Leucocytes such as neutrophils are attracted by N-formylmethionine, but not by methionine. Di-and tripeptides containing formylmethionine are strong attractants for both neutrophils and macrophages, whereas the corresponding nonacylated compounds are not chemotactic. The formylated peptides may be related to an incompletely characterized chemotactic material normally produced by bacteria which attract the same animal cells.

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

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When rabbit peritoneal leukocytes were treated with chemoattractants such as fMet-Leu-Phe, an apparent decrease of [3Hlmethyl incorporation into the lipid fraction from L4methyl-3Hlmethionine was observed. This decrease was a result of increased degradation of methylated phospholipids, not of decreased synthesis. Chemotactic peptides did not affect the metabolism of the phospholipids in which Imethyl-'4Cjcholine was incorporated. The disappearance of the [3Hlmethyl group was associated with the release of (1-14Cjarachidonic acid from phospholipids prelabeled with these compounds. These findings suggested the activation by chemoattractants of phospholipase A2, an enzyme that removes an unsaturated fatty acid from phospholipids. The order of potency of chemoattractants for the stimulated degradation of phospholipids was in good agreement with that for chemotaxis. Mepacrine (quinacrine) and hydrocortisone inhibited and a phorbol ester enhanced both chemotaxis and phospholipase A2 activity. These results, taken together, suggest close association of the metabolism of methylated phospholipids with chemotaxis in rabbit peritoneal leukocytes.Leukocytes respond to various chemoattractants by interaction with specific receptors on the cell surface (1). The biochemical mechanism by which stimulation of chemotactic receptors leads to directed movement of leukocytes is still poorly understood. Recently, our laboratory showed that the activation of protein carboxy-O-methylase in leukocytes is one of the early events in chemotaxis (2). We have also found that phospholipid methylation alters biomembrane structure and functions (3-5). Because the chemotactically responding cells show a marked polarization and have alterations in properties of membranes, we examined the effect of chemoattractants on phospholipid methylation in leukocyte membranes. Here, we report that chemotactic peptides enhance the degradation of phosphatidylcholine synthesized by the transmethylation but not by the choline pathway(s).METHODS AND MATERIALS Cell Preparations. Rabbit leukocytes were obtained by lavage of the peritoneal cavity of rabbits injected with 150 ml of 0.1% glycogen as described (6). When necessary, the peritoneal exudates were exposed to hypotonic saline (0.2%) in the cold for 30 sec to lyse contaminating erythrocytes and then diluted with an equal volume of 1.6% saline. After collection by centrifugation at 600 X g for 10 min, the cells were suspended inGey's balanced salt solution containing 0.1% bovine serum albumin and 0.01 M Hepes buffer at pH 7.4 (modified Gey's solution), at a concentration of 8-11 X 106 cells per ml. A typical cell preparation contained 90% neutrophils and 10% lymphocytes and macrophages. Chemotaxis was measured in modified Boyden chambers as described (7).Assay of Phospholipid Methylation. The phospholipid methylation was assayed by using intact leukocytes and measuring [3H]methyl group from L-[methyl-3H]methionine incorporated into the phospholipid fraction. The cells were preincubated in a total volum...

