Submitted to: Veterinary Immunology International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: 3/15/2004
Publication Date: 4/8/2004
Citation: Bliss, T.W., Emara, M.G., Dohms, J.E., Lillehoj, H.S., Keeler, C.L. 2004. Gene expression patterns in differentially stimulated avian macrophages.. Veterinary Immunology International Symposium, p. 157.
Technical Abstract: Macrophages are important regulators of the innate immune system which are able to differentially respond to various stimuli. We have identified genes expressed by avian macrophage cells derived from the adherent fraction of peripheral blood leukocytes under normal conditions and conditions simulating a bacterial (LPS-induced) and a viral (INF-g induced) infection through the creation and sequencing of expressed sequence tag (EST) libraries. A total of 11,904 clones were sequenced (1,920 from the control library and 4,992 from each of the stimulated libraries). Removal of short, low-quality, and vector sequences left 8,732 total high-quality sequences which were used for later analysis. Individual clustering of these libraries resulted in the identification of 944, 1,875, and 2,014 unique elements in the control, INF-g stimulated, and LPS stimulated libraries, respectively. A combined clustering of all of the unique elements from these three libraries gave 4,152 unique elements. This number includes 489 combined library contigs, 688 elements unique to the control library, 1,406 elements unique to the INF-g stimulated library, and 1,574 elements unique to the LPS library, clearly illustrating differential gene expression patterns according to the type of stimuli used for macrophage activation. Furthermore, this clustering revealed a set of 122 genes that is constitutively expressed regardless of treatment and a core set of 241 genes which are induced following either stimulus. This latter set represents immune genes which are involved in nearly all immune response, including many chemokines and cytokines. BLAST analysis of these libraries against the TIGR Gallus gallus Gene Index and the GenBank non-redundant nucleotide database revealed that these libraries contain mostly ESTs which had previously been identified in chicken (91%). The remaining 9% represented novel chicken ESTs. In silico analysis of the clones represented in these libraries showed that the two stimuli caused differential gene expression in several gene classes. The bacterial (LPS) stimulus significantly induced interleukins and interleukin receptors, including IL-1B, IL-6, IL-8, and the IL-1 receptor, while the INF-g stimulation caused significant up-regulation of many INF related genes, such as the INF-a, B, and g receptors, INF regulatory factors-1 and '2, as well as several other genes including inducible nitric oxide synthase (iNOS). In addition, each stimulus caused specific down-regulation of a unique set of genes. Expression levels of some of the differentially stimulated gene products were confirmed by real-time quantitative RT-PCR, which correlated with the expression patterns observed in silico.