Matrix metalloproteinase 8 contributes to solubilization of IL-13 receptor alpha2 in vivo

Abstract

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.

FIG 1

Levels of soluble IL-13Rα2 in serum and BALF and the expression of MMPs in lungs from PBS- or HDM-treated FVB/N mice. A and B, Soluble IL-13Rα2 in serum and BALF. Data were shown as means ± SD (n = 4–5). *P < .05. The result is representative of 3 experiments. C, RNase protection assay of the RNA from the lungs of PBS- or HDM-treated mice (n = 4). TIMP, Tissue inhibitor of metalloproteinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 2

Acellular cleavage of GST–IL-13Rα2–FLAG fusion protein by MMPs. A, Cleavage of GST–mIL-13Rα2–FLAG. B and C, Cleavage of GST or GST–mIL-13Rα2–FLAG by MMP-8. D and E, Cleavage of GST–mIL-13Rα2–FLAG or human collagen type I by MMP-8 in the presence or absence of an MMP-8 inhibitor or mock MMP-8 inhibitor control (5, 50, or 500 nmol/L). F, Cleavage of GST–hIL-13Rα2–FLAG.

FIG 3

Measurement of soluble IL-13Rα2 in the culture medium and on the cell surface after MMP-8 treatment. Soluble IL-13Rα2 levels are shown in panels A and B. IL-13Rα2 surface expression is shown as mean channel fluorescence (MCF) in panels C and D. Data are shown as means ± SD (n = 3–5). *P < .05. The result is representative of 3 experiments.

FIG 4

IL-13 binding of soluble IL-13Rα2 in culture medium from MMP-8–treated cells. The result is representative of 3 experiments. A, MMP-8 treatment of untransfected or mIL-13Rα2–transfected WEHI cells. The data are shown as means ± SD (n = 5). *P < .05. B, Western blot analysis of MMP-8 treatment of murine GST–IL-13Rα2–FLAG fusion protein for the indicated time (0.5–24 hours). U, Untreated.

FIG 5

AHR, airway inflammation, BALF soluble IL-13Rα2 and its IL-13 binding activity, and BALF IL-13 of PBS- or HDM-treated wild-type and MMP-8–deficient mice. A, APTI (n = 6–8). B, BAL cell counting (n = 6–8). C, Soluble IL-13Rα2 (n = 6–8). D, IL-13 binding activity of soluble IL-13Rα2 (n = 12–17, pooled from 2 experiments). E, Free and IL-13Rα2–bound IL-13 (n = 5). WT, Wild-type mice; KO, MMP-8–deficient mice. The data are shown as means ± SD. *P < .05.