Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50∼70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.

Transforming growth factor (TGF)-β has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-β contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-β/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-β in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-β/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-β was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-β/Smad signaling in mature T cells as a regulatory component of allergic asthma.

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) transmits anti-apoptotic signals in eosinophils and is involved in tissue eosinophilia at the site of allergic inflammation. We determined whether phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAP kinase) are involved in anti-apoptotic signals of GM-CSF in eosinophils. GM-CSF phosphorylated Akt, a downstream component of PI 3-kinase, and MAP kinases (ERK1 and ERK2) at 10 min after stimulation in eosinophils. GM-CSF prevented eosinophil apoptosis and sustained its survival during the 5-day culture. However, neither two PI-3 kinase inhibitors, wortmannin and LY294002, nor MEK inhibitor PD98059 inhibited GM-CSF-induced survival of eosinophils, although wortmannin and PD98059 inhibited GM-CSF-induced Akt phosphorylation and MAP kinase activation in eosinophils, respectively. In contrast, JAK2 inhibitor AG-490 inhibited both GM-CSF-induced JAK2 phosphorylation and cell survival in eosinophils. These results indicate that activation of JAK2, but not activation of PI 3-kinase/Akt and MAP kinase pathways, is critical for anti-apoptotic signals of GM-CSF in human eosinophils. Our findings suggest that manipulation of JAK2 activation would be useful for the treatment of allergic disorders. J. Leukoc. Biol. 65: 700-706; 1999.