2-benzimidazolinone, ERK, Extracellular signal-regulated kinase, GPCR, G-protein–coupled receptor, HLMC, Human lung mast cell, LT, Leukotriene, SCF, Stem cell factor, VASP, Vasodilator-stimulated phosphoprotein

 

Mast cells play a key role in the pathophysiology of asthma through the ongoing release of proinflammatory autacoid mediators, cytokines, and proteases within dysfunctional airway elements such as the airway epithelium, glands, and smooth muscle bundles.1 Unfortunately, there are no good inhibitors of lung mast cell activation currently available for clinical use because so-called mast cell “stabilizers” are of poor efficacy both in vitro and in vivo when administered regularly. For example, although disodium cromoglycate inhibits the degranulation of rat peritoneal mast cells, it is only a weak inhibitor of human lung mast cells (HLMCs) in the high micromolar range, and it is subject to rapid tachyphylaxis.2 β2-Adrenoceptor (β2-AR) agonists are more potent and efficacious at inhibiting IgE-dependent mast cell histamine release when applied acutely in vitro and in vivo,2 but again there is rapid tachyphylaxis. Thus, when they are administered chronically to patients with asthma, not only is their protective effect rapidly lost, but also allergen-induced bronchoconstriction and the accompanying mast cell mediator release are actually enhanced.3, 4 Inhibiting mast cell activation in chronic asthma therefore remains a major therapeutic goal.

In addition to the enhancement of the airway response to allergen challenge, there is evidence that short-acting β2-agonists administered regularly reduce day-to-day asthma control.5, 6 In the absence of an inhaled corticosteroid, there are also concerns over the safety of long-acting β2-agonists,7 although in combination with an inhaled corticosteroid, long-acting β2-agonists improve asthma control and reduce the frequency of asthma exacerbations.8 The mechanism behind these deleterious effects of β2-agonists remains speculative. Agonists such as albuterol are racemic, consisting of both S-enantiomers and R-enantiomers. Although the R-enantiomer is pharmacologically beneficial, the S-enantiomer is proposed to be pharmacologically inactive. However, there are reports that the S-enantiomer may elicit adverse effects by increasing intracellular Ca2+ in airway smooth muscle (ASM) cells and inducing contraction.9 In addition, animal studies have demonstrated that the S-enantiomer of albuterol induces bronchial hyperresponsiveness to histamine and methacholine.10, 11 However, the clinical implications are unclear, and the data are often conflicting.12

Stem cell factor (SCF) is a growth factor critical for mast cell growth, differentiation, and survival,13 but it also enhances IgE-dependent HLMC degranulation and cytokine production.14, 15, 16 SCF expression is increased in the peripheral blood and airways of subjects with asthma, and it is produced by the airway compartments infiltrated by mast cells.17, 18 Previous studies examining the effects of β2-agonists on HLMCs have been performed in the absence of SCF. Because of the profound effects of SCF on mast cell function and the increased expression of SCF in asthma, and because tyrosine kinase receptors modulate G-protein–coupled receptor (GPCR) function, we have examined the efficacy of the short-acting β2-agonist albuterol on HLMC histamine and leukotriene (LT)–C4 secretion in the presence and absence of SCF after activation with IgE alone or IgE/anti-IgE. We demonstrate for the first time that in the presence of SCF, albuterol is ineffective at inhibiting HLMC mediator release, and in the presence of IgE alone, it actually potentiates mediator release.

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Methods 

See this article's Methods in the Online Repository at www.jacionline.org for further details.

Mast cell purification and culture 

Human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee, UK. HLMCs were purified from lung tissue19 and cultured as described previously.15 The human mast cell line HMC-1 was a gift from Dr J. Butterfield.

HLMC activation 

Experiments were performed at 37°C. Twenty-five microliters of control medium or 4x final concentration of albuterol was added to each well of a 96-well V-bottom plate in triplicate. HLMCs were either sensitized with 2.5 μg/mL human myeloma IgE or sham-treated for 45 minutes. Cells were resuspended in Dulbecco's modified Eagle medium (DMEM) at a concentration of 2 × 105 cells/mL before challenge. HLMCs 1 × 104 (50 μL) were added to each well, immediately followed by 25 μL 4x final concentration of IgE alone, anti-IgE, or DMEM control. Plates were incubated for 30 minutes, centrifuged, and the supernatant decanted and stored at –20°C for measurement of mediator content. Control cell pellets were lysed in ultrapure water for the determination of total histamine content.

