fMLP FrancËois Boulay*, Marie-JoseÁphe Rabiet and Marianne Tardif Department of Molecular and Structural Biology, DBMS/BBSI UMR 314 CEA-CNRS CEA, 17 Rue des Martyrs, Grenoble, Cedex 9, F 38054, France * corresponding author tel: (33)04-76-88-31-38, fax: (33)04-76-88-51-85, e-mail: email@example.com DOI: 10.1006/rwcy.2000.12003. SUMMARY N-Formylated peptides are potent activators of phagocytic cells such as neutrophils, monocytes, and macrophages. By interacting with a cell surface receptor (FPR) they induce chemotaxis, granule enzyme secretion, and production of toxic oxygen metabolites. The prototypical tripeptide N formyl-Lmethionyl-L-leucyl-L-phenylalanine is referred to as fMLP and is produced by bacteria (Escherichia coli and Staphylococcus aureus) and may also derive from mitochondria. The methyl ester N-formyl-L-Met-LLeu-L-Phe-OMe was crystallized and its structure was determined. The formylated tripeptide motif is the minimal structure for an optimal bioactivity. The bioactivity can be increased by adding amino acids to the tripeptide module. The action of fMLP can be antagonized by various peptide analogs, including Nter-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe-OH peptide (t-BOC-peptide), the tripeptide Met-Leu-Phe with branched carbamates, and cyclic undecapeptide cyclosporin H (CSH). BACKGROUND Discovery The invention of the Boyden chamber in 1962 (Boyden, 1962) allowed the characterization of a set of substances isolated from bacterial culture supernatants that promote the migration of phagocytic cells. Supernatants of Escherichia coli cultures were found to contain N-formylated di- and tripeptides that chemoattract mammalian phagocytes in vitro (Schiffmann et al., 1975a,b). From the systematic analysis of synthetic peptides, the tripeptide Nformyl-L-methionyl-L-leucyl-L-phenylalanine emerged as the shortest module able to activate phagocyte functions with high potency (Showell et al., 1976; Freer et al., 1980, 1982). A few years later, it was shown that this peptide is the most potent neutrophil chemotactic factor produced by Escherichia coli (Marasco et al., 1984). N-Formylated peptides derived from mitochondria were also found (Carp, 1982; Shawar et al., 1995). Alternative names The prototypical tripeptide N-formyl-L-methionyl-L(N-formyl-L-Met-L-Leu-Lleucyl-L-phenylalanine Phe-OH) is currently referred to as fMLP. It is also known as fMLF (using the single-letter codes for amino acids), as N-formyl-Met-Leu-Phe, CHO-MetLeu-Phe, N-formyl-methionyl-leucyl-phenylalanine, or as N-formyl peptide. Structure Structure±activity studies have allowed the determination of the structural requirements for optimal biological activity of the tripeptide: (1) A formyl group is essential on the -amino group of methionine or norleucine in position 1. Removal or replacement of the formyl group by an acetyl or an ethyl group results in a 1000±10,000-fold loss in bioactivity. (2) An aliphatic residue such as leucine, -aminobutyric acid, valine, or norvaline is required in position 2 but a glycine or an alanine results in a dramatic reduction of the biological activity. (3) Position 3 has a strict requirement for a phenylalanine. A high degree of hydrophobicity appears to be a major determinant for optimal biological activity. Thus, the benzyl ester or the benzylamide derivatives 1310 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif of N-formyl-Met-Leu-Phe-OH are about 10-fold more active than the acid form, but the methyl ester (N-formyl-L-Met-L-Leu-L-Phe-OMe) is slightly less active than the parent peptide (reviewed in Ye and Boulay, 1997). Main activities and pathophysiological roles fMLP is a potent activator of phagocytes such as neutrophils, monocytes, and macrophages. By reacting with a receptor on the cell surface of these cells, it induces not only chemotaxis, but also granule enzyme secretion and the production of toxic oxygen metabolites. N-Formylated peptides and the fMLP receptor are thought to be part of a system involved in inflammation and in host defense against invading