Yasuhiro Tabata

Allergy-Driven Alternative Splicing of IL-13 Receptor α2 Yields Distinct Membrane and Soluble Forms

IL-13 is a key mediator of allergic inflammation. Its diverse functions are mediated by a complex receptor system including IL-4Rα, IL-13Rα1, and IL-13Rα2. IL-4Rα and IL-13Rα1 form a high-affinity signaling heterodimer. IL-13Rα2 binds IL-13 with high affinity and has been found to exist in membrane and soluble forms. Soluble IL-13Rα2 has been postulated as a critical endogenous modulator of IL-13 responses. However, the mechanism of generation for the soluble form remains unclear. We present the initial study that a mechanism for generation of the soluble form is alternative splicing and that alternative splicing yields a distinct form of soluble IL-13Rα2. We found that several mouse organs expressed two IL-13Rα2 transcripts, the 1152-bp transcript encoding the full-length protein and the 1020-bp transcript lacking exon10, which encodes the transmembrane region. Deletion of exon 10 (ΔEx10) caused a frameshift resulting in a different amino acid sequence from position 327 to position 339 and early termination. Constructs encoding both splice variants were transfected into WEHI-274.1 cells. Transfectants expressing the full-length transcript had IL-13Rα2 on the cell surface but produced minimal soluble IL-13Rα2 in the supernatants. In contrast, transfectants expressing the ΔEx10 transcript displayed no membrane IL-13Rα2 but secreted high levels of soluble IL-13Rα2 capable of inhibiting IL-13 signaling. Both variants bound IL-13, but the ΔEx10 variant displayed ∼2-fold increase in IL-13 binding activity. Expression of the two IL-13Rα2 transcripts was differentially regulated in vivo in an experimental allergic asthma model. Thus, alternatively spliced variants of IL-13Rα2 may have a distinct biologic function in vivo.

Level of Expression of IL-13Rα2 Impacts Receptor Distribution and IL-13 Signaling

IL-13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Rα, IL-13Rα1, and IL-13Rα2. IL-4Rα and IL-13Rα1 form a heterodimeric signaling receptor for IL-13. In contrast, IL-13Rα2 binds IL-13 with high affinity but does not signal. IL-13Rα2 exists on the cell surface, intracellularly, and in soluble form, but no information is available regarding the relative distributions of IL-13Rα2 among these compartments, whether the compartments communicate, and how the relative expression levels impact IL-13 responses. Herein, we investigated the distribution of IL-13Rα2 in transfected and primary cells, and we evaluated how the total level of IL-13Rα2 expression impacted its distribution. Our results demonstrate that the distribution of IL-13Rα2 is independent of the overall level of expression. The majority of the IL-13Rα2 protein existed in intracellular pools. Surface IL-13Rα2 was continually released into the medium in a soluble form, yet surface expression remained constant supporting receptor trafficking to the cell surface. IL-13Rα2 inhibited IL-13 signaling proportionally to its level of expression, and this inhibition could be overcome with high concentrations of IL-13.

IL-13Rα2 Membrane and Soluble Isoforms Differ in Humans and Mice

Although mice have nanogram per milliliter serum levels of soluble (s) IL-13Rα2, humans lack sIL-13Rα2 in serum. Our data provide a mechanism for this biological divergence. In mice, discrete transcripts encoding soluble and membrane forms of IL-13Rα2 are generated by alternative splicing. We used small interfering RNA to specifically deplete the transcript encoding membrane (mem) IL-13Rα2 (full-length) or sIL-13Rα2 (ΔEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Rα2 but had no effect on the level of sIL-13Rα2 in cell supernatants at baseline or following cytokine stimulation. Depletion of the ΔEx10 transcript decreased sIL-13Rα2 in supernatants at baseline and following stimulation. In contrast to mice, we were unable to find a transcript encoding sIL-13Rα2 in humans and siRNA-mediated depletion of full-length IL-13Rα2 decreased both sIL-13Rα2 and memIL-13Rα2 in human cells. Inhibition of matrix metalloproteinases (MMP)/MMP-8 abolished production of sIL-13Rα2 from human cells. Thus, sIL-13Rα2 is derived exclusively from the memIL-13Rα2 transcript in humans through MMPs/MMP-8 cleavage of memIL-13Rα2, supporting a limited role for sIL-13Rα2 in humans and highlighting the potential importance of memIL-13Rα2 in human immunity. These observations require consideration when results of murine IL-13 studies are applied to humans.

Matrix metalloproteinase 8 contributes to solubilization of IL-13 receptor α2 in vivo

BACKGROUND IL-13 receptor alpha2 (IL-13R alpha 2) is a high-affinity receptor for IL-13, a central mediator of allergic asthma. It acts predominantly as a decoy receptor but can also contribute to IL-13 responses under certain conditions. IL-13R alpha 2 exists in soluble and membrane forms, which can both bind IL-13 and modulate its activity. Yet the proteolytic processes that contribute to the generation of soluble IL-13R alpha 2 are largely unknown. OBJECTIVE We sought to investigate the role of matrix metalloproteinases (MMPs) in the generation of soluble IL-13R alpha 2. METHODS Acellular cleavage assays by MMPs were performed by using glutathione-S-transferase fusion proteins of murine or human IL-13R alpha 2. IL-13R alpha 2 stable-transfected cells were used for analysis of surface expression and release of soluble IL-13R alpha 2. Wild-type and MMP-8-deficient mice were used for analysis of allergen-induced airway hyperresponsiveness and solubilization of IL-13R alpha 2. RESULTS Among several MMPs tested, only MMP-8 cleaved IL-13R alpha 2. Treatment of transfected human or murine cells expressing high levels of surface IL-13R alpha 2 with MMP-8 resulted in release of soluble IL-13R alpha 2 into the supernatants, with a concomitant decrease in surface IL-13R alpha 2 levels. The IL-13R alpha 2 solubilized by MMP-8 retained IL-13 binding activity. In an asthma model MMP-8-deficient mice displayed increased airway hyperresponsiveness and decreased soluble IL-13R alpha 2 protein levels in bronchoalveolar lavage fluid compared with those seen in wild-type mice after house dust mite challenge. CONCLUSION MMP-8 cleaves IL-13R alpha 2 in vitro and contributes to the solubilization of IL-13R alpha 2 in vivo.

IL-13Rα2 Has a Protective Role in a Mouse Model of Cutaneous Inflammation

IL-13 is expressed in lesions of atopic dermatitis (AD) and has been associated with increased disease severity. IL-13 has two cognate receptors: IL-13Rα1 and IL-13Rα2. Although IL-13Rα2 expression is known to be induced in response to IL-13 in keratinocytes, its function in AD has never been evaluated. We characterized the loss of skin barrier function and the development of cutaneous inflammation in IL-13Rα2–null versus wild-type BALB/c mice following an epicutaneous allergen-sensitization/challenge model that shares similarities with human AD. Mice lacking IL-13Rα2 had significantly increased transepidermal water loss, cutaneous inflammation, peripheral eosinophilia, and IgG1 and IgE levels compared with wild-type mice. The rate of resolution of the cutaneous inflammation was not significantly altered in the IL-13Rα2–null mice. IL-13 induced expression of IL-13Rα2 in keratinocyte cell lines and primary human keratinocytes. Depletion of IL-13Rα2 in a keratinocyte cell line resulted in increased STAT6 signaling in response to IL-13. In conclusion, IL-13Rα2 serves a protective role in the pathogenesis of allergic inflammation and loss of skin barrier function in a mouse model of AD, suggesting that it may be an important endogenous regulator of IL-13–induced cutaneous inflammation in humans.

Real-Time Detection of CTL Function Reveals Distinct Patterns of Caspase Activation Mediated by Fas versus Granzyme B

Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B– and Fas-mediated pathways of killing by CTLs; however, the kinetics of caspase activation remain undefined owing to an inability to monitor target cell–specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease-specific activity in target cells. Biosensors were engineered from a circularly permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Coincubation of murine CTLs with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor used. Additionally, the luciferase signal at 30 min correlated with target cell death, as measured by a 51Cr-release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B– compared with Fas-mediated signal induction in murine CTLs; the latter appeared gradually after a 90-min delay in perforin- or granzyme B–deficient CTLs. Remarkably, the Fas-dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTLs was also detectable after a 90-min delay when measured by redirected killing. Thus, we have used a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTLs.