IL-12 elispot assays to detect and enumerate IL-12 secreting cells.
The cytokine IL-12 promotes Th(1)type immune responses and plays a key role in immune regulation. The complex nature of IL-12 hampered its detection without use of stimulants that might give less relevant information. To detect circulating IL-12 p40, we developed enzyme-linked immunospot (ELISPOT) assays that allow enumeration of IL-12 p40 secreting cells without prior in vitro stimulation of the cells. In parallel, intracellular staining of IL-12 p40 by flow cytometry was performed to compare the two methods. IL-12 p40 secreting cells were detected in healthy subjects at a mean number of 103+/-155 per 10(5)blood mononuclear cells (MNC). Numbers of IL-12 p40 secreting blood MNC correlated with IL-12 p40 positive blood MNC detected by flow cytometry. Bacterial endotoxins and the inflammatory cytokines TNF-alpha and IFN-gamma control IL-12 production by antigen presenting cells. Utilizing IL-12 p40 ELISPOT assays, we could confirm occurrence of elevated numbers of IL-12 p40 secreting blood MNC after stimulation with TNF-alpha, IFN-gamma, LPS, LPS+TNF-alpha or LPS+IFN-gamma, compared to cultures without stimulant. Due to its central role in inflammation and autoimmunity, IL-12 is an attractive target for immunotherapy. IL-12 p40 ELISPOT assays represent a sensitive, specific and reliable tool for investigating the role of IL-12 in both health and disease.
Ozenci
V
V
Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. Volkan.Ozenci@neurotec.ki.se
Kouwenhoven
M
M
Press
R
R
Link
H
H
Huang
Y M
YM
D000328:Adult; D000368:Aged; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005069:Evaluation Studies as Topic; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D007371:Interferon-gamma