Sklar, L. A., Jesaitis, A. J., Painter, R. G. & Cochrane, G. C.. (1981) J. Biol Chem. 256, 9909-9914. 3. Freer, R. J., Day, A. R., Radding, J. A., Schiffimann, E., Aswa-.
Proc. Nati Acad. Sci. USA Vol. 78, No. 12, pp. 7540-7544, December 1981
Cell- Biology
Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation (ligand-receptor interaction/cellular adaptation/hapten-antibody interaction/inflammation)
LARRY A. SKLAR, ZENAIDA G. OADES, ALGIRDAS J. JESAITIS, RICHARD G. PAINTER, CHARLES G. COCHRANE
AND
Department of Immunopathology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037
Communicated by Frank J. Dixon, August 10, 1981
ABSTRACT Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide. When antibody is added to a cell suspension that has been stimulated with the fluoresceinated peptide, we observe that: (i) the apparent membrane depolarizaion response monitored by a fluorescent dye can be inhibited only if antibody is added within 30 sec of stimulation; (ii) the superoxide response can be inhibited even if antibody is added more than 1 min after stimulation and decays with an intrinsic halflife of about 12 sec; (iii) responses to a second dose of nonfluoresceinated peptide are enhanced if the antibody is added within 2 min of stimulation by the fluoresceinated peptide. These results suggest that different neutrophil responses depend in individual ways on the time course and extent of ligand binding to its receptor. A comparison of these data with the time course of binding permits an estimate of the number of receptors involved in these responses.
aspects of the ligand-receptor interaction have been quantified (4, 5), but quantitative information about the connection between the ligand-receptor interaction and the subsequent cell responses is not at all complete. It is our intention to examine the temporal relationships between the ligand-receptor interaction and the cell response and adaptation. The approach we use involves derivatizing with fluorescein the chemotactic hexapeptide (CHP) CHO-Nle-Leu-Phe-NleTyr-Lys (Nle, norleucine), developed by Niedel and Cuatrecasas (5, 6). This peptide has been characterized in binding and functional studies (5). The rhodamine derivative has been used in fluorescence microscopic studies of the organization and internalization ofthe chemotactic peptide receptor (6). Antibodies to fluorescein bind the fluoresceinated peptide within seconds and inhibit further cellular stimulation. By examining both the peptide fluorescence and the cellular response under kinetic spectroscopic conditions, we are able to probe a number of aspects of neutrophil response to this peptide. Part of this work has appeared in abstract form (7).
The elucidation of the responses of cells to ligand interactions with cell surface receptors is a central theme in cellular biochemistry. The description of the mechanism of the response should include (i) a quantitative analysis of the ligand-receptor interaction, (ii) an identification ofmolecular structural changes and the biochemical pathways in the responses, and (iii) a description of cellular adaptation to the stimulus. Neutrophils, or polymorphonuclear leukocytes, are key cells in human pathology. They not only act in the host's defense but also participate in the acute phase of the inflammatory process by contributing to tissue injury. Neutrophils possess a number oftypes ofreceptors, specific for different ligands, which enable these cells to migrate into areas of developing inflammation, where they can be stimulated to release toxic agents (1). The cellular responses depend upon both the particular stimulus and the conditions of stimulation. For example, we have shown that the cellular response to chemotactic agents depends not merely on the dose ofthe chemotactic agent but to a considerable extent on the rate at which it is presented to the cells (2). The neutrophil chemotactic peptides have been extensively characterized; they are N-formylated peptides that are most active when the adjacent residues contain hydrophobic amino acids such as methionine, leucine, and norleucine (3). Some
