4, in the co-culture of paraformaldehyde-fixed basophils and unfixed bronchial epithelial cells, the induction of IL-6, CXCL8 and -defensin 2 with or without MDP stimulation was preserved. and epithelium-derived anti-microbial peptide -defensin 2 in the co-culture. HBE cells were the major SPHINX31 source for the release of IL-6, CXCL8 and -defensin2 upon stimulation by MDP in the co-culture system. The expression of ICAM-1 and VCAM-1 and release of IL-6 and CXCL8 were suppressed by various signalling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be regulated differentially by the mitogen-activated protein kinase pathways and nuclear transcription factors. The results therefore provide a new insight into the functional role of basophils in innate immunity, and the link between respiratory bacteria-mediated innate immunity and subsequent amplification of allergic inflammation in the airway. can induce subsequent allergic inflammation upon the activation by SPHINX31 inhaled allergens through innate immunity-related SPHINX31 dendritic cells [1]. The innate immune system can recognize bacteria through pattern recognition receptors (PRRs), which detect conserved microbial components called pathogen-associated molecular patterns. One of the key intracytosolic PRRs in inflammatory and immune responses is nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs). Two well-characterized NLRs, NOD1 and NOD2, sense the cytosolic presence of the peptidoglycan fragments -D-glutamyl-meso-diaminopimelic acid (iE-DAP) in all Gram-negative and certain Gram-positive bacteria, and peptidoglycan constituent muramyl dipeptide (MDP) in almost all bacteria, respectively [2,3]. The crucial roles of NOD1,2 in immune responses have been proved from the linkage of NOD1,2 polymorphisms with atopic dermatitis, atopic eczema and allergic asthma [4,5]. In fact, NOD1-mediated recognition of bacterial products in the skin or at mucosal interfaces may regulate T helper type 2 (Th2) polarization and immunoglobulin (Ig)E concentrations [4]. NOD2 expression links to innate immune responses against bacterial pathogens [6] and [7]. NOD2 mutations are associated with chronic inflammatory bowel diseases, such as Crohn’s disease [8]. Animal studies have shown that the NOD2 ligand induces the expression of allergic inflammation-related cytokine thymic stromal lymphopoietin, interleukin (IL)-25 and Rabbit Polyclonal to SFRS7 OX40 ligand (OX40L) in lung, resulting in subsequent susceptibility to asthmatic lung inflammation [9]. A recent study showed that NOD2 ligand MDP could induce the secretion of CXCL8, regulate expression of CD11b and CD69 and enhance migration of eosinophils, which were mediated via the nuclear factor (NF)-B signalling pathway [10]. Basophils are rare, circulating granulocytes, representing <1% of the peripheral blood leucocytes. Circulating basophils can be recruited to the inflammatory tissues in allergic disorders including allergic asthma, atopic dermatitis and allergic rhinitis [11C13]. During asthma exacerbation and in response to allergen inhalation challenge, basophils infiltrate markedly into allergic inflammatory sites [14C16]. Studies investigating allergic SPHINX31 late-phase responses have shown that basophils are a significant source of Th2 cytokines IL-4 and IL-13, which are central cytokines for the manifestations of allergic disease [17C19]. Our previous study has shown that the T helper 17 (Th17) cytokine IL-17A cytokine can induce the release of inflammatory cytokines and chemokines upon the interaction of human basophils and bronchial epithelial cells through the differential activation of mitogen-activated protein kinase (MAPK) and NF-B signalling pathways [20]. In our study of the link between infections and the exacerbation of allergic inflammation, we have demonstrated that the induction of cytokines, chemokines, superoxides and eosinophilic cationic protein (ECP), and cell migration by Toll-like receptor (TLR)-2, -5 and -7 ligands, are regulated differentially by intracellular focal adhesion kinase (FAK), NF-B, extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)-Akt and p38 MAPK in eosinophils [21,22]. However, the NLR-mediated activation of basophils in allergic inflammation has not yet been studied. Therefore, we hypothesized that NLR are crucial PRRs in the process of airway inflammation, by linking the innate immune response against respiratory bacterial infection with the subsequent allergic inflammation through the SPHINX31 activation of basophils interacting with bronchial epithelial cells. Materials and methods Reagents The MDP, MDP-negative control D-D isomer, iE-DAP and iE-DAP-negative control -D-Glu-Lys (iE-Lys) were purchased from InvivoGen (San Diego, CA, USA). Recombinant human IL-33 was purchased from R&D Systems (Minneapolis, MN, USA). IB phosphorylation inhibitor BAY11-7082, p38 MAPK inhibitor SB203580 and c-Jun N-terminal protein kinase (JNK) inhibitor SP600125 were purchased from Calbiochem Corporation (San Diego, CA, USA). SB203580 was dissolved.