Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1204-1208, March 1977
Immunology
Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes (chemotaxis/receptors/polypeptide receptors)
LEWIS T. WILLIAMS*, RALPH SNYDERMANtt, MARILYN C. PIKEt, AND ROBERT J. LEFKOWITZt§ t Departments of Medicine, *
North Carolina 27710
Physiology and Pharmacology, t Microbiology and Immunology, and § Biochemistry, Duke University Medical Center, Durham,
Communicated by James B. Wyngaarden, December 3, 1976
for the chemotactic response. This approach is analogous to the recent use of radioligands to study and isolate a variety of hormone receptors in responsive tissues (4-6). These techniques provide a new approach for probing the molecular mechanism of the initiation of the chemotactic response.
Synthetic N-formylmethionyl peptides are ABSTRACT chemotactic attractants for human polymorphonuclear leukocytes. The well-defined structure-activity relationship of these peptides in eliciting a chemotactic response suggests that the interaction of the peptides with a specific cellular binding site may initiate chemotaxis. By using tritiated N-formylmethionyl-leucyl-phenylalanine (fMet-Leu[:3HHPhe), a potent chemotactic peptide with high specific radioactivity, we have directly identified binding sites on human polymorphonuclear leukocytes. Binding of fMet-Leuu:3H]Phe to polymorphonuclear leukocytes is rapid (t1/2 < 2 min) and reversible. The equilibrium dissociation constant (KD) for the interaction of fMet-Leu[:3H]Phe with the binding site is 12-14 nM at 370. The number of binding sites is approximately 2000 per cell. The specificity of the binding sites for a series of N-formylmethionyl peptides exactly reflects the specificity of the chemotactic response to the peptides in that they compete for the binding sites and initiate chemotaxis with the same order of potency (fMet-Leu-Phe > fMet-Met-Met > fMet-Phe > fMet-Leu > fMet). fPhe-Met is a competitive antagonist of the chemotactic activity of Nformylmethionyl peptides and has a calculated KD of 6 X iO-5 M. fPhe-Met also half-maximally inhibits binding of fMetat a concentration of 9 X 10-5 M. Of several cirLeu4:3HJPhe culating cell types tested, the specific activity of fMet-Leu:3H]Phe binding was the highest in polymorphonuclear leukocytes. No binding of fMet-Leu{:3HJPhe to human erythrocytes could be detected. These data indicate that fMet-Leu{[3H]Phe can be used to identify binding sites for chemotactic peptides on human polymorphonuclear leukocytes. It is likely that these binding sites initiate the specific response of motile cells to N-formylmethionyl peptides.
METHODS Compounds. The compound, tritiated N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe), was chosen as the radioligand for these studies because it is known to be a very potent chemotactic agent (3) and because it could be synthesized with tritium atoms substituted for hydrogen in the phenylalanine residue. fMet-Leu-[3H]Phe (specific activity 40 Ci/mmol) was prepared by Andrulis Research Corp. by coupling N-fMet-Leu to [3H]phenylalanine (specific activity 40 Ci/mmol) (New England Nuclear Co.) by a diazotization reaction. The material was purified on Dowex 50 W-X2 and was 95% pure as assessed by high voltage electrophoresis at pH 6.52 and 8.5. The labeled compound was electrophoretically and biologically indistinguishable from authentic unlabeled fMet-Leu-Phe. The composition of the unlabeled compound (melting point 213-213.5°) was documented by amino acid analysis. fMet-Met-Met, fMet-Phe, fPhe-Met, and fMet-Leu were generously supplied by Dr. Elliott Schiffmann. Cell Preparations. Cell preparations were isolated from heparinized (10 units/ml) peripheral blood (100-200 cm3) of healthy human volunteers. Blood was then centrifuged on Ficoll-Hypaque density gradients using the method of Boyum (7). The pellets, containing erythrocytes and PMN, were diluted 1:2 with normal saline and then mixed with an equal volume of 3% (wt/vol) high-molecular-weight dextran (T250, Pharmacia Fine Chemicals Inc., Piscataway, N.J) in normal saline. The erythrocytes were allowed to sediment for 25 min at 250. The supernatant containing at least 98% PMN was exposed to 0.2% NaCl for 20 sec in order to lyse contaminating red cells (8). After restoration of isotonicity with 1.6% NaCl, the PMN were centrifuged at 350 X g for 10 min and again exposed to 0.2% NaCl. After centrifugation the PMN were washed once in incubation buffer (1.7 mM KH2PO4, 8 mM Na2HPO4, 117 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCl2, pH 7.2) and resuspended in incubation buffer at a density of about 108 cells per ml. The mononuclear cell fraction obtained from the FicollHypaque gradient was washed in 0.02 M phosphate-buffered saline (pH 7.2) containing 0.1% gelatin, 5 mM MgCl2, and 0.15 mM CaCl2 and centrifuged at 400 X g for 15 min at 4°.- This pellet was resuspended in incubation buffer (108 cells per ml) for use in the binding assay. Purified lymphocytes were prepared from the mononuclear cell fraction of the Ficoll-Hypaque gradient by two sequential passages over a nylon mesh
The directed migration of cells along a chemical gradient is termed chemotaxis. The activation of this process appears to *be an important mechanism by which immune effector cells localize at sites of inflammation. Phagocytic cells, such as human polymorphonuclear leukocytes (PMN), chemotactically migrate towards a variety of attractants (1). Schiffman et al. (2, 3) have recently demonstrated that certain N-formylmethionyl peptides, resembling chemotactic factors produced by bacteria, are highly potent chemotactic agents. Although it has been postulated that chemotactic attractants such as these peptides interact specifically with the phagocytic cell surface, little is known about the molecular mechanism by which peptides and other chemotactic agents are recognized by the cell as being attractants. Accordingly, in the present report we have studied the first step in the chemotactic response-the interaction of the chemotactic agent with the responding phagocytic cell. We have used a potent, highly purified, radioactively labeled chemotactic agent, tritiated N-formylmethionyl-leucyl-phenylalanine, to directly label specific PMN receptor sites for chemotactic peptides. By using this radioligand it is possible to directly study the interaction of a variety of peptides with specific cellular receptors which may serve as recognition sites Abbreviation: PMN, polymorphonuclear leukocytes. 1204
Immunology: Williams et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
1205
801
0
clc
i 700
060
E) 60 E
I LA0 I
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[PEPTIDE] (M) Chemotactic activity of fMet-Leu-[3H]Phe and fMet-
10S
LOG
FIG. 1.
