Targeted suppression of cytokine production in monocytes but not in T lymphocytes by a tetravalent guanylhydrazone (CNI-1493).

Image analysis was used to study the cytokine-inhibitory effect of the nitric oxide inhibitor tetravalent guanylhydrazone (CNI-1493) in individual immunocytochemically stained human peripheral blood mononuclear cells (PBMC). CNI-1493 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta, IL-6, and IL-8 production whether or not LPS stimulation was enhanced by interferon (IFN)-gamma priming. Addition of TNF-alpha to CNI-1493-exposed LPS-stimulated cells partially restored the incidence of IL-1alpha-, IL-1beta-, and IL-8-producing cells. TNF-alpha production induced by costimulation by ligation of CD3 and CD28 was inhibited by CNI-1493 in monocytes but not in T lymphocytes. The prevalence of IL-2-, IFN-gamma-, and TNF-beta-producing T cells was not reduced by CNI-1493. Phorbol ester and ionomycin activation also resulted in a CNI-1493 -induced inhibition of TNF-alpha in monocytes but resistant production of TNF-alpha, IL-2, and IFN-gamma by T cells. Thus, CNI-1493 preferentially inhibited synthesis of proinflammatory cytokines in monocytes.

Sendai virus-induced IFN-alpha production analysed by immunocytochemistry and computerized image analysis.

IFN-alpha production in Sendai virus-stimulated human buffy coat cultures could readily be demonstrated in individual cells at a protein level by the use of a novel immuno-enzymatic staining procedure. A distinctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN-alpha in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colour camera, which then transfers binary images directly into a computer-controlled operating system. The characteristic appearance of the immunocytochemical staining enabled a computerized image-analysis system to measure IFN-alpha producing cells based on defined criteria set for morphology, intensity, colour and size. The automated system could accurately and reproducibly register a range of 0.1-7.0% of the total cell population as IFN-alpha producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscopy and image analysis concerning the assessment of the incidence of IFN-alpha producing cells in the total cell populations. All IFN-alpha producing cells expressed surface HLA-DR molecules and 95% of these cells belonged to the myelomonocytic lineage. The image analysis system provided, in contrast to conventional microscopy, an opportunity to assess and document differences of signal intensity and cell size of individual IFN-alpha producing as well as non-producing cells.