Mediator assays 

Histamine was measured by radioenzymatic assay and LTC4 by ELISA as described previously.19

Electrophysiology 

The whole-cell variant of the patch-clamp technique was used as described previously.20

Flow cytometry 

HMC-1 cells were treated with 100 ng/mL SCF, 100 ng/mL SCF + 20 μmol/L genistein, or an appropriate control for 2 minutes at 37°C. Samples were then fixed and permeabilized in paraformaldehyde/0.1% saponin for 15 minutes on ice. Cells were then stained with either 20 μg/mL rabbit antihuman phosphorylated β2-AR antibody or appropriate isotype control (rabbit IgG), followed by secondary fluorescein isothiocyanate–goat antirabbit antibody. Staining was examined by flow cytometry.

Confocal microscopy 

To study receptor internalization, HMC-1 cells were transfected with the full-length open reading frame of the β2-AR (Gly16-Glu27-Val34-Thr164 haplotype; a gift from Professor I. Hall, University of Nottingham, UK) tagged with eGFP using the pEGFP-N1 vector. Transfections were carried out using a ratio of 500 ng cDNA to 2 μL FuGENE HD reagent. Cells were imaged live at 37°C for the duration of the recordings.

Immunoprecipitation 

Immunoprecipitation and Western blotting for the validation of the phosphorylated β-AR antibodies was carried out as described previously.21 Antibodies used were anti–β2-AR, pan antiphosphotyrosine mAb, antiphosphorylated β2-AR (P350), and antiphosphorylated β2-AR (P141).

SDS-polyacrylamide gel electrophoresis and Western blot analysis of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase 1/2 phosphorylation 

Immunoblot analysis was performed as described previously.22 HMC-1 cells (4 × 106) were incubated with SCF 100 ng/mL for 2 minutes followed by albuterol 10−6 mol/L or vice versa. Cells were then resuspended in lysis buffer at different time points. The blotted membranes were incubated with a rabbit monoclonal IgG against the phosphorylated form of extracellular signal-regulated kinase (ERK)–1/2 or vasodilator-stimulated phosphoprotein (VASP).

Data presentation and statistical analysis 

Data are expressed as means ± SEMs from all HLMC donors unless otherwise stated. Statistical significance was calculated using a Student paired or unpaired 2-tailed t test as appropriate with P < .05 taken as statistically significant.

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Results 

Anti-IgE–dependent inhibition of HLMC mediator release by albuterol is attenuated by SCF 

Albuterol inhibits mediator release from HLMC in a dose-dependent manner.2, 23 Thus, anti-IgE (1:1000 dilution) induced net HLMC histamine release of 34.3% ± 4.4% of total histamine content, which was reduced dose-dependently to 3.5% ± 1.4% with the addition of 10−5 mol/L albuterol (Fig 1, A; n = 12; P < .0001). However, in the presence of 100 ng/mL SCF, albuterol was not effective at inhibiting histamine release (net anti-IgE–induced histamine release with SCF 39.0% ± 5.3%, with SCF + 10−5 mol/L albuterol 33.5% ± 7.8%; n = 5; P = .1238; Fig 1, A). Therefore, 10−5 mol/L albuterol inhibited anti-IgE–induced histamine release by 87.4% ± 5.5% in the absence of SCF compared with 18.3% ± 8.8% in the presence of SCF (P < .00001). Similar results were seen with the β2-AR agonists terbutaline, formoterol, and salmeterol (see this article's Fig E1 in the Online Repository at www.jacionline.org).

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  • Fig 1. 

    Modification of histamine and LTC4 release by albuterol. A, Albuterol inhibited anti-IgE activated HLMC histamine release in a dose-dependent manner in the absence of SCF (black bars). This was attenuated by 100 ng/mL SCF (gray bars). B, The effect of SCF on albuterol-induced inhibition of mediator release was also evident with LTC4 production. C, Albuterol did not affect histamine release from IgE-stimulated HLMCs in the absence of SCF (black bars) or with SCF in the absence of IgE (gray bars), but potentiated histamine release induced by IgE alone in the presence of SCF (open bars). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. NS, Not significant.

