An objective filter-based, enzymatic method for the in vivo measurement of the migration of human polymorphonuclear leucocytes

Incorporation of control valves into a previously described device enabled us to regulate the formation of 8 suction blisters on the upper surface of the forearm in adult human volunteers. After the removal of the raised epidermis and blister fluid, uniform areas of denuded dermis were obtained by placing hollow adhesive ring reinforcers onto each of the regions of exposed dermis. Single or double nitrocellulose filters were then placed onto each of the areas of moistened, exposed dermis. The chemotactic tripeptide FMLP was incorporated into 1% agarose containing 0.1% bovine serum albumin (BSA) to give a concentration range of 10(-8) M to 10(-6) M FMLP. In control systems the FMLP was omitted. Cylindrical agarose blocks +/- FMLP were then placed onto the filters and encased in individual perspex cups glued firmly onto the skin. The filter(s) and agarose blocks were replaced at 2 h intervals and polymorphonuclear leucocyte (PMNL) migration onto (single filter) and into (double filter) the filters was measured by microscopic enumeration or according to the amount of myeloperoxidase (MPO) and lysozyme in the supernatants of filters immersed in 0.1% Triton-X for 10 min to lyse the PMNL. Microscopic enumeration was found to be unsuitable but the method based on MPO and lysozyme release from filter-associated PMNL was rapid, accurate and reproducible. Detectable PMNL migration (greater than 90%) occurred at 3-4 h and was maximal at 8-10 h. This pattern was observed for both the control and FMLP-containing systems. However, PMNL migration was significantly greater in FMLP-exposed dermis. FMLP at 10(-6) M was found to promote maximal PMNL migration. Significantly greater MPO and lysozyme activities were observed with the double filter system. This method is suitable for the objective quantitation of PMNL migration in vivo.