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

Thirty small molecular weight peptides related to the chemotactic peptide N-formylmethionylleucylphenylalanine (CHO-Met-Leu-Phe-OH) have been prepared by both solidphase and classical peptide synthesis. Compounds were prepared to investigate the structural requirements in the 1 position (N-formylmethionine) and the 3 position (phenylalanine). Each analogue was tested for its ability to induce lysosomal enzyme release from cytochalasin B treated rabbit polymorphonuclear leukocytes in vitro. In addition, some were also tested for their ability to stimulate neutrophil chemotaxis in vitro and for inhibition of specific binding of a 3H-labeled chemotactic peptide, CHO-Nle-Leu-Phe-OH. The results show that the formyl group of CHO-Met-Leu-Phe-OH is essential for good biological activity since N-acetylation, removal of the a-amino group (Le., desamino), or replacement by an ethyl group results in a drastic loss of chemotactic potency (approximately 5000-fold). In addition, the sulfur-

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The addition of the chemotactic peptide Immunoblottlng using anti-phospholipase A2 antibody, does not change upon stimulation of human neutrophils with GM-CSF, fMet-Leu-Phe, or both. In addition, the band that corresponds to phospholipse A2 is shifted upward in membrane isolated from neutrophils stimulated with fMet-Leu-Phe, _ qg t that the enzyme has been altered, possibly phosphorylated, though not on tyrosine residues. A working hypothess is presented. Briefly, stimulation of human neutrophils with GM-CSF, in the absence of an additiona smulus, increases the tyrosine phosphorylation and activation of a mtogen-activated protein kinse, which in turn phosphorylates and activates cytoplasmic phospholipase A2. In the presence of an increased in llar concentration Of free calcium the phospholipse A2 tralocated to the plasma membrane where its substrate is located. GM-CSF also pontites greatly the fMet-Leu-Phe-induced tyrosine phosphorylation and activation of a mi-activated pn kinase and, snce fMetLen-Phe causes an rcalcium rise, the amount of the phospholipase A2 that a ted with the membrane fractfion.

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