Comparable results were seen with LTC4 production (Fig 1, B). Anti-IgE induced the net release of 66.0 ± 37.5 ng LTC4/106 HLMCs in the absence of SCF. With the addition of 10−5 mol/L albuterol, net LTC4 release was reduced to 9.7 ± 9.4 ng LTC4/106 cells (Fig 1, B; n = 4; P = .035; log-transformed data). In the presence of 100 ng/mL SCF, net anti-IgE–induced LTC4 release was greater than in the absence of SCF at 117.3 ± 57.1 ng LTC4/106 cells (P = .0026). With the addition of 10−5 mol/L albuterol in the presence of SCF, anti-IgE–induced LTC4 production was not significantly inhibited (net release 106.2 ± 61.2 ng LTC4/106 cells; Fig 1, B; n = 4; P = .260; log-transformed data). Therefore, 10−5 mol/L albuterol inhibited anti-IgE–induced LTC4 release by 91.0% ± 5.2% in the absence of SCF compared with 18.4% ± 13.7% with the addition of 100 ng/mL SCF (P = .040).

Albuterol potentiates HLMC mediator release in the presence of SCF after exposure to IgE 

IgE alone induces modest but significant histamine release and LTC4 secretion in HLMCs in the presence of SCF.15 HLMCs incubated with IgE alone in the absence of SCF released minimal histamine with net histamine release of 1.6% ± 0.4% (P = .0009), which was not significantly affected with the addition of albuterol (Fig 1, C; n = 12). SCF (100 ng/mL) alone did not induce significant histamine release (net 2.8% ± 1.3%; n = 7; P = .081), and this was not significantly affected by the addition of albuterol (Fig 1, C). However, with the addition of both SCF (100 ng/mL) and IgE (3 μg/mL), net histamine release was 4.1% ± 0.8% (P = .0003) and was potentiated in a dose-dependent manner to 12.8% ± 3.7% with the addition of 10−5 mol/L albuterol (Fig 1, C; n = 11; P = .030).

Leukotriene C4 secretion was relatively low with the addition of both SCF and IgE at 1.2 ± 0.5 ng/106 cells. There was no potentiation of LTC4 release by albuterol under these conditions (data not shown).

SCF attenuates β2-AR–induced closure of the K+ channel KCa3.1 

Albuterol closes KCa3.1 through a mechanism independent of cyclic AMP (cAMP).24 We therefore assessed whether SCF also attenuates β2-AR–dependent KCa3.1 closure. In control cells, whole-cell KCa3.1 current at +40 mV was 103.3 ± 19.1 pA postaddition of the KCa3.1 opener 1-ethyl-2-benzimidazolinone (1-EBIO), and this decreased to 72.0 ± 16.8 pA after the addition of 10−5 mol/L albuterol (n = 6 cells; P = .011; Fig 2, A). In contrast, if SCF was added to the cells just before recording, currents at +40 mV were 145.2 ± 27.9 pA after activation with 1-EBIO, but failed to suppress with the subsequent addition of 10−5 mol/L albuterol (n = 6 cells; P = .637; Fig 2, B). The change in current with albuterol in control cells versus SCF-treated cells was highly significant (P = .014). These results suggest that the effects of SCF on β2-AR function are related to a direct effect on the receptor rather than distal signaling pathways.

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  • Fig 2. 

    SCF specifically attenuates β2-AR-induced closure of the K+ channel KCa3.1. Mean current-voltage (I-V) curves demonstrating (A) induction of typical KCa3.1 currents by 1-EBIO in HLMCs (closed circles) and their attenuation by 10−5 mol/L albuterol (open circles; n = 6). B, In the presence of SCF, albuterol failed to inhibit the 1-EBIO–induced KCa3.1 current (n = 6). C, 10−4 mol/L adenosine inhibited 1-EBIO–induced KCa3.1 currents in HLMCs (n = 6). D, In the presence of SCF, 10−4 mol/L adenosine still inhibited the KCa3.1 currents (n = 5).

Because adenosine also closes KCa3.1 through a Gαs-dependent mechanism,25 we next assessed whether the attenuation of KCa3.1 closure by SCF was specific to the β2-AR. In contrast with the effect with albuterol, SCF did not attenuate adenosine-induced closure of KCa3.1 (Fig 2, C and D). The change in current with adenosine in control cells versus SCF-treated cells was not significantly different (P = .211).