bacteria. PROTEIN Description of protein The most potent neutrophil chemoattractant recovered from E. coli culture supernatants is the tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Its methyl ester form has a bioactivity similar to that of the acid form. Discussion of crystal structure The crystal structure of the ionic form of N-formylL-Met-L-Leu-L-Phe-OH has not been established but the methyl ester N-formyl-L-Met-L-Leu-L-Phe-OMe has been crystallized and its structure determined (Gavuzzo et al., 1989). The backbone of the methylated peptide is folded at the Leu residue without intramolecular hydrogen bonds and is extended at the two external residues. In the solid state, the Leu sidechain is oriented on the same side as the benzene ring of Phe and on the opposite side of the Met side-chain (Figure 1). Although the folded structure found in the solid state is consistent with molecular modeling studies, suggesting that folded conformations of fMLP may be energetically favored, several lines of evidence indicate that the folded conformation may not be the preferred conformation recognized by the receptor. Early studies have tried to define the nature of the biologically active analogs of the tripeptide N-formylL-Met-L-Leu-L-Phe-OH. From studies using NMR spectroscopy, it was concluded that the backbone Figure 1 Perspective view of N-formyl-L-Met-L-Leu-LPhe-OMe. The structure was drawn with the software INSIGHT using the coordinates determined by Gavuzzo et al. (1989). Hydrogen atoms in the amide bonds are shown in gray; carbon atoms are in green; nitrogen atoms are in blue; oxygen atoms are in red; sulfur is in yellow. (Full colour figure may be viewed online.) fMLP 1311 conformation of fMLP in dimethylsulfoxide solution was an extended and semi-rigid sheet conformation (Becker et al., 1979). However, the preference of the fMLP receptor for a -extended conformation was debated since other studies using circular dichroism and infrared spectroscopy demonstrated that the conformation was solvent-dependent. The role of peptide backbone conformation on the biological activity of the tripeptide fMLP has been examined with structurally constrained analogs. The replacement of Leu by 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 in the methyl ester of fMLP yields an analog with a rigid folded type II turn conformation (N-formyl-L-Met-Ac6c-L-Phe-OMe) (Sukumar et al., 1985), whereas incorporation of dipropylglycine (Dpg) in place of Leu gives an analog (N-formyl-L-Met-Dpg-L-Phe-OMe) adopting an extended sheet like structure with a slight conformational flexibility both in solution and in the solid state (Dentino et al., 1991). N-Formyl-L-Met-Dpg-LPhe-OMe was found to be 8-fold more potent than the methylated parent peptide and 16-fold more potent than N-formyl-L-Met-Ac6c-L-Phe-OMe to induce the release of -glucuronidase from human neutrophils. Thus, it appears that the fMLP receptor has a preference for the extended sheet rather than for the stereochemically constrained type II turn folded analog and the unconstrained parent peptide. CELLULAR SOURCES AND TISSUE EXPRESSION Cellular sources that produce The prototypical N-formyl peptide, CHO-L-Met-LLeu-L-Phe-OH, has been purified from natural sources. It is the major neutrophil chemoattractant produced by Escherichia coli (Marasco et al., 1984). A potent chemoattractant for human monocytes which contains equimolar amount of methionine, leucine, isoleucine, and phenylalanine was also purified from Staphylococcus aureus (Rot et al., 1987). This chemoattractant is likely to begin with a N-formylmethionine since it competes with the prototypical tripeptide for binding to human monocytes. The synthetic N-formyl tetrapeptide N-formyl-L-methionyl-L-isoleucyl-L-phenylalanyl-L-leucine emerged as the most potent activator of human monocytes (Rot et al., 1987). Like the bacteria, the mitochondria present in eukaryotic cells use a N-formylmethionine to initiate the biosynthesis of several proteins of the mitochondrial respiratory chain. Several lines of evidence