MATERIALS AND METHODS Chemotactic Peptide. CHP was synthesized by Bachem Fine Chemicals (Torrance, CA). Its identity was verified by thinlayer chromatography and amino acid analysis (2). The fluorescein derivative of the hexapeptide (CHP-Fl) was synthesized from fluorescein isothiocyanate isomer I (Molecular Probes, Plano, TX) under the reaction conditions used for preparing the analogous rhodamine derivatives (6). The CHP-F1 was purified by silica gel thin-layer chromatography in 5:3:1 (vol/vol) chloroform/methanol/triethylamine. Two active bands were obtained. The band with Rf= 0.40, because of its higher reactivity with antifluorescein antibodies (see below), was used in all studies. CHP was quantitated on the basis of its tyrosine absorbance in 1 M NaOH, assuming an extinction coefficient at 294 nm of 2330 M-l cm-1 (8). The fluorescein derivative was quantitated on the basis of its absorbance at pH 7.4 in phosphate-buffered saline with 2% bovine serum albumin, assuming an extinction coefficient for covalently bound fluorescein of 60,000 M-l cm-1 at 495 nm (9) and the observed ratio of the 495 nm peak to the shoulder at 450 nm (10). Antifluorescein Antibodies. Antisera were obtained from a rabbit hyperimmunized with the fluorescein isothiocyanate derivative ofovalbumin (ref. 10; a generous gift ofWalter Dandliker) and from rabbits immunized with the fluorescein isothiocyanate derivative of keyhole limpet hemocyanin according to
The publication costs ofthis article were defrayed in part by page charge payment. This article .must therefore be hereby marked "advertisement" in. accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: AntiFI-IgG, antifluorescein.IgG; 'CHO-NlesLeu-Phe, N-formylnorleucylleucylphenylalanine; CHP (chemotactic hexapeptide), CHO-Nle-Leu-Phe-Nle-Tyr-Lys; CHP-FI, CHP-fluorescein. 7540
Cell Biology: Sklar et al.
Proc. Nati Acad. Sci. USA 78 (1981)
the following protocol. The primary immunization (50 ,ug of protein in complete Freund's adjuvant) was followed by boosters (25 ,ug of protein) at 1 and 2 months, and thereafter at 2month intervals. IgG was obtained from the antisera by precipitation with a final concentration of 50% saturated ammonium sulfate, pH 7.0. The precipitate was dissolved and dialyzed against phosphate-buffered saline or, when further purification was desired, against 0.01 M sodium phosphate buffer (pH 8.0) prior to elution from a DE-52 DEAE-cellulose column (Whatman). Antibody fragments were prepared by standard procedures: F(ab')2 was prepared by pepsin digestion and purification on Sephadex G-200; Fab' was obtained by reduction and alkylation of the F(ab')2 (12). Antibody concentrations and affinity were determined according to the method of Portmann et aL (11). Antisera obtained at 2 months yielded antifluorescein
IgG (antiFl-IgG) with affinity
constant K5
109 M',
and at 4 months the K, was -101o M-'. Spectroscopic Measurements. Fluorescence measurements and time-resolved measurements of neutrophil activity were performed on a computer-interfaced SLM 8000 spectrofluorometer as described (2). Cell suspensions are placed in plastic cuvettes and stirred continuously in a thermostatically controlled sample chamber. Data are acquired continuously without interruption during stimulus addition. The mixing time is less than 1 sec (2). Neutrophils. Neutrophils were obtained from human blood by the method of Henson and Oades (13). Miscellaneous Materials. Cytochrome c was obtained from Sigma. The membrane potential-sensitive dye dipropylthiodicarbocyanine was a gift of Alan Waggoner, Amherst College. Keyhole limpet hemocyanin, grade B, was obtained from Calbiochem.
