A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells

• Published in: Journal of immunological methods 2001-09, Vol.255 (1), p.73-82
• Main Authors: Rainger, G.Ed, Stone, Philip, Morland, Clare M., Nash, Gerard B.
• Format: Article
• Language: English
• Subjects: Arteriosclerosis - etiology; Biological and medical sciences; Blood Circulation; Cell Adhesion; Cells, Cultured; Coculture; Coculture Techniques - instrumentation; Coculture Techniques - methods; Diverse techniques; Endothelial cells; Endothelium, Vascular - cytology; Endothelium, Vascular - physiology; Fibroblasts - cytology; Fibroblasts - physiology; Flow assay; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunobiology; Leukocyte adhesion; Leukocytes - cytology; Leukocytes - physiology; Lymphoid cells: ontogeny, maturation, markers, receptors, circulation and recirculation; Microbiology; Molecular and cellular biology; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - physiology; Phenotype; Smooth muscle cells
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00427-6

Stromal cells may contribute to the inflammatory processes which lead to the recruitment of circulating leukocytes. Here, we describe a multicellular model in which chosen cellular elements of tissue can be cocultured with endothelial cells (EC). Cocultures can be incorporated into a novel parallel plate flow chamber to determine if stromal cells influence the patterns of leukocyte adhesion to the EC. As an example relevant to the pathology of atherosclerosis, EC were cultured with arterial smooth muscle cells (SMC) of the ‘secretory’ phenotype. EC and secretory SMC were cultured on the opposite faces of commercially available porous polyethylene terepthalate (PET) culture inserts, which fitted into a parallel plate flow chamber. Binding of flowing purified lymphocytes, labelled with the fluorochrome calcein-AM, to cocultured EC was assessed by fluorescence microscopy. Lymphocyte adhesion was negligible on unstimulated EC cultured alone or cocultured with SMC. However, when tumour necrosis factor-α (TNF) was added to cocultures, the EC supported greatly increased levels of lymphocyte adhesion compared to TNF-treated EC cultured alone. Additionally, cocultured EC responded to TNF at concentrations far below those at which EC cultured alone responded. This priming was specific in that skin fibroblasts cocultured with EC did not modify lymphocyte adhesion induced by TNF. Thus, we have developed a coculture model to determine the ability of tissue stromal cells to modify leukocyte recruitment. This may have wide applications in the study of the cellular pathology of inflammation by allowing the contribution of the local microenvironment to be assessed.