MIGRATION-ASSOCIATED VOLUME CHANGES IN NEUTROPHILS FACILITATE THE MIGRATORY PROCESS IN-VITRO

Neutrophil granulocytes, while migrating, undergo substantial shape changes from the round, resting state to a polarized phenotype. In the present study, we monitored cell volume changes in neutrophils migrating toward the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (FMLP) in collagen gels. Neutrophil volume was measured through optical sectioning on a confocal microscope and three-dimensional reconstruction. This method correlated well with Coulter counter volume measurements. Migrating neutrophils displayed a significant volume increase of 35-60%. The cell swelling appeared to depend on sodium/proton antiport activity because it was abrogated by amiloride and dimethyl-amiloride, inhibitors of the antiport, and by substitution of sodium in the buffers by choline, which is not transported by the antiport. Neutrophils exposed to FMLP gradients through impenetrable filters also displayed significant volume changes, whereas cells placed on similar filters in homogeneous concentrations of FMLP did not. This supports the notion of a differential volume response depending on the mode of presentation of the chemotactic agent. The role of volume increase in facilitating neutrophil migration was adduced by two lines of evidence: 1) hyposmolar swelling enhanced and hyperosmolar shrinking decreased neutrophil migration toward FMLP in Boyden chambers and 2) sodium/proton antiport inhibitors decreased neutrophil migration; however, this was reversed in hyposmolar buffers, which induced a volume increase similar to that seen in normal migrating cells. The migration-associated volume increase may be a crucial event for the ability of neutrophils to function as the first line of defense at sites of infection.