RESULTS Interaction of CHP-FI with Antifluorescein. Free fluorescein is rapidly bound by antifluorescein antibody and its fragments with a second-order rate constant of greater than 108 M'1 sec&, which does not depend on the overall affinity of the antibody. A reduction of the fluorescein fluorescence to 1/10th1/20th is characteristic when fluorescein binds to the antibody (13). We have examined the binding of CHP-F1 by anti-
l011
8.0
i'6.0
fluorescein antibody and its fragments (Fig. 1). We find that with similar concentrations of fluorescein antibody (=10 nM IgG or fragment, hyperimmune sera, or 2-month antisera) the kinetics of quenching of the fluorescence of CHP-F1 and free fluorescein are very similar and the half-time is -1 second. The rapidity of the interaction of the antibody and hapten indicates that the mixing delay in these and subsequent experiments is less than 1 sec. The Complex of Antifluorescein with CHP-Fl Is Nonstimulatory for Neutrophils. The experiments of Table 1 show that, whereas the antibody itself does not interfere with cellular responsiveness, the complex between the fluoresceinated hexapeptide and antifluorescein antibody is nonstimulatory. The experiments measured the increase in the fluorescence of the dye dipropylthiodicarbocyanine, which signals an apparent membrane depolarization when neutrophils are stimulated by chemotactic peptides and other agents (2). The hexapeptide (CHP) and its fluoresceinated derivative (CHP-Fl) have similar abilities to induce dye responses in neutrophils (Table 1A). However, the complex of CHP-Fl with antiFl-IgG is only about 5% as effective in stimulating neutrophils as CHP-Fl alone (Table 1B). AntiFl-IgG alone neither stimulates cells nor inhibits stimulation by the unlabled CHP (Table 1B). When cells are exposed to chemotactic peptide they are desensitized to a secondary exposure (Table 1C); however, cells exposed to the antibody complex are not desensitized (Table 1B). A similar series of experiments measured superoxide generation of neutrophils Table 1. Effect of antiFl-IgG on neutrophil response A. Comparison of CHP-Fl and CHP in depolarization assay*
Response Dose, nM
to CHP, % 2.5 100 1.0 80 0.25. 13 B. Effect of antiFl or complex on response
Response to CHP-Fl, % 118 73 11
Response to antiFl-IgG % 0 0 0
Response 100 sec later to 2.5 -nM CHP, % 99 104 104
4
107
or complex,
First dose 5 nM antiFl-IgG 10 nM antiFl-IgG 20 nM antiFl-IgG
Complex: 5 nM antiFl-IgG-
10'
2.5 nM CHP-Fl
Complex: 10 nM antiFl-IgG-
4.0
2.0
7541
AntiFItIgG
5
10 15 Time (Seconds)
20
FIG. 1. Kinetics of binding of CHP-Fl at 37°C by antifluorescein. The data are displayed as fluorescein fluorescence vs. time. Each sample contained 2.5 nM CHP-Fl in 2 ml of phosphate-buffered saline (pH 7.4, 0.01 M sodium phosphate/ 150 mM NaCl). At 5 sec, a small volume of antibody (-20 pld) was injected into the stirred CHP-Fl solution. The final antibody concentrations were: 12 nM IgG (hyperimmune sera; K. - 1011 M-'); 11 nM IgG (2-month; K. 109 M-1). The numbers in the figure refer to these affinities. Fab' at 15 nM (2-month; K. 109 M-') and 15 nM 4-month Fab' (K. 10's M-1) gave similar results (data not shown). Fluorescence measurements used excitation at 480 nm (16nm slit) and emission at 520 am (16-nm slit). No cells were present.
2.5 nM CHP-Fl 5 114 Complex: 20 nM antiFl-IgG2.5 nM CHP-F1 10 97 C. Normal desensitization of second response without antibody Response 100 sec First dose, Response to later to 2.5 nM CHP-Fl, nM CHP-Fl, % CHP, % 10 115 0 2.5 100 0 1.0 78 22 0.25 13 91 0 0 100 * Resp~onse to 2.5 nM ClIP is defined to be 100%. Response is defined as AI/AI2.5 ,, in which AI is the fluorescence increase observed when cells are stimulated and Al2.5 M is the response to a stimulus dose of 2.5 nM CHP.