Leu-Phe. Isolated PMN were placed in the upper compartment of modified Boyden chambers and the indicated concentrations of fMet-Leu-Phe (* --- *) or fMet-Leu-H[3JPhe (0-0) were added to the bottom compartments of the chambers. Chemotactic activity is expressed as the average number of PMN per high power field migrating completely through the filter in a 3-hr incubation period.
column at 370 at a rate of 1 ml/min (107 cells per ml) in phosphate-buffered saline. This procedure, designed to remove monocytes that adhere to the nylon mesh, resulted in a purified lymphocyte preparation containing 97% small lymphocytes by morphological criteria. fMet-Leu{3HJPhe Binding Assay. fMet-Leu-[3H]Phe (6 nM) and cells (108 cells per ml) were incubated in 150 ,l of incubation buffer for 12 min (unless specified) at 370 with gentle shaking. Incubations were terminated by rapidly diluting a 125-41 incubation aliquot with 2 ml of ice-cold incubation buffer followed by rapid filtration of the mixture through a Whatman GFC glass fiber filter. After the filters were rapidly washed with 10 ml of ice-cold incubation buffer, they were dried and placed into 15 ml of Triton/toluene scintillation mixture; radioactivity was measured in a liquid scintillation spectrophotometer at an efficiency of 40%. Nonspecific binding was defined as the amount Qf binding not inhibited by 10 AM unlabeled fMet-Leu-Phe and was usually equal to about 2530% of the total counts bound. Specific binding was defined as the total amount of fMet-Leu[3H]Phe bound minus the nonspecific binding. Values of binding in all figures and tables refer to specific binding. E
X
500
4 x 1 W0'M fMet-Leu-Phe -
0~~~~~~~~~~~~~ 0j 20-J C.
0
2
4
6
8
10
12 14 16 TIME (MINUTES)
18
20
22
24 26
FIG. 2. Time course of fMet-Leu-[3H]Phe binding to PMN. fMet-Leu-[3H]Phe (6 nM) was incubated with human PMN for the indicated time intervals at 370 and specific binding was assayed (0). To some incubation mixtures (0) a large excess of unlabeled fMetLeu-Phe (4 X lo-5 M) was added after 12 min of incubation-and fMet-Leu-[3H]Phe binding was assayed at subsequent time intervals as indicated. Each value shown represents the mean of determinations from two separate incubation mixtures.
0
10
20 30 fMet-Leu-[3H]Phe (nM)
40
FIG. 3. Binding of fMet-Leu-[3H]Phe to PMN as a function of concentration of fMet-Leu-[3H]Phe. fMet-Leu-[3HJPhe at the indicated concentrations was incubated with human PMN for 12 min at 370 and specific binding was assayed. Each value shown represents the mean of duplicate determinations.
Chemotaxis Assays. Chemotaxis was measured by described methods (8). Briefly, PMN preparations were obtained using the identical methodology described under Cell Preparations, with the exception that the final PMN pellet was resuspended to a concentration of 3.3 X 106 cells per ml in Gey's balanced salt solution (Flowlabs, Rockville, Md.) containing 2% bovalbumin, and 0.01 M N-2-hydroxyethylpiperazine propane sulfonic acid (Hepes), pH 7.2. The cell suspension (0.4 ml) was placed in the upper well of a modified Boyden chamber (9) separated from the chemotactic stimulant in the lower compartment by a 5.0 Asm pore size nitrocellulose filter (Millipore Corp. Bedford, Mass.). After incubation for 3 hr in 370 humidified air, the chambers were emptied and filters were removed, fixed, and stained with hematoxylin. Chemotaxis was quantified by counting and averaging the number of cells that migrated completely through the filter in 10 microscopic fields (X780) with the aid of an eyepiece grid. Chemotactic activity is expressed as the average number of migrating cells per high power field in triplicate samples. RESULTS
Biological activity of fMet-Leu4[3HJPhe fMet-Leu[3H]Phe had biological activity as a chemotactic attractant that was indistinguishable from the biological activity of unlabeled fMet-Leu-Phe (Fig. 1). Kinetics of fMet-Leu4I3H]Phe binding The specific binding of fMet-Leu-[3H]Phe (6 nM) to human PMN was rapid, with a half-time of less than 2 min at 370 (Fig. 2). Binding reached equilibrium within 12 min. The reversibility of fMet-Leu-[3H]Phe binding was tested by adding a large excess (10,uM) of unlabeled fMet-Leu-Phe to an equilibrated mixture of fMet-Leu-[3H]Phe and PMN. As demonstrated in Fig. 2, fMet-Leu-[3H]Phe binding to PMN is readily reversible.
Saturability of fMet-Leu-3H]Phe binding sites The saturability of the PMN binding sites for fMet-Leu-Phe was tested by adding increasing concentrations of fMet-Leu[3H]Phe to the incubations with PMN (Fig. 3). fMet-Leu[3H]Phe binding was saturable approaching a value of 70-80 fmol/mg of protein (Fig. 3). This value corresponds to about
Immunology: Williams et al.
1206
Proc. Natl. Acad. Sci. USA 74 (1977)
0 z
z 0
i
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60 Z
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F= z
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I
-8
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LOG (EC50 BINDING) (M)
FIG. 5. Correlation of affinity for fMet-Leu-[3H]Phe binding sites with chemotactic potency of N-formylmethionyl peptides. The concentrations of each peptide causing half-maximal inhibition of fMet-Leu-[3H]Phe binding (EC5o binding) was computed from data in Fig. 4A. Similarly, the concentration of each peptide giving a half-maximal chemotactic response (EC50 chemotaxis) was computed from Fig. 4B. No values could be computed for the compound fMet because it neither inhibited binding nor stimulated chemotaxis over the concentration range tested.
