Leukocyte sequestration in pulmonary capillaries is a key step in the inflammatory response to lung infection. P-selectin and Inter Cellular Adhesion Molecule-1 (ICAM-1) have well defined roles in leukocyte (White Blood Cell) adhesion in the systemic venules but their role in pulmonary capillaries is still unclear. Studies using transgenic animals and monoclonal antibodies (mAbs) suggest no role for P-selectin in leukocyte adhesion in pulmonary capillaries while subsequent retention and migration following initial mechanical arrest may be mediated by ICAM-1/β 2-integrins. In this study, a novel in vitroassay Micropipette Cell Adhesion Assay(MCAA) was developed which used P-selectin, ICAM-1 or BSA coated pulmonary capillary-sized glass microvessels as an in vitromodel for a pulmonary capillary. Leukocytes were aspirated into these in vitrocapillaries and the pause times and velocities were determined under pressures representative of pulmonary capillaries.
The average pause time and percent arrest for HL-60 cells in the MCAA was significantly larger on P-selectin than BSA. The HL-60 adhesion to similar densities of P-selectin was greater in MCAA than in a Parallel Plate Flow Assay and increased with increasing contact area of the cell. However, this adhesion to P-selectin was hard to block with mAb and anti-adhesion treatments.
Neutrophil velocities in MCAA were significantly lower on P-selectin than BSA and decreased with increasing P-selectin concentration. Pre-treating P-selectin coated microvessels with an anti-P-selectin mAb resulted in a small but significant increase in velocity. Interestingly, presenting the anti-P-selectin mAb in the fluid phase during the assay caused many neutrophils to arrest. Neutrophil velocities in MCAA were not significantly different on ICAM-1 than BSA. Neutrophil velocities in MCAA were not significantly different on P-selectin and ICAM-1 than P-selectin alone.
These results demonstrate that there is no mechanical force to mediate leukocyte arrest in a non-tapering in vitrocapillary. P-selectin at low density mediates leukocyte adhesion in pulmonary capillary geometry which is hard to block with mAbs and anti-adhesion treatments. The larger contact area of leukocytes in capillary geometry results in greater adhesion in capillaries than venules. ICAM-1, expressed alone or with P-selectin, does not mediate adhesion of unstimulated neutrophils in pulmonary capillary geometry.