SCF trans-phosphorylates Tyr350 on the β2-AR 

G-protein–coupled receptors are phosphorylated by tyrosine kinase receptors with resulting attenuation of GPCR function. This is well described for the β2-AR, which is phosphorylated on tyrosine residues 141 and 350 by the insulin and insulinlike growth factor receptors.26, 27, 28 We therefore used anti–β2-AR Tyr141 and Tyr350 antibodies to examine tyrosine phosphorylation of the β2-AR in the human mast cell line HMC-1. The antibodies were first validated by using Western blotting (see this article's Fig E2 in the Online Repository at www.jacionline.org). The β2-AR was immunoprecipitated from HMC-1 cells, and the resulting SDS gel probed for the β2-AR, β2-AR Tyr141, β2-AR Tyr350, and pan phosphoTyr. A discreet double band of ∼80 kd was evident with all antibodies, indicating they all stain the same protein (Fig E2). This is in keeping with the 80-kd size for the glycosylated and phosphorylated forms of the human β2-AR observed by Bouvier et al.29 It is thus evident that there is constitutive β2-AR phosphorylation in these cells, which might be accounted for by the constitutively activated SCF receptor in HMC-1. To allow accurate and quantitative analysis of β2-AR phosphorylation, we then examined this using flow cytometry. Compared with isotype control, there was some baseline expression of both β2-AR Tyr141 and Tyr350 in HMC-1 cells, which was consistent with the Western blots (Fig 3, A). After preincubation with SCF for 2 minutes, there was a significant increase in staining for Tyr350 but not Tyr141 (Fig 3, B and C). In addition, this increase in Tyr350 phosphorylation was inhibited dose-dependently by the nonspecific tyrosine kinase inhibitor genistein (Fig 3, D). Similar results were seen by using HLMC (see this article's Fig E3 in the Online Repository at www.jacionline.org).

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  • Fig 3. 

    Flow-cytometric analysis of β2 adrenoceptor tyrosine phosphorylation. A, Tyr350 and Tyr141 were constitutively phosphorylated compared with isotype control. (B) The addition of SCF to HMC-1 cells induced the phosphorylation of Tyr350, but not Tyr141 (C), on the β2-AR. D, Effect of SCF and SCF + genistein on β2-AR Tyr350 phosphorylation.

SCF induces β2-AR internalization 

Phosphorylation of Tyr350 on the β2-AR is thought to be a critical step in the internalization of the receptor.28, 30 The addition of 100 ng/mL SCF to HMC-1 cells transfected with a GFP tagged β2-AR chimeric protein induced visible receptor internalization (Fig 4, A). This was evident visually within 10 minutes, but was maximal at 60 minutes, as shown by analyzing the mean fluorescence intensity in the cell cytoplasm with confocal microscopy (Fig 4, B). No receptor internalization was evident in control cells over the same time course.

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  • Fig 4. 

    Confocal microscopy of β2-AR internalization induced by SCF. A, HMC-1 cell transfected with GFP tagged β2-AR. Receptor internalization was evident within 10 minutes after the addition of SCF, but was more marked after 60 minutes. Magnification ×600; representative of 4 separate experiments. B, Mean fluorescence intensity (MFI) of the cell cytoplasm analyzed by using ImageJ (US National Institutes of Health, Bethesda, MD). Means ± SEMs of 4 separate experiments. No receptor internalization was evident in control cells over the same time course. ∗P < .05.

ERK phosphorylation does not contribute to β2-AR counterregulation 

Because phosphorylation of Tyr350 on the β2-AR reveals an Src homology 2 (SH2) binding domain leading to mitogen-activated protein kinase (MAPK) (ERK1 and ERK2) activation through the Ras–Raf–MAPK/Erk Kinase (MEK) pathway31 we tested the effects of SCF and albuterol on ERK1/2 phosphorylation. The addition of SCF (100 ng/mL) to HMC-1 cells led to marked phosphorylation of ERK1/2. There was no induction of ERK1/2 phosphorylation with the addition of 10−5 mol/L albuterol alone. The addition of 10−5 mol/L albuterol to SCF-treated cells inhibited the phosphorylation of ERK1/2 (see this article's Fig E4 in the Online Repository at www.jacionline.org), suggesting that ERK does not contribute to the counterregulation of the β2-AR by SCF.

