Growth hormone secretion by human peripheral blood mononuclear cells detected by an enzyme-linked immunoplaque assay.
In order to evaluate the secretion of GH from human peripheral blood mononuclear cells (PBMC) and its possible role as a modulator of lymphoproliferation, we have developed a hormonal enzyme-linked immunoplaque assay. This assay captures GH between a monoclonal and polyclonal antibody. This is followed by adding substrate and a horseradish peroxidase-conjugated antibody against the polyclonal antibody which produces violet colored plaques where GH has been secreted. This assay is sensitive, specific, highly reproducible, and can detect picogram quantities of GH. Using this assay we have detected GH secretion from approximately 1% of human PBMC under unstimulated conditions. Regression analysis showed a linear relationship between the number of cells plated and the number of GH plaques formed. Therefore, GH plaques were formed by single cells or its progeny and did not represent aggregation of secreting cells. Preincubation of PBMC with cycloheximide, a protein synthesis inhibitor, completely abolished the formation of GH plaques which suggests that the PBMC were responsible for the synthesis of the secreted GH. In addition, we have also observed that stimulation of human PBMC with T-cell mitogens, Concanavalin A and PHA and the cytokine IL-2 led to significant increases in GH plaque area and number whereas stimulation with LPS, a B cell mitogen, was ineffective. The PHA and IL-2 induced increase in plaque number suggests that they can recruit noncommitted lymphocytes to actively secrete GH which raises the possibility that this secreted GH may serve as a growth factor in T cell proliferation. We conclude that this immunoplaque assay may be useful in evaluating the secretion of other peptides from human immune cells.
Varma
S
S
Department of Internal Medicine, Ohio State University Medical Center, Columbus 43210.
Sabharwal
P
P
Sheridan
J F
JF
Malarkey
W B
WB
D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D013006:Growth Hormone / Q000097:blood* / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008934:Mitogens / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D012044:Regression Analysis; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D007376:Interleukin-2; D008070:Lipopolysaccharides; D008934:Mitogens; D011994:Recombinant Proteins; D013006:Growth Hormone; D003513:Cycloheximide