(data not shown). We observed that neither the antiFl-IgG nor the complex of antibody with CHP-F1 stimulated the cells. In addition, neither the antibody nor its complex inhibited subsequent stimulation by the unlabeled peptide. Taken together, these results.indicate that the complex, which is not efficient in stimulating the cells, also does not bind efficiently to the cells. Stimulation of Neutrophils with a "Square Pulse" and Its Relationship to Ligand Binding. A square pulse stimulation may be generated as shown in Fig. 2. CHP-Fl is injected into a cell suspension; at a later time, antiFl-IgG is added to the cell suspension. When the fluorescence intensity of the suspension is monitored, a square pulse profile, which reflects the time dependence of the concentration of the CHP-Fl, is apparent. The addition of antibody quenches nearly all of the fluorescence, except for a residual amount (10-20%) that depends upon the length of time between the addition of CHP-Fl and the addition of antibody (data plots with asterisks). When the chemotactic peptide antagonist Boc-Phe-Leu-Phe-Leu-Phe (25 A.M) is included in the cell suspension (data plots without asterisks), the small amount of the residual fluorescence (=5%) is independent of the length of time prior to antibody addition. The time-dependent increase in the residual fluorescence represents binding of the peptide to the cell receptor where it is inaccessible to antibody. This binding is blocked by antagonist as well as by unlabeled CHP (not shown). The amount of residual fluorescence corresponds to 0.05, 0.08, 0.14, 0.20 nM CHP-Fl bound to cellular receptors at 37.5 (not shown), 75, 150, and 300 sec, respectively. Using a value of 50,000 6hemotactic peptide receptors initially available on a neutrophil (4), we calculate from the initial binding rate a second-order association rate constant of =108 MW' min-'. Assuming a minimal equilibrium value of 0.25 nM bound CHP-F1, we calculate a Kd of =2 nM, a value comparable to the values reported for CHP and CHO-Nle-Leu-Phe. Response of Neutrophils to Square Pulse Stimulation. The responses of cells stimulated by 2.5 nM CHP-Fl, to which antiFl was added at various times, are shown in Figs. 3 and 4. We
8.0 0
Proc. Natl. Acad. Sci. USA 78 (1981)
Cell Biology: Sklar et al.
7542
0) ,.)
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1.5 60 90 Time (Seconds) FIG. 3. Effect of antiFI-IgG on the stimulation of neutrophils by CHP-Fl at 37°C as monitored by the membrane potential-sensitive dye dipropylthiodicarbocyanine. The change in dye fluorescence is measured vs. time after stimulus addition. The cells were stimulated at 0 sec. The cell suspension contained 2 x 106 cells per ml and 2 ,uM equilibrated dye in a modified Gey's buffer as described elsewhere (2). The antibody (10 nM hyperimmune IgG; Ka 1011 M-') was added at 0, 10, or 30 sec or not at all. Representative duplicates are shown. Fluorescence was excited at 610 nm and measured through a red glass Coming filter 2-59. -
noted previously that stimulus doses in the range of 2-5 nM CHP produce responses that are near maximal (2). We observe that the magnitude of the fluorescence response in the membrane potential-sensitive dye is reduced to 10% or less when the antiFl-IgG and the CHP-Fl are mixed prior to their addition to the cell suspension (Table 1). The response is also suboptimal when antibody is added within 10 sec; an optimal response results if antibody is added after 30 sec (Fig. 3). In three such experiments using 2.5 nM CHP-Fl we observed that when antibody was present at the time ofstimulus addition (or added simultaneously), responses in the range of 25-40% resulted. When antibody was added 10 sec after stimulus addition, a response in the 60-80% range resulted. After 25 sec, antibody had essentially no effect on the response. These results indicate that the binding that contributes to the magnitude of the depolarization response is complete within 15-30 sec. However, binding sufficient for activation occurs in a time of 2 sec or less (i.e., the time required for antibody present in the cell
6.0
1= L._
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,
0)
0L.
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0.16 0.16 0.12
~2.52. nMn CHP-FIH -
r
50 Sec. 30 Sec
_ 0.08 Time. (Seconds) FIG. 2. Fluorescence vs. time display of a square pulse interaction of CHP-Fl with neutrophils. The cell suspension contained 107 neutrophils per ml in phosphate-buffered saline (as in Fig. 1) at 25°C. Those samples without asterisks contained 25 ,uM Boc-Phe-Leu-PheLeu-Phe (a chemotactic peptide antagonist). At 50 sec, 1 nM CHP-Fl was added. At later times (125, 200, 350 sec), a small volume of antifluorescein Fab' (2-month; K. - 1010 M-') was added to a final concentration of 10 nM. Fluorescence measurements employed cut-off filters in excitation and cut-on filters in emission to minimize the stray light contribution of the cell suspension. Excitation was at 495 nm (slit 16 nm with two magenta and four blue cut-off filters; Optical Coating Laboratory, Santa Rosa, CA); emission filters (four yellow and four green, Optical Coating Laboratory) were used in place of the emission monochromator.