SCF attenuates β2-AR agonist–induced VASP phosphorylation 

To confirm that the effects of SCF on β2-AR agonist efficacy were indeed through receptor desensitization/uncoupling, we next examined the phosphorylation of VASP, a cAMP-dependent protein kinase A (PKA) substrate downstream of the β2-AR.32 We found that the pretreatment of cells with SCF for 2 minutes significantly reduced albuterol-induced VASP phosphorylation (Fig 5), demonstrating that the desensitization effects of SCF on β2-AR expression were associated with a loss of the β2-AR function. However, the addition of SCF 2 minutes after albuterol had no effect on VASP phosphorylation (Fig 5).

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  • Fig 5. 

    VASP phosphorylation is inhibited by SCF. HMC-1 cells were treated with SCF in the presence or the absence of albuterol for 2 and 10 minutes. SCF was added either 2 minutes before albuterol (A) or 2 minutes after albuterol (B). There was a reduction in VASP phosphorylation only when SCF was added before albuterol. Results are representative of 2 separate blots.

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Discussion 

We have demonstrated for the first time that β2-AR agonist-dependent inhibition of HLMC mediator release and KCa3.1 ion channels is markedly attenuated in the presence of SCF. Remarkably, albuterol actually potentiated IgE-induced histamine release in a dose-dependent manner when both SCF and IgE were present. This was associated with the SCF-dependent phosphorylation of the β2-AR on Tyr350, an effect known to be critical for the counterregulation and internalization of this receptor by the insulin tyrosine kinase receptor.28, 30 These observations have important clinical implications.

Cross-talk between GPCRs and tyrosine kinase receptors is well established (see reviews26, 33). The best studied interaction is between the insulin receptor and the β2-AR. Application of insulin rapidly inhibits β2-AR responses through the inhibition of signaling and internal sequestration of receptors. The C-terminus domain of the β2-AR contains a 15–amino acid sequence between K342 and S356 that is critical for this effect.27 The negative effects of insulin on β2-AR signaling are dependent on both the phosphorylation of Tyr350 and the phosphorylation of S345/S346 indirectly through the phosphatidylinositol-3-kinase (PI3K)-dependent activation of Akt. We have shown that SCF also induces the rapid phosphorylation of Tyr350 on the β2-AR, but not Tyr141, with the latter implicated in counterregulation by insulinlike growth factor.28, 30, 34 The phosphorylation of Tyr350 by SCF occurred within 2 minutes and was paralleled by the rapid attenuation of β2-agonist-induced PKA-dependent phosphorylation of VASP, KCa3.1 channel closure, and inhibition of histamine release, suggesting a functional relationship. This suggests that the pathway by which SCF attenuates β2-AR activity shares common pathways with the insulin receptor.

Although the immediate effects of SCF on β2-AR signaling are most likely caused by receptor uncoupling, the phosphorylation of Tyr350 is significant in that it also creates an SH2 binding site to which Src, Grb2, and dynamin bind to mediate internalization and counterregulation of the β2-AR.26, 28, 30 Thus, the ability of SCF to induce β2-AR internalization suggests a model whereby SCF mediates both the rapid inhibition of β2-AR responses via receptor uncoupling and a more sustained inhibition of β2-AR signaling evoked by receptor internalization.

In keeping with a direct effect of SCF on the β2-AR, VASP phosphorylation, an indicator of cAMP accumulation and PKA activation, was reduced with the addition of SCF 2 minutes before the addition of albuterol. This rapid effect suggests direct linkage between the SCF receptor and the β2-AR leading to uncoupling of the β2-AR and Gαs. In addition, closure of the KCa3.1 K+ channel by albuterol occurs independently of cAMP through a Gαs-dependent mechanism.35 Thus, the attenuation of albuterol-dependent KCa3.1 channel closure by SCF indicates that SCF works proximally on the β2-AR and not on distal β2-AR signaling pathways. Furthermore, this effect of SCF is specific to the β2-AR because KCa3.1 closure by adenosine, which also closes KCa3.1 through Gαs,25 was not affected by SCF.