(U g 100 w
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FIG. 4. Specificity of fMet-Leu-[3H]Phe binding sites towards chemotactically active peptides. (A) Effects of N-formylmethionyl peptides on fMet-Leu-[3H]Phe binding. fMet-Leu-[3H]Phe was incubated with human PMN in the presence of the indicated concentrations of fMet-Leu-Phe (@), fMet-Met-Met (0), fMet-Phe (-), fMet Leu (O), or fMet(*). Specific binding was determined and the percent inhibition of fMet-Leu-[3H]Phe binding caused by each concentration of unlabeled peptide was computed. Each value represents the mean of duplicate determinations from two to four separate experiments. (B) Chemotactic activity of N-formylmethionyl peptides. The chemotactic response was measured for each indicated concentration of fMet-Leu-Phe (0), fMet-Met-Met (0), fMet-Phe (-), fMet-Leu (0), or fMet (*). The maximal response to 10-8 M fMet-Leu-Phe was 102 cells per high power field. Each value represents the mean of triplicate determinations.
2000 binding sites per PMN. Half-maximal saturation occurs at 12-14 nM fMet-Leu-[3H]Phe, providing an estimate of the equilibrium dissociation constant (KD) for the interaction of
fMet-Leu-Phe with the PMN binding sites. Thus, the binding sites for the peptide fMet-Leu-Phe are saturable and of high
affinity. Specificity of fMet-Leu-Phe binding sites If the binding of fMet-Leu-[3H]Phe to PMN is in fact representative of binding to a receptor site that mediates the chemotactic response of PMN to small N-formylated peptides, then the specificity of the chemotactic response to a series of these peptides should be reflected in the specificity of fMet-Leu[3H]Phe binding sites. Accordingly, the relative potencies of several of these peptides as chemotactic agents were compared with their relative abilities to compete for the fMet-Leu[3H]Phe binding sites (Fig. 4). For the binding assays, each of the peptides was added in several concentrations to the incubation mixture of fMet-Leu-[3H]Phe with PMN and the per-
cent of fMet-Leu-[3H]Phe binding inhibited due to the presence of unlabeled peptide was determined. The order of potencies of these peptides in competing for fMet-Leu-[3H]Phe binding sites (Fig. 4A) exactly parallels their orders of potencies as chemotactic agents (Fig. 4B). A plot correlating the concentrations giving half-maximal chemotactic response (EC5o chemotaxis) with the concentration causing half-maximal inhibition of fMet-Leu-[3H]Phe binding (ECso binding) is shown in Fig. 5. The excellent correlation of binding data with chemotactic response data (r = 0.998) indicates that the binding sites labeled by fMet-Leu-[3H]Phe have the specificity expected of receptor sites that mediate the PMN response to chemotactic peptides. C5a, a chemotactic agent that is presumably structurally dissimilar from the small N-formylated peptides, did not compete for fMet-Leu-[3H]Phe binding sites at concentrations 10-fold higher than concentrations that gave a half-maximal chemotactic response. Sodium azide (0.01 M), a compound known to have inhibitory activity for endocytosis in some cell systems, had no effect on binding of fMet-Leu-[3H]Phe. Similarly, tosyl-L-phenylalanyl chloromethane, an irreversible inhibitor of protease activity, did not affect the specific binding of fMet-Leu-[3H]Phe. FPhe-Met, the positional isomer of the chemotactic peptide fMet-Phe, has recently been found to be an antagonist of the chemotactic response to formylmethionyl peptides (10). In order to determine whether this antagonistic effect of fPhe-Met was competitive, we tested the effect of fPhe-Met on the concentration response curve for the chemotactic response to fMet-Leu-Phe (Fig. 6A). Because the presence of 10-4 M fPhe-Met resulted in a 2.6-fold parallel shift in the curve (Fig. 6A), the dissociation constant for fPhe-Met is calculated to be 6 X 10-5 M (see legend to Fig. 6A). In separate experiments it was shown that 10-4 M fPhe-Met has no chemotactic activity of its own. Hence, fPhe-Met is a competitive antagonist of the chemotactic response to peptides and presumably competes with the peptides at the receptor site. This hypothesis was directly tested by binding studies (Fig. 6B) in which it was found that fPhe-Met competes at the fMet-Leu-[3H]Phe binding site with an ECs5 of 9 X 10-5 M, which is in good agreement with the KD calculated from the inhibition of the chemotactic response.
Immunology:
Proc. Natl. Acad. Sci. USA 74 (1977)
Williams et al.
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FIG.
6.
(A) Ability of
fWhe-Met
response to fMet-Leu-Phe. PMN
antagonize the chemotactic placed with medium alone (0)
to
were
with i0~4 M fWhe-Met (0) in the upper compartment of a modified Boyden chamber. The indicated concentrations of fMet-Leu-Phe were placed in the lower compartment and chemotactic activity was meaor
sured. The dissociation constant (K) for the antagonist fPhe-Met was computed from the equation (15): K = [fPhe-Met]/(CR-1), where CR is the ratio of equipotent concentrations of fMet-Leu-Phe in the presence and absence of 1o-4 M fPhe-Met. (B) Competition of fPhe-Met for fMet-Leu-[3H]Phe binding sites. fMet-Leu-[3H]Phe (6 nM) was incubated with human PMN in the presence of the indicated concentrations of fPhe-Met. Specific fMet-Leu-Phe binding was determined; half-maximal inhibition of binding (EC50) was 9 X i0-5 M. Each value represents the mean of duplicate determinations from two separate experiments.