suggest that mitochondria represent a potential source of chemoattractants. Carp (1982) has shown that disrupted mitochondria are able to chemoattract neutrophils and that the chemotactic activity is mediated by mitochondrial N-formylmethioninecontaining proteins but not by the nonformylated proteins. More recently, it has been found that Nformylated peptides that derive from the murine mitochondrially encoded NADP dehydrogenase subunit 1 are potent activators of the release of elastase from rabbit neutrophils (Shawar et al., 1995). Thus, at sites of inflammation or tissue damage, cells that are disrupted may become potential sources of phagocyte activators through the release of mitochondrial N-formylmethionine-containing proteins. It has been recently shown that the bronchial secretions of cystic fibrosis patients contain chemoattractants which activate neutrophil chemotaxis, most likely via the fMLP receptor since the effect is inhibited by the fMLP antagonist N-ter-butoxycarbonylPhe-Leu-Phe-Leu-Phe-OH peptide (Dayer et al., 1998). However, the nature of the chemotactic factor has not been formally characterized. All these findings do not exclude the possibility that N-formylated peptides are only peptidomimetics for an as yet undiscovered natural agonist, for instance a neuropeptide or a lipid. Of interest is the observation that lipoxin A4 binds with high affinity to a receptor highly homologous to the N-formyl peptide receptor also known as FPRL1 (Fiore et al., 1994). RECEPTOR UTILIZATION N-Formylated peptides mediate their effects through a high-affinity plasma membrane receptor known as FMLP-R and FPR. IN VITRO ACTIVITIES In vitro findings The motif CHO-Met-Leu-Phe-OH is the minimal structure for an optimal bioactivity but longer peptides built on this module also proved to be highly potent and not necessarily dependent on the presence of a formyl group at the N-terminus (Table 1). Recent studies on the structure±activity of various synthetic peptides have indicated that the bioactivity can be increased by adding amino acid residues to the tripeptide module. Positions 4, 5, and 6 can accommodate various residues and substituents. The tetrapeptide 1312 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif Table 1 Affinity and bioactivity of N-formylated peptides and derivatives Peptides Kd (nM)a IC50 (nM)b EC50 (nM)c CHO-Met-Leu-Phe-OH 0.3±1 0.3 Freer et al., 1982 CHO-Met-Leu-Phe-NHBzl 0.9 Freer et al., 1982 CHO-Met-Nva-Phe-OH 2.8 Freer et al., 1982 CHO-Nle-Leu-Phe-OH 3.4 Freer et al., 1982 CHO-Met-Gly-Phe-OH 400 Freer et al., 1982 References CHO-Met-Ile-Phe-Leu-OH 0.05 Rot et al., 1987 CHO-Met-Nle-Leu-Phe-Phe-OH 0.1 Gao et al., 1994 Ac-Met-Nle-Leu-Phe-Phe-OH 0.1 Gao et al., 1994 H-Met-Nle-Leu-Phe-Phe-OH 10 Gao et al., 1994 CHO-Met-Leu-Phe-Lys-OH 10 CHO-Nle-Leu-Phe-Nle-Tyr-Lys-OH " CHO-Met-Leu-Phe-N -(Pep12)-Lys-OH d Freer et al., 1980 1.9 Niedel et al., 1980 1 Boulay et al., 1990b CHO-Met-Leu-Phe-N"-(2-( p-azidosalicylamido)ethyl-1,30 -dithiopropionyl)Lys-OH 0.28 Allen et al., 1986 CHO-Met-p-benzoyl-L-phenylalaninePhe-Tyr-N"-(fluorescein)-Lys-OH 39 Mills et al., 1998 CHO-Met-Leu-Phe-Phe-N"-(fluorescein)-Lys 0.03 Fay et al., 1993 a Dissociation constant measured by direct binding of radiolabeled ligands, for instance CHO-Met-Leu-[3H]Phe-OH. Concentration of peptide required to displace 50% of the specifically bound CHO-Met-Leu-[3H]Phe-OH, or fluorescein-labeled CHO-Met-Leu-Phe-Lys-OH. c Concentration of peptide required to produce 50% of the maximum biological response (lysozyme or -glucuronidase release, or Ca2+ mobilization). d Pep12 refers to the hydrophilic dodecapeptide N-acetyl-SDQALSFLKDYC-OH branched to the cysteine residue via the N" amino group of lysine with m-maleimido-N hydroxysuccinimide ester. b CHO-Met-Leu-Phe-Phe-OH is more active than the parent tripeptide (Freer et al., 1982). Insertion of a norleucine between the methionine and the leucine in the latter tetrapeptide yields a pentapeptide (CHOMet-Nle-Leu-Phe-Phe-OH) with a potency in the subnanomolar range (Gao et al., 