COMPUTERIZED ASSESSMENT OF PRODUCTION OF MULTIPLE HUMAN CYTOKINES AT THE SINGLE-CELL LEVEL USING IMAGE-ANALYSIS

A technique using a computerized image analysis system was developed f or evaluating and quantifying human cytokine production, This system r egistered single cells as positive or negative cytokine producers base d on a specific juxtanuclear staining pattern generated by accumulatio n of the proteins in the Golgi-endoplasmatic reticulum compartment. Th e characteristic morphology of the immunocytochemical staining offered the opportunity to register individual producer cells within multicom ponent cell populations. A color camera was then adapted to transfer o n-line images directly into the computer-controlled operating system, In this study cultured human peripheral blood mononuclear cells were p olyclonally stimulated and then analyzed for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necro sis factor-alpha (TNF-alpha), TNF-beta, interferon-gamma, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulatin g factor production. The image-analyzing system detected cytokine-prod ucing cells in a sensitive and reproducible manner, which was in total congruence with enumeration by conventional microscopy. Furthermore, accurate assessments of cell distributions by signal intensity and cel l area were applied at the single-cell level. The image-analyzing syst em allowed the detection of at least 1 in 1,000 events by using unique cytokine-associated morphometric criteria, The results of kinetic stu dies measuring cytokine production following activation and cell trans formation provided data supporting increases in intensity of intracell ular localized specific immunostaining and in cell size within the cyt okine-producing cells.