0.04 30
60 90 Time (Seconds)
120
FIG. 4. Effect of antiFl-IgG on the stimulation of neutrophils by CHP-Fl as measured by the generation of superoxide anion. The change in absorbance of cytochrome c is measured vs. time after stim-
ulus addition. Experimental conditions were as in Fig. 3, except that the stimulus was 2.5 nM CHP-F1 and the antibody (10 nM 2-month Fab'; Ka - 109 M-') was either present in the cell suspension or added after 10, 30, or 50 sec. The reduction of cytochrome c (75 PM) was monitored at 550 nm as described (2). Representative duplicates are shown.
Cell Biology: Sklar et al suspension even at time 0 to remove the free stimulus). The time course of the superoxide response is different. The superoxide-generating system can be inhibited if antibody is added after times as long as 1 min or more (Fig. 4), but the rate at which the superoxide-generating system turns off after the addition of antibody is nearly independent of the time of addition. Thus we observe that the linear region for superoxide production in cells inhibited at 10 see extrapolates to about 40 see; cells inhibited at 30 sec continue until 60 sec; cells inhibited at 50 sec continue until 80 sec. An analysis of these data (not shown) indicates that the half-times of the decay of the superoxide-generating system are ""12-15 sec at 370C. An antagonist to chemotactic peptide binding (carbobenzoxyphenylalanylmethionine) produced the same result (data not shown). These results are not a result of a lag in the reduction of cytochrome c by superoxide, which we showed previously to be essentially instantaneous (2). Cellular Adaptation: Neutrophil Response to a Sequence of Square Pulses. The responses ofcells to a sequence of stimulus doses reflects "desensitization," "adaptation," or both. In principle, a sequence of pulses can be generated by using a series of haptenated CHP and noncrossreacting antihapten antibodies. The simplest experiment of this type (two stimulus doses) has four variables: a primary dose of CHP-FI is followed at a given time by antifluorescein; at a later time, the cells are restimulated with a dose of nonhaptenated CHP. The variables are the two concentrations and the two times. The results of such an experiment are shown in Fig. 5. The doses are 2.5 nM CHP-Fl and 2.5 nM CHP. Antibody is added at 25 sec, a time that does not affect the magnitude of the response of the membrane potential-sensitive probe (Fig. 3). The
c
2.5
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2.0
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i' I=
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Time (Seconds) FIG. 5. Effect of antiFl-IgG on the response of neutrophils to a second stimulus dose as monitored by the membrane potential-sensitive dye. The data are displayed as dye fluorescence vs. time after stimu-
by 2.5 nM CHP-FI; the antibody (10 nM 2-month Fab'; K.109 M-1) is added at 25 sec (B). No antibody is added inA. The second stimulus (2.5 nM CHP) is added at 100, 200, 300, or 400 sec. lation
Proc. Nati Acad. Sci. USA 78 (1981)
7543
variable in the experiment is the time at which the CHP is added. In the absence of antibody (Fig. 5A), no second response is observed when the second dose is given at 100 see, but the response grows in an approximately linear fashion if CHP is given at later times. Full recovery is estimated at ""600 sec. When antifluorescein is added after 25 sec, cells respond when the dose of CHP is given at 100 see and recovery is complete by 400 see (Fig. 5B). In similar experiments we observed that the recovery of the superoxide response appears to lag behind the recovery of the depolarization response (data not shown). A parallel investigation of the recovery of the superoxide responses of neutrophils is complicated by the following facts. While the binding ofCHPFl can be interrrupted without influencing the magnitude of the depolarization response, the magnitude of the superoxide response depends upon the extent of stimulus binding. The recovery of the superoxide response thus involves its dependence on both the extent of stimulus binding and the magnitude of the first response. An examination of this recovery is beyond the scope of this paper. The relationship of the time course ofstimulus binding to the magnitude of the second depolarization response can be demonstrated when the second response is observed at a constant time (i.e., 200 see), but stimulus binding is interrupted by antibody at a variable time (data not shown). The magnitude ofthe second responses decrease as the time interval between the first dose and the antibody addition is increased-i. e., as the period of uninterrupted binding lengthens. We find that the binding that occurs over a period of 2-3 min contributes to the "desensitization" of the second response.
