It has been demonstrated that allergens in the presence of
endotoxins trigger a substantially stronger allergic inflammation, compared with that evoked in the absence of endotoxins.[5-7] After inhalation, endotoxins, such as lipopolysaccharide (LPS), encounter and activate alveolar macrophages, leading to the production and release of pro-inflammatory cytokines, chemokines, adhesion molecules and other mediators. Nasal and lung lavage samples of allergic subjects show increased levels of interleukin-1β (IL-1β), primarily produced by activated macrophages. Production buy Y-27632 of mature IL-1β requires distinct signals, some of which induce gene expression in the so called ‘priming step’, whereas other signals trigger the maturation of pro-IL-1β to IL-1β by a multiprotein complex called inflammasome. The NLRP3 inflammasome complex consists of NLRP3 (NOD-like receptor family pyrin domain-containing 3) sensor, caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) adaptor.[11, 12] NLRP3 inflammasomes play a crucial role in the detection and sensing of exogenous danger signals like pathogen-associated molecular patterns and toxins of microbes, asbestos or silica, as well as endogenous danger signals like monosodium urate and amyloid.[13, 14] Most NLRP3 activators have been shown to induce ROS B-Raf inhibitor drug generation,
and Acyl CoA dehydrogenase inhibitors of ROS production or ROS scavengers attenuate NLRP3 inflammasome activation implying an essential role for ROS in NLRP3 function. As pollen NADPH oxidases are able to generate ROS, and ROS have been implicated in the NLRP3 inflammasome-mediated IL-1β production, we hypothesized that exposure to pollen extract may influence inflammatory responses and IL-1β production of macrophages via NLRP3 inflammasome. Here we report for the first time that ragweed
pollen extract (RWE), typically used as a model for pollen action, significantly elevates LPS-induced IL-1β production of THP-1 or primary macrophages and dendritic cells in an NADPH-dependent manner. We also demonstrate that a caspase-1 inhibitor or NLRP3 silencing abolish this enhancing effect together with the original LPS-triggered inductions. We also show that RWE in the presence of NADPH enhances LPS-induced p38 and Jun N-terminal kinase (JNK) signalling pathways resulting in the activation of AP-1 transcription factors and the subsequent gene transcription/expression of pro-IL-1β and key components of the inflammasome. This effect is mediated by a ROS-dependent mechanism. The THP-1 cell line (ATCC TIB-202) was a generous gift from Professor Laszlo Nagy. THP-1 monocytes were cultured in RPMI-1640 (Gibco BRL Inc., Grand Island, NY) containing 10% heat-inactivated fetal calf serum, penicillin-streptomycin and glutamine, and maintained at 37° under 5% CO2.