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 technique for detecting changes at the mRNA level in circulating monocytes and to find suitable "gene markers" for assessing the hemocompatibility of biomaterials. Human blood was recirculated in a modified Chandler Loop formed of test tubes. Five groups of biomaterials were compared. Monocytes were separated by using magnetic beads, and gene expression analyses were performed using RT-PCR. We investigated the mRNA expression of stress proteins (hsp70) and inflammatory markers (IL-1alpha, IL-1beta, IL-6, TNF-alpha). mRNAs for cytokines were highly upregulated in LPS-stimulated monocytes. Hsp70 transcripts were upregulated after heat shock but were not influenced by treatment with LPS. The gene expression of the cytokines was affected by various biomaterials. The intensity of the inflammatory response increased in the following order: heparin coatings (PVC) < uncoated PVC < silicone, polypeptide-coated PVC. No change was seen in the expression of the hsp70 genes. An inhibitory effect of systemic heparin on surface-activated monocytes was observed for the mRNA expression of the cytokines. The recirculation of human blood in an in vitro model in combination with the immunomagnetic separation of monocytes and the Duplex RT-PCR method is a powerful tool for getting reliable results. Our data demonstrate that hsp70 genes cannot be recommended for short-term, biomaterial-induced mRNA expression studies. Conversely, mRNAs for IL-1alpha, IL-1beta, IL-6, and TNF-alpha were expressed in a material-dependent manner. Thus our model provides an effective tool for assessing the hemocompatibility of biomaterials before their clinical application and it also can serve as a safety control during the industrial manufacturing process. This method can be applied to various blood cell populations and numerous gene expression studies and may enable a more fundamental understanding of the biologic processes involved in blood-material interactions.