The ability of β2-AR activation to enhance IgE-dependent histamine release in the presence of SCF is of great interest but is not readily explained. It is known that the generation of the SH2 binding site on Tyr350 of the β2-AR also leads to MAPK (ERK1/2) activation through the Ras–Raf–mitogen-activated protein kinase kinase pathway.31 Because activation of ERK1 and ERK2 is evident in both SCF-dependent and FcεRI-dependent secretory responses,36 it is possible that synergy occurred through this pathway leading to the enhancement of histamine release in the presence of IgE. However, we were unable to show any potentiation of ERK1/2 phosphorylation by SCF with the addition of albuterol. Indeed, albuterol reduced the SCF-induced ERK1/2 phosphorylation. However, this was in the absence of IgE receptor signaling, which is not possible to study in the HMC-1 cell type. In addition, the fact that albuterol-potentiated secretion was seen only with IgE alone, and not with anti-IgE, suggests important differences in the signaling pathways activated by these 2 stimuli, in keeping with previous studies of monomeric IgE.37, 38

A further mechanism through which albuterol might potentiate histamine release after receptor counterregulation by SCF relates to the stereoenantiomers of this drug.9, 10, 11 Racemic albuterol consists of 50:50 R-enantiomers and S-enantiomers that are nonsuperimposable mirror images. The R-enantiomer is the agonist at the β2-AR leading to the accumulation of cAMP, and this binds with 100-fold greater affinity than the S-enantiomer.39 The S-enantiomer of albuterol is thought to mediate nonreceptor-dependent effects and has been shown to induce both Ca2+ mobilization and contraction in airway smooth muscle cells and to be proinflammatory in animal models.9, 10, 11 It is possible therefore that in HLMCs, the inhibition of antisecretory R-albuterol signaling through β2-AR counterregulation by SCF leaves the harmful effects of the S-enantiomer unchecked, leading to enhanced secretion in the presence of free IgE. An alternative explanation is that albuterol interferes with organic cation transporters expressed in mast cells.40 These transporters can be inhibited by albuterol,41 which might therefore reduce histamine reuptake after exposure to a secretagogue. This would explain why there was potentiation of histamine but not LTC4 release in the presence of SCF.

It has been reported that S-albuterol enhances the IgE/anti-IgE–dependent release of both histamine and IL-4 from mouse mast cells.42 However, this required preincubation with the drug for >6 hours and was not evident after short-term (30-minute) incubation. The increase in histamine release observed was dependent on increased histamine synthesis after the upregulation of histidine decarboxylase. This effect is therefore distinct from the immediate increase in histamine release seen in HLMCs after exposure to IgE alone in the presence of SCF and albuterol.

The ability of SCF to attenuate the effects of β2-agonists on HLMCs is potentially of great clinical significance. SCF expression is markedly increased in asthmatic airways17, 18 and is suppressed by inhaled corticosteroids,18 and in an animal model of asthma, neutralization of SCF attenuated eosinophil accumulation, airway hyperresponsiveness, IL-5 and TNF-α production, and goblet cell hyperplasia.43, 44 Although β2-agonists undoubtedly provide relief from bronchoconstriction in stable asthma, their effects are attenuated with regular treatment and particularly during asthma exacerbations. In fact, during regular treatment, there is evidence that asthma control may deteriorate, and this is particularly evident in patients who do not use an inhaled corticosteroid.5, 6 Our results suggest that the presence of SCF in the airways of patients with asthma may markedly attenuate the potentially beneficial effects of β2-agonists. Targeting the SCF-dependent signaling pathways might therefore increase β2-agonist efficacy and improve disease control. In fact, the suppression of SCF by inhaled corticosteroids18 might represent a further mechanism through which these drugs enhance β2-agonist efficacy in asthma. IgE concentrations are significantly increased in asthma, and there is clear evidence of increased IgE bound to cells in the airway mucosa in both allergic and nonallergic asthma.45 The ability of albuterol to enhance histamine release in the presence of IgE and SCF therefore provides a mechanism through which the regular administration of β2-agonists might reduce asthma control. Again, targeting SCF in the airway might enhance the efficacy of regularly applied β2-agonists.