Specificity of cell type for fMet-Leu[3H]Phe binding The reported biological effects of N-formylmethionyl peptides have been limited to chemotactically active cells such as PMN and monocytes. Using fMet-Leu-[3H]Phe, we tested several cell types (PMN, mononuclear cells, and "purified" lymphocytes) for the presence of specific fMet-Leu-[3H]Phe binding sites. Of the cell populations tested, the highest specific activity of binding was found in the PMN cell preparation that was used throughout this study. The mononuclear cell preparation (81% lymphocytes and 19% monocytes) had only 29% (n = 2) as much binding per mg of protein as the PMN preparation. This amount of binding in the mononuclear cell preparation might be largely accounted for by binding to the monocytes present, because the purified lymphocyte preparation only bound 11% as much radioactivity per mg of protein as the PMN preparation. The binding activity in the purified lymphocyte preparation was too low to characterize and could be due to low affinity binding. Erythrocytes had no detectable binding sites for fMet-Leu-[3H]Phe.
1207
DISCUSSION Several N-formylmethionyl peptides are potent chemotactic agents which elicit the migration of PMN with a distinctive pattern of specificity (Fig. 4B, ref. 3). Using fMet-Leu-[3H]Phe, a radioactively labeled peptide with high affinity as a PMN chemotactic agent, we have developed a method for directly identifying a binding site that has the characteristics expected of a specific PMN receptor for chemotactic peptides. The data presented here suggest that this specific binding site is in fact the biologically important receptor site used by PMN in recognizing the N-formylmethionyl chemotactic peptides. As previously suggested (2), because bacteria initiate protein synthesis with N-formylmethionine, N-formylmethionyl peptides could be selectively detected and used to direct the migration of phagocytic cells towards foreign microorganisms. In view of the low concentrations of these peptides that can elicit chemotaxis and in view of the high degree of specificity of the peptides in eliciting the response, it is not surprising that the recognition of peptide involves a specific high affinity binding site on the PMN. The order of potency of peptides in competing for the fMet-Leu-[3H]Phe binding site is identical to the order of potency of these peptides as chemotactic agents for human PMN, and this in turn is identical to the recently reported order of potency of these compounds as chemotactic factors for guinea pig PMN (3). Previous work (3) has demonstrated an exact correlation between the chemotactic activity of these peptides and their ability to induce lysosomal enzyme release. Hence, the binding sites we have identified may mediate formylmethionyl peptide stimulation of lysosomal enzyme release as well as chemotaxis. Substances other than N-formylated peptides have the capacity to elicit a directed migration of PMN. For example, polypeptides derived from complement (11, 12), denatured proteins (1), oxidized fatty acids (13), and other substances (1, 14) have been reported to be chemotactic. In the present study, the finding that C5a did not compete for the fMet-Leu-[3H]Met binding sites suggests that C5a and N-formylmethionyl peptides elicit the chemotactic response through distinct binding sites. Hence, there may be different cellular sites of interaction for different types of chemotactic substances, and it is possible that some substances may not require specific binding sites for their chemotactic activity. It is of interest that the KD values computed from binding data are about 20-fold higher than the concentrations of peptides that give half-maximal chemotactic response. Thus, full occupancy of the receptors is not required for a maximal response. The existence of "spare receptors" in this system may enhance the sensitivity in the detection of very small concentrations of chemotactic substances and may allow the cell to more easily detect a gradient of chemotactic molecules. Although there are approximately 2000 potential receptors on the responding PMN, only 100 to 200 need be occupied in order to obtain a maximal migratory response. This number of receptors (2000) is in the same range as the number of receptors per cell for several hormones (5). Although chemotaxis is a well-documented phenomenon, very little is known about the molecular mechanism of the chemotactic response. The recognition step of this response to agents such as N-formylmethionyl peptides may involve processes analogous to the recognition of specific hormones by hormonally responsive tissues. The radioligand binding techniques described here provide an approach for probing the molecular nature of directed migration of phagocytic cells elicited by formylmethionyl peptides and may be of general
1208
Immunology: Williams et al.
significance in understanding the chemotactic response to a variety of agents. This work was supported by USPHS Grants NIDR 2R01 DE0373804, Allergy Center Grant AI12026, HEW Gratt HL 16037, and by a grant-in-aid from the American Heart Association with funds contributed in part by the North Carolina Heart Association and by NIH Training Grant 5T 05-7M01675 (to L.T.W.). R.S. and R.J.L. are Investigators of the Howard Hughes Medical Institute. 1. Wilkinson, P. C. (1974) Chemotaxis and Inflammation (Churchill Livingston, Edinburgh, London), p. 54. 2. Schiffman, E., Corcoran, B. A. & Wahl, S. M. (1975) Proc. Natl. Acad. Sci. USA 72,1059-1062. 3. Showell, H. J., Freer, R. J., Zigmond, S. H., Schiffman, E., Aswanikumar, S., Corcoran, B. & Becker, E. L. (1976) J. Exp. Med. 143, 1154-1169. 4. Lefkowitz, R. J., Caron, M. G., Limbird, L. E., Mukherjee, C. & Williams, L. T. (1976) in The Enzymes of Biological Membranes, ed. Martonosi, A. (Plenum Press, New York), Vol. 4, pp. 283310.
Proc. Nati. Acad. Sci. USA 74 (1977) 5. Williams, L. T., Snyderman, R. & Lefkowitz, R. J. (1976) J. Clhn. Invest. 57, 149-155. 6. Williams, L. T. & Lefkowitz, R. J. (1976) Science 21, 791793. 7. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21 Suppl. 97, 77-89. 8. Snyderman, R. & Mergenhagen, S. E. (1972) in Biological Activities of Complement, ed. Ingram, D. G. (Karger, Basel), pp. 117-135. 9. Snyderman, R., Pike, M. C., McCarley, D. & Lang, L. (1975) Infect. Immun. 11, 488-492. 10. Aswanikumar, S., Schiffman, E., Corcoran, B. A. & Wahl, S. M. (1976) Proc. Nati. Acad. Scd. USA 73, 2439-2442. 11. Shin, H. S., Snyderman, R., Friedman, E., Mellors, A. & Mayer, M. M. (1968) Science 162,361-363. 12. Snyderman, R., Gewurz, H. & Mergenhagen, S. E. (1968) J. Exp. Med. 128, 259-275. 13. Turner, S. A., Campbell, J. A. & Lynn, W. S. (1976) J. Exp. Med. 141, 1437. 14. Jensen, J. P. & Esquenazi, V. (1975) Nature 256,215-216. 15. Furchgott, R. F. (1967) Ann. N.Y. Acad. Sci. 139,553-570.
