Location: Livestock Issues Research
Title: Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures Authors
|Ballou, Michael -|
Submitted to: Journal of Animal Science Supplement
Publication Type: Abstract Only
Publication Acceptance Date: July 11, 2010
Publication Date: October 11, 2010
Citation: Hulbert, L.E., Carroll, J.A., Ballou, M. 2010. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures [abstract]. 2010 American Society of Animal Science Meeting, July 11-15, 2010, Denver, CO. Journal of Animal Science. 88(E-Supplement 2):T156. Technical Abstract: Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and whole blood (WB) when stimulated with lipopolysaccharide (LPS from E. coli O111:B4; 1 and 10 microg/mL for WB; 0.01 and 1 microg/mL for PBMC). Thirty-six dairy heifers (12-16 mo. age) free from any signs of disease were analyzed in the study. The PBMCs were isolated using a percoll gradient, washed twice with PBS, counted using a hemacytometer, then resuspended to 2 x 10 exp 6 cells/mL in a cell culture with RPMI medium with 1% antibiotics, 10% autologous plasma and 5 ng/mL of recombinant bovine interferon-gamma. In the WB assay, 200 microliters of whole blood was added to 800 microliters of RPMI with 1% antibiotics. Samples were incubated with their respective LPS doses for 24-h before supernatants were collected and analyzed for TNF-alpha using a commercially available sandwich ELISA. Mean TNF-alpha concentrations from PBMC and WB were moderately correlated (R squared = 0.40). There were strong correlations between the low and high doses of LPS within each assay (R squared = 0.67 and 0.87, for isolated PBMC and WB, respectively). The WB data were not correlated with either the number of leukocytes or the percentages of neutrophils (R squared = 0.15). Inter-heifer coefficients of variation (CV) for the PBMC and WB data were 39.5% and 57.6%, respectively. In another experiment, using samples from 12 Holstein heifers from 3 consecutive days determined that the intra-heifer CVs for PBMC and WB data were 25.33% and 26.51%, respectively. These data indicate the WB assay may serve as a simple but effective ex vivo assay for evaluating bovine pro-inflammatory cytokine synthesis and secretion potential. Additionally, these data elucidate a large population variation, but if a heifer has a reduced response relative to the population at lower LPS concentrations then she will have a reduced response at a higher LPS concentrations, and vice versa.