Role of a Peptidase in Phagocyte Chemotaxis (N-Formylmethionyl Peptides/Neutrophils/Macrophages) S
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Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2439-2442, July 1976 Cell Biology Role of a peptidase in phagocyte chemotaxis (N-formylmethionyl peptides/neutrophils/macrophages) S. ASWANIKUMAR*, E. SCHIFFMANN*, B. A. CORCORAN*, AND S. M. WAHLO * Laboratory of Developmental Biology and Anomalies and t Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, Maryland 20014 Communicated by DeWitt Stetten, Jr., April 30,1976 ABSTRACT The potencies of N-formylmethionyl (fMet) Mr. Henry Showell of the University of Connecticut Health peptides as chemotactic agents for phagocytes are related to the Center, Farniington, Conn., under Contract no. NO DE5 2477 rates at which they are hydrolyzed. Furthermore, chloromethyl with the National Institute of Dental Research and the National ketones inhibit chemotaxis as do the products of hydrolysis of fMet peptides. The directed migration of cells in response to Institutes of Health, Bethesda, Md.; all amino acid residues in such peptides is probably brought about by the binding of the these peptides were of the L configuration. L-(1-tosylamido- peptide to a cell receptor with subsequent cleavage by peptidase 2-phenyl)ethylchloromethyl ketone (Tos-PheCH2Cl), N-a- specific for aromatic residues, a process that allows the chemical p-tosyl-L-lysylchloromethyl ketone(Tos-LysCH2CI), and di- gradient to be detected. isopropylfluorophosphate were obtained from Calbiochem, Bethesda, Md., and carbobenzoxylamide-2-phenylalanyl Chemotaxis is the directed movement of cells along an in- chloromethane was obtained from Fox Chemical Co., Tucson, creasing chemical gradient. Recently we have observed that Ariz. The chemotactic complement derivative C5a and bac- certain N-formylmethionyl peptides attract phagocytes (1). We terial factor were prepared as described (8, 9). studied these peptides in an attempt to identify the chemotactic Assay for Chemotaxis. Neutrophils and macrophages were substances produced by bacteria. Since bacteria initiate protein taken from rabbit and guinea pig peritoneal exudates, respec- synthesis with N-formylmethionine, whereas animal cells use tively. Chemotaxis of each cell type was assayed as previously methionine, N-formylmethionyl peptides could be a selective described (10, 11) using a micropore filter to separate the chemical signal that phagocytes use to detect and guide their compartment in which the cells and chemotactic substances approach to bacteria. Our studies established that whereas the were placed. Briefly, in the case of rabbit neutrophils, the cells formylation of the a-amino group of certain methionyl di- were allowed to incubate in the modified Boyden chamber for peptides conferred chemotactic activity upon them, their rel- 2 hr at 370 (95% 02-5%CO2), after which the 5 gm Millipore ative potencies varied significantly depending upon the C- filter through which the cells had migrated were stained with terminal amino acid. It- was also found that the active fMet hematoxylin and the cells on the underside were counted. The peptides competed with CSa, a peptide of about 15,000 daltons results were expressed as average number of cells per 10 fields (2) produced from the C5 component of the complement sys- at a magnification of 800 for triplicate samples. Guinea pig tem, an observation suggesting similar receptors on the cells for macrophages were incubated for 90 min under similar condi- both materials. Since previous studies have indicated that tions in humidified air. The 5 ,um polycarbonate filters were neutrophils have esterase-like activity that may be involved in processed in a comparable manner, and 20 oil immersion fields chemotaxis (3-6), it was of interest to determine whether there were quantitated per filter in triplicate. is a relationship between the chemotactic activities of simple Peptidase Assay. Cells obtained from intraperitoneal lavage peptide attractants, and the rates of their hydrolysis by the re- were freed of erythrocytes by exposure to ACKt (12) lysing sponding cell. If this were shown to be the case, it might indicate buffer for 1 min at 00, and by subsequent washing in Gey's a general requirement for the metabolism of a variety of pep- balanced salt solution with 2% bovine serum albumin. Control tide chemotoxins during their stimulation of cell migration. In experiments showed that this treatment did not alter the che- this report we present evidence for the involvement of peptidase motactic responsiveness of leukocytes, and was necessary to activity in phagocyte chemotaxis. reduce the amount of contaminating peptidases from red blood cells. Two milliliters of cells (11 X 106 cells per ml) were incu- MATERIALS AND METHODS bated in Gey's solution together with peptides (1 mM), and were Commercially obtained dipeptides were formylated as de- shaken gently for 30 min at 37'. The suspension was centri- scribed by