Immunology
Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes (chemotaxis/receptors/polypeptide receptors)
LEWIS T. WILLIAMS*, RALPH SNYDERMANtt, MARILYN C. PIKEt, AND ROBERT J. LEFKOWITZt§ t Departments of Medicine, *
North Carolina 27710
Physiology and Pharmacology, t Microbiology and Immunology, and § Biochemistry, Duke University Medical Center, Durham,
Communicated by James B. Wyngaarden, December 3, 1976
for the chemotactic response. This approach is analogous to the recent use of radioligands to study and isolate a variety of hormone receptors in responsive tissues (4-6). These techniques provide a new approach for probing the molecular mechanism of the initiation of the chemotactic response.
Synthetic N-formylmethionyl peptides are ABSTRACT chemotactic attractants for human polymorphonuclear leukocytes. The well-defined structure-activity relationship of these peptides in eliciting a chemotactic response suggests that the interaction of the peptides with a specific cellular binding site may initiate chemotaxis. By using tritiated N-formylmethionyl-leucyl-phenylalanine (fMet-Leu[:3HHPhe), a potent chemotactic peptide with high specific radioactivity, we have directly identified binding sites on human polymorphonuclear leukocytes. Binding of fMet-Leuu:3H]Phe to polymorphonuclear leukocytes is rapid (t1/2 < 2 min) and reversible. The equilibrium dissociation constant (KD) for the interaction of fMet-Leu[:3H]Phe with the binding site is 12-14 nM at 370. The number of binding sites is approximately 2000 per cell. The specificity of the binding sites for a series of N-formylmethionyl peptides exactly reflects the specificity of the chemotactic response to the peptides in that they compete for the binding sites and initiate chemotaxis with the same order of potency (fMet-Leu-Phe > fMet-Met-Met > fMet-Phe > fMet-Leu > fMet). fPhe-Met is a competitive antagonist of the chemotactic activity of Nformylmethionyl peptides and has a calculated KD of 6 X iO-5 M. fPhe-Met also half-maximally inhibits binding of fMetat a concentration of 9 X 10-5 M. Of several cirLeu4:3HJPhe culating cell types tested, the specific activity of fMet-Leu:3H]Phe binding was the highest in polymorphonuclear leukocytes. No binding of fMet-Leu{:3HJPhe to human erythrocytes could be detected. These data indicate that fMet-Leu{[3H]Phe can be used to identify binding sites for chemotactic peptides on human polymorphonuclear leukocytes. It is likely that these binding sites initiate the specific response of motile cells to N-formylmethionyl peptides.
METHODS Compounds. The compound, tritiated N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe), was chosen as the radioligand for these studies because it is known to be a very potent chemotactic agent (3) and because it could be synthesized with tritium atoms substituted for hydrogen in the phenylalanine residue. fMet-Leu-[3H]Phe (specific activity 40 Ci/mmol) was prepared by Andrulis Research Corp. by coupling N-fMet-Leu to [3H]phenylalanine (specific activity 40 Ci/mmol) (New England Nuclear Co.) by a diazotization reaction. The material was purified on Dowex 50 W-X2 and was 95% pure as assessed by high voltage electrophoresis at pH 6.52 and 8.5. The labeled compound was electrophoretically and biologically indistinguishable from authentic unlabeled fMet-Leu-Phe. The composition of the unlabeled compound (melting point 213-213.5°) was documented by amino acid analysis. fMet-Met-Met, fMet-Phe, fPhe-Met, and fMet-Leu were generously supplied by Dr. Elliott Schiffmann. Cell Preparations. Cell preparations were isolated from heparinized (10 units/ml) peripheral blood (100-200 cm3) of healthy human volunteers. Blood was then centrifuged on Ficoll-Hypaque density gradients using the method of Boyum (7). The pellets, containing erythrocytes and PMN, were diluted 1:2 with normal saline and then mixed with an equal volume of 3% (wt/vol) high-molecular-weight dextran (T250, Pharmacia Fine Chemicals Inc., Piscataway, N.J) in normal saline. The erythrocytes were allowed to sediment for 25 min at 250. The supernatant containing at least 98% PMN was exposed to 0.2% NaCl for 20 sec in order to lyse contaminating red cells (8). After restoration of isotonicity with 1.6% NaCl, the PMN were centrifuged at 350 X g for 10 min and again exposed to 0.2% NaCl. After centrifugation the PMN were washed once in incubation buffer (1.7 mM KH2PO4, 8 mM Na2HPO4, 117 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCl2, pH 7.2) and resuspended in incubation buffer at a density of about 108 cells per ml. The mononuclear cell fraction obtained from the FicollHypaque gradient was washed in 0.02 M phosphate-buffered saline (pH 7.2) containing 0.1% gelatin, 5 mM MgCl2, and 0.15 mM CaCl2 and centrifuged at 400 X g for 15 min at 4°.- This pellet was resuspended in incubation buffer (108 cells per ml) for use in the binding assay. Purified lymphocytes were prepared from the mononuclear cell fraction of the Ficoll-Hypaque gradient by two sequential passages over a nylon mesh
The directed migration of cells along a chemical gradient is termed chemotaxis. The activation of this process appears to *be an important mechanism by which immune effector cells localize at sites of inflammation. Phagocytic cells, such as human polymorphonuclear leukocytes (PMN), chemotactically migrate towards a variety of attractants (1). Schiffman et al. (2, 3) have recently demonstrated that certain N-formylmethionyl peptides, resembling chemotactic factors produced by bacteria, are highly potent chemotactic agents. Although it has been postulated that chemotactic attractants such as these peptides interact specifically with the phagocytic cell surface, little is known about the molecular mechanism by which peptides and other chemotactic agents are recognized by the cell as being attractants. Accordingly, in the present report we have studied the first step in the chemotactic response-the interaction of the chemotactic agent with the responding phagocytic cell. We have used a potent, highly purified, radioactively labeled chemotactic agent, tritiated N-formylmethionyl-leucyl-phenylalanine, to directly label specific PMN receptor sites for chemotactic peptides. By using this radioligand it is possible to directly study the interaction of a variety of peptides with specific cellular receptors which may serve as recognition sites Abbreviation: PMN, polymorphonuclear leukocytes. 1204
Immunology: Williams et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
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E) 60 E
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[PEPTIDE] (M) Chemotactic activity of fMet-Leu-[3H]Phe and fMet-
10S
LOG
FIG. 1.