In summary, we have shown that SCF attenuates the protective effects of β2-agonists on mast cell function and that this effect occurs immediately on exposure to SCF. The decrease in β2-AR function induced by SCF is associated with β2-AR phosphorylation at Tyr350 and receptor internalization. Interestingly, when IgE is present with SCF, albuterol actually potentiates mediator release from HLMCs. This provides further evidence that SCF is an interesting target for the treatment of asthma. Such a treatment strategy may not only attenuate many of the pathological features but also increase the effectiveness of currently existing treatments.

Clinical implications

Targeting SCF in the airways may not only attenuate many of the pathological features of asthma but also increase the efficacy of currently available treatments.

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The authors thank the Midlands Lung Tissue Consortium for supply of human lung resection tissue.

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Methods 

HLMC purification and culture 

All human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee, United Kingdom (UK). HLMCs were dispersed and purified from lung tissue obtained by resection for bronchial carcinoma as described previously.E1 HLMC purity was >98% with cell viability >97% (monitored by exclusion of Trypan blue). HLMC were cultured as described previously.E2 Before all experiments, cells were washed thoroughly by centrifugation to remove the cytokines added during culture.

Cell lines 

The human mast cell line HMC-1 was a generous gift from Dr J. Butterfield (Mayo Clinic, Rochester, Minn). The cells were cultured as described previously.E3

HLMC activation 

For HLMC activation, all experiments were performed at 37°C. 25 μL of control medium or 4x final concentration of albuterol was added to each well of a 96-well V-bottom plate in triplicate. HLMC were either sensitized with 2.5 μg/mL human myeloma IgE (Calbiochem-Novabiochem, Nottingham, UK) or sham-treated for 45 minutes, centrifuged at 250g for 8 minutes, and resuspended in Dulbecco's modified Eagle medium at a concentration of 2 × 105 cells/mL before challenge. When SCF was added, it was added to the cells 2 minutes before the addition of albuterol. HLMCs 1 × 104 (50 μL) were added to each well, immediately followed by 25 μL 4x final concentration of IgE alone, anti-IgE, or DMEM control. Plates were incubated for 30 minutes, then centrifuged at 250g for 5 minutes and the supernatant decanted and stored at –20°C for measurement of mediator content. Control cell pellets were lysed in ultrapure water for the determination of total histamine content.

Mediator assays 

Histamine was measured by radioenzymatic assay, as described previously.E1 LTC4 (Cayman Chemical Co, Ann Arbor, Mich) was measured by ELISA according to the manufacturer's instructions.

Electrophysiology 

The whole-cell variant of the patch-clamp technique was used as described previously.E4 Experiments were performed with a perfusion system (Automate Scientific, San Francisco, Calif) to allow solution changes, although drugs were added directly to the recording chamber. When SCF was present, it was added 2 minutes before albuterol.

Flow cytometry 

HMC-1 and HLMCs were suspended at 106 cells/mL in DMEM at 37°C. Cells were treated with 100 ng/mL SCF (R&D, Abington, UK), 100 ng/mL SCF + 20 μmol/L genistein (Calbiochem-Novabiochem, Nottingham, UK), or an appropriate control for 2 minutes at 37°C. Samples were then centrifuged and resuspended in 100 μL cold PBS, injected into 1 mL cold 4% paraformaldehyde/0.1% saponin, and incubated for 15 minutes on ice. The cells were then washed before the addition of either 20 μg/mL rabbit antihuman phosphorylated β2-AR antibody (Santa Cruz Biotechnology, Heidelberg, Germany) or the appropriate isotype control (rabbit IgG; BD Biosciences, Oxford, UK). Cells were then labeled with fluorescein isothiocyanate–goat antirabbit secondary antibody (Dako, Cambridge, UK). For the determination of staining, cells were examined by flow cytometry (BD FACScan, BD Biosciences, Oxford, UK).

Confocal microscopy 

To study receptor internalization, HMC-1 cells were used because of the difficulty in transfecting primary HLMCs. HMC-1 cells were transfected with the full-length open reading frame of the β2-AR (Gly16-Glu27-Val34-Thr164 haplotype; a generous gift from Professor Ian Hall, University of Nottingham, UK) tagged with eGFP using the pEGFP-N1 vector (Clontech, Saint-Germain-en-Laye, France). Transfections were carried out by using a ratio of 500 ng cDNA to 2 μL FuGENE HD reagent (Roche Applied Science, Burgess Hill, UK). Cells were incubated for 24 hours at 37°C in a humidified incubator before confocal analysis. Confocal was carried out by using the Olympus FV1000 microscope (Southend on Sea, UK). Cells were imaged live at 37°C for the duration of the recordings. Recordings were taken at the indicated time points by using an oil immersion x60 fluorescent objective and the images analyzed by using Fluoview FV10-ASW 1.6 software. Images were exported into ImageJ 1.37v software (US NIH, Bethesda, Md) for analysis of the mean fluorescence intensity within the cell.

