Introduction he evolutionary ancient innate immune system rep- resents the irst line of host defence against a number of pathogens. Cells of the natural immune response are able to recognize microbial molecular motifs with high speciicity through a series of genetically and sta- ble conserved cell membrane receptors. In particular, receptors related to the Drosophila toll gene, and there- fore referred to as Toll-like receptors (TLRs), seem to play a central role in the initiation of cellular innate immune responses. hese receptors belong to a mul- tigene family encoding important pattern recognition receptors that have been recognized in a number of invertebrate and vertebrate genomes. (1-4) Among these, Toll-like receptor (TLR)-4 is the receptor required for the signal transduction induced by lipopolysaccharide (LPS), the main cell membrane constituent of Gram- negative bacteria. (3,4) It has long been established that lower vertebrates, including ish and amphibians, exhibit resistance to toxic efects induced by LPS, probably due to the lack of a TLR-4 ortholog. In this context, it has recently been reported that in zebraish TLR-4 does not recognize LPS, negatively regulating the TLR signalling pathway, sup- porting the idea of a divergent functional evolution of this receptor. (5) Recent analyses of chicken genome sequences have revealed the presence of TLRs in this animal although their role has not been well clariied. Moreover, a recent report demonstrated that the expression of TLR-4 in peri- toneal macrophages of passerine birds was found to be positively enhanced after Gram-negative treatment. (6) Several authors agree that many efects induced by LPS are mediated by TLR-4 expressed on the surface mem- brane of both immune and nonimmune cell types. (7,8) he cellular signalling events consequent to LPS stimulation by endotoxin receptor engagement consist Immunopharmacology and Immunotoxicology, 2010; 00(00): 000–000 Address for Correspondence: Maria Antonietta Panaro, Dipartimento di Anatomia Umana e Istologia, Università di Bari, P.zza G. Cesare, Bari, Italy. E-mail: ma.panaro@anatomia.uniba.it RESEARCH ARTICLE First identiication of Toll-like receptor-4 in avian brain: evolution of lipopolysaccharide recognition and inlammation-dependent responses Concetta Saponaro 1 , Antonia Cianciulli 1 , Rosa Calvello 2 , Pasqua Cavallo 1 , Vincenzo Mitolo 1 , and Maria Antonietta Panaro 1 1 Dipartimento di Anatomia Umana e Istologia, Università di Bari, P.zza G. Cesare, Bari, Italy, and 2 Dipartimento Farmaco-Biologico, Università di Bari, Via Orabona, Bari, Italy Abstract In this work, we examine the efects of lipopolysaccharide (LPS) treatment on nerve cells of chick embryo used as a universal avian model. We demonstrate that LPS leads to a dramatic cell loss in primary cultures of both glia and neurons, isolated from chick embryos. Toxic efects appear to be mediated by the Toll-like receptor (TLR)-4 complex, expressed in both glial and neuronal cells, since after TLR-4 silencing by RNA interference experiments LPS-induced cytotoxicity was prevented. The role of nitric oxide in LPS-induced cell damage has also been investigated. These results demonstrate, for the irst time in avian nerve cells, the surface expression of TLR-4 and its role as a pattern recognition receptor involved in LPS-induced cell responses in a similar manner to that observed in mammals. Keywords: Glial cells; nervous cells; lipopolysaccharide; nitric oxide; TLR-4 (Received 16 February 2010; accepted 01 March 2010) ISSN 0892-3973 print/ISSN 1532-2513 online © 2010 Informa UK Ltd DOI: 10.3109/08923971003739244 http://www.informahealthcare.com/ipi 593