TY - JOUR
T1 - A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation
AU - Ida, James A.
AU - Shrestha, Niraj
AU - Desai, Seema
AU - Pahwa, Savita
AU - Hanekom, Willem A.
AU - Haslett, Patrick A.J.
N1 - Funding Information:
This research was funded by a grant from the Dana Foundation Human Immunology Program (PAJH/JAI) and the South Florida VA Research Foundation (PAJH).
PY - 2006/3/20
Y1 - 2006/3/20
N2 - We report a practical technique to assess peripheral blood dendritic cell (DC) maturation and function in whole blood (WB), which requires minimal blood volumes and minimizes ex vivo manipulations of clinical specimens. We determined optimal conditions for flow cytometric analysis of markers of DC maturation, including CCR7, CD25, CD80 and CD83, and of the intracellular cytokines, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α), in both myeloid (mDC) and plasmacytoid (pDC) lineages. We demonstrate concentration-dependent production of these cytokines by DC following short-term stimulation with ligands to Toll-like receptors (TLRs) 2/1, 3, 4, 7, 8 and 9. Kinetic studies revealed maximal TNF-α and IFN-α protein expression at 2 to 3 h after stimulation with certain TLR ligands. Finally, utilizing cells from a cohort of eight healthy donors, we compared DC responses to TLR activation in WB, freshly isolated peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. We found that TNF-α responses were essentially preserved, but IFN-α responses were profoundly diminished or entirely abrogated following cryopreservation. In conclusion, we propose that WB analysis of peripheral DC function is a rapid, reliable and simple method to evaluate TLR function in clinical specimens, which obviates artifact-prone cell purification. The major impact of cryopreservation on some DC responses further strengthens the case for a rapid method that uses fresh blood.
AB - We report a practical technique to assess peripheral blood dendritic cell (DC) maturation and function in whole blood (WB), which requires minimal blood volumes and minimizes ex vivo manipulations of clinical specimens. We determined optimal conditions for flow cytometric analysis of markers of DC maturation, including CCR7, CD25, CD80 and CD83, and of the intracellular cytokines, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α), in both myeloid (mDC) and plasmacytoid (pDC) lineages. We demonstrate concentration-dependent production of these cytokines by DC following short-term stimulation with ligands to Toll-like receptors (TLRs) 2/1, 3, 4, 7, 8 and 9. Kinetic studies revealed maximal TNF-α and IFN-α protein expression at 2 to 3 h after stimulation with certain TLR ligands. Finally, utilizing cells from a cohort of eight healthy donors, we compared DC responses to TLR activation in WB, freshly isolated peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. We found that TNF-α responses were essentially preserved, but IFN-α responses were profoundly diminished or entirely abrogated following cryopreservation. In conclusion, we propose that WB analysis of peripheral DC function is a rapid, reliable and simple method to evaluate TLR function in clinical specimens, which obviates artifact-prone cell purification. The major impact of cryopreservation on some DC responses further strengthens the case for a rapid method that uses fresh blood.
KW - Cytokine
KW - Dendritic cell
KW - Toll-like receptor
KW - Whole blood
UR - http://www.scopus.com/inward/record.url?scp=33644853807&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33644853807&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2005.12.008
DO - 10.1016/j.jim.2005.12.008
M3 - Article
C2 - 16455104
AN - SCOPUS:33644853807
VL - 310
SP - 86
EP - 99
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -