Measurement of immunoreactive interleukin-1 beta from human mononuclear cells: optimization of recovery, intrasubject consistency, and comparison with interleukin-1 alpha and tumor necrosis factor.
Numerous studies have reported altered levels of in vitro production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) from blood leukocytes in various human disease states. Most of these studies have used bioassays which are vulnerable to inhibitors produced by these cells. Furthermore in vitro cytokine production is often assessed on a single occasion. The present study was designed to standardize stimulation conditions for in vitro IL-1 beta production and to employ a competitive radioimmunoassay (RIA) to demonstrate reproducibility and long-term variation of in vitro cytokine production in a cohort of healthy human subjects. We also examined relative amounts of immunoreactive IL-1 beta, IL-1 alpha, and TNF induced by the stimuli endotoxin, phytohemagglutinin, or Staphylococcus epidermidis. We show that the RIA can reliably detect IL-1 beta produced from mononuclear cells in concentrations as low as 115 pg/ml. Lysing cells by repeated freeze-thawing yields maximal recovery of total (i.e., secreted plus cell-associated) immunoreactive IL-1 beta, when compared to extraction with the detergent CHAPS or addition of protease inhibitors. Repeated measurement of in vitro cytokine production on different days within 1 week shows good reproducibility for a given individual and a given stimulus (variation coefficient 20 to 30%). Over a long time period (6 months) in vitro cytokine production is stable in some individuals but changes considerably in others. The soluble stimulus endotoxin induces twofold more IL-1 alpha than IL-1 beta or TNF; in contrast the phagocytic stimulus heat-killed S. epidermidis induces fourfold more IL-1 beta and TNF than IL-1 alpha. This distinct pattern of cytokine response indicates differential stimulation of the mononuclear cells by different stimuli. The results form the basis for studying in vitro cytokine production in different human disease states.
Endres
S
S
Department of Medicine, New England Medical Center Hospitals, Boston, Massachusetts.
Ghorbani
R
R
Lonnemann
G
G
van der Meer
J W
JW
Dinarello
C A
CA
D000818:Animals; D007611:Aprotinin; D005615:Freezing; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000592:standards; D007963:Leukocytes, Mononuclear / Q000032:analysis* / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride; D011817:Rabbits; D012016:Reference Values; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000302:isolation & purification / Q000592:standards
D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D010664:Phenylmethylsulfonyl Fluoride; D007611:Aprotinin