In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to naïve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.

The globins and peroxidases, while performing completely different chemistry, share features of the iron heme active site: a protoporphyrin IX prosthetic group is linked to the protein by the proximal histidine residue. X-ray absorption spectroscopy provides a method to determine the local structure of iron heme active sites in proteins. Our previous studies using X-ray absorption spectroscopy revealed a significant difference in the Fe-N epsilon bond length between the peroxidases and the globins [for a review, see Powers, L. (1994) Molecular Electronics and Molecular Electronic Devices, Vol. 3, p 211 CRC Press Inc., Boca Raton, FL]. Globins typically have an Fe-N epsilon distance close to 2.1 A while the Fe-N epsilon distance in the peroxidases is closer to 1.9 A. We have proposed [Sinclair, R., Powers, L., Bumpus, J., Albo, A., & Brock, B. (1992) Biochemistry 31, 4892] that strong hydrogen bonding to the proximal histidine is responsible for the shorter bond length in the peroxidases. Here we use site-specific mutagenesis to eliminate the strong proximal hydrogen bonding in cytochrome c peroxidase and to introduce strong proximal hydrogen bonding in myoglobin. Consistent with our hypothesis, elimination of the Asp235-His175 hydrogen bond in CcP results in elongation of Fe-N epsilon from approximately 1.9 to approximately 2.1 A. Conversely, introduction of a similar strong proximal hydrogen bond in myoglobin shortens Fe-N epsilon from approximately 2.1 to approximately 1.9 A. These results correlate well with other biochemical data.