Leu-Phe. Isolated PMN were placed in the upper compartment of modified Boyden chambers and the indicated concentrations of fMet-Leu-Phe (* --- *) or fMet-Leu-H[3JPhe (0-0) were added to the bottom compartments of the chambers. Chemotactic activity is expressed as the average number of PMN per high power field migrating completely through the filter in a 3-hr incubation period.
column at 370 at a rate of 1 ml/min (107 cells per ml) in phosphate-buffered saline. This procedure, designed to remove monocytes that adhere to the nylon mesh, resulted in a purified lymphocyte preparation containing 97% small lymphocytes by morphological criteria. fMet-Leu{3HJPhe Binding Assay. fMet-Leu-[3H]Phe (6 nM) and cells (108 cells per ml) were incubated in 150 ,l of incubation buffer for 12 min (unless specified) at 370 with gentle shaking. Incubations were terminated by rapidly diluting a 125-41 incubation aliquot with 2 ml of ice-cold incubation buffer followed by rapid filtration of the mixture through a Whatman GFC glass fiber filter. After the filters were rapidly washed with 10 ml of ice-cold incubation buffer, they were dried and placed into 15 ml of Triton/toluene scintillation mixture; radioactivity was measured in a liquid scintillation spectrophotometer at an efficiency of 40%. Nonspecific binding was defined as the amount Qf binding not inhibited by 10 AM unlabeled fMet-Leu-Phe and was usually equal to about 2530% of the total counts bound. Specific binding was defined as the total amount of fMet-Leu[3H]Phe bound minus the nonspecific binding. Values of binding in all figures and tables refer to specific binding. E
X
500
4 x 1 W0'M fMet-Leu-Phe -
0~~~~~~~~~~~~~ 0j 20-J C.
0
2
4
6
8
10
12 14 16 TIME (MINUTES)
18
20
22
24 26
FIG. 2. Time course of fMet-Leu-[3H]Phe binding to PMN. fMet-Leu-[3H]Phe (6 nM) was incubated with human PMN for the indicated time intervals at 370 and specific binding was assayed (0). To some incubation mixtures (0) a large excess of unlabeled fMetLeu-Phe (4 X lo-5 M) was added after 12 min of incubation-and fMet-Leu-[3H]Phe binding was assayed at subsequent time intervals as indicated. Each value shown represents the mean of determinations from two separate incubation mixtures.
0
10
20 30 fMet-Leu-[3H]Phe (nM)
40
FIG. 3. Binding of fMet-Leu-[3H]Phe to PMN as a function of concentration of fMet-Leu-[3H]Phe. fMet-Leu-[3HJPhe at the indicated concentrations was incubated with human PMN for 12 min at 370 and specific binding was assayed. Each value shown represents the mean of duplicate determinations.
Chemotaxis Assays. Chemotaxis was measured by described methods (8). Briefly, PMN preparations were obtained using the identical methodology described under Cell Preparations, with the exception that the final PMN pellet was resuspended to a concentration of 3.3 X 106 cells per ml in Gey's balanced salt solution (Flowlabs, Rockville, Md.) containing 2% bovalbumin, and 0.01 M N-2-hydroxyethylpiperazine propane sulfonic acid (Hepes), pH 7.2. The cell suspension (0.4 ml) was placed in the upper well of a modified Boyden chamber (9) separated from the chemotactic stimulant in the lower compartment by a 5.0 Asm pore size nitrocellulose filter (Millipore Corp. Bedford, Mass.). After incubation for 3 hr in 370 humidified air, the chambers were emptied and filters were removed, fixed, and stained with hematoxylin. Chemotaxis was quantified by counting and averaging the number of cells that migrated completely through the filter in 10 microscopic fields (X780) with the aid of an eyepiece grid. Chemotactic activity is expressed as the average number of migrating cells per high power field in triplicate samples. RESULTS
Biological activity of fMet-Leu4[3HJPhe fMet-Leu[3H]Phe had biological activity as a chemotactic attractant that was indistinguishable from the biological activity of unlabeled fMet-Leu-Phe (Fig. 1). Kinetics of fMet-Leu4I3H]Phe binding The specific binding of fMet-Leu-[3H]Phe (6 nM) to human PMN was rapid, with a half-time of less than 2 min at 370 (Fig. 2). Binding reached equilibrium within 12 min. The reversibility of fMet-Leu-[3H]Phe binding was tested by adding a large excess (10,uM) of unlabeled fMet-Leu-Phe to an equilibrated mixture of fMet-Leu-[3H]Phe and PMN. As demonstrated in Fig. 2, fMet-Leu-[3H]Phe binding to PMN is readily reversible.
Saturability of fMet-Leu-3H]Phe binding sites The saturability of the PMN binding sites for fMet-Leu-Phe was tested by adding increasing concentrations of fMet-Leu[3H]Phe to the incubations with PMN (Fig. 3). fMet-Leu[3H]Phe binding was saturable approaching a value of 70-80 fmol/mg of protein (Fig. 3). This value corresponds to about
Immunology: Williams et al.
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Proc. Natl. Acad. Sci. USA 74 (1977)
0 z
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i
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F= z
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FIG. 5. Correlation of affinity for fMet-Leu-[3H]Phe binding sites with chemotactic potency of N-formylmethionyl peptides. The concentrations of each peptide causing half-maximal inhibition of fMet-Leu-[3H]Phe binding (EC5o binding) was computed from data in Fig. 4A. Similarly, the concentration of each peptide giving a half-maximal chemotactic response (EC50 chemotaxis) was computed from Fig. 4B. No values could be computed for the compound fMet because it neither inhibited binding nor stimulated chemotaxis over the concentration range tested.
