Involvement of phosphatidylinositol-phospholipase C in immune response to Salmonella lipopolysacharide in chicken macrophage cells (HD11)
Introduction
Toll-like receptors (TLRs) are innate immune receptors that recognize structurally conserved pathogen-associated molecular patterns (PAMPs). In mammals, there are at least 11 known TLRs with each member recognizing and responding to different microbial components [1], [2], [3]. For example, TLR4 mediates recognition and immune responses to Gram-negative bacterial cell surface component lipopolysaccharide (LPS). Interaction of microbial agonists with TLRs results in activation of nuclear factors such as NF-κB and AP-1, culminating in transcription of numerous genes involved in innate and adaptive immunities [4]. Several TLRs have been recently identified in chickens through molecular cloning and genome sequencing, including TLR1/6, TLR2, TLR3, TLR4, TLR5, and TLR7 [5], [6], [7], [8], [9]. Functionally, TLR2, 3, 4, 5, 7 and 9 have also been shown to mediate immune responses to various microbial agonists in chicken heterophils, monocytes, and macrophages [10], [11], [12], [13], [14], [15]. TLR signaling pathways are integrated networks involving numerous adaptor proteins and down-stream signaling events. The specificity of TLRs and resulting immune responses often are determined by the participating adaptor protein and its recruited signaling molecules [16].
The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules [17]. Phospholipases function in modulating cellular responses to extracellular stimuli by generating various intracellular lipid signaling molecules through hydrolysis of membrane phospholipids [17], [18]. There are three types of phospholipases: phosphatidylinositol (PI)-phospholipase C (PI-PLC), phosphatidylcholine (PC)-phospholipase C (PC-PLC), and PC-phospholipase D (PC-PLD). PI-PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) and generates signaling molecules inositol-1,4,5-triphosphates (IP3) and diacylglcerol (DAG). IP3 is involved in modulation of intracellular calcium release [19], and DAG is the cofactor of PKC [20]. DAG can also be generated along with phosphocholine (P-Chol) through hydrolysis of choline-containing phospholipids by PC-PLC or formed indirectly by reactions involving PC-PLD and phosphatide phosphohydrolase (PAP) [17]. These intracellular lipid signaling molecules (DAG, IP3, and P-Chol) modulate various cellular responses, including inflammatory responses [21], [22], degranulation [23], [24], phagocytosis [25], respiratory burst [11], [26], [27], [28], nitric oxide (NO) synthesis and cytokine secretion [29], [30], [31], [32], and proliferation [33] in cells through the increase of intracellular [Ca2+] and the activation of PKC, mitogen-activated protein kinases (MAPK), and nuclear factor κB (NF-κB) [29], [34].
LPS, as a major component of the outer membrane of Gram-negative bacteria, is the prime target for recognition by the innate immune system. Recognition of LPS by immune cells involves LPS-binding proteins and TLR4 complex [35]. The LPS-binding protein (LBP) recognizes and binds to the LPS in the circulation and forms a complex with another membrane-bound LPS-binding protein, CD14, where the LPS is then transferred to a LPS receptor complex composed of TLR4 and MD-2. This TLR4/MD-2/CD14 receptor complex transduces the LPS signal through various adaptor proteins that lead to the activation of nuclear factors such as NF-κB and AP-1 and subsequent expression of genes that regulate function in the innate and adaptive immune responses to gram-negative bacterial infection [35].
Avian macrophages, as their counterpart mammalian macrophages, belong to the mononuclear phagocytic system and are originated from bone marrow, entering the blood circulation as monocytes and subsequently differentiating into macrophages upon migration to various tissues. The avian macrophages also perform similar immunological functions as mammalian macrophages, serving as the first line of immune defense as an important cellular component in both innate and adaptive immunity [36], [37]. Their innate immune functions include phagocytosis of infectious agents, generation of microbiocidal reactive oxygen radicals and NO, recognition of PAMPs via innate immune receptors like TLRs, and secretion of pro-inflammatory cytokines and chemokines. Avian macrophages may also involve initiation of adaptive immunity through antigen processing and presentation and expression of costimulatory factors [36]. HD11 is an avian myelocytomatosis virus (MC29) transformed chicken macrophage-like cell line [38]. Previous studies have shown that LPS was able to induce a significant level of NO production in HD11 cells [39], [40], [41]. This LPS-induced NO production in chicken macrophages was shown to be a TLR4/CD14 mediated event [42].
However, it was not clear whether phospholipases play a role in the activation of chicken macrophages by LPS. In this study, selective pharmacological inhibitors were used to identify the role of specific phospholipase activities in TLR4 mediated LPS signaling in chicken macrophages cell HD11 by monitoring the NO induction.
Section snippets
Reagents
PI-PLC inhibitors (U-73122 and ET-18-OCH3), PC-PLC inhibitor (D609), PAP inhibitor (propranolol), and PKC inhibitors (Ro 31-8220 and calphostin C) were obtained from Biomol (Plymouth Meeting, PA, USA). LPS (from Salmonella enteritidis), the selective chelator for intracellular Ca2+ (BAPTA-AM), PC-PLD inhibitor (n-butanol) and all media and additives for cell culture were purchased from Sigma (St. Louis, MO).
Cell culture and stimulation
The HD11, an avian macrophage cell line, was maintained in Dulbecco's Modified Eagles
Induction of NO synthesis in HD11 by LPS from S. enteritidis
TLR4 mediates immune recognition of gram-negative bacteria and associated cell-wall components, such as LPS. Exposure to LPS resulted in activation of HD11 cells and consequential NO production, indicating the involvement of chicken TLR4 in inflammatory response to microbial infections. Stimulation of HD11 cells with LPS led to a dose-dependent induction of NO (Fig. 1). NO synthesis in HD11 cells induced by LPS reached the maximal level when a LPS concentration of 0.1 μg/ml was used, and
Discussion
To evaluate the specific role of phospholipases in LPS stimulated-NO production in chicken macrophages, several selective pharmacological inhibitors (U-73122, ET-18-OCH3, D609, n-butanol, and propranolol) were used to study their inhibitory effects on LPS-induced NO production. In mammalian cells, both U73122, a membrane-permeable aminosteroid, and ET-18-OCH3, an ether lipid analog, selectively inhibit the PI-PLC activity and have been proven useful to evaluate the role of PI-PLC in cell
Acknowledgements
Authors wish to thank Laura Ripley for assistance in cell culture and Drs. Ken Hasson and Loyd Sneed for critical review of the manuscript. Mention of commercial or proprietary products in this paper does not constitute an endorsement of these products by the USDA, nor does it imply the recommendation of products by the USDA to the exclusion of similar products.
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