Sheehan and Yang (7). Met-Phe, Met-Trp, Met-Pro, fuged, and the supernatant was freed of proteins by dilution fMet, p-tosyl-L-arginine methyl ester (Tos-Arg-OMe), N- with alcohol (90% final concentration vol/vol). The supernatant benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), and diethylpy- was subsequently concentrated to a small volume to enable rocarbonate were obtained from the Sigma Chemical Com- characterization of the reaction products by thin-layer chro- pany, St. Louis, Mo.; Met-Asp, Met-Arg, Phe-Met, and Ala-Met matography on silica gel using n-butanol, acetic acid, and water from Research Plus Labs., Denville, N.J.; Met-Tyr, Met-Leu, (4:1:1 vol/vol/vol) as a mobile phase. The cleaved amino acids Met-Ala, Met-Val, and Met-His from Schwarz/Mann, Oran- were visualized after spraying with the cadmium ninhydrin geburg, N.Y. The peptides fMet-Leu-Phe and fMet-Leu-Arg reagent (13) and, in some cases, with the iodoplatinate reagent were prepared by Dr. Richard Freer, Dr. Elmer L. Becker, and for the detection of thioethers (14). The ninhydrin-treated plates were scanned densitometrically Abbreviations: Tos-Arg-OMe, p-tosyl-L-arginine methyl ester; Bz- in a Zeiss spectrophometric scanner at 550 nm. The absorbance Tyr-OEt, N-benzoyl-L-tyrosine ethyl ester; Tos-LysCH2CI, N-a-p- tosyl-L-lysylchloromethyl ketone; Tos-PheCH2CI, L-(1-tosylamido- t Contains per liter: NH4CL 8.29 g; KHCO3 I g; and disodium EDTA, 2-phenyl)ethylchloromethyl ketone. 37.2 mg. 2439 Downloaded by guest on September 25, 2021 2440 Cell Biology: Aswanikumar et al. Proc. Natl. Acad. Sci. USA 73 (1976) Table 1. Effects of protease inhibitors and substrates on chemotaxis Percent inhibition of chemotaxis to*: Cell Additions to cells C5a t Bacterial factor t 0.01 mM fMet-Phe § Neutrophils 0.1 mM Tos-PheCH2Cl 95 ± 2 100 100 ± 3 0.1 mM Tos-LysCH2Cl 75 ± 5 66 ± 1 12 ± 4 0.1mM Bz-Tyr-OEt 42 ± 5 60 ± 1 99 ± 2 0.lmMTos-Arg-OMe 14±6 0±5 18± 2 Macrophages 0.1 mM Tos-PheCH2Cl 91 ± 5 66 ± 3 91 ± 3 0.1 mM Tos-LysCH'Cl 4 ± 0 0 ± 14 12 ± 4 0.1 mM Bz-Tyr-OEt 98 ± 1 90 ± 1 99 ± 2 0.1 mMTos-Arg-OMe 9± 10 0± 30 18± 2 * Results are given as the average of triplicate samples k SEM. Negative control activity (in absence of attractants) was 3 for neutrophils and 7 for macrophages. t Positive control activity was 25 : 4 for neutrophils and 87 4 4 for macrophages. t Positive control activity was 44 i 11 for neutrophils and 52 + 16 for macrophages. § Positive control activity was 31 + 5 for neutrophils and 85 + 8 for macrophages. of known amounts of peptides and the appropriate amino acids diisopropylfluorophosphate, caused a similar degree of inhi- were measured after chromatography under similar conditions. bition of hydrolysis. Macrophages also cleaved fMet-Leu to Radioactive peptides were incubated with cells as described fMet and Leu (not shown). above, the products were chromatographed in the same man- In Fig. 2 are plotted the minimal effective chemotactic ner, and the chromatograms were scanned for radioactivity concentrations of various fMet peptides against their rates of using a Packard radiochromatogram scanner. cleavage. While an exact correspondence is lacking, in general Assay for Cell Proteolytic Activity. Two milliliters of cells the more chemotactically potent the peptides were, the more (11 X 106 cells per ml) were incubated in phosphate-buffered rapidly they were hydrolyzed. A correlation coefficient of 0.72 saline with either Bz-Tyr-OEt or Tos-Arg-OMe (1 mM) at 370 was calculated for the data. The hydrolysis of nonformylated for 30 min. Substrate hydrolysis was determined essentially as peptides was also studied. Met-Phe was cleaved as rapidly as described by Hummel (15). fMet-Phe, even though it is not chemotactic. Met-Asp was readily cleaved, whereas fMet-Asp was not. Neither of these RESULTS peptides are chemotactic. Apparently the correlation is valid Cleavage of fMet peptides by neutrophils for fMet peptides only. Inhibition of chemotaxis by protease inhibitors and fMet-Phe was degraded at least 30% after incubation with substrates neutrophils to N-formylmethione and phenylalanine (Fig. 1). A similar pattern of cleavage by the cells was seen for other Since the protease inhibitors Tos-PheCH2Gl and Tos-LysCH2Cl peptides such as fMet-Trp, fMet-Leu, and fMet-Tyr. This hy- inhibited hydrolysis of chemotactic peptides by cells, it was also drolysis was inhibited at least 90% when the cells were pre- of interest to determine their effects upon chemotaxis. We treated with chloromethyl ketones, Tos-PheCH2Cl, and Tos- compared the effects of these irreversible inhibitors with dif- LysCH2Cl, inhibitors for chymotryptic and tryptic enzymes, ferent specificities on the chemotactic response of phagocytes respectively (not shown). A serine esterase inhibitor, 2.5 mM to C5a, fMet-Phe, and the bacterial chemotactic factor. The results show (Table 1) that in general the response of each type f-MET-PHE of cell to each of the three attractants was more markedly in- 1E -MET-PHE 5 o20000.2 PHE oU.