(U g 100 w
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FIG. 4. Specificity of fMet-Leu-[3H]Phe binding sites towards chemotactically active peptides. (A) Effects of N-formylmethionyl peptides on fMet-Leu-[3H]Phe binding. fMet-Leu-[3H]Phe was incubated with human PMN in the presence of the indicated concentrations of fMet-Leu-Phe (@), fMet-Met-Met (0), fMet-Phe (-), fMet Leu (O), or fMet(*). Specific binding was determined and the percent inhibition of fMet-Leu-[3H]Phe binding caused by each concentration of unlabeled peptide was computed. Each value represents the mean of duplicate determinations from two to four separate experiments. (B) Chemotactic activity of N-formylmethionyl peptides. The chemotactic response was measured for each indicated concentration of fMet-Leu-Phe (0), fMet-Met-Met (0), fMet-Phe (-), fMet-Leu (0), or fMet (*). The maximal response to 10-8 M fMet-Leu-Phe was 102 cells per high power field. Each value represents the mean of triplicate determinations.
2000 binding sites per PMN. Half-maximal saturation occurs at 12-14 nM fMet-Leu-[3H]Phe, providing an estimate of the equilibrium dissociation constant (KD) for the interaction of
fMet-Leu-Phe with the PMN binding sites. Thus, the binding sites for the peptide fMet-Leu-Phe are saturable and of high
affinity. Specificity of fMet-Leu-Phe binding sites If the binding of fMet-Leu-[3H]Phe to PMN is in fact representative of binding to a receptor site that mediates the chemotactic response of PMN to small N-formylated peptides, then the specificity of the chemotactic response to a series of these peptides should be reflected in the specificity of fMet-Leu[3H]Phe binding sites. Accordingly, the relative potencies of several of these peptides as chemotactic agents were compared with their relative abilities to compete for the fMet-Leu[3H]Phe binding sites (Fig. 4). For the binding assays, each of the peptides was added in several concentrations to the incubation mixture of fMet-Leu-[3H]Phe with PMN and the per-
cent of fMet-Leu-[3H]Phe binding inhibited due to the presence of unlabeled peptide was determined. The order of potencies of these peptides in competing for fMet-Leu-[3H]Phe binding sites (Fig. 4A) exactly parallels their orders of potencies as chemotactic agents (Fig. 4B). A plot correlating the concentrations giving half-maximal chemotactic response (EC5o chemotaxis) with the concentration causing half-maximal inhibition of fMet-Leu-[3H]Phe binding (ECso binding) is shown in Fig. 5. The excellent correlation of binding data with chemotactic response data (r = 0.998) indicates that the binding sites labeled by fMet-Leu-[3H]Phe have the specificity expected of receptor sites that mediate the PMN response to chemotactic peptides. C5a, a chemotactic agent that is presumably structurally dissimilar from the small N-formylated peptides, did not compete for fMet-Leu-[3H]Phe binding sites at concentrations 10-fold higher than concentrations that gave a half-maximal chemotactic response. Sodium azide (0.01 M), a compound known to have inhibitory activity for endocytosis in some cell systems, had no effect on binding of fMet-Leu-[3H]Phe. Similarly, tosyl-L-phenylalanyl chloromethane, an irreversible inhibitor of protease activity, did not affect the specific binding of fMet-Leu-[3H]Phe. FPhe-Met, the positional isomer of the chemotactic peptide fMet-Phe, has recently been found to be an antagonist of the chemotactic response to formylmethionyl peptides (10). In order to determine whether this antagonistic effect of fPhe-Met was competitive, we tested the effect of fPhe-Met on the concentration response curve for the chemotactic response to fMet-Leu-Phe (Fig. 6A). Because the presence of 10-4 M fPhe-Met resulted in a 2.6-fold parallel shift in the curve (Fig. 6A), the dissociation constant for fPhe-Met is calculated to be 6 X 10-5 M (see legend to Fig. 6A). In separate experiments it was shown that 10-4 M fPhe-Met has no chemotactic activity of its own. Hence, fPhe-Met is a competitive antagonist of the chemotactic response to peptides and presumably competes with the peptides at the receptor site. This hypothesis was directly tested by binding studies (Fig. 6B) in which it was found that fPhe-Met competes at the fMet-Leu-[3H]Phe binding site with an ECs5 of 9 X 10-5 M, which is in good agreement with the KD calculated from the inhibition of the chemotactic response.
Immunology:
Proc. Natl. Acad. Sci. USA 74 (1977)
Williams et al.
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(A) Ability of
fWhe-Met
response to fMet-Leu-Phe. PMN
antagonize the chemotactic placed with medium alone (0)
to
were
with i0~4 M fWhe-Met (0) in the upper compartment of a modified Boyden chamber. The indicated concentrations of fMet-Leu-Phe were placed in the lower compartment and chemotactic activity was meaor
sured. The dissociation constant (K) for the antagonist fPhe-Met was computed from the equation (15): K = [fPhe-Met]/(CR-1), where CR is the ratio of equipotent concentrations of fMet-Leu-Phe in the presence and absence of 1o-4 M fPhe-Met. (B) Competition of fPhe-Met for fMet-Leu-[3H]Phe binding sites. fMet-Leu-[3H]Phe (6 nM) was incubated with human PMN in the presence of the indicated concentrations of fPhe-Met. Specific fMet-Leu-Phe binding was determined; half-maximal inhibition of binding (EC50) was 9 X i0-5 M. Each value represents the mean of duplicate determinations from two separate experiments.
Specificity of cell type for fMet-Leu[3H]Phe binding The reported biological effects of N-formylmethionyl peptides have been limited to chemotactically active cells such as PMN and monocytes. Using fMet-Leu-[3H]Phe, we tested several cell types (PMN, mononuclear cells, and "purified" lymphocytes) for the presence of specific fMet-Leu-[3H]Phe binding sites. Of the cell populations tested, the highest specific activity of binding was found in the PMN cell preparation that was used throughout this study. The mononuclear cell preparation (81% lymphocytes and 19% monocytes) had only 29% (n = 2) as much binding per mg of protein as the PMN preparation. This amount of binding in the mononuclear cell preparation might be largely accounted for by binding to the monocytes present, because the purified lymphocyte preparation only bound 11% as much radioactivity per mg of protein as the PMN preparation. The binding activity in the purified lymphocyte preparation was too low to characterize and could be due to low affinity binding. Erythrocytes had no detectable binding sites for fMet-Leu-[3H]Phe.
