Attraction of neutrophils (PMN) to a site of inflammation is a result of the integrated response to a variety of inflammatory mediators. Typical chemoattractants of fMLP, interleukin 8 (IL8) and leukotriene B4 (LTB4) have been demonstrated in isolated PMNs to stimulate chemotaxis, cell stiffness and secretory events. Using an in vivo model of chemotaxis, PMNs will chemotax in response to lipopolysacharide (LPS) instillation into the alveolar spaces in rabbit lungs. In similar experiments using fMLP as the chemoattractant, there was no movement of PMNs from the pulmonary vasculature into the alveoli (Control, 0.5 PMNs/μl BAL; LPS, 127.4 PMNs/μl; fMLP, 5.5 PMN/μl). In order to investigate this weak response to the fMLP, we use an isolation procedure that minimized the priming events in rabbit PMNs. PMNs were isolated from peripheral blood of rabbits using NIM2 (Cardinal Associates,Inc) which results in less activation than observed in PMNs isolated using dextran sedimentation. In vitro chemotaxis was measured two ways: visually by the leading front method and fluorometrically using calcein labeled cells. In the leading front technique, rabbit PMNs moved into a filter (3 μm pore, Neuro Probe, Inc.) in response to LTB4 and IL8 (241% and 463% of control), whereas the PMNs were not as responsive to fMLP (123% of control). In the calcein method, the PMNs moved through a filter (5 μm pore, Neuro Probe, Inc.) in response to LTB4 and IL8 (230% and 189% of control), but less so for fMLP (125% of control). As a control, human PMNs used in both the leading front and the calcein techniques moved in response to fMLP, LTB4 and IL8. Deformability of PMNs, isolated with NIM2 from rabbit percheral blood, was determined by forcing them through a 5 μm filter (Poretics Corp.), using back pressure as a measurement of stiffness. PMNs treated with IL8 had a significant increase in back pressure (thus stiffness) over the control PMNs. PMNs pretreated with fMLP did not show a similar increase in stiffness. Human PMNs pretreated with IL8 and fMLP demonstrated increased stiffness over control. The in vitro chemotaxis data in pathogen-free rabbits is supported by similar non-responsiveness by peripheral PMNs in vitro as measured by chemotaxis and deformability.
|State||Published - Mar 20 1998|