DISCUSSION Interaction of Antifluorescein with Fluoresceinated CHONle-Leu-Phe-Nle-Tyr-Lys. Because the binding offluoresceinated peptide by antifluorescein antibody is characterized by a 95% quenching of the fluorescence of fluorescein, conditions are readily identified in which rapid and efficient complex formation is obtained (Fig. 1). With a high-affinity antibody (Ka > 109MI) and the rapid binding rate (kon 108 M ' sec-') of the hapten-antibody interaction it is possible to interrupt binding of the fluoresceinated chemotactic peptide to the cells within seconds. We observe that the preformed complex of CHP-Fl with antiFl-IgG is very inefficient in stimulating cells and does not interfere with subsequent stimulation by CHP (Table 1). These data suggest that the complex does not bind efficiently to the cellular receptor for the chemotactic peptide. Experiments (Fig. 2) in which the residual fluorescence of cell suspensions containing CHP-Fl and antiFI-IgG were analyzed as ligand-receptor binding are consistent with the binding data of other investigators. Sullivan and Zigmond (4) report binding rates for Cho-Nle-Leu-Phe of 2 x 107 M-1 min- at 40C, and rates calculated from data presented by Niedel et al (5) yield a value of 1 x 108 M-1 min1 for CHP at 250C. In each case, binding is partially reversible with half-times for dissociation on the order of several minutes (krff 0.1-0.5 min-). A dissociation rate constant of 0.2 min for CHP-Fl is calculated from the association rate constant (108 M'1 min') and the dissociation constant (2 X 10-9 M) for CHP-Fl. Verification of this rate will be published elsewhere. Structural considerations suggest that antiFl-IgG will not readily bind to CHP-Fl already bound to the receptor. Data of Niedel et aL (S) and Freer et aL (3, 15) suggest that at least 5 amino acids participate in the binding of chemotactic peptide to its surface receptor. The fluorescein rings would protrude at most 15 A from the receptor binding pocket. Because depths of antibody combining sites are esti-
7544
Cell Biology: Sklar et al.
mated to be in the range of 10-13 A (16), only when there is no steric interference from the receptor would it be conceivable that antibody binds the cell-bound CHP-Fl. Experiments using a fluorescence-activated cell sorter demonstrate that the antibody does not bind to the CHP-Fl that is bound to the cell (data not shown). As discussed below, the interpretation of the functional effects of the antibody is not dependent upon whether it binds to the cell. Relationship of the Time Course of Ligand-Receptor Interaction to Cellular Response. Neutrophil response is initiated within 5 sec of the exposure of neutrophils to chemotactic peptide: an apparent depolarization begins in 5 sec and superoxide anion is detected within 8 sec at 37TC (2). Both responses are inhibited when antibody is present at the time of stimulation with CHP-Fl. However, a "depolarization" response is elicited even when the stimulus (2.5 nM CHP-Fl) is removed by antibody within about 2 sec. Assuming a binding rate constant of 108 M'1 min- for the interaction of CHP-Fl with its receptor, only 1-2% of the receptor sites are occupied under these conditions. A maximal dye response for the apparent depolarization is elicited when binding is allowed to proceed beyond 15 sec, at which time roughly 10-20% of the receptor sites are occupied (Fig. 3). In contrast, the full superoxide response is elicited only when binding proceeds for a minute or more (Fig. 4). Thus, it appears that while the superoxide response is initiated by the early binding, continued binding is required to elicit the entire response. When binding is interrupted, the superoxide system turns off at a rate that does not depend on the length of stimulation. This shut-off rate (half-time 15 sec) is apparently intrinsic to the enzyme system and is different from the predicted half-time of ligand-receptor dissociation (3.5 min). Time Course of Cellular Adaptation. We believe that there are at least two components to cellular recovery. Functional recovery could involve resetting a triggering mechanism or replenishing the substrate for an enzyme. Receptor recovery presumably involves both the clearance of occupied receptors and the expression of new or cryptic sites. When 2.5 nM CHP-Fl is administered to cells and followed by 2.5 nM CHP at a later time (Fig. 5A), it appears that the recovery ofboth ofthese components is complete within 10 min, at least for the apparent depolarization response. When antiFl-IgG is added within 25 sec to cells that have been stimulated by CHP-Fl (at a time when the magnitude of the primary response is not diminished by the interruption of binding), the cells can respond to a dose of CHP given at 100 sec and the recovery is complete much earlier than when binding is allowed to proceed in the absence of the antibody. These results imply that the neutrophils begin to be functionally capable of responding within 100 sec and that functional recovery is complete in 400 sec or less. The removal of free ligand is not the primary mechanism by which cell response is enhanced in the presence of antibody because adding antifluorescein antibody just prior to a second dose ofstimulus has no effect on the cell response. Rather, the mechanism involves the inhibition of binding to the cells at short times after exposure of the cells to the stimulus.