1207
DISCUSSION Several N-formylmethionyl peptides are potent chemotactic agents which elicit the migration of PMN with a distinctive pattern of specificity (Fig. 4B, ref. 3). Using fMet-Leu-[3H]Phe, a radioactively labeled peptide with high affinity as a PMN chemotactic agent, we have developed a method for directly identifying a binding site that has the characteristics expected of a specific PMN receptor for chemotactic peptides. The data presented here suggest that this specific binding site is in fact the biologically important receptor site used by PMN in recognizing the N-formylmethionyl chemotactic peptides. As previously suggested (2), because bacteria initiate protein synthesis with N-formylmethionine, N-formylmethionyl peptides could be selectively detected and used to direct the migration of phagocytic cells towards foreign microorganisms. In view of the low concentrations of these peptides that can elicit chemotaxis and in view of the high degree of specificity of the peptides in eliciting the response, it is not surprising that the recognition of peptide involves a specific high affinity binding site on the PMN. The order of potency of peptides in competing for the fMet-Leu-[3H]Phe binding site is identical to the order of potency of these peptides as chemotactic agents for human PMN, and this in turn is identical to the recently reported order of potency of these compounds as chemotactic factors for guinea pig PMN (3). Previous work (3) has demonstrated an exact correlation between the chemotactic activity of these peptides and their ability to induce lysosomal enzyme release. Hence, the binding sites we have identified may mediate formylmethionyl peptide stimulation of lysosomal enzyme release as well as chemotaxis. Substances other than N-formylated peptides have the capacity to elicit a directed migration of PMN. For example, polypeptides derived from complement (11, 12), denatured proteins (1), oxidized fatty acids (13), and other substances (1, 14) have been reported to be chemotactic. In the present study, the finding that C5a did not compete for the fMet-Leu-[3H]Met binding sites suggests that C5a and N-formylmethionyl peptides elicit the chemotactic response through distinct binding sites. Hence, there may be different cellular sites of interaction for different types of chemotactic substances, and it is possible that some substances may not require specific binding sites for their chemotactic activity. It is of interest that the KD values computed from binding data are about 20-fold higher than the concentrations of peptides that give half-maximal chemotactic response. Thus, full occupancy of the receptors is not required for a maximal response. The existence of "spare receptors" in this system may enhance the sensitivity in the detection of very small concentrations of chemotactic substances and may allow the cell to more easily detect a gradient of chemotactic molecules. Although there are approximately 2000 potential receptors on the responding PMN, only 100 to 200 need be occupied in order to obtain a maximal migratory response. This number of receptors (2000) is in the same range as the number of receptors per cell for several hormones (5). Although chemotaxis is a well-documented phenomenon, very little is known about the molecular mechanism of the chemotactic response. The recognition step of this response to agents such as N-formylmethionyl peptides may involve processes analogous to the recognition of specific hormones by hormonally responsive tissues. The radioligand binding techniques described here provide an approach for probing the molecular nature of directed migration of phagocytic cells elicited by formylmethionyl peptides and may be of general
1208
Immunology: Williams et al.
significance in understanding the chemotactic response to a variety of agents. This work was supported by USPHS Grants NIDR 2R01 DE0373804, Allergy Center Grant AI12026, HEW Gratt HL 16037, and by a grant-in-aid from the American Heart Association with funds contributed in part by the North Carolina Heart Association and by NIH Training Grant 5T 05-7M01675 (to L.T.W.). R.S. and R.J.L. are Investigators of the Howard Hughes Medical Institute. 1. Wilkinson, P. C. (1974) Chemotaxis and Inflammation (Churchill Livingston, Edinburgh, London), p. 54. 2. Schiffman, E., Corcoran, B. A. & Wahl, S. M. (1975) Proc. Natl. Acad. Sci. USA 72,1059-1062. 3. Showell, H. J., Freer, R. J., Zigmond, S. H., Schiffman, E., Aswanikumar, S., Corcoran, B. & Becker, E. L. (1976) J. Exp. Med. 143, 1154-1169. 4. Lefkowitz, R. J., Caron, M. G., Limbird, L. E., Mukherjee, C. & Williams, L. T. (1976) in The Enzymes of Biological Membranes, ed. Martonosi, A. (Plenum Press, New York), Vol. 4, pp. 283310.
Proc. Nati. Acad. Sci. USA 74 (1977) 5. Williams, L. T., Snyderman, R. & Lefkowitz, R. J. (1976) J. Clhn. Invest. 57, 149-155. 6. Williams, L. T. & Lefkowitz, R. J. (1976) Science 21, 791793. 7. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21 Suppl. 97, 77-89. 8. Snyderman, R. & Mergenhagen, S. E. (1972) in Biological Activities of Complement, ed. Ingram, D. G. (Karger, Basel), pp. 117-135. 9. Snyderman, R., Pike, M. C., McCarley, D. & Lang, L. (1975) Infect. Immun. 11, 488-492. 10. Aswanikumar, S., Schiffman, E., Corcoran, B. A. & Wahl, S. M. (1976) Proc. Nati. Acad. Scd. USA 73, 2439-2442. 11. Shin, H. S., Snyderman, R., Friedman, E., Mellors, A. & Mayer, M. M. (1968) Science 162,361-363. 12. Snyderman, R., Gewurz, H. & Mergenhagen, S. E. (1968) J. Exp. Med. 128, 259-275. 13. Turner, S. A., Campbell, J. A. & Lynn, W. S. (1976) J. Exp. Med. 141, 1437. 14. Jensen, J. P. & Esquenazi, V. (1975) Nature 256,215-216. 15. Furchgott, R. F. (1967) Ann. N.Y. Acad. Sci. 139,553-570.
