Introduction The intestinal vascular microcirculation plays a pivotal role in the immune cell dysregulation that drives inflammatory bowel disease. T lymphocyte recruitment, adherence and migration are dependent on the integrin heterodimer α4β7, which binds with high affinity to vascular endothelium expressing MAdCAM-1, prior to lymphocyte diapedesis into the lamina propria. This integrin therefore provides a potential therapeutic target.
Methods Using Cellix® technology, a dynamic in vitro model was created, testing the potential of β7 integrin blockade to impair lymphocyte adhesion to MAdCAM-1 under shear flow. The Cellix® system comprises a microfluidic platform and nano pump, with biochip channels that replicate the shear flow and stress found in post-capillary venules. Peripheral blood mononuclear cells (PBMC) were utilised, along with HuT78 T cells that constitutively express the α4β7 integrin and the chemokine receptor CXCR4, known to be upregulated in inflammatory bowel disease. Biochips were coated for 12 h at 4°C with 10 μg/ml MAdCAM-1 Fc, then blocked with 0.1% BSA for 30 min to prevent non-specific adherence to plastic. Adherence of T cells was quantitatively assessed by microscopy at a physiological flow rate of 1 dyne/cm3. The effect of an anti-human β7 integrin monoclonal antibody (clone Fib504, BD Pharmingen) on lymphocyte adherence was measured. 3 nM of the chemokine CXCL12 (ligand for CXCR4) was added to the system to model the pro-inflammatory environment present in inflammatory bowel disease. Experiments were performed at 37°C.
Results HuT78 lymphocytes and PBMC (5×106/ml) provided consistent adherence to MAdCAM-1 under flow, mean ± SE adherent cells/hpf of 32.8±4.5 and 46.2±3 respectively. Adherence was significantly improved with the addition of 3 nM CXCL12 to 45.7±2.8 (p<0.05) and 78.5±1.5 (p<0.001). Incubating the cells with the Fib504 anti-β7 integrin antibody, led to a significant reduction in adherence of unstimulated cells to 17.8±2 (p<0.001) and 18±3.2 (p<0.0001). This reduction was maintained on stimulation with CXCL12, at 17.3±0.9 (p<0.001), and 5.3±0.9 (p<0.0001).
Conclusion This novel in vitro model, demonstrated significant modulation of α4β7 lymphocyte adhesion to the ligand MAdCAM-1 with an anti-β7 antibody. This highly controlled model system provides a more physiological representation of chemokine driven responsiveness than previously published chemotaxis or adhesion assays, and thus the Cellix® platform may serve as a useful tool for the development and validation of future anti-lymphocyte adhesion therapeutics. This research supports the clinical investigation of therapeutics targeting the β7 subunit or α4β7 heterodimer in this disease setting.
Competing interests None declared.
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