Microtubule assembly is enhanced by the addition of 1 M sucrose or 4 M glycerol to the reassembly mixture. Tubulin can be purified from guinea pig brain readily and in good yield by two cycles of assembly in glycerol-containing solutions. The tubules assembled in glycerol and sucrose are more stable than tubules formed in the absence of these compounds. Assembly occurs in glycerol or sucrose in the absence of ATP or GTP, but is greatly accelarated by their presence.
Dendritic cells (DC) mediate airway Ag presentation and play key roles in asthma and infections. Although DC subsets are known to perform different functions, their occurrence in mouse lungs has not been clearly defined. In this study, three major lung DC populations have been found. Two of them are the myeloid and plasmacytoid DC (PDC) well-characterized in other lymphoid organs. The third and largest DC population is the integrin αE (CD103) β7-positive and I-AhighCD11chigh-DC population. This population was found to reside in the lung mucosa and the vascular wall, express a wide variety of adhesion and costimulation molecules, endocytose avidly, present Ag efficiently, and produce IL-12. Integrin αEβ7+ DC (αE-DC) were distinct from intraepithelial lymphocytes and distinguishable from CD11bhigh myeloid and mPDCA-1+B220+Gr-1+ PDC populations in surface marker phenotype, cellular functions, and tissue localization. Importantly, this epithelial DC population expressed high levels of the Langerhans cell marker Langerin and the tight junction proteins Claudin-1, Claudin-7, and ZO-2. In mice with induced airway hyperresponsiveness and eosinophilia, αE-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that αE-DC is a major and distinct lung DC population and a prime candidate APC with the requisite surface proteins for migrating across the airway epithelia for Ag and pathogen capture, transport, and presentation. They exhibit an activated phenotype in allergen-induced lung inflammation and may play significant roles in asthma pathogenesis.
Objective
The NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and puriner-gic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7/ NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN).
Methods
MRL/lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ ASC/caspase 1 inflammasome pathway, and ratio of Thl7 cells to Treg cells were evaluated. P2X7-targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα).
Results
Significant up-regulation of P2X7/NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1β (IL-1β), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1β and IL-17 and the Thl7:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease.
Conclusion
Activation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1β production and enhanced Thl7 cell polarization. Thus, targeting of the P2X7/NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus.
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