Ex vivo simulation of leukocyte function: stimulation of specific subset of leukocytes in whole blood followed by the measurement of function-associated mRNAs.
In order to characterize a wide spectrum of leukocyte functions with clinically applicable procedures, 0.06 ml each of heparinized whole blood was stimulated in triplicate for 4h with phytohemagglutinin (T cell stimulator), heat aggregated IgG (IgG Fc receptor stimulator), lipopolysaccharide (toll-like receptor (TLR)-4 stimulator), zymosan (TLR-2 stimulator), monoclonal antibody against T-cell receptor alpha/beta chain, recombinant interleukin-2, and solvent controls, then 32 different leukocyte function-associated mRNAs were quantified by the method reported previously (Mitsuhashi et al. Clin. Chem. 2006). Two control genes (beta-actin, beta-2-microglobulin) were not affected by these stimulations, whereas the induction of CCL chemokines-2, 4, 8, 20, CXCL chemokines-3, 10, interleukin (IL)-8 (markers of leukocyte accumulation/recruit), granzyme B, perforin 1, tumor necrosis factor superfamily-1, 2, 5, 14, 15, CD16 (markers of cell killing), IL10, transforming growth factor beta 1 (humoral factors of immune suppression), forkhead box P3, CD25, arginase (cellular markers of immune suppression), IL2, IL4, interferon-gamma, IL17 (markers of various subsets of T helper cells), granulocyte-macrophage colony-stimulating factor (marker of antigen presenting cells), immunoglobulin heavy locus (marker of B-cells), vascular endothelial growth factor (marker of angiogenesis), pro-opiomelanocortin (marker of local pain), and CD11a mRNA (marker of leukocyte adherence to endothelium) were identified by these stimulations. The blood volume in this assay was 1.44 ml, and 4 h' incubation in whole blood was physiological. Using triplicate aliquots of whole blood for both stimulant and solvent control, statistical conclusion was drawn for each stimulant for each mRNA. The method introduced in this study will be a new paradigm for clinical cellular immunology.
Mitsuhashi
Masato
M
Hitachi Chemical Research Center, Inc. Irvine, CA, United States. mmitsuhashi@hcrcenter.com
D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis* / Q000276:immunology; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology*; D007962:Leukocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000276:immunology
D000943:Antigens, Differentiation; D016207:Cytokines; D007155:Immunologic Factors; D012333:RNA, Messenger