QUANTIFICATION OF SUPERANTIGEN INDUCED IFN-GAMMA PRODUCTION BY COMPUTERIZED IMAGE-ANALYSIS - INHIBITION OF CYTOKINE PRODUCTION AND BLAST-TRANSFORMATION BY POOLED HUMAN-IGG

A quantitative image analysis technique was developed to assess the cy tokine content of immunocytochemically stained cytokine producing cell s. Peripheral blood mononuclear cells were stimulated to induce cytoki ne production with the superantigen streptococcal pyrogenic exotoxin-A . We have developed a method based on indirect immunocytochemistry whi ch identifies IFN-gamma producing cells by a characteristic morphology generated by the accumulation of IFN-gamma in the Golgi organelle. An image analysis technique permitted discrimination between these produ cer cells and IFN-gamma binding target cells, which showed a different appearance, with staining restricted to the cell surface membrane. A semi-automated routine programme allowed the signal from a video camer a to be processed by computerised image analysis methodology. This ena bled us to measure the number of cytokine producing cells, the cytokin e staining intensity in individual cells and the cell size expressed i n actual cell area. The incidence of IFN-gamma producing cells determi ned by image analysis measurement was compared to results obtained usi ng manual microscopy. Cell size was assessed by the image analysis sys tem as well as by now cytometry. Administration of pooled human IgG fo r intravenous use (IVIg) to the superantigen stimulated cells signific antly down-regulated IFN-gamma production, both in terms of the number s of producer cells and in terms of cytokine stainingintensity in indi vidual cells. In addition blast transformation of cells was substantia lly reduced. These effects, mediated by IVIg, were also evident follow ing delayed IVIg administration 24 h after the initial cell stimulatio n.