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We have studied the events that occur during the development of chemotaxis in HL60, a promyelocytic leukemia cell line that acquires the features of mature neutrophils when exposed to dimethylformamide (DMF). Chemotactic function first appears between 48 and 96 hr of DMF induction and is associated not only with the coincidental development of deformability, spontaneous motility, greatly increased binding of fMet-Leu-Phe, and orientation but also with decreasing cell size and pleomorphism of nuclei. Surface adhesiveness develops earlier (3648 hr) and is coincident with a 10-fold increase in protein synthesis not seen in other DMFinducible cell lines. This burst of protein synthesis precedes the expression of chemotactic function. These studies show that the HL6O cell line can provide a useful model for delineating control mechanisms responsible for the development of complex cellular functions present in differentiated myeloid cells in humans. During their development in the marrow, neutrophils acquire a variety of complex cellular functions such as chemotaxis, phagocytosis, and microbial killing. Little is known about the development of these functions in myeloid precursor cells, largely due to the difficulty in obtaining pure populations of these cells at different stages of maturation. Therefore studies of the functional differentiation have been limited to those individual precursor cells in a mixed cell population that may be recognized histologically (1-4).Recently a human leukemia cell line (HL60) has been established that when exposed to polar compounds is induced to differentiate along recognizable myeloid lines, acquiring both morphologic and functional characteristics of postmitotic neutrophils (5-7). We have used this cell line to study the sequential development of functional attributes intrinsic to neutrophil chemotaxis. Our studies indicate that while the development of chemotactic factor receptors is coincident with the development of full chemotactic responsiveness, the presence of receptors is not sufficient of itself for the chemotactic response. MATERIALS AND METHODSCell Cultures. HL60 cells were kindly provided by R. C. Gallo (National Institutes of Health, Bethesda, MD). HL60 and mouse erythroleukemia cells were grown as described (7,8). Both cell lines were free of mycoplasma. Induction of HL60 cells and 585 cells was performed by inoculating cells in their respective media at a concentration of 5 X 104 cells per ml and following 2 generations (60 hr), dimethylformamide (DMF; Sigma) was added to a final concentration of 120 mM. Leukocyte suspensions were prepared as described (9,10

The potencies of N-formylmethionyl (fMet) peptides as chemotactic agents for phagocytes are related to the rates at which they are hydrolyzed. Furthermore, chloromethyl ketones inhibit chemotaxis as do the products of hydrolysis of fMet peptides. The directed migration of cells in response to such peptides is probably brought about by the binding of the peptide to a cell receptor with subsequent cleavage by peptidase specific for aromatic residues, a process that allows the chemical gradient to be detected.Chemotaxis is the directed movement of cells along an increasing chemical gradient. Recently we have observed that certain N-formylmethionyl peptides attract phagocytes (1). We studied these peptides in an attempt to identify the chemotactic substances produced by bacteria. Since bacteria initiate protein synthesis with N-formylmethionine, whereas animal cells use methionine, N-formylmethionyl peptides could be a selective chemical signal that phagocytes use to detect and guide their approach to bacteria. Our studies established that whereas the formylation of the a-amino group of certain methionyl dipeptides conferred chemotactic activity upon them, their relative potencies varied significantly depending upon the Cterminal amino acid. It-was also found that the active fMet peptides competed with CSa, a peptide of about 15,000 daltons (2) produced from the C5 component of the complement system, an observation suggesting similar receptors on the cells for both materials. Since previous studies have indicated that neutrophils have esterase-like activity that may be involved in chemotaxis (3-6), it was of interest to determine whether there is a relationship between the chemotactic activities of simple peptide attractants, and the rates of their hydrolysis by the responding cell. If this were shown to be the case, it might indicate a general requirement for the metabolism of a variety of peptide chemotoxins during their stimulation of cell migration. In this report we present evidence for the involvement of peptidase activity in phagocyte chemotaxis. MATERIALS AND METHODSCommercially obtained dipeptides were formylated as described by Sheehan and Yang (7). Met-Phe, Met-Trp, Met-Pro, fMet, p-tosyl-L-arginine methyl ester (Tos-Arg-OMe), Nbenzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), and diethylpyrocarbonate were obtained from the Sigma Chemical Company, St (8, 9). Assay for Chemotaxis. Neutrophils and macrophages were taken from rabbit and guinea pig peritoneal exudates, respectively. Chemotaxis of each cell type was assayed as previously described (10, 11) using a micropore filter to separate the compartment in which the cells and chemotactic substances were placed. Briefly, in the case of rabbit neutrophils, the cells were allowed to incubate in the modified Boyden chamber for 2 hr at 370 (95% 02-5%CO2), after which the 5 gm Millipore filter through which the cells had migrated were stained with hematoxylin and the cells on the underside were counted. The results were expressed as average number of cells...

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