A large number of different kinds of substances are reported to be chemotactic for neutrophils (1). The size, complexity, and unknown structure of most of these have precluded any definitive analysis of the structural or molecular basis of their chemotactic activity. Recently, Schiffmann et al. have reported that simple, synthetic N-formyl methionyl peptides are chemotactic for neutrophils and macrophages (2). This has made possible the beginning of a systematic study of the relation of the structure of simple peptides to their chemotactic activity and thus, eventually to directly investigate the primary interaction of chemotactic agents with the neutrophil surface. In addition to their chemotactic activity, substances such as C5a or low molecular weight peptides isolated from Escherichia coli culture filtrates induce lysosomal enzyme release from rabbit or human neutrophils in the presence of cytochalasin B (3, 4) or, when the neutrophils are on a suitable surface, in its absence (5, 6).We have synthesized a series of 24 peptides, all but 2 of them methionyl or Nformyl methionyl derivatives. Most of them are di-and tripeptides, with a few tetrapeptides. We shall show that these substances not only increase random movement but are chemotactic as well, that is, they also induce directed movement. In addition, a systematic study of the relation of structure to activity has demonstrated a highly specific dependence of the activity of these peptides upon their structure. The ability of a given peptide to induce migration strictly correlates with its ability to induce lysosomal enzyme secretion from cytochalasin B-treated cells suggesting that the same primary interaction of peptide and cell initiates both activities. Materials and MethodsRabbit polymorphononuclear leukocytes (neutrophils) were obtained 12-14 h after the intraperitoneal injection of 0.1% glycogen, as described (7). They were washed in Hanks' balanced salt

The transport properties of the rabbit peritoneal polymorphonuclear leukocyte (PMN) plasma membrane to Na § K § and Ca .' § have been characterized. The use of a silicone oil centrifugation technique provided a rapid and reliable method for measuring ion fluxes in these cells. Na § and K + movements across PMN membranes were found to be rapid. The value for the unidirectional steady-state fluxes (in meq/liter cell x min) were of the order of 3.0 for Na § and 7.4 for K § Ouabain inhibited both K § influx and Na § efflux, the latter being also dependent on the presence of extracellular potassium. The rate constant (in min -1) for 45Ca influx was found to be .05 and that for 45Ca efflux .04. The synthetic chemotactic factor formyl-methionyl-leucyl-phenylalanine (FMLP) was found to affect the fluxes of Na § K § and Ca .' § at concentrations as low as 10 -l~ M. FMLP induced a large and rapid increase in the permeability of the PMN plasma membrane to 2ZNa. Smaller and delayed enhancements of 4ZK influx and 22Na efflux were also noted. Some evidence that the latter findings are a consequence of the increased 22Na influx is presented. 4~Ca influx and efflux were also stimulated by FMLP. In the presence of 0.25 mM extracellular calcium, FMLP induced an increase in the steady-state level of cell-associated 45Ca. In the presence of .01 mM extracellular calcium, however, a transient decrease in the steady-state level of cell-associated 45Ca was induced by FMLP. The curves relating the concentration of FMLP to its effects on cation fluxes are very similar to those found for its enhancement of migration.The presence of actin-and myosin-like proteins has been demonstrated in almost all mammalian cells that have been investigated (19, 16) including polymorphonuclear leukocytes (PMN) from various species (33, 28). Various investigators have postulated the participation of these contractile elements in the control of cellular shape change, secretion and movement (11, 1). In 1954, Fukushima et al. (13) suggested that the movement of polymorphonuclear leukocyte cells involved contractile mechanisms similar to those found in muscle cells. Recently, various lines of indirect evidence have been presented in support of this idea. For example, glycerinated rabbit peritoneal PMN have been shown to contract upon addition of ATP in a calcium-dependent manner (15); actin and myosin have been isolated and characterized from leukocytes (33, 28); human neutrophils with 428

Changes in the movements of Na § K § and Ca +2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na § and 45Ca +2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and ~Ca +z in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.

The interaction of chemotactic factors (fMet-Leu-Phe and C5a) with rabbit neutrophils leads to rapid and specific release of membrane calcium, as evidenced by changes in the fluorescence of cell-associated chlorotetracycline. These two structurally different stimuli appear to interact with the same pool of membrane calcium.

We have utilized the fluorescent chelate probe chlorotetracycline to investigate the possible involvement of membrane calcium in the response of rabbit peritoneal neutrophils to chemotactic factors . Two chemotactic factors, the small molecular weight fragment of the fifth component of complement C5a and the synthetic peptide formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe), were tested and found to decrease the fluorescence of cell-associated chlorotetracycline in a manner strongly suggesting stimulus-induced displacement of membrane calcium . The time-course, concentration dependence, and receptor specificity of the calcium redistribution induced by the stimuli are consistent with its early role in the initiation of the various neutrophil functions. F-Met-Leu-Phe and C5a appear to interact with the same pool of membrane calcium and to release it to the cytoplasmic side of the plasma membrane . Intracellular calcium then binds back to the membrane(s) from where it can be displaced by additional stimulation . The release of membrane calcium, experimentally defined here, appears to play a central role in the initiation of the various neutrophil functions .