Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE, which is not blocked by ACE inhibitors. High amounts of ACE2 are present in the proximal tubule, and ACE2 catalyzes generation of angiotensin 1-7 (Ang-(1-7)) by this segment. Ang-(1-7) binds to a receptor distinct from the AT1 or AT2 Ang II receptor, identified as the mas receptor. We studied the effects of Ang-(1-7) on Ang II-mediated cell signaling pathways in proximal tubule. In primary cultures of rat proximal tubular cells, activation of mitogen-activated protein kinases (MAPK) was detected by immunoblotting, in the presence or absence of agonists/antagonists. Transforming growth factor-beta1 (TGF-beta1) was measured by enzyme-linked immunosorbent assay. Ang II (5 min, 10(-7) M) stimulated phosphorylation of the three MAPK (p38, extracellular signal-related kinase (ERK 1/2), and c-Jun N-terminal kinase (JNK)). While incubation of proximal tubular cells with Ang-(1-7) alone did not significantly affect MAPK phosphorylation, Ang-(1-7) (10(-7) M) completely inhibited Ang II-stimulated phosphorylation of p38, ERK 1/2, and JNK. This inhibitory effect was reversed by the Ang-(1-7) receptor antagonist, D-Ala7-Ang-(1-7). Ang II significantly increased production of TGF-beta1 in proximal tubular cells, an effect that was partly inhibited by Ang-(1-7). Ang-(1-7) had no significant effect on cyclic 3',5'-adenosine monophosphate production in these cells. In summary, Ang-(1-7) inhibits Ang II-stimulated MAPK phosphorylation in proximal tubular cells. Generation of Ang-(1-7) by proximal tubular ACE2 could thereby serve a protective role by counteracting the effects of locally generated Ang II.
Background Allergic diseases affect large population. Pollen, a ubiquitous allergen, is the trigger of seasonal rhinitis, conjunctivitis and asthma, as well as an exacerbating factor of atopic dermatitis. However, the underlying mechanism by which pollen induces thymic stromal lymphopoietin (TSLP) triggered allergic inflammation via epithelial innate immunity is largely unknown. Objective To explore whether short ragweed (SRW) pollen induces TSLP/OX40L/OX40 signaling via Toll-like receptor (TLR) 4-dependent pathways in allergic disease. Methods Three models were used for this study, a well-characterized murine model of allergic conjunctivitis induced by SRW pollen, a topical challenge model on mouse ocular surface, and a culture model of primary human corneal epithelium exposed to aqueous extract of defatted SRW pollen (SRWe). Results The topical challenges with SRW pollen generated a typical allergic conjunctivitis in BALB/c mice. Clinical signs, stimulated TSLP/OX40L/OX40 signaling and Th2 cytokines in ocular mucosa and draining cervical lymph nodes were significantly reduced or eliminated in TLR4 deficient (Tlr4-d) or MyD88 knockout (MyD88−/−) mice, compared with their wild type littermates. SRWe stimulated TSLP production by ocular epithelia in wild type, but not Tlr4-d or MyD88−/− mice. SRWe stimulated TSLP was blocked by TLR4 antibody and NF-kB inhibitor in mouse and human corneal epithelia. Conclusion We for the first time uncovered a novel phenomenon that SRW pollen, acting as a functional TLR4 agonist, initiates TLR4-dependent TSLP/OX40L/OX40 signaling that triggers Th2-dominant allergic inflammation. These findings shed light on the understanding of mucosal epithelial innate immunity, and create new therapeutic targets to cure allergic diseases.
Evidence suggests that the cytokine interferon (IFN)-γ released by natural killer and CD4+ T cells contributes to the conjunctival goblet cell (GC) loss in dry eye. The purpose of this study was to investigate if topical neutralization of IFN-γ prevents or alleviates GC loss in an experimental desiccating stress (DS) model of dry eye. In this study, we found that topical IFN-γ neutralization significantly decreased DS-induced conjunctival GC loss. This was accompanied by decreased epithelial apoptosis, and increased IL-13 and decreased FoxA2 expression in the forniceal conjunctiva. To establish that IFN-γ produced by pathogenic CD4+ T cells contributes to DS-induced GC loss, adoptive transfer of CD4+ T cells isolated from DS exposed donors to naïve RAG-1−/− recipient mice was performed. Similar to the donor mice, topical IFN-γ neutralization decreased conjunctival GC loss, suppressed apoptosis and increased IL-13 expression in adoptive transfer recipients. In summary, this study demonstrated that topical neutralization of IFN-γ prevents GC loss via modulating apoptosis and maintaining IL-13 signaling.
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