Gene monitoring of surface-activated monocytes in circulating whole blood using duplex RT-PCR

Previous studies have shown that blood-contacting materials activate blood monocytes. The aim of this study is to evaluate a highly sensitive techniqu e for detecting changes at the mRNA level in circulating monocytes and to f ind suitable "gene markers" for assessing the hemocompatibility of biomater ials. Human blood was recirculated in a modified Chandler Loop formed of te st tubes. Five groups of biomaterials were compared. Monocytes were separat ed by using magnetic beads, and gene expression analyses were performed usi ng RT-PCR. We investigated the mRNA expression of stress proteins (hsp70) a nd inflammatory markers (IL-1 alpha, IL-1 beta, IL-6, TNF-alpha). mRNAs for cytokines were highly upregulated in LPS-stimulated monocytes. Hsp70 trans cripts were upregulated after heat shock but were not influenced by treatme nt with LPS. The gene expression of the cytokines was affected by various b iomaterials. The intensity of the inflammatory response increased in the fo llowing order: heparin coatings (PVC) < uncoated PVC < silicone, polypeptid e-coated PVC. No change was seen in the expression of the hsp70 genes. An i nhibitory effect of systemic heparin on surface-activated monocytes was obs erved for the mRNA expression of the cytokines. The recirculation of human blood in an in vitro model in combination with the immunomagnetic separatio n of monocytes and the Duplex RT-PCR method is a powerful tool for getting reliable results. Our data demonstrate that hsp70 genes cannot be recommend ed for short-term, biomaterial-induced mRNA expression studies. Conversely, mRNAs for IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha were expressed in a m aterial-dependent manner. Thus om model provides an effective tool for asse ssing the hemocompatibility of biomaterials before their clinical applicati on and it also can serve as a safety control during the industrial manufact uring process. This method can be applied to various blood cell populations and numerous gene expression studies and may enable a more fundamental und erstanding of the biologic processes involved in blood-material interaction s. (C) 2001 John Wiley & Sons, Inc.