Proc. Natl. Acad. Sci. USA 78 (1981)
In conclusion, we find that overlapping time domains describe neutrophil response. Cellular functions are initiated by peptide binding, which occurs over the first few seconds. Whereas less than 30 sec of binding is required for the depolarization response, continued binding over a minute or more is required to elicit the complete superoxide response. While functional recovery begins within 100 sec, the complete recovery depends strongly on the extent of ligand-receptor occupancy: the binding that occurs over several minutes contributes to receptor occupancy, and receptor modulation is not complete for 10 min or more. From fluorescence microscopy (6), it appears that receptor reorganization occurs on a time scale ofseveral minutes, apparently much longer than required for the primary cellular response. Direct binding studies show that receptor modulation occurs on a time scale of 10-20 min (4). Finally, we would like to emphasize that the experimental strategies developed here should prove useful in examining ligand-receptor interaction and cellular response in other systems. There are numerous examples of fluorescein derivatives of peptide and steroid hormones with high-affinity receptors, and we anticipate that these present approaches will be applicable to some of these systems. We thank Mrs. Monica Bartlett for assistance in the preparation of the manuscript. This work was supported in part by National Institutes of Health Grants AI-17354 and HL-16411. R.G.P. is recipient of Research Career Development Award AM-00437 from the National Institutes of Health. This is publication no. 2470 from the Department of Immunopathology. 1. Niedel, J. E. (1980) Curr. Top. CelL Regul 17, 137-170. 2. Sklar, L. A., Jesaitis, A. J., Painter, R. G. & Cochrane, G. C. (1981) J. Biol Chem. 256, 9909-9914. 3. Freer, R. J., Day, A. R., Radding, J. A., Schiffimann, E., Aswanikumar, S., Showell, H. J. & Becker, E. L. (1980) Biochemistry
19, 2404-2410. 4. Sullivan, S. J. & Zigmond, S. H. (1980)]. Cell Biol 85, 703-711. 5. Niedel, J., Wilkinson, S. & Cuatrecasas, P. (1979)J. Biol Chem.
254, 10700-10706. 6. Niedel, J. E., Kahane, I. & Cuatrecasas, P. (1979) Science 205, 1412-1414. 7. Sklar, L. A., Oades, Z. G., Jesaitis, A. J., Painter, R. G. & Cochrane, C. G., (1981) Fed. Proc. Fed. Am. Soc. Exp. Biol. 40, 790
(abstr.).
8. Little, J. R. & Donahue, H. (1968) in Methods in Immunology and Immunochemistry, eds. Williams, C. A. & Chase, M. A. (Academic, New York), Vol. 2, p. 362. 9. Kawamura, A. (1977) Fluorescent Antibody Techniques and Their Applications (University Park, Baltimore, MD), p. 58. 10. Mercola, D. A., Morris, J. W. S. & Arguilla, E. R. (1972) Biochemistry 11, 3860-3874. 11. Levison, S. A., Hicks, A. N., Portmann, A. J. & Dandliker, W. B. (1975) Biochemistry 14, 3778-3786. 12. Nisonoff, A., Wissler, F. C., Lipman, L. N. & Woernly, D. F. (1960) Arch. Biochem. Biophys. 89, 230-244. 13. Portmann, A. J., Levison, S. A. & Dandliker, W. B. (1975) Immunochemistry 12, 461-466. 14. Henson, P. M. & Oades, Z. G. (1975) J. Clin. Invest. 56, 1053-1061. 15. Freer, R. J., Day, A. R., Muthukumaraswamy, N., Pinon, D., Wu, A., Showell, H. J. & Becker, E. L. (1981) Fed. Proc. Fed. Am. Soc. Exp. Biol 40, 790 (abstr.). 16. Wilder, R. L., Green, G. & Shumaker, V. M. (1975) Immunochemistry 12, 49-54.
