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Binding of different forms of lipopolysaccharide and gene expression in bovine blood neutrophils
- Worku, M., Morris, A.
- Journal of dairy science 2009 v.92 no.7 pp. 3185-3193
- gene expression, neutrophils, cattle, lipopolysaccharides, receptors, tumor necrosis factors, interleukin-8, interleukin-1, transcription (genetics), antigens, macrophages, endotoxins, chemical structure, transcriptional activation
- The objective of this study was to evaluate the effect of smooth (S) and rough (R) forms of lipopolysaccharide (LPS) on gene expression in bovine blood neutrophils. Isolated neutrophils (10⁷ cells/mL) were treated with Escherichia coli LPS serotype O111:B4 (S+R) and R forms (Ra, Rd, or Rc). Flow cytometry was used to assess surface expression of toll-like receptor 4 (TLR-4). Specific primers for IL-8, tumor necrosis factor-α (TNF-α), IL-1β, cluster of differentiation antigen 14 (CD14), and natural resistance associated macrophage protein 1 (Nramp1) were used to assess transcription. Secretion of IL-8, TNF-α, or IL-1β was determined using ELISA kits. Both S and R forms of LPS bound to neutrophils. Exposure induced increased surface expression of TLR-4. No TLR-4 or CD14 mRNA was detected but transcripts for IL-8, TNF-α, IL-1β, and Nramp1 were detected. Secretion of IL-8 and TNF-α but not IL-1β was observed following treatment with R forms of LPS. The longest R form tested (Ra) increased RNA purity and IL-8 and TNF-α secretion in bovine neutrophils. The Rd form increased TLR-4 expression and reduced IL-8 and TNF-α secretion. Exposure to LPS induced increased cell surface expression of TLR-4 and enhanced expression of IL-8 genes. Enhanced TLR-4 activity by LPS was not dependent on transcriptional activation. Recruitment of TLR-4 to the cell membrane may account for increased cell surface expression. A CD14-independent, TLR-4-dependent pathway may be important in the response to different forms of LPS by bovine neutrophils.