1994). In this pentapeptide the N-formylation of methionine is no longer a prerequisite. Its N-acetylated form is as active as the N-formylated counterpart and the unacetylated H-Met-Nle-Leu-Phe-Phe-OH pentapeptide retains a high potency, indicating that the bioactivity is not entirely dependent on the presence of a formyl at the N-terminus but can be modulated by the side-chain of internal amino acid residues. The tetrapeptide CHO-Met-Leu-Phe-Lys-OH, the pentapeptide CHO-Met-Leu-Phe-Phe-Lys-OH, and the hexapeptide CHO-Nle-Leu-Phe-Nle-Tyr-Lys-OH are as active as the prototypical tripeptide fMLP. This feature proved to be very useful for studying FPR because the lysine residue could be derivatized either with a hydrophilic dodecapeptide which renders the entire molecule highly water soluble (Boulay et al., 1990b), with bulky hydrophobic photoactivatable moieties (Niedel et al., 1980; Schmitt et al., 1983; Allen et al., 1986; Boulay et al., 1990a), or with fluorescent chromophores (Fay et al., 1991, 1993) without loss of specificity and biological activity. Regulatory molecules: Inhibitors and enhancers The leukocyte responses to fMLP are modulated by a surface membrane endopeptidase that cleaves the N-formylated peptide. The neutral endopeptidase known as CD10/NEP (EC 22.214.171.124) is a cell surface enzyme that hydrolyzes fMLP and, thereby, reduces its local concentration and availability for receptor binding and signal transduction as illustrated in fMLP 1313 Figure 2 Downregulation of fMLP by CD10/NEP on leukocyte plasma membrane. Table 2 Bioassays used to determine the bioactivity of Nformyl peptides Bioassays Sensitivity to fMLP IC50 (nM) EC50 (nM) Chemotaxis 0.1±1 Enzyme release 0.3 Lysozyme, -glucuronidase, N-acetyl--D-glucosaminidase GTPase activity 5±10 Superoxide production 20±50 Calcium mobilization 1±10 Binding inhibition (CHO-Met-Leu-(3H)Phe-OH) 10±30 Bioassays used Figure 2 (Connelly et al., 1985; Yuli and Lelkes, 1991; Painter and Aiken, 1995). Cell surface CD10/NEP enzymatic activity is increased by fMLP and other inflammatory mediators such as TNF, GM-CSF, and C5a (Shipp et al., 1991; Werfel et al., 1991). Thus, fMLP appears to regulate its proinflammatory potential by controlling its own degradation. More information on CD10/NEP can be obtained in a review by Shipp and Look (1993). In vitro, the action of fMLP can be antagonized by various peptide analogs. For years, the N-ter-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe-OH peptide (t-BOC peptide) was known as the most potent competitive antagonist commercially available. It inhibits lysozyme release with an IC50 in the range of 0.2±0.3 mM (Freer et al., 1980). Specific antagonists with IC50 values ranging between 0.25 and 2 mM were engineered by substituting the N-terminus of the tripeptide Met-Leu-Phe with branched carbamates, such as iso- and ter-butyloxycarbonyl (Derian et al., 1996). Unbranched carbamates, such as methoxycarbonyl and ethoxycarbonyl, resulted in agonist activity. A completely unrelated peptide, the cyclic undecapeptide cyclosporin H (CSH), an analog of cyclosporin A, has been described as a very selective antagonist that is about 5-fold more potent than the t-BOC peptides in inhibiting fMLP-induced cellular responses (Wenzel-Seifert and Seifert, 1993). CSH inhibits fMLP-induced calcium mobilization, superoxide production, and -glucuronidase release in differentiated HL-60 cells with EC50 values of 80 nM, 240 nM, and 450 nM, respectively. The bioactivity of N-formylated peptides can be measured by several means, as summarized in Table 2. Neutrophil or monocyte chemotaxis and granule enzyme release are by far the most sensitive assays to estimate the potency of N-formylated peptides, most likely because the triggering of these two responses requires a low level of receptor occupancy. The release of N-acetyl--D-glucosaminidase from rabbit neutrophils is a particularly reproducible method that is about 100-fold more sensitive than the binding competition assay (Kermode et al., 1988). Superoxide anion release is assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome C at 550 nm (Cohen and Chovaniec, 1978). The main advantage of this assay is its simplicity, but it requires a maximal level of receptor occupancy and it is less sensitive than the release of N-acetyl--D-glucosaminidase. Transient increases of intracellular calcium can be determined after loading FPR-expressing cells with the fluorescent dye Fura 2. As with the superoxide assay, the N-formylpeptide-mediated calcium transients are monitored continuously. The determination of GTPase activity in membrane preparation has been widely used for many agonists. Several variations of the method have recently been described and discussed (Gierschik et al., 1994). Measurement of binding inhibition is a reliable approach which is very useful when the specificity of a new peptide chemoattractant and its relative affinity for FPR have to be determined. However, this approach is inappropriate when minute amounts of peptide chemoattractants are isolated from biological fluids. 1314 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS Normal physiological roles The prevailing model is that N-formylated peptides initiate neutrophil responses to bacterial invasion of the host. At sites of tissue necrosis, N-formyl peptides may be liberated by mitochondria and may trigger the accumulation of phagocytic cells at these sites. Pharmacological effects The effects of the chemotactic factor fMLP have been mainly examined in rabbits. Early studies suggested that inhalation of fMLP by rabbits causes a bronchoconstriction and airway inflammation most likely mediated via the activation of inflammatory cells (Berend et al., 1986; Peters et al., 1991). In guinea pigs, aerosol inhalation or intratracheal injection of fMLP causes a significant infiltration of eosinophils in tracheal mucosa, which is not prevented by PAF antagonists and 5-lipoxygenase inhibitors (Amagai et al., 1992). In rabbit trachea, fMLP has been shown to increase microvascular leakage (Matheson et al., 1997). This effect appears to be mediated by PAF and leukotrienes C4 and D4 produced upon fMLP inhalation. Intravenous administration of fMLP in normal rabbits induces transient and dose-dependent hypotension, neutropenia, and thrombocytopenia (Jonsson et al., 1997). It is unclear, however, whether these effects result simply from neutrophil activation or from the activation of other cell types that express FPR or the FPR homolog FPRL1. Interactions with cytokine network fMLP triggers the production of a variety of proinflammatory cytokines and chemokines, including IL-1, IL-6, GM-CSF, and IL-8. Endogenous inhibitors and enhancers Cytokines such as TNF and GM-CSF render granulocytes more responsive to fMLP, resulting in increased superoxide production in vitro. This phenomenon is known as `priming', but the underlying mechanisms are still unclear (Hallett and Lloyds, 1995). This priming phenomenon is observed in mice challenged with staphylococcal enterotoxin B. The treated animals present an acute inflammatory lung injury, a high level of TNF in serum, and their peripheral blood granulocytes respond with an increased production of toxic oxygen metabolites upon fMLP exposure. This priming effect may increase the tissue-damaging potential of granulocytes when recruited in lungs of staphylococcal enterotoxin B-treated mice (Neumann et al., 1997). References Allen, R. A., Tolley, J. O., and Jesaitis, A. J. (1986). Preparation and properties of an improved photoaffinity ligand for the Nformylpeptide receptor. Biochim. Biophys. Acta 882, 271±280. Amagai, M., Ohashi, Y., and Makino, S. (1992). Eosinophil infiltration and enhancement of airway reactivity by leukocyte chemotactic factor, formyl-methionyl-leucyl-phenylalanine (fMLP), in guinea pigs. Aerugi 41, 1547±1560. Becker, E. L., Bleich, H. E., Day, A. R., Freer, R. J., Glasel, J. A., and Visintainer, J. (1979). Nuclear Magnetic resonance conformation studies on the chemotactic tripeptide formyl-L-methionyl-L-leucyl-L-phenylalanine. A small sheet. Biochemistry 18, 4656±4668. Berend, N., Armour, C. L., and Black, J. L. (1986). Formylmethionyl-leucyl-phenylalanine causes bronchoconstriction in rabbits. Agents Actions 17, 466±471. Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990a). The human N-formylpeptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein-coupled receptors. Biochemistry 29, 11123±11133. Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990b). Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNA. Biochem. Biophys. Res. Commun. 168, 1103±1109. Boyden, S. E. J. (1962). The chemotactic effects of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453±466. Carp, H. (1982). Mitochondrial N-formylmethionyl protein as chemoattractants for neutrophils. J. Exp. Med. 155, 264±275. Cohen, H. J., and Chovaniec, M. E. (1978). Superoxide generation by digitonin-stimulated guinea pig granulocytes. A basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system. J. Clin. Invest. 61, 1081±1087. Connelly, J. C., Skidgel, R. A., Schulz, W. W., Johnson, A. R., and ErdoÈs, E. G. (1985). Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. Proc. Natl Acad. Sci. USA 82, 8737±8741. Dayer, P. D., Schlegel-Haueter, S. E., Belli, D. C., Rochat, T., Dudez, T. S., and Suter, S. (1998). Chemotactic factors in bronchial secretions of cystic fibrosis patients. J. Infect. Dis. 177, 1413±1417. Dentino, A. R., Raj, P. A., Bhandary, K. K., Wilson, M. E., and Levine, M. J. (1991). Role of peptide backbone conformation on biological activity of chemotactic peptides. J. Biol. Chem. 266, 18460±18468. Derian, C. K., Solomon, H. F., Higgins III, J. D., Beblavy, M. J., Santulli, R. J., Bridger, G. J., Pike, M. C., Kroon, D. J., and Fischman, A. J. (1996). Selective inhibition of N-formylpeptideinduced neutrophil activation by carbamate-modified peptide analogues. Biochemistry 35, 1265±1269. Fay, S. P., Posner, R. G., Swann, W. N., and Sklar, L. A. (1991). Realtime analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry. Biochemistry 30, 5066±5075. fMLP 1315 Fay, S. P., Domaleswski, M. D., and Sklar, L. A. (1993). Evidence for protonation in human neutrophil formyl peptide receptor binding pocket. Biochemistry 32, 1627±1631. Fiore, S., Maddox, J. F., Perez, H. D., and Serhan, C. N. (1994). Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor. J. Exp. Med. 180, 253±260. Freer, R. J., Day, A. R., Radding, J. A., Schiffmann, E., Aswanikumar, S., Showell, H. J., and Becker, E. L. (1980). Further studies on the structural requirements for synthetic peptide chemoattractants. Biochemistry 19, 2404±2410. Freer, R. J., Day, A. R., Muthukumaraswamy, N., Pinon, D., Wu, A., Showell, H. J., and Becker, E. L. (1982). Formyl peptide chemoattractants: a model of the receptor on rabbit neutrophils. Biochemistry 21, 257±263. Gao, J. L., Becker, E. L., Freer, R. J., Muthukumaraswamy, N., and Murphy, P. M. (1994). A high potency nonformylated peptide agonist for the phagocyte N-formylpeptide chemotactic receptor. J. Exp. Med. 180, 2191±2197. Gavuzzo, E., Mazza, F., Pochetti, G., and Scatturin, A. (1989). Crystal structure, conformation, and potential energy calculations of the chemotactic peptide N-formyl-L-Met-L-Leu-L-PheOMe. Int. J. Peptide Protein Res. 34, 409±415. Gierschik, P., Bouillon, T., and Jakobs, K. H. (1994). In ``Methods in Enzymology, Vol 237'' (ed R. Iyengar), Receptor-stimulated hydrolysis of guanosine 50 triphosphate in membrane preparation, pp. 13±26. Academic Press, New York. Hallett, M. B., and Lloyds, D. (1995). Neutrophil priming: the cellular signals that say `amber' and not `green'. Immunol. Today 16, 264±268. Jonsson, M., Tzanela, M., Kolbeck, R. C., and McCormick, J. R. (1997). Hemodynamic and metabolic effects of intravenous formyl-methionyl-leucyl-phenylalanine (FMLP) in rabbits. In Vivo 11, 133±139. Kermode, J. C., Muthukumaraswamy, N., and Freer, R. J. (1988). Characteristics of binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanine, to the receptors on rabbit neutrophils. J. Leukocyte Biol. 43, 420±428. Marasco, W. A., Phan, S. H., Krutzsch, H., Showell, H. J., Feltner, D. E., Nairn, R., Becker, E. L., and Ward, P. A. (1984). Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. J. Biol. Chem. 259, 5430±5439. Matheson, M. J., Rynell, A. C., McLean, M. A., and Berend, N. (1997). Role of platelet activating factor, leukotrienes and polymorphs in the FMLP induced increase in vascular leakage in rabbit trachea. Respirology 2, 57±61. Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer, M. S., Dratz, E. A., Sunner, J., and Jesaitis, A. J. (1998). Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectroscopy. J. Biol. Chem. 273, 10428±10435. Neumann, B., Engelhardt, B., Wagner, H., and Holzmann, B. (1997). Induction of acute inflammatory lung injury by staphylococcal enterotoxin B. J. Immunol. 158, 1862±1871. Niedel, J., Davis, J., and Cuatrecasas, P. (1980). Covalent affinity labeling of the formyl peptide chemotactic receptor. J. Biol. Chem. 255, 7063±7066. Painter, R. G., and Aiken, M. L. (1995). Regulation of N-formylmethionyl-leucyl-phenylalanine receptor recycling by surface membrane neutral endopeptidase-mediated degradation of ligand. J. Leukocyte Biol. 58, 468±476. Peters, M. J., Panaretto, K., Breslin, A. B., and Berend, N. (1991). Effects of prolonged inhalation of N-formyl-methionyl-leucylphenylalanine in rabbits. J. Appl. Physiol. 70, 2448±2454. Rot, A., Henderson, L. E., Copeland, T. D., and Leonard, E. J. (1987). A series of six ligands for the human formyl peptide receptor: tetrapeptides with high chemotactic potency and efficacy. Proc. Natl Acad. Sci. USA 84, 7967±7971. Schiffmann, E., Corcoran, B. A., and Wahl, S. (1975a). N-formylmethionyl peptides as chemoattractants for leucocytes. Proc. Natl Acad. Sci. USA 72, 1059±1062. Schiffmann, E., Showell, H. V., Corcoran, B. A., Ward, P. A., Smith, E., and Becker, E. L. (1975b). The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli. J. Immunol. 114, 1831±1837. Schmitt, M., Painter, R. G., Jesaitis, A. J., Preissner, K., Sklar, L. A., and Cochrane, C. G. (1983). Photoaffinity labeling of the N-formyl peptide receptor binding site of intact human polymorphonuclear leukocytes. A label suitable for following the fate of the receptor-ligand complex. J. Biol. Chem. 258, 649± 654. Shawar, S. M., Rich, R. R., and Becker, E. L. (1995). Peptides from the amino-terminus of mouse mitochondrially encoded NADH dehydrogenase subunit 1 are potent chemoattractants. Biochem. Biophys. Res. Commun. 211, 812±188. Shipp, M. A., and Look, A. T. (1993). Hematopoietic differentiation antigens that are membrane-associated enzymes: Cutting is the key. Blood 82, 1052±1070. Shipp, M. A., Stefano, G. B., Switzer, S. N., Griffin, J. D., and Reinherz, E. L. (1991). CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation. Blood 78, 1834±1841. Showell, H. J., Freer, R. J., Zigmond, S. H., Schiffmann, E., Aswanikumar, S., Corcoran, B., and Becker, E. L. (1976). The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J. Exp. Med. 143, 1155±1169. Sukumar, M., Raj, A. P., Balaram, P., and Becker, E. L. (1985). A highly active chemotactic peptide analog incorporating the unusual residue 1-aminocyclohexanecarboxylic acid at position 2. Biochem. Biophys. Res. Commun. 128, 339±344. Wenzel-Seifert, K., and Seifert, R. (1993). Cyclosporin H is a potent and selective formylpeptide receptor antagonist. J. Immunol. 150, 4591±4599. Werfel, T., Sonntag, G., Weber, M. H., and GoÈtze, O. (1991). Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a. J. Immunol. 147, 3909±3914. Ye, R., and Boulay, F. (1997). Structure and function of leukocyte chemoattractant receptors. Adv. Pharmacol. 39, 221±290. Yuli, I., and Lelkes, P. I. (1991). Neutral endopeptidase activity in the interaction of N-formyl-L-methionyl-L-leucyl-L-phenylalanine with human polymorphonuclear leukocytes. Eur. J. Biochem. 201, 421±430. LICENSED PRODUCTS N-Formyl peptides can be purchased from Sigma; Cyclosporin H can be obtained from Novartis Pharmacia AG, Research, CH 4002 Basel, Switzerland (contact Anna Maria Suter, Anna_Maria.Suter@pharma.novartis.com).