Immunoprecipitation 

Immunoprecipitation and Western blotting for the validation of the phosphorylated β-AR antibodies were carried out as described previously.E5 Antibodies used were anti–β2-AR (Abcam, Cambridge, UK), antiphosphotyrosine mAb (PY99), anti-phosphorylated β2-AR (P350), and antiphosphorylated β2-AR (P141; all from Santa Cruz Biotechnology). These studies were performed on HMC-1 cells because of the number of cells required.

SDS-polyacrylamide gel electrophoresis and Western blot analysis of VASP and ERK1/2 phosphorylation 

Immunoblot analysis was performed as described previously.E6, E7 Briefly, HMC-1 cells (4 × 106) were incubated with SCF 100 ng/mL for 2 minutes followed by albuterol 10−6 mol/L or vice versa. Cells were then washed with cold PBS and resuspended in lysis buffer at different time points. The blotted membranes were incubated with a rabbit monoclonal IgG against the phosphorylated form of ERK1/2 or VASP (both from New England Biolabs, Herts, UK), detected by peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), visualized by the enhanced chemiluminescence system (Thermo-Scientific, Cramlington, UK), and then autoradiographed. These studies were performed on HMC-1 cells because of the number of cells required.

Data presentation and statistical analysis 

Unless otherwise stated, data are expressed as the means ± SEMs from all HLMC donors. Histamine data were expressed as the net percentage of histamine release from the total histamine content calculated from the cell pellets. Statistical significance was calculated by using a Student paired or unpaired 2-tailed t test as appropriate with P <.05 taken as statistically significant. Data from patch clamping was log-transformed for statistical analysis.

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Fig E1. 

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  • SCF attenuates β2-AR responsiveness to long-acting and short-acting β-agonists. A, Albuterol. B, Albuterol + 10 μmol/L propranolol. C, Terbutaline. D, Salmeterol. E, Formoterol. Means ± SEMs from 4 separate donors. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

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Fig E2. 

  • View full-size image.

  • Validation of anti–β2-AR and phospho-specific antibodies. A, HMC-1 immunoprecipitation with Abcam (Cambridge, UK) anti–β2-AR, probed with same antibody. B, Same sample as A, but probed with antiphosphotyrosine. C, Same sample as A, probed with Santa Cruz anti–β2-AR. D, Same sample as A, probed with antiphosphorylated β2-AR, P350. E, Same sample as A, probed with antiphosphorylated β2-AR, P141. Ab, Probed sample; M, kd marker.

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Fig E3. 

  • View full-size image.

  • SCF induces phosphorylation of Tyr350 on the β2-AR, but not Tyr141 in HLMCs. The addition of SCF induced phosphorylation of Tyr350 which was attenuated with the addition of 30 μmol/L genistein. Tyr141 was unaffected by SCF.

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Fig E4. 

  • View full-size image.

  • SCF-inducedERK 1/2 activation is partially suppressed by albuterol. HMC-1 cells were treated with SCF in the presence or the absence of albuterol for 2 and 10 minutes. Cells were lysed and cytoplasmic extracts were prepared and assayed for the nonactivated and activated (phosphorylated) forms of ERK1/2 (p42/44) by immunoblot analysis as described in Methods. Results are representative of 2 separate blots.

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References 

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 Supported by Asthma UK grant no. 04/026.

 Disclosure of potential conflict of interest: P. Bradding receives research funding from Genentech, Novartis, and Boehringer-Ingelheim. The rest of the authors have declared that they have no conflict of interest.

PII: S0091-6749(09)01261-5

doi:10.1016/j.jaci.2009.08.020

The Journal of Allergy and Clinical Immunology
Volume 125, Issue 1 , Pages 257-263.e5, January 2010

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